Xenopus Heterochronic Presumptive Primordial Germ Cells (pPGCs) Implanted in the Correct Position in Host Neurula Embryos can Differentiate into PGCs

Presumptive primordial germ cells (pPGCs) in explants, derived from single germ plasm-bearing cells of Xenopus 32-cell embryos, at the equivalent of neurula stage (stage 20) in control embryos (designated as 'stage-20' explants) were demonstrated to be able to differentiate into PGCs, when implanted into a prospective place of pPGCs in host embryos (stage 20) (Ikenishi & Tsuzaki, 1988). According to a recent proposal that individual early embryonic cells in Xenopus , at both in vivo and in vitro , are able to measure elapsed time since fertilization (Cooke and Smith, 1990), the result means that the implanted pPGCs having the same elapsed time as the host embryos (isochronic pPGCs) could differentiate into PGCs. In the present study, in order to know whether the compatibility in elapsed times of implanted pPGCs and host embryos is necessary for the differentiation of PGCs, labelled, heterochronic pPGCs in 'stages 12–33/34' explants were implanted into unlabelled, host neurulae (stage 19).
Those heterochronic pPGCs could differentiate into PGCs like isochronic pPGCs in 'stage-19' explants as the control. By comparing the average diameters and yolk contents of labelled PGCs with those of unlabelled, host ones in experimental tadpoles, the possibility that a certain mechanism modulating the elapsed time of heterochronic pPGCs to that of host pPGCs is present in host embryos was also suggested.     

Ectopic germline cells in embryos of Xenopus laevis

Whether all descendants of germline founder cells inheriting the germ plasm can migrate correctly to the genital ridges and differentiate into primordial germ cells (PGCs) at tadpole stage has not been elucidated in Xenopus. We investigated precisely the location of descendant cells, presumptive primordial germ cells (pPGCs) and PGCs, in embryos at stages 23-48 by whole-mount in situ hybridization with the antisense probe for Xpat RNA specific to pPGCs and whole-mount immunostaining with the 2L-13 antibody specific to Xenopus Vasa protein in PGCs. Small numbers of pPGCs and PGCs, which were positively stained with the probe and the antibody, respectively, were observed in ectopic locations in a significant number of embryos at those stages. A few of the ectopic PGCs in tadpoles at stages 44-47 were positive in TdT-mediated dUTP digoxigenin nick end labeling (TUNEL) staining. By contrast, pPGCs in the embryos until stage 40, irrespective of their location and PGCs in the genital ridges of the tadpoles at stages 43-48 were negative in TUNEL staining. Therefore, it is evident that a portion of the descendants of germline founder cells cannot migrate correctly to the genital ridges, and that a few ectopic PGCs are eliminated by apoptosis or necrosis at tadpole stages.     

Direct Evidence for the Presence of Germ Cell Determinant in Vegetal Pole Cytoplasm of Xenopus laevis and in a Subcellular Fraction of It

To test for the presence of germ cell determinant in Xenopus embryos, vegetal pole cytoplasm containing the "germ plasm", or a subcellular fraction of it, was microinjected into single somatic blastomeres isolated from 32-cell embryos. Injected or non-injected (control) blastomeres were cultured in ³H-thymidine until normal control embryos reached the neurula stage. The labeled explants were then implanted into unlabeled host neurulae, which were allowed to develop to the tadpole stage. Labeled PGCs of explant origin in the genital ridges of the experimental tadpoles were examined by autoradiography.
Isolated blastomeres were injected with vegetal pole cytoplasm of 32-cell embryos or with a 20,000 g pellet made from vegetal pole cytoplasm of 2-cell embryos. Labeled PGCs were found in 7.6% and 2.3% of the experimental tadpoles, respectively. No labeled PGCs were found in the control tadpoles, except for one tadpole in the first experiment. These results strongly suggest that the vegetal pole cytoplasm and its subcellular fractions act as germ cell determinant.     

Presumptive Primordial Germ Cells (pPGCs) and PGCs in Tadpoles from UV-irradiated embryos of Xenopus

In order to determine whether or not tadpoles that once lacked primordial germ cells (PGCs) in the genital ridges and dorsal mesentery as a result of ultraviolet (UV) irradiation subsequently contained germ cells at more advanced stages of larval development, the numbers of presumptive PGCs or PGCs were carefully examined in Xenopus tadpoles at Nieuwkoop and Faber's stage 35/36–52 that developed normally from UV-irradiated eggs.
No late-appearing germ cells were observed in almost all the UV-irradiated tadpoles examined at stages 49–52. This same population had completely lacked PGCs at about stage 46. Moreover, presumptive PGCs (pPGCs) or cells with granular cytoplasm that reacted with a monoclonal antibody specific for the germ plasm of cleaving Xenopus eggs stayed in the central part of the endoderm cell mass in the irradiated tadpoles at stage 35/36, when the majority of those cells were located in the dorsal part of the endoderm in unirradiated controls. Furthermore, in the irradiated embryos pPGCs were demonstrated to decrease in number with development and eventually to disappear in tadpoles at about stage 40. The results strongly suggest that UV irradiation under the conditions used here totally eliminated germline cells from the irradiated animals.     

