Tbx1 stabilizes differentiation of the cardiopharyngeal mesoderm and drives morphogenesis in the pharyngeal apparatus
Lanzetta, Olga
;
Bilio, Marchesa
;
Liebig, Johannes
;
Jechow, Katharina
;
Wei Ten, Foo
;
Ferrentino, Rosa
;
Aurigemma, Ilaria
;
Illingworth, Elizabeth
;
Conrad, Christian
;
Lukassen, Soeren
;
Angelini, Claudia
;
Baldini, Antonio
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.
TBX1 is a key regulator of pharyngeal apparatus (PhAp) development. Vitamin B12 (vB12) treatment partially rescues aortic arch patterning defects of Tbx1+/- embryos. Here, we show that it also improves cardiac outflow tract septation and branchiomeric muscle anomalies of Tbx1 hypomorphic mutants. At the molecular level, in vivo vB12 treatment enabled us to identify genes that were dysregulated by Tbx1 haploinsufficiency and rescued by treatment. We found that SNAI2, also known as SLUG, encoded by the rescued gene Snai2, identified a population of mesodermal cells that was partially overlapping with, but distinct from, ISL1+ and TBX1+ populations. In addition, SNAI2+ cells were mislocalized and had a greater tendency to aggregate in Tbx1+/- and Tbx1-/- embryos, and vB12 treatment restored cellular distribution. Adjacent neural crest-derived mesenchymal cells, which do not express TBX1, were also affected, showing enhanced segregation from cardiopharyngeal mesodermal cells. We propose that TBX1 regulates cell distribution in the core mesoderm and the arrangement of multiple lineages within the PhAp.
