Preface

Preface

Upon the death of Francis Maitland Balfour, a desire very naturally arose among his friends and admirers to provide some memorial of him. And, at a public meeting held at Cambridge in October 1882, the Vice-Chancellor presiding, and many distinguished men of science being present, it was decided to establish a 'Balfour Fund' the proceeds of which should be applied: firstly to maintain a studentship, the holder of which should devote himself to original research in Biology, especially in Animal Morphology, and secondly, 'by occasional grants of money, to further in other ways original research in the same subject'. The sum of £8446 was subsequently raised; this was, under certain conditions, entrusted to and accepted by the University of Cambridge; and the first 'Balfour student' was appointed in October 1883.

The publication of Balfour's works in a collected form was not proposed as an object on which part of the fund should be expended, since his family had expressed their wish to take upon themselves the charge of arranging for a memorial edition of their brother's scientific writings. That edition, with no more delay than circumstances have rendered necessary, is now laid before the public. It comprises four volumes.

The first volume contains, in chronological order, all Balfour's scattered original papers, including those published by him in conjunction with his pupils, as well as the Monograph on the Elasmobranch Fishes. The last memoir in the volume, that on the Anatomy and Development of Peripatus Capensis, was published after his death, from his notes and drawings, with additions by Prof. Moseley and Mr Adam Sedgwick, who prepared the manuscript for publication. To the volume is prefixed an introductory biographical notice.

The second and third volumes are the two volumes of the Comparative Embryology reprinted from the original edition without alteration, save the correction of obvious misprints and omissions.

The fourth volume contains the plates illustrating the memoirs contained in Vol. 1. We believe that we are consulting the convenience of readers in adopting this plan, rather than in distributing the plates among the memoirs to which they belong. To assist the reader the explanations of these plates have been given twice: at the end of the memoir to which they belong (in the case of the Monograph on Elasmobranch Fishes at the end of each separate chapter), and in the volume of plates.

All the figures of these plates had to be redrawn on the stone, and our best thanks are due to the Cambridge Scientific Instrument Company for the pains which they have taken in executing this work. We are also indebted to the Committee of Publication of the Zoological Society for the gift of electrotypes of the woodcuts illustrating memoir no. XX. of Vol. 1.

Several photographs of Balfour, taken at different times of his life, the last shortly before his death, are in the possession of his relatives and friends; but these, in the opinion of many, leave much to be desired.

There is also a portrait of him in oils painted since his death by Mr John Collier, A.R.A., and Herr Hildebrand of Florence has executed a posthumous bust in bronze[1]. The portrait which forms the frontispiece of Vol. 1. has been drawn on stone by Mr E. Wilson of the Cambridge Scientific Instrument Company, after the latest photograph. Should it fail, in the eyes of those who knew Balfour well, to have reproduced with complete success his features and expression, we would venture to ask them to bear in mind the acknowledged difficulties of posthumous portraiture.

Footnote 1: In possession of the family. Copies also exist in the Library of Trinity College, and in the Morphological Laboratory, at Cambridge.

I. On some points in the Geology of the East Lothian Coast. By G. W. and F. M. BALFOUR 25

1873

II. The development and growth of the layers of the blastoderm. With Plate 1 29

III. On the disappearance of the Primitive Groove in the Embryo Chick. With Plate 1 41

IV. The development of the blood-vessels of the Chick. With Plate 2 47

1874

V. A preliminary account of the development of the Elasmobranch Fishes. With Plates 3 and 4 60

1875

VI. A comparison of the early stages in the development of Vertebrates. With Plate 5 112

VII. On the origin and history of the urinogenital organs of Vertebrates 135

VIII. On the development of the spinal nerves in Elasmobranch Fishes. With Plates 22 and 23 168

1876

IX. On the spinal nerves of Amphioxus 197

1876-78

X. A Monograph on the development of Elasmobranch Fishes. With Plates 6-21 203

1878

XI. On the phenomena accompanying the maturation and impregnation of the ovum 521

XII. On the structure and development of the vertebrate ovary. With Plates 24, 25, 26 549

1879

XIII. On the existence of a Head-kidney in the Embryo Chick, and on certain points in the development of the Müllerian duct. By F. M. BALFOUR and A. SEDGWICK. With Plates 27 and 28 618

XIV. On the early development of the Lacertilia, together with some observations on the nature and relations of the primitive Streak. With Plate 29 644

XV. On certain points in the Anatomy of Peripatus Capensis 657

XVI. On the morphology and systematic position of the Spongida 661

1880

XVII. Notes on the development of the Araneina. With Plates 30, 31, 32 668

XVIII. On the spinal nerves of Amphioxus 696

XIX. Address to the Department of Anatomy and Physiology of the British Association for the Advancement of Science 698

1881

XX. On the development of the skeleton of the paired fins of Elasmobranchii, considered in relation to its bearings on the nature of the limbs of the Vertebrata. With Plate 33 714

XXI. On the evolution of the Placenta, and on the possibility of employing the characters of the Placenta in the classification of the Mammalia 734

1882

XXII. On the structure and development of Lepidosteus. By F. M. BALFOUR and W. N. PARKER. With Plates 34-42 738

XXIII. On the nature of the organ in Adult Teleosteans and Ganoids which is usually regarded as the Head-kidney or Pronephros 848

XXIV. A renewed study of the germinal layers of the Chick. By F. M. BALFOUR and F. DEIGHTON. With Plates 43, 44, 45 854

POSTHUMOUS, 1883

XXV. The Anatomy and Development of Peripatus Capensis. Edited by H. N. MOSELEY and A. SEDGWICK. With Plates 46-53 871

Francis Maitland Balfour, the sixth child and third son of James Maitland Balfour of Whittinghame, East Lothian, and Lady Blanche, daughter of the second Marquis of Salisbury, was born at Edinburgh, during a temporary stay of his parents there, on the 10th November, 1851. He can hardly be said to have known his father, who died of consumption in 1856, at the early age of thirty-six, and who spent the greater part of the last two years of his life at Madeira, separated from the younger children who remained at home. He fancied at one time that he had inherited his father's constitution; and this idea seems to have spurred him on to achieve early what he had to do. But, though there was a period soon after he went to College, during which he seemed delicate, and the state of his health caused considerable anxiety to his friends, he eventually became fairly robust, and that in spite of labours which greatly taxed his strength.

The early years of his life were spent chiefly at Whittinghame under the loving care of his mother. She made it a point to attempt to cultivate in all her children some taste for natural science, especially for natural history, and in this she was greatly helped by the boys' tutor, Mr J. W. Kitto. They were encouraged to make collections and to form a museum, and the fossils found in the gravel spread in front of the house served as the nucleus of a geological series. Frank soon became greatly interested in these things, and indeed they may be said to have formed the beginnings of his scientific career. At all events there was thus awakened in him a love for geology, which science continued to be his favorite study all through his boyhood, and interested him to the last. He was most assiduous in searching for fossils in the gravel and elsewhere, and so great was his love for his collections that while as yet quite a little boy the most delightful birthday present he could think of was a box with trays and divisions to hold his fossils and specimens. His mother, thinking that his fondness for fossils was a passing fancy and that he might soon regret the purchase of the box, purposely delayed the present. But he remained constant to his wish and in time received his box. He must at this time have been about seven or eight years old. In the children's museum, which has been preserved, there are specimens labelled with his childish round-hand, such as a piece of stone with the label "marks of some shels;" and his sister Alice, who was at that time his chief companion, remembers discussing with him one day after the nursery dinner, when he was about nine years old, whether it were better to be a geologist or a naturalist, he deciding for the former on the ground that it was better to do one thing thoroughly than to attempt many branches of science and do them imperfectly.

Besides fossils, he collected not only butterflies, as do most boys at some time or other, but also birds; and he with his sister Alice, being instructed in the art of preparing and preserving skins, succeeded in making a very considerable collection. He thus acquired before long not only a very large but a very exact knowledge of British birds.

In the more ordinary work of the school-room he was somewhat backward. This may have been partly due to the great difficulty he had in learning to write, for he was not only left-handed but, in his early years, singularly inapt in acquiring particular muscular movements, learning to dance being a great trouble to him. Probably however the chief reason was that he failed to find any interest in the ordinary school studies. He fancied that the family thought him stupid, but this does not appear to have been the case.

In character he was at this time quick tempered, sometimes even violent, and the energy which he shewed in after life even thus early manifested itself as perseverance, which, when he was crossed, often took on the form of obstinacy, causing at times no little trouble to his nurses and tutors. But he was at the same time warm-hearted and affectionate; full of strong impulses, he disliked heartily and loved much, and in his affections was wonderfully unselfish, wholly forgetting himself in his thought for others, and ready to do things which he disliked to please those whom he loved. Though, as we have said, somewhat clumsy, he was nevertheless active and courageous; in learning to ride he shewed no signs of fear, and boldly put his pony to every jump which was practicable.

In 1861 he was sent to the Rev. C. G. Chittenden's preparatory school at Hoddesden in Hertfordshire, and here the qualities which had been already visible at home became still more obvious. He found difficulty not only in writing but also in spelling, and in the ordinary school-work he took but little interest and made but little progress.

In 1865 he was moved to Harrow and placed in the house of the Rev. F. Rendall. Here, as at Hoddesden, he did not show any great ability in the ordinary school studies, though as he grew older his progress became more marked. But happily he found at Harrow an opportunity for cultivating that love of scientific studies which was yearly growing stronger in him. Under the care of one of the Masters, Mr G. Griffith, the boys at Harrow were even then taught the elements of natural science. The lessons were at that time, so to speak, extra-academical, carried on out of school hours; nevertheless, many of the boys worked at them with diligence and even enthusiasm, and among these Balfour became conspicuous, not only by his zeal but by his ability. Griffith was soon able to recognize the power of his new pupil, and thus early began to see that the pale, earnest, somewhat clumsy-handed lad, though he gave no promise of being a scholar in the narrower sense of the word, had in him the makings of a man of science. Griffith chiefly confined his teaching to elementary physics and chemistry with some little geology, but he also encouraged natural history studies and began the formation of a museum of comparative anatomy. Balfour soon began to be very zealous in dissecting animals, and was especially delighted when the Rev. A. C. Eaton, the well-known entomologist, on a visit to Harrow, initiated Griffith's pupils in the art of dissecting under water. The dissection of a caterpillar in this way was probably an epoch in Balfour's life. Up to that time his rough examination of such bodies had revealed to him nothing more than what in school-boy language he spoke of as "squash;" but when under Eaton's deft hands the intricate organs of the larval Arthropod floated out under water and displayed themselves as a labyrinth of threads and sheets of silvery whiteness a new world of observation opened itself up to Balfour, and we may probably date from this the beginning of his exact morphological knowledge.

While thus learning the art of observing, he was at the same time developing his power of thinking. He was by nature fond of argument, and defended with earnestness any opinions which he had been led to adopt. He was very active in the Harrow Scientific Society, reading papers, taking part in the discussions, and exhibiting specimens. He gained in 1867 a prize for an essay on coal, and when, in 1868, Mr Leaf offered a prize (a microscope) "for the best account of some locality visited by the writer during the Easter Holidays," two essays sent in, one by Balfour, the other by his close friend, Mr Arthur Evans, since well known for his researches in Illyria, were found to be of such unusual merit that Prof. Huxley was specially requested to adjudicate between them. He judged them to be of equal merit, and a prize was given to each. The subject of Balfour's essay was "The Geology and Natural History of East Lothian." When biological subjects were discussed at the Scientific Society, Balfour appears to have spoken as a most uncompromising opponent of the views of Mr Charles Darwin, little thinking that in after life his chief work would be to develop and illustrate the doctrine of evolution.

The years at Harrow passed quickly away, Balfour making fair, but perhaps not more than fair, progress in the ordinary school learning. In due course however he reached the upper sixth form, and in his last year, became a monitor. At the same time his exact scientific knowledge was rapidly increasing. Geology still continued to be his favorite study, and in this he made no mean progress. During his last years at Harrow he and his brother Gerald worked out together some views concerning the geology of their native county. These views they ultimately embodied in a paper, which was published in their joint names in the Geological Magazine for 1872, under the title of "Some Points in the Geology of the East Lothian Coast," and which was in itself a work of considerable promise. Geology however was beginning to find a rival in natural history. Much of his holiday time was now spent in dredging for marine animals along the coast off Dunbar. Each specimen thus obtained was carefully determined and exact records were kept of the various 'finds,' so that the dredgings (which were zealously continued after he had left Harrow and gone to Cambridge) really constituted a serious study of the fauna of this part of the coast. They also enabled him to make a not inconsiderable collection of shells, in the arrangement of which he was assisted by his sister Evelyn, of crustacea and of other animals.

Both to the masters and to his schoolfellows he became known as a boy of great force of character. Among the latter his scrupulous and unwavering conscientiousness made him less popular perhaps than might have been expected from his bright kindly manner and his unselfish warmheartedness. In the incidents of school life a too strict conscience is often an inconvenience, and the sternness and energy with which Balfour denounced acts of meanness and falsehood were thought by some to be unnecessarily great. He thus came to be feared rather than liked by many, and comparatively few grew to be sufficiently intimate with him to appreciate the warmth of his affections and the charm of his playful moments.

At the Easter of 1870 he passed the entrance examination at Trinity College, Cambridge, and entered into residence in the following October. His college tutor was Mr J. Prior, but he was from the first assisted and guided in his studies by his friend, Mr Marlborough Pryor, an old Harrow boy, who in the same October had been, on account of his distinction in Natural Science, elected a Fellow of the College, in accordance with certain new regulations which then came into action for the first time, and which provided that every three years one of the College Fellowships should be awarded for excellence in some branch or branches of Natural Science, as distinguished from mathematics, pure or mixed. During the whole of that year and part of the next Mr Marlborough Pryor remained in residence, and his influence in wisely directing Balfour's studies had a most beneficial effect on the latter's progress.

During his first term Balfour was occupied in preparation for the Previous Examination; and this he successfully passed at Christmas. After that he devoted himself entirely to Natural Science, attending lectures on several branches. During the Lent term he was a very diligent hearer of the lectures on Physiology which I was then giving as Trinity Prælector, having been appointed to that post in the same October that Balfour came into residence. At this time he was not very strong, and I remember very well noticing among my scanty audience, a pale retiring student, whose mind seemed at times divided between a desire to hear the lecture and a feeling that his frequent coughing was growing an annoyance to myself and the class. This delicate-looking student, I soon learnt, was named Balfour, and when the Rev. Coutts Trotter, Mr Pryor and myself came to examine the candidates for the Natural Science Scholarships which were awarded at Easter, we had no difficulty in giving the first place to him. In point of knowledge, and especially in the thoughtfulness and exactitude displayed in his papers and work, he was very clearly ahead of his competitors.

During the succeeding Easter term and the following winter he appears to have studied physics, chemistry, geology and comparative anatomy, both under Mr Marlborough Pryor and by means of lectures. He also continued to attend my lectures, but though I gradually got to know him more and more we did not become intimate until the Lent term of 1872. He had been very much interested in some lectures on embryology which I had given, and, since Marlborough Pryor had left or was about to leave Cambridge, he soon began to consult me a good deal about his studies. He commenced practical histological and embryological work under me, and I remember very vividly that one day when we were making a little excursion in search of nests and eggs of the stickleback in order that he might study the embryology of fishes, he definitely asked my opinion as to whether he might take up a scientific career with a fair chance of success. I had by this time formed a very high opinion of his abilities, and learning then for the first time that he had an income independent of his own exertions, my answer was very decidedly a positive one. Soon after, feeling more and more impressed with his power and increasingly satisfied both with his progress in biological studies and his sound general knowledge of other sciences, anxious also, it may be, at the same time that as much original inquiry as possible should be carried on at Cambridge in my department, I either suggested to him or acquiesced in his own suggestion that he should at once set to work on some distinct research; and as far as I remember the task which I first proposed to him was an investigation of the layers of the blastoderm in the chick. It must have been about the same time that I proposed to him to join me in preparing for publication a small work on Embryology, the materials for this I had ready to hand in a rough form as lectures which I had previously given. To this proposal he enthusiastically assented, and while the lighter task of writing what was to be written fell to me, he undertook to work over as far as was possible the many undetermined points and unsatisfactory statements across which we were continually coming.

During his two years at College his health had improved; though still hardly robust and always in danger of overworking himself, he obviously grew stronger. He rejoiced exceedingly in his work, never tiring of it, and was also making his worth felt among his fellow students, and especially perhaps among those of his own college whose studies did not lie in the same direction as his own. At this time he must have been altogether happy, but a sorrow now came upon him. His mother, to whom he was passionately attached, and to whose judicious care in his early days not only the right development of his strong character but even his scientific leanings were due, had for some time past been failing in health, though her condition caused no immediate alarm. In May 1872, however, she died quite suddenly from unsuspected heart disease. Her loss was a great blow to him, and for some time afterward I feared his health would give way; but he bore his grief quietly and manfully and threw himself with even increased vigour into his work.

During the academic session of 1872-3, he continued steadily at work at his investigations, and soon began to make rapid progress. At the beginning he had complained to me about what he considered his natural clumsiness, and expressed a fear that he should never be able to make satisfactory microscopic sections; as to his being able to make drawings of his dissections and microscopical preparations, he looked upon that at first as wholly impossible. I need hardly say that in time he acquired great skill in the details of microscopical technique, and that his drawings, if wanting in so-called artistic finish, were always singularly true and instructive. While thus struggling with the details which I could teach him, he soon began to manifest qualities which no teacher could give him. I remember calling his attention to Dursy's paper on the Primitive Streak, and suggesting that he should work the matter over, since if such a structure really existed, it must, most probably, have great morphological significance. I am free to confess that I myself rather doubted the matter, and a weaker student might have been influenced by my preconceptions. Balfour, however, thus early had the power of seeing what existed and of refusing to see what did not exist. He was soon able to convince me that Dursy's streak was a reality, and the complete working out of its significance occupied his thoughts to the end of his days.

The results of these early studies were made known in three papers which appeared in the Quarterly Journal of Microscopical Science for July 1873, and will be found in the beginning of this volume. The summer and autumn of that year were spent partly in a visit to Finland, in company with his friend and old school-fellow Mr Arthur Evans, and partly in formal preparation for the approaching Tripos examination. Into this preparation Balfour threw himself with characteristic energy, and fully justified my having encouraged his spending so much of the preceding time in original research, not only by the rapidity with which he accumulated the stock of knowledge of various kinds necessary for the examination but also by the manner in which he acquitted himself at the trial itself. At that time the position of the candidates in the Natural Sciences Tripos was determined by the total number of marks, and Balfour was placed second, the first place being gained by H. Newell Martin of Christ's College, now Professor at Baltimore, U.S.A. In the examination, in which I took part, Balfour did not write much, and he had not yet learnt the art of putting his statements in the best possible form; he won his position chiefly by the firm thought and clear insight which was present in almost all his answers.

The examination was over in the early days of Dec. 1873 and Balfour was now free to devote himself wholly to his original work. Happily, the University had not long before secured the use of two of the tables at the then recently founded Stazione Zoologica at Naples. And upon the nomination of the University, Balfour, about Christmas, started for Naples in company with his friend Mr A. G. Dew-Smith, also of Trinity College. The latter was about to carry on some physiological observations; Balfour had set himself to work out as completely as he could the embryology of Elasmobranch fishes, about which little was at that time known, but which, from the striking characters of the adult animals could not help proving of interest and importance.

From his arrival there at Christmas 1873 until he left in June 1874, he worked assiduously, and with such success, that as the result of the half-year's work he had made a whole series of observations of the greatest importance. Of these perhaps the most striking were those on the development of the urogenital organs, on the neurenteric canal, on the development of the spinal nerves, on the formation of the layers and on the phenomena of segmentation, including a history of the behaviour of nuclei in cell division. He returned home laden with facts and views both novel and destined to influence largely the progress of embryology.

In August of the same year he attended the meeting of the British Association for the Advancement of Science at Belfast; and the account he then gave of his researches formed one of the most important incidents at the Biological Section on that occasion.

In the September of that year the triennial fellowship for Natural Science was to be awarded at Trinity College, and Balfour naturally was a candidate. The election was, according to the regulations, to be determined partly by the result of an examination in various branches of science, and partly by such evidence of ability and promise as might be afforded by original work, published or in manuscript. He spent the remainder of the autumn in preparation for this examination. But when the examination was concluded it was found that in his written answers he had not been very successful; he had not even acquitted himself so well as in the Tripos of the year before, and had the election been determined by the results of the examination alone, the examiners would have been led to choose the gentleman who was Balfour's only competitor. The original work however which Balfour sent in, including a preliminary account of the discoveries made at Naples, was obviously of so high a merit and was spoken of in such enthusiastic terms by the External Referee Prof. Huxley, that the examiners did not hesitate for a moment to neglect altogether the formal written answers (and indeed the papers of questions were only introduced as a safeguard, or as a resource in case evidence of original power should be wanted) and unanimously recommended him for election. Accordingly he was elected Fellow in the early days of October.

Almost immediately after, the little book on Embryology appeared, on which he and I had been at work, he doing his share even while his hands and mind were full of the Elasmobranch inquiry. The title-page was kept back some little time in order that his name might appear on it with the addition of Fellow of Trinity, a title of which he was then, and indeed always continued to be, proud. He also published in the October number of the Quarterly Journal of Microscopical Science a preliminary account of his Elasmobranch researches.

He and his friends thought that after these almost incessant labours, and the excitement necessarily contingent upon the fellowship election, he needed rest and change. Accordingly on the 17th of October he started with his friend Marlborough Pryor on a voyage to the west coast of South America. They travelled thither by the Isthmus of Panama, visited Peru and Chili, and returned home along the usual route by the Horn; reaching England some time in Feb. 1875.

Refreshed by this holiday, he now felt anxious to complete as far as possible his Elasmobranch work, and very soon after his return home, in fact in March, made his way again to Naples, where he remained till the hot weather set in in May. On his return to Cambridge, he still continued working on the Elasmobranchii, receiving material partly from Naples, partly from the Brighton Aquarium, the then director of which, Mr Henry Lee, spared no pains to provide him both with embryo and adult fishes. While at Naples, he communicated to the Philosophical Society at Cambridge a remarkable paper on "The Early Stages of Vertebrates," which was published in full in the Quarterly Journal of Microscopical Science, July, 1875; he also sent me a paper on "The Development of the Spinal Nerves", which I communicated to the Royal Society, and which was subsequently published in the Philosophical Transactions of 1876. He further wrote in the course of the summer and published in the Journal of Anatomy and Physiology in October, 1875, a detailed account of his "Observations and Views on the Development of the Urogenital Organs."

Some time in August of the same year he started in company with Mr Arthur Evans and Mr J. F. Bullar for a second trip to Finland, the travellers on this occasion making their way into regions very seldom visited, and having to subsist largely on the preserved provisions which they carried with them, and on the produce of their rods and guns. From a rough diary which Balfour kept during this trip it would appear that while enjoying heartily the fun of the rough travelling, he occupied himself continually with observations on the geology and physical phenomena of the country, as well as on the manners, antiquities, and even language of the people. It was one of his characteristic traits, a mark of the truly scientific bent of his mind, of his having, as Dohrn soon after Balfour's first arrival at Naples said, 'a real scientific head,' that every thing around him wherever he was, incited him to careful exact observation, and stimulated him to thought.

In the early part of the Long Vacation of the same year he had made his first essay in lecturing, having given a short course on Embryology in a room at the New Museums, which I then occupied as a laboratory. Though he afterwards learnt to lecture with great clearness he was not by nature a fluent speaker, and on this occasion he was exceedingly nervous. But those who listened to him soon forgot these small defects as they began to perceive the knowledge and power which lay in their new teacher.

Encouraged by the result of this experiment, he threw himself, in spite of the heavy work which the Elasmobranch investigation was entailing, with great zeal into an arrangement which Prof. Newton, Mr J. W. Clark and myself had in course of the summer brought about, that he and Mr A. Milnes Marshall, since Professor at Owens College, Manchester, should between them give a course on Animal Morphology, with practical instruction, Prof. Newton giving up a room in the New Museums for the purpose.

In the following October (1875) upon Balfour's return from Finland, these lectures were accordingly begun and carried on by the two lecturers during the Michaelmas and Lent Terms. The number of students attending this first course, conducted on a novel plan, was, as might be expected, small, but the Lent Term did not come to an end before an enthusiasm for morphological studies had been kindled in the members of the class.

The ensuing Easter term (1876) was spent by Balfour at Naples, in order that he might carry on towards completion his Elasmobranch work. He had by this time determined to write as complete a monograph as he could of the development of these fishes, proposing to publish it in instalments in the Journal of Anatomy and Physiology, and subsequently to gather together the several papers into one volume. The first of these papers, dealing with the ovum, appeared in Jan. 1876; most of the numbers of the Journal during that and the succeeding year contained further portions; but the complete monograph did not leave the publisher's hands until 1878.

He returned to England with his pupil and friend Mr J. F. Bullar some time in the summer; on their way home they passed through Switzerland, and it was during the few days which he then spent in sight of the snow-clad hills that the beginnings of a desire for that Alpine climbing, which was destined to be so disastrous, seem to have been kindled in him.

In October, 1876, he resumed the lectures on Morphology, taking the whole course himself, his colleague, Mr Marshall, having meanwhile left Cambridge. Indeed, from this time onward, he may be said to have made these lectures, in a certain sense, the chief business of his life. He lectured all three terms, devoting the Michaelmas and Lent terms to a systematic course of Animal Morphology, and the Easter term to a more elementary course of Embryology. These lectures were given under the auspices of Prof. Newton; but Balfour's position was before long confirmed by his being made a Lecturer of Trinity College, the lectures which he gave at the New Museums, and which were open to all students of the University, being accepted in a liberal spirit by the College as equivalent to College Lectures. He very soon found it desirable to divide the morphological course into an elementary and an advanced course, and to increase the number of his lectures from three to four a week. Each lecture was followed by practical work, the students dissecting and examining microscopically, an animal or some animals chosen as types to illustrate the subject-matter of the lecture; and although Balfour had the assistance at first of one[2], and ultimately of several demonstrators, he himself put his hand to the plough, and after the lecture always spent some time in the laboratory among his pupils. Had Balfour been only an ordinary man, the zeal and energy which he threw into his lectures, and into the supervision of the practical work, added to the almost brotherly interest which he took in the individual development of every one of the pupils who shewed any love whatever for the subject, would have made him a most successful teacher. But his talents and powers were such as could not be hid even from beginners. His extensive and exact knowledge, the clearness with which in spite of, or shall I not rather say, by help of a certain want of fluency, he explained difficult and abstruse matters, the trenchant way in which he lay bare specious fallacies, and the presence in almost his every word of that power which belongs only to the man who has thought out for himself everything which he says, these things aroused and indeed could hardly fail to arouse in his hearers feelings which, except in the case of the very dullest, grew to be those of enthusiasm. His class, at first slowly, but afterwards more rapidly, increased in numbers, and, what is of more importance, grew in quality. The room allotted to him soon became far too small and steps were taken to provide for him, for myself, whose wants were also urgent, and for the biological studies generally, adequate accommodation; but it was not until Oct. 1877 that we were able to take possession of the new quarters.

Footnote 2: His first Demonstrator up to Christmas 1877, was Mr J. F. Bullar. In Jan. 1878, Mr Adam Sedgwick took the post of Senior Demonstrator, and held it until Balfour's death.

Even this new accommodation soon became insufficient, and in the spring of 1882 a new morphological laboratory was commenced in accordance with plans suggested by himself. He was to have occupied them in the October term, 1883, but did not live to see them finished.

As might have been expected from his own career, he regarded the mere teaching of what is known as a very small part of his duties as Lecturer; and as soon as any of his pupils became sufficiently advanced, he urged or rather led them to undertake original investigations; and he had the satisfaction before his death of seeing the researches of his pupils (such as those by Messrs. Bullar, Sedgwick, Mitzikuri, Haddon, Scott, Osborne, Caldwell, Heape, Weldon, Parker, Deighton and others) carried to a successful end. In each of these inquiries he himself took part, sometimes a large part, generally suggesting the problem to be solved, indicating the methods, and keeping a close watch over the whole progress of the study. Hence in many cases the published account bears his name as well as that of the pupil.

In the year 1878 his Monograph on Elasmobranch Fishes was published as a complete volume, and in the same year he received the honour of being elected a Fellow of the Royal Society, a distinction which now-a-days does not often fall to one so young. No sooner was the Monograph completed than in spite of the labours which his lectures entailed, he set himself to the great task of writing a complete treatise on Comparative Embryology. This not only laid upon him the heavy burden of gathering together the observations of others, enormous in number and continually increasing, scattered through many journals and books, and recorded in many different languages, as well as of putting them in orderly array, and of winnowing out the grain from the chaff (though his critical spirit found some relief in the latter task), but also caused him much labour, inasmuch as at almost every turn new problems suggested themselves, and demanded inquiry before he could bring his mind to writing about them. This desire to see his way straight before him, pursued him from page to page, and while it has resulted in giving the book an almost priceless value, made the writing of it a work of vast labour. Many of the ideas thus originated served as the bases of inquiries worked out by himself or his pupils, and published in the form of separate papers, but still more perhaps never appeared either in the book or elsewhere and were carried with him undeveloped and unrecorded to the grave.

The preparation of this work occupied the best part of his time for the next three years, the first volume appearing in 1880, the second in 1881.

In the autumn of 1880, he attended the Meeting at Swansea of the British Association for the Advancement of Science, having been appointed Vice-President of the Biological Section with charge of the Department of Anatomy and Physiology. At the Meetings of the Association, especially of late years, much, perhaps too much, is expected in the direction of explaining the new results of science in a manner interesting to the unlearned. Popular expositions were never very congenial to Balfour, his mind was too much occupied with the anxiety of problems yet to be solved; he was therefore not wholly at his ease, in his position on this occasion. Yet his introductory address, though not of a nature to interest a large mixed audience, was a luminous, brief exposition of the modern development and aims of embryological investigation.

During these years of travail with the Comparative Embryology the amount of work which he got through was a marvel to his friends, for besides his lectures, and the researches, and the writing of the book, new labours were demanded of him by the University for which he was already doing so much. Men at Cambridge, and indeed elsewhere as well, soon began to find out that the same clear insight which was solving biological problems could be used to settle knotty questions of policy and business. Moreover he united in a remarkable manner, the power of boldly and firmly asserting and maintaining his own views, with a frank courteousness which went far to disarm opponents. Accordingly he found himself before long a member of various Syndicates, and indeed a very great deal of his time was thus occupied, especially with the Museums and Library Syndicates, in both of which he took the liveliest interest. Besides these University duties his time and energy were also at the service of his College. In the preparation of the New Statutes, with which about this time the College was much occupied, the Junior Fellows of the College took a conspicuous share, and among these Junior Fellows Balfour was perhaps the most active; indeed he was their leader, and he threw himself into the investigation of the bearings and probable results of this and that proposed new statute with as much zeal as if he were attacking some morphological problem.

While he was in the midst of these various labours, his friends often feared for his strength, for though gradually improving in health after his first year at Cambridge, he was not robust, and from time to time he seemed on the point of breaking down. Still, hard as he was working, he was in reality wisely careful of himself, and as he grew older, paid more and more attention to his health, daily taking exercise in the form either of bicycle rides or of lawn-tennis. Moreover he continued to spend some part of his vacations in travel. Combining business with pleasure, he made frequent visits to Germany and France, and especially to Naples. The Christmas of 1876-7 he spent in Greece, that of 1878-9 at Ragusa, where his old school-fellow and friend Mr Arthur Evans was at that time residing, and the appointment of his friend Kleinenberg to a Professorship at Messina led to a journey there. Early in the long vacation of 1880, he went with his sister, Mrs H. Sidgwick, and her husband to Switzerland, and was joined there for a short time by his friend and pupil Adam Sedgwick. During this visit he took his first lessons in Alpine climbing, making several excursions, some of them difficult and dangerous; and the love of mountaineering laid so firm a hold upon him, that he returned to Switzerland later on in the autumn of the same year, in company with his brother Gerald, and spent some weeks near Zermatt in systematic climbing, ascending, among other mountains, the Matterhorn and the Weisshorn. In the following summer, 1881, he and his brother Gerald again visited the Alps, dividing their time between the Chamonix district and the Bernese Oberland. On this occasion some of the excursions which they made were of extreme difficulty, and such as needed not only great presence of mind and bodily endurance, but also skilful and ready use of the limbs. As a climber indeed Balfour soon shewed himself fearless, indefatigable, and expert in all necessary movements as well as full of resources and expedients in the face of difficulties, so much so that he almost at once took rank among the foremost of distinguished mountaineers. In spite of his apparent clumsiness in some matters, he had even as a lad proved himself to be a bold and surefooted climber. Moreover he had been perhaps in a measure prepared for the difficulties of Alpine climbing by his experience in deer-stalking. This sport he had keenly and successfully pursued for many years at his brother's place in Rosshire. When however about the year 1877, the question of physiological experiments on animals became largely discussed in public, he felt that to continue the pursuit of this or any other sport involving, for the sake of mere pleasure, the pain and death of animals, was inconsistent with the position which he had warmly taken up, as an advocate of the right to experiment on animals; and he accordingly from that time onward wholly gave it up.

His fame as an investigator and teacher, and as a man of brilliant and powerful parts, was now being widely spread. Pupils came to him, not only from various parts of England, but from America, Australia and Japan. At the York Meeting of the British Association for the Advancement of Science, in August, 1881, he was chosen as one of the General Secretaries. In April, 1881, the honorary degree of LL.D. was conferred upon him by the University of Glasgow, and in November of the same year the Royal Society gave him one of the Royal Medals in recognition of his embryological discoveries, and at the same time placed him on its Council.

At Cambridge he was chosen, in the autumn of 1880, President of the Philosophical Society, and in the December of that year a brilliant company were gathered together at the Annual Dinner to do honour to their new young President. Otherwise nothing as yet had been done for him in his own University in the way of recognition of his abilities and services; and he still remained a Lecturer of Trinity College, giving lectures in a University building. An effort had been made by some of his friends to urge the University to take some step in this direction; but it was thought at that time impossible to do anything. In 1881 a great loss fell upon the sister University of Oxford in the death of Prof. George Rolleston; and soon after very vigorous efforts were made to induce Balfour to become a candidate for the vacant chair. The prospect was in many ways a tempting one, and Balfour seeing no very clear way in the future for him at his own University, was at times inclined to offer himself, but eventually he decided to remain at Cambridge. Hardly had this temptation if we may so call it been overcome when a still greater one presented itself. Through the lamented death of Sir Wyville Thomson in the winter of 1881-2, the chair of Natural History at Edinburgh, perhaps the richest and most conspicuous biological chair in the United Kingdom, became vacant. The post was in many ways one which Balfour would have liked to hold. The teaching duties were it is true laborious, but they had in the past been compressed into a short time, occupying only the summer session and leaving the rest of the year free, and it seemed probable that this arrangement might be continued with him. The large emolument would also have been grateful to him inasmuch as he would have felt able to devote the whole of it to scientific ends; and the nearness to Whittinghame, his native place and brother's home, added to the attractions; but what tempted him most was the position which it would have given him, and the opportunities it would have afforded, with the rich marine Fauna of the north-eastern coast close at hand, to develop a large school of Animal Morphology. The existing Professors at Edinburgh were most desirous that he should join them, and made every effort to induce him to come. On the part of the Crown, in whose hands the appointment lay, not only were distinct offers made to him, but he was repeatedly pressed to accept the post. Nor was it until after a considerable struggle that he finally refused, his love for his own University in the end overcoming the many inducements to leave; he elected to stay where he was, trusting to the future opening up for him some suitable position. In this decision he was undoubtedly influenced by the consideration that Cambridge, besides being the centre of his old friendships, had become as it were a second home for his own family. By the appointment of Lord Rayleigh to the chair of Experimental Physics his sister Lady Rayleigh had become a resident, his sister Mrs Sidgwick had lived there now for some years, and his brother Gerald generally spent the summer there; their presence made Cambridge doubly dear to him.

At the close of the Michaelmas term, with feelings of relief at having completed his Comparative Embryology, the preparation of the second volume of which had led to almost incessant labour during the preceding year, he started to spend the Christmas vacation with his friend Kleinenberg at Messina. Stopping at Naples on his way thither he found his pupil Caldwell, who had been sent to occupy the University table at the Stazione Zoologica, lying ill at Capri, with what proved to be typhoid fever. The patient was alone, without any friend to tend him, and his mother who had been sent for had not yet arrived. Accordingly Balfour (with the kindness all forgetful of himself which was his mark all his life through) stayed on his journey to nurse the sick man until the mother came. He then went on to Messina, and there seemed to be in good health, amusing himself with the ascent of Etna. Yet in January, soon after his return home, he complained of being unwell, and in due time distinct symptoms of typhoid fever made their appearance. The attack at first promised to be severe, but happily the crisis was soon safely passed and the convalescence was satisfactory.

While yet on his sick bed, a last attempt was made to induce him to accept the Edinburgh offer, and for the last time he refused. These repeated offers, and the fact that the dangers of his grave illness had led the University vividly to realize how much they would lose if Balfour were taken away from them, encouraged his friends to make a renewed effort to gain for him some adequate position in the University. This time the attempt was successful, and the authorities took a step, unusual but approved of by the whole body of resident members of the University; they instituted a new Professorship of Animal Morphology, to be held by Balfour during his life or as long as he should desire, but to terminate at his death or resignation unless it should be otherwise desirable. Accordingly in May, 1882, he was admitted into the Professoriate as Professor of Animal Morphology.

During his illness his lectures had been carried on by his Demonstrator, Mr Adam Sedgwick, who continued to take his place during the remainder of that Lent Term and during the ensuing Easter Term. The spring Balfour spent partly in the Channel Islands with his sister Alice, partly in London with his eldest brother, but in the course of the Easter Term returned to Cambridge and resumed his work though not his lectures. His recovery to health was steady and satisfactory, the only drawback being a swelling over the shin-bone of one leg, due to a blow on the rocks at Sark; otherwise he was rapidly becoming strong. He himself felt convinced that a visit to the Alps, with some mountaineering of not too difficult a kind, would complete his restoration to health. In this view many of his friends coincided; for the experience of former years had shewn them what a wonderfully beneficial effect the Alpine air and exercise had upon his health. He used to go away pale, thin and haggard, to return bronzed, clear, firm and almost stout; nor was there anything in his condition which seemed to forbid his climbing, provided that he was cautious at the outset. Accordingly, early in June he left Cambridge for Switzerland, having long ago, during his illness in fact, engaged his old guide, Johann Petrus, whom he had first met in 1880, and who had always accompanied him in his expeditions since.

His first walking was in the Chamonix district; and here he very soon found his strength and elasticity come back to him. Crossing over from Montanvert to Courmayeur, by the Col du Géant, he was attracted by the peak called the Aiguille Blanche de Peuteret, a virgin peak, the ascent of which had been before attempted but not accomplished. Consulting with Petrus he determined to try it, feeling that the fortnight, which by this time he had spent in climbing, had brought back to him his old vigour, and that his illness was already a thing of the past.

There is no reason to believe that he regarded the expedition as one of unusual peril; and an incident which at the time of his death was thought by some to indicate this was in reality nothing more than a proof of his kindly foresight. The guide Petrus was burdened by a debt on his land amounting to about £150. In the previous year Balfour and his brother had come to know of this debt; and, seeing that no Alpine ascent is free from danger, that on any expedition some accident might carry them off, had conceived the idea of making some provision for Petrus' family in case he might meet with sudden death in their service. This suggestion of the previous year Balfour carried out on this occasion, and sent home to his brother Gerald a cheque of £150 for this purpose. But the cheque was sent from Montanvert before he had even conceived the idea of ascending the Aiguille Blanche. It was not a provision for any specially dangerous ascent, and must be regarded as a measure prompted not by a sense of coming peril but rather by the donor's generous care for his servant.

On Tuesday afternoon, July 18, he and Petrus, with a porter to carry provisions and firing to their sleeping-place on the rocks, set out from Courmayeur, the porter returning the same night. They expected to get back to Courmayeur some time on the Thursday, but the day passed without their appearing. This did not cause any great anxiety because it was supposed that they might have found it more convenient to pass over to the Chamonix side than to return to Courmayeur. When on Friday however telegrams dispatched to Chamonix and Montanvert brought answers that nothing had been seen of them, it became evident that some accident had happened, and an exploring party set out for the hills. It was not until early on the Sunday morning that this search party found the bodies, both partly covered with snow, lying on the Glacier de Fresney, below the impassable icefall which separates the upper basin of the glacier from the lower portion, and at the foot of a couloir which descends by the side of the icefall. Their tracks were visible on the snow at the top of the couloir. Balfour's neck was broken, and his skull fractured in three places; Petrus' body was also fractured in many places. The exact manner of their death will never be known, but there can be no doubt that, in Balfour's case at all events, it was instantaneous, and those competent to form a judgment are of opinion that they were killed by a sudden fall through a comparatively small height, slipping on the rocks as they were descending by the side of the ice-fall, and not precipitated from the top of the couloir. There is moreover indirect evidence which renders it probable that in the fatal fall Petrus slipped first and carried Balfour with him. Whether they had reached the summit of the Aiguille and were returning home after a successful ascent or whether they were making their way back disheartened and wearied with failure, is not and perhaps never will be known. Since the provisions at the sleeping-place were untouched, the deaths probably took place on Wednesday the 19th. The bringing down the bodies proved to be a task of extreme difficulty, and it was not till Wednesday the 26th that the remains reached Courmayeur, where M. Bertolini, the master of the hotel, and indeed everyone, not least the officers of a small body of Italian troops stationed there, shewed the greatest kindness and sympathy to Balfour's brothers, Gerald and Eustace, who hastened to the spot as soon as the news of the terrible disaster was telegraphed home. Mr Walter Leaf also and Mr Conway, friends of Balfour, the former a very old one, who had made their way to Courmayeur from other parts of Switzerland as soon as they heard of the accident, rendered great assistance. The body was embalmed, brought to England, and buried at Whittinghame on Saturday, Aug. 5, the Fellows of Trinity College holding a service in the College Chapel at the same time.

In person he was tall, being fully six feet in height, well built though somewhat spare. A broad forehead overhanging deeply set dark brown eyes whose light shining from beneath strongly marked eye-brows told all the changes of his moods, slightly prominent cheek-bones, a pale skin, at times inclined to be even sallow, dark brown hair, allowed to grow on the face only as a small moustache, and slight whiskers, made up a countenance which bespoke at once strength of character and delicacy of constitution. It was an open countenance, hiding nothing, giving sign at once, both when his body was weary or weak, and when his mind was gladdened, angered or annoyed.

