YAP1 Regulates the Self-organized Fate Patterning of hESCs-Derived Gastruloids

During gastrulation, the coordinated activity of BMP, WNT and NODAL signaling pathways guide the differentiation of the pluripotent epiblast into the three germinal layers. Recent studies underline the role of the Hippo-effector YAP1 regulating WNT and NODAL signaling pathways and repressing mesoendodermal differentiation in human embryonic stem cells (hESCs). However, the contribution of YAP1 to the cell-fate patterning decisions that transform the epiblast in a three-germ layer gastrula remains unknown. We address this question by analyzing micropatterned 2D-gastruloids derived from hESCs, in the presence and absence of YAP1. Our findings show that YAP1 is necessary for gastrulation. YAP1 KO-gastruloids display reduced ectoderm layer and enlarged mesoderm and endoderm layers, compared to WT. Furthermore, YAP1 regulates the self-organized patterning of the hESCs, as the discrete position of the three germ layers is altered in the YAP1 KO-gastruloids. Our epigenome (single-nuclei ATACseq) and transcriptome (RNA-seq) analysis revealed that YAP1 directly represses the chromatin accessibility and transcription of key genes in the NODAL pathway, including the NODAL and FOXH1 genes. In WT gastruloids, a gradient of NODAL: SMAD2.3 signaling from the periphery to the center of the colony regulates the exit of pluripotency toward endoderm, mesoderm and ectoderm, respectively. Hence, in the absence of YAP1, a hyperactive NODAL signaling retains SMAD2.3 in the nuclei impeding the self-organized differentiation of hESCs. Accordingly, the partial inhibition of NODAL signaling is sufficient to rescue the differentiation and pattern -defective phenotypes of the YAP1 KO gastruloids. Our work revealed that YAP1 is a master regulator of NODAL signaling, essential to instruct germ layer fate patterning in human gastruloids.