Origin of primordial germ cells in the prestreak chick embryo

The temporal and spatial pattern of segregation of the avian germline from the formation of the area pellucida to the beginning of primitive streak formation (stages VII–XIV, EG&K) was investigated using the culture of whole embryos and central and peripheral embryo fragments on vilelline membranes at stages VII–IX, immunohistological analysis of whole mount embryos and sections with monoclonal antibodies MC-480 against stage-specific embryonic antigen-1 (SSEA-1) and EMA-1, and with the culture of dispersed blastoderms at stages IX–XIV with and without an STO feeder layer. Whole embryos at intrauterine stages developed up to the formation of the primitive streak despite the absence of area pellucida expansion. Primordial germ cells (PGCs) appeared in the cultures of whole embryos and only in central fragments containing a partially formed area pellucida at stages VII–IX. When individual stage IX–XIV embryos were dispersed and cultured without a feeder layer, 25–45 PGCs/embryo were detected only with stage X–XIV, but not with stage IX blastoderms. However, the culture of dispersed cells from the area pellucida of stages IX–XIII on STO feeder layers yielded about 150 PGCs/embryo. The carbohydrate epitopes recognized by anti-SSEA-1 and EMA-1 first appeared at stage X on cells in association with polyingressing cells on the ventral surface of the epiblast and later on the dorsal surface of the hypoblast. The SSEA-1-positive hypoblast cells gave rise to chicken PGCs when cultured on a feeder layer of quail blastodermal cells. From these observations, we propose that the segregation and development of avian germline is a gradual, epigenetic process associated with the translocation of SSEA-1/EMA-1-positive cells from the ventral surface of the area pellucida at stage X to the dorsal side of the hypoblast at stages XI–XIV. © 1996 Wiley-Liss, Inc.     

Primordial germ cell development in cultures of dispersed central disks of stage X chick blastoderms

Central disk fragments cut from stage X chick blastoderms were dispersed and cultured on glass coverslips. After 48 hr of incubation the cultures showed various degrees of organization into three-layered aggregates in which no axis development was observed. Primordial germ cells (PGCs) were detected in ail cultures. The number of PGCs was found to be correlated to the initial cell concentration in the suspension. By regression analysis it was found that in cultures initiated from 10 central disks or more, the mean number of PGCs per fragment was constant and matched the number of PGCs found for intact control central disks incubated for the same length of time. It appears that in cultures of stage X, the morphologic expression of PGCs is related to the level of differentiation and organization of the somatic cells in the culture, which, in turn, is dependent on the initial concentration of cells in the culture.     

The developmental origin of primordial germ cells and the transmission of the donor-derived gametes in mixed-sex germline chimeras to the offspring in the chicken

A novel system has been developed to determine the origin and development of primordial germ cells (PGCs) in avian embryos directly. Approximately 700 cells were removed from the center of the area pellucida, the outer of the area pellucida, and the area opaca of the stage X blastoderm (Eyal-Giladi and Kochav, 1976; Dev Biol 49:321–337). When the cells were removed from the center of the area pellucida, the mean number of circulating PGCs per 1 μl of blood was significantly decreased to 13 (P < 0.05) in the embryo at stage 15 (Hamburger and Hamilton, 1951: J Morphol 88:49–92) as compared to intact embryos of 51. When the removed recipient cells from the center of the area pellucida were replenished with 500 donor cells, no reduction in the PGC number was observed. The removal of cells from the outer of area pellucida or from the area opaca had no effect on the number of PGCs. When another set of the manipulated embryos were cultured ex vivo to hatching and reared to sexual maturity, the absence of germ cells and the degeneration of seminiferous tubules were observed in resulting chickens derived from the blastoderm from which the cells were removed from the center of the area pellucida. Chimeric embryos produced by the male donor cells and the female recipient contained the female-derived cells at 97.2% in the whole embryo and 94.3% in the erythrocytes at 5 days of incubation. At 5–7 days of incubation, masculinization was observed in about one half of the mixed-sex embryos. The proportions of the female-derived cells in the whole embryo and in the erythrocytes were 76.5% and 80.2% at 7 days to 55.7% and 62.5% at 10 days of incubation, respectively. When the chimeras reached their sexual maturity, they were test mated to assess donor contribution to their germline. Five of six male chimeras (83%) and three of five female chimeras (60%) from male donor cells and a female recipient embryo from which 700 cells at the center of area pellucida were removed were germline chimeras. Three of the five male germline chimeras (60%) and one of the three female germline chimeras (33%) transmitted exclusively (100%) donor-derived gametes into the offspring. When embryonic cells were removed from the outer of area pellucida or area opaca, regardless of the sex combination of the donor and the recipient, the transmission of the donor-derived gametes was essentially null. The findings in the present studies demonstrated, both in vivo and in vitro, that the PGCs originate in the central part of the area pellucida and that the developmental fate to germ cell (PGCs) had been destined at stage X blastoderm in chickens. Mol. Reprod. Dev. 48:501–510, 1997. © 1997 Wiley-Liss, Inc.     

