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

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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.