Early Depletion of Primordial Germ Cells in Zebrafish Promotes Testis Formation.
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ABSTRACT:
SUBMITTER: Tzung KW
PROVIDER: S-EPMC4618446 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:As complete absence of germ cells leads to sterile males in zebrafish, we explored the relationship between primordial germ cell (PGC) number and sexual development. Our results revealed dimorphic proliferation of PGCs in the early zebrafish larvae, marking the beginning of sexual differentiation. We applied morpholino-based gene knockdown and cell transplantation strategies to demonstrate that a threshold number of PGCs is required for the stability of ovarian fate. Using histology and transcriptomic analyses, we determined that zebrafish gonads are in a meiotic ovarian stage at 14 days postfertilization and identified signaling pathways supporting meiotic oocyte differentiation and eventual female fate. The development of PGC-depleted gonads appears to be restrained and delayed, suggesting that PGC number may directly regulate the variability and length of gonadal transformation and testicular differentiation in zebrafish. We propose that gonadal transformation may function as a developmental buffering mechanism to ensure the reproductive outcome.
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Project description:The requirement for primordial germ cells (PGCs) during sexual differentiation is variable among vertebrates. It has been shown that in zebrafish complete loss of PGCs in the embryos causes exclusive male development. Further, transplantation of a single PGC into a germline deficient zebrafish embryos generates male exclusively, suggesting PGC number might be important for the ovarian fate.To explore how PGC number might regulate sexual development in zebrafish, we experimentally manipulated its number by injecting dnd MO into embryos to generate fish containing a spectrum of PGC number. The experiment was designed to compare transcriptomes between the developing trunk regions of wild type and dnd morphants at different developmental stages. The dnd MO was microinjected into one cell stage embryos to generate zebrafish with a range of PGC numbers. Transcriptomes of developing trunk regions of wild type and dnd morphants at 14 dpf and 22 dpf were analyzed.
2015-05-08 | E-GEOD-57046 | biostudies-arrayexpress
Project description:The requirement for primordial germ cells (PGCs) during sexual differentiation is variable among vertebrates. It has been shown that in zebrafish complete loss of PGCs in the embryos causes exclusive male development. Further, transplantation of a single PGC into a germline deficient zebrafish embryos generates male exclusively, suggesting PGC number might be important for the ovarian fate.To explore how PGC number might regulate sexual development in zebrafish, we experimentally manipulated its number by injecting dnd MO into embryos to generate fish containing a spectrum of PGC number. The experiment was designed to compare transcriptomes between the developing trunk regions of wild type and dnd morphants at different developmental stages.
2015-05-08 | GSE57046 | GEO
Project description:The combination of genome editing and primordial germ cell (PGC) transplantation has enormous significance in the study of developmental biology and genetic breeding, despite its low efficiency due to limited number of donor PGCs. Here, we employ a combination of germplasm factors to convert blastoderm cells into induced PGCs (iPGCs) in zebrafish and obtain functional gametes either through iPGC transplantation or via the single blastomere overexpression of germplasm factors. Zebrafish-derived germplasm factors convert blastula cells of Gobiocypris rarus into iPGCs, and Gobiocypris rarus spermatozoa can be produced by iPGC-transplanted zebrafish. Moreover, the combination of genome knock-in and iPGC transplantation perfectly resolves the contradiction between high knock-in efficiency and early lethality during embryonic stages and greatly improves the efficiency of genome knock-in. Together, we present an efficient method for generating PGCs in a teleost, a technique that will have a strong impact in basic research and aquaculture.
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Project description:Primordial germ cells (PGCs) are the precursors to the adult germline stem cells that are set aside early during embryogenesis and specified through the inheritance of the germ plasm, which contains the mRNAs and proteins that function as the germline fate determinants. In Drosophila melanogaster, formation of the PGCs requires the microtubule and actin cytoskeletal networks to actively segregate the germ plasm from the soma and physically construct the pole buds (PBs) that protrude from the posterior cortex. Of emerging importance is the central role of centrosomes in the coordination of microtubule dynamics and actin organization to promote PGC development. We previously identified a requirement for the centrosome protein Centrosomin (Cnn) in PGC formation. Cnn interacts directly with Pericentrin-like protein (PLP) to form a centrosome scaffold structure required for pericentriolar material recruitment and organization. In this study, we identify a role for PLP at several discrete steps during PGC development. We find PLP functions in segregating the germ plasm from the soma by regulating microtubule organization and centrosome separation. These activities further contribute to promoting PB protrusion and facilitating the distribution of germ plasm in proliferating PGCs.
