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

点击翻译按钮获取中文摘要

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.