Proliferation, fusion and cell migration to establish the musculature of the male reproductive system.

增殖、融合和细胞迁移以建立男性生殖系统的肌肉组织。

• 批准号: 237549245
• 负责人: Professorin Dr. Renate Renkawitz-Pohl
• 金额: --
• 依托单位: Arbeitsgruppe Kommunikation und Dynamik tierischer Zellen
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2013
• 资助国家: 德国
• 起止时间: 2012-12-31 至 2016-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/237549245?language=en
• 关键词: Proliferation; fusion; cell; migration; establish

This project aims to understand the establishment of a complex organ from different primordia, a question of high general importance. We consider the Drosophila male reproductive system as an excellent, experimentally well-accessible system to address communication between myoblasts and their cellular environment and its consequences for the organ establishment. The somatic parts of the male reproductive system originate from the genital disc, to which the stem cells of the musculature (adepithelial cells) are recruited during third instar larvae. Our preliminary work revealed unconventional mononucleated striated and multinucleated smooth muscles in the male reproductive system. During metamorphosis muscles develop on the genital disc in coordination with the other somatic parts of the reproductive tissue or guidance cues from the testis. We identified a number of in-vivo markers, in-vivo assays by RNAi-mediated knock-down and by injection into pupae and in vitro organ cultures combined with inhibitors of enzymes. The objectives of this work are to elucidate in time and space 1) myoblast determination and estab-lishment of multinucleated myotubes and 2) signalling cascades and involment of F-Actin for my-oblast/nascent myotube migration.The first workpackage will address, how distinct myoblasts are specified to form the different muscle types and to investigate when, where and how multinucleated muscles arise. This will be approached functionally by RNAi-mediated knock-down controlled in time and space. We plan to identify new genes of relevance in an unbiased approach. We will isolate myoblasts at different developmental stages by sorting mCD8-marked myoblasts with anti-mCD8-coupled magnetic beads and then perform comparative, genome-wide transcriptome analysis. New genes of interest will be analysed in this part with respect to their relevance for myoblast determination and creating multinucleated myotubes. The second workpackage will address the migration of myoblasts. We asked which signalling cas-cades cause myoblasts and nascent myotubes to move from the genital disc towards their distinct destination on the male reproductive tract? And how is F-actin involved? This will be approached by applying inhibitors and activators of signalling cascades in vivo and/or by a large collection of hypo-morph alleles as well as constitutive-active, dominant-negative versions of signalling molecules and F-Actin regulators. We will analyse our transcriptome data for components of signalling cascades, new F-Actin regulators and for molecules which induce F-Actin modifications in response to the investigated signalling cascades. Methods: Transcriptome of myoblasts, genetic tools such as transgenic Drosophilae lines (protein trap lines; UAS/GAL4 system, GAL80ts, Lifeact-eGFP, reporter contructs, RNAi lines) and hypomorph or temperature sensitive alleles, GFP and RFP marker in vivo, inhibitor application and labbeling of replication in vivo.

该项目旨在了解不同原基的复杂器官的建立，这是一个具有高度普遍重要性的问题。我们认为果蝇雄性生殖系统是一个极好的，在实验上可很好地访问的系统，以解决成肌细胞与其细胞环境之间的通信及其对器官建立的影响。雄性生殖系统的体细胞部分起源于生殖盘，肌肉系统的干细胞(上皮细胞)在三龄幼虫期间被招募到生殖盘。我们的初步工作揭示了男性生殖系统中非常规的单核、条纹和多核平滑肌肉。在变态过程中，生殖盘上的肌肉与生殖组织的其他躯体部分或来自睾丸的引导信号协调发展。我们鉴定了一些体内标记物，通过RNAi介导的击倒和注射入蛹和结合酶抑制剂的体外器官培养进行体内检测。这项工作的目的是在时间和空间上阐明1)成肌细胞的确定和多核肌管的建立，2)F-肌动蛋白在我的成肌细胞/新生肌管迁移中的信号级联和参与。第一个工作包将解决如何指定不同的成肌细胞形成不同的肌肉类型，并调查何时、何地和如何出现多核肌肉。这将通过RNAi介导的在时间和空间上控制的击倒来实现。我们计划以一种不偏不倚的方式确定新的相关基因。我们将通过使用抗mCD8偶联磁珠分选mCD8标记的成肌细胞来分离不同发育阶段的成肌细胞，然后进行比较的全基因组转录组分析。新的感兴趣的基因将在这一部分中分析它们与成肌细胞决定和创造多核肌管的相关性。第二个工作包将解决成肌细胞的迁移问题。我们询问了哪些信号通路导致成肌细胞和新生肌管从生殖盘向其在男性生殖道上的不同目的地移动？F-肌动蛋白是如何参与的？这将通过在体内应用信号级联的抑制剂和激活剂和/或通过大量的亚型等位基因集合以及信号分子和F-肌动蛋白调节剂的结构活性、显性负向版本来实现。我们将分析我们的转录组数据，以寻找信号级联的组件，新的F-肌动蛋白调节器，以及诱导F-肌动蛋白修饰的分子，以响应所研究的信号级联。方法：成肌细胞转录组，转基因果蝇系(蛋白质陷阱系，UAS/GAL4系统，GAL80ts，Lifeact-EGFP，报告结构，RNAi系)，低形态或温度敏感等位基因，体内GFP和RFP标记，抑制剂应用和体内复制标记等遗传工具。