Molecular Regulation of the Perinatal Male Germ Cell Niche

描述(申请人提供)：性细胞(或成熟原细胞)是精原干细胞(SSCs)的前体，SSCs通过其自我更新和生成子细胞的能力为精子发生提供基础。尽管它们相对重要，但对胎儿性腺细胞维持和出生后向SSCs过渡的调控机制知之甚少。利用转基因小鼠，我们证实了支持细胞中NOTCH1信号的结构性激活会导致性细胞丢失--这是这一信号通路在睾丸中潜在作用的第一个暗示。然后，我们抑制了小鼠支持细胞中的缺口激活，并观察到生殖细胞数量和睾丸大小的增加。因此，NOTCH信号的失调会导致不育(NOTCH过度激活)或增殖，从而增加睾丸癌的易感性(NOTCH下调)。这项提议将检验如下假设：通过其靶标效应物HEY1和Heyl下调两个维持生殖细胞未分化状态的关键分子：GDNF和CYP26B1。我们将使用NOTCH过度激活、NOTCH功能缺乏和野生型小鼠模型来测试转录抑制因子HEY1和/或HeYL在NOTCH被激活时是否直接影响GDNF和CYP26B1的表达。在Aim1中，我们将通过qPCR来研究Hey1和Heyl转录因子的瞬时表达，并使用芯片-PCR来证明这些抑制物直接与GDNF启动子结合。此外，我们将测试在我们的缺口功能缺失模型中维持性腺细胞静止的失败是否会导致类似原位癌(CIS-like)的表型。在目标2中，我们将研究Noch信号对细胞色素P26B1表达的作用，这是一种阻止生殖细胞分化的酶。利用芯片-聚合酶链式反应分析，我们将证明HEY1/HeY1转录因子直接与Cyp26b1启动子结合，下调其表达。最后，我们将测试Noch信号的过度表达是否真的通过抑制CYP26B1而导致仅支持细胞综合征。在目标3中，使用生殖细胞-支持细胞共培养，我们将检验这样一个假设，即生殖细胞调节支持细胞中的缺口活动，因此可以 控制自己的人数。总之，这项提议将首次证明，Noch信号调节生殖细胞增殖和维持未分化状态所必需的两个分子的表达，是正常生殖细胞动态平衡的组成部分。这一途径的失调会导致不育或生殖细胞增殖。

DESCRIPTION (provided by applicant): Gonocytes (or prospermatogonia) are the precursors to spermatogonial stem cells (SSCs), which provide the foundation for spermatogenesis through their ability to both self-renew and generate daughter cells. Despite their relative importance, th regulatory mechanisms that govern gonocyte maintenance in the fetus and transition to SSCs after birth are poorly understood. Using transgenic mice, we established that constitutive activation of NOTCH1 signaling in Sertoli cells causes gonocyte loss-the first suggestion of the potential role of this signaling pathway in the testis. We then inhibited NOTCH activation in mouse Sertoli cells and observed an increase in germ cell numbers and testicular size. Therefore dysregulation of NOTCH signaling induces either sterility (NOTCH overactivation) or hyperplasia that could enhance predisposition to testicular cancer (NOTCH downregulation). This proposal will test the hypotheses that NOTCH activity, through its target effectors HEY1 and HEYL, downregulates two crucial molecules that maintain the undifferentiated states of germ cells: GDNF and CYP26B1. We will use NOTCH overactivation, NOTCH lack of function and wild type mouse models to test whether the transcriptional repressors HEY1 and/or HEYL directly influence the expression of GDNF and CYP26B1 when NOTCH is activated. In Aim1, we will investigate the temporal expression of Hey1 and HeyL transcription factors by qPCR, and use ChIP-PCR to demonstrate direct binding of these repressors to the Gdnf promoter. Further, we will test whether failure of maintaining gonocyte quiescence in our NOTCH lack-of-function model leads to a carcinoma-in-situ-like (CIS-like) phenotype. In Aim 2, we will investigate the role of NOTCH signaling on the expression of CYP26B1, an enzyme that blocks germ cell differentiation. Using ChIP-PCR analysis, we will demonstrate that HEY1/HEYL transcription factors directly bind to the Cyp26b1 promoter to downregulate its expression. Finally, we will test whether overexpression of NOTCH signaling truly leads to a Sertoli cell-only syndrome through inhibition of CYP26B1. In Aim 3, using germ cell-Sertoli cells co-cultures, we will test the hypothesis that germ cells regulate NOTCH activity in Sertoli cells and therefore can regulate their own numbers. Altogether, this proposal will demonstrate for the first time that NOTCH signaling modulates the expression of two molecules essential for germ cell proliferation and maintenance of the undifferentiated state, and is a component of normal germ cell homeostasis. Dysregulation of this pathway will induce sterility or germ cell hyperplasia.