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

生殖细胞的迁移、分化和命运维持在细胞和分子水平上是保守的。在种系发育的不同阶段需要的一个关键分子是rna结合蛋白死端。在没有Dead end蛋白的情况下，种系丧失，导致动物不育。我们可以证明死角在生殖细胞中通过抑制体细胞分化来保护其单能分化潜能，从而形成配子。Dead end对生殖细胞转录组影响的全球分析表明，Dead end缺失的细胞中表达了一系列体细胞分化因子，同时与生殖细胞发育相关的rna转录水平降低。重要的是，在被Dead end抑制的基因中有编码多能因子的基因，如Nanog和Oct4同源基因。在下一个资助期，我们建议将Dead end的功能扩展到生殖细胞发育的后期阶段。建议的工作首先涉及建立不同阶段抑制死胡同的实验系统。这个过程依赖于通过表达针对标记蛋白的特定纳米体来诱导死端蛋白降解。在生殖细胞发育的不同阶段被敲除的转录组和染色质结构将被分析，以揭示死端在以前未探讨过的阶段的作用。Dead end对多能性蛋白的调控作用将在实验中进行研究，其中Dead end功能与多能性促进基因的操作（基因突变和过表达）相结合被抑制。总之，这些研究的结果将加强对指导种系发育潜力到配子形成的分子机制的理解。

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.