Deciphering Heterogeneity in Drosophila multi-nucleated Muscles by decoding Gene Regulatory Networks active in Single Muscle Nuclei

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The growth and development of muscles is a carefully coordinated process that leads to the formation of a balanced and functional musculature capable of performing its specific functions. In Drosophila embryos and larvae, there is a repeating pattern of 30 different muscle fibers in each abdominal segment. Although these muscle fibers share similarities, each differs in size, shape, orientation, number of nuclei, innervation, and tendon attachment sites. This diversity of muscle fibers is controlled by muscle identity genes that frequently encode homeodomain (HD) transcription factors (TFs) expressed in subsets of muscle precursors. Moreover, it has become increasingly clear over the years that not only are individual muscles diverse, but also nuclei within multinucleated muscles are heterogeneous. However, much is still unknown about the molecular mechanisms by which diversity between different muscles as well as between nuclei within individual muscle fibers is controlled. We have used single-cell RNA sequencing of Drosophila embryonic muscles and identified specific combinations of HD TF and immunoglobulin (Ig) cell surface molecules expressed in individual muscles. Our data suggest that Ig as well as other realizer genes may be controlled by HD TFs to mediate their specific functions like the interaction with other cell types. However, it is unclear whether and how HD and other TFs active in individual muscles can precisely control transcriptional programs. To address these issues, we will apply multi-omic approaches to identify gene regulatory networks (GRNs) active in individual muscle nuclei. To this end, we will develop computational methods for GRN inference across heterogeneous cell populations. We will use this information to establish tools to study the contribution of HD and other TFs to target gene regulation and will carefully characterize one muscle regulatory region to deduce features critical for GRN activity in vivo. Our results will demonstrate how highly conserved HD and other TFs control muscle properties with utmost precision in single cells, and provide novel resources for systematically deciphering the regulatory programs active in single muscles. In addition, the single nucleus sequencing approach will allow us to dissect the heterogeneity of nuclei within individual muscles and thus provide a starting point for elucidating the underlying mechanisms of sub-functionalization within individual muscles. In sum, our results will be critical in understanding how specific combinations of TFs control muscle diversity and sub-functionalization, which will be highly relevant for other cell types and organisms.