Fig. 5: YAP1 mechanotransduction-mediated WLS activation.

a, Images show cross-sections of organoids stained with antibodies labelling YAP1 (day 16) and WLS (day 15) from Matrigel and no-matrix conditions. Scale bars, 100 µm. b, Violin plots showing protein expression distribution of nuclear YAP1 (day 16) and cytoplasmic WLS (day 15) from Matrigel and no-matrix conditions. *P < 0.05, calculated using a Wilcoxon rank-sum test (two-sided) between conditions corrected for multiple testing using the Benjamini–Hochberg method. The boxes of the violin plots show the interquartile range, the line at the centre is the median and the whiskers extend to the data range excluding outliers. For P values, see Supplementary Table 13. c, Schematic showing the developing brain with distinct regions along the rostrocaudal axis: prosencephalon (telencephalon (Tel.) + diencephalon (Die.)), mesencephalon (Mes.) and rhombencephalon (Rh.). The dotted lines show coronal sections to illustrate lumen (brain ventricle) size differences. A schematic summarizing the morphological distinctions between Matrigel and no-matrix organoids with corresponding YAP1 and WLS expression differences is also shown (right). d, Signal tracks of bulk CUT&Tag sequencing data showing the enrichment intensity of YAP1 binding to the WLS gene, profiled with two different YAP1 antibodies. Tracks are shown for IgG and Tn5 control and the repressive and active marks profiled with H3K27me3, H3K4me3 and H3K9ac antibodies. Chr. 1, chromosome 1. e, Schematic of the light-sheet imaging and scRNA-seq experiment with control and YAP1 activator-treated organoids (top). EBs were cultured in NIM with Matrigel embedding on day 4. YAP1 activator (Py-60) or DMSO (control) was added to the imaging sub-chamber on day 5 or day 7. Imaging was terminated on day 10, and corresponding organoids from all three conditions were profiled with scRNA-seq on day 10. UMAP embeddings of scRNA-seq data from day 10 organoids in control and YAP1 treatment conditions are also shown (bottom left). A stacked barplot showing the cluster proportion of each cell population is also shown (bottom right). *P < 0.05, calculated using a Fisher’s exact test between the cluster proportions of the control and day 5-treated or day 7-treated conditions corrected for multiple testing using the Benjamini–Hochberg method. For P values, see Supplementary Table 13. The number of cells recovered after pre-processing of the scRNA-seq experiment: n = 2,085 for control, n = 763 for Py-60 on day 5 and n = 1,955 for Py-60 on day 7. f, Dotplot showing average expression and percentage of cells expressing selected regional marker genes per cell population. g, Maximum intensity projections (left) and cross-sections (right) at day 8, showing control organoids and YAP1 activator (given on day 5) treated organoids imaged with light-sheet microscopy. Sparse and multi-mosaic organoids contain cells labelled with nuclear membrane (lamin, RFP in orange), actin (GFP in cyan) and tubulin (RFP in orange) and unlabelled cells. Scale bars, 100 µm. Organoids imaged per condition, n = 4. h, Schematic of the scRNA-seq experiment with organoids generated from control and WLS-knockout (WLS-KO) iPS cell lines with five treatments. EBs were cultured in Matrigel or no-matrix conditions starting at day 4. WNT (CHIR99021) or YAP1 (Py-60) activators were added to a subset of organoids cultured with Matrigel from day 10 to day 12 and day 10 to day 16, respectively. Organoids were hashed and profiled with scRNA-seq on day 55. i, UMAP embeddings of scRNA-seq data coloured by cell population (top), genetic status (bottom left) or condition (bottom right). Mes./rhomb., mesencephalon or rhombencephalon. j, Stacked barplot showing the cluster proportion of each cell population in the different treatment conditions. k, Dotplot showing the average expression and the percentage of cells expressing selected regional marker genes per cell populations.