The physiology of twinning

Front matter

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Preface

This book deals primarily with the causes and consequences of twinning. In 191 7 was published a volume belonging to this series and written by the present author, entitled The Biology of Twins. That volume was limited to twinning in mammals and was largely morphological in character. The word " twins" was viewed broadly so as to include one-egg (monozygotic) as well as two-egg (dizygotic) twins. We now realize that true twinning is essentially a matter of the division of a single egg at various stages of development and that dizygotic twinning is only a case of plural offspring born simultaneously. The present volume is confined to one- egg twinning and, instead of being limited to mammals, includes all known types of complete or partial one-egg twinning in the animal kingdom. In The Biology of Twins very little attention was paid to theories as to the causes of twins or to the interinfluences of twins upon each other. One definite theory, however, was advanced by the writer to account for the curiously unique process of twinformation in the armadillos. This theory was the first real causal theory of twinning based on facts. Inasmuch as this theory is now supported by a great body of evidence from many sources, the writer, at the risk of appearing too solicitous about matters of priority, feels impelled to lay definite claim to its authorship. This theory is the center and substance of the present book. When used as a working hypothesis it bids fair to explain a long array of peculiar twinning processes in diverse viii PREFACE

groups of animals. A considerable number of interesting situations, such as hemihypertrophy in man, bilaterality in echinoderm larvae, symmetry reversal, double limbs and tails, supernumerary organs, and certain types of tumors, turn out to be phases of twinning. This all seems to mean that twinning is a much more general phenomenon than we have previously supposed. Whether it occurs in worms or in man, it expresses itself in the same series of types; and there is a deep-seated coherency and consistency about its varied expressions. It is because twinning is believed to be a consequence of one of the most fundamental of biological processes that the present volume has been written, in the hope that some of the riddles of life may at least partially be answered.

I wish to express my thanks to my friends, Professor F. R. Lillie and Dr. A. W. Bellamy, for their help in reading the manuscript and for valuable suggestions, and to Mr. Kenji Toda, who has so skilfully drawn or redrawn the illustrations.

H. H. Newman

Contents

PAGE

List of Illustrations xi

CHAPTER

Chapter I The Nature, Scope, And Causes Of Twinning

THE NATURE OF TWINNING

Strictly speaking, twinning is twaining or two-ing — ■ the division of an individual or an organ into two equivalent and more or less completely separate individuals or organs. The term dichotomy is almost a perfect synonym for twinning, for it means literally a process of division into two parts. When, as in the specific twinning of the armadillos and in the sporadic twinning of other species, more than two offspring are produced from one egg, the condition may be still called twinning because the process of dichotomy is simply repeated two or more times. Thus we may have single twinning, double, triple, or even quadruple twinning. The most advanced phase of twinning is that seen in the South American armadillo Dasypus hybridus, which undergoes always as many as three twinning processes and often proceeds well into a fourth, so as to produce up to twelve embryos from a single egg. I do not consider polyembryony as exhibited among the parasitic hymenoptera an instance of true twinning: this, for reasons that will be made clear later. Furthermore, since twinning is essentially a division of one into two, we are not justified in retaining within the category of twins any cases in which two individuals have originated from two germ cells originally separate. In this narrower sense, therefore, twinning proper is always monozygotic or one-egg twinning.

Though thus limited, twinning is still a multifarious process: there are many kinds of true twinning. If the blastomeres of the two-cell stage of cleavage, destined to develop into the right and left primordia of a single individual, become physically or physiologically isolated so as to develop independently of each other, two half-sized, but otherwise normal, individuals result: these are twins. If a young blastoderm becomes bilaterally separated into two more or less equivalent blastoderms and two independent individuals result, these are twins. If a single blastoderm loses its axiate organization so that two equivalent secondary points of gastrulation arise instead of one, and two embryonic axes result, this is twinning. If a single embryonic axis exhibits a tendency for the two bilateral halves to grow independently and a double-headed or double-tailed individual results, this, no matter how complete or incomplete the isolation of the two sides, is twinning. If an appendage such as a hand or a foot becomes double instead of single, this is also a phase of twinning.

