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. 2022 Mar 16;32(7):670–686. doi: 10.1038/s41422-022-00643-9

Fig. 1. Lepr+ MCs surround intestinal crypts and expand upon irradiation.

Fig. 1

a Schematic diagram of MC isolation from WT intestinal crypts and capture of single cells by droplet-based device, 3ʹ-scRNA-seq, and downstream query analyses. b t-SNE plot reveals cellular heterogeneity of intestinal crypt MCs. Fourteen distinct clusters are identified and annotated (k = 7511 viable cells). The putative identity of each cell cluster is defined on the right. c Relative expression of selected cluster-specific genes shown as high-density bar charts (n = 2 genes/cluster). d Feature plots of expression distribution for Lepr. Expression levels are color-coded as in t-SNE. e Quantification of the percentage of Lepr+ cells per cluster. f, g In situ hybridization for Lepr in intestinal crypts and villi from 8-week-old mice (f, n = 3) and human (g, n = 2). Arrowheads point to Lepr+ signal in single cells. The dashed line indicates the border between intestinal epithelium and mesenchyme in crypts. Scale bar, 50 μm (f); 10 μm (g). h Fluorescence images for GFP (refers to Lepr+ cells) and td-Tomato in cross sections of jejunum and colon from Lepr-Cre;mTmG mice (n = 3). Scale bar, 200 μm. i Fluorescence images for td-Tomato (refers to Lepr+ cells) and GFP (refers to Lgr5+ cells) in intestine and colon from Lepr-Cre;td-Tomato;Lgr5-eGFP-CreERT2 mice (n = 3). Arrowheads point to Lepr+ cells. Scale bar, 25 μm. j Schematic diagram showing defined base, middle and top regions in jejunum. Graph showing quantification for Lepr+ cells that are spatially distributed at base, middle and top regions of jejunum. Lepr+ cells were counted in defined areas along the crypt–villus axis (n = 120 intestinal architectures, n = 3 mice). k Fluorescence images for GFP (refers to Lepr+ cells) and td-Tomato in cross sections of jejunum from Lepr-Cre;mTmG mice after 12 Gy of irradiation at the indicated time points. Graph represents quantification of Lepr+ cells per crypt at indicated time points. n = 3 at each time point. Scale bar, 50 μm. l In situ hybridization for Lepr in 8-week-old mice before and 3 days post-irradiation (3 dpi, n = 3). Scale bar, 50 μm. m In situ hybridization for LEPR in intestinal crypts from normal and enteritis human tissues (n = 2). Scale bar, 10 μm. Values in the graphs represent means ± SD. Unpaired Student’s t-test was used for calculating P values in j, k. **P < 0.01; ***P < 0.001.