Transitional basal cells at the squamous-columnar junction generate Barrett's oesophagus

Abstract

In several organ systems, the transitional zone between different types of epithelium is a hotspot for pre-neoplastic metaplasia and malignancy, but the cells of origin for these metaplastic epithelia and subsequent malignancies remain unknown. In the case of Barrett's oesophagus, intestinal metaplasia occurs at the gastro-oesophageal junction, where stratified squamous epithelium transitions into simple columnar cells. On the basis of a number of experimental models, several alternative cell types have been proposed as the source of this metaplasia but in all cases the evidence is inconclusive: no model completely mimics Barrett's oesophagus in terms of the presence of intestinal goblet cells. Here we describe a transitional columnar epithelium with distinct basal progenitor cells (p63⁺KRT5⁺KRT7⁺) at the squamous-columnar junction of the upper gastrointestinal tract in a mouse model. We use multiple models and lineage tracing strategies to show that this squamous-columnar junction basal cell population serves as a source of progenitors for the transitional epithelium. On ectopic expression of CDX2, these transitional basal progenitors differentiate into intestinal-like epithelium (including goblet cells) and thereby reproduce Barrett's metaplasia. A similar transitional columnar epithelium is present at the transitional zones of other mouse tissues (including the anorectal junction) as well as in the gastro-oesophageal junction in the human gut. Acid reflux-induced oesophagitis and the multilayered epithelium (believed to be a precursor of Barrett's oesophagus) are both characterized by the expansion of the transitional basal progenitor cells. Our findings reveal a previously unidentified transitional zone in the epithelium of the upper gastrointestinal tract and provide evidence that the p63⁺KRT5⁺KRT7⁺ basal cells in this zone are the cells of origin for multi-layered epithelium and Barrett's oesophagus.

Extended Data Figure 1. Multiple models have been proposed to explain the cell of origin for BE

a, Transdifferentiation of the stratified squamous oesophageal epithelium into Barrett’s epithelium. b, Transdifferentiation of circulating bone marrow cells into Barrett’s epithelium. c, Expansion of the oesophageal submucosal gland leads to BE. d, Stem/progenitor cells (Lgr5⁺) in the cardia mucosa differentiate into BE. e, Expansion of the quiescent residual embryonic cells (RECs) at the SCJ leads to BE formation. Note that none of the studies recapitulates the pathological changes characteristically associated with BE in humans, e.g. presence of intestinal goblet cells.

Extended Data Figure 2. Expansion of the columnar epithelium at the SCJ in Krt5-CreER;R26^Sox2-GFP mutants

a, Schematic depicts generation of Krt5-CreER;R26^Sox2-GFP (SOX2 overexpression) mutants. b, The columnar epithelium secretes mucin as indicated by Alcian blue and periodic acid-Schiff staining. n=7 per group. c, The mucin secreting cells (AGR2⁺) are derived from KRT5⁺ basal progenitor cells as verified by the lineage tracing tag GFP. n=7. d, High magnification picture of Figure 1b to show that expanded GFP⁺KRT7⁺ basal progenitor cells invade underneath of the cardia mucosa upon SOX2 overexpression. n=7. e, High magnification picture of Figure 1c to show that expanded basal cells (KRT5⁺) invade and intercalate with the cardia mucosal epithelium (CLDN18⁺) upon SOX2 overexpression. Note KRT5⁺ cells (arrow and arrowheads) do not express CLDN18. Conversely, CLDN18⁺ cell does not express KRT5 (star). n=7. f, The columnar epithelium does not express the squamous cell marker Loricrin. n=7. The white and blue dotted lines indicate the amplified columnar epithelium and the stratified squamous epithelium, respectively. g, Co-staining of X-gal with KRT5 and p63 indicates Lgr5⁺ cardia progenitor cells do not contribute to KRT5⁺ or p63⁺ basal cells in Lgr5-CreER; R26^lacZ mice with both short-term tracing and long-term tracing. n=3 per group. Scale bar, 20 μm.

Extended Data Figure 3. Basal progenitor cells contribute to columnar metaplasia following anastomosis surgery-induced bile acid reflux

a, Schematic depicts the use of oesophageal-duodenal anastomosis and lineage tracing in p63-CreER;R26^tdTomato mice. b, Metaplasia does not occur at the distal oesophagus where the anastomosis surgery site is located (arrow). Note that Alcian blue, PAS and CDX2 label the intestinal but not the squamous oesophageal epithelium. n=5. c, Lineage-labeled transitional epithelial cells (tdT⁺) expand and resemble the multilayered epithelium, expressing the columnar markers (KRT7, KRT8) and basal cell markers (KRT5 and p63). The expanded epithelium secretes mucin as indicated by Alcian blue and PAS staining. n=5. Scale bar, 20 μm.

