Molecular Regulation of the Perinatal Male Germ Cell Niche

描述（由申请人提供）：性腺细胞（或促生母细胞）是精原干细胞（ssc）的前体，它通过自我更新和产生子细胞的能力为精子发生提供基础。尽管它们相对重要，但在胎儿中控制卵泡细胞维持和出生后向ssc过渡的调节机制尚不清楚。利用转基因小鼠，我们确定了支持细胞中NOTCH1信号的组成性激活会导致gonocyte丢失——这是该信号通路在睾丸中的潜在作用的第一个建议。然后，我们抑制NOTCH在小鼠支持细胞中的激活，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）或增生，从而增加睾丸癌的易感性（NOTCH下调）。该提案将验证NOTCH活性通过其靶效应物HEY1和HEYL下调维持生殖细胞未分化状态的两个关键分子：GDNF和CYP26B1的假设。我们将使用NOTCH过激活、NOTCH功能缺失和野生型小鼠模型来测试当NOTCH被激活时，转录抑制因子HEY1和/或HEYL是否直接影响GDNF和CYP26B1的表达。在Aim1中，我们将通过qPCR研究Hey1和HeyL转录因子的时间表达，并使用ChIP-PCR证明这些抑制因子与Gdnf启动子的直接结合。此外，我们将在NOTCH功能缺失模型中测试维持淋巴细胞静止的失败是否会导致原位癌样（CIS-like）表型。在Aim 2中，我们将研究NOTCH信号在CYP26B1表达中的作用，CYP26B1是一种阻断生殖细胞分化的酶。通过ChIP-PCR分析，我们将证明HEY1/HEYL转录因子直接结合Cyp26b1启动子下调其表达。最后，我们将通过抑制CYP26B1来测试NOTCH信号的过表达是否真的会导致Sertoli细胞综合征。在Aim 3中，使用生殖细胞-支持细胞共培养，我们将测试生殖细胞调节支持细胞中NOTCH活性的假设，因此可以

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.