Fig 1: WNT10A/ß-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).
Fig 2: 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).
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: Neuronal infection is required for lethal SARS-CoV-2 infection in hACE2fl mice.(A, B) Weight loss and survival of hACE2fl/y and Baf53b-Cre; hACE2fl/y mice after infection with 104 PFU of SARS-CoV-2. N = 10 (hACE2fl/y) and 12 (Baf53b-Cre; hACE2fl/y), three independent experiments. (C) Pulse oximetry measured in WT, hACE2fl/y, and Baf53b-Cre; hACE2fl/y mice 6 days after exposure to 104 PFU of SARS-CoV-2 virus. (D) Immunoblotting of whole brain lysates from hACE2fl/y and Baf53b-Cre; hACE2fl/y and WT mice was performed using anti-panACE2, anti-hACE2, and anti-ß-actin antibodies. Each lane represents a single animal. n = 4, two independent experiments. (E) Immunohistochemistry of SARS-CoV-2 nucleocapsid, epithelial cell Krt8, and OSN OMP in the OE of WT, hACE2fl/y, and Baf53b-Cre; hACE2fl/y mice 6 DPI. Representative of n = 4 animals per genotype. (F) Immunohistochemistry of SARS-CoV-2 nucleocapsid, AT1 cell PDPN, and AT2 cell DC-LAMP in the lungs of WT, hACE2fl/y, and Baf53b-Cre; hACE2fl/y mice 6 DPI. (G) Immunohistochemistry of SARS-CoV-2 nucleocapsid, neuronal NeuN, and glial cell GFAP in the cerebral cortex at low and high magnification of the same tissue section of WT, hACE2fl/y, and Baf53b-Cre; hACE2fl/y mice 6 DPI. Representative of N = 5–6 animals per genotype and time point. Scale bars in all images 50 µm. ****p < 0.0001 by unpaired two-tailed t test, one-way ANOVA with Holm–Sidak correction for multiple comparisons, or log-rank Mantel Cox test. Numerical data in corresponding S1 Metadata tab. AT1, alveolar type 1; AT2, alveolar type 2; DPI, days postinfection; OE, olfactory epithelium; OMP, olfactory marker protein; OSN, olfactory sensory neuron; PDPN, Podoplanin; PFU, plaque-forming unit; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2; WT, wild-type.
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