Translational repression restricts expression of the C. elegans Nanos homolog NOS-2 to the embryonic germline

Members of the nanos gene family are evolutionarily conserved regulators of germ cell development. In several organisms, Nanos protein expression is restricted to the primordial germ cells (PGCs) during early embryogenesis. Here, we investigate the regulation of the Caenorhabditis elegans nanos homolog nos-2. We find that the nos-2 RNA is translationally repressed. In the adult germline, translation of the nos-2 RNA is inhibited in growing oocytes, and this inhibition depends on a short stem loop in the nos-2 3'UTR. In embryos, nos-2 translation is repressed in early blastomeres, and this inhibition depends on a second region in the nos-2 3'UTR. nos-2 RNA is also degraded in somatic blastomeres by a process that is independent of translational repression and requires the CCCH finger proteins MEX-5 and MEX-6. Finally, the germ plasm component POS-1 activates nos-2 translation in the PGCs. A combination of translational repression, RNA degradation, and activation by germ plasm has also been implicated in the regulation of nanos homologs in Drosophila and zebrafish, suggesting the existence of conserved mechanisms to restrict Nanos expression to the germline.     

Zebrafish Staufen1 and Staufen2 are required for the survival and migration of primordial germ cells

In sexually reproducing organisms, primordial germ cells (PGCs) give rise to the cells of the germ line, the gametes. In many animals, PGCs are set apart from somatic cells early during embryogenesis. Work in Drosophila, C. elegans, Xenopus, and zebrafish has shown that maternally provided localized cytoplasmic determinants specify the germ line in these organisms (Raz, E., 2003. Primordial germ-cell development: the zebrafish perspective. Nat. Rev., Genet. 4, 690--700; Santos, A.C., Lehmann, R., 2004. Germ cell specification and migration in Drosophila and beyond. Curr. Biol. 14, R578-R589). The Drosophila RNA-binding protein, Staufen is required for germ cell formation, and mutations in stau result in a maternal effect grandchild-less phenotype (Schupbach,T., Weischaus, E., 1989. Female sterile mutations on the second chromosome of Drosophila melanogaster:1. Maternal effect mutations. Genetics 121, 101-17). Here we describe the functions of two zebrafish Staufen-related proteins, Stau1 and Stau2. When Stau1 or Stau2 functions are compromised in embryos by injecting antisense morpholino modified oligonucleotides or dominant-negative Stau peptides, germ layer patterning is not affected. However, expression of the PGC marker vasa is not maintained. Furthermore, expression of a green fluorescent protein (GFP):nanos 3'UTR fusion protein in germ cells shows that PGC migration is aberrant, and the mis-migrating PGCs do not survive in Stau-compromised embryos. Stau2 is also required for survival of neurons in the central nervous system (CNS). These phenotypes are rescued by co-injection of Drosophila stau mRNA. Thus, staufen has an evolutionarily conserved function in germ cells. In addition, we have identified a function for Stau proteins in PGC migration.     

Repression of primordial germ cell differentiation parallels germ line stem cell maintenance

In Drosophila, primordial germ cells (PGCs) are set aside from somatic cells and subsequently migrate through the embryo and associate with somatic gonadal cells to form the embryonic gonad. During larval stages, PGCs proliferate in the female gonad, and a subset of PGCs are selected at late larval stages to become germ line stem cells (GSCs), the source of continuous egg production throughout adulthood. However, the degree of similarity between PGCs and the self-renewing GSCs is unclear. Here we show that many of the genes that are required for GSC maintenance in adults are also required to prevent precocious differentiation of PGCs within the larval ovary. We show that following overexpression of the GSC-differentiation gene bag of marbles (bam), PGCs differentiate to form cysts without becoming GSCs. Furthermore, PGCs that are mutant for nanos (nos), pumilio (pum) or for signaling components of the decapentaplegic (dpp) pathway also differentiate. The similarity in the genes necessary for GSC maintenance and the repression of PGC differentiation suggest that PGCs and GSCs may be functionally equivalent and that the larval gonad functions as a "PGC niche".     

