Visualizing the molecular basis for stem cell niche function and Hh signal transduction

该研究的目的是将果蝇遗传学与细胞生物学和先进的基于显微镜的生物物理学方法相结合，以提高我们对干细胞生态位功能和关键潜在信号通路组织的理解。在所有后生动物性腺中，生殖系和体细胞系的细胞都是器官发育和体内平衡所必需的，因此必须由各自的干细胞库来维持。在果蝇睾丸中，生殖系干细胞（GSCs）和体细胞囊肿干细胞（CySCs）被排列在一组称为hub的有丝分裂后体细胞周围，这是干细胞研究的一个既定模型系统。这些产生了多种生长因子，有助于调节这两个干细胞池的生态位微环境。最初，大多数研究都集中在生殖系方面，在睾丸中，由中枢产生的BMP家族生长因子抑制GSC分化（Losick et al., 2011）。然而，近年来，一些研究小组已经开始研究hubgenerated信号在体细胞CySCs的维持和两个干细胞池的交叉调节中的作用。中心细胞分泌细胞因子样配体Upd。随后体细胞中Jak/Stat信号级联的激活积极调节CySC的干性、与中枢的粘附以及与GSC维持相关的次级信号的产生（isigonis等人，2009；Flaherty等人，2010；Leatherman和Dinardo, 2010； Singh等人，2010）。此外，我们最近可以证明，CySC的维持严格依赖于从中枢到体细胞的Hh信号。Hh信号缺陷的CySC克隆在分化过程中丢失，而Hh通路激活导致体细胞干细胞过度增殖（Michel et al., 2012）。因此，在第一个子项目中，我们将确定参与CySC维持的Hh靶基因，并阐明Hh信号与参与体细胞生态位功能的其他途径的关系。然而，与其他信号通路相比，Hh通路的功能知之甚少。具体来说，目前尚不清楚作为重要途径组分的7个跨膜结构域蛋白Smoothened的磷酸化、聚类和亚细胞定位是如何被连接和调控的（Ingham等，2011）。这使得Hh级联和其他信号通路之间交叉调控相互作用的机制分析变得难以分析。基于我们之前的BMP受体激活报告（Michel et al., 2011），我们因此生成了一种基于荧光的激活状态特异性传感器，用于平滑磷酸化，保留信号功能并准确反映Hh通路激活。在第二个子项目中，我们将使用该报告来剖析Hh信号转导的细胞生物学基础，并可视化Hh信号在生态位中的时空模式。

The aim of the proposed study is to combine Drosophila genetics with cell biology and advanced, microscopy based biophysical methods to improve our understanding of both stem cell niche function and the organization of a key underlying signalling pathway.In all metazoan gonads, cells from both germ line and somatic lineages are required for organ development and homeostasis and must therefore be maintained by their respective stem cell pools. In the Drosophila testis, an established model system for stem cell research, germ line stem cells (GSCs) and the somatic cyst stem cells (CySCs) are arranged around a group of postmitotic somatic cells termed hub. These produce a variety of growth factors that contribute to the niche microenvironment regulating both stem cell pools. Initially, most research was focussed on the germline side, where in the testis BMP family growth factors generated by the hub suppress GSC differentiation (Losick et al., 2011).However, in recent years several groups have begun addressing the role of hubgenerated signals in maintenance of the somatic CySCs and the cross-regulation of the two stem cell pools. The hub cells secrete the cytokine-like ligand Upd. Subsequent activation of the Jak/Stat signalling cascade in the somatic cells positively regulates CySC stemness, adhesion to the hub, and the production of secondary signals involved in GSC maintenance (Issigonis et al., 2009; Flaherty et al., 2010; Leatherman and Dinardo, 2010; Singh et al., 2010). Furthermore, we could recently show that CySC maintenance is in addition strictly dependent on Hh signalling from the hub to the somatic cells. CySC clones defective for Hh signalling are lost by differentiation, while Hh pathway activation leads to somatic stem cell overproliferation (Michel et al., 2012).In the first subproject we will therefore identify the Hh target genes involved in CySC maintenance, and clarify the relationship of the Hh signal with the other pathways involved in somatic niche function.However, in comparison with other signalling pathways, the function of the Hh pathway is poorly understood. Specifically, it is unclear how phosphorylation, clustering, and subcellular localization of an essential pathway component, the seven transmembrane domain protein Smoothened, are connected and regulated (Ingham et al., 2011). This makes the mechanistic analysis of cross-regulatory interactions between the Hh cascade and other signalling pathways difficult to analyze. Based on our previous reporter for BMP receptor activation (Michel et al., 2011) we have therefore generated a fluorescence based, activation state specific sensor for Smoothened phosphorylation that retains signalling function and accurately reflects Hh pathway activation.In the second subproject we will use this reporter both to dissect the cell biological basis of Hh signal transduction, and to visualize the spatiotemporal pattern of Hh signalling in the niche.