Fig 1: Thbs1 is expressed by Lgr5+ cancer stem cells in vivo and induces YAP activation in neighbouring epithelial cells.(A, C) Representative section of Apc mutant intestinal tumours analysed by single-molecule fluorescence in situ hybridisation (smFISH) for Thbs1 (pThbs1, red dots) and Lgr5 (pLgr5, green dots in A) or the YAP target Sca1 (pSca1, green dots in C). Examples of segmented and processed region of interest (ROI) that were automatically counted as co-localisation (Thbs1+/Lgr5+ in A or Thbs1+/Sca1+ cells in C outlined in yellow and indicated by yellow arrows) or single-probe expression (outlined in red or green and indicated by arrows of the corresponding colour) are shown. E-cadherin demarcates epithelial cells in white and DAPI labels nuclei in blue in (A) and (C). (B, D) Quantification of the frequency of tumour regions expressing exclusively one probe or co-expressing two probes (yellow): Thbs1 only in red or Lgr5 only in green (B); Thbs1 only in red or Sca1 only in green (D). The observed frequencies of co-localisation (yellow in B) or mutual exclusion (yellow in D) are statistically significant compared to the calculated probability of random co-expression (blue columns) (n = 22 sections from two tumours in B and n = 51 sections from five tumours in D). (E) Correlation of the number of RNA molecules (dots/mm²) detected by single-molecule RNA fluorescence in situ hybridisation (smRNA FISH) for the YAP target CTGF and Thbs1 in mouse intestinal tumours. Red dots indicate large tumours (= 8 mm), orange dots small tumours (<8 mm). Dashed lines indicate 95% confidence intervals. (F, G) Representative sections of tumours derived from VillinCreERT2;Apcflox/+ (Apc+/- in F) or VillinCreERT2;Apcflox/flox (Apc-/- in G) immunostained for YAP1 (in red) and ß-catenin (in green). Wildtype (WT) glands displaying membrane-bound ß-catenin, adjacent to mutant areas presenting diffuse cytoplasmic/nuclear ß-catenin expression are demarcated by dashed lines. White arrows indicate examples of cells showing high levels of nuclear YAP. Scale bars = 50 µm and 10 µm in insets. Statistical analysis was performed with Wilcoxon test in (B–D) and linear regression test with 95% confidence in (E). Figure 5—source data 1.Source data related to Figure 5B. Figure 5—source data 2.Source data related to Figure 5D. Figure 5—source data 3.Source data related to Figure 5E.