Decoding Gene Regulatory Networks in single Drosophila Motoneurons

在大多数动物中，神经回路是固定连接的，以确保功能任务的精确执行。为了确保这种精确度，神经回路的结构和连接是遗传指定的。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，仍然没有完全了解。为了解决这些重要问题，我们对果蝇运动神经元（MNs）进行了单细胞RNA测序，发现了同源结构域转录因子（HD TF）和免疫球蛋白（Ig）基因在单个MNs中表达的细胞特异性组合，并在控制分化的MNs与其靶肌肉的匹配中发挥重要作用。重要的是，我们的数据表明，Ig基因作用于HD tf的下游，介导其在突触连接中的功能。然而，HD tf是否直接控制细胞表面分子（如Igs和其他效应分子）的表达，以确定对其功能至关重要的MNs的特异性，仍未得到解决。此外，还不清楚在单个MNs中活跃的HD tf是否以及如何以细胞特异性的方式控制转录程序/子程序。其中一个问题是，尽管我们和其他研究表明，HD tf在MNs中起着重要作用，但它们在分子水平上是如何起作用的尚不清楚。这有几个原因：首先，基因组控制区，即所谓的增强子是未知的，HD和其他tf与之相互作用以控制MNs中靶基因的表达；其次，HD TF与高度相似的DNA序列相互作用，提出了这类TF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，需要对单个MNs中活跃的调控程序和对程序元件的细胞特异性操作进行系统分析。因此，在本项目的背景下，我们将应用单细胞多重混合来识别在单个MNs中活跃的基因调控网络（grn），并利用这些信息建立细胞特异性增强子，作为探索HD tf对靶基因调控的贡献的工具。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基序）对GRN活性至关重要的特征之间的相互作用。总之，我们的研究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特性，并为系统地解码单个MNs中活跃的调节程序提供宝贵的资源。由于HD tf在神经系统发育中的一般功能，我们的研究结果将对我们理解细胞特异性“同源编码”如何以最高的准确性控制神经元规范、分化和电路形成至关重要。

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.