Human pedigree with a novel loss-of-function WNT10A mutation

Here we report a 41-year-old woman of Indian descent who contacted our dermatology clinic complaining of thinning hair (Fig. 1a), onychodystrophy (Fig. 1b), palmoplantar scaling (Fig. 1c,d) and decreased palmoplantar sweating (Fig. 1e,f). The patient’s tongue surface was abnormally smooth (Fig. 1g,h). Taste testing did not reveal decreased sensitivity to salt, sweet and bitter tastes (Fig. 1i,j); however, her affective (like versus dislike) taste response was blunted compared with her affective response to odors. Her low ability to taste quinine was concordant with genotyping for a TAS2R19 allele associated with quinine sensitivity (heterozygous A:G for rs10772420)9 and homozygosity for the non-taster diplotype (AVI/AVI) for bitter receptor gene TAS2R38 (ref. 10). She had low alveolar bone density and a history of severe dental defects including small, conical primary teeth with taurodontism, and complete failure of secondary dentition (Fig. 1k).

Figure 1: Clinical features associated with human WNT10A mutation. (a) Thinning hair. (b) Nail dystrophy. (c,d) Fissures and scaling on palms and soles. (e,f) Starch-iodine sweat testing. Note brown grains on control palm indicating sweat production, and decreased sweating in patient (arrows). Insets show higher magnification of areas indicated by the lower arrow in each photograph. (g,h) Smooth tongue surface. (i) Taste testing. Patient data (red line) is similar to comparison group except for quinine and PTC1 (bitter). DB, denatonium benzoate (bitter). Higher y axis values indicate greater intensity (scale, 1–12). Patient was tested twice; 1=trial 1; 2=trial 2. (j) Taste quality assessment. NComp, number of comparison subjects (N total =41) who gave same response as patient. (k) Oral X-ray. Note decreased alveolar bone density and tooth root and cusp defects. (l) Splice donor site mutation and predicted truncated protein. (m) qPCR with primers indicated in l confirms aberrant splicing. Full size image

Genetic testing ruled out mutation of the ectodermal dysplasia-associated genes EDA, EDAR and EDARRAD11. Instead, the patient was homozygous for a single nucleotide G>A transversion at position c.756+1 (c.756+1 G>A) of WNT10A, which mutates a conserved mRNA splice donor site for intron 3 (Fig. 1l). The proband’s younger brother was homozygous for the same mutation and had similar symptoms including conical primary teeth, failure to develop permanent teeth, alopecia and palmoplantar scaling. Both parents were heterozygous carriers, and a male second cousin on her paternal side also reported dental abnormalities.

qPCR of WNT10A transcripts isolated from the patient’s plucked scalp hairs revealed the presence of normally spliced exon 1 and exon 2 transcripts at levels comparable to those detected in a similarly aged control female of Indian descent. However, transcripts resulting from splicing of intron 3 were present at <10% of control levels (Fig. 1m). The predicted translation product is truncated after amino acid 252 (Gln), resulting in absence of 16 of the 24 conserved C-terminal cysteine residues necessary for disulfide bridge formation, Wnt protein secondary structure12 and binding to Frizzled receptors13. As human patients with a wide range of different WNT10A mutations including homozygous missense mutations and mutations predicted to truncate the protein at nine amino acids3 display overlapping phenotypes, these likely result from loss of WNT10A function.

Localization of Wnt signalling and Wnt10a expression

Wnt/β-catenin signalling stabilizes cytoplasmic β-catenin, allowing it to accumulate and enter the nucleus where it associates with TCF/LEF family DNA-binding factors, and activates target gene expression. Wnt/β-catenin signalling is active in embryonic ectodermal appendages and is required for their formation14. In adult life, Wnt/β-catenin signalling localizes to interfollicular epidermis, hair follicles (HFs), and tongue filiform papillae and taste buds (TBs)15,16,17. Immunofluorescence detection of nuclear β-catenin, and analysis of Wnt/β-catenin reporter expression in Axin2lacZ, Axin2-CreERT2/tdT(Axin2-tdT) and TCF/Lef-H2B-GFP (TL-GFP) mice16,18,19 revealed signalling in additional tissues that show defects in WNT10A patients, including sweat gland germs; adult sweat gland ducts, but not secretory cells; footpad epidermis; and basal and differentiated TB cells in adult fungiform and circumvallate taste papillae (Fig. 2a–h″). In tongue filiform papillae, Axin2-tdT expression localized to basal cells and both anterior HOXC13− and posterior HOXC13+ differentiating cells (Fig. 2i–i″), while TL-GFP was expressed in LEF1+ basal and HOXC13− differentiating cells but not in HOXC13+ cells (Fig. 2j–k″), indicating differential sensitivity of these reporters to Wnt signalling. In addition to Wnt activity in HF matrix, pre-cortical and dermal papilla (DP) cells, we also detected Wnt reporter expression in the HF dermal sheath, isthmus and sebaceous gland peripheral cells (Fig. 2l–n).

