Analysis of SDF-1/CXCR4 signaling in primordial germ cell migration and survival or differentiation in Xenopus laevis

Directional migration of primordial germ cells (PGCs) toward future gonads is a common feature in many animals. In zebrafish, mouse and chicken, SDF-1/CXCR4 chemokine signaling has been shown to have an important role in PGC migration. In Xenopus, SDF-1 is expressed in several regions in embryos including dorsal mesoderm, the target region that PGCs migrate to. CXCR4 is known to be expressed in PGCs. This relationship is consistent with that of more well-known animals. Here, we present experiments that examine whether chemokine signaling is involved in PGC migration of Xenopus. We investigate: (1) Whether injection of antisense morpholino oligos (MOs) for CXCR4 mRNA into vegetal blastomere containing the germ plasm or the precursor of PGCs disturbs the migration of PGCs? (2) Whether injection of exogenous CXCR4 mRNA together with MOs can restore the knockdown phenotype? (3) Whether the migratory behavior of PGCs is disturbed by the specific expression of mutant CXCR4 mRNA or SDF-1 mRNA in PGCs? We find that the knockdown of CXCR4 or the expression of mutant CXCR4 in PGCs leads to a decrease in the PGC number of the genital ridges, and that the ectopic expression of SDF-1 in PGCs leads to a decrease in the PGC number of the genital ridges and an increase in the ectopic PGC number. These results suggest that SDF-1/CXCR4 chemokine signaling is involved in the migration and survival or in the differentiation of PGCs in Xenopus.     

Maternally inherited intron coordinates primordial germ cell homeostasis during Drosophila embryogenesis

Primordial germ cells (PGCs) give rise to the germline stem cells (GSCs) in the adult Drosophila gonads. Both PGCs and GSCs need to be tightly regulated to safeguard the survival of the entire species. During larval development, a non-cell autonomous homeostatic mechanism is in place to maintain PGC number in the gonads. Whether such germline homeostasis occurs during early embryogenesis before PGCs reach the gonads remains unclear. We have previously shown that the maternally deposited sisRNA sisR-2 can influence GSC number in the female progeny. Here we uncover the presence of a homeostatic mechanism regulating PGCs during embryogenesis. sisR-2 represses PGC number by promoting PGC death. Surprisingly, increasing maternal sisR-2 leads to an increase in PGC death, but no drop in PGC number was observed. This is due to ectopic division of PGCs via the de-repression of Cyclin B, which is governed by a genetic pathway involving sisR-2, bantam and brat. We propose a cell autonomous model whereby germline homeostasis is achieved by preserving PGC number during embryogenesis.Subject terms: Development, Gene regulation     

Fatty acid degradation plays an essential role in proliferation of mouse female primordial germ cells via the p53-dependent cell cycle regulation

Primordial germ cells (PGCs) are embryonic founders of germ cells that ultimately differentiate into oocytes and spermatogonia. Embryonic proliferation of PGCs starting from E11.5 ensures the presence of germ cells in adulthood, especially in female mammals whose total number of oocytes declines after this initial proliferation period. To better understand mechanisms underlying PGC proliferation in female mice, we constructed a proteome profile of female mouse gonads at E11.5. Subsequent KEGG pathway analysis of the 3,662 proteins profiled showed significant enrichment of pathways involved in fatty acid degradation. Further, the number of PGCs found in in vitro cultured fetal gonads significantly decreased with application of etomoxir, an inhibitor of the key rate-limiting enzyme of fatty acid degradation carnitine acyltransferase I (CPT1). Decrease in PGCs was further determined to be the result of reduced proliferation rather than apoptosis. The inhibition of fatty acid degradation by etomoxir has the potential to activate the Ca²⁺/CamKII/5′-adenosine monophosphate-activated protein kinase (AMPK) pathway; while as an upstream activator, activated AMPK can function as activator of p53 to induce cell cycle arrest. Thus, we detected the expressional level of AMPK, phosphorylated AMPK (P-AMPK), phosphorylated p53 (P-p53) and cyclin-dependent kinase inhibitor 1 (p21) by Western blots, the results showed increased expression of them after treatment with etomoxir, suggested the activation of p53 pathway was the reason for reduced proliferation of PGCs. Finally, the involvement of p53-dependent G1 cell cycle arrest in defective proliferation of PGCs was verified by rescue experiments. Our results demonstrate that fatty acid degradation plays an important role in proliferation of female PGCs via the p53-dependent cell cycle regulation.     

