Lifting regenerative barriers promotes epithelial cell fate plasticity supporting lineage conversion

Domain: physics
Published: 2025-11-27
Editorial outcome: major_revision

Abstract
The ability of adult epithelial cells to rewire their cell fate programme in response to injury has emerged as a new paradigm in stem cell biology. This plasticity supersedes the concept of strict stem cell hierarchies, granting cells access to a wider repertoire of fate choices. Yet, in order to prevent a disordered cellular response, this process must be finely regulated. Here we investigate the little-known regulatory processes that restrict fate permissibility in adult cells, and keep plasticity in check. Using a 3D regenerative culture system, that enables co-culturing epithelium and stroma of different origins, we demonstrate that oesophageal cells exposed to the ectopic signals of the dermis are capable of switching their identity towards skin. Lineage tracing experiments and histological analysis, however, reveal that the oesophageal-to-skin lineage conversion process is highly inefficient, pointing to the existence of barriers limiting cell fate re-specification. Single-cell RNA sequencing capturing the temporality of this process shows that cells transitioning towards skin identity resist the natural progression towards tissue maturation by remaining in a persistent regenerative state marked by a particularly strong hypoxic signature. Gain and loss of function experiments demonstrate that the HIF1a-SOX9 axis acts as a key modulator of epithelial cell fate plasticity, restricting changes in identity during tissue regeneration. Taken together, our results reveal the existence of lineage conversion barriers that must be resolved for cells to respond to signals instructing alternative fate choices, shedding light on the principles underlying the full regenerative capacity of adult epithelial cells.

Introduction
Traditionally, each adult cell type has been thought to emerge from a designated pool of lineage-restricted stem/progenitor cells (SCs)1–7. This model has been challenged by mounting evidence suggesting that cell fate, particularly in an epithelial context, is more dynamic than originally thought8–11. It has become widely accepted that, in response to certain tissue perturbations, epithelial cells retain the ability to reacquire developmental features, rewiring their programme of cell fate towards alternative outcomes/identities in a process known as cell fate plasticity12–22. However, despite its unique potential for regenerative medicine, the mechanisms governing cell fate plasticity remain largely unknown.

Under physiological conditions, epithelial plasticity represents a well-established mechanism activated in response to tissue injury. Cells in the damaged area are granted access to a wider range of cell fate choices than those available under normal homeostasis13–17,19–22. Fate plasticity may include the reactivation of stem and regenerative properties in committed cells via de-differentiation14–16,23,24, or enable cells to transdifferentiate into alternative identities in order to replenish adjacent tissue structures that may have been damaged13,19–21. Ultimately, this process expands the pool of cells that can respond to tissue injury beyond traditional SCs, providing a mechanism of defence that ensures the rapid and efficient replacement of lost or damaged tissue.

Plasticity, however, is a non-trivial process; increasing permissibility to acquire other fates predisposes epithelial tissues to disease and abnormal growth8,9. Hence, in order to safeguard integrity, tissues must be equipped with finely regulated mechanisms limiting the range of fates accessible to cells during regeneration. Unveiling the unknown processes that limit plasticity represents an outstanding challenge in stem cell biology, critical to unearthing the basic rules that dictate the full regenerative capacity of epithelial cells.

In an attempt to explore the limits of cell plasticity, previous studies have shown that changes in fate can go well beyond physiological constraints. Epithelial cells artificially exposed to stromal cues instructing alternative fates are able to switch their identity, giving rise to ectopic structures (such as hair, feathers, and secretory glands), not typically found in the native tissue25–31. These tissue recombination approaches, which combine epithelium and stroma of different origin, represent an ideal experimental model to investigate regenerative fate plasticity. However, the restricted number of re-specification events and limited sample availability in those assays represent a constraint32. The recent advent of single-cell sequencing technologies33 has now opened the possibility to revisit tissue recombination methods to study cell fate plasticity with single-cell resolution at an unprecedented level of detail.

Here we develop an ex vivo regenerative assay that models oesophageal-to-skin lineage conversion to investigate the mechanisms regulating one of the most remarkable modes of adult cell fate plasticity, i.e., transdifferentiation. In this heterotypic system, based on a tissue recombination assay7, we expose the squamous epithelium of the mouse oesophagus (OE; of endodermal origin) to the stroma of the murine skin, which contains niches for specific stem cell populations including hair follicles (HFs) and sebaceous glands (all of them ectodermal origin). The uncomplicated architecture of the OE, lacking epithelial appendages (such as hair follicles or glands), makes it a particularly suitable model to investigate fate re-specification (Fig. 1A)2,7,34–40. Our data demonstrate that, although oesophageal cells are able to switch towards skin identity in response to dermal cues, this process is highly inefficient. Single-cell transcriptional profiling reveals that the majority of transitioning cells remain stalled between the two lineages in an unresolved regenerative state marked by the overexpression of HIF1a-regulated genes. Lifting the regenerative blockage, by inhibiting HIF1a or its downstream effector SOX9, favours cell fate re-specification, enabling further progression from oesophageal towards skin identity. Notably, SOX9, known to play a key role in hair follicle stem cell specification and maintenance in the skin41–45, exhibited a context-dependent activity, regulating the capacity of oesophageal cells to undergo cell fate plasticity in response to perturbations. Our results reveal the existence of lineage barriers, inherent to tissue regeneration. We propose that these barriers may represent a mechanism to prevent cells from responding prematurely to inadequate or incomplete instructive cues during tissue repair.

Results
Adult oesophageal cells re-epithelialise the denuded dermis forming typical skin appendages
In order to investigate the initial mechanisms triggering the activation of adult cell fate plasticity in response to regenerative signals, we developed a heterotypic wound healing assay7 to co-culture tissues of different origins. This enabled us to grow adult oesophageal tissue over the living denuded stroma of the skin. Based on earlier tissue recombination studies25–31, we hypothesised that this heterotypic system would prompt cell fate re-specification towards skin identity, enabling us to capture early plasticity events.

Briefly, pieces of intact oesophageal tissue (including epithelium and stroma from the middle half of the oesophagus)7 were placed over a larger piece of denuded skin dermis (Fig. 1B and Supplementary Fig. 1A) that had previously been peeled off from its overlying epidermis. The peeled dermis consistently retained the architectural footprint of its appendages and associated stromal niches (Supplementary Fig. 1B, C; see Methods), exposing the oesophagus to different dermal compartments that constitute niches to specific skin stem cell populations. These heterotypic tissue composites were cultured for up to 10 days under minimal media conditions (lacking any added growth factors)7, over which period the emerging signs of oesophageal-to-skin lineage conversion events were explored. To be able to track the tissue of origin (OE vs skin), different reporter mouse lines were used; oesophageal tissues constitutively expressed tdTomato (red; mTmG or nTnG mouse strains), while skin derivatives were EGFP+ (green; H2B-EGFP mouse strain), unless otherwise stated (Fig. 1B). Similar tissue constructs where oesophageal tissue and skin were grown over their respective denuded stroma were used as controls (Supplementary Fig. 1A).

Over time, the OE growing over the dermis recapitulated the initial steps of a wound healing response46, migrating out of their original tissue, progressively replacing the missing skin, and forming a de novo epithelium (Oesophageal-derived-skin; Fig. 1C and Supplementary Fig. 1D; see Methods). This newly formed epithelium was amenable for whole-mounting techniques (Fig. 1D), which enabled the plastic behaviour of cells to be studied at whole tissue level7,35. The emerging oesophageal-derived skin showed typical in vivo features, being sustained by basal cell proliferation and retaining low Caspase 3 activity (Supplementary Fig. 1E–I), which confirmed the integrity of the in vitro derived tissues. Interestingly, adult OE cells (red) were able to fully re-epithelialise the bare dermis and empty skin HF sockets, creating a de novo stratified epithelium (Fig. 1E) and forming structures reminiscent of in vivo HFs, hereafter referred as oesophageal-derived HFs (oeHFs; Fig. 1C–F). Re-epithelialisation of the denuded skin and HF niches by oesophageal cells in heterotypic cultures represented a highly efficient process, with 91.4±1.8% of the original EGFP+ HFs being replaced by oesophageal-derived HFs-like structures (tdTomato; red) at day (D) 10 post-culture (Supplementary Fig. 1J).

Remarkably, oesophageal-derived hair follicles oeHFs (red) showed features reminiscent of the in vivo appendages. These included pigmented fibre-like structures (Fig. 1G and Supplementary Fig. 1K) positive for Fontana-Masson stain, which labels melanin (Supplementary Fig. 1L). Oesophageal-derived HFs were also positive for Oil Red O staining, indicating the accumulation of sebum in the proximal area where sebaceous glands are typically found (Supplementary Fig. 1M).

These results showed that the squamous epithelium of the adult oesophagus has the ability to grow and re-epithelialise the ectopic niche of the adult dermis, restoring the skin architecture and forming HF-like structures.

Oesophagus-to-skin re-specification ex vivo; heterogeneous lineage conversion response
To further assess whether the adult OE cells re-epithelialising the adult dermis were actively switching their identity towards skin lineage, heterotypic epithelial whole mounts (oeSKIN; OE grown over denuded dermis) were immunostained for typical oesophageal and HF markers and compared to their respective in vivo tissue whole mounts (OE and SKIN) as well as the individual tissues cultured under comparable ex vivo conditions (oeOE and sSKIN; oesophagus and skin reconstituted over their own native stroma; Fig. 2A).

Confocal analysis assessing skin marker expression in oeSKIN whole mounts showed extensive signs of lineage conversion towards epidermal fate (Fig. 2B and Supplementary Fig. 2A–C). HF markers, such as Keratin 15 (KRT15), Keratin 17 (KRT17) and Keratin 24 (KRT24)47–49, as well as the well-known HF stem cell marker CD3443,50 were specifically localised to the oeHFs in oeSKIN samples, supporting the notion of an active oesophageal-to-skin cell fate switch taking place. Other upregulated markers included SCD1 (which marks sebaceous glands), as well as Keratin 10 (KRT10), and SCA-1 (typically labelling the infundibulum and interfollicular epidermis) (Fig. 2B and Supplementary Fig. 2A–D).

Of note, oeHFs showed atypical morphology and ectopic expression, with markers localised outside their anatomical position within in vivo skin HFs. This, however, was observed to be a recurrent feature of ex vivo reconstituted HFs regardless of their skin or oesophageal origin (Compare sSKIN and oeSKIN versus Skin in Fig. 2B and Supplementary Fig. 2A) in line with previous reports51–55. Reassuringly, oesophageal control samples in vivo (OE) and in vitro (oeOE) confirmed the lineage specificity of skin markers (Supplementary Fig. 2B, C)56. Additionally, oeHFs were positive for the basal epithelial marker KRT14, while failing to express the fibroblast marker PDGFRα, further verifying the epithelial origin of the CD34+ cells in oeSKIN (Supplementary Fig. 2E).

The upregulation of epidermal markers in the oeSKIN (oeIFE and oeHF) was accompanied by the downregulation of oesophageal markers, including Keratin 4 (KRT4, differentiation marker in the oesophagus; Supplementary Fig. 2F) and SOX2 (a basal oesophageal marker; Supplementary Fig. 2G, H). Overall, our marker characterisation analysis demonstrates that adult epithelial cells retain the ability to change their identity when exposed to an adult foreign niche, indicating that fate re-specification is not restricted to developing tissues25,32.

Additional heterotypic experiments, where the adult OE was grown over the dermis of developing skin, one day post birth (P1), showed the specific upregulation of the marker LHX2, known to label developing HFs in vivo (Fig. 2C), in oeHFs. This observation revealed the critical role that the stroma plays in the identity switch, being able to rewire the oeHF fate towards developmental stages. In line with this, dermal papillae derived from the host skin (EGFP) were found in heterotypic cultures (Supplementary Fig. 2I, J). This indicates that the instructive signals promoting lineage conversion originate in the dermal niche, reminiscent of normal skin physiology57,58.

Next, we sought to determine whether oesophageal plasticity was restricted to the skin or reflected a more widespread phenomenon. To this end, we repeated our heterotypic cultures, exposing oesophageal cells to the ectopic stroma of other epithelial tissues, i.e., tongue and bladder. At the culture end-point, oesophageal cells expressed Keratin 8 (KRT8), a marker typically expressed in simple epithelial tissues such as the bladder and glandular regions of the tongue (Supplementary Fig. 2K–M). Intriguingly, epidermal epithelium cultured on oesophageal stroma did not acquire oesophageal markers, suggesting intrinsic differences in plastic potential across epithelial tissues (Supplementary Fig. 2N). Despite tangible evidence proving the ability of oesophageal cells to undergo environmental reprogramming and switch towards skin identity, further inspection of the oeHF data revealed the limited nature of the fate conversion process. In particular, the expression of HF markers was highly variable across oeHFs appendages, ranging from widespread expression in the case of KRT17 and KRT24 (~78% and ~91% oeHFs expressing these markers, respectively), to only a reduced proportion of oeHFs (~28%) expressing CD34 (Fig. 2D). The expression was similarly variable within individual oeHFs, with the number of cells expressing CD34 and KRT15 representing just a minor proportion when compared to native skin (Fig. 2E). These results unveiled the inefficiency of the OE-to-skin lineage conversion process.

We conclude that adult oesophageal epithelial cells exposed to the ectopic stromal signals during tissue re-epithelialisation are able to actively switch towards alternative epithelial identities, as instructed by the stromal niche. The heterogeneous and limited expression of HF markers in oesophageal-derived HFs, however, revealed the restricted nature of the cell fate plasticity programme during epithelial regeneration.

Polyclonal contribution of oesophageal cells to de novo hair follicle formation
To better understand the lineage conversion process, we went on to investigate the OE contribution to oeHFs formation. To this end, we traced the behaviour of individual cells in heterotypic cultures using a genetic lineage tracing approach40. R26CreERT/R26Confetti mice were treated with tamoxifen resulting in OE clonal cell labelling 48h post-induction (Fig. 3A); approximately 5 in 100 cells expressed one of the four fluorescent reporters (YFP, CFP, RFP or GFP)59. Labelled oesophagi were collected and cultured over wild-type denuded dermis for 8 days. Clonal labelling was subsequently analysed by confocal microscopy to track the behaviour of the cells forming the oeSKIN. The data revealed that over the 8-day time course, OE labelled cells experienced a marked expansion (Fig. 3B). Starting at an initial clonal labelling covering just ~5% of the basal layer, fluorescent OE cells expanded and re-epithelialised the dermis colonising on their way two-thirds of all the oeHF structures present in the tissues (66.37%±6.07; mean±SD). As a result, upon clonal expansion, approximately 66% of all oeHFs formed contained at least one or more labelled cells, revealing that, in line with previous observations in oesophageal wound healing7, OE cells undergo a widespread recruitment during oeHF formation.

To further understand the cellular dynamics behind oeHF formation, we next asked whether these structures were of monoclonal or polyclonal origin. We anticipated that if oeHFs emerged from a discrete cell population in the tissue, monoclonal oeHFs derived from single cells (i.e., entirely labelled by a single fluorescent reporter) would be found. The results, however, showed that all labelled oeHFs analysed comprised a minimum of two and up to five clonal units (Fig. 3C, D), arguing in favour of the polyclonal origin of newly formed oeHFs.

Collectively, our results indicated that, during the lineage conversion process, oesophageal cells expand contributing polyclonally and in a widespread manner to de novo oeHF formation.

Lineage conversion in oesophageal-derived HFs is dictated by the dermal niche
So far, our data demonstrates that, despite the extensive oesophageal contribution to dermal regeneration, the ability of OE cells to switch their identity towards skin remains strictly limited. Given the inefficiency of the process and the emerging recognition of heterogeneity within the oesophageal basal cell population60–62, it is important to consider the dynamics of basal cell contribution to the lineage conversion process. To determine whether the basis of the inefficient OE-to-skin fate re-specification was associated with restricted competency of a heterogeneous cell population, we analysed the KRT15 and CD34 expression within oeHF confetti labelled clones. We reasoned that if the conversion process was linked to a particular cell population, cells originally primed to undergo lineage conversion would produce clones preferentially enriched for cells expressing HF markers (KRT15 and CD34), indicative of their clonal origin. Interestingly, neither KRT15, nor CD34 expression were linked to particular clonal events, with areas enriched for KRT15 or CD34 being largely polyclonal instead (Fig. 3E, F). Additionally, clones largely negative for CD34 were seen to express this marker in sporadic cells positioned in close proximity to other CD34+ cells, forming clusters of cells undergoing lineage conversion independent of their clonal origin (Fig. 3G). A similar level of marker polyclonality was also observed in in vitro skin controls (sSKIN; Fig. S3A–C), where cell fate plasticity in response to tissue repair is well established13,20,63. Overall, these results suggest that, in our regenerative 3D assay, marker expression is context dependent, i.e. relies on signals from the dermal niche, rather than being a clone-specific process.

To demonstrate that marker expression, indeed, relied on niche signals, we repeated our heterotypic culture assay using decellularised dermis devoid of stromal cells (Supplementary Fig. 3D). The results showed that OE cells cultured over the acellular dermal scaffold exhibited an impaired re-epithelialisation, oeHF formation, and lacked CD34 expression (out of 82 oeHFs analysed, none expressed CD34; Supplementary Fig. 3E–H), supporting the instructive role of dermal signals. Interestingly, this data also shows that, under the right experimental conditions, adult stromal cells retain the ability to instruct identity changes in adult epithelia, beyond developmental stages25,32.

Together, these results indicate that the change in identity depends on niche signals from the skin dermis, rather than on the oesophageal cell of origin. While we cannot discard that multiple oesophageal basal cell pools may contribute to lineage conversion, our lineage tracing data suggest no single basal subpopulation is solely responsible for this process. Remarkably, the observation that, out of all the OE cells exposed to the dermal HF niche, only a reduced proportion were able to acquire CD34 and KRT15 expression (Fig. 2D, E) suggests the existence of inherent barriers limiting the lineage conversion process.

Single-cell RNA sequencing identifies a regenerative blockage limiting oesophageal-to-skin lineage conversion
To investigate the nature of any existing barriers affecting cell fate re-specification, we first explored transcriptional changes in cells exposed to different niches, (endogenous versus ectopic), using our heterotypic model. For this, single-cell RNA sequencing (scRNA-seq) was performed on tdTomato OE (from nTnG mice) growing over its own denuded stroma (oeOE) or the skin dermis (oeSKIN) at two different timepoints; early during epithelial regeneration at day (D) 3, and after full re-epithelialisation at D10 (experimental end-point; Fig. 4A). Sequenced samples were spiked with 5% EGFP epidermal sSKIN cells (i.e. ex vivo skin grown over its endogenous stroma; Fig. 4C and Supplementary Fig. 3I). The sSKIN cells served as a reference control to pinpoint OE cells transitioning towards skin identity (Fig. 4A–C; Source Data). The EGFP sSKIN reference cells were found in a unique cluster (C), C18 (Fig. 4B–F and Supplementary Fig. 3J, K); defined as cells presenting high levels of Egfp and Gt(ROSA)26Sor (Fig. 4D and Supplementary Fig. 3J) that lack of Sox2 expression (Fig. 4E, F)64–66. The identity of C18 cells was further validated by implementing a tdTomato+/EGFP+ semi-supervised gene classifier approach (Supplementary Fig. 3K; see Methods).

Having identified the sSKIN reference population, we went on to annotate oesophageal-derived cells. Consistent with in vivo observations7,36, there were three distinctive oesophageal cell populations in all sample types (Fig. 4G; Source Data; Methods): i) actively cycling basal progenitors; ii) resting basal cells; and iii) differentiated cells (Supplementary Fig. 3L, M).

The UMAP distribution and differential gene expression analysis of the single-cell data revealed the existence of three distinctive transcriptional patterns across the experimental conditions analysed (Fig. 4H): oeOE/oeSKIN-D3 denoted an early re-epithelialisation state at D3 post-culture (Supplementary Fig. 4A, left panels), oeOE-D10 and oeSKIN-D10 represented oesophageal cells grown over their own denuded stroma or that of the skin dermis at D10 post-culture, respectively (Supplementary Fig. 4A, right panels). Interestingly, although D3 samples showed an indistinguishable profile independent of the origin of the stroma used as substrate (oeOE versus oeSKIN), there were marked differences at D10 (Fig. 4H and Supplementary Fig. 4A). Further analysis of the basal cell compartment, where progenitor cells reside7, showed that OE cells grown over the skin niche, oeSKIN-D10, showed a gene expression pattern more closely related to early timepoints, oeOE/oeSKIN-D3, than their timepoint counterpart oeOE-D10 (Fig. 4I). Indeed, gene enrichment analysis of the associated gene expression patterns revealed that both, D3 and oeSKIN-D10, shared a strong regenerative signature, being particularly enriched for pathways involved in active wound healing and tissue repair (Fig. 4J, K and Supplementary Fig. 4B, D). oeOE-D10 samples instead showed a natural progression away from a regenerative state marked by an increased expression of genes associated with epithelial differentiation, keratinisation and tissue maturation (Fig. 4K and Supplementary Fig. 4B–D).

We concluded that oesophageal-derived skin fails to progress to the same extent as oeOE-D10 samples, remaining locked in an unresolved regenerative state where cells become permissive to environmental cues instructing an identity switch. Indeed, marker gene expression analysis showed that, as oeSKIN-D10 transitions towards skin identity, oesophageal marker expression drops, while epidermal genes become upregulated when compared to their endogenous control oeOE-D10 (Fig. 4L, M).

