Extended Data Fig. 3: Characterization of the fetal small intestinal epithelium.
a, t-distributed stochastic neighbor embedding (t-SNE) plots from scRNA-seq of epithelial cells from the proximal small intestine showing expression of intestinal stem-cell (Lgr5) and differentiation markers (Muc2, Lyz1, ChgA and Alpi). Darker colour indicates higher normalized gene expression. Each dot represents a cell; a total of 3,509 cells is shown. b, Detection of differentiation markers in E16.5 and adult small intestine. Tissue is counterstained with haematoxylin and eosin. Scale bar, 250 μm. Samples from n = 3 mice were analysed at each time point and representative images are shown. c, Cartoon depicting that adult villi are transcriptionally zonated in five regions numbered from bottom to top (z, zone). d, t-SNE plots showing enrichment of villi clusters in the scRNA-seq from E16.5 small intestine. e, t-SNE plot showing the enrichment of a proliferation signature in specific cell populations (left) and fraction of cells in each subpopulation scoring positive for the proliferation gene signature (right). A darker colour in the t-SNE plot indicates higher expression levels of the proliferation gene signature. f, t-SNE plot showing expression of keratin 19 (Krt19). Darker colour indicates higher normalized gene expression levels. g, Detection of KRT19 (red) and GFP (Lgr5-DTR-eGFP) at E16.5 in proximal small intestine. Tissue is counterstained with DAPI (cyan). Scale bars, 50 μm. Samples from three mice were analysed and a representative image is shown. h, Detection of KRT19 (red) at different time points in tissue from the small intestine. Tissue is counterstained with DAPI (cyan). Scale bars, 50 μm. Samples from three mice were analysed per time point and representative images are shown. i, Relative volume (projected) of KRT19 clones and epithelium based on E-cadherin. E-cadherin is also shown in Fig. 1c and KRT19 clones are also shown in Fig. 2. KRT19 clones, number of biologically independent samples: P0 n = 1; P5 n = 3, P11 n = 6, adult n = 3; E-cadherin, number of biologically independent samples: P0 n = 9, P5 n = 9, P11 n = 9, adult n = 9. j, Quantification of the localization of labelled clones at P0 following administration of 4-hydroxytamoxifen at E16.5 in Rosa26-lsl-Confetti;Krt19-creERT mice. Villi containing clones were divided in three equal regions (T, top; M, middle; B, bottom) based on the z projection in 3D to determine where clones were located at P0 (n = 27). k, Detection of GFP (green) and RFP (red) in whole mounts from the proximal part of the small intestine isolated from mT/mG Krt19-creERT mice at P0 following induction at E16.5. A representative image of n = 3 biologically independent samples is shown. Scale bars, 25 μm. l, Apoptotic cells were detected by cleaved caspase-3. Arrowheads demarcate positive cells in inserts. Samples from three mice were analysed per time point and representative pictures are shown. Scale bars, 250 μm.