Figure 2: Wnt/β-catenin signalling and generation of Wnt10a mutant mice. (a–c) TL-GFP localizes to sweat gland (SG) germs at P1 (a), and KRT8+ SG luminal cells at P10 (b), but not adult SG secretory cells (c). (d) Axin2lacZ localizes to sweat ducts (SD) and footpad epidermis (FE), but not SG secretory cells in adult footpad. (e) Nuclear and cytoplasmic β-catenin localizes to basal (white arrow) and suprabasal (yellow arrow) cells in adult sweat ducts. (f) Nuclear β-catenin localizes to basal (white arrow) and differentiated (yellow arrow) adult fungiform TB cells. (g–g″) TL-GFP localizes to KRT14+ basal (white arrows) and KRT14− differentiated (yellow arrows) TB cells. (h–h″) tdT expression (pink) in p63+ basal (white arrows) and p63− differentiating (yellow arrows) cells in Axin2-CreERT2/tdT (Axin2-tdT) circumvallate papillae. (i–i″) tdT expression in posterior HOXC13+ (yellow arrows), anterior HOXC13− differentiating (red arrows) and HOXC13− basal (white arrows) filiform papilla cells in Axin2-tdT mice. (j–k″) TL-GFP localizes to anterior HOXC13− differentiating (j–j″, red arrows) and LEF1+ basal (k–k″, white arrows) but not posterior HOXC13+ differentiating (j–j″, yellow arrows) filiform papilla cells. (l) TL-GFP localizes to matrix, pre-cortex and dermal sheath in P35 anagen HFs. (m,n) TL-GFP (m) and Axin2lacZ (n) localize to HF isthmus (yellow arrows) and sebaceous gland peripheral cells (white arrows) at P50 (telogen). (o–t) Wnt10a expression (in situ hybridization, purple) in adult plantar epidermis (o); adult footpad epidermis (FE) and sweat ducts (SD) (p); neonatal (q) and P100 (r–t) filiform and fungiform papillae in anterior (s) and posterior (t) dorsal tongue; and myoepithelial cells of adult SG (lower red arrow) (p). (u–w) Generation of Wnt10a-floxed mice. (u) A conditional Wnt10a allele was generated by recombination using a cassette with loxP sites flanking exons 3–4. Correctly targeted ES cells were confirmed by Southern blotting of Sca1-digested genomic DNA (v); probe is indicated in u. (w) PCR-genotyping with primers P1+P2 confirmed germ-line transmission. Wnt10afl/fl mice were crossed with CMV-Cre mice to generate a null allele. PCR-genotyping with primers P1+P3 confirmed deletion of exons 3–4, encoding amino acids 126–528. Primer positions are indicated in u. Scale bar, 10 μm (g–g″) or 25 μm (other panels). Full size image

Wnt10a expression coincides with β-catenin signalling in molar tooth development20,21, in embryonic anlagen for HFs and taste papillae, in adult interfollicular epidermis, and in HF epithelial cells and DP16,22,23,24. We detected Wnt10a expression in plantar and footpad epidermis at similar levels to those in haired skin epidermis, and in regenerating adult epithelia including filiform and fungiform papillae and sweat ducts (Fig. 2o–t). Wnt10a localized to sweat gland myoepithelial cells, but was not detectable in sweat gland mesenchyme (Fig. 2p).

WNT10A/β-catenin signalling in molar tooth development

To study the functions and mechanisms of action of WNT10A in vivo, we generated conditional Wnt10afl/fl mutant mice with loxP sites flanking exons 3 and 4, which encode 20 of the 24 conserved, functionally required cysteine residues (Fig. 2u–w). Wnt10afl/fl mice were crossed with CMV-Cre, Krt14-Cre or Krt5-rtTA tetO-Cre transgenic mice to generate null, constitutive epithelial, and doxycycline (dox)-inducible epithelial mutants. Approximately 50% of Wnt10a−/− mice developed loosely anchored ectopic molar M4 teeth (Supplementary Fig. 1a,b). Maxillary and mandibular molar teeth had flattened cusps, reduced size and defective root bifurcation and extension compared with littermate controls (Supplementary Fig. 1a,b and Fig. 3a–c), consistent with a previous report25. Cusp and size abnormalities were observed by E17.5 (Fig. 3d), indicating their morphogenetic origin, and mimicked the effects of Wnt/β-catenin inhibition after tooth initiation20. By 1 year of age, alveolar bone density was markedly decreased in Wnt10a−/− mice compared with controls, in line with human patient phenotypes (Fig. 1k), and molar teeth were frequently missing (Fig. 3b,c). Krt14-Cre Wnt10afl/fl mutants displayed molar cusp and root defects, but not decreased bone density (Fig. 3e,j), and a subset of these mice formed ectopic M4 molars. Thus, epithelial Wnt10a is required for normal tooth morphogenesis and suppression of ectopic molar formation, and mesenchymal Wnt10a maintains alveolar bone. β-catenin signalling in incisor region mesenchyme prevents ectopic incisor development by stimulating expression of Bmp4 and Shh, which then act to limit Wnt activity26. Wnt10a may be involved in a similar feedback mechanism to suppress ectopic molar formation by signalling to molar mesenchyme. In line with this possibility, Wnt10a−/− mandibles displayed reduced expression of Shh at E14.5 (Fig. 3f).

Figure 3: Tooth defects in Wnt10a mutant mice. (a–c) Micro-CT analysis of mandibles (a,b) and maxillae (c) from control and Wnt10a−/− mice at the stages indicated. (d) Serial H&E-stained sections reveal smaller molar M1 size and blunted cusp development in E17.5 Wnt10a−/− mutant. (e) Micro-CT analysis of Krt14-Cre Wnt10afl/fl and control littermate mandibles at 12 months. (f) Whole-mount in situ hybridization of E14.5 control and Wnt10a−/− mandibles with Shh probe. (g,h) Analysis of whole mount (g) or frozen sections (h) of E17.5 Wnt10a−/− TL-GFP and control TL-GFP developing molars shows decreased GFP signal in mutant cusp precursors. (i) Immunofluorescence for KRT14 (green) and DLX3 (red) in P15 mandibular molar from control and Wnt10a−/− littermates. Dlx3 expression is reduced in the mutant. (j) Diagram summarizing Wnt10a mutant tooth phenotypes at 12 months of age. Scale bar, 50 μm (h), 250 μm (i) or 1 mm (all other panels). Full size image

TL-GFP expression was reduced in E17.5 Wnt10a−/− mutant molar cusps compared with controls (Fig. 3g,h), indicating that Wnt10a regulates cusp formation through the β-catenin pathway. Interactions between Hertwig’s epithelial root sheath, a proliferative structure that expresses Wnt10a (ref. 21), and adjacent mesenchymal cells control molar root size and shape. The molar root defects in Wnt10a mutants phenocopied those caused by deletion of the direct Wnt/β-catenin target gene Dlx3 in dental mesenchyme27,28. DLX3 expression was markedly reduced in mutant root-forming mesenchyme at P15 (Fig. 3i), indicating that Wnt10a promotes molar root formation by activating mesenchymal Dlx3 expression.

