Mouse primordial germ cells: a reappraisal.

CHAPTER ONE

Mouse Primordial Germ Cells: A Reappraisal Maria M. Mikedis, Karen M. Downs1 Department of Cell and Regenerative Biology, University of Wisconsin–Madison School of Medicine and Public Health, Madison, Wisconsin, USA 1 Corresponding author: e-mail address: [email protected]

Contents 1. Introduction 2. Current Model of the Origin of Primordial Germ Cells (PGCs) and Segregation from Soma 3. Flaws in Current Model of PGC Origin and Segregation 3.1 Lineage continuity and segregation of PGCs from soma: Criteria and evidence 3.2 PGC trajectory and “markers” of PGCs 3.3 Errant PGCs 4. Comparison of Mammalian Germline Program to That Across Metazoa 5. Embryonic–Extraembryonic Interface and Fetal–Umbilical Connection: PGCs and ACD 6. Loss/Mislocalization of PGCs and Associated Posterior Defects 6.1 Mir-290–295 6.2 Prdm14 7. Perspectives 7.1 Alternative models 7.2 Where we are now 8. Conclusions Acknowledgments References

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Abstract Current dogma is that mouse primordial germ cells (PGCs) segregate within the allantois, or source of the umbilical cord, and translocate to the gonads, differentiating there into sperm and eggs. In light of emerging data on the posterior embryonic– extraembryonic interface, and the poorly studied but vital fetal–umbilical connection, we have reviewed the past century of experiments on mammalian PGCs and their relation to the allantois. We demonstrate that, despite best efforts and valuable data on the pluripotent state, what is and is not a PGC in vivo is obscure. Furthermore, sufficient experimental evidence has yet to be provided either for an extragonadal origin of mammalian PGCs or for their segregation within the posterior region. Rather, most evidence points to an alternative hypothesis that PGCs in the mouse allantois are part of a stem/

International Review of Cell and Molecular Biology, Volume 309 ISSN 1937-6448 http://dx.doi.org/10.1016/B978-0-12-800255-1.00001-6

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Maria M. Mikedis and Karen M. Downs

progenitor cell pool that exhibits all known PGC “markers” and that builds/reinforces the fetal–umbilical interface, common to amniotes. We conclude by suggesting experiments to distinguish the mammalian germ line from the soma.

ABBREVIATIONS ACD Allantoic Core Domain AP alkaline phosphatase IPS intraembryonic posterior primitive streak EG cell embryonic germ cell ES cell embryonic stem cell PGC primordial germ cell PGCLC primordial germ cell-like cell PVE posterior visceral endoderm T Brachyury VER ventral ectodermal ridge XPS extraembryonic posterior primitive streak

1. INTRODUCTION The current model of the origin and segregation of the mammalian germ line is based upon alkaline phosphatase (AP) activity (Chiquoine, 1954; Witschi, 1948). According to this model, mouse primordial germ cells (PGCs) form a small cluster of about 40 AP-positive cells in the base of the allantois, or precursor umbilical cord (Ginsburg et al., 1990; Lawson and Hage, 1994; Ozdzenski, 1967) (Fig. 1.1A1–2). The PGCs then translocate to the hindgut (Fig. 1.1A3) and migrate toward the developing gonads (Molyneaux and Wylie, 2004), which they colonize a few days later, subsequently completing their differentiation as sperm and eggs (not shown). This view of mammalian germline development has dominated the field for the past 60 years, silencing early calls by many scientists for an experimental demonstration that so-called extragonadal mammalian PGCs actually contribute to the gonads and are not generalized stem cells that build the conceptus (Simkins, 1923). However, to this day, scientists have not sufficiently demonstrated lineage continuity along the PGC trajectory. Specifically, they have not shown that putative PGCs in the posterior region of the mouse conceptus give rise to definitive germ cells in the gonads. Thus, whether the allantois contains a germ line distinct from the surrounding soma remains obscure.

Mouse Primordial Germ Cells: A Reappraisal

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Figure 1.1 Localization of PGCs before and after discovery of ACD, based on alkaline phosphatase (AP) staining. All images and schematics are oriented with anterior to the left and posterior to the right. (A1–3) The PGC trajectory is based on staining for AP activity (red). Images were modified, with the publisher's permission, from Chuva de Sousa Lopes and Roelen (2008) and are magnifications of the posterior region; panel lettering has been changed, embryonic days removed, and the abbreviations “PVE” and “hg” included. (A1) PGCs form a cluster within the base of the allantoic bud (al). (A2) As the allantois elongates into the exocoelom (x), PGCs persist in the allantois while also expanding into the underlying embryonic region and overlying posterior visceral endoderm (PVE). (A3) PGCs localize to the invagination of the hindgut (hg). (B1–3) In the current model, in which PGCs are thought to be lineage restricted, the posterior end of the (Continued)

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Part of the problem in studying the extragonadal germ line is that its relationship to the allantois has been largely ignored. Recent evidence has unexpectedly revealed that the mouse allantois is not a mass of extraembryonic mesoderm but, rather, an architecturally complex structure (Daane et al., 2011; Downs et al., 2009) whose proximal region contains a stem/progenitor cell pool, the Allantoic Core Domain (ACD), defined by Brachyury (T) (Fig. 1.2A1, B1). The ACD is a condensation of cells in the posterior end of the primitive streak, or embryonic body axis, that bears striking functional and molecular similarity to the streak’s anterior condensation, Hensen’s node (Downs, 2009; Downs et al., 2009). Like the node, which extends the body axis anteriorly via the notochord (Fig. 1.2A1), the ACD extends the body axis further posteriorly through the allantoic midline (Fig. 1.2A2, B2). In this way, the primitive streak creates an axial continuum between the embryo and its vital connection to the mother. To reinforce the fetal–umbilical connection, ACD cells contribute substantially not only to the allantois (Downs et al., 2009; Inman and Downs, 2006) but also to derivatives of all three germ layers of the embryo proper (Mikedis and Downs, Figure 1.1—Cont'd primitive streak, or embryonic anteroposterior (A-P) axis, is thought to terminate at the embryonic–extraembryonic junction, identified via the site of insertion of the amnion into the posterior region. (B1) PGCs forming in the allantoic bud are thought to be posterior to the primitive streak. These PGCs originate from Prdm1expressing cells on the right side of the proximal epiblast (epi), which also forms the future posterior end of the anteroposterior axis (inset). (B2) Some PGCs persist extraembryonically in the allantois while also expanding into the embryonic primitive streak as well as to the overlying embryonic and extraembryonic posterior visceral endoderm. (B3) PGCs outside of the hindgut, whose localization in this figure is based on that reported by Mintz and Russell (1957), were thought to be “ectopic” PGCs that have lost their way along the PGC trajectory. (C1–3) In the revised model, the PGCs are part of the posterior end of the primitive streak and function there as a pluripotent progenitor population that forms the fetal–umbilical connection in the posterior region of the conceptus; they are not a lineage-restricted germ line. (C1) The posterior end of the primitive streak extends into the extraembryonic region, where it forms the extraembryonic primitive streak (XPS), to which the “PGCs” localize. These cells, expressing Prdm1, originate from the right side of the proximal epiblast, the future posterior end of the primitive streak (inset). (C2) The XPS then expands to form the ACD, a stem/progenitor cell pool. “PGCs” in the ACD and intraembryonic posterior primitive streak (IPS) are part of the anteroposterior body axis and contribute to multiple somatic lineages within the surrounding posterior embryo (Mikedis and Downs, 2012). (C3) The posterior end of the primitive streak has regressed/differentiated and is confined to the embryo. Some descendants of the ACD form a midline extension similar to the notochordal extension of the anterior node (Fig. 1.2). “PGCs” contribute to the hindgut and other somatic tissues. Other abbreviations: vys, visceral yolk sac.


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Figure 1.2 ACD defines primitive streak's posterior terminus. Panels A1–2 are sagittally oriented with anterior to the left and posterior to the right; panels B1–2 are frontal views from the posterior end. This depiction of the posterior end of the streak is based on a combination of morphological, fate mapping, and immunohistochemical analysis (Downs et al., 2009). (A1, B1) The ACD, which forms at headfold stages (E7.75–8.0), is continuous with the primitive streak (yellow). The ACD caps the posterior end of the primitive streak, similar to the node (yellow circle), which caps the anterior end of the streak. The ACD persists during stages of allantoic elongation (headfold—6somite pairs, E7.75–8.5). (A2, B2) By 8–12-somite pairs (E8.5–9.0), allantoic elongation is complete, the allantois has fused with the chorion, and the ACD has regressed and/or differentiated. Some ACD descendants within the allantois form a midline file of cells (black line within allantois), similar to the anterior node-derived notochord (black line extending from the yellow circle representing the node) (Downs et al., 2009).

2012). Thus, the ACD appears to be a major posterior stem/progenitor cell pool that builds and weaves together embryonic and extraembryonic tissues in the posterior region of the conceptus. Intriguingly, the ACD exhibits all of the so-called “markers” of PGCs: AP activity (Chiquoine, 1954; Ginsburg et al., 1990; Ozdzenski, 1967), tissue nonspecific AP (Tnap) expression (Macgregor et al., 1995), OCT-3/4 (OCT-3, OCT-4, POU5F1) (Downs, 2008; Rosner et al., 1990;

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Scholer et al., 1990), PRDM1 (BLIMP1) (Mikedis and Downs, unpublished; Ohinata et al., 2005; Vincent et al., 2005), STELLA (DPPA3, PGC7) (Mikedis and Downs, 2012; Saitou et al., 2002), and IFITM3 (FRAGILIS, MIL1) (Mikedis and Downs, 2013; Saitou et al., 2002). Thus, in light of the existence of the ACD, we investigated the evidence for an extragonadal germ line within the allantois, and balanced this question by turning it on its head, asking whether PGCs might actually be part of a pluripotent stem cell pool that builds the understudied fetal–umbilical connection. This question necessitated an objective review of (i) the evidence for an extragonadal origin of mouse PGCs, (ii) the properties of PGC markers, and (iii) their relation to the allantois. Based on the published literature, which spans almost a century, we conclude that key experiments have not been carried out: there is no evidence for either continuity of extragonadal PGCs with the gonads or for lineage segregation of a mammalian PGC population within any given tissue at any given moment in the trajectory to the gonads. This is not to imply the absence of such a lineage, but rather, that there is no definitive evidence for it. Based on the data we uncovered and their limitations, the more likely scenario appears to be one in which so-called PGCs are part of a pool of posterior stem/progenitor cells that builds the fetal–umbilical connection of the placental mammal. We finish by suggesting experiments to elucidate the true whereabouts of the mammalian germ line and by emphasizing the urgency for study of the posterior embryonic–extraembryonic interface, whose importance in amniote development has been overlooked. For morphological staging, readers are encouraged to consult Downs and Davies (1993), and for development of the allantois, please see Inman and Downs (2007).

