Wnt-responding stem/progenitor cells are essential for tympanic membrane repair

Wnt-responding stem/progenitor cells are essential for tympanic membrane repair | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Wnt-responding stem/progenitor cells are essential for tympanic membrane repair Abigail Tucker, Olivia Dunwoodie, Juan Fons This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9587608/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Tympanic membrane (eardrum) perforations are common, with the majority healing, suggesting an innate ability to regenerate after damage. Epithelial cells expressing putative stem cell markers have been shown to reside in niches confined to specific regions of the membrane. Utilising Axin2 reporter mice, we show that label-retaining and long-lived, Wnt-responding cells are located in these presumptive stem cell niches. scRNAseq analysis and lineage tracing revealed that these Wnt-responding epithelial cells had hallmarks of quiescent cells and did not contribute significantly to homeostasis. In contrast, during wound healing, these cells were activated and contributed to substantial proportions of the repaired membrane. Loss of Wnt signaling in the Axin2 population in vivo and in vitro, led to impaired healing as cells failed to bridge the gap and create the normal tri-layered membrane structure. Our results reveal a reserve stem cell population that is crucial for repair, providing a target for future therapeutics to treat chronic perforations. Biological sciences/Stem cells/Adult stem cells Health sciences/Medical research/Experimental models of disease repair wnt signalling ear quiescence Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction The mammalian ear is divided into three parts, each essential for hearing: the outer, middle, and inner ears. The external, or outer, ear serves as the first point of contact for sound vibrations in the process of hearing. Sound waves transmitted from the pinna enter the ear canal where they are funneled to the tympanic membrane (TM), the structure at the end of the outer ear (Anthwal & Thompson, 2016 ; Ugarteburu et al., 2022 ). The TM consists of two main parts, the superior pars flaccida (PF) and inferior pars tensa (PT), and is made up of three layers, each originating from a separate germ layer. The outer epidermal layer (OEL), derived from the ectoderm, consists of keratinocytes, the neural crest-derived mesenchymal layer, which is also known as the lamina propria (LP), additionally accommodates the blood vessels and nerves of the TM, while the inner mucosal layer (IML) is endoderm derived (Dinwoodie et al., 2024 ). Tympanic membrane perforations (TMPs) are common, affecting 2.1% of the population in the US each year (Kim et al., 2022 ). In the majority of TMPs, the TM will spontaneously heal within 10 days, and further medical intervention is not required (Berger, 1989 ). In 6–10% of cases, the TMP will not heal and becomes chronic (Dolhi & Weimer, 2022 ). Chronic TMPs are serious and can result in recurrent infections, middle ear cysts, otalgia, tinnitus, and conductive hearing loss (Dolhi & Weimer, 2022 ). The use of growth factors, stem cells and scaffolds have been researched as alternative therapies to treat TMPs and move past the need for surgical intervention (currently the only treatment option). However, the mechanisms altering healing rates due to the application of these factors is poorly understood, and until this knowledge is expanded the use of these approaches is limited (Akkoca et al., 2022 ; Parekh et al., 2009 ; Rajkhowa et al., 2011 ; Rohanizadeh et al., 2008 ; Shen et al., 2012 ). Given the impressive regenerative capacity of the TM, it has been postulated that a stem/progenitor population must reside in the membrane to contribute to its repair after injury (Chari et al., 2019 ; S. W. Kim et al., 2015 ; Knutsson et al., 2011 ; Tucker et al., 2018 ). BrDU-retaining cells have been localized to the manubrium of the malleus and the annulus, highlighting these cells of interest as a potential stem/progenitor population (Tucker et al., 2018 ). The manubrium inserts into the middle of the TM, while the annulus connects the membrane to the supporting tympanic membrane (Tucker et al., 2018 ). Cells expressing epithelial stem cell markers such as α 6 -integrin, β 1 -integrin and cytokeratin 19 have also been localized to these two regions, adding further weight that these locations are the niche regions of the TM (S. W. Kim et al., 2015 ; Knutsson et al., 2011 ). Finally, recent work involving Edu-incorporation has suggested epithelial cell proliferative zones to be located at the handle of the malleus and the mallear folds (the border region between the PF and PT), providing an epidermal specific stem cell niche responsible for homeostatic turnover of the outer layer of the TM (Frumm et al., 2021 ). Here we investigate the role of Wnt-responding cells in homeostasis and repair of the tympanic membrane using the mouse as a model. Canonical Wnt signalling has been shown to be essential for stem-cell renewal, proliferation, and differentiation in a number of organs (Clevers et al., 2014 ) but has not been previously assessed in the TM. Axin2 is a direct intracellular target of canonical Wnt signalling and has been shown to label both epithelial and mesenchymal stem cells populations (Syed et al., 2020 ; Lav et al., 2023 ). We show that Axin2 expressing cells, are located in the putative TM stem cell niches, contribute to the repair process, and play essential roles during repair of the membrane. Results Wnt-responding cells are found in the presumptive stem cell regions of the tympanic membrane To identify the location of Wnt responding cells in the TM, Axin2LacZ heterozygous mice were utilised. This reporter shows current canonical Wnt activity (Lohi et al., 2010 ). Wnt responding cells were observed around the annulus and the manubrial regions, the two sites previously identified as putative stem cell niches (Fig. 1 A-C). A similar expression pattern was observed using Axin2creERT2;TdTomato mice analyzed 5 days after 3 days of tamoxifen injection (Fig. 1 D-F), confirming that Axin2 labelled cells (tdTomato positive) reside in the presumptive stem cell regions of the TM under homeostatic conditions. In addition to the region around the annulus and manubrium, Axin2 positive cells were also evident at the border between the pars flaccida and pars tensa (Fig. 1 D), a region from which the epithelial cells have been shown to turn over during homeostasis (Frumm et al., 2021 ). To discern the location of the cells within the three layers of the tympanic membrane (outer epithelium, middle mesenchyme, inner mucosa), the TMs were sectioned, and immunofluorescent analysis performed. Vimentin was used as a marker of the mesenchymal layer of the TM, while Pan-cytokeratin (PCK) was used as a marker of the outer epithelial layer of the TM (Fig. 1 G,H,I). The majority of Axin2 labelled cells (RFP positive) co-expressed vimentin, highlighting their location in the mesenchyme of the TM at the manubrium and annulus (Fig. 1 G). In contrast, only a few Axin2 labelled cells were located in the endoderm and ectoderm layers (Fig. 1 G-I). Wnt responding cells in the TM are label retaining and long-lasting To discern whether Axin2cre labelled cells contributed to homeostasis of the TM, three consecutive injections of tamoxifen were given to Axin2creERT2;TdTomato mice and the labeled Wnt-responding cells were traced for 2- and 10- months. The outer epithelial layer of the tympanic membrane has been shown to have a turnover of 3 weeks (Frumm et al., 2021 ). At both 2 and 10 months the original Axin2 cells and any progeny were still located around the annulus and the manubrium, highlighting their longevity (Fig. 2 A,B). The majority of the Axin2 -labelled cells were situated within the mesenchyme (Fig. 2 C,E), with occasional RFP positive lineage traced cells in the outer epidermal layer (Fig. 2 E, white box arrowhead) and inner mucosal layer (Fig. 2 C, arrowhead). The rare epithelial Axin2 cells, therefore, did not appear to play any significant role in homeostasis. In contrast, the Axin2 -labelled cells in the mesenchymal layer, were more prominent across the membrane at 2 and 10 months post tamoxifen (Fig. 2 C,E), when compared to 15 days post tamoxifen injection (15dpi) (Fig. 2 D), suggesting the cells had contributed to turnover of the middle layer of the membrane during homeostasis. Quantification of the number of Axin2 labelled cells in the central portions of the TM (defined as 200µm away from the annular and manubrial regions), confirmed significantly higher numbers of Axin2 -labelled cells in the mesenchyme in this region over time (Fig. 2 F). Interesting, the turnover of the mesenchymal layer appearing much slower than has been reported for the outer ectodermal layer, suggesting independent turnover rates in the different layers (Frumm et al., 2021 ). To further investigate whether the Axin2- positive cells are a stem/progenitor population, the label retaining potential of the Axin2 -positive population was analyzed. BrDU was injected into a pregnant dam (tdTom female crossed to Axin2creERT2;TdTomato male) at E13.5 and E15.5. The majority of cells were positive for BrDU at P0, confirming successful incorporation of BrDU as a “pulse” (Fig. 2 G). When the offspring were 6–8 weeks old (7–9 week chase), three consecutive injections of tamoxifen were given to label Axin2 expressing cells. Eardrums were quantified 5 days post injection to determine the proportion of a) BrDU-positive, b) Axin2 -positive, and c) both Axin2- positive and BrDU-positive and d) BrDU-positive but Axin2- negative cells (N = 6 eardrums in total). Label retaining cells made up roughly 10% of the total cells in the TM and were identified in all layers of the membrane, concentrated around the annulus and manubrium (Fig. 2 H-K), agreeing with previous reports (Tucker et al., 2018 ). Importantly, > 99% of the label retaining cells expressed Axin2 (Fig. 2 H”-J”,K). The label retaining cells in the TM are, therefore, Wnt-responding cells. The Axin2- positive population made up a higher proportion than the label-retaining population (around 35–40%) (Fig. 2 H’,I’,J’,K), and, therefore, not all Axin2 expressing cells were BrDU positive, particularly in the mesenchyme (Fig. 2 H”-J”,K). This suggests that the Axin2 expressing cells label distinct subsets in the membrane. To further investigate the epithelial Axin2 -positive population in the TM, a previously published single cell RNAseq dataset was analyzed (Frumm et al., 2021 ). Epithelial Axin2 -positive cells were analyzed for their expression of epithelial stem cell markers (Fig. 2 L) and markers of cellular quiescence (Fig. 2 M). The Axin2 positive population in the OEL strongly expressed α 6 -integrin ( Itga6 ) and β 1 -integrin ( Itgb1 ), epithelial stem cell markers that have previously been identified in the TM (Kim et al., 2015 ), as well as the basal keratinocyte maker, keratin 5 ( Krt5 ) (Fig. 2 L). As the Axin2 positive population was not involved in the homeostatic turnover of the OEL, it was hypothesized that these cells therefore might be a quiescent population. The population was found to express Trp53 , Smarca2 and Cdkn1a (Fig. 2 M), all markers of cellular quiescent. Overall, the epithelial Axin2 -positive population in the ear drum was shown to be label retaining, long-lasting, and positive for stem and quiescent cell markers, highlighting this subset of cells as an epithelial stem cell population. Wnt-responding cells are involved in the repair of tympanic membrane perforations To discern whether the Axin2-postive LRCs could be activated after injury, the Wnt-responding population was followed after TM perforation. A perforation was made 5 days after 3 consecutive rounds of tamoxifen injections and the Axin2creERT2;TdTomato mice were sacrificed at three timepoints, 5-, 10- and 30-days post perforation (dpp). The murine TM is able to robustly repair small perforations over this time frame (Scaria et al., 2023 ; Dinwoodie et al., 2024 ). Perforations of uniform size were made at uniform positions under the manubrium using an otoscope for visualisation (Dinwoodie et al., 2024 ). The left ear of each mouse was left uninjured and was used as an internal control. Whole-mount imaging of the TMs confirmed that in the unperforated ears the Axin2 labelled population was restricted to the annulus and manubrium (Fig. 3 A) at 5dpp (days post perforation of the contralateral side), 10 dpp (Fig. 3 B) and 30dpp (Fig. 3 C). In contrast, in the injury model the labelled Axin2 population, and any progeny, moved towards the wound site as a sheet of cells from the surrounding tissues (Fig. 3 D, G) evident at 5dpp. At 10dpp, the Axin2 population was observed covering the site of the original perforation (Fig. 3 E, H circle). By 30dpp, when the tissue had nearly completed the remodelling phase, the labelled population were present in the healed wound site (Fig. 3 F, I)(N = 6/timepoint). To decipher whether the Axin2 cells were poised for activation in response to injury, the Axin2- positive cell population was labelled by injection of tamoxifen, but the mice were left for 5 months prior to perforation. After perforation, the long-lineage traced cells were observed in and around the healed perforation at 10dpp (Fig. 3 J,K), in an identical fashion to the shorter lineage traces (Fig. 3 E,H). Axin2 labelled cells contribute extensively to the epithelial layer of the healing membrane During the first phases of repair the epithelium initially retracts away from the wound site before proliferating and extending to form a scaffold that bridges the gap around day 7. The mesenchyme and endodermal layers then migrate along the epithelium to recreate the three-tissue layer structure (Dinwoodie et al., 2024 ). Sections through the TM at 5 days post perforation (5dpp), highlighted the presence of a large number of Axin2 labelled cells (red) in the forming epithelial scaffold (labelled in green with pan-cytokeratin) (Fig. 4 A arrow-asterix), and in the mesenchymal layer (Fig. 4 A arrowhead) prior to closure of the perforation. These cells were still evident at the wound site in all three layers (ectoderm, mesenchyme, endoderm) at 10dpp (Fig. 4 B). A similar arrangement of Axin2 labelled cells was observed in the long-lineage trace experiments where the membrane was perforated 5 months after tamoxifen activation (Supplementary Fig. 1), illustrating that the population labelled 5 months prior to perforation contribute to the healing of all layers of the TM at 10dpp. Many of the epithelial and mesenchymal Axin2 labelled cells were positive for the proliferation marker PCNA (Fig. 4 B' arrowhead), with proliferation in the epithelium at this stage mainly observed in the basal layer (Fig. 4 B'). By 30 days post perforation, the tissue had almost fully healed and was undergoing remodelling to regain its thin structure (Scaria et al.,2023). The Axin2 population at this timepoint made up much of the epithelial and mesenchymal layers (Fig. 4 D, D’), compared to the unperforated contralateral ear, particularly for the epithelial population (Fig. 4 C). Quantification of the proportions of Axin2 -positive cells in the epithelium under control conditions and after healing of a perforated drum at 30dpp, showed a significant increase in the ratio of Axin2 -labelled