In order to determine the specific mechanisms underlying the lineage conversion process, it was first necessary to define the set of oesophageal cells undergoing the transition towards skin/HF lineages and establish the transcriptional changes that set them apart. To this end, we searched for OE-derived clusters enriched in epidermal gene expression, as defined by the reference clusters SKIN-C18. Cluster 8 (C8) emerged as a cluster enriched in cells transitioning towards skin identity (Fig. 4N). Indeed, the sSKIN-C18 reference cluster was found to partially merge with C8 of oesophageal origin, marking the UMAP area where cells undergoing fate conversion are located (Fig. 4N). In line with our histological characterisation (Fig. 2), the number of oesophageal cells showing signs of conversion was limited, illustrating the inefficiency of the lineage conversion process at the molecular level.

Given that C8, which contained transitioning cells potentially representing a plastic state, was present in both oesophageal cells grown on dermis (oeSKIN) as well as on their native stroma (oeOE), we went on to explore the existence of any potential physiological counterparts in the in vivo oesophageal epithelium. To this end, we integrated the current in vitro dataset with our previous in vivo scRNA-seq data, including adult homeostatic tissue as well as earlier postnatal stages36. This analysis revealed that, although cell clusters were largely comparable between the two datasets, the signature of C8, marked by the upregulation of tissue repair genes, was unique to in vitro conditions (Fig. 4O, P). This indicates that C8 constitutes a state not present under normal homeostatic conditions, that is instead associated with the active regenerative state imposed by the ex vivo system.

To investigate whether the oesophageal-to-skin lineage conversion process involved epigenetic reprogramming, we performed single-cell assay for transposase-accessible chromatin using sequencing (scATAC-seq) to profile genome-wide chromatin accessibility in in vitro oesophageal-derived skin (oeSKIN), alongside control samples of in vitro oesophagus (oeOE) and in vitro skin (sSKIN) (Fig. 5A and Supplementary Fig. 4E, F). The data revealed that, upon exposure to ectopic skin stroma, oesophageal cells exhibited altered chromatin accessibility at 29.9% of genomic regions compared to oeOE. Notably, 38.7% of these differentially accessible regions align with those characteristic of the sSKIN chromatin landscape (Supplementary Fig. 4G; Source Data). In line with the observed lineage conversion, we detected increased accessibility at loci associated with epidermal identity and decreased accessibility at loci linked to oesophageal identity (Fig. 5B, C and Supplementary Fig. 4G). Additionally, the scATAC-seq analysis further revealed the inefficiency of the lineage conversion process, with only a limited number of oesophageal cells transitioning towards skin identity (see cluster 6; Supplementary Fig. 4E, F). This supports the existence of inherent barriers to cell fate plasticity.

Since our data, so far, suggested that oeSKIN cells remain locked at a regenerative state, we next asked whether altering the in vitro regenerative state, associated with dermal re-epithelialisation and de novo skin reconstitution, influences the lineage conversion process. To this end, we adapted our ex vivo system to limit the ability of the OE to re-epithelialise the dermis. This was achieved by using peeled OE lacking the underlying stroma, which is known to promote cell migration and epithelial expansion during wound healing46,67 (Supplementary Fig. 4H, I). Indeed, when growing OE under impaired re-epithelialisation conditions, the OE formed oeHFs that contained a significantly higher number of CD34+ cells compared to our standard heterotypic approach (Supplementary Fig. 4J, K). These results reveal that, by partially restricting the re-epithelialisation capacity of epithelial cells, the fate conversion process becomes more efficient.

Together our data shows that, in line with other studies on epithelial cell fate plasticity9,68, the oesophageal-to-skin fate transition is also associated with cells displaying a regenerative signature. However, we further demonstrate that, although tissue regeneration is known to promote epithelial plasticity9,10, it also bears intrinsic fate barriers preserving cell identity and restricting re-specification events.

Oesophageal cells transitioning towards skin identity present a marked hypoxic signature
With the aim to define the specific mechanisms dictating the permissibility of OE cells to signals instructing alternative fates, we focused on the transition between C8 and C18 marking the switch from oesophageal to epidermal identity at D10 (Fig. 5D). The new re-clustered UMAP segregated cells by tissue of origin, showing cells of different origin at the UMAP boundaries. Pseudotime analysis further revealed the trajectory along the cell fate conversion process; with cells of different identity plotting at opposite ends of the pseudotime path (i.e., sSKIN-C18 and oeOE-C8), and transitioning oeSKIN cells arranged between the two (oeSKIN-C8; Fig. 5D–F). To further validate this fate trajectory, and avoid any potential confounding effects as a result of including cells derived from different tissues, the pseudotime analysis was repeated only including cells of oesophageal origin this time (Supplementary Fig. 5A–C). The UMAP representation of the oesophageal pseudotime also showed a clear segregation between OE cells exposed to their own stroma (oeOE, blue; Supplementary Fig. 5A–C), and those exposed to the ectopic stroma of the skin (oeSKIN, magenta; Supplementary Fig. 5A–C), reproducing the trajectory obtained when the 3 experimental conditions were included (Fig. 5D–F).

Next, we examined the transcriptional profile along the three main in vitro states defined by the pseudotime trajectory, namely Oesophagus, Transitioning Cells (TC) and Skin (Fig. 5F; see Methods). We identified four major patterns defining changes in the gene expression profile across states (Fig. 5G and Supplementary Fig. 5D). Pattern (P) 2 marked the switch in cell identity by showing a sustained increase in the expression of typical HF genes (Cd200, Apoe, Sparc, BC100530, Gas1, Sostdc, Cst3, Aqp3)69–72 (Fig. 5H). In contrast, P4 revealed a progressive reduction in those epithelial genes found to be enriched in oesophageal cells (oeOE; Krt7, Krt13, Pitx1, S100a4, Wfdc18, Wfdc2; Fig. 5I)73. P1 and P3 represented the most relevant patterns, containing genes being specifically modulated in TCs undergoing cell fate conversion (Fig. 5J and Supplementary Fig. 5E–H). Downregulated genes in P1 were related to processes associated with epithelial differentiation74, including protein biogenesis (Ddx21, Gnl3, Ncl, Nop56, Nop58, Rexo2)75 and oxidative metabolism (Mgst1, Mgst2, Prdx6)76 (Supplementary Fig. 5E). P3, instead, contained genes upregulated in TCs. Gene enrichment analysis for these genes revealed processes known to be associated with cell fate plasticity77,78 (Fig. 5J and Supplementary Fig. 5F–H). This included terms defining an active tissue remodelling signature i.e., wound healing, invasion, EMT and tumour progression (Hbegf, Serpine1, Bsg, Plod2, Pthlh, Cited1)79–84. Other genes denoted the active regenerative state of TCs, including those associated with ECM reorganisation (Adam8, Crim1, Itgb1, Mmp3, Mmp9, Mmp13, Pthlh)85,86 and glycolytic metabolism (Aldoc, Gapdh, Gpi1, Pgam1, Pkm, Tpi1) (Supplementary Fig. 5F–H). Interestingly, one of the most significant transcriptional features defining transitioning cells was their marked hypoxic profile, expressing particularly high levels of genes regulated by HIF1a (Adm, Bsg, Cited1, Ddit4, Ero1l, Espn, Hbegf, Pgf, Bnip3, Cav1, Egln3, P4hb, Prelid2, Ptges, Slc2a1, Vegfa; Fig. 5J, K)87–91. This emerged as a common hallmark, shown to underlie all cells in C8, that was notoriously more accentuated in cells transitioning towards skin identity (TC; Fig. 5J, K and Supplementary Fig. 5G–I; Source Data). In keeping with these findings, we observed that chromatin regions associated with identity switch loci, including Hif1a, present a more open state in oeSKIN cells at the transition between oesophagus and skin (along the scATAC-seq pseudotime trajectory; Fig. 5L–N), supporting its potential involvement in the lineage conversion process.

To histologically characterise the HIF1a signature observed within the regenerative cluster C8, tissue whole mounts derived from heterotypic cultures were immunostained against HIF1a (Supplementary Fig. 5J, K). The results showed that, indeed, HIF1a expression was associated with an active regenerative state, revealing a marked HIF1a expression early during tissue re-epithelialisation at D3, independent of whether the oesophagus was grown over their own or ectopic stroma. At D10, however, HIF1a levels dropped in the maturing in vitro oesophagus, while remained persistently high in oesophageal-derived skin (sSKIN), including oeHFs, suggesting a persistent regenerative state in oeSKIN samples.

In light of our scRNA-seq analysis, we conclude that oesophageal cells transitioning towards skin identity remain in an unresolved regenerative state marked by a sustained hypoxic signature, denoted by the overexpression of HIF1a downstream target genes.

Inhibition of the HIF1a-SOX9 axis lifts the lineage conversion barrier
Next, we set out to investigate the impact of HIF1a on the lineage conversion process. HIF1a downregulation has been shown to be necessary for the adequate resolution of skin wounds92,93. Thus, we hypothesised that its persistent activation in transitioning cells may be locking cells in a regenerative state, limiting their ability to respond to ectopic niche signals and preventing the identity switch. To explore this possibility, we treated heterotypic cultures with pharmacological compounds known to inhibit (KC7F2) or activate (DMOG) HIF1a94,95 (Supplementary Fig. 6A–C). Remarkably, HIF1a inhibition favoured differentiation towards the skin lineage, showing a significant increase in the percentage (%) of CD34+ and KRT15+ cells in the newly formed oeHFs (Fig. 5O–Q). Conversely, activation of HIF1a pathway resulted in the opposite effect, leading to a significant reduction in CD34+ and KRT15+ cells per oeHF (Fig. 5O–Q). These results revealed that HIF1a inhibition enabled an increase in the number of cells expressing HF markers (CD34 and KRT15) per oeHFs. We concluded that the persistent regenerative state limiting the lineage conversion process was modulated, at least in part, by HIF1a pathway. Moreover, inhibition of HIF1a signalling pathway promotes the escape from the regenerative blockage and favours fate re-specification towards skin as instructed by dermal signals.

Further mechanistic insight was obtained when analysing SOX9 expression, a known HF stem cell marker in the skin essential for HF formation and maintenance13,41–44,96. Remarkably, oesophageal-derived HFs showed that SOX9 featured the opposite profile to that observed for other HF markers, such as CD34. SOX9 was instead positively regulated by HIF1a; being downregulated upon HIF1a inhibition and increased in response to HIF1a activation (Fig. 5O, R). Previous work already established a connection between SOX9 and HIF1a, demonstrating the direct regulation of SOX9 expression by HIF1a in bone morphogenesis97,98. In line with this, SOX9 expression was also found to positively correlate with HIF1a expression in oeHFs (Supplementary Fig. 6D). These results suggest that SOX9 may be acting in a context/tissue dependent manner, having a different role to that described during HF development and maintenance41,42,45,99. To test whether SOX9 expression is HIF1a-dependent in an oesophageal context, we first performed whole organ ex vivo treatments36, exposing the oesophagi to hypoxic conditions and/or KC7F2 (HIF1a inhibitor). The results showed that SOX9 expression was indeed induced by hypoxic conditions and that this induction could be prevented by inhibiting HIF1a (Fig. 6A, B). The potential co-regulation of these two transcription factors was supported by the increased HIF1a expression observed upon genetic inactivation of Sox9 expression in epithelial oeHF cells (Krt14CreERT/Sox9fl/fl; Supplementary Fig. 6E, F). Taken together, these intriguing results revealed that, in our model, SOX9 is regulated by HIF1a, playing a role different to that previously established in skin HFs.

Previous work had identified SOX9 as a transcription factor associated with regenerative cell fate plasticity, becoming ectopically expressed by the interfollicular epidermis in response to skin injury13,96. Hence, to better understand the context in which SOX9 becomes expressed in the oesophagus, we analysed SOX9 expression in oesophageal whole mounts under different physiological and non-physiological conditions. These included adult homeostasis, early postnatal development, oesophageal excisional wound healing, as well as in two different 3D organ culture systems (Epithelioids100) and oeOE re-epithelialisation assays7. The results revealed that, although SOX9 is typically absent in the adult homeostatic epithelium, its expression is notably upregulated in all non-homeostatic conditions tested (Supplementary Fig. 6G, H), denoting a link between an active regenerative/developmental response and SOX9 expression.

Next, we asked whether SOX9 may be acting as an effector of HIF1a in the lineage conversion process. To this end, we explored whether the ablation of Sox9 expression reproduced the HIF1a effect on cell identity. For this, we made use of the Krt14CreERT/Sox9fl/fl conditional mouse model, whose in vivo induction resulted in a significant reduction of SOX9 expression specifically in epithelial cells (Supplementary Fig. 6E). Heterotypic cultures derived from induced Krt14CreERT/Sox9fl/fl oesophagi showed an increased expression of HF markers (CD34 and KRT15) in the oeHF (Fig. 6C–F). Interestingly, this boost in lineage conversion happened despite the increase in HIF1a levels seen upon SOX9 inhibition (Supplementary Fig. 6F), further supporting the notion that SOX9 is contributing to the lineage conversion barrier downstream of HIF1a. Taken together, these intriguing results revealed that, in our model, SOX9 is regulated by HIF1a, playing a role different to that previously established in skin HFs.

Finally, to further demonstrate the mechanistic role of the HIF1a-SOX9 axis in the lineage conversion process, we examined the interplay between hypoxic signals and SOX9 expression in oeHFs. Krt14CreERT/Sox9fl/fl derived heterotypic cultures were exposed to hypoxic conditions (5% O2), in order to simultaneously inactivate SOX9 and stimulate HIF1a (Fig. 6G). We anticipated that if HIF1a and SOX9 expression were acting as independent barriers blocking the lineage conversion process, the hypoxic treatment would rescue the Krt14CreERT/Sox9fl/fl phenotype, bringing HF markers back to control levels. Marker quantification showed that this was not the case, the hypoxic treatment did not restore HF markers in oeHFs (Fig. 6G–I), indicating that, in this context, HIF1a is acting via SOX9 to modulate the lineage conversion process. Again, these observations appeared to conflict with findings in the skin, where SOX9 is known to play a key role for the specification and maintenance of HF stem cells41–45. Hence, to specifically test whether the role of SOX9 was context dependent, we repeated the experiment in Krt14CreERT/Sox9fl/fl derived sSKIN (Supplementary Fig. 6J–L). Indeed, SOX9 inactivation in in vitro skin led to the opposite effect, reducing the number of CD34+ and KRT15+ cells in the sSKIN (Supplementary Fig. 6J–L). HIF1a stimulation via hypoxia had no significant effect on sHF marker expression (Supplementary Fig. 6J–L). These results evidence the context dependent role of SOX9, driving different biological outputs in various biological processes. This is in line with its widely known involvement in numerous developmental, regenerative and pathological processes where the common denominator is tissue remodelling13,101–103. Overall, these data demonstrate that HIF1a and its downstream effector SOX9 establish a regenerative barrier that prevents cell plasticity during tissue repair in oesophageal cells.

HIF1a-SOX9 axis prevents cell fate re-specification in vivo
Next, we investigated the lineage conversion process in vivo and its underlying regulation. To this end, we implemented two different grafting approaches. First, we developed a heterotypic transplantation technique that recreates our in vitro strategy. Skin punches were excised from the back of nude mice, and replaced by peeled tail dermis (expressing EGFP) overlaid with a strip of OE (expressing tdTomato; Supplementary Fig. 7A), both tissues collected from adult mice. One to two weeks following the procedure, OE cells generated proliferating HF-like structures that expressed the HF marker KRT24, and contained a central fibre resembling the HF shaft (Supplementary Fig. 7B–F). We conclude that adult OE cells are able to change their identity and form oeHF structures also in an in vivo context.

This heterotypic grafting approach served its purpose to characterise in vivo cell fate switching. However, grafts were ultimately lost, preventing longer-term assessment of OE-derived hair formation. To address this, we made use of a well-established in vivo HF reconstitution assay104–106. Dissociated cells from adult OE cells (tdTomato+) and neonate skin (EGFP+) were combined and grafted onto the back skin of nude mice using a silicon chamber (Fig. 7A).

This resulted in the emergence of skin-derived HFs (EGFP; Supplementary Fig. 7G), as well as chimeric oesophageal/epidermal HFs (tdTomato/EGFP; Supplementary Fig. 7H) that generated functional hair shaft-like structures 6 weeks post-grafting (Fig. 7B). To improve the oesophageal HF formation efficiency, we increased the ratio of oesophageal cells by 4 times. This led to the emergence of HFs fully derived from oesophageal cells (tdTomato; Fig. 7C, D and Supplementary Fig. 7I). Oesophageal-derived HFs formed adjacent to EGFP skin-derived HFs (arrowhead, Fig. 7C and Supplementary Fig. 7I), and were surrounded by supportive skin-derived EFGP mesenchymal cells (Fig. 7C, D). Additionally, the HF marker P-cadherin was expressed in HF-like structures showing its typical in vivo expression pattern, being primarily localised in the outer root sheath of the hair follicle (Fig. 7E)107–109. Overall, these results are indicative of a switch from oesophageal-to-skin identity.

Consistent with the limited lineage conversion observed in our in vitro system, the OE contribution to HF formation in vivo was highly sporadic. We identified 12 HFs events out of 14×106 OE cells in a total of 34 grafts. Next, we tested whether targeting the HIF1a-SOX9 axis would improve the efficiency of the lineage conversion in vivo. HF reconstitution assays where oesophageal cells were either exposed to KC7F2 (to inhibit HIF1a) or undergone conditional SOX9 inactivation revealed a significant increase in the HF forming efficiency, showing multiple oesophageal-derived HF structures per graft (Fig. 7F–H). KC7F2 treatment produced ~5.4 times more oeHFs than control conditions (14 HFs events out of 3×106 OE cells in a total of 6 grafts), while SOX9 inactivation led to ~9.2 times more oeHF than control conditions (55 HFs events out of 7×106 OE cells in a total of 14 grafts; Supplementary Fig. 7J).

Finally, 3D heterotypic cultures derived from tracheal epithelium (tdTomato) and denuded skin dermis (EGFP) revealed that the HIF1a-SOX9 axis operates as a fate plasticity barrier beyond the oesophageal epithelium. The expression of HF markers, CD34 and KRT15, was significantly increased in tracheal-derived skin upon genetic inactivation of Sox9 expression (Krt14CreERT/Sox9fl/fl; Supplementary Fig. 7K–M). Overall, our data show that the oesophageal-to-skin lineage conversion process involves a series of events. These include i) the re-epithelialisation of the denuded skin dermis by oesophageal cells. Here oesophageal cells repopulate the empty epidermal stroma forming HF-like structures as they fill the empty HF sockets. ii) This process exposes oesophageal cells to the ectopic HF niche. iii) The instructive niche signals enable the lineage conversion process in competent oesophageal cells in a HIF1a-SOX9 dependent manner. We go on to demonstrate that blocking HIF1a or its downstream target SOX9 lifts the regenerative blockage and favours cell fate plasticity by promoting the transition from oesophageal to skin lineage both in vivo and in vitro (Proposed model Fig. 7I), and that this may represent a more universal mechanism operating beyond oesophageal epithelium.

Methods
Mousestrains and allele induction
All mouse experiments were approved by the local ethical review committees at the University of Cambridge, and conducted according to Home Office project license PPL70/8866 and PP7037913 at the Cambridge Stem Cell Institute, Cambridge University.

To identify the tissue-of-origin in 3D heterotypic cultures following fluorescent reporter mouse lines were used: mTmG (R26mTmG; stock #007676, Jackson Laboratory130) and nTnG (R26nTnG; stock #023537, Jackson Laboratory131), which constitutively express tdTomato localised in the cell membrane and nuclei, respectively; H2B-EGFP (CAG::H2B-EGFP; kindly provided by J. Nichols132) with constitutive EGFP nuclear expression; and Lgr5-EGFP (Lgr5-EGFP-IRES-creERT2; stock #008875, Jackson Laboratory133) with an EGFP cassette targeted to the 3′ untranslated region of the Lgr5 gene.

Fucci2a mouse line (R26Fucci2a, kindly provided by Ian J. Jackson134), which incorporates constitutive expression of cell cycle reporter proteins (G1 marked by mCherry-hCdt1, and S/G2/M marked by mVenus-hGem), was used to visualise cycling cells. For lineage tracing analysis, R26-CreERT2 animals (stock #008463, Jackson laboratory135) were crossed onto R26R-Confetti (stock #017492, Jackson laboratory59) to generate inducible R26CreERT/ R26Confetti mice. The resulting mice express one of four fluorescent reporter proteins (YFP, GFP, RFP, or CFP) in sporadic cells following tamoxifen-mediated Cre recombination. For Sox9 conditional knockout experiments, Sox9 flox/flox mice (obtained from MRC-Harwell, which distributes these mice on behalf of the European Mouse Mutant Archive https://www.infrafrontier.eu136) were crossed onto K14-CreERT mice (stock #005107, Jackson laboratory137) to generate the K14CreERT/ Sox9flox/flox enabling genetic inactivation of Sox9 expression in epithelial cells upon tamoxifen induction. C57BL/6J mice (strain code, 632) and nude athymic mice (strain code, 490) were purchased from Charles River, UK.

Recombination of R26CreERT2/ R26RConfetti mice was induced by a single intraperitoneal (i.p.) tamoxifen injection (3 mg per 20 g of body weight in sunflower seed oil). K14CreERT/ Sox9flox/flox received two subcutaneous and one intraperitoneal tamoxifen injections (5 mg per 20 g of body weight in sunflower seed oil) in 3 consecutive days. The oesophagi of treated animals were collected 72h after the first TAM administration.

