Derivation in culture of primordial germ cells from cells of the mouse epiblast: phenotypic induction and growth control by Bmp4 signalling

Primordial germ cells (PGCs) are the embryonic precursors of the gametes of the adult. PGCs derive from cells of the most proximal part of the cup-shaped epiblast corresponding to the presumptive region of the extraembryonic mesoderm. At 7.2 days post coitum (dpc) a small group of PGCs located at the base of the allantois can be recognised due to a strong alkaline phosphatase activity. Thus far, scant information was available on the mechanism(s) controlling the lineage of PGCs in the mouse embryo. However, results obtained in mice defective for bone morphogenetic protein-4 (Bmp4) secreted molecule revealed that this growth factor has important functions for the derivation of PGCs from extraembryonic mesoderm cells. In this paper, we have studied the effects in culture of Bmp4 on epiblast cells obtained from egg-cylinder stage mouse embryos (5.5-6.0 dpc) and PGCs from 11.5 dpc embryos. We found that Bmp4 treatment enables recruitment of pluripotent cells to a PGC phenotype by a multi-step process involving an initial pre-commitment of epiblast cells and a following stage of PGC phenotypic determination. We further provide evidences that Bmp4 may promote the growth of gonadal PGCs through a Smad1/4 signalling.     

Stage-specific tissue and cell interactions play key roles in mouse germ cell specification

Primordial germ cells (PGCs) in mice have been recognized histologically as alkaline phosphatase (AP) activity-positive cells at 7.2 days post coitum (dpc) in the extra-embryonic mesoderm. However, mechanisms regulating PGC formation are unknown, and an appropriate in vitro system to study the mechanisms has not been established. Therefore, we have developed a primary culture of explanted embryos at pre- and early-streak stages, and have studied roles of cell and/or tissue interactions in PGC formation. The emergence of PGCs from 5.5 dpc epiblasts was observed only when they were co-cultured with extra-embryonic ectoderm, which may induce the conditions required for PGC formation within epiblasts. From 6.0 dpc onwards, PGCs emerged from whole epiblasts as did a fragment of proximal epiblast that corresponds to the area containing presumptive PGC precursors without neighboring extra-embryonic ectoderm and visceral endoderm. Dissociated epiblasts at these stages, however, did not give rise to PGCs, indicating that interactions among a cluster of a specific number of proximal epiblast cells is needed for PGC differentiation. In contrast, we observed that dissociated epiblast cells from a 6.5-b (6.5+15-16 hours) to 6.75 dpc embryo that had undergone gastrulation gave rise to PGCs. Our results demonstrate that stage-dependent tissue and cell interactions play key roles in PGC determination.     

The different roles of cyclinD1-CDK4 in STP and mGluR-LTD during the postnatal development in mice hippocampus area CA1

Background

The extraembryonic tissues, visceral endoderm (VE) and extraembryonic ectoderm (ExE) are known to be important for the induction of primordial germ cells (PGCs) in mice via activation of the bone morphogenetic protein (BMP) signalling pathway. We investigated whether the VE and ExE have a direct role in the specification of PGCs, or in an earlier event, namely the induction of the PGC precursors in the proximal posterior epiblast cells.

Results

We cultured embryonic day (E) 5.75 to E7.0 mouse embryos in an explant-assay with or without extraembryonic tissues. The reconstituted pieces of embryonic and extraembryonic tissues were assessed for the formation of both PGC precursors and specified PGCs. For this, Blimp1:gfp and Stella:gfp transgenic mouse lines were used to distinguish between PGC precursors and specified PGC, respectively. We observed that the VE regulates formation of an appropriate number of PGC precursors between E6.25–E7.25, but it is not essential for the subsequent specification of PGCs from the precursor cells. Furthermore, we show that the ExE has a different role from that of the VE, which is to restrict localization of PGC precursors to the posterior part of the embryo.

Conclusion

We show that the VE and ExE have distinct roles in the induction of PGC precursors, namely the formation of a normal number of PGC precursors, and their appropriate localization during early development. However, these tissues do not have a direct role during the final stages of specification of the founder population of PGCs.     

Primordial germ cells: what does it take to be alive?