Wnt10a in embryonic development of non-dental epithelia

Primary and secondary HF placodes, taste papilla placodes and sweat gland germs developed normally in Wnt10a mutants (Fig. 4a–h). Constitutive epithelial β-catenin deletion in Krt14-Cre Ctnnb1fl/fl mice caused defective formation of tongue filiform papillae and the tongue barrier, and loss of expression of Pax9, a transcriptional factor critical for filiform papilla development and barrier establishment29 (Fig. 4m–t). By contrast, Wnt10a deletion did not grossly affect embryonic development of filiform or fungiform papillae (Fig. 4i–l). Thus, other Wnts may compensate for WNT10A in non-dental embryonic development.

Figure 4: Wnt10a mutation does not affect embryonic development of HFs or sweat glands or tongue papillae. (a–d) Whole-mount in situ hybridization of control and Wnt10a mutant embryos at the indicated time points using DIG-labelled probe for β-catenin, an HF placode marker (purple signal), reveals that early stages of HF development are unaffected by constitutive epithelial (a,b) or global (c,d) deletion of Wnt10a. (e,f) TL-GFP expression in tongue whole mounts at E14.5 shows that fungiform taste papilla placodes and Wnt/β-catenin signalling (arrows) are not affected by global loss of Wnt10a. (g–j) H&E staining at E18.5 reveals normal initiation of sweat gland (g,h, yellow arrows) and filiform papilla (i,j, red arrows) development in Wnt10a mutants. (k,l) P1.5 Wnt10a mutant tongues display normal fungiform and filiform papillae, assayed by SEM. (m–p) Constitutive epithelial β-catenin deletion causes failure of filiform papilla formation at P0.5: H&E staining (m,n); SEM analysis (o,p). (q,r) Constitutive epithelial β-catenin deletion results in defective tongue barrier formation, revealed by dye penetration (dark blue staining), which is confined to fungiform papillae in controls (q, arrow) but is broadly apparent in mutants (r, arrow). (s,t) In situ hybridization for Pax9 reveals its decreased expression in epithelial β-catenin mutant dorsal tongue (purple/brown signal). N=3 mutants and n=3 control mice were analysed in all experiments. Scale bar, 25 μm (g,h), 40 μm (s,t), 50 μm (i,j,m–p), 100 μm (k,l) or 200 μm (q,r). Full size image

Wnt10a mutant HFs show progressive defects in adult life

Adult mice with global or constitutive epithelial Wnt10a deletion displayed increasingly sparse hair with age. HFs undergo periodic cycles of growth (anagen), regression (catagen) and rest (telogen), driven by rarely proliferating epithelial stem cells in the permanent KRT15+ CD34+ bulge region, and rapidly proliferating progenitors in the adjacent KRT15+ CD34− secondary hair germ (SHG)30. Immunofluorescence for pan-hair shaft keratins produced a signal in mutant HFs, and cuticle structure appeared grossly normal by scanning electron microscope (SEM); however, mutant hair shafts were shorter and thinner than controls, with disorganized internal structures (Supplementary Fig. 2a–j). To investigate the mechanisms underlying these defects, we induced epithelial Wnt10a deletion at successive time points in K5-rtTA tetO-Cre Wnt10afl/fl mice. Wnt10a loss from P9 (embryonic anagen) caused premature HF regression, cessation of matrix cell proliferation and decreased cyclin D1 expression (Fig. 5a–b′ and Supplementary Fig. 2k,l). Deletion at P18 delayed initiation of anagen, indicated by histology and absent SHG proliferation (Fig. 5c–d′), and prevented timely TL-GFP activation and external hair growth (Fig. 5k–m). These phenotypes mimicked the effects of Wnt/β-catenin inhibition16. Mutant HFs eventually entered anagen by P29 (Fig. 5e,f), but proliferation and cyclin D1 expression remained lower than in controls (Fig. 5e′ and Supplementary Fig. 2m–p). Wnt10a deletion in full postnatal anagen slightly reduced proliferation but did not cause HF regression (Fig. 5g–i and Supplementary Fig. 2q,r), suggesting compensatory activity of other Wnts. Thus, epithelial WNT10A/β-catenin signalling maintains embryonic anagen and promotes anagen onset.

Figure 5: Wnt10a deletion causes altered hair cycle progression. (a–h′) H&E stained sections (a–h) and BrdU immunofluorescence (a′–h′) of Krt5-rtTA tetO-Cre Wnt10fl/fl mutant and littermate control dorsal skin dox induced from P9, P18 or P25 and analysed at the stages indicated (a–b′, n=3 controls, 3 mutants; c–d′, n=6 controls, 6 mutants; e–f′, n=4 controls, 4 mutants; g–h′, n=5 controls, 5 mutants). (i) Quantification reveals statistically significantly reduced proliferation of Wnt10a mutant HF compared with controls. >20 control and >20 mutant HF were counted from n=4 mutants and 4 controls (P18-29) or 5 mutants and 5 controls (P25-35). Significance was calculated with two-tailed Student’s t-test. Error bars indicate s.e.m. (j) Accelerated catagen following Wnt10a deletion from P9 (photographed at P14 after hair clipping). (k) Delayed anagen entry following Wnt10a deletion from P20 (photographed at P26 after hair clipping). (l,m) Reduced TL-GFP expression in HFs (arrows) following Wnt10a deletion from P18. (n,o) Wnt10a mutant HFs are miniaturized and sebaceous glands (arrows) enlarged compared to controls at P180. (p–s) Oil-red O (p,q) and FABP5 (r,s) staining reveal increased lipid in Wnt10a mutant HFs compared with controls (arrows). (t–w) Bulge stem cell markers KRT15 and CD34 are retained in Wnt10a mutant HFs at P180. Scale bar, 25 μm (l,m,r,s), 50 μm (a′-h′,n–q,t–w) or 200 μm (a–h). Full size image

By 6 months of age, Wnt10a mutant HFs became miniaturized with enlarged sebaceous glands and elevated lipid production (Fig. 5n–s). Dominant negative Lef1 also causes sebaceous gland expansion31, consistent with decreased Wnt/β-catenin signalling in Wnt10a mutant HFs. Despite HF miniaturization in Wnt10a mutants, CD34+ KRT15+ bulge stem cells were retained (Fig. 5t–w). Miniaturized HFs in human androgenetic alopecia similarly display enlarged sebaceous glands and bulge stem cell retention32. As data from genome-wide association studies indicate association of a WNT10A variant with androgenetic alopecia6, decreased WNT10A/β-catenin signalling may contribute to this condition.