All experiments comprised mixed male and female animals, with no gender specific differences observed. For RNA sequencing experiments, only male animals were used in order to avoid confounding effects due to oestrous cycle. All animal cohorts were adults between 8–25 weeks of age. Mice were bred and maintained under specific-pathogen-free conditions at the Cambridge Stem Cell Institute and Gurdon Institute of the University of Cambridge. All animals were housed between 20–24°C, 45–65% humidity, and a 12-h light-dark cycle.

3D Oesophageal-Dermal Heterotypic and control cultures
In order to investigate the initial mechanisms modulating adult cell fate plasticity in response to regenerative signals, we adapted our ex vivo organotypic wound healing assay7 to co-culture adult epithelia and stroma of different origins, in this case, mouse tail skin and oesophagus. To this end, tissues were freshly dissected out and processed to separate epithelial and stromal compartments. Epithelium and stroma were obtained from the middle half of the oesophagus to avoid confounding effects due to cellular heterogeneity along the longitudinal axis of the oesophagus7. Oesophagi were opened longitudinally, the muscle layer removed, and the tissue flattened under a dissecting microscope. Skin and oesophageal stroma – referred to as dermis and submucosa, respectively – were carefully peeled away from their overlying epithelium with fine forceps after a 3–4 h incubation in 5 mM EDTA at 37°C. The stroma was cut into 7x9 mm pieces for the dermis and 5 x 10 mm pieces for the submucosa and placed onto transparent ThinCertTM inserts (0.4 μm pore size; Greiner Bio-One Ltd; Cat#657641). Immediately afterwards, two strips of oesophageal tissue (5x1 mm; lacking the muscle layer) were laid on top of the denuded/peeled stroma. To generate in vitro skin controls (sSKIN; sHF, Hair Follicle; sIFE, Interfollicular Epidermis; s-prefix denotes in vitro skin), the skin from the ear pinna was dissected, cut in strips (5x1 mm) and laid on top of the denuded tail dermis following the same procedure as with the oesophagus. Heterotypic tissue composites were then allowed to settle in the ThinCertTM for 5 min at 37°C to ensure attachment, and covered up to tissue level with minimal medium (mFAD; lacking added growth factors) containing one-part DMEM (4.5 g/L D-Glucose, Pyruvate, L-Glutamine), one-part DMEM/F12, supplemented with 5% foetal calf serum, 5 μg/ml insulin, 0.18 mM adenine, 5–10 μg/ml transferrin and 1% Penicillin-Streptomycin. Heterotypic constructs are viable for up to 15 days of culture7. Here, cultures were grown for up to 10 days at 37°C and 5% CO2, replacing the medium on alternate days. During this period of time, the denuded/peeled stroma was re-epithelialised by oesophageal cells. Oesophageal epithelium was grown over its native stroma as a control (oeOE; prefix e- denotes oesophageal re-epithelialisation in vitro). OE grown on top of denuded dermis was considered oesophageal-derived skin (oeSKIN), and associated compartments (oeHF, Hair Follicle; oeIFE, Interfollicular Epidermis). Unless otherwise stated, oesophageal tissues were tdTomato+ (mTmG or nTnG mouse lines), while tissues of skin origin were EGFP+ (from H2B-EGFP line).

At the end-point, organotypic cultures were either embedded in optimal cutting temperature compound (OCT; Fisher Scientific Ltd; 12678646) for cryosectioning, or the epithelium was whole-mounted by peeling away from the stroma following a 2-h incubation in 5 mM EDTA at 37°C.

For the limited re-epithelialisation assay (Supplementary Fig. 4H, I), heterotypic cultures were performed using peeled OE, lacking oesophageal stroma. For this, the oesophagus epithelium was peeled from the underlying submucosa following a 3-h incubation in 5 mM EDTA at 37°C. The freshly peeled epithelium was cut into 5 × 8 mm pieces and laid on the denuded dermis (as above). For controls, 5 x 8 mm pieces of unpeeled epithelium were used instead.

For explant cultures pieces of oesophageal tissue (5 × 5 mm) were grown directly onto ThinCertTM inserts under mFAD media conditions (as above), replacing the medium on alternate days. 10 days post-culture tissues were processed as epithelial whole mounts (see below) for immunofluorescence analysis.

Oesophageal-tongue and oesophageal-bladder heterotypic constructs
To investigate whether the exposure of the oesophageal epithelium to an ectopic stroma other than the dermis could trigger cell fate rewiring, we followed the procedure described above for oesophageal-dermis heterotypic constructs with some modifications as follows. Bladders and tongues were dissected from EGFP+ mice. The muscle layer was removed, and bladder and tongue tissues were incubated in EDTA 5 mM for 2 h and EDTA 50 mM for 90 min, respectively. The epithelial compartments were then peeled off and discarded. The bladder or tongue stroma was placed in inserts and oesophageal strips laid on top.

Tissue whole mount preparation and immunofluorescence
Whole mounts from the mouse tail skin and the middle third of the oesophagus were obtained as follows. Tissues were dissected as indicated above and cut into pieces of approximately 5x8 mm, followed by a 3-4 h incubation in 5 mM EDTA at 37°C. The epithelium was then gently peeled away from the underlying stroma and fixed in 4% paraformaldehyde (PFA) in PBS for 30 min. For immunostaining, whole-mounts were incubated in blocking buffer (0.5% Bovine Serum Albumin, 0.25% Fish skin gelatin, 0.5% Triton X-100 in PBS and 10% donkey serum) for 1 h at 37°C prior to staining with primary antibodies (diluted in blocking buffer) overnight at 4°C (see below). This was followed by four 30-min washes with 0.2% Tween-20 in PBS. Samples were subsequently incubated with secondary antibodies (diluted in blocking buffer) for 3 h at RT. Samples were washed as above, cell nuclei counterstained with 1 μg/ml DAPI in PBS, and samples mounted in 1.52 RapiClear mounting media.

For staining of tissue cryosections, fixed samples were embedded in OCT, cut at 150 μm and placed onto poly-L-lysine coated glass slides. Cryosections were stained and mounted following the whole-mount protocol (as above).

Primary antibodies used included: Caspase-3 active (Rabbit, 1:200, Abcam #ab2302), CD34 (Rat, 1:100, BD Bioscience #553731), CD49f (integrin α6, Rat, 1:200, BioLegend #313610), HIF1a (Rabbit, 1:100, Novus Biological #NB100-479), KRT4 (Mouse, 1:2000, Vector Laboratories #VP-C399), KRT10 (Rabbit, 1:200, Abcam #ab76318), KRT14 (Rabbit, 1:1000, BioLegend #905301), KRT15 (Mouse, 1:1000, Abcam #ab80522), KRT17 (Rabbit, 1:250, Cell Signalling #4543S), KRT24 (Rabbit, 1:500, Atlas #HPA022978), LHX2 (Mouse, 1:200, Santa Cruz #SC-517243), MMP9 (Goat, 1:50, R&D Systems #AF909-SP), PDGFRA (Goat, 1:200, R&D Systems #AF1062), SCA-1 (Rat, 1:100, Invitrogen #14-5981-82), SCD1 (Rat, 1:100, R&D Systems #MAB4404), SOX2 (Rat, 1:200, eBioscience #14-9811-82) and SOX9 (Rabbit, 1:1000, Millipore #AB5535).

Secondary antibodies used included: Goat IgG 647 (Millipore #AP180SA6), Goat IgG 750 (Abcam #ab175745), Mouse IgG 647 (Invitrogen #A-31571), Mouse IgG 750 (Abcam #ab175738), Rabbit IgG 488 (Invitrogen #A-21206), Rabbit IgG 555 (Invitrogen #A-31572), Rabbit IgG 647 (Invitrogen #A-31573), Rabbit IgG 750 (Abcam #ab175728), Rabbit IgG (Abcam #ab171870), Rat IgG 647 (Abcam #ab150155), and Rat IgG 750 (Abcam #ab175750). All secondary antibodies were diluted 1:500.

Confocal imaging and analysis
Images were acquired using either inverted Leica SP5, or SP8 confocal microscopes (Leica Microsystems) with standard laser configuration or a Stellaris 8 FALCON FLIM microscope with a white light laser. Typical settings for z-stack image acquisition included optimal pinhole, line average 3, optimal step size, bi-directional scan with speed of 400 Hz and a resolution of 1024 × 1024 pixels. Images were acquired using a 10x, 20x or 40x objective. For the latter, a digital zoom of 3x for OE and IFE epithelium, and 1x for HFs was typically used. Confocal images were reconstructed and analysed using Volocity 5.3.3 software (PerkinElmer) and arivis 3.5.0 (Vision4D). Fluorescence intensity analysis of individual cells was performed in a minimum of 18 HF units (from a minimum of n=3 animals). To calculate HF markers expression rate, a minimum of 150 oeHF units (from n=3 animals) were examined on live scanning mode. Figures show representative images.

Histology
Haematoxylin and Eosin (H&E) and Fontana-Masson (Sigma; Cat#HT200) staining were performed in 4.5 μm paraffin-embedded sections by the Histology Core Service at Cambridge Stem Cell Institute. Fontana-Masson silver staining was used to visualise melanin in skin tissues in vivo (skin and denuded dermis) and in vitro (oeSKIN), based on melanin-mediated reduction of silver nitrate to metallic silver.

Oil Red O staining was performed in oesophageal-derived skin (oeSKIN) peeled whole mounts to visualise lipid accumulation. Briefly, epithelial whole mounts were fixed with 4% paraformaldehyde (PFA) in PBS for 30 min. Tissues were incubated in 60% isopropanol for 5 min at RT and immediately after, incubated in a filtered solution of 1% Oil Red O for 30 min at RT. Tissues were rinsed with 60% isopropanol, washed twice in PBS and mounted on slides. Stained sections and whole-mounts were imaged using Apotome Imaging System (Zeiss).

Dermal decellularisation assay
The decellularisation method was adapted from previously published protocols138,139. Briefly, freshly denuded dermis (as above) were incubated for 4 h in hypertonic solution (0.1 M NaOH, 1 M NaCl and 25 mM EDTA in DI water) to lyse cells, followed by an overnight incubation in 100 mM EDTA at 4°C. DNA was enzymatically digested for 6 h in a solution containing 2 U/ml Benzonase, 47 mM Tris, 1.4 mM MgCl2, 19 mM NaCl in deionised water at pH 8-9.5. Dermises were washed overnight in 100 mM EDTA at 4°C, followed by a 2-h incubation in a solution containing 8 mM CHAPS, 1 M NaCl and 25 mM EDTA in PBS at pH 8, and a final wash in a 100 mM EDTA solution overnight. The decellularised dermis was then thoroughly washed with PBS for 30 min and sterilised by an overnight incubation in 1% P/S PBS at 4°C. Unless otherwise specified, incubation steps were done in an orbital shaker at room temperature (RT). Between different solutions, dermis were equilibrated in PBS (3 washes of 10 min on each occasion).

Oesophageal-derived HF formation efficiency
To analyse the ability of oesophageal cells to colonise the HF niche and form structures resembling HFs, 7x9 mm tail skin pieces from Lgr5-EGFP reporter mice were incubated for 30 min in 4 μg/ml DAPI in PBS, tile-scanned on a confocal microscope (Leica SP5), and the number of HFs initially present in the tissue quantified. Immediately after imaging, the quantified tail pieces were used to perform heterotypic cultures (as above). In short, skin pieces were washed in PBS and incubated in 5 mM EDTA for 4 h at 37°C. The dermis was dissected away from the epidermis and oesophageal tissue from mTmG mice laid on top of the former. Heterotypic cultures were grown for 10 days, after which period the resulting tissue was fixed in full thickness in 4% PFA for 1 h. Whole mounts were then washed three times in PBS and mounted in 1.52 RapiClear mounting media for imaging. The number of oesophageal-derived HF structures (mTomato+) were quantified using xyz rendered views of tiled confocal z-stack images. oeHF formation efficiency in vitro was calculated as the percentage of oeHF formed in vitro compared to the number of original HFs in the in vivo skin piece. A total of 3 independent samples were quantified (n=3 animals).

Single-cell RNA isolation and library preparation
Heterotypic 3D cultures were prepared as indicated above using oesophagus from nTnG mice and denuded tail skin dermis from H2B-EGFP mice. As controls samples, oesophagus from nTnG was grown over peeled oesophageal stroma from H2B-EGFP mice. The resulting epithelia (oesophageal control, oeOE; and oesophageal-derived skin, oeSKIN) were harvested 3 and 10 days post-culture (D3 and D10, respectively), at which point the epithelia were carefully peeled from their underlying stroma following a 50 mM EDTA incubation for 15 min at 37°C. A single-cell suspension was obtained by rinsing the peeled epithelia with PBS, and incubating it with 0.5 mg/ml Dispase (Sigma) for 5 min. EDTA was then added to the samples at a final concentration of 5 mM, and the suspension was diluted 1/5 by adding FACS Buffer (FB; 2% heat-inactivated Foetal bovine serum (Life Technologies; 26140079), 25 mM HEPES (Life Technologies; 15630056)) in order to inhibit Dispase activity. The cells suspension was filtered through a 30 μm cell strainer, and centrifuged at 300 g for 10 min at 4°C. Cells were finally resuspended in FB containing 1 U/μl RNAse Inhibitor. Single viable epithelial cells were sorted (using a BD FACSAria™ III cell sorter) by tissue of origin: tdTomato (oesophageal origin) and EGFP (in vitro skin). Sorted tdTomato cells from Batch 1 were spiked in with EGFP in vitro skin cells derived from skin organotypic cultures grown under the same conditions as the oeSKIN and oeOE as an internal control to define epidermal skin identity. Batch 2 was kept free of EGFP in vitro skin cells to avoid confounding effects (see details in Source Data). scRNA-seq libraries were generated and sequenced using 10X Genomics kits (Single Cell 3′ v3) at the CRUK-CI Genomics Core Facility of the CRUK Cambridge Institute. Libraries were processed in two different batches/dates. Batch 1 included 3 biological replicates for oeOE at D3, oeSKIN at D3, oeOE at D10, and oeSKIN at D10, rendering 12 libraries; Batch 2 included 2 biological replicates for oeSKIN at D10, rendering 2 libraries. Each biological replicate consisted of pooled cultures from 4-5 wells with a total of 9,000 sorted cells per sample. The libraries were multiplexed and sequenced on 4 lanes Illumina NovaSeq 6000 S2 flow cells.

Single-cell RNA-seq analysis
Pre-processing quality checks were performed on the R2 reads using FastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc). The data was then processed using the Cell Ranger software (v3.1.0) with standard alignment (genome assembly GRCm38.97 of M musculus) and default filtering parameters. Upper and lower bounds on the distributions of counts, features, mitochondrial and ribosomal RNA were used to remove outlier cells. Cells were included in the analysis if their sequencing depth was over 8750, the number of expressed genes was between 2000 and 8000, and the percentage of mitochondrial and ribosomal DNA was lower than 15% and 45%, respectively. Mitochondrial and ribosomal genes were subsequently removed from the matrix. As a result, a total of 107,445 cells, discarding doublets, were selected for further analysis. The raw expression matrix was normalised using sctransform140; and the downstream analysis was performed using the Seurat R package (v3.2.2)141 (see Source Data). To filter out non-epithelial cells, cells expressing fibroblast or immune marker genes including Col1a2, Pdgfra or Ptprc were excluded from the analysis (these were cells with a normalised read count value higher than the first local minimum of the density distribution in all cells for each of them markers).

Dimensionality reductions (PCA and UMAP), as well as clustering, were performed using Seurat, with the number of unsupervised clusters being the optimal selected by the software, with default parameters. The biological replicates for each timepoint overlapped well with each other, confirming negligible batch effects between samples and conditions; therefore, no batch effect correction was necessary.

Based on the characteristics of the dataset (stability of partitioning assessed in an incremental manner), we focused on the 1000 most abundant genes to identify the main transcriptional features for each cluster. After the gene expression analysis (identified by testing for differential gene expression using the Wilcoxon Rank Sum test on the Bonferroni-adjusted p-value, with a natural log-fold change threshold of 0.25 and a p-value threshold of 0.01), we defined 19 relevant clusters in the present dataset. Clustering stability was evaluated using a PAC analysis142. This resulted in a total of 75,519 cells used in the subsequent analysis.

The EGFP in vitro skin reference cells were initially identified in the UMAP space and localised to C18 by the expression of genes known to specifically discriminate EGFP in vitro skin versus tdTomato oesophageal cells. EGFP epidermal cells showed an enriched expression of the reporter gene Egfp and Gt(ROSA)26Sor (blocked in Rosa26nTnG oesophageal cells due to the targeting of the nTnG construct to the Rosa26 locus131; Fig. 4D and Supplementary Fig. 3J), as well as the negligible expression of the oesophageal basal marker Sox265,66 (Fig. 4E, F). The skin origin of C18 was further confirmed by the implementation of a semi-supervised gene classifier method, which infers identity based on the transcriptomics signatures of the detected tdTomato+ and EGFP+ cells, allowing us to set C18 as a cluster of reference to discriminate conversion events. In short, to avoid high similarity regions between EGFP and tdTomato nucleotide sequences, we selected, based on a Clustal Omega alignment143, 3 and 5 specific sub-transcripts for each (see Source Data), with less than 20% identity between the two markers. Using STAR aligner (v2.5.2a)144, with relaxed mapped length parameters, we aligned the reads across all samples to the selected sub-transcripts; the expression levels on the sub-transcripts were defined as the algebraic sum of incident UMIs. Next, we selected ~70 cells with the highest, independent expression of red or green markers, respectively; these two sets were used to train a classifier, part of a semi-supervised model that extends the transcriptomics signatures of the EGFP and tdTomato cells to all unlabelled cells i.e., cells with no or little quantification of the fluorescent markers. The discriminative genes were selected using a random forest model (the top 50 genes with the highest variation of gini index) and standard statistical tests i.e., t-test, Kolmogorov-Smirnov and Mann–Whitney U; genes with adjusted p-value (on a Benjamini-Hochberg multiple testing correction) lower than 0.05 were selected as discriminative. The final set of markers, used subsequently for inferring labels, was obtained by intersecting the two approaches. For each unlabelled cell, we predicted the label by correlating the selected markers to true tdTomato+ and EGFP+ cells used for the original classifier. Two different correlation-based distance approaches (Pearson and Spearman) yielded comparable results. The two distributions of distances were compared using a minimum IQR approach i.e., if the distance between the tdTomato and EGFP distributions on the proximal quartiles was larger than the average standard deviations, then the cell was labelled according to the distribution closer to 0, otherwise the cell was labelled “unknown”.

After identifying cluster 18 as in vitro skin EGFP expressing cells (Fig. 4E and Supplementary Fig. 3K, L), the remaining clusters were manually annotated based on gene expression of known cell cycle, epithelial basal and differentiated marker genes (Fig. 4G, Supplementary Fig. 3L, M and Source Data). We then focused on differences in gene expression of the basal cells in the three major transcriptional patterns (D3, oeOE-D10 and oeSKIN-D10 as in Fig. 4H, I). To this end, clusters of annotated basal cells were assigned to one branch if >75% of its cells belonged to the correspondent sample origin. Clusters 3 and 8 had an equivalent contribution from the three sample types and therefore cells in these clusters were split in the three branches depending on their origin (Source Data). Next, we identified differentially expressed genes (DEGs) in pair-wise comparisons between branches. The total number of DEGs was 567. We then scaled the expression value for each gene in the [0, 100] range using an affine transformation and summarised the scaled expression levels in a clustered heatmap. The genes were clustered using hierarchical clustering with 1 minus Pearson correlation as the distance measure, which allowed us to identify 6 major patterns: Pattern 1, 50 genes; Pattern 2, 66 genes; Pattern 3, 133 genes; Pattern 4, 54 genes; Pattern 5, 32 genes; Pattern 6, 232 genes (Supplementary Fig. 4D and Source Data). We performed gene enrichment analysis of the resulting 6 patterns using g:Profiler145 (https://biit.cs.ut.ee/gprofiler/gost; R package (v0.1.8)), against the standard GO terms, and the KEGG146 and Reactome147 pathway databases (Source Data). The background set comprised all genes expressed in the dataset.

The expression profile of skin-related genes pointed to a region of the UMAP where two clusters of different identity merged. In vitro skin cluster C18 of epidermal origin and C8 of oesophageal origin marked the area where cells undergoing fate conversion resided. First, we investigated the hallmark genes of oesophageal C8. To this end, we performed a differential gene expression analysis between C8 and each of the remaining basal cell clusters. Next, we performed a gene enrichment analysis using those genes that were differentially expressed uniquely in C8 (Source Data). To explore whether the cell population defined by C8 had an equivalent population in vivo, we integrated our data with our in vivo scRNA-seq dataset36 (ArrayExpress EMTAB-8662) using the Harmony single cell data integration package (v0.1.0) with default parameters148.