Twinning is a matter of great defmiteness and depends on the bilateral organization of the embryo. Rarely does a division into more than two individuals occur, unless one of the two redivides. Triplets are a hundred times as rare as are twins. This very fact emphasizes the essential feature of twinning: its two-ness. Moreover, not merely two individuals, but two equivalent individuals are formed, which are nearly always symmetrically placed with reference to each other. Neither one is the original individual; neither one is secondary or subordinate to the other; but both are equivalent individuals regenerated from a half-embryo.

Twinning is essentially a process of regulation or regeneration of a whole individual out of a prospective half individual. All that is necessary in order to get two individuals instead of one is to accomplish the division of an embryo into two equivalent regions. Each region is totipotent and soon reorganizes its own complete axiate relations and becomes a whole individual. When the separation of halves is incomplete, we get, in the case of whole organisms, double monsters; and in the case of special organs, merely double organs.

THE DISTRIBUTION OF TWINNING THROUGHOUT THE ANIMAL KINGDOM AND ITS SIGNIFICANCE

In his book, Materials for the Study of Variation, Bateson surveys the incidence of double monstrosity in the animal kingdom. We find that it is found most commonly among vertebrates, not infrequently among annelids and arthropods, occasionally in echinoderms of various classes, rarely in coelenterates, cestodes, brachiopods, protozoans. I have never seen a reference to a case of twins or double monstrosity in Mollusca, Nemertinea, Nemathelminthes, Rotifera, Ctenophora, or Tunicata. These latter groups are characterized by determinate cleavage in its most definite form. In such groups the uncleaved egg is already highly organized, each part of the egg having a definite prospective value as an organ-forming region. Hence, if the blastomeres are isolated in two-, four-, or eight-cell stages they are not able to produce whole individuals, but merely parts of individuals. If divided in the two-cell stage, one cell will develop into a left-half and the other cell into a right-half embryo. It is no wonder then that in groups

IT with strictly determinate cleavage we find no examples of twinning, for twinning requires a totipotency of blastomeres or regions of the blastoderm.

Equally significant is the fact that twinning is especially characteristic of the vertebrates, a group in which cleavage is most clearly indeterminate, in the sense that the early cleavage cells appear to be totipotent, i.e., each able, if isolated, to produce a whole individual. In the vertebrates, although the axes of polarity and symmetry are already laid down in the undivided egg and the axes of the embryo tend to coincide with those of the egg, it appears to be relatively easy to break down the axiate relations either in the egg or in various embryonic stages, and then to establish new axes. This all means that, apart from their axiate integration, the blastomeres, until a relatively late period of cleavage, are potentially equivalent, no blastomere containing any unique organ-forming material, but each being essentially germinal in character and able under the proper conditions to become a new apical point and to establish new axes of polarity and symmetry. This extreme versatility of the elements of the vertebrate blastoderm is, in my opinion, the secret of its twinning capacity. There is a good reason then why the vertebrates are par excellence the favored group for twins.

The other group in which twinning occurs readily is that of the echinoderms, in which cleavage is also decidedly indeterminate. In the annelids and arthropods, where partial twinning occurs only occasionally or is confined to a few types, we find that cleavage is strictly intermediate between the extreme determinate and the extreme indeterminate types. Thus again the parallel-

NATURE, SCOPE, AND CAUSES OF TWINNING 5

ism between the mode of cleavage and the incidence of twinning is confirmed.

THE CAUSES OF TWINNING

For a long time I have held the view that twinning is essentially a phenomenon involving a physiological isolation of equivalent parts of the blastoderm and a regulation of the isolated or twinned regions into complete embryos. As to the cause of physiological isolation, I have maintained, for the armadillo at least, that the essential feature is temporary cessation or radical retardation of development at a critical period. It is not especially important for the theory to determine the exact cause or causes of the lowered developmental rate, for we know that a great many agencies give the same end-result so long as they accomplish a retardation. What retardation evidently does to the egg or embryo is more or less completely to disorganize its integrational relations, or, in a word, to deaxiate it. If through retardation the polarity of the egg or embryo becomes so weakened that there are no relatively high or low metabolic regions, all parts of the embryo are left on a parity; so that, when normal developmental conditions once more appear, any part of the embryo may become the apical point and a new gradient will be established. What usually happens, however, is that enough of the original axis of polarity persists to permit just two equivalent points on the axis, at equal distances from the original apical point, to remain in such a condition that they are favored when developmental conditions return, and these become the growing points or apical ends of twin embryos. Very commonly the developmental retardation does not seriously affect the blastoderm until the process of gastrulation begins, a process of extreme delicacy, as has often been pointed out. Once fairly started the cleavage process seems to be easily accomplished even under great difficulties. This was clearly brought out by the writer (Newman, 191 5) in his work with teleost hybrids.

