Tbx1stabilizes differentiation of the cardiopharyngeal mesoderm and drives morphogenesis in the pharyngeal apparatus

Abstract

ABSTRACTBackgroundTBX1 is required for the development of the pharyngeal apparatus. In the mouse, fish, and ascidian,Tbx1is a marker of cardiopharyngeal mesoderm (CPM), a cell population that provides progenitors to the heart and branchiomeric muscles. However, in mammals: a) the molecular cascade that drives the diversification of this multipotent cell population, and b) the role ofTbx1therein, are not well defined.Material and methodsWe usedin vitrodifferentiation of WT andTbx1−/−mouse embryonic stem cells into precardiac mesoderm, and performed single cell RNA-seq and ATAC-seq at two differentiation stages. We then used WT andTbx1−/−mouse embryos forin vivovalidation of the key findings.Results and conclusionsWe found that the response to loss of TBX1 is cell sub-population-specific, both in terms of gene expression and chromatin remodeling. We show thatTbx1regulates chromatin accessibility and gene expression of an ancient transcriptional module that orchestrates the development of the trunk, pharynx and heart across evolution. This module is co-regulated and includes genes encoding the conserved transcription factor families of Tea Shirt (Tshz), Sine Oculis (Six), Eye absent (Eya), and Ebf/Collier. Analysis of putative regulatory regions of these genes, which were selected using a machine-learning computational procedure, predicted a feed-forward regulatory relationship between TBX1 and SIX factors that drives or stabilizes the module. Most surprisingly, we found a drift in the differentiation trajectory of theTbx1mutant CPM that led to a relative expansion of cells with epithelial-like transcriptional features in the cell culture model and in mouse embryos. We conclude that TBX1 is a critical factor for maintaining the transcriptional profile of the CPM.