2. CURRENT MODEL OF THE ORIGIN OF PRIMORDIAL GERM CELLS (PGCs) AND SEGREGATION FROM SOMA In this section, Chiquoine’s model of PGC development, which has been expanded upon over the past decade, is summarized (Fig. 1.1B). In subsequent sections, we highlight deficiencies (Section 3.1) and other concerns (Sections 3.2 and 3.3) regarding this model, and propose a revised one, based on new evidence concerning the posterior embryonic– extraembryonic interface (Section 5; Fig. 1.1C). In the current model, the PGCs are defined by expression of at least two hallmark genes, Stella (Saitou et al., 2002; Sato et al., 2002) and Prdm1 (Ohinata et al., 2005; Vincent et al., 2005), both of which were isolated from


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subpopulations of cells expressing Tnap from the posterior region of the mouse gastrula. It is therefore not surprising that these proteins localize to the same posterior tissues that exhibit AP activity. First, Prdm1-expressing PGCs segregate asymmetrically from somatic precursor cells within the proximal epiblast at E6.25, which is prior to formation of the primitive streak and the onset of gastrulation (Ohinata et al., 2005) (Fig. 1.1B1, inset). Moreover, this site of segregated PGCs, which are also thought to exhibit Ifitm3 expression, corresponds to the presumptive posterior end of the anteroposterior embryonic axis (Ohinata et al., 2005). Then, as gastrulation is initiated (E6.5), the Prdm1/Ifitm3 population translocates posteriorly, traversing the primitive streak and exiting it alongside extraembryonic mesoderm of the allantois. The Prdm1/Ifitm3 PGCs then settle into the base of the allantois (Fig. 1.1B1), where they further acquire Tnap and Stella expression (Ohinata et al., 2005; Saitou et al., 2002). However, some cells that express Tnap fail to acquire Stella and thereby fail to become PGCs (Saitou et al., 2002). Based on colocalization with AP activity (Anderson et al., 2000), Oct-3/4 reporter expression has also been associated with the PGCs in this early posterior region (Anderson et al., 2000; Scholer et al., 1990). However, some of these AP-positive, Oct-3/4-expressing cells may contribute to the allantois and are therefore not PGCs (Anderson et al., 2000). Furthermore, where Oct-3/4 fits into the context of these other “marker” proteins is obscure, as Oct-3/4 has never been colocalized with the more recently characterized PGC proteins. Shortly thereafter, the Prdm1/Ifitm3/Tnap/Stella population moves into adjacent posterior visceral endoderm (PVE) and the embryonic streak (Fig. 1.1B2), at which point NANOG colocalizes to a minority of cells with STELLA protein (Yamaguchi et al., 2005). Once the hindgut invagination appears approximately 12 h later, cells exhibiting this group of PGC markers colonize its ventral component (Fig. 1.1B3). By this point, the majority of, but not all, cells exhibiting STELLA protein colocalize NANOG (Yamaguchi et al., 2005). After several days of migration, PGCs exit the hindgut, traverse the dorsal mesentery, and enter the gonads, where they complete their differentiation into sperm and eggs (not shown). While the movement of the PGCs from the posterior region to the gonads is referred to as “migration,” the data may be insufficient to distinguish between active movement versus passive carriage along the PGC trajectory (Freeman, 2003); migration of PGCs to the gonad will not be emphasized in this review. Why would the PGCs take such a circuitous route, moving from the embryonic proximal epiblast into the extraembryonic allantois only to

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return to the embryo? McLaren suggested that “perhaps cell populations within the embryo are subjected to pressures of determination and differentiation during this crucial period, that would threaten the totipotency that primordial germ cells need to preserve” (McLaren, 1992). Unfortunately, this statement reflects the notion that the primitive streak, whose presence and activity defines amniote gastrulation (Beddington, 1983), is limited to the embryo proper (reviewed in Downs, 2009). Indeed, nearly every figure depicting the posterior end of the streak in textbooks, reviews, and original research articles shows a primitive streak that terminates within the embryo, beneath the site of amniotic insertion into the posterior region (Fig. 1.1B1–2). The allantois is depicted as a mesodermal outcropping of the primitive streak, and the PGCs are a small cluster nestled within proximal allantoic mesoderm (Fig. 1.1B1–2). However, only a handful of researchers have previously described or depicted an “embryonic component” of the allantois (Anderson et al., 2000; Dalcq, 1957; Downs, 2009; Ozdzenski, 1967) which is consistent with recent experimental evidence in mouse (Fig. 1.1C1–2) (Downs, 2009; Downs et al., 2009), discussed in Section 5. Indeed, that the embryonic body axis extends into the allantois was observed in some studies of Hox gene expression but was not immediately appreciated (Deschamps et al., 1999; discussed in Downs, 2009). This discovery bears directly upon the reach of gastrulation within the extraembryonic compartment of the conceptus, the identity of the PGCs, and the source of cells that builds the posterior region of the mammal and its connection to the mother. In the next section, we will systematically scrutinize the evidence for the current model of PGC formation and segregation, focusing on relevant gene products in a case-by-case basis. Then, in Section 5, we will place the current model of PGC development within the context of recent studies on the embryonic–extraembryonic interface.

3. FLAWS IN CURRENT MODEL OF PGC ORIGIN AND SEGREGATION 3.1. Lineage continuity and segregation of PGCs from soma: Criteria and evidence The name “primordial germ cells” itself implies a segregated cell type, which should therefore accord with the definition put forth by Seydoux and Braun (2006), that “the primordial germ cells are the founder cells for the germline [sic]. They divide symmetrically and all their descendants are germ cells.”


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Moreover, the operational definition of differentiation is retention of identity in ectopic sites (Gardner, 1993). Thus, at least two properties of PGCs must be demonstrated: (i) continuity of the germ line between the allantois and gonads by fate mapping; and (ii) segregation of PGCs from the soma, either by clonal fate mapping to show that daughter cells are identical to each other or by grafting into ectopic sites to demonstrate maintenance of germ cell phenotype despite the pressures of a foreign environment. As discussed here, and throughout Section 3, neither of these requirements has been met. The absence of critical lineage tracing data is due to the technical limitations of whole embryo culture (Nagy et al., 2003) as well as to limitations of prospectively identifying PGCs in postimplantation stage conceptuses, which are embedded in the uterus. Conceptuses labeled at E7.5, when the PGCs are found in the base of the allantois, cannot be cultured in test tubes through gonad formation at E10.5. Studies that have attempted to fate map the PGCs via whole embryo culture (Lawson and Hage, 1994; discussed in detail in Section 3.2.1) had, by necessity, to use experimental end points prior to gonad formation and thereby could not provide definitive results regarding germline lineage restriction. Moreover, targeted cell labeling cannot be carried out in utero at early stages of gastrulation (Mu et al., 2008). New technology for inducible genetic lineage tracing in vivo can potentially overcome these limitations; however, it has not been fully harnessed to address the question of PGC lineage segregation (discussed in further detail in Section 7.2). While ectopic grafting experiments have used PGC markers to identify the PGCs, cells exhibiting these marker proteins have never been experimentally verified as a lineage-restricted germ line. Thus, these ectopic grafting experiments cannot provide meaningful results regarding PGC segregation. For example, small clumps of proximal epiblast, where the PGCs are thought to form, were grafted to distal sites prior to gastrulation (Chuva de Sousa Lopes et al., 2007; Tam and Zhou, 1996). Results of these experiments failed to identify PGCs in ectopic sites, either by AP activity (Tam and Zhou, 1996) or by Prdm1 reporter expression (Chuva de Sousa Lopes et al., 2007); thus, the interpretation, based on the current model, was that PGCs had not segregated from the soma at the time of grafting. However, in light of the fact that lineage restriction has not been shown, these results can only indicate that AP-positive or Prdm1-expressing cells were not a segregated population at the time of manipulation. By contrast, when E9.5 mouse hindgut PGCs were ectopically transplanted into chick

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embryos, AP-positive cells were observed in chick tissue up to 3 days posttransplantation (Rogulska et al., 1971). While the goals of this study were to assess attraction of mouse PGCs to chick gonads and conservation of homing to the gonads among amniotes (Rogulska et al., 1971), in light of the definitions above, results might also be taken as demonstration that E8.5 PGCs had segregated from the soma. Again, based on the limitations of PGC markers, these results only suggest that the AP-positive population, rather than PGCs, has segregated. Whether the AP-positive cells found at the end point of the transplantation were descended from the initially grafted AP-positive cells, or whether they were a new population that arose independently in mouse tissue in response to their new environment, was not addressed. Thus, because the allantois’ PGCs have not been lineage traced to demonstrate their exclusive contribution to gonadal germ cells, and because maintenance of PGC identity in ectopic sites has not been unequivocally demonstrated, the identity of PGCs and their timing of segregation from the soma remain unknown. Therefore, mouse PGCs do not merit their PGC appellation, regardless of whether the PGCs are identified via AP activity or via the expression of other PGC markers. For purposes of clarity within this review, we will continue to use the term “PGCs” as defined by the broader mammalian germline field (see Section 2), but we emphasize that whether these cells are truly the precursors of the mammalian germ line remains obscure.

3.2. PGC trajectory and “markers” of PGCs Below, we summarize the data that have led the field to claim specific gene products as “markers” of the PGCs. None of these data show confinement of PGC “marker” proteins to Chiquoine’s PGC trajectory, and none demonstrate that the PGCs within the trajectory form a lineage-restricted population. In light of emerging data on the largely understudied embryonic–extraembryonic posterior interface, it remains possible that so-called PGCs function as a pluripotent progenitor population that contributes to the somatic tissues of the posterior region (Section 5). 3.2.1 AP activity and PGC trajectory AP activity, first noted in the gonads (Chiquoine, 1954), forms the basis for the current model of mouse germline development. APs are orthophosphoric monoester phosphohydrolases, with alkaline optima (Coleman, 1992). Several APs are capable of dephosphorylating both protein and DNA substrates


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in vitro, and thus would be expected to localize to both the nucleus and extranuclear sites. Chiquoine found that gonadal germ cells exhibited high cytoplasmic AP activity while surrounding cells showed minimal, if any (Chiquoine, 1954). In the original model of PGC development, Chiquoine traced the history of AP-positive cells back to embryonic days E6–10 and ignored cells with nuclear activity. He reported the presence of so-called PGCs in the caudal end of the primitive streak, followed by their appearance in the base of the allantois and associated visceral endoderm (Fig. 1.1A1–2). From there, AP-positive PGCs moved into the hindgut (Fig. 1.1A3), “migrating” through ventral hindgut epithelium (Chiquoine, 1954). These AP-positive cells increased in number at an exponential rate, which is consistent with a lineage-segregated population that is dividing (Tam and Snow, 1981). However, whether AP-positive cells entered and left the population at a rate that mimics exponential growth was never addressed. Eventually, AP-positive PGCs exited the hindgut, traversed the dorsal mesentery, and colonized the gonads. That PGCs are extragonadal and take a defined route to the gonads was supported at the time by genetic analyses in Dominant White Spotting (W) mutants. While homozygous mutant AP-positive cells apparently formed in correct numbers in the base of the allantois, they gradually diminished in number en route to the gonads (Mintz and Russell, 1957). There are several problems with these conclusions. First, embryos could not be genotyped in the 1950s to distinguish wildtype, heterozygotes, and homozygous mutants. Second, the numbers of AP-positive cells at early stages varied enormously from embryo to embryo. Third, Dominant White Spotting homozygous animals exhibited not only infertility defects but also defects in other stem cell populations, including hematopoietic and neural crest stem cells (Fleischman, 1993). Because AP activity is now recognized as a component of many stem cells (Benham et al., 1983; Bernstine et al., 1973), the need to clarify the whereabouts of each AP-positive stem cell pool vis-a`-vis each affected lineage in these mutants is critical. For example, when PGCs localize to the hindgut and mesentery prior to colonization of the gonads, they spatiotemporally coincide with another pluripotent progenitor population there, the neural crest, which forms the future enteric nervous system there (Tjaden and Trainor, 2013; Young and Newgreen, 2001). Defects in the migration, proliferation, differentiation, survival, and apoptosis of progenitor neural crest can result in a congenital absence of enteric neurons in a portion of the intestinal tract,