cells (Fig. 4 E), with numbers increasing 5-fold to contribute to almost 60% of the epithelium of the repaired part of the membrane. The increase in Axin2 -lineage cells in the epithelium was likely due to proliferation of the progeny of the rare label retaining epithelial Axin2- cells of the manubrium and annulus. Alternatively, it was possible some cells from the mesenchyme were able to undergo a mesenchyme-epithelial transformation during repair and thereby contribute cells to the repaired epithelium. In keeping with this, repair of the membrane has been described as forming by creation of a multi-lineage blastema-like cell mass (Scaria et al., 2023 ). To test this, perforations were performed in Wnt1cre;TdTomato mouse line where the middle mesenchymal layer is labelled with RFP (Dinwoodie et al., 2024 ). Robust expression of RFP was evident in the middle layer after healing at 30dpp, with no contribution to the epithelial layer (Fig. 4 F,F’). MET can, therefore, be ruled out. The Wnt pathway is upregulated in the mesenchyme layer during repair In addition, to its role in regulating stem cells, Wnt signalling is upregulated in response to injury in a number of tissues (reviewed in Whyte et al., 2012 ). To observe whether cells were actively responding to Wnt signalling as a consequence of the injury, the expression of Lef-1 was investigated at 5dpp. Lef-1, like Axin2 , is a downstream target of canonical Wnt signalling (Behrens et al., 1996 ), and, therefore, its expression marks cells actively responding to Wnt signalling. Robust Lef-1 expression was observed in the mesenchymal layer at the injury site, suggesting activation of Wnt signalling in response to injury (Fig. 5 A). In contrast, the large epithelial scaffold was entirely Lef-1 negative (Fig. 5 A). In keeping with this, the Axin2 cells labelled prior to injury showed overlapping expression with Lef-1 in the mesenchyme but not epithelium (Fig. 5 A’,A”). At 10 days post perforation, the repairing tissue has already undergone mesenchymal and endodermal proliferation and migration over the wound site (Dinwoodie et al., 2024 ). At this stage, Lef-1 expression in the mesenchyme remained high, with numerous RFP positive and Lef-1 positive cells (Fig. 5 B). To confirm that Wnt signalling was activated in the mesenchymal layer as part of the repair response the Wnt response was analysed in Axin2LacZ mice at 10 and 17dpp. High levels of LacZ were observed in the mesenchyme and inner mucosal layer (Fig. 5 C,D). In contrast, only minimal labelling of LacZ was observed in the recovering outer epithelial layer (Fig. 5 C,D arrow). Loss-of-function of β -catenin in Axin2 cells results in a higher turnover of the mesenchymal layer. In order to investigate the impact of loss of the Axin2 population on homeostasis, the Axin2creERT2; β-catlox(ex2-6);TdTomato mouse model was utilised, where Cre-mediates the excision of exon 2–6 of β-catenin is disrupt canonical Wnt signalling (Brault et al., 2001 ). To confirm the effectiveness of the tamoxifen induced recombination, sections from the model were stained at 5-days post injection for non-phosphorylated (active) β-catenin. In control TMs (Fig. 6 A), active β-catenin was found in the nucleus of cells found at the annular region of the TM. In the loss-of-function model, however, no β-catenin staining was observed in these annular cells (Fig. 6 B). As shown at 5 days post injection (Fig. 2 D,F), at 15 days post injection (15dpi), only a few Axin2 labelled cells were observed in central regions of the TM at a distance from the stem cell niches of the annulus and manubrium in control mice ( Axin2creERT2 ; tdTomato ) (Fig. 6 C). In the loss-of-function model however, at 15 dpi, Axin2cre labelled cells were observed in the mesenchyme in the central TM (Fig. 6 D,F). Quantification revealed that a statistically higher proportion of cells in the central mesenchymal layer (defined as 200µm away from the annular and manubrial regions) of the TM were Axin2cre labelled in the loss-of-function compared to controls (Fig. 6 E). As the Axin2cre labelled population is responsible for the mesenchymal turnover of the TM (Fig. 2 ), this process was accelerated in the loss-of-function model, suggesting a disruption to the turnover of this population. Loss-of-function of Wnt signaling in Axin2cre labelled cells results in TMs that cannot repair at 10 or 15 dpp. The impact of loss of Wnt signalling in the Axin2cre population on repair was then tested using the perforation model. At 5dpp, the loss-of-function mouse appeared very similar morphologically in its early response to a perforation when compared to the reporter mouse line Axin2creERT2;Tomato (Fig. 7 A-C). Wnt signalling in the Axin2 population was, therefore, not required for blastema formation. In contrast at 10dpp, 80% of the perforations were still open in the loss of function eardrums (N = 4/5 LOF TMs), while the Axin2creERT2;TdTomato controls showed closure in 100% of cases (N = 8/8 control TMs) (Fig. 7 C-F). Interestingly, although the wounds failed to close completely, Axin2cre labelled lineage traced cells had still been recruited to around the wound site in the mutants (Fig. 7 E-F). In section, defects in layer structure were evident in the one ear drum that had managed to close, while in the others, epithelial cells were observed pushing through the ear drum into the middle ear cavity (Supplementary Fig. 2A,B). By 15dpp, the hole had healed in 100% of control eardrums (N = 8/8 control TMs), however, the perforation failed to heal in 66% of cases in the mutants (N = 2/6 LOF TMs) (Fig. 7 C,G-I), with histology showing a persistent gap in the membrane (Fig. 7 J,K). Interestingly, the ear drums that had healed showed defects, such as creation of a bilayered structure with a missing mesenchymal layer (Supplementary Fig. 2C,D). Therefore, disrupting Wnt signaling in the Axin2 population prior to TM perforation impairs wound repair at 10- and 15 dpp. Discussion Tympanic membrane perforation is a common condition which affects 1 in 50 people worldwide, of which, 6–10% develop into debilitating and painful chronic perforations (Dolhi & Weimer, 2022 ; A. S. Kim et al., 2022 ). Despite the need for effective and readily available treatments for chronic perforations, research on tympanic membrane repair is limited, hindering general understanding of the organ and possible treatment development. The possibility of a stem cell population residing in the TM had been touched on by previous researchers via immunohistochemistry for stem cell markers, although more generally as a hypothetical assumption due to the acute ability of the TM to regenerate after injury (Dinwoodie et al., 2024 ; Frumm et al., 2021 ; S. W. Kim et al., 2015 ). We have shown that Axin2cre labelled cells were located in the previously described stem cell regions of the eardrum (Frumm et al., 2021 ; Kim et al., 2015 ; Tucker et al., 2018 ), and importantly all BrDU label-retaining cells were also Axin2 positive. The Axin2cre labelled population was long lived, being retained over a 10 month period, suggesting these cells represent a stem cell population. The Axin2cre labelled cells in the epithelium were rare and did not contribute extensively to homeostasis. In contrast, the Axin2cre labelled cells in the mesenchyme were found to populate the ear drum over time, suggesting lineage specific roles for these cells (Fig. 8 ). The Axin2cre labelled cells located in the mesenchymal layer may be stem/progenitor cells, or long-lasting quiescent fibroblasts, such as those found in the dermis of the skin (Zorina et al., 2023 ). Although, the Axin2cre labelled cells in the epithelium did not contribute to homeostasis, they contributed to a large part of the ear drum during repair. This population, is therefore, able to exit quiescence, proliferate, and help reform the membrane. The TM’s outer layer may therefore have separate populations of epithelial cells that are a) Wnt-independent and capable of maintaining the outer layer during homeostasis with a turnover of 3 weeks (Frumm et al., 2021 ), and b) Wnt-dependent and quiescent but are activated after injury and responsible for its repair. This is reminiscent of the distinct roles of different stem cell populations in the cranial sutures (Maruyama et al., 2016 ). The Axin2-labelled cells in the mesenchyme and endoderm layers also contributed to repair, generating a new membrane that was largely derived of Axin2-lineage cells (Fig. 8 ). Although the TM has been proposed to be a blastema (Scaria et al., 2023 ), we saw no evidence of mesenchymal cells contributing to the epithelial or endodermal layers of the repaired membrane, highlighting that during repair the layered structure of the membrane is maintained. Importantly, the contribution of Axin2-labelled cells to repair was evident, even when the injury was created 5 months after lineage labelling of these cells. As is common during many repair processes, Wnt signalling was upregulated in the TM after injury. Interestingly, this upregulation was highest in the mesenchymal layer, with minimal upregulation in the epithelial layer, not all layers, therefore, responded to injury in the same way. When the ability of the Axin2cre labelled cells to respond to Wnt signaling was impaired via use of the Axin2creERT2;Bcatlox(ex2-6);TdTomato mouse model, mesenchymal homeostasis was affected with a high number of positive cells in the centre of the membrane after 15 days. It is possible therefore that Wnt signaling in the presumptive stem cell areas of the drum functions to keep cells naive, as in other systems such as the adult mammary gland, where Wnt signals promote self-renewal of the Axin2 positive stem cells(Zeng & Nusse, 2010, Lim et al., 2013 ). The importance of the Axin2cre labelled cell population to repair was highlighted using the perforated Axin2creERT2;Bcatlox(ex2-6);TdTomato mouse model. At 10 days post perforation, in control TMs, all perforations had covered the wound site. In loss-of-function TMs at 10dpp, 80% had not managed to do so, and, furthermore, the samples that had appeared to have healed, in fact, had defects in creation of the trilaminar structure. The formation of middle ear cholesteatomas has been proposed to result from the proliferation of epithelial cells in the middle ear cavity, resulting in collections of epithelial cells that have the ability to grow to large sizes (Sudhov & Tos, 2007 ). In the Axin2creERT2;Bcatlox(ex2-6);TdTomato mouse line, the outer epithelial cell layer was observed pushing into the middle ear cavity, potentially indicating the beginnings of cholesteatoma formation. The mechanisms underlying why choleastomas form are not well understood, although recent work has suggested canonical Wnt signaling to be implicated in human cholesteatoma, due to the increased expression of Wnt related genes in cholesteatoma tissue compared to that of the middle ear, as well as elevated nuclear b-catenin in the abnormal tissue (Lin et al., 2019 ). While increased Wnt is observed in fully formed cholesteatoma, it is interesting that loss of Wnt may be involved in cholesteatoma formation . This could be linked to the lack of the ability of the mesenchymal layer to cover the perforation. Without the stability provided by this layer, the epithelium could enter the middle ear cavity, leading to cholesteatoma formation (Stenfeldt et al., 2006 , 2013 ). At 15dpp, bilayer TMs were evident in the LOF mutants. Bi-layered TMs have been described in the literature, whereby, as the name suggests, the TM after repair of a perforation only contains two-layers. Electron microscopy suggested that the fibroblasts of the mesenchymal layer were lacking(Govaerts et al., 1988 ). Overall, our work has identified a marker for putative stem/progenitor cell populations in the TM, with the epithelial population reserved for repair. These finds suggest that the development of therapies augmenting Wnt signaling may have positive effects on chronic TM repair. Materials and Methods Mouse Models All mice were kept in the Biological Services Unit (BSU) at King’s College London (KCL-BSU, New Hunt’s House Guy’s Campus, KCL, UK). All animal husbandry and procedures were carried out in accordance with King’s College ethical guidelines and Home Office (HO) guidelines. Axin2creERT2- (van Amerongen et al., 2012); Wnt1Cre - (Danielian et al., 1998) expressing males and Rosa26;Tomato- ( tdTomato [ Gt(ROSA)26 Sor tm14(CAG-tdTomato) Hze /J]) expressing females were bred to create the reporter mouse lines Axin2creErt2;TdTomato and Wnt1cre;TdTomato . Axin2creErt2;TdTomato mice were bred with Bcatlox(ex2-6) (Brault et al., 2001) mouse lines to create Axin2creERT2;B-catlox(ex2-6);TdTomato mouse models. Axin2LacZ mouse lines (Lustig et al., 2002) were also used. Mice were bred on the C57/Bl6 background. Tamoxifen and TM perforation When mice were 6-8 weeks old, three rounds of intraperitoneal injection of tamoxifen (Sigma-Adrich T5648-1g) were administered at 75mg/kg (approx. 100µl of 20mg/ml tamoxifen solution) over three consecutive days. Ear drum perforation was performed in 6-8 weeks old mice as previously described in Dinwoodie et al., 2024. At specific time points mice were sacrificed and the ear drums, attached to the tympanic ring, dissected out under a Leica MZFLIII dissection scope. Dissected tissue was fixed in 4% paraformaldehyde for one hour and DAPI stained prior to whole-mount imaging. Histology and Immunofluorescence TMs were decalcified, dehydrated, and embedded in paraffin wax. 6mm sections were cut using a Leica microtome RM2245 and mounted onto superfrost slides (FisherScientific 11976299). Sections were rehydrated followed by antigen retrieval using 0.1M citrate buffer (pH 6) in a water bath at 95°C. Sections were blocked using blocking buffer (10% FBS, 1% BSA and 0.0125% tween20 in PBS) for 1-2 hours. Primary antibodies were applied overnight at 4°C (See supplementary material for table of antibodies) and washed off before secondary antibodies were applied. Finally, sections were washed and mounted using fluoroshield (Sigma F6182). TM sections are fragile and optimisations of the IHC protocol included not preheating the antigen retrieval buffer and using 0.0125% Tween20 in PBT when washing and in blocking solution. β - galactosidase Staining For Axin2LacZ mice, TMs were fixed with glutaraldehyde fixative (0.2% glutaraldehyde, 5mM EGTA and 2mM MgCl2 in 0.1M PBS). TMs were then washed with Solution B (MgCl2, deoxycholate and IGEPAL). The color was developed with a solution containing Solution B with K4Fe(CN)6, K3Fe(Ca)6 and X-gal. The blue stain developed between 3-4hrs at which time TMs were fixed overnight at 4% PFA. For Axin2LacZ sections, paraffin sections were dewaxed and co-stained for eosin to allow histological analysis. BrDU Pulse-Chase Labelling All dividing cells in the embryos of Axin2creERT2;TdTomato mice were labelled with BrDU (via intraperitoneal injection of the mother at E13.5 and E15.5). At 6-8 weeks old, the mice were given 3 consecutive injections of tamoxifen (20mg/ml), and TM samples collected 5 days after the final injection. BrDU detection was achieved by immunofluorescence as previously described. Imaging Whole ear drums on FluoroDishes (FD35-100), or immunofluorescent stained slides were imaged on a Leica TCS SP5 confocal microscope with LAS AF software or a ZEISS LSM 980 microscope. Trichrome stained slides were imaged on the Nikon