Post-nodal mesoblast caudalizes the host axis and inhibits cell population growth, and induces new primitive streaks in chick embryos

One to eight post-nodal fragments (PN) or Hensen's nodes (HN) from full primitive streak stage chick embryos were transplanted onto the area pellucida or area opaca of stage 4 embryos and cultured for 20 h. Thirteen morphological and numerical parameters were affected in the host embryos and analyzed by multiple logistic regression for parametric hierarchy. In the area pellucida, both PN and HN transplants inhibited cell population growth while only PN caudalized the host axis and induced supernumerary primitive streaks expressing the mesoderm-specific gene Brachyury. In the area opaca, neither grafts influenced host axis morphogenesis, but PN inhibited the cell population growth significantly. Tracking [(3)H]TdR labeled grafts showed that PN cells migrated towards the host axis and participated in the formation of supernumerary somites and hearts. When placed near the host axis, PN caudalized it and inhibited cell population growth.     

Axis formation in half blastoderms of the chick: Stage at separation and the relative positions of fused halves influence axis development

The blastoderm of the avian embryo acts during the early stages of development as an integrative system programmed to form a single embryonic axis. Isolated parts of the blastoderm are known to each form an axis, owing to the system's properties. In the work reported here, the regulative capability of the right and left halves of chick blastoderms to form an embryonic axis was examined systematically at different stages. This revealed a progressive change in the developing blastoderm. After early separation, the axis in each half will form at some distance from the blastoderm's original midline, while with late separation the axis will form next to the original midline and may even lack one row of somites at the medial rim. Since development stops in culture after about 2 days, axis development after early separation ceases before somites are formed, whereas after late separation somites and brain vesicles can develop. In addition, an attempt was made to learn whether the two halves of blastoderm, when shifted along the midline and then reunited in staggered fashion, act as a single or two separate embryonic fields. When reunion of the right and left halves was achieved so that the posterior end of one half was adjoining the posterior area pellucida region of the other half, a single embryonic axis developed. When, on the other hand, the shift was larger so that the posterior end was fused to the central area pellucida of the other half, two separated embryonic axes developed.     

Inhibitory and stimulatory influences on mesodermal erythropoiesis in the early chick blastoderm

We use a standing-drop culturing method to investigate the effect on mesodermal erythropoiesis of ectoderm and endoderm from the area opaca vasculosa (AOV) and area pellucida (AP) of stage-4 chick blastoderms. We find that ectoderm from the AOV and ectoderm and endoderm from the AP exert an inhibitory influence on mesodermal erythropoiesis. This inhibitory influence is coupled with the tendency of the explants to spread out and become flattened in culture. In contrast, endoderm from the AOV is found to be stimulatory, in agreement with previous studies. We correlate these in vitro inhibitory and stimulatory influences with the morphogenetic patterns that occur during normal development.     