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such as that which occurs in Hirschsprung disease. Initially confined to the midgut, these enteric neural crest cells cross the mesentery to enter the hindgut at E10.5–11.5 (Nishiyama et al., 2012), the timepoint at which the PGCs are entering the dorsal mesentery and completing their colonization of the gonads. Nowadays, enteric neural crest cells can be identified via expression of genes such as Ret, which promotes proliferation, differentiation, and survival of this population (Tjaden and Trainor, 2013). It would therefore be important to elucidate the relationship between AP-positive hindgut cells and neural crest stem cells. Later experiments expanded on the use of AP activity to identify the earliest time at which PGCs segregated from the soma. By the latter part of the last century, results of tour-de-force clonal fate mapping had shown that cells in the base of the allantois were derived from proximal epiblast that translocated anisotropically toward the primitive streak as the latter formed (Lawson et al., 1991). Clonal fate mapping of proximal epiblast in conjunction with staining for AP activity further revealed that when a daughter cell of the labeled clone was AP-positive, the other daughter cell always contributed to AP-negative soma in the allantois; in other words, a labeled clone never produced two AP-positive daughter cells, which would demonstrate segregation (Lawson and Hage, 1994). Rather, based on an extrapolative argument, the authors concluded that AP-positive PGCs segregated from soma within the allantois at E7.25 (Lawson and Hage, 1994). However, the developmental end point for these clonal studies was prior to the formation of, and PGC translocation into, the hindgut (Lawson and Hage, 1994). Therefore, the conclusions were based on the assumption that AP activity in the allantois identifies a lineage-restricted germ line, something that has never been experimentally demonstrated. A later study using a different PGC “marker,” Prdm1, refuted Lawson and Hage’s conclusions (1994) with Prdm1-Cre genetic lineage tracing, suggesting that PGC lineage segregation occurs in the prestreak epiblast as early as E6.25 (Ohinata et al., 2005). Suspending judgment for a moment that neither study traced these so-called PGCs to the gonads, the disparate conclusions may be reconciled by (i) the possibility that segregated Prdm1expressing PGCs in the proximal epiblast were missed in the clonal fate mapping studies (Lawson and Hage, 1994) owing to the population’s very small size (Ohinata et al., 2005) and/or (ii) a model in which cells continue to initiate Prdm1 expression and become segregated PGCs between E6.25 and E7.25 (McLaren and Lawson, 2005). The details and limitations of this Prdm1-based study will be discussed in Section 3.2.4.


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AP staining protocols are temperamental; the efficiency of hydrolysis is dependent upon pH, buffer, and salts; for fixed tissues, activity is further dependent upon the fixative used, temperature, and time of exposure to the substrate (Gomori, 1946, 1951). Thus, because protocols used among laboratories have been inconsistent, descriptions of the whereabouts of AP activity in the mouse gastrula lack uniformity. For example, a modification to the AP staining protocol enabled Ginsburg et al. (1990) to distinguish a small cluster of AP-positive cells within the posterior-most tip of the primitive streak at a stage just prior to allantoic bud formation, which was earlier than with previous methods. Moreover, wide variation in numbers of AP-positive cells has been reported in many studies (Chiquoine, 1954; Ginsburg et al., 1990; Lawson and Hage, 1994; Lawson et al., 1999; Mintz and Russell, 1957; Ozdzenski, 1967; Snow, 1981; Tam and Snow, 1981); whether these can be attributed to varied staining conditions is not known. Alternatively, fluctuations in AP number may be indicative of stem/progenitor cells in varying states of differentiation (Mikedis and Downs, 2012). 3.2.1.1 AP activity: Embryonic germ cells

AP activity has been used to isolate embryonic germ (EG) cells (Labosky et al., 1994; Matsui et al., 1992). EG cells derived from the posterior region at E8.5 (e.g., Fig. 1.2A2) and injected into the blastocyst contribute to derivatives of the three primary germ layers (Matsui et al., 1992) or to the germ line (Labosky et al., 1994), but not to both within the same study. Germline transmission was also observed when E8.5-derived EG cells were injected into 8-cell embryos, just prior to blastocyst formation (Durcova-Hills et al., 1999). The methods of isolating the posterior cells that created EG cell lines varied significantly. Two of these studies used the entire E8.5 posterior region between the last somite and base of the allantois, essentially encompassing most of the primitive streak, but excluding the allantois (e.g., Fig. 1.2A2) (Labosky et al., 1994; Matsui et al., 1992), while a third study immunomagnetically separated SSEA-1positive cells, which are thought to be pluripotent cells and PGCs (Wu and Chow, 2005), from negative ones within the E8.5 allantois (e.g., Fig. 1.2A2) (Durcova-Hills et al., 1999). Whether each EG cell line was derived from the same PGC population remains obscure, particularly as studies did not examine whether posterior somatic cells exclusive of PGCs are similarly pluripotent. Regardless, results of these experiments highlight the potency of the isolated populations, rather than their fate, during normal development.

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3.2.1.2 AP activity: Cytoplasmic “spot”

While PGCs are thought to constitute a posterior AP-positive cell population, scientists after Chiquoine nevertheless recognized that AP is not restricted to the PGC trajectory (Clark and Eddy, 1975; Ginsburg et al., 1990; Hahnel et al., 1990; Macgregor et al., 1995; Mintz and Russell, 1957). To justify AP as a PGC-specific “marker,” investigators have claimed that an intracellular “spot” of AP activity, attributed to the Golgi apparatus (Clark and Eddy, 1975), distinguishes PGCs from other AP-positive cells (e.g., Ginsburg et al., 1990; Lawson and Hage, 1994) (Fig. 1.1A). However, to the best of our knowledge, evidence that this spot identifies a lineagerestricted germline population is not available. 3.2.2 Tnap Of the multiple genes that encode AP proteins, only Tnap (also known as liver/bone/kidney AP; Akp2) expression was detected, via RT-PCR, in the mouse conceptus at E7.5, 8.5, and 9.5 (Hahnel et al., 1990). Based on a lacZ reporter construct, Tnap was not limited to the PGC population but was found broadly in the conceptus, particularly within many posterior tissues, including the epiblast, primitive streak, and amniotic ectoderm, as well as within the trophoblast-derived chorion (Macgregor et al., 1995). Therefore, Tnap is not specific to the germ line (Macgregor et al., 1995). Furthermore, loss of Tnap did not affect germline development or function, as the Tnap knockout mice were fertile and grossly normal (Macgregor et al., 1995). As the epiblast (Beddington, 1981, 1982; Brons et al., 2007; Tesar et al., 2007), primitive streak (Kinder et al., 2001a), amnion (Dobreva et al., 2010), and chorion (Uy et al., 2002) all contain stem cell populations, Tnap in these tissues might be indicative of such stem cell pools. Surprisingly, considering Tnap’s wide expression and absent role in germline development, a Tnap-Cre construct employing the Cre/LoxP system “specifically” activated reporter expression in the PGC lineage as early as E9.0 (Lomeli et al., 2000). Though Cre activity was not detected in nonPGC populations (Lomeli et al., 2000) that normally express Tnap (Macgregor et al., 1995), the analysis relied on whole-mount, rather than sectioned, specimens. Therefore, tissues with less robust reporter expression would not have been detected, and it is likely that Tnap-Cre-mediated deletion is occurring in non-PGC populations. This Tnap-Cre has subsequently been used to “specifically” knock out genes of interest in the PGCs (specific experiments discussed in Sections 3.2.3 and 3.2.7). It is possible that the PGC phenotypes observed in such experiments reflect a requirement for


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the gene of interest not in the PGC population but in the surrounding somatic tissue. Therefore, caution is needed in interpreting results from Tnap-Cre conditional deletion experiments. 3.2.3 OCT-3/4 The POU domain transcription factor OCT-3/4 was first associated with the germ line when Oct-3/4 transcripts and OCT-3/4 protein were detected in the germ cell-rich gonads at E11.0 and 12.0, respectively (Scholer et al., 1989, 1990). Oct-3/4 expression was also found at E8.5 in the posterior region of the mouse conceptus in a pattern similar to that of AP activity (Scholer et al., 1990), though AP/Oct-3/4 colocalization was not shown at the time. Later immunostaining at closely spaced 2–4-h intervals demonstrated that OCT-3/4 protein is not specific to the PGC trajectory but is found in myriad sites outside of it until hindgut formation (Downs, 2008) when, intriguingly, the only OCT-3/4-positive cells present at 16-somite pairs (E9.5) were in the hindgut. Unfortunately, the molecular nature of these cells has not yet been further characterized. In Gata4 conditional knockout mice, in which Gata4 is deleted via the tamoxifen-inducible Cre/LoxP system after E8.75, the gonad never forms from the coelomic epithelium, but Oct-3/4 reporter-expressing cells localize normally to this region of the embryo (Hu et al., 2013). Therefore, the gonad is not necessary for the localization of these Oct-3/4-expressing cells near the pregonadal coelomic epithelium (Hu et al., 2013). However, whether these Oct-3/4-expressing cells are PGCs that originate from the base of the allantois remains obscure. Oct-3/4-null mice die around the time of implantation, precluding an analysis of the germ line. However, a conditional knockout of Oct-3/4 (Kehler et al., 2004) mediated by Tnap-Cre (Lomeli et al., 2000; described in Section 3.2.2.) has shown that loss of Oct-3/4 in the Tnap-expressing population results in both a decrease in the size of the AP-positive PGC population after E9.5 and reduced numbers of AP-positive germ cells in the gonads at E12.5 (Kehler et al., 2004). As Cre-mediated excision did not occur in the Tnap-expressing population until E9.0 (Lomeli et al., 2000), after the PGCs have translocated to the hindgut endoderm, these data do not reveal anything about the role of Oct-3/4 prior to E9.0 nor about germ line continuity. Additional studies in which reporter expression was driven from the distal, but not proximal, enhancer of the Oct-3/4 promoter (Oct-3/ 4DPE:LacZ) (Yeom et al., 1996) showed, by whole-mount analysis of lacZ

16


expression, that Oct-3/4 was exclusive to the PGC trajectory (hindgut and gonads) from E9.25 through E12.5. Therefore, it was concluded that the Oct-3/4DPE:LacZ reporter identifies the PGCs. However, and inexplicably, additional reporter signal was also identified at E9.0 near the posterior neuropore, which is not part of the PGC trajectory; also, at odds with the localization of reportedly segregated PGCs in the allantois (Lawson and Hage, 1994; Ohinata et al., 2005), reporter signal was not detected there (Yeom et al., 1996). In a later study, the lacZ gene of Oct-3/4DPE:LacZ, above, was replaced with GFP (Oct-3/4DPE:GFP) (Anderson et al., 2000; Yeom et al., 1996). For reasons that are not clear, GFP reporter signal, localizing throughout the epiblast and primitive streak, was observed at earlier stages than lacZ reporter signal. The PGCs were thought to be identifiable from surrounding somatic signal as early as allantoic bud stages (designated as E7.5–8.0 by the authors), when GFP-positive cells within the posterior primitive streak and overlying visceral endoderm colocalized with AP activity (Anderson et al., 2000). Why the GFP reporter exhibited signal at stages when the lacZ reporter did not remains obscure.

