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

In the nascent mesoderm, Brachyury (TBXT) expression must be precisely regulated to ensure cells exit the primitive streak and pattern the anterior-posterior axis, but how varying dosage informs morphogenesis is not well understood. In this study, we define the transcriptional consequences of TBXT dosage reduction during early human gastrulation using human induced pluripotent stem cell (hiPSC) models of gastrulation and mesoderm differentiation. Multiomic single-nucleus RNA and single-nucleus ATAC sequencing of 2D gastruloids comprised of WT, TBXT heterozygous (TBXT-Het), or TBXT null (TBXT-KO) hiPSCs reveal that varying TBXT dosage does not compromise a cell's ability to differentiate into nascent mesoderm, but instead directly influences the temporal progression of the epithelial to mesenchymal transition (EMT) with WT transitioning first, followed by TBXT-Het, and then TBXT-KO. By differentiating cells into nascent mesoderm in a monolayer format, we further illustrate that TBXT dosage directly impacts the persistence of junctional proteins and cell-cell adhesions. These results demonstrate that EMT progression can be decoupled from the acquisition of mesodermal identity in the early gastrula and shed light on the mechanisms underlying human embryogenesis.