How Does Germline Regenerate?

Front matter

How Does Germline Regenerate?

CONVENING SCIENCE

DISCOVERY AT THE MARINE BIOLOGICAL LABORATORY

Contents

INTRODUCTION | 1

1 Uncovering Assumptions That Have Shaped Germ Cell Science | 9 2 Backgrounding Conflicts within Germ Cell Science | 42 3 Challenging Assumptions in Germline Science | 61 4 Implications of Reenvisioning Germline

Regeneration | 108

Conclusion

Throughout this chapter, we have seen how scientists thought about the origins and nature of germ cells, beginning with Leeuwenhoek’s discovery of spermatozoa in 1677 and going on through the establishment of the Weismann Barrier as a cornerstone of the geneticists’ view of heredity in the twentieth century. We have also seen how the Weismann Barrier was not established from biological facts per se—it was established by misinterpreting the theoretical work of its namesake, August Weismann. But this history is only part of the story of how the Weismann Barrier became entrenched within many scientists’ thinking and remained a pervasive assumption within considerations of germline. So far, I have traced how the Weismann Barrier was absorbed by the fledgling fields of transmission genetics and propagated in the context of thinking about heredity. The other part of the story involves understanding how the Weismann Barrier—a concept that indicates a relationship between cells—became established within embryology and how the other two assumptions that underlie thinking about germline regeneration that I laid out in the introduction arose. For this part of the story, we need to move to the next chapter.

We have now seen how transmission geneticists absorbed the Weismann Barrier as a main tenet of their thinking during the early twentieth century: Germ cells can become somatic cells, but somatic cells can never become germ cells. Embracing this assumption within transmission genetics was not based on careful investigations of the origins and nature of germ cells; rather it was a convenient way of doing exactly the opposite — setting the details of cells and development to the side. In effect, these early geneticists treated what should have been an empirical claim about the relationship between germ cells and somatic cells as a normative claim. An empirical claim can be determined to be true or false via evidence; whereas a normative claim defines the way in which something ought to operate.

By treating the Weismann Barrier as a normative claim, early transmission geneticists could simply point to the germline and say that germ cells held the keys to heredity because they operated in the way that the Weismann Barrier dictated. But the problems of the origins of germ cells and germinal continuity didn’t go away simply because transmission geneticists refused to take them into account. Instead, they became the purview of embryologists, who were not content simply to accept the relationship between germ cells and somatic cells that the Weismann Barrier dictated.

As we'll see, for some embryologists, the entire premise of the Weismann Barrier was absurd— there was no fundamental difference between germ cells and somatic cells; it was a matter of differentiation as with any other kinds of cells. For others, the distinction was the cornerstone of their understanding of development and organismal biology.’

In the end, the diversity of perspectives coalesced around a more unified embrace of the Weismann Barrier during the middle of the twentieth century. This shift was not a simple one. Disagreements about the nature and origins of germ cells, like most problems within the sciences, did not disappear overnight. Rather, as the century wore on, the assumptions that drove earlier investigators into conflict gradually faded into the background. These assumptions have lingered in the background of scientific research and thinking about germ cells, germline, and germline regeneration and still shape our understanding of these things.

THERE IS NOTHING SPECIAL ABOUT GERM CELLS

In 1931, American embryologist Florence Heys provided an extensive review of the search for the origins of germ cells and their fates in which she laid out exactly how disparate views were at this time.” According to her analysis, scientific thinking about germ cells in the first few decades of the twentieth century existed along a spectrum. At one end were those who didn’t think that there was anything special about germ cells — they were simply another type of cell—therefore, there was no Weismann Barrier, and no continuity of germ cells within organisms or between generations. At the other end were those who saw germ cells as unique, separate, and distinct from somatic cells, as a continuous lineage, and the relationship between germ cells and somatic cells as upholding the Weismann Barrier. Others held some combination of these views.

American embryologist and zoologist, George T. Hargitt, was one of the researchers who thought there was nothing special about germ cells. Throughout his research on germ cells, Hargitt investigated a wide array of species and was one of just a few scientists who studied germ cells in both vertebrates and invertebrates. From his earliest research concerning germ cells, Hargitt became convinced that methodological assumptions about identifying germ cells had tainted the conclusions that many of his contemporaries had reached.

