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

这个项目旨在了解从不同的原基中建立一个复杂的器官，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很好的，实验上很容易接近的系统，可以解决成肌细胞和细胞环境之间的交流及其对器官建立的影响。雄性生殖系统的躯体部分起源于生殖盘，在第三龄幼虫期间，肌肉组织的干细胞（上皮细胞）被招募到生殖盘。我们的初步工作揭示了非传统的单核横纹肌和多核平滑肌在男性生殖系统。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指导信号协调发展。我们通过rnai介导的敲除、注射到蛹和结合酶抑制剂的体外器官培养中鉴定了许多体内标记物。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)成肌细胞/新生肌管迁移的信号级联和F-Actin的参与。第一个工作包将解决，如何不同的成肌细胞被指定形成不同的肌肉类型，并调查何时，何地以及如何多核肌肉出现。这将通过在时间和空间上控制的rnai介导的敲除在功能上接近。我们计划以公正的方式确定新的相关基因。我们将用抗mcd8偶联磁珠对mcd8标记的成肌细胞进行分选，分离不同发育阶段的成肌细胞，然后进行全基因组转录组分析。本部分将分析新基因与成肌细胞测定和产生多核肌管的相关性。第二个工作包将解决成肌细胞的迁移问题。我们的问题是，是哪一种信号通路导致成肌细胞和新生肌管从生殖盘向男性生殖道上的特定目的地移动？f -肌动蛋白是如何参与的呢？这将通过在体内应用信号级联的抑制剂和激活剂和/或大量低形态等位基因以及信号分子的构成活性，显性阴性版本和F-Actin调节因子来实现。我们将分析我们的转录组数据信号级联的组成部分，新的f -肌动蛋白调节因子和诱导f -肌动蛋白修饰的分子响应所研究的信号级联。方法：利用成肌细胞转录组、转基因果蝇系（蛋白诱捕系、UAS/GAL4系统、GAL80ts、Lifeact-eGFP、报告基因构建、RNAi系）和低形或温敏等位基因等遗传工具、体内GFP和RFP标记、抑制剂应用和体内复制标记。

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.