Delay in primordial germ cell migration in adamts9 knockout zebrafish.
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ABSTRACT:
SUBMITTER: Carver JJ
PROVIDER: S-EPMC8058341 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundAs a global environmental concern, hypoxia is known to be associated with many biological and physiological impairments in aquatic ecosystems. Previous studies have mainly focused on the effect of hypoxia in adult animals. However, the effect of hypoxia and the underlying mechanism of how hypoxia affects embryonic development of aquatic animals remain unclear.Methodology/principal findingsIn the current study, the effect of hypoxia on primordial germ cell (PGC) migration in zebrafish embryos was investigated. Hypoxic embryos showed PGC migration defect as indicated by the presence of mis-migrated ectopic PGCs. Insulin-like growth factor (IGF) signaling is required for embryonic germ line development. Using real-time PCR, we found that the mRNA expression levels of insulin-like growth factor binding protein (IGFBP-1), an inhibitor of IGF bioactivity, were significantly increased in hypoxic embryos. Morpholino knockdown of IGFBP-1 rescued the PGC migration defect phenotype in hypoxic embryos, suggesting the role of IGFBP-1 in inducing PGC mis-migration.Conclusions/significanceThis study provides novel evidence that hypoxia disrupts PGC migration during embryonic development in fish. IGF signaling is shown to be one of the possible mechanisms for the causal link between hypoxia and PGC migration. We propose that hypoxia causes PGC migration defect by inhibiting IGF signaling through the induction of IGFBP-1.
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Project description: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).
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Project description: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.
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Project description:In many lower animals, germ cell formation, migration, and maintenance depend on maternally provided determinants in germ plasm. In zebrafish, these processes have been extensively studied in terms of RNA-binding proteins and other coding genes. The role of small non-coding RNAs in the regulation of primordial germ cell (PGC) development remains largely unknown and poorly investigated, even though growing interests for the importance of miRNAs involved in a wide variety of biological processes. Here, we reported the role and mechanism of the germ plasm-specific miRNA miR-202-5p in PGC migration: (i) both maternal loss and knockdown of miR-202-5p impaired PGC migration indicated by the mislocalization and reduced number of PGCs; (ii) cdc42se1 was a direct target gene of miR-202-5p, and overexpression of Cdc42se1 in PGCs caused PGC migration defects similar to those observed in loss of miR-202-5p mutants; (iii) Cdc42se1 not only interacted with Cdc42 but also inhibited cdc42 transcription, and overexpression of Cdc42 could rescue PGC migration defects in Cdc42se1 overexpressed embryos. Thus, miR-202-5p regulates PGC migration by directly targeting and repressing Cdc42se1 to protect the expression of Cdc42, which interacts with actin to direct PGC migration.
| S-EPMC7493028 | biostudies-literature
Project description:Long noncoding RNAs (lncRNAs) are regulatory transcripts in various biological processes. However, the role of lncRNAs in germline development remains poorly understood, especially for fish primordial germ cell (PGC) development. In this study, the lncRNA profile of zebrafish PGC was revealed by single cell RNA-sequencing and bioinformatic prediction. We established the regulation network of lncRNA-mRNA associated with PGC development, from which we identified three novel lncRNAs-lnc172, lnc196, and lnc304-highly expressing in PGCs and gonads. Fluorescent in situ hybridization indicated germline-specific localization of lnc196 and lnc304 in the cytoplasm and nucleus of spermatogonia, spermatocyte, and occyte, and they were co-localized with vasa in the cytoplasm of the spermatogonia. By contrast, lnc172 was localized in the cytoplasm of male germline, myoid cells and ovarian somatic cells. Loss- and gain-of-function experiments demonstrated that knockdown and PGC-specific overexpression of lnc304 as well as universal overexpression of lnc172 significantly disrupted PGC development. In summary, the present study revealed the lncRNA profile of zebrafish PGC and identified two novel lncRNAs associated with PGC development, providing new insights for understanding the regulatory mechanism of PGC development.
| S-EPMC9954370 | 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:The regulated migration of cells is essential for development and tissue homeostasis, and aberrant cell migration can lead to an impaired immune response and the progression of cancer. Primordial germ cells (PGCs), precursors to sperm and eggs, have to migrate across the embryo to reach somatic gonadal precursors, where they carry out their function. Studies of model organisms have revealed that, despite important differences, several features of PGC migration are conserved. PGCs require an intrinsic motility programme and external guidance cues to survive and successfully migrate. Proper guidance involves both attractive and repulsive cues and is mediated by protein and lipid signalling.