Between 1913 and 1919, Hargitt produced a series of research articles that tracked the origins of germ cells within a series of hydrozoan species— small, marine invertebrates related to jellyfish and coral—not coincidentally the same group of animals that led Weismann to his germ plasm theory.° Hargitt wanted to know where and when the germ cells originated within these different hydrozoans, and so he made careful studies of their embryos and larvae. From these years of staring at hydrozoan embryos, larvae, and adults, Hargitt determined that the germ cells differentiate at the time just before sexual maturity, and that they appear to arise from functional somatic cells. He concluded about hydrozoans that “there is no definite migration of germ cells and no germ-track; there is no invisible germ plasm in the body cells.’* Hargitt’s investigations of hydrozoans led him to believe that there was nothing unique about germ cells—they were simply differentiated somatic cells —and that there was no continuity of a germ cell lineage or an invisible and unobservable germ plasm. Following the publications of his results, several researchers intimated that Hargitt’s conclusions about germ cells and germ plasm might well apply to hydrozoans but that such evidence could not be used to dismiss this idea within vertebrates." In turn, Hargitt extended his studies to vertebrates. In 1924, he published his findings on the origins of germ cells in the salamander, Diemyctylus viridescens, also known as the crimsonspotted newt.° Due to the differences between the life cycles of hydrozoans and vertebrates, when examining Diemyctylus, Hargitt divided the problem of the origin of germ cells in vertebrates into two phases. The first phase was concerned with the earliest appearance and source of primordial germ cells during development and their subsequent fate. The second phase dealt with the relationship in adults between these primordial germ cells and subsequent germ cells. Hargitt focused his attention on the second phase in Diemyctylus. He collected and observed tissues of the testes and noticed that germ cells were always found in the supportive tissue surrounding the structures of the testes, called stroma (a somatic structure). The consistent location of these germ cells implied to Hargitt that they were differentiated from the surrounding somatic tissue. Because he could not find evidence that germ cells that had segregated early during development led to these germ cells that he witnessed in the gonads, he believed that germ cells arose from the somatic cells of the gonads. These observations led Hargitt to conclude that his findings from hydrozoans were true for vertebrates (at least Diemyctylus).

Hargitt continued his studies of germ cell origins in vertebrates by investigating the earliest appearance and source of primordial germ cells during development and their subsequent fate in rats.” Hargitt studied embryos in stages from just before germ layer formation (the germ layers are three tissues that give rise to all subsequent parts of the organism) to the beginning of gonad formation. He carefully cultivated embryos, fixed them in solution to preserve them, cut them into sections, and then applied various stains to the sections.® He began by observing the later-stage embryos in which the gonads had begun to form and germ cells could be definitively identified, and worked his way backward, reasoning that this approach would allow him to trace any germ cells that contributed to the population in and around the presumptive gonad. From his observations, Hargitt concluded that the germ cells do not appear early during development, and that there is no germ track in the rat. Thus, through his studies of rats, Hargitt again confirmed the conclusions that he had reached about the origins and nature of germ cells from his early work on hydrozoans: that there was nothing unique about germ cells — they were simply differentiated somatic cells —and that there was no continuity of a germ cell lineage or an invisible and unobservable germ plasm.

There were two underlying and intertwined methodological assumptions that Hargitt observed in previous scientists’ work on the origins of germ cells. The first was that scientists could accurately identify primordial germ cells within the developing embryo. Primordial germ cells are the first type of germ cell in the body, which are supposed to differentiate well before the gonads form. In other words, scientists thought they knew what primordial germ cells looked like. But Hargitt tells his reader that “the fact is there is no single character which will distinguish germ cells from other cells, nor is there any combination of characters which may be used indiscriminately for such a test”? He meant that none of the methods or criteria that scientists had developed to identify primordial germ cells was specific to these cells, and thus they were not reliably accurate. Let’s take a closer look at these identification methods.