The record of some of his thoughts and work, all that he had given to the world will be found in the following pages. But who can tell the ideas which had passed into his quick brain, but which as yet were known only to himself, of which he had given no sign up to that sad day on which he made the fatal climb? And who can say whither he might not have reached had he lived, and his bright young life ripened as years went on? This is not the place to attempt any judgment of his work: that may be left to other times, and to other hands; but it may be fitting to place here on record a letter which shews how much the greatest naturalist of this age appreciated his younger brother. Among Balfour's papers was found a letter from Charles Darwin, acknowledging the receipt of Vol. II. of the Comparative Embryology in the following words:

"July 6, 1881. DOWN, BECKENHAM, KENT.

MY DEAR BALFOUR,

I thank you heartily for the present of your grand book, and I congratulate you on its completion. Although I read almost all of Vol. I, I do not feel that I am worthy of your present, unless indeed the fullest conviction that it is a memorable work makes me worthy to receive it.

* * * * *

Once again accept my thanks, for I am proud to receive a book from you, who, I know, will some day be the chief of the English Biologists.

Believe me, Yours sincerely, CHARLES DARWIN."

The loss of him was a manifold loss. He is mourned, and will long be mourned, for many reasons. Some miss only the brilliant investigator; others feel that their powerful and sympathetic teacher is gone; some look back on his memory and grieve for the charming companion whose kindly courtesy and bright wit made the hours fly swiftly and pleasantly along; and to yet others is left an aching void when they remember that they can never again lean on the friend whose judgment seemed never to fail and whose warm-hearted affection was a constant help. And to some he was all of these. At the news of his death the same lines came to the lips of all of us, so fittingly did Milton's words seem to speak our loss and grief--

"For Lycidas is dead, dead ere his prime, Young Lycidas, and hath not left his peer."

M. FOSTER.

I. ON SOME POINTS IN THE GEOLOGY OF THE EAST LOTHIAN COAST[3].

Footnote 3: From the Geological Magazine, Vol. IX. No. 4. April, 1872.

By G. W. and F. M. BALFOUR, Trinity College, Cambridge.

The interesting relation between the Porphyrite of Whitberry Point, at the mouth of the Tyne, near Dunbar, and the adjacent sedimentary rocks, was first noticed, we believe, by Professor Geikie, who speaks of it in the Memoirs of the Geological Survey of East Lothian, pages 40 and 31, and again in the new edition of Jukes's Geology, p. 269. The volcanic mass which forms the point consists of a dark felspathic base with numerous crystals of augite: it is circular in form, and is exposed for two-thirds of its circumference in a vertical precipice facing the sea, about twenty feet in height.

The rock is traversed by numerous joints running both in a horizontal and in a vertical direction. The latter are by far the most conspicuous, and give the face of the cliff, when seen from a distance, a well-marked columnar appearance, though the columns themselves are not very distinct or regular. They are quadrangular in form, and are evidently produced by the intersection at right-angles of the two series of vertical joints.

It is clear that the face of the precipice has been gradually receding in proportion as it yielded to the action of the waves; and that at a former period the volcanic rock extended considerably further than at present over the beds which are seen to dip beneath it. These latter consist of hard fine-grained calcareous sandstones belonging to the Lower Carboniferous formation. Their colour varies from red to white, and their prevailing dip is in a N.W. direction, with an average inclination of 12-20°. If the volcanic mass is a true intrusive rock, we should naturally expect the strata which surround it to dip away in all directions, the amount of their inclination diminishing in proportion to their distance from it. We find, however, that the case is precisely the reverse: as the beds approach the base of the cliff, they dip towards it from every side at perpetually increasing angles, until at the point of junction the inclination amounts in places to as much as 55 degrees. The exact amount of dip in the various positions will be seen on referring to the accompanying map.

We conceive that the phenomenon is to be explained by supposing the orifice through which the lava rose and overflowed the surface of the sedimentary strata to have been very much smaller in area than the extent of igneous rock at present visible; and that the pressure of the erupted mass on the soft beds beneath, aided perhaps by the abstraction of matter from below, caused them to incline towards the central point at a gradually increasing angle. The diagram, fig. 2, will serve further to illustrate this hypothesis. A is the neck or orifice by which the melted matter is supposed to ascend. C shews the sheet of lava after it has overspread the surface of the sandstone beds B, so as to cause them to assume their present inclination. The dotted lines represent the hypothetical extension of the igneous mass and sandstones previous to the denudation which they have suffered from the action of the waves.

Professor Geikie, in his admirable treatise on the Geology of the county[4], adopts a view on this subject which is somewhat different from that which is suggested in this paper. He considers that the whole mass is an intrusive neck of rock with perpendicular sides; and that it once filled up an orifice through the surrounding sedimentary strata, of which it is now the only remnant.

Footnote 4: Memoirs of Geological Survey of Scotland, sheet 33, pp. 40, 41.

He admits that the inclination of the sandstone beds towards the igneous mass in the centre is a phenomenon that is somewhat difficult to explain, and suggests that a subsequent contraction of the column may have tended to produce such a result. To use his own words: "In the case of a solid column of felstone or basalt, the contraction of the melted mass on cooling may have had some effect in dragging down the sides of the orifice[5]."

Footnote 5: Note on p. 41 of _Mem. Geol. Survey of East Lothian_.

But, apart from other objections, it is scarcely conceivable that this result should have been produced by the contraction of the column.

In his recent edition of Jukes's Manual of Geology (p. 269), in which he also refers to this instance, he states that in other cases of "necks" it is found to be an almost invariable rule, "that strata are bent down so as to dip into the neck all round its margin." We are not aware to what other instances Prof. Geikie may allude; but on referring to his Memoir on the Geology of East Lothian, we find that he states in the cases of 'North Berwick Law' and 'Traprain' (which he compares with the igneous mass at Whitberry Point), that the beds at the base of these two necks, where exposed, dip away from them, and that at a high angle.

In support of the hypothesis which we have put forward, the following arguments may be urged:

(1) That in one place at least the sedimentary strata are seen to be actually dipping beneath the superincumbent basalt; and that the impression produced by the general relation of the two rocks is, that they do so everywhere.

(2) Since the columns into which the lava is split are vertical, the cooling surface must have been horizontal: the mass must, therefore, have formed a sheet, and not a dyke; for, in the latter case, the cooling surfaces would have been vertical.

(3) It is difficult to conceive, on the supposition that the volcanic rock is a neck with perpendicular sides, that the marine denudation should have uniformly proceeded only so far as to lay bare the junction between the two formations. We should have expected that in many places the igneous rock itself would have been cut down to the general level, whereas the only signs of such an effect are shown in a few narrow inlets where the rock was manifestly softer than in the surrounding parts.

The last objection is greatly confirmed by the overhanging cliffs and numerous blocks of porphyrite which lie scattered on the beach, as if to attest the former extension of that ancient sheet of which these blocks now form but a small remnant. Indeed, the existence of such remains appears sufficient of itself to condemn any hypothesis which presumes the present face of the cliff to have formed the original boundary of the mass.

It may be fairly objected to our theory, as Prof. Geikie himself has suggested, that the high angle at which the strata dip is difficult to account for. But, in fact, this steep inclination constitutes the very difficulty which any hypothesis on the subject must be framed to explain; and it is a difficulty which is not more easily solved by Prof. Geikie's theory than by our own.

II. THE DEVELOPMENT AND GROWTH OF THE LAYERS OF THE BLASTODERM[6].

Footnote 6: From the _Quarterly Journal of Microscopical Science_, Vol. XIII., 1873.

With Plate 1, figs. 1-5 and 9-12.

The following paper deals with the changes which take place in the cells of the blastoderm of the hen's egg during the first thirty or forty hours of incubation. The subject is one which has, as a general rule, not been much followed up by embryologists, but is nevertheless of the greatest interest, both in reference to embryology itself, and to the growth and changes of protoplasm exhibited in simple embryonic cells. I am far from having exhausted the subject in this paper, and in some cases I shall be able merely to state facts, without being able to give any explanation of their meaning.

My method of investigation has been the examination of sections and surface views. For hardening the blastoderm I have employed, as usual, chromic acid, and also gold chloride. It is, however, difficult to make sections of blastoderms hardened by this latter reagent, and the sections when made are not in all cases satisfactory. For surface views I have chiefly used silver nitrate, which brings out the outlines of the cells in a manner which leaves nothing to be desired as to clearness. If the outlines only of the cells are to be examined, a very short immersion (half a minute) of the blastoderm in a half per cent. solution of silver nitrate is sufficient, but if the immersion lasts for a longer period the nuclei will be brought out also. For studying the latter, however, I have found it better to employ gold chloride or carmine in conjunction with the silver nitrate.

My observations begin with the blastoderm of a freshly laid egg. The appearances presented by sections of this have been accurately described by Peremeschko, "Ueber die Bildung der Keimblätter im Hühnerei," Sitzungsberichte der K. Akademie der Wissenschaften in Wien, 1868. Oellacher, "Untersuchung über die Furchung und Blatterbildung im Hühnerei," Studien aus dem Institut für Experim. Pathologie in Wien, 1870 (pp. 54-74), and Dr Klein, lxiii. Bande der Sitz. der K. Acadamie der Wiss. in Wien, 1871.

The unincubated blastoderm (Pl. 1, fig. 1) consists of two layers. The upper layer is composed of a single row of columnar cells. Occasionally, however, the layer may be two cells thick. The cells are filled with highly refracting spherules of a very small size, and similar in appearance to the finest white yolk spherules, and each cell also contains a distinct oval nucleus. This membrane rests with its extreme edge on the white yolk, its central portion covering in the segmentation cavity. From the very first it is a distinct coherent membrane, and exhibits with silver nitrate a beautiful hexagonal mosaic of the outlines (Pl. 1, fig. 6) of the cells. The diameter of the cells when viewed from above is from 1/2000 - 1/3000 of an inch. The under layer is very different from this: it is composed of cells which are slightly, if at all, united, and which vary in size and appearance, and in which a nucleus can rarely be seen. The cells of which it is composed fill up irregularly the segmentation cavity, though a distinct space is even at this time occasionally to be found at the bottom of it. Later, when the blastoderm has spread and the white yolk floor has been used as food, a considerable space filled with fluid may generally be found.

The shape of the floor of the cavity varies considerably, but it is usually raised in the middle and depressed near the circumference. In this case the under layer is perhaps only two cells deep at the centre and three or four cells deep near the circumference.

The cells of which this layer is composed vary a good deal in size; the larger cells being, however, more numerous in the lower layers. In addition, there are usually a few very large cells quite at the bottom of the cavity, occasionally separated from the other cells by fluid. They were called formative cells (Bildungselemente) by Peremeschko (loc. cit.); and, according to Oellacher's observations (loc. cit.), some of them, at any rate, fall to the bottom of the segmentation cavity during the later stages of segmentation. They do not differ from the general lower layer cells except in size, and even pass into them by insensible gradations. All the cells of the lower layer are granular, and are filled with highly refracting spherules precisely similar to the smaller white yolk spherules which line the bottom of the segmentation cavity.

The size of the ordinary cells of the lower layer varies from 1/2000 - 1/1000 of an inch. The largest of the formative cells come up to 1/300 of an inch. It will be seen from this description that, morphologically speaking, we cannot attach much importance to the formative cells. The fact that they broke off from the blastoderm, towards the end of the segmentation--even if we accept it as a normal occurrence, rather than the result of manipulation--is not of much importance, and, except in size, it is impossible to distinguish these cells from other cells of the lower layer of the blastoderm.

Physiologically, however, as will be afterwards shewn, they are of considerable importance.

The changes which the blastoderm undergoes during the first three or four hours of incubation are not very noticeable. At about the sixth or eighth hour, or in some cases considerably earlier, changes begin to take place very rapidly. These changes result in the formation of a hypoblast and mesoblast, the upper layer of cells remaining comparatively unaltered as the epiblast.

To form the hypoblast a certain number of the cells of the lower layer begin to undergo remarkable changes. From being spherical and, as far as can be seen, non-nucleated, they become (vide fig. 2, h) flattened and nucleated, still remaining granular, but with fewer spherules.

Here, then, is a direct change, of which all the stages can be followed, of a cell of one kind into a cell of a totally different character. The new cell is not formed by a destruction of the old one, but directly from it by a process of metamorphosis. These hypoblast cells are formed first at the centre and later at the circumference, so that from the first the cells at the circumference are less flattened and more granular than the cells at the centre. A number of cells of the original lower layer are enclosed between this layer and the epiblast; and, in addition to these, the formative cells (as has been shewn by Peremeschko, Oellacher, and Klein, whose observations I can confirm) begin to travel towards the circumference, and to pass in between the epiblast and hypoblast.

Both the formative cells, and the lower layer cells enclosed between the hypoblast and epiblast, contribute towards the mesoblast, but the mode in which the mesoblast is formed is very different from that in which the hypoblast originates.

It is in this difference of formation that the true distinction between the mesoblast and hypoblast is to be looked for, rather than in the original difference of the cells from which they are derived.

The cells of the mesoblast are formed by a process which seems to be a kind of free cell formation. The whole of the interior of each of the formative cells, and of the other cells which are enclosed between the epiblast and the hypoblast, become converted into new cells. These are the cells of the mesoblast. I have not been able perfectly to satisfy myself as to the exact manner in which this takes place, but I am inclined to think that some or all of the spherules which are contained in the original cells develop into nuclei for the new cells, the protoplasm of the new cells being formed from that of the original cells.

The stages of formation of the mesoblast cells are shewn in the section (Pl. 1, fig. 2), taken from the periphery of a blastoderm of eight hours.

The first formation of the mesoblast cells takes place in the centre of the blastoderm, and the mass of cells so formed produces the opaque line known as the primitive streak. This is shown in Pl. 1, fig. 9.

One statement I have made in the above description in reference to the origin of the mesoblast cells, viz. that they are only partly derived from the formative cells at the bottom of the segmentation cavity, is to a certain extent opposed to the statements of the three investigators above mentioned. They state that the mesoblast is entirely derived from the formative cells. It is not a point to which I attach much importance, considering that I can detect no difference between these cells and any other cells of the original lower layer except that of size; and even this difference is probably to be explained by their proximity to the white yolk, whose spherules they absorb. But my reason for thinking it probable that these cells alone do not form the mesoblast are: 1st. That the mesoblast and hypoblast are formed nearly synchronously, and except at the centre a fairly even sprinkling of lower layer cells is from the first to be distinguished between the epiblast and hypoblast. 2nd. That if some of the lower layer cells are not converted into mesoblast, it is difficult to see what becomes of them, since they appear to be too numerous to be converted into the hypoblast alone. 3rd. That the chief formation of mesoblast at first takes place in the centre, while if the formative cells alone took part in its formation, it would be natural to expect that it would begin to be formed at the periphery.

Oellacher himself has shewn (Zeitschrift für wissenschaftliche Zoologie, 1873, "Beiträge zur Entwick. Gesch. der Knochenfische") that in osseous fishes the cells which break away from the blastoderm take no share in the formation of the mesoblast, so that we can derive no argument from the formation of the mesoblast in these animals, for believing that in the chick it is derived only from the formative cells.

In the later stages, however, from the twelfth to the twenty-fifth hour, the growth of the mesoblast depends almost entirely on these cells, and Peremeschko's discovery of the fact is of great value.

Waldeyer (Henle und v. Pfeufer's Zeitschrift, xxxiv. Band, für 1869) has given a different account of the origin of the layers. There is no doubt, however, in opposition to his statements and drawings, that from the very first the hypoblast is distinct from the mesoblast, which is, indeed, most conspicuously shewn in good sections; and his drawings of the derivation of the mesoblast from the epiblast are not very correct.

The changes which have been described are also clearly shewn by means of silver nitrate. Whereas, at first this reagent brought out no outline markings of cells in the lower layer, by the eighth to the twelfth hour the markings (Pl. 1, fig. 3) are very plain, and shew that the hypoblast is a distinct coherent membrane.

In section, the cells of the hypoblast appear generally very thin and spindle shaped, but the outlines brought out by the silver nitrate shew that they are much expanded horizontally, but very irregular as to size, varying even within a small area from 1/4000 - 1/400 of an inch in the longest diameter.

At about the twelfth hour they are uniformly smaller a short way from each extremity of its longer axis than over the rest of the blastoderm.

It is, perhaps, fair to conclude from this that growth is most rapid at these parts.

At this time the hypoblast, both in sections and from a surface view after treatment with silver nitrate, appears to end abruptly against the white yolk. The surface view also shews that its cells are still filled with highly refractive globules, making it difficult to see the nucleus. In some cases I thought that I could (fig. 3, a) make out that it was hour-glass shaped, and some cells certainly contain two nuclei. Some of the cells (fig. 3, b) shew re-entrant curves, which prove that they have undergone division.

The cells of the epiblast, up to the thirteenth hour, have chiefly undergone change in becoming smaller.

In surface views they are about 1/4000 of an inch in diameter over the centre of the pellucid area, and increase to 1/2000 of an inch over the opaque area.

In the centre of the pellucid area the form of the epiblast cells is more elongated vertically and over the opaque area more flattened than was the case with the original upper layer cells. In the centre the epiblast is two or three cells deep.

Before going on to the further changes of the blastodermic cells it will be well to say a few words in reference to the origin of the mesoblast.

From the description given above it will be clear that in the chick the mesoblast has an independent origin; it can be said neither to originate from the epiblast nor from the hypoblast. It is formed coincidently with the latter out of apparently similar segmentation cells. The hypoblast, as has been long known, shews in the chick no trace of its primitive method of formation by involution, neither does the mesoblast shew any signs of its primitive mode of formation. In so excessively highly differentiated a type as birds we could hardly expect to find, and certainly do not find, any traces of the primitive origin of the mesoblast, either from the epiblast or hypoblast, or from both. In the chick the mesoblast cells are formed directly from the ultimate products of segmentation. From having a secondary origin in most invertebrates the mesoblast comes to have, in the chick, a primary origin from the segmentation spheres, precisely as we find to be the case with the nervous layer in osseous fishes. It is true we cannot tell which segmentation-cells will form the mesoblast, and which the hypoblast; but the mesoblast and hypoblast are formed at the same time, and both of them directly from segmentation spheres.

The process of formation of the mesoblast in Loligo, as observed by Mr Ray Lankester (Annals and Magazine of Natural History, February, 1873), is still more modified. Here the mesoblast arises independently of the blastoderm, and by a process of free cell-formation in the yolk round the edge of the blastoderm. If Oellacher's observations in reference to the origin of formative cells are correct, then the modes of origin of the mesoblast in Loligo and the chick would have nothing in common; but if the formative cells are in reality derived from the white yolk, and also are alone concerned in the formation of the mesoblast, then the modes of formation of the mesoblast in the chick would be substantially the same as that observed by Mr Ray Lankester in Loligo.

No very important changes take place in the actual forms of the cells during the next few hours. A kind of fusion takes place between the epiblast and the mesoblast along the line of the primitive streak forming the axis-string of His; but the line of junction between the layers is almost always more or less visible in sections. In any case it does not appear that there is any derivation of mesoblast cells from the epiblast; and since the fusion only takes place in the region of the primitive groove, and not in front, where the medullary groove arises (see succeeding paper), it cannot be considered of any importance in reference to the possible origin of the Wolffian duct, &c., from the epiblast (as mooted by Waldeyer, Eierstock und Ei, Leipzig, 1870). The primitive groove, as can be seen in sections, begins to appear very early, generally before the twelfth hour. The epiblast spreads rapidly over the white yolk, and the area pellucida also increases in size.

From the mesoblast forming at first only a small mass of cells, which lies below the primitive streak, it soon comes to be the most important layer of the blastoderm. Its growth is effected by means of the formative cells. These cells are generally not very numerous in an unincubated blastoderm, but rapidly increase in numbers, probably by division; at the same time they travel round the edge of, and in some cases through, the hypoblast, and then become converted in the manner described into mesoblast cells. They act as carriers of food from the white yolk to the mesoblast till, after the formation of the vascular area, they are no longer necessary. The numerous cases in which two nucleoli and even two nuclei can be seen in one cell prove that the mesoblast cells also increase by division.

The growth of the hypoblast takes place in a very different way. It occurs by a direct conversion, cell for cell, of the white yolk spheres into hypoblast cells. This interpretation of the appearances, which I will describe presently, was first suggested to me by Dr Foster, from an examination of some of my specimens of about thirty-six hours, prepared with silver nitrate. Where there is no folding at the junction between the pellucid and opaque areas, there seems to be a perfect continuity in the silver markings and a gradual transition in the cells, from what would be undoubtedly called white yolk spheres, to as undoubted hypoblast cells (vide Pl. 1, fig. 5). In passing from the opaque to the pellucid areas the number of white yolk spherules in each cell becomes less, but it is not till some way into the pellucid area that they quite cease to be present. I at first thought that this was merely due to the hypoblast cells feeding on the white yolk sphericles, but the perfect continuity of the cells, and the perfect gradation in passing from the white yolk cells to the hypoblast, proves that the other interpretation is the correct one, viz. that the white yolk spheres become directly converted into the hypoblast cells. This is well shewn in sections (vide Pl. 1, fig. 4) taken from embryos of all ages from the fifteenth to the thirty-sixth hour and onwards. But it is, perhaps, most easily seen in embryos of about twenty hours. In such an embryo there is a most perfect gradation: the cells of the hypoblast become, as they approach the edge of the pellucid area, broader, and are more and more filled with white yolk sphericles, till at the line of junction it is quite impossible to say whether a particular cell is a white-yolk cell (sphere) or a hypoblast cell. The white-yolk cells near the line of junction can frequently be seen to possess nuclei. At first the hypoblast appears to end abruptly against the white yolk; this state of things, however, soon ends, and there supervenes a complete and unbroken continuity between the hypoblast and the white yolk.

Of the mode of increase of the epiblast I have but little to say. The cells undoubtedly increase entirely by division, and the new material is most probably derived directly from the white yolk.

Up to the sixth hour the cells of the upper layer retain their early regular hexagonal pattern, but by the twelfth hour they have generally entirely lost this, and are irregularly shaped and very angular. The cells over the centre of the pellucid area remain the smallest up to the twenty-fifth hour or later, while those over the rest of the pellucid area are uniformly larger.

In the hypoblast the cells under the primitive groove, and on each side as far as the fold which marks off the exterior limit of the protovertebræ are at the eighteenth hour considerably smaller than any other cells of this layer.

In all the embryos between the eighteenth and twenty-third hour which I have examined for the purpose, I have found that at about two-thirds of the distance from the anterior end of the pellucid area, and just external to the side fold, there is a small space on each side in which the cells are considerably larger than anywhere else in the hypoblast. These larger cells, moreover, contain a greater number of highly refractive spherules than any other cells. It is not easy to understand why growth should have been less rapid here than elsewhere, as the position does not seem to correspond to any feature in the embryo. In some specimens the hypoblast cells at the extreme edge of the pellucid area are smaller than the cells immediately internal to them. At about the twenty-third hour these cells begin rapidly to lose the refractive spherules they contained in the earlier stages of incubation, and come to consist of a nucleus surrounded simply by granular protoplasm.

At about this period of incubation the formative cells are especially numerous at the periphery of the blastoderm, and, no doubt, become converted into the mass of mesoblast which is found at about the twenty-fifth hour in the region of the vascular area. Some of them are lobate, and appear as if they were undergoing division. At this time also the greatest number of formative cells are to be found at the bottom of the now large segmentation cavity.

In embryos of from thirty to forty hours the cells of the hypoblast have, over the central portion of the pellucid area, entirely lost their highly refractive spherules, and in the fresh state are composed of the most transparent protoplasm. When treated with reagents they are found to contain an oval nucleus with one or sometimes two nucleoli, imbedded in a considerable mass of protoplasm. The protoplasm appears slightly granular and generally contains one or two small vacuoles. I have already spoken of the gradation of the hypoblast at the edge of the blastoderm into white yolk. I have, therefore, only to mention the variations in the size of its cells in different parts of the pellucid area. The points where the cells are smallest seem generally to coincide with the points of maximum growth. Over the embryo the cells are more regular than elsewhere. They are elongated and arranged transversely to the long axis of the embryo. They are somewhat hexagonal in shape, and not unlike the longer pieces in the dental plate of a Myliobatis (Pl. 1, fig. 10). This regularity, however, is much more marked in some specimens than in others. These cells are about 1/4000th of an inch in breadth, and 1/1000th in length. On each side of the embryo immediately external to the protovertebræ the cells are frequently about the same size as those over the embryo itself. In the neck, however, and near the end of the sinus rhomboidalis, they are considerably smaller, about 1/4000th inch each way. The reason of this small size is not very clear, but probably shews that the greatest growth is taking place at these two points. The cells, again, are very small at the head fold, but are very much larger in front of this--larger, in fact, than any other cells of the hypoblast. Outside the embryo they gradually increase in size towards the edge of the pellucid area. Here they are about 1/1000th of an inch in diameter, irregular in shape and rather angular.

The outlines of the cells of the epiblast at this time are easily distinguished from the cells of the hypoblast by being more elongated and angular; they are further distinguished by the presence of numerous small oval cells, frequently at the meeting point of several cells, at other times at points along the lines of junction of two cells (Pl. 1, fig. 12). These small cells look very like the smaller stomata of endothelial membranes, but are shewn to be cells by possessing a nucleus. There is considerable variation in size in the cells in different parts of the epiblast. Between the front lobes of the brain the cells are very small, 1/4000th inch, rising to 1/2000th on each side. They are about the latter size over the greater part of the embryo. But over the sinus rhomboidalis they fall again to from 1/3000th to 1/4000th inch. This is probably to be explained by the growth of the medullary fold at this point, which pushes back the primitive groove. At the sides of the head the cells are larger than anywhere else in the epiblast, being here about 1/1000th inch in diameter. I at present see no explanation of this fact. At the periphery of the pellucid area and over the vascular area the cells are 1/1500th to 1/2000th inch in diameter, but at the periphery of the opaque area they are smaller again, being about the 1/3000th of an inch. This smaller size at the periphery of the area opaca is remarkable, since in the earlier stages the most peripheral epiblast cells were the largest. It, perhaps, implies that more rapid growth is at this time taking place in that part of the epiblast which is spreading over the yolk sac.

EXPLANATION OF PLATE 1, Figs. 1-5 and 9-12.

Fig. 1. Section through an unincubated blastoderm, shewing the upper layer, composed of a single row of columnar cells, and the lower layer, composed of several rows of rounded cells in which no nucleus is visible. Some of the "formative cells," at the bottom of the segmentation cavity, are seen at (b).

Fig. 2. Section through the periphery of an eight hours' blastoderm, shewing the epiblast (p), the hypoblast (h), and the mesoblast commencing to be formed (c), partly by lower-layer cells enclosed between the epiblast and hypoblast, and partly by formative cells. Formative cells at the bottom of the segmentation cavity are seen at b. At s is one of the side folds parallel to the primitive groove.

Fig. 3. Portion of the hypoblast of a thirteen hours' blastoderm, treated with silver nitrate, shewing the great variation in the size of the cells at this period. An hour-glass shaped nucleus is seen at a.

Fig. 4. Periphery of a twenty-three hours' blastoderm, shewing cell for cell the junction between the hypoblast (h) and white-yolk spheres (w).

Fig. 5. Junction between the white-yolk spheres and the hypoblast cells at the passage from the area pellucida to the area opaca. The specimen was treated with silver nitrate to bring out the shape of the cells. The line of junction between the opaque and pellucid areas passes diagonally.

Fig. 9. Section through the primitive streak of an eight hours' blastoderm. The specimen shews the mesoblast very much thickened in the immediate neighbourhood of the primitive streak, but hardly formed at all on each side of the streak. It also shews the primitive groove just beginning to be formed (pr), and the fusion between the epiblast and the mesoblast under the primitive groove. The hypoblast is completely formed in the central part of the blastoderm. At f is seen one of the side folds parallel to the primitive groove. Its depth has been increased by the action of the chromic acid.

Fig. 10. Hypoblast cells from the hinder end of a thirty-six hours' embryo, treated with silver nitrate, shewing the regularity and elongated shape of the cells over the embryo and the smaller cells on each side.

Fig. 11. Epiblast cells from an unincubated blastoderm, treated with silver nitrate, shewing the regular hexagonal shape of the cells and the small spherules they contain.

Fig. 12. Portion of the epiblast of a thirty-six hours' embryo, treated with silver nitrate, shewing the small rounded cells frequently found at the meeting-points of several larger cells which are characteristic of the upper layer.

III. ON THE DISAPPEARANCE OF THE PRIMITIVE GROOVE IN THE EMBRYO CHICK[7].

Footnote 7: From the _Quarterly Journal of Microscopical Science_, Vol. XIII, 1873.

With Plate 1, Figs. 6-8 and 13-19.

The investigations of Dursy (Der Primitivstreif des Hühnchens, von Dr E. Dursy. Lahr, 1866) on the primitive groove, shewing that it is a temporary structure, and not connected with the development of the neural canal, have in this country either been ignored or rejected. They are, nevertheless, perfectly accurate; and had Dursy made use of sections to support his statements I do not think they would so long have been denied. In Germany, it is true, Waldeyer has accepted them with a few modifications, but I have never seen them even alluded to in any English work. The observations which I have made corroborating Dr Dursy may, perhaps, under these circumstances be worth recording.

After about twelve hours of incubation the pellucid area of a hen's egg has become somewhat oval, with its longer axis at right angles to the long axis of the egg. Rather towards the hinder (narrower) end of this an opaque streak has appeared, with a somewhat lighter line in the centre. A section made at the time shews that the opaque streak is due partly to a thickening of the epiblast, but more especially to a large collection of the rounded mesoblast cells, which along this opaque line form a thick mass between the epiblast and the hypoblast. The mesoblast cells are in contact with both hypoblast and epiblast, and appear to be fused with the latter. The line of junction between them can, however, almost always be made out.

Soon after the formation of this primitive streak a groove is formed along its central line by a pushing inwards of the epiblast. The epiblast is not thinner where it lines the groove, but the mass of mesoblast below the groove is considerably thinner than at its two sides. This it is which produces the peculiar appearance of the primitive groove when the blastoderm is viewed by transmitted light as a transparent line in the middle of an opaque one.

This groove, as I said above, is placed at right angles to the long axis of the egg, and nearer the hind end, that is, the narrower end of the pellucid area. It was called "the primitive groove" by the early embryologists, and they supposed that the neural canal arose from the closure of its edges above. It is always easy to distinguish this groove, in transverse sections, by several well-marked characters. In the first place, the epiblast and mesoblast always appear more or less fused together underneath it; in the second place, the epiblast does not become thinner where it lines the groove; and in the third place, the mesoblast beneath it never shews any signs of being differentiated into any organ.

As Dursy has pointed out, there is frequently to be seen in fresh specimens, examined as transparent objects, a narrow opaque line running down the centre of this groove. I do not know what this line is caused by, as there does not appear to be any structural feature visible in sections to which it can correspond.

From the twelfth to the sixteenth hour the primitive groove grows rapidly, and by the sixteenth hour is both absolutely and considerably longer than it was at the twelfth hour, and also proportionately longer as compared with the length of the pellucid area.

There is a greater interval between its end and that of the pellucid area in front than behind.

At about the sixteenth hour, or a little later, a thickening of the mesoblast takes place in front of the primitive groove, forming an opaque streak, which in fresh specimens looks like a continuation from the anterior extremity of the primitive groove (vide Pl. 1, fig. 8). From hardened specimens, however, it is easy to see that the connection of this streak with the primitive groove is only an apparent one. Again, it is generally possible to see that in the central line of this streak there is a narrow groove. I do not feel certain that there is no period when this groove may not be present, but its very early appearance has not been recognized either by Dursy or by Waldeyer. Moreover, both these authors, as also His, seem to have mistaken the opaque streak spoken of above for the notochord. This, however, is not the case, and the notochord does not make its appearance till somewhat later. The mistake is of very minor importance, and probably arose in Dursy's case from his not sufficiently making use of sections. At about the time the streak in front of the primitive groove makes its appearance a semicircular fold begins to be formed near the anterior extremity of the pellucid area, against which the opaque streak, or as it had, perhaps, better be called, "the medullary streak," ends abruptly.

This fold is the head fold, and the groove along the medullary streak is the medullary groove, which subsequently forms the cavity of the medullary or neural canal.

Everything which I have described above can without difficulty be made out from the examination of fresh and hardened specimens under the simple microscope; but sections bring out still more clearly these points, and also shew other features which could not have been brought to light without their aid. In Pl. 1, figs. 6 and 7, two sections of an embryo of about eighteen hours are shewn. The first of these passes through the medullary groove, and the second of them through the extreme anterior end of the primitive groove. The points of difference in the two sections are very obvious.

From fig. 6 it is clear that a groove has already been formed in the medullary streak, a fact which was not obvious in the fresh specimen. In the second place the mesoblast is thickened both under the groove and also more especially in the medullary folds at the sides of the groove; but shews hardly a sign of the differentiation of the notochord. So that it is clear that the medullary streak is not the notochord, as was thought to be the case by the authors above mentioned. In the third place there is no adhesion between the epiblast and the mesoblast. In all the sections I have cut through the medullary groove I have found this feature to be constant; while (for instance, as in Pl. 1, figs. 7, 9, 17) all sections through the primitive groove shew most clearly an adhesion between the epiblast and mesoblast. This fact is both strongly confirmatory of the separate origins of the medullary and primitive grooves, and is also important in itself, as leaving no loophole for supposing that in the region of embryo there is any separation of the cells from the epiblast to form the mesoblast.

By this time the primitive groove has attained its maximum growth, and from this time begins both absolutely to become smaller, and also gradually to be pushed more and more backwards by the growth of the medullary groove.

The specimen figured in Pl. 1, fig. 18, magnified about ten diameters, shews the appearance presented by an embryo of twenty-three hours. The medullary groove (mc) has become much wider and deeper than it was in the earlier stage; the medullary folds (A) are also broader and more conspicuous. The medullary groove widens very much posteriorly, and also the medullary folds separate far apart to enclose the anterior end of the primitive groove (pr).

All this can easily be seen with a simple microscope, but the sections taken from the specimen figured also fully bear out the interpretations given above, and at the same time shew that the notochord has at this age begun to appear. The sections marked 13-17 pass respectively through the lines with corresponding numbers in fig. 18. Section 1 (fig. 13) passes through the middle of the medullary canal.

In it the following points are to be noted. (1) That the epiblast becomes very much thinner where it lines the medullary canal (mc), a feature never found in the epiblast lining the primitive groove. (2) That the mesoblast is very much thickened to form the medullary folds at A, A, while there is no adherence between it and the epiblast, below the primitive groove. (3) The notochord (ch) has begun to be formed, though its separation from the rest of the mesoblast is not as yet very distinct[8].

Footnote 8: In the figure the notochord has been made too distinct.

In fig. 14 the medullary groove has become wider and the medullary folds broader, the notochord has also become more expanded: the other features are the same as in section 1. In the third section (fig. 15) the notochord is still more expanded; the bottom of the now much expanded medullary groove has become raised to form the ridge which separates the medullary from the primitive groove. The medullary folds are also flatter and broader than in the previous section. Section 4 (fig. 16) passes through the anterior end of the primitive groove. Here the notochord is no longer visible, and the adherence between the mesoblast and epiblast below the primitive groove comes out in marked contrast with the entire separation of the two layers in the previous sections.

The medullary folds (A) are still visible outside the raised edges of the primitive groove, and are as distinctly as possible separate and independent formations, having no connection with the folds of the primitive groove. In the last section (fig. 17), which is taken some way behind section 4, no trace of the medullary folds is any longer to be seen, and the primitive groove has become deeper. This series of sections, taken in conjunction with the specimen figured in fig. 18, must remove all possible doubt as to the total and entire independence of the primitive and medullary grooves. They arise in different parts of the blastoderm; the one reaches its maximum growth before the other has commenced to be formed; and finally, they are distinguished by almost every possible feature by which two such grooves could be distinguished.

Soon after the formation of the notochord, the protovertebræ begin to be formed along the sides of the medullary groove (Pl. 1, fig. 19, pv). Each new protovertebra (of those which are formed from before backwards) arises just in front of the anterior end of the primitive groove. As growth continues, the primitive groove becomes pushed further and further back, and becomes less and less conspicuous, till at about thirty-six hours only a very small and curved remnant is to be seen behind the sinus rhomboidalis; but even up to the forty-ninth Dursy has been able to distinguish it at the hinder end of the embryo.

The primitive groove in the chick is, then, a structure which appears very early, and soon disappears without entering directly into the formation of any part of the future animal, and without, so far as I can see, any function whatever. It is clear, therefore, that the primitive groove must be the rudiment of some ancestral feature; but whether it is a rudiment of some structure which is to be found in reptiles, or whether of some earlier form, I am unable to decide. It is just possible that it is the last trace of that involution of the epiblast by which the hypoblast is formed in most of the lower animals. The fact that it is formed in the hinder part of the pellucid area perhaps tells slightly in favour of this hypothesis, since the point of involution of the epiblast not unfrequently corresponds with the position of the anus.

EXPLANATION OF PLATE 1, Figs. 6-8 and 13-19.

Figs. 6 and 7 are sections through an embryo rather earlier than the one drawn in fig. 8. Fig. 6 passes through the just commencing medullary groove (md), which appears in fresh specimens, as in fig. 8, merely as an opaque streak coming from the end of the primitive groove. The notochord is hardly differentiated, but the complete separation of mesoblast and hypoblast under the primitive groove is clearly shewn. Fig. 7 passes through the anterior end of the primitive groove (pr), and shews the fusion between the mesoblast and epiblast, which is always to be found under the primitive groove.

Fig. 8 is a view from above of a twenty hours' blastoderm, seen as a transparent object. Primitive groove (pr). Medullary groove (md), which passes off from the anterior end of the primitive groove, and is produced by the thickening of the mesoblast. Head fold (pf).

Figs. 13-17 are sections through the blastoderm, drawn in fig. 18 through the lines 1, 2, 3, 4, 5 respectively.

The first section (fig. 13) passes through the true medullary groove (mc); the two medullary folds (A, A) are seen on each side with the thickened mesoblast, and the mesoblast cells are beginning to form the notochord (nc) under the medullary groove. There is no adherence between the mesoblast cells and the epiblast under the medullary groove.

The second (fig. 14) section passes through the medullary groove where it has become wider. Medullary folds, A, A; notochord, ch.

In the third section (fig. 15) the notochord (ch) is broader, and the epiblast is raised in the centre, while the medullary folds are seen far apart at A.

In section fig. 16 the medullary folds (A) are still to be seen enclosing the anterior end of the primitive groove (pr). Where the primitive groove appears there is a fusion of the epiblast and mesoblast, and no appearance of the notochord.

In the last section, fig. 17, no trace is to be seen of the medullary folds.

Figs. 18 and 19 are magnified views of two hardened blastoderms. Fig. 18 is twenty-three hours old; fig. 19 twenty-five hours. They both shew how the medullary canal arises entirely independently of the primitive groove and in front of it, and also how the primitive groove gets pushed backwards by the growth of the medullary groove. pv, Protovertebræ; other references as above. Fig. 18 is the blastoderm from which sections figs. 13-17 were cut.

IV. THE DEVELOPMENT OF THE BLOOD-VESSELS OF THE CHICK[9].

Footnote 9: From the _Quarterly Journal of Microscopical Science_, Vol. XIII, 1873.

With Plate 2.

The development of the first blood-vessels of the yolk-sac of the chick has been investigated by a large number of observers, but with very discordant results. A good historical résumé of the subject will be found in a paper of Dr Klein (liii. Band der K. Akad. der Wissensch. Wien), its last investigator.

The subject is an important one in reference to the homologies of the blood-vascular system of the vertebrata. As I shall shew in the sequel (and on this point my observations agree with those of Dr Klein), the blood-vessels of the chick do not arise as spaces or channels between the cells of the mesoblast; on the contrary, they arise as a network formed by the united processes of mesoblast-cells, and it is through these processes, and not in the spaces between them, that the blood flows. It is, perhaps, doubtful whether a system of vessels arising in this way can be considered homologous with any vascular system which takes its origin from channels hollowed out in between the cells of the mesoblast.

My own researches chiefly refer to the development of the blood-vessels in the pellucid area. I have worked but very slightly at their development in the vascular area; but, as far as my observations go, they tend to prove that the mode of their origin is the same, both for the pellucid and the vascular area.

The method which I have principally pursued has been to examine the blastoderm from the under surface. It is very difficult to obtain exact notions of the mode of development of the blood-vessels by means of sections, though these come in as a valuable confirmation of the other method.

For the purpose of examination I have employed (1) fresh specimens; (2) specimens treated with spirit, and then mounted in glycerine; (3) specimens treated with chloride of gold for about half a minute, and then mounted in glycerine; and (4) specimens treated with osmic acid.

All these methods bring out the same appearances with varying clearness; but the successful preparations made by means of the gold chloride are the best, and bring out the appearances with the greatest distinctness.

The first traces of the blood-vessels which I have been able to distinguish in the pellucid area are to be seen at about the thirtieth hour or slightly earlier, at about the time when there are four to five protovertebræ on each side.

Fig. 1 shews the appearance at this time. Immediately above the hypoblast there are certain cells whose protoplasm sends out numerous processes. These processes vary considerably in thickness and size, and quickly come in contact with similar processes from other cells, and unite with them.

I have convinced myself, by the use of the hot stage, that these processes continually undergo alteration, sometimes uniting with other processes, sometimes becoming either more elongated and narrower or broader and shorter. In this way a network of somewhat granular protoplasm is formed with nuclei at the points from which the processes start.

From the first a difference may be observed in the character of this network in different parts of the pellucid area. In the anterior part the processes are less numerous and thicker, the nuclei fewer, and the meshes larger; while in the posterior part the processes are generally very numerous, and at first thin, the meshes small, and the nuclei more frequent. As soon as this network commences to be formed the nuclei begin to divide. I have watched this take place with the hot stage. It begins by the elongation of the nucleus and division of the nucleolus, the parts of which soon come to occupy the two ends of the nucleus. The nucleus becomes still longer and then narrows in the centre and divides. By this means the nuclei become much more numerous, and are found in almost all the larger processes. Whether they are carried out into the processes by the movement of the surrounding protoplasm, or whether they move through the protoplasm, I have been unable to determine; the former view, however, seems to be the most probable.

It is possible that some nuclei arise spontaneously in the protoplasm, but I am much more inclined to think that they are all formed by the division of pre-existing nuclei--a view favoured by the number of nuclei which are seen to possess two nucleoli. Coincidently with the formation of the new nuclei the protoplasm of the processes, as well as that surrounding the nuclei at the starting-points of the processes, begins to increase in quantity.

At these points the nuclei also increase more rapidly than elsewhere, but at first the resulting nuclei seem to be all of the same kind.