In normal aged mice (18–34 months), HFs miniaturize via loss of stem cells due to COL17A1 proteolysis33. Miniaturized HFs of 6-month-old Wnt10a mutants expressed COL17A1 (Supplementary Fig. 2s,t), consistent with maintenance of stem cell markers and suggesting that miniaturization was not caused by accelerated aging. In line with this, levels of Axin2 expression are similar in young and aged HFs33.

WNT10A/β-catenin signalling in early postnatal appendages

Constitutive global or epithelial-specific Wnt10a deletion caused progressive defects in tongue papillae structure from ∼P7 (Fig. 6a,b and Supplementary Fig. 3a–b′). TBs were miniaturized, and TL-GFP reporter activity was decreased in filiform papillae and TBs compared with controls (Supplementary Fig. 3c–h). Inducible epithelial β-catenin deletion in early postnatal life caused similar defects (Supplementary Fig. 3i,j). Sweat gland ducts failed to extend in Wnt10a−/− mutants (Fig. 6r,s), or following postnatal epithelial β-catenin deletion (Supplementary Fig. 3m,n), and starch-iodine tests revealed a functional inability to sweat (Supplementary Fig. 3k–l′). Thus, WNT10A/β-catenin signalling is required to complete postnatal oral appendage and sweat duct development. In line with this, our patient, and several other human WNT10A pedigrees5,34, display palmoplantar hypohidrosis. However, palmoplantar hyperhidrosis has also been described in some WNT10A patients5,35. We speculate that variable compensatory mechanisms, for instance upregulation of other Wnt genes during development, could account for these disparate findings. Approximately 20% of epithelial Wnt10a mutants also displayed defects in nail growth (Supplementary Fig. 3o,p), consistent with onychodystrophy in human patients (Fig. 1b).

Figure 6: WNT10A/β-catenin signalling is required for postnatal development and maintenance of epidermal appendages. (a–d) SEM shows fungiform (FuP) and filiform (FiP) papilla defects in adult global (a,b) and inducible epithelial (c,d) Wnt10a mutants. (e,f) SEM reveals loss of TBs following inducible Wnt10a deletion in adult tongue epithelium. (g–i) Decreased expression of TB markers KRT8 (red) and SOX2 (green) (g,h) and reduced percentage of KTR8+ TB cells (i) in sectioned circumvallate papillae following inducible Wnt10a deletion. KRT8+ and total DAPI+ cells were counted in 10 sections from 3 controls and 10 sections from 3 mutants. (j–n″,o,p) Induced Wnt10a deletion at the stages indicated causes decreased basal cell proliferation in filiform (j–k′), fungiform (m–n″) and circumvallate (o,p) papillae. (l,q) Quantification of proliferation in filiform (l), and fungiform and circumvallate (q) papillae. FiP: BrdU+/KRT14+ and total KRT14+ cells counted in 10 fields at 20 × from 3 controls and the same for 3 mutants. Fungiform TBs: Ki67+/KRT14+ and total KRT14+ cells counted in 10 TBs from 3 control and 3 mutant (P9-14), 6 control and 6 mutant (P18-26) or 5 control and 5 mutant (P25-25) mice. Circumvallate TBs: Ki67+/KRT14+ and total KRT14+ cells counted in 30 TBs from 3 controls and 30 TBs from 3 mutants. (r,s) SEM reveals failure of postnatal sweat duct development in P16 Wnt10a−/− mutant footpad. (t–w) Inducible Wnt10a deletion in adults prevents sweat duct maintenance (Nile blue staining) (t,u) and decreases sweating (starch-iodine staining, purple dots) (v,w). (x–y′) Inducible Wnt10a deletion in early postnatal or adult life causes decreased sweat duct basal cell proliferation. (z) Quantification of sweat duct proliferation. Ki67+/KRT14+ or BrdU+/KRT14+ and total KRT14+ cells counted in 10 ducts from 3 controls and 10 ducts from 3 mutants (P9-14) or 10 ducts from 4 controls and 10 ducts from 4 mutants (P25-160). ≥3 control and 3 mutants used for other analyses. Significance was calculated with two-tailed t-test. Error bars indicate s.e.m. Dox induction periods are indicated; mice were analysed at the end of the induction period. Scale bar, 25 μm (j–p,x–y′), 50 μm (r,s), 100 μm (a,b,g,h, insets in c,d), 500 μm (c,d) or 2 mm (e,f). Full size image

WNT10A maintains adult oral appendages and sweat ducts

To determine whether WNT10A/β-catenin signalling is required for regeneration of adult non-hair bearing epithelia, we examined the effects of inducing epithelial Wnt10a or β-catenin deletion, or expression of the Wnt/β-catenin inhibitor DKK1 and its receptor Kremen1, in adult K5-rtTA tetO-Cre Wnt10afl/fl, K5-rtTA tetO-Cre Ctnnb1fl/fl and K5-rtTA tetO-Dkk1 K14-Krm1 mice16. In each case, SEM revealed progressively abnormal filiform and fungiform papilla structures, causing a flattened tongue surface (Fig. 6c,d and Supplementary Fig. 3q,r). SEM or whole-mount immunofluorescence for the TB marker KRT8 revealed decreased TB numbers in mutant fungiform and circumvallate papillae (Fig. 6e–i and Supplementary Fig. 3s). TBs contain Type I, II and III taste receptor cells, marked, respectively, by expression of ENTPD2, PLCβ2 and SNAP-25, and required for glial-like supporting function, and detection of sweet, umami, bitter and sour tastes. While forced β-catenin activation preferentially induces Type I fate36, Wnt10a or β-catenin deletion reduced marker expression for all three cell types (Supplementary Fig. 3s). However, we did not detect significant differences in the taste responses of adult mutants versus controls for sweet, sour, salt or bitter tastes, or for the irritant, capsaicin (Supplementary Fig. 4), indicating that, as in our human patient, residual taste function is sufficient to discriminate these compounds. Interestingly, Wnt10a−/− mutants had higher water intakes than controls following mild, but not more severe, water restriction. This could reflect increased dehydration, possibly caused by a slight epidermal barrier defect.

