Fig. 4: Multiplexed immunohistochemistry (4i) reveals spatial region emergence in organoid development.

a, Overview of the 4i data acquisition pipeline. Organoids from a timecourse were fixed and sectioned followed by mounting on a glass coverslip. b, Image showing an example organoid section with segmented compartments (extracellular, cytoplasmic and nuclear) used for downstream quantitative analysis. c, Selected images showing protein stainings on day 21 on organoid slices in Matrigel (n = 4) and no-matrix (n = 3) conditions. d, UMAP embedding based on the combined cellular (nuclear + cytoplasmic) protein expression, with each dot representing a cell, clustered and annotated as distinct cell types. e, Example organoids (day 21) from each condition (Matrigel and no-matrix) with cell clusters projected back to the image. f, UMAP embedding based on the combined protein expression in the extracellular compartment showing individual clusters. g, Example organoids (day 21) from each condition (Matrigel and no-matrix) with extracellular cell clusters projected back to the image. h, Stacked barplot showing the cluster proportion of each cell population from all days in Matrigel and no-matrix conditions. *P < 0.05, calculated using a Fisher's exact test (two-sided) between the cluster proportions of the conditions corrected for multiple testing using the Benjamini–Hochberg method. For P values, see Supplementary Table 13. i, Stacked barplot showing the ECM cluster proportion per cell population. The violin plots show the major axis of the lumen that have been assigned to each cell cluster. Dienceph, diencephalic; NC, neural crest; NCC, neural crest cell; prog., progenitor; prosenceph., prosencephalic; tel., telencephalic. j, Violin plot showing the protein expression in extracellular and cellular compartments in Matrigel and no-matrix conditions. *P < 0.05, calculated using a one-way analysis of variance (ANOVA) across the timepoints for a condition corrected for multiple testing using the Benjamini–Hochberg method. n = 5,137 for Matrigel and n = 3,958 for no-matrix for the extracellular quantifications, and n = 17,140 for Matrigel and n = 16,007 for no-matrix for the cytoplasmic quantifications. 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 (i,j). For P values, see Supplementary Table 13. Scale bars, 100 µm (all panels).