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.     

Conserved Mechanisms for Germ Cell-Specific Localization of nanos3 Transcripts in Teleost Species with Aquaculture Significance

The importance of the aquaculture production is increasing with the declining global fish stocks, but early sexual maturation in several farmed species reduces muscle growth and quality, and escapees could have a negative impact on wild populations. A possible solution to these problems is the production of sterile fish by ablation of the embryonic primordial germ cells (PGCs), a technique developed in zebrafish. Cell-specific regulation of mRNA stability is crucial for proper specification of the germ cell lineage and commonly involves microRNA (miRNA)-mediated degradation of targeted mRNAs in somatic cells. This study reports on the functional roles of conserved motifs in the 3′ untranslated region (UTR) of the miRNA target gene nanos3 identified in Atlantic cod, Atlantic salmon, and zebrafish. The 3′UTR of cod nanos3 was sufficient for targeting the expression of green fluorescent protein (GFP) to the presumptive PGCs in injected embryos of the three phylogenetically distant species. 3′UTR elements of importance for PGC-specific expression were further examined by fusing truncated 3′UTR variants of cod nanos3 to GFP followed by injections in zebrafish embryos. The expression patterns of the GFP constructs in PGCs and somatic cells suggested that the proximal U-rich region is responsible for the PGC-specific stabilization of the endogenous nanos3 mRNA. Morpholino-mediated downregulation of the RNA-binding protein Dead end (DnD), a PGC-specific inhibitor of miRNA action, abolished the fluorescence of the PGCs in cod and zebrafish embryos, suggesting a conserved DnD-dependent mechanism for germ cell survival and migration.     

The mechanism for primordial germ-cell migration is conserved between Japanese eel and zebrafish

Primordial germ cells (PGCs) are segregated and specified from somatic cells during early development. These cells arise elsewhere and have to migrate across the embryo to reach developing gonadal precursors. Several molecules associated with PGC migration (i.e. dead-end, nanos1, and cxcr4) are highly conserved across phylum boundaries. However, since cell migration is a complicated process that is regulated spatially and temporally by multiple adaptors and signal effectors, the process is unlikely to be explained by these known genes only. Indeed, it has been shown that there are variations in PGC migration pattern during development among teleost species. However, it is still unclear whether the actual mechanism of PGC migration is conserved among species. In this study, we studied the migration of PGCs in Japanese eel (Anguilla japonica) embryos and tested the migration mechanism between Japanese eel and zebrafish (Danio rerio) for conservation, by transplanting eel PGCs into zebrafish embryos. The experiments showed that eel PGCs can migrate toward the gonadal region of zebrafish embryos along with endogenous PGCs, even though the migration patterns, behaviors, and settlements of PGCs are somewhat different between these species. Our results demonstrate that the migration mechanism of PGCs during embryonic development is highly conserved between these two distantly related species (belonging to different teleost orders).     

Spatiotemporal localization of germ plasm RNAs during zebrafish oogenesis

In zebrafish, primordial germ cells (PGCs) are determined by a specialized maternal cytoplasm, the germ plasm, which forms at the distal ends of the cleavage furrows in 4-cell embryos. The germ plasm includes maternal mRNAs from the germline-specific genes such as vasa and nanos1, and vegetally localized dazl RNA is also incorporated into the germ plasm. However, little is known about the distributions and assembly mechanisms of germ plasm components, especially during oogenesis. Here we report that the germ plasm RNAs vasa, nanos1, and dazl co-localize with the mitochondrial cloud (MC) and are transported to the vegetal cortex during early oogenesis. We found that a mitochondrial cloud localization element (MCLE) previously identified in the 3' untranslated region (3'UTR) of Xenopus Xcat2 gene can direct RNA localization to the vegetal cortex via the MC in zebrafish oocytes. In addition, the RNA-binding protein Hermes is a component of the MC in zebrafish oocytes, as is the case in Xenopus. Moreover, we provide evidence that the dazl 3'UTR possesses at least three types of cis-acting elements that direct multiple steps in the localization process: MC localization, anchorage at the vegetal cortex, and localization at the cleavage furrows. Taken together, the data show that the MC functions as a conserved feature that participates in transport of the germ plasm RNAs in Xenopus and zebrafish oocytes. Furthermore, we propose that the germ plasm components are assembled in a stepwise and spatiotemporally-regulated manner during oogenesis and early embryogenesis in zebrafish.     

nos-1 and nos-2, two genes related to Drosophila nanos, regulate primordial germ cell development and survival in Caenorhabditis elegans.