nanos1 is required to maintain oocyte production in adult zebrafish

Development of the germline requires the specification and survival of primordial germ cells (PGCs) in the embryo as well as the maintenance of gamete production during the reproductive life of the adult. These processes appear to be fundamental to all Metazoans, and some components of the genetic pathway regulating germ cell development and function are evolutionarily conserved. In both vertebrates and invertebrates, nanos-related genes, which encode RNA-binding zinc finger proteins, have been shown to play essential and conserved roles during germ cell formation. In Drosophila, maternally supplied nanos is required for survival of PGCs in the embryo, while in adults, nanos is required for the continued production of oocytes by maintaining germline stem cells self-renewal. In mice and zebrafish, nanos orthologs are required for PGC survival during embryogenesis, but a role in adults has not been explored. We show here that nanos1 in zebrafish is expressed in early stage oocytes in the adult female germline. We have identified a mutation in nanos1 using a reverse genetics method and show that young female nanos mutants contain oocytes, but fail to maintain oocyte production. This progressive loss of fertility in homozygous females is not a phenotype that has been described previously in the zebrafish and underlines the value of a reverse genetics approach in this model system.     

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.     

BMP and Hh signaling affects primordial germ cell division in Drosophila

The germline is segregated from the remainder of the soma during early embryonic development in metazoan species. In Drosophila, female primordial germ cells (PGCs) continue to proliferate during larval development, and become germline stem cells at the early pupal stage. To elucidate the roles of growth factors in larval PGC division, we examined expression patterns of a bone morphogenetic protein (BMP) growth factor, Decapentaplegic (Dpp), and Hedgehog (Hh), along with factors downstream of each, in the ovary during larval development. Dpp signaling appeared in the ovarian soma from early larval development, and was prominent in the terminal filament cells at late larval stage, whereas Hh appeared in the ovarian soma and PGCs from the third instar larval stage. The number of PGCs decreased when components of these signal transduction pathways were abrogated by RNAi in the PGCs, indicating that both Dpp and Hh signals directly regulate PGC proliferation. Experiments on the up- and down-regulation of Dpp and Hh with a tissue-specific Gal4 driver indicated that Dpp and Hh act as extrinsic and autocrine growth factors. Furthermore, heat-pulse experiments with hs-Gal4 showed that Dpp is active in PGC proliferation throughout larval development, whereas Hh has effects only during late larval development. In addition to Dpp, the reduction of Glass bottom boat (Gbb), another BMP molecule, caused a decrease in the number of PGCs and initiation of larval PGCs differentiation into cystocytes, indicating that Gbb functions to promote PGC division and repress differentiation.     

C. elegans meg-1 and meg-2 differentially interact with nanos family members to either promote or inhibit germ cell proliferation and survival

The closely related C. elegans MEG-1 and MEG-2 proteins localize to P granules during a brief period of embryogenesis when the germ lineage is being separated from the soma. Embryonic primordial germ cells still develop in the absence of MEG activity, but major defects emerge during larval stages when germ cells fail to proliferate or differentiate normally, resulting in sterility. To investigate meg-1 function, we conducted a targeted RNAi screen for enhancers and suppressors of meg-1 sterility. Here, we show that meg-1 interacts with multiple pathways that promote germ cell proliferation and survival. Surprisingly, we found that two nanos family members had opposing effects on the meg-1 phenotype. Loss of nos-3 suppressed the meg-1 phenotype, restoring fertility, while loss of nos-2 enhanced the meg-1 phenotype, abolishing proliferation and causing early and pronounced germ cell degeneration. Together, our analyses suggest that, under circumstances that favor proliferation, MEG function is not essential for germ cells to proliferate, although it is important for optimal proliferation. Additionally, MEG activity is likely more directly involved in germ cell survival than previously thought. genesis 49:380-391, 2011.     

dead end,a novel vertebrate germ plasm component,is required for zebrafish primordial germ cell migration and survival