Eclipse 80i and brightfield images were taken using the Leica FlexaCam A5. Images were processed and quantified in Image J (version 1.0) and figures (including schematics) were made using Adobe Illustrator (2021). Graphs were made using GraphPad Prism (Version 9.0.2). RNAsequencing Analysis The accession number for the scRNA-seq data reported in this paper is GEO: GSE196692. Quantification and Statistics An a priori power analysis was conducted using G*Power version 3.1.9.7 to determine the minimum biological sample size for the perforation experiments (Faul et al., 2007). Statistical analysis was carried out using un-paired two tailed student t-tests between control and perforated samples. Statistical analysis was conducted using GraphPad Prism (Version 9.0.2). Nnumbers for each experiment are shown in the figure legends. N numbers refer to 'mice' rather than a single tympanic membrane unless otherwise specified. Declarations Ethical approval King’s College London, under UK Home Office licenses and regulations in line with the regulations set out under the United Kingdom Animals (Scientific Procedures) Act 1986. Availability of data and materials The RNAseq datasets generated during this study were from Frumm et al., 2021. GEO: GSE196692. Competing interests The authors declare no conflict of interest. Author contributions Conceptualization: A.S.T.; Methodology: O.M.D., J.M.F.; Formal analysis: O.M.D.; Investigation: O.M.D., J.M.F.; Writing - original draft: O.M.D.; Writing - review & editing: A.S.T., J.M.F.; Supervision: A.S.T., J.M.F.; Project administration: A.S.T.; Funding acquisition: A.S.T. Funding This work was supported by the Medical Research Council (MRC), project grant awarded to A.S.T. (MR/R023719/1). O.D. was funded by the King’s College London MRC doctoral training programme. References Akkoca Ö, Kargın Kaytez S, Kaptan Z (2022) The effectiveness of repairment of traumatic tympanic membrane perforations with cigarette paper. https://doi.org/10.14744/tjtes.2020.98968 Anthwal N, Thompson H (2016) The development of the mammalian outer and middle ear. J Anat 228(2):217–232. https://doi.org/10.1111/JOA.12344 Aragona M, Dekoninck S, Rulands S, Lenglez S, Mascré G, Simons BD, Cédric Blanpain (2017) Defining stem cell dynamics and migration during wound healing in mouse skin epidermis. https://doi.org/10.1038/ncomms14684 Behrens J, Von Kries JP, Kühl M, Bruhn L, Wedlich D, Grosschedl R, Birchmeier W (1996) Functional interaction of β-catenin with the transcription factor LEF-1. Nat 1996 382(6592):6592. 638–642. https://doi.org/10.1038/382638a0 Berger GB (1989) Nature of spontaneous tympanic membrane perforation in acute otitis media in children. J Laryngology Otology 103(12):1150–1153. https://doi.org/10.1017/S0022215100111247 Bowmana AN, Van Amerongen R, Palmer TD, Nusse R (2013) Lineage tracing with Axin2 reveals distinct developmental and adult populations of Wnt/β-catenin-responsive neural stem cells. Proc Natl Acad Sci USA 110(18):7324–7329. https://doi.org/10.1073/PNAS.1305411110/SUPPL_FILE/PNAS.201305411SI.PDF Brault V, Moore R, Kutsch S, Ishibashi M, Rowitch DH, McMahon AP, Sommer L, Boussadia O, Kemler R (2001) Inactivation of the β-catenin gene by Wnt1-Cre-mediated deletion results in dramatic brain malformation and failure of craniofacial development. Development 128(8):1253–1264. https://doi.org/10.1242/DEV.128.8.1253 Chai Y, Jiang X, Ito Y, Bringas P, Han J, Rowitch DH, Soriano P, McMahon AP, Sucov HM (2000) Fate of the mammalian cranial neural crest during tooth and mandibular morphogenesis. Development 127(8):1671–1679. https://doi.org/10.1242/DEV.127.8.1671 Chari DA, Frumm SM, Akil O, Tward AD (2019) Cellular Dynamics in Early Healing of Mouse Tympanic Membranes. Otology & Neurotology: Official Publication of the American Otological Society, American Neurotology Society [and] European Academy of Otology and Neurotology, 40(2), E160–E166. https://doi.org/10.1097/MAO.0000000000002060 Clevers H, Loh KM, Nusse R (2014) Stem cell signalling: An integral program for tissue renewal and regeneration: Wnt signaling and stem cell control. Science 346(6205):1248012 Danielian PS, Muccino D, Rowitch DH, Michael SK, McMahon AP (1998) Modification of gene activity in mouse embryos in utero by a tamoxifen-inducible form of Cre recombinase. Curr Biology: CB 8(24):1323–1326. https://doi.org/10.1016/S0960-9822(07)00562-3 De Calisto J, Araya C, Marchant L, Riaz CF, Mayor R (2005) Essential role of non-canonical Wnt signalling in neural crest migration. Development 132(11):2587–2597. https://doi.org/10.1242/DEV.01857 Dinwoodie OM, Tucker AS, Fons-Romero J (2024) Tracking cell layer contribution during repair of the tympanic membrane. Dis Models Mech. https://doi.org/10.1242/DMM.050466 Dolhi N, Weimer AD (2022) Tympanic Membrane Perforations. In StatPearls. StatPearls Publishing. https://www.ncbi.nlm.nih.gov/books/NBK557887/ Faul F, Erdfelder E, Lang AG, Buchner A (2007) G*Power 3: a flexible statistical power analysis program for the social, behavioral, and biomedical sciences. Behav Res Methods 39(2):175–191. https://doi.org/10.3758/BF03193146 Frumm SM, Yu SK, Chang J, Artichoker JA, Scaria SM, Lee KP, Byrnes LE, Sneddon JB, Tward AD (2021) A Hierarchy of Proliferative and Migratory Keratinocytes Maintains the Tympanic Membrane. Cell Stem Cell 28(2):315–330e5. https://doi.org/10.1016/J.STEM.2020.10.006 Govaerts PJ, Jacob WA, Marquet J (1988) Histological study of the thin replacement membrane of human tympanic membrane perforations. Acta Otolaryngol 105(3–4):297–302. https://doi.org/10.3109/00016488809097011 Kim AS, Betz JF, Reed NS, Ward BK, Nieman CL (2022) Prevalence of Tympanic Membrane Perforations Among Adolescents, Adults, and Older Adults in the United States. Otolaryngology-Head and Neck Surgery, 2022(2), 356–358. https://doi.org/10.1177/01945998211062153 Kim SW, Kim J, Seonwoo H, Jang KJ, Kim YJ, Lim HJ, Lim KT, Tian C, Chung JH, Choung YH (2015) Latent progenitor cells as potential regulators for tympanic membrane regeneration. Sci Rep 2015 5(1):1. 1–8. https://doi.org/10.1038/srep11542 Knutsson J, Von Unge M, Rask-Andersen H (2011) Localization of Progenitor/Stem Cells in the Human Tympanic Membrane. Audiol Neurotology 16(4):263–269. https://doi.org/10.1159/000320612 Lav R, Krivanek J, Anthwal N, Tucker AS (2023) Wnt signaling from Gli1-expressing apical stem/progenitor cells is essential for the coordination of tooth root development. Stem Cell Rep 18:1015–1029 Lim X, Tan SH, Koh WLC, Chau RMW, Yan KS, Kuo CJ, Van Amerongen R, Klein AM, Nusse R (2013) Interfollicular epidermal stem cells self-renew via autocrine Wnt signaling. Science 342(6163):1226–1230. https://doi.org/10.1126/SCIENCE.1239730/SUPPL_FILE/LIM.SM.PDF Lin J, Ye Q, Wang Y, Wang Y, Zeng Y (2019) Original Article Wnt/β-catenin signaling regulates pathogenesis of human middle ear cholesteatoma. Int J Clin Exp Pathol 12(4):1154–1162. Lohi M, Tucker AS, Sharpe PT (2010) Expression of Axin2 indicates a role for canonical Wnt signaling in development of the crown and root during pre- and postnatal tooth development. Dev Dynamics: Official Publication Am Association Anatomists 239(1):160–167. https://doi.org/10.1002/DVDY.22047 Lustig B, Jerchow B, Sachs M, Weiler S, Pietsch T, Karsten U, Van De Wetering M, Clevers H, Schlag PM, Birchmeier W, Behrens J (2002) Negative Feedback Loop of Wnt Signaling through Upregulation of Conductin/ Axin2 in Colorectal and Liver Tumors. Mol Cell Biol 22(4):1184–1193. https://doi.org/10.1128/MCB.22.4.1184-1193.2002 Maruyama T, Jeong J, Sheu T-J, Hsu W (2016) ARTICLE Stem cells of the suture mesenchyme in craniofacial bone development, repair and regeneration. https://doi.org/10.1038/ncomms10526 Nusse R (2008) Wnt signaling and stem cell control. Cell Res 2008 18(5):523–527. https://doi.org/10.1038/cr.2008.47 Parekh A, Mantle B, Banks J, Douglas Swarts J, Badylak SF, Dohar JE, Hebda PA, Medicine R (2009) Repair of the Tympanic Membrane with Urinary Bladder Matrix. Laryngoscope 119(6):1206–1213. https://doi.org/10.1002/lary.20233 Park S, Gonzalez DG, Guirao B, Boucher JD, Cockburn K, Marsh ED, Mesa KR, Brown S, Rompolas P, Haberman AM, Bellaïche Y, Greco V (2017) Tissue-scale coordination of cellular behaviour promotes epidermal wound repair in live mice. Nat Cell Biol 19(2):155–163. https://doi.org/10.1038/NCB3472 Rajkhowa R, Levin B, Redmond SL, Li LH, Wang L, Kanwar JR, Atlas MD, Wang X (2011) Structure and properties of biomedical films prepared from aqueous and acidic silk fibroin solutions. J Biomed Mater Res A 97(1):37–45. https://doi.org/10.1002/JBM.A.33021 Reijnen CJH, Kuijpers W (1971) Chapter II: Experimental study on the healing process of the drum membrane. Acta Otolaryngol 72(S287):11–37. https://doi.org/10.3109/00016487109127860 Rohanizadeh R, Swain MV, Mason RS (2008) Gelatin sponges (Gelfoam®) as a scaffold for osteoblasts. J Mater Science: Mater Med 19(3):1173–1182. https://doi.org/10.1007/S10856-007-3154-Y/FIGURES/8 Santa Maria PL, Redmond SL, Atlas MD, Ghassemifar R (2010a) Keratinocyte growth factor 1, fibroblast growth factor 2 and 10 in the healing tympanic membrane following perforation in rats. https://doi.org/10.1007/s10735-010-9306-2 Santa Maria PL, Redmond SL, Atlas MD, Ghassemifar R (2010b) The role of epidermal growth factor in the healing tympanic membrane following perforation in rats. J Mol Hist 41:309–314. https://doi.org/10.1007/s10735-010-9287-1 Scaria SM, Frumm SM, Vikram EP, Easow SA, Sheth AH, Shamir ER, Yu SK, Tward AD (2023) Epimorphic regeneration in the mammalian tympanic membrane. Npj Regenerative Med 2023 8(1):1–15. https://doi.org/10.1038/s41536-023-00332-0 Shen Y, Redmond SL, Dip A, Sci A, Teh BM, Yan S, Wang Y, Zhou L, Budgeon CA, Eikelboom RH, David M, Dilley RJ, Zheng M, Marano RJ (2012) Scaffolds for Tympanic Membrane Regeneration in Rats. Tissue Engineering Part A, 19(5–6). https://doi.org/10.1089/ten.tea.2012.0053 Stenfeldt K, Johansson C, Eriksson PO, Hellström S (2013) Collagen type II is produced in healing pars tensa of perforated tympanic membranes: An experimental study in the rat. Otology Neurotology 34(7). https://doi.org/10.1097/MAO.0b013e3182908b51 Stenfeldt K, Johansson C, Hellström S (2006) The collagen structure of the tympanic membrane: collagen types I, II, and III in the healthy tympanic membrane, during healing of a perforation, and during infection. Archives Otolaryngology–Head Neck Surg 132(3):293–298. https://doi.org/10.1001/ARCHOTOL.132.3.293 Sudhov H, Tos M (2007) Pathogenesis of sinus cholesteatoma. Eur Arch Otorhinolaryngol 264:1137–1143. https://doi.org/10.1007/s00405-007-0340-y Syed SM, Kumar M, Ghosh A, Tomasetig F, Ali A, Whan RM, Alterman D, Tanwar P (2020) Endometrial Axin2 + cells drive epithelial homeostasis, regeneration and cancer following oncogenic transformation. Cell Stem Cell 26(1):64–80 Tucker AS, Dyer CJ, Romero JMF, Teshima THN, Fuchs JC, Thompson H (2018) Mapping the distribution of stem/progenitor cells across the mouse middle ear during homeostasis and inflammation. Dev (Cambridge) 145(1). https://doi.org/10.1242/DEV.154393/264412 . /AM/MAPPING-THE-DISTRIBUTION-OF-STEM-PROGENITOR-CELLS Ugarteburu M, Withnell RH, Cardoso L, Carriero A, Richter CP (2022) Mammalian middle ear mechanics: A review. Front Bioeng Biotechnol 10. https://doi.org/10.3389/FBIOE.2022.983510 Van Amerongen R, Bowman AN, Nusse R (2012) Developmental stage and time dictate the fate of Wnt/β-catenin-responsive stem cells in the mammary gland. Cell Stem Cell 11(3):387–400. https://doi.org/10.1016/J.STEM.2012.05.023 Wang B, Zhao L, Fish M, Logan CY, Nusse R (2015) Self-renewing diploid Axin2 + cells fuel homeostatic renewal of the liver. Nature 524(7564):180. https://doi.org/10.1038/NATURE14863 Whyte JL, Smith AA, Helms JA (2012) Wnt Signaling and Injury Repair. Perspectives in Biology. https://doi.org/10.1101/cshperspect.a008078 Yuan X, Xu Q, Zhang X, Van Brunt LA, Ticha P, Helms JA (2019) Wnt-Responsive Stem Cell Fates in the Oral Mucosa. IScience 21:84–94. https://doi.org/10.1016/J.ISCI.2019.10.016 Zorina A, Zorin V, Isaev A, Kudlay D, Vasileva M, Kopnin P (2023) Dermal Fibroblasts as the Main Target for Skin Anti-Age Correction Using a Combination of Regenerative Medicine Methods. Curr Issues Mol Biol 45(5):3829. https://doi.org/10.3390/CIMB45050247 Additional Declarations There is NO Competing Interest. Supplementary Files Supplementarymaterial.docx Suplementary methods and data Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9587608","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":634942260,"identity":"0d1e1b30-45be-408f-9665-3df928422c22","order_by":0,"name":"Abigail Tucker","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAUlEQVRIiWNgGAWjYDACCcYGBgYDCxCTGYhtDBjYIRIGBLRIMPBAtKQZgCn8WqAkVMthwlr4Zzc3v/hRIMFgL5H82ODjnvPG/M0MjB9+MBw2xmnJnYNtlj0gh0mkGSfOeHbbTOIwA7NkD8NhM5wOu5HYZsAD1pLDfJjnwG0bA6DDpIEutMGlQx6oxfAPTMufA+dAWph/49NicCOx+THMlmSGAwfMgFrYQLbgdJgh0C/MMgYSPDxnnhkb9hxINpY4zAjyXTpO78vdbn/88c0fGzn29uTHEj8O2Bn2tzcfvvGjwtqwAaf/GdhAUcODJACOXNzqgYD5A17pUTAKRsEoGAUAa/lMg0P+9LoAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0001-8871-6094","institution":"King's College London","correspondingAuthor":true,"prefix":"","firstName":"Abigail","middleName":"","lastName":"Tucker","suffix":""},{"id":634942261,"identity":"00b694b9-656d-45c3-a955-bb7ef8a7312a","order_by":1,"name":"Olivia Dunwoodie","email":"","orcid":"","institution":"King's College London","correspondingAuthor":false,"prefix":"","firstName":"Olivia","middleName":"","lastName":"Dunwoodie","suffix":""},{"id":634942262,"identity":"39cdc5c1-cb11-4a95-9593-8999afab699d","order_by":2,"name":"Juan Fons","email":"","orcid":"","institution":"King's College London","correspondingAuthor":false,"prefix":"","firstName":"Juan","middleName":"","lastName":"Fons","suffix":""}],"badges":[],"createdAt":"2026-05-01 15:50:42","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9587608/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9587608/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108678351,"identity":"0f742181-7f3c-4d01-808b-25d58287851f","added_by":"auto","created_at":"2026-05-07 08:57:19","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":590471,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eWnt-responding cells are located in the presumptive stem cell regions of the tympanic membrane\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A-C) Whole mount imaging of the Axin2LacZ mouse TMs (A) show positive LacZ staining around the manubrial (B) and annular regions (C). Boxed regions in A denote images B and C. LacZ labelled cells in blue. Scale bar in A = 500um, Scale bar in C=200um (same scale as in B). N = 8.\u003c/p\u003e\n\u003cp\u003e(D-F) Whole-mount imaging of the Axin2creERT2;TdTomato at 5 days post tamoxifen injection (dpi). Axin2 cells in red. DAPI in blue. Boxed regions in D denote images E (manubrium) and F (annulus). Scale bar in D= 500µm. Scale bar in E and F = 200µm. N=8.\u003c/p\u003e\n\u003cp\u003e(G-I) TM sections at 5 days post injection (5dpi). (G) Red fluorescent protein, RFP (red), vimentin (white), Dapi (blue), showing presence of Axin2cre labelled cells in the lamina propria. (H-I) section through the manubrial (H) and annular (I) regions stained for RFP (red), pan-cytokeratin (PCK) (green) show little colocalization, highlighting low levels of Axin2cre labelled cells in the outer epithelial layers of the TM. Axin2cre labelled cells can be observed in the mesenchymal region (I, arrow). Scale bar H and I = 50um. N=6, immuno repeated 3 times.