Specification of primordial germ cells (PGCs) in the proximal epiblast enables about 45 founder PGCs clustered at the base of the allantoic bud to enter the embryo by active cell movement. Specification of the PGC lineage depends on paracrine signals derived from the somatic cell neighbors in the extraembryonic ectoderm. Secretory bone morphogenetic proteins (BMP) 4, BMP8b, and BMP2 and components of the Smad signaling pathway participate in the specification of PGCs. Cells in the extraembryonic ectoderm induce expression of the gene fragilis in the epiblast in the presence of BMP4, targeting competence of PGCs. The fragilis gene encodes a family of transmembrane proteins presumably involved in homotypic cell adhesion. As PGCs migrate throughout the hindgut, they express nanos3 protein. In the absence of nanos3 gene expression, no germ cells are detected in ovary and testis. During migration and upon arrival at the genital ridges, the population of PGCs is regulated by a balanced proliferation/programmed cell death or apoptosis. Paracrine and autocrine mechanisms, involving transforming growth factor-beta1 and fibroblast growth factors exert stimulatory or inhibitory effects on PGCs proliferation, modulated in part by the membrane-bound form of stem cell factor. Apoptosis requires the participation of the pro-apoptotic family member Bax, whose activity is balanced by the anti-apoptotic family member Bcl21/Bcl-x. In addition, a loss of cell-cell contacts in vitro results in the apoptotic elimination of PGCs. It needs to be determined whether apoptosis is triggered by a failure of PGC to establish and maintain appropriate cell-cell contacts with somatic cells or whether undefined survival factors released by adjacent somatic cells cannot reach physiological levels to satisfy needs of the expanding population of PGCs.     

Collagen and tenascin-C expression along the migration pathway of mouse primordial germ cells

Primordial germ cells (PGCs) are the progenitor cells of the vertebrate germ line. These cells originate outside of the embryo and, through separation, migration, and colonization, arrive at the genital ridge, contributing to gonad development. Diverse extracellular matrix molecules are present along the PGC migratory pathway, permitting or inhibiting PGC displacement. Collagens and tenascin form the substratum for in vitro migration of neural crest cells and PGCs. However, little is known about the expression and distribution of these molecules during in situ PGC migration. Using immunohistochemistry, we identified tenascin-C and types I, III, and V collagen along the mouse PGC migration pathway. These molecules were spatiotemporally expressed in basement membranes of hindgut, coelomic epithelia, and mesonephric tubules and mesenchyme throughout the study. Our results complement previous data from our laboratory and contribute to building comprehension of the composition of the mouse PGC migratory pathway extracellular matrix, thereby enhancing understanding of the process.     

Cooperation of endoderm-derived BMP2 and extraembryonic ectoderm-derived BMP4 in primordial germ cell generation in the mouse

The primordial germ cells (PGCs) of the mouse are derived from proximal epiblast cells that are adjacent to the extraembryonic ectoderm during gastrulation. Previous studies have demonstrated that extraembryonic ectoderm-derived BMP4 and BMP8B are both required for PGC generation. Here we show that Bmp2, a member of the Dpp class of the Bmp superfamily, also plays a role in PGC generation. PGC number is significantly reduced in Bmp2 heterozygous and homozygous embryos at the N2 generation onto C57BL/6 background. Bmp2 homozygous embryos also have a short allantois and about 50% of them do not undergo normal chorioallantoic fusion. Using whole-mount in situ hybridization, we show that Bmp2 is primarily expressed in the endoderm of mouse pregastrula and gastrula embryos. Using a genetic approach, we further show that Bmp2 and Bmp4, but not Bmp2 and Bmp8b, have an additive effect on PGC generation. These results suggest that PGC generation in the mouse embryo is regulated not only by extraembryonic ectoderm-derived BMP4 and BMP8B, but also by endoderm-derived BMP2.     

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.     