In the anterior part of the pellucid area (fig. 4) the increase in the number of nuclei and in the amount of protoplasm at the starting-points of the protoplasm is not very great, but in the posterior part the increase in the amount of the protoplasm at these points is very marked, and coincidently the increase in number of the nuclei is also great. This is shewn in figs. 2 and 3. These are both taken from the tail end of an embryo of about thirty-three hours, with seven or eight protovertebræ. Fig. 3 shews the processes beginning to increase in thickness, and also the protoplasm at the starting-points increasing in quantity; at the same time the nuclei at these points are beginning to become more numerous. Fig. 3 is taken from a slightly higher level, i.e. slightly nearer the epiblast. In it the protoplasm is seen to have increased still more in quantity, and to be filled with nuclei. These nuclei have begun to be slightly coloured, and one of them is seen to possess two nucleoli.

Very soon after this a change in the nuclei begins to be observed, more especially in the hinder part of the embryo. While before this time they were generally elongated, some of them now become more nearly circular. In addition to this, they begin to have a yellowish tinge, and the nuclei, when treated with gold (for in the fresh condition it is not easy to see them distinctly), have a more jagged and irregular appearance than the nucleoli of the other nuclei.

This change takes place especially at the starting-points of the processes, so that the appearance presented (fig. 5) is that of spherical masses of yellowish nuclei connected with other similar spherical masses by protoplasmic processes, in which nuclei of the original type are seen imbedded. These masses are surrounded by a thin layer of protoplasm, at the edge of which a normal nucleus may here and there be detected, as at fig. 5, a and a´, the latter possessing two nucleoli. Some of these processes are still very delicate, and it is exceedingly probable that they undergo further changes of position before the final capillary system is formed.

These differentiated nuclei are the first stage in the formation of the blood-corpuscles. From their mode of formation it is clear that the blood-corpuscles of the Sauropsida are to be looked upon as nuclei containing nucleoli, rather than as cells containing nuclei; indeed, they seem to be merely ordinary nuclei with red colouring matter.

This would make them truly instead of only functionally homologous with the red corpuscles of the Mammalia, and would well agree with the fact that the red corpuscles of Mammalia, in their embryonic condition, possess what have previously been called nuclei, but which might perhaps more properly be called nucleoli.

In the anterior part of the blastoderm the processes, as I have stated, are longer and thinner, and the spaces enclosed between them are larger. This is clearly brought out in Pl. 2, fig. 4. But, besides these large spaces, there are other smaller spaces, such as that at v. It is, on account of the transparency of the protoplasm, very difficult to decide whether these are vacuoles or simply spaces enclosed by the processes, but I am inclined to think that they are merely spaces. The difficulty of exactly determining this point is increased by the presence of numerous white-yolk spherules in the hypoblast above, which considerably obscure the view. At about the same time that the blood-corpuscles appear in the posterior end of the pellucid area, or frequently a little later, they begin to be formed in the anterior part also. The masses of them are, however, far smaller and far fewer than in the posterior part of the embryo. It is at the tail end of the pellucid area that the chief formation of blood-corpuscles takes place.

The part of the pellucid area intermediate in position between the anterior and posterior ends of the embryo is likewise intermediate as regards the number of corpuscles formed and the size of the spaces between the processes; the spaces being here larger than at the posterior extremity, but smaller than the spaces in front. Close to the sides of the embryo the spaces are, however, smaller than in any other part of the pellucid area. It is, however, in this part that the first formation of blood-corpuscles takes place, and that the first complete capillaries are formed.

We have then somewhat round protoplasmic masses filled with blood-corpuscles and connected by means of processes, a few of which may begin to contain blood-corpuscles, but the majority of which only contain ordinary nuclei. The next changes to be noticed take place in the nuclei which were not converted into blood-corpuscles, but which were to be seen in the protoplasm surrounding the corpuscles. They become more numerous and smaller, and, uniting with the protoplasm in which they were imbedded, become converted into flat cells (spindle-shaped in section), and in a short time form an entire investment for the masses of blood-corpuscles. The same change also occurs in the protoplasmic processes which connect the masses of corpuscles. In the case of those processes which contain no corpuscles the greater part of their protoplasm seems to be converted into the protoplasm of the spindle-shaped cells. The nuclei arrange themselves so as completely to surround the exterior of the protoplasmic processes. In this way each process becomes converted into a hollow tube, completely closed in by cells formed from the investment of the original nuclei by the protoplasm which previously formed the solid processes. The remainder of the protoplasm probably becomes fluid, and afterwards forms the plasma in which the corpuscles float. While these changes are taking place the formation of the blood-corpuscles does not stand still, and by the time a system of vessels, enclosed by cellular walls, is formed out of the protoplasmic network, a large number of the connecting processes in this network have become filled with blood-corpuscles. The appearances presented by the network at a slightly later stage than this is shewn in Pl. 2, fig. 6, but in this figure all the processes are seen to be filled with blood-corpuscles.

This investment of the masses of corpuscles by a cellular wall occurs much earlier in some specimens than in others, both in relation to the time of incubation and to the completion of the network. It is generally completed in some parts by the time there are eight or nine protovertebræ, and is almost always formed over a great part of the pellucid area by the thirty-sixth hour. The formation of the corpuscles, as was pointed out above, occurs earliest in the central part of the hour-glass shaped pellucid area, and latest in its anterior part. In the hinder part of the pellucid area the processes, as well as their enlarged starting-points, become entirely filled with corpuscles; this, however, is by no means the case in its anterior part. Here, although the corpuscles are undoubtedly developed in parts as shewn in fig. 7, yet a large number of the processes are entirely without them. Their development, moreover, is in many cases very much later. When the development has reached the stage described, very little is required to complete the capillary system. There are always, of course, a certain number of the processes which end blindly, and others are late in their development, and are not by this time opened; but, as a general rule, when the cellular investment is formed for the masses of corpuscles, there is completed an open network of tubes with cellular walls, which are more or less filled with corpuscles. These become quickly driven into the opaque area in which at that time more corpuscles may almost always be seen than in the pellucid area.

By the formation of a network of this kind it is clear that there must result spaces enclosed between the walls of the capillaries; these spaces have under the microscope somewhat the appearance of being vesicles enclosed by walls formed of spindle-shaped cells. In reality they are only spaces enclosed at the sides, and, as a general rule, not above and below. They have been mistaken by some observers for vesicles in which the corpuscles were supposed to be developed, and to escape by the rupture of the walls into the capillary spaces between. This mistake has been clearly pointed out by Klein (loc. cit.).

At the time when these spaces are formed, and especially in the hinder two-thirds of the pellucid area, and in the layer of blood-vessels immediately above the hypoblast, a formation takes place which forms in appearance a secondary investment of the capillaries. Dr Klein was the first to give a correct account of this formation. It results from the cells of the mesoblast in the meshes of the capillary system. Certain of these cells become flattened, and send out fine protoplasmic processes. They arrange themselves so as completely to enclose the spaces between the capillaries, forming in this way vesicles.

Where seen on section (vide fig. 6) at the edge of the vesicles these cells lining the vesicles appear spindle-shaped, and look like a secondary investment of the capillaries. This investment is most noticeable in the hinder two-thirds of the pellucid area; but, though less conspicuous, there is a similar formation in its anterior third, where there would seem to be only veins present. Dr Klein (loc. cit., fig. 12) has also drawn this investment in the anterior third of the pellucid area. He has stated that the vessels in the mesoblast between the splanchnopleure and the somatopleure, and which are enclosed by prolongations from the former, do not possess this secondary investment; he has also stated that the same is true for the sinus terminalis; but I am rather doubtful whether the generalisation will hold, that veins and arteries can from the first be distinguished by the latter possessing this investment. I am also rather doubtful whether the spaces enclosed by the protoplasmic threads between the splanchnopleure and somatopleure are the centres of vessels at all, since I have never seen any blood-corpuscles in them.

It is not easy to learn from sections much about the first stages in the formation of the capillaries, and it is impossible to distinguish between a completely-formed vessel and a mere spherical space. The fine protoplasmic processes which connect the masses of corpuscles can rarely be seen in sections, except when they pass vertically, as they do occasionally (vide Pl. 2, fig. 9) in the opaque area, joining the somatopleure and the splanchnopleure. Dr Klein considers these latter processes to be the walls of the vessels, but they appear rather to be the processes which will eventually become new capillaries.

From sections, however, it is easy to see that the appearances of the capillaries in the vascular area are similar to the appearances in the pellucid area, from which it is fair to conclude that their mode of formation is the same in both. It is also easy to see that the first formation of vessels occurs in the splanchnopleure, and that even up to the forty-fifth hour but few or no vessels are found in the somatopleure. The mesoblast of the somatopleure is continued into the opaque area as a single layer of spindle-shaped cells.

Sections clearly shew in the case of most of the vessels that the secondary investment of Klein is present, even in the case of those vessels which lie immediately under the somatopleure.

In reference to the origin of particular vessels I have not much to say. Dr Klein's account of the origin of the sinus terminalis is quite correct. It arises by a number of the masses of blood-corpuscles, similar to those described above, becoming connected together by protoplasmic processes. The whole is subsequently converted into a continuous vessel in the usual way.

From the first the sinus terminalis possesses cellular walls, as is clear from its mode of origin. I am inclined to think that Klein is right in saying that the aortæ arise in a similar manner, but I have not worked out their mode of origin very fully.

It will be seen from the account given above that, in reference to the first stages in the development of the blood-vessels, my observations differ very considerably from those of Dr Klein; as to the later stages, however, we are in tolerable agreement. We are in agreement, moreover, as to the fundamental fact that the blood-vessels are formed by a number of cells becoming connected, and by a series of changes converted into a network of vessels, and that they are not in the first instance merely channels between the cells of the mesoblast.

By the forty-fifth hour colourless corpuscles are to be found in the blood whose exact origin I could not determine; probably they come from the walls of the capillaries.

In the vessels themselves the coloured corpuscles undergo increase by division, as has already been shewn by Remak. Corpuscles in the various stages of division may easily be found. They do not appear to show very active amoeboid movements in the vessels, though their movements are sometimes very active when removed from the body.

To recapitulate--some of the cells of the mesoblast of the splanchnopleure send out processes, these processes unite with the processes from other cells, and in this way a network is formed. The nuclei of the original cells divide, and at the points from which the processes start their division is especially rapid. Some of them acquire especially at these points a red colour, and so become converted into blood-corpuscles; the others, together with part of the protoplasm in which they are imbedded, become converted into an endothelium both for the processes and the masses of corpuscles; the remaining protoplasm becomes fluid, and thus the original network of the cells becomes converted into a network of hollow vessels, filled with fluid, in which corpuscles float.

In reference to the development of the heart, my observations are not quite complete. It is, however, easy to prove from sections (vide figs. 10 and 11, Pl. 2) that the cavity of the heart is produced by a splitting or absorption of central cells of the thickened mesoblast of the splanchnopleure, while its muscular walls are formed from the remaining cells of this thickened portion. It is produced in the following way:--When the hypoblast is folded in to form the alimentary canal the mesoblast of the splanchnopleure follows it closely, and where the splanchnopleure turns round to assume its normal direction (fig. 11) its mesoblast becomes thickened. This thickened mass of mesoblast is, as can easily be seen from figs. 10 and 11, Pl. 2, entirely distinct from the mesoblast which forms the outside walls of the alimentary canal. At the point where this thickening occurs an absorption takes place to form the cavity of the heart. The method in which the cavity is formed can easily be seen from figs. 10 and 11. It is in fig. 11 shewn as it takes place in the mesoblast on each side, the folds of the splanchnopleure not having united in the middle line; and hence a pair of cavities are formed, one on each side. It is, however, probable that, in the very first formation of the heart, the cavity is single, being formed after the two ends of the folded mesoblast have united (vide hz, fig. 10). In some cases the two folds of the mesoblast appear not at first to become completely joined in the middle line; in this case the cavity of the heart is still complete from side to side, but the mesoblast-cells which form its muscular walls are deficient above. By the process of absorption, as I said, a cavity is produced in the thickened part of the mesoblast of the splanchnopleure, a cavity which is single in front, but becomes divided further behind, where the folds of the mesoblast have not united, into two cavities, to form the origin of the omphalomeseraic veins. As the folding proceeds backwards the starting-point of the omphalomeseraic veins is also pushed backwards, and the cavities which were before separated become joined together. From its first formation the heart is lined internally by an endothelium; this is formed of flattened cells, spindle-shaped in section. The exact manner of the origin of this lining I have not been able to determine; it is, however, probable that some of the central mesoblast-cells are directly converted into the cells of the endothelium.

I have obtained no evidence enabling me to determine whether Dr Klein is correct in stating that the cells of the mesoblast in the interior of the heart become converted partly into blood-corpuscles and partly into a cellular lining forming the endothelium of the heart, in the same way that the blood-vessels in the rest of the blastoderm are formed. But I should be inclined to think that it is very probable--certainly more probable than that the cavity of the heart is formed by a process of splitting taking place. Where I have used the word "absorption" in speaking of the formation of the cavity of the heart, I must be understood as implying that certain of the interior cells become converted into the endothelium, while others either form the plasma or become blood-corpuscles.

The originally double formation of the hinder part of the heart probably explains Dr Afanassiev's statement (Bullétin de l'Académ. Impériale de St Petersb., tom. xiii, pp. 321-335), that he finds the endothelium of the heart originally dividing its interior into two halves; for when the partition of the mesoblast which separated at first the two halves of the heart became absorbed, the endothelium lining of each of the originally separate vessels would remain complete, dividing the cavity of the heart into two parts. The partition in the central line is, however, soon absorbed.

The account given above chiefly differs from that of Remak by not supposing that the mesoblast-cells which form the heart are in any way split off from the wall of the alimentary canal.

There can be no doubt that His is wrong in supposing that the heart originates from the mesoblast of the splanchnopleure and somatopleure uniting to form its walls, thus leaving a cavity between them in the centre. The heart is undoubtedly formed out of the mesoblast of the splanchnopleure only.

Afanassiev's observations are nearer to the truth, but there are some points in which he has misinterpreted his sections.

Sections Pl. 2, figs. 10 and 11, explain what I have just said about the origin of the heart. Immediately around the notochord the mesoblast is not split, but a very little way outside it is seen to be split into two parts so and sp; the former of these follows the epiblast, and together with it forms the somatopleure, which has hardly begun to be folded at the line where the sections are taken. The latter (sp) forms with the hypoblast (hy) the splanchnopleure, and thus has become folded in to form the walls of the alimentary canal (d). In fig. 11 the folds have not united in the central line, but in fig. 10 they have so united. In fig. 11, where the mesoblast, still following the hypoblast, turns back to assume its normal direction, it is seen to be thickened and to have become split, so that a cavity (of) (of the omphalomeseraic vein) is formed in it on each side, lined by endothelium.

In the section immediately behind section fig. 11 the mesoblast was thickened, but had not become split.

In fig. 10 the hypoblast folds are seen to have united in the centre, so as to form a completely closed digestive canal (d); the folds of the mesoblast have also united, so that there is only a single cavity in the heart (hz), lined, as was the case with the omphalomeseraic veins, by endothelium.

In conclusion, I have to thank Dr Foster for his assistance and suggestions throughout the investigations which have formed the subject of these three short papers, and which were well carried on in the apartments used by him as a Physiological Laboratory.

EXPLANATION OF PLATE 2.

Fig. 1 is taken from the anterior part of the pellucid area of a thirty hours' chick, with four protovertebræ. At n is a nucleus with two nucleoli.

Figs. 2 and 3 are taken from the posterior end of the pellucid area of a chick with eight protovertebræ. In fig. 3 the nuclei are seen to have considerably increased in number at the points of starting of the protoplasmic processes. At n is seen a nucleus with two nucleoli.

Fig. 4 is taken from the anterior part of the pellucid area of an embryo of thirty-six hours. It shews the narrow processes characteristic of the anterior part of the pellucid area, and the fewer nuclei. Small spaces, which have the appearance of vacuoles, are shewn at v.

Fig. 5 is taken from the posterior part of the pellucid area of a thirty-six hours' embryo. It shews the nuclei, with somewhat irregular nucleoli, which have begun to acquire the red colour of blood-corpuscles; the protoplasmic processes containing the nuclei; the nuclei in the protoplasm surrounding the corpuscles, as shewn at a, a´.

Fig. 6 shews fully formed blood-vessels, in part filled with blood-corpuscles and in part empty. The walls of the capillaries, formed of cells, spindle-shaped in section, are shewn, and also the secondary investment of Klein at k, and at b is seen a narrow protoplasmic process filled with blood-corpuscles.

Fig. 7 is taken from the anterior part of the pellucid area of a thirty-six hours' embryo. It shews a collection of nuclei which are beginning to become blood-corpuscles.

Figs. 1-5 are drawn with an 1/8 object-glass. Fig. 6 is on a much smaller scale. Fig. 7 is intermediate.

Fig. 8.--A transverse section through the dorsal region of a forty-five hours' embryo; ao, aorta with a few blood-corpuscles. v, Blood-vessels, all of them being formed in the splanchnopleure, and all of them provided with the secondary investment of Klein; pe, pellucid area; op, opaque area.

Fig. 9.--Small portion of a section through the opaque area of a thirty-five hours' embryo, showing protoplasmic processes, with nuclei passing from the somatopleure to the splanchnopleure.

Fig. 10.--Section through the heart of a thirty-four hours' embryo. a. Alimentary canal; hb, hind brain; nc, notochord; e, epiblast; so, mesoblast of the somatopleure; sp, mesoblast of the splanchnopleure; hy, hypoblast; hz, cavity of the heart.

Fig. 11.--Section through the same embryo as fig. 10, and passing through the orifice of the omphalomeseraic vein. of, Omphalomeseraic vein; other references as above.

These two sections shew that the heart is entirely formed from the mesoblast of the splanchnopleure, and that it is formed by the splitting of that part of the mesoblast which has turned to assume its normal direction after being folded in to form the muscular wall of the alimentary canal. In fig. 11 the cavities so formed on each side have not yet united, but in fig. 10 they have united. When the folding becomes more complete the cavities (of, of) in fig. 11 will unite, and in this way the origin of the omphalomeseraic veins will be carried further backwards. In the section immediately behind section 11 the mesoblast had become thickened, but had not split.

V. A PRELIMINARY ACCOUNT OF THE DEVELOPMENT OF THE ELASMOBRANCH FISHES[10].

Footnote 10: From the Quarterly Journal of Microscopical Science, Vol. XIV. 1874. Read in Section D, at the Meeting of the British Association at Belfast.

With Plates 3 and 4.

During the spring of the present year I was studying at the Zoological Station, founded by Dr Dohrn at Naples, and entirely through its agency was supplied with several hundred eggs of various species of Dog-fish (Selachii)--a far larger number than any naturalist has previously had an opportunity of studying. The majority of the eggs belonged to an oviparous species of Mustelus, but in addition to these I had a considerable number of eggs of two or three species of Scyllium, and some of the Torpedo. Moreover, since my return to England, Professor Huxley has most liberally given me several embryos of Scyllium stellare in a more advanced condition than I ever had at Naples, which have enabled me to fill up some lacunæ in my observations.

On many points my investigations are not yet finished, but I have already made out a number of facts which I venture to believe will add to our knowledge of vertebrate embryology; and since it is probable that some time will elapse before I am able to give a complete account of my investigations, I have thought it worth while preparing a preliminary paper in which I have briefly, but I hope in an intelligible manner, described some of the more interesting points in the development of the Elasmobranchii. The first-named species (Mustelus sp.?) was alone used for the early stages, for the later ones I have also employed the other species, whose eggs I have had; but as far as I have seen at present, the differences between the various species in early embryonic life are of no importance.

Without further preface I will pass on to my investigations.

The Egg-shell.

In the eggs of all the species of Dog-fishes which I have examined the yolk lies nearest that end of the quadrilateral shell which has the shortest pair of strings for attachment. This is probably due to the shape of the cavity of the shell, and is certainly not due to the presence of any structures similar to chalazæ.

The Yolk.

The yolk is not enclosed in any membrane comparable to the vitelline membrane of Birds, but lies freely in a viscid albumen which fills up the egg-capsule. It possesses considerable consistency, so that it can be removed into a basin, in spite of the absence of a vitelline membrane, without falling to pieces. This consistency is not merely a property of the yolk-sphere as a whole, but is shared by every individual part of it.

With the exception of some finely granular matter around the blastoderm, the yolk consists of rather small, elliptical, highly refracting bodies, whose shape is very characteristic and renders them easily recognizable. A number of striæ like those of muscle are generally visible on most of the spherules, which give them the appearance of being in the act of breaking up into a series of discs; but whether these striæ are normal, or produced by the action of water I have not determined.

Position of the Blastoderm.

The blastoderm is always situated, immediately after impregnation, near the pole of the yolk which lies close to the end of the egg-capsule. Its position varies a little in the different species and is not quite constant in different eggs of the same species. But this general situation is quite invariable. It is of about the same proportional size as the blastoderm of a bird.

Segmentation.

In a fresh specimen, in which segmentation has only just commenced, the blastoderm or germinal disc appears as a circular disc, distinctly marked off by a dark line from the rest of the yolk. This line, as is proved by sections, is the indication of a very shallow groove. The appearance of sharpness of distinction between the germ and the yolk is further intensified by their marked difference of colour, the germ itself being usually of a darker shade than the remainder of the yolk; while around its edge, and apparently sharply separated from it by the groove before mentioned, is a ring of a different shade which graduates at its outer border into the normal shade of the yolk.

These appearances are proved by transverse sections to be deceptive. There is no sharp line either at the sides or below separating the blastoderm from the yolk. In the passage between the fine granular matter of the germ to the coarser yolk-spheres every intermediate size of granule is present; and, though the space between the two is rather narrow, in no sense of the word can there be said to be any break or line between them.

This gradual passage stands in marked contrast with what we shall find to be the case at the close of the segmentation. In the youngest egg which I had, the germinal disc was already divided into four segments by two furrows at right angles. These furrows, however, did not reach its edge; and from my sections I have found that they were not cut off below by any horizontal furrow. So that the four segments were continuous below with the remainder of the germ without a break.

In the next youngest specimen which I had, there were already present eighteen segments, somewhat irregular in size, but which might roughly be divided into an outer ring of larger spheres, separated, as it were, by a circular furrow from an inner series of smaller segments. The furrows in this case reached quite to the edge of the germinal disc.

The remarks I made in reference to the earlier specimen about the separation of the germ from the yolk apply in every particular to the present one. The external limit of the blastoderm was not defined by a true furrow, and the segmentation furrows still ended below without meeting any horizontal furrows, so that the blastoderm was not yet separated by any line from the remainder of the yolk, and the segments of which it was composed were still only circumscribed upon five sides. In this particular the segmentation in these animals differs materially from that in the Bird, where the horizontal furrows appear very early.

In each segment a nucleus was generally to be seen in sections. I will, however, reserve my remarks upon the nature of the nuclei till I discuss the nuclei of the blastoderm as a whole.

For some little time the peripheral segments continue larger than the more central ones, but this difference of size becomes less and less marked, and before the segments have become too small to be seen with the simple microscope, their size appears to be uniform over the whole surface of the blastoderm.

In the blastoderms somewhat older than the one last described the segments have already become completely separate masses, and each of them already possesses a distinct nucleus. They form a layer one or two segments deep. The limits of the blastoderm are not, however, defined by the already completed segments, but outside these new segments continue to be formed around nuclei which appear in the yolk. At this stage there is, therefore, no line of demarcation between the germ and the yolk, but the yolk is being bored into, so to speak, by a continuous process of fresh segmentation.

The further segmentation of the already existing spheres, and the formation of new ones from the yolk below and to the sides, continues till the central cells acquire their final size, the peripheral ones being still large, and undefined towards the yolk. These also soon reach the final size, and the blastoderm then becomes rounded off towards the yolk and sharply separated from it.

The Nuclei of the Yolk.

Intimately connected with the segmentation is the appearance and history of a number of nuclei which arise in the yolk surrounding the blastoderm.

When the horizontal furrows appear which first separate the blastoderm from the yolk, the separation does not occur along the line of passage from the fine to the coarse yolk, but in the former at some distance from this line.

The blastoderm thus rests upon a mass of finely granular material, from which, however, it is sharply separated. At this time there appear in this finely granular material a number of nuclei of a rather peculiar character.

They vary immensely in size--from that of an ordinary nucleus to a size greater than the largest blastoderm-cell.

In Pl. 3, fig. 1, n, is shewn their distribution in this finely granular matter and their variation in size. But whatever may be their size, they always possess the same characteristic structure. This is shewn in Pl. 3, figs. 1 and 2, n.

They are rather irregular in shape, with a tendency when small to be roundish, and are divided by a number of lines into distinct areas, in each of which a nucleolus is to be seen. The lines dividing them into these areas have a tendency (in the smaller specimens) to radiate from the centre, as shewn in Pl. 3, fig. 1.

These nuclei colour red with hematoxylin and carmine and brown with osmic acid, while the nucleoli or granules contained in the areas also colour very intensely with all the three above-named reagents.

With such a peculiar structure, in favourable specimens these nuclei are very easily recognised, and their distribution can be determined without difficulty. They are not present alone in the finely granular yolk, but also in the coarsely granular yolk adjoining it. They form very often a special row, sometimes still more markedly than in Pl. 3, fig. 1, along the floor of the segmentation cavity. They are not, however, found alone in the yolk. All the blastoderm-cells in the earlier stages possess precisely similar nuclei! From the appearance of the first nucleus in a segmentation-sphere till a comparatively late period in development, every nucleus which can be distinctly seen is found to be of this character. In Pl. 3, fig. 2, this is very distinctly shewn.

(1) We have, then, nuclei of this very peculiar character scattered through the sub-germinal granular matter, and also universally present in the cells of the blastoderm. (2) These nuclei are distributed in a special manner under the floor of the segmentation cavity on which new cells are continually appearing. Putting these two facts together, there would be the strongest presumption that these nuclei do actually become the nuclei of cells which enter the blastoderm, and such is actually the case. In my account of the segmentation I have, indeed, already mentioned this, and I will return to it, but before doing so will enter more fully into the distribution of these nuclei in the yolk.

They appear in small numbers around the blastoderm at the close of segmentation, and round each one of them there may at this time be seen in osmic acid specimens, and with high powers, a fine network similar to but finer than that represented in Pl. 3, fig. 2. This network cannot, as a general rule, be traced far into the yolk, but in some exceptionally thin specimens it may be seen in any part of the fine granular yolk around the blastoderm, the meshes of the network being, however, considerably coarser between than around the nuclei. This network may be seen in the fine granular material around the germ till the latest period of which I have yet cut sections of the blastoderm. In the later specimens, indeed, it is very much more distinctly seen than in the earlier, owing to the fact that in parts of the blastoderm, especially under the embryo, the yolk-granules have disappeared partly or entirely, leaving only this fine network with the nuclei in it.

A specimen of this kind is represented in Pl. 3, fig. 2, where the meshes of the network are seen to be finer immediately around the nuclei, and coarser in the intervals. The specimen further shows in the clearest manner that this network is not divided into areas, each representing a cell and each containing a nucleus. I do not know to what extent this network extends into the yolk. I have never yet seen the limits of it, though it is very common to see the coarsest yolk-granules lying in its meshes. Some of these are shewn in Pl. 3, fig. 2, yk.

This network of lines[11] (probably bubbles) is characteristic of many cells, especially ova. We are, therefore, forced to believe that the fine granular and probably coarser granular yolk of this meroblastic egg consists of an active organized basis with passive yolk-spheres imbedded in it. The organized basis is especially concentrated at the germinal pole of the egg, but becomes less and less in quantity, as compared with the yolk-spheres, the further we depart from this.

Footnote 11: The interpretation of this network is entirely due to Dr Kleinenberg, who suggested it to me on my shewing him a number of specimens exhibiting the nuclei and network.

Admitting, as I think it is necessary to do, the organized condition of the whole yolk-sphere, there are two possible views as to its nature. We may either take the view that it is one gigantic cell, the ovum, which has grown at the expense of the other cells of the egg-follicle, and that these cells in becoming absorbed have completely lost their individuality; or we may look upon the true formative yolk (as far as we can separate it from the remainder of the food-yolk) as the remains of one cell (the primitive ovum), and the remainder of the yolk as a body formed from the coalescence of the other cells of the egg-follicle, which is adherent to, but has not coalesced with, the primitive ovum, the cells in this case not having completely lost their individuality; and to these cells, the nuclei, I have found, must be supposed to belong.

The former view I think, for many reasons, the most probable. The share of these nuclei in the segmentation, and the presence of similar nuclei in the cells of the germ, both support it, and are at the same time difficulties in the way of the other view. Leaving this question which cannot be discussed fully in a preliminary paper like the present one, I will pass on to another important question, viz.:

How do these nuclei originate? Are they formed by the division of the pre-existing nuclei, or by an independent formation? It must be admitted that many specimens are strongly in favour of the view that they increase by division. In the first place, they are often seen "two together;" examples of this will be seen in Pl. 3, fig. 1. In the second place, I have found several specimens in which five or six appear close together, which look very much as if there had been an actual division into six nuclei. It is, however, possible in this case that the nuclei are really connected below and only appear separate, owing to the crenate form of the mass. Against this may be put the fact that the division of a nucleus is by no means so common as has been sometimes supposed, that in segmentation it has very rarely been observed that the nucleus of a sphere first divides[12], and that then segmentation takes place, but segmentation generally occurs and then a new nucleus arises in each of the newly formed spheres. Such nuclei as I have described are rare; they have, however, been observed in the egg of a Nephelis (one of the Leeches), and have in that case been said to divide. Dr Kleinenberg, however, by following a single egg through the whole course of its development, has satisfied himself that this is not the case, and that, further, these nuclei in Nephelis never form the nuclei of newly developing cells.

Footnote 12: Kowalevsky ("Beiträge zur Entwicklungsgeschichte der Holothurien," Mémoires de l'Ac. Imp. de St Petersbourg, vii ser., Vol. XI. 1867) describes the division of nuclei during segmentation in the Holothurians, and other observers have described it elsewhere.

I must leave it an open question, and indeed one which can hardly be solved from sections, whether these nuclei arise freely or increase by division, but I am inclined to believe that both processes may possibly take place. In any case their division does not appear to determine the segmentation or segregation of the protoplasm around them.

As was mentioned in my account of the segmentation, these nuclei first appear during that process, and become the nuclei of the freshly formed segmentation spheres. At the close of segmentation a few of them are still to be seen around the blastoderm, but they are not very numerous.

From this period they rapidly increase in number, up to the commencement of the formation of the embryo as a body distinct from the germ. Though before this period they probably become the nuclei of veritable cells which enter the germ, it is not till this period, when the growth of the blastoderm becomes very rapid and it commences to spread over the yolk, that these new cells are formed in large numbers. I have many specimens of this age which shew the formation of these new cells with great clearness. This is most distinctly to be seen immediately below the embryo, where the yolk-spherules are few in number. At the opposite end of the blastoderm I believe that more of these cells are formed, but, owing to the presence of numerous yolk-spherules, it is much more difficult to make certain of this.

As to the final destination of these cells, my observations are not yet completed. Probably a large number of them are concerned in the formation of the vascular system, but I will give reasons later on for believing that some of them are concerned in the formation of the walls of the digestive canal and of other parts.

I will conclude my account of these nuclei by briefly summarizing the points I have arrived at in reference to them.

A portion, or more probably the whole, of the yolk of the Dog-fish consists of organized material, in which nuclei appear and increase either by division or by a process of independent formation, and a great number of these subsequently become the nuclei of cells formed around them, frequently at a distance from the germ, which then travel up and enter it.

The formation of cells in the yolk, apart from the general process of segmentation, has been recognised by many observers. Kupffer (Archiv. für Micr. Anat., Bd. IV. 1868) and Owsjannikow ("Entwicklung der Coregonus," Bulletin der Akad. St Petersburgh, Vol. XIX.) in osseous fishes[13], Ray Lankester (Annals and Mag. of Nat. Hist. Vol. XI. 1873, p. 81) in Cephalopoda, Götte (Archiv. für Micr. Anat. Vol. X.) in the chick, have all described a new formation of cells from the so-called food-yolk. The organized nature of the whole or part of this, previous to the formation of the cells from it, has not, however, as a rule, been distinctly recognised. In the majority of cases, as, for instance, in Loligo, the nucleus is not the first thing to be formed, but a plastide is first formed, in which a nucleus subsequently makes its appearance.

Footnote 13: Götte, at the end of a paper on "The Development of the Layers in the Chick" (Archiv. für Micr. Anat., Vol. X. 1873, p. 196), mentions that the so-called cells in Osseous fishes which Oellacher states to have migrated into the yolk, and which are clearly the same as those mentioned by Owsjannikow, are really not cells, but large nuclei. If this statement is correct the phenomena in Osseous fishes are precisely the same as those I have described in the Dog-fish.

Formation of the Layers.

Leaving these nuclei, I will now pass on to the formation of the layers.

At the close of segmentation the surface of the blastoderm is composed of cells of a uniform size, which, however, are too small to be seen by the aid of the simple microscope.

The cells of this uppermost layer are somewhat columnar, and can be distinguished from the remainder of the cells of the blastoderm as a separate layer. This layer forms the epiblast; and the Dog-fish agree with Birds, Batrachians, and Osseous fish in the very early differentiation of it.

The remainder of the cells of the blastoderm form a mass, many cells deep, in which it is impossible as yet or till a very considerably later period to distinguish two layers. They may be called the lower layer cells. Some of them near the edge of this mass are still considerably larger than the rest, but they are, as a whole, of a fairly uniform size. Their nuclei are of the same character as the nuclei in the yolk.

There is one point to be noticed in the shape of the blastoderm as a whole. It is unsymmetrical, and a much larger number of its cells are found collected at one end than at the other. This absence of symmetry is found in all sections which are cut parallel to the long axis of the egg-capsule. The thicker end is the region where the embryo will subsequently appear.

This very early appearance of distinction in the blastoderm between the end at which the embryo will appear, and the non-embryonic end is important, especially as it shews the affinity of the modes of development of Osseous fishes and the Elasmobranchii. Oellacher (Zeitschrift für Wiss. Zoologie, Vol. XXXIII. 1873) has shewn, and, though differing from him on many other points, on this point Götte (Arch. für Micr. Anat. Vol. IX. 1873) agrees with him, that a similar absence of symmetry by which the embryonic end of the germ is marked off, occurs almost immediately after the end of segmentation in Osseous fishes. In the early stages of development there are a number of remarkable points of agreement between the Osseous fish and the Dog-fish, combined with a number of equally remarkable points of difference. Some of these I shall point out as I proceed with my description.

The embryonic end of the germ is always the one which points towards the pole of the yolk farthest removed from the egg-capsule.

The germ grows, but not very rapidly, and without otherwise undergoing any very appreciable change, for some time.

The growth at these early periods appears to be particularly slow, especially when compared with the rapid manner in which some of the later stages of the development are passed through.

The next important change which occurs is the formation of the so-called "segmentation cavity."

This forms a very marked feature throughout the early stages. It appears, however, to have somewhat different relations to the blastoderm than the homologous structure in other vertebrates. In its earliest stage which I have observed, it appears as a small cavity in the centre of the lower layer cells. This grows rapidly, and its roof becomes composed of epiblast and only a thin lining of "lower layer" cells, while its floor is formed by the yolk (Pl. 3, fig. 3, sg). In the next and third stage (Pl. 3, fig. 4, sg) its floor is formed by a thin layer of cells, its roof remaining as before. It has, however, become a less conspicuous formation than it was; and in the last (fourth) stage in which it can be distinguished it is very inconspicuous, and almost filled up by cells.

What I have called the second stage corresponds to a period in which no trace of the embryo is to be seen. In the third stage the embryonic end of the blastoderm projects outwards to form a structure which I shall speak of as the "embryonic rim," and in the fourth and last stage a distinct medullary groove is formed. For a considerable period during the second stage the segmentation cavity remains of about the same size; during the third stage it begins to be encroached upon, and becomes smaller both absolutely, and relatively to the increased size of the germ.

The segmentation cavity of the Dog-fish most nearly agrees with that of Osseous fishes in its mode of formation and relation to the embryo.

Dog-fish resemble Osseous fish in the fact that their embryos are entirely formed from a portion of the germ which does not form part of the roof of the segmentation cavity, so that the cells forming the roof of the segmentation cavity take no share at any time in the formation of their embryos. They further agree with Osseous fish (always supposing that the descriptions of Oellacher, loc. cit., and Götte, Archiv. für Micr. Anat. Bd. IX. are correct) in the floor of the segmentation cavity being formed at one period by yolk. Together with these points of similarity there are some important differences.

(1) The segmentation cavity in the Osseous fish from the first arises as a cavity between the yolk and the blastoderm, and its floor is never at any period covered with cells. In the Dog-fish, as we have said above, both in the earlier and later periods the floor is covered with cells.

(2) The roof in the Dog-fish is invariably formed by the epiblast and a row of flattened lower layer cells.

According to both Götte and Oellacher the roof of the segmentation cavity in Osseous fishes is in the earlier stages formed alone of the two layers which correspond with the single layer forming the epiblast in the Dog-fish. In Osseous fishes it is very difficult to distinguish the various layers, owing to the similarity of their component cells. In Dog-fish this is very easy, owing to the great distinctness of the epiblast, and it appears to me, on this account, very probable that the two above-named observers may be in error as to the constitution of its roof in the Osseous fish. With both the Bird and the Frog the segmentation cavity of the Dog-fish has some points of agreement, and some points of difference, but it would take me too far from my present subject to discuss them.

When the segmentation cavity is first formed, no great changes have taken place in the cells forming the blastoderm. The upper layer--the epiblast--is composed of a single layer of columnar cells, and the remainder of the cells of blastoderm, forming the lower layer, are of a fairly uniform size, and polygonal from mutual pressure. The whole edge of the blastoderm is thickened, but this thickening is especially marked at its embryonic end.

This thickened edge of the blastoderm is still more conspicuous in the next and second stage (Pl. 3, fig. 3).

In the second stage the chief points of progress, in addition to the increased thickness of the edge of the blastoderm, are--

(1) The increased thickness and distinctness of the epiblast, caused by its cells becoming more columnar, though it remains as a one-cell-thick layer.

(2) The disappearance of the cells from the floor of the segmentation cavity.

The lower layer cells have undergone no important changes, and the blastoderm has increased very little if at all in size.

From Pl. 3, fig. 3, it is seen that there is a far larger collection of cells at the embryonic than at the opposite end.

Passing over some rather unimportant stages, I will come to the next important one.

The general features of this (the third) stage in a surface view are--

(1) The increase in size of the blastoderm.

(2) The diminution in size of the segmentation cavity, both relatively and absolutely.

(3) The appearance of a portion of the blastoderm projecting beyond the rest over the yolk. This projecting rim extends for nearly half the circumference of the yolk, but is most marked at the point where the embryo will shortly appear. I will call it the "embryonic rim."

These points are still better seen from sections than from surface views, and will be gathered at once from an inspection of Pl. 3, fig. 4.

The epiblast has become still more columnar, and is markedly thicker in the region where the embryo will appear. But its most remarkable feature is that at the outer edge of the "embryonic rim" (er) it turns round and becomes continuous with the lower layer cells. This feature is most important, and involves some peculiar modifications in the development. I will, however, reserve a discussion of its meaning till the next stage.

The only other important feature of this stage is the appearance of a layer of cells on the floor of the segmentation cavity.

Does this layer come from an ingrowth from the thickened edge of the blastoderm, or does it arise from the formation of new cells in the yolk?

It is almost impossible to answer this question with certainty. The following facts, however, make me believe that the newly formed cells do play an important part in the formation of this layer.

(1) The presence at an earlier date of almost a row of nuclei under the floor of the segmentation cavity (Pl. 3, fig. 1).

(2) The presence on the floor of the cavity of such large cells as those represented in fig. 1, bd, cells which are very different, as far as the size and granules are concerned, from the remainder of the cells of the blastoderm.

On the other hand, from this as well as other sections, I have satisfied myself that there is a distinct ingrowth of cells from the embryonic swelling. It is therefore most probable that both these processes, viz. a fresh formation and an ingrowth, have a share in the formation of the layer of cells on the floor of the segmentation cavity.

In the next stage we find the embryo rising up as a distinct body from the blastoderm, and I shall in future speak of the body, which now becomes distinct as the embryo. It corresponds with what Kupffer (loc. cit.) in his paper on the "Osseous Fishes" has called the "embryonic keel." This starting-point for speaking of the embryo as a distinct body is purely arbitrary and one merely of convenience. If I wished to fix more correctly upon a period which could be spoken of as marking the commencing formation of the embryo, I should select the time when structures first appear to mark out the portion of the germ from which the embryo becomes formed; this period would be in the Elasmobranchii, as in the Osseous fish, at the termination of segmentation, when the want of symmetry between the embryonic end of the germ and the opposite end first appears.

I described in the last stage the appearance of the "embryonic rim." It is in the middle point of this, where it projects most, that the formation of the embryo takes place. There appear two parallel folds extending from the edge of the blastoderm towards the centre, and cut off at their central end by another transverse fold. These three folds raise up, between them, a flat broadish ridge, "the embryo" (Pl. 3, fig. 5). The head end of the embryo is the end nearest the centre of the blastoderm, the tail end being the one formed by its (the blastoderm's) edge.

Almost from its first appearance this ridge acquires a shallow groove--the medullary groove (Pl. 3, fig. 5, mg)--along its middle line, where the epiblast and hypoblast are in absolute contact (vide fig. 6_a_, 7_a_, 7_b_, &c.) and where the mesoblast (which is already formed by this stage) is totally absent. This groove ends abruptly a little before the front end of the embryo, and is deepest in the middle and wide and shallow behind.

On each side of it is a plate of mesoblast equivalent to the combined vertebral and lateral plates of the Chick. These, though they cannot be considered as entirely the cause of the medullary groove, may perhaps help to make it deeper. In the parts of the germ outside the embryo the mesoblast is again totally absent, or, more correctly, we might say that outside the embryo the lower layer cells do not become differentiated into hypoblast and mesoblast, and remain continuous only with the lower of the two layers into which the lower layer cells become differentiated in the body of embryo. This state of things is not really very different from what we find in the Chick. Here outside the embryo (i.e. in the opaque area) there is a layer of cells in which no differentiation into hypoblast and mesoblast takes place, but the layer remains continuous rather with the hypoblast than the mesoblast.