\u003c/p\u003e\n\u003cp\u003eA = annulus. M = manubrium. OEL = outer epidermal layer, IML = inner mucosal layer.\u003c/p\u003e","description":"","filename":"image1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9587608/v1/a4ab20a9767b63946b52b9f6.jpeg"},{"id":108678358,"identity":"4501928c-d6cb-4ab5-bb20-db99e4006d30","added_by":"auto","created_at":"2026-05-07 08:57:21","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":601566,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eWnt responding cells in the TM are long lasting and label retaining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A,B) Whole-mount imaging of the Axin2creERT2;TdTomato mouse at 2 months (A) and 10 months (B) post tamoxifen injection shows that Axin2cre labelled cells remain in the putative stem cell areas of the TM over this timeframe. Scale bar in B = 500um, same scale in A. N=3 long trace.\u003c/p\u003e\n\u003cp\u003e(C-E) Sections of Axin2creErt2;TdTomato mouse. RFP (red), pan-cytokeratin (green), dapi (blue). (C) 10 month chase. Arrow showing location of RFP (red) cells in the LP around the manubrium. In contrast, the PCK-positive epithelial layer does not show any labelled cells. (D) Short lineage trace (15dpi). Only a few RFP-positive cells are evident in the central region of the membrane between the annulus and manubrium. (E) 10 month chase. Compared to C, the membrane has many more RFP-positive cells (Arrow). Insert shows rare RFP-positive cell in the outer epithelium. Scale bar in C=50um, Scale bar in D = 100um. Scale bar in E= 200um. (F) Graph showing the percentage of total cells in the LP which are Axin2cre positive in the Axin2creERT2;Tdtomato mouse line at 5 days post injection (DPI) vs 2-10 month post injection (mpi), N=3 per age. Counts were conducted in 200um away from the annulus up to 200um away from the manubrium. Statistical analyses was performed via an unpaired students t-test. P value=0.038. Error bars show SD.\u003c/p\u003e\n\u003cp\u003e(G-J) BrDU labelling of the TM. Injections at E13.5 and E15.5. (G) Section through P0 TM stained for BrDU (green). All cells are labelled after the pulse (Scale bar = 50um). (H-J) BrDU sections at the annulus (H-I''), and manubrial regions (J-J'') 6-8 weeks after the chase. I'' scale bar =50um (same scale as in H-J'') N=3 mice (6TMs). Arrows in H'', I'' and J'' point to double positive (yellow) RFP (red) and BrDU (green) cells. (K) Quantification of BrDU and Axin2 positive cells. All BrDU positive cells are RFP positive. Statistics used to compare two bars (Axin2 and BrDU positive vs BrDU positive and Axin2 negative was a student’s unpaired t-test). P value = 0.002, error bars = SD.\u003c/p\u003e\n\u003cp\u003eA= annulus, M= manubrium. OEL = outer epidermal layer, IML = inner mucosal layer.\u003c/p\u003e\n\u003cp\u003e(L) U-maps showing high expression of epithelial stem cell markers (Itga6, Krt5 and Itgb1) in Axin2 positive epithelial cells in the unwounded TM. (M) U-maps showing the expression of cellular quiescence markers (Trp53, Smarca2, Cdkn1a) in Axin2 positive epithelial cells.\u003c/p\u003e","description":"","filename":"image2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9587608/v1/e8cca5999e5263d318ada4ac.jpeg"},{"id":108678320,"identity":"a3c63dbb-b46e-42b2-a0a2-234cfa62ce72","added_by":"auto","created_at":"2026-05-07 08:57:14","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":894816,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eWnt-responding cells are involved in the repair of tympanic membrane perforations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A-K) Whole-mount imaging of \u003cem\u003eAxin2creERT2;TdTomato\u003c/em\u003e TMs (schematic for protocol showing perforation 5 days after tamoxifen injection). (A-C) Wnt-responding cells are located at the manubrium and annulus in control TMs (contralateral ear to perforation) at 5dpp (A), 10dpp (B) and 30dpp (C). \u0026nbsp;(D-I) Perforated TMs at 5dpp (D,G), 10dpp (E,H) and 30dpp (F,I). \u0026nbsp;Scale bar in F = 500um, same scale in A-E. (G-I) Higher magnification view of the perforated regions in D,E,F (Scale bar in I= 100um, same as in G and H). White circles in D-I denote area of injury. N=6/timepoint.\u003c/p\u003e\n\u003cp\u003e(J,K) Whole-mount imaging of \u003cem\u003eAxin2creERT2;TdTomato\u003c/em\u003e TMs (schematic for protocol showing perforation 5 months after tamoxifen injection, imaged 10 days after perforation (10dpp)). K showed high power of dashed region in J. N= 2. Scale bar in J =500um, scale bar in K = 200um.\u003c/p\u003e","description":"","filename":"image3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9587608/v1/8dae43fe9c5f5251cefa904d.jpeg"},{"id":108678410,"identity":"f33797bd-b3bc-4247-819f-24a53f55f6d9","added_by":"auto","created_at":"2026-05-07 08:57:38","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":633443,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLineage traced Wnt-responding cells make a major contribution to the healed membrane\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A-G) Sections through the \u003cem\u003eAxin2creERT2;TdTomato\u003c/em\u003e TM. RFP (red), Pan-cytokeratin (green), PCNA (white) dapi (blue). (A,B,B’,D,D’) Perforated TM. Arrowheads point to RFP positive cells. IML= inner mucosal layer, OEL= outer epithelial layer. LP = lamina propria. (A) 5dpp (5 days post perforation). The PCK positive outer epithelial layer has proliferated and contains many RFP-positive cells (arrowhead with asterix). RFP positive cells are abundant in the middle layer (LP) (arrowhead). (B,B’) 10dpp. The perforation is bridged at this stage. RFP positive cells are observed in all layers of the repairing membrane. Arrowheads in B point to positive cells in the outer epithelial layer. \u0026nbsp;(B’) Serial section to B. PCNA positive proliferating cells in all layers of the repairing membrane. Arrowhead points to co-localisation of PCNA in the RFP positive basal cells of the outer epithelium. 10dpp timepoint N=6. PCK/PCNA/RFP immunos N=3. (C) Unperforated ear drum (contralateral to perforated at 30 days post perforation 30dpp). (D) Perforated ear drum 30dpp. The membrane is still thickened at the site of repair. Arrowhead points to RFP positive cell in the inner endoderm layer. (D’) magnification of boxed area in D. Arrows point to multiple RFP positive cells in the PCK positive outer epithelium.\u003c/p\u003e\n\u003cp\u003eScale bar in D =50um, same scale as in C.\u003c/p\u003e\n\u003cp\u003e(E) Graph showing the proportion of \u003cem\u003eAxin2cre\u003c/em\u003elabelled cells present in the outer epithelial layer of the TM at unperforated and 30dpp timepoints. Statistical analysis via unpaired students t-tests. RFP/PCK immuno repeated 6 times/ timepoint. N=6 TMs for each condition. p value \u0026gt;0.0001.\u003c/p\u003e\n\u003cp\u003e(F,F’) \u003cem\u003eWnt1cre;TdTomato\u003c/em\u003e mouse line. Sections through membrane 30dpp. Scale bar in F = 100um. (F’) Magnification boxed area in F. \u003cem\u003eWnt1cre;TdTomato\u003c/em\u003e30dpp N=2.\u003c/p\u003e\n\u003cp\u003eIML= inner mucosal layer, OEL= outer epithelial layer. LP = lamina propria.\u003c/p\u003e","description":"","filename":"image4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9587608/v1/2f13afc735b3080c90ecc759.jpeg"},{"id":108678355,"identity":"a2087838-b045-45ee-9a06-00ac03ef932d","added_by":"auto","created_at":"2026-05-07 08:57:20","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":469900,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eWnt signaling is upregulated in the mesenchymal layer during repair\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A-D) Sections through the TM after perforation. (A-A’’) 5dpp. (A) Lef-1 staining (yellow) as a read out of Wnt activity is found in the mesenchyme at the site of repair but is absent from the expanded outer epithelium. (A’) RFP (red) in same section. (A’’) Merged channels to highlight lack of overlap in the epithelium. Inset shows that not all Lef1 cells are also RFP labelled in the mesenchyme (arrow-asterix). Scale bars = 50um. 5dpp timepoint N=6. Lef1/RFP immuno N=3. (B) 10dpp. RFP (red) and Lef1 (yellow). \u0026nbsp;Wnt signaling is still active in the mesenchymal layer at this timepoint. Insert. Arrow points to Lef1 and RFP positive cell, arrow-asterisk points to Lef-1, RFP negative cell. Scale bar = 100um. 10dpp timepoint N=6. Lef1/RFP immuno N=3.\u003c/p\u003e\n\u003cp\u003e(C,D) Axin2LacZ mouse at 10dpp (C) and 17dpp (D). Axin2 staining (blue) to indicate active Wnt signaling in the mesenchymal and inner layers with limited label (arrow) in the outer epidermal layer. Scale bars in C and D = 100um. N=3 Axin2LacZ/timepoint.\u003c/p\u003e\n\u003cp\u003eIML= inner mucosal layer, OEL= outer epithelial layer. M = Mesenchymal layer.\u003c/p\u003e","description":"","filename":"image5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9587608/v1/ee4dc82c08c894c670cfb3d0.jpeg"},{"id":108678411,"identity":"2cc003b3-f710-4433-8399-aa775577c1c5","added_by":"auto","created_at":"2026-05-07 08:57:38","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":425320,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLoss-of-function of B-catenin in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAxin2cre\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e labelled cells in homeostatic conditions changes the distribution of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAxin2cre\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e labelled cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A,B) Non-phosphorylated (active) β-catenin staining (cyan) of the cells at the annular region of the TM. Dapi (dark blue). (A) Control (\u003cem\u003eAxin2creERT2;TdTomato\u003c/em\u003e) mouse line, showing expression of β-catenin in the nucleus (overlapping with DAPI). Insert shows expression in mesenchymal cells at annulus. (B) Loss-of-function (\u003cem\u003eAxin2creERT2;Bcatlox(ex2-6);TdTomato\u003c/em\u003e) mouse line shows loss of expression of active β-catenin at 5 days post tamoxifen injection (5dpi). Scale bar in B = 50um, same scale in A. N=2/condition.\u003c/p\u003e\n\u003cp\u003e(C,D,F) RFP (red), PCK (green), Dapi (blue). (C) \u003cem\u003eAxin2creERT2;Tomato\u003c/em\u003emouse TM 15dpi. Limited RFP (red) cells are evident in the main body of the membrane. (D,F) \u003cem\u003eAxin2creERT2;Bcatlox(ex2-6);Tomato\u003c/em\u003e loss-of-function mouse TM 15dpi. White arrowhead in F showing presence of RFP positive cells in the middle layer in the centre of the membrane. Boxed region in D = area zoomed in in F. Ctrl N=6, LOF N=3 (mice). (E) Graph showing the percentage of cells in the central TM middle layer (200um away from annular and manubrial regions) which are \u003cem\u003eAxin2cre\u003c/em\u003e positive at 15 dpi. Error bars show SEM, statistical analysis performed via an unpaired students t-test to give p= 0.0002 (N=3/ condition, mice).\u003c/p\u003e\n\u003cp\u003eIML = inner mucosal layer, OEL = outer epithelial layer\u003c/p\u003e","description":"","filename":"image6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9587608/v1/bd3025784747170c990eac4d.jpeg"},{"id":108678421,"identity":"a1dc0ed2-02ce-4ab8-a108-c56daaf6d73d","added_by":"auto","created_at":"2026-05-07 08:57:42","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":876394,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLoss-of-function of Wnt signaling in Axin2cre labelled cells results in TMs that fail to repair by 15 dpp.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A,B) Trichrome stained sections of control (A) and LOF (B) mouse models at 5 days post perforation (dpp) showing extensive epithelial scaffold formation in both conditions. Scale bar in A= 100um, same as in B. C) Table comparing healing between control and LOF Wnt signaling mouse models at 5 days post perforation (D5), 10 days post perforation (D10) and 15 days post perforation (D15). Red circles denote control TMs, blue triangles denote LOF TM. (Controls D5 N=8, D10 N=8, D15 N=8, LOF D5 N=2, D10 N=5, D15 N=6.\u003c/p\u003e\n\u003cp\u003e(D-I) Whole-mount imaging of TM perforations. (D, G) Control \u003cem\u003eAxin2creERT2;TdTomato\u003c/em\u003e shows perforation closure and coverage by the \u003cem\u003eAxin2Cre\u003c/em\u003elabelled population at 10dpp (D) and 15dpp (G). (E,F,H,I) Whole-mount imaging of \u003cem\u003eAxin2creERT2;Bcatlox;TdTomato\u003c/em\u003e TM perforation. (E, F) 10dpp. (H,I) 15dpp. A hole in the membrane is still apparent at these stages. White arrows in D and E indicate air bubbles. White boxes in E and H denote regions in F and I. Scale bars in E and H = 500um, same scale in D and G.\u003c/p\u003e\n\u003cp\u003e(J,K) Trichrome stained TM 15dpp. (J) Control \u003cem\u003eAxin2creERT2;TdTomato\u003c/em\u003emouse showing healed perforation. (K) LOF \u003cem\u003eAxin2creERT2;Bcatlox;TdTomato\u003c/em\u003e mouse showing unhealed perforation. Scale bar in K= 200um, same scale as in J.\u003c/p\u003e\n\u003cp\u003eIML = inner mucosal layer, OEL = outer epithelial layer\u003c/p\u003e","description":"","filename":"image7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9587608/v1/b19aa241e8161070f1aad634.jpeg"},{"id":108678423,"identity":"7cb11193-181c-4bac-92f8-3ce3d7174cc5","added_by":"auto","created_at":"2026-05-07 08:57:45","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":224171,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic of the roles of Axin2cre labelled cells in TM homeostasis and repair.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAxin2 labelled cells (red dots) are located in all layers of the TM at the manubrium and annulus. In homeostasis, the epithelial Axin2 population remains quiescent, but the mesenchymal population contributes to turn over in the middle layer.\u003c/p\u003e\n\u003cp\u003eDuring repair the epithelial Axin2 labelled cells proliferate and contribute to creation of the repaired TM. In homeostasis active Wnt signalling (light blue areas) is localised to the manubrium and annulus, but during repair is upregulated in the mesenchyme and endoderm layers of the repairing TM.\u003c/p\u003e\n\u003cp\u003eIML= inner mucosal layer (yellow), LP= lamina propria (pink), OEL= outer epithelial layer (green), M= manubrium, A= annulus.\u003c/p\u003e","description":"","filename":"image8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9587608/v1/52c3617108d49bee9c3ed59f.jpeg"},{"id":108805546,"identity":"cdbdfd04-a5b5-451c-a18c-170f9f7021c0","added_by":"auto","created_at":"2026-05-08 15:26:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5090296,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9587608/v1/62207d81-1e7c-4fad-9c77-e48b0e95410b.pdf"},{"id":108678391,"identity":"9fba02d0-4ef7-41aa-b3b0-5c3cabb6037e","added_by":"auto","created_at":"2026-05-07 08:57:32","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":6840730,"visible":true,"origin":"","legend":"Suplementary methods and data","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-9587608/v1/c467557eb57c1ac2fc29d706.