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.     

Mouse embryos lacking Smad1 signals display defects in extra-embryonic tissues and germ cell formation

The Smad proteins are important intracellular mediators of the transforming growth factor beta (TGFbeta) family of secreted growth factors. Smad1 is an effector of signals provided by the bone morphogenetic protein (BMP) sub-group of TGFbeta molecules. To understand the role of Smad1 in mouse development, we have generated a Smad1 loss-of-function allele using homologous recombination in ES cells. Smad1-/- embryos die by 10.5 dpc because they fail to connect to the placenta. Mutant embryos are first recognizable by 7.0 dpc, owing to a characteristic localized outpocketing of the visceral endoderm at the posterior embryonic/extra-embryonic junction, accompanied by a dramatic twisting of the epiblast and nascent mesoderm. Chimera analysis reveals that these two defects are attributable to a requirement for Smad1 in the extra-embryonic tissues. By 7.5 dpc, Smad1-deficient embryos show a marked impairment in allantois formation. By contrast, the chorion overproliferates, is erratically folded within the extra-embryonic space and is impeded in proximal migration. BMP signals are known to be essential for the specification and proliferation of primordial germ cells. We find a drastic reduction of primordial germ cells in Smad1-deficient embryos, suggesting an essential role for Smad1-dependent signals in primordial germ cell specification. Surprisingly, despite the key involvement of BMP signaling in tissues of the embryo proper, Smad1-deficient embryos develop remarkably normally. An examination of the expression domains of Smad1, Smad5 and Smad8 in early mouse embryos show that, while Smad1 is uniquely expressed in the visceral endoderm at 6.5 dpc, in other tissues Smad1 is co-expressed with Smad5 and/or Smad8. Collectively, these data have uncovered a unique function for Smad1 signaling in coordinating the growth of extra-embryonic structures necessary to support development within the uterine environment.     

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.     

Anterior visceral endoderm SMAD4 signaling specifies anterior embryonic patterning and head induction in mice

SMAD4 serves as a common mediator for signaling of TGF-β superfamily. Previous studies illustrated that SMAD4-null mice die at embryonic day 6.5 (E6.5) due to failure of mesoderm induction and extraembryonic defects; however, functions of SMAD4 in each germ layer remain elusive. To investigate this, we disrupted SMAD4 in the visceral endoderm and epiblast, respectively, using a Cre-loxP mediated approach. We showed that mutant embryos lack of SMAD4 in the visceral endoderm (Smad4(Co/Co);TTR-Cre) died at E7.5-E9.5 without head-fold and anterior embryonic structures. We demonstrated that TGF-β regulates expression of several genes, such as Hex1, Cer1, and Lim1, in the anterior visceral endoderm (AVE), and the failure of anterior embryonic development in Smad4(Co/Co);TTR-Cre embryos is accompanied by diminished expression of these genes. Consistent with this finding, SMAD4-deficient embryoid bodies showed impaired responsiveness to TGF-β-induced gene expression and morphological changes. On the other hand, embryos carrying Cre-loxP mediated disruption of SMAD4 in the epiblasts exhibited relatively normal mesoderm and head-fold induction although they all displayed profound patterning defects in the later stages of gastrulation. Cumulatively, our data indicate that SMAD4 signaling in the epiblasts is dispensable for mesoderm induction although it remains critical for head patterning, which is significantly different from SMAD4 signaling in the AVE, where it specifies anterior embryonic patterning and head induction.     

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.     

Differential requirements for Smad4 in TGFbeta-dependent patterning of the early mouse embryo

