Identifying novel regulators of specification, migration, and differentiation of muscle precursor cells in Drosophila
Final Report Abstract
We have performed a saturating EMS mutagenesis screen for the 2nd chromosome in Drosophila in order to obtain mutations affecting various processes of heart, somatic muscle, and gut muscle development and to characterize the functions of the affected genes. The screen employed embryos in which the heart tube was marked with GFP, and the org-1-positive muscles (M5, M25, SBM and alary muscles) plus the longitudinal midgut muscles were simultaneously marked with RFP. We retained over 450 independent mutant lines in which these muscle types were affected, either individually or in combination, and until now have mapped ~170 of them to ~50 different genes. Subsequently we utilized particular mutants from this collection to analyze three different aspects of muscle and heart development and the roles of the affected genes in depth. Firstly, we studied the specification of the caudal visceral mesoderm and development of longitudinal midgut muscles with the help of mutations in HLH54F, which encodes a basic helix-loop-helix transcription factor. We showed that the spatial expression of HLH54F mRNA is determined by the combinatorial action of mesodermal snail and terminal genes during blastoderm stage, which defines the primordium of the caudal visceral mesoderm. The activity of HLH54F is crucial for the specification of longitudinal gut muscle progenitors from this primordium and this function is also instrumental in maintaining the normal morphology of the midgut. We defined the Dorsocross genes of the T-box gene family as examples of genes that are directly regulated by HLH45F through a tissue specific enhancer element during this process. Secondly, mutations in the FGF-encoding gene pyr allowed us to study the role of FGF signals during the long-distance, anterior migration of the longitudinal gut muscle progenitors along the trunk visceral mesoderm. We showed that the ligand Pyr (and its cognate Ths) released from the trunk visceral mesoderm and endoderm activate the FGF receptor signaling cascade within the longitudinal gut muscle progenitors. Notably, these signals are required for the tight adherence of the migrating cells to their substratum, which in turn is essential for protecting the cells from programmed cell death. Through this mechanism, FGF signals can guide the longitudinal gut muscle progenitors along the trunk visceral mesoderm or, in artificial situations created by us, along ectopic paths. Thirdly, with mutations in a collagen IV encoding gene, Cg25C, and in a laminin-β chain encoding gene, LanB1, we dissected the role of the extracellular matrix in the stable attachment of alary muscles to the heart tube. We showed that the ECM is particularly important for the stable attachment of the cardioblasts to pericardial cells, to which the alary muscles are tightly attached. Based upon the specific mutations obtained we defined the LN domain of laminin-β as a particularly important domain for securing the stability of this specific ECM interface as well as for the formation of the heart lumen. In addition, we found that normally-structured collagen IV is needed for the efficient secretion and incorporation of ECM components such as perlecan. We also observed that certain collagen glycine repeat mutations cause intracellular collagen accumulation, thus paralleling observations made in other organisms and in connection with collagen-related diseases. In ongoing studies we examine the precise effects of additional mutations obtained in the screen and their assignments to specific genes.
Publications
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(2010) Genetic and genomic dissection of cardiogenesis in the Drosophila model. Pediatr Cardiol 31, 325-334
Reim, I., Frasch, M.
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(2010) HLH54F is required for the specification and migration of longitudinal gut muscle founders from the caudal mesoderm of Drosophila. Development 137, 3107-3117
Ismat, A., Schaub, C., Reim, I., Kirchner, K., Schultheis, D., Frasch, M.
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(2012) The FGF8-related signals Pyramus and Thisbe promote pathfinding, substrate adhesion, and survival of migrating longitudinal gut muscle founder cells. Dev Biol 368, 28-43
Reim, I., Hollfelder, D., Ismat, A., Frasch, M.
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Distinct functions of the laminin β LN domain and collagen IV during cardiac extracellular matrix formation and stabilization of alary muscle attachments revealed by EMS mutagenesis in Drosophila. BMC Developmental Biology, 2014, 14:26
Hollfelder, D., Frasch, M., Reim, I.