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Wnt-responding stem/progenitor cells are essential for tympanic membrane repair","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe mammalian ear is divided into three parts, each essential for hearing: the outer, middle, and inner ears. The external, or outer, ear serves as the first point of contact for sound vibrations in the process of hearing. Sound waves transmitted from the pinna enter the ear canal where they are funneled to the tympanic membrane (TM), the structure at the end of the outer ear (Anthwal \u0026amp; Thompson, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Ugarteburu et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The TM consists of two main parts, the superior pars flaccida (PF) and inferior pars tensa (PT), and is made up of three layers, each originating from a separate germ layer. The outer epidermal layer (OEL), derived from the ectoderm, consists of keratinocytes, the neural crest-derived mesenchymal layer, which is also known as the lamina propria (LP), additionally accommodates the blood vessels and nerves of the TM, while the inner mucosal layer (IML) is endoderm derived (Dinwoodie et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTympanic membrane perforations (TMPs) are common, affecting 2.1% of the population in the US each year (Kim et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In the majority of TMPs, the TM will spontaneously heal within 10 days, and further medical intervention is not required (Berger, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1989\u003c/span\u003e). In 6\u0026ndash;10% of cases, the TMP will not heal and becomes chronic (Dolhi \u0026amp; Weimer, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Chronic TMPs are serious and can result in recurrent infections, middle ear cysts, otalgia, tinnitus, and conductive hearing loss (Dolhi \u0026amp; Weimer, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The use of growth factors, stem cells and scaffolds have been researched as alternative therapies to treat TMPs and move past the need for surgical intervention (currently the only treatment option). However, the mechanisms altering healing rates due to the application of these factors is poorly understood, and until this knowledge is expanded the use of these approaches is limited (Akkoca et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Parekh et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Rajkhowa et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Rohanizadeh et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Shen et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eGiven the impressive regenerative capacity of the TM, it has been postulated that a stem/progenitor population must reside in the membrane to contribute to its repair after injury (Chari et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; S. W. Kim et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Knutsson et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Tucker et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). BrDU-retaining cells have been localized to the manubrium of the malleus and the annulus, highlighting these cells of interest as a potential stem/progenitor population (Tucker et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The manubrium inserts into the middle of the TM, while the annulus connects the membrane to the supporting tympanic membrane (Tucker et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Cells expressing epithelial stem cell markers such as α\u003csub\u003e6\u003c/sub\u003e-integrin, β\u003csub\u003e1\u003c/sub\u003e-integrin and cytokeratin 19 have also been localized to these two regions, adding further weight that these locations are the niche regions of the TM (S. W. Kim et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Knutsson et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Finally, recent work involving Edu-incorporation has suggested epithelial cell proliferative zones to be located at the handle of the malleus and the mallear folds (the border region between the PF and PT), providing an epidermal specific stem cell niche responsible for homeostatic turnover of the outer layer of the TM (Frumm et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHere we investigate the role of Wnt-responding cells in homeostasis and repair of the tympanic membrane using the mouse as a model. Canonical Wnt signalling has been shown to be essential for stem-cell renewal, proliferation, and differentiation in a number of organs (Clevers et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) but has not been previously assessed in the TM. \u003cem\u003eAxin2\u003c/em\u003e is a direct intracellular target of canonical Wnt signalling and has been shown to label both epithelial and mesenchymal stem cells populations (Syed et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Lav et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). We show that \u003cem\u003eAxin2\u003c/em\u003e expressing cells, are located in the putative TM stem cell niches, contribute to the repair process, and play essential roles during repair of the membrane.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eWnt-responding cells are found in the presumptive stem cell regions of the tympanic membrane\u003c/h2\u003e \u003cp\u003eTo identify the location of Wnt responding cells in the TM, \u003cem\u003eAxin2LacZ\u003c/em\u003e heterozygous mice were utilised. This reporter shows current canonical Wnt activity (Lohi et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Wnt responding cells were observed around the annulus and the manubrial regions, the two sites previously identified as putative stem cell niches (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-C). A similar expression pattern was observed using \u003cem\u003eAxin2creERT2;TdTomato\u003c/em\u003e mice analyzed 5 days after 3 days of tamoxifen injection (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD-F), confirming that \u003cem\u003eAxin2\u003c/em\u003e labelled cells (tdTomato positive) reside in the presumptive stem cell regions of the TM under homeostatic conditions. In addition to the region around the annulus and manubrium, \u003cem\u003eAxin2\u003c/em\u003e positive cells were also evident at the border between the pars flaccida and pars tensa (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD), a region from which the epithelial cells have been shown to turn over during homeostasis (Frumm et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo discern the location of the cells within the three layers of the tympanic membrane (outer epithelium, middle mesenchyme, inner mucosa), the TMs were sectioned, and immunofluorescent analysis performed. Vimentin was used as a marker of the mesenchymal layer of the TM, while Pan-cytokeratin (PCK) was used as a marker of the outer epithelial layer of the TM (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG,H,I). The majority of \u003cem\u003eAxin2\u003c/em\u003e labelled cells (RFP positive) co-expressed vimentin, highlighting their location in the mesenchyme of the TM at the manubrium and annulus (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). In contrast, only a few \u003cem\u003eAxin2\u003c/em\u003e labelled cells were located in the endoderm and ectoderm layers (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG-I).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eWnt responding cells in the TM are label retaining and long-lasting\u003c/h3\u003e\n\u003cp\u003eTo discern whether \u003cem\u003eAxin2cre\u003c/em\u003e labelled cells contributed to homeostasis of the TM, three consecutive injections of tamoxifen were given to \u003cem\u003eAxin2creERT2;TdTomato\u003c/em\u003e mice and the labeled Wnt-responding cells were traced for 2- and 10- months. The outer epithelial layer of the tympanic membrane has been shown to have a turnover of 3 weeks (Frumm et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). At both 2 and 10 months the original \u003cem\u003eAxin2\u003c/em\u003e cells and any progeny were still located around the annulus and the manubrium, highlighting their longevity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA,B). The majority of the \u003cem\u003eAxin2\u003c/em\u003e-labelled cells were situated within the mesenchyme (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC,E), with occasional RFP positive lineage traced cells in the outer epidermal layer (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, white box arrowhead) and inner mucosal layer (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, arrowhead). The rare epithelial Axin2 cells, therefore, did not appear to play any significant role in homeostasis. In contrast, the \u003cem\u003eAxin2\u003c/em\u003e-labelled cells in the mesenchymal layer, were more prominent across the membrane at 2 and 10 months post tamoxifen (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC,E), when compared to 15 days post tamoxifen injection (15dpi) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), suggesting the cells had contributed to turnover of the middle layer of the membrane during homeostasis. Quantification of the number of \u003cem\u003eAxin2\u003c/em\u003e labelled cells in the central portions of the TM (defined as 200\u0026micro;m away from the annular and manubrial regions), confirmed significantly higher numbers of \u003cem\u003eAxin2\u003c/em\u003e-labelled cells in the mesenchyme in this region over time (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). Interesting, the turnover of the mesenchymal layer appearing much slower than has been reported for the outer ectodermal layer, suggesting independent turnover rates in the different layers (Frumm et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further investigate whether the \u003cem\u003eAxin2-\u003c/em\u003epositive cells are a stem/progenitor population, the label retaining potential of the \u003cem\u003eAxin2\u003c/em\u003e-positive population was analyzed. BrDU was injected into a pregnant dam (tdTom female crossed to \u003cem\u003eAxin2creERT2;TdTomato\u003c/em\u003e male) at E13.5 and E15.5. The majority of cells were positive for BrDU at P0, confirming successful incorporation of BrDU as a \u0026ldquo;pulse\u0026rdquo; (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). When the offspring were 6\u0026ndash;8 weeks old (7\u0026ndash;9 week chase), three consecutive injections of tamoxifen were given to label \u003cem\u003eAxin2\u003c/em\u003e expressing cells. Eardrums were quantified 5 days post injection to determine the proportion of a) BrDU-positive, b) \u003cem\u003eAxin2\u003c/em\u003e-positive, and c) both \u003cem\u003eAxin2-\u003c/em\u003epositive and BrDU-positive and d) BrDU-positive but \u003cem\u003eAxin2-\u003c/em\u003enegative cells (N\u0026thinsp;=\u0026thinsp;6 eardrums in total). Label retaining cells made up roughly 10% of the total cells in the TM and were identified in all layers of the membrane, concentrated around the annulus and manubrium (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH-K), agreeing with previous reports (Tucker et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Importantly, \u0026gt;\u0026thinsp;99% of the label retaining cells expressed \u003cem\u003eAxin2\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH\u0026rdquo;-J\u0026rdquo;,K). The label retaining cells in the TM are, therefore, Wnt-responding cells. The \u003cem\u003eAxin2-\u003c/em\u003epositive population made up a higher proportion than the label-retaining population (around 35\u0026ndash;40%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH\u0026rsquo;,I\u0026rsquo;,J\u0026rsquo;,K), and, therefore, not all \u003cem\u003eAxin2\u003c/em\u003e expressing cells were BrDU positive, particularly in the mesenchyme (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH\u0026rdquo;-J\u0026rdquo;,K). This suggests that \u003cem\u003ethe Axin2\u003c/em\u003e expressing cells label distinct subsets in the membrane.\u003c/p\u003e \u003cp\u003eTo further investigate the epithelial \u003cem\u003eAxin2\u003c/em\u003e-positive population in the TM, a previously published single cell RNAseq dataset was analyzed (Frumm et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Epithelial \u003cem\u003eAxin2\u003c/em\u003e-positive cells were analyzed for their expression of epithelial stem cell markers (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eL) and markers of cellular quiescence (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eM). The \u003cem\u003eAxin2\u003c/em\u003e positive population in the OEL strongly expressed α\u003csub\u003e6\u003c/sub\u003e-integrin (\u003cem\u003eItga6\u003c/em\u003e) and β\u003csub\u003e1\u003c/sub\u003e-integrin (\u003cem\u003eItgb1\u003c/em\u003e), epithelial stem cell markers that have previously been identified in the TM (Kim et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), as well as the basal keratinocyte maker, keratin 5 (\u003cem\u003eKrt5\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eL). As the \u003cem\u003eAxin2\u003c/em\u003e positive population was not involved in the homeostatic turnover of the OEL, it was hypothesized that these cells therefore might be a quiescent population. The population was found to express \u003cem\u003eTrp53\u003c/em\u003e, \u003cem\u003eSmarca2\u003c/em\u003e and \u003cem\u003eCdkn1a\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eM), all markers of cellular quiescent. Overall, the epithelial \u003cem\u003eAxin2\u003c/em\u003e-positive population in the ear drum was shown to be label retaining, long-lasting, and positive for stem and quiescent cell markers, highlighting this subset of cells as an epithelial stem cell population.\u003c/p\u003e\n\u003ch3\u003eWnt-responding cells are involved in the repair of tympanic membrane perforations\u003c/h3\u003e\n\u003cp\u003eTo discern whether the Axin2-postive LRCs could be activated after injury, the Wnt-responding population was followed after TM perforation. A perforation was made 5 days after 3 consecutive rounds of tamoxifen injections and the \u003cem\u003eAxin2creERT2;TdTomato\u003c/em\u003e mice were sacrificed at three timepoints, 5-, 10- and 30-days post perforation (dpp). The murine TM is able to robustly repair small perforations over this time frame (Scaria et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Dinwoodie et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Perforations of uniform size were made at uniform positions under the manubrium using an otoscope for visualisation (Dinwoodie et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The left ear of each mouse was left uninjured and was used as an internal control. Whole-mount imaging of the TMs confirmed that in the unperforated ears the \u003cem\u003eAxin2\u003c/em\u003e labelled population was restricted to the annulus and manubrium (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) at 5dpp (days post perforation of the contralateral side), 10 dpp (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB) and 30dpp (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). In contrast, in the injury model the labelled \u003cem\u003eAxin2\u003c/em\u003e population, and any progeny, moved towards the wound site as a sheet of cells from the surrounding tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, G) evident at 5dpp. At 10dpp, the \u003cem\u003eAxin2\u003c/em\u003e population was observed covering the site of the original perforation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, H circle). By 30dpp, when the tissue had nearly completed the remodelling phase, the labelled population were present in the healed wound site (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF, I)(N\u0026thinsp;=\u0026thinsp;6/timepoint).