Inducible Wnt10a deletion or forced Dkk1 expression after sweat glands reached maturity at P20 caused sweat duct regression and impaired sweating ability compared with controls (Fig. 6t–w and Supplementary Fig. 3t–u′), revealing a previously unknown role for β-catenin signalling in sweat duct maintenance.

Stratified skin epidermis and tongue epithelium regenerate continuously from keratin 14 (KRT14)+ basal cells every 8–10 and 3–4 days, respectively37,38, while TB cells in fungiform and circumvallate papillae arise from KRT14+ SOX2+ basal cells and have slower turnover rates of up to 3 weeks39. Inducible Wnt10a deletion in either the early postnatal period, or in adult life, caused significantly decreased basal cell proliferation in filiform papillae and plantar epithelium (Fig. 6j–l and Supplementary Fig. 3v,w,z), similar to the effects of inducible β-catenin deletion16. Basal proliferation was also reduced in fungiform and circumvallate TBs (Fig. 6m–q).

Sweat gland ducts are composed of proliferating basal and non-proliferative suprabasal populations, both of which renew at least every 6 weeks40. Inducible Wnt10a or β-catenin deletion caused decreased sweat duct basal cell proliferation (Fig. 6x–z and Supplementary Fig. 3x–z). Thus, WNT10A/β-catenin signalling is required for progenitor cell proliferation and adult renewal in non-hairy epithelia, as well as for normal hair growth.

Axin2 marks self-renewing stem cells in adult epithelia

Wnt-activated self-renewing stem cells are present in filiform papillae, interfollicular epidermis and resting (telogen) HFs16,41,42. However, whether Wnt activity marks self-renewing stem cells in anagen HFs is unknown. The Wnt/β-catenin target Lgr5 marks stem cells in a subset of TBs in posterior tongue43, but it is unclear whether Wnt activity is a universal characteristic of TB stem cells. The related gene Lgr6 marks self-renewing stem cells for the sebaceous gland and nail44,45, but it is unknown whether Lrg6 is Wnt regulated at these sites, or whether Wnt-activated self-renewing cells are present in sweat gland ducts or dental epithelia. As epithelial progenitor cell proliferative defects contribute to WNT10A mutant phenotypes in these tissues we used lineage tracing to ask whether Wnt activity marks their stem cells.

To ensure that the time between labelling and long-term analysis was sufficient to ensure loss of initially labelled cells that were committed to terminal differentiation rather than self-renewal, we dox-induced R26-rtTA tetO-H2B-GFP mice from P14 to efficiently label histones in proliferating cells with H2B-GFP. Dox was withdrawn at P70 and tissues examined after 2 weeks or 3.5 months (P84 or P174). H2B-GFP label was retained in rarely cycling KRT15+HF bulge stem cells at P84 and P174, but by P174 was absent from the SHG, upper follicle and sebaceous gland, and from sweat ducts and fungiform and circumvallate TBs (Fig. 7a–f). Thus, with the exception of the HF bulge, epithelial cells in all these tissues turned over within 104 days.

Figure 7: Pulse-chase estimates of epithelial tissue turnover rates and lineage tracing of Axin2+ cells in anagen HFs. (a–f) Pulse-chase analysis of epithelial turnover times. (a) Labelling strategy: R26R-rtTA tetO-H2B-GFP mice were placed on doxcycline (dox) chow for 8 weeks (P14-P70) to induce expression of H2BGFP and its incorporation into the chromatin of dividing cells (Pulse). Mice were removed from dox treatment at P70 and analysed at successive time points following dox withdrawal (Chase). Label is gradually diluted out in cells that continue to proliferate. (b,c) Most HF epithelial cells, including sebaceous gland cells and some KRT15+ bulge stem cells, were H2BGFP positive at P70 (b). By P174, label had been lost from the epithelium, with the exception of KRT15+ bulge LRCs (c). (d,e) In fungiform (d) and circumvallate (e) taste papillae, epithelial cells including KRT14+ TB basal cells and KRT8+ differentiated TB cells were H2BGFP+ at P70, but lost label by P174 indicating that they turned over within 104 days. (f) In sweat ducts, KRT14+ basal cells and KRT6+ luminal cells were H2BGFP+ at P70, but had completely lost label by P174. (g–v) Lineage tracing of Axin2-expressing cells in HFs. (g) Schematic of lineage-tracing strategy. (h–t) Paraffin sectioned dorsal skin from AxinCreERT2/tdT R26RmTmG (h–q,s) or AxinCreERT2/tdT R26RConfetti (r,t) mice tamoxifen treated at P20-21 and analysed at the time points indicated. DAPI-stained sections were co-stained with markers for bulge and SHG (KRT15), proliferation (Ki67), inner root sheath (IRS) (AE15), hair shaft (HS) precursors (AE13), dermal sheath (SMA), outer root sheath (ORS) (KRT14), isthmus (LRIG1), sebaceous gland (SG) (FAS, FABP) as indicated. (u,v) Whole-mounted tail skin epidermis from AxinCreERT2/tdT R26RConfetti mice induced at P28-29 and analysed at 11.5 months. At least three mice were analysed for each tracing condition; ≥20 labelled HFs were analysed for each mouse. Scale bar, 10 μm (b–f) or 25 μm (h–t). Full size image

Axin2lacZ and TL-GFP Wnt reporters are expressed in differentiated KRT8+ as well as basal TB cells. Lineage tracing in adult Krt8-CreERT2 R26REYFP R26R-EYFP mice tamoxifen treated for 2 days and examined after 4 days or 7 months revealed EYFP-labelled differentiated, but not basal, TB cells at 4 days, and complete loss of label by 7 months, indicating that KRT8+ cells are unable to self renew (Supplementary Fig. 5a,b).