Heparan sulfate glycosaminoglycans modulate migration and survival in zebrafish primordial germ cells

Early in embryonic development, primordial germ cells (PGCs) are specified and migrate from the site of their origin to where the gonad develops, following a specific route. Heparan sulfate glycosaminoglycans (HS-GAGs) are ubiquitous in extracellular matrix and the cell surface and have long been speculated to play a role during the migration of PGCs. In line with this speculation, whole-mount immunohistochemistry revealed the existence of HS-GAGs in the vicinity of migrating PGCs in early zebrafish embryos. To examine the roles of HS-GAGs during PGC migration, zebrafish heparanase 1 (hpse1), which degrades HS-GAGs, was cloned and overexpressed specifically in PGCs. The guidance signal for the migration of PGCs was disrupted with the overexpression of hpse1, as cluster formation and marginal localization at the blastoderm were significantly perturbed at 6 hours postfertilization. Furthermore, the number of PGCs was significantly decreased with the lack of vicinal HS-GAGs, as observed in the whole-mount in situ hybridization and quantitative PCR of the PGC marker gene vasa. Terminal deoxynucleotidyl transferase dUTP nick-end labeling indicated significantly increased apoptosis in PGCs overexpressing hpse1, suggesting that HS-GAGs contribute to the maintenance of PGC survival. In conclusion, HS-GAGs play multifaceted roles in PGCs during migration and are required both for guidance signals and multiplication of PGCs.     

Nanos3 maintains the germ cell lineage in the mouse by suppressing both Bax-dependent and -independent apoptotic pathways

Cell death in the germ line is controlled by both positive and negative mechanisms that maintain the appropriate number of germ cells and that prevent the possible formation of germ cell tumors. In the mouse embryo, Steel/c-Kit signaling is required to prevent migrating primordial germ cells (PGCs) from undergoing Bax-dependent apoptosis. In our current study, we show that migrating PGCs also undergo apoptosis in Nanos3-null embryos. We assessed whether the Bax-dependent apoptotic pathway is responsible for this cell death by knocking out the Bax gene together with the Nanos3 gene. Differing from Steel-null embryos, however, the Bax elimination did not completely rescue PGC apoptosis in Nanos3-null embryos, and only a portion of the PGCs survived in the double knockout embryo. We further established a mouse line, Nanos3-Cre-pA, to undertake lineage analysis and our results indicate that most of the Nanos3-null PGCs die rather than differentiate into somatic cells, irrespective of the presence or absence of Bax. In addition, a small number of surviving PGCs in Nanos3/Bax-null mice are maintained and differentiate as male and female germ cells in the adult gonads. Our findings thus suggest that heterogeneity exists in the PGC populations and that Nanos3 maintains the germ cell lineage by suppressing both Bax-dependent and Bax-independent apoptotic pathways.     

Formation and cultivation of medaka primordial germ cells

Primordial germ cell (PGC) formation is pivotal for fertility. Mammalian PGCs are epigenetically induced without the need for maternal factors and can also be derived in culture from pluripotent stem cells. In egg-laying animals such as Drosophila and zebrafish, PGCs are specified by maternal germ plasm factors without the need for inducing factors. In these organisms, PGC formation and cultivation in vitro from indeterminate embryonic cells have not been possible. Here, we report PGC formation and cultivation in vitro from blastomeres dissociated from midblastula embryos (MBEs) of the fish medaka (Oryzias latipes). PGCs were identified by using germ-cell-specific green fluorescent protein (GFP) expression from a transgene under the control of the vasa promoter. Embryo perturbation was exploited to study PGC formation in vivo, and dissociated MBE cells were cultivated under various conditions to study PGC formation in vitro. Perturbation of somatic development did not prevent PGC formation in live embryos. Dissociated MBE blastomeres formed PGCs in the absence of normal somatic structures and of known inducing factors. Most importantly, under culture conditions conducive to stem cell derivation, some dissociated MBE blastomeres produced GFP-positive PGC-like cells. These GFP-positive cells contained genuine PGCs, as they expressed PGC markers and migrated into the embryonic gonad to generate germline chimeras. Our data thus provide evidence for PGC preformation in medaka and demonstrate, for the first time, that PGC formation and derivation can be obtained in culture from early embryos of medaka as a lower vertebrate model.     

