Control over phase-separated germ-granules morphology, molecular patterning, positioning and function.

某些脊椎动物和无脊椎动物生殖细胞的发育依赖于胚质——RNA和蛋白质的相分离凝聚体，它们没有被膜包围。胚质和其中的成分被称为胚粒，它们对生殖细胞的发育和后期发育非常重要。因此，除了指导细胞承担生殖细胞的命运外，种质成分对于细胞向配子、精子和卵子的迁移和分化也是必不可少的。在这项研究计划中，我们使用斑马鱼胚胎作为一个可访问的通用模型来研究活脊椎动物生物中胚芽颗粒的基本生物学，并最终将研究结果与人类不育联系起来。提出的研究目标是- 1。利用显微镜、新型图像分析工具和不同的生物物理方法，详细描述活胚胎中颗粒的观察数量、分布和形态的演变。2. 确定相分离物质与细胞内其他组分之间的空间关系，以及不同细胞器和细胞结构对颗粒形状和位置的影响。3. 开发新的工具，研究Tdrd7蛋白的功能，这是获得正确数量和大小的胚芽颗粒所必需的，从而了解控制这些特性的机制。4. 研究相分离物质的分子分布规律，考察颗粒大小对细胞器内组织的影响。5. 确定控制Tdrd7功能的蛋白质结构域，并确定这些结构域内的突变是否会影响生殖细胞的发育，并可能与人类的生育能力有关。

The development of germ cells in certain vertebrate and invertebrate organisms depends on germ plasm - RNA and protein-containing phase-separated condensates that are not surrounded by a membrane. Germ plasm and components that reside within it are termed germ granules and these are important for the specification of the germ cells and their development at later stages. Thus, in addition to directing cells to assume the germ-cell fate, germ plasm components are also essential for the migration and differentiation of the cells into gametes, sperm, and egg. In this research proposal, we use zebrafish embryos as an accessible general model for studying the basic biology of germ granules in the live vertebrate organism and eventually link the findings to infertility in humans. The objectives of the proposed research are- 1. To provide a detailed description of the evolution of the observed number, distribution, and morphology of the granules employing microscopy, novel image analysis tools, and different biophysical methods in the live embryo. 2. To determine the spatial relationships between the phase-separated material and other components within the cell and the effect of the different organelles and cellular structures on the granule shape and position. 3. Development of new tools which will allow for investigating the function of the Tdrd7 protein, which is required for attaining the correct number and size of the germ granules, thereby learning about the mechanisms controlling these features of the germ plasm. 4. Study the patterning of the phase-separated material concerning the molecules' distribution and examine the effect granule size has on the intra-organelle organization. 5. Define protein domains that control the function of Tdrd7 and determine if mutations within those domains affect the development of germ cells and can be linked to fertility in humans.