A Possibility of an in Vitro Differentiation of Primordial Germ Cells (PGCs) from Blastomeres Containing "Germinal Plasm" of Early Cleavage Stage in Xenopus laevis

The blastomeres containing the "germinal plasm" were isolated from 32-cell stage Xenopus embryos and cultured in vitro for various periods of time till the control embryos developed to stage 28, 33/34, 40 and 45, respectively. The cells containing the plasm in the 'stage-28', '33/34' and '40' explants were similar in external shape, and in distribution in the spherical endodermal cell mass to the presumptive primordial germ cells (pPGCs) in normal embryos of the corresponding stages. In addition, the cells in explants as well as the pPGCs were separated by a large intercellular space from the surrounding endodermal cells. The change in proportion of the compact or the loosely structured germinal granules and the irregularly shaped-stringlike bodies (ISBs) occurred in the cells of the explants with the prolongation of the culture period. In the cells of the 'stage-45' explant as well as in the PGCs of normal stage-45 tadpoles the ISBs and "granular materials" replace those germinal granules. These facts lead to the conclusion that the change of the germinal granules through the ISBs, to the "granular materials", noticed in the normal course of differentiation of pPGCs into PGCs (see (1)), also takes place in the cells of the explants during the culture. Therefore, it is likely that the cells in the explants are genuine pPGCs or PGCs. This is the first demonstration of a possibility of the in vitro differentiation of PGCs from the blastomeres containing the "germinal plasm" of early cleavage stage.     

Members of the ErbB receptor tyrosine kinases are involved in germ cell development in fetal mouse gonads.

To isolate the genes involved in mouse primordial germ cell (PGC) development, we carried out subtraction cDNA cloning between PGC-derived embryonic germ (EG) cells and inner cell mass-derived embryonic stem cells. Among the genes preferentially expressed in EG cells, we found a gene encoding a receptor tyrosine kinase ErbB3. By in situ hybridization and immunohistochemical staining, the expression of ErbB3 as well as that of ErbB2, a coreceptor for ErbB3, was detected in PGCs in genital ridges at 12.5 dpc (days postcoitum). The expression was, however, downregulated at 14.5 dpc when the PGCs underwent growth cessation. Neuregulin-beta, a ligand for ErbB2 and ErbB3, was also expressed in genital ridges. In addition, a recombinant Neuregulin-beta enhanced the number of PGCs in 12.5-dpc embryos in culture. Taken together, these observations suggest that ErbB signaling controls the growth or survival of PGCs in genital ridges.     

Genital ridges exert long-range effects on mouse primordial germ cell numbers and direction of migration in culture

The functional gametes of all vertebrates first arise in the early embryo as a migratory population of cells, the primordial germ cells (PGCs). These migrate to, and colonise, the genital ridges (GR) during the early organogenesis period, giving rise to the complete differentiating gonad. PGCs first become visible by alkaline phosphatase staining in the root of the developing allantois at 8.5 days post coitum (dpc). At 9.5 dpc they are found in the wall of the hind-gut and, during the following three days, they migrate along the hind-gut mesentery to the dorsal body wall, and then to the genital ridges. By 12.5 dpc, the great majority of PGCs have colonised the genital ridges. During this period the number of PGCs increases from less than 100 to approximately 4000. In a previous paper (Donovan et al. 1986), we showed that 10.5 dpc PGCs can be explanted from the hind-gut mesentery, and will spread and migrate on feeder cell layers. We showed also that the intrinsic ability of PGCs to spread and migrate changes as they colonise the genital ridges. In this paper, we examine extrinsic factors that control PGC behaviour in vitro. Using PGCs taken from 8.5 dpc embryos, at the beginning of their migratory phase, we show that culture medium conditioned by 10.5 dpc genital ridges causes an increase in the number of PGCs in these cultures. We also show that PGCs migrate towards 10.5 dpc genital ridges in preference to other explanted organs. These experiments show that genital ridges exert long-range effects on the migrating population of PGCs.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Xdsg protein in presumptive primordial germ cells (pPGCs) is essential to their differentiation into PGCs in Xenopus

