Fig. 1: HFHSD alters lineage allocation from ISCs and shifts the regional identity of cells.

a, Experimental design for FVF-based SI crypt-cell enrichment by flow cytometry and scRNA-seq. Single FVF⁺ and whole crypt cells from SI crypts of CD- and HFHSD-fed FVF mice were isolated in equal numbers by flow cytometry and combined for each sample. FVF-enriched single-cell samples were then transcriptionally profiled by scRNA-seq. b, Uniform manifold approximation and projection (UMAP) plot of 27,687 profiled single SI crypt cells. Colours highlight clustering into major intestinal cell types based on the expression of previously published marker genes. One cluster of cells could not be assigned owing to a missing marker gene signature (NA). c, Heatmap depicting scaled expression of cell-type-specific gene signatures in CD- and HFHSD-derived cells. Cells are represented in columns and genes are represented in rows. Colour bars indicate cell types assigned to both cells and genes. Selected known marker genes for every lineage are indicated. d,e, Cell-type composition differences in CD- and HFHSD-derived single-cell samples visualized by cell density projected onto the two-dimensional UMAP embedding (d) and quantified as proportions over cell types (mean ± s.e.m. of biologically independent samples, n = 3 mice per group, Dirichlet multinomial model) (e). Densities were estimated using Gaussian kernels. * Indicates a credible shift (95% highest posterior density interval of the frequency shift modelled by a Dirichlet multinomial model does not overlap 0). n = 3 CD mice, n = 3 HFHSD mice. f, UMAP coloured by the regional identities of cells in ISCs and goblet cell and enterocyte cell lineage. Subclusters were classified on the basis of expression of regional marker genes. g, Proportions of cells with proximal or distal identity in ISCs and enterocyte and goblet cell lineage (mean ± s.e.m. of biologically independent samples). n = 3 CD mice, n = 3 HFHSD mice.

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