A whole-organism approach to uncover gene regulatory mechanisms in Drosophila melanogaster: towards a regulatory map at single-cell resolution

Single-cell transcriptomics and single-cell epigenomics provide a powerful means of cataloging known cell types, discovering novel ones, and deciphering their underlying regulatory principles. Here, we propose to generate a single-cell multi-omics atlas, combining scRNA-seq and scATAC-seq, across the whole adult fruit fly Drosophila melanogaster. The scATAC-seq atlas will be annotated using bridge integration with the previously published, and fully annotated Fly Cell Atlas. We will then use this multi-ome atlas to infer gene regulatory networks at the whole-organism level, possibly with the focus on the fly brain, using the recently developed method SCENIC+. Based on these predictions we will study transcription factor and enhancer pleiotropy; we will determine the optimal combinations of TF per cell type; and we will study network motifs (e.g., feedback and feedforward loops) and gene repression. Importantly, we will combine our computational predictions with genetic experiments in an iterative fashion, leading both to new biological insight and better computational models. For the in vivo validations, we will take advantage of the advanced fly genetics toolbox. This will include the generation of transgenic flies for enhancer-reporter assays, and the use of CRISPR-Cas9 to study the effect of enhancer and TF mutations.