We then focused on identifying cell fate conversion events which appeared within cluster 8 and cluster 18 (sSKIN) (Fig. 4N). To dissect the transcriptional evolution of the identity switch events identified within C8 and C18, we re-clustered cells from C18 and C8 in 10-day samples. The new UMAP distribution segregated cells by the tissue, with oeSKIN showing a partial overlap (Fig. 5D). To identify genes changing along the cell fate conversion trajectory, we performed a pseudotime analysis, using the monocle3 R package (v0.2.3.0)149. Since C18 in vitro skin cells served as a reference for the skin identity switch, these were, therefore, fixed as the endpoint of the pseudotime trajectory. Pseudotime analysis ordered cells along the fate conversion trajectory; with cells of different origin plotting at opposite ends of the differentiation path (i.e., C18 sSKIN and C8 oeOE), and oeSKIN cells transitioning between the two (Fig. 5F). To further discern the differences between Oesophagus and TC cells, we repeated the analysis focusing on C8. We re-clustered the C8 cells and performed a pseudotime analysis on the resulting UMAP (Supplementary Fig. 5A, B). For consistency, we selected the start-point identified in the C8-C18 combined pseudotime as a starting point for this pseudotime analysis. The ordering of cells closely aligned with that found in the C8-C18 pseudotime, showing a notable separation between oesophageal control cells (C8 oeOE) and oesophageal-derived skin cells (oeSKIN). TCs remained separated from the rest at the end of the trajectory (Supplementary Fig. 5C). To examine in more detail the changes occurring in the Transitioning cells (TC), we defined three equidistant groups along the pseudotime axis: Oesophagus, TC, and Skin and identified the DEGs (Differentially Expressed Genes) for each pair-wise comparison (1,173 genes; Source Data). To focus on the exclusive features that drive the TC away from their original identity (Oesophagus), we selected the genes that had >0.2 log FC between the Oesophagus and the TC (357 genes). We then scaled the expression value for each gene in the [0, 100] range using an affine transformation and summarised the scaled expression levels in a clustered heatmap using the Pearson correlation for the clustering to reveal patterns of expression. The nine unsupervised clusters were then grouped in four distinctive patterns of expression: Pattern 1, 94 genes; Pattern 2, 26 genes; Pattern 3, 162 genes; Pattern 4, 75 genes (Fig. 5G and Supplementary Fig. 5D; Source Data). We run gene enrichment analysis for each of the observed patterns using g:Profiler145, against the standard GO terms, the KEGG146 and Reactome147 pathway databases, and TRANSFAC150 as transcription-factor-binding motifs prediction database (Source Data). The background set comprised of all genes expressed in clusters 8 and 18. Terms and genes were manually curated in Supplementary Fig. 5H, in accordance with the biological context of this work. We then displayed relevant DEGs resulting from the analysis along the pseudotime trajectory (Fig. 5H–J and Supplementary Fig. 5E–G); for each gene, auto-scaled expression was plotted using a generalised additive model for smoothing.

Single-nuclei isolation and library preparation for ATAC-seq
Epithelia (oesophageal control, oeOE; oesophageal-derived skin, oeSKIN; and skin control, sSKIN) were harvested 10 days post-culture, at which point the epithelia were carefully peeled from their underlying stroma following a 50 mM EDTA incubation for 15 min at 37°C. The peeled epithelia were rinsed with PBS, and incubated with 0.5 mg/ml Dispase (Sigma) for 5 min. EDTA was then added to the samples at a final concentration of 5 mM, and the suspension was diluted 1/5 by adding PBS+0.04% BSA buffer. A cell suspension was obtained by filtering through a 30 um cell strainer, centrifuging at 300 g for 5 min at 4 degrees and resuspending in PBA+0.04% BSA. After assessing viability and cell number, nuclei were isolated following 10X CG000169 protocol. Libraries were prepared following the manufacturer’s instructions for Chromium Next GEM Single Cell ATAC Kit v2, multiplexed, and sequenced in one lane Illumina NovaSeq X Plus 25B flow cell.

Single-cell ATAC-seq processing and analysis
Single-cell ATAC-seq libraries were processed using cellranger-atac count (v2.2.0)151, aligning reads to the reference genome (mm10 2024-A) and generating fragment files along with filtered peak-barcode matrices. Downstream analysis was conducted in R (v4.4.3) using the Signac (v1.14.0)152 and Seurat (v5.3.0) packages. Cells were filtered based on quality control metrics, including the number of fragments in peaks and number of features to remove outliers. Only cells with ≥40% of fragments in peak regions and a Transcription Start Site (TSS) enrichment score >1 were retained, resulting in a total of 32,499 high-quality cells for downstream analysis.

Chromatin accessibility was normalised using Term Frequency–Inverse Document Frequency (TF-IDF) transformation, followed by Singular Value Decomposition (SVD) on the peak-cell matrix. Dimensionality reduction was performed via Latent Semantic Indexing (LSI), and top LSI components (excluding the first) were used to construct a Shared Nearest Neighbour (SNN) graph. Louvain clustering identified distinct cell populations, and Uniform Manifold Approximation and Projection (UMAP) was used for visualisation in two dimensions.

To visualise aggregate chromatin accessibility at selected loci, coverage plots were generated using the CoveragePlot function in Signac, displaying fragment density and annotated gene structures. Trajectory inference was performed using Cicero (v1.3.9)153, which was used to construct an ATAC-specific cell_data_set object from the peak-cell matrix. Monocle3 (v1.4.26) functions were then applied to learn the trajectory graph and order cells in pseudotime based on chromatin accessibility dynamics. Pseudotime-dependent changes in accessibility at individual peaks were visualised using the plot_accessibility_in_pseudotime function.

Differentially accessible (DA) chromatin regions were identified across samples using the FindAllMarkers function in Seurat with a logistic regression test. Peaks were considered significant if they exhibited a log2 fold-change greater than 0.5 and an adjusted p-value (p_val_adj) < 0.05. To prioritise region-specific differences, the top 100 DA peaks per sample were selected based on the highest average log2 fold-change.

Average chromatin accessibility for the selected peaks was computed per sample using the AverageExpression function in Seurat. Normalised accessibility values were used for heatmap visualisation, preserving absolute differences in chromatin accessibility between peaks and samples. Heatmap colour scaling was based on quantiles (1st, 25th, 50th, 75th, and 99th percentiles) of the accessibility distribution.

HIF1a modulation treatments
Heterotypic cultures were treated with the HIF1a translation inhibitor KC7F2 (10 μM; SML1043, Merck) or the HIF1a protein stabiliser DMOG (1 mM; 71210, Cayman) in mFAD medium (as above) during a period of 8 days. Control cultures were treated with DMSO vehicle alone in mFAD medium. Cultures were grown in a normoxic humidified incubator at 37°C, 5% CO2, and medium was changed on alternate days. Experiments modulating HIF1a signalling pathway via hypoxia were performed by exposing the oesophageal tissue/cultures to low oxygen conditions for 4 h in a hypoxic humidified incubator at 37°C, 5% CO2, 5% O2.

Oesophageal biopsy wounding
Oesophageal wounding was performed by controlled micro-endoscopic biopsying of the mouse oesophagus as previously described7. Briefly, C57BL/6J mice undergoing endoscopy were anesthetised using a combination of 100 mg/kg Ketamine (Pfizer Animal Health) and 10 mg/kg Xylazine administered intraperitoneally. Animals without a complete loss of righting reflex after anesthetic induction were topped up with inhaled isoflurane prior to intervention. A 9.5 Fr diagnostic miniature endoscope with a 3 Fr instrument channel was used in conjunction with an AIDA COM II image capture system for visualisation (Karl Storz GmbH). 3 Fr diameter biopsy forceps with double action jaws (Karl Storz GmBH) were used to create one superficial wound in the middle third of the mouse oesophagus of between 0.4–0.9 mm diameter. Anesthesia was reversed using Atipamezole (Pfizer Animal Health) given at 1 mg/kg subcutaneously at least 20 min after induction. Animals were euthanised 24 h after wounding.

Oesophageal-dermal heterotypic in vivo grafting assay
Heterotypic oesophageal-dermal tissue composites were prepared for in vivo grafting using the strategy described for our in vitro assay. Dermis was obtained from H2B-EGFP animals and oesophageal tissue from mTmG or nTnG mice, unless otherwise stated. To reduce tissue dehydration prior to grafting, oesophageal explants were laid on the dermis and kept in a humidified environment until transplantation. Female nude athymic mice were used as recipients. In a typical experiment, recipient mice anesthetised by isoflurane inhalation underwent a single excision on the dorsal shoulder using a sterile 8 mm diameter biopsy punch. Immediately afterwards, a heterotypic graft (8 mm diameter) was fitted and adhered to the surrounding tissue with Vetbond tissue adhesive (3M). The area was further sutured using steri-strip wound closure strips (3M) and covered with Tegaderm dressing film (3M) to protect the tissue implant. Animals were monitored thereafter, and skin tissue was harvested up to 4 weeks post-transplant.

In vivo hair reconstitution assay
Hair reconstitution studies were adapted from previously described protocols105. In short, back skin was obtained from postnatal day 2 (P2) H2B-EGFP mice and placed epidermal side up, floating in 10 ml 0.25% trypsin overnight at 4°C. After washing with PBS, the epithelium was peeled off using fine forceps and thoroughly minced with a scalpel. The epidermal cell suspension was repeatedly pipetted to disaggregate clumps, filtered through a 30 μm strainer and span down at 500 g for 8 min at RT. Epidermal cells were resuspended in cFAD medium, containing one-part DMEM (4.5 g/L D-Glucose, Pyruvate, L-Glutamine), one-part DMEM/F12, supplemented with 5% foetal calf serum, 5 μg/ml insulin, 5-10 μg/ml transferrin, 0.5 μg/ml hydrocortisone, 1 nM cholera enterotoxin, 10 ng/ml EGF and 1% Penicillin-Streptomycin. The neonatal dermis was separately minced and incubated in 0.25% collagenase for 25 min at 37°C. The dermal cell suspension was then filtered through a 100 μm strainer and the HF buds were pelleted by centrifugation at 100 g for 5 min at 4°C. The supernatant was transferred to a new tube, centrifuged at 300 g for 5 min at 4°C and resuspended in cFAD medium.

Oesophageal epithelial cells were isolated from adult nTnG or induced K14CreERT/ Sox9flox/flox mice. Longitudinally opened oesophagi were cut in 4 pieces and incubated in 0.5 mg/ml Dispase in PBS for 15 min at 37°C. The epithelium was peeled, minced with a scalpel and transferred to a new tube with fresh Dispase solution. After a 5 min incubation at RT, EDTA was added at a final concentration of 5 mM to inhibit Dispase activity. Cells were mixed by pipetting and filtered through 30 μm strainers. OE cells suspensions were centrifuged at 300 g for 5 min at 4°C, and resuspended in cFAD medium. All cell suspensions were kept on ice until transplantation.

On the first set of cell transplantations, a cell suspension typically containing 5 × 105 oesophageal epithelial cells, 5 × 106 neonatal dermal cells and 106 neonatal epidermal cells was prepared and surgically implanted onto a PDMS silicon chamber secured on the dorsal fascia of recipient nude mice105, following a full-thickness punch biopsy (8 mm diameter). As these experiments only generated oesophageal/epidermal chimeric hair follicles (Supplementary Fig. 7H), we increased four times the oesophageal: epidermal cell ratio. Following assays (Fig. 7C–H) were performed by implanting a cell suspension containing 5 × 105 oesophageal epithelial cells, 2.5 × 106 neonatal dermal cells and 2.5 × 105 neonatal epidermal cells onto a PDMS silicon chamber in nude mice. In the HIF1a modulation group (Fig. 7G), HIF1a inhibitor KC7F2 (10 μM) was added to the cell suspension prior injection into the silicon chamber. Grafted mice were monitored and tissue harvested for immunofluorescence analysis 6 weeks post-grafting.

Statistics and reproducibility
All experiments were performed independently at least three times with similar results, unless otherwise stated. The reproducibility of all key findings was confirmed in independent experiments conducted on different days and using independent biological samples.

The number of animals used for each experiment is indicated in the figure legends (“n” in legends refer to number of independent replicates per timepoint and/or condition). For oesophageal-derived skin characterisation, in particular, similar results were observed across more than 50 independent experiments. No data were excluded.

Representative images shown in the figures were selected from experiments that were repeated multiple times across independent biological replicates. All figures show representative images of a minimum of 3 mice/tissue cultures. Experimental data are expressed as mean values ± SD, unless otherwise indicated.