| S-EPMC8091889 | biostudies-literature
Project description:Teleost sex differentiation largely depends on the number of undifferentiated germ cells. Here, we describe the generation and characterization of a novel transgenic zebrafish line, Tg(piwil1:egfp-UTRnanos3)ihb327Tg, which specifically labels the whole lifetime of germ cells, i.e., from primordial germ cells (PGCs) at shield stage to the oogonia and early stage of oocytes in the ovary and to the early stage of spermatogonia, spermatocyte, and spermatid in the testis. By using this transgenic line, we carefully observed the numbers of PGCs from early embryonic stage to juvenile stage and the differentiation process of ovary and testis. The numbers of PGCs became variable at as early as 1 day post-fertilization (dpf). Interestingly, the embryos with a high amount of PGCs mainly developed into females and the ones with a low amount of PGCs mainly developed into males. By using transient overexpression and transgenic induction of PGC-specific bucky ball (buc), we further proved that induction of abundant PGCs at embryonic stage promoted later ovary differentiation and female development. Taken together, we generate an ideal transgenic line Tg(piwil1:egfp-UTRnanos3)ihb327Tg which can visualize zebrafish germline for a lifetime, and we have utilized this line to study germ cell development and gonad differentiation of teleost and to demonstrate that the increase of PGC number at embryonic stage promotes female differentiation.
| S-EPMC6441407 | biostudies-literature
Project description:A crucial event in animal development is the specification of primordial germ cells (PGCs), which become the stem cells that create sperm and eggs. How PGCs are created provides a valuable paradigm for understanding stem cells in general. We find that the PGCs of the sea urchin Strongylocentrotus purpuratus exhibit broad transcriptional repression, yet enrichment for a set of inherited mRNAs. Enrichment of several germline determinants in the PGCs requires the RNA-binding protein Nanos to target the transcript that encodes CNOT6, a deadenylase, for degradation in the PGCs, thereby creating a stable environment for RNA. Misexpression of CNOT6 in the PGCs results in their failure to retain Seawi transcripts and Vasa protein. Conversely, broad knockdown of CNOT6 expands the domain of Seawi RNA as well as exogenous reporters. Thus, Nanos-dependent spatially restricted CNOT6 differential expression is used to selectively localize germline RNAs to the PGCs. Our findings support a 'time capsule' model of germline determination, whereby the PGCs are insulated from differentiation by retaining the molecular characteristics of the totipotent egg and early embryo.
| S-EPMC4197533 | biostudies-literature
Project description:Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene affect fertility in both sexes. However, the involvement of CFTR in regulating germ cell development remains largely unknown. Here, we used zebrafish model to investigate the role of CFTR in primordial germ cells (PGCs) development. We generated a cftr frameshift mutant zebrafish line using CRISPR/Cas9 technique and investigated the migration of PGCs during early embryo development. Our results showed that loss of Cftr impairs the migration of PGCs from dome stages onward. The migration of PGCs was also perturbed by treatment of CFTRinh-172, a gating-specific CFTR channel inhibitor. Moreover, defected PGCs migration in cftr mutant embryos can be partially rescued by injection of WT but not other channel-defective mutant cftr mRNAs. Finally, we observed the elevation of cxcr4b, cxcl12a, rgs14a and ca15b, key factors involved in zebrafish PGCs migration, in cftr-mutant zebrafish embryos. Taken together, the present study revealed an important role of CFTR acting as an ion channel in regulating PGCs migration during early embryogenesis. Defect of which may impair germ cell development through elevation of key factors involved in cell motility and response to chemotactic gradient in PGCs.
| S-EPMC6106808 | biostudies-literature
Project description:Production of all-male and sterile fish may not only substantially improve yield but also be crucial for the application of genome modified species in aquaculture. Previously, it was reported that the fish lacking primordial germ cells (PGCs) becomes infertile, and nitroreductase, an enzyme converting non-toxic metronidazole (MTZ) into toxic metabolites, induces targeted toxicity to kill the cells expressing it. In this study, we generated a transgenic zebrafish line of Tg(nanos3:nfsB-mCherry-nanos3 3'UTR) in which the NfsB nitroreductase is solely expressed in PGCs. Treating the embryos derived from the female transgenic zebrafish with MTZ from 0 through 2 dpf (days post fertilization), we found that the germ cells were completely eliminated in the ones older than 2.5 dpf. At 20 dpf, the MTZ-treated juvenile had no germ cells in their gonads. At 100 dpf, the MTZ-treated adult exhibited male-like morphology and showed normal mating behaviors although they had no germ cells but only supporting cells in their gonads. Taken together, our results demonstrated that conditional elimination of PGCs during early development make the zebrafish male-like and infertile. It may provide an alternative strategy to make sterile and all-male farmed fish that is good for increasing aquaculture yield and preventing the genome modified species from potential ecological risks.
| S-EPMC5789895 | biostudies-literature
Project description:Primordial germ cells (PGCs) are the stem cells of the germline. Generally, germline induction occurs via zygotic factors or the inheritance of maternal determinants called germ plasm (GP). GP is packaged into ribonucleoprotein complexes within oocytes and later promotes the germline fate in embryos. Once PGCs are specified by either mechanism, GP components localize to perinuclear granular-like structures. Although components of zebrafish PGC germ granules have been studied, the maternal factors regulating their assembly and contribution to germ cell development are unknown. Here, we show that the scaffold protein Dazap2 binds to Bucky ball, an essential regulator of oocyte polarity and GP assembly, and colocalizes with the GP in oocytes and in PGCs. Mutational analysis revealed a requirement for maternal Dazap2 (MDazap2) in germ-granule maintenance. Through molecular epistasis analyses, we show that MDazap2 is epistatic to Tdrd7 and maintains germ granules in the embryonic germline by counteracting Dynein activity.
| S-EPMC4497839 | biostudies-literature