There is one peculiarity in the formation of the mesoblast which I wish to call attention to, i.e. its formation as two lateral masses, one on each side of the middle line, but not continuous across this line (vide figs. 6_a_ and 6_b_, and 7_a_ and 7_b_). Whether this remarkable condition is the most primitive, i.e. whether, when in the stage before this the mesoblast is first formed, it is only on each side of the middle line that the differentiation of the lower layer cells into hypoblast and mesoblast takes place, I do not certainly know, but it is undoubtedly a very early condition of the mesoblast. The condition of the mesoblast as two plates, one on each side of the neural canal, is precisely similar to its embryonic condition in many of the Vermes, e.g. Euaxes and Lumbricus. In these there are two plates of mesoblast, one on each side of the nervous cord, which are known as the Germinal streaks (Keimstreifen) (vide Kowalevsky "Würmern u. Arthropoden"; Mém. de l'Acad. Imp. St Petersbourg, 1871).

From longitudinal sections I have found that the segmentation cavity has ceased by this stage to have any distinct existence, but that the whole space between the epiblast and the yolk is filled up with a mass of elongated cells, which probably are solely concerned in the formation of the vascular system. The thickened posterior edge of the blastoderm is still visible.

At the embryonic end of the blastoderm, as I pointed out in an earlier stage, the epiblast and the lower layer cells are perfectly continuous.

Where they join the epiblast, the lower layer cells become distinctly divided, and this division commenced even in the earlier stage, into two layers; a lower one, more directly continuous with the epiblast, consisting of cells somewhat resembling the epiblast-cells, and an upper one of more flattened cells (Pl. 3, fig. 4, m). The first of these forms the hypoblast, and the latter the mesoblast. They are indicated by hy and m in the figures. The hypoblast, as I said before, remains continuous with the whole of the rest of lower layer cells of the blastoderm (vide fig. 7_b_). This division into hypoblast and mesoblast commences at the earlier stage, but becomes much more marked during this one.

In describing the formation of the hypoblast and mesoblast in this way I have assumed that they are formed out of the large mass of lower layer cells which underlie the epiblast at the embryonic end of the blastoderm. But there is another and, in some ways, rather a tempting view, viz. to suppose that the epiblast, where it becomes continuous with the hypoblast, in reality becomes involuted, and that from this involuted epiblast are formed the whole mesoblast and hypoblast.

In this case we would be compelled to suppose that the mass of lower layer cells which forms the embryonic swelling is used as food for the growth of the involuted epiblast, or else employed solely in the growth over the yolk of the non-embryonic portion of the blastoderm; but the latter possibility does not seem compatible with my sections.

I do not believe that it is possible, from the examination of sections alone, to decide which of these two views (viz. whether the epiblast is involuted, or whether it becomes merely continuous with the lower layer cells) is the true one. The question must be decided from other considerations.

The following ones have induced me to take the view that there is no involution, but that the mesoblast and hypoblast are formed from the lower layer cells.

(1) That it would be rather surprising to find the mass of lower layer cells which forms the "embryo swelling" playing no part in the formation of embryo.

(2) That the view that it is the lower layer cells from which the hypoblast and mesoblast are derived agrees with the mode of formation of these two layers in the Bird, and also in the Frog; since although, in the latter animal, there is an involution, this is not of the epiblast, but of the larger cells of the lower pole of the yolk, which in part correspond with what I have called the lower layer cells in the Dog-fish.

If the view be accepted that it is from the lower layer cells that the hypoblast and mesoblast are formed, it becomes necessary to explain what the continuity of the hypoblast with the epiblast means.

The explanation of this is, I believe, the keystone to the whole position. The vertebrates may be divided as to their early development into two classes, viz. those with holoblastic ova, in which the digestive canal is formed by an involution with the presence of an "anus of Rusconi."

This class includes "Amphioxus," the "Lamprey," the "Sturgeon," and "Batrachians."

The second class are those with meroblastic ova and no anus of Rusconi, and with an alimentary canal formed by the infolding of the sheet of hypoblast, the digestive canal remaining in communication with the food-yolk for the greater part of embryonic life by an umbilical canal.

This class includes the "Elasmobranchii," "Osseous fish," "Reptiles," and "Aves."

The mode of formation of the alimentary canal in the first class is clearly the more primitive; and it is equally clear that its mode of formation in the second class is an adaptation due to the presence of the large quantity of food-yolk.

In the Dog-fish I believe that we can see, to a certain extent, how the change from the one to the other of these modes of development of the alimentary canal took place.

In all the members of the first class, viz. "Amphioxus," the "Lamprey," the "Sturgeon," and the "Batrachians," the epiblast becomes continuous with the hypoblast at the so-called "anus of Rusconi," and the alimentary canal, potentially in all and actually in the Sturgeon (vide Kowalevsky, Owsjannikow, and Wagner, Bulletin der Acad. d. St Petersbourg, Vol. XIV. 1870, "Entwicklung der Störe"), communicates freely at its extreme hind end with the neural canal. The same is the case in the Dog-fish. In these, when the folding in to form the alimentary canal on the one hand, and the neural on the other, takes place, the two foldings unite at the corner, where the epiblast and hypoblast are in continuity, and place the two tubes, the neural and alimentary, in free communication with each other[14].

Footnote 14: This has been already made out by Kowalevsky, "Würmern u. Arthropoden," loc. cit.

There is, however, nothing corresponding with the "anus of Rusconi," which merely indicates the position of the involution of the digestive canal, and subsequently completely closes up, though it nearly coincides in position with the true anus in the Batrachians, &c.

This remarkable point of similarity between the Dog-fish's development and the normal mode of development in the first class (the holoblastic) of vertebrates, renders it quite clear that the continuity of the epiblast and hypoblast in the Dogfish is really the remnant of a more primitive condition, when the alimentary canal was formed by an involution. Besides the continuity between neural and alimentary canals, we have other remnants of the primitive involution. Amongst these the most marked is the formation of the embryonic rim, which is nothing less than the commencement of an involution. Its form is due to the flattened, sheet-like condition of the germ. In the mode in which the alimentary canal is closed in front I shall shew there are indications of the primitive mode of formation of the alimentary canal; and in certain peculiarities of the anus, which I shall speak of later, we have indications of the primitive anus of Rusconi; and finally, in the general growth of the epiblast (small cells of the upper pole of the Batrachian egg) over the yolk (lower pole of the Batrachian egg), we have an example of the manner in which the primitive involution, to form the alimentary canal, invariably disappears when the quantity of yolk in an egg becomes very great.

I believe that in the Dog-fish we have before our eyes one of the steps by which a direct mode of formation comes to be substituted for an _in_direct one by involution. We find, in fact, in the Dog-fish, that the cells from which are derived the mesoblast and hypoblast come to occupy their final position in the primitive arrangement of the cells during segmentation, and not by a subsequent and secondary involution.

This change in the mode of formation of the alimentary canal is clearly a result of change of mechanical conditions from the presence of the large food-yolk.

Excellent parallels to it will be found amongst the Mollusca. In this class the presence or absence of food-yolk produces not very dissimilar changes to those which are produced amongst vertebrates from the same cause.

The continuity of the hypoblast and epiblast at the embryonic rim is a remnant which, having no meaning or function, except in reference to the earlier mode of development, is likely to become lost, and in Birds no trace of it is any longer to be found.

I will not in the present preliminary paper attempt hypothetically to trace the steps by which the involution gradually disappeared, though I do not think it would be very difficult to do so. Nor will I attempt to discuss the question whether the condition with a large amount of food-yolk (as seems more probable) was twice acquired--once by the Elasmobranchii and Osseous fishes, and once by Reptiles and Birds--or whether only once, the Reptiles and Birds being lineal descendants of the Dog-fish.

In reference to the former point, however, I may mention that the Batrachians and Lampreys are to a certain extent intermediate in condition between the Amphioxus and the Dog-fishes, since in them the yolk becomes divided during segmentation into lower layer cells and epiblast, but a modified involution is still retained, while the Dog-fish may be looked upon as intermediate between Birds and Batrachians, the continuity at the hind end between the epiblast and hypoblast being retained by them, though not the involution.

It may be convenient here to call attention to some of the similarities and some of the differences which I have not yet spoken of between the development of Osseous fish and the Dog-fish in the early stages. The points of similarity are--(1) The swollen edge of the blastoderm. (2) The embryo-swelling. (3) The embryo-keel. (4) The spreading of the blastoderm over the yolk-sac from a point corresponding with the position of the embryo, and not with the centre of the germ. The growth is almost nothing at that point, and most rapid at the opposite pole of the blastoderm, being less and less rapid along points of the circumference in proportion to their proximity to the embryonic swelling. (5) The medullary groove.

In external appearance the early embryos of Dog-fish and Teleostei are very similar; some of my drawings could almost be substituted for those given by Oellacher. This similarity is especially marked at the first appearance of the medullary groove. In the Dog-fish the medullary groove becomes converted into the medullary canal in the same way as in Birds and all other vertebrates, except Osseous fishes, where it comes to nothing, and is, in fact, a rudimentary structure. But in spite of Oellacher's assertions to the contrary, I am convinced from the similarity of its position and appearance to the true medullary groove in the Dog-fish, that the groove which appears in Osseous fishes is the true medullary groove; although Oellacher and Kuppfer appear to have conclusively proved that it does not become converted into the medullary canal. The chief difference between the Dog-fish and Osseous fish, in addition to the point of difference about the medullary groove, is that the epiblast is in the Dog-fish a single layer, and not divided into nervous and epidermic layers as in Osseous fish, and this difference is the more important, since, throughout the whole period of development till after the commencement of the formation of the neural canal, the epiblast remains in Dog-fish as a one-cell-deep layer of cells, and thus the possibility is excluded of any concealed division into a neural and epidermic layer, as has been supposed to be the case by Stricker and others in Birds.

Development of the Embryo.

After the embryo has become definitely established, for some time it grows rapidly in length, without externally undergoing other important changes, with the exception of the appearance of two swellings, one on each side of its tail.

These swellings, which I will call the Caudal lobes (figs. 8 and 9, ts), are also found in Osseous fishes, and have been called by Oellacher the Embryonal saum. They are caused by a thickening of mesoblast on each side of the hind end of the embryo, at the edge of the embryonic rim, and form a very conspicuous feature throughout the early stages of the development of the Dog-fish, and are still more marked in the Torpedo (Pl. 3, fig. 9). Although from the surface the other changes which are visible are very insignificant, sections shew that the notochord is commencing to be formed.

I pointed out that beneath the medullary groove the epiblast and hypoblast were not separated by any interposed mesoblast. Along the line (where the mesoblast is deficient) which forms the long axis of the embryo, a rod-like thickening of the hypoblast appears (Pl. 3, figs. 7_a_ and 7_b_, ch and ch´), first at the head end of the embryo, and gradually extends backwards. This is the rudiment of the notochord; it remains attached for some time to the hypoblast, and becomes separated from it first at the head end of the embryo, and the separation is then carried backwards. This thickening of the hypoblast projects up and comes in contact with the epiblast, and in the later stages with bad (especially chromic-acid) specimens the line of separation between the epiblast and the thickening may become a little obscured, and might possibly lead to the supposition that a structure similar to that which has been called the "axis cord" was present. In all my best (osmic-acid) specimens the line of junction is quite clear; and any one who is aware how easily two separate masses of cells may be made indistinguishably to fuse together from simple pressure will not be surprised to find the occasional obscurity of the line of junction between the epiblast and hypoblast. In the earlier stage of the thickening there is never in the osmic-acid preparations any appearance of fusion except in very badly prepared ones. Its mode of formation will be quite clear without further description from an inspection of Pl. 3, figs. 7_a_ and 7_b_, ch and ch´. Both are taken from one embryo. In fig. 7_b_, the most anterior of the two, the notochord has become quite separated from the hypoblast. In fig. 7_a_, ch, there is only a very marked thickening of hypoblast, which reaches up to the epiblast, but the thickening is still attached to the hypoblast. Had I had space to insert a drawing of a third section of the same embryo there would only have been a slight thickening of the hypoblast. In the earlier stage it will be seen, by referring to figs. 6_a_ and 6_b_, that there is no sign of a thickening of the hypoblast. My numerous sections (all made from embryos hardened in osmic acid) shewing these points are so clear that I do not think there can be any doubt whatever of the notochord being formed as a thickening of the hypoblast. Two interpretations of this seem possible.

I mentioned that the mesoblast appeared to be primitively formed as two independent sheets, split off, so to speak, from the hypoblast, one on each side of the middle line of the embryo. If we looked upon the notochord as a third median sheet of mesoblast, split off from the hypoblast somewhat later than the other two, we should avoid having to admit its hypoblastic origin.

Professor Huxley, to whom I have shewn my specimens, strongly advocates this view.

The other possibility is that the notochord is primitively a true hypoblastic structure which has only by adaptation become an apparently mesoblastic one in the higher vertebrates. In favour of this view are the following considerations:

(1) That this is the undoubtedly natural interpretation of the sections. (2) That the notochord becomes separated from the hypoblast after the latter has acquired its typical structure, and differs in that respect from the two lateral sheets of mesoblast, which are formed coincidently with the hypoblast by a homogeneous mass of cells becoming differentiated into two distinct layers. (3) That the first mode of looking at the matter really proves too much, since it is clear that by the same method of reasoning we could prove the mesoblastic origin of any organ derived from the hypoblast and budded off into the mesoblast. We would merely have to assert that it was really a mass of mesoblast budded off from the hypoblast rather later than the remainder of the mesoblast. Still, it must be admitted that the first view I have suggested is a possible, not to say a probable one, though the mode of arguing by which it can be upheld may be rather dangerous if generally applied. We ought not, however, for that reason necessarily to reject it in the present case. As Mr Ray Lankester pointed out to me, if we accept the hypoblastic origin of the notochord, we should find a partial parallel to it in the endostyle of Tunicates, and it is perhaps interesting to note in reference to it that the notochord is the only unsegmented portion of the axial skeleton.

Whether the strong à priori difficulties of the hypoblastic origin of the notochord are sufficient to counterbalance the natural interpretation of my sections, cannot, I think, be decided from the single case of the Dog-fish. It is to be hoped that more complete investigations of the Lamprey, &c., may throw further light upon the question.

Whichever view of the primitive origin of the notochord is the true one, its apparent origin is very instructive as illustrating the possible way in which an organ might come to change the layer to which it primarily belonged.

If the notochord is a true mesoblastic structure, it is easy to be seen how, by becoming separated from the hypoblast a little later than is the case with the Dog-fish, its mesoblastic origin would become lost; while if, on the other hand, it is primitively a hypoblastic structure, we see from higher vertebrates how, by becoming separated from the hypoblast rather earlier than in the Dog-fish, viz. at the same time as the rest of the mesoblast, its primitive derivation from the hypoblast has become concealed.

The view seemingly held by many embryologists of the present day, that an organ, when it was primitively derived from one layer, can never be apparently formed in another layer, appears to me both unreasonable on à priori grounds, and also unsupported by facts.

I see no reason for doubting that the embryo in the earliest periods of development is as subject to the laws of natural selection as is the animal at any other period. Indeed, there appear to me grounds for the thinking that it is more so. The remarkable differences in allied species as to the amount of food-yolk, which always entail corresponding alterations in the development--the different modes of segmentation in allied species, such as are found in the Amphipoda and Isopoda--the suppression of many stages in freshwater species, which are retained in the allied marine species--are all instances of modifications due to natural selection affecting the earliest stages of development. If such points as these can be affected by natural selection I see no reason why the arrangement of individual cells (or rather primitive elements) should not also be modified; why, in fact, a mass of cells which was originally derived from one layer, but in the course of development became budded off from that layer and entered another layer, should not by a series of small steps cease ever to be attached to the original layer, but from the first moment it can be distinguished should be found as a separate mass in the second layer.

The change of layers will, of course, only take place where some economy is effected by it. The variations in the mode of development of the nervous system may probably be explained in this way.

If we admit that organs can undergo changes, as to the primitive layer from which they are derived, important consequences must follow.

It will, for instance, by no means be sufficient evidence of two organs not being homologous that they are not developed from the same layer. It renders the task of tracing out the homologies from development much more difficult than if the ordinary view of the invariable correspondence of the three layers throughout the animal kingdom be accepted. Although I do not believe that this correspondence is invariable or exact, I think that we both find and should expect to find that it is, roughly speaking, fairly so.

Thus, the muscles, internal skeleton, and connective tissue are always placed in the adult between the skin (epidermis) and the epithelium of the alimentary canal.

We should therefore expect to find them, and, as a matter of fact, we always do find them, developed from a middle layer when this is present.

The upper layer must always and does always form the epidermis, and similarly the lower layer or hypoblast must form a part of the epithelium of the alimentary canal. A full discussion of this question would, however, lead me too far away from my present subject.

The only other point of interest which I can touch on in this stage is the commencing closure of the alimentary canal in the region of the head. This is shewn in Pl. 3, figs. 6_a_, 6_b_, 7_b_, n.a. From these figures it can be seen that the closing does not take place as much by an infolding as by an ingrowth from the side walls of the alimentary canal towards the middle line. In this abnormal mode of closing of the alimentary canal we have again, I believe, an intermediate stage between the mode of formation of the alimentary canal in the Frog and the typical folding in which occurs in Birds. There is, however, another point in reference to it which is still more interesting. The cells to form the ingrowth from the bottom (ventral) wall of the alimentary canal are derived by a continuous fresh formation from the yolk, being formed around the nuclei spoken of above (vide p. 63 et seq.). All my sections shew this with more or less clearness, especially those a little later than fig. 6_b_, in which the lower wall of the alimentary canal is nearly completed. This is the more interesting since, from the mode of formation of the alimentary canal in the Batrachians, &c., we might expect that the cells from the yolk would take a share in its formation in the Dog-fish. I have not as yet made out for certain the share which is taken by these freshly formed cells of the yolk in the formation of any other organ.

By the completion of its lower wall in the way described, the throat early becomes a closed tube, its closing taking place before any other important changes are visible in the embryo from surface views.

A considerable increase in length is attained before other changes than an increase in depth of the medullary groove and a more complete folding off of the embryo from the blastoderm take place. The first important change is the formation of the protovertebræ.

These are formed by the lateral plates of mesoblast, which I said were equivalent at once to the vertebral and lateral plates in the Bird, becoming split by transverse divisions into cubical masses.

At the time when this occurs, and, indeed, up till a considerably later period, the mesoblast is not split into somatopleure and splanchnopleure, and it is not divided into vertebral and lateral plates. The transverse lines of division of the protovertebræ do not, however, extend to the outer edge of the undivided lateral plates.

The differences between this mode of formation of the protovertebræ and that occurring in Birds are too obvious to require pointing out. I will speak of them more fully when I have given the whole history of the protovertebræ of the Dog-fish.

I will only now say that I have had in the early stages to investigate the formation of the protovertebræ entirely by means of sections, the objects being too opaque to be otherwise studied.

The next change of any importance is the commencement of the formation of the head. The region of the head first becomes distinguishable by the flattening out of the germ at its front end.

The flattened-out portion of the germ grows rapidly, and forms a spatula-like termination to the embryo (Pl. 3, fig. 8).

In the region of the head the medullary groove is at first totally absent (vide section, Pl. 3, fig. 8_a_).

Indeed, as can be seen from fig. 8_b_, the laminæ dorsales, so far from bending up at this stage, actually bend down in the opposite direction.

I am at present quite unable even to form a guess what this peculiar feature of the brain means. It, no doubt, has some meaning in reference to the vertebrate ancestry if we could only discover it. The peculiar spatula-like flattened condition of the head is also (vide loc. ant. cit.) apparently found in the Sturgeons; it must therefore almost undoubtedly be looked upon as not merely an accidental peculiarity.

While these changes have been taking place in the head not less important changes have occurred in the remainder of the body. In the first place the two caudal lobes have increased in size, and have become, as it were, pushed in together, leaving a groove between them (fig. 8, ts). They are very conspicuous objects, and, together with the spatula-like head, give the whole embryo an almost comical appearance. The medullary canal has by this time become completely closed in the region of the tail (figs. 8 and 8_b_).

It is still widely open in the region of the back, and, though more nearly closed again in the neck, is, as I have said, flattened out to nothing in the head.

The groove[15] between the two caudal lobes must not be confused (as may easily be done) with the medullary groove, which by the time the former groove has become conspicuous is a completely closed canal.

Footnote 15: This groove is the only structure which it seems possible to compare with the so-called "primitive groove" of Birds. It is, however, doubtful whether they are really homologous.

The vertebral plates are not divided (vide fig. 7) into a somatopleuric and splanchnopleuric layer by this stage, except in the region of the head (vide fig. 8_b_, pp), where there is a distinct space between the two layers, which is undoubtedly homologous with the pleuro-peritoneal cavity of the hinder portion of the body.

It is probably the same cavity which Oellacher (loc. cit.) calls in Osseous fishes the pericardial cavity. In the Dog-fish, at least, it has no connection with the pericardium. Of its subsequent history I shall say a few words when I come to speak of the later stages.

The embryo does not take more than twenty-four hours in passing from this stage, when the head is a flat plate, to the stage when the whole neural canal (including the region of the head) is closed in. The other changes, in addition to the closing in of the neural canal, are therefore somewhat insignificant. The folding off of the embryo from the germ has, however, progressed considerably, and a portion of the hind gut is closed in below. This is accomplished, not by a tail-fold, as in Birds, but by two lateral folds, which cause the sides of the body to meet and coalesce below. At the extreme hind end, where the epiblast is continuous with the hypoblast, the lateral folds turn round, so to speak, and become continuous with the medullary folds, so that when the various folds meet each other an uninterrupted canal is found passing round from the neural into the alimentary canal, and placing these two in communication at the tail end of the body. Since I have already mentioned this, and spoken of its significance, I will not dwell on it further here.

The cranial flexure commences coincidently with the closing in of the neural canal in the region of the brain, and the division into fore, mid, and hind brain becomes visible at the same time as or even before the closing of the canal occurs. The embryo has now become more or less transparent, and protovertebræ, of which about twenty are present, can now be seen in the fresh specimens. The heart, however, is not yet formed.

Up to this period, a period at which the embryo becomes very similar in external appearance to any other vertebrate embryo, I have followed in my description a chronological order. I shall now cease to do so, since it would be too long for a preliminary notice of this kind, but shall confine myself to the history of a few organs whose development is either more important or more peculiar than that of the others.

The Protovertebræ.

I have thought it worth while to give a short history of the development of the protovertebræ, firstly, because it is very easy to follow this in the Dog-fish, and, secondly, because I believe that the Dog-fish have more nearly retained the primitive condition of the protovertebræ than any other vertebrate whose embryology has hitherto been described with sufficient detail.

I intend to describe, at the same time, the development of the spinal nerves.

I left each lateral mass of mesoblast in my last stage as a plate which had not yet become split into a somatic and a splanchnic sheet (Pl. 3, fig. 8_a_, vp), but which had become cut by transverse lines (not, indeed, extending to the outer limit of the sheet, but as yet not cut off by longitudinal lines of cleavage) into segments, which I called protovertebræ.

This sheet of mesoblast is fairly thick at its proximal (upper) end, but thins off laterally to a sheet two cells deep, and its cells are so arranged as to foreshadow its subsequent splitting into somatic and splanchnic sheets. Its upper (proximal) end is at this stage level with the bottom of the neural canal, but soon begins to grow upwards, and at the same time the splitting into somatopleure and splanchnopleure commences (Pl. 3, fig. 10, so and sp).

The separation between the two sheets is first visible in its uppermost part, and thence extends outwards. By this means each of the protovertebræ becomes divided into two sheets, which are only connected at their upper ends and outside the region of the body. I speak of the whole lateral sheet as being composed of protovertebræ, because at this time no separation into vertebral and lateral plates can be seen; but I may anticipate matters by saying that only the upper portion of the sheet from the level of the top of the digestive canal, becomes subsequently the true protovertebræ. From this it is clear that the pleuro-peritoneal cavity extends primitively quite up to the top of the protovertebræ; and that thus a portion of a sheet of mesoblast, at first perfectly continuous with the splanchnic sheet from which is derived the muscular wall of the alimentary canal, is converted into a part of the voluntary muscular system of the body, having no connection whatever with the involuntary muscular system of the digestive tract.

The pleuro-peritoneal cavity is first distinctly formed at a time when only two visceral clefts are present. Before the appearance of a third visceral cleft in a part of the innermost layer of each protovertebræ (which may be called the splanchnic layer, from its being continuous with the mesoblast of the splanchnopleure), opposite the bottom of the neural tube, some of the cells commence to become distinguishable from the rest, and to form a separate mass. This mass becomes much more distinct a little later, its cells being characterised by being spindle-shaped, and having an elongated nucleus which becomes deeply stained by reagents (Pl. 4, fig. 11, mp´). Coincidently with its appearance the young Dog-fish commences spontaneously to move rapidly from side to side with a kind of serpentine motion, so that, even if I had not traced the development of this differentiated mass of cells till it becomes a band of muscles close to the notochord, I should have had little doubt of its muscular nature. It is indicated in figs. 11, 12, 13, by the letters mp´. Its early appearance is most probably to be looked upon as an adaptation consequent upon the respiratory requirements of the young Dog-fish necessitating movements within the egg.

Shortly after this date, at a period when three visceral clefts are present, I have detected the first traces of the spinal nerves.

At this time they appear in sections as small elliptical masses of cells, entirely independent of the protovertebræ, and closely applied to the upper and outer corners of the involuted epiblast of the neural canal (Pl. 4, fig. 11, spn). These bodies are far removed from any mesoblastic structures, and at the same time the cells composing them are not similar to the cells composing the walls of the neural canal, and are not attached to these, though lying in contact with them. I have not, therefore, sufficient evidence at present to enable me to say with any certainty where the spinal nerves are derived from in the Dog-fish. They may be derived from the involuted epiblast of the neural canal, and, indeed, this is the most natural interpretation of their position.

On the other hand, it is possible that they are formed from wandering cells of the mesoblast--a possibility which, with our present knowledge of wandering cells, must not be thrown aside as altogether improbable.

In any case, it is clear that the condition in the Bird, where the spinal nerves are derived from tissue of the protovertebræ, is not the primitive one. Of this, however, I will speak again when I have concluded my account of the development of the protovertebræ.

About the same time that the first rudiments of the nerves appear, the division of the mesoblast of the sides of the body into a vertebral and a lateral portion occurs. This division first appears in the region where the oviduct (Müller's duct) is formed (Pl. 4, fig. 11, ov).

At this part opposite the level of the dorsal aorta the two sheets, viz. the splanchnic and the somatic, unite together, and thus each lateral sheet of mesoblast becomes divided into an upper portion (fig. 11, mp), split up by transverse partitions into protovertebræ, and a lower portion not so split, but consisting of an outer layer, the true somatopleure, and an inner layer, the true splanchnopleure. These two divisions of the primitive plate are thus separated by the line at which a fusion between the mesoblast of the somatopleure and splanchnopleure takes place. The mass of cells resulting from the fusion at this point corresponds with the intermediate cell-mass of Birds (vide Waldeyer, Eierstock und Ei).

At the same time, in the upper of these two sheets (the protovertebræ), the splanchnic layer sends a growth of cells inwards towards the notochord and the neural canal. This growth is the commencement of the large quantity of mesoblastic tissue around the notochord, which is in part converted into the axial skeleton, and in part into the connective tissue adjoining this.

This mass of cells is at first quite continuous with the splanchnic layer of the protovertebræ, and I see no reason for supposing that it is not derived from the growth of the cells of this layer. The ingrowth to form it first appears a little after the formation of the dorsal aorta; but, as far as I have been able to see, its cells have no connection with the walls of the aorta.

What I have said as to the development of the skeleton-forming layer will be quite clear from figs. 11 and 12_a_; and from these it will also be clear, especially from fig. 11_a_, that the outermost layer of this mass of cells, which was the primitive splanchnic layer of the protovertebræ, still retains its epithelial character, and so can easily be distinguished from those cells which will form the skeleton. In the next stage which I have figured (fig. 12_a_), this outer portion of the splanchnic layer is completely separated from the skeleton-forming cells, and at the same time, having united below as well as above with the outer (somatic) layer of the two layers of which the protovertebræ are formed, the two together form an independent mass (fig. 12, mp), similar in appearance and in every way homologous with the muscle-plate of Birds.

On the inner side of this, which we may now call the muscle-plate, is seen the bundle of earlier-developed muscles (fig. 12, mp´) which I spoke of before.

The section represented in fig. 12 is from a very considerably later embryo than that represented in fig. 11, so that the skeleton-forming cells, few in number in the earlier section, have become very numerous in the later one, and have grown up above the neural canal, and also below the notochord, between the digestive canal and the aorta. They have, moreover, changed their character; they were round before, now they have become stellate. As to their further history, it need only be said that the layer of them immediately around the notochord and neural canal forms the cartilaginous centra and arches of the vertebræ, and that the remaining portion of them, which becomes much more insignificant in size as compared with the muscles, forms the connective tissue of the skeleton and of the parts around and between the muscles.

A muscle-plate itself is at this stage (shewn in fig. 12) composed of an inner and an outer layer of columnar cells (splanchnic and somatic) united at the upper and lower ends of the plate, and on the inner of the two lies the more developed mass of muscles before spoken of (mp´).

Each of these plates now grows both upwards and downwards; and at the same time connective-tissue cells appear between the plates and epidermis; but from where they come I do not know for certain; very probably they are derived from the somatic layer of the muscle-plate.

While the muscle-plates continue to grow both upwards and downwards, the cells of which they are composed commence to become elongated and soon acquire an unmistakably muscular character (Pl. 4, fig. 13, mp).

Before this has occurred the inner mass of muscles has also undergone further development and become a large and conspicuous band of muscles close to the notochord (fig. 13, mp´).

At the same time that the muscle-plates acquire the true histological character of muscle, septa of connective tissue grow in and divide them into a number of distinct segments which subsequently form separate bands of muscle. I will not say more in reference to the development of the muscular system than that the whole of the muscles of the body (apart from the limbs, the origin of whose muscular system I have not yet investigated) are derived from the muscle-plates which grow upwards above the neural canal and downwards to the ventral surface of the body.

During the time the muscle-plates have been undergoing these changes the nerve masses have also undergone developmental changes.

They become more elongated and fibrous, their main attachment to the neural tube being still at its posterior (dorsal) surface, near which they first appeared. Later still they become applied closely to the sides of the neural tube and send fibres to it below as well as above. Below (ventral to) the neural tube a ganglion appears, forming only a slight swelling, but containing a number of characteristic nerve-cells. The ganglion is apparently formed just below the junction of the anterior and posterior roots, though probably the fibres of the two roots do not mix till below it.

The main points which deserve notice in the development of the protovertebræ are--

(1) That at the time when the mesoblast becomes split horizontally into somatopleure and splanchnopleure the vertebral and lateral plates are one, and the splitting extends to the very top of the vertebral or muscle-plate, so that the future muscle-plates are divided into a splanchnic and somatic layer, the space between which is at first continuous with the pleuro-peritoneal cavity.

(2) That the following parts are respectively formed by the vertebral and lateral plates:

(a) Vertebral plate. From the splanchnic layer of this, or from cells which appear close to and continuous with it, the skeleton, and connective tissue of the upper part of the body, are derived.

The remainder of the plate, consisting of a splanchnic and somatic layer, is entirely converted into the muscles of the trunk, all of which are derived from it.

(b) Between the vertebral plate and the lateral plate is a mass of cells where, as I mentioned above, the mesoblast of the somatopleure and splanchnopleure fuse together. This mass of cells is the equivalent of the intermediate cell mass of Birds (vide Waldeyer, Eierstock und Ei).

From it are derived the Wolffian bodies and duct, the oviduct, the ovaries and the testis, and the connective tissue of the parts adjoining these.

(c) The lateral plate. From the somatic layer of this is derived the connective tissue of the ventral half of the body; the mesoblast of the limbs, including probably the muscles, and certainly the skeleton. From its splanchnic layer are derived the muscles and connective tissue of the alimentary canal.

(3) The spinal nerves are developed independently of the protovertebræ, so that the protovertebræ of the Elasmobranchii do not appear to be of such a complicated structure as the protovertebræ of Birds.

The Digestive Canal.

I do not intend to enter into the whole history of the digestive canal, but to confine myself to one or two points of interest connected with it. These fall under two heads:

(1) The history of the portion of the digestive canal between the anus and the end of the tail where the digestive canal opens into the neural canal.

(2) Certain less well-known organs derived from the digestive canal.

The anus is a rather late formation, but its position becomes very early marked out by the hypoblast of the digestive canal approaching at that point close to the surface, whilst receding to some little distance from it on either side. The portion of the digestive tract I propose at present dealing with is that between this point, which I will call, for the sake of brevity, the anus and the hind end of the body. This portion of the canal is at first very short; it is elliptical in section, and of rather a larger bore than the remainder of the canal. Its diameter becomes, however, slightly less as it approaches the tail, dilating again somewhat at its extreme end. It is lined by a markedly columnar epithelium. Though at first very short, its length increases with the growth of the tail, but at the same time its calibre continually becomes smaller as compared with the remainder of the alimentary canal.

It commences to become smaller, first of all, near, though not quite, at its extreme hind end, and thus becomes of a conical shape; the base of the cone being just behind the anus, while the apex of the cone is situated a short distance from the hind end of the embryo. The extreme hind end, however, at the same time does not diminish in size, and becomes relatively (if not also absolutely) much larger in diameter than it was at first, as compared with the remainder of the digestive canal. It becomes, in fact, a vesicle or vesicular dilatation at the end of a conical canal.

Just before the appearance of the external gills this part of the digestive canal commences to atrophy. It begins to do so close to the terminal vesicle, which, however, still remains as or more conspicuous than it was before. The lumen of the canal becomes smaller and smaller, and finally it becomes a solid string of cells, and these also soon become indistinguishable and not a trace of the canal is left.

Almost the whole of it has disappeared before the vesicle begins to atrophy, but very shortly after all trace of the rest of the canal has vanished the terminal vesicle also vanishes. This occurs just about the time or shortly after the appearance of the external gills--there being slight differences probably in this respect in the different species.

In this history there are two points of especial interest:

(1) The terminal vesicle.

(2) The disappearance of a large and well-developed portion of the alimentary canal.

The interest in the terminal vesicle lies in the possibility of its being some rudimentary structure.

In Osseous fishes Kupffer has described the very early appearance of a vesicle near the tail end, which he doubtfully speaks of as the "allantois." The figure he gives of it in his earlier paper (Archiv. für Micro. Anat. Vol. II. pl. xxiv, fig. 2) bears a very strong resemblance to my figures of this vesicle at the time when the hind end of the alimentary canal is commencing to disappear; and I feel fairly confident that it is the same structure as I have found in the Dog-fish: but until the relations of the Kupffer's vesicle to the alimentary canal are known, any comparison between it and the terminal vesicle in the Dog-fish must be to a certain extent guess-work.

I have, however, been quite unsuccessful in finding any other vesicular structure which can possibly correspond to the so-called allantoic vesicle of Osseous fish.

The disappearance of a large portion of the alimentary canal behind the anus is very peculiar. In order, however, to understand the whole difficulties of the case I shall be obliged to speak of the relations of the anus of the Dog-fish to the anus of Rusconi in the Lamprey, &c.

In those vertebrates whose alimentary canal is formed by an involution, the anus of Rusconi represents the opening of this involution, and therefore the point where the alimentary canal primitively communicates with the exterior. When, however, the "anus of Rusconi" becomes closed, the wall of the alimentary canal still remains at that point in close juxtaposition to the surface, and the new and final anus is formed at or close to that point. In the Dog-fish, although the anus of Rusconi is not present, still, during the closing of the alimentary canal, the point which would correspond with this becomes marked out by the alimentary canal there approaching the surface, and it is at this point that the involution to form the true anus subsequently appears.

The anus in the Dog-fish has thus, more than a mere secondary significance. It corresponds with the point of closing of the primitive involution. If it was not for this peculiarity of the vertebrate anus we would naturally suppose, from the disappearance of a considerable portion of the alimentary canal lying behind its present termination, that in the adult the alimentary canal once extended much farther back than at present, and that the anus we now find was only a secondary anus, and not the primitive one. It is perhaps possible that this hinder portion of the alimentary canal is a result of the combined growth of the tail and the persisting continuity (at the end of the body) of the epiblast with the hypoblast.

Whichever view is correct, it may be well to mention, in order to shew that the difficulty about the anus of Rusconi is no mere visionary one, that Götte ("Untersuchung über die Entwicklung der Bombinator igneus," Archiv. für Micro. Anat., vol. V. 1869) has also described the disappearance of the hind portion of the alimentary canal in Batrachians, a rudiment (according to him) remaining in the shape of a lymphatic trunk.

It is, perhaps, possible that we have a further remnant of this "hind portion" of the alimentary canal amongst the higher vertebrates in the "allantois."

Organs developed from the Digestive Canal.

In reference to the development of the liver, pancreas, &c., as far as my observations have at present gone, the Dog-fish presents no features of peculiar interest. The liver is developed as in the Bird, and independently of the yolk.

There are, however, two organs derived from the hypoblast which deserve more attention. Immediately under the notochord, and in contact with it (vide Pl. 3, fig. 10; Pl. 4, figs. 11 and 12, x), a small roundish (in section) mass of cells is to be seen in most of the sections.

Its mode of development is shewn in fig. 10, x. That section shows a mass of cells becoming pinched off from the top of the alimentary canal. By this process of pinching off from the alimentary canal a small rod-like body close under the notochord is formed. It persists till after the appearance of the external gills, but later than that I have not hitherto succeeded in finding any trace of it.

It was first seen by Götte (loc. cit.) in the Batrachians, and he gave a correct account of its development, and added that it became the thoracic duct.

I have not myself worked out the later stages in the development of this body with sufficient care to be in a position to judge of the correctness of Götte's statements as to its final fate. If it is true that it becomes the thoracic duct it is very remarkable, and ought to throw some light upon the homologies of the lymphatic system.

Some time before the appearance of the external gills another mass of cells becomes, I believe, constricted off from the part of the alimentary canal in the neighbourhood of the anus, and forms a solid rod composed at first of dark granular cells lying between the Wolffian ducts. I have not followed out its development quite completely, but I have very little doubt that it is really constricted off from a portion of the alimentary canal chiefly in front of the point where the anus appears, but also, I believe, from a small portion behind this.

Though the cells of which it is composed are at first columnar and granular (fig. 12, su, r), they soon begin to become altered, and in the latter stage of its development the body forms a conspicuous rounded mass of cells with clear protoplasm, and each provided with a large nucleus. Later still it becomes divided into a number of separate areas of cells by septa of connective tissue, in which (the septa) capillaries are also present. Since I have not followed it to its condition in the adult, I cannot make any definite statements as to the fate of this body; but I think that it possibly becomes the so-called suprarenal organ, which in the Dog-fish forms a yellowish elongated body lying between the two kidneys.

The development of the Wolffian Duct and Body and of the Oviduct.

The development of the Wolffian duct and the Oviduct in the various classes of vertebrates is at present involved in some obscurity, owing to the very different accounts given by different observers.

The manner of development of these parts in the Dog-fish is different from anything that previous investigators have met with in other classes, but I believe that it gives a clearer insight into the true constitution of these parts than vertebrate embryology has hitherto supplied, and at the same time renders easier the task of understanding the differences in the modes of development in the different classes.

I shall commence with a simple description of the observed facts, and then give my view as to their meaning. At about the time of the appearance of the third visceral cleft, and a short way behind the point up to which the alimentary canal is closed in front, the splanchnopleure and somatopleure fuse together opposite the level of the dorsal aorta.

From the mass of cells formed by this fusion a solid knob rises up towards the epiblast (Pl. 4, fig. 11_b_, ov), and from this knob a solid rod of cells grows backwards towards the tail (fig. 11_c_, ov) very closely applied to the epiblast. This description will be rendered clear by referring to figs. 11_b_ and c. Fig. 11_b_ is a section at the level of the knob, and fig. 11_c_ is a section of the same embryo a short way behind this point. So closely does the rod of cells apply itself to the epiblast that it might very easily be supposed to be derived from it. Such, indeed, was at first my view till I cut a section passing through the knob. In order, however, to avoid all possibility of mistake I made sections of a large number of embryos of about the age at which this appears, and invariably found the large knob in front, and from it the solid string growing backwards.

This string is the commencement of the Oviduct or Müller's duct, which in the Dog-fish as in the Batrachians is the first portion of the genito-urinary system to appear, and is in the Dog-fish undoubtedly at first solid. All my specimens have been hardened with osmic acid, and with specimens hardened with this reagent it is quite easy to detect even the very smallest hole in a mass of cells.

As a solid string or rod of cells the Oviduct remains for some time; it grows, indeed, rapidly in length, the extreme hind end of the rod being very small and the front end continuing to remain attached to the knob. The knob, however, travels inwards and approaches nearer and nearer to the true pleuro-peritoneal cavity, always remaining attached to the intermediate cell mass.

At about the time when five visceral clefts are present the Oviduct first begins to get a lumen and to open at its front end into the pleuro-peritoneal cavity. The cells of the rod are first of all arranged in an irregular manner, but gradually become columnar and acquire a radiating arrangement around a central point. At this point, where the ends of all the cells meet, a very small hole appears, which gradually grows larger and becomes the cavity of the duct (fig. 12, ov). The hole first makes its appearance at the anterior end of the duct, and then gradually extends backwards, so that the hind end is still without a lumen, when the lumen of the front end is of a considerable size.

At the front knob the same alteration in the cells takes place as in the rest of the duct, but the cells become deficient on the side adjoining the pleuro-peritoneal cavity, so that an opening is formed into the pleuro-peritoneal cavity, which soon becomes of a considerable size. Soon after its first formation, indeed, the opening becomes so large that it may be met in from two to three consecutive sections if these are very thin.

Thus is formed the lumen of the Oviduct. The duct still, at this age, ends behind without having become attached to the cloaca, so that at this time the Oviduct is a canal closed behind, but communicating in front by a large opening with the pleuro-peritoneal cavity.

It has during this time been travelling downwards, and is now much nearer the pleuro-peritoneal cavity than the epiblast.

It may be well to point out that the mode of development which I have described is really not very different from an involution, and must, in fact, be only looked upon as a modification of an involution. Many examples from all classes in the animal kingdom could be selected to exemplify how an involution may become simply a solid thickening. In the Osseous fish nearly all the organs which are usually formed by an involution have undergone this change in their mode of development. I shall attempt to give reasons later on for the solid form having been acquired in this particular case of the Oviduct.

At about the time when a lumen appears in the Oviduct the first traces of the Wolffian duct become visible.