Axin2 is a ubiquitous Wnt/β-catenin target gene that provides a reliable indicator of Wnt/β-catenin pathway activity42. To determine whether Wnt signalling marks self-renewing stem cells, we utilized Axin2-CreERT2/tdT mice16 crossed with R26REYFP, R26RmTmG or R26RConfetti Cre reporter lines (Fig. 7g). Tissues were examined shortly after Cre induction to reveal cells active for Wnt signalling, and after several cycles of epithelial renewal to identify self-renewing stem cells and their progeny. Un-induced Axin2-CreERT2/tdT R26REYFP mice displayed sporadic EYFP labelling in palatal rugae and tongue muscle, but not in skin or tongue epithelia (Supplementary Fig. 5c). We did not detect leakiness in any of these tissues when Axin2-CreERT2/tdT was used with R26RmTmG or R26RConfetti; however, as in any lineage tracing experiment, we cannot absolutely exclude that some clones resulted from leaky Cre activity.

In Axin2-CreERT2/tdT R26RmTmG mice induced in early anagen (P20-21) and examined 40 h after the first tamoxifen injection, mG+ cells were detected in the HF SHG, KRT15+ bulge, DP and dermal sheath (Fig. 7h–j). After 15 days (P35, full anagen), mG+ cells were present in both epithelial and dermal HF lineages (Fig. 7k–m). Cells in all regions of the permanent HF were labelled in telogen (Fig. 7n), and all lineages contained mG+ cells in the subsequent anagen (Fig. 7o–q). Similar results were obtained using the R26RConfetti reporter (Fig. 7r). Thus, self-renewing Axin2-expressing cells labelled in early anagen contribute to all epithelial and dermal HF components.

Sebaceous gland stem cells reside in the upper HF and sebaceous gland peripheral layer and are marked by LRIG1 and LGR6 (refs 44, 46). After long-term lineage tracing with Axin2-CreERT2/tdT R26RmTmG, the isthmus and an entire lobe of the sebaceous gland was labelled in some HFs that lacked labelling of the bulge and lower HF (Fig. 7s). Examination of Axin2-CreERT2/tdT R26RConfetti lineage-traced skin, which permits clonal analysis, confirmed this result (Fig. 7t–v). Thus, Axin2-expressing cells in the isthmus and/or sebaceous gland peripheral layer repopulate the sebaceous gland during homoeostasis.

Axin2-CreERT2/tdT R26REYFP fungiform and circumvallate papillae from mice induced at P20-P21 displayed EYFP expression in KRT14+ basal cells and KRT14− differentiated TB cells 40 h after Cre induction (Fig. 8a,c,e,g,i). After 6 or 9 months, KRT14+ basal cells remained labelled, indicating that they self-renew (Fig. 8b,d), and we also detected EYFP in differentiated Type I and II cells in fungiform and circumvallate papillae and Type III cells in circumvallate papillae (Fig. 8b,d,f,h,j). Similar data were obtained using the R26RmTmG reporter (Fig. 8k–n). Thus, self-renewing Axin2-expressing basal progenitors can produce all three taste cell types.

Figure 8: Lineage tracing of Axin2+ cells in taste papillae and sweat ducts and nails. (a–j) Adult Axin2-CreERT2/tdT R26REYFP mice were tamoxifen treated at P60 and P61 and EYFP expression analysed in fungiform (a,b,e,f) and circumvallate (c,d,g–j) papillae after 40 h, 6 months or 9 months as indicated. KRT14 marks basal cells (a–d); SOX2 marks basal and Type I TB cells (b,d); PLCβ2 marks Type II TB cells (e–h); SNAP-25 marks Type III TB cells (i,j). Yellow arrows indicate EYFP+ basal cells; red arrows indicate EYFP+ differentiated TB cells. Both basal and differentiated TB cells remain labelled over 6–9 months. (k–n) Adult Axin2-CreERT2/tdT R26RmTmG mice were treated with tamoxifen on 2 successive days and mGFP expression analysed in cryosectioned circumvallate papillae at 40 h (k,m) and 4.5 months (l,n) as indicated. KRT14 marks basal cells (k,l); ENTP2 marks differentiated Type I TB cells (m,n). Both basal KRT14+ cells (yellow arrows) and differentiated KRT14− cells, including ENTP2+ Type I cells, (red arrows) were labelled at 40 h and maintained label after 4.5 months. (o–r) Sectioned sweat ducts (o,p) and nail (q,r) from AxinCreERT2/tdT R26RmTmG mice tamoxifen treated at P20-21 and analysed at the stages indicated. Yellow arrows indicate KRT14+ basal cells; red arrows indicate KRT6+ sweat duct suprabasal cells (o,p) or KRT14− differentiating nail plate cells (q,r). Insets in q are magnified images of boxed region; lower image in r is a higher magnification photomicrograph of the arrowed region in the upper image. At least three mice were analysed for each tracing condition; ≥10 labelled taste papillae or sweat gland ducts and ≥3 nails were analysed for each condition. Scale bar, 10 μm (a–n), 25 μm (o,p), 50 μm (insets in q), 100 μm (q; magnified region (lower image) in r) or 250 μm (upper image in r). Full size image

In Axin2-CreERT2/tdT R26RmTmG sweat gland ducts at 40 h after Cre induction, mG+ cells were present in both basal (KRT6−) and suprabasal (KRT6+) layers (Fig. 8o), and both populations remained labelled after 4.5 months (Fig. 8p). As we did not detect significant proliferation of suprabasal cells, these likely originate from self-renewing Axin2-expressing basal cells.

Terminally differentiated cells of the nail arise from self-renewing KRT14+ LGR6+ stem cells in the proximal nail matrix45,47. Consistent with Axin2lacZ expression in this region47, we observed mG+ KRT14+ proximal matrix cells (Fig. 8q, yellow arrows) as well as mG+ differentiating cells (Fig. 8q, red arrow) 4 days after induction of Axin2-CreERT2/tdT R26RmTmG mice. Clones of mG+ cells emanating from the KRT14+ proximal matrix and giving rise to the nail bed and nail plate persisted after 6 months (Fig. 8r, arrows). Thus, self-renewing Axin2-expressing basal cells contribute to nail growth.

