Fig. 2. Single cell analysis reveals hierarchical organization of the anal TZ cells.

a Main cell populations visualized on Uniform Manifold Approximation and Projection (UMAP) dimensional reduction from scRNA sequencing of anorectal epithelial cells. Nine hundred and thirty-nine cells are visualized and colored by clustering. b–d Expression of key genes expressed in rectum, differentiated anal cells and TZ populations respectively. Darker color in the UMAP plot indicates higher expression level of the selected gene. e Expression of Krt6 and nectin-4 genes defines the suprabasal TZ population. f Immunofluorescence with Krt6 (red), nectin-4 (green) antibodies confirms that TZ population can be separated into basal (expressing the polarized α6-integrin in white) and suprabasal clusters (n = 3 independent experiments from three mice). Scale bar is 50 μm. fi, fii are zoom 2,5-fold into the TZ closed to the anal canal and the rectum respectively. g Immunofluorescence with Krt6 (red), and CD34 (white) shows that TZ basal cells closed to the rectum express the stem cell marker CD34 (n = 3 independent experiments from three mice). Scale bar is 50 μm. Boxed areas gi and gii are shown at higher magnification (2.5-fold). h Immunofluorescence with Gpc3 (red), Krt6 (green) and α6-integrin (white) shows the specific expression of Gpc3 in basal cells of the anal canal (n = 3 independent experiments from three mice). Scale bar is 50 μm. Boxed areas hi and hii are shown at higher magnification (2.5-fold). i Representation of the TZ heterogeneity depending on cell positioning. i’ Zoom into the TZ.