Decoding Gene Regulatory Networks in single Drosophila Motoneurons

在大多数动物中，神经元回路通常是连线的，以确保功能任务的准确执行。为了确保这种精确度，神经回路的结构和连通性是由基因指定的。然而，这些复杂的相互关联的过程是如何在基因组中编码并由细胞蛋白质机制执行的，仍然不完全清楚。为了解决这些重要问题，我们使用了果蝇运动神经元(MNS)的单细胞RNA测序，它确定了同源域转录因子(HD Tf)和免疫球蛋白(Ig)基因在单个MN中表达的细胞特异性组合，并在控制分化的MN与其靶肌肉的匹配方面发挥了重要作用。重要的是，我们的数据表明，免疫球蛋白基因在HD TF下游发挥作用，调节它们在突触连接中的功能。然而，HD转录因子是否直接控制细胞表面分子，如免疫球蛋白和其他效应分子的表达，以确定对其功能至关重要的MN的特定性质，仍未得到解决。此外，目前还不清楚活跃在单个MN中的HD TF是否以及如何以细胞特有的方式控制转录程序/子程序。问题之一是，尽管我们和其他研究表明HD TF在MNS中发挥着重要作用，但它们如何在分子水平上发挥作用还不太清楚。这有几个原因：第一，基因组控制区，即所谓的增强子，HD和其他TF相互作用来控制MNS中的靶基因表达，是未知的；第二，HD TF与高度相似的DNA序列相互作用，提出了这个TF类如何在单个细胞中提供靶基因调控的特异性的问题。为了解决这些问题，需要对活跃在单个MN中的调控程序和对程序元素的特定细胞操作进行系统分析。因此，在这个项目的背景下，我们将应用单细胞多聚体来鉴定活跃在单个MN中的基因调控网络(GRN)，并利用这些信息来建立细胞特异性增强子，作为工具来探索HD TF对靶基因调控的贡献。此外，我们将通过解剖一个运动神经元增强子来定义顺式(HD TF基序)和反式(活性HD TF)对GRN活性至关重要的相互作用。总之，我们的结果将揭示高度保守的HD TF如何在单个细胞中以最高的精度控制运动神经元属性，并产生宝贵的资源来系统地解码单个MN中活跃的调节程序。由于HD转录因子在神经系统发育中的一般功能，我们的发现将对我们理解细胞特有的“同源码”如何以最高精度控制神经元的指定、分化和电路形成至关重要，这对生物体功能至关重要。

In most animal neuronal circuits are wired stereotypically to ensure the precise execution of functional tasks. To ensure such precision, the structure and connectivity of neural circuits is genetically specified. However, how these complex interconnected processes are encoded in the genome and executed by the cellular protein machinery is still not fully understood. To tackle these important questions, we used single-cell RNA sequencing of Drosophila motoneurons (MNs), which identified cell specific combinations of homeodomain transcription factors (HD TF) and Immunglobulin (Ig) genes to be expressed in individual MNs and to play important roles in controlling the matching of differentiated MNs and their target muscles. Importantly, our data suggested that Ig genes act downstream of HD TFs to mediate their function in synaptic wiring. However, it remains unsolved whether HD TFs directly control the expression of cell surface molecules like Igs and other effector molecules to determine the specific properties of MNs critical for their functioning. Furthermore, it is also unclear whether and how HD TFs active in individual MNs can control transcriptional programs/sub-routines in a cell specific manner. One of the problems is that although our and other studies have shown that HD TFs play an important role in MNs, it is less clear how they do so on the molecular level. There are several reasons for this: first, genomic control regions, so-called enhancers, with which HD and other TFs interact to control target gene expression in MNs, are unknown; and second, HD TFs interact with highly similar DNA sequences, raising the question how this TF class provides specificity in target gene regulation in individual cells. To tackle these problems, a systematic analysis of the regulatory programs active in single MNs and cell-specific manipulation of program elements is required. In the context of this project, we will therefore apply single cell multiomix to identify Gene Regulatory Networks (GRNs) active in single MNs and use this information to establish cell-specific enhancers as tools to probe the contribution of HD TFs to target gene regulation. Furthermore, we will define the interplay of cis (HD TF motifs) and trans (active HD TFs) features critical for GRN activity by dissecting one motoneuronal enhancer. In sum, our results will reveal how highly conserved HD TFs control motoneuronal properties with highest precision in individual cells and generate invaluable resources to systematically decode the regulatory programs active in individual MNs. Due to the general function of HD TFs in nervous system development, our findings will be critical for our understanding how cell-specific “homeo-codes” control neuronal specification, differentiation and circuit formation with highest accuracy critical for organismal function.