Control Spermatogonial Stem Cell Fate Decisions by bHLH Transcription Regulators

描述(申请人提供)：精原干细胞(SSCs)的自我更新和分化为精子发生提供基础。在世界范围内，数以百万计的男性经历不育症或亚生育，这可能是由于性干细胞功能受损。此外，精子发生是一个典型的干细胞依赖过程，可以作为其他组织特异性干细胞系统的模型。因此，破译监管SSC命运决定的机制至关重要。与其他成体干细胞群体相似，SSC的功能受生态位微环境的影响和特定分子网络的激活而受到外部控制。已有研究证实，胶质细胞源性神经营养因子(GDNF)是调节哺乳动物SSCs自我更新的重要生长因子。生长因子对干细胞命运决定的影响是通过调节特定的内在分子网络来调节的，目前在SSCs中对此知之甚少。我们发现GDNF能刺激由SSCs和非干细胞精原细胞组成的小鼠睾丸Thy1+生殖细胞群体中转录抑制因子分化抑制因子4(Id4)的表达。此外，我们确定体内缺乏Id4表达会扰乱小鼠正常的精子发生，导致增殖的精原细胞数量枯竭。此外，野生型SSCs中Id4表达的降低使其在体外的自我更新能力丧失。这些观察表明，Id4是决定SSC命运的重要内在调节因子；然而，其作用机制尚不清楚。ID4是碱性螺旋-环-螺旋(BHLH)转录调控家族的成员，但缺乏DNA结合域。在其他组织中，ID蛋白家族通过结合和抑制刺激分化的特定bHLH转录因子的活性来维持祖细胞处于未分化状态。在小鼠雄性生殖系中，Id4是唯一由精原细胞表达的ID家族蛋白，bHLH转录因子Neurogenin 3(Ngn3)和精子发生和卵子发生螺旋螺旋1(Sohlh1)是精原细胞早期分化的调节因子。Id4和这些bHLH转录因子在SSCs中的相互作用还没有被研究过。我们的中心假设是，小鼠SSCs的自我更新和分化之间的平衡是由一条内在的分子途径控制的，在该途径中，Id4通过抑制诱导精原细胞分化的特定bHLH转录因子的活性来促进自我更新，如Ngn3和Sohlh1。这一假设将通过以下几个方面得到验证：1)确定Id4是否具有控制SSC自我更新的独特能力；2)确定Ngn3和Sohlh1是否是SSC分化的调节因子；3)确定Id4是否抑制Ngn3和Sohlh1的转录因子活性。公共卫生相关性：破译Id4、Ngn3和Sohlh1在控制SSC命运决定中的作用将增强对调控精子发生和男性生育的基础过程的全面理解。这些信息可能有助于诊断和治疗人类男性生殖失败的特定事件。此外，这项研究将扩大干细胞生物学的一般知识，这些知识可能适用于对维持生命至关重要的其他组织特定干细胞群体。

DESCRIPTION (provided by applicant): Self-renewal and differentiation of spermatogonial stem cells (SSCs) provides the foundation for spermatogenesis. Worldwide, millions of men experience infertility or sub-fertility which may be due to impairment of SSC functions. Also, spermatogenesis is a classic stem cell-dependent process which can serve as a model for other tissue-specific stem cell systems. Thus, deciphering the mechanisms that regulate SSC fate decisions is of critical importance. Similar to other adult stem cell populations, SSC functions are controlled extrinsically by influence of a niche microenvironment and intrinsically via activation of specific molecular networks. Previous studies have determined that glial cell line-derived neurotrophic factor (Gdnf) is an essential growth factor regulating self-renewal of mammalian SSCs. The influence of growth factors on stem cell fate decisions is mediated via regulating specific intrinsic molecular networks, which are currently poorly understood in SSCs. We showed that Gdnf stimulates expression of the transcriptional repressor, inhibitor of differentiation 4 (Id4) in the Thy1+ germ cell population of mouse testes which is composed of SSCs and non-stem cell spermatogonia. Additionally, we determined that lack of Id4 expression in vivo disrupts normal spermatogenesis in mice resulting in depletion of the proliferating spermatogonia population. Furthermore, reduction of Id4 expression in wild-type SSCs abolishes their ability to self-renew in vitro. These observations indicate that Id4 is an essential intrinsic regulator of SSC fate decisions; however, its mechanism of action is unknown. Id4 is a member of the basic helix-loop-helix (bHLH) family of transcription regulators, but lacks a DNA binding domain. In other tissues, the Id family of proteins maintain progenitor cells in an undifferentiated state by binding to and repressing the activity of specific bHLH transcription factors that stimulate differentiation. In the mouse male germline, Id4 is the only Id family protein expressed by spermatogonia and the bHLH transcription factors neurogenin 3 (Ngn3) and spermatogenesis and oogenesis helix-loop-helix 1 (Sohlh1) are regulators of early spermatogonial differentiation. Interaction between Id4 and these bHLH transcription factors in SSCs has not been examined. Our central hypothesis is that balance between self-renewal and differentiation of mouse SSCs is controlled by an intrinsic molecular pathway in which Id4 promotes self-renewal by repressing the activity of specific bHLH transcription factors that induce spermatogonial differentiation such as Ngn3 and Sohlh1. This hypothesis will be tested by; 1) determining whether Id4 has the singular capacity to control SSC self-renewal, 2) determining whether Ngn3 and Sohlh1 are regulators of SSC differentiation, and 3) determining whether Id4 represses the transcription factor activity of Ngn3 and Sohlh1. PUBLIC HEALTH RELEVANCE: Deciphering the role of Id4, Ngn3, and Sohlh1 in control of SSC fate decisions will enhance overall understanding of foundation processes regulating spermatogenesis and male fertility. This information may be useful in diagnosing and treating specific incidences of reproductive failure in human males. Also, this research will expand general knowledge of stem cell biology that may be applicable to other tissue-specific stem cell populations important for sustaining life.