Fig 2: Cellular characteristics of LSCs cultured on 3T3 cells overexpressing Wnt6. LSCs were cultured on GFP-3T3, Wnt6-3T3, and uninfected 3T3 cells for 10–14 days. (A) Morphology of LSCs was comparable under all conditions. (B) CFEs of cultured LSCs were comparable. (C) Quantitation of CFE percentages. (D) Quantitation of cell proliferation. (E) Proportions of small cells with diameters = 12 mm, (F) Immunostainings of p63 cells in the different groups and proportions of p63abright cells, (G) Immunostainings of K12 and K14 cells in the different groups and proportions of undifferentiated K14+ cells and differentiated cells determined by expression of K12. *P = 0.05 in comparison with the corresponding GFP-3T3 controls. Data are means ± SEM of 4 experiments.

Fig 3: Wnt/ß-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 ß-catenin localizes to basal (white arrow) and suprabasal (yellow arrow) cells in adult sweat ducts. (f) Nuclear ß-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).

Fig 4: Organ-like structures of the NBOs and LCOs. a Bright-field microscopy images of a NBO, NBO-90. NBO-90 was cultured in MBM and MBM + WNA (W; Wnt3A, N; Noggin, A; A83-01). Scale bars, 200 µm. b H&E staining image of NBO-90. Scale bar, 100 µm. c–e Immunofluorescence images showing the expression of pancytokeratin, p63, MUC1, KRT5, KRT7, CC10, and cilia co-stained with acetylated a-tubulin (Ac-Tub) and Arl13b (The enlarged images in yellow boxes showed cilia co-stained with Ac-Tub and Arl13b). Nuclei (blue) were stained with DAPI. Scale bar, 20 µm

Fig 5: WNT10A/ß-catenin signalling is required for region-specific differentiation.(a–d) Filiform papillae are present in Wnt10a-/- and inducible ß-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 ß-catenin (g–h?,k,l) induced from P25, P110 or P15 as indicated causes decreased expression of nuclear ß-catenin, LEF1 and HOXC13 (white arrows, LEF1+ proliferating cells; yellow arrows, HOXC13+ differentiating cells). (m) qPCR shows significantly decreased Hoxc13 levels in Wnt10a and ß-catenin mutant tongue epithelium. (n–r) IF and qPCR reveal reduced levels of KRT9 protein (n–q) and mRNA (r) in Wnt10a-/- and inducible ß-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 ß-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?).