Mouse epiblasts change responsiveness to BMP4 signal required for PGC formation through functions of extraembryonic ectoderm

Mouse primordial germ cells (PGCs) are initially identified as a cluster of alkaline phosphatase (AP)-positive cells within the extraembryonic mesoderm near the posterior part of the primitive streak at embryonic day (E) 7.25. Clonal analysis of epiblast cells has revealed that the putative precursors of PGCs are localized in the proximal epiblast, and we demonstrated that the conditions required for PGC formation are induced in the proximal region of epiblasts by extraembryonic ectoderm. Bone morphogenetic protein (BMP) 4 and BMP8b, which belong to the transforming growth factor-beta (TGF-beta) superfamily, might generate induction signals from extraembryonic ectoderm. Smad1 and Smad5, which are intracellular signaling molecules for BMP4, might also play a critical role in stimulating epiblasts to form PGC. However, how pluripotential epiblasts temporally and spatially respond to BMP signals to form PGCs remains unclear. The present study examines changes of responsiveness to BMP4 for PGC formation in epiblasts and their molecular mechanisms. We initially examined the effect of recombinant human (rh) BMP4 upon cultured epiblasts at different developmental stages, and found that they acquire the ability to respond to BMP4 signals for PGC formation between E5.25 and E5.5. In addition, such competence was conferred upon epiblasts by the extraembryonic ectoderm. We also showed that the increased expression of Smad1 and the onset of Smad5 expression induced by extraembryonic ectoderm might be responsible for quick acquisition of this competence. Furthermore, we show that only proximal epiblast cells maintain responsiveness to BMP4 for PGC formation at E6.0, and that this is associated with the proximal epiblast-specific expression of Smad5. These results explain why only the proximal region of epiblasts can sustain the ability to form PGCs.     

X chromosome activity in mouse XX primordial germ cells

In the early epiblast of female mice, one of the two X chromosomes is randomly inactivated by a Xist-dependent mechanism, involving the recruitment of Ezh2-Eed and the subsequent trimethylation of histone 3 on lysine 27 (H3K27me3). We demonstrate that this random inactivation process applies also to the primordial germ cell (PGC) precursors, located in the proximal region of the epiblast. PGC specification occurs at about embryonic day (E)7.5, in the extraembryonic mesoderm, after which the germ cells enter the endoderm of the invaginating hindgut. As they migrate towards the site of the future gonads, the XX PGCs gradually lose the H3K27me3 accumulation on the silent X chromosome. However, using a GFP transgene inserted into the X chromosome, we observed that the XX gonadal environment (independently of the gender) is important for the substantial reactivation of the inactive X chromosome between E11.5 and E13.5, but is not required for X-chromosome reactivation during the derivation of pluripotent embryonic germ cells. We describe in detail one of the key events during female PGC development, the epigenetic reprogramming of the X chromosome, and demonstrate the role of the XX somatic genital ridge in this process.     

Analysis of germ line development in the chick embryo using an anti-mouse EC cell antibody

We have found that EMA-1, a monoclonal antibody originally raised against mouse embryonal carcinoma (Nulli SCC1) cells (Hahnel & Eddy, 1982), also labels chick primordial germ cells (PGCs). We have used this antibody in immunohistological studies to follow the development of PGCs in the chick embryo from the time of their initial appearance beneath the epiblast, through their migratory phase and subsequent colonization of the germinal epithelium. During hypoblast formation, individual EMA-1-labelled cells appeared to separate from the basal surface of the epiblast and enter the blastocoel, coincident with the appearance of morphologically identifiable PGCs in this same area. EMA-1 continued to label germ cells until the initiation of gametogenesis in each sex; specifically, labelling was absent by 7-8 days of incubation in females and started to decrease at 11 days of incubation in males. There was a recurrence of the epitope on oogonia at 15 days of incubation, but not on spermatogonia during the remainder of development through hatching. These observations are consistent with an epiblast origin for the avian germ line, and are strikingly similar to those reported for the early mouse embryo using the same antibody (Hahnel & Eddy, 1986).     

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.     

