Specification, migration, and morphogenesis of arthropod muscles
Final Report Abstract
Among the 103 dsRNAs that were recognized to cause embryonic muscle phenotypes in the second round of the iBeetle screen we selected 50 for rescreening. 25 of these showed the same phenotype upon reinjections of the same dsRNA fragment and 13 were confirmed with our most stringent verification criteria. The gene products of the verified genes from this screening round have diverse predicted molecular functions, including RNA binding and processing, signaling (e.g., FGF8 and TGFβ-receptor), enzymes and transporters potentially involved in regulating signaling activities, chromatin regulators, and the transcription factor Crocodile (Tc-Croc). 12 of these genes have direct orthologs in Drosophila and the 13th has several fly homologs as well. So far we have followed up on functional studies on the forkhead domain gene croc. Pupal Tc-croc dsRNA injections cause specific disruptions of the ventral set of body wall muscles in Tribolium, which we showed to correlate with specific ventral somatic mesodermal mRNA expression of Tc-croc in Tribolium embryos and, likewise, of Dm-croc in Drosophila embryos. Ongoing analyses of the role of Dm-croc in Drosophila muscle development involve the analysis of double mutants for croc and another forkhead domain gene expressed in the somatic mesoderm, ches-1-like, as the absence of any muscle phenotypes in croc mutants invoked the possibility of functional redundancy of forkhead domain genes in this process. Apart from the work on these newly-identified genes, we performed in-depth studies on genes identified in the first round of screening. One gene, TC011075/Sclp, encodes leucine-rich repeat domains and was identified in the larval injection screen, where dsRNAs caused the absence of a specific subset of indirect flight muscles in the pupal thorax. In Drosophila embryos, Sclp is specifically expressed in the developing somatic muscles, and reports on both Drosophila and Manduca Sclp had implicated this gene in certain physiological or developmental changes in the musculature. We have produced Drosophila Sclp gene-trap mutations and are currently investigating their phenotypes to gain insight into the function of this gene. Our major focus was directed towards the analysis of the role of genes encoding proteins of the F-Bar domain family. The encoded proteins contain an F-Bar domain, which interacts with the inner surface of the plasma membrane to introduce membrane bending, as well as a C-terminal SH3 domain, which binds regulators of the actin cytoskeleton. RNAi knock-downs of the Tribolium F-Bar gene TC01374 caused a phenotype of very thin body wall muscles. Similar phenotypes were observed upon knock-downs of several genes in the iBeetle screen whose Drosophila counterparts are important for myoblast fusion. The Drosophila ortholog of TC01374, termed nostrin, is expressed specifically in the fusion-competent myoblasts of the somatic and visceral muscles. Two related genes encoding both F-Bar and SH3 domains, cip4 and syndapin (synd), are co-expressed with nostrin in the somatic and visceral mesoderm, in addition to their expression in epidermal and neuronal tissues. After producing nostrin mutations we went on to demonstrate that these F-Bar domain genes, particularly cip4 and nostrin, have functionally-redundant roles in somatic and visceral muscle development. Double mutants for cip4 and nostrin as well as triple mutants for cip4, nostrin, and synd showed myoblast fusion defects that were evident from the abnormal presence of unfused myoblasts in late stage embryos, the concomitant absence of muscles at these positions, and the lower number of myonuclei in syncytial muscles that are present. Adult flies lacking the functions of both cip4 and nostrin also showed severe gut muscle phenotypes. In particular, the longitudinal midgut muscles were shorter and thinner, highly branched, and disarranged. As these defects develop only during metamorphosis, we undertook a detailed analysis of normal adult midgut muscle development during pupariation in order to connect these events with the observed mutant phenotypes and obtain insight into the possible roles of cip4 and nostrin during normal gut muscle development. These analyses yielded the interesting and unexpected observation that the syncytial longitudinal midgut muscles completely dedifferentiate and fragment into mostly mononucleated myoblasts during metamorphosis, which then fuse again with one another and finally redifferentiate into the adult longitudinal gut muscles. Parallel studies in Tribolium pupae showed similar events of dedifferentiation, fragmentation and rediffentiation of longitudinal gut muscles in this species, and an association of visceral muscles with the intestinal stem cells in the crypts of the midgut epithelium, indicating a large degree of conservation of arthropod gut muscle metamorphosis and midgut homeostasis. Taken altogether, we propose that the investigated F-Bar domain proteins are involved in coordinating the extensive events of membrane bending and cytoskeletal rearrangements that are required during embryonic myoblast fusion as well as during the cell fusion and redifferentiation of longitudinal midgut muscles during metamorphosis. In sum, the iBeetle screen revealed novel players in muscle development, which in Drosophila were not found due to functional redundancies. Follow-up studies revealed novel processes of muscle de- and redifferentiation in the insect gut, thus validating Tribolium as a gainful screening platform for basic cell biological questions.
Publications
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(2015) Large scale RNAi screen in Tribolium reveals novel target genes for pest control and the proteasome as prime target. BMC Genomics 16, 674
Ulrich, J., Dao, V. A., Majumdar, U., Schmitt-Engel, C., Schwirz, J., Schultheis, D., Ströhlein, N., Troelenberg, N., Grossmann, D., Richter, T., Dönitz, J., Gerischer, L., Leboulle, G., Vilcinskas, A., Stanke, M., Bucher, G.
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(2015) The iBeetle large-scale RNAi screen reveals gene functions for insect development and physiology. Nat Commun 6, 7822
Schmitt-Engel, C., Schultheis, D., Schwirz, J., Ströhlein, N., Troelenberg, N., Majumdar, U., Dao, V. A., Grossmann, D., Richter, T., Tech, M., Dönitz, J., Gerischer, L., Theis, M., Schild, I., Trauner, J., Koniszewski, N. D., Kuster, E., Kittelmann, S., Hu, Y., Lehmann, S., Siemanowski, J., Ulrich, J., Panfilio, K. A., Schröder, R., Morgenstern, B., Stanke, M., Buchhholz, F., Frasch, M., Roth, S., Wimmer, E. A., Schoppmeier, M., Klingler, M., Bucher, G.
