Regulation of Germ Cell Pluripotency Through The RNA-Binding Protein, DND1

描述（由申请人提供）：睾丸畸胎瘤发生于人类，是一种良性、非腺瘤性生殖细胞肿瘤，其特征是分化出多种特定细胞和组织类型。这些不寻常的肿瘤反映了生殖细胞、胚胎干细胞和肿瘤细胞之间的密切关系，并为编程这些细胞命运和调节它们之间的转换的机制提供了重要线索。虽然畸胎瘤在啮齿动物模型中几乎是未知的，但在20世纪70年代发现了一种自发的小鼠突变Ter，该突变导致睾丸畸胎瘤的高发病率，特别是在129/SvJ遗传背景上，而不是在其他菌株如C57 BL/6 J上。在大多数遗传背景下，Ter-/- XY生殖细胞在出生时完全消失。最近，Ter突变被定位到Dnd 1，这是一个参与斑马鱼生殖细胞调控的基因的小鼠直系同源物。DND 1是一种RNA结合蛋白，靶向参与生殖细胞分化和细胞周期控制的几种已知转录物，并保护它们免受miRNA介导的降解。基于我们的初步数据，我们假设DND 1调节许多在PGC发育的过渡阶段控制生殖细胞编程的靶点。我们建议使用一个公正的Y3 H屏幕，以确定DND 1的mRNA客户端，和一个计算方法，以确定相互作用的miRNA和可能的应变差异，可能占畸胎瘤形成的不同敏感性。我们还计划创建Dnd 1的条件标记等位基因，并在（1）生殖细胞特化/迁移阶段，（2）睾丸中生殖细胞向前精原细胞转变的胎儿阶段和（3）出生后动物中删除它。我们将使用这个标记的等位基因，以确定细胞定位的DND 1在发展过程中，并免疫沉淀DND 1结合其在体内的mRNA目标。我们还将从易感（129）和非易感（B6）菌株中分离E14.5时的野生型和突变型生殖细胞，以获得全局表达谱。这些数据将与DND 1靶数据相关，以阐明控制生殖细胞周期进展和菌株对肿瘤形成敏感性的转录和转录后机制。整合的翻译机制与转录网络潜在的应变敏感性，以肿瘤的形成有直接的临床相关性，人类生殖细胞肿瘤。 公共卫生相关性：生殖细胞对生殖健康至关重要。发育中断会导致不育或生殖细胞肿瘤的形成。通过整合转录和转录后信息的水平，我们将确定DND 1，RNA结合蛋白，控制生殖细胞的细胞周期和编程转换的机制。虽然DND 1是生殖细胞特有的，但我们预计所涉及的调控机制可能是许多多能干细胞群体所共有的。

DESCRIPTION (provided by applicant): Testicular teratomas arise in humans as benign, non-seminomatous germ cell tumors characterized by the differentiation of a diverse array of specific cell and tissue types. These unusual tumors reflect the close relationship between germ cells, embryonic stem cells, and tumor cells, and hold important clues about the mechanisms that program these cell fates and regulate the transitions between them. Although teratomas were virtually unknown in rodent models, a spontaneous mouse mutation, Ter, was discovered in the 1970s that led to a high incidence of testicular teratomas, specifically on the 129/SvJ genetic background but not on other strains such as C57BL/6J. On most genetic backgrounds Ter-/- XY germ cells disappear completely by the time of birth. Recently, the Ter mutation was mapped to Dnd1, the mouse ortholog of a gene involved in germ cell regulation in zebrafish. DND1 is an RNA-binding protein that targets several known transcripts involved in germ cell differentiation and cell cycle control and protects them from miRNA-mediated degradation. Based on our preliminary data, we hypothesize that DND1 regulates many targets that control germ cell programming at transitional stages of PGC development. We propose to use an unbiased Y3H screen to identify the mRNA clients of DND1, and a computational approach to identify interacting miRNAs and possible strain differences that may account for the different sensitivity to teratoma formation. We also plan to create a conditional tagged allele of Dnd1 and delete it (1) at germ cell specification/migration stages, (2) during the fetal stage of germ cell transition to pro- spermatogonia in the testis and (3) in postnatal animals. We will use this tagged allele to determine the cellular localization of DND1 during development, and to immunoprecipitate DND1 bound to its in vivo mRNA targets. We will also isolate wild type and mutant germ cells at E14.5 from both the susceptible (129) and non-susceptible (B6) strains to obtain global expression profiles. These data will be correlated with DND1 target data to elucidate the transcriptional and post-transcriptional mechanisms governing germ cell cycle progression and strain sensitivity to tumor formation. The integration of translational mechanisms with transcriptional networks underlying strain sensitivity to tumor formation has immediate clinical relevance to human germ cell tumors. PUBLIC HEALTH RELEVANCE: Germ cells are crucial to reproductive health. Disruptions of their development lead to sterility or the formation of germ cell tumors. By integrating levels of transcriptional and post-transcriptional information, we will identify the mechanisms through which DND1, an RNA-binding protein, controls the cell cycle and programming transitions of germ cells. Although DND1 is specific to germ cells, we anticipate that the regulatory mechanisms involved may be common to many pluripotent stem cell populations.