The germ cell determinant Blimp1 is not required for derivation of pluripotent stem cells

Blimp1 (Prdm1), the key determinant of primordial germ cells (PGCs), plays a combinatorial role with Prdm14 during PGC specification from postimplantation epiblast cells. They together initiate epigenetic reprogramming in early germ cells toward an underlying pluripotent state, which is equivalent to embryonic stem cells (ESCs). Whereas Prdm14 alone can?promote reprogramming and is important for the propagation of the pluripotent state, it is not known whether Blimp1 is similarly involved. By using a genetic approach, we demonstrate that Blimp1 is?dispensable for the derivation and maintenance of ESCs and postimplantation epiblast stem cells (epiSCs). Notably, Blimp1 is also dispensable for reprogramming epiSCs to ESCs. Thus, although Blimp1 is obligatory for PGC specification, it is not required for the reversion of epiSCs to ESCs and for their maintenance thereafter. This study suggests that reprogramming, including that of somatic cells to ESCs, may not entail an obligatory route through a Blimp1-positive PGC-like state.     

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.     

The yeast putative transcriptional repressor RGM1 is a proline-rich zinc finger protein

I have cloned a yeast gene, RGM1, which encodes a proline-rich zinc, finger protein. rgm1 mutants do not show any obvious phenotype but overexpression of RGM1 gene greatly impairs cell growth. The proline-rich region of RGM1 attached to a heterologous DNA binding domain is able to repress the expression of the target gene. RGM1 shares similar zinc finger motifs with the mammalian Egr (early growth response) proteins as well as proline-rich sequences with a high serine and threonine content, suggesting that RGM1 and Egr proteins could have functional similarities.     

The Sm protein methyltransferase PRMT5 is not required for primordial germ cell specification in mice

PRMT5 is a type II protein arginine methyltransferase with roles in stem cell biology, reprograming, cancer and neurogenesis. During embryogenesis in the mouse, it was hypothesized that PRMT5 functions with the master germline determinant BLIMP1 to promote primordial germ cell (PGC) specification. Using a Blimp1‐Cre germline conditional knockout, we discovered that Prmt5 has no major role in murine germline specification, or the first global epigenetic reprograming event involving depletion of cytosine methylation from DNA and histone H3 lysine 9 dimethylation from chromatin. Instead, we discovered that PRMT5 functions at the conclusion of PGC reprograming I to promote proliferation, survival and expression of the gonadal germline program as marked by MVH. We show that PRMT5 regulates gene expression by promoting methylation of the Sm spliceosomal proteins and significantly altering the spliced repertoire of RNAs in mammalian embryonic cells and primordial cells.     

Endoglin is dispensable for angiogenesis, but required for endocardial cushion formation in the midgestation mouse embryo

Vascular patterning depends on precisely coordinated timing of endothelial cell differentiation and onset of cardiac function. Endoglin is a transmembrane receptor for members of the TGF-β superfamily that is expressed on endothelial cells from early embryonic gestation to adult life. Heterozygous loss of function mutations in human ENDOGLIN cause Hereditary Hemorrhagic Telangiectasia Type 1, a vascular disorder characterized by arteriovenous malformations that lead to hemorrhage and stroke. Endoglin null mice die in embryogenesis with numerous lesions in the cardiovascular tree including incomplete yolk sac vessel branching and remodeling, vessel dilation, hemorrhage and abnormal cardiac morphogenesis. Since defects in multiple cardiovascular tissues confound interpretations of these observations, we performed in vivo chimeric rescue analysis using Endoglin null embryonic stem cells. We demonstrate that Endoglin is required cell autonomously for endocardial to mesenchymal transition during formation of the endocardial cushions. Endoglin null cells contribute widely to endothelium in chimeric embryos rescued from cardiac development defects, indicating that Endoglin is dispensable for angiogenesis and vascular remodeling in the midgestation embryo, but is required for early patterning of the heart.     