The pluripotency of the extraembryonic mesodermal cells of the early chick blastoderm: effects of the AP and AOV environments

This study investigates the developmental potential of the extraembryonic mesodermal cells of the early chick blastoderm. [3H]Thymidine-labeled mesodermal fragments from the extraembryonic area pellucida (AP) and area opaca vasculosa (AOV) were transplanted into the AP or AOV of nonlabeled host blastoderms in culture, and their fate followed autoradiographically. All the homotopically transplanted mesodermal cells differentiated in accordance with their normal fates. However, not all the heterotopically transplanted mesodermal cells did so, for some of the stage 8 AP extraembryonic mesodermal cells (normally nonerythropoietic) gave rise to blood cells when transplanted into the AOV. We also observed that the stage 4-5 AOV mesoderm continues to migrate peripherally when heterotopically transplanted into the AP, at a time when the AP mesodermal cells are nonmigratory. In support of our premise that the stage 8-9 AP extraembryonic mesoderm has the potential to form blood, we observed a clear-cut production of hemoglobin when the latter mesoderm was co-cultured on coverslips with stage 4 AOV endoderm.     

Ring lethal: an early embryonic failure in medium white turkeys

Ring lethal denotes an early embryonic failure of developing blastoderms in medium white turkeys that can be recognized macroscopically in situ after 48 hours of incubation. The condition is characterized by a white ring of amorphous cells in the area opaca with or without the presence of cells in the area pellucida. The disorder is inherited as an autosomal recessive trait that is expressed in the homozygous condition. Attempts to elucidate the cause of the ring lethal gene's expression have been unsuccessful. The symbol rl is proposed for the gene.     

Hepten inhibitors of the endogenous galactose binding lectins and anti-lectin antibodies inhibit primitive streak formation in the early chick embryo

The early chick blastoderm expresses two endogenous galactose-bindinglectins of 14 kDa and 16 kDa. We have studied the effect thelectin hapten inhibitors thiodigalactoside and the syntheticneoglycoprotein lactosyl-bovine serum albumin as well as polyclonalanti-lectin antibodies on the development of early chick embryoscultured in a defined medium. Controls consisted of maltose,maltosyl bovine serum albumin and rabbit IgG. Embryos treatedat the onset of cell migration during early gastrulation underwentblastoderm retraction with decrease in surface area. In addition,they exhibited a lack of demarcation between the presumptiveembryonic area (area pellucida) and the presumptive extraembryonicarea (area opaca). These blastoderms also lacked a primitivestreak, that is, the structure that forms in the area pellucidaduring gastrulation as cell migrate to form the endodermal andmesodermal layers of the embryo. Embryos treated at later stagesof gastrulation showed development similar to that of controlsin that they were able to undergo early organogenesis. The resultssuggest that lectin mediated mechanisms are essential for themigratory movements of early gastrulation and that, at lategastrulation, other mechanisms exist in the embryo to compensatefor lectin function. blastoderm chick embryo galectin     

Effect of spatial position of uterine quail blastoderms cultured in vitro on bilateral symmetry formation

Summary A method of in vitro culture for uterine quail blastoderms has been developed, which allows them to develop from cleavage throughout gastrulation and further: stages 4–10 of Hamburger and Hamilton (1951). The method consists of cultivating the blastoderms on egg albumen in a vertical position; this permits about 50% of the blastoderms explanted before area pellucida formation to develop bilateral symmetry and to form normal primitive streak, somites and head structures. Development of the blastoderms explanted after their area pellucida was already formed, occurred normally and was not influenced by their spatial position in the culture.This work was performed as part of project no. 09.7.1.5.2 of the Polish Academy of Sciences     

Experimental study of the precocious formation of the endothelial wall in bird embryos]

Pieces of ectomesoderm from the area pellucida of primitive streak stages don't give normal endothelium when transplanted on ectoderm of the area opaca. Endothelium is able to differenciate from mesoderm transplanted with endoderm. Mesenchyme from the primitive streak migrating between the endoderm and the ectoderm of the host always gives endothelial tubes.     

Nuclear beta-catenin and the development of bilateral symmetry in normal and LiCl-exposed chick embryos.

