Modeling Gene Regulatory Networks for Early Cardiopharyngeal Development

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Congenital heart defects affect ~1% of newborn infants and are often associated with craniofacial defects as is the case in the DiGeorge/CardioVeloFacial syndrome (DGS/CVFS). The latter is often caused by spontaneous de novo 22q11.2 deletions, which result in TBX1 haploinsufficiency, a major cause of the disease. TBX1 is thought to function in multipotent progenitors for the second heart field and branchiomeric/pharyngeal head muscles in early embryos, and thus combined cardiac and craniofacial malformations emerge from early defects in the cardiopharyngeal mesoderm. The existence of multiple interacting genetic modifiers of DGS penetrance hints at the complex gene regulatory network (GRN) controlling early cardiopharyngeal development. Despite progress in identifying key genetic determinants, the relative complexity of vertebrate embryos has hindered progress in modeling cardiopharyngeal networks. The tunicate Ciona is a tractable model where early cardiopharyngeal development can be studied with unprecedented spatial and temporal resolution in chordates, using functional genomics methods. In previous studies, comprehensive gene expression and chromatin accessibility profiles were obtained by lineage-specific whole genome assays, including single cell RNA-seq (scRNA-seq) and ATAC-seq. A method combining sample barcoding, CRISPR/Cas9-mediated mutagenesis, and scRNA-seq was developed to systematically interrogate the function of coding and non-coding genetic elements using high-content scRNA-seq assays. First, this approach will be used to profile loss-of-function perturbations for candidate transcription regulators expressed in the cardiopharyngeal lineage. Ciona and available mouse datasets will be integrated into cross-species models to jointly learn conserved and divergent features of cardiopharyngeal GRNs. Next, perturbations of accessible non-coding elements for selected transcription regulators (plus high-content scRNA-seq assays and new computational methods) will permit the explicit integration of non-coding elements in the context of our GRN models. Finally, perturbing regulators of cardiopharyngeal-specific chromatin accessibility followed by lineage-specific ATAC-seq will further disentangle the impact of transcription regulators on accessibility vs. activity. Integrating these datasets into lasting and evolving GRN models will support comprehensive understanding of early cardiopharyngeal development and the etiology of congenital cardio-craniofacial syndromes.