Differences between groups/conditions were assessed by using two-tailed unpaired t-test, one-way analysis of variance (ANOVA), and Kruskal-Wallis ANOVA test as indicated in figure legends. ANOVA-based analysis was followed by Tukey’s test, and Kruskal-Wallis was followed by Dunn’s test for multiple comparisons. All tests were two-sided. Exact p-values are indicated in relevant figures up to ten decimal places. Statistical differences between groups were assessed using GraphPad Prism software. No statistical method was used to predetermine sample size. Experiments were performed without randomisation or blinding.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This manuscript by Bejar and colleagues investigates the molecular mechanisms that restrict cell fate plasticity in adult\nepithelial cells, with a focus on the HIF1α-SOX9 axis as a key barrier to esophageal-to-skin lineage conversion. Using an\ninnovative 3D regenerative culture system, lineage tracing, and single-cell RNA sequencing (scRNA-seq), the authors\ndemonstrate that while esophageal cells exhibit some degree of plasticity, they encounter significant barriers in fully\nadopting a skin epidermal identity. Importantly, the study reveals that inhibiting HIF1α or SOX9 enhances cell fate\nconversion, suggesting promising avenues for regenerative medicine applications.\nStrengths :\n- The study challenges traditional concepts of rigid stem cell hierarchies by demonstrating that adult epithelial cells retain\nlatent plasticity.\n- The heterotypic culture system (esophageal epithelium cultured on skin dermis) provides an elegant model to study cell\nfate conversion.\n- The use of scRNA-seq offers high-resolution insights into cell identity transitions and intermediate states.\n- The concept of removing regenerative barriers has important implications for enhancing tissue repair and cell\nreprogramming strategies.\nOverall, this is a well-conceived and experimentally robust study that advances our understanding of epithelial cell plasticity\nand regenerative barriers. The identification of HIF1α/SOX9 as key restrictive factors is significant, but the manuscript would\nbe strengthened by addressing the following comments to provide a more comprehensive understanding of the mechanisms\ngoverning cell fate plasticity in adult epithelial tissues.\nMajor comments :\n1. A study from the group of Yann Barrandon (Claudinot et al, 2020), has already demonstrated that p63-expressing\nepithelial stem cells from various tissues (including esophagus) have hairy skin competence. The main novelty of the\npresent study is that the HIF1α-SOX9 axis is a key barrier to esophageal-to-skin lineage conversion. While this axis is well-\ncharacterized, the study would benefit from exploring additional regulatory mechanisms. A single cell multi-omics approach\ncombining scRNA-seq with scATAC-seq would provide deeper insights into the molecular basis of lineage restriction and\nthe epigenetic landscape changes during reprogramming. Indeed, Sox9 has been reported as a pioneer factor in mouse\nepidermis (Adam et al, 2015 ; Larsimont et al, 2015) and Sox9 conditional knockout in a model of esophageal cell\nreprogramming in vivo modifies chromatin accessibility (Vercauteren Drubbel et al, 2021)\n2. It remains unclear whether lineage rewiring involves a dedifferentiation step and whether this process involves a fetal-like\nintermediate phenotype, as observed in regenerating stomach (Vallone et al, 2016), intestine (Nusse et al, 2018; Yui et al,\n2018) and transcommitted esophageal keratinocytes (Vercauteren Drubbel et al, 2021). This deserves further investigation.\nAnalysis of scRNA-seq data and/or multi-omics data could help addressing this question.\n3. The inefficiency of lineage rewiring raises questions about whether all basal progenitors possess the intrinsic competence\nto transdifferentiate. To what extent are esophageal progenitors homogenous in the scRNA-seq analysis? As suggested in\nthe first point, scATAC-seq could help determine if certain progenitor subpopulations have an epigenetic background more\npermissive for transdifferentiation.\n4. In the same line, recent studies have highlighted a cephalocaudal gradient of Troy expression in basal epithelial cells of\nmouse esophagus (Grommisch et al, 2024). In addition, some basal progenitors from the proximal part of the esophagus can\ndifferentiate into taste buds (epithelial appendages) (Vercauteren Drubbel & Beck, 2023). The competence of basal\nprogenitors to change lineage may therefore be heterogeneously distributed in the esophagus. Discussing these regional\ndifferences would provide better context for the observations.\nAdditional Suggestions (discussion)\n1. While molecular changes associated with lineage conversion are demonstrated, the study lacks assessment of whether\nconverted cells are functionally equivalent to native skin cells (e.g., skin barrier function, response to mechanical stress,\nwound healing capacity). This should be discussed.\n2. The study focuses solely on esophagus-to-skin conversion. Testing other transitions (e.g., esophagus-to-intestine) would\nreveal whether similar barriers exist in embryologically related tissues. This is really important in the field as esophageal-to-\nintestinal transdifferentiation may occur under pathological conditions (metaplasia). This is obviously beyond the scope of\nthe manuscript, but this should be discussed.\n3. The potential oncogenic implications merit further discussion. While inhibiting HIF1α-SOX9 increases plasticity\n(potentially beneficial for regeneration), this could prove risky in cancer contexts. The authors should consider discussing\nhow modulating lineage restriction barriers might affect tumor initiation or progression in epithelial tissues.\nReferences\nAdam RC, Yang H, Rockowitz S, Larsen SB, Nikolova M, Oristian DS, Polak L, Kadaja M, Asare A, Zheng D, et al (2015)\nPioneer factors govern super-enhancer dynamics in stem cell plasticity and lineage choice. Nature 521: 366–370\nClaudinot S, Sakabe J-I, Oshima H, Gonneau C, Mitsiadis T, Littman D, Bonfanti P, Martens G, Nicolas M, Rochat A, et al\n(2020) Tp63-expressing adult epithelial stem cells cross lineages boundaries revealing latent hairy skin competence. Nat\nCommun 11: 5645\nGrommisch D, Wang M, Eenjes E, Svetličič M, Deng Q, Giselsson P & Genander M (2024) Defining the contribution of Troy-\npositive progenitor cells to the mouse esophageal epithelium. Dev Cell 59: 1269-1283.e6\nLarsimont J-C, Youssef KK, Sánchez-Danés A, Sukumaran V, Defrance M, Delatte B, Liagre M, Baatsen P, Marine J-C,\nLippens S, et al (2015) Sox9 Controls Self-Renewal of Oncogene Targeted Cells and Links Tumor Initiation and Invasion.\nCell Stem Cell 17: 60–73\nNusse YM, Savage AK, Marangoni P, Rosendahl-Huber AKM, Landman TA, de Sauvage FJ, Locksley RM & Klein OD\n(2018) Parasitic helminths induce fetal-like reversion in the intestinal stem cell niche. Nature 559: 109–113\nVallone VF, Leprovots M, Strollo S, Vasile G, Lefort A, Libert F, Vassart G & Garcia M-I (2016) Trop2 marks transient gastric\nfetal epithelium and adult regenerating cells after epithelial damage. Development: dev.131490\nVercauteren Drubbel A & Beck B (2023) Single-cell transcriptomics uncovers the differentiation of a subset of murine\nesophageal progenitors into taste buds in vivo. Sci Adv 9: eadd9135\nVercauteren Drubbel A, Pirard S, Kin S, Dassy B, Lefort A, Libert F, Nomura S & Beck B (2021) Reactivation of the\nHedgehog pathway in esophageal progenitors turns on an embryonic-like program to initiate columnar metaplasia. Cell\nStem Cell: S1934-5909(21)00152–1\nYui S, Azzolin L, Maimets M, Pedersen MT, Fordham RP, Hansen SL, Larsen HL, Guiu J, Alves MRP, Rundsten CF, et al\n(2018) YAP/TAZ-Dependent Reprogramming of Colonic Epithelium Links ECM Remodeling to Tissue Regeneration. Cell\nStem Cell 22: 35-49.e7", "reviewer_2": "Review of “Lifting regenerative barriers promotes epithelial cell fate plasticity supporting lineage conversion”\nBy Bejar et al.\nSummery:\nThis manuscript addresses a potentially compelling question, whether esophageal epithelial cells transdifferentiate into\nskin/hair‐follicle lineages under specific in vitro and in vivo conditions-yet it falls short of providing robust data to substantiate\nits main claims. In particular, the central assertions that full hair‐follicle‐like structures are derived from esophageal\nepithelium and that the HIF1a–SOX9 axis is a key mechanistic barrier are not convincingly supported by the limited\nimmunostaining, insufficient marker validation, and sparse quantitative data presented. As currently written, the paper leaves\ncritical gaps in experimental detail and does not demonstrate that the new structures are fully functional or definitively\nesophageal in origin, weakening confidence in the broader conclusions.\nOn a more personal note, I like the idea of the paper and believe it can have broad implications, especially in the context of\nadult epithelial plasticity and regenerative medicine. However, as currently presented, the manuscript does not sufficiently\nsupport its central claims.\nMajor Comments\n1. Extent of Bona Fide Hair‐Follicle Formation: The authors provide preliminary evidence (e.g., CD34, KRT17/24 staining, partial pigmentation) that esophageal cells may become hair‐follicle‐like. However, further morphological and molecular\nvalidation (for instance, K15, or other keratins; staining for melanin, basal layer maker) would strengthen the claim that\ncomplete hair follicles are formed rather than partial or rudimentary structures or cells simply filling in the molds left upon\nepidermal/HF removal.\n• There is no clear architectural evidence is provided that the follicles formed are organized into bulge, matrix, ORS, etc.\n• Pigmentation is vaguely mentioned; Fontana-Masson staining is needed.\n• If sebaceous glands are suggested, perform Oil Red O or equivalent lipid staining to validate. Also Ki67 or proliferation\nmarkers would help show proper biology of the SG and basal layer HG, bulge.\n• More than anything, I would like to see clear data showing RFP cells in a GFP setting (dermal cells) with markers of HFSC\nas well as IFE differentiation markers. More high quality multi-color stainings are needed to be convincing. I am not certain\nthat the HFs I see are indeed novel HFs. It appears that is some scenarios these are cells filling in the molds left in the\ndermis and assume a shape similar to a hair follicle. In such a case would it be surprising that they share some similarity\nwith HF kerationcytes/HF cellular populations as well as esophageal. Wouldn’t it be expected that the dermal mold/scaffold\nwould generate such an effect?\n2. Markers Confirming Epithelial and Appendage Identities: Because CD34 can also mark hematopoietic or stromal cells,\nrelying on CD34 alone as the readout for hair‐follicle stem‐cell identity is limiting. Also, the cd34 staining (and others) are far\nfrom ideal. It is over exposed and appears somewhat non-specific. In general figure 2B is far from ideal. As stated above a\nlot more images with GFP and RFP together with markers (wholemounts and/or sections) are required. Again, the\nmanuscript should include additional hair follicle stem cell markers (e.g., K15 ) to show that esophageal‐derived cells fully\nintegrate into different compartments of the follicle. Co‐staining for epithelial keratins (e.g., K14) would also clarify that the\nsame esophageal‐derived cells are indeed adopting a skin/HF phenotype.\n3. No evidence of function: Are the HF structures producing hair shafts? Cycling? Integrating with the skin niche? Grafting of\nthe ex vivo system would be very informative. There is no data on whether these structures are dynamic or terminally\ndifferentiated.\n4. It is confusing whether this process occurs rarely or frequently. In figures 1D it is very frequent. Also Sup1G states it is\nmore than 90%. Next the authors state: “ The limited expression of the HF stem cell marker CD34 in eHFs became even\nmore apparent when compared to native skin (Fig. 2E), unveiling the inefficiency of the EE-to-skin lineage conversion\nprocess”. Was this the foundation for the rare assumption? The CD34 stains look far from ideal and over exposed. At\nminimum I would stain using a panel of markers or utilize genetic tools (such as: lgr5, lgr6, K15, Lrig, Sox9) before drawing\nthis conclusion.\nAlso it is key to recall that CD34+ HFSCs grown in vitro also lose cd34 (except when grown as organoids in Matrigel) but\nmaintain their SC features. Could it be that simply the CD34 mark is not normal but that it is not indicative?\nIf the authors are correct and it’s a rare event what are the 90% Hfs seen? Is that just cells filling in the molds the maintain\nthe semi eosopehsgal/HF signature? If so why not examine the effect of such a signature or the heterogeneity within the\nformed HFs in correlation with function/morphology/cycling/etc?\nThis point is a critical point of the manuscript and has to be made a lot clearer.\n5. Mechanistic Evidence for HIF1a–SOX9 Axis: The proposed HIF1a–SOX9 axis is underdeveloped. Pharmacological and\ngenetic manipulation (Sox9 deletion) show shifts in CD34 expression. Still, additional immunofluorescence panels (e.g.,\ndouble labeling for SOX9 and HIF1a) and a more direct demonstration that Sox9‐null/HIF1a‐inhibited cells adopt stable hair‐\nfollicle gene signatures is required.\n6. Imaging and presentation: Multiple figures lack clarity and proper zoom-in insets (e.g., Fig 1C). Stratification is mentioned,\nbut not clearly shown or validated.\n• Some immunostainings (e.g., Supplementary Fig. 1E) should be repeated with basal and proliferation markers to better\ncharacterize the tissue structure.\n• Controls are inconsistently presented—e.g., peeled vs. unpeeled dermis, esophagus-only grafts, skin-only grafts. This\ncomparison must be rigorously controlled and quantified.\nAdditional specific comments organized by Figure\nIntroduction:\nRationale for Esophagus–Skin Model: The manuscript would benefit from a more compelling explanation of why\nesophageal tissue was chosen to test epithelial fate switching in a skin‐dermis environment. Although the esophagus is\nsquamous and devoid of adnexal structures, it would be helpful to articulate how this setup advances the concept of adult\nstem cell plasticity or potential clinical applications.\nFigure 1\n• Include additional details on culture duration and viability, how long the peeled dermis and esophageal strips remain\nviable in the 3D culture?\n• Panel 1C could benefit from zoomed‐in insets (e.g., progressive days of re‐epithelialization) to visualize early stages of\nesophageal cell expansion over the dermis.\n• 1C-need clearer zoomed in images of the process.\n• What stages are required for the formation of the hair follicles arise from esophogeal tissue? Author shows the appearance\nof the follicles without clear indication and discussion on how they are formed. Not clear if mCherry cells only fill out empty\nareas. Are the follicles in specific stage? What are the stages of hair formation and how are they similar to embryonic\nformation of hair follicle?\n• Confirm the endogenous staining in figure 1c with epidermal markers, basal layer markers, to confirm that mCherry cells\nindeed convert to epidermal cells.\n• Supplementary figure 1E- cp3 and ki67—need to show the staining in the tissue couples with basal layer marker\n• Need farther verification to confirm epidermal tissues, staining with different markers and zoom in images to show\nstratification, is there epidermal turnover? Is it functional? The author mentions the formation of squamous stratified epithilum\nbut there are no evidence for it.\n• Image 1F is not enough to show pigmentation. Perform fontana mason analysis in order to indicate pigmentation. Are there\nmelanocyte stem cells in the follicles?\n• Are the structures that appear to be the sebaceous glands, actually the sebaceous glands? Perform oil staining and\nzoomed in images of the cells.\n• Is that the hair shaft in 1F middle and right HF?\nFigure 2\n• Use additional markers for hair‐follicle identity including K15\n• Use conventional epithelial markers (K14 for basal cells, K10 or others for differentiation) would confirm that the newly\nformed structures exhibit proper stratification rather than partial epithelial overgrowth.\n• In figure supplementary figure 1C, positive staining of sox 2 indicate the presence of the dermal papilla in the peeled skin,\nis that true for every area where hair formed? Is the presence of the DP required for hair formation in this case? Is there any\ndata regarding the specific fibroblasts populations contributing?\n• You mention low efficiency of hair formation, yet in images such as supp 1 D we see hair formation via esophageal tissue\nthat is almost all over the tissue.\nFigure 3\n• Please also show GFP channel in all the images in order to see the cells originated from skin stromal origin.\n• The conclusion that based on cd34 marker only the regeneration depends on dermal niche is insufficient. Need to check in\nother stem cell populations and cell types.\n• The author shows polyclonal contribution for the esophageal cells make up the hair follicle, yet it will be valuable to add if\ndifferent clones give rise to different populations and their actual contribution to hair follicle formation\nFigure 6\n• The text states there is a SOX9–HIF1a Co‐localization : “The data showed a positive correlation between SOX9 and HIF1a\nlevels; with HIF1a expressing cells being more likely to be positive for SOX9, as measured by immunostaining\n(Supplementary Fig. 6C)” , but panels explicitly showing co-staining are not shown\n• Need proof of concept for sox9 floxed model, are the knocked out cells the ones express HIF1a?\nFigure 7\n• The paper mentions a low incidence of complete HF formation in vivo. A bar chart or table quantifying “follicles per graft”\nacross several mice would help interpret these data.\n• The image in 7G does not seem to be representative to the data mentioned in the text regarding number of hair follicles per\ngrafts.\n• The low frequency of the HF formation event raises questions regarding the validity and the relevance of the mechanism", "reviewer_3": "Bejar et al. dissect the molecular mechanism of fate permissibility in adult cells using a 3D regenerative culture system\nwhere TdT-marked esophageal tissue is placed on epithelium-less GFP skin dermis and the esophageal epithelial (EE)\ncells crawl out and on to the skin dermal support. They nicely show that esophageal to skin lineage conversion of the\nmigrating EE is polyclonal and inefficient (using CD34+) showing mixed EE and skin epithelial (SE) markers and that\nconversion is dermal niche cell-dependent, as removal of the LGR5-GFP dermal cells prevents CD34+ conversion in TdT+\nmigratory cells. scRNA seq of in vitro transitioning TdT cells indicates reactivation of the embryologically expressed HIF1a\nand Sox9 (Fig. S6F) among other gene expression changes. Addition of HIF1a small molecular inducers/inhibitors\nor K14Cre-ERT2 conditional Sox9 mutants cells allows more efficient conversion within the denuded skin microenvironment,\nproviding evidence for an epigenetic block for fate conversion. Overall the methodology follows the lab's previous published\nin vitro wound healing model and standard chamber regeneration methods.\nThe work follows pioneering work from the Barrandon lab and others (refs. 25-31) showing p63+ non-skin epithelium retains\nplasticity to convert to skin in the presence of skin dermal cues, and that of the Fuchs lab showing that Sox9 can reprogram\nskin IFE to HF. While the present work presents an interesting observation, it is still preliminary and not at the level for this\njournal. The concept of plasticity is not new and the authors focus only on unidirectional plasticity of one tissue in the present\nwork. The conversion requires additional characterization (see below) to distinguish loss of esophageal markers as well as\ngain of hair follicle vs. interfollicular epithelial fates. The present work provides a new mechanistic insight by showing\nHIF1a/SOX9 prevent efficient conversion in this system, but need to demonstrate generalizability and reversibility to\nenhance the overall impact of the work. In addition previous work clearly shows the role for Sox9 as a pioneer factor for hair\nfollicle lineage (Yang PMID: 37488435 ; Kadaja PMID: 24532713; Pyczek PMID: 31867038 ). The present work showing\nSOX9 removal enhances contribution to skin HF lineage is quite surprising and contradicts much previous\nwork and so additional controls and experimental data is required to clarify this issue prior to publication.\nMajor Issues:\n1. Generalizability of the work. Barrandon showed cornea, bladder, thymus niches are instructive so would help to confirm\nthis as well in other K14+; p63+ tissues, not just esophagus. Using the authors wound migration assay they could implant\none or more of these other tissues using the same TdT-lineage tracing mouse allele and demonstrate the universality of the\nHIF1a/SOX9 universal block using DMOG/KC7F2/Sox9 fl/fl.\n2. Reversibility. Similarly, Barrandon elegantly showed that plasticity is reversable (Bonfanti PMID: 20725041) and the\nauthors should show whether skin to EE transdifferentiation occurs and is stromal dependent. Again this could be done\nusing the same assay performed in reverse with skin epidermal cells migrating onto esophageal mucosa.\n3. Stromal Dependence. The authors show dermal dependence in Fig. 3B-F. However a critical control is required and the\nauthors should show that replacing the skin substrate with esophageal dermal cells maintains esophageal fate and prevent\nskin transdifferentiation, and whether prostate, bladder corneal stromal cells regulate transdifferentiation to the cognate\nepithelial state rather than skin.\n4. Role of Pioneer Sox 9 in HF lineage. Previous work mentioned above indicates Sox9 is required for HF lineage\ncommitment from skin IFE. The authors show that wound EE reactivates embryonic Sox9 expression and K14 CreERT2\nSox9 fl/ fl enhances rather than blocks transdifferentiation into the HF compartment. This is surprising and an apparent\ncontradiction from previous work. In particular the quantitation of Sox 9 KO CD34+ in Fig 6B-E looks similar to that in the\ncontrol and quantitation from Fig 2B, arguing against a true difference. The authors should perform scRNA seq on Sox9 fl/fl\nTdT eHF (or in Fig. 7H) and compare it to published datasets.For example SOX9 is a major regulator of the hedgehog\npathway critical for HF growth and the so if HF growth is occurring via hedgehog /WNT signaling then the results would\nsuggest an alternative HF pioneer pathway distinct from skin IFE to skin HF. This would be of interest to the broader\naudience.\nMinor Critiques:\n5. It would be interesting to perform immunofluorescence for some key esophageal and skin transcription factors, like Sox2,\nPax9, Lhx2. Whether the CD34+ cells exhibit reduced levels of esophageal transcription factors and elevated skin\ntranscription factors, compared to the CD34- cells or interfollicular cells?\n6. In Figure 4H, what are the cells in grey color? From Figure 4G, these cells are annotated as differentiated cells. Which\ntissue origin are they from?\n7. In Figure 4I and 4J, the authors first generated transcriptional signatures for D3, eSkin-D10, and eEE-D10 cells, and then\nchecked how many genes were overlapped in a pairwise way. Can the authors compare the similarity among these three\ntypes of cells based on whole transcriptome using PCA or Pearson’s correlation analysis?\n8. In the scRNA-seq, can the authors distinguish hair follicle cells versus interfollicular cells in the eSkin samples? From the\ndata presented here, these cells should be quite distinguishable.\n9. The authors used % CD34+ cells per eHF and % CD34+ HF as index for fate conversion, and found that pharmaceutical\ninhibiting Hif1a or knockout of Sox9 increased CD34+ percentage. How about cells in the IFE, do they exhibit more\nconversion towards skin IFE fate?\nMinor edits:\n10. HIF1 has been recently shown to be the major driver for the skin wound healing response, including vascularization and\nepithelial and dermal migration. It would be of interest for the authors to discuss the relationship between wound healing and\nlineage fidelity in light of their work. In particular clusters C8 / C18 at D10 of the assay from this and their previous paper.\n11. Pg. 10. Line 8-10 Fig S4F, G) These results reveal that, by partially restricting the regenerative capacity of epithelial\ncells, the fate conversion process becomes more efficient. The experimental design the authors term “less regenerative” is\nremoving EE dermis and showing that it can transdifferentiate more. The term regenerative is quite vague and should be\nbetter defined here. Moreover an alternative explanation is that EE dermis contains inhibitory signals acting on the\nunderlying skin dermis.\n12. There are numerous typographic errors.\nLine 7 “maintain plasticity at check” should be “in check”.\nPg 8 line 1 “…that maker expression” misspelled"}, "author_response": ""}, {"round": 2, "reviewer_comments": {"reviewer_1": "Thank you for giving me the opportunity to re-evaluate the revised manuscript by Bejar and colleagues. I have carefully\nreviewed both the authors’ detailed rebuttal and the revised version of the manuscript. I am satisfied that the authors have\naddressed my initial concerns in a comprehensive and convincing manner.