In most animals, primordial germ cell (PGC) specification and development depend on maternally provided cytoplasmic determinants that constitute the so-called germ plasm. Little is known about the role of germ plasm in vertebrate germ cell development, and its molecular mode of action remains elusive. While PGC specification in mammals occurs via different mechanisms, several germ plasm components required for early PGC development in lower organisms are expressed in mammalian germ cells after their migration to the gonad and are involved in gametogenesis. Here we show that the RNA of dead end, encoding a novel putative RNA binding protein, is a component of the germ plasm in zebrafish and is specifically expressed in PGCs throughout embryogenesis; Dead End protein is localized to perinuclear germ granules within PGCs. Knockdown of dead end blocks confinement of PGCs to the deep blastoderm shortly after their specification and results in failure of PGCs to exhibit motile behavior and to actively migrate thereafter. PGCs subsequently die, while somatic development is not effected. We have identified dead end orthologs in other vertebrates including Xenopus, mouse, and chick, where they are expressed in germ plasm and germ-line cells, suggesting a role in germ-line development in these organisms as well.     

Coactivation of STAT and Ras is required for germ cell proliferation and invasive migration in Drosophila

Primordial germ cells (PGCs) undergo proliferation, invasion, guided migration, and aggregation to form the gonad. Here we show that in Drosophila, the receptor tyrosine kinase Torso activates both STAT and Ras during the early phase of PGC development, and coactivation of STAT and Ras is required for PGC proliferation and invasive migration. Embryos mutant for stat92E or Ras1 have fewer PGCs, and these cells migrate slowly, errantly, and fail to coalesce. Conversely, overactivation of these molecules causes supernumerary PGCs, their premature transit through the gut epithelium, and ectopic colonization. A requirement for RTK in Drosophila PGC development is analogous to the mouse, in which the RTK c-kit is required, suggesting a conserved molecular mechanism governing PGC behavior in flies and mammals.     

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.     

Analysis of hypomorphic KitlSl mutants suggests different requirements for KITL in proliferation and migration of mouse primordial germ cells

Germ cell development in mice is initiated when a small number of primordial germ cells (PGCs) are set aside from somatic cells during gastrulation. In the subsequent 4 to 5 days, PGCs enter the hindgut, undergo a directed migration away from the hindgut into the developing gonads, and undergo a massive increase in cell number. It is well established that Kit ligand (KITL, also known as stem cell factor and mast cell growth factor) is required for the survival and proliferation of PGCs. However, there is little information on a direct role for KITL in PGC migration. By comparing the effects of multiple Kitl mutations, including two N-ethyl-N-nitrosourea-induced hypomorphic mutations, we were able to distinguish stages of PGC development that are preferentially affected by certain mutations. We provide evidence that the requirements for KITL in proliferation are different in PGCs before and after they start migrating, and different levels of KITL function are required to support PGC proliferation and migration. This study illustrates the usefulness of an allelic series of mutations to dissect developmental processes and suggests that these mutants may be useful for further studies of molecular mechanisms of KITL functions in gametogenesis.     

Time-lapse analysis reveals different modes of primordial germ cell migration in the medaka Oryzias latipes

Previous studies have shown that medaka primordial germ cells (PGC) are first distinguishable by olvas expression during late gastrulation, and that they migrate to the gonadal region through the lateral plate mesoderm. Here, we demonstrate that medaka nanos expression marks the germ line at early gastrulation stage. By marking the germ line with green fluorescent protein (GFP) fused to the nanos 3' untranslated region, we were able to visualize the behavior of PGC using time-lapse imaging. We show that there are three distinct modes of PGC migration that function at different stages of development. At early gastrulation stage, PGC actively migrate towards the marginal zone, a process that requires the function of a chemokine receptor, CXCR4. However, at late gastrulation stage, PGC change the mode and direction of their movement, as they are carried towards the midline along with somatic cells undergoing convergent movements. After aligning bilaterally, PGC actively migrate to the posterior end of the lateral plate mesoderm. This posterior movement depends on the activity of both HMGCoAR and a ligand of CXCR4, SDF-1a. These results demonstrate that PGC undergo different modes of migration to reach the prospective gonadal region of the embryo.     