| S-EPMC4521894 | biostudies-literature
Project description:BackgroundExploring the molecular mechanisms of primordial germ cell (PGC) migration and the involvement of gonadal somatic cells in gonad development is valuable for comprehending the origins and potential treatments of reproductive-related diseases.MethodsDiaphanous related formin 1 (Diaph1, also known as mDia1) was screened by analyzing publicly available datasets (ATAC-seq, DNase-seq, and RNA-seq). Subsequently, the CRISPR-Cas9 technology was used to construct Diaph1 knockout mice to investigate the role of Diaph1 in gonad development.ResultsBased on data from public databases, a differentially expressed gene Diaph1, was identified in the migration of mouse PGC. Additionally, the number of PGCs was significantly reduced in Diaph1 knockout mice compared to wild type mice, and the expression levels of genes related to proliferation (Dicer1, Mcm9), adhesion (E-cadherin, Cdh1), and migration (Cxcr4, Hmgcr, Dazl) were significantly decreased. Diaph1 knockout also inhibited Leydig cell proliferation and induced apoptosis in the testis, as well as granulosa cell apoptosis in the ovary. Moreover, the sperm count in the epididymal region and the count of ovarian follicles were significantly reduced in Diaph1 knockout mice, resulting in decreased fertility, concomitant with lowered levels of serum testosterone and estradiol. Further research found that in Diaph1 knockout mice, the key enzymes involved in testosterone synthesis (CYP11A1, 3β-HSD) were decreased in Leydig cells, and the estradiol-associated factor (FSH receptor, AMH) in granulosa cells were also downregulated.ConclusionsOverall, our findings indicate that the knockout of Diaph1 can disrupt the expression of factors that regulate sex hormone production, leading to impaired secretion of sex hormones, ultimately resulting in damage to reproductive function. These results provide a new perspective on the molecular mechanisms underlying PGC migration and gonadal development, and offer valuable insights for further research on the causes, diagnosis, and treatment of related diseases.
| S-EPMC11247884 | biostudies-literature
Project description:Although embryonic germ (EG) cell-mediated gene transfer has been successful in the mouse for more than a decade, this approach is limited in other species due to the difficulty of isolating the small numbers of progenitors of germ cell lineage (PGCs) from early-stage embryos and the lack of information on the in vitro culture requirements of the cells. In this study, methods were established for the culture of PGCs obtained from zebrafish embryos. Transgenic embryos that express the red fluorescent protein (RFP) under the control of the PGC-specific vasa promoter were used, making it possible to isolate pure populations of PGCs by fluorescence-activated cell sorting (FACS) and to optimize the culture conditions by counting the number of fluorescent PGC colonies produced in different media. Cultures initiated from 26-somite-stage embryos contained the highest percentage of PGCs that proliferated in vitro to generate colonies. The effect of growth factors, including Kit ligand a and b (Kitlga and Kitlgb) and stromal cell-derived factor 1a and 1b (Sdf-1a and Sdf-1b), on PGC proliferation was studied. Optimal in vitro growth and survival of the zebrafish PGCs was achieved when recombinant Kitlga and Sdf-1b were added to the culture medium through transfected feeder cells, resulting in a doubling of the number of PGC colonies. Results from RT-PCR and in situ hybridization analysis demonstrated that PGCs maintained in culture expressed the kita receptor, even though receptor expression was not detected in PGCs isolated by FACS directly from dissociated embryos. In optimal growth conditions, the PGCs continued to proliferate for at least 4 months in culture. The capacity to establish long-term PGC cultures from zebrafish will make it possible to conduct in vitro studies of germ cell differentiation and EG cell pluripotency in this model species and may be valuable for the development of a cell-mediated gene transfer approach.
| S-EPMC2741329 | biostudies-literature
Project description:BackgroundSmall micromeres are produced at the fifth cleavage of sea urchin development. They express markers of primordial germ cells (PGCs), and are required for the production of gametes. In most animals, PGCs migrate from sites of formation to the somatic gonad. Here, we investigated whether they also exhibit similar migratory behaviors using live-cell imaging of small micromere plasma membranes.ResultsEarly in gastrulation, small micromeres transition from non-motile epithelial cells, to motile quasi-mesenchymal cells. Late in gastrulation, at 43 hr post fertilization (HPF), they are embedded in the tip of the archenteron, but remain motile. From 43-49 HPF, they project numerous cortical blebs into the blastocoel, and filopodia that contact ectoderm. By 54 HPF, they begin moving in the plane of the blastoderm, often in a directed fashion, towards the coelomic pouches. Isolated small micromeres also produced blebs and filopodia.ConclusionsPrevious work suggested that passive translocation governs some of the movement of small micromeres during gastrulation. Here we show that small micromeres are motile cells that can traverse the archenteron, change position along the left-right axis, and migrate to coelomic pouches. These motility mechanisms are likely to play an important role in their left-right segregation.
| S-EPMC4164171 | biostudies-literature