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

肌肉的生长和发展是一个精心协调的过程，它导致形成一个能够执行其特定功能的平衡和功能肌肉体系。在果蝇胚胎和幼虫中，每一腹部节段有30种不同的肌肉纤维组成的重复模式。虽然这些肌肉纤维有相似之处，但在大小、形状、方向、核数量、神经支配和肌腱附着位置上各有不同。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，多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不同的。然而，对于控制不同肌肉之间以及单个肌肉纤维内细胞核之间差异的分子机制仍然知之甚少。我们使用了果蝇胚胎肌肉的单细胞RNA测序，并鉴定了在单个肌肉中表达的HD、Tf和免疫球蛋白(Ig)细胞表面分子的特定组合。我们的数据表明，免疫球蛋白和其他实现基因可能受HD转录因子的控制，以调节它们的特定功能，如与其他类型细胞的相互作用。然而，目前还不清楚HD和其他活跃在个体肌肉中的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活跃在单个肌肉核中的基因调控网络(GRN)。为此，我们将开发跨异质细胞群体的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和其他转录因子对靶基因调控的贡献，并将仔细描述一个肌肉调控区域，以推断体内GRN活动的关键特征。我们的结果将展示高度保守的HD和其他TF如何在单细胞中以最高的精度控制肌肉属性，并为系统地破译活跃在单个肌肉中的调控程序提供新的资源。此外，单核测序方法将使我们能够剖析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。总而言之，我们的结果将对理解特定的TF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.