Developmentally regulated expression of mil-1 and mil-2, mouse interferon-induced transmembrane protein like genes,during formation and differentiation of primordial germ cells

In all multicellular organisms, germ cells originating from a fertilized egg have the highly specialized role of transmitting genetic information to the next generation. In many animal species, the establishment of the germ cell lineage is regulated by the maternally inherited germplasm. In mammals, however, germline determination is not based on the unequal distribution of maternal determinants. In the processes of mammalian germ cell formation and subsequent differentiation, the molecular basis of the acquisition of germ cell status is not well understood. Since migrating primordial germ cells (PGCs) are lineage-restricted to the germline, they have already acquired a germ cell specific fate distinct from that of pluri/multi-potent stem cells. However, there have been no molecules known to be expressed in migrating PGCs but not in the inner cell mass of blastocysts. Such molecules should be involved in early germ cell development, and they should make good markers for following the process of PGC formation. To identify such molecules, we performed a subtracted cDNA screening with migrating PGCs and blastocysts in mice, and isolated 11 clones preferentially expressed in PGCs. Here, we report the identification of two genes with similarity to human interferon-induced transmembrane protein (Ifitm) genes, and expression patterns of these genes in forming and in differentiating PGCs. During germ cell formation, mouse Ifitm like (mil)-1 was expressed in putative PGC ancestors in embryos at 6.5-7.5 days post coitum. In migrating PGCs, mil-1 expression was continuously observed and mil-2 expression was first detected during germ cell differentiation.     

Developmentally regulated expression of mil-1 and mil-2, mouse interferon-induced transmembrane protein like genes, during formation and differentiation of primordial germ cells

In all multicellular organisms, germ cells originating from a fertilized egg have the highly specialized role of transmitting genetic information to the next generation. In many animal species, the establishment of the germ cell lineage is regulated by the maternally inherited germplasm. In mammals, however, germline determination is not based on the unequal distribution of maternal determinants. In the processes of mammalian germ cell formation and subsequent differentiation, the molecular basis of the acquisition of germ cell status is not well understood. Since migrating primordial germ cells (PGCs) are lineage-restricted to the germline, they have already acquired a germ cell specific fate distinct from that of pluri/multi-potent stem cells. However, there have been no molecules known to be expressed in migrating PGCs but not in the inner cell mass of blastocysts. Such molecules should be involved in early germ cell development, and they should make good markers for following the process of PGC formation. To identify such molecules, we performed a subtracted cDNA screening with migrating PGCs and blastocysts in mice, and isolated 11 clones preferentially expressed in PGCs. Here, we report the identification of two genes with similarity to human interferon-induced transmembrane protein (Ifitm) genes, and expression patterns of these genes in forming and in differentiating PGCs. During germ cell formation, mouse Ifitm like (mil)-1 was expressed in putative PGC ancestors in embryos at 6.5-7.5 days post coitum. In migrating PGCs, mil-1 expression was continuously observed and mil-2 expression was first detected during germ cell differentiation.     

Cadherin-mediated cell interaction regulates germ cell determination in mice

The germ cell lineage segregates from the somatic cell lineages in early embryos. Germ cell determination in mice is not regulated by maternally inherited germplasm, but is initiated within the embryo during gastrulation. However, the mechanisms of germ cell specification in mice remain unknown. We located precursors to primordial germ cells (PGCs) within early embryos, and show here that cell-cell interaction among these precursors is required for germ cell specification. We found that the expression of a calcium-dependent cell adhesion molecule, E-cadherin, is restricted to the proximal region of extra-embryonic mesoderm that contains PGC precursors, and that blocking the functions of E-cadherin with an antibody inhibits PGC formation in vitro. These results showed that E-cadherin-mediated cell-cell interaction among cells containing PGC precursors is essential to directing such cells to the germ cell fate.