A critical role for Xdazl, a germ plasm-localized RNA, in the differentiation of primordial germ cells in Xenopus

Xdazl is an RNA component of Xenopus germ plasm and encodes an RNA-binding protein that can act as a functional homologue of Drosophila boule. boule is required for entry into meiotic cell division during fly spermatogenesis. Both Xdazl and boule are related to the human DAZ and DAZL, and murine Dazl genes, which are also involved in gamete differentiation. As suggested from its germ plasm localization, we show here that Xdazl is critically involved in PGC development in Xenopus. Xdazl protein is expressed in the cytoplasm, specifically in the germ plasm, from blastula to early tailbud stages. Specific depletion of maternal Xdazl RNA results in tadpoles lacking, or severely deficient in, primordial germ cells (PGCs). In the absence of Xdazl, PGCs do not successfully migrate from the ventral to the dorsal endoderm and do not reach the dorsal mesentery. Germ plasm aggregation and intracellular movements are normal indicating that the defect occurs after PGC formation. We propose that Xdazl is required for early PGC differentiation and is indirectly necessary for the migration of PGCs through the endoderm. As an RNA-binding protein, Xdazl may regulate translation or expression of factors that mediate migration of PGCs.     

Xenogenesis in teleost fish through generation of germ-line chimeras by single primordial germ cell transplantation

Primordial germ cells (PGCs) are the only cells in developing embryos with the potential to transmit genetic information to the next generation. PGCs therefore have the potential to be of value for gene banking and cryopreservation, particularly via the production of donor gametes with germ-line chimeras. Currently, it is not clear how many PGCs are required for germ-line differentiation and formation of gonadal structures. In the present study, we achieved complete germ-line replacement between two related teleost species, the pearl danio (Danio albolineatus) and the zebrafish (Danio rerio), with transplantation of a single PGC into each host embryo. We isolated and transplanted a single PGC into each blastula-stage, zebrafish embryo. Development of host germ-line cells was prevented by an antisense dead end morpholino oligonucleotide. In many host embryos, the transplanted donor PGC successfully migrated toward the gonadal anlage without undergoing cell division. At the gonadal anlage, the PGC differentiated to form one normally sized gonad rather than the pair of gonads usually present. Offspring were obtained from natural spawning of these chimeras. Analyses of morphology and DNA showed that the offspring were of donor origin. We extended our study to confirm that transplanted single PGCs of goldfish (Carassius auratus) and loach (Misgurnus anguillicaudatus) can similarly differentiate into sperm in zebrafish host embryos. Our results show that xenogenesis is realistic and practical across species, genus, and family barriers and can be achieved by the transplantation of a single PGC from a donor species.     

Molecular and biological aspects of early germ cell development in interspecies hybrids between chickens and pheasants

Interspecific hybrids provide insights into fundamental genetic principles, and may prove useful for biotechnological applications and as tools for the conservation of endangered species. In the present study, interspecies hybrids were generated between the Korean ring-necked pheasant (Phasianus colchicus) and the White Leghorn chicken (Gallus gallus domesticus). We determined whether these hybrids were good recipients for the production of germline chimeric birds. PCR-based species-specific amplification and karyotype analyses showed that the hybrids inherited genetic material from both parents. Evaluation of biological function indicated that the growth rates of hybrids during the exponential phase (body weight/week) were similar to those of the pheasant but not the chicken, and that the incubation period for hatching was significantly different from that of both parents. Primordial germ cells (PGCs) of hybrids reacted with a pheasant PGC-specific antibody and circulated normally in blood vessels. The peak time of hybrid PGC migration was equivalent to that of the pheasant. In late embryonic stages, germ cells were detected by the QCR1 antibody on 15 d male gonads and were normally localized in the seminiferous cords. We examined the migration ability and developmental localization of exogenous PGCs transferred into the blood vessels of 63 h hybrid embryos. Donor-derived PGCs reacted with a donor-specific antibody were detected on 7 d gonads and the seminiferous tubules of hatchlings. Therefore, germ cell transfer into developing embryos of an interspecies hybrid can be efficiently used for the conservation of threatened animals and endangered species, and many biotechnological applications.     