Extended Data Figure 4. The transitional columnar epithelium is present at the SCJ of normal mice

a, The transitional epithelium (star) does not express Involucrin and Loricrin, markers labeling the stratified squamous epithelium. n=11. b, Basal cells of the transitional epithelium express KRT5 but not the cardia epithelial marker CLDN18. n=11. c, The KRT7⁺ transitional basal cells are highly proliferative (Ki67⁺, arrowheads). n=3. d, The transitional basal cells of at the SCJ express p75, KRT7 and KRT5, but not KRT8. Note that p75 and KRT5 are also expressed in the neighboring squamous basal cells. n=5. e, FACS analysis reveals p75⁺ basal cells include two subpopulations, squamous basal cells (p63⁺ KRT7⁻) and transitional basal cells (p63⁺ KRT7⁺). n=3 independent experiments. f, A representative culture of p63⁺KRT7⁻ and p63⁺KRT7⁺ basal progenitor cells. Note that p63⁺ KRT7⁺ transitional basal cells in the colony are loose unlike the cobblestone characteristic of squamous basal cell colony (p63⁺ KRT7⁻). n=5 per group. g, p63⁺KRT7⁻ and p63⁺KRT7⁺ basal progenitor cells generate keratinized (Loricrin⁺, KRT7⁻) and non-keratinized (Loricrin⁻, KRT7⁺) epithelium in organoid, respectively. n=5 per group. h, i, The transitional epithelium is also present at the SCJ of the anus (h) and cervix (i). Lineage trancing with the Krt7-CreER mouse line confirmed KRT7⁺ cells serve as progenitors for the transitional epithelium. n=3 per group. Abbreviation: Sq, stratified squamous epithelium; Co, columnar epithelium. Scale bar, 20 μm.

Extended Data Figure 5. Loss of p63 prevents the stratification of KRT7⁺ columnar epithelium during embryonic development

a, Diagram shows the proposed REC model of downward expansion of p63⁺ KRT7⁻ basal cells and retreatment of p63⁻KRT7⁺ embryonic cells. b, The columnar epithelium lining the mouse forestomach and the SCJ expresses p63, KRT7 and KRT8 from E11.5 to E16.5. Note Claudin18 expression is limited to the hindstomach epithelium. n=5. c, Expression of KRT7 is restricted to the SCJ transitional epithelium at E18.5, and basal cells (arrowheads) express p63, KRT5, KRT7 and low levels of KRT8 but not CLDN18. n=5. d, Schematic depicts the gradual restriction of KRT7 expression to the SCJ transitional epithelium during development. Initially the simple columnar epithelium lining the forestomach and SCJ expresses both p63 and KRT7. Upon stratification the forestomach epithelium loses KRT7 expression, while basal cells at the SCJ maintain expression of both p63 and KRT7. e, Lineage tracing of epithelial progenitor cells (p63promoter active) in p63^CreER/CreER; R26^lacZ (p63 null) mutants. f, Whole-mount x-gal staining of the oesophagus and stomach isolated from p63^CreER/CreER; R26^LacZ mutants and p63^CreER/+; R26^LacZ controls. n=3. g, h, The simple columnar epithelium lining the forestomach (g) and oesophagus (h) of mutants is derived from basal progenitor cells (p63 promotor active) as indicated by x-gal staining. n=3 per group. i, Normal oesophagus and forestomach is lined by simple columnar epithelium at E11.5. n=3. Abbreviation: Es, oesophagus; FS, forestomach; HS, hindstomach; Tr, trachea. Scale bar, 20 μm.

Extended Data Figure 6. Ectopic CDX2 expression promotes intestinal metaplasia of the transitional epithelium at the SCJ

a, Targeted expression of CDX2 in basal progenitor cells of Krt5-CreER; otet-CDX2-T2A-mCherry mice. Doxycycline (Dox) water was given every other week for 3 months to induce CDX2 and mCherry expression. b, CDX2 overexpression leads to the expansion of the transitional epithelium at the SCJ at the first week of Dox treatment. n=3 per group. c, 4-week Dox treatment promotes further expansion of the transitional epithelium at the SCJ, and the epithelium presents multilayered epithelium characteristics with the co-expression of columnar markers KRT8, KRT7 and squamous marker p63. Note some basal cells start to lose p63 expression while gaining the expression of the intestinal marker AGR2. n=3 per group. d, Intestinal metaplasia is apparent following 8-week Dox treatment and some metaplastic cells lose the expression of p63. n=4 per group. Scale bar, 20 μm.