At intervals along the whole length, between the front and hind ends of the Oviduct, involutions arise from the pleuro-peritoneal cavity (fig. 12_a_, pwd) on the inside (nearer the middle line) of the Oviduct. The upper ends of these numerous involutions unite together and form a string of cells, at first solid, but very soon acquiring a lumen, and becoming a duct which communicates (as it clearly must from its mode of formation), at numerous points with the pleuro-peritoneal cavity. It is very probable that there is one involution to each segment of the body between the front and hind ends of the Oviduct. This duct is the Wolffian duct, which thus, together with the Oviduct, is formed before the appearance of the external gills.

For a considerable period the front end of the Oviduct does not undergo important changes; the hind end, however, comes into connection with the extreme end of the alimentary canal. The two Oviducts do not open together into the cloaca, though, as my sections prove, their openings are very close together. The whole Oviduct, as might be expected, shares in the general growth, and its lumen becomes in both sexes very considerably greater than it was before.

It is difficult to define the period at which I find these changes accomplished without giving drawings of the whole embryo. The stage is one considerably after the external gills have appeared, but before the period at which the growth of the olfactory bulbs renders the head of an elongated shape.

During the same period the Wolffian duct has undergone most important changes. It has commenced to bud off diverticula, which subsequently become the tubules of the Wolffian body (vide fig. 13, wd). I am fairly satisfied that the tubules are really budded off, and are not formed independently in the mesoblast. The Dog-fish agrees so far with Birds, where I have also no doubt the tubules of the Wolffian body are formed as diverticula from the Wolffian duct.

The Wolffian ducts have also become much longer than the Oviduct, and are now found behind the anus, though they do not extend as far forward as does the Oviduct.

They have further acquired a communication with the Oviduct, in the form of a narrow duct passing from each of them into an Oviduct a short way before the latter opens into the cloacal dilatation of the alimentary canal.

The canals formed by the primitive involution leading from the pleuro-peritoneal cavity into the Wolffian duct have become much more elongated, and at the same time narrower. One of these is shewn in fig. 13, pwd.

Any doubt which could possibly be entertained as to the true character of the ducts whose development I have described is entirely removed by the development of the tubules of the Wolffian body. In the still later stage than this further proofs are furnished involving the function of the Oviduct. At the period when the olfactory lobes have become so developed as to render the head of the typical elongated shape of the adult, I find that the males and females can be distinguished by the presence in the former of the clasping appendages[16]. I find at this stage that in the female the front ends of the Oviducts have approached the middle line, dilated considerably, and commenced to exhibit at their front ends the peculiarities of the adult. In the male they are much less conspicuous, though still present.

Footnote 16: For the specimens of this age I am indebted to Professor Huxley.

At the same time the tubules of the Wolffian body become much more numerous, the Malpighian tufts appear, and the ducts cease almost, if not entirely, to communicate with the pleuro-peritoneal cavity. I have not made out anything very definitely as to the development of the Malpighian tufts, but I am inclined to believe that they arise independently in the mesoblast of the intermediate cell mass.

The facts which I have made out in reference to the development of the Wolffian duct, especially of its arising as a series of involutions from the pleuro-peritoneal cavity, will be found, I believe, of the greatest importance in understanding the true constitution of the Wolffian body. To this I will return directly, but first wish to clear the ground by insisting upon one preliminary point.

From their development the Oviduct and Wolffian body appear to stand to each other in the relation of the Wolffian duct being the equivalent to a series, so to speak, of Oviducts.

I pointed out before that the mode of development of the Oviduct could only be considered as a modification of a simple involution from the pleuro-peritoneal cavity. Its development, both in the Birds and in the Batrachians as an involution, still more conclusively proves the truth of this view.

The explanation of its first appearing as a solid rod of cells which keeps close to the epiblast is, I am inclined to think, the following. Since the Oviduct had to grow a long way backwards from its primitive point of involution, it was clearly advantageous for it not to bore its way through the mesoblast of the intermediate cell mass, but to pass between this and the epiblast. This modification having been adopted, was followed by the knob forming the origin of the duct coming to be placed at the outside of the intermediate cell mass rather than close to the pleuro-peritoneal cavity, a change which necessitated the mode of development by an involution being dropped and the solid mode of development substituted for it, a lumen being only subsequently acquired.

In support of the modification in the development being due to this cause is the fact that in Birds a similar modification has taken place with the Wolffian duct. The Wolffian duct there arises differently from its mode of development in all the lower vertebrates as a solid rod close to the epiblast[17], instead of as an involution.

Footnote 17: If Romiti's observations (Archives für Mikr. Anatom. Vol. IX. p. 200) are correct, then the ordinary view of the Wolffian duct arising in Birds as a solid rod at the outer corner of the protovertebræ will have to be abandoned.

If the above explanation about the Oviduct be correct, then it is clear that similar causes have produced a similar modification in development (only with a different organ) in Birds; while, at the same time, the primitive mode of origin of the Oviduct (Müller's duct) has been retained by them.

The Oviduct, then, may be considered as arising by an involution from the pleuro-peritoneal cavity.

The Wolffian duct arises by a series of such involutions, all of which are behind (nearer the tail) the involution to form the Oviduct.

The natural interpretation of these facts is that in the place of the Oviduct and Wolffian body there were primitively a series of similar bodies (probably corresponding in number with the vertebral segments), each arising by an involution from the pleuro-peritoneal cavity; and that the first of these subsequently became modified to carry eggs, while the rest coalesced to form the Wolffian duct.

If we admit that the Wolffian duct is formed by the coalescence of a series of similar organs, we shall only have to extend the suggestion of Gegenbaur as to the homology of the Wolffian body in order to see its true nature. Gegenbaur looks upon the whole urinogenital system as homologous with a pair of segmental organs. Accepting its homology with the segmental organs, its development in Elasmobranchii proves that it is not one pair, but a series of pairs of segmental organs with which the urinogenital system is homologous. The first of these have become modified so as to form the Oviducts, and the remainder have coalesced to form the Wolffian ducts.

The part of a segmental organ which opens to the exterior appears to be lost in the case of all but the last one, where this part is still retained, and serves as the external opening for all.

Whether the external opening of the first segmental organ (Oviduct) is retained or not is doubtful. Supposing it has been lost, we must look upon the external opening for the Wolffian body as serving also for the Oviduct. In the case of all other vertebrates whose development has been investigated (but the Elasmobranchii), the Wolffian duct arises by a single involution, or, what is equivalent to it, the other involutions having disappeared. This even appears to be the case in the Marsipobranchii. In the adult Lamprey the Wolffian duct terminates at its anterior end by a large ciliated opening into the pleuro-peritoneal cavity. It will, perhaps, be found, when the development of the Marsipobranchii is more carefully studied, that there are primitively a number of such openings[18]. The Oviduct, when present, arises in other vertebrates as a single involution, strongly supporting the view that its mode of formation in the Dog-fish is fundamentally merely an involution.

Footnote 18: While correcting the proofs of this paper I have come across a memoir of W. Müller ("Ueber die Persistenz der Urniere bei Myxine Glutinosa," Jenaische Zeitschrift, Vol. VII. 1873), in which he mentions that in Myxine the upper end of the Wolffian duct communicates by numerous openings with the pleuro-peritoneal cavity; this gives to the suggestion in the text a foundation of fact.

The duct of the testes is, I have little doubt, derived from the anterior part of the Wolffian body; if so, it must be looked upon as not precisely equivalent to the Oviduct, but rather to a series of coalesced organs, each equivalent to the Oviduct. The Oviduct is in the Elasmobranchii, as in other vertebrates, primitively developed in both sexes. In the male, however, it atrophies. I found it still visible in the male Torpedos, though much smaller than in the females near the close of intra-uterine life.

Whether or not these theoretical considerations as to the nature of the Wolffian body and Oviduct are correct, I believe that the facts I have brought to light in reference to the development of these parts in the Dog-fish will be found of service to every one who is anxious to discover the true relations of these parts.

Before leaving the subject I will say one or two words about the development of the Ovary. In both sexes the germinal epithelium (fig. 13) becomes thickened below the Oviduct, and in both sexes a knob (in section but really a ridge) comes to project into the pleuro-peritoneal cavity on each side of the mesentery (fig. 13, pov). In both sexes, but especially the females, the epithelium on the upper surface of this ridge becomes very much thickened, whilst subsequently it elsewhere atrophies. In the females, however, the thickened epithelium on the knob grows more and more conspicuous, and develops a number of especially large cells with large nuclei, precisely similar to Waldeyer's (loc. cit.) "primitive ova" of the Bird. In the male the epithelium on the ridge, though containing primitive ova, is not as conspicuous as in the female. Though I have not worked out the matter further than this at present, I still have no doubt that these projecting ridges become the Ovaries.

The Head.

The study of the development of the parts of the head, on account of the crowding of organs which occurs there, always presents greater difficulties to the investigator than that of the remainder of the body. My observations upon it are correspondingly incomplete. I have, however, made out a few points connected with it in reference to some less well-known organs, which I have thought it worth while calling attention to in this preliminary account.

The continuation of the Pleuro-peritoneal Cavity into the Head.

In the earlier part of this paper (p. 86) I called attention to the extension of the separation between somatopleure and splanchnopleure into the head, forming a space continuous with the pleuro-peritoneal cavity (Pl. 3, fig. 8_a_, pp); this becomes more marked in the next stage, and, indeed, the pleuro-peritoneal cavity is present for a considerable time in the head before it becomes visible elsewhere. At the time of the appearance of the second visceral cleft it has become for the most part atrophied, but there persist two separated portions of it in front of the first cleft, and also remnants of it less well marked between and behind the two clefts. The visceral clefts necessarily divide it into separate parts.

The two portions in front of the first visceral cleft remain very conspicuous till the appearance of the external gills, and above the hinder one of the two the fifth nerve bifurcates.

These two are shewn as they appear in a surface view in fig. 14, pp. They are in reality somewhat flattened spaces, lined by a mesoblastic epithelium; the epithelium on the inner surface of the space corresponding to the splanchnopleure, and that on the outer to the somatopleure.

I have not followed the history of these later than the time of the appearance of the external gills.

The presence of the pleuro-peritoneal cavity in the head is interesting, as shewing the fundamental similarity between the head and the remainder of the body.

The Pituitary Body.

All my sections seem to prove that it is a portion of the epiblastic involution to form the mouth which is pinched off to form the pituitary body, and not a portion of the hypoblast of the throat. Since Götte (Archiv. für Micr. Anat. Bd. IX.) has also found that the same is the case with the Batrachians and Mammalia, I have little doubt it will be found to be universally the case amongst vertebrates.

Probably the observations which lead to the supposition that it was the throat which was pinched off to form the pituitary body were made after the opening between the mouth and throat was completed, when it would naturally be impossible to tell whether the pinching off was from the epiblast of the mouth involution or the hypoblast of the throat.

The Cranial Nerves.

The cranial nerves in their early condition are so clearly visible that I have thought it worth while giving a figure of them, and calling attention to some points about their embryonic peculiarities.

From my figure (14) it will be seen that there is behind the auditory vesicle a nervous tract, from which four nerves descend, and that each of these nerves is distributed to the front portion of a visceral arch. When the next and last arch (in this species) is developed, a branch from this nervous mass will also pass down to it. That each of these is of an equal morphological value can hardly be doubted.

The nerve to the third arch becomes the glosso-pharyngeal (fig. 14, gl), the nerves to the other arches become the branchial branches of the vagus nerve (fig. 14, vg). Thus the study of their development strongly supports Gegenbaur's view of the nature of the vagus and glosso-pharyngeal, viz. that the vagus is a compound nerve, each component part of it which goes to an arch being equivalent to one nerve, such as the glosso-pharyngeal.

Of the nerves in front of the auditory sac the posterior is the seventh nerve (fig. 14, VII). Its mode of distribution to the second arch leaves hardly a doubt that it is equivalent to one such nerve as those distributed to the posterior arches. Subsequently it acquires another branch, passing forwards towards the arch in front.

The most anterior nerve is the fifth (fig. 14, V), of which two branches are at this stage developed. The natural interpretation of its present condition is, that it is equivalent to two nerves, but the absence of relation in its branches to any visceral clefts renders it more difficult to determine the morphology of the fifth nerve than of the other nerves. The front branch of the two is the ophthalmic branch of the adult, and the hind branch the inferior maxillary branch. The latter branch subsequently gives off low down, i.e. near its distal extremity, another branch, the superior maxillary branch.

In its embryonic condition this latter branch does not appear like a third branch of the fifth, equivalent to the seventh or the glosso-pharyngeal nerves, but rather resembles the branch of the seventh nerve which passes to the arch in front, which also is present in all the other cranial nerves.

Modes of Preparation.

Before concluding I will say one or two words as to my modes of preparation.

I have used picric and chromic acids, both applied in the usual way; but for the early stages I have found osmic acid by far the most useful reagent. I placed the object to be hardened, in osmic acid (half per cent.) for two hours and a half, and then for twenty four in absolute alcohol.

I then embedded and cut sections of it in the usual way, without staining further.

I found it advantageous to cut sections of these embryos immediately after hardening, since if kept for long in the absolute alcohol the osmic acid specimens are apt to become brittle.

LIST OF WORKS REFERRED TO.

Gegenbaur. Anat. der Wirbelthiere, III Heft, Leipzig, 1873.

A. Götte. Archiv. für Micr. Anat., Vol. X. 1873. "Der Keim der Forelleneies," Archiv. für. Micr. Anat., Vol. IX. 1873. "Untersuchung über die Entwicklung der Bombinator igneus," Archiv. für Micr. Anat., Vol. V. 1869. "Kurze Mittheilungen aus der Entwicklungsgeschichte der Unke," Archiv. für Micr. Anat., Vol. IX. 1873.

Kupffer. Archiv. für Micr. Anat., Vol. II. 1866, p. 473. Ibid. Vol. IV. 1868, p. 209.

Kowalevsky. "Entwicklungsgeschichte der Holothurien," Mémoires de l'Acad. Impér. des Sciences de St Petersbourg, vii ser. Vol. XI. 1867.

Kowalevsky, Owsjannikow, und Wagner. "Entwicklung der Störe," Bulletin der K. Acad. St Petersbourg, Vol. XIV. 1873.

Kowalevsky. "Embryologische Studien an Würmern und Arthropoden," Mémoirs de l'Acad. Impér. des Sciences de St Petersbourg, Vol. XVI. 1871.

E. Ray Lankester. Annals and Mag. of Nat. History, Vol. XI. 1873, p. 81.

W. Müller. "Ueber die Persistenz der Urniere bei Myxine Glutinosa," Jenaische Zeitschrift, Vol. VII. 1873.

Oellacher. Zeitschrift für Wiss. Zoologie, Vol. XXIII. 1873.

Owsjannikow. "Entwicklung der Coregonus," Bul. der K. Akad. St Petersbourg, Vol. XIX.

Romiti. Archiv. für Micr. Anat., Vol. IX. 1873.

Waldeyer. Eierstock u. Eie.

EXPLANATION OF PLATES 3 AND 4.

COMPLETE LIST OF REFERENCE LETTERS.

al. Alimentary canal. ao. Dorsal aorta. auv. Auditory vesicle. bd. Formative cell probably derived from the yolk. cav. Cardinal vein. ch. Notochord. ch´. Thickening of hypoblast to form the notochord. eb. Line indicating the edge of the blastoderm. ep. Epiblast. ep´. Epidermis. er. Embryonic rim. es. Embryonic swelling. gl. Glosso-pharyngeal nerve. h. Head. ht. Heart. hy. Hypoblast. ll. Lower layer cells. ly. Line of separation between the blastoderm and the yolk. m. Mesoblast. mc. Medullary canal. mg. Medullary groove. mp. Muscle-plate. mp´. Early formed mass of muscles. n. Peculiar nuclei formed in the yolk. n´. Similar nuclei in the cells of the blastoderm. na. Cells which help to close in the alimentary canal, and which are derived from the yolk. ny. Network of lines present in the food-yolk. ol. Olfactory pit. op. Eye. ov. Oviduct. pn. Pineal gland. pov. Projection which becomes the ovary. pp. Pleuro-peritoneal cavity. pp´. Remains of pleuro-peritoneal cavity in the head. prv. Protovertebræ. pwd. Primary points of involution from the pleuro-peritoneal cavity by the coalescence of which the Wolffian duct is formed. sg. Segmentation cavity. so. Somatopleure. sos. Stalk connecting embryo with yolk-sac. sp. Splanchnopleure. spn. Spinal nerve. sur. Suprarenal body. ts. Caudal lobes. v. Blood-vessel. vg. Vagus nerve. V. Fifth nerve. VII. Seventh nerve. vc, 1, 2, 3, &c. 1st, 2nd and 3rd &c. visceral clefts. vp. Vertebral plates. wd. Wolffian duct. x. Peculiar body underlying the notochord derived from the hypoblast. yk. Yolk spherules.

All the figures were drawn with the Camera Lucida.

Plate 3.

Fig. 1. Section parallel with the long axis of the embryo through a blastoderm, in which the floor of the segmentation cavity (sg) is not yet completely lined by cells. The roof of the segmentation cavity is broken. (Magnified 60 diam.) The section is intended chiefly to illustrate the distribution of nuclei (n) in the yolk under the blastoderm. One of the chief points to be noticed in their distribution is the fact that they form almost a complete layer under the floor of the segmentation cavity. This probably indicates that the cells whose nuclei they become take some share in forming the layer of cells which subsequently (vide fig. 4) forms the floor of the cavity.

Fig. 2. Small portion of blastoderm and subjacent yolk of an embryo at the time of the first appearance of the medullary groove. (Magnified 300 diam.)

The specimen is taken from a portion of the blastoderm which will form part of the embryo. It shews two large nuclei of the yolk (n) and the network in the yolk between them; this network is seen to be closer around the nuclei than in the intervening space. The specimen further shews that there are no areas representing cells around the nuclei.

Fig. 3. Section parallel with the long axis of the embryo through a blastoderm, in which the floor of the segmentation cavity is not yet covered by a complete layer of cells. (Magnified 60 diam.)

It illustrates (1) the characters of the epiblast, (2) the embryonic swelling (es), (3) the segmentation cavity (sg). It should have been drawn upon the same scale as fig. 4; the line above it represents its true length upon this scale.

Fig. 4. Longitudinal section through a blastoderm at the time of the first appearance of the embryonic rim, and before the formation of the medullary groove. (Magnified 45 diam.)

It illustrates (1) the embryonic rim, (2) the continuity of epiblast and hypoblast at edge of this, (3) the continual differentiation of the lower layer cells, to form, on the one hand, the hypoblast, which is continuous with the epiblast, and on the other the mesoblast, between this and the epiblast; (4) the segmentation cavity, whose floor of cells is now completed.

N.B. The cells at the embryonic end of the blastoderm have been made rather too large.

Fig. 5. Surface view of the blastoderm shortly after the appearance of the medullary groove. To shew the relation of the embryo to the blastoderm.

Fig. 6_a_ and b. Two transverse sections of the same embryo, shortly after the appearance of the medullary groove. (Magnified 96 diam.)

a. In the region of the groove. It shews (1) the two masses of mesoblast on each side, and the deficiency of the mesoblast underneath the medullary groove; (2) the commencement of the closing in of the alimentary canal below, chiefly from cells (na) derived from the yolk.

b. Section in the region of the head where the medullary groove is deficient, other points as above.

Fig. 7_a_ and b. Two transverse sections of an embryo about the age or rather younger than that represented in fig. 5. (Magnified 96 diam.)

a. Section nearer the tail; it shews the thickening of the hypoblast to form the notochord (ch´).

In b the thickening has become completely separated from the hypoblast as the notochord. In a the epiblast and hypoblast are continuous at the edge of the section, owing to the section passing through the embryonic rim.

Fig. 8. Surface view of a spatula-shaped embryo. The figure shews (1) the flattened head (h) where the medullary groove is deficient, (2) the caudal lobes, with a groove between them; it also shews that at this point, the medullary groove has become roofed over and converted into a canal.

Fig. 8_a_. Transverse section of fig. 8, passing through the line a. (Magnified 90 diam.) The section shews (1) the absence of the medullary groove in the head and the medullary folds turning down at this time instead of upwards; (2) the presence of the pleuro-peritoneal cavity in the head (pp); (3) the completely closed alimentary canal (al).

Fig. 8_b_. Transverse section of fig. 8, through the line b. (Magnified 90 diam.) It shews (1) the neural canal completely formed; (2) the vertebral plates of mesoblast not yet split up into somatopleure and splanchnopleure.

Fig. 9. Side view of an embryo of the Torpedo, seen as a transparent object a little older than the embryo represented in fig. 8. (Magnified 20 diam.) The internal anatomy has hardly altered, with the exception of the medullary folds having closed over above the head and the whole embryo having become more folded off from the germ.

The two caudal lobes, and the very marked groove between them, are seen at ts. The front end of the notochord became indistinct, and I could not see its exact termination. The epithelium of the alimentary canal (al) is seen closely underlying the notochord and becoming continuous with the epiblast at the hind end of the notochord.

The first visceral cleft (1_vc_) and eye (op) are just commencing to be formed, and the cranial flexure has just appeared.

Fig. 10. Section through the dorsal region of an embryo somewhat older than the one represented in fig. 9. (Magnified 96 diam.)

It shews (1) the formation by a pinching off from the top of the alimentary canal of a peculiar body which underlies the notochord (x); (2) the primitive extension of the pleuro-peritoneal cavity up to the top of the vertebral plates.

Plate 4.

Fig. 11_a_, b, and c. Three sections closely following each other from an embryo in which three visceral clefts are present; a is the most anterior of the three. (Magnified 96 diam.) In all of these the muscle-plates are shewn at mp. They have become separated from the lateral plates in b and c, but are still continuous with them in a. The early formed mass of muscles is also shewn in all the figures (mp´).

The figures further shew (1) the formation of the spinal nerves (spn) as small bodies of cells closely applied to the upper and outer edge of the neural canal.

(2) The commencing formation of the cells which form the axial skeleton from the inner (splanchnopleuric) layer of the muscle-plate. Sections b and c are given more especially to shew the mode of formation of the oviduct (ov).

In b it is seen as a solid knob (ov), arising from the point where the somatopleure and splanchnopleure unite, and in c (the section behind b) as a solid rod (ov) closely applied to the epiblast, which has grown backwards from the knob seen in b.

N.B. In all three sections only one side is completed.

Fig. 12_a_ and b. Two transverse sections of an embryo just before the appearance of the external gills. (Magnified 96 diam.)

In a there is seen to be an involution on each side (pwd), while b is a section from the space between two involutions from the pleuro-peritoneal cavity, so that the Wolffian duct (at first solid) (wd) is not connected as in a with the pleuro-peritoneal cavity. The further points shewn in the sections are--

(1) The commencing formation of the spiral valve (al). (2) The suprarenal body (sur). (3) The oviduct (ov), which has acquired a lumen. (4) The increase in length of the muscle-plates, the spinal nerves, &c.

Fig. 13. Section through the dorsal region of an embryo in which the external gills are of considerable length. (Magnified 40 diam.) The chief points to be noticed:

(1) The formation of the Wolffian body by outgrowths from the Wolffian duct (wd). (2) One of the still continuing connections (primitive involutions) between the Wolffian duct and the pleuro-peritoneal cavity (pwd). (3) The oviduct largely increased in size (ov). N.B. On the left side the oviduct has been accidentally made too small. (4) The growth downwards of the muscle-plate to form the muscles of the abdomen. (5) The formation of an outgrowth on each side of the mesentery (pov), which will become the ovary. (6) The spiral valve (al).

Fig. 14. Transparent view of the head of an embryo shortly before the appearance of the external gills. (Magnified 20 diam.) The chief points to be noticed are--

(1) The relation of the cranial nerves to the visceral clefts and the manner in which the glosso-pharyngeal (gl) and vagus (vg) are united. (2) The remnants of the pleuro-peritoneal cavity in the head (pp). (3) The eye (op). The stalk, as well as the bulb of the eye, are supposed to be in focus, so that the whole eye has a somewhat peculiar appearance.

VI. A COMPARISON OF THE EARLY STAGES IN THE DEVELOPMENT OF VERTEBRATES[19].

Footnote 19: From the _Quarterly Journal of Microscopical Science_, Vol. XV. 1875.

With Plate 5.

If the genealogical relationships of animals are to be mainly or largely determined on embryological evidence, it becomes a matter of great importance to know how far evidence of this kind is trustworthy.

The dependence to be placed on it has been generally assumed to be nearly complete. Yet there appears to be no à priori reason why natural selection should not act during the embryonic as well as the adult period of life; and there is no question that during their embryonic existence animals are more susceptible to external forces than after they have become full grown: indeed, an immense mass of evidence could be brought to shew that these forces do act upon embryos, and produce in them great alterations tending to obscure the genealogical inferences to be gathered from their developmental histories. Even the time-honoured layers form to this no exception. In Elasmobranchii, for instance, we find the notochord derived from the hypoblast and the spinal ganglia derived from the involuted epiblast of the neural canal, whilst in the higher vertebrates both of these organs are formed in the mesoblast. Such instances are leading embryologists to recognise the fact that the so-called layers are not quite constant and must not be absolutely depended upon in the determination of homologies. But though it is necessary to recognise the fact that great changes do occur in animals during their embryonic life, it is not necessary to conclude that all embryological evidence is thereby vitiated; but rather it becomes incumbent on us to attempt to determine which embryological features are ancestral and which secondary. For this purpose it is requisite to ascertain what are the general characters of secondary features and how they are produced. Many vertebrates have in the first stages of their development a number of secondary characters which are due to the presence of food material in the ovum; the present essay is mainly an attempt to indicate how those secondary characters arose and to trace their gradual development. At the same time certain important ancestral characters of the early phases of the development of vertebrates, especially with reference to the formation of the hypoblast and mesoblast, are pointed out and their meaning discussed.

There are three orders of vertebrates of which no mention has been made, viz., the Mammals, the Osseous fishes, and the Reptiles. The first of these have been passed over because the accounts of their development are not sufficiently satisfactory, though as far as can be gathered from Bischoff's account of the dog and rabbit there would be no difficulty in shewing their relations with other vertebrates.

We also require further investigations on Osseous fishes, but it seems probable that they develop in nearly the same manner as the Elasmobranchii.

With reference to Reptiles we have no satisfactory investigations.

* * * * *

Amphioxus is the vertebrate whose mode of development in its earliest stages is simplest, and the modes of development of other vertebrates are to be looked upon as modifications of this due to the presence of food material in their ova. It is not necessary to conclude from this that Amphioxus was the ancestor of our present vertebrates, but merely that the earliest stages of development of this vertebrate ancestor were similar to those of Amphioxus.

The ovum of Amphioxus contains very little food material and its segmentation is quite uniform. The result of segmentation is a vesicle whose wall is formed of a single layer of cells. These are all of the same character, and the cavity of the vesicle called the segmentation cavity is of considerable size. A section of the embryo, as we may now call the ovum, is represented in Plate 5, fig. A I.

The first change which occurs is the pushing in of one half of the wall of the vesicle towards the opposite half. At the same time by the narrowing of its mouth the hollow hemisphere so formed becomes again a vesicle[20].

Footnote 20: I have been able to make at Naples observations which confirm the account of the invagination of Amphioxus as given by Kowalevsky, though my observations are not nearly so complete as those of the Russian naturalist.

Owing to its mode of formation the wall of this secondary vesicle is composed of two layers which are only separated by a narrow space, the remnant of the segmentation cavity.

Two of the stages in the formation of the secondary vesicle by this process of involution are shewn in Plate X, fig. A II, and A III. In the second of these the general growth has been very considerable, rendering the whole animal much larger than before. The cavity of this vesicle, A III, is that of the commencing alimentary canal whose final form is due to changes of shape undergone by this primitive cavity. The inner wall of the vesicle becomes converted into the wall of the alimentary canal or hypoblast, and also into part or the whole of the mesoblast.

During the involution the cells which are being involuted undergo a change of form, and before the completion of the process have acquired a completely different character to the cells forming the external wall of the secondary vesicle or epiblast. This change of character in the cells is already well marked in fig. A II. It is of great importance, since we shall find that some of the departures from this simple mode of development, which characterise other vertebrates, are in part due to the distinction between the hypoblast and epiblast cells appearing during segmentation, and not subsequently as in Amphioxus during the involution of the hypoblast.

Kowalevsky (Entwicklungsgeschichte des Amphioxus) originally believed that the narrow mouth of the vesicle (according to Mr Lankester's terminology blastopore) became the anus of the adult. He has since, and certainly correctly, given up this view. The opening of the involution becomes closed up and the adult anus is no doubt formed as in all other vertebrates by a pushing in from the exterior, though it probably corresponds in position very closely with the point of closing up of the original involution.

The mode of formation of the mesoblast is not certainly known in Amphioxus; we shall find, however, that for all other vertebrates it arises from the cells which are homologous with the involuted cells of this animal.

Since food material is a term which will be very often employed, it will be well to explain exactly the sense in which it will be used. It will be used only with reference to those passive highly refractive particles which are found embedded in most ova.

In some eggs, of which the hen's egg may be taken as a familiar example, the yolk-spherules or food material form the larger portion of the ovum, and a distinction is frequently made between the germinal disc and the yolk.

This distinction is, however, apt to lead to a misconception of the true nature of the egg. There are strong grounds for believing that the so-called yolk, equally with the germinal disc, is composed of an active protoplasmic basis endowed with the power of growth, in which passive yolk-spherules are embedded; but that the part ordinarily called the yolk contains such a preponderating amount of yolk-spherules that the active basis escapes detection, and does not exhibit the same power of growth as the germinal disc.

With the exception of mammals, whose development requires to be more completely investigated, Amphioxus is as far as we know the only vertebrate whose ovum does not contain a large amount of food material.

In none of these (vertebrate) yolk-containing ova is the food material distributed uniformly. It is always concentrated much more at one pole than at the other, and the pole at which it is most concentrated may be conveniently called the lower pole of the egg.

In eggs in which the distribution of food material is not uniform segmentation does not take place with equal rapidity through all parts of the egg, but its rapidity is, roughly speaking, inversely proportional to the quantity of food material.

When the quantity of food material in a part of the egg becomes very great, segmentation does not occur at all; and even in those cases where the quantity of food yolk is not too great to prevent segmentation the resulting segmentation spheres are much larger than where the yolk-granules are more sparsely scattered.

The Frog is the vertebrate whose development comes nearest to that of Amphioxus, as far as the points we are at present considering are concerned. But it will perhaps facilitate the understanding of their relations shortly to explain the diagrammatic sections which I have given of an animal supposed to be intermediate in its development between the Frog and Amphioxus. Plate 5, fig. B I, represents a longitudinal section of this hypothetical egg at the close of segmentation. The lower pole, coloured yellow, represents the part containing more yolk material, and the upper pole, coloured blue, that with less yolk. Owing to the presence of this yolk the lower pole even at the close of segmentation is composed of cells of a different character to those of the upper pole. In this respect this egg can already be distinguished from that of Amphioxus, in which no such difference between the two poles is apparent at the corresponding period (Plate 5, fig. A I).

The segmentation cavity in this ovum is not quite so large proportionately as in Amphioxus, and the encroachment upon it is due to the larger bulk of the lower pole of the egg. In fig. B II the involution of the lower pole has already commenced; this involution is (1) not quite symmetrical, and (2) on the ventral side (the left side) the epiblast cells forming the upper part of the egg are growing round the cells of the lower pole of the egg or lower layer cells. Both of these peculiarities are founded upon what happens in the Frog and the Selachian, but it is to be noticed that the change from the lower layer cells being involuted towards the epiblast cells, to the epiblast cells growing round the lower layer cells, is a necessary consequence of the increased bulk of the latter.

In this involution not only are the cells of the lower pole pushed on, but also some of those of the upper or yellow portion; so that in this as in all other cases the true distinction between the epiblast and hypoblast does not appear till the involution to form the latter is completed. In the next stage, B III, the involution has become nearly completed and the opening to the exterior or blastopore quite constricted.

The segmentation cavity has been entirely obliterated, as would have been found to be the case with Amphioxus had the stage a little older than that on Plate 5, A III, been represented. The cavity marked (al), as was the case with Amphioxus, is that of the alimentary canal.

The similarities between the mode of formation of the hypoblast and alimentary canal in this animal and in Amphioxus are so striking and the differences between the two cases so slight that no further elucidation is required. One or two points need to be spoken of in order to illustrate what occurs in the Frog. When the involution to form the alimentary canal occurs, certain of the lower layer cells (marked hy) become distinguished from the remainder of the lower layer cells as a separate layer and form the hypoblast which lines the alimentary canal. It is to be noticed that the cells which form the ventral epithelium of the alimentary canal are not so soon to be distinguished from the other lower layer cells as those which form its dorsal epithelium. This is probably a consequence of the more active growth, indicated by the asymmetry of the involution, on the dorsal side, and is a fact with important bearings in the ova with more food material. The cells marked m and coloured red also become distinguished as a separate layer from the remainder of the hypoblast and form the mesoblast. The remainder of the lower layer cells form a mass equivalent to the yolk-sac of many vertebrates, and are not converted directly into the tissues of the animal.

Another point to be noticed is the different relation of epiblast cells to the hypoblast cells at the upper and lower side of the mouth of the involution. Above it, on its dorsal side, the epiblast and hypoblast are continuous with one another. On its ventral side they are primitively not so continuous. This is due to the epiblast, as was before mentioned, growing round the lower layer cells on the ventral side, vide B II, and merely remaining continuous with them on the dorsal. The importance of these two points will appear when we come to speak of other vertebrates.

The next animal whose development it is necessary to speak of is the Frog, and its differences from the mode of development are quite easy to follow and interpret. Segmentation is again not uniform, and results in the formation of an upper layer of smaller cells and a lower one of larger; in the centre is a segmentation cavity. The stage at the close of segmentation is represented in C I. From the diagram it is apparent that the lower layer cells occupy a larger bulk than they did in the previous animal (Plate 5, B I), and tend to encroach still more upon the segmentation cavity, otherwise the differences between the two are unimportant. There are, however, two points to be noted. In the first place, although the cells of the upper pole are distinguished in the diagrams from the lower by their colour, it is not possible at this stage to say what will become epiblast and what hypoblast. In the second place the cells of the upper pole or epiblast consist of two layers--an outer called the epidermic layer and an inner called the nervous. In the previous cases the epiblast consisted of a single layer of cells. The presence of these two layers is due to a distinction which, arising in most other vertebrates late, in the Frog arises early. In most other vertebrates in the later stages of development the epiblast consists of an outer layer of passive and an inner of active cells. In the Frog and other Batrachians these two layers become distinguished at the commencement of development.

In the next stage (C II) we find that the involution to form the alimentary canal has commenced (al), but that it is of a very different character to the involution in the previous case. It consists in the growing inwards of a number of cells from the point x (C I) towards the segmentation cavity. The cells which grow in this way are partly the blue cells and partly the smaller yellow ones. At first this involuted layer of cells is only separated by a slit from the remainder of the lower layer cells; but by the stage represented in C II this has widened into an elongated cavity (al). In its formation this involution pushes backwards the segmentation cavity, which finally disappears in the stage C III. The point x remains practically stationary, but by the general growth of the epiblast, mesoblast and hypoblast, becomes further removed from the segmentation cavity in C II than in C I. On the opposite side of the embryo to that at which the involution occurs the epiblast cells as before, grow round the lower layer cells. The commencement of this is already apparent in C I, and in C II the process is nearly completed, though there is still a small mass of yolk filling up the blastopore. The features of this involution are in the main exaggerations of what was supposed to occur in the previous animal. The asymmetry of the involution is so great that it is completely one-sided and results, in the first instance, in a mere slit; and the whole process of enclosing the yolk by epiblast is effected by the epiblast cells on the side of the egg opposite to the involution.

The true mesoblast and hypoblast are formed precisely as in the previous case. The involuted cells become separated into two layers, one forming the dorsal epithelium of the alimentary canal, and a layer between this and the epiblast forming the mesoblast. There is also a layer of mesoblast accompanying the epiblast which encloses the yolk, which is derived from the smaller yellow cells at y (C I). The edge of this mesoblast, m´, forms a thickened ridge, a feature which persists in other vertebrates.

It is a point of some importance for understanding the relation between the mode of formation of the alimentary canal in the Frog and other vertebrates to notice that on the ventral surface the cells which are to form the epithelium of the alimentary canal become distinguished as such very much later than do those to form its dorsal epithelium, and are derived not from the involuted cells but from the primitive large yolk-cells. It is indeed probable that only a very small portion of epithelium of the ventral wall of the mid-gut is in the end derived from these larger yolk-cells. The remainder of the yolk-cells (C III, and C II, yk) form the yolk mass and do not become directly formed into the tissues of the animal.

In the last stage I have represented for the frog, C III, there are several features to be noticed.

The direct connection at their hind-ends between the cavities of the neural and alimentary canals is the most important of these. This is a result of the previous continuity of the epiblast and hypoblast at the point x, and is a feature almost certainly found in Amphioxus, but which I will speak of more fully in my account of the Selachian's development. The opening of the blastopore called the anus of Rusconi is now quite narrowed, it does not become the anus of the adult. It may be noticed that at the front end of the embryo the primitive dorsal epithelium of the alimentary canal is growing in such a way as to form the epithelium both of the dorsal and ventral surfaces of the fore-gut.

In spite of various features rendering the development of the Frog more difficult of comprehension than that of most other vertebrates, it is easy to see that the step between it and Amphioxus is not a very great one, and will very likely be bridged over at some future time, when our knowledge of the development of other forms becomes greater.

From the Frog to the Selachian is a considerable step, but I have again hypothetically sketched a type intermediate between them whose development agrees in some important points with that of Pelobates fuscus as described by Bambeke. The points of agreement, though not obvious at first sight, I shall point out in the course of my description.

The first stage (D I), at the close of segmentation, deserves careful attention. The segmentation cavity by the increase of the food yolk is very much diminished in size, and, what is still more important, has as it were sunk down so as to be completely within the lower layer cells. The roof of the segmentation cavity is thus formed of epiblast and lower layer cells, a feature which Bambeke finds in Pelobates fuscus and which is certainly found in the Selachians. In the Frog we found that the segmentation cavity began to be encroached on by the lower layer cells, and from this it is only a small step to find these cells creeping still further up and forming the roof of the cavity. In the lower layer cells themselves we find an important new feature, viz. that during segmentation they become divided in two distinct parts--one of these where the segments owing to the presence of much food yolk are very large, and the other where the segments are much smaller.

The separation between these two is rather sharp. Even this separation was foreshadowed in the Frog's egg, in which a number of lower layer cells were much smaller and more active at the two sides of the segmentation cavity than elsewhere. The segmentation cavity at first lies completely within the region of the small spheres. The larger cells serve almost entirely as food yolk. The epiblast, as is normal with vertebrates, consists of a single layer of columnar cells.

In the next stage (D II) the formation of the alimentary canal (al) has commenced, but it is to be observed that there is in this case no true involution.

As an accompaniment to the encroachment upon the segmentation cavity, which was a feature of the last stage, the cells to form the walls of the alimentary canal have come to occupy their final position during segmentation and without the intermediation of an involution, and traces only of the involution, are to be found in (1) a split in the lower layer cells which passes along the line separating the small and the large lower layer cells; and (2) in the epiblast becoming continuous with the hypoblast on the dorsal side of the mouth of this split. It is even possible that at this point a few cells (though certainly only a very small number) of those marked blue in D I become involuted. This point in this, as in all other cases, is the tail end of the embryo. The other features of this stage are as follows:--(1) The segmentation cavity has become smaller and less conspicuous than it was. (2) The epiblast cells have begun to grow round the yolk even in a more conspicuous manner than they did in the Frog, and are accompanied by a layer of mesoblast cells which again becomes thickened at its edge. The mesoblast cells in the region of the body are formed in the same way as before, viz. by the separation of a layer to form the epithelium of the alimentary canal, the other cells remaining as mesoblast; and as in the Frog, or in a more conspicuous manner, we find that the dorsal surface only of the alimentary cavity has a wall formed of a distinct layer of cells, but on the ventral side the cavity is at first closed in by the large spheres of the yolk only. The formation of the alimentary canal by a split and not by an involution is exactly what Bambeke finds in Pelobates.

The next stage, D III, is about an equivalent age to C III in the Frog. It exhibits the same connection between the neural and the alimentary canals as was found there.

The alimentary canal is beginning to become closed in below, and this occurs near the two ends earlier than in the middle. The cells to form the ventral wall are derived from the large yolk-cells. The non-formation of the ventral wall of the alimentary canal so soon in the middle as at the ends is an early trace of the umbilical canal found in Birds and Selachians, by which the alimentary tract is placed in communication with the yolk-sac. The segmentation cavity has by this stage completely vanished, and the epiblast with its accompanying mesoblast has spread completely round the yolk material so as to form the ventral wall of the body.

Though in some points this manner of development may seem to differ from that of the Frog, there is really a fundamental agreement between the two, and between this mode of development and that of the Selachians we shall find the agreement to be very close.

After segmentation we find that the egg of a Selachian consists of two parts--one of these called the germinal disc or blastoderm, and the other the yolk. The former of these corresponds with the epiblast and the part of the lower pole composed of smaller segments in the last-described egg, and the latter to the larger segments of the lower pole. This latter division, owing to the quantity of yolk which it contains, has not undergone segmentation, but its homology with the larger segments of the previous eggs is proved (1) by its containing a number of nuclei (E I, n), which become the nuclei of true cells and enter the blastoderm, and (2) by the presence in it of a number of lines forming a network similar to that of many cells. The segmentation cavity, as before, lies completely within the lower layer cells.

The next stage, E II, is almost precisely similar to the second stage of the last egg. As there, the primitive involution is merely represented by a split separating the yolk and the germinal disc, and on the dorsal side alone is there a true cellular wall for this split, and at the dorsal mouth of the split the alimentary epithelium becomes continuous with the epiblast.

The segmentation cavity has become diminished, and round the yolk the epiblast, accompanied by a layer of mesoblast, is commencing to grow. In this growth all parts of the blastoderm take a share except that part where the epiblast and hypoblast are continuous. This manner of growth is precisely what occurs in the Frog, though there it is not so easily made out; and not all the investigators who have studied the Frog have understood the exact meaning of the appearances they have seen and drawn. This similarity of relation of the epiblast to the yolk in the two cases is a further confirmation of the identity of the Selachian's yolk with the large yolk-spheres of the previous eggs.

The next stage, E III, is in many ways identical with the corresponding stage in the last-described egg, and in the same way as in that case the neural and alimentary canals are placed in communication with each other.

The mode in which this occurs will be easily gathered from a comparison of E II and E III. It is the same for the Selachians and Batrachians. The neural canal (nc) is by the stage figured E III, completely formed in the way so well known in the Bird, and between the roof of the canal and the external epiblast a layer of mesoblast has already grown in. The floor of the neural canal is the same layer marked ep in E II, and therefore remains continuous with the hypoblast at x; and when by a simultaneous process the roof of the neural canal and the ventral wall of the alimentary become formed by the folding over of one continuous layer (the epiblast and hypoblast continuous at the point x), the two canals, viz. the neural and alimentary, are necessarily placed in communication at their hind-ends, as is seen in the diagram.

