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

干细胞生态位功能和 Hh 信号转导的分子基础可视化

• 批准号: 232508456
• 负责人: Dr. Christian Bökel
• 金额: --
• 依托单位: Core Facility Konfokale und Multiphotonen Mikroskopie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2012
• 资助国家: 德国
• 起止时间: 2011-12-31 至 2015-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/232508456?language=en
• 关键词: Visualizing; molecular; basis; stem; cell

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.

拟议的研究的目的是联合收割机果蝇遗传学与细胞生物学和先进的，显微镜为基础的生物物理学方法，以提高我们的理解干细胞的利基功能和组织的一个关键的潜在signalingpathway.In所有后生动物性腺，从生殖系和体细胞谱系的细胞是必需的器官发育和稳态，因此必须保持各自的干细胞池。在果蝇睾丸中，生殖系干细胞（germinlinestemcells，GSCs）和体细胞包囊干细胞（somatocysteemcells，CySCs）围绕着一组有丝分裂后的体细胞（称为hub）。这些产生各种生长因子，有助于调节两个干细胞库的生态位微环境。最初，大多数研究集中在生殖系方面，其中在睾丸中，由中枢产生的BMP家族生长因子抑制GSC分化（Losick等人，然而，近年来，几个研究小组已经开始研究中心产生的信号在维持体细胞CySC和两个干细胞库的交叉调节中的作用。中枢细胞分泌类胡萝卜素配体Upd。体细胞中Jak/Stat信号级联的随后激活正调节CySC干性、对中枢的粘附以及参与GSC维持的次级信号的产生（Issigonis等人，2009; Flaherty等人，2010; Leatherman和Dinardo，2010; Singh等人，2010年）。此外，我们最近可以表明，CySC的维持另外严格依赖于从中枢到体细胞的Hh信号传导。Hh信号传导缺陷的CySC克隆因分化而丢失，而Hh途径活化导致体干细胞过度增殖（Michel et al.，因此，在第一个子项目中，我们将鉴定参与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信号通路的关系。然而，与其他信号通路相比，Hh通路的功能知之甚少。具体而言，尚不清楚磷酸化、聚簇和必需途径组分（七跨膜结构域蛋白Smoothened）的亚细胞定位是如何连接和调节的（Ingham等人，2011年）。这使得Hh级联和其他信号通路之间的交叉调节相互作用的机制分析难以分析。基于我们先前的BMP受体活化的报道者（Michel等人，2011）因此，我们产生了一种基于荧光的、激活状态特异性的Smoothened磷酸化传感器，它保留了信号功能并准确反映了Hh途径的激活。在第二个子项目中，我们将使用这种报告基因来剖析Hh信号转导的细胞生物学基础，并可视化利基中Hh信号的时空模式。

项目成果

Phosphorylation of the Smo tail is controlled by membrane localisation and is dispensable for clustering

• DOI: 10.1242/jcs.128926
• 发表时间: 2013-10
• 期刊: Journal of Cell Science
• 影响因子: 4
• 作者: Adam P. Kupinski;I. Raabe;Marcus Michel;Divya Ail;L. Brusch;T. Weidemann;Christian Bökel