\nFirst, in response to my request for deeper mechanistic insights beyond the HIF1α–SOX9 axis, the authors performed single-\ncell ATAC-seq experiments. These new data (Fig. 5A–C; Supplementary Fig. 4E–G) convincingly demonstrate chromatin\naccessibility changes during lineage conversion, with +/-30% of genomic regions altered in oesophageal-derived skin\ncompared to controls. Importantly, they highlight increased accessibility at epidermal identity loci (Lhx2) and decreased\naccessibility at oesophageal loci (Krt4), providing clear epigenetic support for the conclusions. In addition, the pseudotime\nanalysis (Fig. 5E–N) strengthens the evidence that subsets of cells move toward an epidermal trajectory. This new dataset\nsignificantly enhances the mechanistic weight of the manuscript.\nSecond, I asked whether lineage conversion involves a fetal-like or developmental intermediate state. The authors show that\ntheir scRNA-seq analysis indeed detects developmental signatures in certain clusters, particularly in the oesophagus-to-skin\ntransition cluster. Although the number of transitioning cells limited the resolution of this analysis, I appreciate the authors’\ntransparency about these technical challenges and their commitment to address this in future work. I wish these data could\nbe integrated version of the manuscript but I understand that this specific point may be the ground of a future manuscript.\nThird, I raised the issue of basal progenitor heterogeneity and regional differences along the esophagus, including the\npotential contribution of Troy+ cells. The authors have clarified that lineage tracing demonstrates a polyclonal origin of\nconversion events, and they show that Tnfrsf19 (Troy) is only expressed in scattered basal cells without enrichment in the\nconversion cluster (Supplementary Fig. 3M). Their new UMAP analyses and discussion (pp. 8–9) strengthen the argument\nthat no single progenitor subpopulation exclusively drives lineage conversion. They also clarified their methodological\nchoice to use the middle portion of the esophagus and provided new data showing no differences across proximal, middle,\nor distal regions (Response Fig. 3). Together, these additions resolve my earlier concerns about progenitor heterogeneity\nand anatomical bias.\nFourth, I had suggested further exploration of functional equivalence of converted cells. While the authors appropriately\nacknowledge that their in vitro model is not intended to achieve complete functional identity, they have added a much more\ndetailed characterization of the converted structures (Fig. 2; Supplementary Fig. 1L–M, 2A–J), including pigmentation,\nsebaceous-like lipid accumulation, and expression of epidermal markers. These data convincingly reinforce the extent of\nconversion achieved and are a substantial improvement over the original submission.\nFinally, I asked whether the findings generalize to other epithelial contexts. The authors now include new heterotypic culture\ndata with oesophagus, tongue, bladder, and tracheal epithelia (Supplementary Figs. 2K–M and 7K–M), and show that SOX9\nacts as a barrier in tracheal epithelium as well. Although they were unable to establish oesophagus-to-intestinal conversions\nin their culture system, they openly discuss this limitation and highlight the translational importance of this question (pp. 18–\n20). The revised discussion also explicitly considers the oncogenic risks of modulating plasticity (p. 20), which I view as an\nessential addition.\nIn summary, the authors have provided extensive new data, thoughtful clarifications, and expanded discussion. Their\nrevisions substantially strengthen the manuscript, particularly through the addition of scATAC-seq and heterotypic culture\nexperiments. I am satisfied that all my initial concerns have been adequately addressed, and I have no further comments.", "reviewer_2": "The authors have done an elegant job in thoroughly addressing all of my comments. Their revisions are clear, thoughtful,\nand have substantially improved the manuscript.\nI have no further comments, and I'm pleased to recommend this paper for publication.", "reviewer_3": "Bejar et al. dissect the molecular mechanism of fate permissibility in adult cells using a 3D regenerative culture system\nwhere TdT-marked esophageal tissue is placed on epithelium-less GFP skin dermis and the esophageal epithelial (EE)\ncells crawl out and on to the skin dermal support. In the revised version, they have expanded the plasticity observation using\nstroma of other stratified epithelial tissues, showing local partial cell fate acquisition that is blocked by Sox9 and HIF1.\nThey include a more detailed single cell analysis of the fate transition and additional histological characterization of the eeHF\nconversion. I appreciate the authors expanded discussion of HIF and Sox9, and additional explanation for the HIF/Sox9\nbarriers and their relationship to committed skin lineages and wound healing. Overall the additional data in this revised\nversion supports a general stratified epithelial signaling environment in the skin dermis that will be of general interest to\njournal readers.\nThis reviewer suggests including the intriguing negative data of the inability of skin epithelium to convert to esophageal\n(Response Fig. 5) and at least discussing the delayed re-epithelialization of esophageal stroma. With the data suggesting\nthe skin stroma provides the local information to convert esophgeal mucosa to skin, these additional data provide clues to\nthe strong skin environmental determinants."}, "author_response": "Response to reviewers’ NCOMMS-25-11438\nAuthors’ point-by-point response to reviewers (NCOMMS-25-11438)\nBejar et. al. entitled “Lifting regenerative barriers promotes epithelial cell fate plasticity supporting\nlineage conversion”\nText from reviewers’ comments presented in blue italics, our responses to reviewers’ questions in black,\nand quoted references to the revised manuscript text are highlighted in orange italics.\nPlease note, American English has been changed to British English; hence, the acronyms have been\nupdated accordingly. Most notably, the reference to oesophageal epithelium is now referred to as OE.\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nThis manuscript by Bejar and colleagues investigates the molecular mechanisms that restrict cell fate\nplasticity in adult epithelial cells, with a focus on the HIF1α-SOX9 axis as a key barrier to esophageal-\nto-skin lineage conversion. Using an innovative 3D regenerative culture system, lineage tracing, and\nsingle-cell RNA sequencing (scRNA-seq), the authors demonstrate that while esophageal cells exhibit\nsome degree of plasticity, they encounter significant barriers in fully adopting a skin epidermal identity.\nImportantly, the study reveals that inhibiting HIF1α or SOX9 enhances cell fate conversion, suggesting\npromising avenues for regenerative medicine applications.\nStrengths:\n- The study challenges traditional concepts of rigid stem cell hierarchies by demonstrating that adult\nepithelial cells retain latent plasticity.\n- The heterotypic culture system (esophageal epithelium cultured on skin dermis) provides an elegant\nmodel to study cell fate conversion.\n- The use of scRNA-seq offers high-resolution insights into cell identity transitions and intermediate\nstates.\n- The concept of removing regenerative barriers has important implications for enhancing tissue repair\nand cell reprogramming strategies.\nOverall, this is a well-conceived and experimentally robust study that advances our understanding of\nepithelial cell plasticity and regenerative barriers. The identification of HIF1α/SOX9 as key restrictive\nfactors is significant, but the manuscript would be strengthened by addressing the following comments\nto provide a more comprehensive understanding of the mechanisms governing cell fate plasticity in\nadult epithelial tissues.\nReviewer #1 - Author response: We thank the reviewer for the overall positive comments, highlighting\nthe “innovative”, “well-conceived” and “robust” nature of our work, which offers “promising avenues\nfor regenerative medicine applications”. We are also grateful to the reviewer for pointing us towards\naspects of the data that needed further consideration in order to strengthen the claims of our study.\nWe have addressed each of the comments as described below.\nMajor comments:\n1. A study from the group of Yann Barrandon (Claudinot et al, 2020), has already demonstrated that\np63-expressing epithelial stem cells from various tissues (including esophagus) have hairy skin\ncompetence. The main novelty of the present study is that the HIF1α-SOX9 axis is a key barrier to\nesophageal-to-skin lineage conversion. While this axis is well-characterized, the study would benefit\nfrom exploring additional regulatory mechanisms. A single cell multi-omics approach combining\nscRNA-seq with scATAC-seq would provide deeper insights into the molecular basis of lineage restriction\nResponse to reviewers’ NCOMMS-25-11438\nand the epigenetic landscape changes during reprogramming. Indeed, Sox9 has been reported as a\npioneer factor in mouse epidermis (Adam et al, 2015 ; Larsimont et al, 2015) and Sox9 conditional\nknockout in a model of esophageal cell reprogramming in vivo modifies chromatin accessibility\n(Vercauteren Drubbel et al, 2021)\nQuestion 1. Reviewer #1\nWe thank the reviewer for acknowledging the novelty of our study and for their insightful suggestions\nwhich certainly enhance the message of our study. Following the reviewer’s comments, we have now\nperformed a single-cell ATAC sequencing experiment (scATAC-seq), including in vitro oesophageal-\nderived skin (oeSKIN), as well as in vitro oesophagus (oeOE) and in vitro skin (sSKIN) as controls. The\nnew data and analysis can be found in the revised version of the manuscript (Fig. 5A, B and\nSupplementary Fig. 4E-G).\nThe UMAP projection of the scATAC-seq data recapitulates the overall scRNA-seq profile, revealing that\na subset of oeSKIN cells acquires a chromatin accessibility landscape more closely resembling that of\nthe epidermal control (sSKIN cells; see cluster 6; Supplementary Fig. 4E-G). This, as suggested by the\nscATAC-seq pseudotime analysis, indicates a shift in chromatin accessibility trajectory consistent with\nacquisition of epidermal identity (Fig. 5L).\nOverall, the data demonstrate that oesophageal-derived skin cells at the culture end-point exhibited\nchanges in chromatin accessibility at ~30% of genomic regions compared to oesophageal control\n(oeOE). Supporting the skin-to-oesophageal lineage conversion process, we find increased accessibility\nat loci associated with epidermal identity and decreased accessibility at loci linked to oesophageal\nidentity (Fig. 5B, C and Supplementary Fig. 4G). In particular, we observe that chromatin regions\nassociated with identity switch loci, including Hif1a (plasticity barrier-related), present a more open\nstate in oeSKIN cells at the transition between oesophagus and skin (along the scATAC-seq pseudotime\ntrajectory; Fig. 5M,N), further reinforcing our overall claim.\nAccordingly, in line with our histological characterisation, as well as existing scRNA-seq data, the\nscATAC-seq analysis also highlights the inefficiency of the lineage conversion process, with only a\nlimited subset of oesophageal cells showing a shift in chromatin accessibility consistent with epidermal\nidentity (see cluster 6; Supplementary Fig. 5E-G; Fig. 5L). This reinforces the main message of our\nstudy highlighting the existence of inherent barriers to cell fate plasticity. However, this inefficiency\nalso comes with technical limitations, rendering very few transitioning cells available for in-depth\nmolecular analysis; an aspect we have found challenging when performing the current scATAC-seq\nanalysis. Despite this, we hope the reviewer agrees that our new data robustly supports the lineage\nconversion process and reinforces our mechanistic observations.\n2. It remains unclear whether lineage rewiring involves a dedifferentiation step and whether this\nprocess involves a fetal-like intermediate phenotype, as observed in regenerating stomach (Vallone et\nal, 2016), intestine (Nusse et al, 2018; Yui et al, 2018) and transcommitted esophageal keratinocytes\n(Vercauteren Drubbel et al, 2021). This deserves further investigation. Analysis of scRNA-seq data\nand/or multi-omics data could help addressing this question.\nQuestion 2. Reviewer #1\nWe fully appreciate the reviewer’s comment. Indeed, during our initial scRNA-seq analysis we had\nspotted a foetal-like/developmental signature in our in vitro re-epithelisation assay. In particular, we\nResponse to reviewers’ NCOMMS-25-11438\nobserved that, as anticipated for a regenerative ex vivo model, basal cells show a heterogeneous foetal-\nlike/developmental signature; with some cell clusters presenting a more prominent developmental\nsignature than others (see below Response Fig. 1). Interestingly, Cluster 8, which contains\noesophageal-to-skin transitioning cells, emerged as a cluster with a variable developmental signature,\ndepending on study used as reference signature1-10 (see below Response Fig. 1). We presume this\nvariability may be linked to technical limitations associated with the reduced number of cells found to\nundergo lineage conversion in our system. Since we agree on the importance of this point, we are\ncurrently working on scaling up our approach to be able to dissect this interesting point in-depth as\npart of a follow up project. This represents future efforts that are currently undergoing in my\nlaboratory. We hope the reviewer agrees with us that this does not preclude or impact the\nobservations presented in the current manuscript.\nResponse Figure 1: Violin plots showing the distribution of previously published gene signatures scores\nacross basal cell clusters in our single-cell RNA-seq dataset (as in Fig. 4B). Signature scores were\ncalculated per cell using Seurat’s AddModuleScore function, which normalises for expression levels\nusing matched control genes to provide a relative measure of gene set activity.\nEmbryonic/developmental oesophageal signatures were derived from Yang et al. (2025)4, Kumar et al.\n(2024)3 , Magaletta et al. (2022)1, McGinn et al. (2021)5, and Kuwahara et al. (2020)2. Foetal-like\ntransitioning cell signatures were obtained from Chen et al. (2023, intestine)6, Vercauteren et al. (2021,\noesophagus)9, Yui et al. (2018, intestine)10, Nusse et al. (2018, intestine)7, and Vallone et al. (2016,\nstomach)8.\nResponse to reviewers’ NCOMMS-25-11438\n3. The inefficiency of lineage rewiring raises questions about whether all basal progenitors possess the\nintrinsic competence to transdifferentiate. To what extent are esophageal progenitors homogenous in\nthe scRNA-seq analysis? As suggested in the first point, scATAC-seq could help determine if certain\nprogenitor subpopulations have an epigenetic background more permissive for transdifferentiation.\nQuestion 3. Reviewer #1 Please consider this response in conjunction with Question 4. Reviewer #1,\nas both are related.\nWe agree with Reviewer #1 that, given the inefficiency of the lineage conversion process and the\nemerging recognition of heterogeneity within the oesophageal basal cell population9,11,12, the\npossibility that oeHFs emerge from a specific cellular subpopulation warrants further discussion. We\napologise for the lack of clarity; this was the intended purpose of sections 3 and 4 of results (lineage\ntracing sections). Using unbiased lineage tracing of epithelial oesophageal cells, we conclude that the\nchange in identity depends on niche signals from the skin dermis, rather than on the oesophageal cell\nof origin (see revised manuscript for further details; section 4 of results).\nAlthough, we cannot rule out the involvement of diverse equipotent basal cell populations (known or\nyet to be described). Our scRNA-seq and newly added scATAC-seq data do not indicate any apparent\nbasal heterogeneity. However, low-abundance populations like may not be readily detectable unless\nthey form a distinct and cohesive cluster with a unique transcriptional and/or chromatin accessibility\nprofile.\nAs rightly indicated by the reviewer in question 5, a recent paper (Grommisch et al., 202411) has\ndescribed a Troy basal cell subpopulation in the mouse oesophagus that shows a proximo-distal\ngradient of expression. The authors showed that this population restricts progenitor proliferation and\n⁺\nenables commitment to differentiation. To address the reviewer’s point, we confirm that we also find\nTnfrsf19 (Troy) to be expressed in scattered cells across the basal population both in vivo (postnatal\ndevelopment and adult samples; McGinn et al., 2021)5, as well as in our heterotypic culture model\n(see scRNA-seq UMAPs below Response Fig. 2). Consistent with the findings by Grommisch et al., Troy\ncells represent a small disperse fraction of basal cells, failing to form a cohesive cluster. We also\n⁺\nobserve a modest enrichment in the resting basal population transitioning towards differentiation\n(Response Fig. 2). Notably, the lineage conversion cluster (C8) associated with the oesophageal\nidentity switch does not express Tnfrsf19. While we cannot entirely exclude the contribution of Troy+\ncells, the low frequency of Tnfrsf19-expressing cells in our system and the polyclonal nature of eHF\nformation (as shown by our lineage tracing analysis, Figs. 3 and 4) make it improbable that Troy cells\nare the sole contributors to lineage conversion.\n⁺\nIn summary, while multiple basal cell pools may contribute to lineage conversion, our lineage tracing\ndata suggest that no single basal subpopulation is solely responsible of this process, with oeHF\nformation being a widespread and polyclonal process.\nWe have now included in the revised manuscript UMAP plots showing Tnfrsf19 expression\n(Supplementary Fig. 3M) and have added a reference in the revised manuscript to acknowledge the\npotential contribution of Troy and other basal cell populations to the lineage conversion process. The\ntext reads:\n⁺\n“Given the inefficiency of the process and the emerging recognition of heterogeneity within the\noesophageal basal cell population9,11,12, it is important to consider the dynamics of basal cell\nResponse to reviewers’ NCOMMS-25-11438\ncontribution to the lineage conversion process. To determine whether the basis of the inefficient OE-to-\nskin fate re-specification was associated with restricted competency of a heterogeneous cell\npopulation, we analysed the KRT15 and CD34 expression within oeHF confetti labelled clones.”… (p8;\nl18-23)\n…”While we cannot discard that multiple oesophageal basal cell pools may contribute to lineage\nconversion, our lineage tracing data suggest no single basal subpopulation is solely responsible for this\nprocess.”…. (p9; l11-12).\nGrommisch et al., 2024 McGinn et al., 2021 Bejar et.al (manuscript)\nResponse Figure 2: Cell distribution in the UMAP dimensionality reduction space showing Tnfrsf19\n(TROY) expression as per Grommisch et al., 202411 (adult oesophagus), McGinn et al., 20215 (Postnatal\nand adult oesophagus), and Bejar et.al. (current manuscript, Supplementary Fig. 3M) from left to right.\n4. In the same line, recent studies have highlighted a cephalocaudal gradient of Troy expression in basal\nepithelial cells of mouse esophagus (Grommisch et al, 2024). In addition, some basal progenitors from\nthe proximal part of the esophagus can differentiate into taste buds (epithelial appendages)\n(Vercauteren Drubbel & Beck, 2023). The competence of basal progenitors to change lineage may\ntherefore be heterogeneously distributed in the esophagus. Discussing these regional differences would\nprovide better context for the observations.\nQuestion 4. Reviewer #1 Please consider this response in conjunction with Question 3. Reviewer #1,\nas both are related.\nWe thank the reviewer for raising this point and apologise for the lack of clarity. In our study we use\nthe middle half of the oesophagus rather than the entire oesophagus, we have clarified this in the\nrevised manuscript (refer to Results section 1 and Methods section; 3D Oesophageal-Dermal\nHeterotypic and control cultures). Given the obvious anatomical and architectural differences, as well\nas emerging cellular heterogeneity11,12 between the main body of the oesophagus and the most\nproximal and distal areas, we focused our work on the main body of the oesophagus to minimise any\npotential cephalocaudal complexity. See below Response Fig. 3 for the details on oesophageal\npreprocessing; the most proximal and distal parts of the oesophagus are cut out prior any further\ndownstream assay, including heterotypic cultures. This allows us to refer back to our original study on\noesophageal cell fate dynamics where a similar approach was used13.\nResponse to reviewers’ NCOMMS-25-11438\nHowever, given the importance of this point, we have repeated our heterotypic cultures further\nseparating tissue samples (middle half of the oesophagus) by their anatomical position along the\nlongitudinal axis of the oesophagus (proximal, middle, distal, see below Response Fig. 3). The aim here\nis to determine whether our original observations may have been hindered by artefacts related to the\nlongitudinal origin of the samples used. Interestingly, we did not see any differences either in the re-\nepithelialisation efficiency or lineage conversion process, as assessed by the emergence of HF markers,\nCD34 and KRT15 (Response Fig. 3).\nResponse Figure 3: (A) Representative images oesophageal tissue pre-processing method. Upon\ndissection, the most proximal and distal quarters of the oesophagus were discarded. Only the middle\nhalf of the oesophagus was used to generate 3D organ cultures on denuded oesophageal or skin\nstroma (dermis).\n(B) Image showing samples used for new regional heterotypic cultures assays. Oesophagi were further\ncut into defined proximal, middle and distal regions, and used to assess regional differences in our\nheterotypic organ cultures.\n(C) Re-epithelialisation of dermis after 8 days of culture, expressed as percentage of area covered.\n(D-E) Quantification of HF marker expression in oesophageal-derived HFs derived from different regions\nalong the longitudinal axis of the oesophagus. CD34 and KRT15 intensity showed no significant\ndifferences across samples. (n=3 biological replicates, 3 technical replicates each).\nResponse to reviewers’ NCOMMS-25-11438\nAdditional Suggestions (discussion)\n1. While molecular changes associated with lineage conversion are demonstrated, the study lacks\nassessment of whether converted cells are functionally equivalent to native skin cells (e.g., skin barrier\nfunction, response to mechanical stress, wound healing capacity). This should be discussed.\nMinor point 1. Reviewer #1\nWe fully appreciate the reviewer’s point. In response to this point, we have expanded our analysis with\na more comprehensive characterisation of oesophageal-derived hair follicles (oeHFs). Specifically, we\nhave included staining of oeSKIN wholemounts for various epidermal markers, such as LHX2, KRT15,\nKRT10, SCD1, and SCA-1 (Fig. 2 and Supplementary Fig. 2A-J), which further support the conversion\nfrom oesophageal to skin lineage. Additionally, we utilised Oil Red O staining to highlight lipid\naccumulation in oesophageal cells (akin of sebaceous glands; Supplementary Fig. 1M), and Fontana\nMasson staining to track melanin incorporation within the oeHFs (Supplementary Fig. 1L). These new\ndata strengthen the evidence for the lineage conversion process. The text has been amended in line\nwith the new data provided:\n“Remarkably, oesophageal-derived hair follicles oeHFs (red) showed features reminiscent of the in vivo\nappendages. These included pigmented fibre-like structures (Fig. 1G and Supplementary Fig. 1K)\npositive for Fontana-Masson stain, which labels melanin (Supplementary Fig. 1L). Oesophageal-\nderived HFs were also positive for Oil Red O staining, indicating the accumulation of sebum in the\nproximal area where sebaceous glands are typically found (Supplementary Fig. 1M).”(p5, l24-28).\nFinally, while our data provide compelling evidence for early lineage conversion events, we wish to\nreiterate that the central novelty of this study lies in demonstrating the presence of lineage barriers\nthat inherently constrain inappropriate lineage conversion (regulated, at least in part, via the HIF1a-\nSOX9 axis). In this context, we do not anticipate observing a complete identity switch in our in vitro\nmodel. And, hence, attempting a direct functional comparison between oesophageal-derived HFs and\nskin would be beyond reach in the context of our observations. We hope the reviewer will be satisfied\nby the deeper marker oeHF characterisation provided in the revised manuscript.\n2. The study focuses solely on esophagus-to-skin conversion. Testing other transitions (e.g., esophagus-\nto-intestine) would reveal whether similar barriers exist in embryologically related tissues. This is really\nimportant in the field as esophageal-to-intestinal transdifferentiation may occur under pathological\nconditions (metaplasia). This is obviously beyond the scope of the manuscript, but this should be\ndiscussed.\nMinor point 2. Reviewer #1\nWe thank the reviewer for their insightful comment. Following the reviewer’s recommendation, we\nhave now included heterotypic cultures combining epithelium and stromal from additional epithelial\ntissues. These include oesophageal cells exposed to the ectopic stroma of the tongue and bladder\n(Supplementary Fig. 2K-M), tracheal epithelium exposed to denuded dermis (Supplementary Fig. 7K-\nM), as well as relevant controls. As anticipated, epithelial cells expressed ectopic markers as instructed\nby the stroma they were exposed to; KRT8 for denuded tongue and bladder stroma (Supplementary\nFig. 2K-M), CD34 and KRT15 for denuded dermis (Supplementary Fig. 7K-M). We have further shown\nthat the SOX9 lineage conversion barrier also operates in the tracheal epithelium (Supplementary Fig.