From the late nineteenth century through well into the twentieth century, scientists relied on two means of identifying primordial germ cells: (1) morphological criteria, and (2) selective staining criteria. There were a lot of morphological criteria that were supposed to distinguish germ cells from somatic cells: for example, the presence of granules in the cytoplasm, the appearance of the mitochondria (a subcellular structure), the large size of the cell, the round shape of the cell, and finally, well-defined cellular membranes.’° Hargitt believed that each of these morphological criteria was problematic. For instance, the presence of a specific type of mitochondria in germ cells was definitively disproved just a few years after it was identified in 1910.'' Meanwhile, granules were not found in the cytoplasm of germ cells in all species. And, while germ cells like ova are certainly large, round, and have well-defined cell membranes, these characteristics apply to other cells as well. Most cells tend to round out when approaching mitosis (cell division), and they acquire a more well-defined cell membrane. As for the size of the cell, Hargitt tells his reader that “it seems pretty clear that practically every cell of large size in any part of the embryo has been called a germ cell???

Selective staining criteria refers to applying chemicals to cells and or embryos. These chemicals are supposed to color or highlight specific subcellular parts, which allow scientists to differentiate between cell types. Researchers developed quite a few selective staining criteria for germ cells during the late nineteenth and early twentieth centuries. For example, in 1912, Russian physician and biologist W. Rubaschkin described staining his mouse embryos with a mixture of chemicals called azure II (which gives a blue tint) and eosin (which gives a red or pinkish tint).'* According to Rubaschkin, primordial germ cells have a different reaction to this combination of cell stains than all other cells, so primordial germ cells can be identified by their red-tinted nuclei and pale blue cytoplasms. In 1923, American embryologist Cleveland Simkins described how inaccurate Rubaschkin’s method was, highlighting how cell cycle phase heavily affects the way in which cells take up stains: many different types of cells that are preparing for mitosis will absorb eosin and present with a red nucleus." In 1925, Hargitt had this to say about selective staining criteria: “The staining reaction of germ cells is sometimes stated to be different from other cells, but this is a very uncertain test, for the germ cells even in the same section do not by any means stain constantly. ... All staining reactions depend upon the physical and chemical conditions, and these are correlated with the metabolic state of the cells; we have no right to expect any positive or constant difference in staining reactions of germ cells and other cells.”

Thus, Hargitt found many of the selective staining criteria that earlier scientists had developed to be dubious and inaccurate, and he maintained that they could not be used to definitively identify primordial germ cells. In Hargitt’s estimation, then, the morphological and selective staining criteria that scientists used to identify primordial germ cells were so nonspecific that it led them to assume the presence of germ cells during the pre-gonadal stages of development. In other words, identifications of germ cells before the gonads formed were based on faulty assumptions.

The second methodological assumption that Hargitt saw is interconnected with the first: many of his predecessors and contemporaries believed that they could identify primordial germ cells without extensive investigations of development. If, Hargitt reasoned, someone wanted to make a claim about where and when germ cells originate before the formation of the gonads, they would need to trace the lineages of the germ cells that could be definitively identified within the gonads, because there was no reliable means of identifying germ cells before this stage. In other words, not many studies of germ cell origins were exhaustive cell lineage studies, and this was especially true for vertebrates. You can see how these two assumptions build on each other—without a means of accurately identifying primordial germ cells before the formation of the gonads, scientists could not just point to cells in early-stage embryos and call them germ cells. They would need to trace the cell lineages from the germ cells within the gonads backward through development to identify primordial germ cells definitively, and this was not a common practice.

Hargitt found these two methodological assumptions to be particularly problematic because they led many of his contemporaries to claim that germ cells and germ plasm were continuous—a proposition that Hargitt found no evidence to support.

THE WEISMANN BARRIER REIGNS

At the opposite end of Heys’s spectrum of thinking about germ cells in the early twentieth century is American invertebrate zoologist Robert Hegner, whose driving question was: What makes a germ cell a germ cell?

Hegner’s first attempt at addressing this question came in 1908, when he spent several months experimenting on leaf beetles.* He identified a dark, granular substance in the posterior end of freshly fertilized embryos that he dubbed the “pole-disc.” Hegner watched as these embryos developed and tracked how the cells divided and migrated around the embryo. He noticed that some cells migrated through the pole-disc, giving them a characteristic dark color and the appearance of granules within their cytoplasm (recall my prior discussion of morphological identification criteria), and they came to reside as a clump at the posterior end of the embryo. He called these “pole cells.’ Following the development of the pole cells further, Hegner determined that they became the primordial germ cells (the first type of germ cell in the body). He noticed that during cell migration within these early embryos, other cells came into contact with the pole-disc, but did not become pole cells —they didn’t take on the dark appearance or have granules within their cytoplasm. Hegner reasoned that the pole-disc granules bore some responsibility for the formation of the primordial germ cells, and introduced the term “germ cell determinants” to describe these granules.

