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Abstract

Gene regulatory networks control the precise spatial and temporal execution of developmental gene expression programs during embryogenesis. Enhancers are core components of these regulatory networks and they serve as integration platforms for the developmental signals delivered by transcriptions factors (TFs). Dissecting the function of gene regulatory networks and their underlying components is a major goal of modern biology. In this thesis, I leverage the recent advances in single-cell genomics methods to assess the impact of perturbations on developmental regulatory networks, using Drosophila melanogaster embryogenesis as a well-studied model system.

I started by optimizing a protocol to profile single-cell chromatin accessibility by sci- ATAC-seq in Drosophila embryos and applied it to profile a dense time-course of over 20,000 cells during mesoderm development. The time-course comprised eight overlapping time-points spanning half of embryogenesis and captured a continuum of regulatory transitions as cells move from multipotency to different developmental lineages. I used this dataset to reconstruct developmental trajectories of all major celltypes, and uncover both the TFs and enhancers involved.

I then present two approaches that integrate the single-cell resolution of sci-ATAC-seq with perturbations of TFs and enhancers, as a means to dissect regulatory networks. First, I perturbed regulatory networks in trans, by mutating four key developmental TFs that drive Drosophila mesoderm development and used sci-ATAC-seq to jointly assess the regulatory outcome at both the cellular and molecular level. I demonstrate that this approach not only recovers previously described high-level phenotypes, but also more subtle alterations in cell fate, while simultaneously providing information on the TF’s input at enhancers. Second, I perturbed regulatory networks in cis by exploiting natural sequence variation as a means for large-scale enhancer disruption. By profiling chromatin accessibility in hybrid embryos obtained from mating genetically diverse parent lines, I show how sci-ATAC-seq can be used to discover the cellular context affected by genetic variants and I discuss upcoming in vivo experiments to assess their impact on enhancer activity.