Tbx16 and mesogenin 1 promote presomitic mesoderm differentiation by repressing the mesodermal progenitor cell state.

During zebrafish embryonic body elongation, differentiation of mesodermal progenitors into presomitic mesoderm requires the transcription factors tbx16 and mesogenin 1. Here, by using temporally controlled tbx16 and mesogenin 1 overexpression and RNAseq to identify immediate downstream changes in gene expression, we elucidate how these genes promote presomitic mesoderm differentiation. Using machine learning and game theory, we integrated differentially expressed genes with wild-type scRNAseq data and identified genes downstream of tbx16 and mesogenin 1 during mesoderm differentiation. This data-driven analysis indicates that mesogenin 1 and tbx16 primarily repress expression of genes as mesodermal progenitors differentiate. Strikingly, the genes that are most important for defining transcriptional cell states during mesoderm differentiation are most strongly repressed by tbx16 and mesogenin 1. Moreover, these downstream effectors are enriched for genes with known roles in mesoderm development and body elongation such as Fgf, Wnt and Bmp pathways and the transcription factors tbxta, eve1, hoxd12a, hoxd13b, lef1, cdx4, tbx16l, ved, vent and vox. Gradients of Fgf and Wnt specify the mesodermal progenitor state in the posterior tailbud and activate many of these transcription factors indicating that tbx16 and mesogenin 1 promote mesoderm differentiation by repressing this progenitor state.