Die Analyse einer Ribokinase als neue myogenese-relevante Komponente und erste Hinweise auf eine Beteiligung von Ca2+-abhängigen Faktoren in der Myogenese von Drosophila melanogaster

The EMS-induced mutant E832 (P3-106/2) shows strong defects in the development of the somatic and visceral larval musculature in the embryonic development of Drosophila melanogaster (Reichert, 2004; Wierach, 2005). The goal of this thesis was to further analyze this mutant and the characterization and identification of the responsible gene for the mutant phenotype. After complementation analysis by using characterised deletions to narrow down the genomic region it was shown that likely three lethal mutations in myogenesis-relevant genes on the E832 chromosome are present. Because of this assumptions an uncoupling experiment has been done to isolate and separate these mutations by recombination. The resulting fly strains were analysed by immunohistology and complementation experiments. Two regions (32F1-33A1 and 33E1-33E3) were shown to be responsible for the somatic muscle phenotype. The visceral phenotype was difficult to examine and so the priority was set on the analysis of the somatic-relevant mutation in the region 33E1-33E3. Most of the genes located to this region has been cloned to carry out rescue experiments with the help of the UAS-Gal4 system. Additionally the mRNA expression was analysed by In situ-hybridisations and in comparison to the rescue experiments a few of these genes were excluded due of their expression in other tissues like trachea (CG31862) and the garland cells (CG31763 and Pkd2). Only one of the candidate genes (CG17010) is expressed during mesoderm development. CG17010 has a coding capacity for a ribokinase and so the protein is renamed to D-Rbks33E. The ectopic expression of D-Rbks33E leads to a rescue of the mutant somatic phenotype in 33E1-33E3. The mRNA of D-Rbks33E is ubiquitously expressed, but a slightly higher transcript level can be observed in the somatic mesoderm. Sequencing of the mutated allele of D-Rbks33E revealed a base pair deletion. This leads to the loss of the ATP-binding site on the protein level of D-Rbks33E. First immunohistological analysis showed, that cell adhesion in these mutants is normal and so there seems to be a defect in the following process during myoblast fusion. Furthermore, in search for additional fusion-relevant proteins we took a closer look at Ca2+ dependent factors and here especially at Ca2+-binding proteins. The protein Kosh has been identified as a first candidate. In situ-hybridizations of kosh resulted in first clues for a function in fusion competent myoblasts during fusion with founder cells. So Kosh is one of the first proteins which indicate a role for calcium in developmental biology beside its function in physiology. Therefore two possibly new components has been identified, which has to be integrated in the signalling cascades of myogenesis.