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

这个项目的目的是了解从不同的原基建立一个复杂的器官，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优秀的，实验上很容易接近的系统，以解决成肌细胞和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，在三龄幼虫期间，肌肉组织的干细胞（adepithelial细胞）被募集到生殖盘。我们的初步工作揭示了非传统的单核横纹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线索协调发展。我们鉴定了许多体内标记物，通过RNAi介导的敲低和通过注射到脓中的体内测定以及与酶抑制剂组合的体外器官培养物。本研究的目的是从时间和空间上阐明1）成肌细胞的决定和多核肌管的形成; 2）成肌细胞/新生肌管迁移的信号级联和F-肌动蛋白的参与。第一个工作包将阐述不同的成肌细胞是如何形成不同的肌肉类型的，并研究多核肌肉何时、何地和如何产生。这将通过在时间和空间上控制的RNAi介导的敲低在功能上接近。我们计划以无偏见的方法确定新的相关基因。我们将通过用抗mCD 8偶联磁珠分选mCD 8标记的成肌细胞来分离不同发育阶段的成肌细胞，然后进行比较性的全基因组转录组分析。新的感兴趣的基因将在这一部分进行分析，其相关性成肌细胞的决定和创造多核肌管。第二个工作包将涉及成肌细胞的迁移。我们询问是哪些信号级联导致成肌细胞和新生肌管从生殖盘移向男性生殖道上的不同目的地？F-actin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以及信号分子和F-肌动蛋白调节剂的组成型活性、显性阴性版本来实现。我们将分析我们的转录组数据的组件信号级联，新的F-肌动蛋白调节剂和分子诱导F-肌动蛋白的修改，以响应调查的信号级联。研究方法：成肌细胞的转录组、遗传工具如转基因果蝇系（蛋白质捕获系; UAS/GAL 4系统、GAL 80 ts、Lifeact-eGFP、报告构建体、RNAi系）和亚型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的标记。

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.