Inter-species transplantation and migration of primordial germ cells in cyprinid fish

Primordial germ cells (PGCs) are the only cells in developing embryos that can transmit genetic information to the next generation. PGCs therefore have considerable potential value for gene banking and cryopreservation, particularly via production of donor gametes using germ-line chimeras. In some animal species, including teleost fish, the feasibility of using PGC transplantation to obtain donor-derived offspring, within and between species, has been demonstrated. Successful use of PGC transplantation to produce germ-line chimeras is absolutely dependent on the migration of the transplanted cells from the site of transplantation to the host gonadal region. Here, we induced germ-line chimeras between teleost species using two different protocols: blastomere transplantation and single PGC transplantation. We evaluated the methods using the rate of successful migration of transplanted PGCs to the gonadal region of the host embryo. First, we transplanted blastomeres from zebrafish, pearl danio, goldfish, or loach into blastula-stage zebrafish embryos. Some somatic cells, derived from donor blastomeres, were co-transplanted with the PGCs and formed aggregates in the host embryos; a low efficiency of PGC transfer was achieved. Second, a single PGC from the donor species was transplanted into a zebrafish embryo. In all inter-species combinations, the donor PGC migrated toward the gonadal region of the host embryo at a comparatively high rate, regardless of the phylogenetic relationship of the donor and host species. These transplantation experiments showed that the mechanism of PGC migration is highly conserved beyond the family barrier in fish and that transplantation of a single PGC is an efficient method for producing inter-species germ-line chimeras.     

Effects of specific and prolonged expression of zebrafish growth factors,Fgf2 and Lif in primordial germ cells in vivo

Primordial germ cells (PGCs), specified early in development, proliferate and migrate to the developing gonad before sexual differentiation occurs in the embryo and eventually give rise to spermatogonia or oogonia. In this study, we discovered that nanos3 3′UTR, a common method used to label PGCs, not only directed PGC-specific expression of DsRed but also prolonged this expression up to 26 days post fertilization (dpf) when DsRed-nanos3 3′UTR hybrid mRNAs were introduced into 1- to 2-cell-stage embryos. As such, we employed this knowledge to express zebrafish leukemia inhibitory factor (Lif), basic fibroblast growth factor (Fgf2) and bone morphogenetic protein 4 (Bmp4) in the PGCs and evaluate their effects on PGC development in vivo for over a period of 3 weeks. The results show that expression of Fgf2 significantly increased PGC number at 14- and 21-dpf while Bmp4 resulted in severe ventralization and death of the embryos by 3 days. Expression of Lif resulted in a significant disruption of PGC migration. Mopholino knockdown experiments indicated that Lif illicited its effect on PGC migration through Lif receptor a (Lifra) but not Lifrb. The general approach described in this study could be used to achieve prolonged PGC-specific expression of other proteins to investigate their roles in germ cell and gonad development. The results also indicate that zebrafish PGCs have a mechanism to stabilize and prolong the expression of mRNA that carries nanos3 3′UTR. Understanding this mechanism may make it possible to achieve prolonged RNA expression in other cell types.     

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

Nuclear transfer embryonic stem cells (ntESCs) show stem cell characteristics such as pluripotency but cause no immunological disorders. Although ntESCs are able to differentiate into somatic cells, the ability of ntESCs to differentiate into primordial germ cells (PGCs) has not been examined. In this work, we examined the capacity of mouse ntESCs to differentiate into PGCs in vitro. ntESCs aggregated to form embryoid bodies (EB) in EB culture medium supplemented with bone morphogenetic protein 4(BMP4) as the differentiation factor. The expression level of specific PGC genes was compared at days 4 and 8 using real time PCR. Flow cytometry and immunocytochemical staining were used to detect Mvh as a specific PGC marker. ntESCs expressed particular genes related to different stages of PGC development. Flow cytometry and immunocytochemical staining confirmed the presence of Mvh protein in a small number of cells. There were significant differences between cells that differentiated into PGCs in the group treated with Bmp4 compared to non-treated cells. These findings indicate that ntESCs can differentiate into putative PGCs. Improvement of ntESC differentiation into PGCs may be a reliable means of producing mature germ cells.     