In Drosophila, the posterior determinant nanos is required for embryonic patterning and for primordial germ cell (PGC) development. We have identified three genes in Caenorhabditis elegans that contain a putative zinc-binding domain similar to the one found in nanos, and show that two of these genes function during PGC development. Like Drosophila nanos, C. elegans nos-1 and nos-2 are not generally required for PGC fate specification, but instead regulate specific aspects of PGC development. nos-2 is expressed in PGCs around the time of gastrulation from a maternal RNA associated with P granules, and is required for the efficient incorporation of PGCs into the somatic gonad. nos-1 is expressed in PGCs after gastrulation, and is required redundantly with nos-2 to prevent PGCs from dividing in starved animals and to maintain germ cell viability during larval development. In the absence of nos-1 and nos-2, germ cells cease proliferation at the end of the second larval stage, and die in a manner that is partially dependent on the apoptosis gene ced-4. Our results also indicate that putative RNA-binding proteins related to Drosophila Pumilio are required for the same PGC processes as nos-1 and nos-2. These studies demonstrate that evolutionarily distant organisms utilize conserved factors to regulate early germ cell development and survival, and that these factors include members of the nanos and pumilio gene families.     

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".     

The Role of DNA Methylation Reprogramming During Sex Determination and Transition in Zebrafish

DNA methylation is a prevalent epigenetic modification in vertebrates, and it has been shown to be involved the regulation of gene expression and embryo development. However, it remains unclear how DNA methylation regulates sexual development, especially in species without sex chromosomes. To determine this, we utilized zebrafish to investigate DNA methylation reprogramming during juvenile germ cell development and adult female-to-male sex transition.We reveal that primordial germ cells(PGCs) undergo significant DNA methylation reprogramming during germ cell development, and the methylome of PGCs is reset to an oocyte/ovary-like pattern at 9 days post fertilization(9 dpf). When DNA methyltransferase(DNMT) activity in juveniles was blocked after 9 dpf, the zebrafish developed into females. We also show that Tet3 is involved in PGC development. Notably, we find that DNA methylome reprogramming during adult zebrafish sex transition is similar to the reprogramming during the sex differentiation from 9 dpf PGCs to sperm. Furthermore, inhibiting DNMT activity can prevent the female-to-male sex transition, suggesting that methylation reprogramming is required for zebrafish sex transition. In summary, DNA methylation plays important roles in zebrafish germ cell development and sexual plasticity.     

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.     

Insulin-like growth factor receptor 1b is required for zebrafish primordial germ cell migration and survival

Insulin-like growth factor (IGF) signaling is a critical regulator of somatic growth during fetal and adult development, primarily through its stimulatory effects on cell proliferation and survival. IGF signaling is also required for development of the reproductive system, although its precise role in this regard remains unclear. We have hypothesized that IGF signaling is required for embryonic germline development, which requires the specification and proliferation of primordial germ cells (PGCs) in an extragonadal location, followed by directed migration to the genital ridges. We tested this hypothesis using loss-of-function studies in the zebrafish embryo, which possesses two functional copies of the Type-1 IGF receptor gene (igf1ra, igf1rb). Knockdown of IGF1Rb by morpholino oligonucleotides (MO) results in mismigration and elimination of primordial germ cells (PGCs), resulting in fewer PGCs colonizing the genital ridges. In contrast, knockdown of IGF1Ra has no effect on PGC migration or number despite inducing widespread somatic cell apoptosis. Ablation of both receptors, using combined MO injections or overexpression of a dominant-negative IGF1R, yields embryos with a PGC-deficient phenotype similar to IGF1Rb knockdown. TUNEL analyses revealed that mismigrated PGCs in IGF1Rb-deficient embryos are eliminated by apoptosis; overexpression of an antiapoptotic gene (Bcl2l) rescues ectopic PGCs from apoptosis but fails to rescue migration defects. Lastly, we show that suppression of IGF signaling leads to quantitative changes in the expression of genes encoding CXCL-family chemokine ligands and receptors involved in PGC migration. Collectively, these data suggest a novel role for IGF signaling in early germline development, potentially via cross-talk with chemokine signaling pathways.     

