Identification of epithelial label-retaining cells at the transition between the anal canal and the rectum in mice

Abstract

In certain regions of the body, transition zones exist where stratified squamous epithelia directly abut against other types of epithelia. Certain transition zones are especially prone to tumorigenesis an example being the anorectal junction, although the reason for this is not known. One possibility is that the abrupt transition of the simple columnar epithelium of the colon to the stratified squamous epithelium of the proximal portion of the anal canal may contain a unique stem cell niche. We investigated whether the anorectal region contained cells with stem cell properties relative to the adjacent epithelium. We utilized a tetracycline-regulatable histone H2B-GFP transgenic mice model, previously used to identify hair follicle stem cells, to fluorescently label slow-cycling anal epithelial cells (e.g. prospective stem cells) in combination with a panel of putative stem cell markers. We identified a population of long-term GFP label-retaining cells concentrated at the junction between the anal canal and the rectum. These cells are BrdU-retaining cells and expressed the stem cell marker CD34. Moreover, tracking the fate of the anal label-retaining cells in vivo revealed that the slow-cycling cells only gave rise to progeny of the anal epithelium. In conclusion, we identified a unique population of cells at the anorectal junction which can be separated from the other basal anal epithelial cells based upon the expression of the stem cell marker CD34 and integrin a6, and thus represent a putative anal stem cell population.

Acknowledgements

We thank Dr. James Lessard for valuable discussions, Drs. Elaine Fuchs, Pierre Coulombe and Jeffrey Whittsett for providing their antibodies. We thank Dr. Adrian McNairn for valuable criticism of the manuscript. We also thank Dr. Amalia Pasolli for her advice on the electron microscopy procedures. This work was funded by CCHMC Trustee Grant Award and part by PHS Grant P30 DK 078392 (G.G.).

Figures and Tables

Figure 1 Characterization of the adult mouse anal canal and the anorectal junction. (A) Semi-thin section stained with toluidine blue of the anorectal transitional epithelium from 4–6 weeks old CD-1 mice. The dashed line indicates the basement membrane. Like the epidermis, the anal canal is composed of a basal layer and differentiating spinous, granular and stratum corneum layers (Fig. S1A). (B) Ultrastructural analysis of the anorectal transitional epithelium. The dentate line separates the anal transition zone from the rectal epithelium (Fig. S1D). The dotted line denotes the presence of the basement membrane. Boxed areas B′ and B″ are shown below at higher magnification. (B″) Intercellular junctions between basal anal keratinocytes are rich in desmosomes (Fig. S1B and C). The anorectal junction possesses fewer desmosomes. (B″) Basal anal keratinocytes display numerous hemidesmosomes that connect with the basement membrane. TZ, transition zone; BL, basal layer; Sp, spinous layer; Gr, granular layer; Sc, stratum corneum; Dm, Desmosome; M, Mitochondria; Hm, Hemidesmosome.

Figure 2 Differentiation markers of the adult mouse anal canal and the anorectal junction. (A–D) Immunofluorescence analysis for the indicated markers. Differentiation markers of the anal canal include Keratin 6, Keratin 10, Loricrin and Filagrin. Keratin 8 marks the simple epithelium of the rectum. DAPI labels all the nuclei in blue. BL, basal layer; Gr, granular layer, Sc; stratum corneum; K6, Keratin 6; K8, Keratin 8; K10, Keratin 10; α6, α6 integrin; K5, Keratin 5.

Figure 3 In vivo detection of label-retaining cells in the anorectal junction. (A–D) Anorectal sections of pTRE-H2B-GFPxK5tTA mice before (26 days old) and after 4 week chase (56 days old). Shown are epifluorescence of H2B-GFP (green) and 4′,6″-diamidino-2-phenylindole (DAPI) (blue) and indirect immunofluorescence with indicated antibodies (Rhodamine Red). (A and B) Before the chase, all Keratin 5 expressing cells and their progeny show nuclear GFP expression in the perianal skin, hair follicles (A) and anal epithelium. (B) Note that the rectum is GFP-negative. (C and D) GFP-positive cells are retained in the anal region adjacent to the dentate line (anal transition zone). Keratin 17,58 is found expressed at the anal transition zone in adult mice. P26, 26 days old; P56, 56 days old; K17, Keratin 17. The asterisk denotes autofluorescence.

Figure 4 H2B-GFP retaining cells colocalize with BrdU-retaining cells. (A) Schematic of BrdU and doxy pulse-chase experiments. Small arrows represent ten intraperitoneal BrdU injections at the indicated time points. (B–E) Colocalization of BrdU and H2B-GFP before (B and C) and after the chase (D and E). Before the chase, at 26 days old (P26), all the epithelial cells expressed H2B-GFP and most of the cells (including the cells in the rectum) are labeled with an anti-BrdU antibody showing the efficiency of the BrdU pulse (B and C). After the chase, white arrows indicated label-retaining cells in the bulge of the perianal hair follicle (D) and in the basal layer of the anal TZ (E). (E′) Higher magnification of the anal TZ, showing colocalization of H2B-GFP and BrdU-retaining cells. SG, sebaceous gland; Bu, Bulge; TZ, transition zone; The asterisk denotes autofluorescence.

Figure 5 Expression of stem cell markers at the anorectal junction. (A) CD34 is expressed at a low level in the H2B-GFP retaining cells (LRCs). High expression of CD34 is detected in the stroma surrounding the anal region. Boxed area is magnified and shown to the right, demonstrating CD34 expression in the GFP-retaining cells. (B) p63 is expressed through the entire basal layer of the anal epithelium and the anal glands, colocalizing with CD34 in a small group of cells at the anorectal transitional zone (indicated with white arrows). Boxed area enlarged in inset, showing the expression of CD34. (C) Keratin 19 is expressed in the simple epithelium of the rectum and not in the anal transitional cells. (D and E) Sox2 is expressed in the basal cells of the anal epithelium (including the anal transition zone) and its expression decreases as cells differentiate. Sox2 and p63 are not expressed in the rectum. K19, Keratin 19; LRCs, H2B-GFP-retaining cells; α6, α6-integrin.

Figure 6 Monitoring the fate of anal H2B-GFP retaining cells in vivo. (A–D) 4 weeks chase (P56) anorectal sections overexposed for GFP and double-labeled with the indicated antibodies against each differentiation cell type, indicating that the LRCs give rise to differentiated anal epithelium. TZ, transition zone; BL, basal layer; SupraBL, suprabasal layer; K6, Keratin 6; K10, Keratin 10. The asterisk denotes autofluorescence.