Studies in Xenopus laevis and zebrafish suggest a key role for beta-catenin in the specification of the axis of bilateral symmetry. In these organisms, nuclear beta-catenin demarcates the dorsalizing centers. We have asked whether beta-catenin plays a comparable role in the chick embryo and how it is adapted to the particular developmental constraints of chick development. The first nuclear localization of beta-catenin is observed in late intrauterine stages of development in the periphery of the blastoderm, the developing area opaca and marginal zone. Obviously, this early, radially symmetric domain does not predict the future organizing center of the embryo. During further development, cells containing nuclear beta-catenin spread under the epiblast and form the secondary hypoblast. The onset of hypoblast formation thus demarcates the first bilateral symmetry in nuclear beta-catenin distribution. Lithium chloride exposure also causes ectopic nuclear localization of beta-catenin in cells of the epiblast in the area pellucida. Embryos treated before primitive streak formation become completely radialized, as shown by the expression of molecular markers, CMIX and GSC. Lithium treatments performed during early or medium streak stages cause excessive development of the anterior primitive streak, node and notochord, and lead to a degeneration of prospective ventral and posterior structures, as shown by the expression of the molecular markers GSC, CNOT1, BMP2 and Ch-Tbx6L. In summary, we found that in spite of remarkable spatiotemporal differences, beta-catenin acts in the chick in a manner similar to that in fish and amphibia.     

Restriction of proliferation of primordial germ cells by the irradiation of Japanese quail embryos with soft X-rays.

Primordial germ cells (PGCs) are the progenitor cells for the gametes. Avian PGCs are located in the central region of the area pellucida at the blastoderm stage. Shortly after further incubation, they migrate to the extra-embryonic germinal crescent, and then as soon as the blood vessels form, they enter the circulation and finally settle in the gonadal primordium. We have developed a simple method using soft X-ray irradiation (18 kV power, 20 cm distance) to reduce the number of PGCs in Japanese quail embryos, which should be useful in preparing recipient embryos for PGC-transfer studies. When embryos were exposed to the soft X-rays for 40 s before incubation, the concentration of circulating PGCs was less than one-fifth that in controls after 2 days of incubation. Embryos at day 6 of incubation contained approximately half the number of PGCs compared to controls when they were exposed before or at day 2 of incubation. Irradiation for 40 s is recommended taking into consideration the restriction of proliferation of PGCs, and viability and hatchability.     

Response to fibronectin of mouse primordial germ cells before, during and after migration.

The adhesive extracellular matrix glycoprotein fibronectin is thought to play a central role in cell migration during embryogenesis. In order to define this role, we have examined the response to fibronectin in cell culture of mouse primordial germ cells (PGCs) before, during and after their migration from the hindgut into their target tissue, the genital ridges. Using an explant culture system, we show that PGCs will emigrate from tissue fragments containing hindgut, and that fibronectin stimulates this migration. Adhesion assays show that the start of PGC migration is associated with a fall in adhesion to fibronectin. Double-labelling studies using in situ hybridization and histochemistry demonstrate that migrating PGCs do not contain detectable fibronectin mRNA, suggesting that they do not synthesize and secrete the fibronectin within their migratory substratum. Taken together, these findings are consistent with an important role for fibronectin in stimulating PGC migration. In addition, however, they suggest that the interaction between PGCs and fibronectin may be important in timing the start of migration, with the fall in adhesion allowing the PGCs to commence their migration towards the genital ridges.     

The formation of primordial germ cells from germline cells in spherical embryos derived from the blastodisc of 2-cell embryos in goldfish, Carassius auratus

The property of primordial germ cells (PGCs) in fragmented goldfish embryos was investigated. When 1- and 2- cell embryos were cut at several perpendicular levels at the animal-vegetal axis, cells expressing vas mRNA were observed in the resultant embryos derived from all kinds of animal fragments. Blastodisc fragments from the 1- to 2-cell stage developed to spherical embryos containing yolk body with a yolk syncytial layer (YSL). Germ ring and no tail expression were not observed in the spherical embryo. When the spherical embryo labeled with tracer dye or GFP-nos1 3'UTR mRNA was transplanted onto the animal part of the blastoderm in a host embryo at the blastula stage, PGCs of spherical embryo origin were detected around the gonadal ridges in the resultant embryos which developed normally. These results suggest that small animal fragments should contain factors sufficient for PGC differentiation and that PGCs differentiate without mesoderm induction, since mesoderm is not induced in a spherical embryo.     