Over a series of experiments, Hegner tested his reasoning. He tried tying off the posterior end of freshly laid eggs with silk thread to remove the pole-disc; it didn’t work—the eggs burst. He then came up with a way to puncture freshly laid eggs on the posterior end such that they would extrude the pole-disc material (and often a lot else). The results of these early experiments were not definitive, but they gave a good indication that when the pole-disc was removed, the primordial germ cells would not form. Over the next few years, Hegner continued to refine his experiments on removing the pole-disc from freshly laid insect eggs.!” He gradually got more definitive results until 1911, when he declared that, “the pole-disc granules are necessary for the formation of germ cells, and that they are really ‘germ cell determinants.”"®

The contribution of cytoplasmic granules to primordial germ cells was well documented before Hegner’s experiments, if not underappreciated. In 1863, Weismann noticed the contribution of cytoplasmic granules to what he called “Polzellen” (German for “pole cells”) during his observations of two species of insect, but did not yet recognize that the pole cells became the primordial germ cells.'? In 1865, the Russian- French zoologist (and later a groundbreaking immunologist), Elie Metschnikoff, as well as the German botanist and illustrator Rudolf Leuckart, independently verified that the pole cells became primordial germ cells.” In the intervening years between Metschnikoff, Leuckart, and Hegner, a whole host of other scientists recognized the presence of granules within pole cells and also noted that primordial germ cells tended to contain a lot of dense, yolk-like globules in their cytoplasm. What Hegner proposed in 1908, and later confirmed, was that the cytoplasmic granules within the pole cells, what he called “germ cell determinants,’ was “the material which fixes the character of the cells”?! Hegner thus believed that germ cell determinants were the substance that made a germ cell a germ cell. In 1914, Hegner switched from calling the cytoplasmic material “germ cell determinant” to “keimbahn-determinant.’”” Recall from the previous chapter that keimbahn was the German term Weismann coined, which became “germ-track.” In renaming the cytoplasmic materials that Hegner thought gave germ cells their identities, he was explicitly referencing Weismann and his germ plasm theory. But, he came to some very un-Weismann-like conclusions about the nature of germ cells. Weismann had thought of the germ-track as the lineage of cells that leads from the single cell of the fertilized egg to the emergence of the first germ cell and believed that it was composed of somatic cells that carried a full complement of unaltered germ plasm. For Weismann, germ cells were a specialized type of somatic cells and were especially important for their unique roles in heredity; they were not special because of a fundamental difference in their structure or their substance. After all, unaltered germ plasm was also present in cells in areas of the body that were susceptible to regeneration. Hegner’s thinking about the keimbahn aligned with Weismann’s only insofar as he, too, envisioned a lineage of cells. However, for Hegner, the cells in this lineage were all germ cells, because they contained keimbahn-determinants —a cytoplasmic material that gave germ cells their identities.

In his 1914 book called The Germ-Cell Cycle in Animals, which was a massive review of research on germ cells, almost entirely based on studies of invertebrates, Hegner evaluated the two theories of continuity that had been ascribed to germ cells: germinal continuity (germ plasm continuity) and morphological continuity (cellular continuity). Regarding germinal continuity, Hegner agreed with Weismann—there was a continuity of germ plasm. But, relying on his own studies and evidence from other invertebrate studies, Hegner sought to expand on Weismann’s understanding of the germ plasm. In considering germ plasm continuity, Hegner wrote, “If, then, we accept germinal continuity as a fact and consider the germ-plasm to be a substance that is not contaminated by the body in which it lies, but remains inviolate generation after generation, we should next inquire as to the nature of this substance”? Although Hegner mused that the relationship between his keimbahn-determinants and the germ plasm was “not yet definitely known,”* given his experimental evidence, he concluded that the keimbahn-determinants located within the cytoplasm were made of this substance. After all,

Hegner reasoned, if Weismann’s germ plasm theory simply required a substance to be reserved for germ cell formation, the keimbahn-determinants certainly fit this description. I should mention that while Hegner did have reasonable evidence to believe that his keimbahn-determinants in some way were connected with the identity of primordial germ cells, he also had evidence that they (1) were not the same in all species in which they were found and (2) were not found throughout all species examined. He didn’t consider either of these points problematic for his conclusions.

