Fig 1: COL17 deletion induces transient IFE hyperproliferation in neonates.(a) Hematoxylin and eosin (H&E) staining and E-cadherin (E-cad) labeling (with PI nuclear counterstain) of Col17a1-/- and control IFE skin samples from Col17a1+/- or Col17a1+/+ littermates (Control) at P1 (n = 5) and P20 (n = 4). Scale bar: 20 µm. Quantitation of the number of epidermal layers and epidermal cell counts. The values are shown as relative ratios to the controls. (b) PH3 staining at P1 and P20. Scale bar: 20 µm. The number of epidermal basal cells positively labeled for PH3 per mm epidermis (n = 4). BM, basement membrane. (c) PCNA and BrdU labeling at P1. Scale bar: 20 µm. Quantitation of PCNA- (n = 5) and BrdU-positive basal cells (n = 4). The values are shown as relative ratios to the controls. (d) Quantitative RT-PCR (qRT-PCR) of Itga6, Itgb1, Tgm1, Ppl and Ivl mRNAs (n = 5). (e) Loricrin and cleaved caspase-3 staining (representative images from 3 mice). Scale bar: 20 µm. BM, basement membrane. (f) An in silico model of the epidermal cell proliferation upon the reduced adhesion of committed progenitor cells to the BMZ. The details are described in the Material and Methods. The data in all of the histograms are the means ± SE. *0.01
Fig 2: Proliferative ability of 3D reconstituted human epidermis treated with COL17A1 siRNAsDOI: http://dx.doi.org/10.7554/eLife.26635.022
Fig 3: Physical aging affects epidermal proliferation and COL17 distribution.(a) H&E, E-cad, PH3, BrdU and PCNA staining of IFE skin from young (6–10 weeks old) and aged (19–27 months old) adult C57BL/6 wild-type (WT) mice. Scale bar: 20 µm. The numbers of epidermal layers, epidermal cell counts, and PH3-, BrdU- and PCNA-positive basal cells (n = 5). Student’s t-test. (b) The gene expression levels of Itga6, Itgb1, Tgm1, Ppl, Evpl, and Col17a1 in IFE skin samples from young and aged WT mice (n = 5 for Itga6, Itgb1, Tgm1 and Col17a1; n = 3 for Ppl and Evpl). Student’s t-test. (c) COL17 staining (antibodies to the juxtamembranous portion) in IFE skin samples from the following groups: young and aged WT mice (n = 5), young (<15 years old) and aged (>85 years old) normal human individuals (representative images from three human samples), and Klotho+/- and Klotho-/- littermates at 6 weeks (representative images from three mice). Scale bar: 20 µm. The quantitative fluorescent intensity of lateral membrane of IFE basal cells from young and aged WT mice (n = 5). Mann-Whitney test. (d) COL17 labeling following the Triton X-100 treatment of IFE skin from young/aged WT mice and human individuals (representative images from three samples). Scale bar: 20 µm. (e) The optical sectioning of 3D reconstructed whole mount COL17-stained skin from young and aged murine WT IFE. The IFE cell membrane was visualized with wheat germ agglutinin (WGA). DAPI (4',6-diamidino-2-phenylindole) was used for nuclear staining. Scale bar: 10 µm. The quantitative fluorescent intensity of COL17 in lateral membrane of IFE basal cells from young and aged WT mice (n = 6). Mann-Whitney test. (f) The distributions of COL17 and desmogleins 1 and 2 in young murine WT IFE using N-SIM (structured illumination microscopy) image reconstruction (representative images from two mice). Basal keratinocytes were depicted by white lines. Scale bar: 5 µm. BM, basement membrane. The data are the means ± SE. *0.01
Fig 4: Overexpression of human COL17 ablates hyperproliferation in aged IFE.(a) COL17 labeling and its quantitative fluorescent intensity in IFE skin from WT and K14-hCOL17 aged mice (>19 months old) (n = 5). The antibody used in this assay detects both human and murine COL17. Scale bar: 20 µm. Mann-Whitney test. (b) H&E-, E-cad-, PH3- and PCNA-stained skin samples from WT and K14-hCOL17 aged IFE. The numbers of epidermal layers, total epidermal cell counts, and PH3- and PCNA-positive basal cell counts (n = 5 for H&E, E-cad, and PCNA staining; n = 4 for PH3 in aged K14-hCOL17 mice; n = 3 for PH3 of aged WT mice). Student’s t-test. Scale bar: 20 µm. (c) The gene expression levels of Itga6 and Itgb1 in IFE skin samples from WT and K14-hCOL17 aged IFE (n = 5). Student’s t-test. (d) H&E-, E-cad-, PH3- and PCNA-staining; quantifications of epidermal layers; total epidermal cells; and PH3-, PCNA-, and BrdU-positive basal cells in the IFE of Col17a1-/- mice and littermate controls (3 months old) (n = 4). Student’s t-test. Scale bar: 20 µm. The data are the means ± SE. *0.01
Fig 5: Induction of human COL17 abrogates epidermal hyperproliferation and the expression of Wnt-ß-catenin signaling molecules in neonatal Col17a1-/- IFE.(a) H&E, E-cad, PH3 and PCNA staining of IFE skin specimens from Col17a1+/+ or Col17a1+/- (as hCOL17-; CTL (control)) and hCOL17+; Col17a1-/- littermate mice at P1. Quantification of epidermal layers, epidermal cell counts, and PH3- and PCNA-positive cells (n = 4). Scale bar: 20 µm. (b) Gene expression of Wnt-related molecules in IFE skin samples from hCOL17-; CTL and hCOL17+; Col17a1-/- littermate mice at P1 (n = 4). (c) LEF1 staining of IFE skin samples from hCOL17-; CTL and hCOL17+; Col17a1-/- littermates at P1 (n = 4). Scale bar: 20 µm. (d) ß-catenin staining of IFE skin samples from hCOL17-; CTL and hCOL17+; Col17a1-/- littermates at P1 (n = 4). Nuclear ß-catenin is indicated with arrows. The number of nuclear ß-catenin-positive cells. Scale bar: 20 µm. The data are the means ± SE. Student’s t-tests.DOI: http://dx.doi.org/10.7554/eLife.26635.010
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