Fig. 2: Extrinsic ECM affects brain organoid morphogenesis.

a, UMAP embedding of scRNA-seq data with cells coloured by diffusion component ranking. b, Density plots showing cell distributions arranged in a pseudotime prediction (diffusion component 1 (DC1)) for time point (left) and cell type (right) labels. c, Heatmap showing normalized gene expression over DC1 ranking. d, Top DAVID Gene Ontology analysis terms calculated for genes that change over pseudotime from day 5 to day 11. A Fisher’s exact test was used to assess the significance of enrichment. e, Feature plots showing normalized expression of example ECM-related genes that show an increase in expression over time. f, Schematic representation of the extracellular microenvironment and the corresponding brightfield image for organoids grown with Matrigel (extrinsic ECM), without any external embedding (no-matrix) and with a low-melting agarose embedding (diffusion barrier). N = 3, n = 4 organoids. Scale bars, 100 µm. g, Images show cross-sections of sparse and multi-mosaic organoids containing cells labelled with nuclear membrane (lamin, RFP in orange), actin (GFP in cyan), tubulin (RFP in orange) and unlabelled cells from a light-sheet imaging experiment where organoids were embedded in Matrigel (n = 4), no-matrix (n = 8) or 0.6% agarose diffusion barrier (n = 4). The dashed lines outline the lumen. Scale bars, 100 µm. h, 3D renderings of segmented lumen in the organoids shown in panel g, colour coded for lumen axis measurements. i, Graphs showing total lumen number (top) measured per day from day 4 to day 9 for all imaged organoids, and total volume of all lumen (bottom) over time. j, Graph showing the number of lumen fusions over time. The shading indicates standard deviation and the centre line denotes the mean (i,j).