Primordial Development in Avians

MIKA GINSBURG Department of Cell and Animal Biology, The Silverman Institute of Life Sciences, Hebrew University of Jerusalem 91904, Israel

ABSTRACT The origin of the is studied in pushed anteriorly by the invading endodermal cells. In avians by tracing primordial germ cells from the stage of contrast, it has been shown that in the mouse the germ the germinal crescent backwards to earlier developmen- cells are allocated during in a cluster of tal stages. It has been demonstrated that during cells. Our results demonstrate that in avians the Downloaded from https://academic.oup.com/ps/article-abstract/76/1/91/1624140 by guest on 07 March 2019 formation, the germline has already allocation takes place according to a different mode. The been segregated. However, during this stage, the cells close association between the germ cells and the extra- are seen gradually migrating from the to the embryonic indicates that the germline in . The vertical migration is followed by a avians, as in the mouse, develops from a subset of cells horizontal translocation to the extra-embryonic germinal that have already segregated from the epiblast as extra- crescent, being carried out by the hypoblast that is embryonic mesoderm. (Key words: primordial germ cells, avian blastoderm, avian gastrulation, extra-embryonic mesoderm) 1997 Poultry Science 76:91–95

INTRODUCTION mechanisms that enable germ cells to give rise to the next generation. Where and when does the germline arise in the avians? Is it set aside immediately, before embryonic cells become committed along specific pathways, or THE PERIOD OF PRIMITIVE STREAK does it appear de novo from other somatic cells at some FORMATION point during development? Weismann’s (1885) idea Primordial germ cells were first detected in the about the continuity of the germ plasma, hence, a chicken embryos by Swift (1914) at the end of the “preformistic” mode of germ cell development, has been gastrulation process. Nevertheless, they are better distin- the paradigm for vertebrate investigators in the past. guished at the 10- stage as a dispersed population However, new technologies have paved the way to a of approximately 200 cells located within an extra- different analysis, demonstrating that, at least in the embryonic area, the germinal crescent, anterior to the mouse (Lawson and Hage, 1994), the germ cells differentiate from a somatic lineage. For elucidating the end of the embryo. At this stage, the cells are mode of the germline allocation in the other vertebrates, characterized by their size, by the eccentric position of a detailed analysis is needed, defining the primary their large nucleus, and by their large glycogen content, population of the germ cells, its location, and involve- thus allowing their detection by histochemical methods ment with other developmental events. such as the periodic acid Schiff staining (Meyer, 1960). In the avian embryos, no germplasma has ever been From this area, they migrate into the embryo with the found. The germ cells are detected relatively late in circulating blood. development, toward the end of gastrulation, in an Considering the area in which the detection has been extra-embryonic area. Since they were first detected by made, two questions arise. 1) Do the primordial germ Swift in 1914, the study of their earlier history has been cells form at the germinal crescent or are they formed very limited. Nevertheless, a wealth of factual informa- earlier elsewhere? 2) Does the formation or the migra- tion has accumulated during the last decade and there tion of the primordial germ cells depend upon the has been some success in tracing primordial germ cells gastrulation process? backwards to earlier developmental stages. Elucidating During gastrulation, cells are seen leaving the epiblast the events concerned with germ cell determination and through the primitive streak to form the middle layer, differentiation would allow the study of the molecular the mesoderm, as well as replacing the hypoblast with proper (Nicholet, 1971). As a result, the system is transformed from a two- into a three-layered Received for publication August 13, 1995. system. Consequently, the hypoblast, invaded by the Accepted for publication April 25, 1996. endoderm, is pushed anteriorly to become an extra-

91 92 GINSBURG translocation of the primordial germ cells into the extra- embryonic area. We have used a polyclonal antiserum produced against quail gonadal primordial germ cells for a direct and specific detection of primordial germ cells in the quail hypoblast during gastrulation (Ginsburg et al., 1989). Using this technique, our observations indicate that: 1) In avians, the germline exists as a separate lineage during primitive streak formation; 2) The primordial germ cells are carried into the germinal crescent mainly by the hypoblast; 3) The translocation into the extra-embryonic area is gradual and terminates

only at the end of gastrulation. Downloaded from https://academic.oup.com/ps/article-abstract/76/1/91/1624140 by guest on 07 March 2019

