Dissection of the transcriptional network of human primordial germ cells

项目摘要 人类的发育始于受精，经过一系列的卵裂分裂， 胚胎和胚泡的形成;然后就在原肠胚形成的早期阶段之前或期间，一些 细胞被搁置或分配成为下一代的生殖细胞，并传递一个人的DNA。 下一代。生殖细胞分化缺陷是人类不育的常见原因， 10-15%的夫妇。然而，至少部分由于缺乏早期人类生殖细胞发育的模型， 大多数不孕症的潜在细胞和分子相关性仍然未知。这个项目的总体目标是 项目是填补这一知识空白，至少部分，通过解剖内在的转录网络，在早期 人类生殖细胞。这一建议的假设是，一个独特的TF网络，由人类组成， 体细胞谱系特化因子与多能性基因合作，控制着最早的发育转变 在生殖细胞从多能干细胞向人原始生殖细胞（hPGCs）的分化中， 体细胞基因表达，同时激活生殖细胞程序;此外，我们假设，同样的， 网络功能，以维持生殖细胞的身份，并促进进展到精原阶段， 超越。我们的初步研究发现了人类生殖细胞中独特的转录网络， 由OCT 4-PAX 5-PRDM 1电路调节。为了解决我们的假设，我们提出了三个具体目标：1） 在真正的hPGC中，TF的诊断性全基因组定位图。2)剖析协同性和上位性 通过功能获得和功能丧失分析的hPGC中的TF网络（OCT 4、SOX 17、T和PAX 5）。3)诱导 人生殖细胞通过定义的TF。本研究在假设、初步数据和研究方法等方面具有创新性。 干细胞生物学、分化、人类基因组编辑、全基因组转录、 表观遗传分析和计算生物学，我们用来实现我们的总体目标。这个项目是 重要的是，它将增加我们对人类生殖细胞发育遗传学的了解， 调控从多能性到生殖细胞谱系的细胞命运获得的转录网络， 建立一个强大的遗传系统，可以平行的果蝇和小鼠方面， 能够检查复杂的基因型和表型，也可能大大有助于潜在的新的 在不孕症的诊断和开发新的治疗方法的临床应用策略。

PROJECT SUMMARY Human development begins at fertilization, progresses through a series of cleavage divisions, compaction of the embryo and formation of the blastocyst; then just prior to or during the early stages of gastrulation, a few cells are set aside or allocated to become the germ cells of the next generation and pass the DNA of one generation to the next. Defects in germ cell differentiation are a common cause of human infertility that afflicts 10-15% of couples. However, at least in part due to a lack of models of early human germ cell development, most of the underlying cellular and molecular correlates of infertility remain unknown. The overall goal of this project is to fill this knowledge void, at least in part, by dissecting the intrinsic transcriptional network in early human germ cells. The hypothesis underlying this proposal is that a unique TF network, comprised of human somatic lineage specifiers in cooperation with pluripotency genes, controls the earliest developmental transition in germ cell differentiation from pluripotent stem cells to human primordial germ cells (hPGCs) by repressing somatic gene expression while activating germ cell programs; moreover, we hypothesize that the same network functions to maintain germ cell identity and promote progression to the spermatogonial stage and beyond. Our preliminary studies have uncovered a unique transcriptional network in human germ cells that is regulated by an OCT4-PAX5-PRDM1 circuit. To address our hypothesis, we propose three specific aims to: 1) Map diagnostic genome-wide localization of TFs in bona fide hPGCs. 2) Dissect cooperativity and epistasis of the TF network (OCT4, SOX17, T and PAX5) in hPGCs by gain- and loss-of-function analysis. 3) Induce human germ cells via defined TFs. The study is innovative in terms of the hypothesis, preliminary data and the combination of tools of stem cell biology, differentiation, human genome editing, genome-wide transcriptional, epigenetic analysis, and computational biology, that we use to accomplish our overall goal. This project is significant in that it will increase our knowledge of human germ cell developmental genetics by illuminating the transcriptional network governing acquisition of cell fate from pluripotency to the germ cell lineage, enable establishment of a robust genetic system that may parallel that of Drosophila and the mouse in terms of the ability to examine complex genotypes and phenotypes, and may also contribute substantially to potential novel strategies in clinical applications in diagnosis and development of novel therapeutics for infertility.