Interestingly, we were unable to detect labelling of the epithelial incisor tooth cervical loop, the site of stem cells that generate enamel-secreting ameloblasts of the continuously growing incisor. This is consistent with absence of enamel defects in Wnt10a mutant incisors, and previous reports documenting lack of Wnt/β-catenin signalling in the cervical loop48,49. Thus, Axin2 promoter activity is not a universal characteristic of self-renewing epithelial stem cells.

WNT10A controls region-specific epithelial differentiation

Patients and mice with WNT10A mutations exhibit severe tongue and palmoplantar abnormalities that are highly region specific, and cannot entirely be explained by decreased progenitor cell proliferation. We therefore asked whether WNT10A loss also affects specialized tissue differentiation. Remarkably, we found that expression of the hard keratin genes Krt36 (Krt1-5) and Krt84 (Krt2-16) that specifically characterize tongue filiform papillae was reduced or absent in Wnt10a mutant tongue, or following inducible epithelial β-catenin deletion, even though rudimentary papilla structures were present and included differentiating cells (Fig. 9a–d). This result is consistent with co-expression of Axin2-tdT with HOXC13, a transcription factor required for Krt36 and Krt84 expression50 (Fig. 2i–i″). Nuclear β-catenin and TCF4 also localize to HOXC13+ cells, which are non-overlapping with LEF1+ proliferating progenitors (Supplementary Fig. 6a–d). Inducible epithelial Wnt10a or β-catenin deletion caused absence of nuclear, or all, β-catenin, respectively (Fig. 9e–h), and loss of LEF1 and HOXC13 expression (Fig. 9e′–m).

Figure 9: WNT10A/β-catenin signalling is required for region-specific differentiation. (a–d) Filiform papillae are present in Wnt10a−/− and inducible β-catenin mutant dorsal tongue (yellow arrows), but horny structures and expression of hard keratins (in situ hybridization, purple signals) are decreased (red arrows). (e–l) Epithelial deletion of Wnt10a (e–f″,i,j) or β-catenin (g–h″,k,l) induced from P25, P110 or P15 as indicated causes decreased expression of nuclear β-catenin, LEF1 and HOXC13 (white arrows, LEF1+ proliferating cells; yellow arrows, HOXC13+ differentiating cells). (m) qPCR shows significantly decreased Hoxc13 levels in Wnt10a and β-catenin mutant tongue epithelium. (n–r) IF and qPCR reveal reduced levels of KRT9 protein (n–q) and mRNA (r) in Wnt10a−/− and inducible β-catenin mutant footpad epidermis. (s–v″) Co-IF for KRT9 and KRT10 in plantar epidermis from patients homozygous for WNT10A c.756+1G>A (s–t″) or WNT10A c.391G>A (u–v″) compared with similarly aged sex-matched controls. For qPCR, RNA levels were quantified in six control and six mutant (P40) or four control and four mutant (P20-100) samples with three technical replicates for each, and normalized to β-actin mRNA. Significance was calculated with two-tailed Student’s t-test. Error bars indicate s.e.m. Scale bar, 25 μm (e–l) or 50 μm (a–d,n–q,s–v″). Full size image

Human WNT10A patients display skin scaling and cracking of skin in palmoplantar, but not other, skin regions. These defects overlap with the effects of mutation or deletion of KRT9, whose expression is confined to suprabasal palmoplantar epidermis in humans51 and footpad epidermis in mice52. KRT9 is the most highly differentially expressed gene in human palmoplantar versus dorsal hand or foot epidermis, and in vitro studies suggest that Wnt/β-catenin signalling enhances its expression53,54. Consistent with this, we observed nuclear-localized β-catenin and TCF3 in mouse footpad epidermis (Supplementary Figs 6e,f and 7r). LEF1 expression was undetectable in this tissue, and TCF1 and TCF4 were mainly associated with sweat ducts (Supplementary Fig. 6g–i). Global Wnt10a loss or inducible epithelial β-catenin deletion caused decreased KRT9 protein and mRNA levels (Fig. 9n–r). By contrast, the suprabasal keratin gene Krt10 and the terminal differentiation proteins filaggrin and involucrin, which locate to epidermis in all body regions, were unaffected by Wnt10a or β-catenin deletion (Supplementary Fig. 9j–r). Similarly, while KRT10 and loricrin expression was comparable in WNT10A patient and sex- and age-matched control plantar epidermis (Fig. 9s′–t″ and Supplementary Fig. 6s,t), KRT9 protein was decreased in our WNT10A c.756+1 G>A patient, and in an unrelated patient homozygous for a WNT10A c.391 G>A mutation4 (Fig. 9s–v″), and KRT9 mRNA levels were lower in patient plantar skin than in control (Supplementary Fig. 6u).

β-catenin complexes in differentiation and proliferation

Our surprising finding that WNT10A/β-catenin signalling is required for region-specific differentiation as well as progenitor proliferation suggested that β-catenin may complex with transcriptional co-activators that are specifically expressed in differentiating versus proliferating cells, allowing its localization to distinct sets of target genes. We therefore examined potential transcriptional partners that are differentially expressed in suprabasal versus basal epithelial cells. A strong candidate was KLF4, which localizes to suprabasal epidermis, but is low or absent in the basal layer, and is required for embryonic tongue filiform papillae differentiation7. KLF4 expression overlapped with nuclear β-catenin and HOXC13 in adult tongue filiform papillae (Fig. 10a and Supplementary Fig. 7a), but was unaffected by loss of either Wnt10a (Fig. 10a) or β-catenin, indicating that it is not regulated by WNT10A/β-catenin signalling. Inducible epithelial Klf4 deletion in adults caused defects in filiform papilla structure and loss of HOXC13, but did not affect LEF1 expression or basal proliferation (Supplementary Fig. 7b–e and Fig. 10b,c). Nuclear β-catenin remained detectable in LEF1-, as well as LEF1+, filiform papilla cells in Klf4 mutants (Fig. 10d); thus, nuclear β-catenin is insufficient for HOXC13 expression in the absence of KLF4. Consistent with this, mutation of epithelial β-catenin to a constitutively active form in Krt5-rtTA tetO-Cre Ctnnb1fl(Ex3)/+ mice enhanced HOXC13 levels in differentiating filiform papilla cells, but did not induce its ectopic expression in basal cells (Supplementary Fig. 7f–i).