Extended Data Figure 7. Ectopic CDX2 expression promotes intestinal metaplasia of the transitional epithelium

a, Intestinal metaplasia occurs in Krt5-rtTA;otet-CDX2 mutants fed with Dox-containing water for 3 months as shown by PAS staining. n=5 per group. b, Goblet cell enriched with vesicles (stars) present in the SCJ of Krt5-rtTA;otet-CDX2 mutants following 3-month Dox treatment as shown by electron microscope. n=3 per group. c, KRT7 is expressed by both BE and MLE but not the neighboring squamous epithelium. Residual CDX2 is expressed in a subpopulation of the metaplastic columnar cells although KRT5 expression is not detectable. n=5 per group. d, Reduced expression of basal cell genes (p63, Krt5) and increased expression of the intestinal genes (Villin1, Agr2, Muc2, Muc4, TFF3) during intestinalization of the SCJ following CDX2 overexpression. (ns, p>0.05; *p<0.05; **p<0.001; two-tailed Student’s t-test; n=3 independent experiments) e, Normal SCJ structure is maintained in control mice (otet-CDX2-T2A-mCherry) following 3-month Dox treatment. n=5. f, CDX2 overexpression does not promote columnar metaplasia in the oesophagus and forestomach. Squamous hyperplasia is present in the forestomach (dotted black line). Note that CDX2 expression does not induce ectopic expression of KRT7 in the stratified squamous epithelium. n=5. Abbreviation: Sq, stratified squamous epithelium; Co, columnar epithelium; G, goblet; N, nucleus. Scale bar, 20 μm.

Extended Data Figure 8. Intestinal metaplasia is maintained even after withdrawal of Dox in Krt5-rtTA;otet-CDX2-T2A-mCherry mutants

a, Severe metaplasia develops at the SCJ of mutants that have been treated with Dox for the first 13weeks and chased for another 11weeks. n=3 per group. b, The metaplastic cells remain at the SCJ even after withdrawal of Dox containing water. The metaplastic cells express KRT7 and KRT8. Note that mCherry which indicates CDX2 expression is not detected. n=3 per group. c, Metaplastic cells maintain AGR2 and Villin1 expression. n=3 per group. Scale bar, 20 μm.

Extended Data Figure 9. Ectopic CDX2 expression promotes intestinal metaplasia of the transitional epithelium in Krt7-CreER; R26^rtTA; otet-CDX2-T2A-mCherry mutants

a, Schematic shows induction of CDX2 overexpression by combined treatment of Tamoxifen and Doxycycline. b, 30-day CDX2 expression drives the differentiation of the transitional basal cells into intestinal-like epithelium including goblet cells in Krt7-CreER; R26^rtTA; otet-CDX2-T2A-mCherry mice. n=3 per group. c, 90-day CDX2 overexpression leads to prominent intestinal metaplasia of the transitional basal progenitor cells as indicated by intestinal markers. n=4 per group. Abbreviation: Sq, stratified squamous epithelium; Co, columnar epithelium. Scale bar, 20 μm.

Extended Data Figure 10. Intestinal metaplasia develops in air-liquid culture of the transitional (p63⁺ KRT7⁺) but not the squamous (p63⁺ KRT7⁻) basal progenitor cells upon CDX2 expression

a, Schematic depicts Doxycycline-induced CDX2 expression. b, Intestinal metaplasia occurs in Dox-treated transitional (p63⁺ KRT7⁺) but not squamous basal (p63⁺ KRT7⁻) progenitor cells. Note that p63⁺ KRT7⁻ basal cells generate a thick keratin layer in the ALI culture, and the differentiated cells express Loricrin. n=5 per group. Scale bar, 20 μm.

Extended Data Figure 11. Different response to CDX2 overexpression in the transitional (p63⁺ KRT7⁺) and the squamous (p63⁺ KRT7⁻) basal progenitor cells in vitro. Human and mouse MLE present similar gene expression