Interactions between Wnt and Vg1 signalling pathways initiate primitive streak formation in the chick embryo

The posterior marginal zone (PMZ) of the chick embryo has Nieuwkoop centre-like properties: when transplanted to another part of the marginal zone, it induces a complete embryonic axis, without making a cellular contribution to the induced structures. However, when the PMZ is removed, the embryo can initiate axis formation from another part of the remaining marginal zone. Chick Vg1 can mimic the axis-inducing ability of the PMZ, but only when misexpressed somewhere within the marginal zone. We have investigated the properties that define the marginal zone as a distinct region. We show that the competence of the marginal zone to initiate ectopic primitive streak formation in response to cVg1 is dependent on Wnt activity. First, within the Wnt family, only Wnt8C is expressed in the marginal zone, in a gradient decreasing from posterior to anterior. Second, misexpression of Wnt1 in the area pellucida enables this region to form a primitive streak in response to cVg1. Third, the Wnt antagonists Crescent and Dkk-1 block the primitive streak-inducing ability of cVg1 in the marginal zone. These findings suggest that Wnt activity defines the marginal zone and allows cVg1 to induce an axis. We also present data suggesting some additional complexity: first, the Vg1 and Wnt pathways appear to regulate the expression of downstream components of each other's pathway; and second, misexpression of different Wnt antagonists suggests that different classes of Wnts may cooperate with each other to regulate axis formation in the normal embryo.     

Appearance of Primordial Germ Cells in Young Chick Blastoderms Cultured in vitro

Appearance of primordial germ cells (PGCs) in young chick blastoderms was investigated by the cultivation of only the epiblast or hypoblast. Presumptive PGCs exist in the epiblast before primitive-streak formation. They translocate gradually to the lower layer during early stages of primitive-streak formation, though substantial number of presumptive PGCs remain in the upper layer. The existing primary hypoblast under the epiblast is dispensable for the further differentiation of the PGCs.     

Effects of L-tryptophan on morphogenesis and growth in the early chick blastoderm

Summary Chick blastoderms, suppliedin vitro andin ovo with L-tryptophan at the primitive streak stage, showed in their continued development typical retardation of brain formation and somitogenesis in the embryo, whereas heart formation remained unaffected. In contrast to an overall reduction in size observed at the higher L-tryptophan concentrations, a moderate enlargement of the area opaca, compared with the controls, was found at the lower concentrations. This enlargement was combined with an increased flattening of the ectodermal area opaca cells and a reduction of the number of microvilli covering these cells. As a simultaneous supply of glucose could reduce, to some extent, the morphogenetic disturbances, these might partly be ascribed to a blocking of gluconeogenesis from L-tryptophan, but the overall reduction in size mentioned, together with the observation of a reduced decomposition of intracellular yolk granules in the L-tryptophan-treated blastoderms, indicates that impairment of intracellular yolk granule decomposition was the principal disturbance. The possible role of serotonin—probably formed from the L-tryptophan supplied—is suggested as a regulating factor in this connexion.     

Immunohistochemical analysis of the segregation process of the quail germ cell lineage

An antiserum against quail 7 day gonadal germ cells was found to react specifically with gonadal germ cells of both sexes. Transverse sections from a range of early quail developmental stages were submitted to the antibody PAP reaction. Blastodiscs from the earliest uterine stages (II to X E.G. & K) reacted very strongly, while the overall reaction gradually decreased in older blastoderms. At stage XIII both epiblast and hypoblast were weakly stained, but some large, PGC-like cells stained intensively. During gastrulation (PS formation) the reaction of the epiblast disappears quicker than that of the hypoblast. The newly formed mesoderm and entoderm do not react at all and the reaction gradually becomes limited mainly to the PGCs and somewhat to the primary hypoblast which is moving into the germinal crescent. The widely spread reaction at the early stages is thus gradually being restricted to the PGCs.     

On the Origin of Primordial Germ Cells in the Chick Embryo

An attempt was made to re-examine the location of the primordial germ cells (PGCs) in very young chick embryos. Freshly laid blastoderms, prior to hypoblast formation, of a known anterio-posterior axis, were transversely bisected and each half was separately grown in vitro. Both anterior and posterior halves were shown to be fertile and each was shown to contain roughly the same amount of PGCs as a normal control embryo. It has been concluded that in the chick as well as in the duck there is no concentration of cells containing germinal plasm in the posterior part of the blastoderm.
Two other possibilities should be investigated:
1. A concentric arrangement of cells containing germinal plasm. 2. The absence of a germinal plasm and a relatively late appearance of PGCs as a result of induction.