Spermatogonial Stem Cell Establishment

 描述（由申请方提供）：精原干细胞（SSC）对于维持整个成年期的精子发生至关重要。虽然我们对精原干细胞的了解已经取得了相当大的进展，但对最初在哺乳动物中建立这些细胞的调控机制知之甚少。在本申请中，我们建议通过研究以下内容来填补这一空白： 我们发现同源异型盒基因Rhox 10对小鼠睾丸中精原干细胞的正常初始建立至关重要。Rhox 10是我的实验室几年前发现的一个大型X连锁同源异型盒基因簇的成员。在这个Rhox基因簇中的基因优先在生殖道中表达，提高了它们编码对生殖至关重要的转录因子的可能性，但对此几乎没有直接证据作为测试其作用的一种手段，我们最近产生了整个Rhox簇敲除（KO）小鼠，它们缺乏Rhox簇中的所有33个同源框基因。这些小鼠具有“进行性生精下降”表型，其非常类似于具有SSC缺陷的良好建立的小鼠模型。我们认为最有可能负责的单个Rhox基因的条件性KO-Rhox 10-产生了与整个Rhox簇的KO基本相同的表型。对Rhox 10-KO小鼠的深入分析表明，它们在出生后阶段（通常是首次建立SSC）产生SSC的能力存在很大缺陷。这种缺陷可能是由于前体细胞的分化缺陷导致的，这些前体细胞产生SCC-Pro-精原细胞（ProSG）-基于几条证据，包括单细胞（SC）-RNAseq分析。本提案的目的1是阐明RHOX 10促进ProSG分化和SSC建立的机制。我们将首先使用SC-RNAseq分析和其他方法精确定义SSC建立期间运行的细胞亚群和发育途径。然后，我们将对Rhox 10-KO小鼠进行相同的分析，以确定RHOX 10转录因子在ProSG分化和SSC建立中的特定作用。作为这项分析的一部分，我们将验证我们最近发现的候选ProSG标记基因，并筛选新的基因。这对于该领域是关键的，因为据我们所知，先前尚未鉴定出良好建立的ProSG标记基因。本提案的目的2是描述RHOX 10在生殖细胞中的分子作用模式。我们初步的SC-RNAseq实验已经在生殖细胞亚群中鉴定出了候选的RHOX 10调控基因。我们将验证这些候选靶点，并将我们的分析扩展到我们显示RHOX 10在体内起作用的特定生殖细胞亚群。RHOX 10直接靶标将通过一系列方法进行鉴定，包括ChIP-seq分析。将进行功能研究以筛选对RHOX 10在ProSG和SSC建立中的功能重要的靶基因。鉴于对配子发生的这一关键时期知之甚少，这些实验将填补该领域的一个巨大空白，并将为体内转录因子的作用提供前所未有的视角。

 DESCRIPTION (provided by applicant): Spermatogonial stem cells (SSCs) are critical for maintaining spermatogenesis throughout adult life. While considerable progress has been made in our understanding of SSCs, little is known about the regulatory mechanisms that initially establish these cells in mammals. In this application, we propose to fill this gap through study of a homeobox gene-Rhox10-that we find is essential for the normal initial establishment of SSCs in the mouse testis. Rhox10 is a member of a large X-linked homeobox gene cluster that my laboratory discovered some years ago. The genes in this Rhox gene cluster are preferentially expressed in the reproductive tract, raising the possibility that they encode transcription factors critical for reproduction, but there has been little direct evidence for this As one means to test their role, we recently generated whole Rhox cluster knockout (KO) mice that lack all 33 homeobox genes in the Rhox cluster. These mice have a "progressive spermatogenic decline" phenotype that strongly resembles well-established mouse models harboring SSC defects. Conditional KO of the single Rhox gene that we regarded as most likely to be responsible - Rhox10 - yielded essentially the same phenotype as KO of the entire Rhox cluster. In-depth analysis of Rhox10-KO mice revealed they have a strong defect in the ability to generate SSCs during the postnatal period when SSCs are normally first established. This deficit likely results from a differentiation defect in the precursor cells that give rise to SCCs-Pro- spermatogonia (ProSG)-based on several lines of evidence, including single-cell (SC)-RNAseq analysis. Aim 1 of this proposal is to elucidate the mechanisms by which RHOX10 promotes ProSG differentiation and SSC establishment. We will first precisely define the cell subsets and developmental pathways operating during the SSC establishment period using SC-RNAseq analysis and other methods. We will then perform the same analyses on Rhox10-KO mice to define the specific roles of the RHOX10 transcription factor in ProSG differentiation and SSC establishment. As part of this analysis, we will verify candidate ProSG marker genes we have recently identified, and screen for new ones. This is critical for the field, as, to our knowledge, no well- established ProSG marker genes have previously been identified. Aim 2 of this proposal is to delineate the molecular mode of action of RHOX10 in germ cells. Our preliminary SC-RNAseq experiments have already identified candidate RHOX10-regulated genes in germ cell subsets. We will verify these candidate targets and expand our analysis to the specific germ cell subsets that we show RHOX10 acts in in vivo. RHOX10 direct targets will be identified by a battery of approaches, including ChIP-seq analysis. Functional studies will be performed to screen for target genes important for RHOX10's function in ProSG and SSC establishment. Given that little is known about this critical period of gametogenesis, these experiments will fill a large gap in the field and will provide an unprecedented view of the role o a transcription factor in vivo.