There are several important points of difference between E III and D III. In the first place, owing to the larger size of the yolk mass in E III, the epiblast, accompanied by mesoblast, has not proceeded nearly so far round it as in the previous case. It is also worth notice that at the right as well as at the left end of the germinal disc the epiblast is commencing to grow round the yolk. The yolk has, however, become surrounded to a much smaller extent on the right hand than on the left. Since, in the earlier stage, the epiblast became continuous with the hypoblast at x, it is not from sections obvious how this occurs. I have therefore appended a diagram to explain it (E´). The blastoderm rests like a disc on the yolk and grows over it on all sides, except at the point where the epiblast and hypoblast are continuous (x). This point becomes as it were left in a bay. Next the two sides of the bay coalesce, the bay becomes obliterated, and the effect produced is exactly as if the blastoderm had grown round the yolk at the point x (corresponding with the tail of the embryo) as well as everywhere else. It thus comes about that the final point where the various parts of the blastoderm meet and completely enclose the yolk mass does not correspond with the anus of Rusconi of the Frog, but is at some little distance from the hind-end of the embryo. In other words, the position of the blastopore in the Selachian is not the same as in the Frog.

Another point deserving attention is the formation of the ventral wall of the alimentary canal. This takes place in two ways--partly by a folding-in at the sides and end, and partly from cells formed around the nuclei (n) in the yolk. From these a large portion of the ventral wall of the mid-gut is formed.

The folding-in of the sheet of hypoblast to assist in the closing-in of the ventral wall of the alimentary canal is a consequence of the flattened form of the original alimentary slit which is far too wide to form the cavity of the final canal. In the Bird whose development must next be considered this folding-in is a still more prominent feature in the formation of the alimentary canal. As in the last case, the alimentary canal is widely open in the middle to the yolk at the time when its two ends are closed below and shut off from it; still later this opening becomes very narrow and forms the duct of the so-called umbilical cord which places the yolk-sac in communication with the alimentary canal. As the young animal becomes larger the yolk-sac ceases to communicate directly with the alimentary canal, and is carried about by it for some time as an appendage and only at a later period shrivels up.

The mesoblast is formed in a somewhat different way in the Sharks than in other vertebrates. It becomes split off from the hypoblast, not in the form of a single sheet as in other vertebrates, but as two lateral sheets, one on each side of the middle line and separated from one another by a considerable interval; whilst the notochord is derived not as in other vertebrates from the mesoblast, but from the hypoblast (vide F. M. Balfour, "Development of Selachians[21]," Journal of Microscopical Science, Oct., 1874).

Footnote 21: Paper No. V, p. 82 et seq. in this edition.

Between the Selachians and the Aves there is a considerable gulf, which it is more difficult satisfactorily to bridge over than in the previous cases; owing to this I have not attempted to give any intermediate stage between them.

The first stage of the Bird (F I) is very similar in many respects to the corresponding stage in the Selachian. The segmentation cavity is, however, a less well-defined formation, and it may even be doubted whether a true segmentation cavity, homologous with the segmentation cavity in the previously described eggs, is present. On the floor of the cavity which is formed by the yolk are a few larger cells known as formative cells which, according to Götte's observations, are derived from the yolk, in a somewhat similar manner to the cells which were formed around the nuclei in the Selachian egg, and which helped to form the ventral wall of the alimentary canal. Another point to be noticed is that the segmentation cavity occupies a central position, and not one to the side as in the Selachian.

The yolk is proportionately quite as large as in the Selachian's egg, but, as in that case, there can be little or no doubt of its being homologous with the largest of the segmentation spheres of the previous eggs. It does not undergo segmentation. The epiblast is composed of columnar cells, and extends a short way beyond the edge of the lower layer cells.

In the next stage the more important departures from the previous type of development become visible.

The epiblast spreads uniformly over the yolk-sac and not on the one side only as in the former eggs.

This is due to the embryo (indicated in F II by a thickening of the cells) lying in the centre and not at the edge of the blastoderm. A necessary consequence of this is, that the epiblast does not, as in the previous cases, become continuous with the hypoblast at the tail end of the embryo. This continuity, being of no functional importance, could easily be dispensed with, and the central position of the embryo may perhaps be explained by supposing the process, by which in the Selachian egg the blastopore ceases to correspond in position with the opening of the alimentary slit or anus of Rusconi (vide E´), to occur quite early during segmentation instead of at a late period of development. For the possibility of such a change in the date of formation, the early appearance of the nervous and epidermic layers in the Frog affords a parallel.

The epiblast in its growth round the yolk is only partially accompanied by mesoblast, which, however, is thickened at its extreme edge as in the Frog. Owing to the epiblast not becoming continuous with the hypoblast at the tail end of the embryo, the alimentary slit is not open to the exterior. The hypoblast is formed by some of the lower layer cells becoming distinguished as a separate layer; the remainder of the lower layer cells become the mesoblast.

The formation of the mesoblast and hypoblast out of the lower layer cells has been accepted for the Bird by most observers, but has been disputed by several, and recently by Kölliker. These have supposed that the mesoblast is derived from the epiblast. I feel convinced that these observers are in the wrong, and that the mesoblast is genuinely derived from the lower layer cells.

The greater portion of the alimentary cavity consists of the original segmentation cavity (vide diagrams). This feature of the segmentation cavity of Birds sharply distinguishes it from any segmentation cavity of other eggs, and renders it very doubtful whether the similarly named cavities of the Bird and of other vertebrates are homologous. On the floor of the cavity are still to be seen some of the formative cells, but observers have not hitherto found that they take any share in forming the ventral wall of the alimentary canal.

The features of the next stage are the necessary consequences of those of the last.

The ventral wall of the alimentary canal is entirely formed by a folding-in of the sheet of hypoblast.

The more rapid folding-in at the head still indicates the previous more vigorous growth there, otherwise there is very little difference between the forms of the fold at the head and tail. The alimentary canal does not of course, at this or any period, communicate with the neural tube, since the epiblast and hypoblast are never continuous. The other features, such as the growth of the epiblast round the yolk-sac, are merely continuations of what took place in the last stage.

In the development of a yolk-sac as a distinct appendage, and its absorption within the body, at a later period, the bird fundamentally resembles the Dog-fish.

Although there are some difficulties in deriving the type of development exhibited by the Bird directly from that of the Selachian, it is not very difficult to do so directly from Amphioxus. Were the alimentary involution to remain symmetrical as in Amphioxus, and the yolk-containing part of the egg to assume the proportions it does in the Bird, we should obtain a mode of development which would not be very dissimilar to that of the Bird. The epiblast would necessarily overgrow the yolk uniformly on all sides and not in the unsymmetrical fashion of the Selachian egg. A confirmation of this view might perhaps be sought for in the complete difference between the types of circulation of the yolk-sac in Birds and Selachians; but this is not so important as might at first sight appear, since it is not from the Selachian egg but from some Batrachian that it would be necessary to derive the Reptiles' and Birds' eggs.

If this view of the Bird's egg be correct, we are compelled to suppose that the line of ancestors of Birds and Reptiles did not include amongst them the Selachians and the Batrachians, or at any rate Selachians and Batrachians which develop on the type we now find.

The careful investigation of the development of some Reptiles might very probably throw light upon this important point. In the meantime it is better to assume that the type of development of Birds is to be derived from that of the Frog and Selachians.

Summary.--If the views expressed in this paper are correct, all the modes of development found in the higher vertebrates are to be looked upon as modifications of that of Amphioxus. It is, however, rather an interesting question whether it is possible to suppose that the original type was not that of Amphioxus, but of some other animal, say, for instance, that of the Frog, and that this varied in two directions,--on the one hand towards Amphioxus, in the reverse direction to the course of variation presupposed in the text; and on the other hand in the direction towards the Selachians as before.

The answer to this question must in my opinion be in the negative. It is quite easy to conceive the food material of the Frog's egg completely vanishing, but although this would entail simplifications of development and possibly even make segmentation uniform, there would, as far as I can see, be no cause why the essential features of difference between the Frog's mode of development and that of Amphioxus should change. The asymmetrical and slit-like form of involution on the one side and the growth of the epiblast over the mesoblast on the other side, both characteristics of the present Frog's egg, would still be features in the development of the simplified egg.

In the Mammal's egg we probably have an example of a Reptile's egg simplified by the disappearance of the food material; and when we know more of Mammalian embryology it will be very interesting to trace out the exact manner in which this simplification has affected the development. It is also probable that the eggs of Osseous fish are fundamentally simplified Selachian eggs; in which case we already know that the diminution of food material has affected but very slightly the fundamental features of development.

One common feature which appears prominently in reviewing the embryology of vertebrates as a whole is the derivation of the mesoblast from the hypoblast; in other words, we find that it is from the layer corresponding to that which becomes involuted in Amphioxus so as to line the alimentary cavity that the mesoblast is split off.

That neither the hypoblast or mesoblast can in any sense be said to be derived from the epiblast is perfectly clear. When the egg of Amphioxus is in the blastosphere stage we cannot speak of either an epiblast or hypoblast. It is not till the involution or what is equivalent has occurred, converting the single-walled vesicle into a double-walled one, that we can speak of these two layers. It might seem scarcely necessary to insist upon this point, so clear is it without explanation, were it not that certain embryologists have made a confusion about it.

The derivation of the mesoblast from the hypoblast is the more interesting, since it is not confined to the vertebrates, but has a very wide extension amongst the invertebrates. In the cases (whose importance has been recently insisted upon by Professor Huxley), of the Asteroids, the Echinoids, Sagitta, and others, in which the body-cavity arises as an outgrowth of the alimentary canal and the somatopleure and splanchnopleure are formed from that outgrowth, it is clear without further remark that the mesoblast is derived from the hypoblast. For the Echinoderms in which the water-vascular system and muscular system arise as a solid outgrowth of the wall of the alimentary canal there can also be no question as to the derivation of the mesoblast from the hypoblast.

Amongst other worms, in addition to Sagitta, the investigations of Kowalevsky seem to shew that in Lumbricus the mesoblast is derived from the hypoblast.

Amongst Crustaceans, Bobretsky's[22] observations on Oniscus (Zeitschrift für wiss. Zoologie, 1874) lead to the same conclusion.

Footnote 22: He says, p. 182: "Bevor aber die Hälfte der Eioberfläche von den Embryonalzellen bedeckt ist, kommt die erste gemeinsame Anlage des mittleren und unteren Keimblattes zum Vorschein."

In Insects Kowalevsky's observations lead to the conclusion that mesoblast and hypoblast arise from a common mass of cells; Ulianin's observations bring out the same result for the abnormal Poduridæ, and Metschnikoff's observations shew that this also holds for Myriapods.

In Molluscs the point is not so clear.

In Tunicates, even if we are not to include them amongst vertebrates[23], the derivation of mesoblast from hypoblast is without doubt.

Footnote 23: Anton Dohrn, Der Ursprung des Wirbelthieres. Leipzig, 1875.

Without going further into details it is quite clear that the derivation of the mesoblast from the hypoblast is very general amongst invertebrates.

It will hardly be disputed that primitively the muscular system of the body-wall could not have been derived from the layer of cells which lines the alimentary canal. We see indeed in Hydra and the Hydrozoa that in its primitive differentiation, as could have been anticipated beforehand, the muscular system of the body is derived from the epiblast cells. What, then, is the explanation of the widespread derivation of the mesoblast, including the muscular system of the body, from the hypoblast?

The explanation of it may, I think, possibly be found, and at all events the suggestion seems to me sufficiently plausible to be worth making, in the fact that in many cases, and probably this applies to the ancestors of the vertebrates, the body-cavity was primitively a part of the alimentary.

Mr Lankester, who has already entered into this line of speculation, even suggests (Q. J. of Micr. Science, April, 1875) that this applies to all higher animals. It might then be supposed that the muscular system of part of the alimentary canal took the place of the primitive muscular system of the body; so that the whole muscular system of higher animals would be primitively part of the muscular system of the digestive tract.

I put this forward merely as a suggestion, in the truth of which I feel no confidence, but which may perhaps induce embryologists to turn their attention to the point. If we accept it for the moment, the supplanting of the body muscular system by that of the digestive tract may hypothetically be supposed to have occurred in the following way.

When the diverticulum or rather paired diverticula were given off from the alimentary canal they would naturally become attached to the body-wall, and any contractions of their intrinsic muscles would tend to cause movements in the body-wall. So far there is no difficulty, but there is a physiological difficulty in explaining how it can have happened that this secondary muscular system can have supplanted the original muscular system of the body.

The following suggestions may lessen this difficulty, though perhaps they hardly remove it completely. If we suppose that the animal in which these diverticula appeared had a hard test and was not locomotive, the intrinsic muscular system of the body would naturally completely atrophy. But since the muscular system of the diverticula from the stomach would be required to keep up the movement of the nutritive fluid, it would not atrophy, and were the test subsequently to become soft and the animal locomotive, would naturally form the muscular system of the body. Or even were the animal locomotive in which the diverticula appeared, it is conceivable that the two systems might at first coexist together; that either (1) subsequently owing to the greater convenience of early development, the two systems might acquire a development from the same mass of cells and those the cells of the inner or hypoblast layer, so that the derivation of the body muscles from the hypoblast would only be apparent and not real, or (2) owing to their being better nourished as they would necessarily be, and to their possibly easier adaptability to some new form of movement of the animal, the muscle-cells of the alimentary canal might become developed exclusively whilst the original muscular system atrophied.

I only hold this view provisionally till some better explanation is given of the cases of Sagitta and the Echinoderms, as well as of the nearly universal derivation of the mesoblast from the hypoblast. The cases of this kind may be due to some merely embryonic changes and have no meaning in reference to the adult condition, but I think that we have no right to assume this till some explanation of the embryonic can be suggested.

For vertebrates, I have shewn that in Selachians the body-cavity at first extends quite to the top of what becomes the muscle plate, so that the line or space separating the two layers of the muscle plate (vide Balfour, 'Development of Elasmobranch Fishes[24],' Quart. Journ. of Micro. Science for Oct., 1874. Plate XV, fig. 11,a, 11_b_, 12_a_, mp.) is a portion of the original body-cavity. If this is a primitive condition, which is by no means certain, we have a condition which we might expect, in which both the inner and the outer wall of the primitive body-cavity assists in forming the muscular system of the body.

Footnote 24: Paper No. V, p. 60 et seq. of this edition, pl. 4, figs. 11_a_, 11_b_, 12_a_, mp.

It is very possible that the formation of the mesoblast as two masses, one on each side of the middle line as occurs in Selachians, and which as I pointed out in the paper quoted above also takes place in some worms, is a remnant of the primitive formation of the body-cavity as paired outgrowth of the alimentary canal. This would also explain the fact that in Selachians the body-cavity consists at first of two separate portions, one on each side of the alimentary canal, which only subsequently become united below and converted into a single cavity (vide loc. cit.[25], Plate XIV, fig. 8_b_, pp).

Footnote 25: Pl. 3 of this edition, fig. 8_b_, pp.

In the Echinoderms we find instances where the body-cavity and water-vascular system arise as an outgrowth from the alimentary canal, which subsequently becomes constricted off from the latter (Asteroids and Echinoids), together with other instances (Ophiura, Synapta) where the water-vascular system and body-cavity are only secondarily formed in a solid mass of mesoblast originally split off from the walls of the alimentary canal.

These instances shew us how easily a change of this kind may take place, and remove the difficulty of understanding why in vertebrates the body-cavity never communicates with the alimentary.

The last point which I wish to call attention to is the blastopore or anus of Rusconi.

This is the primitive opening by which the alimentary canal communicates with the exterior, or, in other words, the opening of the alimentary involution. It is a distinctly marked structure in Amphioxus and the Batrachians, and is also found in a less well-marked form in the Selachians; in Birds no trace of it is any longer to be seen. In all those vertebrates in which it is present, it closes up and does not become the anus of the adult. The final anus nevertheless corresponds very closely in position with the anus of Rusconi. Mr Lankester has shewn (Quart. Journ. of Micro. Science for April, 1875) that in invertebrates as well as vertebrates the blastopore almost invariably closes up. It nevertheless corresponds as a rule very nearly in position either with the mouth or with the anus.

If this opening is viewed, as is generally done, as really being the mouth in some cases and the anus in others, it becomes very difficult to believe that the blastopore can in all cases represent the same structure. In a single branch of the animal kingdom it sometimes forms the mouth and sometimes the anus: thus for instance in Lumbricus it is the mouth (according to Kowalevsky), in Palæmon (Bobretzky) the anus. Is it credible that the mouth and anus have become changed, the one for the other?

If, on the other hand, we accept the view that the blastopore never becomes either the one or the other of these openings, it is, I think, possible to account for its corresponding in position with the mouth in some cases or the anus in others.

That it would soon come to correspond either with the mouth or anus (probably with the earliest formed of these in the embryo), wherever it was primitively situated, follows from the great simplification which would be effected by its doing so. This simplification consists in the greater facility with which the fresh opening of either mouth or anus could be made where the epiblast and hypoblast were in continuity than elsewhere. Even a change of correspondence from the position of the mouth to that of the anus or vice versa could occur. The mode in which this might happen is exemplified by the case of the Selachians. I pointed out in the course of this paper how the final point of envelopment of the yolk became altered in Selachians so as to cease to correspond with the anus of Rusconi; in other words, how the position of the blastopore became changed. In such a case, if the yolk material again became diminished, the blastopore would correspond in position with neither mouth nor anus, and the causes which made it correspond in position with the anus before, would again operate, and make it correspond in position perhaps with the mouth. Thus the blastopore might absolutely cease to correspond in position with the anus and come to correspond in position with the mouth.

It is hardly possible to help believing that the blastopore primitively represented a mouth. It may perhaps have lost this function owing to an increase of food yolk in the ovum preventing its being possible for the blastopore to develop directly into a mouth, and necessitating the formation of a fresh mouth. If such were the case, there would be no reason why the blastopore should ever again serve functionally as a mouth in the descendants of the animal which developed this fresh mouth.

EXPLANATION OF PLATE 5.

COMPLETE LIST OF REFERENCES.

al. Cavity of alimentary canal. bl. Blastoderm. ch. Notochord. ep. Epiblast. em. Embryo. f. Formative cells. hy. Hypoblast. ll. Lower layer cells. m. Mesoblast. n. Nuclei of yolk of Selachian egg. nc. Neural canal. sg. Segmentation cavity. x. Point where epiblast and hypoblast are continuous at the mouth of the alimentary involution. This point is always situated at the tail end of the embryo. yk. Yolk.

Epiblast is coloured blue, mesoblast red, and hypoblast yellow. The lower layer cells before their separation into hypoblast and mesoblast are also coloured green.

A I, A II, A III. Diagrammatic sections of Amphioxus in its early stages (founded upon Kowalevsky's observations).

B I, B II, B III. Diagrammatic longitudinal sections of an hypothetical animal, intermediate between Amphioxus and Batrachians, in its early stages.

C I, C II, C III. Diagrammatic longitudinal sections of Bombinator igneus in its early stages (founded upon Götte's observations). In C III the neural canal is completed, which was not the case in B III. The epiblast in C III has been diagrammatically represented as a single layer.

D I, D II, D III. Diagrammatic longitudinal sections of an animal, intermediate between Batrachians and Selachians, in its early stages.

E I, E II, E III. Diagrammatic longitudinal sections of a Selachian in its early stages.

E´. Surface view of the yolk of a Selachian's egg to shew the manner in which it is enclosed by the Blastoderm. The yolk is represented yellow and the Blastoderm blue.

F I, F II, F III. Diagrammatic longitudinal sections of a Bird in its early stages.

VII. ON THE ORIGIN AND HISTORY OF THE URINOGENITAL ORGANS OF VERTEBRATES[26].

Footnote 26: From the Journal of Anatomy and Physiology, Vol. X. 1875.

Recent discoveries[27] as to the mode of development and anatomy of the urinogenital system of Selachians, Amphibians, and Cyclostome fishes, have greatly increased our knowledge of this system of organs, and have rendered more possible a comparison of the types on which it is formed in the various orders of vertebrates.

Footnote 27: The more important of these are:--

Semper--Ueber die Stammverwandtschaft der Wirbelthiere u. Anneliden. Centralblatt f. Med. Wiss. 1874, No. 35.

Semper--Segmentalorgane bei ausgewachsenen Haien. Centralblatt f. Med. Wiss. 1874, No. 52.

Semper--Das Urogenitalsystem der höheren Wirbelthiere. Centralblatt f. Med. Wiss. 1874, No. 59.

Semper--Stammesverwandtschaft d. Wirbelthiere u. Wirbellosen. Arbeiten aus Zool. Zootom. Inst. Würzburg. II Band.

Semper--Bildung u. Wachstum der Keimdrüsen bei den Plagiostomen. Centralblatt f. Med. Wiss. 1875, No. 12.

Semper--Entw. d. Wolf. u. Müll. Gang. _Centralblatt f. Med. Wiss._ 1875, No. 29.

Alex. Schultz--Phylogenie d. Wirbelthiere. _Centralblatt f. Med. Wiss._ 1874, No. 51.

Spengel--Wimpertrichtern i. d. Amphibienniere. _Centralblatt f. Med. Wiss._ 1875, No. 23.

Meyer--Anat. des Urogenitalsystems der Selachier u. Amphibien. Sitzb. Naturfor. Gesellschaft. Leipzig, 30 April, 1875.

F. M. Balfour--Preliminary Account of development of Elasmobranch fishes. Quart. Journ. of Micro. Science, Oct. 1874. (This edition, Paper V. p. 60 et seq.)

W. Müller--Persistenz der Urniere bei Myxine glutinosa. Jenaische Zeitschrift, 1873.

W. Müller--Urogenitalsystem d. Amphioxus u. d. Cyclostomen. Jenaische Zeitschrift, 1875.

Alex. Götte--Entwicklungsgeschichte der Unke (Bombinator igneus).

The following paper is an attempt to give a consecutive history of the origin of this system of organs in vertebrates and of the changes which it has undergone in the different orders.

For this purpose I have not made use of my own observations alone, but have had recourse to all the Memoirs with which I am acquainted, and to which I have access. I have commenced my account with the Selachians, both because my own investigations have been directed almost entirely to them, and because their urinogenital organs are, to my mind, the most convenient for comparison both with the more complicated and with the simpler types.

On many points the views put forward in this paper will be found to differ from those which I expressed in my paper (loc. cit.) which give an account of my original[28] discovery of the segmental organs of Selachians, but the differences, with the exception of one important error as to the origin of the Wolffian duct, are rather fresh developments of my previous views from the consideration of fresh facts, than radical changes in them.

Footnote 28: These organs were discovered independently by Professor Semper and myself. Professor Semper's preliminary account appeared prior to my own which was published (with illustrations) in the Quarterly Journal of Mic. Science. Owing to my being in South America, I did not know of Professor Semper's investigations till several months after the publication of my paper.

* * * * *

In Selachian embryos an intermediate cell-mass, or middle plate of mesoblast is formed, as in birds, from a partial fusion of the somatic and splanchnic layers of the mesoblast at the outer border of the protovertebræ. From this cell-mass the whole of the urinogenital system is developed.

At about the time when three visceral clefts have appeared, there arises from the intermediate cell-mass, opposite the fifth protovertebra, a solid knob, from which a column of cells grows backwards to opposite the position of the future anus (Fig. 1. pd.).

This knob projects outwards toward the epiblast, and the column lies at first between the mesoblast and epiblast. The knob and column do not long remain solid. The knob becoming hollow acquires a wide opening into the pleuro-peritoneal or body-cavity, and the column a lumen; so that by the time that five visceral clefts have appeared, the two together form a duct closed behind, but communicating in front by a wide opening with the pleuro-peritoneal cavity.

Before these changes are accomplished, a series of solid[29] outgrowths of elements of the 'intermediate cell-mass' appear at the uppermost corner of the body-cavity. These soon become hollow and appear as involutions from the body-cavity, curling round the inner and dorsal side of the previously formed duct.

Footnote 29: These outgrowths are at first solid in both Pristiurus, Scyllium and Torpedo, but in Torpedo attain a considerable length before a lumen appears in them.

One involution of this kind makes its appearance for each protovertebra, and the first belongs to the protovertebra immediately behind the anterior end of the duct whose development has just been described. In Pristiurus there are in all 29 of these at this period. The last two or three arise from that portion of the body-cavity, which at this stage still exists behind the anus. The first-formed duct and the subsequent involutions are the rudiments of the whole of the urinary system. The duct is the primitive duct of the kidney[30]; I shall call it in future the segmental duct; and the involutions are the commencements of the segmental tubes which constitute the body of the kidney. I shall call them in future segmental tubes.

Footnote 30: This duct is often called either Müller's duct, the oviduct, or the duct of the primitive kidneys 'Urnierengang.' None of these terms are very suitable. A justification of the name I have given it will appear from the facts given in the later parts of this paper. In my previous paper I have always called it oviduct, a name which is very inappropriate.

Soon after their formation the segmental tubes become convoluted, and their blind ends become connected with the segmental duct of the kidney. At the same time, or rather before this, the blind posterior termination of each of the segmental ducts of the kidneys unites with and opens into one of the horns of the cloaca. At this period the condition of affairs is represented in Fig. 2.

There is at pd, the segmental duct of the kidneys, opening in front (o) into the body-cavity, and behind into the cloaca, and there are a series of convoluted segmental tubes (st), each opening at one end into the body-cavity, and at the other into the duct (pd).

The next important change which occurs is the longitudinal division of the segmental duct of the kidneys into Müller's duct, or the oviduct, and the duct of the Wolffian bodies or Leydig's duct. The splitting[31] is effected by the growth of a wall of cells which divides the duct into two parts (fig. 3, wd. and md.). It takes place in such a way that the front end of the segmental duct, anterior to the entrance of the first segmental tube, together with the ventral half of the rest of the duct, is split off from its dorsal half as an independent duct (vide fig. 2, x).

Footnote 31: This splitting was first of all discovered and an account of it published by Semper (Centralblatt f. Med. Wiss. 1875, No. 29). I had independently made it out for the female a few weeks before the publication of Semper's account--but have not yet made observations about the point for the male.

My own previous account of the origin of the Wolffian duct (Quart. Journ. of Micros. Science, Oct. 1874, and this edition, Paper V.), is completely false, and was due to my not having had access to a complete series of my sections when I wrote the paper.

The dorsal portion also forms an independent duct, and into it the segmental tubes continue to open. Such at least is the method of splitting for the female--for the male the splitting is according to Professor Semper, of a more partial character, and consists for the most part in the front end of the duct only being separated off from the rest. The result of these changes is the formation in both sexes of a fresh duct which carries off the excretions of the segmental involutions, and which I shall call the Wolffian duct--while in the female there is formed another complete and independent duct, which I shall call the Müllerian duct, or oviduct, and in the male portions only of such a duct.

The next change which takes place is the formation of another duct from the hinder portion of the Wolffian duct, which receives the secretion of the posterior segmental tubes. This secondary duct unites with the primary or Wolffian duct near its termination, and the primary ducts of the two sides unite together to open to the exterior by a common papilla.

Slight modifications of the posterior terminations of these ducts are found in different genera of Selachians (vide Semper, Centralblatt für Med. Wiss. 1874, No. 59), but they are of no fundamental importance.

These constitute the main changes undergone by the segmental duct of the kidneys and the ducts derived from it; but the segmental tubes also undergo important changes. In the majority of Selachians their openings into the body-cavity, or, at any rate, the openings of a large number of them, persist through life; but the investigations of Dr Meyer[32] render it very probable that the small portion of each segmental tube adjoining the opening becomes separated from the rest and becomes converted into a sort of lymph organ, so that the openings of the segmental tubes in the adult merely lead into lymph organs and not into the gland of the kidneys.

Footnote 32: Sitzun. der Naturfor. Gesellschaft, Leipzig, 30 April, 1875.

These constitute the whole changes undergone in the female, but in the male the open ends of a varying number (according to the species) of the segmental tubes become connected with the testis and, uniting with the testicular follicles, serve to carry away the seminal fluid[33]. The spermatozoa have therefore to pass through a glandular portion of the kidneys before they enter the Wolffian duct, by which they are finally carried away to the exterior.

Footnote 33: We owe to Professor Semper the discovery of the arrangement of the seminal ducts. Centralblatt f. Med. Wiss. 1875, No. 12.

In the adult female, then, there are the following parts of the urinogenital system (fig. 4):

(1) The oviduct, or Müller's duct (fig. 4, md.), split off from the segmental duct of the kidneys. Each oviduct opens at its upper end into the body-cavity, and behind the two oviducts have independent communications with the cloaca. The oviducts serve simply to carry to the exterior the ova, and have no communication with the glandular portion of the kidneys.

(2) The Wolffian ducts (fig. 4, wd.) or the remainder of the segmental ducts of the kidneys. Each Wolffian duct ends blindly in front, and the two unite behind to open by a common papilla into the cloaca.

This duct receives the secretion of the whole anterior end of the kidneys[34], that is to say, of all the anterior segmental tubes.

Footnote 34: This upper portion of the kidneys is called Leydig's gland by Semper. It would be better to call it the Wolffian body, for I shall attempt to shew that it is homologous with the gland so named in Sauropsida and Mammalia.

(3) The secondary duct (fig. 4, d.) belonging to the lower portion of the kidneys opening into the former duct near its termination.

(4) The segmental tubes (fig. 4, st) from whose convolutions and outgrowths the kidney is formed. They may be divided into two parts, according to the duct by which their secretion is carried off.

In the male the following parts are present:

(1) The Müllerian duct (fig. 5, md.), consisting of a small remnant, attached to the liver, which represents the foremost end of the oviduct of the female.

(2) The Wolffian duct (fig. 5, wd), which precisely corresponds to the Wolffian duct of the female, except that, in addition to functioning as the duct of the anterior part of the kidneys, it also serves to carry away the semen. In the female it is straight, but has in the adult male a very tortuous course (vide fig. 5).

(3) the duct (fig. 5, d.) of the posterior portion of the kidneys, which has the same relations as in the female.

(4) The segmental tubes (fig. 5, st.). These have the same relations as in the female, except that the most anterior two, three or more, unite with the testicular follicles, and carry away the semen into the Wolffian duct.

* * * * *

The mode of arrangement and the development of these parts suggest a number of considerations.

In the first place it is important to notice that the segmental tubes develop primitively as completely independent organs[35], one of which appears in each segment. If embryology is in any way a repetition of ancestral history, it necessarily follows that these tubes were primitively independent of each other. Ancestral history, as recorded in development, is often, it is true, abridged; but it is clear that though abridgement might prevent a series of primitively separate organs from appearing as such, yet it would hardly be possible for a primitively compound organ, which always retained this condition, to appear during development as a series of separate ones. These considerations appear to me to prove that the segmented ancestors of vertebrates possessed a series of independent and segmental excretory organs.

Footnote 35: Further study of my sections has shewn me that the initial independence of these organs is even more complete than might be gathered from the description in my paper (loc. cit.). I now find, as I before conjectured, that they at first correspond exactly with the muscle-plates, there being one for each muscle-plate. This can be seen in the fresh embryos, but longitudinal sections shew it in an absolutely demonstrable manner.

Both Professor Semper and myself, on discovering these organs, were led to compare them and state our belief in their identity with the so-called segmental organs of Annelids.

This view has since been fairly generally accepted. The segmental organs of annelids agree with those of vertebrates in opening at one end into the body-cavity, but differ in the fact that each also communicates with the exterior by an independent opening, and that they are never connected with each other.

On the hypothesis of the identity of the vertebrate segmental tubes with the annelid segmental organs, it becomes essential to explain how the external openings of the former may have become lost.

This brings us at once to the origin of the segmental duct of the kidneys, by which the secretion of all the segmental tubes was carried to the exterior, and it appears to me that a right understanding of the vertebrate urinogenital system depends greatly upon a correct view of the origin of this duct. I would venture to repeat the suggestion which I made in my original paper (loc. cit.) that this duct is to be looked upon as the most anterior of the segmental tubes which persist in vertebrates. In favour of this view are the following anatomical and embryological facts. (1) It develops in nearly the same manner as the other segmental tubes, viz. in Selachians as a solid outgrowth from the intermediate cell-mass, which subsequently becomes hollowed so as to open into the body-cavity: and in Amphibians and Osseous and Cyclostome fishes as a direct involution from the body-cavity. (2) In Amphibians, Cyclostomes and Osseous fishes its upper end develops a glandular portion, by becoming convoluted in a manner similar to the other segmental tubes. This glandular portion is often called either the head-kidney or the primitive kidney. It is only an embryonic structure, but is important as demonstrating the true nature of the primitive duct of the kidneys.

We may suppose that some of the segmental tubes first united, possibly in pairs, and that then by a continuation of this process the whole of them coalesced into a common gland. One external opening sufficed to carry off the entire secretion of the gland, and the other openings therefore atrophied.

This history is represented in the development of the dog-fish in an abbreviated form, by the elongation of the first segmental tube (segmental duct of the kidney) and its junction with each of the posterior segmental tubes. Professor Semper looks upon the primitive duct of the kidneys as a duct which arose independently, and was not derived from metamorphosis of the segmental organs. Against this view I would on the one hand urge the consideration, that it is far easier to conceive of the transformation by change of function (comp. Dohrn, Functionswechsel, Leipzig, 1875) of a segmental organ into a segmental duct, than to understand the physiological cause which should lead, in the presence of so many already formed ducts, to the appearance of a totally new one. By its very nature a duct is a structure which can hardly arise de novo. We must even suppose that the segmental organs of Annelids were themselves transformations of still simpler structures. On the other hand I would point to the development in this very duct amongst Amphibians and Osseous fishes of a glandular portion similar to that of a segmental tube, as an à posteriori proof of its being a metamorphosed segmental tube. The development in insects of a longitudinal tracheal duct by the coalescence of a series of transverse tracheal tubes affords a parallel to the formation of a duct from the coalescence of a series of segmental tubes.

Though it must be admitted that the loss of the external openings of the segmental organs requires further working out, yet the difficulties involved in their disappearance are not so great as to render it improbable that the vertebrate segmental organs are descended from typical annelidan ones.

The primitive vertebrate condition, then, is probably that of an early stage of Selachian development while there is as yet a segmental duct,--the original foremost segmental tube opening in front into the body-cavity and behind into the cloaca; with which duct all the segmental tubes communicate. Vide Fig. 2.

The next condition is to be looked upon as an indirect result of the segmental duct serving as well for the products of the generative organs as the secretions of the segmental tubes.

As a consequence of this, the segmental duct became split into a ventral portion, which served alone for the ova, and a dorsal portion which received the secretion of the segmental tubes. The lower portion, which we have called the oviduct, in some cases may also have received the semen as well as the ova. This is very possibly the case with Ceratodus (vide Günther, Trans. of Royal Society, 1871), and the majority of Ganoids (Hyrtl, Denkschriften Wien, Vol. VIII.). In the majority of other cases the oviduct exists in the male in a completely rudimentary form; and the semen is carried away by the same duct as the urine.

In Selachians the transportation of the semen from the testis to the Wolffian duct is effected by the junction of the open ends of two or three or more segmental tubes with the testicular follicles, and the modes in which this junction is effected in the higher vertebrates seem to be derivatives from this. If the views here expressed are correct it is by a complete change of function that the oviduct has come to perform its present office. And in the bird and higher vertebrates no trace, or only the very slightest (vide p. 165) of the primitive urinary function is retained during embryonic or adult life.

The last feature in the anatomy of the Selachians which requires notice is the division of the kidney into two portions, an anterior and posterior. The anatomical similarity between this arrangement and that of higher vertebrates (birds, &c.) is very striking. The anterior one precisely corresponds, anatomically, to the Wolffian body, and the posterior one to the true permanent kidney of higher vertebrates: and when we find that in the Selachians the duct for the anterior serves also for the semen as does the Wolffian duct of higher vertebrates, this similarity seems almost to amount to identity. A discussion of the differences in development in the two cases will come conveniently with the account of the bird; but there appear to me the strongest grounds for looking upon the kidneys of Selachians as equivalent to both the Wolffian bodies and the true kidneys of the higher vertebrates.

The condition of the urinogenital organs in Selachians is by no means the most primitive found amongst vertebrates.

The organs of both Cyclostomous and Osseous fishes, as well as those of Ganoids, are all more primitive; and in the majority of points the Amphibians exhibit a decidedly less differentiated condition of these organs than do the Selachians.

In Cyclostomous fishes the condition of the urinary system is very simple. In Myxine (vide Joh. Müller Myxinoid fishes, and Wilhelm Müller, Jenaische Zeitschrift, 1875, Das Urogenitalsystem des Amphioxus u. d. Cyclostomen) there is a pair of ducts which communicate posteriorly by a common opening with the abdominal pore. From these ducts spring a series of transverse tubules, each terminating in a Malpighian corpuscle. These together constitute the mass of the kidneys. About opposite the gall-bladder the duct of the kidney (the segmental duct) narrows very much, and after a short course ends in a largish glandular mass (the head-kidney), which communicates with the pericardial cavity by a number of openings.

In Petromyzon the anatomy of the kidneys is fundamentally the same as in Myxine. They consist of the two segmental ducts, and a number of fine branches passing off from these, which become convoluted but do not form Malpighian tufts. The head-kidney is absent in the adult.

W. Müller (loc. cit.) has given a short but interesting account of the development of the urinary system of Petromyzon. He finds that the segmental ducts develop first of all as simple involutions from the body-cavity. The anterior end of each then develops a glandular portion which comes to communicate by a number of openings with the body-cavity. Subsequently to the development of this glandular portion the remainder of the kidneys appears in the posterior portion of the body-cavity; and before the close of embryonic life the anterior glandular portion atrophies.

The comparison of this system with that of a Selachian is very simple. The first developed duct is the segmental duct of a Selachian, and the glandular portion developed at its anterior extremity, which is permanent in Myxine but embryonic in Petromyzon, is, as W. Müller has rightly recognized, equivalent to the head-kidney of Amphibians, which remains undeveloped in Selachians. It is, according to my previously stated view, the glandular portion of the first segmental organ or the segmental duct. The series of orifices by which this communicates with the body-cavity are due to the division of the primary opening of the segmental duct. This is shewn both by the facts of their development in Petromyzon given by Müller, as well as by the occurrence of a similar division of the primary orifice in Amphibians, which is mentioned later in this paper. In a note in my original paper (loc. cit.) I stated that these openings were equivalent to the segmental involutions of Selachians. This is erroneous, and was due to my not having understood the description given in a preliminary paper of Müller (Jenaische Zeitschrift, 1873). The large development of this glandular mass in the Cyclostome and Osseous fishes and in embryo Amphibians, implies that it must at one time have been important. Its earlier development than the remainder of the kidneys is probably a result of the specialized function of the first segmental organ.

The remainder of the kidney in Cyclostomes is equivalent to the kidney of Selachians. Its development from segmental involutions has not been recognized. If these segmental involutions are really absent it may perhaps imply that the simplicity of the Cyclostome kidneys, like that of so many other of their organs, is a result of degeneration rather than a primitive condition.

In Osseous fishes the segmental duct of the kidneys develops, as the observations of Rosenberg[36] ("Teleostierniere," Inaug. Disser. Dorpat, 1867) and Oellacher (Zeitschrift für Wiss. Zool. 1873) clearly prove, by an involution from the body-cavity. This involution grows backwards in the form of a duct and opens into the cloaca. The upper end of this duct (the most anterior segmental tube) becomes convoluted, and forms a glandular body, which has no representative in the urinary apparatus of Selachians, but whose importance, as indicating the origin of the segmental duct of the kidneys, I have already insisted upon.

Footnote 36: I am unfortunately only acquainted with Dr Rosenberg's paper from an abstract.

The rest of the kidney becomes developed at a later period, probably in the same way as in Selachians; but this, as far as I know, has not been made out.

The segmental duct of the kidneys forms the duct for this new gland, as in embryo Selachians (Fig. 2), but, unlike what happens in Selachians, undergoes no further changes, with the exception of a varying amount of retrogressive metamorphosis of its anterior end. The kidneys of Osseous fish usually extend from just behind the head to opposite the anus, or even further back than this. They consist for the most part of a broader anterior portion, an abdominal portion reaching from this to the anus, and, as in those cases in which the kidneys extend further back than the anus, of a caudal portion.

The two ducts (segmental ducts of the kidneys) lie, as a rule, in the lower part of the kidneys on their outer borders, and open almost invariably into a urinary bladder. In some cases they unite before opening into the bladder, but generally have independent openings.

This bladder, which is simply a dilatation of the united lower ends of the primitive kidney-ducts, and has no further importance, is almost invariably present, but in many cases lies unsymmetrically either to the right or the left. It opens to the exterior by a very minute opening in the genito-urinary papilla, immediately behind the genital pore. There are, however, a few cases in which the generative and urinary organs have a common opening. For further details vide Hyrtl, Denk. der k. Akad. Wien, Vol. II.

It is possible that the generative ducts of Osseous fishes are derived from a splitting from the primitive duct of the kidney, but this is discussed later in the paper.

In Osseous fishes we probably have an embryonic condition of the Selachian kidneys retained permanently through life.

* * * * *

In the majority of Ganoids the division of the segmental duct of the kidney into two would seem to occur, and the ventral duct of the two (Müllerian duct), which opens at its upper end into the body-cavity, is said to serve as an excretory duct for both male and female organs.

The following are the more important facts which are known about the generative and urinary ducts of Ganoids.

In Spatularia (vide Hyrtl, Geschlechts u. Harnwerkzeuge bei den Ganoiden, Denkschriften der k. Akad. Wien, Vol. VIII.) the following parts are found in the female.

(1) The ovaries stretching along the whole length of the abdominal cavity.

(2) The kidneys, which are separate and also extend along the greater part of the abdominal cavity.

(3) The ureters lying on the outer borders of the kidneys. Each ureter dilates at its lower end into an elongated wide tube, which continues to receive the ducts from the kidneys. The two ureters unite before terminating and open behind the anus.

(4) The two oviducts (Müllerian ducts). These open widely into the abdominal cavity, at about two-thirds of the distance from the anterior extremity of the body-cavity. Each opens by a narrow pore into the dilated ureter of its side.

In the male the same parts are found as in the female, but Hyrtl found that the Müllerian duct of the left side at its entrance into the ureter became split into two horns, one of which ended blindly. On the right side the opening of the Müllerian duct was normal.

In the Sturgeon (vide J. Müller, Bau u. Grenzen d. Ganoiden, Berlin Akad. 1844; Leydig, Fischen u. Reptilien, and Hyrtl, Ganoiden) the same parts are found as in Spatularia.