When reviewing morphological continuity, Hegner began by telling his reader that “No case of a complete morphological continuity of germ cells has ever been described”? Despite this admission, Hegner assumed that it existed and that germ cells were continuous—he contended that the evidence just hadn't caught up to fully justifying this view. We can see how Hegner could come to such a conclusion, given his assumptions of germ plasm continuity and the keimbahn-determinants as in some way connected with Weismann’s germ plasm. For him, primordial germ cells were defined by the presence of keimbahn-determinants, which were somehow connected to germ plasm, and germ plasm was continuous. Therefore, germ cells must be continuous.

Hegner thus connected his keimbahn-determinants with Weismann’s germ plasm, creating a conceptual shift in the material from the nucleus to the cytoplasm. In doing so, Hegner inadvertently conflated Weismann’s view of germ plasm continuity with a view of germ cell continuity. He also shifted the role and function of the germ plasm from the material that could account for heredity and all cellular differentiation to a material that defined the identity of germ cells. These views proved influential with subsequent scientists.

In 1934, French biologist Louis Bounoure published the results of his studies of the origins of germ cells in the frog, Rana pipiens. While observing developing frog embryos, Bounoure noticed the presence of cytoplasmic granules that segregated into the primordial germ cells. Referencing Hegner’s earlier work, Bounoure called these granules “cytoplasme germinale; which translates to “cytoplasmic germ plasm,’ or simply “germ plasm,””° Five years later, Bounoure published a massive review of germ cell research in a book titled, Lorigine des cellules reproductrices et le problème de la lignée germinale.” In this text, Bounoure suggests that the germ plasm that he had described in the cytoplasm of frog eggs years earlier was responsible for determining the germ cell lineage (Hegner’s keimbahn).

Bounoure’s book had a large impact on embryologists and, later, developmental biologists interested in germ cells. It was the first extensive review of germ cell research since Hegner’s 1914 text. Other reviews appeared within this interval, such as Heys’s 1931 article, but they were far less extensive than Bounoure’s book and tended to shy away from theoretical claims. Bounoure had no such compunction, and he declared both that the continuity of the germ plasm was a general law of organismal reproduction and that the cellular continuity and early differentiation of the germ cells could not be doubted.”

Bounoure saw the germ plasm—the cytoplasmic substance that Hegner had connected with germ cell identity—as the material that defined the germ cells and made them continuous within and between generations.

CONFLICTING ASSUMPTIONS

Looking at the research programs of George Hargitt and Robert Hegner and the conclusions they reached gives us good insight into where the conflicts about the origins and nature of germ cells and germinal continuity lay in the first few decades of the 20th century, and what assumptions underpinned these conflicts. Let’s briefly look at these assumptions.

First, as Hargitt and others pointed out, there were methodological assumptions around identifying germ cells. Hargitt laid out in detail the issues that surrounded the morphological and selective staining criteria that scientists used to identify primordial germ cells. He showed how problems with identifying these earliest germ cells led to problems with reaching conclusions about the early origin or continuity of germ cells. This in turn made scientists who used these methods without considered diligence vulnerable to introducing a lot of error into their understanding of the origin of germ cells. Hargitt was not the only scientist at the time who was concerned with the methods of identifying germ cells and the conclusions to which these methods led. However, these concerns were not pervasive in the literature.

Second, as we saw in the case of Hegner and Bounoure, a lot of assumptions surrounded the problem of continuity (of germ plasm and of germ cells) and the nature of the germ plasm.

Hegner’s movement of the germ plasm from the nucleus to the cytoplasm allowed him to call the germ plasm continuous, and we saw how his reasoning about the role of the germ plasm led him to conclude that germ cells were also continuous. Hegner did not have evidence to support a continuous germ cell lineage, and as I pointed out, there were some issues with conceiving of a continuous germ plasm. As Hargitt noted in 1944, Hegner’s proposal to shift the germ plasm to the cytoplasm and change its role from heredity to germ cell fate was a major leap in understanding the germ plasm. Hargitt wrote, “What a strange reversal: from the view that germ cells differ from tissue cells because of diverse nuclear composition to the view that all nuclei contain complete hereditary material of the species, and the differences between cells are due to the kind of cytoplasm in which the nuclei are located!””?