3.2.4 PRDM1 Prdm1 is expressed and plays a role in the development of a range of cell types (John and Garrett-Sinha, 2009). After translocating from the proximal epiblast through the primitive streak, Prdm1-expressing cells were reportedly confined to the posterior region; while they were not detected in “allantoic mesoderm” (Ohinata et al., 2005), the published whole-mount specimens appeared to contain Prdm1 in situ hybridization signal in the base of the allantoic bud, a region that is typically annotated as “allantoic mesoderm” (Kaufman, 1992; Lawson and Hage, 1994). Within the posterior region, almost all (>90%) Prdm1-expressing cells colocalized with STELLA protein (N ¼ 3; early headfold stage, E7.75; e.g., Fig. 1.1B2) (Ohinata et al., 2005). Similarly, genetic lineage tracing, in which expression of a Prdm1-Cre construct ultimately activates a constitutively active GFP reporter (N ¼ 4; late bud to 3-somite stage range; E7.5–8.25), indicated that almost all of the descendants of Prdm1-expressing cells localized STELLA protein. These analyses were carried out in optical confocal sections, from which it was concluded that Prdm1 identifies lineage-restricted PGCs in the proximal epiblast (Ohinata et al., 2005). However, studies beyond the 3-somite stage have yet to be carried out.


17

It has since become clear that when Prdm1 is expressed in the posterior region, it is also found in nonposterior sites of the mouse conceptus (Vincent et al., 2005); therefore, when Prdm1 is found in posterior sites, it is thought to uniquely identify PGCs in the early posterior embryonic–extraembryonic junction, but not in the entire conceptus. Prdm1-null mutants exhibit fewer posterior TNAP-positive cells (stages designated as early bud to late headfold or E7.5, Ohinata et al., 2005; stages designated as early headfold stage or E7.75, Vincent et al., 2005) as well as fewer Stella-expressing cells as early as late streak/no (allantoic) bud stages (E7.0, Kurimoto et al., 2008) (Table 1.1). Analysis of this remnant population revealed that Prdm1 upregulates expression of pluripotency factor Sox2 and downregulates expression of Hoxb1, associated with somatic differentiation (Kurimoto et al., 2008). While PRDM1 was the predominant factor responsible for the repression of so-called somatic genes in the posterior PGC population during the allantoic elongation phase (E8.0–8.5), additional PRDM1-independent pathways complement this PRDM1mediated regulation. Indeed, from allantoic bud stages (E7.25) through E8.25, PGCs downregulate 16% of so-called PGC specification genes and upregulate 32% of somatic genes (Kurimoto et al., 2008). These results are consistent with a model in which at least some of the PGC population differentiates and contributes to surrounding somatic tissues. Intriguingly, because Prdm1 contributes to anteroposterior patterning in Xenopus (de Souza et al., 1999) and zebrafish (Wilm and Solnica-Krezel, 2005), one study specifically sought, but failed to find, anteroposterior axis defects in the mouse mutants (Vincent et al., 2005) (Table 1.1). While the allantois was reported as normal (Ohinata et al., 2005), the Prdm1-null placental vascular labyrinth failed to expand (Vincent et al., 2005). This is consistent with Prdm1 in the progenitors of embryonic and umbilical endothelium (Mould et al., 2012; Vincent et al., 2005), and with the widespread hemorrhaging observed in the mutants (Vincent et al., 2005) (Table 1.1). The initiation of labyrinth expansion requires successful chorioallantoic fusion (Inman and Downs, 2007), which was never demonstrated in the mutants (Vincent et al., 2005). However, while Prdm1-null pups die between E10.5 and E11.5 (Robertson et al., 2007; Vincent et al., 2005), pups in which Prdm1 was conditionally knocked out only in the epiblast via Sox2-Cre survived until E18.5, though no newborn pups were recovered (Robertson et al., 2007). While this suggests that labyrinth expansion occurred to the degree needed for physiological function through E18.5, and that Prdm1 in the epiblast-derived allantois is not required up to this timepoint, whether Prdm1

Table 1.1 Genes required for the development of PGCs in the posterior region also affecting the development of surrounding soma Posterior tissue(s) concurrently Earliest stage affected at which PGC PGC “markers” defect is A-P Hg Al Details of Gene Gene product used reporteda PGC defect axis posterior defects Reference

Bmp2

Intracellular signaling molecule

AP activity

NP and HF stages (E7.0–8.0)

PGC population n/d þ is reduced. At later stages, when wild-type PGCs are in the hindgut, mutant PGCs mislocalize to posterior streak and base of allantois

þ

Zhang and Bradley Extension of primitive streak (1996); Ying and into allantois not Zhao (2001) examined; absence of clear hindgut endoderm or an open hindgut with obvious endoderm layer; developmental delay of allantoic elongation with the allantois in some specimens failing to reach chorion

Bmp4


AP activity

E7.2–7.5

PGCs fail to form þ

n/d þ

Bmp8b


AP activity

LS stage (E7.0)

n/d n/d þ In 50% of specimens, PGCs fail to form; in other specimens, PGCs exhibit delayed formation at reduced numbers. PGCs that do form localize normally

Posterior truncation of anteroposterior axis with disorganized posterior structures; allantois absent

Winnier et al. (1995); Lawson et al. (1999)

Ying et al. (2000) Extension of primitive streak into allantois not examined; hindgut development not analyzed but we note that the hindgut appears normal in morphological sections in Fig. 1B and C; delayed allantoic elongation; some null pups survive postnatally Continued

Table 1.1 Genes required for the development of PGCs in the posterior region also affecting the development of surrounding soma—cont'd Posterior tissue(s) concurrently Earliest stage affected PGC at which PGC “markers” defect is A-P Hg Al Details of Gene Gene product used reported PGC defect axis posterior defects Reference

Cdx2b

Homeobox transcription factor

AP activity; EB stage PRDM1 and (E7.25) IFITM3 double IF

þ Initial PGC population is similar in size to wild type but does not increase at the same rate as the wild-type population. No apoptosis was detected, suggesting that the mutant PGCs are not proliferating normally

þ

Eed

Polycomb protein

AP activity

þ Mutant PGCs exhibit reduced clustering and are more anteriorly distributed

n/d þ

E8.5

þ

Chawengsaksophak Truncated et al. (2004); posterior axis; Bialecka et al. (2012) formation of hindgut invagination delayed from 4-s stage (E8.25) to 8-s stage (E8.5); truncated allantois that fails to fuse with chorion Thickened and Faust et al. (1995) kinked primitive streak (anteroposterior axis); allantois is enlarged, shifted dorsally, and continuous with amniotic mesoderm; definitive endoderm forms

Forkhead Foxa2 (Hnf3b; transcription Tcf-3b) factor

AP activity

E8.0

PGC population þ is absent or severely reduced

n/d þ

Anterior truncated primitive streak (anteroposterior axis) without a node or notochord; abnormal allantois; disorganized posterior region

Tsang et al. (2001); Ang and Rossant (1994); McKnight et al. (2010)

Lhx1 (Lim1)


AP activity

E8.0

PGC population þ is absent or severely reduced

n/d þ

Misaligned primitive streak (anteroposterior axis); abnormal allantois

Kinder et al. (2001b); Tsang et al. (2001)

Otx2


AP activity

Not specified Ectopic localization in the yolk sac

þ

n/d þ

Misaligned primitive streak (anteroposterior axis); abnormal allantoic development

Unpublished data from K. Lawson, cited in Lawson et al. (1999); Kinder et al. (2001b); unpublished data from K. Lawson, cited in Bosman et al. (2006) Continued

Table 1.1 Genes required for the development of PGCs in the posterior region also affecting the development of surrounding soma—cont'd Posterior tissue(s) concurrently Earliest stage affected at which PGC PGC A-P Hg Al Details of “markers” defect is Gene Gene product used reported PGC defect axis posterior defects Reference

Prdm1 Transcriptional AP activity; (Blimp1) repressor Stella expression; STELLA protein localization

LS/OB stage Reduced mutant (E7.0) PGC population in the posterior region

Ror2

E9.0

Receptor tyrosine kinase

AP activity

Mutant PGCs mislocalize to allantois, tail mesoderm, and caudal hindgut

n/d n/d Disrupted labyrinth formation at site of chorioallantoic fusion; whether the defect is in the chorion and/or allantois has not been investigated

n/d þ

Ohinata et al. (2005); Vincent et al. (2005); Kurimoto et al. (2008)

n/d Shortened and Laird et al. (2011) widened hindgut

Smad1

Intracellular signaling molecule for BMP pathway

AP activity

EB-LB stages PGCs drastically þ (E7.25–7.5) reduced; about 50% of mutants have no PGCs

Smad2


AP activity

E8.5

þ 5/25 mutants lacked PGCs; the rest of the mutants formed “abundant PGCs,” though precise numbers were not reported

n/d þ

þ

Twisted primitive streak (anteroposterior axis); stunted allantois that fails to fuse with chorion

Lechleider et al. (2001); Tremblay et al. (2001); Hayashi et al. (2002)

Waldrip et al. n/d Failure of primitive streak (1998); Tremblay (anteroposterior et al. (2001) axis) formation; failure of definitive endoderm formation, thereby disrupting hindgut development; mutant allantois identified but morphology and development not assessed Continued

Table 1.1 Genes required for the development of PGCs in the posterior region also affecting the development of surrounding soma—cont'd Posterior tissue(s) concurrently Earliest stage affected at which PGC PGC A-P Hg Al Details of “markers” defect is axis posterior defects Reference reported PGC defect Gene Gene product used

Smad4


AP activity

E8.5

The majority of mutants lacked PGCs; the remaining mutants had fewer than 10 PGCs

þ

þ

Chu et al. (2004) n/d Broadened primitive streak (anteroposterior axis) with disorganized posterior region; failure of definitive endoderm formation, thereby disrupting hindgut development; mutant allantois identified but morphology and development not assessed

Smad5


AP activity; Oct-3/4, Stella, and Ifitm3 expression

LS stage (E7.0)

n/d þ No PGCs in about 20% of null embryos; remaining mutants exhibit reduced PGC population with mislocalization, many into the amnion

þ

Extension of primitive streak into allantois not examined; delayed formation of hindgut invagination; delayed allantoic elongation; most mutant allantoises fuse with chorion but remain short and irregularly shaped

Sox17

HMG box transcription factor

AP activity; Ifitm3 expression; STELLA protein localization

7–8-s stage (E8.5)

n/d þ Mutant PGC population is reduced and mislocalizes to extraembryonic endoderm when wild-type PGCs are primarily in the hindgut

Kanai-Azuma et al. Extension of primitive streak (2002); Hara et al. into allantois not (2009) examined; failure of hindgut to expand; development of allantois not examined

Chang et al. (1999); Chang and Matzuk (2001); Bosman et al. (2006)

Continued

Table 1.1 Genes required for the development of PGCs in the posterior region also affecting the development of surrounding soma—cont'd Posterior tissue(s) concurrently Earliest stage affected PGC at which PGC “markers” defect is A-P Hg Al Details of axis posterior defects Reference Gene Gene product used reported PGC defect

Wnt3a

a


AP activity; 4–6-s stages Mutant PGCs PRDM1 and (E8.25–8.5) form a reduced population IFITM3 double IF

þ

n/d

Posterior axis truncation; chorioallantoic fusion with circulation established by E9.5

Takada et al. (1994); Bialecka et al. (2012)

Staging listed as reported; where morphological staging was reported, approximate embryonic day stage has been included. Because Cdx2-null mutants die prior to gastrulation due to defects in trophoblast development (Chawengsaksophak et al., 2004), analysis of PGCs, anteroposterior axis, hindgut, and allantois occurred in tetraploid-rescued mutants with wild-type trophectoderm and Cdx2-null inner cell mass, which contributes to future epiblast, (Chawengsaksophak et al., 2004) or in Sox2-Cre transgenic mice that conditionally knock out Cdx2 in the epiblast only (Bialecka et al., 2012). Abbreviations: Al, allantois; A-P, anteroposterior; AP, alkaline phosphatase; EB, early (allantoic) bud; HF, headfold; Hg, hindgut; IF, immunofluorescence; LB, late (allantoic) bud; LS, late streak; NP, neural plate; OB, no (allantoic) bud; s, somites. b