The kidneys extend along the whole length of the body-cavity; and the ureter, which does not reach the whole length of the kidneys, is a thin-walled wide duct lying on the outer side. On laying it open the numerous apertures of the tubules for the kidney are exposed. The Müllerian duct, which opens in both sexes into the abdominal cavity, ends, according to Leydig, in the cases of some males, blindly behind without opening into the ureter, and Müller makes the same statement for both sexes. It was open on both sides in a female specimen I examined[37], and Hyrtl found it invariably so in both sexes in all the specimens he examined.

Footnote 37: For this specimen I am indebted to Dr Günther.

Both Rathke and Stannius (I have been unable to refer to the original papers) believed that the semen was carried off by transverse ducts directly into the ureter, and most other observers have left undecided the mechanism of the transportation of the semen to the exterior. If we suppose that the ducts Rathke saw really exist they might perhaps be supposed to enter not directly into the ureter, but into the kidney, and be in fact homologous with the vasa efferentia of the Selachians. The frequent blind posterior termination of the Müllerian duct is in favour of the view that these ducts of Rathke are really present.

In Polypterus (vide Hyrtl, Ganoiden) there is, as in other Ganoids, a pair of Müllerian ducts. They unite at their lower ends. The ureters are also much narrower than in previously described Ganoids and, after coalescing, open into the united oviducts. The urinogenital canal, formed by coalescence of the Müllerian ducts and ureters, has an opening to the exterior immediately behind the anus.

In Amia (vide Hyrtl) there is a pair of Müllerian ducts which, as well as the ureters, open into a dilated vesicle. This vesicle appears as a continuation of the Müllerian ducts, but receives a number of the efferent ductules of the kidneys. There is a single genito-urinary pore behind the anus.

In Ceratodus (Günther, Phil. Trans. 1871) the kidneys are small and confined to the posterior extremity of the abdomen. The generative organs extend however along the greater part of the length of the abdominal cavity. In both male and female there is a long Müllerian duct, and the ducts of the two sides unite and open by a common pore into a urinogenital cloaca which communicates with the exterior by the same opening as the alimentary canal. In both sexes the Müllerian duct has a wide opening near the anterior extremity of the body-cavity. The ureters coalesce and open together into the urinogenital cloaca dorsal to the Müllerian ducts. It is not absolutely certain that the semen is transported to the exterior by the Müllerian duct of the male, which is perhaps merely a rudiment as in Amphibia. Dr Günther failed however to find any other means by which it could be carried away.

The genital ducts of Lepidosteus differ in important particulars from those of the other Ganoids (vide Müller, loc. cit. and Hyrtl, loc. cit.).

In both sexes the genital ducts are continuous with the investments of the genital organs.

In the female the dilated posterior extremities of the ureters completely invest for some distance the generative ducts, whose extremities are divided into several processes, and end in a different way on the two sides. A similar division and asymmetry of the ducts is mentioned by Hyrtl as occurring in the male of Spatularia, and it seems not impossible that on the hypothesis of the genital ducts being segmental tubes these divisions may be remnants of primitive glandular convolutions. The ureters in both sexes dilate as in other Ganoids at their posterior extremities, and unite with one another. The unpaired urinogenital opening is situated behind the anus. In the male the dilated portion of the ureters is divided into a series of partitions which are not present in the female.

Till the embryology of the secretory system of Ganoids has been worked out, the homologies of their generative ducts are necessarily a matter of conjecture. It is even possible that what I have called the Müllerian duct in the male is functionless, as with Amphibians, but that, owing to the true ducts of the testis having been overlooked, it has been supposed to function as the vas deferens. Günther's (loc. cit.) injection experiments on Ceratodus militate against this view, but I do not think they can be considered as conclusive as long as the mechanism for the transportation of the semen to the exterior has not been completely made out. Analogy would certainly lead us to expect the ureter to serve in Ganoids as the vas deferens.

The position of the generative ducts might in some cases lead to the supposition that they are not Müllerian ducts, or, in other words, the most anterior pair of segmental organs but a pair of the posterior segmental tubes.

What are the true homologies of the generative ducts of Lepidosteus, which are continuous with the generative glands, is somewhat doubtful. It is very probable that they may represent the similarly functioning ducts of other Ganoids, but that they have undergone further changes as to their anterior extremities.

It is, on the other hand, possible that their generative ducts are the same structures as those ducts of Osseous fishes, which are continuous with the generative organs. These latter ducts are perhaps related to the abdominal pores, and had best be considered in connection with these; but a completely satisfactory answer to the questions which arise in reference to them can only be given by a study of their development.

In the Cyclostomes the generative products pass out by an abdominal pore, which communicates with the peritoneal cavity by two short tubes[38], and which also receives the ducts of the kidneys.

Footnote 38: According to Müller (Myxinoiden, 1845) there is in Myxine an abdominal pore with two short canals leading into it, and Vogt and Pappenheim (An. Sci. Nat. Part IV. Vol. XI.) state that in Petromyzon there are two such pores, each connected with a short canal.

Gegenbaur suggests that these are to be looked upon as Müllerian ducts, and as therefore developed from the segmental ducts of the kidneys. Another possible view is that they are the primitive external openings of a pair of segmental organs. In Selachians there are usually stated to be a pair of abdominal pores. In Scyllium I have only been able to find, on each side, a large deep pocket opening to the exterior, but closed below towards the peritoneal cavity, so that in it there seem to be no abdominal pores[39]. In the Greenland Shark (Læmargus Borealis) Professor Turner (Journal of Anat. and Phys. Vol. VIII.) failed to find either oviduct or vas deferens, but found a pair of large open abdominal pores, which he believes serve to carry away the generative products of both sexes. Whether the so-called abdominal pores of Selachians usually end blindly as in Scyllium, or, as is commonly stated, open into the body-cavity, there can be no question that they are homologous with true abdominal powers.

Footnote 39: My own rough examination of preserved specimens was hardly sufficient to enable me to determine for certain the presence or absence of these pores. Mr Bridge, of Trinity College, has, however, since then commenced a series of investigations on this point, and informs me that these pores are certainly absent in Scyllium as well as in other genera.

The blind pockets of Scyllium appear very much like the remains of primitive involutions from the exterior, which might easily be supposed to have formed the external opening of a pair of segmental organs, and this is probably the true meaning of abdominal pores. The presence of abdominal pores in all Ganoids in addition to true genital ducts and of these pockets or abdominal pores in Selachians, which are almost certainly homologous with the abdominal pores of Ganoids and Cyclostomes, and also occur in addition to true Müllerian ducts, speak strongly against the view that the abdominal pores have any relation to Müllerian ducts. Probably therefore the abdominal pores of the Cyclostomous fishes (which seem to be of the same character as other abdominal pores) are not to be looked on as rudimentary Müllerian ducts.

We next come to the question which I reserved while speaking of the kidneys of Osseous fishes, as to the meaning of their genital ducts.

In the female Salmon and the male and female Eel, the generative products are carried to the exterior by abdominal pores, and there are no true generative ducts. In the case of most other Osseous fish there are true generative ducts which are continuous with the investment of the generative organs[40] and have generally, though not always, an opening or openings independent of the ureter close behind the rectum, but no abdominal pores are present. It seems, therefore, that in Osseous fish the generative ducts are complementary to abdominal pores, which might lead to the view that the generative ducts were formed by a coalescence of the investment of the generative glands with the short duct of abdominal pore.

Footnote 40: The description of the attachment of the vas deferens to the testis in the Carp given by Vogt and Pappenheim (Ann. Scien. Nat. 1859) does not agree with what I found in the Perch (Perca fluvialis). The walls of the duct are in the Perch continuous with the investment of the testis, and the gland of the testis occupies, as it were, the greater part of the duct; there is, however, a distinct cavity corresponding to what Vogt and P. call the duct, near the border of attachment of the testis into which the seminal tubules open. I could find at the posterior end of the testis no central cavity which could be distinguished from the cavity of this duct.

Against this view there are, however, the following facts:

(1) In the cases of the salmon and the eel it is perfectly true that the abdominal pore exactly corresponds with the opening of the genital duct in other Osseous fishes, but the absence of genital ducts in these cases must rather be viewed, as Vogt and Pappenheim (loc. cit.) have already insisted, as a case of degeneration than of a primitive condition. The presence of genital ducts in the near allies of the Salmonidæ, and even in the male salmon, are conclusive proofs of this. If we admit that the presence of an abdominal pore in Salmonidæ is merely a result of degeneration, it obviously cannot be used as an argument for the complementary nature of abdominal pores and generative ducts.

(2) Hyrtl (Denkschriften der k. Akad. Wien, Vol. 1) states that in Mormyrus oxyrynchus there is a pair of abdominal pores in addition to true generative ducts. If his statements are correct, we have a strong argument against the generative ducts of Osseous fishes being related to abdominal pores. For though this is the solitary instance of the presence of both a genital opening and abdominal pores known to me in Osseous fishes, yet we have no right to assume that the abdominal pores of Mormyrus are not equivalent to those of Ganoids and Selachians. It must be admitted, with Gegenbaur, that embryology alone can elucidate the meaning of the genital ducts of Osseous fishes.

In Lepidosteus, as was before mentioned, the generative ducts, though continuous with the investment of the generative bodies, unite with the ureters, and in this differ from the generative ducts of Osseous fishes. The relation, indeed, of the generative ducts of Lepidosteus to the urinary ducts is very similar to that existing in other Ganoid fishes; and this, coupled with the fact that Lepidosteus possesses a pair of abdominal pores on each side of the anus[41], makes it most probable that its generative ducts are true Müllerian ducts.

Footnote 41: This is mentioned by Müller (Ganoid fishes, Berlin Akad. 1844), Hyrtl (loc. cit.), and Günther (loc. cit.), and through the courtesy of Dr Günther I have had an opportunity of confirming the fact of the presence of the abdominal pores on two specimens of Lepidosteus in the British Museum.

* * * * *

In the Amphibians the urinary system is again more primitive than in the Selachians.

The segmental duct of the kidneys is formed[42] by an elongated fold arising from the outer wall of the body-cavity, in the same position as in Selachians. This fold becomes constricted into a canal, closed except at its anterior end, which remains open to the body-cavity. This anterior end dilates, and grows out into two horns, and at the same time its opening into the body-cavity becomes partly constricted, and so divided into three separate orifices, one for each horn and a central one between the two. The horns become convoluted, blood channels appearing between their convolutions, and a special coil of vessels is formed arising from the aorta and projecting into the body-cavity near the openings of the convolutions. These formations together constitute the glandular portion[43] of the original anterior segmental tube or segmental duct of the kidneys. I have already pointed out the similarity which this organ exhibits to the head-kidneys of Cyclostome fishes in its mode of formation, especially with reference to the division of the primitive opening. The lower end of the segmental duct unites with a horn of the cloaca.

Footnote 42: My account of the development of these parts in Amphibians is derived for the most part from Götte, _Die Entwicklungsgeschichte der Unke_.

Footnote 43: It is called Kopfniere (head-kidney), or Urniere (primitive kidney), by German authors. Leydig correctly looks upon it as together with the permanent kidney constituting the Urniere of Amphibians. The term Urniere is one which has arisen in my opinion from a misconception; but certainly the Kopfniere has no greater right to the appellation than the remainder of the kidney.

After the formation of the gland just described the remainder of the kidney is formed.

This arises in the same way as in Selachians. A series of involutions from the body-cavity are developed; these soon form convoluted tubes, which become branched and interlaced with one another, and also unite with the primitive duct of the kidneys. Owing to the branching and interlacing of the primitive segmental tubes, the kidney is not divided into distinct segments in the same way as with the Selachians. The mode of development of these segmental tubes was discovered by Götte. Their openings are ciliated, and, as Spengel (loc. cit.) and Meyer (loc. cit.) have independently discovered, persist in most adult Amphibians. As both these investigators have pointed out, the segmental openings are in the adult kidneys of most Amphibians far more numerous than the vertebral segments to which they appertain. This is due to secondary changes, and is not to be looked upon as the primitive state of things. At this stage the Amphibian kidneys are nearly in the same condition as the Selachian, in the stage represented in Fig. 2. In both there is the segmental duct of the kidneys, which is open in front, communicates with the cloaca behind, and receives the whole secretion from the kidneys. The parallelism between the two is closely adhered to in the subsequent modifications of the Amphibian kidney, but the changes are not completed so far in Amphibians as in Selachians. The segmental duct of the Amphibian kidney becomes, as in Selachians, split into a Müllerian duct or oviduct, and a Wolffian duct or duct for the kidney.

The following points about this are noteworthy:

(1) The separation of the two ducts is never completed, so that they are united together behind, and for a short distance, blend and form a common duct; the ducts of the two sides so formed also unite before opening to the exterior.

(2) The separation of the two ducts does not occur in the form of a simple splitting, as in Selachians. But the efferent ductules from the kidney gradually alter their points of entrance into the primitive duct. Their points of entrance become carried backwards further and further, and since this process affects the anterior ducts proportionally more than the posterior, the efferent ducts finally all meet and form a common duct which unites with the Müllerian duct near its posterior extremity. This process is not always carried out with equal completeness. In the tailless Amphibians, however, the process is generally[44] completed, and the ureters (Wolffian ducts) are of considerable length. Bufo cinereus, in the male of which the Müllerian ducts are very conspicuous, serves as an excellent example of this.

Footnote 44: In Bombinator igneus, Von Wittich stated that the embryonic condition was retained. Leydig, Anatom. d. Amphib. u. Reptilien, shewed that this is not the case, but that in the male the Müllerian duct is very small, though distinct.

In the Salamander (Salamandra maculosa), Figs. 6 and 7, the process is carried out with greater completeness in the female than in the male, and this is the general rule in Amphibians. In the male Proteus, the embryonic condition would seem to be retained almost in its completeness so that the ducts of the kidney open directly and separately into the still persisting primitive duct of the kidney. The upper end of the duct nevertheless extends some distance beyond the end of the kidney and opens into the abdominal cavity. In the female Proteus, on the other hand, the separation into a Müllerian duct and a ureter is quite complete. The Newt (Triton) also serves as an excellent example of the formation of distinct Müllerian and Wolffian ducts being much more complete in the female than the male. In the female Newt all the tubules from the kidney open into a duct of some length which unites with the Müllerian duct near its termination, but in the male the anterior segmental tubes, including those which, as will be afterwards seen, serve as vasa efferentia of the testis, enter the Müllerian duct directly, while the posterior unite as in the female into a common duct before joining the Müllerian duct. For further details as to the variations exhibited in the Amphibians, the reader is referred to Leydig, Anat. Untersuchung, Fischen u. Reptilien. Ditto, Lehrbuch der Histologie, Menschen u. Thiere. Von Wittich, Siebold u. Kölliker, Zeitschrift, Vol. IV. p. 125.

The different conditions of completeness of the Wolffian ducts observable amongst the Amphibians are instructive in reference to the manner of development of the Wolffian duct in Selachians. The mode of division in the Selachians of the segmental duct of the kidney into a Müllerian and Wolffian duct is probably to be looked upon as an embryonic abbreviation of the process by which these two ducts are formed in Amphibians. The fact that this separation into Müllerian and Wolffian ducts proceeds further in the females of most Amphibians than in the males, strikingly shews that it is the oviductal function of the Müllerian duct which is the indirect cause of its separation from the Wolffian duct. The Müllerian duct formed in the way described persists almost invariably in both sexes, and in the male sometimes functions as a sperm reservoir; e.g. Bufo cinereus. In the embryo it carries at its upper end the glandular mass described above (Kopfniere), but this generally atrophies, though remnants of it persist in the males of some species (e.g. Salamandra). Its anterior end opens, in most cases by a single opening, into the perivisceral cavity in both sexes, and is usually ciliated. As the female reaches maturity, the oviduct dilates very much; but it remains thin and inconspicuous in the male.

The only other developmental change of importance is the connection of the testes with the kidneys. This probably occurs in the same manner as in Selachians, viz. from the junction of the open ends of the segmental tubes with the follicles of the testes. In any case the vessels which carry off the semen constitute part of the kidney, and the efferent duct of the testis is also that of the kidney. The vasa efferentia from the testis either pass through one or two nearly isolated anterior portions of the kidney (Proteus, Triton) or else no such special portion of the kidney becomes separated from the rest, and the vasa efferentia enter the general body of the kidney.

* * * * *

In the male Amphibian, then, the urinogenital system consists of the following parts (Fig. 6):

(1) Rudimentary Müllerian ducts, opening anteriorly into the body-cavity, which sometimes carry aborted Kopfnieren.

(2) The partially or completely formed Wolffian ducts (ureters) which also serve as the ducts for the testes.

(3) The kidneys, parts of which also serve as the vasa efferentia, and whose secretion, together with the testicular products, is carried off by the Wolffian ducts.

(4) The united lower parts of Wolffian and Müllerian ducts which are really the lower unsplit part of the segmental ducts of the kidneys.

In the female, there are (Fig. 7)

(1) The Müllerian ducts which function as the oviducts.

(2) The Wolffian ducts.

(3) The kidneys.

(4) The united Müllerian and Wolffian ducts as in the male.

The urinogenital organs of the adult Amphibians agree in almost all essential particulars with those of Selachians. The ova are carried off in both by a specialized oviduct. The Wolffian duct, or ureter, is found both in Selachians and Amphibians, and the relations of the testis to it are the same in both, the vasa efferentia of the testes having in both the same anatomical peculiarities.

The following points are the main ones in which Selachians and Amphibians differ as to the anatomy of the urinogenital organs; and in all but one of these, the organs of the Amphibian exhibit a less differentiated condition than do those of the Selachian.

(1) A glandular portion (Kopfniere) belonging to the first segmental organ (segmental duct of the kidneys) is found in all embryo Amphibians, but usually disappears, or only leaves a remnant in the adult. It has not yet been found in any Selachian.

(2) The division of the primitive duct of the kidney into the Müllerian duct and the Wolffian duct is not completed so far in Amphibians as Selachians, and in the former the two ducts are confluent at their lower ends.

(3) The permanent kidney exhibits in Amphibians no distinction into two glands (foreshadowing the Wolffian bodies and true kidneys of higher vertebrates), as it does in the Selachians.

(4) The Müllerian duct persists in its entirety in male Amphibians, but only its upper end remains in male Selachians.

(5) The openings of the segmental tubes into the body-cavity correspond in number with the vertebral segments in most Selachians, but are far more numerous than these in Amphibians. This is the chief point in which the Amphibian kidney is more differentiated than the Selachian.

* * * * *

The modifications in development which the urinogenital system has suffered in higher vertebrates (Sauropsida and Mammalia) are very considerable; nevertheless it appears to me to be possible with fair certainty to trace out the relationship of its various parts in them to those found in the Ichthyopsida. The development of urinogenital organs has been far more fully worked out for the bird than for any other member of the amniotic vertebrates; but, as far as we know, there are no essential variations except in the later periods of development throughout the division. These later variations, concerning for the most part the external apertures of the various ducts, are so well known and have been so fully described as to require no notice here. The development of these parts in the bird will therefore serve as the most convenient basis for comparison.

In the bird the development of these parts begins by the appearance of a column of cells on the upper surface of the intermediate cell-mass (Fig. 8, W.d). As in Selachians, the intermediate cell-mass is a group of cells between the outer edge of the protovertebræ and the upper end of the body-cavity. The column of cells thus formed is the commencement of the duct of the Wolffian body. Its development is strikingly similar to that of the segmental duct of the kidney in Selachians. I shall attempt when I have given an account of the development of the Müllerian duct to speak of the relations between the Selachian duct and that of the bird.

Romiti (Archiv f. Micr. Anat. Vol. X.) has recently stated that the Wolffian duct develops as an involution from the body-cavity. The fact that the specimens drawn by Romiti to support this view are too old to determine such a point, and the inspection of a number of specimens made by my friend Mr Adam Sedgwick of Trinity College, who, at my request, has been examining the urinogenital organs of the fowl, have led me to the conclusion that Romiti is in error in differing from his predecessors as to the development of the Wolffian duct. The solid string of cells to form the Wolffian duct lies at first close to the epiblast, but, by the alteration in shape which the protovertebræ undergo and the general growth of cells around it, becomes gradually carried downwards till it lies close to the germinal epithelium which lines the body-cavity. While undergoing this change of position it also acquires a lumen, but ends blindly both in front and behind. Towards the end of the fourth day the Wolffian duct opens into a horn of the cloaca. The cells adjoining its inner border commence, as it passes down on the third day, to undergo histological changes, which, by the fourth day, result in the formation of a series of ducts and Malpighian tufts which form the mass of the Wolffian body[45].

Footnote 45: This account of the origin of the Wolffian body differs from that given by Waldeyer, and by Dr Foster and myself (Elements of Embryology, Foster and Balfour), but I have been led to alter my view from an inspection of Mr Sedgwick's preparations, and I hope to shew that theoretical considerations lead to the expectation that the Wolffian body would develop independently of the duct.

The Müllerian duct arises in the form of an involution, whether at first solid or hollow, of the germinal epithelium, and, as I am satisfied, quite independently of the Wolffian duct. It is important to notice that its posterior end soon unites with the Wolffian duct, from which however it not long after becomes separated and opens independently into the cloaca. The upper end remains permanently open to the body-cavity, and is situated nearly opposite the extreme front end of the Wolffian body.

Between the 80th and 100th hour of incubation the ducts of the permanent kidneys begin to make their appearance. Near its posterior extremity each Wolffian duct becomes expanded, and from the dorsal side of this portion a diverticulum is constricted off, the blind end of which points forwards. This is the duct of the permanent kidneys, and around its end the kidneys are found. It is usually stated that the tubules of the permanent kidneys arise as outgrowths from the duct, but this requires to be worked over again.

The condition of the urinogenital system in birds immediately after the formation of the permanent kidneys is strikingly similar to its permanent condition in adult Selachians. There is the Müllerian duct in both opening in front into the body-cavity and behind into the cloaca. In both the kidneys consist of two parts--an anterior and posterior--which have been called respectively Wolffian bodies and permanent kidneys in birds and Leydig's glands and the kidneys in Selachians.

The duct of the permanent kidney, which at first opens into that of the Wolffian body, subsequently becomes further split off from the Wolffian duct, and opens independently into the cloaca.

The subsequent changes of these parts are different in the two sexes.

In the female the Müllerian ducts[46] persist and become the oviducts. Their anterior ends remain open to the body-cavity. The changes in their lower ends in the various orders of Sauropsida and Mammalia are too well known to require repetition here. The Wolffian body and duct atrophy: there are left however in many cases slight remnants of the anterior extremity of the body forming the parovarium of the bird, and also frequently remnants of the posterior portion of the gland as well as of the duct. The permanent kidney and its duct remain unaltered.

Footnote 46: The right oviduct atrophies in birds, and the left alone persists in the adult.

In the male the Müllerian duct becomes almost completely obliterated. The Wolffian duct persists and forms the vas deferens, and the anterior so-called sexual portion of the Wolffian body also persists in an altered form. Its tubules unite with the seminiferous tubules, and also form the epididymis. Unimportant remnants of the posterior part of the Wolffian body also persist, but are without function. In both sexes the so-called permanent kidneys form the sole portion of the primitive uriniferous system which persists in the adult.

In considering the relations between the modes of development of the urinogenital organs of the bird and of the Selachians, the first important point to notice is, that whereas in the Selachians the segmental duct of the kidneys is first developed and subsequently becomes split into the Müllerian and Wolffian ducts; in the bird these two ducts develop independently. This difference in development would be accurately described by saying that in birds the segmental duct of the kidneys develops as in Selachians, but that the Müllerian duct develops independently of it.

Since in Selachians the Wolffian duct is equivalent to the segmental duct of the kidneys with the Müllerian removed from it, when in birds the Müllerian duct develops independently of the segmental kidney duct, the latter becomes the same as the Wolffian duct.

The second mode of stating the difference in development in the two cases represents the embryological facts of the bird far better than the other method.

It explains why the Wolffian duct appears earlier than the Müllerian and not at the same time, as one might expect according to the other way of stating the case. If the Wolffian duct is equivalent to the segmental duct of Selachians, it must necessarily be the first duct to develop; and not improbably the development of the Müllerian duct would in birds be expected to occur at the time corresponding to that at which the primitive duct in Selachians became split into two ducts.

It probably also explains the similarity in the mode of development of the Wolffian duct in birds and the primitive duct of the kidneys in Selachians.

This way of stating the case is also in accordance with theoretical conclusions. As the egg-bearing function of the Müllerian duct became more and more confirmed we might expect that the adult condition would impress itself more and more upon the embryonic development, till finally the Müllerian duct ceased to be at any period connected with the kidneys, and the history of its origin ceased to be traceable in its development. This seems to have actually occurred in the higher vertebrates, so that the only persisting connection between the Müllerian duct and the urinary system is the brief but important junction of the two at their lower ends on the sixth or seventh day. This junction justly surprised Waldeyer (Eierstock u. Ei, p. 129), but receives a complete and satisfactory explanation on the hypothesis given above.

The original development of the segmental tubes is in the bird solely retained in the tubules of the Wolffian body arising independently of the Wolffian duct, and I have hitherto failed to find that there is a distinct division of the Wolffian bodies into segments corresponding with the vertebral segments.

I have compared the permanent kidneys to the lower portion of the kidneys of Selachians. The identity of the anatomical condition of the adult Selachian and embryonic bird which has been already pointed out speaks strongly in favour of this view; and when we further consider that the duct of the permanent kidneys is developed in nearly the same way as the supposed homologous duct in Selachians, the suggested identity gains further support. The only difficulty is the fact that in Selachians the tubules of the part of the kidneys under comparison develop as segmental involutions in point of time anteriorly to their duct, while in birds they develop in a manner not hitherto certainly made out but apparently in point of time posteriorly to their duct. But when the immense modifications in development which the whole of the gland of the excretory organ has undergone in the bird are considered, I do not think that the fact I have mentioned can be brought forward as a serious difficulty.

The further points of comparison between the Selachian and the bird are very simple. The Müllerian duct in its later stages behaves in the higher vertebrates precisely as in the lower. It becomes in fact the oviduct in the female and atrophies in the male. The behaviour of the Wolffian duct is also exactly that of the duct which I have called the Wolffian duct in Ichthyopsida, and in the tubules of the Wolffian body uniting with the tubuli seminiferi we have represented the junction of the segmental tubes with the testis in Selachians and Amphibians. It is probably this junction of two independent organs which led Waldeyer to the erroneous view that the tubuli seminiferi were developed from the tubules of the Wolffian body.

With the bird I conclude the history of the origin of the urinogenital system of vertebrates. I have attempted, and I hope succeeded, in tracing out by the aid of comparative anatomy and embryology the steps by which a series of independent and simple segmental organs like those of Annelids have become converted into the complicated series of glands and ducts which constitute the urinogenital system of the higher vertebrates. There are no doubt some points which require further elucidation amongst the Ganoid and Osseous fishes. The most important points which appear to me still to need further research, both embryological and anatomical, are the abdominal pores of fishes, the generative ducts of Ganoids, especially Lepidosteus, and the generative ducts of Osseous fishes.

The only further point which requires discussion is the embryonic layer from which these organs are derived.

I have shewn beyond a doubt (loc. cit.) that in Selachians these organs are formed from the mesoblast. The unanimous testimony of all the recent investigators of Amphibians leads to the same conclusion. In birds, on the other hand, various investigators have attempted to prove that these organs are derived from the epiblast. The proof they give is the following: the epiblast and mesoblast appear fused in the region of the axis cord. From this some investigators have been led to the conclusion that the whole of the mesoblast is derived from the upper of the two primitive embryonic layers. To these it may be replied that, even granting their view to be correct, it is no proof of the derivation of the urinogenital organs from the epiblast, since it is not till the complete formation of the three layers that any one of them can be said to exist. Others look upon the fusion of the two layers as a proof of the passage of cells from the epiblast into the mesoblast. An assumption in itself, which however is followed by the further assumption that it is from these epiblast cells that the urinogenital system is derived! Whatever may have been the primitive origin of the system, its mesoblastic origin in vertebrates cannot in my opinion be denied.

Kowalewsky (Embryo. Stud. an Vermen u. Arthropoda, Mem. Akad. St Petersbourg, 1871) finds that the segmental tubes of Annelids develop from the mesoblast. We must therefore look upon the mesoblastic origin of the excretory system as having an antiquity greater even than that of vertebrates.

VIII. ON THE DEVELOPMENT OF THE SPINAL NERVES IN ELASMOBRANCH FISHES[47].

Footnote 47: [From the _Philosophical Transactions of the Royal Society of London_, Vol. CLXVI. Pt. 1. Received October 5, Read December 16, 1875.]

With Plates 22 and 23.

In the course of an inquiry into the development of Elasmobranch Fishes, my attention has recently been specially directed to the first appearance and early stages of the spinal nerves, and I have been led to results which differ so materially from those of former investigators, that I venture at once to lay them before the Society. I have employed in my investigations embryos of Scyllium canicula, Scyllium stellare, Pristiurus, and Torpedo. The embryos of the latter animal, especially those hardened in osmic acid, have proved by far the most favourable for my purpose, though, as will be seen from the sequel, I have been able to confirm the majority of my conclusions on embryos of all the above-mentioned genera.

A great part of my work was done at the Zoological Station founded by Dr Dohrn at Naples; and I have to thank both Dr Dohrn and Dr Eisig for the uniformly obliging manner in which they have met my requirements for investigation. I have more recently been able to fill up a number of lacunæ in my observations by the study of embryos bred in the Brighton Aquarium; for these I am indebted to the liberality of Mr Lee and the directors of that institution.

The first appearance of the Spinal Nerves in Pristiurus.

In a Pristiurus-embryo, at the time when two visceral clefts become visible from the exterior (though there are as yet no openings from without into the throat), a transverse section through the dorsal region exhibits the following features (Pl. 22, fig. A):--

The external epiblast is formed of a single row of flattened elongated cells. Vertically above the neural canal the cells of this layer are more columnar, and form the rudiment of the primitively continuous dorsal fin.

The neural canal (nc) is elliptical in section, and its walls are composed of oval cells two or three deep. The wall at the two sides is slightly thicker than at the ventral and dorsal ends, and the cells at the two ends are also smaller than elsewhere. A typical cell from the side walls of the canal is about 1/1900 inch in its longest diameter. The outlines of the cells are for the most part distinctly marked in the specimens hardened in either chromic or picric acid, but more difficult to see in those prepared with osmic acid; their protoplasm is clear, and in the interior of each is an oval nucleus very large in proportion to the size of its cell. The long diameter of a typical nucleus is about 1/3000 inch, or about two-thirds of that of the cell.

The nuclei are granular, and very often contain several especially large and deeply stained granules; in other cases only one such is present, which may then be called a nucleolus.

In sections there may be seen round the exterior of the neural tube a distinct hyaline membrane: this becomes stained of a brown colour with osmic acid, and purple or red with hæmatoxylin or carmine respectively. Whether it is to be looked upon as a distinct membrane differentiated from the outermost portion of the protoplasm of the cells, or as a layer of albumen coagulated by the reagents applied, I am unable to decide for certain. It makes its appearance at a very early period, long before that now being considered; and similar membranes are present around other organs as well as the neural tube. The membrane is at this stage perfectly continuous round the whole exterior of the neural tube as well on the dorsal surface as on the ventral.

The section figured, whose features I am describing, belongs to the middle of the dorsal region. Anteriorly to this point the spinal cord becomes more elliptical in section, and the spinal canal more lanceolate; posteriorly, on the other hand, the spinal canal and tube become more nearly circular in section. Immediately beneath the neural tube is situated the notochord (ch). It exhibits at this stage a central area rich in protoplasm, and a peripheral layer very poor in protoplasm; externally it is invested by a distinct cuticular membrane.

Beneath the notochord is a peculiar rod of cells, constricted from the top of the alimentary canal[48]. On each side and below this are the two aortæ, just commencing to be formed, and ventral to these is the alimentary canal.

Footnote 48: Vide Balfour, "Preliminary account of the Development of Elasmobranch Fishes," _Quart. Journ. of Microsc. Science_, Oct. 1874, p. 33. [This edition, p. 96.]

On each side of the body two muscle-plates are situated; their upper ends reach about one-third of the way up the sides of the neural tube. The two layers which together constitute the muscle-plates are at this stage perfectly continuous with the somatic and splanchnic layers of the mesoblast, and the space between the two layers is continuous with the body-cavity. In addition to the muscle-plates and their ventral continuations, there are no other mesoblast-cells to be seen. The absence of all mesoblastic cells dorsal to the superior extremities of the muscles is deserving of special notice.

Very shortly after this period and, as a rule, before a third visceral cleft has become visible, the first traces of the spinal nerves make their appearance.

First Stage.--The spinal nerves do not appear at the same time along the whole length of the spinal canal, but are formed first of all in the neck and subsequently at successive points posterior to this.

Their mode of formation will be most easily understood by referring to Pl. 22, figs. B I, B II, B III, which are representations of three sections taken from the same embryo. B I is from the region of the heart; B II belongs to a part of the body posterior to this, and B III to a still posterior region.

In most points the sections scarcely differ from Pl. 22, fig. A, which, indeed, might very well be a posterior section of the embryo to which these three sections belong.

The chief point, in addition to the formation of the spinal nerves, which shews the greater age of the embryo from which the sections were taken is the complete formation of the aortæ.

The upper ends of the muscle-plates have grown no further round the neural canal than in fig. A, and no scattered mesoblastic connective-tissue cells are visible.

In fig. A the dorsal surface of the neural canal was as completely rounded off as the ventral surface; but in fig. B III this has ceased to be the case. The cells at the dorsal surface of the neural canal have become rounder and smaller and begun to proliferate, and the uniform outline of the neural canal has here become broken (fig. B III, pr). The peculiar membrane completely surrounding the canal in fig. A now terminates just below the point where the proliferation of cells is taking place.

The prominence of cells which springs in this way from the top of the neural canal is the commencing rudiment of a pair of spinal nerves. In fig. B II, a section anterior to fig. B III, this formation has advanced much further (fig. B II, pr). From the extreme top of the neural canal there have now grown out two club-shaped masses of cells, one on each side; they are perfectly continuous with the cells which form the extreme top of the neural canal, and necessarily also are in contact with each other dorsally. Each grows outwards in contact with the walls of the neural canal; but, except at the point where they take their origin, they are not continuous with its walls, and are perfectly well separated by a sharp line from them.

In fig. B I, though the club-shaped processes still retain their attachment to the summit of the neural canal, they have become much longer and more conspicuous.

Specimens hardened in both chromic acid (Pl. 22, fig. C) and picric acid give similar appearances as to the formation of these bodies.

In those hardened in osmic acid, though the mutual relations of the masses of cells are very clear, yet it is difficult to distinguish the outlines of the individual cells.

In the chromic acid specimens (fig. C) the cells of these rudiments appear rounded, and each of them contains a large nucleus.

I have been unable to prepare longitudinal sections of this stage, either horizontal or vertical, to shew satisfactorily the extreme summit of the spinal cord; but I would call attention to the fact that the cells forming the proximal portion of the outgrowth are seen in every transverse section at this stage, and therefore exist the whole way along, whereas the distal portion is seen only in every third or fourth section, according to the thickness of the sections. It may be concluded from this that there appears a continuous outgrowth from the spinal canal, from which discontinuous processes grow out.

In specimens of a very much later period (Pl. 23, fig. I) the proximal portions of the outgrowth are unquestionably continuous with each other, though their actual junctions with the spinal cord are very limited in extent. The fact of this continuity at a later period is strongly in favour of the view that the posterior branches of the spinal nerves arise from the first as a continuous outgrowth of the spinal cord, from which a series of distal processes take their origin. I have, however, failed to demonstrate this point absolutely. The processes, which we may call the nerve-rudiments, are, as appears from the later stages, equal in number to the muscle-plates.

It may be pointed out, as must have been gathered from the description above, that the nerve-rudiments have at this stage but one point of attachment to the spinal cord, and that this one corresponds with the dorsal or posterior root of the adult nerve.

The rudiments are, in fact, those of the posterior root only.

The next or second stage in the formation of these structures to which I would call attention occurs at about the time when three to five visceral clefts are present. The disappearance from the notochord in the anterior extremity of the body of a special central area rich in protoplasm serves as an excellent guide to the commencement of this epoch.

Its investigation is beset with far greater difficulties than the previous one. This is owing partly to the fact that a number of connective-tissue cells, which are only with great difficulty to be distinguished from the cells which compose the spinal nerves, make their appearance around the latter, and partly to the fact that the attachment of the spinal nerves to the neural canal becomes much smaller, and therefore more difficult to study.

Fortunately, however, in Torpedo these peculiar features are not present to nearly the same extent as in Pristiurus and Scyllium.

The connective-tissue cells, though they appear earlier in Torpedo than in the two other genera, are much less densely packed, and the large attachment of the nerves to the neural canal is retained for a longer period.

Under these circumstances I consider it better, before proceeding with this stage, to give a description of the occurrences in Torpedo, and after that to return to the history of the nerves in the genera Pristiurus and Scyllium.

The development of the Spinal Nerves in Torpedo.

The youngest Torpedo-embryo in which I have found traces of the spinal nerves belongs to the earliest part of what I called the second stage.

The segmental duct[49] is just appearing, but the cells of the notochord have not become completely vacuolated. The rudiments of the spinal nerves extend half of the way towards the ventral side of the spinal cord; they grow out in a most distinct manner from the dorsal surface of the spinal cord (Pl. 22, fig. D a, pr); but the nerve-rudiments of the two sides are no longer continuous with each other at the dorsal median line, as in the earlier Pristiurus-embryos. The cells forming the proximal portion of the rudiment have the same elongated form as the cells of the spinal cord, but the remaining cells are more circular.

Footnote 49: Vide Balfour, "Origin and History of Urinogenital Organs of Vertebrates," Journal of Anatomy and Physiology, Oct. 1875. [This edition, No. VII.]

From the summit of the muscle-plates (mp) an outgrowth of connective tissue has made its appearance (c), which eventually fills up the space between the dorsal surface of the cord and the external epiblast. There is not the slightest difficulty in distinguishing the connective-tissue cells from the nerve-rudiment. I believe that in this embryo the origin of the nerves from the neural canal was a continuous one, though naturally the peripheral ends of the nerve-rudiments were separate from each other.

The most interesting feature of the stage is the commencing formation of the anterior roots. Each of these arises (Pl. 22, fig. D a, ar) as a small but distinct outgrowth from the epiblast of the spinal cord, near the ventral corner of which it appears as a conical projection. Even from the very first it has an indistinct form of termination and a fibrous appearance, while the protoplasm of which it is composed becomes very attenuated towards its termination.

The points of origin of the anterior roots from the spinal cord are separated from each other by considerable intervals. In this fact, and also in the nerves of the two sides never being united with each other in the ventral median line, the anterior roots exhibit a marked contrast to the posterior.

There exists, then, in Torpedo-embryos by the end of this stage distinct rudiments of both the anterior and posterior roots of the spinal nerves. These rudiments are at first quite independent of and disconnected with each other, and both take their rise as outgrowths of the epiblast of the neural canal.

The next Torpedo-embryo (Pl. 22, fig. D b), though taken from the same female, is somewhat older than the one last described. The cells of the notochord are considerably vacuolated; but the segmental duct is still without a lumen. The posterior nerve-rudiments are elongated, pear-shaped bodies of considerable size, and, growing in a ventral direction, have reached a point nearly opposite the base of the neural canal. They still remain attached to the top of the neural canal, though the connexion has in each case become a pedicle so narrow that it can only be observed with great difficulty.

It is fairly certain that by this stage each posterior nerve-rudiment has its own separate and independent junction with the spinal cord; their dorsal extremities are nevertheless probably connected with each other by a continuous commissure.

The cells composing the rudiments are still round, and have, in fact, undergone no important modifications since the last stage.

The important feature of the section figured (fig. D b), and one which it shares with the other sections of the same embryo, is the appearance of connective-tissue cells around the nerve-rudiment. These cells arise from two sources; one of these is supplied by the vertebral rudiments, which at the end of the last stage (Pl. 22, fig. C, vr) become split off from the inner layer of the muscle-plates. The vertebral rudiments have in fact commenced to grow up on each side of the neural canal, in order to form the mass of cells out of which the neural arches are subsequently developed.

The dorsal extremities of the muscle-plates form the second source of these connective-tissue cells. These latter cells lie dorsal and external to the nerve-rudiments.

The presence of this connective tissue, in addition to the nerve-rudiments, removes the possibility of erroneous interpretations in the previous stages of the Pristiurus-embryo.

It might be urged that the two masses which I have called nerve-rudiments are nothing else than mesoblastic connective tissue commencing to develop around the neural canal, and that the appearance of attachment to the neural canal which they present is due to bad preparation or imperfect observation. The sections of both this and the last Torpedo-embryo which I have been describing clearly prove that this is not the case. We have, in fact, in the same sections the developing connective tissue as well as the nerve-rudiments, and at a time when the latter still retains its primitive attachment to the neural canal. The anterior root (fig. D b, ar) is still a distinct conical prominence, but somewhat larger than in the previously described embryo; it is composed of several cells, and the cells of the spinal cord in its neighbourhood converge towards its point of origin.

In a Torpedo-embryo (Pl. 22, fig. D c) somewhat older than the one last described, though again derived from the oviduct of the same female, both the anterior and the posterior rudiments have made considerable steps in development.

In sections taken from the hinder part of the body I found that the posterior rudiments nearly agreed in size with those in fig. D b.

It is, however, still less easy than there to trace the junction of the posterior rudiments with the spinal cord, and the upper ends of the rudiments of the two sides do not nearly meet.

In a considerable series of sections I failed to find any case in which I could be absolutely certain that a junction between the nerve and the spinal cord was effected; and it is possible that in course of the change of position which this junction undergoes there may be for a short period a break of continuity between the nerve and the cord. This, however, I do not think probable. But if it takes place at all, it takes place before the nerve becomes functionally active, and so cannot be looked upon as possessing any physiological significance.

The rudiment of the posterior nerve in the hinder portion of the body is still approximately homogeneous, and no distinction of parts can be found in it.

In the same region of the body the anterior rudiment retains nearly the same condition as in the previous stage, though it has somewhat increased in size.

In the sections taken from the anterior part of the same embryo the posterior rudiment has both grown in size and also commenced to undergo histological changes by which it has become divided into a root, a ganglion, and a nerve.

The root (fig. D c, pr) consists of small round cells which lie close to the spinal cord, and ends dorsally in a rounded extremity.

The ganglion (g) consists of larger and more elongated cells, and forms an oval mass enclosed on the outside by the downward continuation of the root, having its inner side nearly in contact with the spinal cord.

