斑马鱼生殖质microRNA miRNA-202调控原始生殖细胞发育的作用机制研究

Gametogenesis is one of the most important developmental events in sexual reproduction. In animals, germ cell development begins with the formation of the primordial germ cells (PGCs) in the early embryos. It is still a major challenge in developmental biology and medical sciences to clearly understand the molecular mechanism under the germ cell development. Zebrafish is an ideal vertebrate model for researches in reproductive biology due to its advantages. The fate of zebrafish PGCs is determined by the maternal germ plasm which mainly consists of proteins and RNAs. Many functional proteins and mRNAs have been identified from the germ plasm, but no any microRNA specific to germ plasm and/or PGC has been characterized in zebrsfish although it has been believed that microRNAs are involved in almost all biological processes including reproductive development. In our precious work, we identified a microRNA miR-202-5p in the zebrsfish germ plasm for the first time, and subsequently found it was specifically localized in the developing PGCs. Further studies indicated that miR-202-5p played a key role in PGC development because knockdown of miR-202-5p in early zebrafish embryos led to reduce of PGC number and ectopic localization of PGCs. In this project, we propose to illuminate the detailed regulatory role of miR-202-5p in PGC formation, proliferation, survival and/or migration and the molecular mechanisms involved in these processes in zebrafish. We will (1)fruther confirm the functional role of miR-202-5p in zebrafish PGC developement, (2) identify the target genes of miR-202-5p by bioinformatic methods, transcriptome analysis, and other biological technologies, (3) investigate the potential roles of the target genes of miR-202-5p in the germ cell and gonad development in zebrafish, (4) explore the factors interacting with the target genes of miR-202-5p, and (4) summarize the molecular network consisting of miR-202-5p, its target genes and other possible factors which are related to PGC development. The results and findings in this project will contribute new insights to the mechanisms involved in the germ cell development of zebrafish, and also are valuable to the researches of reproductive development in other animals.

配子发生是动物有性生殖的重要发育事件。配子发生始于原始生殖细胞(PGC)的形成。PGC的形成、增殖、维持、分化和迁移是受到精确调控的复杂过程，阐明该过程的调控机制是发育和医学领域的重要前沿研究课题。斑马鱼是研究生殖细胞发育的理想模式，其PGC的发育受到母源生殖质的调控，但生殖质调控PGC发育的内在机制尚未阐明。在前期工作中，我们首次鉴定到斑马鱼生殖质特异microRNA miR-202-5p，并初步证明其异常表达会导致胚胎中PGC数量下降和分布位置异常。本研究拟(1)进一步明确miR-202-5p在斑马鱼生殖细胞发育中的作用；(2)预测并鉴定其靶基因；(3)研究关键靶基因在斑马鱼生殖细胞和性腺发育中的作用；(4)研究miR-202-5p及相关因子间的调控关系，以阐明miR-202-5p调控斑马鱼PGC发育过程的具体环节和作用机制。本项目研究结果将提供理解斑马鱼生殖细胞发育遗传机制的新认识。

配子发生是动物有性生殖的重要发育事件。配子发生始于原始生殖细胞(PGC)的形成。PGC的形成、增殖、维持、分化和迁移是受到精确调控的复杂过程，阐明该过程的调控机制是发育和医学领域的重要前沿研究课题。斑马鱼是研究生殖细胞发育的理想模式，其PGC的发育受到母源生殖质的调控，但生殖质调控PGC发育的内在机制尚未阐明。本项目围绕生殖特异miRNA miR-202-5p在斑马鱼PGC迁移过程中的作用和机制开展了一系列的研究，取得以下进展：（1）描述了miR-202-5p与其他生殖标记在斑马鱼性腺和组织中的表达和定位，证明其实斑马鱼生殖质特异的miRNA。通过敲降、过表达和敲除等实验方法，证明敲低或者母源缺失miR-202-5p将导致斑马鱼PGC迁移出现异常，但是miR-202-5p纯合突变体斑马鱼的繁殖能力没有显著影响。初步阐明miR-202-5p在斑马鱼生殖细胞发育中的功能。（2）利用单细胞转录组测序揭示了WT和MmiR-202 PGC的转录表达谱，预测miR-202-5p调控PGC迁移的生物信息学网络。通过生物信息学预测，结合qPCR、双荧光素酶和荧光蛋白报告系统等实验验证，进而鉴定了4个miR-202-5p靶基因cdc42se1、rab10、fsta和trim25。（3）通过一系列的获得和缺失功能研究，证明两种miR-202-5p调控斑马鱼PGC迁移的分子机制：miR-202-5p负调控cdc42se1维持cdc42的正常表达来确保斑马鱼PGC的迁移；miR-202-5p通过维持rab10的稳态来调控斑马鱼PGC迁移。上述研究为初步阐明miRNA在斑马鱼PGC发育中的功能和分子机制，为理解斑马鱼生殖细胞发育遗传机制的新认识。