Medaka dead end encodes a cytoplasmic protein and identifies embryonic and adult germ cells

dead end (dnd) was identified in zebrafish as a gene encoding an RNA-binding protein essential for primordial germ cell (PGC) development and gametogenesis in vertebrates. The adult dnd RNA expression has been restricted to the ovary in Xenopus or to the testis in mouse. Its protein product is nuclear in chicken germ cells but both cytosolic and nuclear in mouse cell cultures. Here we report the cloning and expression pattern of Odnd, the medakafish (Oryzias latipes) dnd gene. Sequence comparison, gene structure, linkage analysis and expression demonstrate that Odnd encodes the medaka Dnd orthologue. A systematic comparison of Dnd proteins from five fishes and tetrapod representatives led to the identification of five previously unidentified conserved regions besides the RNA recognition motif. The Odnd RNA is maternally supplied and preferentially segregated with PGCs. Its adult expression occurs in both sexes and is restricted to germ cells. In the testis, Odnd is abundant in spermatogonia and meiotic cells but absent in sperm. In the ovary, Odnd RNA persists throughout oogenesis. Furthermore, we developed a dual color fluorescent in situ hybridization procedure allowing for precise comparisons of expression and distribution patterns between two genes in medaka embryos and adult tissues. Importantly, this procedure co-localized Odnd and Ovasa in testicular germ cells and PGCs. Surprisingly, by cell transfection and embryo RNA injection we show that ODnd is cytoplasmic in cell cultures, cleavage embryos and PGCs. Therefore, medaka dnd encodes a cytoplasmic protein and identifies embryonic and adult germ cells of both sexes.     

Complexities in Bombyx germ cell formation process revealed by Bm-nosO (a Bombyx homolog of nanos) knockout

Inheritance (sequestration of a localized determinant: germplasm) and zygotic induction are two modes of metazoan primordial germ cell (PGC) specification. vasa and nanos homologs are evolutionarily conserved germline marker genes that have been used to examine the ontogeny of germ cells in various animals. In the lepidopteran insect Bombyx mori, although the lack of vasa homolog (BmVLG) protein localization as well as microscopic observation suggested the lack of germplasm, classical embryo manipulation studies and the localization pattern of Bm-nosO (one of the four nanos genes in Bombyx) maternal mRNA in the egg raised the possibility that an inheritance mode is operating in Bombyx. Here, we generated Bm-nosO knockouts to examine whether the localized mRNA acts as a localized germ cell determinant. Contrary to our expectations, Bm-nosO knockout lines could be established. However, these lines frequently produced abnormal eggs, which failed to hatch, to various extent depending on the individuals. We also found that Bm-nosO positively regulated BmVLG expression at least during embryonic stage, directly or indirectly, indicating that these genes were on the same developmental pathway for germ cell formation in Bombyx. These results suggest that these conserved genes are concerned with stable germ cell production. On the other hand, from the aspect of BmVLG as a PGC marker, we showed that maternal Bm-nosO product(s) as well as early zygotic Bm-nosO activity were redundantly involved in PGC specification; elimination of both maternal and zygotic gene activities (as in knockout lines) resulted in the apparent lack of PGCs, indicating that an inheritance mechanism indeed operates in Bombyx. This, however, together with the fact that germ cells are produced at all in Bm-nosO knockout lines, also suggests the possibility that, in Bombyx, not only this inheritance mechanism but also an inductive mechanism acts in concert to form germ cells or that loss of early PGCs are compensated for by germline regeneration: mechanisms that could enable the evolution of preformation. Thus, Bombyx could serve as an important organism in understanding the evolution of germ cell formation mechanisms; transition between preformation and inductive modes.     

