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

• Applicant: Professorin Dr. Renate Renkawitz-Pohl
• Subject Area: Developmental Biology
• Term: from 2013 to 2017
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 237549245

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.

• DFG Programme: Research Grants