Almost any cross, even those between the most distantly related species, will go through cleavage normally or nearly so, but, because of the relative slowness of the cleavage process, the important crisis of gastrulation is the usual halting-point for such hybrid embryos: their developmental momentum is insufficient for them to accomplish gastrulation. Now gastrulation results in the establishment of the embryonic axes, including the axis of polarity and the axis of symmetry. The result is that if gastrulation is only temporarily halted the process of formation of the axis of symmetry, or the bilateral axis, may be so interfered with that the primordia of the two equivalent halves of the symmetrical embryonic axis may become more or less completely isolated physiologically, and each half may produce a more or less perfect whole embryo in the region where the isolation has occurred.

INTERPRETATION OF DOUBLE MONSTERS (COSMOBIA)

My interpretation, therefore, of double monsters, at least of those of a symmetrical sort which have certain of their central or axial structures united, is that they are products of a partial twinning process involving a separation of equivalent right and left parts of an originally single embryonic axis. This is opposed to the

NATURE, SCOPE, AND CAUSES OF TWINNING 7

prevailing theory that double monsters are products of the fusion of two originally complete and separate embryos. This question is quite crucial for our general interpretation of the nature and causes of twinning. It is a time-honored question and has been discussed pro and con by leaders in the history of zoology for nearly two hundred years. Important names such as those of Caspar Frederick Wolff, Meckel, the two Saint- Hillaires, Knoch, Dareste, Rauber, Panum, and others are associated with the earlier phases of the problem. Within recent times we find such men as Windle, Gemmill, Kaestner, Wilder, Stockard, and others still taking sides on the vexed question of whether double monsters are derived from the division of one or the fusion of two embryonic axes. Gemmill and Stockard, among recent writers, stand for the fusion of separate embryos; Wilder and myself, on the other hand, hold to the view that double monsters are incompletely divided single embryos. All of these views will be discussed in detail because, to my mind, an acceptable theory of the morphology and the physiology of twinning depends on a correct interpretation of the mode of origin of double monsters.

If double monsters are merely the incompletely separated and regenerated bilateral halves of an originally single embryo, we have a rational interpretation of situs inversus viscerum and mirror-image symmetry. From this point of view we can proceed to an understanding of various kinds of partial twinning and of such allied phenomena as symmetry reversal in various asymmetrical forms such as echinoderm larvae. The conception that double monsters are products of a partial fission is an integrating thread running through the whole fabric of our twinning theories.

THE ORDER OF PRESENTATION OF DATA

ON TWINS

In general, the order of chapters is based on the relative simplicity or the fundamental character of the twinning processes. The simpler and more fully understood cases of twinning, such as those in the starfishes and earthworms, come first because it is believed that there the phenomenon of twinning is less obscured by developmental complexities. The most obscure and least understood cases of twinning, as that in man, are reserved for later consideration in order that we may have as much information as possible about the modes, causes, and consequences of twinning before tackling the most difficult of our problems. In dealing with twinning in the vertebrate classes the phylogenetic order is followed because this order seems to lead from the simpler to the more complex phases of the subject. The causes of twinning in the armadillos of the genus Dasypus seem to me to be more definitely known than those of any other vertebrate, but the restatement of this situation is postponed until after the consideration of the twinning conditions in the birds and in the reptiles.

