Fig. 6. Post-wound single cell RNA sequencing reveals a TZ population enriched in the transcription factor JunB.

a Main cell populations visualized on UMAP from scRNA-seq of anal epithelial cells 1 week of post-EDTA wound b Structure of the differentiation trajectory reconstruction using the slingshot R package suggests three trajectories. Pseudotime values show an elongated distribution on each trajectory suggesting that there are more intermediate phenotypes and therefore a differentiation process. c Hierarchical relationship between clusters 4, 5, and 6 and expression of key genes expressed in TZ hybrid state cells. d In tissue, JunB (white) is specifically expressed by transition zone cells and is absent in Krt8 (red) glandular epithelium (n = 3 independent experiments from three mice). e Similar to in vivo, CD34 (red) and JunB (white) expressions are specific to TZ-derived organoids but not organoids from the rectum (n = 3 independent experiments from three TZ and rectum organoids). f One week of post-EDTA wound, few GFP+ (green) cells participating in the regenerative crypt are JunB+ (white) (n = 3 independent experiments from three mice). Insets are zoom in 1.7-fold. g TZ organoids infected with control shRNA-RFP and shJunB-RFP were FACS-sorted based on double expression of GFP and RFP (n = 3 independent FACS from three independent samples). h Brightfield and RFP images of TZ and rectum organoids infected with control shRNA or shJunB (red). JunB staining (white) revealed downregulation in TZ organoids infected with shJunB compared to control shRNA. Rectum organoids were used as negative control (n = 4 independent experiments from four independent samples). i, j JunB knockdown impairs TZ cells differentiation properties. i Western Blot analysis showing JunB downregulation in TZ organoids infected with sh2 and sh3 JunB compared to control shRNA and sh1JunB infections (n = 6 independent experiments from six biologically independent samples). Differentiation marker Krt10 expression is decreased following JunB downregulation in organoids infected with sh2 and sh3 JunB (n = 2 independent experiments from two biologically independent samples). GAPDH was used as a loading control. (Source data are provided as a source data file). j Immunofluorescence confirms the downregulation of Krt10 in sh2 and sh3 JunB organoids (n = 5 independent experiments from 5 independent samples). The defect in differentiation is also detected using antibody against Loricrin (n = 5 independent experiments from five independent samples). Scale bars are 50 µm (d, e) (TZ organoid) (f, j), and 20 µm (e) (TZ rectum).