TBX1, a T-box transcription factor, is essential for pharyngeal apparatus development and marks cardiopharyngeal mesoderm (CPM) in various species. However, in mammals, we have an incomplete knowledge of the molecular pathways driving CPM diversification and of the role of TBX1 in this context. Using CPM-relevant in vitro differentiation of wild-type and Tbx1−/− mouse embryonic stem cells, we performed simultaneous single-nucleus RNA-seq and ATAC-seq at two stages, validated findings in embryos, and found that TBX1 loss affects gene expression and chromatin remodeling in a cell subpopulationspecific manner. TBX1 regulates chromatin accessibility and gene expression of distinct and evolutionarily conserved transcriptional modules for branchiomeric and cardiac development, and for tissue morphogenesis. Computational analyses predicted a feed-forward regulatory relationship between TBX1 and SIX factors. Notably, selected Tbx1 mutant CPM cell populations showed an altered differentiation trajectory, exhibiting activation of a mesothelial-like transcriptional program. We also observed cell death later in development. Thus, TBX1 is crucial for maintaining CPM transcriptional identity.