a, Two distinct basal progenitor populations (p63⁺ KRT7⁻ Vs p63⁺ KRT7⁺) are present at the human SCJ as indicated by flow cytometric analysis. n=3 independent experiments. b, Schematic depicts the induction of CDX2 overexpression with Doxycycline treatment of CDX2 virus-infected human SCJ basal progenitor cells. c, CDX2 overexpression promotes intestinal metaplasia of p63⁺ KRT7⁺ cells. The metaplastic columnar cells are PAS⁺ and express Villin1, Muc2 and TFF3. n=6 per group. d, Ectopic CDX2 expression does not promote intestinal metaplasia of the stratified squamous epithelium in organoids formed by p63⁺KRT7⁻ squamous basal cells. n=4 per group. e, The transitional epithelium with underlying basal cells is dramatically expanded in patients with long-term gastro-esophageal acid reflux. Dotted lines indicate the basement membrane. n=3. f, The transitional epithelium with basal cells is amplified in BE mixed with MLE. n=5. g, Similar phenotypic presentation of human MLE and mouse MLE developed at the SCJ following CDX2 overexpression and oesophageal-duodenal anastomosis surgery. human MLE n=10; Krt5-rtTA; otet-CDX2 mutants n=5; surgical mice n=5. h, Goblet cells in human BE is positive for Alcian blue and PAS staining. BE epithelium loses the expression of KRT5 and p63 while maintaining the expression of KRT7. Note BE gains the expression of CDX2, MUC2, AGR2 and Villin1. n=12. Scale bar, 20 μm.

Figure 1. An expanded columnar epithelium consisting of basal and luminal cells at the esophageal squamous-columnar junction (SCJ) in Krt5-CreER;R26^Sox2-GFP mutants

a, SOX2 overexpression leads to the formation of multilayered epithelium (MLE, dotted line) with basal (p63⁺, arrowheads) and luminal cells (KRT8⁺). n=7 per group. b, Basal progenitors at the SCJ generate the MLE (KRT7⁺GFP⁺SOX2^hi). n=7. c, Basal cells in the MLE express KRT5 (white line) but not Claudin18 which labels the cardia mucosa (yellow line). n=7. d, The MLE is located between the stratified squamous epithelium and the simple columnar cardia. Note two distinct basal progenitors at the SCJ. Sq, stratified squamous; Co, columnar. Scale bar, 20μm.

Figure 2. A unique basal progenitor population (p63⁺ KRT7⁺) maintains the transitional epithelium at the SCJ

a, The transitional epithelium containing basal progenitors (p63⁺ KRT7⁺ KRT8⁻, arrowheads) is located between the stratified squamous and simple columnar cardia epithelium. n=11. b, The transitional basal cells serve as progenitors for the transitional epithelium as shown by lineage tracing with the p63-CreER and Krt7-CreER alleles. n=3. c, Schematic depicts culture of the transitional basal progenitor cells. d, Distinct epithelium reconstituted by p63⁺KRT7⁻ and p63⁺KRT7⁺ basal progenitors in air-liquid interface (ALI) culture. Note the thick keratin layer (star) and Loricrin⁺ cells are not present in the epithelium reconstituted by the transitional basal progenitors. n=5 per group. e, Diagram shows the transitional basal progenitor cells (p63⁺ KRT7⁺) and luminal cells (KRT7⁺) in the transitional epithelium. Scale bar, 20 μm.

Figure 3. CDX2 overexpression promotes intestinal metaplasia of the transitional basal progenitor cells at the SCJ

a, CDX2 overexpression in basal cells leads to metaplasia (insert) in the SCJ of Krt5-rtTA; otet-CDX2-T2A-mCherry mice. n=5 per group. b-c, CDX2 overexpression leads to intestinal metaplasia with goblet cells (black arrows) and the loss of p63 protein in some metaplastic cells (white arrows), while the MLE contains p63⁺ and KRT8⁺ cells. n=5 per group. d, Metaplastic cells express the intestinal proteins Villin1 and AGR2, and the goblet cell markers MUC2 and TFF3. n=5 per group. e, CDX2 overexpression in KRT7⁺ cells leads to metaplasia (insert) at the SCJ of Krt7-CreER; R26^rtTA; otet-CDX2-T2A-mCherry mice. n=4 per group. f, CDX2 overexpression leads to the loss of p63 protein in the metaplastic epithelium (arrows). Note the presence of MLE with underlying p63⁺ basal cells and KRT8⁺ luminal cells. n=4 per group. Scale bar, 50 μm.

Figure 4. The transitional epithelium is also present in the human SCJ and amplified during BE pathogenesis

a, The transitional epithelium consists of basal progenitors (arrows) and luminal cells. n=5. b, Basal progenitor cells in the transitional epithelium express p63, KRT5 and KRT7 (arrowheads). Note that basal cells of the stratified squamous (sq) cells are KRT7⁻ (arrows). n=5. c, Schematic shows two types of basal progenitor cells in the human SCJ. d, CDX2 overexpression promotes intestinal metaplasia of p63⁺KRT7⁺ cells in 3D organoid culture. n=6 per group. e, The transitional epithelium with underlying basal cells is dramatically expanded in patients with gastro-oesophageal acid reflux. Dotted lines indicate the basement membrane. n=3. f, The transitional epithelium with basal cells is amplified in BE mixed with MLE. n=5. Me, mesenchyme. Scale bar, 50 μm.