The study of the literature on twins and double monsters in man brought to light two rather unexpected and extraordinary situations that seem to me to throw much light on matters formerly quite obscure to biologists. The first of these situations has to do with the various developmental hazards of human twins, especially one-egg twins, due largely to interinfluences of the two

NATURE, SCOPE, AND CAUSES OF TWINNIXd 9

individuals. The second situation, which is technically called hemihypertrophy, or a unilateral gigantism of one half of the body, strongly suggests partial physiological isolation of the bilateral primordia, or minimal twinning. The consideration of double monsters of all classes has brought to light the existence of the phenomenon known as situs inversus viscerum and other sorts of mirror-image symmetry between the two components. This phenomenon strikes deep at the roots of the physiology of symmetry and asymmetry in the vertebrates; hence a chapter is devoted to an analysis of the observed conditions. After the subject of twinning of whole organisms has been concluded the discussion shifts to twinning in tails and limbs. Duplicity in tails turns out to be merely a phase of bilateral twinning involving a posterior growing region, the tail-bud. Twinning in appendages, however, seems to involve certain complexities that are not present in bilateral twinning. An understanding of the sources of complexity, however, seems to indicate that the same fundamental causes and consequences of twinning hold good for twinning or duplicity in limbs as for twinning of the whole body. There occur, moreover, comparable phenomena of symmetry reversal and mirror-imaging that seem to aid in an analysis of the general laws of symmetry.

In a final chapter twinning as a mode of reproduction is discussed. It is shown that twinning is a form of axiate reproduction, that twinning is not a reminiscence of a lost ancestral asexual phase in an ideal alternation of generations, and that twinning and polyembryony are two entirely different phenomena and should never be confused.

io THE PHYSIOLOGY OF TWINNING

Throughout the book an attempt is made to put the various types of twinning in their proper place in the system of biological phenomena and to show in what ways a knowledge of the causes and consequences of twinning helps to advance our knowledge of several obscure and difficult but none the less fundamental aspects of animal biology.

Introduction

Since The Biology of Twins was written much new light has been thrown on the nature and causes of twinning and perhaps the clearest analysis of the fundamentals of twinning has been obtained through the experiments of the present writer on the eggs and embryos of the Pacific Coast starfish, Patiria miniata. It was felt that an experimental analysis of twinning was almost impossible in the case of mammals and it was therefore decided to make use of some species whose development was well known to be simple, readily controlled, and capable of easy observation. One would naturally select for such work either the bony fishes or the echinoderms: two groups which for a long time have been favorite materials for experimental biology. The echinoderms were chosen in preference to the fishes partly because the processes of cleavage and gastrulation are simpler and less open to controversy. For a long time I have felt that twinning was intimately associated with the process of gastrulation and on that account it seemed wise to study twinning in a form in which this whole process is so nearly diagrammatic.

When I went to the Pacific Grove laboratory in the spring of 1920, one of my chief concerns was to study experimentally the process of twinning in some echinoderm, but I hardly expected to find so favorable a form as Patiria y a form that produces abundant twins almost without any effort on the part of the investigator. Under ordinary laboratory conditions, and especially under conditions of crowding, one gets a high percentage of twins of many sorts. It then becomes the task of the investigator to discover the factors responsible for this high incidence of twins. Such an analysis has been made and is here to be presented.

THE CONDITIONS UNDER WHICH TWINS ARE PRODUCED

A. Partheno genetic twins. — It was a matter of some surprise to note in certain control cultures of Patiria eggs, that had never been fertilized, considerable numbers of actively swimming larvae. Many of these larvae were of dwarf size, others were almost formless, wrinkled ciliated masses; but there were always some distinctly twinned larvae, in the sense that they had two or more archentera or primitive alimentary tracts. Such twins lived a long time but were always quite subnormal with respect to the development of typical larval characters. The percentage of parthenogenetically developing eggs in Patiria ranges from o in some cultures to as high as 75 in one culture. The usual percentage ranges from i to 10 and is more commonly about 2 or 3. Of the eggs developing parthenogenetically as many as a third, or possibly a half in some cases, show some phase or other of twinning.

