YAP levels regulate anteroposterior elongation of hESC-derived gastruloids

Abstract

Human embryonic stem cells (hESCs) can self-organize into anteroposterior patterned three-dimensional structures, characterized by polarized expression of CDX2 and GATA6, known as human 3D gastruloids (h3D-gastruloids). This patterning emerges through hESC-intrinsic mechanisms that remain poorly understood. Here, we demonstrate that the formation and elongation of h3D-gastruloids is modulated by the activity levels of the Hippo pathway effector YAP. Using complementary chemical and genetic perturbations, we show that elevated nuclear YAP activity inhibits gastruloid elongation, whereas YAP inhibition enhances axial elongation relative to controls. Single-cell RNA sequencing (scRNA-seq) analyses reveal that YAP activation disrupts the establishment of distinct anterior GATA6 and posterior CDX2 poles. Conversely, YAP inhibition promotes clearer segregation of these domains, facilitating symmetry breaking and elongation. Finally, we developed a high-throughput platform for h3D-gastruloid generation, enabling the production of over 500 gastruloids per well. Together, our findings uncover a role for YAP in orchestrating the three-dimensional organization of human gastruloids.