\n7L, M), revealing the relevance of our mechanistic insights beyond the oesophageal epithelium.\nResponse to reviewers’ NCOMMS-25-11438\nRegarding the oesophagus-to-intestine transition, we have indeed attempted to perform our\nheterotypic cultures by growing oesophageal epithelium over the stroma of glandular tissues,\nincluding the stomach and the intestine. However, we found that our in vitro assay does not allow for\nepithelial cells to repopulate glandular tissue stroma. We have tested a number of different culture\nmedia conditions, including our standard conditions as well as intestinal and stomach organoid media.\nHowever, epithelial cells were not able to efficiently grow out of the original tissue explant under any\nof the conditions tested. Given its translational relevance, follow-up work, beyond this study, will focus\non optimizing culture conditions to address this important question.\nWe have revised the manuscript to highlight the translational relevance of understanding epithelial\ncell fate plasticity in the context of oesophageal metaplasia. The revised text now reads: “It has now\nbecome widely accepted that cell fate decisions are not as predetermined as originally thought. In\nresponse to injury or environmental perturbations, adult epithelial cells present a remarkable fate\nplasticity allowing them to respond to changes in tissue needs 14-16. Yet, if dysregulated, this\nadaptability can promote epithelial disease and cancer 15,17-19. Particularly, in the context of the\noesophagus, columnar metaplasia has been associated with the development of oesophageal\ncancer9,20-22. Hence, understanding the largely unclear mechanisms balancing cell fate plasticity is\ncritical to unveil its regenerative potential while preventing inherent tumorigenic risks 23.” (p18; l2-8).\n3. The potential oncogenic implications merit further discussion. While inhibiting HIF1α-SOX9 increases\nplasticity (potentially beneficial for regeneration), this could prove risky in cancer contexts. The authors\nshould consider discussing how modulating lineage restriction barriers might affect tumor initiation or\nprogression in epithelial tissues.\nMinor point 2. Reviewer #1\nWe completely agree with the reviewer and thank them for this word of caution. Understanding the\nmechanisms modulating cell fate plasticity indeed holds great promise to promote tissue repair and\nunleash the true regenerative nature of mammalian tissues. However, it does come at a cost. The\nactivation of plasticity cell fate programs or the disruption of lineage barriers carries inherent risks,\nincluding aberrant tissue growth and malignant transformation15,17-19,23. Hence, although diving into\nthe mechanisms modulating cell fate plasticity are of utmost importance, they should underscore the\nneed for caution. A major challenge lies ahead of us to precisely understanding how to fine-tune\nepithelial plasticity; maximizing its reparative potential while minimizing unintended pathological\nconsequences. We have highlighted this in the revised version of the manuscript, which now reads:\n“This study opens exciting new avenues to decipher the inner workings dictating cell fate plasticity,\nproviding a benchmark to identify the principles underlying the full regenerative capacity of epithelial\ncells. However, given the potential risk of abnormal tissue growth and malignancy15,17-19,23, it also\nunderscores the need for caution. Careful consideration must be given when developing strategies to\neither promote tissue plasticity or disrupt plastic barriers in order to minimise unintended pathological\nconsequences. Much work is still needed to understand how to finely balance the plastic response of\nour epithelial tissues or therapeutic gain.” (p20; l4-10).\nReferences\nAdam RC, Yang H, Rockowitz S, Larsen SB, Nikolova M, Oristian DS, Polak L, Kadaja M, Asare A, Zheng\nD, et al (2015) Pioneer factors govern super-enhancer dynamics in stem cell plasticity and lineage\nchoice. Nature 521: 366–370\nResponse to reviewers’ NCOMMS-25-11438\nClaudinot S, Sakabe J-I, Oshima H, Gonneau C, Mitsiadis T, Littman D, Bonfanti P, Martens G, Nicolas\nM, Rochat A, et al (2020) Tp63-expressing adult epithelial stem cells cross lineages boundaries\nrevealing latent hairy skin competence. Nat Commun 11: 5645\nGrommisch D, Wang M, Eenjes E, Svetličič M, Deng Q, Giselsson P & Genander M (2024) Defining the\ncontribution of Troy-positive progenitor cells to the mouse esophageal epithelium. Dev Cell 59: 1269-\n1283.e6\nLarsimont J-C, Youssef KK, Sánchez-Danés A, Sukumaran V, Defrance M, Delatte B, Liagre M, Baatsen\nP, Marine J-C, Lippens S, et al (2015) Sox9 Controls Self-Renewal of Oncogene Targeted Cells and Links\nTumor Initiation and Invasion. Cell Stem Cell 17: 60–73\nNusse YM, Savage AK, Marangoni P, Rosendahl-Huber AKM, Landman TA, de Sauvage FJ, Locksley RM\n& Klein OD (2018) Parasitic helminths induce fetal-like reversion in the intestinal stem cell niche. Nature\n559: 109–113\nVallone VF, Leprovots M, Strollo S, Vasile G, Lefort A, Libert F, Vassart G & Garcia M-I (2016) Trop2\nmarks transient gastric fetal epithelium and adult regenerating cells after epithelial damage.\nDevelopment: dev.131490\nVercauteren Drubbel A & Beck B (2023) Single-cell transcriptomics uncovers the differentiation of a\nsubset of murine esophageal progenitors into taste buds in vivo. Sci Adv 9: eadd9135\nVercauteren Drubbel A, Pirard S, Kin S, Dassy B, Lefort A, Libert F, Nomura S & Beck B (2021)\nReactivation of the Hedgehog pathway in esophageal progenitors turns on an embryonic-like program\nto initiate columnar metaplasia. Cell Stem Cell: S1934-5909(21)00152–1\nYui S, Azzolin L, Maimets M, Pedersen MT, Fordham RP, Hansen SL, Larsen HL, Guiu J, Alves MRP,\nRundsten CF, et al (2018) YAP/TAZ-Dependent Reprogramming of Colonic Epithelium Links ECM\nRemodeling to Tissue Regeneration. Cell Stem Cell 22: 35-49.e7\nReviewer #2 (Remarks to the Author):\nReview of “Lifting regenerative barriers promotes epithelial cell fate plasticity supporting lineage\nconversion”\nBy Bejar et al.\nReviewer comments:\nSummery:\nThis manuscript addresses a potentially compelling question, whether esophageal epithelial cells\ntransdifferentiate into skin/hair follicle lineages under specific in vitro and in vivo conditions-yet it falls\nshort of providing robust data to substantiate its main claims. In particular, the central assertions that\n-\nfull hair follicle like structures are derived from esophageal epithelium and that the HIF1a–SOX9 axis\nis a key mechanistic barrier are not convincingly supported by the limited immunostaining, insufficient\n- -\nmarker validation, and sparse quantitative data presented. As currently written, the paper leaves\ncritical gaps in experimental detail and does not demonstrate that the new structures are fully\nfunctional or definitively esophageal in origin, weakening confidence in the broader conclusions.\nOn a more personal note, I like the idea of the paper and believe it can have broad implications,\nespecially in the context of adult epithelial plasticity and regenerative medicine. However, as currently\npresented, the manuscript does not sufficiently support its central claims.\nResponse to reviewers’ NCOMMS-25-11438\nReviewer #2 - Author response: We are most grateful to the reviewer for appreciating the “compelling\nquestion” addressed in our study and recognizing the “broad implications” of our work “especially in\nthe context of adult epithelial plasticity and regenerative medicine”.\nIn light of the reviewer’s detailed and insightful assessment of our study, we have implemented the\nproposed recommendations, which further enhance the robustness of our results and strengthen the\nmanuscript.\nMajor Comments\n1. Extent of Bona Fide Hair Follicle Formation: The authors provide preliminary evidence (e.g., CD34,\nKRT17/24 staining, partial pigmentation) that esophageal cells may become hair follicle like. However,\n-\nfurther morphological and molecular validation (for instance, K15, or other keratins; staining for\n- -\nmelanin, basal layer maker) would strengthen the claim that complete hair follicles are formed rather\nthan partial or rudimentary structures or cells simply filling in the molds left upon epidermal/HF\nremoval.\n• There is no clear architectural evidence is provided that the follicles formed are organized into bulge,\nmatrix, ORS, etc.\n• Pigmentation is vaguely mentioned; Fontana-Masson staining is needed.\n• If sebaceous glands are suggested, perform Oil Red O or equivalent lipid staining to validate. Also\nKi67 or proliferation markers would help show proper biology of the SG and basal layer HG, bulge.\n• More than anything, I would like to see clear data showing RFP cells in a GFP setting (dermal cells)\nwith markers of HFSC as well as IFE differentiation markers. More high quality multi-color stainings are\nneeded to be convincing. I am not certain that the HFs I see are indeed novel HFs. It appears that is\nsome scenarios these are cells filling in the molds left in the dermis and assume a shape similar to a\nhair follicle. In such a case would it be surprising that they share some similarity with HF\nkerationcytes/HF cellular populations as well as esophageal. Wouldn’t it be expected that the dermal\nmold/scaffold would generate such an effect?\nQuestion 1. Reviewer #2\nWe thank the reviewer and acknowledge the importance of their comments and suggestions. As\nrequested, the revised manuscript now includes a deeper characterisation of oesophageal derived hair\nfollicles (oeHFs), including additional markers that reinforce the oesophageal-to-skin lineage\nconversion notion. New data is summarised below.\na) To further support the oesophageal switch towards HF identity, we have included\nimmunofluorescent images showing additional epidermal markers in oesophageal-derived\nskin (oeSKIN), including LHX2, KRT15, KRT10, SCD1 and SCA-1 (Fig. 2 and Supplementary Figs.\n2A-E).\nTo strengthen the oesophageal-to-skin identity switch, we have also included a more\ncomprehensive marker analysis showing the downregulation of key oesophageal markers,\nincluding Krt4 and Sox2, within oesophageal-derived skin (oeSKIN; Supplementary Figs. 2F-H).\nTo add additional depth to this point, we have made use of our scRNA-seq data to identify\ndifferentially expressed genes (DEG) between oesophagus and skin, and used them to illustrate\nt illustrate\nthe cell fate transition. As anticipated, the data show that genes enriched in oeOE versus sSKIN\nare expressed at lower levels in oesophageal-derived skin (oeSKIN; Figs. 4L-N).\nAdditionally, regarding the request of more markers, we would like to note that there is a\nlimited repertoire of markers known to be specific to skin, i.e. not expressed in the\nResponse to reviewers’ NCOMMS-25-11438\noesophagus. Despite this, we hope the reviewer agrees that our newly added data robustly\nsupports the lineage conversion process.\nb) Single-cell ATAC sequencing experiment (scATAC-seq); we have performed scATAC-seq,\nincluding in vitro oesophageal-derived skin (oeSKIN), as well as in vitro oesophagus (oeOE) and\nin vitro skin (sSKIN) as controls. The new data and analysis can be found in the revised version\nof the manuscript (Fig. 5A and Supplementary Fig. 4E-G).\nThe UMAP projection of the scATAC-seq data recapitulates the overall scRNA-seq profile,\nrevealing that a subset of oeSKIN cells acquires a chromatin accessibility landscape more\nclosely resembling that of the epidermal control (sSKIN cells; see cluster 6; Supplementary\nFig. 4E-G). This, as suggested by the scATAC-seq pseudotime analysis, indicates a shift in\nchromatin accessibility trajectory consistent with acquisition of epidermal identity (Fig. 5L).\nc) In relation to oeHF pigmentation, the Fontana Mason staining now included in the revised\nmanuscript shows that the denuded dermis retains melanin after peeling, which may be taken\nup by oesophageal keratinocytes during oeHF formation (Supplementary Fig. 1L). We further\nsupport this notion by showing how pigmented structures in Fig. 1G and Supplementary Fig.\n1K are indeed of oesophageal origin (i.e. express Tomato reporter).\nd) To confirm the conversion towards a sebaceous gland identify we show that, besides the\nacquisition of the sebaceous gland morphology at the upper region, oeHFs also contain cells\nexpressing the sebaceous gland marker stearoyl-CoA desaturase 1 (SCD1; Figs. 2B and\nSupplementary Fig. 2A), and can accumulate lipid droplets (as shown by Oil Red O staining in\nSupplementary Fig. 1M).\ne) In relation to the request of images showing GFP and RFP together with markers, it’s\nimportant to mention that, to enable good antibody penetration and 3D confocal\nreconstruction of the tissue, oeSKIN wholemounts are peeled (dermal composite was removed\nprior immunostaining). Hence, epithelial stainings in most cases only show the epithelial\ncompartment post-wholemount peeling. Please note that EGFP confocal channel (green\nchannel) is always ON (as stated in the figure caption). Hence, the lack of EGFP signal merely\nshows the lack of EGFP skin-derived cells in the oeSKIN wholemounts. Where possible images\nshowing tdTomato (OE) and EGFP (dermal) composites are shown (Figs. 1C, E and\nSupplementary Figs. 1F, 2D and 7E-I).\nAs per oeHF architecture, we agree with the reviewer that emerging oeHFs do not yet show a fully\nmature HF morphology and marker compartmentalisation. However, we have not been surprised by\nthis observation given the current challenges associated with recreating hair follicle-like structures in\nin vitro systems24-28. As far as we are aware, the only approach able to fully reproduce this in vitro has\nbeen achieved by recapitulating the HF developmental programme in iPSCs derived hair follicle\norganoids26,27. Otherwise, studies have traditionally relied on in vivo grafting assays to show the\nemergence of relevant markers in lineage conversion models underscoring the challenge of hair follicle\nmarker detection in in vitro systems29,30. To further support the notion that this is a feature associated\nwith ex vivo conditions, we have now included additional controls, including skin-derived in vitro HFs\n(sHF) in relevant figures, which show that the unusual morphology and ectopic marker expression are\nfound in in vitro reconstituted HFs regardless of their skin or oesophageal origin. We have alluded to\nthis important point in the revised manuscript: “Of note, oeHFs showed atypical morphology and\nectopic expression, with markers localised outside their anatomical position within in vivo skin HFs.\nThis, however, was observed to be a recurrent feature of ex vivo reconstituted HFs regardless of their\nskin or oesophageal origin (Compare sSKIN and oeSKIN versus Skin in (Fig. 2B and Supplementary Fig.\nResponse to reviewers’ NCOMMS-25-11438\n2A) in line with previous reports26,27,31-33. Reassuringly, oesophageal control samples in vivo (OE) and in\nvitro (oeOE) confirmed the lineage specificity of these skin markers (Supplementary Figs 2B, C)34.” (p6;\nl15-22).\nFinally, despite the compelling evidence pointing towards early lineage conversion events, as provided\nby existing and newly presented experiments, we would like to reinforce the point that the novelty of\nthis study centres on the existence of lineage barriers, which naturally limit inadequate lineage\nconversion events. Hence, under this premise, we would not anticipate a complete identity switch in\nour in vitro model system. On the contrary, we believe that this highly reproducible and scalable system\nof partial lineage conversion offers an unprecedented model where to start elucidating the largely\nunknown mechanisms regulating the transition states associated with cell fate plasticity.\n2. Markers Confirming Epithelial and Appendage Identities: Because CD34 can also mark hematopoietic\nor stromal cells, relying on CD34 alone as the readout for hair follicle stem cell identity is limiting. Also,\nthe cd34 staining (and others) are far from ideal. It is over exposed and appears somewhat non-specific.\n- -\nIn general figure 2B is far from ideal. As stated above a lot more images with GFP and RFP together\nwith markers (wholemounts and/or sections) are required. Again, the manuscript should include\nadditional hair follicle stem cell markers (e.g., K15 ) to show that esophageal derived cells fully\nintegrate into different compartments of the follicle. Co staining for epithelial keratins (e.g., K14) would\n-\nalso clarify that the same esophageal derived cells are indeed adopting a skin/HF phenotype.\n- - \nQuestion 2. Reviewer #2\n- \nWe acknowledge the reviewer’s point regarding HF markers. To address this comment, we have now\nadded a deeper characterisation of oesophageal derived hair follicles (oeHFs). New data includes:\na) CD34- We acknowledge the fact that CD34 may potentially come from hematopoietic or\nstromal cells. For this reason, we originally included the co-staining of KRT14 (basal epithelial\nmarker), PDGFRa (fibroblast markers) and CD34 (HF putative marker) in oeSKIN, see\nSupplementary Fig. 2E. Our results show that CD34 expression indeed colocalises with KRT14\nin oesophageal epithelial cells within oeHFs. And, hence, CD34 expression in oeHFs is not the\nresult of stromal contamination.\nb) Keratin 15- Additionally, to address the reviewer’s concerns regarding the fidelity of CD34 as\nHF marker, we have now included in relevant experiments (including HIF1a-SOX9 functional\nassays), not only CD34, but also KRT15, as HF markers (Figs. 2, 3E, 5O-R and 6, Supplementary\nFigs. 2A-C, 3A-C and 6J-L). Our new data further supports the inefficiency of the lineage\nconversion process, as shown the KRT15 expression quantification, and demonstrates that this\nis not limited to CD34 expression (Figs. 2D, E). Overall, these new results reinforce our claims\nregarding the lineage conversion process, as well as the proposed regulatory mechanism via\nthe HIF1a-SOX9 axis.\nc) Hair reconstitution assays- Additionally, we would like to reinforce that existing functional\nexperiments also show that, in in vivo hair reconstitution assays, oeHFs were entirely derived\nfrom oesophageal cells (exclusively tomato+ oeHFs; Fig. 7A-H and Supplementary Fig. 7A-I).\nAnd, what is more, in vivo oeHF formation may be modulated by targeting the HIF1a-SOX9\naxis. Both KC7F2 treatment, which inhibits HIF1a, and genetic SOX9 loss of function\nexperiments led to an increased formation of oesophageal derived HFs in grafting experiments\nusing primary cells (Fig 7F-H). We believe that these results robustly support the existence of\nResponse to reviewers’ NCOMMS-25-11438\nlineage conversion barriers, and show that the lineage conversion in this context is a regulated\nprocess and not a mere experimental artefact.\nd) HF Markers (as indicated above)- Staining of oeSKIN wholemounts for additional markers\nsupporting the conversion from oesophageal to skin lineage (Fig. 2 and Supplementary Figs.\n1L and 2A-E).\n3. No evidence of function: Are the HF structures producing hair shafts? Cycling? Integrating with the\nskin niche? Grafting of the ex vivo system would be very informative. There is no data on whether these\nstructures are dynamic or terminally differentiated.\nQuestion 3. Reviewer #2\nWe thank the reviewer for their comment and fully acknowledge the importance of assessing hair\nfollicle (HF) cycling in reconstitution assays. Understandably, the current heterotypic approach\npresents limitations, as do most existing reconstitution systems. To date, and to the best of our\nknowledge, even state-of-the-art iPSC-derived HF organoids have not yet been shown to cycle26,27.\nHence, it seems rather unremarkable that we have been unable to follow hair cycling in our 3D\noesophageal-derived skin cultures.\nNonetheless, we do show that epithelial cells in oeHFs proliferate in vitro (Supplementary Figs. 1E, F)\nand contribute to rudimentary hair shafts in in vivo skin reconstitution assays (Fig 7), thereby\ndemonstrating the ability of oeHFs to generate functional hair structures (to a certain extent). Whether\nsuch ex vivo-generated HFs are capable of undergoing long-term cycling remains a major unresolved\nchallenge in the field.\n4. It is confusing whether this process occurs rarely or frequently. In figures 1D it is very frequent. Also\nSup1G states it is more than 90%. Next the authors state: “ The limited expression of the HF stem cell\nmarker CD34 in eHFs became even more apparent when compared to native skin (Fig. 2E), unveiling\nthe inefficiency of the EE-to-skin lineage conversion process”. Was this the foundation for the rare\nassumption? The CD34 stains look far from ideal and over exposed. At minimum I would stain using a\npanel of markers or utilize genetic tools (such as: lgr5, lgr6, K15, Lrig, Sox9) before drawing this\nconclusion.\nAlso it is key to recall that CD34+ HFSCs grown in vitro also lose cd34 (except when grown as organoids\nin Matrigel) but maintain their SC features. Could it be that simply the CD34 mark is not normal but\nthat it is not indicative?\nIf the authors are correct and it’s a rare event what are the 90% Hfs seen? Is that just cells filling in the\nmolds the maintain the semi eosopehsgal/HF signature? If so why not examine the effect of such a\nsignature or the heterogeneity within the formed HFs in correlation with\nfunction/morphology/cycling/etc?\nThis point is a critical point of the manuscript and has to be made a lot clearer.\nQuestion 4. Reviewer #2\nWe agree with the reviewer that, in retrospective, the point referring to the “efficiency” of the process\nwas not properly conveyed and appears rather misleading. The statement referring to 90% efficiency\n(Supplementary Fig. 1J) indeed refers to re-epithelialisation or, as the reviewer refers to “cells filling\nthe molds” of pre-existing HFs. We have now amended the text to make a clear distinction between\nthe two statements. This has no connection to the lineage conversion process per se, and merely\nacknowledges that cells are able to efficiently repopulate the denuded dermis, hence this should not\nResponse to reviewers’ NCOMMS-25-11438\nrepresent a limitation for cells to receive instructive cues from the stroma. See amended text. The text\nreads: “Re-epithelialisation of the denuded skin and HF niches by oesophageal cells in heterotypic\ncultures represented a highly efficient process, with 91.4 ± 1.8% of the original EGFP+ HFs being\nreplaced by oesophageal-derived HFs-like structures (tdTomato; red) at day (D) 10 post-culture\n(Supplementary Fig. 1J).” (p5; l20-23).\nThe poor efficiency of the lineage conversion process, instead, was initially based on CD34 expression.\nHowever, we completely take the reviewers point regarding the fact that CD34 expression can be\nsignificantly impacted by experimental conditions. Hence, to support our claim that the lineage\nconversion process is inefficient, we have now also included the quantification of KRT15 expression in\noesophageal-derived HFs (Figs 2D, E). Regarding the expression of CD34 in vitro, we can only elaborate\non the fact that in our 3D culture system cells are grown directly over alive stroma in order to more\nclosely mimic in vivo conditions.\nRegarding the heterogeneous competency of oeHFs to acquire certain HF markers, we believe this is\ncovered, at least in part, by the epidermal marker characterisation (extended now in the new version\nof the manuscript; Fig 2 and Supplementary Fig. 2). Our data shows that certain epidermal markers\nsuch as KRT24 and KRT17, become widely expressed. While others such as CD34, KRT15, and KRT10\nexpression is only acquired by certain oeSKIN cells (Fig. 2D). We have now added a text remark for\nclarity (See amended text below). In relation to the characterisation of this heterogeneity, as shown\nby our lineage conversion trajectory analysis (scRNA-seq and scATAC-seq; Figs. 4 and 5; Supplementary\nFigs. 4 and 5) as well as mechanistic studies (Figs. 6 and 7; Supplementary Figs. 6 and 7), we associate\nthis to the existence of barriers to lineage conversion. In particular, we define the HIF1a-SOX9 axis as\na critical regulatory barrier. To further support his notion, we have now included new mechanistic data\n(Fig. 5O-R, Fig. 6 and Supplementary Fig. 6J-L). For further details, please refer to our answer to the\nnext point (Question 5. Reviewer #2).\nAmended text:\n“Despite tangible evidence proving the ability of oesophageal cells undergo environmental\nreprogramming and switch towards skin identity oeHF, further inspection of the oeHF data revealed\nthe limited nature of the fate conversion process. In particular, the expression of HF markers was highly\nvariable across oeHFs appendages, ranging from widespread expression in the case of KRT17 and\nKRT24 (~78% and ~91% oeHFs expressing these markers, respectively), to only a reduced proportion of\neHFs (~28%) expressing CD34 (Fig. 2D). The expression was similarly variable within individual oeHFs,\nwith the number of cells expressing CD34 and KRT15 representing just a minor proportion when\ncompared to native skin (Fig. 2E). These results unveiled the inefficiency of the OE-to-skin lineage\nconversion process.” (p7; l9-17).\n5. Mechanistic Evidence for HIF1a–SOX9 Axis: The proposed HIF1a–SOX9 axis is underdeveloped.\nPharmacological and genetic manipulation (Sox9 deletion) show shifts in CD34 expression. Still,\nadditional immunofluorescence panels (e.g., double labeling for SOX9 and HIF1a) and a more direct\ndemonstration that Sox9 null/HIF1a inhibited cells adopt stable hair follicle gene signatures is\nrequired.