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo decipher whether the \u003cem\u003eAxin2\u003c/em\u003e cells were poised for activation in response to injury, the \u003cem\u003eAxin2-\u003c/em\u003epositive cell population was labelled by injection of tamoxifen, but the mice were left for 5 months prior to perforation. After perforation, the long-lineage traced cells were observed in and around the healed perforation at 10dpp (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ,K), in an identical fashion to the shorter lineage traces (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE,H).\u003c/p\u003e\n\u003ch3\u003eAxin2 labelled cells contribute extensively to the epithelial layer of the healing membrane\u003c/h3\u003e\n\u003cp\u003eDuring the first phases of repair the epithelium initially retracts away from the wound site before proliferating and extending to form a scaffold that bridges the gap around day 7. The mesenchyme and endodermal layers then migrate along the epithelium to recreate the three-tissue layer structure (Dinwoodie et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Sections through the TM at 5 days post perforation (5dpp), highlighted the presence of a large number of \u003cem\u003eAxin2\u003c/em\u003e labelled cells (red) in the forming epithelial scaffold (labelled in green with pan-cytokeratin) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA arrow-asterix), and in the mesenchymal layer (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA arrowhead) prior to closure of the perforation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThese cells were still evident at the wound site in all three layers (ectoderm, mesenchyme, endoderm) at 10dpp (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). A similar arrangement of \u003cem\u003eAxin2\u003c/em\u003e labelled cells was observed in the long-lineage trace experiments where the membrane was perforated 5 months after tamoxifen activation (Supplementary Fig.\u0026nbsp;1), illustrating that the population labelled 5 months prior to perforation contribute to the healing of all layers of the TM at 10dpp. Many of the epithelial and mesenchymal \u003cem\u003eAxin2\u003c/em\u003e labelled cells were positive for the proliferation marker PCNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB' arrowhead), with proliferation in the epithelium at this stage mainly observed in the basal layer (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB'). By 30 days post perforation, the tissue had almost fully healed and was undergoing remodelling to regain its thin structure (Scaria et al.,2023). The \u003cem\u003eAxin2\u003c/em\u003e population at this timepoint made up much of the epithelial and mesenchymal layers (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, D\u0026rsquo;), compared to the unperforated contralateral ear, particularly for the epithelial population (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Quantification of the proportions of \u003cem\u003eAxin2\u003c/em\u003e-positive cells in the epithelium under control conditions and after healing of a perforated drum at 30dpp, showed a significant increase in the ratio of \u003cem\u003eAxin2\u003c/em\u003e-labelled cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE), with numbers increasing 5-fold to contribute to almost 60% of the epithelium of the repaired part of the membrane. The increase in \u003cem\u003eAxin2\u003c/em\u003e-lineage cells in the epithelium was likely due to proliferation of the progeny of the rare label retaining epithelial \u003cem\u003eAxin2-\u003c/em\u003ecells of the manubrium and annulus. Alternatively, it was possible some cells from the mesenchyme were able to undergo a mesenchyme-epithelial transformation during repair and thereby contribute cells to the repaired epithelium. In keeping with this, repair of the membrane has been described as forming by creation of a multi-lineage blastema-like cell mass (Scaria et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). To test this, perforations were performed in \u003cem\u003eWnt1cre;TdTomato\u003c/em\u003e mouse line where the middle mesenchymal layer is labelled with RFP (Dinwoodie et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Robust expression of RFP was evident in the middle layer after healing at 30dpp, with no contribution to the epithelial layer (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF,F\u0026rsquo;). MET can, therefore, be ruled out.\u003c/p\u003e\n\u003ch3\u003eThe Wnt pathway is upregulated in the mesenchyme layer during repair\u003c/h3\u003e\n\u003cp\u003eIn addition, to its role in regulating stem cells, Wnt signalling is upregulated in response to injury in a number of tissues (reviewed in Whyte et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). To observe whether cells were actively responding to Wnt signalling as a consequence of the injury, the expression of Lef-1 was investigated at 5dpp. Lef-1, like \u003cem\u003eAxin2\u003c/em\u003e, is a downstream target of canonical Wnt signalling (Behrens et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1996\u003c/span\u003e), and, therefore, its expression marks cells actively responding to Wnt signalling. Robust Lef-1 expression was observed in the mesenchymal layer at the injury site, suggesting activation of Wnt signalling in response to injury (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). In contrast, the large epithelial scaffold was entirely Lef-1 negative (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). In keeping with this, the \u003cem\u003eAxin2\u003c/em\u003e cells labelled prior to injury showed overlapping expression with Lef-1 in the mesenchyme but not epithelium (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA\u0026rsquo;,A\u0026rdquo;). At 10 days post perforation, the repairing tissue has already undergone mesenchymal and endodermal proliferation and migration over the wound site (Dinwoodie et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). At this stage, Lef-1 expression in the mesenchyme remained high, with numerous RFP positive and Lef-1 positive cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). To confirm that Wnt signalling was activated in the mesenchymal layer as part of the repair response the Wnt response was analysed in \u003cem\u003eAxin2LacZ\u003c/em\u003e mice at 10 and 17dpp. High levels of LacZ were observed in the mesenchyme and inner mucosal layer (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC,D). In contrast, only minimal labelling of LacZ was observed in the recovering outer epithelial layer (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC,D arrow).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eLoss-of-function of\u003c/b\u003e \u003cb\u003eβ\u003c/b\u003e\u003cb\u003e-catenin in Axin2 cells results in a higher turnover of the mesenchymal layer.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn order to investigate the impact of loss of the \u003cem\u003eAxin2\u003c/em\u003e population on homeostasis, the \u003cem\u003eAxin2creERT2; β-catlox(ex2-6);TdTomato\u003c/em\u003e mouse model was utilised, where Cre-mediates the excision of exon 2\u0026ndash;6 of β-catenin is disrupt canonical Wnt signalling (Brault et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). To confirm the effectiveness of the tamoxifen induced recombination, sections from the model were stained at 5-days post injection for non-phosphorylated (active) β-catenin. In control TMs (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA), active β-catenin was found in the nucleus of cells found at the annular region of the TM. In the loss-of-function model, however, no β-catenin staining was observed in these annular cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). As shown at 5 days post injection (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD,F), at 15 days post injection (15dpi), only a few Axin2 labelled cells were observed in central regions of the TM at a distance from the stem cell niches of the annulus and manubrium in control mice (\u003cem\u003eAxin2creERT2\u003c/em\u003e;\u003cem\u003etdTomato\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). In the loss-of-function model however, at 15 dpi, \u003cem\u003eAxin2cre\u003c/em\u003e labelled cells were observed in the mesenchyme in the central TM (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD,F). Quantification revealed that a statistically higher proportion of cells in the central mesenchymal layer (defined as 200\u0026micro;m away from the annular and manubrial regions) of the TM were Axin2cre labelled in the loss-of-function compared to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). As the \u003cem\u003eAxin2cre\u003c/em\u003e labelled population is responsible for the mesenchymal turnover of the TM (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), this process was accelerated in the loss-of-function model, suggesting a disruption to the turnover of this population.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eLoss-of-function of Wnt signaling in Axin2cre labelled cells results in TMs that cannot repair at 10 or 15 dpp.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe impact of loss of Wnt signalling in the Axin2cre population on repair was then tested using the perforation model. At 5dpp, the loss-of-function mouse appeared very similar morphologically in its early response to a perforation when compared to the reporter mouse line \u003cem\u003eAxin2creERT2;Tomato\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-C). Wnt signalling in the Axin2 population was, therefore, not required for blastema formation. In contrast at 10dpp, 80% of the perforations were still open in the loss of function eardrums (N\u0026thinsp;=\u0026thinsp;4/5 LOF TMs), while the \u003cem\u003eAxin2creERT2;TdTomato\u003c/em\u003e controls showed closure in 100% of cases (N\u0026thinsp;=\u0026thinsp;8/8 control TMs) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC-F). Interestingly, although the wounds failed to close completely, \u003cem\u003eAxin2cre labelled\u003c/em\u003e lineage traced cells had still been recruited to around the wound site in the mutants (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE-F). In section, defects in layer structure were evident in the one ear drum that had managed to close, while in the others, epithelial cells were observed pushing through the ear drum into the middle ear cavity (Supplementary Fig.\u0026nbsp;2A,B).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBy 15dpp, the hole had healed in 100% of control eardrums (N\u0026thinsp;=\u0026thinsp;8/8 control TMs), however, the perforation failed to heal in 66% of cases in the mutants (N\u0026thinsp;=\u0026thinsp;2/6 LOF TMs) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC,G-I), with histology showing a persistent gap in the membrane (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eJ,K). Interestingly, the ear drums that had healed showed defects, such as creation of a bilayered structure with a missing mesenchymal layer (Supplementary Fig.\u0026nbsp;2C,D). Therefore, disrupting Wnt signaling in the Axin2 population prior to TM perforation impairs wound repair at 10- and 15 dpp.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eTympanic membrane perforation is a common condition which affects 1 in 50 people worldwide, of which, 6\u0026ndash;10% develop into debilitating and painful chronic perforations (Dolhi \u0026amp; Weimer, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; A. S. Kim et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Despite the need for effective and readily available treatments for chronic perforations, research on tympanic membrane repair is limited, hindering general understanding of the organ and possible treatment development.\u003c/p\u003e \u003cp\u003eThe possibility of a stem cell population residing in the TM had been touched on by previous researchers via immunohistochemistry for stem cell markers, although more generally as a hypothetical assumption due to the acute ability of the TM to regenerate after injury (Dinwoodie et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Frumm et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; S. W. Kim et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). We have shown that \u003cem\u003eAxin2cre\u003c/em\u003e labelled cells were located in the previously described stem cell regions of the eardrum (Frumm et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Kim et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Tucker et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), and importantly all BrDU label-retaining cells were also \u003cem\u003eAxin2\u003c/em\u003e positive. The \u003cem\u003eAxin2cre\u003c/em\u003e labelled population was long lived, being retained over a 10 month period, suggesting these cells represent a stem cell population. The \u003cem\u003eAxin2cre\u003c/em\u003e labelled cells in the epithelium were rare and did not contribute extensively to homeostasis. In contrast, the \u003cem\u003eAxin2cre\u003c/em\u003e labelled cells in the mesenchyme were found to populate the ear drum over time, suggesting lineage specific roles for these cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). The \u003cem\u003eAxin2cre\u003c/em\u003e labelled cells located in the mesenchymal layer may be stem/progenitor cells, or long-lasting quiescent fibroblasts, such as those found in the dermis of the skin (Zorina et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAlthough, the \u003cem\u003eAxin2cre\u003c/em\u003e labelled cells in the epithelium did not contribute to homeostasis, they contributed to a large part of the ear drum during repair. This population, is therefore, able to exit quiescence, proliferate, and help reform the membrane. The TM\u0026rsquo;s outer layer may therefore have separate populations of epithelial cells that are a) Wnt-independent and capable of maintaining the outer layer during homeostasis with a turnover of 3 weeks (Frumm et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), and b) Wnt-dependent and quiescent but are activated after injury and responsible for its repair. This is reminiscent of the distinct roles of different stem cell populations in the cranial sutures (Maruyama et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe Axin2-labelled cells in the mesenchyme and endoderm layers also contributed to repair, generating a new membrane that was largely derived of Axin2-lineage cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). Although the TM has been proposed to be a blastema (Scaria et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), we saw no evidence of mesenchymal cells contributing to the epithelial or endodermal layers of the repaired membrane, highlighting that during repair the layered structure of the membrane is maintained. Importantly, the contribution of Axin2-labelled cells to repair was evident, even when the injury was created 5 months after lineage labelling of these cells.\u003c/p\u003e \u003cp\u003eAs is common during many repair processes, Wnt signalling was upregulated in the TM after injury. Interestingly, this upregulation was highest in the mesenchymal layer, with minimal upregulation in the epithelial layer, not all layers, therefore, responded to injury in the same way.