Protocadherin-1 is expressed in the notochord of mouse embryo but is dispensable for its formation

Notochord is an embryonic midline structure that serves as mechanical support for axis elongation and the signaling center for the surrounding tissues. Precursors of notochord are initially induced in the dorsal most mesoderm region in gastrulating embryo and separate from the surrounding mesoderm/endoderm tissue to form an elongated rod-like structure, suggesting that cell adhesion molecules may play an important role in this step. In Xenopus embryo, axial protocadherin (AXPC), an orthologue of mammalian Protocadherin-1 (PCDH1), is indispensable for the assembly and separation from the surrounding tissue of the notochord cells. However, the role of PCDH1 in mammalian notochord remains unknown. We herein report that PCDH1 is expressed in the notochord of mouse embryo and that PCDH1-deficient mice form notochord normally. First, we examined the temporal expression pattern of pcdh1 and found that pcdh1 mRNA was expressed from embryonic day (E) 7.5, prior to the stage when notochord cells detach from the surrounding endoderm tissue. Second, we found that PCDH1 protein is expressed in the notochord of mouse embryos in addition to the previously reported expression in endothelial cells. To further investigate the role of PCDH1 in embryonic development, we generated PCDH1-deficient mice using the CRISPR-Cas9 system. In PCDH1-deficient embryos, notochord formation and separation from the surrounding tissue were normal. Structure and marker gene expression of notochord were also unaffected by loss of PCDH1. Major vascular patterns in PCDH1-deficient embryo were essentially normal. These results suggest that PCDH1 is dispensable for notochord formation, including the tissue separation process, in mammalian embryos. We successfully identified the evolutionary conserved expression of PCDH1 in notochord, but its function may differ among species.     

The pro-apoptotic gene Bax is required for the death of ectopic primordial germ cells during their migration in the mouse embryo

In the mouse embryo, significant numbers of primordial germ cells (PGCs) fail to migrate correctly to the genital ridges early in organogenesis. These usually die in ectopic locations. In humans, 50% of pediatric germ line tumors arise outside the gonads, and these are thought to arise from PGCs that fail to die in ectopic locations. We show that the pro-apoptotic gene Bax, previously shown to be required for germ cell death during later stages of their differentiation in the gonads, is also expressed during germ cell migration, and is required for the normal death of germ cells left in ectopic locations during and after germ cell migration. In addition, we show that Bax is downstream of the known cell survival signaling interaction mediated by the Steel factor/Kit ligand/receptor interaction. Together, these observations identify the major mechanism that removes ectopic germ cells from the embryo at early stages.     

The expression of platelet endothelial cell adhesion molecule-1 in mouse primordial germ cells during their migration and early gonadal formation

The platelet endothelial cell adhesion molecule-1 (PECAM-1), or CD31, a member of the immunoglobulin superfamily, is located on the plasma membrane of endothelial and hematopoietic cells and involved in vascular development and inflammation. In this study, by use of immunohistochemistry at light and electron microscopic levels in combination with enzyme histochemistry for alkaline phosphatase, we demonstrated that PECAM-1/CD31 is expressed in the mouse primordial germ cell (PGC). Up to 8 days postcoitum (dpc), PGCs with alkaline phosphatase activity showed no PECAM-1/CD31 immunoreactivity. At 9 dpc, PECAM-1/CD31 immunoreactivity was first detected with low intensity in some PGCs located in the hindgut. Between 10 and 11 dpc, intense immunoreactivity was shown on the entire surface of PGCs migrating along the dorsal wall. After arrival and settlement of PGCs in the genital ridges around 11.5 dpc, the intense immunoreactivity was maintained on the entire surface of PGCs. By electron microscopy, the immunoreactivity was localized exclusively on the plasma membrane of PGCs, being as strong at the portions adjacent to neighboring PGCs as those adjacent to somatic cells. As the male and female gonads began to differentiate, PECAM-1/CD31 immunoreactivity remained strong in germ cells until 13 dpc, after which it gradually decreased in intensity and disappeared by 16 dpc. These results suggested that cell-to-cell interaction through PECAM-1/CD31 plays roles in the development of PGCs during their migration on the dorsal wall and homing in the gonads.     

