Analysis of the formation and regulation of quiescence of the adult muscle precursor cells (AMPs) during embryonic development by using the GFP-Labeled marker genes twist and Him in Drosophila melanogaster
Final Report Abstract
AMPs - adult muscle precursor cells - are the source of adult musculature, formed within the pupae during the transition from larval to adult stage of Drosophila melanogaster. To date, the regulation of the development and the specific properties of AMPs are not very well understood. In Aim1 the role of the mesodermal fate gene twist, which is expressed in the AMPs, was analyzed. Therefore, a rescue construct was brought n a twi null mutant background, where the late Twi expression within AMPs is eliminated without affecting its early panmesodermal expression. This construct was able to weakly rescue during the early Twi expression (stage 5). In later stages, Twi expression was no longer detectable. According to the weak rescue potential. It might be possible that the integration occurred into less transcribed chromatin. New experiments using the new ϕC31-based Integrafion system should lead to a safisfying result. The identification of the AMP enhancer element of the twist gene and the generation of AMP-GFP reporter lines was the prerequisite for the performance of the deficiency screen proposed in Aim2. The two selected deficiency collections cover ≈85% of the Drosophila genome. During the screening of both available kits, the DrosDel Deficiency Kit and the Exelixis Deficiency Collection, 652 lines were analyzed concerning their influence on AMP development. In 34 lines ofthe DrosDel deficiency Kit AMPs were altered in number and shape. Out of these 34 lines, 5 lines affect AMPs formation specifically, while the remaining deficiencies also showed a change in the somatic musculature and the heart. In 110 lines of the Exelixis Deficiency Collection the AMP formation was changed. The influence of these lines on the remaining musculature, somatic and heart, will be further investigated by antibody stainings against Twi, mef2 and Tropomyosin. Lines that exclusively show an effect on AMPs will be analyzed closer. The genetic analysis of two genes based on their phenotype concerning AMPs development proposed in Aim3 is in progress. From five promising lines identified so far, gene expression and gene silencing experiments will rule out genes not affecting AMPs in their formation and development. Characterization of the remaining genes will shed new light on AMP development.
