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

肌肉的生长和发育是一个精心协调的过程，导致形成一个平衡和功能性的肌肉组织，能够执行其特定的功能。在果蝇的胚胎和幼虫中，在每个腹部节中有30种不同的肌肉纤维的重复模式。虽然这些肌纤维有相似之处，但在大小、形状、方向、核数、神经支配和肌腱附着部位上各不相同。肌肉纤维的这种多样性是由肌肉识别基因控制的，这些基因经常编码在肌肉前体亚群中表达的同源结构域（HD）转录因子（tf）。此外，近年来越来越清楚的是，不仅单个肌肉是多样化的，而且多核肌肉中的细胞核也是异质的。然而，关于不同肌肉之间以及单个肌纤维内细胞核之间的多样性是如何被控制的分子机制，我们仍然知之甚少。我们使用果蝇胚胎肌肉的单细胞RNA测序，鉴定了HD TF和免疫球蛋白（Ig）细胞表面分子在单个肌肉中表达的特定组合。我们的数据表明，Ig以及其他实现基因可能由HD tf控制，以介导其特定功能，如与其他细胞类型的相互作用。然而，目前尚不清楚HD和其他在个别肌肉中活跃的tf是否以及如何精确地控制转录程序。为了解决这些问题，我们将应用多组学方法来识别在单个肌肉核中活跃的基因调控网络（grn）。为此，我们将开发跨异质细胞群的GRN推断的计算方法。我们将利用这些信息建立工具来研究HD和其他tf对靶基因调控的贡献，并将仔细表征一个肌肉调控区域，以推断体内GRN活性的关键特征。我们的研究结果将展示高度保守的HD和其他tf如何在单个细胞中以最高的精度控制肌肉特性，并为系统地破译单个肌肉中活跃的调节程序提供新的资源。此外，单核测序方法将使我们能够解剖单个肌肉内细胞核的异质性，从而为阐明单个肌肉内亚功能化的潜在机制提供一个起点。总之，我们的结果对于理解tf的特定组合如何控制肌肉多样性和亚功能化至关重要，这将与其他细胞类型和生物体高度相关。

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.