Decoding Gene Regulatory Networks in single Drosophila Motoneurons

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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.