Fig. 3: Oesophageal-to-skin lineage conversion is polyclonal and instructed by skin dermal signals.

A R26^CreERT/ R26R^Confetti mice were administered tamoxifen (TAM) and oesophagi were harvested 48 h post-induction to establish 3D heterotypic cultures. Inset (right) shows 3D rendered basal view of an OE wholemount at the time of collection. Initial recombination efficiency is indicated as percentage of labelled cells for each of the Confetti reporters (CFP (cyan), GFP (green), YFP (yellow) and RFP (red)) relative to OE basal cells. Data expressed as mean ± SEM; n = 4920 basal cells out of 3 animals. Created in BioRender. Alcolea, M. (2025) https://BioRender.com/2gfnch4. B Epithelial wholemount 3D rendered images of oesophageal epithelium grown over native stroma (oeOE; left) or over the denuded skin dermis (oeSKIN; right) 8 days post-culture. Images show widespread oesophageal cell contribution during the re-epithelialisation process. Dotted lines mark original oesophageal tissue strips. C Representative 3D rendered side views showing polyclonal contribution to oeHF. D Number of reporters found to be labelling individual oeHF structures. “Not labelled” indicates that no reporter was found to be expressed. Due to clonal fragmentation, clonal events are reported as the “minimum number of clonal units”. Not labelled areas were considered minimal clonal units if coexisting with another reporter. Data is shown as absolute number of oeHF. A total of 178 oeHF structures from 4 animals were analysed. E, F 3D rendered side views of confocal z-stacks showing polyclonal contribution within KRT15+ (E) and CD34+ (F) cells in oeHFs. KRT15+ and CD34+ cells were not associated to clonal events. G Representative confocal images showing side views of oeHF depict CD34- clonal unit (in yellow) containing an individual CD34+ cell (marked by asterisk; dotted lines, insets to the right). Dashed lines delimit hair follicles borders. Fluorescent labels. Blue, DAPI; Cyan, Green, Red and Yellow depict Confetti clones; Greyscale, CD34 or KRT15. Scale bars. 20 µm (A,C, E, F, and G), 200 µm (B). Mouse lines. C57BL/6 wild-type mice were used as source of tail dermis. 48h-induced R26^CreERT/ R26^Confetti mice were the source of oesophageal tissue. See also Supplementary Fig. 3A–C. Source data are provided as a Source Data file.