\u003c/p\u003e \u003cp\u003eWhen the ability of the \u003cem\u003eAxin2cre\u003c/em\u003e labelled cells to respond to Wnt signaling was impaired via use of the \u003cem\u003eAxin2creERT2;Bcatlox(ex2-6);TdTomato\u003c/em\u003e mouse model, mesenchymal homeostasis was affected with a high number of positive cells in the centre of the membrane after 15 days. It is possible therefore that Wnt signaling in the presumptive stem cell areas of the drum functions to keep cells naive, as in other systems such as the adult mammary gland, where Wnt signals promote self-renewal of the \u003cem\u003eAxin2\u003c/em\u003e positive stem cells(Zeng \u0026amp; Nusse, 2010, Lim et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe importance of the \u003cem\u003eAxin2cre\u003c/em\u003e labelled cell population to repair was highlighted using the perforated \u003cem\u003eAxin2creERT2;Bcatlox(ex2-6);TdTomato\u003c/em\u003e mouse model. At 10 days post perforation, in control TMs, all perforations had covered the wound site. In loss-of-function TMs at 10dpp, 80% had not managed to do so, and, furthermore, the samples that had appeared to have healed, in fact, had defects in creation of the trilaminar structure. The formation of middle ear cholesteatomas has been proposed to result from the proliferation of epithelial cells in the middle ear cavity, resulting in collections of epithelial cells that have the ability to grow to large sizes (Sudhov \u0026amp; Tos, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). In the \u003cem\u003eAxin2creERT2;Bcatlox(ex2-6);TdTomato\u003c/em\u003e mouse line, the outer epithelial cell layer was observed pushing into the middle ear cavity, potentially indicating the beginnings of cholesteatoma formation. The mechanisms underlying why choleastomas form are not well understood, although recent work has suggested canonical Wnt signaling to be implicated in human cholesteatoma, due to the increased expression of Wnt related genes in cholesteatoma tissue compared to that of the middle ear, as well as elevated nuclear b-catenin in the abnormal tissue (Lin et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). While increased Wnt is observed in fully formed cholesteatoma, it is interesting that loss of Wnt may be involved in cholesteatoma \u003cem\u003eformation\u003c/em\u003e. This could be linked to the lack of the ability of the mesenchymal layer to cover the perforation. Without the stability provided by this layer, the epithelium could enter the middle ear cavity, leading to cholesteatoma formation (Stenfeldt et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2006\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). At 15dpp, bilayer TMs were evident in the LOF mutants. Bi-layered TMs have been described in the literature, whereby, as the name suggests, the TM after repair of a perforation only contains two-layers. Electron microscopy suggested that the fibroblasts of the mesenchymal layer were lacking(Govaerts et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1988\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOverall, our work has identified a marker for putative stem/progenitor cell populations in the TM, with the epithelial population reserved for repair. These finds suggest that the development of therapies augmenting Wnt signaling may have positive effects on chronic TM repair.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003ch2\u003e\u003cstrong\u003eMouse Models\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eAll mice were kept in the Biological Services Unit (BSU) at King’s College London (KCL-BSU, New Hunt’s House Guy’s Campus, KCL, UK). All animal husbandry and procedures were carried out in accordance with King’s College ethical guidelines and Home Office (HO) guidelines. \u003cem\u003eAxin2creERT2-\u003c/em\u003e (van Amerongen et al., 2012);\u0026nbsp;\u003cem\u003eWnt1Cre\u003c/em\u003e- (Danielian et al., 1998) expressing males and \u003cem\u003eRosa26;Tomato-\u003c/em\u003e (\u003cem\u003etdTomato\u003c/em\u003e [\u003cem\u003eGt(ROSA)26 Sor tm14(CAG-tdTomato) Hze\u003c/em\u003e/J]) expressing females were bred to create the reporter mouse lines \u003cem\u003eAxin2creErt2;TdTomato\u003c/em\u003e and \u003cem\u003eWnt1cre;TdTomato\u003c/em\u003e. \u003cem\u003eAxin2creErt2;TdTomato\u003c/em\u003e mice were bred with \u003cem\u003eBcatlox(ex2-6)\u003c/em\u003e (Brault et al., 2001) mouse lines to create \u003cem\u003eAxin2creERT2;B-catlox(ex2-6);TdTomato\u003c/em\u003e mouse models. \u003cem\u003eAxin2LacZ\u003c/em\u003e mouse lines (Lustig et al., 2002) were also used. Mice were bred on the C57/Bl6 background.\u0026nbsp;\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eTamoxifen and TM perforation\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eWhen mice were 6-8 weeks old, three rounds of intraperitoneal injection of tamoxifen (Sigma-Adrich T5648-1g) were administered at 75mg/kg (approx. 100µl of 20mg/ml tamoxifen solution) over three consecutive days.\u0026nbsp;Ear drum perforation was performed in 6-8 weeks old mice as previously described in Dinwoodie et al., 2024. At specific time points mice were sacrificed and the ear drums, attached to the tympanic ring, dissected out under a Leica MZFLIII dissection scope. Dissected tissue was fixed in 4% paraformaldehyde for one hour and DAPI stained prior to whole-mount imaging. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistology and Immunofluorescence\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTMs were decalcified, dehydrated, and embedded in paraffin wax. 6mm sections were cut using a Leica microtome RM2245 and mounted onto superfrost slides (FisherScientific 11976299).\u0026nbsp;Sections were rehydrated followed by antigen retrieval using 0.1M citrate buffer (pH 6) in a water bath at 95°C. Sections were blocked using blocking buffer (10% FBS, 1% BSA and 0.0125% tween20 in PBS) for 1-2 hours. Primary antibodies were applied overnight at 4°C (See supplementary material for table of antibodies) and washed off before secondary antibodies were applied. Finally, sections were washed and mounted using fluoroshield (Sigma F6182).\u003c/p\u003e\n\u003cp\u003eTM sections are fragile and optimisations of the IHC protocol included not preheating the antigen retrieval buffer and using 0.0125% Tween20 in PBT when washing and in blocking solution.\u0026nbsp;\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003e\u003cem\u003eβ\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e-\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003egalactosidase Staining\u003c/em\u003e\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eFor \u003cem\u003eAxin2LacZ\u003c/em\u003e mice, TMs were fixed with glutaraldehyde fixative (0.2% glutaraldehyde, 5mM EGTA and 2mM MgCl2 in 0.1M PBS). TMs were then washed with Solution B (MgCl2, deoxycholate and IGEPAL). The color was developed with a solution containing Solution B with K4Fe(CN)6, K3Fe(Ca)6 and X-gal. The blue stain developed between 3-4hrs at which time TMs were fixed overnight at 4% PFA. \u0026nbsp;For \u003cem\u003eAxin2LacZ\u003c/em\u003e sections, paraffin sections were dewaxed and co-stained for eosin to allow histological analysis.\u0026nbsp;\u003c/p\u003e\n\u003ch3 id=\"_Toc156914964\"\u003e\u003cstrong\u003e\u003cem\u003eBrDU Pulse-Chase Labelling\u003c/em\u003e\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eAll dividing cells in the embryos of \u003cem\u003eAxin2creERT2;TdTomato\u003c/em\u003e mice were labelled with BrDU (via intraperitoneal injection of the mother at E13.5 and E15.5). At 6-8 weeks old, the mice were given 3 consecutive injections of tamoxifen (20mg/ml), and TM samples collected 5 days after the final injection. BrDU detection was achieved by immunofluorescence as previously described.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003e\u003cem\u003eImaging\u003c/em\u003e\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eWhole ear drums on FluoroDishes (FD35-100), or immunofluorescent stained slides were imaged on a Leica TCS SP5 confocal microscope with LAS AF software or a ZEISS LSM 980 microscope. Trichrome stained slides were imaged on the Nikon Eclipse 80i and brightfield images were taken using the Leica FlexaCam A5. Images were processed and quantified in Image J (version 1.0) and figures (including schematics) were made using Adobe Illustrator (2021). \u0026nbsp;Graphs were made using GraphPad Prism (Version 9.0.2).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eRNAsequencing Analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe accession number for the scRNA-seq data reported in this paper is GEO: GSE196692.\u0026nbsp;\u003c/p\u003e\n\u003ch2 id=\"_Toc156914938\"\u003e\u003cstrong\u003e\u003cem\u003eQuantification and Statistics\u003c/em\u003e\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eAn a priori power analysis was conducted using G*Power version 3.1.9.7 to determine the minimum biological sample size for the perforation experiments\u0026nbsp;(Faul et al., 2007).\u0026nbsp;Statistical analysis was carried out using un-paired two tailed student t-tests between control and perforated samples. Statistical analysis was conducted using GraphPad Prism (Version 9.0.2). Nnumbers for each experiment are shown in the figure legends. N numbers refer to 'mice' rather than a single tympanic membrane unless otherwise specified.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKing’s College London, under UK Home Office licenses and regulations in line with the regulations set out under the United Kingdom Animals (Scientific Procedures) Act 1986.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe RNAseq datasets generated during this study were from Frumm et al., 2021.\u0026nbsp;GEO: GSE196692.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: A.S.T.; Methodology: O.M.D., J.M.F.; Formal analysis: O.M.D.;\u003c/p\u003e\n\u003cp\u003eInvestigation: O.M.D., J.M.F.; Writing - original draft: O.M.D.; Writing - review \u0026amp;\u003c/p\u003e\n\u003cp\u003eediting: A.S.T., J.M.F.; Supervision: A.S.T., J.M.F.; Project administration: A.S.T.;\u003c/p\u003e\n\u003cp\u003eFunding acquisition: A.S.T.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Medical Research Council (MRC), project grant\u003c/p\u003e\n\u003cp\u003eawarded to A.S.T. (MR/R023719/1). O.D. was funded by the King’s College London\u003c/p\u003e\n\u003cp\u003eMRC doctoral training programme.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAkkoca \u0026Ouml;, Kargın Kaytez S, Kaptan Z (2022) The effectiveness of repairment of traumatic tympanic membrane perforations with cigarette paper. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.14744/tjtes.2020.98968\u003c/span\u003e\u003cspan address=\"10.14744/tjtes.2020.98968\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAnthwal N, Thompson H (2016) The development of the mammalian outer and middle ear. J Anat 228(2):217\u0026ndash;232. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/JOA.12344\u003c/span\u003e\u003cspan address=\"10.1111/JOA.12344\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAragona M, Dekoninck S, Rulands S, Lenglez S, Mascr\u0026eacute; G, Simons BD, C\u0026eacute;dric Blanpain (2017) Defining stem cell dynamics and migration during wound healing in mouse skin epidermis. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/ncomms14684\u003c/span\u003e\u003cspan address=\"10.1038/ncomms14684\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBehrens J, Von Kries JP, K\u0026uuml;hl M, Bruhn L, Wedlich D, Grosschedl R, Birchmeier W (1996) Functional interaction of β-catenin with the transcription factor LEF-1. Nat 1996 382(6592):6592. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e638\u0026ndash;642. https://doi.org/10.1038/382638a0\u003c/span\u003e\u003cspan address=\"638\u0026ndash;642. 10.1038/382638a0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBerger GB (1989) Nature of spontaneous tympanic membrane perforation in acute otitis media in children. J Laryngology Otology 103(12):1150\u0026ndash;1153. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1017/S0022215100111247\u003c/span\u003e\u003cspan address=\"10.1017/S0022215100111247\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBowmana AN, Van Amerongen R, Palmer TD, Nusse R (2013) Lineage tracing with Axin2 reveals distinct developmental and adult populations of Wnt/β-catenin-responsive neural stem cells. Proc Natl Acad Sci USA 110(18):7324\u0026ndash;7329. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1073/PNAS.1305411110/SUPPL_FILE/PNAS.201305411SI.PDF\u003c/span\u003e\u003cspan address=\"10.1073/PNAS.1305411110/SUPPL_FILE/PNAS.201305411SI.PDF\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrault V, Moore R, Kutsch S, Ishibashi M, Rowitch DH, McMahon AP, Sommer L, Boussadia O, Kemler R (2001) Inactivation of the β-catenin gene by Wnt1-Cre-mediated deletion results in dramatic brain malformation and failure of craniofacial development. Development 128(8):1253\u0026ndash;1264. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1242/DEV.128.8.1253\u003c/span\u003e\u003cspan address=\"10.1242/DEV.128.8.1253\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChai Y, Jiang X, Ito Y, Bringas P, Han J, Rowitch DH, Soriano P, McMahon AP, Sucov HM (2000) Fate of the mammalian cranial neural crest during tooth and mandibular morphogenesis. Development 127(8):1671\u0026ndash;1679. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1242/DEV.127.8.1671\u003c/span\u003e\u003cspan address=\"10.1242/DEV.127.8.1671\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChari DA, Frumm SM, Akil O, Tward AD (2019) Cellular Dynamics in Early Healing of Mouse Tympanic Membranes. Otology \u0026amp; Neurotology: Official Publication of the American Otological Society, American Neurotology Society [and] European Academy of Otology and Neurotology, 40(2), E160\u0026ndash;E166. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1097/MAO.0000000000002060\u003c/span\u003e\u003cspan address=\"10.1097/MAO.0000000000002060\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eClevers H, Loh KM, Nusse R (2014) Stem cell signalling: An integral program for tissue renewal and regeneration: Wnt signaling and stem cell control. Science 346(6205):1248012\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDanielian PS, Muccino D, Rowitch DH, Michael SK, McMahon AP (1998) Modification of gene activity in mouse embryos in utero by a tamoxifen-inducible form of Cre recombinase. Curr Biology: CB 8(24):1323\u0026ndash;1326. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/S0960-9822(07)00562-3\u003c/span\u003e\u003cspan address=\"10.1016/S0960-9822(07)00562-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDe Calisto J, Araya C, Marchant L, Riaz CF, Mayor R (2005) Essential role of non-canonical Wnt signalling in neural crest migration. Development 132(11):2587\u0026ndash;2597. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1242/DEV.01857\u003c/span\u003e\u003cspan address=\"10.1242/DEV.01857\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDinwoodie OM, Tucker AS, Fons-Romero J (2024) Tracking cell layer contribution during repair of the tympanic membrane. Dis Models Mech. