Fig. 7

Microenvironmental oncogenesis drives heterogenous premalignant clones in adults. a Single cells were isolated from PND16 WT and ROBO^VilCre by fractionation to enrich for crypt epithelium. scRNAseq and tSNE visualization was performed for WT and ROBO^VilCre cells. (SC: stem cell, TA: transiently amplifying cell, ENT: enterocytes that included bottom (b1: S/G2M phase and b2: G1), middle (m), and top (t), and were recently described⁴⁵, SECR: secretory Paneth and Goblet cells, IMMU: immune, ENTEND: enteroendocrine, FIBR: fibroblast). For cell type markers, see also Supplementary Fig. 14. b Heatmap visualization of cell types present in WT and ROBO^VilCre isolates as a percentage of total cells. c Heat map visualization of cell cycle phase as a percentage of each cell type. d–g ROBO^CreER/T2 (N = 9 mice, 6–15 weeks of age) were I.P. injected with 200 mg/kg of tamoxifen and sacrificed 3 days (N = 4) or 8 weeks later (N = 5), and the small intestine was vibratome sectioned and imaged by tiling confocal microscopy. Confocal imaged vibratome sections of ROBO^CreER/T2 small intestines at d 3 days or e 8 weeks post tamoxifen injection. f Nuclei were segmented and counted and normalized to the total volume imaged. Clone spread was determined by comparing the total recombined nuclei for each fate at 8 weeks relative to day 3. The asterisk denotes statistical significance by mixed effects modelling and Sidak multicomparison correction (RSPO3: <0.000001, PTPRK^e1:RSPO3^e2–5 (P1:R2-5): p = 0.31 and PTPRK^e1–7:RSPO3^e2–5 (P1-7:R2-5): p = 0.94). g Evidence for attenuated crypt fixation 8 weeks post tamoxifen injection as revealed by chimeric crypts in whole-mount confocal imaging. (SEM included for each graph). Scale bars = 100 µm in d, e, g. Source data are provided as a “Source Data file”.