\n- - -\nQuestion 5. Reviewer #2\nWe apologise for the lack of depth in our mechanistic validation of the HIF1a-SOX9 axis. Please find\nbelow new experiments, included in the revised manuscript, further demonstrating the role of the\nHIF1a-SOX9 axis as a barrier in the lineage conversion process:\nResponse to reviewers’ NCOMMS-25-11438\na) Hypoxic cues induce SOX9 expression to establish the lineage conversion barrier– We have\nincluded additional experiments that add functional depth to the proposed mechanism. To\ndirectly test the interplay between hypoxic signals and SOX9 during the lineage conversion\nprocess, Krt14CreERT;Sox9fl/fl derived heterotypic cultures were exposed to a hypoxic treatment (4h)\nto simultaneously block SOX9 expression and stimulate HIF1a (Fig. 6G-I). We anticipated that if\nHIF1a and SOX9 expression were acting as independent barriers blocking the lineage conversion\nprocess, the hypoxic treatment would rescue the Sox9fl/fl phenotype, bringing HF markers back to\ncontrol levels. However, this was not the case, the hypoxic treatment did not restore HF markers\n(Fig. 6G-I), indicating that, in this context, HIF1a is acting via SOX9.\nIn line with these results, we now also include experiments showing that SOX9 expression is\nindeed directly regulated by hypoxic signals. New data from hypoxic ex vivo oesophagi reveal an\nincrease in SOX9 expression that can be rescued by treating them with the HIF1a inhibitor KC7F2\n(Fig. 6A,B), indicating that SOX9 is indeed downstream HIF1a.\nOverall, these new observations further demonstrate that the cell identify barrier is exerted via\nthe HIF1a-SOX9 axis and that SOX9 is downstream HIF1a, as originally proposed.\nb) SOX9 promotes HF identity in the skin– Interestingly, and in line with the reviewer’s comment\nregarding the discrepant role of SOX9 in the skin, new experiments on sSKIN (in vitro skin-derived\nfrom epidermis) reassuringly showed the opposite trend to oeSKIN (oesophageal derived skin);\nwith Sox9fl/fl epidermal cells expressing less CD34 and KRT15 than controls (Supplementary Fig.\n6J-L). Accordingly, in vivo oeHFs derived from Krt14CreERT;SOX9fl/fl hair reconstitution assays were\nnot viable long-term, suggesting that once the HF identity is established, SOX9 expression is\nrequired for oeHF maintenance. These observations reveal that SOX9 is having the opposite effect\nin endogenous skin cells and oesophageal cells, and demonstrate the context specific role of\nSOX9. This is not an entirely surprising observation, given that SOX9 is a transcription factor that\ndoes not merely modulate HF morphogenesis and maintenance. SOX9 has been shown to be a\npioneering factor, acting as a master regulator that governs cell fate during development, injury\nand cancer in multiple tissues35-41, often showing context dependent roles in epithelial biology.\nAll the results described above have now been included and are discussed in an extended revised\n-\nversion of the manuscript, our observations support a role of the HIF1a-SOX9 axis as a barrier to\nlineage conversion.\n6. Imaging and presentation: Multiple figures lack clarity and proper zoom-in insets (e.g., Fig 1C).\nStratification is mentioned, but not clearly shown or validated.\n• Some immunostainings (e.g., Supplementary Fig. 1E) should be repeated with basal and proliferation\nmarkers to better characterize the tissue structure.\n• Controls are inconsistently presented—e.g., peeled vs. unpeeled dermis, esophagus-only grafts, skin-\nonly grafts. This comparison must be rigorously controlled and quantified.\nQuestion 6. Reviewer #2\nThank you for this suggestion. Images showing stratification and zoomed in insets have been added to\nFigure 1 as requested. Apologies for missing to depict this feature in the original version of the\nmanuscript.\nAdditionally, we have significantly reconfigured figures in order to enhance clarity across the revised\nversion of the manuscript, including new controls. Please note that most antibodies used required\npeeling (removal of the underlying stroma) in order to achieve adequate penetration and staining of\nepithelial wholemounts. Indeed, wholemount immunostaining was a necessary technique to study\nResponse to reviewers’ NCOMMS-25-11438\noesophageal-to-skin lineage conversion, given the limited number of fate switch events found in\nheterotypic cultures, which, in itself, represents part of the main message of the presented work.\nAdditional specific comments organized by Figure\nIntroduction:\nRationale for Esophagus–Skin Model: The manuscript would benefit from a more compelling\nexplanation of why esophageal tissue was chosen to test epithelial fate switching in a skin dermis\nenvironment. Although the esophagus is squamous and devoid of adnexal structures, it would be\n-\nhelpful to articulate how this setup advances the concept of adult stem cell plasticity or potential\nclinical applications.\nGeneral comment. Reviewer #2\nWe agree with the reviewer that highlighting the relevance of studying epithelial cells fate plasticity in\nthe oesophagus is important, particularly given that columnar metaplasia (transdifferentiation) in the\noesophagus has been associated with clinical complications, including oesophageal cancer9,20-23. We\nhave revised the manuscript to highlight the translational relevance of understanding epithelial cell\nfate plasticity in the context of the oesophagus. The revised text now reads:\n“It has now become widely accepted that cell fate decisions are not as predetermined as originally\nthought. In response to injury or environmental perturbations, adult epithelial cells present a\nremarkable fate plasticity allowing them to respond to changes in tissue needs 14-16. Yet, if dysregulated,\nthis adaptability can promote epithelial disease and cancer 15,17-19. Particularly, in the context of the\noesophagus, columnar metaplasia has been associated with the development of oesophageal\ncancer9,20-23. Hence, understanding the largely unclear mechanisms balancing cell fate plasticity is\ncritical to unveil its regenerative potential while preventing inherent tumorigenic risks 23.” (p18; l2-8).\nFigure 1\n• Include additional details on culture duration and viability, how long the peeled dermis and\nesophageal strips remain viable in the 3D culture?\nHeterotypic culture details. Reviewer #2\nWe thank the reviewer for pointing this out. We have now added additional technical details about\nour heterotypic culture approach in the methods section (please refer to 3D Oesophageal-Dermal\nHeterotypic and control cultures section of Methods). For the reviewer’s reference the maximum time\nwe have been able to grow cells using tissue recombination assays under the presented experimental\nconditions has been 15 days, as characterised in (Doupe & Alcolea et al. Science 2012; Supplementary\nFig 9 in Science paper)13. Beyond that time the tissue constructs start shrinking and altering the\narchitecture.\n• Panel 1C could benefit from zoomed in insets (e.g., progressive days of re epithelialization) to\nvisualize early stages of esophageal cell expansion over the dermis.\n- -\n• 1C-need clearer zoomed in images of the process.\nRe-epithelialisation. Reviewer #2\nAs suggested by the reviewer, new Fig. 1E includes zoomed in images to visualise the re-\nepithelialisation process.\n• What stages are required for the formation of the hair follicles arise from esophogeal tissue? Author\nshows the appearance of the follicles without clear indication and discussion on how they are formed.\nResponse to reviewers’ NCOMMS-25-11438\nNot clear if mCherry cells only fill out empty areas. Are the follicles in specific stage? What are the\nstages of hair formation and how are they similar to embryonic formation of hair follicle?\noeHF formation. Reviewer #2\nWe agree that this point needs to be reinforced to avoid confusion. Our data suggests that the steps\nof oeHF formation, in heterotypic cultures, involves:\ni) The re-epithelialisation of the denuded skin dermis by oesophageal cells. Here oesophageal\ncells acquire a HF-like structure that resembles the stage of the HF that was originally\noccupying a given niche.\nii) This process exposes oesophageal cells to the ectopic HF niche.\nThe instructive niche signals enable the lineage conversion process in competent oesophageal cells.\nWe have shown that this depends on the HIF1a-SOX9 axis, which is now further supported with new\nfunctional data (Fig. 6).\nWe have amended the original text to clarify the steps of oeHF formation. However, it is worth noting\nthe fact that oesophageal cells undergo lineage conversion in vivo (in grafting experiments), where\noesophageal cells receive instructive signals of developing skin without the “architectural footprint”\nof existing HFs in the dermis. This suggests that access to instructive niche signals represents the key\nstep to initiating the lineage conversion process. The text now reads:\n“Interestingly, adult OE cells (red) were able to fully re-epithelialise the bare dermis and empty skin HF\nsockets, creating a de novo stratified epithelium (Fig. 1E) and forming structures reminiscent of in vivo\nHFs, hereafter referred as oesophageal-derived HFs (oeHFs; Figs. 1C-F).” (p5; l17-20).\n“Overall, our data shows that the oesophageal-to-skin lineage conversion process involves series of\nevents. These includes i) the re-epithelialisation of the denuded skin dermis by oesophageal cells. Here\noesophageal cells repopulate the empty epidermal stroma forming HF-like structures as they fill the\nempty HF sockets. ii) This process exposes oesophageal cells to the ectopic HF niche. iii) The instructive\nniche signals enable the lineage conversion process in competent oesophageal cells in a HIF1a-SOX9\ndependent manner. We go on to demonstrate that blocking HIF1a or its downstream target SOX9 lifts\nthe regenerative blockage and favours cell fate plasticity by promoting the transition from oesophageal\nto skin lineage both in vivo and in vitro (Proposed model Fig. 7I), and that this may represent a more\nuniversal mechanism operating beyond oesophageal epithelium.”(p17; l14-22).\n• Confirm the endogenous staining in figure 1c with epidermal markers, basal layer markers, to confirm\nthat mCherry cells indeed convert to epidermal cells.\nEpithelial origin of oeHFs. Reviewer #2\nWe are grateful to the reviewer for this suggestion, which greatly enhances the clarity of the skin the\nheterotypic re-epithelialisation assay. Supplementary Fig. 2 already contained a panel showing Krt14-\nCD34-PDGFRa staining to demonstrate the epithelial origin of the oeHF CD34 cells (Supplementary\nFig. 2 E). For further transparency, we have now also included a panel in Supplementary Fig. 1F to\nshow that tdTomato oesophageal-derived skin expresses P63 in the basal progenitor proliferative\ncompartment.\n• Supplementary figure 1E- cp3 and ki67—need to show the staining in the tissue couples with basal layer marker\nCaspase 3. Reviewer #2\nResponse to reviewers’ NCOMMS-25-11438\nIn line with the point above, we now include in Supplementary Fig. 1F a panel to show that basal\nproliferation (Ki67 staining) coincides with basal marker p63 expression. We additionally add\nrepresentative active Caspase 3 images Supplementary Figs. 1G-H.\n• Need farther verification to confirm epidermal tissues, staining with different markers and zoom in\nimages to show stratification, is there epidermal turnover? Is it functional? The author mentions the\nformation of squamous stratified epithilum but there are no evidence for it.\nStratification and proliferation. Reviewer #2\nIn line with the reviewer’s suggestion, new Fig. 1E and Supplementary Fig. 1F include zoomed in\nimages to show the stratification of oesophageal-derived skin (oeSKIN). New data includes basal\nsquamous epithelial marker p63, as well as Ki67 to reflect the active proliferation of basal cells in the\nnewly formed squamous tissue.\n• Image 1F is not enough to show pigmentation. Perform fontana mason analysis in order to indicate\npigmentation. Are there melanocyte stem cells in the follicles?\nPigmentation. Reviewer #2\nIn relation to oeHF pigmentation, the newly added Fontana Mason staining shows that the denuded\ndermis retains melanin after peeling, which may be taken up by oesophageal keratinocytes during\noeHF formation (Supplementary Fig. 1L). We further support this notion by showing how pigmented\nstructures in Fig. 1G and Supplementary Fig. 1K are indeed of oesophageal origin (i.e. express\ntdTomato reporter).\n• Are the structures that appear to be the sebaceous glands, actually the sebaceous glands? Perform\noil staining and zoomed in images of the cells.\nOil red. Reviewer #2\nAs requested by the reviewer, we can confirm oeHFs contain cells expressing the sebaceous gland\nmarker stearoyl-CoA desaturase 1 (SCD1; Fig. 2B and Supplementary Fig. 2A), and can accumulate\nlipid droplets (as shown by Oil Red O staining in Supplementary Fig. 1M).\n• Is that the hair shaft in 1F middle and right HF?\nHair shaft. Reviewer #2\nIndeed, the stratified cells inside the oeHFs create what resembles a hair shaft-like structure\n(Supplementary Fig. 7D). However, since we are not able to keep heterotypic cultures for more than\n15 days, we have been unable to follow hair cycling long-term. Notwithstanding this, we do show that\nepithelial cells in oeHF are contributing to rudimentary oeHF shafts in in vivo skin hair reconstitution\nassays (Fig. 7).\nFigure 2\n• Use additional markers for hair follicle identity including K15\nAdditional markers 1. Reviewer #2\n-\nIn response to the reviewer’s request, we have stained oeSKIN wholemounts for additional markers\nsupporting the conversion from oesophageal to skin lineage (LHX2, KRT15, KRT10, SCD1 and SCA-1;\nFig. 2 and Supplementary Fig. 2A-E). Additionally, Oil Red O has been used to mark lipid accumulation\nin oesophageal cells (akin of sebaceous glands; Supplementary Fig. 1M), and Fontana Masson as a\nResponse to reviewers’ NCOMMS-25-11438\nproxy to study melanin incorporation in oeHFs (Supplementary Fig. 1L). These data included in the\nrevised version of the manuscript further supports the lineage conversion notion.\n• Use conventional epithelial markers (K14 for basal cells, K10 or others for differentiation) would\nconfirm that the newly formed structures exhibit proper stratification rather than partial epithelial\novergrowth.\nAdditional markers 2. Reviewer #2\nTo address this point additional images showing basal and differentiation markers KRT14, p63, KRT10\nhave been added to Supplementary Fig. 1F and Supplementary Fig. 2A-E.\n• In figure supplementary figure 1C, positive staining of sox 2 indicate the presence of the dermal\npapilla in the peeled skin, is that true for every area where hair formed? Is the presence of the DP\nrequired for hair formation in this case? Is there any data regarding the specific fibroblasts populations\ncontributing?\nDermal Papillae. Reviewer #2\nApologies for the misunderstanding, we can confirm that in denuded (peeled) dermis there is\nwidespread retention of dermal papillae (DP) of skin origin (EGFP positive; Supplementary Fig. 1C).\nAt the culture end-point, oeSKIN wholemounts were peeled from the dermis, in order to enable good\nantibody penetration and 3D confocal reconstruction of the tissue. Hence, the dermal papilla was not\npart of our systematic analytical pipeline. Despite this, the DP was found in a number of oeHFs at the\nend-point cultures even after peeling (Supplementary Fig. 2I, J), supporting its potential role in the\nlineage conversion process. Additionally, our lineage tracing data highlights the importance of the skin\nniche in the lineage conversion process (Fig. 3). The text has been amended to clarify this point:\n…“The peeled dermis consistently retained the architectural footprint of its appendages and associated\nstromal niches (Supplementary Figs. 1B, C; see methods)”…(p4; l31-32).\n…”In line with this, dermal papillae derived from the host skin (EGFP) were found in heterotypic cultures\n(Supplementary Fig. 2I,J). This indicates that the instructive signals promoting lineage conversion\noriginate in the dermal niche, reminiscent of normal skin physiology 42,43.”… (p6-7; l32-3).\n• You mention low efficiency of hair formation, yet in images such as supp 1 D we see hair formation\nvia esophageal tissue that is almost all over the tissue.\noeHF efficiency. Reviewer #2\nThis point has already been addressed in our response to Question 4 of this reviewer. In short, we\nacknowledged the lack of clarity on this point. We have now explained in the text the difference\nbetween re-epithelialisation efficiency (Supplementary Fig. 1J) which denotes the high efficiency at\nwhich the oesophagus re-epithelialised the denuded dermis, versus the low efficiency of the lineage\nconversion process (which involves HF marker expression and architectural features; Fig. 2D, E).\nFigure 3\n• Please also show GFP channel in all the images in order to see the cells originated from skin stromal\norigin.\nEGFP. Reviewer #2\nOur apologies for the lack of clarity. Please note that epithelial wholemounts were peeled before\nimaging to enable better antibody penetration, facilitate visualisation, and 3D reconstruction of the\nResponse to reviewers’ NCOMMS-25-11438\ntissues. This is particularly needed for this study due to limited oesophageal-to-skin lineage conversion.\nDue to the peeling process, EGFP+ signal from the host epidermis can only be visualised in samples\nthat have been sectioned (where tissue composites retain EGFP-derived dermis Fig. 1C,E and\nSupplementary Figs. 1F, 2D and 7E-I). As already mentioned, all our images show all the channels\nindicated in the associated image caption. This is in all cases without exception. Hence, if a colour is\nnot present, this means that given labelling/reporter is not there or undetectable; in the case of dermal\nEGFP due to the peeling process. In summary, the reason why EGFP is not detected in oeSKIN samples\nis because there was little to no contribution of skin cells (EGFP+) to the newly formed oeSKIN once\nthe dermis is peeled away from stroma for wholemount purposes.\n• The conclusion that based on cd34 marker only the regeneration depends on dermal niche is\ninsufficient. Need to check in other stem cell populations and cell types.\nNiche. Reviewer #2\nThe lineage tracing experiments (Fig. 3) investigate the cell dynamics involved in dermal re-\nepithelialisation and oeHF formation, revealing the polyclonal nature of the process. The profile of\nCD34 expression originally suggested that niche signal instructed the oeHF identity switch. New data\nincluding KRT15 expression, as an additional HF marker, now shows a much less apparent\ncompartmentalisation. Hence, we have decided to underemphasise the potential relevance of specific\nanatomical cues within the HF dermal niche. We have amended the text referring to stromal signals in\ngeneral:\n“Additional heterotypic experiments, where the adult OE was grown over the dermis of developing\nskin, one day post birth (P1), showed the specific upregulation of the marker LHX2, known to label\ndeveloping HFs in vivo (Fig. 2C), in oeHFs. This observation revealed the critical role that the stroma\nplays in the identity switch, being able to rewire the oeHF fate towards developmental stages. In line\nwith this, dermal papillae derived from the host skin (EGFP) were found in heterotypic cultures\n(Supplementary Fig. 2I,J). This indicates that the instructive signals promoting lineage conversion\noriginate in the dermal niche, reminiscent of normal skin physiology 42,43.” (p6-7; l29-32; l1-3).\n• The author shows polyclonal contribution for the esophageal cells make up the hair follicle, yet it will\nbe valuable to add if different clones give rise to different populations and their actual contribution to\nhair follicle formation\nLineage tracing. Reviewer #2\nWe apologise for the lack of clarity. We have now amended the text providing a more detailed account\nof the data.\nGiven that clonal induction is under the control of the ubiquitous Rosa26 promoter (R26CreERT), the\ncontribution of labelled clones would be expected to be the same compared to other unlabelled clones\npresent in the tissue, and by no means is selective of specific cell populations/cell states that may\nemerge during the re-epithelialisation process. In that vein, we observe that the emerging oeHFs are\nof polyclonal nature along the entire structure, with no clonal preferential enrichment or expansion in\nany specific areas. Given that these experiments were done under minimal (clonal) recombination, the\ndata therefore argues against a specific cell subpopulation being responsible for oeHF formation.\nPlease see newly generated images for reference (Fig. 3E and Supplementary Fig. 3A-C). Please refer\nResponse to reviewers’ NCOMMS-25-11438\nto main text for further clarity on this point: “Starting at an initial clonal labelling covering just ~5% of\nthe basal layer, fluorescent OE cells expanded and re-epithelialised the dermis colonizing in their way\ntwo thirds of all the oeHF structures present in the tissues (66.37% ± 6.07; mean ± SD). As a result,\nupon clonal expansion, approximately 66% of all oeHFs formed contained at least one or more labelled\ncells, revealing that, in line with previous observations in oesophageal wound healing 13, OE cells\nundergo a widespread recruitment during oeHF formation.“(p8; l1-6).\nFigure 6\n• The text states there is a SOX9–HIF1a Co localization : “The data showed a positive correlation\nbetween SOX9 and HIF1a levels; with HIF1a expressing cells being more likely to be positive for SOX9,\n-\nas measured by immunostaining (Supplementary Fig. 6C)” , but panels explicitly showing co-staining\nare not shown.\n• Need proof of concept for sox9 floxed model, are the knocked out cells the ones express HIF1a?\nFigure 7\nSOX9-HIF1a axis. Reviewer #2\nWe appreciate the reviewer’s comment. As they will be able to infer from Supplementary Fig. 5K and\nas well-established, the HIF1a staining profile shows a diffuse staining pattern (punctate staining).\nHence, we believe that a single image would not be representative of the data and would not be able\nto convey the message (see example below Response Fig. 4). For quantification purposes, we made\nuse of an unbiased automated nuclear segmentation pipeline (Arivis 3.5.1. segmentation), which\nenabled us to quantify both HIF1a and SOX9 staining in oeSKIN. This is the data shown in\nSupplementary Fig. 6D.\nResponse Figure 4: oeSKIN immunostained for HIF1α and SOX9. The image shows a representative\nHIF1α punctate staining pattern, illustrating the need for quantification across multiple oeHF units to\naccurately reflect the expression profile.\n• The low frequency of the HF formation event raises questions regarding the validity and the relevance\nof the mechanism\noeHF inefficiency. Reviewer #2\nWe would like to note that our intention in the presented study is not to prove that oesophageal cells\ncan be reprogrammed into fully functional HF structures using our environmental reprogramming\napproach. The novelty of this study centres around identifying the mechanisms that naturally protect\nour tissues from inadequate cell fate plasticity. We believe that identifying those will offer a benchmark\nto develop strategies to improve tissue regeneration and potentially prevent fate promiscuous\nbehaviour in certain pathologies, such as cancer. We have noted this in the text:\n…“Overall, these data demonstrate that HIF1a and its downstream effector SOX9 establish a\nregenerative barrier that prevents cell plasticity during tissue repair in oesophageal cells.” (p16; l6-8).\nThe revised manuscript also centres the narrative around barriers to lineage conversion rather than\nthe lineage conversion process itself. Using robust cellular and molecular approaches, we have\nthoroughly established the existence of barriers protecting the identity of epithelial cells both in vivo\nas well as ex vivo. And we have gone one step further, unveiling the role of the HIF1a-SOX9 axis in the\nprocess, not only in the oesophagus, but new data also show that this barrier may be operating in\nother epithelial tissues such that of the trachea (Supplementary Fig. 7K-M). We believe that this work\ncontributes to our understanding of what mechanisms limit adult cell fate plasticity preventing an\naberrant and uncontrolled response that could lead to abnormal growth and tumorigenesis upon\ntissue perturbations. Our data also opens exciting new avenues to decipher the inner workings\ndictating cell fate plasticity, providing a benchmark to identify the principles underlying the full\nregenerative capacity of epithelial cells.\nReviewer #3 (Remarks to the Author):\nBejar et al. dissect the molecular mechanism of fate permissibility in adult cells using a 3D regenerative\nculture system where TdT-marked esophageal tissue is placed on epithelium-less GFP skin dermis and\nthe esophageal epithelial (EE) cells crawl out and on to the skin dermal support. In the revised version,\nthey have expanded the plasticity observation using stroma of other stratified epithelial tissues, showing\nlocal partial cell fate acquisition that is blocked by Sox9 and HIF1. They include a more detailed single\ncell analysis of the fate transition and additional histological characterization of the eeHF\nconversion. I appreciate the authors expanded discussion of HIF and Sox9, and additional explanation for the HIF/Sox9\nbarriers and their relationship to committed skin lineages and wound healing. Overall the additional data in this revised\nversion supports a general stratified epithelial signaling environment in the skin dermis that will be of general interest to\njournal readers.\nThis reviewer suggests including the intriguing negative data of the inability of skin epithelium to convert to esophageal\n(Response Fig. 5) and at least discussing the delayed re-epithelialization of esophageal stroma. With the data suggesting\nthe skin stroma provides the local information to convert esophgeal mucosa to skin, these additional data provide clues to\nthe strong skin environmental determinants."}]

Decision letter