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1242/DMM.050466\u003c/span\u003e\u003cspan address=\"10.1242/DMM.050466\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDolhi N, Weimer AD (2022) Tympanic Membrane Perforations. In StatPearls. StatPearls Publishing. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/books/NBK557887/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/books/NBK557887/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFaul F, Erdfelder E, Lang AG, Buchner A (2007) G*Power 3: a flexible statistical power analysis program for the social, behavioral, and biomedical sciences. Behav Res Methods 39(2):175\u0026ndash;191. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3758/BF03193146\u003c/span\u003e\u003cspan address=\"10.3758/BF03193146\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFrumm SM, Yu SK, Chang J, Artichoker JA, Scaria SM, Lee KP, Byrnes LE, Sneddon JB, Tward AD (2021) A Hierarchy of Proliferative and Migratory Keratinocytes Maintains the Tympanic Membrane. Cell Stem Cell 28(2):315\u0026ndash;330e5. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/J.STEM.2020.10.006\u003c/span\u003e\u003cspan address=\"10.1016/J.STEM.2020.10.006\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGovaerts PJ, Jacob WA, Marquet J (1988) Histological study of the thin replacement membrane of human tympanic membrane perforations. Acta Otolaryngol 105(3\u0026ndash;4):297\u0026ndash;302. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3109/00016488809097011\u003c/span\u003e\u003cspan address=\"10.3109/00016488809097011\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim AS, Betz JF, Reed NS, Ward BK, Nieman CL (2022) Prevalence of Tympanic Membrane Perforations Among Adolescents, Adults, and Older Adults in the United States. Otolaryngology-Head and Neck Surgery, 2022(2), 356\u0026ndash;358. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1177/01945998211062153\u003c/span\u003e\u003cspan address=\"10.1177/01945998211062153\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim SW, Kim J, Seonwoo H, Jang KJ, Kim YJ, Lim HJ, Lim KT, Tian C, Chung JH, Choung YH (2015) Latent progenitor cells as potential regulators for tympanic membrane regeneration. Sci Rep 2015 5(1):1. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e1\u0026ndash;8. https://doi.org/10.1038/srep11542\u003c/span\u003e\u003cspan address=\"1\u0026ndash;8. 10.1038/srep11542\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKnutsson J, Von Unge M, Rask-Andersen H (2011) Localization of Progenitor/Stem Cells in the Human Tympanic Membrane. Audiol Neurotology 16(4):263\u0026ndash;269. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1159/000320612\u003c/span\u003e\u003cspan address=\"10.1159/000320612\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLav R, Krivanek J, Anthwal N, Tucker AS (2023) Wnt signaling from Gli1-expressing apical stem/progenitor cells is essential for the coordination of tooth root development. Stem Cell Rep 18:1015\u0026ndash;1029\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLim X, Tan SH, Koh WLC, Chau RMW, Yan KS, Kuo CJ, Van Amerongen R, Klein AM, Nusse R (2013) Interfollicular epidermal stem cells self-renew via autocrine Wnt signaling. Science 342(6163):1226\u0026ndash;1230. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1126/SCIENCE.1239730/SUPPL_FILE/LIM.SM.PDF\u003c/span\u003e\u003cspan address=\"10.1126/SCIENCE.1239730/SUPPL_FILE/LIM.SM.PDF\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLin J, Ye Q, Wang Y, Wang Y, Zeng Y (2019) Original Article Wnt/β-catenin signaling regulates pathogenesis of human middle ear cholesteatoma. Int J Clin Exp Pathol 12(4):1154\u0026ndash;1162. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e\u003c/span\u003e\u003cspan address=\"http://www.ijcep.com/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLohi M, Tucker AS, Sharpe PT (2010) Expression of Axin2 indicates a role for canonical Wnt signaling in development of the crown and root during pre- and postnatal tooth development. Dev Dynamics: Official Publication Am Association Anatomists 239(1):160\u0026ndash;167. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/DVDY.22047\u003c/span\u003e\u003cspan address=\"10.1002/DVDY.22047\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLustig B, Jerchow B, Sachs M, Weiler S, Pietsch T, Karsten U, Van De Wetering M, Clevers H, Schlag PM, Birchmeier W, Behrens J (2002) Negative Feedback Loop of Wnt Signaling through Upregulation of Conductin/ Axin2 in Colorectal and Liver Tumors. Mol Cell Biol 22(4):1184\u0026ndash;1193. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/MCB.22.4.1184-1193.2002\u003c/span\u003e\u003cspan address=\"10.1128/MCB.22.4.1184-1193.2002\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMaruyama T, Jeong J, Sheu T-J, Hsu W (2016) ARTICLE Stem cells of the suture mesenchyme in craniofacial bone development, repair and regeneration. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/ncomms10526\u003c/span\u003e\u003cspan address=\"10.1038/ncomms10526\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNusse R (2008) Wnt signaling and stem cell control. Cell Res 2008 18(5):523\u0026ndash;527. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/cr.2008.47\u003c/span\u003e\u003cspan address=\"10.1038/cr.2008.47\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eParekh A, Mantle B, Banks J, Douglas Swarts J, Badylak SF, Dohar JE, Hebda PA, Medicine R (2009) Repair of the Tympanic Membrane with Urinary Bladder Matrix. Laryngoscope 119(6):1206\u0026ndash;1213. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/lary.20233\u003c/span\u003e\u003cspan address=\"10.1002/lary.20233\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePark S, Gonzalez DG, Guirao B, Boucher JD, Cockburn K, Marsh ED, Mesa KR, Brown S, Rompolas P, Haberman AM, Bella\u0026iuml;che Y, Greco V (2017) Tissue-scale coordination of cellular behaviour promotes epidermal wound repair in live mice. Nat Cell Biol 19(2):155\u0026ndash;163. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/NCB3472\u003c/span\u003e\u003cspan address=\"10.1038/NCB3472\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRajkhowa R, Levin B, Redmond SL, Li LH, Wang L, Kanwar JR, Atlas MD, Wang X (2011) Structure and properties of biomedical films prepared from aqueous and acidic silk fibroin solutions. J Biomed Mater Res A 97(1):37\u0026ndash;45. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/JBM.A.33021\u003c/span\u003e\u003cspan address=\"10.1002/JBM.A.33021\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eReijnen CJH, Kuijpers W (1971) Chapter II: Experimental study on the healing process of the drum membrane. Acta Otolaryngol 72(S287):11\u0026ndash;37. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3109/00016487109127860\u003c/span\u003e\u003cspan address=\"10.3109/00016487109127860\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRohanizadeh R, Swain MV, Mason RS (2008) Gelatin sponges (Gelfoam\u0026reg;) as a scaffold for osteoblasts. J Mater Science: Mater Med 19(3):1173\u0026ndash;1182. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/S10856-007-3154-Y/FIGURES/8\u003c/span\u003e\u003cspan address=\"10.1007/S10856-007-3154-Y/FIGURES/8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSanta Maria PL, Redmond SL, Atlas MD, Ghassemifar R (2010a) Keratinocyte growth factor 1, fibroblast growth factor 2 and 10 in the healing tympanic membrane following perforation in rats. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s10735-010-9306-2\u003c/span\u003e\u003cspan address=\"10.1007/s10735-010-9306-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSanta Maria PL, Redmond SL, Atlas MD, Ghassemifar R (2010b) The role of epidermal growth factor in the healing tympanic membrane following perforation in rats. J Mol Hist 41:309\u0026ndash;314. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s10735-010-9287-1\u003c/span\u003e\u003cspan address=\"10.1007/s10735-010-9287-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eScaria SM, Frumm SM, Vikram EP, Easow SA, Sheth AH, Shamir ER, Yu SK, Tward AD (2023) Epimorphic regeneration in the mammalian tympanic membrane. Npj Regenerative Med 2023 8(1):1\u0026ndash;15. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41536-023-00332-0\u003c/span\u003e\u003cspan address=\"10.1038/s41536-023-00332-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShen Y, Redmond SL, Dip A, Sci A, Teh BM, Yan S, Wang Y, Zhou L, Budgeon CA, Eikelboom RH, David M, Dilley RJ, Zheng M, Marano RJ (2012) Scaffolds for Tympanic Membrane Regeneration in Rats. Tissue Engineering Part A, 19(5\u0026ndash;6). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1089/ten.tea.2012.0053\u003c/span\u003e\u003cspan address=\"10.1089/ten.tea.2012.0053\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStenfeldt K, Johansson C, Eriksson PO, Hellstr\u0026ouml;m S (2013) Collagen type II is produced in healing pars tensa of perforated tympanic membranes: An experimental study in the rat. Otology Neurotology 34(7). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1097/MAO.0b013e3182908b51\u003c/span\u003e\u003cspan address=\"10.1097/MAO.0b013e3182908b51\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStenfeldt K, Johansson C, Hellstr\u0026ouml;m S (2006) The collagen structure of the tympanic membrane: collagen types I, II, and III in the healthy tympanic membrane, during healing of a perforation, and during infection. Archives Otolaryngology\u0026ndash;Head Neck Surg 132(3):293\u0026ndash;298. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1001/ARCHOTOL.132.3.293\u003c/span\u003e\u003cspan address=\"10.1001/ARCHOTOL.132.3.293\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSudhov H, Tos M (2007) Pathogenesis of sinus cholesteatoma. Eur Arch Otorhinolaryngol 264:1137\u0026ndash;1143. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00405-007-0340-y\u003c/span\u003e\u003cspan address=\"10.1007/s00405-007-0340-y\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSyed SM, Kumar M, Ghosh A, Tomasetig F, Ali A, Whan RM, Alterman D, Tanwar P (2020) Endometrial Axin2\u0026thinsp;+\u0026thinsp;cells drive epithelial homeostasis, regeneration and cancer following oncogenic transformation. Cell Stem Cell 26(1):64\u0026ndash;80\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTucker AS, Dyer CJ, Romero JMF, Teshima THN, Fuchs JC, Thompson H (2018) Mapping the distribution of stem/progenitor cells across the mouse middle ear during homeostasis and inflammation. Dev (Cambridge) 145(1). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1242/DEV.154393/264412\u003c/span\u003e\u003cspan address=\"10.1242/DEV.154393/264412\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e/AM/MAPPING-THE-DISTRIBUTION-OF-STEM-PROGENITOR-CELLS\u003c/span\u003e\u003cspan address=\"http:///AM/MAPPING-THE-DISTRIBUTION-OF-STEM-PROGENITOR-CELLS\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUgarteburu M, Withnell RH, Cardoso L, Carriero A, Richter CP (2022) Mammalian middle ear mechanics: A review. Front Bioeng Biotechnol 10. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/FBIOE.2022.983510\u003c/span\u003e\u003cspan address=\"10.3389/FBIOE.2022.983510\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVan Amerongen R, Bowman AN, Nusse R (2012) Developmental stage and time dictate the fate of Wnt/β-catenin-responsive stem cells in the mammary gland. Cell Stem Cell 11(3):387\u0026ndash;400. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/J.STEM.2012.05.023\u003c/span\u003e\u003cspan address=\"10.1016/J.STEM.2012.05.023\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang B, Zhao L, Fish M, Logan CY, Nusse R (2015) Self-renewing diploid Axin2\u0026thinsp;+\u0026thinsp;cells fuel homeostatic renewal of the liver. Nature 524(7564):180. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/NATURE14863\u003c/span\u003e\u003cspan address=\"10.1038/NATURE14863\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWhyte JL, Smith AA, Helms JA (2012) Wnt Signaling and Injury Repair. Perspectives in Biology. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1101/cshperspect.a008078\u003c/span\u003e\u003cspan address=\"10.1101/cshperspect.a008078\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYuan X, Xu Q, Zhang X, Van Brunt LA, Ticha P, Helms JA (2019) Wnt-Responsive Stem Cell Fates in the Oral Mucosa. IScience 21:84\u0026ndash;94. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/J.ISCI.2019.10.016\u003c/span\u003e\u003cspan address=\"10.1016/J.ISCI.2019.10.016\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZorina A, Zorin V, Isaev A, Kudlay D, Vasileva M, Kopnin P (2023) Dermal Fibroblasts as the Main Target for Skin Anti-Age Correction Using a Combination of Regenerative Medicine Methods. Curr Issues Mol Biol 45(5):3829. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/CIMB45050247\u003c/span\u003e\u003cspan address=\"10.3390/CIMB45050247\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"repair, wnt signalling, ear, quiescence","lastPublishedDoi":"10.21203/rs.3.rs-9587608/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9587608/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTympanic membrane (eardrum) perforations are common, with the majority healing, suggesting an innate ability to regenerate after damage. Epithelial cells expressing putative stem cell markers have been shown to reside in niches confined to specific regions of the membrane. Utilising \u003cem\u003eAxin2\u003c/em\u003e reporter mice, we show that label-retaining and long-lived, Wnt-responding cells are located in these presumptive stem cell niches. scRNAseq analysis and lineage tracing revealed that these Wnt-responding epithelial cells had hallmarks of quiescent cells and did not contribute significantly to homeostasis. In contrast, during wound healing, these cells were activated and contributed to substantial proportions of the repaired membrane. Loss of Wnt signaling in the \u003cem\u003eAxin2\u003c/em\u003e population in vivo and in vitro, led to impaired healing as cells failed to bridge the gap and create the normal tri-layered membrane structure. Our results reveal a reserve stem cell population that is crucial for repair, providing a target for future therapeutics to treat chronic perforations.\u003c/p\u003e","manuscriptTitle":"Wnt-responding stem/progenitor cells are essential for tympanic membrane repair","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-07 08:55:35","doi":"10.21203/rs.3.rs-9587608/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"b13ad895-2e2c-42b9-965b-a90035049464","owner":[],"postedDate":"May 7th, 2026","published":true,"recentEditorialEvents":[{"type":"reviewersInvited","content":"6","date":"2026-05-05T16:01:36+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-05-05T09:17:26+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-05-01T20:55:51+00:00","index":"","fulltext":""},{"type":"submitted","content":"Nature Communications","date":"2026-05-01T15:47:55+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":67576617,"name":"Biological sciences/Stem cells/Adult stem cells"},{"id":67576618,"name":"Health sciences/Medical research/Experimental models of disease"}],"tags":[],"updatedAt":"2026-05-07T08:55:36+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-07 08:55:35","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9587608","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9587608","identity":"rs-9587608","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}