THE PERIOD OF HYPOBLAST FORMATION The hypoblast is the ventral layer of the blastoderm. It starts to form as individual cells ingress from the ventral surface of the one-layered area pellucida and FIGURE 1. Vertical distribution of primordial germ cells between spread as islands beneath this surface. It gradually the upper and the lower layers of blastoderms at Stage XII to XIII merges into a continuous layer, into which a front of (Eyal-Giladi and Kochav, 1976) to Stage 4 (Hamburger and Hamilton, cells, advancing from the posterior marginal zone, is 1951). Embryos at each stage were separated into lower and upper layers. These were incubated to complete 48 h of incubation. The integrated (Weinberger and Brick, 1982). The presence of primordial germ cells detected in each pair of layers from the same primordial germ cells in the hypoblast has led some embryo were counted and the percentages calculated. Data presented investigators to conclude that the primordial germ cells are means of 7 to 10 embryos. originate in the hypoblast (Dubois, 1969). However, Eyal-Giladi et al. (1981), investigating chimeras of quail hypoblast and chicken epiblast (or vice versa), estab- embryonic tissue (Vakaet, 1962). This area constitutes lished that the avian primordial germ cells are of the germinal crescent, where the primordial germ cells epiblastic origin. This finding must mean that they are located. Moreover, an extra-embryonic mesoderm migrate to the hypoblast during development. However, that gives rise to extra-embryonic blood circulation is the close association between the hypoblast and the always associated with the primordial germ cells in this primordial germ cells, which has not yet been analyzed, area. Because reliable specific markers for early avian may reveal that the growing hypoblast is more signifi- primordial germ cells are not yet available, our ability to cant to the development of the germ cells than that of a mere vehicle of translocation. trace their history is limited to strategies that rely The presence of a few primordial germ cells under mainly on morphological criteria. Hence, the various the area pellucida at Stages X to XIII (Eyal-Giladi and manipulations that have been performed on early Kochav, 1976), was reported by several investigators gastrulating embryos could be analyzed only after who used various antibodies, none of which were further incubation for a period equivalent to that which specific for avian primordial germ cells (Urven et al., allowed a control blastoderm to reach Stage 10 (Ham- 1988, with EMA-1; Pardanaud et al., 1987, with QH1 in burger and Hamilton, 1951). the quail; Loveless et al., 1990, with FC10.2). However, Separating the lower layer from embryos at stages of even if the labeled cells were in fact primordial germ primitive streak formation and culturing it in parallel cells, immunohistochemistry could detect only those with the embryos from which it was taken (Ginsburg that had already segregated. Hence, the existence and and Eyal-Giladi, 1986) has demonstrated that 1) The distribution of the primordial germ cell progenitors primordial germ cell population at the germinal crescent within the area pellucida at those developmental stages increases during gastrulation due to migration of remains obscure. Dissecting blastoderms at Stages X to additional cells from the lower layer. 2) The removal of XII to small fragments and allowing those fragments to the hypoblast prevents this additional population from grow in culture for 2 d as tiny explants (Ginsburg and reaching the germinal crescent. 3) The migration is a Eyal-Giladi, 1987) demonstrated the presence of primor- gradual process: the later the stage at which the dial germ cells in all the fragments that originated from separation was performed, the larger the proportion of the area pellucida. No primordial germ cells could be the primordial germ cell population that appeared in the detected in fragments that were excised from the area lower layer (Figure 1). Because the hypoblast moves opaca. As was demonstrated in whole mounts prepara- anteriorly to become the major component of the tions stained with periodic acid Schiff and in histological germinal crescent, it could serve as a vehicle for the sections each tiny explant derived from the area SYMPOSIUM: CURRENT ADVANCES IN AVIAN EMBRYOLOGY AND INCUBATION 93 Downloaded from https://academic.oup.com/ps/article-abstract/76/1/91/1624140 by guest on 07 March 2019

FIGURE 2. Schematic representations of three forms of germline development: 1) The “preformistic” mode of germ cell formation. Primordial germ cell progenitors are sparsely located in the area pellucida already at Stage X (Eyal-Giladi and Kochav, 1976). 2) The germ cells segregate from the pluripotent area pellucida as a result of a stochastic signal. 3) The “epigenetic” mode: germ cells arise from a particular subset of cells that appear to be relatively differentiated part of the soma. ap = area pellucida; e = epiblast; pgc = primordial germ cell; ppgc = primordial germ cell progenitors; cl = a subset of somatic cells.