What is there about parthenogenesis that favors twinning? This was, of course, the most obvious question that presented itself, and it was not to remain long unanswered. One needed only to watch carefully a batch of unfertilized eggs simultaneously with another batch of fertilized eggs to note certain obvious differences: (a) Both sets of eggs undergo maturation at the same time, (b) Numerous eggs in the fertilized lot produce well-elevated fertilization membranes; while in parthenogenetic cultures this happens in only a few eggs, (c) In two hours after insemination the fertilized eggs begin cleavage, which goes forward at a rapid rate; while in the unfertilized lot those eggs that had formed a membrane fail to cleave and disintegrate after a few hours. (d) Some of the maturated eggs in the unfertilized lot, without having formed any distinctly lifted-up fertilization membranes, begin cleavage after about six hours, nearly four hours later than in the fertilized eggs, and cleavage goes on relatively much more slowly than in the latter.

It is obvious from these observations that the development of parthenogenetic eggs has been greatly slowed down. The stimulus afforded by the entrance of the spermatozoon has been lacking, as is evidenced by the absence of cortical changes and the failure of a fertilization membrane to be elevated from the vitelline membrane. Moreover, all of the developmental changes go on at an abnormally slow rate.

B. Hybrid twins. — Twins of another sort, rather better developed and more viable than parthenogenetic twins, were frequently found in cross-bred cultures in which the eggs of Patiria had been fertilized by the sperm of various other species of starfishes and sea urchins. As compared with pure-bred Patiria Larvae, hybrid larvae always showed a very distinctly slower developmental rate at all stages. It seems obvious, however, that though foreign sperm less completely stimulates the egg than own sperm, yet some stimulus is given, for hybrid embryos develop more rapidly and more normally than do parthenogenetic embryos.

C. Twins due to crowding.— Varying percentages of twins of an even more advanced type were frequently found in control batches of eggs that had been fertilized by own sperm, but had been allowed to remain in much too large numbers in relatively small containers. Some of the most advanced twin larvae were discovered under these conditions, and such twin larvae, when transferred to new quarters, where they had plenty of room and oxygen, developed farther than any other twin larvae observed. In some cases they practically recovered the single condition through the absorption of the smaller twin by the larger. It seems obvious that these larvae in the crowded cultures had suffered a period of growth depression due to the low oxygen and high C02 content of the sea water. It was my intention to perform some experiments involving lack of oxygen, but the work was interrupted before there was opportunity to do so.

Retarded development, the primary cause of twinning. — In all three of these methods of twinning the common factor is undoubtedly a retardation, more or less severe, of the normal rate of development. This was not at all an unlooked-for result; quite the contrary. One who, like the writer, had for years realized the fact that most, if not all developmental anomalies, are, in last analysis, the result of interferences with the normal developmental rate of organisms, and who had offered a theory of armadillo twinning based on the idea that twinning was the direct result of interrupted development, would naturally be on the lookout for just this type of result.

THREE TYPES OF ONE-EGG TWINS

From the point of view of their mode of origin three distinct types of twins were readily distinguishable: (1) dwarf larvae resulting from the physiological isolation, followed by physical isolation, of the cleavage products of separate blastomeres; (2) double, triple, or multiple individuals resulting from the formation of two or more archentera; (3) " two-headed" larvae resulting from a dichotomy of the anterior end of the archenteron.

In addition to these three primary types certain secondary types appeared as the result of fusion of adjacent archentera to form individuals with a A -shaped archenteron, single in front and divided behind (Fig. 5) . Each of these types will be discussed separately.

1. Dwarf larvae. — -These larvae always appeared in parthenogenetic cultures. When the very much belated process of cleavage begins, it proceeds with so little energy that many eggs start the first cleavage and lose their developmental momentum before the two daughter-cells are fully separated. In others the first cleavage completes itself, but the second cleavage takes place in only one of the blastomeres. The belated blastomere either remains permanently undivided or else resumes division at a slower rate than the first. There is obvious physiological discoordination between the two sets ol cells thus produced. Each produces a blastula, oiu- less normal than the other, and before long they rupture the membrane and become swimming, half-sized larvae.

Neither one is fully normal, but both at least begin gastrulation and some of them form fairly normal gastrulae. Never, however, do these dwarfs attain a condition that approximates that of a bipennaria larva. They all stop at the gastrula stage. We may denominate this mode of origin of twins physiological blastotomy; for it is the result of the physiological isolation of the first two blastomeres, so that they act as though they were independent eggs. Such a condition is equivalent to the occurrence of two separate blastoderms on a fish or a chick egg.

