Extended Data Fig. 9: Differentiated progeny transition through CLU+ cells to produce de novo LGR5+ CBCs. | Nature

Extended Data Fig. 9: Differentiated progeny transition through CLU+ cells to produce de novo LGR5+ CBCs.

From: Single-cell transcriptomes of the regenerating intestine reveal a revival stem cell

Extended Data Fig. 9

a, Gene correlation matrix for the indicated genes was calculated from the crypt scRNA-seq data (left) and used to generate a GCN (middle) with genes as nodes (labelled and coloured according to legend on the left) and correlations as edge colours (scale). The minimum spanning tree of the GCN is shown on the right. b, Illustration of experimental strategy and interpretation. (see Methods for detailed discussion). c, Related to b (subpanel (ii); top right), representative images of small intestines treated as indicated and co-stained for tdTomato (red) and GFP (green) (left: yellow arrows show crypts expressing Lgr5-GFP and tdTomato, and white arrows show crypts expressing Lgr5-GFP but not expressing tdTomato). Right, quantification of green and yellow crypt frequency (n = 32 crypts across two mice each, two-tailed Mann–Whitney test; data are mean ± s.e.m., two independent experiments performed with similar results). d, Related to b (subpanel (iii); bottom right), representative images of small intestines co-stained for tdTomato (red) and GFP (green) and labelled as in c. Flow cytometry plots to quantify co-expression are shown on the right. Note that tracing LGR5+ cells and their offspring at 7 and 6 dpt shows 98% of Lgr5-GFP+ cells also express tdTomato. Each data point represents a mouse; n = 18 mice analysed; two-tailed Mann–Whitney test; box plot shows the median, box edges represent the first and third quartiles, and the whiskers show minimum and maximum values; pooled data from two biologically independent experiments with similar results are shown. e, Representative images of Lgr5GFP-iDTR intestines stained for Lgr5-GFP (green), cleaved-caspase-3 (red) and DAPI (blue), 6 h after DT treatment, as indicated. White arrows show dying cells losing GFP expression and gaining cleaved caspase-3 activity (n = 2 mice). f, Experimental scheme for single versus continuous ablation (left). Cells expressing tdTomato (white arrowheads) or endogenous Clu (yellow arrows) were identified using smFISH (RNAScope) (n = 3 mice). Scale bars, 70 μm (c, d); 30 μm (e, f).

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