TBXT dose sensitivity and the decoupling of mesoderm specification from EMT progression in 2D human gastruloids

In the nascent mesoderm, levels of Brachyury (TBXT) expression must be precisely regulated to ensure cells exit the primitive streak and pattern the anterior-posterior axis, but how this varying dosage informs morphogenesis is not well understood. In this study, we define the transcriptional consequences of TBXT dose during early gastrulation using human induced pluripotent stem cell (hiPSC)-based models of early gastrulation and mesoderm differentiation. Multiomic single-nucleus RNA and single-nucleus ATAC sequencing reveal that TBXT is required for the temporal progression of epithelial-to-mesenchymal transition (EMT) in a dose-dependent manner and that this transition occurs independently of the acquisition of mesodermal identity. These results demonstrate that EMT can be decoupled from mesoderm development in the early gastrula and shed light on the mechanisms underlying human embryogenesis. Overall design: 2D gastruloids derived from WT, TBXT-Het, or TBXT-KO human iPSCs were isolated after 48hrs of BMP4 treatment for multiomic snRNA-seq and snATAC-seq. Nuclei from two bioreplicates of about 288 2D gastruloids per genotype per replicate were sequenced.