Genetic and biochemical data have identified Smad4 as a key intracellular effector of the transforming growth factor beta (TGFbeta superfamily of secreted ligands. In mouse, Smad4-null embryos do not gastrulate, a phenotype consistent with loss of other TGFbeta-related signaling components. Chimeric analysis reveals a primary requirement for Smad4 in the extra-embryonic lineages; however, within the embryo proper, characterization of the specific roles of Smad4 during gastrulation and lineage specification remains limited. We have employed a Smad4 conditional allele to specifically inactivate the Smad4 gene in the early mouse epiblast. Loss of Smad4 in this tissue results in a profound failure to pattern derivatives of the anterior primitive streak, such as prechordal plate, node, notochord and definitive endoderm. In contrast to these focal defects, many well-characterized TGFbeta- and Bmp-regulated processes involved in mesoderm formation and patterning are surprisingly unaffected. Mutant embryos form abundant extra-embryonic mesoderm, including allantois, a rudimentary heart and middle primitive streak derivatives such as somites and lateral plate mesoderm. Thus, loss of Smad4 in the epiblast results not in global developmental abnormalities but instead in restricted patterning defects. These results suggest that Smad4 potentiates a subset of TGFbeta-related signals during early embryonic development, but is dispensable for others.     

Confinement and clearance of OCT4 in the porcine embryo at stereomicroscopically defined stages around gastrulation

In the areas of developmental biology and embryonic stem cell research, reliable molecular markers of pluripotency and early lineage commitment are sparse in large animal species. In this study, we present morphological and immunohistochemical findings on the porcine embryo in the period around gastrulation, days 8-17 postinsemination, introducing a stereomicroscopical staging system in this species. In embryos at the expanding hatched blastocyst stage, OCT4 is confined to the inner cell mass. Following detachment of the hypoblast, and formation of the embryonic disk, this marker of pluripotency was selectively observed in the epiblast. A prominent crescent-shaped thickening at the posterior region of the embryonic disk marked the first polarization within this structure reflecting incipient cell ingression. Following differentiation of the epiblast, clearance of OCT4 from the three germ layers was observed at defined stages, suggesting correlations to lineage specification. In the endoderm, clearance of OCT4 was apparent from early during its formation at the primitive streak stage. The endoderm harbored progenitors of the "fourth germ layer," the primordial germ cells (PGCs), the only cells maintaining expression of OCT4 at the end of gastrulation. In the ectodermal and mesodermal cell lineages, OCT4 became undetectable at the neural groove and somite stage, respectively. As in the mouse, PGCs showed onset of c-kit expression when located in extraembryonal compartments. They appeared to follow the endoderm during extraembryonal allocation and the mesoderm on return to the genital ridge.     

Smad5 determines murine amnion fate through the control of bone morphogenetic protein expression and signalling levels

Smad5 is an intracellular mediator of bone morphogenetic protein (Bmp) signalling. It is essential for primordial germ cell (PGC) development, for the development of the allantois and for amnion closure, as demonstrated by loss of Bmp signalling. By contrast, the appearance of ectopic PGC-like cells and regionalized ectopic vasculogenesis and haematopoiesis in thickened Smad5(m1/m1) amnion are amnion defects that have not been associated with loss of Bmp signalling components. We show that defects in amnion and allantois can already be detected at embryonic day (E) 7.5 in Smad5 mutant mice. However, ectopic Oct4-positive (Oct4(+)) and alkaline phosphatase-positive (AP(+)) cells appear suddenly in thickened amnion at E8.5, and at a remote distance from the allantois and posterior primitive streak, suggesting a change of fate in situ. These ectopic Oct4(+), AP(+) cells appear to be Stella negative and hence cannot be called bona fide PGCs. We demonstrate a robust upregulation of Bmp2 and Bmp4 expression, as well as of Erk and Smad activity, in the Smad5 mutant amnion. The ectopic expression of several Bmp target genes in different domains and the regionalized presence of cells of several Bmp-sensitive lineages in the mutant amnion suggest that different levels of Bmp signalling may determine cell fate. Injection of rBMP4 in the exocoelom of wild-type embryos can induce thickening of amnion, mimicking the early amnion phenotype in Smad5 mutants. These results support a model in which loss of Smad5 results paradoxically in gain of Bmp function defects in the amnion.     