In order to know the role of the Xdsg gene in presumptive PGCs (pPGCs) of Xenopus, we attempted to inhibit the translation of Xdsg mRNA in pPGCs by injecting antisense morpholino oligo (asMO), together with Fluorescein Dextran-Lysine (FDL), into single germ plasm-bearing cells of 32-cell embryos. Among three types of asMOs complementary to different parts of the 5'-untranslated region of Xdsg mRNA tested, only one asMO, designated as Xdsg-3, inhibited the translation of the mRNA in FDL-labeled pPGCs, resulting in the absence of labeled PGCs in experimental tadpoles. On the other hand, two other asMOs, Xdsg-1 and -2, did not inhibit the translation, so that a similar number of labeled PGCs found in FDL-injected but asMO-uninjected control tadpoles were observed in experimental tadpoles derived from asMO-injected embryos. Surprisingly, use of Xdsg-3 asMO resulted in the disappearance of the protein of Xenopus vasa homolog (Xenopus vasa-like gene 1, XVLG1) from FDL-labeled pPGCs by inhibiting the translation of XVLG1 mRNA. However, the effect of Xdsg-3 asMO on the translation of Xdsg and XVLG1 mRNAs and PGC formation could be canceled by the coinjection with Xdsg mRNA. Consequently, the Xdsg protein in pPGCs may play an important role in the formation of PGCs by regulating the production of XVLG1 protein.     

ULTRASTRUCTURE OF THE 'GERMINAL PLASM' IN XENOPUS EMBRYOS AFTER CLEAVAGE

The endodermal location of 'germinal plasm'-bearing cells (GPBCs) and the ultrastructure of the 'germinal plasm' were studied in Xenopus laevis embryos at gastrula, neurula, tailbud and younger tadpole stages. Primordial germ cells (PGCs) of feeding tadpoles were also observed ultrastructurally.
GPBCs were found in the inner endoderm and in the yolk plug region at the late gastrula stage, in the middle and in the dorsal part of the endoderm cell mass at the late neurula and late tailbud stages, respectively. At the younger tadpole stage they were observed in the uppermost dorsal part of the endoderm. Germinal granules were always present in GPBCs at all stages examined but were not found in PGCs of feeding tadpoles. Irregularly shaped-stringlike bodies (ISBs) which seemed to have changed from germinal granules were first noticed in GPBCs at the late neurula stage, and were still present in PGCs of tadpoles, while 'granular materials' were not seen in GPBCs until the feeding tadpole stages. These facts and ultrastructural similarities shared by these organelles lead us to conclude that the change of the germinal granule through ISB, to the 'granular material' takes place during the differentiation of GPBCs into PGCs.     

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.     

Germ line chimera produced by transfer of cultured chick primordial germ cells.

Intrinsic primordial germ cells (PGCs) from stage 27 (5-day-old) chick embryonic germinal ridges were cultured in vitro for a further 5 days, and shown to proliferate on stroma cells derived from the germinal ridge. To determine whether these cultured PGCs could colonize and contribute to the germ-line, PGCs were isolated by gentle pipetting, labeled with PKH26 fluorescent dye and injected into the blood stream of stage 17 (2.5-day-old) chick embryos. The recipient embryos were incubated until they reached stage 28. Thin sections of these embryos were analysed by fluorescent confocal laser microscopy. These analyses showed that the labeled donor PGCs had migrated into the germinal ridges of the recipient embryos, and transplanted PGCs had undergone at least 3-7 divisions. These results suggest that PGCs that had passed far beyond the migration stage in vivo were still able to migrate, colonize and proliferate in recipient chick embryonic gonads.     

mRNA translation during oocyte maturation plays a key role in development of primordial germ cells in<Emphasis Type="Italic">Xenopus</Emphasis> embryos

It is believed that cytoplasmic localization in the egg is necessary for development of primordial germ cells (PGCs) inXenopus embryos. In this study, we sought to determine if translation of maternal mRNA during oocyte maturation is involved in the development of PGCs. Donor oocytes were collected from both stimulated (those who receive gonadotropin) and unstimulated females, artificially matured and fertilized using a host transfer technique. Using chloramphenicol (50 μM and 500 μM RNA), RNA translation was inhibited during oocyte maturation. Our results showed that in unstimulated embryos treated with 50 μM chloramphenicol, there was a significant reduction in the number of PGCs reaching genital ridges. In stimulated embryos, however, the number of PGCs was unchanged unless a higher concentration (500 (μM) of chloramphenicol was used. From these results it is suggested that maternal mRNA translation during oocyte maturation plays a key role in development of PGCs.     