Implication of DNA Demethylation and Bivalent Histone Modification for Selective Gene Regulation in Mouse Primordial Germ Cells

Primordial germ cells (PGCs) sequentially induce specific genes required for their development. We focused on epigenetic changes that regulate PGC-specific gene expression. mil-1, Blimp1, and Stella are preferentially expressed in PGCs, and their expression is upregulated during PGC differentiation. Here, we first determined DNA methylation status of mil-1, Blimp1, and Stella regulatory regions in epiblast and in PGCs, and found that they were hypomethylated in differentiating PGCs after E9.0, in which those genes were highly expressed. We used siRNA to inhibit a maintenance DNA methyltransferase, Dnmt1, in embryonic stem (ES) cells and found that the flanking regions of all three genes became hypomethylated and that expression of each gene increased 1.5- to 3-fold. In addition, we also found 1.5- to 5-fold increase of the PGC genes in the PGCLCs (PGC-like cells) induced form ES cells by knockdown of Dnmt1. We also obtained evidence showing that methylation of the regulatory region of mil-1 resulted in 2.5-fold decrease in expression in a reporter assay. Together, these results suggested that DNA demethylation does not play a major role on initial activation of the PGC genes in the nascent PGCs but contributed to enhancement of their expression in PGCs after E9.0. However, we also found that repression of representative somatic genes, Hoxa1 and Hoxb1, and a tissue-specific gene, Gfap, in PGCs was not dependent on DNA methylation; their flanking regions were hypomethylated, but their expression was not observed in PGCs at E13.5. Their promoter regions showed the bivalent histone modification in PGCs, that may be involved in repression of their expression. Our results indicated that epigenetic status of PGC genes and of somatic genes in PGCs were distinct, and suggested contribution of epigenetic mechanisms in regulation of the expression of a specific gene set in PGCs.     

Detection and characterization of primordial germ cells in pheasant (Phasianus colchicus) embryos

The developmental similarity between the chicken and pheasant (Phasianus colchicus) allows the novel biotechnologies developed in the chicken to be applied to the production of transgenic pheasants and interspecies germline chimeras. To detect pheasant primordial germ cells (PGCs) efficiently, which is important for inducing germline transmission, the ultrastructure of PGCs and their reactivity to several antibodies (2C9, QB2, anti-SSEA-1, and QCR1) and periodic acid-Schiff's solution (PAS) were examined. To obtain PGCs, blood was taken from embryos incubated for 62-72 h or from gonads from embryos incubated for 156-216 h. The PGCs collected from both sources had the typical ultrastructure of pluripotent cells: a large nucleus with a distinct nucleolus, a high ratio of nuclear to cytoplasmic volume, and a distinct cytoplasmic membrane. In comparing the morphology of PGCs collected from different sites, more mitochondria and better-developed membrane microvilli were found in gonadal PGCs than in circulating PGCs. The nucleus of gonadal PGCs was flattened and had a large eccentrically positioned nucleolus. Of the antibodies tested, only QCR1 antibody reacted with an epitope in pheasant PGCs, and no specific signal was detected to other antibodies. The temporal change in the PGC populations in the blood and gonads of embryos was examined. In blood, the population was greater (P < 0.0001) in embryos incubated for 64 h than in embryos incubated for 62 or 66-72 h (31.4 versus 5.6-16.2 microL(-1)). In embryonic gonads, the number of PGCs increased continuously from 156 to 216 h of incubation (193-2,718 cells/embryo), although the ratio of PGCs to total gonadal cells did not change significantly (0.50-0.61%). In conclusion, pheasant PGCs have typical germ cell morphology and possess the QCR1 epitope. Circulating blood and the gonads of embryos incubated for 64 and 216 h, respectively, are good sources of PGCs.     

Medaka vasa is required for migration but not survival of primordial germ cells

vasa is essential for germline development. However, the precise processes in which vasa involves vary considerably in diverse animal phyla. Here we show that vasa is required for primordial germ cell (PGC) migration in the medakafish. vasa knockdown by two morpholinos led to the PGC migration defect that was rescued by coinjection of vasa RNA. Interestingly, vasa knockdown did not alter the PGC number, identity, proliferation and motility even at ectopic locations. We established a cell culture system for tracing PGCs at the single cell level in vitro. In this culture system, control and morpholino-injected gastrulae produced the same PGC number and the same time course of PGC survival. Importantly, vasa-depleted PGCs in culture had similar motility and locomotion to normal PGCs. Expression patterns of wt1a, sdf1b and cxcr4b in migratory tissues remained unchanged by vasa knockdown. By chimera formation we show that PGCs from vasa-depleted blastulae failed to migrate properly in the normal environment, whereas control PGCs migrated normally in vasa-disrupted embryos. Furthermore, ectopic PGCs in vasa-depleted embryos also retained all the PGC properties examined. Taken together, medaka vasa is cell-autonomously required for PGC migration, but dispensable to PGC proliferation, motility, identity and survival.