From its ventral end is continued the nerve, which is of considerable length, and has a course approximately parallel to that of the muscle-plate. It forms a continuation of the root rather than of the ganglion.

Further details in reference to the histology of the nerve-rudiment at this stage are given later in this paper, in the description of Pristiurus-embryos, of which I have a more complete series of sections than of the Torpedo-embryos.

When compared with the nerve-rudiment in the posterior part of the same embryo, the nerve-rudiment last described is, in the first place, considerably larger, and has secondly undergone changes, so that it is possible to recognize in it parts which can be histologically distinguished as nerve and ganglion.

The developmental changes which have taken place in the anterior root are not less important than those in the posterior. The anterior root now forms a very conspicuous cellular prominence growing out from the ventral corner of the spinal cord (fig. D c, ar). It has a straight course from the spinal cord to the muscle-plate, and there shews a tendency to turn downwards at an open angle: this, however, is not represented in the specimen figured. The cells of which it is composed each contain a large oval nucleus, and are not unlike the cells which form the posterior rudiment. The anterior and posterior nerves are still quite unconnected with each other; and in those sections in which the anterior root is present the posterior root of the same side is either completely absent or only a small part is to be seen. The cells of the spinal cord exhibit a slight tendency to converge towards the origin of the anterior nerve-root.

In the spinal cord itself the epithelium of the central canal is commencing to become distinguished from the grey matter, but no trace of the white matter is visible.

I have succeeded in making longitudinal vertical sections of this stage, which prove that the ends of the posterior roots adjoining the junction with the cord are all connected with each other (Pl. 22, fig. D d).

If the figure representing a transverse section of the embryo (fig. D c) be examined, or better still the figure of a section of the slightly older Scyllium-embryo (Pl. 23, fig. H I or I I), the posterior root will be seen to end dorsally in a rounded extremity, and the junction with the spinal cord to be effected, not by the extremity of the nerve, but by a part of it at some little distance from this.

It is from these upper ends of the rudiments beyond the junction with the spinal cord that I believe the commissures to spring which connect together the posterior roots.

My sections shewing this for the stage under consideration are not quite as satisfactory as is desirable; nevertheless they are sufficiently good to remove all doubt as to the presence of these commissures.

A figure of one of these sections is represented (Pl. 22, fig. D d). In this figure pr points to the posterior roots and x to the commissures uniting them.

In a stage somewhat subsequent to this I have succeeded in making longitudinal sections, which exhibit these junctions with a clearness which leaves nothing to be desired.

It is there effected (Pl. 23, fig. L) in each case by a protoplasmic commissure with imbedded nuclei[50]. Near its dorsal extremity each posterior root dilates, and from the dilated portion is given off on each side the commissure uniting it with the adjoining roots.

Footnote 50: This commissure is not satisfactorily represented in the figure. Vide Explanation of Plate 23.

Considering the clearness of this formation in this embryo, as well as in the embryo belonging to the stage under description, there cannot be much doubt that at the first formation of the posterior rudiments a continuous outgrowth arises from the spinal cord, and that only at a later period do the junctions of the roots with the cord become separated and distinct for each nerve.

I now return to the more complete series of Pristiurus-embryos, the development of whose spinal nerves I have been able to observe.

Second Stage of the Spinal Nerves in Pristiurus.

In the youngest of these (Pl. 22, fig. E) the notochord has undergone but very slight changes, but the segmental duct has made its appearance, and is as much developed as in the Torpedo-embryo from which fig. D b was taken.

(The embryo from which fig. E a was derived had three visceral clefts.)

There have not as yet appeared any connective-tissue cells dorsal to the top of the muscle-plates, so that the posterior nerve-rudiments are still quite free and distinct.

The cells composing them are smaller than the cells of the neural canal; they are round and nucleated; and, indeed, in their histological constitution the nerve-rudiments exhibit no important deviations from the previous stage, and they have hardly increased in size. In their mode of attachment to the neural tube an important change has, however, already commenced to be visible.

In the previous stage the two nerve-rudiments met above the summit of the spinal cord and were broadly attached to it there; now their points of attachment have glided a short distance down the sides of the spinal cord[51].

Footnote 51: [May 18, 1876.--Observations I have recently made upon the development of the cranial nerves incline me to adopt an explanation of the change which takes place in the point of attachment of the spinal nerves to the cord differing from that enunciated in the text. I look upon this change as being apparent rather than real, and as due to a growth of the roof of the neural canal in the median dorsal line, which tends to separate the roots of the two sides more and more, and cause them to assume a more ventral position.]

The two nerve-rudiments have therefore ceased to meet above the summit of the canal; and in addition to this they appear in section to narrow very much before becoming united with its walls, so that their junctions with these appear in a transverse section to be effected by at most one or two cells, and are, comparatively speaking, very difficult to observe.

In an embryo but slightly older than that represented in Fig. E a the first rudiment of the anterior root becomes visible. This appears, precisely as in Torpedo, in the form of a small projection from the ventral corner of the spinal cord (fig. E b, ar).

The second step in this stage (Pl. 22, fig. F) is comparable, as far as the connective-tissue is concerned, with the section of Torpedo (Pl. 22, fig. D d). The notochord (the histological details of whose structure are not inserted in this figure) is rather more developed, and the segmental duct, as was the case with the corresponding Torpedo-embryo, has become hollow at its anterior extremity.

The embryo from which the section was taken possessed five visceral clefts, but no trace of external gills.

In the section represented, though from a posterior part of the body, the dorsal nerve-rudiments have become considerably larger than in the last embryo; they now extend beyond the base of the neural canal. They are surrounded to a great extent by mesoblastic tissue, which, as in the case of the Torpedo, takes its origin from two sources, (1) from the commencing vertebral bodies, (2) from the summits of the muscle-plates.

It is in many cases very difficult, especially with chromic-acid specimens, to determine with certainty the limits of the rudiments of the posterior root.

In the best specimens a distinct bordering line can be seen, and it is, as a rule, possible to state the characters by which the cells of the nerve-rudiments and vertebral bodies differ. The more important of these are the following:--(1) The cells of the nerve-rudiment are distinctly smaller than those of the vertebral rudiment; (2) the cells of the nerve-rudiment are elongated, and have their long axis arranged parallel to the long axis of the nerve-rudiment, while the cells surrounding them are much more nearly circular.

The cells of the nerve-rudiment measure about 1/1600 × 1/4500 to 1/1600 × 1/3200 inch, those of the vertebral rudiment 1/1600 × 1/1900 inch. The greater difficulty experienced in distinguishing the nerve-rudiment from the connective-tissue in Pristiurus than in Torpedo arises from the fact that the connective-tissue is much looser and less condensed in the latter than in the former.

The connective-tissue cells which have grown out from the muscle-plates form a continuous arch over the dorsal surface of the neural tube (vide Pl. 22, fig. F): and in some specimens it is difficult to see whether the arch is formed by the rudiment of the posterior root or by connective-tissue. It is, however, quite easy with the best specimens to satisfy one's self that it is from the connective-tissue, and not the nerve-rudiment, that the dorsal investment of the neural canal is derived.

As in the previous case, the upper ends of each pair of posterior nerve-rudiments are quite separate from one another, and appear in sections to be united by a very narrow root to the walls of the neural canal at the position indicated in fig. F[52].

Footnote 52: The artist has not been very successful in rendering this figure.

The cells forming the nerve-rudiments have undergone slight modifications; they are for the most part more distinctly elongated than in the earlier stage, and appear slightly smaller in comparison with the cells of the neural canal.

They possess as yet no distinctive characters of nerve-cells. They stain more deeply with osmic acid than the cells around them, but with hæmatoxylin there is but a very slight difference in intensity between their colouring and that of the neighbouring connective-tissue cells.

The anterior roots have grown considerably in length, but their observation is involved in the same difficulties with chromic-acid specimens as that of the posterior rudiments.

There is a further difficulty in observing the anterior roots, which arises from the commencing formation of white matter in the cord. This is present in all the anterior sections of the embryo from which fig. F is taken. When the white matter is formed the cells constituting the junction of the anterior nerve-root with the spinal cord undergo the same changes as the cells which are being converted into the white matter of the cord, and become converted into nerve-fibres; these do not stain with hæmatoxylin, and thus an apparent space is left between the nerve-root and the spinal cord. This space by careful examination may be seen to be filled up with fibres. In osmic acid sections, although even in these the white matter is stained less deeply than the other tissues, it is a matter of comparative ease to observe the junction between the anterior nerve root and the spinal cord.

I have been successful in preparing satisfactory longitudinal sections of embryos somewhat older than that shewn in fig. F, and they bring to light several important points in reference to the development of the spinal nerves. Three of these sections are represented in Pl. 22, figs. G1, G2, and G3.

The sections are approximately horizontal and longitudinal. G1 is the most dorsal of the three; it is not quite horizontal though nearly longitudinal. The section passes exactly through the point of attachment of the posterior roots to the walls of the neural canal.

The posterior rudiments appear as slight prominences of rounded cells projecting from the wall of the neural canal. From transverse sections the attachment of the nerves to the wall of the neural canal is proved to be very narrow, and from these sections it appears to be of some length in the direction of the long axis of the embryo. A combination of the sections taken in the two directions leads to the conclusion that the nerves at this stage thin out like a wedge before joining the spinal cord.

The independent junctions of the posterior rudiments with the spinal cord at this stage are very clearly shewn, though the rudiments are probably united with each other just dorsal to their junction with the spinal cord.

The nerves correspond in number with the muscle-plates, and each arises from the spinal cord, nearly opposite the middle line of the corresponding muscle-plates (figs. G1 and G2).

Each nerve-rudiment is surrounded by connective-tissue cells, and is separated from its neighbours by a considerable interval.

At its origin each nerve-rudiment lies opposite the median portion of a muscle-plate (figs. G1 and G2); but, owing to the muscle-plate acquiring an oblique direction, at the level of the dorsal surface of the notochord it appears in horizontal sections more nearly opposite the interval between two muscle-plates (figs. G2 and G3).

In horizontal sections I find masses of cells which make their appearance on a level with the ventral surface of the spinal cord. I believe I have in some sections successfully traced these into the spinal cord, and I have little doubt that they are the anterior roots of the spinal nerves; they are opposite the median line of the muscle-plates, and do not appear to join the posterior roots (vide fig. G3, ar).

At the end of this period or second stage the main characters of the spinal nerves in Pristiurus are the following:--

(1) The posterior nerve-rudiments form somewhat wedge-shaped masses of tissue attached dorsally to the spinal cord.

(2) The cells of which they are composed are typical undifferentiated embryonic cells, which can hardly be distinguished from the connective-tissue cells around them.

(3) The nerves of each pair no longer meet above the summit of the spinal canal, but are independently attached to its sides.

(4) Their dorsal extremities are probably united by commissures.

(5) The anterior roots have appeared; they form small conical projections from the ventral corner of the spinal cord, but have no connexion with the posterior rudiments.

The Third Stage of the Spinal Nerves in Pristiurus.

With the third stage the first distinct histological differentiations of the nerve-rudiments commence. Owing to the changes both in the nerves themselves and in the connective-tissue around them, which becomes less compact and its cells stellate, the difficulty of distinguishing the nerves from the surrounding cells vanishes; and the difficulties of investigation in the later stages are confined to the modes of attachment of the nerves to the neural canal, and the histological changes which take place in the rudiments themselves.

The stage may be considered to commence at the period when the external gills first make their appearance as small buds from the walls of the visceral clefts. Already, in the earliest rudiments of the posterior root of this period now figured, a number of distinct parts are visible (Pl. 23, fig. H I).

Surrounding nearly the whole structure there is present a delicate investment similar to that which I mentioned as surrounding the neural canal and other organs; it is quite structureless, but becomes coloured with all staining reagents. I must again leave open the question whether it is to be looked upon as a layer of coagulated protoplasm or as a more definite structure. This investment completely surrounds the proximal portion of the posterior root, but vanishes near its distal extremity.

The nerve-rudiment itself may be divided into three distinct portions:--(1) the proximal portion, in which is situated the pedicle of attachment to the wall of the neural canal; (2) an enlarged portion, which may conveniently, from its future fate, be called the ganglion; (3) a distal portion beyond this. The proximal portion presents a fairly uniform diameter, and ends dorsally in a rounded expansion; it is attached remarkably enough, not by its extremity, but by its side, to the spinal cord. The dorsal extremities of the posterior nerves are therefore free; as was before mentioned, they probably serve as the starting-point of the longitudinal commissures between the posterior roots.

The spinal cord at this stage is still made up of fairly uniform cells, which do not differ in any important particulars from the cells which composed it during the last stage. The outer portion of the most peripheral layer of cells has already begun to be converted into the white matter.

The delicate investment spoken of before still surrounds the whole spinal cord, except at the points of junction of the cord with the nerve-rudiments. Externally to this investment, and separated from it for the most part by a considerable interval, a mesoblastic sheath (Pl. 23, fig. H I, i) for the spinal cord is beginning to be formed.

The attachment of the nerve-rudiments to the spinal cord, on account of its smallness, is still very difficult to observe. In many specimens where the nerve is visible a small prominence may be seen rising up from the spinal cord at a point corresponding to x (Pl. 23, fig. H I). It is, however, rare to see this prominence and the nerve continuous with each other: as a rule they are separated by a slight space, and frequently one of the cells of the mesoblastic investment of the spinal cord is interposed between the two. In some especially favourable specimens, similar to the one figured, there can be seen a distinct cellular prominence (fig. H I, x) from the spinal cord, which becomes continuous with a small prominence on the lateral border of the nerve-rudiment near its free extremity. The absence of a junction between the two in a majority of sections is only what might be expected, considering how minute the junction is.

Owing to the presence of the commissure connecting the posterior roots, some part of a nerve is present in every section.

The proximal extremity of the nerve-rudiment itself is composed of cells, which, by their smaller size and a more circular form, are easily distinguished from cells forming the ganglionic portion of the nerve.

The ganglionic portion of the nerve, by its externally swollen configuration, is at once recognizable in all the sections in which the nerve is complete. The delicate investment before mentioned is continuous around it. The cells forming it are larger and more elongated than the cells forming the upper portion of the nerve-rudiment: each of them possesses a large and distinct nucleus.

The remainder of the nerve rudiment forms the commencement of the true nerve. It can in this stage be traced only for a very small distance, and gradually fades away, in such a manner that its absolute termination is very difficult to observe.

The connective-tissue cells which surround the nerve-rudiment are far looser than in the last stage, and are commencing to throw out processes and become branched.

The anterior root-nerve has grown very considerable since the last stage. It projects from the same region of the cord as before, but on approaching the muscle-plate takes a sudden bend downwards (fig. H II, ar).

I have failed to prove that the anterior and posterior roots are at this stage united.

Fourth Stage.

In an embryo but slightly more advanced than the one last described, important steps have been made in the development of the nerve-rudiment. The spinal cord itself now possesses a covering of white matter; this is thickest at the ventral portion of the cord, and extends to the region of the posterior root of the spinal nerve.

The junction of the posterior root with the spinal cord is easier to observe than in the last stage.

It is still effected by means of unaltered cells, though the cells which form the projection from the cord to the nerve are commencing to undergo changes similar to those of the cells which are being converted into white matter.

In the rudiment of the posterior root itself there are still three distinct parts, though their arrangement has undergone some alteration and their distinctness has become more marked (Pl. 23, fig. I I).

The root of the nerve (fig. I I, pr) consists, as before, of nearly circular cells, each containing a nucleus, very large in proportion to the size of the cell. The cells have a diameter of about 1/3000 of an inch. This mass forms not only the junction between the ganglion and the spinal canal, but is also continued into a layer investing the outer side of the ganglion and continuous with the nerve beyond the ganglion.

The cells which compose the ganglion (fig. I I, sp.g) are easily distinguished from those of the root. Each cell is elongated with an oval nucleus, large in proportion to the cell; and its protoplasm appears to be continued into an angular, not to say fibrous process, sometimes at one and more rarely at both ends. The processes of the cells are at this stage very difficult to observe: figs. I a, I b, I c represent three cells provided with them and placed in the positions they occupied in the ganglion.

The relatively very small amount of protoplasm in comparison to the nucleus is fairly represented in these figures, though not in the drawing of the ganglion as a whole. In the centre of each nucleus is a nucleolus.

Fig. I b, in which the process points towards the root of the nerve, I regard as a commencing nerve-fibre: its more elongated shape seems to imply this. In the next stage special bundles of nerve-fibres become very conspicuous in the ganglion. The long diameter of an average ganglion-cell is about 1/1600 of an inch. The whole ganglion forms an oval mass, well separated both from the nerve-root and the nerve, and is not markedly continuous with either. On its outer side lies the downward process of the nerve-root before mentioned.

The nerve itself is still, as in the last case, composed of cells which are larger and more elongated than either the cells of the root or the ganglion.

The condition of the anterior root at this stage is hardly altered from what it was; it is composed of very small cells, which with hæmatoxylin stain more deeply than any other cell of the section. A figure of it is given in I II.

Horizontal longitudinal sections of this stage are both easy to make and very instructive. On Pl. 23, fig. K I is represented a horizontal section through a plane near the dorsal surface of the spinal cord: each posterior root is seen in this section to lie nearly opposite the anterior extremity of a muscle-plate.

In a more ventral plane (fig. K II) this relation is altered, and the posterior roots lie opposite the hinder parts of the muscle-plates.

The nerves themselves are invested by the hyaline membrane spoken of above; and surrounding this again there is present a delicate mesoblastic investment of spindle-shaped cells.

Longitudinal sections also throw light upon the constitution of the anterior nerve roots (vide fig. K II, ar). In the two segments on the left-hand side in this figure the anterior roots are cut through as they are proceeding, in a more or less horizontal course, from the spinal cord to the muscle-plates.

Where the section (which is not quite horizontal) passes through the plane of the notochord, as on the right-hand side, the anterior roots are cut transversely. Each root, in fact, changes its direction, and takes a downward course.

The anterior roots are situated nearly opposite the middle of the muscle-plates: their section is much smaller than that of the posterior roots, and with hæmatoxylin they stain more deeply than any of the other cells in the preparation.

The anterior roots, so far as I have been able to observe, do not at this stage unite with the posterior; but on this point I do not speak with any confidence.

The period now arrived at forms a convenient break in the development of the spinal nerves; and I hope to treat the remainder of the subject, especially the changes in the ganglion, the development of the ganglion-cells, and of the nerve-fibres, in a subsequent paper.

I will only add that, not long after the stage last described, the posterior root unites with the anterior root at a considerable distance below the cord: this is shewn in Pl. 23, fig. L. Still later the portion of the root between the ganglion and the spinal cord becomes converted into nerve-fibres, and the ganglion becomes still further removed from the cord, while at the same time it appears distinctly divided into two parts.

As regards the development of the cranial nerves, I have made a few observations, which, though confessedly incomplete, I would desire to mention here, because, imperfect as they are, they seem to shew that in Elasmobranch Fishes the cranial nerves resemble the spinal nerves in arising as outgrowths from the central nervous system.

I have given a figure of the development of a posterior root of a cranial nerve in fig. M I. The section is taken from the same embryo as figs. B I, B II, and B III.

It passes through the anterior portion of a thickening of the external epiblast, which eventually becomes involuted as the auditory vesicle.

The posterior root of a nerve (VII) is seen growing out from the summit of the hind brain in precisely the same manner that the posterior roots of the spinal nerves grow out from the spinal cord: it is the rudiment of the seventh or facial nerve. The section behind this (fig. M II), still in the region of the ear, has no trace of a nerve, and thus serves to shew the early discontinuity of the posterior nerve-rudiments which arise from the brain.

I have as yet failed to detect any cranial anterior roots like those of the spinal nerves[53]. The similarity in development between the cranial and spinal nerves is especially interesting, as forming an important addition to the evidence which at present exists that the cranial nerves are only to be looked on as spinal nerves, especially modified in connexion with the changes which the anterior extremity of the body has undergone in existing vertebrates.

Footnote 53: [May 18, 1876.--Subsequent observations have led me to the conclusion that no anterior nerve-roots are to be found in the brain.]

* * * * *

My results may be summarized as follows:--

Along the extreme dorsal summit of the spinal cord there arises on each side a continuous outgrowth.

From each outgrowth processes corresponding in number to the muscle-plates grow downwards. These are the posterior nerve-rudiments.

The outgrowths, at first attached to the spinal cord throughout their whole length, soon cease to be so, and remain in connexion with it in certain spots only, which form the junctions of the posterior roots with the spinal cord.

The original outgrowth on each side remains as a bridge, uniting together the dorsal extremities of all the posterior rudiments. The points of junction of the posterior roots with the spinal cord are at first situated at the extreme dorsal summit of the latter, but eventually travel down, and are finally placed on the sides of the cord.

After these events the posterior nerve-rudiments grow rapidly in size, and become differentiated into a root (by which they are attached to the spinal canal), a ganglion, and a nerve.

The anterior roots, like the posterior, are outgrowths from the spinal cord; but the outgrowths to form them are from the first discontinuous, and the points from which they originally spring remain as those by which they are permanently attached to the spinal cord, and do not, as in the case of the posterior roots, undergo a change of position. The anterior roots arise, not vertically below, but opposite the intervals between the posterior roots.

The anterior roots are at first quite separate from the posterior roots; but soon after the differentiation of the posterior rudiment into a root, ganglion, and nerve, a junction is effected between each posterior nerve and the corresponding anterior root. The junction is from the first at some little distance from the ganglion.

* * * * *

Investigators have hitherto described the spinal nerves as formed from part of the mesoblast of the protovertebræ. His alone, so far as I know, takes a different view.

His's[54] observations lead him to the conclusion that the posterior roots are developed as ingrowths from the external epiblast into the space between the protovertebræ and the neural canal. These subsequently become constricted off, unite with the neural canal and form spinal nerves.

Footnote 54: Erste Anlage des Wirbelthier-Leibes.

These statements, which have not been since confirmed, diverge nearly to the same extent from my own results as does the ordinary account of the development of these parts.

Hensen (Virchow's Archiv, Vol. XXXI. 1864) also looks upon the spinal nerves as developed from the epiblast, but not as a direct result of his own observations[55].

Footnote 55: [May 18, 1876.--Since the above was written Hensen has succeeded in shewing that in mammals the rudiments of the posterior roots arise in a manner closely resembling that described in the present paper; and I have myself, within the last few days, made observations which incline me to believe that the same holds good for the chick. My observations are as yet very incomplete.]

Without attempting, for the present at least, to explain this divergence, I venture to think that the facts which I have just described have distinct bearings upon one or two important problems.

One point of general anatomy upon which they throw considerable light is the primitive origin of nerves.

So long as it was admitted that the spinal and cerebral nerves developed in the embryo independently of the central nervous system, their mode of origin always presented to my mind considerable difficulties.

It never appeared clear how it was possible for a state of things to have arisen in which the central nervous system, as well as the peripheral terminations of nerves, whether motor or sensory, were formed independently of each other, while between them a third structure was developed which, growing in both directions (towards the centre and towards the periphery), ultimately brought the two into connexion.

That such a condition could be a primitive one seemed scarcely possible.

Still more remarkable did it appear, on the supposition that the primitive mode of formation of these parts was represented in the developmental history of vertebrates, that we should find similar structural elements in the central and in the peripheral nervous systems.

The central nervous system arises from the epiblast, and yet contains precisely similar nerve-cells and nerve-fibres to the peripheral nervous system, which, if derived, as is usually stated, from the mesoblast, was necessarily supposed to have a completely different origin from the central nervous system.

Both of these difficulties are to a great extent removed by the facts of the development of these parts in Elasmobranchii.

If it be admitted that the spinal roots develop as outgrowths from the central nervous system in Elasmobranch Fishes, the question arises, how far can it be supposed to be possible that in other vertebrates the spinal roots and ganglia develop independently of the spinal cord, and only subsequently become united with it.

I have already insisted that this cannot be the primary condition; and though I am of opinion that the origin of the nerves in higher vertebrates ought to be worked over again, yet I do not think it impossible that, by a secondary adaptation, the nerve-roots might develop in the mesoblast[56].

Footnote 56: [May 18, 1876.--Hensen's observations, as well as those recently made by myself on the chick, render it almost certain that the nerves in all Vertebrates spring from the spinal cord.]

The presence of longitudinal commissures connecting the central ends of all the posterior roots is very peculiar. The commissures may possibly be looked on as outlying portions of the cord, rather than as parts of the nerves.

I have not up to this time followed their history beyond a somewhat early period in embryonic life, and am therefore unacquainted with their fate in the adult.

As far as I am aware, no trace of similar structures has been met with in other vertebrates.

The commissures have a very strong resemblance to those by which in Elasmobranch Fishes the glossopharyngeal nerve and the branches of the pneumogastric are united in an early embryonic stage[57].

Footnote 57: Balfour, "A Preliminary Account of the Development of Elasmobranch Fishes," Q. J. Micros. Sc. 1874, plate XV. fig. 14, v.g. [This edition, Pl. 4, fig. 14, vg].

I think it not impossible that the commissures in the two cases represent the same structures. If this is the case, it would seem that the junction of a number of nerves to form the pneumogastric is not a secondary state, but the remnant of a primary one, in which all the spinal nerves were united, as they embryonically are in Elasmobranchii.

One point brought out in my investigations appears to me to have bearings upon the origin of the central canal of the Vertebrate nervous system, and in consequence upon the origin of the Vertebrate group itself.

The point I allude to is the posterior nerve-rudiments making their first appearance at the extreme dorsal summit of the spinal cord.

The transverse section of the ventral nervous cord of an ordinary segmented worm consists of two symmetrical halves placed side by side.

If by a mechanical folding the two lateral halves of the nervous cord became bent towards each other, while into the groove formed between the two the external skin became pushed, we should have an approximation to the Vertebrate spinal cord. Such a folding might take place to give extra rigidity to the body in the absence of a vertebral column.

If this folding were then completed in such a way that the groove, lined by external skin and situated between the two lateral columns of the nervous system, became converted into a canal, above and below which the two columns of the nervous system united, we should have in the transformed nervous cord an organ strongly resembling the spinal cord of Vertebrates.

This resemblance would even extend beyond mere external form. Let the ventral nervous cord of the common earthworm, Lumbricus agricola, be used for comparison[58], a transverse section of which is represented by Leydig[59] and Claparède. In this we find that on the ventral surface (the Annelidan ventral surface) of the nervous cord the ganglion-cells (grey matter) (k) are situated, and on the dorsal side the nerve-fibres or white matter (h). If the folding that I have supposed were to take place, the grey and white matters would have very nearly the relative situations which they have in the Vertebrate spinal cord.

Footnote 58: The nervous cords of other Annelids resemble that of Lumbricus in the relations of the ganglion-cells of the nerve-fibres.

Footnote 59: Tafeln zur vergleichenden Anatomie, Taf. iii. fig. 8.

The grey matter would be situated in the interior and surround the epithelium of the central canal, and the white matter would nearly surround the grey and form the anterior white commissure. The nerves would then arise, not from the sides of the nervous cord as in existing Vertebrates, but from its extreme ventral summit.

One of the most striking features which I have brought to light with reference to the development of the posterior roots, is the fact of their growing out from the extreme dorsal summit of the neural canal--a position analogous to the ventral summit of the Annelidan nervous cord. Thus the posterior roots of the nerves in Elasmobranchii arise in the exact manner which might have been anticipated were the spinal cord due to such a folding as I have suggested. The argument from the nerves becomes the stronger, from the great peculiarity in the position of the outgrowth, a feature which would be most perplexing without some such explanation as I have proposed. The central epithelium of the neural canal according to this view represents the external skin; and its ciliation is to be explained as a remnant of the ciliation of the external skin now found amongst many of the lower Annelids.

I have, however, employed the comparison of the Vertebrate and Annelidan nervous cords, not so much to prove a genetic relation between the two as to shew the à priori possibility of the formation of a spinal canal and the à posteriori evidence we have of the Vertebrate spinal canal having been formed in the way indicated.

I have not made use of what is really the strongest argument for my view, viz. that the embryonic mode of formation of the spinal canal, by a folding in of the external epiblast, is the very method by which I have supposed the spinal canal to have been formed in the ancestors of Vertebrates.

My object has been to suggest a meaning for the peculiar primitive position of the posterior roots, rather than to attempt to explain in full the origin of the spinal canal.

EXPLANATION OF THE PLATES[60].

Footnote 60: The figures on these Plates give a fair general idea of the appearance presented by the developing spinal nerves; but the finer details of the original drawings have in several cases become lost in the process of copying.

The figures which are tinted represent sections of embryos hardened in osmic acid; those without colour sections of embryos hardened in chromic acid.

PLATE 22.

Fig. A. Section through the dorsal region of an embryo of Scyllium stellare, with the rudiments of two visceral clefts. The section illustrates the general features at a period anterior to the appearance of the posterior nerve-roots.

nc. neural canal. mp. muscle-plate. ch. notochord. x. subnotochordal rod. ao. rudiment of dorsal aorta. so. somatopleure. sp. splanchnopleure. al. alimentary tract. All the parts of the section except the spinal cord are drawn somewhat diagrammatically.

Figs. B I, B II, B III. Three sections of a Pristiurus-embryo. B I is through the heart, B II through the anterior part of the dorsal region, and B III through a point slightly behind this. Drawn with a camera. (Zeiss CC ocul. 2.)

In B III there is visible a slight proliferation of cells from the dorsal summit of the neural canal.

In B II this proliferation definitely constitutes two club-shaped masses of cells (pr), both attached to the dorsal summit of the neural canal. The masses are the rudiments of the posterior nerve-roots.

In B I the rudiments of the posterior roots are of considerable length.

pr. rudiment of posterior roots. nc. neural canal. mp. muscle-plate. ch. notochord. x. subnotochordal rod. ao. dorsal aorta. so. somatopleure. sp. splanchnopleure. al. alimentary canal. ht. heart.

Fig. C. Section from a Pristiurus-embryo, slightly older than B. Camera. (Zeiss CC ocul. 2.) The embryo from which this figure was taken was slightly distorted in the process of removal from the blastoderm.

vr. rudiment of vertebral body. Other reference letters as in previous figures.

Fig. D a. Section through the dorsal region of a Torpedo-embryo with three visceral clefts. (Zeiss CC ocul. 2.) The section shews the formation of the dorsal nerve-rudiments (pr) and of a ventral anterior nerve-rudiment (ar), which at this early stage is not distinctly cellular.

ar. rudiment of an anterior nerve-root. y. cells left behind on the separation of the external skin from the spinal cord. c. connective-tissue cells springing from the summit of the muscle-plates. Other reference letters as above.

Fig. D b. Section from dorsal region of a Torpedo-embryo somewhat older than D a. Camera. (Zeiss CC ocul. 2.) The posterior nerve-rudiment is considerably longer than in fig. Da, and its pedicle of attachment to the spinal cord is thinner. The anterior nerve-rudiment, of which only the edge is present in the section, is distinctly cellular.

m. mesoblast growing up from vertebral rudiment. sd. segmental duct.

Fig. D c. Section from a still older Torpedo-embryo. Camera. (Zeiss CC ocul. 2.) The connective-tissue cells are omitted. The rudiment of the ganglion (g) on the posterior root has appeared. The rudiment of the posterior nerve is much longer than before, and its junction with the spinal cord is difficult to detect. The anterior root is now an elongated cellular structure.

g. ganglion.

Fig. D d. Longitudinal and vertical section through a Torpedo-embryo of the same age as D c.

The section shews the commissures (x) uniting the posterior roots.

Fig. E a. Section of a Pristiurus-embryo belonging to the second stage. Camera. (Zeiss CC ocul. 2.) The section shews the constriction of the pedicle which attaches the posterior nerve-rudiments to the spinal cord.

pr. rudiment of posterior nerve-root. nc. neural canal. mp. muscle-plate. vr. vertebral rudiment. sd. segmental duct. ch. notochord. so. somatopleure. sp. splanchnopleure. ao. aorta. al. alimentary canal.

Fig. E b. Section of a Pristiurus-embryo slightly older than Ea. Camera. (Zeiss CC ocul. 2.) The section shews the formation of the anterior nerve-root (ar).

ar. rudiment of the anterior nerve-root.

Fig. F. Section of a Pristiurus-embryo with the rudiments of five visceral clefts. Camera. (Zeiss CC ocul. 2.)

The rudiment of the posterior root is seen surrounded by connective-tissue, from which it cannot easily be distinguished. The artist has not been very successful in rendering this figure.

Figs. G1, G2, G3. Three longitudinal and horizontal sections of an embryo somewhat older than F. The embryo from which these sections were taken was hardened in osmic acid, but the sections have been represented without tinting. G1 is most dorsal of the three sections. Camera. (Zeiss CC ocul. 1.)

nc. neural canal. sp.c. spinal cord. pr. rudiment of posterior root. ar. rudiment of anterior root. mp. muscle-plate. c. connective-tissue cells. ch. notochord.

PLATE 23.

Fig. H I. Section through the dorsal region of a Pristiurus-embryo in which the rudimentary external gills are present as very small knobs. Camera. (Zeiss CC ocul. 2.)

The section shews the commencing differentiation of the posterior nerve-rudiment into root (pr), ganglion (sp.g), and nerve (n), and also the attachment of the nerve-root to the spinal cord (x). The variations in the size and shape of the cells in the different parts of the nerve-rudiment are completely lost in the figure.

pr. posterior nerve-root. sp.g. ganglion of posterior root. n. nerve of posterior root. x. attachment of posterior root to spinal cord. w. white matter of spinal cord. i. mesoblastic investment to the spinal cord.

Fig. H II. Section through the same embryo as H I. (Zeiss CC ocul. 1.)

The section contains an anterior root, which takes its origin at a point opposite the interval between two posterior roots.

The white matter has not been very satisfactorily represented by the artist.

Figs. I I, I II. Two sections of a Pristiurus-embryo somewhat older than H. Camera. (Zeiss CC ocul. 1.)

The connective-tissue cells are omitted.

Figs. I a, I b, I c. Three isolated cells from the ganglion of one of the posterior roots of the same embryo.

Figs. K I, K II. Two horizontal longitudinal sections through an embryo in which the external gills have just appeared. K I is the most dorsal of the two sections. Camera. (Zeiss CC ocul. 1.)

The sections shew the relative positions of the anterior and posterior roots at different levels.

pr. posterior nerve-rudiment. ar. anterior nerve-rudiment. sp.c. spinal cord. n.c. neural canal. mp. muscle-plate. mp´. first-formed muscles.

Fig. L. Longitudinal and vertical section through the trunk of a Scyllium-embryo after the external gills have attained their full development. Camera. (Zeiss CC ocul. 1.)

The embryo was hardened in a mixture of chromic acid and osmic acid.

The section shews the commissures which dorsally unite the posterior roots, and also the junction of the anterior and posterior roots. The commissures are unfortunately not represented in the figure with great accuracy; their outlines are in nature perfectly regular, and not, as in the figure, notched at the junctions of the cells composing them. Their cells are apparently more or less completely fused, and certainly not nearly so clearly marked as in the figure. The commissures stain very deeply with the mixture of osmic and chromic acid, and form one of the most conspicuous features in successful longitudinal sections of embryos so hardened. In sections hardened with chromic acid only they cannot be seen with the same facility.

sp.c. spinal cord. gr. grey matter. w. white matter. ar. anterior root. pr. posterior root. x. commissure uniting the posterior roots.

Figs. M I, M II. Two sections through the head of the same embryo as fig. B. M I, the foremost of the two, passes through the anterior part of the thickening of epiblast, which becomes involuted as the auditory vesicle. It contains the rudiment of the seventh nerve, VII. Camera. (Zeiss CC ocul. 2.)

VII. rudiment of seventh nerve. au. thickening of external epiblast, which becomes involuted as the auditory vesicle. n.c. neural canal. ch. notochord. pp. body-cavity in the head. so. somatopleure. sp. splanchnopleure. al. throat exhibiting an outgrowth to form the first visceral cleft.

IX. ON THE SPINAL NERVES OF AMPHIOXUS[61].

Footnote 61: From the Journal of Anatomy and Physiology, Vol. X. 1876.

During a short visit to Naples in January last, I was enabled, through the kindness of Dr Dohrn, to make some observations on the spinal nerves of Amphioxus. These were commenced solely with the view of confirming the statements of Stieda on the anatomy of the spinal nerves, which, if correct, appeared to me to be of interest in connection with the observations I had made that, in Elasmobranchii, the anterior and posterior roots arise alternately and not in the same vertical plane. I have been led to conclusions on many points entirely opposed to those of Stieda, but, before recording these, I shall proceed briefly to state his results, and to examine how far they have been corroborated by subsequent observers.

Stieda[62], from an examination of sections and isolated spinal cords, has been led to the conclusion that, in Amphioxus, the nerves of the opposite sides arise alternately, except in the most anterior part of the body, where they arise opposite each other. He also states that the nerves of the same side issue alternately from the dorsal and ventral corners of the spinal cord. He regards two of these roots (dorsal and ventral) on the same side as together equivalent to a single spinal nerve of higher vertebrates formed by the coalescence of a dorsal and ventral root.

Footnote 62: Mém. Acad. Pétersbourg, Vol. XIX.

Langerhans[63] apparently agrees with Stieda as to the facts about the alternation of dorsal and ventral roots, but differs from him as to the conclusions to be drawn from those facts. He does not, for two reasons, believe that two nerves of Amphioxus can be equivalent to a single nerve in higher vertebrates: (1) Because he finds no connecting branch between two succeeding nerves, and no trace of an anastomosis. (2) Because he finds that each nerve in Amphioxus supplies a complete myotome, and he considers it inadmissible to regard the nerves, which in Amphioxus together supply two myotomes, as equivalent to those which in higher vertebrates supply a single myotome only.

Footnote 63: Archiv f. mikr. Anatomie, Vol. XII.

Although the agreement as to facts between Langerhans and Stieda is apparently a complete one, yet a critical examination of the statements of these two authors proves that their results, on one important point at least, are absolutely contradictory. Stieda, Pl. III. fig. 19, represents a longitudinal and horizontal section through the spinal cord which exhibits the nerves arising alternately on the two sides, and represents each myotome supplied by one nerve. In his explanation of the figure he expressly states that the nerves of one plane only (i.e. only those with dorsal or only those with ventral roots) are represented; so that if all the nerves which issue from the spinal cord had been represented double the number figured must have been present. But since each myotome is supplied by one nerve in the figure, if all the nerves present were represented, each myotome would be supplied by two nerves.

Since Langerhans most emphatically states that only one nerve is present for each myotome, it necessarily follows that he or Stieda has made an important error; and it is not too much to say that this error is more than sufficient to counterbalance the value of Langerhans' evidence as a confirmation of Stieda's statements.

I commenced my investigations by completely isolating the nervous system of Amphioxus by maceration in nitric acid according to the method recommended by Langerhans[64]. On examining specimens so obtained it appeared that, for the greater length of the cord, the nerves arose alternately on the two sides, as was first stated by Owsjannikow, and subsequently by Stieda and Langerhans; but to my surprise not a trace could be seen of a difference of level in the origin of the nerves of the same side.

Footnote 64: Loc. cit.

The more carefully the specimens were examined from all points of view, the more certainly was the conclusion forced upon me, that nerves issuing from the ventral corner of the spinal cord, as described by Stieda, had no existence.

Not satisfied by this examination, I also tested the point by means of sections. I carefully made transverse sections of a successfully hardened Amphioxus, through the whole length of the body. There was no difficulty in seeing the dorsal roots in every third section or so, but not a trace of a ventral root was to be seen. There can, I think, be no doubt, that, had ventral roots been present, they must, in some cases at least, have been visible in my sections.

In dealing with questions of this kind it is no doubt difficult to prove a negative; but, since the two methods of investigation employed by me both lead to the same result, I am able to state with considerable confidence that my observations lend no support to the view that the alternate spinal nerves of Amphioxus have their roots attached to the ventral corner of the spinal cord.

How a mistake on this point arose it is not easy to say. All who have worked with Amphioxus must be aware how difficult it is to conserve the animal in a satisfactory state for making sections. The spinal cord, especially, is apt to be distorted in shape, and one of its ventral corners is frequently produced into a horn-like projection terminating in close contact with the sheath. In such cases the connective tissue fibres of the sheath frequently present the appearance of a nerve-like prolongation of the cord; and for such they might be mistaken if the sections were examined in a superficial manner. It is not, however, easy to believe that, with well conserved specimens, a mistake could be made on this point by so careful and able an investigator as Stieda, especially considering that the histological structure of the spinal nerves is very different from that of the fibrous prolongations of the sheath of the spinal cord.

It only remains for me to suppose that the specimens which Stieda had at his disposal, were so shrunk as to render the origin of the nerves very difficult to determine.

The arrangement of the nerves of Amphioxus, according to my own observations, is as follows.

The anterior end of the central nervous system presents on its left and dorsal side a small pointed projection, into which is prolonged a diverticulum from the dilated anterior ventricle of the brain. This may perhaps be called the olfactory nerve, though clearly of a different character to the other nerves. It was first accurately described by Langerhans[65].

Footnote 65: Loc. cit.

Vertically below the olfactory nerve there arise two nerves, which issue at the same level from the ventral side of the anterior extremity of the central nervous system. These form the first pair of nerves, and are the only pair which arise from the ventral portion of the cerebro-spinal cord. The two nerves, which form the second pair, arise also opposite each other but from the dorsal side of the cord. The first and second pair of nerves have both been accurately drawn and described by Langerhans: they, together with the olfactory nerve, can easily be seen in nervous systems which have been isolated by maceration.

In the case of the third pair of nerves, the nerve on the right-hand side is situated not quite opposite but slightly behind that on the left. The right nerve of the fourth pair is situated still more behind the left, and, in the case of the fifth pair, the nerve to the right is situated so far behind the left nerve that it occupies a position half-way between the left nerves of the fifth and sixth pairs. In all succeeding nerves the same arrangement holds good, so that they exactly alternate on two sides.

Such is the arrangement carefully determined by me from one specimen. It is possible that it may not be absolutely constant, but the following general statement almost certainly holds good.

All the nerves of Amphioxus, except the first pair, have their roots inserted in the dorsal part of the cord. In the case of the first two pairs the nerves of the two sides arise opposite each other; in the next few pairs, the nerves on the right-hand side gradually shift backwards: the remaining nerves spring alternately from the two sides of the cord.

For each myotome there is a single nerve, which enters, as in the case of other fishes, the intermuscular septum. This point may easily be determined by means of longitudinal sections, or less easily from an examination of macerated specimens. I agree with Langerhans in denying the existence of ganglia on the roots of the nerves.

X.

A MONOGRAPH

ON THE

DEVELOPMENT OF ELASMOBRANCH FISHES.

PUBLISHED 1878.

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