Reprint of "Unsaturated diacylglycerol as a possible messenger for the activation of calcium-activated, phospholipid-dependent protein kinase system"

Murine embryonic stem cells (ESCs) are pluripotent cells that differentiate into multiple cell lineages. It was recently observed that all-trans retinoic acid (RA) provides instructive signals for the commitment of the germ cell lineage from ESCs. However, little is known about the molecular mechanisms by which RA signals lead to germ cell commitment. In this study, we determined if RA induced ESC differentiation to the germ lineage through modulation of the (bone morphogenetic protein) BMP/Smad pathway activity. In a monolayer culture, RA significantly induced both the expression of the early germ-specific genes, Stra8, Dazl and Mvh, and prolonged activation of Smad1/5 (for at least 24h). Meanwhile, dorsomorphin (a BMP-Smad1/5 specific inhibitor) significantly reduced the RA-induced germ-specific gene expression and completely blocked the RA-induced activation of Smad1/5. Moreover, RA-induced germ-specific gene expression was significantly increased by treatment with the potential activator of Smad1/5, SB431542. Furthermore, the biochemical manipulation of Smad1/5 expression through shRNA knockdown significantly reduced RA-mediated up-regulation of germ-specific gene expression. Our results clearly demonstrate that the Smad1/5 pathway is specifically required at an early stage of germ cell differentiation, corresponding to the RA-dependent commitment of ESCs.     

Paracrine effects of embryo-derived FGF4 and BMP4 during pig trophoblast elongation

The crosstalk between the epiblast and the trophoblast is critical in supporting the early stages of conceptus development. FGF4 and BMP4 are inductive signals that participate in the communication between the epiblast and the extraembryonic ectoderm (ExE) of the developing mouse embryo. Importantly, however, it is unknown whether a similar crosstalk operates in species that lack a discernible ExE and develop a mammotypical embryonic disc (ED). Here we investigated the crosstalk between the epiblast and the trophectoderm (TE) during pig embryo elongation. FGF4 ligand and FGFR2 were detected primarily on the plasma membrane of TE cells of peri-elongation embryos. The binding of this growth factor to its receptor triggered a signal transduction response evidenced by an increase in phosphorylated MAPK/ERK. Particular enrichment was detected in the periphery of the ED in early ovoid embryos, indicating that active FGF signalling was operating during this stage. Gene expression analysis shows that CDX2 and ELF5, two genes expressed in the mouse ExE, are only co-expressed in the Rauber's layer, but not in the pig mural TE. Interestingly, these genes were detected in the nascent mesoderm of early gastrulating embryos. Analysis of BMP4 expression by in situ hybridisation shows that this growth factor is produced by nascent mesoderm cells. A functional test in differentiating epiblast shows that CDX2 and ELF5 are activated in response to BMP4. Furthermore, the effects of BMP4 were also demonstrated in the neighbouring TE cells, as demonstrated by an increase in phosphorylated SMAD1/5/8. These results show that BMP4 produced in the extraembryonic mesoderm is directly influencing the SMAD response in the TE of elongating embryos. These results demonstrate that paracrine signals from the embryo, represented by FGF4 and BMP4, induce a response in the TE prior to the extensive elongation. The study also confirms that expression of CDX2 and ELF5 is not conserved in the mural TE, indicating that although the signals that coordinate conceptus growth are similar between rodents and pigs, the gene regulatory network of the trophoblast lineage is not conserved in these species.     

Heparan sulfate facilitates FGF and BMP signaling to drive mesoderm differentiation of mouse embryonic stem cells

Heparan sulfate (HS) has been implicated in regulating cell fate decisions during differentiation of embryonic stem cells (ESCs) into advanced cell types. However, the necessity and the underlying molecular mechanisms of HS in early cell lineage differentiation are still largely unknown. In this study, we examined the potential of EXT1(-/-) mouse ESCs (mESCs), that are deficient in HS, to differentiate into primary germ layer cells. We observed that EXT1(-/-) mESCs lost their differentiation competence and failed to differentiate into Pax6(+)-neural precursor cells and mesodermal cells. More detailed analyses highlighted the importance of HS for the induction of Brachyury(+) pan-mesoderm as well as normal gene expression associated with the dorso-ventral patterning of mesoderm. Examination of developmental cell signaling revealed that EXT1 ablation diminished FGF and BMP but not Wnt signaling. Furthermore, restoration of FGF and BMP signaling each partially rescued mesoderm differentiation defects. We further show that BMP4 is more prone to degradation in EXT1(-/-) mESCs culture medium compared with that of wild type cells. Therefore, our data reveal that HS stabilizes BMP ligand and thereby maintains the BMP signaling output required for normal mesoderm differentiation. In summary, our study demonstrates that HS is required for ESC pluripotency, in particular lineage specification into mesoderm through facilitation of FGF and BMP signaling.     