2. Twins with two or more archentera.- — -Much the commonest mode of twinning in the starfish is one in which two or more points of invagination occur, resulting in two or more archentera. Many varieties of this type of twin occur. A very common type is that in which the original archenteron seems to persist and a secondary one (often two or three) arise at the opposite or apical end of the larva. Such a larva may subsequently rid itself of these accessory archentera by closing their blastopores and pinching off the small archentera so as to leave one or more small internal vesicles or cysts that remain attached to the body wall for some time, only to be ultimately resorbed. Some biaxiate larvae, however, such as that shown in Figures i and 2, have the secondary archenteron nearly as large as the primary and the animal exists as a biaxiate bipennaria for a long time. In swimming, such a larva moves in a direction determined by the position of the primary blastopore, i.e., with this structure posteriorly directed. The anterior component of this twin seems to be under the dominance of the posterior or primary component.

Another common type of twin larva is one in which there seems to have been a partial obliteration of the primary symmetry relations and two secondary equivalent symmetries result. In such larvae both archentera develop symmetrically and are quite equivalent, so that it wrould not be possible to call one the

Figs. 1-3. — Outline drawings of three types of twin larvae of the starfish Patiria miniata, showing plural archentera. Fig. 1 . an advanced bipennaria with primary archenteron at the original basal pole and a welldeveloped secondary archenteron at the site of the original apical poK . Fig. 2, a similar bipennaria with two supernumerary archentera, b secondary and a tertiary. Fig. 3, a true twin larva with two symmetrically placed archentera, neither of which is the primary one. (From Newman.)

i8 primary and the other the secondary component. Such twin larvae develop symmetrically throughout their lives and reach fairly advanced stages. Typical examples of the relations of the two components are shown in

Figs. 4-6. — Three very common types of symmetrical twin larvae of Patiria miniata. Scores of such twins were found in almost every batch of larvae which were in any way retarded in development. (Original.)

Figures 3, 4, 5, 6, 7. When in larvae of this sort one of the archentera is originally smaller or secondarily comes to be less active in its growth than the other, it quite commonly happens that the smaller one comes into contact with the larger, fuses with it at its anterior end, and, after closing its blastopore, becomes first a pouch of the larger archenteron and then merely a thickening in the wall of the latter. This is a very striking example of the way in which a stronger twin may dominate over and ultimately rid itself of a weaker twin so as to come to be practically a normal individual. Occasionally also two quite equivalent paired archentera grow toward one another at such an angle that they meet

Figs. 7, 8. — Two advanced larvae of Patiria. Fig. 7, a symmetrical twin with the two separate alimentary tracts considerably differentiated and exhibiting striking mirror-image symmetry. Fig. 8, a twin of the same age with the anterior ends of the two alimentary tracts fused to form a common pharynx. (From Newman.)

in the middle of the cleavage cavity and fuse at their anterior ends. Such larvae come to have one anterior and two posterior archentera and two blastopores. In several cases such larvae as this (Fig. 8) went so far as to differentiate two stomach enlargements, two oesophagi and two intestines, communicating with a common pharynx.

3. Twins with bifurcated archcntcron. — A relatively rare type of retarded larva undergoes dichotomy of the anterior end of the archenteron, which results in a sort of "double-headed" condition. This phenomonen is discussed in a subsequent section under the caption An adidymi among starfishes (pp. 23, 24).

The mode of origin of these twins resulting from plural gastrulation processes is of considerable interest. One can easily trace the twinning back to the blastula

Figs. 9-14.— Types of blastulae with appropriate gastrulae of Patiria. Fig. 9, a typical blastula, with the typical gastrula (Fig. 10) that arises from it. Fig. 11, a blastula that has entirely lost its polarity, with the very abnormal multiple gastrula (Fig. 12) that arises from it. Fig. 13^ a bipolar blastula, the original polarity of which has been only partially lost, with the bipolar gastrula (Fig. 14) that arises from it. (From Newman.)