More than two decades later, Bounoure approached continuity with an even more dogmatic style. While Hegner couched his conclusions in qualifying language, like noting that the relationship between Weismann’s nuclear germ plasm and his own cytoplasmic keimbahn-determinants were “not yet definitely known,” Bounoure made no such concessions.*° As the prominent British evolutionary biologist Julian Huxley, who along with George Gaylord Simpson and Ernst Mayr founded the Modern Synthesis, put it in his review of Bounoure’s book, “For him [Bounoure], germinal continuity is not a problem to be investigated, but a doctrine, or rather a dogma, to be proclaimed and supported”?! In other words, Bounoure assumed that there was germ cell and germ plasm continuity, even when he did not have direct evidence to support such an assumption.

We could ask why Hegner and Bounoure were so drawn to the idea of continuity that they would make strong claims about it without definitive evidence, or why many investigators ignored Hargitt’s call to question their methods of identifying Primordial Germ Cells. Looking at the state of germ cell research in 1948, embryologists John Normal Berrill and Chien-Kang Liu give us a good answer to these questions: “The weight of authority . . . of the Weismann-Nussbaum combination [germ cell continuity] convinced many later workers of the existence of facts they could not observe, and much subsequent argument has arisen over the identity of so-called primordial germ cells and the existence of a germ-track in developmental stages younger than those in which germ cells can be safely recognized.”*” Some scientists, then, may have been so tied to the idea of continuity that they were willing to overlook both the lack of evidence to support it and the potential problems with the evidence that they did have. This way of thinking was not tied solely to Hegner and Bounoure; it was widespread within studies of germ cells at the time.

THE FALSE VITALITY OF THE WALKING DEAD?

COALESCING AROUND GERM PLASM AND CONTINUITY

In 1948, Berrill and Liu evaluated the evidence and thinking behind research on germ cell origins and claims of germ cell and germ plasm continuity. They noted how investigators like Hargitt and Cleveland Simkins made strong and condemnatory attacks against the notion of the continuity of germ cells and germ plasm, but despite these attacks, the idea always regained strength. This led Berrill and Liu to ponder whether “this vitality [of the continuity of germ cells and germ plasm] may of course flow from truth itself incorporated in the germplasm concept, or it may be a false vitality akin to the walkingdead habits of Dracula.”*? It’s worth taking Berrill and Liu’s point seriously.

Less than thirty years after Berrill and Liu’s paper was published, developmental studies of the origins and nature of germ cells had coalesced around the conceptual work of Hegner that Bounoure had dogmatized. In 1976, American biologist Edward Mitchell Eddy published a massive review article, titled “Germ plasm and the differentiation of the germ cell line,” which has proven influential, as evidenced by its citation record.** In this paper, Eddy laid out the state of thinking about germ cells. By this point, germ plasm was considered to be “a substance present in the cytoplasm of gametes, which is segregated into specific cells during blastulation and determines that those cells shall become the progenitors of the germ cell line during subsequent development.’** Eddy made reference to Weismann’s concept of germ plasm continuity, but indicated that this concept had shifted to something else entirely: “Although when Weismann wrote of the continuity of germ plasm he was referring to genetic material of the nucleoplasm, this account forms the basis of the present germ plasm hypothesis.”*° The germ plasm hypothesis, indicating that the germ cell line is determined by an agent present in mature germs cells, suggests that the agent contains stored information capable of influencing the formation of primordial germ cells.”>” By 1976, then, Hegner’s vision of the germ plasm and the conflation of germ-plasm and germ cell continuity that it had initiated, had become standard within germ cell research. The germ plasm was now understood as a cytoplasmic material that determined the germ cell lineage. Germ plasm and the germ cells were continuous. Eddy’s portrayal of thinking about germ cells reflected a much broader acceptance of the germ cells and germ plasm along these lines. Such thinking about the origins and nature of germ cells has continued in this vein into quite a bit of current germ cell research.