Requirement of Bmp8b for the generation of primordial germ cells in the mouse

In the mouse embryo, the generation of primordial germ cells (PGCs) from the epiblast requires a bone morphogenetic protein-4 (BMP4) signal from the adjacent extraembryonic ectoderm. In this study, we report that Bmp8b, a member of the Gbb-60A class of the BMP superfamily, is expressed in the extraembryonic ectoderm in pregastrula and gastrula stage mouse embryos and is required for PGC generation. A mutation in Bmp8b on a mixed genetic background results in the absence of PGCs in 43% null mutant embryos and severe reduction in PGC number in the remainder. The heterozygotes are unaffected. On a largely C57BL/6 background, Bmp8b null mutants completely lack PGCs, and Bmp8b heterozygotes have a reduced number of PGCs. In addition, Bmp8b homozygous null embryos on both genetic backgrounds have a short allantois, and this organ is missing in some more severe mutants. Since Bmp4 heterozygote embryos have reduced numbers of PGCs, we used a genetic approach to generate double-mutant embryos to study interactions of Bmp8b and Bmp4. Embryos that are double heterozygotes for the Bmp8b and Bmp4 mutations have similar defects in PGC number as Bmp4 heterozygotes, indicating that the effects of the two BMPs are not additive. These findings suggest that BMP4 and BMP8B function as heterodimers and homodimers in PGC specification in the mouse.     

Migration,proliferation and potency of primordial germ cells

In most species, the cells allocated to the germ line, the primordial germ cells (PGCs) arise very early in embryo-genesis, and migrate to join the somatic cells at the site where the gonad will form. In three widely studied animals; the mouse, the frog and Drosophila, the PGCs associate with the developing gut, from which they migrate during the period of organogenesis to the gonads. During this migration, the germ cell population increases by an amount which is more or less constant for a particular species. Genes important in the control of PGC migration and population are being identified in two ways. In invertebrates, and to a lesser extent in mice, genetic approaches have identified important loci or gene products. Culturing PGCs in a variety of conditions has been an alternative approach in mouse embryos. From these latter studies, it is now known that a number of growth factors, released from surrounding tissues, control many aspects of PGC behaviour, including their proliferation, migration, potency, and survival. Attention is also focusing on changes in PGC adhesiveness during migration.     

Production of germ line chimera by transfer of primordial germ cells in the domestic chicken (Gallus domesticus)

Blood was collected from Stage 13 to 14 (1) chick embryos. Primordial germ cells (PGCs) were separated from blood cells by Ficoll density gradient centrifugation. One hundred Rhode Island Red PGCs per embryo were transferred to the blood stream of Stage 14 to 15 White Leghorn embryos. Also, one hundred White Leghorn PGCs per embryo were transferred to the blood stream of Stage 14 to 15 Rhode Island Red embryos. Hatched male and female chicks were raised until sexual maturity, and progeny tests were performed by mating these PGC recipients with Rhode Island Red chickens of the opposite sex. Chicks apparently derived from the transferred PGCs, based on the feather color of the chicks, were produced from all 4 possible mating combinations. The present results indicate that the germ line of PGC recipient chickens consists of 2 distinct populations of germ cells.     

Spermatogenesis from epiblast and primordial germ cells following transplantation into postnatal mouse testis

Primordial germ cells (PGCs) are derived from a population of pluripotent epiblast cells in mice. However, little is known about when and how PGCs acquire the capacity to differentiate into functional germ cells, while keeping the potential to derive pluripotent embryonic germ cells and teratocarcinomas. In this investigation, we show that epiblast cells and PGCs can establish colonies of spermatogenesis after transfer into postnatal seminiferous tubules of surrogate infertile mice. Furthermore, we obtained normal fertile offspring by microinsemination using spermatozoa or spermatids derived from PGCs harvested from fetuses as early as 8.5 days post coitum. Thus, fetal male germ cell development is remarkably flexible, and the maturation process, from epiblast cells through PGCs to postnatal spermatogonia, can occur in the postnatal testicular environment. Primordial germ cell transplantation techniques will also provide a novel tool to assess the developmental potential of PGCs, such as those manipulated in vitro or recovered from embryos harboring lethal mutations.     

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.     

Cellular and molecular aspects of mouse primordial germ cell migration and proliferation in culture.

The development of mouse primordial germ cells is followed from their first appearance in the extraembryonic mesoderm of the posterior amniotic fold (7 dpc embryo) to their settlement in the genital ridges (12.5 dpc embryo). The role of fibronectin as adhesive substrate and/or in stimulating cell motility during PGC migration is discussed. Recent papers showing how PGCs migrate when cultured in vitro on cellular monolayers are reviewed. The process of PGC homing is proposed to be controlled by chemotaxis as well by developmentally regulated cell-to-cell interactions. Finally, evidence that survival and proliferation of PGCs is strictly dependent on growth factors such as LIF and MGF, and possibly on a cAMP-dependent mechanism is reported.