Extended Data Fig. 9: scRNA-seq analysis of phenotypes produced by RXRi treatment.

a, Left, tSNE maps from scRNA-seq. Each data point represents a single cell, colour-coded by sample (see key). n = 4,787, 3,570, 5,365, 4,963, 5,576 and 5,304 single cells for day 4 DMSO, RXRi, atRA and vitamin A-free, and day 1 DMSO control samples 1 and 2 respectively. Right, expression (log₂ normalized transcript counts) of indicated genes in samples of the scRNA-seq experiment (Fig. 3); sampling of n = 3,000 cells. b, Cell-type composition of scRNA-seq samples. Confidence cutoffs for cell-type assignment were set individually for each cell type on the basis of the mean expression levels of marker genes from ref. ²⁹; n = 29,565 single cells. c–e, tSNE maps of our scRNA-seq experiment, colour coded by mean normalized expression (log₂-transformed counts) of gene groups. c, Expression of cell-type signature genes from ref. ²⁹. The numbers of genes per cell-type signature are shown in individual plots. d, Expression of genes that are differentially regulated following intestinal helminth infection³⁴. e, Expression of genes with biased expression in tissues (GTEX database; https://gtexportal.org/home/). f, Left, partial tSNE maps (top) and density map (bottom) of single cells from the RXR-antagonist-treated sample of the scRNA-seq experiment. In the density map, the x- and y-axes show the mean expression (mean of log₂-transformed counts) of genes from the indicated categories. Colours show cell counts in respective bins. Red dashed lines show cutoffs used to assign populations. Right, tSNE maps of the scRNA-seq experiment, showing assigned identities in the RXR-antagonist-treated sample. g, Left, average expression of genes in populations of cells as assigned in f. Colours depict the log₂-transformed fold change (log₂FC) in the ‘early progenitor’ population compared with the ‘regenerative identity’ population. Every data point represents a gene. Centre, log₂FC values of the indicated genes. Green, absorptive-lineage-specific genes; blue, secretory-lineage-specific genes. Right, tSNE maps of the scRNA-seq experiment colour-coded by mean normalized expression (log₂-transformed counts) of genes with log₂FC values of greater than 0.5 between ‘early progenitors’ and ‘regenerative’ populations. a, b, atRA, 10 μM all-trans retinoic acid; DMSO, DMSO control; RXRi, 5 μM RXR antagonist; w/o VitA, vitamin-A-depleted medium. a–f, In tSNE maps each data point represents a single cell; sampling of 5,000 single cells. c–e, g, Ranges of colour mapping and numbers of genes per category (n) are indicated in each plot.

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