A new cell population regulating cell fate and morphogenesis in the mouse testis

描述(由申请人提供)：我们的目标是了解细胞命运指定和细胞重组的机制，这些机制将胎儿性腺转变为有功能的睾丸。睾丸器官发生的第一步是性腺原基中大约一半的体细胞对支持细胞命运的承诺。支持细胞包围生殖细胞，经历新生脐带形成过程。这种基本的细胞分选过程定义了睾丸的两个部分：睾丸索和间质。睾丸索将成为生精小管，在成年生活中精子在这里产生，而产生雄激素的间质细胞和其他特征较差的群体在间质中分化。虽然脐带形成的过程对胎儿的男性化和成人的生育能力是必不可少的，但人们对此还不太了解。它被认为是支持细胞驱动的过程；然而，一些证据表明间质细胞在形成睾丸索的过程中起着关键作用。在果蝇性腺形态发生过程中，一个对细胞分选至关重要的基因是交通堵塞(TJ)。TJ编码Maf转录因子家族成员，并通过细胞黏附分子(CaM)的调节促进果蝇性腺的器官发生。TJ的哺乳动物同源基因MafB在一群未鉴定的祖细胞中表达，这些祖细胞在胎儿睾丸形成开始时从支持细胞中分离出来，并形成睾丸间质。MafB的表达与血管系统密切相关，后来变得仅限于胎儿间质细胞。我们推测，MafB在间质细胞类型的确定和睾丸索的形成中发挥作用，可能是通过控制CaM或细胞外基质(ECM)蛋白来发挥作用。我们最近发现，Notch信号对于调节间质中的祖细胞数量和间质细胞分化是必需的。在特定的目标1中，我们将通过免疫细胞化学、原位杂交和定量PCR，使用体内和体外的Notch增益和Ibss功能分析，来研究Notch信号参与MafB表达细胞的规范的假设。在特定的目标2，我们建议阐明MafB表达细胞在睾丸形态发生中的作用，利用聚合酶链式反应、微阵列技术和遗传学方法来确定哪些CaM受MafB的调控。在这两个目标中，我们将利用几个转基因小鼠系进行活体成像和荧光激活细胞分选，以在细胞水平上研究睾丸的形态发生。这种对细胞命运和细胞-细胞相互作用的研究将有助于我们对器官发生的基本理解，并为不孕不育和生殖障碍的发育基础提供洞察。

DESCRIPTION (provided by applicant): Our goal is to understand the mechanisms of cell fate specification and cellular reorganization that transform the fetal gonad into a functional testis. The first step of testis organogenesis is the commitment of approximately half of the somatic cells in the gonad primordium to the Sertoli cell fate. Sertoli cells surround germ cells and undergo the process of de novo cord formation. This basic process of cell sorting defines two compartments of the testis: the testis cords and the interstitium. The testis cords will become the seminiferous tubules, where sperm are produced in adult life, whereas the androgen-producing Leydig cells and other less well-characterized populations differentiate in the interstitium. Although the process of cord formation is essential for masculinization of the fetus and fertility of the adult, it is not well understood. It has been viewed as a Sertoli-cell-driven process; however, several lines of evidence suggest that interstitial cells play a critical role in shaping the process of testis cord formation. One gene that is essential for cell sorting during gonad morphogenesis in Drosophila is traffic jam (tj). tj encodes a member of the Maf transcription factor family, and promotes organogenesis of the Drosophila gonad via the regulation of cell adhesion molecules (CAMs). The mammalian ortholog of tj, MafB, is expressed in an uncharacterized population of progenitor cells that sort out from Sertoli cells at the beginning of fetal testis formation and give rise to the interstitium of the testis. MafB expression is closely associated with the vasculature, and later becomes restricted to fetal Leydig cells. We hypothesize that MafB plays a role in specification of the cell types of the interstitium and in the formation of testis cords, likely acting through control of CAMs or extracellular matrix (ECM) proteins. We have recently shown that Notch signaling is required for regulation of the progenitor population in the interstitium and for Leydig cell differentiation. In Specific Aim 1, we will investigate the hypothesis that Notch signaling is involved in the specification of MafB-expressing cells, using in vivo and ex vivo Notch gain- and Ibss-of-function analysis via immunocytochemistry, in situ hybridization, and quantitative PCR. In Specific Aim 2, we propose to elucidate the role of MafB-expressing cells during testis morphogenesis, employing PCR, microarray techniques, and genetic methods to determine which CAMs are regulated by MafB. In both aims, we will utilize several transgenic mouse lines for live imaging and fluorescence-activated cell sorting to study testis morphogenesis at the cellular level. This study of cell fate specification and cell-cell interactions will contribute to our basic understanding of organogenesis and provide insight into the developmental basis for infertility and reproductive disorders.