27

conditional knockouts exhibited rescued labyrinth expansion was not reported (Robertson et al., 2007). It is possible that an allantoic defect precludes the birth of Prdm1 conditional knockout pups, or that conditional deletion of Prdm1 in the chorion, but not the allantois, rescues labyrinth expansion. Further, the status of the fetal-umbilical connection in the conditional knockouts was not reported. Finally, regarding the role of PRDM1 in PGC fate, the key experiment, gonadal analysis in these mutants, was not carried out (Robertson et al., 2007). 3.2.5 STELLA Single-cell transcriptome analysis of the allantoic Tnap-expressing population within the early allantoic bud revealed that cells with strong Tnap expression also exhibited expression of Ifitm3 (Section 3.2.6) and Stella (Saitou et al., 2002). While Ifitm3 was more broadly expressed in the posterior region during allantoic elongation (Section 3.2.6), Stella was claimed to be expressed in a more spatially restricted posterior population, first in the base of the precursor allantois (E7.0), followed by the base of the elongating allantois (E7.25–8.25), and then exclusively in the developing hindgut by E8.5 (Saitou et al., 2002). However, this study was carried out via wholemount analysis, rather than sectional analysis, the latter of which offers a much more detailed report of tissue-specific localization. Because (i) this Ifitm3/Stella population in the allantois/posterior region exhibited repression of homeobox genes relative to adjacent somatic cells (Saitou et al., 2002), (ii) the Ifitm3-expressing population failed to form in the proximal epiblast of Bmp4-null mutants (Saitou et al., 2002), which do not form AP-positive PGCs (Lawson et al., 1999) (Table 1.1), and (iii) STELLA protein also localized to dissociated germ cells positive for SSEA-1 (Wu and Chow, 2005) from the E12.5 gonad (Saitou et al., 2002), it was concluded that the Ifitm3/Stella population represented segregated PGCs. However, no lineage tracing experiments were done to support or negate this conclusion. Moreover, the repression of homeobox gene expression also occurs in ES cells (Lee et al., 2006); thus, while repression of homeobox gene expression indicates pluripotency, it does not necessarily indicate a PGC population. Therefore, there was no evidence that Stella-positive cells, like the Tnappositive cells from which they were derived, identified PGCs, either their precursors or their segregated descendants, within the allantois. Stella-null mutants are fertile (Bortvin et al., 2004; Payer et al., 2003), though STELLA is required as a maternally inherited factor to protect against DNA demethylation in the preimplantation conceptus (Nakamura

28


et al., 2007; Payer et al., 2003). If STELLA plays a similar role in the germ line, then its activity against DNA demethylation is not required for germline formation or function (Nakamura et al., 2007; Payer et al., 2003). Later studies of STELLA and their relevance to the germ line will be discussed in the context of emerging insight into the allantois (Section 5) and in an analysis of where the field stands now (Section 7.2). 3.2.6 IFITM3 Ifitm3 is expressed broadly within the conceptus, and its expression alone is not thought to be unique to PGCs (Lange et al., 2003; Tanaka and Matsui, 2002). Prior to gastrulation, Ifitm3 is initially expressed weakly throughout the epiblast (E6.0) but subsequently becomes restricted to the proximal epiblast (E6.25–6.5), to which the PGCs are thought to localize (Saitou et al., 2002). However, while Prdm1-expressing PGCs localize to only the right side or presumptive posterior end of the anteroposterior embryonic axis, Ifitm3 appears simultaneously on both left and right sides of the proximal epiblast and is therefore not thought to be specific to segregated PGCs (Ohinata et al., 2005). With the formation of the primitive streak (E6.5), Ifitm3-expressing cells translocate posteriorly. Based on single-cell transcriptome analysis at the late streak (E7.0) and early (allantoic) bud stages (E7.25), Ifitm3 is expressed in the same cells that express Prdm1 (Ohinata et al., 2005); these same cells also express Stella by the early (allantoic) bud stage (E7.25) (Ohinata et al., 2005; Saitou et al., 2002). However, Ifitm3 expression is not confined to the PGC population, as Stella-negative somatic cells within the allantois also express Ifitm3 (Saitou et al., 2002). While Ifitm3 is thought to identify PGCs in the hindgut, the extent to which it localizes with Stella and other PGC markers remains obscure. Intriguingly, previous reports demonstrated that IFITM3 localizes as a cytoplasmic spot with cell surface staining within PGCs of the elongating allantois (Matsui and Okamura, 2005; Saitou et al., 2002). Recent systematic analysis revealed that this IFITM3 protein profile was unique to the posterior region, as IFITM3 in other sites of the conceptus exhibited other profiles (Mikedis and Downs, 2013). However, the cytoplasmic spot of IFITM3 with accompanying cell surface staining was not unique to the PGC trajectory; it was also found throughout the allantois, as well as in posterior mesoderm, surface ectoderm, and the ventral ectodermal ridge (VER) (Mikedis and Downs, 2013), a stem cell pool created by the remnant primitive streak (Goldman et al., 2000; Gru¨neberg, 1956). In several


29

of these posterior tissues, including the allantois, IFITM3 colocalized with Flk1 and Runx1 (Mikedis and Downs, 2013), which identify endothelial (Shalaby et al., 1995) and hematopoietic (North et al., 2002) progenitor cells, respectively. This is consistent with the hematopoietic potential of so-called PGCs (Rich, 1995) and the allantois (Corbel et al., 2007; Zeigler et al., 2006). In addition, in the ventral hindgut, cells with the round morphology associated with PGCs exhibited IFITM3 as a cytoplasmic spot with cell surface staining or as a cytoplasmic spot alone, a subcellular localization profile that was found in a variety of non-PGC tissues of the conceptus (Mikedis and Downs, 2013). Thus, IFITM3 cannot be used to identify the PGC lineage (Mikedis and Downs, 2013). Ifitm3-null mutants are fertile (Lange et al., 2008), but ectopic expression has suggested that IFITM3 may play a role in facilitating PGC localization from mesoderm to hindgut endoderm within the posterior region (Tanaka et al., 2005). This conclusion came from experiments in which vectors constitutively expressing Ifitm3 were electroporated into somatic cells of the visceral endoderm at neural plate stages (no (allantoic) bud and early bud stages, designated as E7.5; Tanaka et al., 2005). After culture, cell populations that had received the Ifitm3 expression vectors were reported to localize more frequently to the hindgut endoderm compared to those that had received GFPexpressing control vectors (Tanaka et al., 2005). Unfortunately, the control specimen displayed in the manuscript (Fig. 4V of Tanaka et al., 2005) was at an earlier developmental stage than the experimental specimen and had not yet formed a hindgut, thereby calling into question the validity of this conclusion. 3.2.7 NANOG NANOG, a homeodomain transcription factor found in many pluripotent cells (Saunders et al., 2013), was not detected in the posterior region of the mouse conceptus until E7.75, when, based on sectional analysis, it localized to 50% of cells exhibiting STELLA protein (Yamaguchi et al., 2005). By E8.5, however, when the PGC population had translocated into the hindgut endoderm, 90% of STELLA-positive endodermal cells colocalized NANOG (Yamaguchi et al., 2005). Analysis at every embryonic day thereafter revealed that this degree of colocalization persisted along the PGC trajectory through E12.5, when the germ cells have colonized the gonads (Yamaguchi et al., 2005). While the authors claimed that all STELLA-positive cells exhibited NANOG, their data presented a different conclusion (Table 1 from Yamaguchi et al., 2005), clearly showing that many (10%) STELLA-positive cells did not (Yamaguchi et al., 2005).

30


The presence of a STELLA-positive, NANOG-negative population within the hindgut would probably represent a different lineage from the STELLAand NANOG-positive cells, again calling into question what is and is not a PGC within the PGC trajectory. Conditional knockdown of Nanog (Yamaguchi et al., 2009) via short hairpin RNA, which was expressed upon Cre-mediated recombination driven either by activation of the estrogen receptor from tamoxifen injection of pregnant females at E7.5 or by endogenous activation of the Tnap promoter, resulted in fewer SSEA1-positive germ cells in E12.5 gonads (Yamaguchi et al., 2009). In addition, tamoxifen-mediated deletion of Nanog resulted in increased apoptosis of SSEA1-positive PGCs as early as E9.5 (Yamaguchi et al., 2009). Therefore, Nanog is required for the survival and maintenance of the hindgut PGCs during later stages of migration to the gonads (Yamaguchi et al., 2009). As mentioned in a previous section, Tnapdriven Cre recombination activity was not detected until E9.0 (Lomeli et al., 2000; described in Section 3.2.2). In addition, tamoxifen injections at E7.5 are not expected to induce estrogen receptor-driven Cre activity until E8.5 (Hayashi and McMahon, 2002; Yamaguchi et al., 2009). Therefore, as neither system conditionally knocked-out/knocked-down Nanog expression before the PGCs translocated into the hindgut endoderm, the role of Nanog in the development of the PGC population during localization to the posterior region remains obscure.

3.3. Errant PGCs Cells exhibiting PGC markers outside of the PGC trajectory (e.g., allantois, hindgut, and gonads) are generally considered to be errant PGCs that have lost their way to the gonads (e.g., Tres et al., 2004). Within the adrenal glands or within tissues near, but outside of, the gonads, “off-piste” PGCs develop into cells that appear morphologically similar to differentiating male and female germ cells (Francavilla and Zamboni, 1985; Upadhyay and Zamboni, 1982). Many of these “ectopic germ cells” were observed degenerating (Francavilla and Zamboni, 1985) and were not detectable after birth (Upadhyay and Zamboni, 1982). In other tissues, ectopic PGCs can, on rare occasion, form extragonadal germ cell tumors (Runyan et al., 2008). Based on live imaging observations of PGCs identified via the Oct-3/ 4DPE:GFP reporter (Anderson et al., 2000; Yeom et al., 1996), other errant PGCs are thought to die rather than differentiate into ectopic germ cells or extragonadal germ cell tumors (Anderson et al., 2000; Molyneaux et al., 2001; Stallock et al., 2003). “Ectopic” PGCs persist in the dorsal mesentery


31

without migrating toward the gonads (Molyneaux et al., 2001; Stallock et al., 2003). These Oct-3/4-expressing cells fragment and disappear. By contrast, in mutants that lack the proapoptotic gene Bax, these cells remain in the dorsal mesentery (Stallock et al., 2003). Unfortunately, lacking in these studies were controls showing the presence of such fragmented cells in uncultured, ex vivo material that was immediately fixed after dissection. Thus, whether apoptosis is an artifact of the tissue culture conditions used during imaging remains obscure. Results of the aforementioned studies do not preclude the possibility that “ectopic” PGCs contribute to surrounding tissues prior to apoptosis, especially as recent evidence suggests that proapoptotic proteins play a role in a pluripotent cell’s ability to differentiate. Specifically, mouse embryonic stem (ES) cells nullizygous for Caspase-3, which encodes a protease that mediates apoptosis, exhibited defects in differentiation (Fujita et al., 2008). Caspase-3 can be upregulated through a Bax-mediated pathway (Cregan et al., 1999); moreover, Bax-null germ cells from E14.5 gonads formed EG cell colonies at a greater frequency than wild type (Runyan et al., 2008). Though not discussed by the authors, these results suggest that loss of Bax may maintain germ cells in a more pluripotent state than their wild-type counterparts. If Bax does play a role in the balance between pluripotency and differentiation, and the PGCs are, in reality, a pluripotent population that contributes to multiple lineages (see Section 5), then the increase in ectopic PGCs observed in Bax-null mutants (Runyan et al., 2008; Stallock et al., 2003) may be the result of disrupted differentiation and/or loss of Bax-mediated apoptosis. Therefore, it remains possible that “ectopic” PGCs differentiate into somatic cells.