Ultraviolet effects on presumptive primordial germ cells (pPGCs) in Xenopus laevis after the cleavage stage

The presumptive primordial germ cell (pPGC) number with development after the cleavage stage and the fate of pPGCs damaged by uv irradiation were studied in successive Epon sections (0.5 μm thick) with the light microscope in both uv-irradiated and unirradiated Xenopus embryos. taking survival rate and sterility into consideration. The pPGCs of the uv-irradiated embryos occupy nearly the same location in the embryos as those of the unirradiated embryos at stages 12, 17, 23, and 28 [see Ikenishi, K., and Kotani, M. (1975). Develop. Growth Different. 17, 101–110]. At stage $\text{33}\text{34}$ they are found in the central part of the endoderm cell mass in the uv-irradiated embryos, while they are situated in the lateral or dorsal part of the endoderm cell mass in the unirradiated. In the uv-irradiated embryos, a cavity which was never found in the unirradiated embryos was observed in the endoderm cell mass beneath the archenteron cavity and in the almost-median part of the posterior endoderm cell mass at stages 17 and 23, respectively, and some vacuoles in pPGCs as well as in somatic cells around those pPGCs were noticed at stages $\text{17–}\text{33}\text{34}$. The number of pPGCs of the unirradiated enbryos increases about three- or fourfold during stages 12–46, while the pPGCs of the uv-irradiated embryos slowly increase in number from stage 17 to stage 28, indicating that the division occurs in pPGCs, then decrease with development and finally disappear from the tadpole.     

Early loss of Xist RNA expression and inactive X chromosome associated chromatin modification in developing primordial germ cells

BACKGROUND: The inactive X chromosome characteristic of female somatic lineages is reactivated during development of the female germ cell lineage. In mouse, analysis of protein products of X-linked genes and/or transgenes located on the X chromosome has indicated that reactivation occurs after primordial germ cells reach the genital ridges. PRINCIPAL FINDINGS/METHODOLOGY: We present evidence that the epigenetic reprogramming of the inactive X-chromosome is initiated earlier than was previously thought, around the time that primordial germ cells (PGCs) migrate through the hindgut. Specifically, we find that Xist RNA expression, the primary signal for establishment of chromosome silencing, is extinguished in migrating PGCs. This is accompanied by displacement of Polycomb-group repressor proteins Eed and Suz(12), and loss of the inactive X associated histone modification, methylation of histone H3 lysine 27. CONCLUSIONS/SIGNIFICANCE: We conclude that X reactivation in primordial germ cells occurs progressively, initiated by extinction of Xist RNA around the time that germ cells migrate through the hindgut to the genital ridges. The events that we observe are reminiscent of X reactivation of the paternal X chromosome in inner cell mass cells of mouse pre-implantation embryos and suggest a unified model in which execution of the pluripotency program represses Xist RNA thereby triggering progressive reversal of epigenetic silencing of the X chromosome.     

Expression of low density lipoprotein receptor-related protein 4 (Lrp4) gene in the mouse germ cells

The low density lipoprotein receptor-related protein 4 gene (Lrp4) was identified by subtractive screening of cDNAs of the migratory primordial germ cells (PGCs) of E8.5-9.5 embryo and E3.5 blastocysts. Lrp4 is expressed in PGCs in the hindgut and the dorsal mesentery of E9.5 embryos, and in germ cells in the genital ridges of male and female E10.5-13.5 embryos. Lrp4 is also expressed in spermatogonia of the neonatal and adult testes and in the immature oocytes and follicular cells of the adult ovary. The absence of Lrp4 expression in the blastocyst, embryonic stem cells and embryonic germ cells suggests the Lrp4 is a molecular marker that distinguishes the germ cells from embryo-derived pluripotent stem cells.     

Migration and proliferation of primordial germ cells in the rat

Information about early primordial germ cell (PGC) formation and migration in rats is lacking. In utero developed and in vitro cultivated whole rat embryos were studied on days 10-13 postcoitum (p.c.). The development of the PGCs was investigated in serial sections stained for alkaline phosphatase activity. On postcoital day 10, PGCs were found in the invaginating visceral yolk sac endoderm and at the base of the allantois. At day 11 p.c. PGCs were mostly found in the ventral and lateral gut wall or in the mesenchyme between the gut and the future genital ridges. At day 12 p.c. most of the PGCs (94%) could be localised in the mesenchyme or in the future genital ridges. On postcoital day 13 almost all PGCs had reached the now-well-developed genital ridges. Quantitative measurements showed an increase in the number of PGCs from 84 at day 10 p.c. up to 2,768 at day 13 p.c. Only slight differences were found between in vivo and in vitro embryos with respect to the number of PGCs and their developmental pattern. The in vitro culture of whole rat embryos enables the discrimination between the effects of indirect (maternal) and direct action of PGC-toxic agents.