Loss of heterochromatin protein 1 gamma reduces the number of primordial germ cells via impaired cell cycle progression in mice

Signals from extraembryonic tissues in mice determine which proximal epiblast cells become primordial germ cells (PGCs). After their specification, approximately 40 PGCs appear at the base of the allantoic bud and migrate to the genital ridges, where they expand to about 25?000 cells by Embryonic Day (E)13.5. The heterochromatin protein 1 (HP1) family members HP1alpha, HP1beta, and HP1gamma (CBX5, CBX1, and CBX3, respectively) are thought to induce heterochromatin structure and to regulate gene expression by binding methylated histone H3 lysine 9. We found a dramatic loss of germ cells before meiosis in HP1gamma mutant (HP1gamma(-/-)) mice that we generated previously. The reduction in PGCs in HP1gamma(-/-) embryos was detectable from the early bud stage (E7.25), and the number of HP1gamma(-/-) PGCs was gradually reduced thereafter. Bromodeoxyuridine incorporation into PGCs was significantly reduced in E7.25 and E12.5 HP1gamma(-/-) embryos. Furthermore, a lower proportion of HP1gamma(-/-) PGCs than wild-type PGCs was in S phase, and a higher proportion, respectively, was in G1 phase at E12.5. Moreover, the proportion of p21 (Cip, official symbol CDKN1A)-positive HP1gamma(-/-) PGCs was increased, suggesting that the G1/S phase transition was inhibited. However, no differences were detected between fate determination, migration, apoptosis, or histone modification of PGCs of control embryos and those of HP1gamma(-/-) embryos. Therefore, the reduction in PGCs in HP1gamma(-/-) embryos could be caused by impaired cell cycle in PGCs. These results suggest that HP1gamma plays an important role in keeping enough germ cells by regulating the PGC cell cycle.     

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.     

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.     

The onset of germ cell migration in the mouse embryo

Mouse primordial germ cells (PGCs) are specified between embryonic day 6.5 (E6.5) and E7.5, when they have been visualized as an alkaline phosphatase-positive (AP+) cell population in the developing allantois. By E8.5, they are embedded in the hind-gut epithelium. Previous experiments have suggested different sites for PGCs' origin, and it is unclear how they reach the gut epithelium. We have used transgenic mice expressing GFP under a truncated Oct4 promoter to visualize living PGCs. We find GFP+/AP+ cells in the posterior end of the primitive streak as a dispersed population of cells actively migrating into the allantois, and directly into the adjacent embryonic endoderm. Time-lapse analysis shows these cells to be actively migratory from the time they exit the primitive streak.     

BMP receptor IA is required in the mammalian embryo for endodermal morphogenesis and ectodermal patterning

BMPRIA is a receptor for bone morphogenetic proteins with high affinity for BMP2 and BMP4. Mouse embryos lacking Bmpr1a fail to gastrulate, complicating studies on the requirements for BMP signaling in germ layer development. Recent work shows that BMP4 produced in extraembryonic tissues initiates gastrulation. Here we use a conditional allele of Bmpr1a to remove BMPRIA only in the epiblast, which gives rise to all embryonic tissues. Resulting embryos are mosaics composed primarily of cells homozygous null for Bmpr1a, interspersed with heterozygous cells. Although mesoderm and endoderm do not form in Bmpr1a null embryos, these tissues are present in the mosaics and are populated with mutant cells. Thus, BMPRIA signaling in the epiblast does not restrict cells to or from any of the germ layers. Cells lacking Bmpr1a also contribute to surface ectoderm; however, from the hindbrain forward, little surface ectoderm forms and the forebrain is enlarged and convoluted. Prechordal plate, early definitive endoderm, and anterior visceral endoderm appear to be expanded, likely due to defective morphogenesis. These data suggest that the enlarged forebrain is caused in part by increased exposure of the ectoderm to signaling sources that promote anterior neural fate. Our results reveal critical roles for BMP signaling in endodermal morphogenesis and ectodermal patterning.