Germ line development in the grasshopper Schistocerca gregaria: vasa as a marker

Vasa is a widely conserved germline marker, both in vertebrates and invertebrates. We identify a vasa orthologue, Sgvasa, and use it to study germline development in the grasshopper Schistocerca gregaria, a species in which no germ plasm has been identified. In adults, Sgvasa is specifically expressed in the ovary and testis. It is expressed at high levels during early oogenesis, but no detectable vasa RNA and little Vasa protein are present in mature unlaid eggs. None appears to be localized to any defined region of the egg cortex, suggesting that germline specification may not depend on maternal germ plasm expressing vasa. Vasa protein is expressed in most cleavage energids as they reach the egg surface and persists at high levels in most cells aggregating to form the embryonic primordium. However, after gastrulation, Vasa protein persists only in extraembryonic membranes and in cells at the outer margin of the late heart-stage embryo. In the embryo, it then become restricted to cells at the dorsal margin of the forming abdomen. In older embryos, these Vasa-positive cells move toward the midline; Vasa protein accumulates asymmetrically in their cytoplasm, a pattern closely resembling that of germ cells in late embryonic gonads. Thus, we suggest that the Vasa-stained cells in the abdominal margin are germ cells, as proposed by Nelson (1934), and not cardioblasts, as has been proposed by others.     

Improving the efficiency for generation of genome-edited zebrafish by labeling primordial germ cells

Although CRISPR/Cas, a new versatile genome-editing tool, has been widely used in a variety of species including zebrafish, an important vertebrate model animal for biomedical research, the low efficiency of germline transmission of induced mutations and particularly knockin alleles made subsequent screening for heritable offspring tedious, time-consuming, expensive and at times impossible. In this study, we reported a method for improving the efficiency of germline transmission screening for generation of genome-edited zebrafish mutants. Co-microinjecting yfp-nanos3 mRNA with Cas9 mRNA, sgRNA and single strand DNA donor to label the distribution of microinjected nucleotides in PGCs (primordial germ cells), we demonstrated that founders carrying labeled PGCs produced much higher numbers of knockin and knockout progeny. In comparison with the common practice of selecting founders by genotyping fin clips, our new strategy of selecting founders with tentatively fluorescent-labeled PGCs significantly increase the ease and speed of generating heritable knocking and knockout animals with CRISPR/Cas9.     

Marker genes identify three somatic cell types in the fetal mouse ovary

The two main functions of the ovary are the production of oocytes, which allows the continuation of the species, and secretion of female sex hormones, which control many aspects of female development and physiology. Normal development of the ovaries during embryogenesis is critical for their function and the health of the individual in later life. Although the adult ovary has been investigated in great detail, we are only starting to understand the cellular and molecular biology of early ovarian development. Here we show that the adult stem cell marker Lgr5 is expressed in the cortical region of the fetal ovary and this expression is mutually exclusive to FOXL2. Strikingly, a third somatic cell population can be identified, marked by the expression of NR2F2, which is expressed in LGR5- and FOXL2 double-negative ovarian somatic cells. Together, these three marker genes label distinct ovarian somatic cell types. Using lineage tracing in mice, we show that Lgr5-positive cells give rise to adult cortical granulosa cells, which form the follicles of the definitive reserve. Moreover, LGR5 is required for correct timing of germ cell differentiation as evidenced by a delay of entry into meiosis in Lgr5 loss-of-function mutants, demonstrating a key role for LGR5 in the differentiation of pre-granulosa cells, which ensure the differentiation of oogonia, the formation of the definitive follicle reserve, and long-term female fertility.