pellucida has developed a lower layer. The primordial number of primordial germ cells exist and they are germ cells were found scattered within a middle layer located between the mesenchyme-like tissue, which comprised of mesenchyme-like cells, which start to form constitutes the middle layer, and the ventral layer. This the blood system. The total number of primordial germ result implies that three lineages do not need the cells counted in all pieces derived from the same primitive streak for their differentiation: the hypoblast, blastoderm was always in the same range (150 to 200) as the mesenchyme-like mesoderm that develops into found in control blastoderms incubated for 2 d. blood tissue, and the primordial germ cells. These three At this stage of the investigation, the following lineages also do not require the area opaca because this models were considered (Figure 2): 1) Primordial germ had been previously discarded. cells progenitors are sparsely located in the area The observed association of the avian primordial pellucida already at Stage X. 2) There is no lineage germ cells with the mesenchyme cells that are not part restriction to the germline at Stage X. However, a of the gastrulated has focused our stochastic event transmits a signal to the pluripotent attention on the extra-embryonic mesoderm, a cell epiblast cells, causing some of them to differentiate into lineage that has been rather neglected in studies of early primordial germ cells. 3) The differentiation into primor- embryogenesis of the avians. In all the various manipu- dial germ cells is induced within a subpopulation of lations that we have performed on early stages, cells that has already been segregated from the epiblast. primordial germ cells were always associated with an extra-embryonic mesoderm. Support for our observation CIRCUMVENTING THE PRIMITIVE STREAK came from a recent study in the mouse (Lawson and Hage, 1994). By labeling cells in the proximal epiblast in Isolation of the area pellucida from the area opaca both pregastrulation and early-streak embryos, these and the subsequent dissociation and culture of the area investigators have demonstrated that the precursors of pellucida-derived cells as an aggregate (Ginsburg and the primordial germ cells constitute a part of the Eyal-Giladi, 1989) would not support gastrulation be- presumptive extra-embryonic mesoderm and are not cause polarity was destroyed. However, the expected lineage-restricted while in the epiblast. They are allo- 94 GINSBURG cated during gastrulation as a cluster of approximately 40 cells already segregated in the extra-embryonic mesoderm posterior to the primitive streak. The mean- ing of this observation is that during gastrulation in the mouse embryo, cells are segregating from the epiblast, circumventing the primitive streak, to form the extra- embryonic mesoderm. The descendants of this lineage that are located in a specific area at the base of the allantois are differentiated by an unknown mechanism into germ cells. In the avian embryo, no cluster of primordial germ cells has ever been found. Compared to the mouse, the avian germline already exists as a separate lineage of Downloaded from https://academic.oup.com/ps/article-abstract/76/1/91/1624140 by guest on 07 March 2019 scattered cells prior to primitive streak formation. We hypothesize that in the avian embryo, the extra- embryonic mesoderm segregates from the epiblast earlier than the formation of the primitive streak. We assume that the first cells that leave the ventral surface of the area pellucida form clusters that spread and merge to form the hypoblast (Figure 3). However, cell ingression is continuous and results in the accumulation of additional cells between the epiblast and the hypob- last. This population of cells constitutes the extra- embryonic mesoderm from which some cells differenti- ate into the germline. Being located on the hypoblast, both the extra-embryonic mesoderm and the primordial germ cells are later carried anteriorly, away from the gastrulating area. There is evidence in avians for a middle layer of cells between epiblast and hypoblast prior to primitive streak formation (Izpisua-Belmonte et al., 1993; Ruiz i. Altaba et al., 1995). Hatada and Stern (1994) have demonstrated by carbocyanine dyes labeling, that cells contributing to the yolk sac and its stalk are very widely distributed over the entire surface of the prestreak blastoderm. Neverthe- less, no detailed cell lineage analysis for the extra- embryonic mesoderm has ever been performed in avians. We therefore think that the location of the FIGURE 3. Schematic illustration of the ventral ingression of cells of prestreak stages. The first wave of ingressed cells form the presumptive extra-embryonic mesoderm as being hypoblast. The cells that ingress later develop into extra-embryonic described by Stern (1992), in the fate map for Stages X to mesoderm. Primordial germ cells form by an unknown mechanism XIII at the posterior end of the blastoderm, should be from the subset of cells committed to develop to extra-embryonic revised. mesoderm. ap = area pellucida; e = epiblast; h = hypoblast; exm = extra-embryonic mesoderm; pgc = primordial germ cell. Does the germline in the avians segregate from the epiblast earlier than in the mouse? Izpisua-Belmonte et al. (1993) have demonstrated an early small population of middle layer cells, associated with Koller’s sickle, that tive streak, and only later start to populate the embryo express the gene goosecoid during stages of hypoblast (Ginsburg et al., 1990). However, in avians these cells formation. If the expression of goosecoid is indeed a segregate from the entire surface of the epiblast in a marker for an organizer, it means that the process of scattered mode and translocate onto an extra-embryonic gastrulation starts in avians earlier than the visible tissue, the hypoblast. The latter carries them anteriorly, appearance of the primitive streak. Based on these into an extra-embryonic location, away from the primi- criteria, it is most likely that the segregation of the avian tive streak that starts to advance from the posterior area germline takes place, similar to the mouse, during early of the blastoderm. gastrulation. In a study that is currently underway, we are In both the mouse embryo and in avians, the attempting to analyze the different populations of cells primordial germ cells seem to escape the developing that are found between the epiblast and the hypoblast of primitive streak, circumventing it in two different blastoderms at Stages X to XIII. For this purpose we are modes. In the mouse they segregate as a cluster of cells using various monoclonal antibodies labeling and carob- within an extra-embryonic area posterior to the primi- cyanine dyes labeling. We hope that a detailed analysis SYMPOSIUM: CURRENT ADVANCES IN AVIAN EMBRYOLOGY AND INCUBATION 95 of the segregation mode of the extra-embryonic meso- Izpisua-Belmonte, J. C., E. M. De Robertis, K. G. Storey, and C. derm from the epiblast, and the detection of the D. Stern, 1993. The homeobox gene goosecoid and the primordial germ cells within this tissue, will allow us to origin of organizer cells in the early chick blastoderm. Cell define the exact location and association of the germline 74:645–659. allocation in the avians. Lawson, K. A., and J. Hage, 1994. Clonal analysis of the origin of primordial germ cells in the mouse, Page 68–91 in: Germline Development. Ciba Foundation Symposium 182, REFERENCES John Wiley and Sons Ltd., Chichester, UK. Loveless, W., R. Bellairs, S. J. Thorpe, M. Page, and T. Feizi, Dubois, R., 1969. Le mechanisme d’entree des cellules 1990. 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