Figure 10: β-catenin interacts differentially with TCF/LEF1 and KLF4 to control proliferation and specialized differentiation. (a) Nuclear β-catenin and KLF4 overlap in differentiating filiform papillae cells (yellow arrows). Nuclear β-catenin, but not KLF4, is depleted following inducible Wnt10a deletion. (b–d) Inducible epithelial Klf4 deletion causes decreased HOXC13 (b, green) but does not affect LEF1 (b,d, pink), proliferation (Ki67) (c, pink) or nuclear β-catenin (d, green). (e–h) PLA (pink signals) with adult dorsal tongue sections and anti-β-catenin and KLF4 (e), TCF4 and KLF4 (f), β-catenin and TCF4 (g), or β-catenin and LEF1 (h) reveals direct interactions of β-catenin, KLF4 and TCF4 in differentiating (yellow arrows) and β-catenin and LEF1 in proliferating (white arrows) cells. Signals were lacking following inducible Klf4 or β-catenin deletion (right panels). Asterisks indicate autofluorescence. (i) Overlap of nuclear β-catenin and KLF4 in suprabasal footpad epidermis. KLF4 is unaffected by inducible β-catenin deletion. (j–l) Inducible epithelial Klf4 deletion causes decreased expression of KRT9 (j) but not KRT10 (k) or nuclear β-catenin (l). (m–p) PLA in adult footpad with anti-TCF3 and KLF4 (m), β-catenin and KLF4 (n) or β-catenin and TCF3 (o,p) shows KLF4, β-catenin and TCF3 directly interact in suprabasal cells (yellow arrows); β-catenin and TCF3 interact in basal cells (white arrows). Signals are absent in tissues with inducible Klf4 (m) or β-catenin (n,o) deletion (right panels), and are enhanced by inducible stabilizing mutation of β-catenin (p; right panel shows single antibody control). (q) qPCR shows significant reduction of Krt9 but not Krt10 mRNA levels in P93 footpad epidermis following inducible epithelial Klf4 deletion. mRNA levels normalized to β-actin. (r) ChiP-qPCR with adult footpad epidermis shows specific enrichment for KLF4, TCF3 and β-catenin in a region containing conserved KLF4 and LEF/TCF-binding motifs, 16 kb downstream of Krt9 TSS. Samples from ≥3 mutant and ≥3 control mice analysed in all assays. For q,r, three technical replicates were additionally performed for each sample; significance calculated with two-tailed Student’s t-test; error bars indicate s.e.m. (s) Model showing distinct nuclear β-catenin complexes regulating proliferation versus differentiation in tongue and footpad epithelia. Scale bar, 25 μm (a–i,m–p) or 75 μm (j–l). Full size image

These data suggested that KLF4, β-catenin and TCF4 might co-regulate differentiation genes in filiform papillae. To test whether β-catenin, KLF4 and TCF4 form a complex in differentiating filiform papilla cell nuclei, we used proximity ligation assay (PLA), which detects interacting epitopes that are separated by less than 30–40 nm (ref. 55). PLA with antibodies to β-catenin and KLF4, TCF4 and KLF4 or β-catenin and TCF4 produced signals in differentiating cell nuclei but not in progenitors (Fig. 10e–g, yellow arrows). Conversely, PLA for β-catenin and LEF1 produced positive signals in proliferating basal cell nuclei (Fig. 10h, white arrows). Signals were not detected in tissue lacking β-catenin or Klf4 (Fig. 10e–h and Supplementary Fig. 7j,k), or using only one antibody (Supplementary Fig. 7l,m), indicating specificity of the assay. β-catenin/LEF1 complexes formed in the absence of KLF4 (Supplementary Fig. 7n,o), and, interestingly, TCF4/KLF4 complex formation was unaffected by β-catenin deletion (Supplementary Fig. 7p,q).

To determine whether KLF4 plays a similar role in region-specific footpad epidermal differentiation, we examined its localization in this tissue. Immunofluorescence revealed KLF4 expression in suprabasal footpad epidermis where it co-localized with TCF3 (Supplementary Fig. 7r,s), and was unaffected by β-catenin deletion (Fig. 10i). Inducible epithelial Klf4 deletion in adults caused enhanced pigmentation and flaking of footpad skin (Supplementary Fig. 7t,u), a phenotype overlapping with the effects of loss of KRT9 (ref. 56). In line with this, KRT9 protein and mRNA expression were significantly reduced in Klf4 mutant footpad epidermis compared with controls, while Krt10 expression was unaffected (Fig. 10j,k,q). As in dorsal tongue epithelium, nuclear and cytoplasmic β-catenin localization was unaffected by Klf4 deletion (Fig. 10l). PLA revealed direct interactions of TCF3 and KLF4, and β-catenin and KLF4, in suprabasal footpad epidermal nuclei (Fig. 10m,n), and direct interactions of β-catenin and TCF3 in both basal and suprabasal cells (Fig. 10o). Gain-of-function mutation of epithelial β-catenin enhanced β-catenin/TCF3 interactions (Fig. 10p). A consensus TCF/LEF-binding motif conserved in dog, horse and human localizes 16,185 bp downstream of the mouse Krt9 transcription start site (TSS), close to a predicted KLF4-binding site. ChIP assays confirmed binding of KLF4, TCF3 and β-catenin to this region in footpad epidermis (Fig. 10r).

Our data support a model in which, in KLF4- basal cells, β-catenin promotes proliferation via interactions with LEF1 in filiform papillae and TCF3 in footpad epidermis. In suprabasal cells, which express KLF4 in a non-β-catenin-dependent manner, β-catenin instead complexes with KLF4 and TCF4 in filiform papillae, and with KLF4 and TCF3 in footpad epidermis, to activate specialized suprabasal differentiation programmes (Fig. 10s). The regional specificity of LEF1, TCF3 and TCF4 expression may underlie the distinct transcriptional programmes activated by WNT10A/β-catenin signalling in filiform papillae versus footpad epidermis.