4. COMPARISON OF MAMMALIAN GERMLINE PROGRAM TO THAT ACROSS METAZOA Many of the “markers” used to identify early PGCs in the posterior region of the mouse conceptus are not conserved components of germline development across metazoa. Neither AP nor Tnap activity has been associated with any nonmammalian germ line. Stella homologs have only been identified in mammals (Flicek et al., 2013); Ifitm3 homologs have only been identified in vertebrates (Flicek et al., 2013; Hickford et al., 2012; Siegrist et al., 2011); and Nanog homologs have only been identified in jawed vertebrates (Camp et al., 2009; Flicek et al., 2013; Schuff et al., 2012). Prdm1 has homologs throughout bilateral animals, but its putative roles in germline development

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appear to be unique to mice/mammals (Flicek et al., 2013; John and GarrettSinha, 2009). This is in contrast to genes such as Nanos, Ddx4 (Vasa, Mvh), and Dazl. While the specific functions of these gene products are not conserved across species, these genes are conserved components of germline development (Ewen-Campen et al., 2010; Johnson et al., 2003). Intriguingly, in mouse, Nanos3 (Tsuda et al., 2003), Ddx4 (Toyooka et al., 2000), and Dazl (Hackett et al., 2013; Seligman and Page, 1998; Yamaguchi et al., 2013) have been associated with the germ line as early as E9.5–E10.5, developmental stages that are well past the point of PGC localization to the posterior region and the completion of allantoic elongation. Because lineage restriction of mouse PGCs has not been demonstrated, it is possible that they only become restricted once expression of the evolutionarily conserved components of germline development has been initiated. Prior to that, they may function as a pluripotent population that contributes to both somatic tissues (see Section 5) and the germ line. This is similar to axolotl, in which the posterior region contains a pluripotent population that can be induced to form blood or PGCs, depending on the protein signals received (Johnson et al., 2011). The axolotl germ line is thought to become restricted at later stages (axolotl late tailbud stages; approximately equivalent to E9.0–9.5 in mouse), when Dazl and Ddx4 homologs begin to be expressed (Bachvarova et al., 2004). Intriguingly, mouse PGC proteins PRDM1 (Turner et al., 1994) and IFITM3 (Mikedis and Downs, 2013; Smith et al., 2006) have also been associated with hematopoietic cells, suggesting that mouse blood cells and PGCs exhibit a close developmental lineage within the posterior region. Another conserved characteristic of the germ line across species is nuage, or germ granules, a collection of perinuclear fibrillar material and dense core vesicles unique to the germ line (Voronina et al., 2011). Nuage is also conserved in its components, which include proteins of germline development, DDX4 and NANOS (Voronina et al., 2011). Intriguingly, nuage is not sufficient for inducing germline fate, even in organisms in which the germline fate is inherited via germ plasm, as C. elegans embryos which mispartition nuage (called P granules) do not form extra germ cells (Gallo et al., 2010). In mouse, dense core vesicles are associated with nuage in the PGCs of the hindgut at E9.0–9.5 (Clark and Eddy, 1975). Presomitic embryos (designated as E8.0–8.5 by authors) exhibit small vesicles that contain “a suggestion of a dense core” in AP-positive PGCs, but similar structures were also found in surrounding somatic cells (Clark and Eddy, 1975). Thus, in mouse, perhaps nuage distinguishes a lineage-restricted germ line from soma


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after E9.0. However, whether nuage is required for germ cell development in the mouse remains to be seen.

5. EMBRYONIC–EXTRAEMBRYONIC INTERFACE AND FETAL–UMBILICAL CONNECTION: PGCs AND ACD Most of the aforementioned PGC “markers” are associated with pluripotency (Benham et al., 1983; Bernstine et al., 1973; Leitch and Smith, 2013). It has been suggested that PGC proteins indicate a latent pluripotent state, in which pluripotency is dormant or poised during normal development and revealed only during EG cell derivation or teratocarcinogenesis (Leitch and Smith, 2013). However, this hypothesis is based on the assumption that PGCs do not give rise to any somatic cell types during development (Leitch and Smith, 2013). Since, as discussed above (Section 3), cells exhibiting these proteins cannot be claimed to be a segregated germ line without rigorous experimental analysis, an alternative view, consistent with all of the data, is that PGCs are part of a pool of progenitor cells for somatic lineages, including the extraembryonic allantois, that build the posterior region. The rationale is described below. In their transition to life on land, amniotes (reptiles, birds, and mammals) evolved two major features not found in anamiotes (fish and amphibians): the primitive streak and extraembryonic tissues (also called fetal membranes), the latter of which encompass the amnion, vascular yolk sac, trophoblast (chorion), and allantois (Stern and Downs, 2012). The primitive streak is not only the overt manifestation of the anteroposterior axis, generating bilateral symmetry in the embryo, but it is also the conduit through which epiblast is transformed into two of the primary germ layers, mesoderm and definitive endoderm (Tam and Beddington, 1987). Mesoderm is the source of the entire circulatory system, both embryonic and extraembryonic, allowing the embryo/fetus to develop either within an egg or the maternal reproductive tract. Eutherian, or placental mammals, exhibit an extreme form of viviparity in which the fetus is wholly dependent on the mother throughout gestation for its supply of nutrients, gases, and the elimination of toxic wastes. In these mammals, the allantois gives rise to the placenta’s umbilical component, whose vessels carry fetal blood to and from the chorionic disk, where fetal-maternal exchange takes place. The three main circulatory systems of amniotes, derived from the allantois, yolk sac, and fetus, are established

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independently of each other (Downs, 2003). At a specific developmental timepoint, they amalgamate and become confluent at a precise midline site in register with the primitive streak (Daane and Downs, 2011; Downs et al., 1998; Inman and Downs, 2006). So as to achieve maximally efficient exchange with the environment, it would seem that the streak provides the necessary spatial coordinates to ensure vascular confluence at the embryonic–extraembryonic interface (discussed in Daane and Downs, 2011). Recently, we have unexpectedly discovered in mouse that, once the exocoelom forms, the posterior end of the embryonic streak extends into it (Fig. 1.1C1), creating the allantois and a stem/progenitor cell pool, the ACD (Figs. 1.1C2, 1.2A1, and 1.2B1) (Downs et al., 2009). The ACD encompasses the population of so-called segregated PGCs and contains all known PGC proteins, including AP, STELLA, OCT-3/4, PRDM1, IFITM3, and NANOG. With the exception of IFITM3, these proteins also play established roles in pluripotency. Retrospective analysis of clonal fate maps clearly demonstrates that the ACD originates from the cell population in the proximal epiblast claimed to be the progenitors of the PGCs (Lawson and Hage, 1994; Lawson et al., 1991). While fate and potency mapping have demonstrated that the distal end of the allantois contributes only to mesodermal derivatives and is thus limited in its developmental potential (Downs and Harmann, 1997; Mikedis and Downs, 2012), the ACD exhibits properties of a broader stem/progenitor cell pool. In particular, it resembles Hensen’s node, or the anterior condensation of the primitive streak (Fig. 1.2). First, DiI applied to the ACD (Fig. 1.3A1) resulted in a midline file of cells that extended through the allantois (Fig. 1.3A2) (Downs et al., 2009), similar to the node and its notochordal extension (Beddington, 1994) (Fig. 1.2A). At the same time, some of the DiI persisted in the ACD, suggesting the presence of a self-maintaining stem cell pool there (Fig. 1.3A2) (Downs et al., 2009), also similar to the node (Beddington, 1994). By contrast, labeling the midline of the caudal region of the intraembryonic posterior primitive streak (IPS) (Fig. 1.3B1) revealed no midline contributions typical of the anterior node, but rather only contribution to lateral mesoderm in the allantois (Fig. 1.3B2) (Downs et al., 2009). Contribution to laterally displaced mesoderm is typical of the nonnodal components of the streak (Tam and Gad, 2004). In addition, label placed on either side of the allantoic midline (Fig. 1.3C1) was displaced toward the distal allantois by IPS-derived mesoderm (Fig. 1.3C2). Microsurgical removal of the ACD (Fig. 1.4A1) resulted in truncated allantoic


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Figure 1.3 Unique contribution of ACD to the allantoic midline and distal allantois, based on fate mapping via vital dye (red). Based on experiments from Downs et al., 2009. Panels are frontally oriented, with the posterior end up, and thus left is to the left, and right is to the right. (A) Labeling of the proximal midline of the allantois (A1), (Continued)

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regenerates composed of IPS-generated mesoderm (Fig. 1.4A2). Similarly, genetic loss of the T-defined ACD in various Brachyury (T) mutants (Fig. 1.4B1) resulted in a stunted allantois that failed to elongate (Fig. 1.4B2) (Gluecksohn-Schoenheimer, 1944; Inman and Downs, 2006; Shedlovsky et al., 1988). Grafts of wild-type ACD rescued allantoic elongation in homozygous T-curtailed (T C/T C) mutants and in those embryos whose ACD had been microsurgically removed (Fig. 1.4C–E) (Downs et al., 2009). Intriguingly, fate mapping the ACD and IPS by grafting revealed distinct and overlapping contributions to derivatives of all three primary germ layers at the fetal–umbilical interface (Fig. 1.5) (Mikedis and Downs, 2012). Thus, the ACD and IPS are two distinct domains of the primitive streak. A compartmentalized primitive streak is consistent with previous experiments in which segments of the streak cultured in isolation exhibited autonomous development of embryonic body structures (Snow, 1981). Thus, the streak is capped at both ends by structures, Hensen’s node and the ACD, that extend the body axis anteriorly and posteriorly, respectively. Although the specific organizational properties of the ACD have not yet been described, the evidence suggests that this element plays a major role in patterning the fetal-umbilical connection. Our interpretation of the ACD and its relation to the PGCs is consistent with Ozdzenski’s conclusion that “the embryonic rudiment of the allantois represents an extension of the primitive streak and undoubtedly originates from it. It seems justifiable, therefore, to consider the caudal end of the primitive streak and the embryonic rudiment of allantois jointly as a region of formation of PGCs” (Ozdzenski, 1967). While Ozdzenski was not aware of the ACD and the significance of this posterior end of the streak in terms of its developmental potential and contribution to the fetal–umbilical region, he nevertheless considered the posterior region as a unified embryonic–extraembryonic interface, the significance of which is only just coming to light (Downs, 2011). Therefore, mouse PGCs localizing to the ACD may

Figure 1.3—Cont'd followed by 20 h of whole embryo culture, resulted in labeled descendants both remaining in place and forming a midline file up the allantois and expanding into the distal allantois (A2). (B) Labeling of the posterior embryonic midline, or intraembryonic primitive streak (IPS), below the allantois (B1), resulted in lateral-labeled descendants in the posterior region of the embryo and in the flanks of the proximal allantois (B2), all exclusive of the midline. Labeling of the flanks of the elongating allantois (C1) resulted in labeled descendants that were displaced to the midregion of the allantois, but confined to its lateral flanks (C2).


