Figure 4. Transit amplifying-like proliferating cells predominate in the absence of Lgr5⁺ cells upon HNF4 loss.

(A) WT organoids exhibit branched structures and express robust Lgr5-GFP, whereas Hnf4αɣ^DKO organoids form spherical structures and exhibit little Lgr5-GFP fluorescence (see arrows). 1 μM tamoxifen or vehicle control was added to culture medium of primary organoids on Day 2 after seeding. After passage, Hnf4αɣ^DKO organoids failed to survive (n=6 independent experiments). Scale bar, 50 μm. Tamoxifen-induced Cre-only controls were also tested and shown in Fig. S4F. (B) WT and Hnf4αɣ^DKO organoids were passaged three times, and the number of surviving cells of organoids (n=4 independent organoid cultures) was counted at each passage, from primary (P0) passage to the third passage (P3). While HNF4 double-mutant organoids grow initially as proliferative spheres, they cannot be passaged indefinetly, such as their control counterparts. (C) Co-staining of EdU and Ki67 (proliferative markers). Organoids were treated with 10 μM EdU 6 hours prior to fixation. Scale bar, 50 μm. qRT-PCR shows a significant decrease of Lgr5⁺ stem cell markers (D) but a significant increase of proliferative cell markers (E) in Hnf4αɣ^DKO organoids. 1 μM tamoxifen or vehicle control was added to culture medium of primary organoids on Day 2 after seeding. All the primary organoids were harvested at Day 6 after seeding. Data are presented as mean ± SEM (n=3 independent organoid cultures, Student’s t-test, two-sided at P < 0.001***). (F) Crypt elongation is observed in Hnf4αɣ^DKO at day 4 post tamoxifen-induced knockout as shown in Ki67 immunofluorescent staining (representative of 4 biological replicates). (G) RNA-seq data reveal an elevation of proliferative cell markers in Hnf4αɣ^DKO compared to their littermate controls (n=3 biological replicates). Statistical tests were embedded in Cuffdiff at FDR < 0.001***.