The role of cadherins during primordial germ cell migration and early gonad formation in the mouse

Primordial germ cells (PGCs) are the founder cells of the gametes. In mammals, PGCs migrate from the hindgut to the genital ridges, where they coalesce with each other and with somatic cells to form the primary sex cords. We show here that, in both sexes, PGCs express P- and E-cadherins during and after migration, and N-cadherin at post-migratory stages. E-Cadherin is not expressed by PGCs whilst in the hindgut, but is upregulated as they leave. Blocking antibodies against E-, but not P-cadherin cause defective PGC-PGC coalescence, and in some cases, ectopic PGCs.     

On the fate of primordial germ cells injected into early mouse embryos

Primordial germ cells (PGCs) are the founder cells of the germline. Via gametogenesis and fertilisation this lineage generates a new embryo in the next generation. PGCs are also the cell of origin of multilineage teratocarcinomas. In vitro, mouse PGCs can give rise to embryonic germ (EG) cells – pluripotent stem cells that can contribute to primary chimaeras when introduced into pre-implantation embryos. Thus, PGCs can give rise to pluripotent cells in the course of the developmental cycle, during teratocarcinogenesis and by in vitro culture. However, there is no evidence that PGCs can differentiate directly into somatic cell types. Furthermore, it is generally assumed that PGCs do not contribute to chimaeras following injection into the early mouse embryo. However, these data have never been formally published. Here, we present the primary data from the original PGC-injection experiments performed 40 years ago, alongside results from more recent studies in three separate laboratories. These results have informed and influenced current models of the relationship between pluripotency and the germline cycle. Current technologies allow further experiments to confirm and expand upon these findings and allow definitive conclusions as to the developmental potency of PGCs.     

Gamma-secretase activity is dispensable for mesenchyme-to-epithelium transition but required for podocyte and proximal tubule formation in developing mouse kidney

Notch signaling is involved in pronephros development in Xenopus and in glomerulogenesis in mice. However, owing to early lethality in mice deficient for some Notch pathway genes and functional redundancy for others, a role for Notch signaling during early stages of metanephric development has not been defined. Using an antibody specific to the N-terminal end of gamma-secretase-cleaved Notch1, we found evidence for Notch1 activation in the comma and S-shaped bodies of the mouse metanephros. We therefore cultured mouse metanephroi in the presence of a gamma-secretase inhibitor, N-S-phenyl-glycine-t-butyl ester (DAPT), to block Notch signaling. We observed slightly reduced ureteric bud branching but normal mesenchymal condensation and expression of markers indicating that mesenchyme induction had occurred. However, fewer renal epithelial structures were observed, with a severe deficiency in proximal tubules and glomerular podocytes, which are derived from cells in which activated Notch1 is normally present. Distal tubules were present but in reduced numbers, and this was accompanied by an increase in intervening, non-epithelial cells. After a transient 3-day exposure to DAPT, proximal tubules expanded, but podocyte differentiation failed to recover after removal of DAPT. These observations suggest that gamma-secretase activity, probably through activation of Notch, is required for maintaining a competent progenitor pool as well as for determining the proximal tubule and podocyte fates.     

Fanconi anemia complementation group C is required for proliferation of murine primordial germ cells

Fanconi anemia is a polygenic trait hypothesized to be a DNA damage repair disease. We show that all three Fanconi anemia loci that have been cloned are expressed in the embryonic gonad during the period of primordial germ cell proliferation. Mice mutant for the Fanconi anemia complementation group C locus (Fancc) have reduced germ cell numbers as early as embryonic day E12.5, suggesting the Fancc protein functions prior to meiosis in both sexes. Depletion in the mutant occurs at a time when all three loci would be expressed in a wild-type gonad, implying a function in the early germline. Determination of the mitotic index of primordial germ cells by BrdU incorporation shows that germ cells in Fancc(-/-) mice proliferate significantly more slowly than littermate controls. This study demonstrates Fancc is required for mitotic proliferation of primordial germ cells.