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Figure 1.4 T-positive ACD required for elongation of allantois, based on microsurgical and genetic analyses. Panels are sagittally oriented with anterior to the left and posterior to the right. (A) When the wild-type ACD was microsurgically removed at headfold stages (WT ACD; A1), followed by 20 h of whole embryo culture, the allantois did not elongate, forming a stunted allantoic remnant (A2) (Downs et al., 2009). (B) Similarly, genetic loss of the ACD in T-curtailed (T c) mutants (B1) resulted in a stunted and misshapen allantois that failed to elongate, based on analysis of control ex vivo specimens that developed entirely in utero (B2) (Inman and Downs, 2006). (C–E) A wild-type ACD graft isolated from a lacZ-labeled donor conceptus at headfold stages (C, blue) and grafted into a wild-type host whose ACD has been removed (WT ACD, þACD graft; D1) or into a Tc/Tc homozygous mutant (Tc/Tc þ ACD graft; E1) rescued allantoic elongation in both scenarios (D2, E2) (Downs et al., 2009). Chorioallantoic union was rescued by the wild-type ACD graft in the wild-type conceptus (D2) but not in the Tc/Tc homozygous mutant (E2).

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Figure 1.5 “PGC”-containing ACD and fetal–umbilical connection, based on fate mapping via grafting. Based on experiments from Mikedis and Downs, 2012. Panels are sagittally oriented with anterior to the left and posterior to the right. (A) Grafts were


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function not as PGCs but as a progenitor cell pool that contributes to the derivatives of all three primary germ layers. This hypothesis is supported by systematic analysis of STELLA protein in sections of mouse gastrulae at 2–4-h intervals. STELLA first appeared in the extraembryonic extension of the primitive streak (XPS) or precursor tissue of the ACD (neural plate/no (allantoic) bud stage; E7.0) and persisted there as the allantoic bud formed (early and late bud stages; E7.25–7.5) (Mikedis and Downs, 2012). Shortly after ACD formation at headfold stages (E7.75–8.0), STELLA expanded across the embryonic–extraembryonic junction into the IPS and also appeared within the PVE overlying the embryonic–extraembryonic interface (Mikedis and Downs, 2012). By 6-somite pairs (E8.5), when the T-defined ACD appeared to regress/differentiate, STELLA-positive cells localized primarily to the hindgut. However, small populations persisted outside of the PGC trajectory, specifically in the allantois, posterior surface ectoderm, posterior mesoderm, and VER (Mikedis and Downs, 2012). While the localization of STELLA at later timepoints, before PGC colonization of the gonads, is based on limited analysis at E9.5 (Sato et al., 2002; Yamaguchi et al., 2005), these data demonstrate that STELLA protein alone cannot distinguish a PGC from surrounding soma within the mouse conceptus. To discover the contribution of tissues that contained STELLA to the posterior region, the latter (headfold stage, E7.75–8.0) was subdivided into

isolated from the posterior region of lacZ-labeled donor embryos at headfold stages (E7.75–8.0). (B–E) All grafts were inserted into the posterior region of wild-type host conceptuses at headfold stages (E7.75–8.0), cultured for 20 h, and analyzed for lacZpositive graft contribution (blue) and STELLA at 8–12-somites (E8.5–9.0). (B) Approximate synchronous orthotopic grafting of the distal allantois into the proximal ACD (WT þ distal allantois graft; B1) revealed contribution to the distal allantois (B2). All contribution was STELLA-negative. (C) Approximate synchronous orthotopic grafting of the distal ACD into the proximal ACD (WT þ distal ACD graft; C1) revealed contribution to the allantois and posterior vasculature (C2). All contribution was STELLA-negative. (D) Synchronous orthotopic grafting of the proximal ACD (WT þ proximal ACD graft; D1) revealed contribution to the allantois and multiple lineages within the embryo proper (D2). Contribution included STELLA-positive cells in the allantois and hindgut (not shown). (E) Synchronous orthotopic grafting of the intraembryonic posterior primitive streak (WT þ IPS graft; E1) revealed contribution to the proximal allantois and multiple lineages within the posterior embryo (E2). Contribution included STELLA-positive cells to the allantois, hindgut, posterior mesoderm, and VER (not shown).

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four components: IPS, proximal ACD, distal ACD, and distal allantois (Fig. 1.5). The majority of the posterior region’s STELLA-positive cells were evenly distributed to the former two regions, while STELLA rarely localized to the distal ACD and never to the distal allantois. The distal allantois (Fig. 1.5A and B1) and distal ACD (Fig. 1.5A and C1) were approximately orthotopically grafted into the proximal ACD, while the proximal ACD (Fig. 1.5A and D1) and IPS (Fig. 1.5A and E1) were orthotopically grafted into host conceptuses. Host conceptuses were then cultured, after which the resulting chimeras were colocalized for both the lineage tracer (lacZ expression based on b-galactosidase activity) and for STELLA. The distal allantois and distal ACD never produced STELLA-positive cells (Fig. 1.5B2 and C2). By contrast, while some ACD- and IPS-derived STELLA-positive cells localized to the hindgut, additional STELLAexhibiting cells were found in other posterior sites, including the allantois, posterior mesoderm, and VER (Fig. 1.5D2 and E2) (Mikedis and Downs, 2012). These results revealed that STELLA is not confined to a single lineage; rather, STELLA contributes to multiple cell types in the posterior region (Mikedis and Downs, 2012). Results further revealed that the total number of graft-derived STELLA-positive cells at the end of the study was significantly less than those at the beginning. As cell death was ruled out, this suggests that many STELLA-positive cells differentiated into STELLA-negative somatic cells, further underscoring the conclusion that STELLA does not identify a single lineage across multiple stages of embryonic development (Mikedis and Downs, 2012). Based on the aforementioned new evidence concerning (i) the whereabouts of the posterior end of the streak, (ii) systematic localization of PGC “marker” proteins to tissues outside of the PGC trajectory, and (iii) results of fate mapping this region, we have adjusted the current model of PGC segregation (Fig. 1.1B) to account for the presence of the ACD (Fig. 1.1C). We propose that those cells designated “PGCs” on the basis of AP activity and/or exhibition of the proteins described in Section 2 are progenitor cells that build the posterior region of the mouse conceptus. Translocation of some of these cells from the base of the allantois into the distal allantois, associated PVE, and underlying IPS (Fig. 1.1C2) is coordinated with the timing of appearance of the ACD at the headfold stage (7.75–8.0; Downs, 2008; Mikedis and Downs, 2012, 2013). Dispersal of these cells knits together the embryonic-extraembryonic interface, thereby ensuring a path to the mother’s bloodstream via a robust fetal– umbilical connection (Fig. 1.1C3).


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This revised model is also consistent with recent reports that ES cells can be differentiated into so-called primordial germ cell-like cells (PGCLCs) that express Stella and Prdm1 (Hayashi et al., 2011; Hayashi et al., 2012). Furthermore, PGCLCs sorted for Prdm1 reporter expression can give rise to functional sperm (Hayashi et al., 2011) and oocytes (Hayashi et al., 2012), which can contribute to normal offspring. While exciting from a broader scientific standpoint, these results do not provide information regarding the PGC lineage in vivo: they demonstrate only potency, rather than fate, of the Stella- and Prdm1-expressing PGCLC population. Intriguingly, Prdm1-enriched PGCLCs differed from the unsorted PGCLC population in that when injected into the seminiferous tubules, only the latter formed teratomas, suggesting different potencies for these populations (Hayashi et al., 2011). However, to obtain a more complete understanding of the potency of the enriched PGCLCs, it would be interesting to assess whether this population can contribute to somatic lineages outside of the context of the gonad, as signaling pathways within the gonad may cause the enriched PGCLCs to favor germ cell, rather than somatic, differentiation.

6. LOSS/MISLOCALIZATION OF PGCs AND ASSOCIATED POSTERIOR DEFECTS If, as suggested by the fate mapping results discussed above, the ACD’s PGCs are progenitor cells for the posterior region, we would predict that genetic mutations that affect the so-called PGCs would also simultaneously affect the allantois, anteroposterior axis, hindgut, and other posterior somatic tissues to which the ACD contributes (Fig. 1.5D2). Indeed, of all known mutations that affect the PGCs within the posterior region (up to E8.5–9.0; Tables 1.1 and 1.2), the vast majority also affects development of the surrounding posterior soma (Table 1.1). Specifically, these mutants exhibited defects in embryonic axis formation, allantoic development, and/or hindgut development. Defects in these tissues are consistent with defects in the ACD, whose cells contribute to all of them. These mutants include knockouts for components of the TGFb/BMP signaling pathway, thought to “induce” formation of the PGCs in the proximal epiblast (Lawson et al., 1999; Ying and Zhao, 2001; Ying et al., 2000). As the current model of PGC origin (Section 2) places the PGCs specifically on the right side of the proximal epiblast, or presumptive posterior end of the anteroposterior axis, an alternative model is that the TGFb/BMP signaling

Table 1.2 Genes required for the development of PGCs in the posterior region but not for the development of surrounding soma Earliest stage at which PGC defect is PGC defect Noted abnormalities Reference reporteda Gene Gene product PGC “markers” used

Mir-290 MicroRNAs –mir-295

Prdm14

a

Oct-3/4:GFP reporter E8.5

EB stage Transcriptional AP activity; Prdm1repressor mVenus reporter; Stella (E7.25) transcription; STELLA protein localization

Mutant PGCs mislocalize to base of allantois when wild-type PGCs localize to hindgut

Medeiros Homozygous mutants et al. result in partially (2011) penetrant embryonic lethality, with resorptions observed as early as E9.5; of surviving homozygous mutants, females are sterile while males are fertile

Reduced mutant PGC population in the posterior region; Stella transcription is reduced and protein is undetectable in this population

Mutants lack germ cells in Yamaji et al. the gonads; otherwise, (2008) pups appear grossly normal and are born at expected Mendelian ratios

Staging listed as reported; where morphological staging was reported, approximate embryonic day stage has been included. Abbreviations: EB, early (allantoic) bud.


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pathway induces the formation of this posterior end of the primitive streak, rather than the PGCs per se. Of the many mutations that affect the extragonadal posterior PGC population (Tables 1.1 and 1.2), only two are specific to PGCs without (apparently) affecting the surrounding soma: mir-290–295 and Prdm14 (Table 1.2).

6.1. Mir-290–295 Mir-290–295 (Table 1.2) is a mammalian-specific microRNA cluster required for localization of the Oct-3/4:GFP-expressing PGCs as early as E8.5, when the majority of wild-type reporter-expressing PGCs are in the hindgut but the majority of mir-290–295 mutant PGCs are in the base of the allantois (Medeiros et al., 2011). However, mislocalization is not particularly severe, as some mutant PGCs do successfully colonize the gonads by E11.5 (Medeiros et al., 2011). Therefore, if the PGCs are actually a pluripotent progenitor population rather than lineage-restricted germ line, it is not surprising that surrounding somatic tissues are apparently unaffected (Medeiros et al., 2011).

6.2. Prdm14 Prdm14 is also required for PGC development (Table 1.2). PRDM14 protein colocalizes with Prdm1 reporter expression at the late streak/no (allantoic) bud stage (E7.0) in what has now been identified as the extraembryonic component of the primitive streak (XPS) that extends into the exocoelom (E7.0–7.5; Fig. 1.1C1) before expanding into the ACD (E7.75–8.0; Fig. 1.1C2) (Downs et al., 2009). Prdm14 transcripts have also been detected in Prdm1-expressing cells at mid-to-late streak (E6.75–7.0) and (allantoic) bud (E7.25–7.5; Fig. 1.1C1) stages (Yamaji et al., 2008). In the absence of Prdm14, the PGCs, which were visualized either via TNAP activity or via Prdm1 reporter expression, were reduced as early as the neural plate/early (allantoic) bud stage (E7.25; Table 1.2) (Yamaji et al., 2008), presumably in the XPS. In addition, within the PGC population that did form, Stella transcription was reduced based on single-cell transcript analysis, and STELLA protein was not immunofluorescently detectable in the posterior region (Yamaji et al., 2008). Prdm14 nullizygous mice were grossly normal and born at expected Mendelian ratios, but their gonads were devoid of germ cells (Yamaji et al., 2008). While Prdm14’s extragonadal localization and exclusive requirement in the germ line suggests the existence of an extragonadal germ line, it does not mean that the PGCs are lineage

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restricted. Other explanations are possible. First, as Prdm14 was not carefully spatiotemporally localized, it is not clear whether its requirement in germ cell formation might actually occur at a later stage elsewhere; thus, Prdm14-expressing, lineage-restricted PGCs may form outside of the allantois. Second, based on the model of the PGCs as a pluripotent population, loss of Prdm14 in the PGCs may prevent maintenance of germline competence, allowing the PGCs to contribute normally to surrounding somatic tissues but not to germ line. Therefore, even mutations that are claimed exclusively to affect the PGC population provide no insight into what is and is not a PGC.

