Fig. 5. Activation of YAP induces fetal-like features in aOrg cultures.

(A) Left, representative images of TetON-YAP aOrg cultures in ENR medium with the absence or presence of DOX (±DOX). Right, bar plot depicting quantification of organoid circularity. Scale bar, 200 μm. (B) Analysis of SCA1 protein expression by flow cytometry in tetON-YAP aOrg cultures. (C) Volcano plot showing differentially expressed genes upon DOX treatment. Representative adult- and fetal-specific genes are highlighted in blue and red, respectively. Representative known YAP target genes are highlighted in yellow. (D) Principal components analysis (PCA) from RNA-seq profiling data from FEnS and TetON-YAP aOrg (±DOX) cultures in ENR medium. PCA was carried out on the basis of the top 1% most variable genes (i.e., 137) across all samples. (E) Venn plot depicting fraction of shared up-regulated genes in FEnS (versus aOrg −DOX) and aOrg +DOX (versus aOrg −DOX) cultures. (F) GSEA showing enrichment of FEnS-associated gene signature upon YAP induction (+DOX). (G) Uniform Manifold Approximation and Projection (UMAP) visualization of single cells from FEnS and aOrg cultures colored by sample (left) or cell type (right). Cell-type clusters were annotated on the basis of expression of known marker genes. (H) Average expression of YAP-associated gene signature overlaid on the UMAP cell-type plot. (h) Violin plot depicting enrichment of the YAP signature in each cell type cluster. See Materials and Methods and associated manuscript for further details on the single-cell RNA analyses in (G) and (H).