The role of the Dead end protein in controlling germ cell fate

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The migration, specification and fate maintenance of germ cells is a conserved at the cellular and molecular levels. A key molecule that is required during different steps of germline development is the RNA-binding protein Dead end.In the absence of the Dead end protein, the germline is lost giving rise to sterile animals. We could show that Dead end functions in germ cells to protect their unipotent differentiation potential by inhibiting somatic differentiation such that gametes can be formed. Global analysis of the effect of Dead end on the transcriptome of germ cells revealed that a range of somatic differentiation factors become expressed in Dead end depleted cells, concomitantly with a reduction in the transcription level of RNAs linked to germline development. Importantly, among the genes suppressed by Dead end are genes encoding for pluripotency factors such as Nanog and the Oct4 homolog.In the next funding period we propose to expand the scope of understanding the function of Dead end to later stages of germline development. The proposed work first involves the establishment of an experimental system for inhibiting Dead end at different stages. This procedure relies on inducing Dead end protein degradation by expressing specific nanobodies directed against the tagged protein. The transcriptome and chromatin structure of germ cells knocked down at different stages of their development will be assayed to reveal the role of the Dead end at stages previously not explored regarding this issue. The role of the regulation of pluripotency proteins by Dead end will be studied in experiments where Dead end function is inhibited in combination with manipulations (genetic mutations of and over-expression) in pluripotency-promoting genes. Together, the results of these studies will enhance the understanding of the molecular mechanisms that direct the developmental potential of the germline to the formation of gametes.