7. PERSPECTIVES 7.1. Alternative models Chiquoine’s model of PGC development has been so widely accepted that, to our knowledge, only one study prior to recent papers (Mikedis and Downs, 2012, 2013) has challenged it and attempted to put forth an alternative theory (Soriano and Jaenisch, 1986). The origin of the germ line was queried by infecting blastomeres with RNA retroviruses at the 4- to 16-cell stages of preimplantation mouse conceptuses. Analysis of the resulting adult mice revealed that some proviral insertions found in the germ line were not found in the soma, and vice versa, leading the authors to conclude that the germ line became lineage restricted by the 64-cell stage. Unfortunately, the allantois-derived umbilical cord, with which, as described above, the germ cells are thought to share a common lineage (Lawson and Hage, 1994), was not examined. Furthermore, contribution to tissues that were replaced/ remodeled/degenerated prior to provirus analysis, such as the yolk sac’s omphalomesenteric artery (Zovein et al., 2010), would not have been detected. In addition, it is possible that the assay used was not sensitive enough to detect low levels of mosaicism present in analyzed tissues.

7.2. Where we are now Study of PGCs forms an essential foundation for rational translational applications in the treatment of infertility. Moreover, because changes in potency are implicated in most normal biological processes during early stages of development and in regeneration, as well as in abnormal ones such as tumorigenesis and metastasis, the importance of understanding PGC identity can scarcely be overemphasized. Despite the profound importance of the


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allantois in the PGC life cycle, investigators have largely ignored its biology and, especially, its developmental relationship to the early germ line. Unfortunately, Chiquoine set a low standard of proof in the study of the origin of the germ line by basing the identity of the PGCs not only on a single “marker,” which is wholly insufficient evidence for such a claim, but on one whose expression is widespread. In addition, we know now that AP activity is also a hallmark of pluripotent cells. Sixty years later, there is still a lack of definitive evidence for lineage continuity with the gonads and/or segregation of the mammalian germ line. The reason, as suggested in Section 3.1, may be one of practicality: conceptuses at stages of allantoic elongation can only be cultured for a maximum 48 h (Nagy et al., 2003), precluding them from reaching stages when the germ cells have colonized the gonads. Thus, classical embryological techniques using vital dyes or grafting, while powerful during early stages of gastrulation, cannot be used at later ones. Instead, genetic lineage tracing systems using transgenic mice with a drug-inducible, conditional reporter (Bockamp et al., 2008) may provide the only viable alternative. For example, in a system using a modified Cre recombinase (Danielian et al., 1998; Hayashi and McMahon, 2002), Cre expression is driven by the promoter from the gene of interest. The modified Cre protein normally localizes to the cytoplasm, but in the presence of tamoxifen or its derivative, hydroxytamoxifen, Cre recombinase translocates to the nucleus, where it mediates the excision of a LoxP-flanked early stop codon and thereby creates a constitutively active reporter. Therefore, only those cells expressing the gene of interest during, but not prior to or after, the drug induction period will be genetically labeled by the reporter, after which embryos can continue to develop in utero. Although it is highly unlikely that a single gene product will be restricted to the germ line, nevertheless, genetic lineage tracing of cells exclusively exhibiting Mir-290–295 and/or Prdm14 (Sections 6.2 and 6.3) might be most useful. Of the gene products best studied thus far, Stella-expressing cells may be most promising, as STELLA protein identifies a smaller, more specific population in the posterior region than Tnap (Saitou et al., 2002), OCT-3/4 (Downs, 2008), IFITM3 (Mikedis and Downs, 2013; Saitou et al., 2002), and possibly PRDM1 (Mikedis and Downs, unpublished). Indeed, one group has developed an inducible Stella genetic lineage tracing system and has demonstrated that induction during stages of PGC migration can result in labeling of germ cells in the gonads (Hirota et al., 2011). Specifically, the earliest administration of hydroxytamoxifen was E7.0, just prior

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to allantoic bud formation when Stella expression is found in the posterior end of the extraembryonic primitive streak (XPS) (Mikedis and Downs, 2012). Analysis at E12.5 revealed a small number of labeled germ cells in the gonads. Administration of hydroxytamoxifen at E8.5 and 9.5, when the Stella-expressing PGCs are in the hindgut, resulted in increasingly greater numbers of labeled germ cells in the E12.5 gonads (Hirota et al., 2011). Within the gonads, reporter expression colocalized with DDX4 (Hirota et al., 2011), which is detected in gonadal germ cells as early as E10.5 (Toyooka et al., 2000). However, as tantalizing as these results are, they do not provide solid evidence of PGC lineage restriction or even of lineage continuity between the posterior region and gonadal germ cells. First, whether the genetically labeled germ cells at E12.5 are derived from Stella-expressing cells along the PGC trajectory remains obscure because STELLA is not specific to the PGC trajectory at E8.5–8.75 (Mikedis and Downs, 2012). Furthermore, STELLA has not been systematically localized throughout the conceptus after E8.75 to investigate other potential sites of emergence. Second, whether a Stella-expressing population at any time point exclusively contributes to the germ line is unknown, as the conditional Stella reporter system was not analyzed to its full capacity: only a single timepoint (E12.5) was examined, and a systematic analysis of extragonadal regions was not performed. Third, the potential negative effects of Cre activity in specimens were not assessed. Because Cre recombinase activity in some Cre lines can cause DNA damage (Schmidt-Supprian and Rajewsky, 2007) and apoptosis (Naiche and Papaioannou, 2007), each Cre line must be appropriately monitored to determine whether some of the genetically fate mapped cells are dying, thereby obfuscating the fate map. Finally, the induction period of this Stella-Cre system has not been defined; therefore, it is unclear for how long after hydroxytamoxifen administration Cre is mediating reporter recombination and genetically labeling Stella-expressing cells. Even though tamoxifen and hydroxytamoxifen in mouse serum have half-lives of 12 and 6 h, respectively (Robinson et al., 1991), some drug-induced Cre systems used to study embryonic development have exhibited recombination up to at least 48 h after drug administration (Gu et al., 2002; Hayashi and McMahon, 2002). Consistent with this, Cre recombinase can persist in the nucleus of a minority of cells even 48 h after tamoxifen injection (Hayashi and McMahon, 2002). While the Cre protein from the conditional Stella reporter system has been modified to include the PEST sequence, a degradation signal (Rechsteiner and Rogers, 1996), so that the protein


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presumably does not persist in the nucleus long after drug administration (Hirota et al., 2011), this has not been experimentally demonstrated. As such, it remains possible that the small number of genetically labeled germ cells resulting from hydroxytamoxifen administration at E7.0 (Hirota et al., 2011) was derived from a small number of Stella-expressing cells with Cre activity as late as E9.0 but not from those at earlier stages. Therefore, genetic lineage tracing tools such as the Stella-Cre system must be fully analyzed in order to provide reliable results regarding PGC lineage continuity and segregation.

8. CONCLUSIONS The chance finding in the middle of the last century that cells in the base of the allantois are rich in AP activity appeared to settle the question of an extragonadal origin of the mammalian germ line. Despite the lack of AP specificity to the germ line, all that we know—or think we know—about PGCs and their movement in the intact embryo is based on the model established by AP activity. However, as discussed in this review, the major criteria for lineage continuity with the gonads and timing of PGC segregation from the soma have not yet been experimentally fulfilled. Moreover, almost all papers focused on the biology of the PGCs have ignored their relation to the allantois, and the possibility that PGCs are actually part of a posterior pool of cells that knits together the fetal–umbilical interface, thereby ensuring the vital vascular continuum of the conceptus with its mother. Thus, while study of the battery of PGC “marker” proteins has led to unprecedented new insight into the molecular control of pluripotency, scientists may be studying cells that have the potential to colonize the gonads, but not the lineage-restricted PGCs themselves. Until PGCs can be distinguished from the soma in vivo, and conclusively lineage traced to demonstrate their contributions during development, alternative hypotheses concerning the nature of cells bearing these proteins must be considered. Other amniote species should be examined, as the posterior end of the primitive streak may be less conserved among species than the node at the anterior end of the streak and would surely offer expanded insight into this interface (Downs, 2009). For example, studies in the rabbit suggested that the allantois is not required for a germ cell niche (Hopf et al., 2011). The data collected on the mouse PGC lineage, discussed in this review article, are entirely compatible with the alternative hypothesis that so-called

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PGCs are pluripotent cells that build the fetal–umbilical connection. Challenging dogma, especially when the evidence is weak in light of new discoveries, is essential for fully understanding the origin of the germ line in vivo.

ACKNOWLEDGMENTS K. M. D. is supported by grants from the March of Dimes (1-FY09-511) and National Institutes of Child Health and Development (R01 HD042706). M. M. M. is a National Science Foundation Graduate Research Fellow and was further supported by a predoctoral fellowship from the Stem Cell and Regenerative Medicine Center at the University of Wisconsin–Madison.

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In vitro differentiation of germ cells from stem cells: a comparison between primordial germ cells and in vitro derived primordial germ cell-like cells.

Stem cells. Human primordial germ cells in a dish.

Primordial Germ Cells: Current Knowledge and Perspectives.

The ability of mouse nuclear transfer embryonic stem cells to differentiate into primordial germ cells.

Induction of primordial germ cell-like cells from mouse embryonic stem cells by ERK signal inhibition.

Functional Concentrations of BMP4 on Differentiation of Mouse Embryonic Stem Cells to Primordial Germ Cells.

Pluripotent stem cells derived from mouse primordial germ cells by small molecule compounds.

Induction of dedifferentiated male mouse adipose stromal vascular fraction cells to primordial germ cell-like cells.

Hematopoietic activity in putative mouse primordial germ cell populations.

Leukemia inhibitory factor sustains the survival of mouse primordial germ cells cultured on TM4 feeder layers.

Histochemical identification of primordial germ cells in diandric and digynic triploid mouse embryos.

Complete in vitro generation of fertile oocytes from mouse primordial germ cells.

Differentiation of Mouse Primordial Germ Cells into Functional Oocytes In Vitro.

Generation of mouse functional oocytes in rat by xeno-ectopic transplantation of primordial germ cells.

Effects of the steel gene product on mouse primordial germ cells in culture.

How to make a primordial germ cell.

Deadenylase depletion protects inherited mRNAs in primordial germ cells.

Sex Specification and Heterogeneity of Primordial Germ Cells in Mice.

Glycoconjugate unique to migrating primordial germ cells differs with genera.

The origin and migration of primordial germ cells in sturgeons.

Cryopreservation of specialized chicken lines using cultured primordial germ cells.

Erasure of DNA methylation, genomic imprints, and epimutations in a primordial germ-cell model derived from mouse pluripotent stem cells.

From primordial germ cells to primordial follicles: a review and visual representation of early ovarian development in mice.

Primordial Germ Cell Specification and Migration.