Functional Characterization of CG9650 in development of the Indirect Flight Muscles of Drosophila melanogaster

Abstract

Muscle development is a complex and multifactorial process involving assembly of thousands of proteins in a precise and synchronized manner over the course of development of an organism. Multiple genes and signalling pathways have been identified to have pivotal roles in the development and function of a healthy and functional muscle. However, the role of many genes in muscle development remains unknown. One such gene are the BCL11A/B genes. BCL11A and BCL11B are paralogous genes which belong to the kruppel-like C2H2 type zinc finger transcription factors. The BCL11A and BCL11B protein sequences are 58% identical and 68% similar to each other. Both, BCL11A and BCL11B mainly have non-overlapping functions in neurogenesis and immune cell development. Recent studies have reported mutations in BCL11A to be associated with muscle-related defects like hypotonia, speech disorder and gross motor impairments, while mutations in BCL11B have been shown to be associated with muscle-related defects like hypertrophic cardiomyopathy and aortic stiffness. However, there are no studies which have addressed the molecular function of BCL11A and BCL11B in muscle development and function. CG9650 is the ortholog of BCL11A and BCL11B in Drosophila melanogaster. Overall, CG9650 bears 84% similarity to the vertebrate BCL11A and BCL11B proteins. The Indirect Flight Muscles (IFMs) of Drosophila melanogaster occupy a majority of the thorax of the adult fly and are responsible for powering the wing stroke during flight. These muscles consist of two opposing sets of muscles, namely the dorso-longitudinal muscles (DLMs; six in number) oriented anterior to posterior, and the dorso-ventral muscles (DVMs; three in number) oriented in a dorsal -to-ventral manner. The DLMs are formed by fusion of myoblasts with 3 pre-existing templates called Larval Oblique Muscles (LOMs), and subsequent splitting to form the 6 DLMs. The DVMs are formed by de novo fusion of myoblasts. Due to various advantages like the spatially and temporally distinct time-course of the development, dispensability to survival, similarity in development of the IFMs and vertebrate myogenesis, etc, the Indirect Flight Muscles (IFMs) of Drosophila melanogaster are an excellent model system to study the function of genes in muscle development. In this study, we have attempted to determine the role of CG9650 in development of the IFMs of Drosophila melanogaster. Our experiments show CG9650 is expressed during the specification (embryonic), proliferation (larval), and migration & fusion (pupal) stages of IFM development and depletion of CG9650 leads to a defect in the pattern of the DLMs (i.e. reduced number of DLMs). The expression of CG9650 during the proliferation, migration and fusion stages is crucial for patterning of the DLMs. This patterning defect could be rescued by transgenic expression of CG9650 during IFM development. The CG9650-depleted flies were compromised in their flight ability; walking ability of these flies remained unaffected. At the fascicular level, these DLMs have a larger cross-sectional area, more fibers per fascicle, but a decreased packing density of myofibrils compared to control flies. The sarcomeres of CG9650-depleted flies are thinner and longer, and expressed of the Z-disc protein Actinin was reduced compared to that control flies. These fascicular and sarcomeric defects are believed to cause reduced flight ability of the CG9650-depleted flies. CG9650 is also shown to affect Notch signalling in a context-dependant manner. Loss of CG9650 leads to upregulation of Notch signalling in myoblasts at the proliferation (larval) stage of IFM development. This leads to increased proliferation of the myoblasts. Additionally, loss of CG9650 leads to reduced Armadillo levels, which in turn leads to reduced wingless signalling, thus the stratification of myoblasts on the notum region of the wing disc. During the migration & fusion stages of IFM development, SnS, a (Ig-domain containing protein) is required for fusion of the migrating myoblast with the developing fiber. Migrating myobalsts show a diffuse expression of SnS. Notch signalling induces the formation of SnS puncta required for fusion of the myoblast with developing fiber. Perturbation of CG9650 during IFM development leads to decreased Notch signalling and a decrease in the formation of SnS puncta. This leads to decreased myoblast fusion and hence, arrested splitting of the developing LOMs. A transcriptomics approach was used to determine the genes/pathways regulated by CG9650. An RNA-seq using DLMs depleted for CG9650 revealed misregulation of genes mainly involved in myogenesis and neurogenesis. Aret, Act88F, thor, CanA, myofilin, Mlp60A, Zasp52 were among the differentially regulated genes with known functions in myogenesis. These genes have functions during the myofibrillogenesis stage of IFM development and their differential expression could account for the fascicular and sarcomeric defects seen in CG9650-depleted DLMs. Notably, expression of Hibris (a gene known to function in conjunction with SnS to regulate myoblast fusion) was also differentially regulated. Multiple genes known to be targets of Notch signalling were also found to be differentially regulated, thus confirming CG9650 as a regulator of the Notch pathway. Differential regulation of expression of genes regulating in cell division, channel proteins, immunity, cuticle formation and gene expression was also observed. In summary, our study highlights CG9650 as a novel modulator of Notch signalling output and novel regulator of DLM patterning during IFM development.