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Figure 1 Generation of mice with a keratinocyte-restricted ablation of the  1 integrin gene. (A) Schematic presentation of the floxed 1 integrin gene and the cre-mediated deletion of the gene. After deletion of the 1 integrin gene, the 1 integrin promoter will transcribe the lacZ cDNA. (E, exons in arbitrary numbering; D, splice variant; pA, polyadenylation sequence of 1 integrin gene). Epidermis of back skin of 9-day-old control (B and C) and mutant mice (D and E) stained for 1 integrin (B and D) and lacZ activity (C and E) (bar, 50  m). Note the loss of 1 integrin expression in the mutant basal keratinocytes (arrows in B and D). Bulb region of hair follicles of back skin of 9-day-old control (F and H) and mutant mice (G and I) double-stained for 1 (red) and  6 integrin (green) (F and G) and for lacZ activity (H and I) (bar, 50 m). Note the loss of 6 and 1 integrin in the hair matrix and the thin unstained region visible between the 6 integrin expressing ORS cells and 1 integrin expressing cells surrounding the hair follicle (G). Control (J) and mutant (K) mice at 4 weeks of age. Mutant mice were approximately half the weight of control littermates, lost nearly all hair, and had frequent wounds, especially in mechanically stressed regions of the skin.
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 | Figure 2 Aberrant hair follicle morphogenesis and loss of subcutaneous fat layer in keratinocyte-restricted 1-deficient mice. Hematoxylin–eosin stained sections of back skin of 9-day (A and B) and 7-week-old (C and D) control (A and C) and mutant mice (B and D). At 9 days of age the mutant hair follicles have grown less deeply into the subcutis than normal hair follicles (A, B). Seven-week-old mutant mice had lost all hair follicles and had no subcutaneous fat layer (C, D; bar, 200 m). Hematoxylin–eosin staining of hair follicles derived from 9-day-old control (E) and mutant mice (F, G). Hair follicles in mutant mice showed gross abnormalities, ranging from multilayered ORS (F, arrow) to severely malformed follicles with peripheral hair deposition (G, arrow; bar, 100 m).
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Figure 3 Reduced proliferation of hair matrix cells lacking 1 integrin. Back skin sections of 16-day-old control (A, C and E) and mutant mice (B, D and F) were stained for Ki67 (A and B), TUNEL (C and D) and nidogen (E and F). Hair matrix cells in the hair bulb (arrows) of the mutant mice showed no Ki67-positive proliferating cells (B), in contrast to normal mice (A). Ki67-positive cells between the mutant hair follicles were proliferating dermal fibroblasts. No increased apoptosis was observed in the matrix cells of mutants (D). Nidogen deposition around the 1-deficient hair follicles was thickened but continuous (F; bar, 100 m).
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 | Figure 4 Abnormalities in keratinocyte morphology and differentiation. Back skin sections of 9-day-old control (A, C, E, G and I) and mutant mice (B, D, F, H and J) were stained with hematoxylin–eosin (A and B; bar, 30 m) and with antibodies for keratin-6 (K6; C and D; bar, 50 m), keratin-10 (K10; green; E, F, I and J), keratin-14 (K14; green; G and H), nidogen (red; E–H) and loricrin (red; I and J). Basal keratinocytes from normal mice (A, arrow) attach to a nidogen-containing basement membrane (E and G, arrows). Mutant mice showed basal keratinocytes with an aberrant morphology, a thickened epidermis and blistering between epidermis and dermis (arrow and arrowheads in B, F, H and J). Keratin-6 is upregulated in mutant epidermis (D). Basal cells did not express keratin-10 either in normal or mutant skin (E, F, I and J). Suprabasal cells were positive for keratin-10 and keratin-14 (E–J). Most of the keratin-10 positive cells in the mutant skin showed no expression of loricrin (J), indicating a delayed terminal differentiation (E–J; bar, 40 m). The barrier function of the skin was tested using a hematoxylin dye penetration assay (for details see Materials and methods). Sections of back skin of 4-week-old control (K) and mutant mice (L) were counterstained with eosin. Hematoxylin penetrated the superficial layers of the stratum corneum (violet), but neither the lower layers (L, arrows) nor any underlying tissue in the mutant mice, indicating an intact barrier function of the skin (bar, 50 m).
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Figure 5 Reduced expression of 64 integrin in mutant epidermis. Back skin sections of 16-day-old control (A) and mutant (B) mice were stained for 4 integrin. In mutants, skin expression of 4 integrin by the basal keratinocytes was reduced and discontinuous (B) (bar, 50 m). (C) FACS analysis of keratinocyte preparations from back skin of 3-week-old normal and mutant mice double stained for 4 and 1 integrin, and for 4 and 6 integrin, respectively. Basal keratinocytes expressing high amounts of 1, 6 and 4 integrin were found in normal, but hardly in mutant skin. Instead, mutant skin preparations contained a population of 1-null cells expressing low amounts of 4 and 6 integrin.
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 | Figure 6 Reduced Sos expression of basal keratinocytes in mutant skin. Back skin sections of 9-day-old control (A) and mutant (B) mice were stained for Sos. Sos expression was significantly reduced in basal keratinocytes of mutant skin compared with normal (bar, 50 m).
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Figure 7 Aberrant processing and deposition of laminin-5 and collagen VII in mutant skin. Back skin sections of 16-day-old control (A, C and E) and mutant mice (B, D and F) were stained for the  2 chain of laminin-5 using antibodies recognizing both the unprocessed and the processed form (2LE4-6; A and B), and antibodies specific for a fragment present only in the unprocessed form (2L4m; C and D). 2LE4-6 showed diffuse staining in the dermis of mutant skin (B, arrow). Significantly increased amounts of unprocessed laminin 2 were detected with the 2L4m specific antibody around the basal keratinocytes of mutant mice, while dermally deposited laminin-5 was processed normally (D). Note the virtual absence of unprocessed laminin 2 in the basement membrane of normal epidermis (C) and the normal 2LE4-6 staining around the hair follicle in mutant skin (B; bar, 50 m). Staining for the anchoring fibril component collagen VII (E, F) revealed a diffuse, dermal staining in mutant mice (F). In blisters (F, arrow), small amounts of collagen VII could be detected at the epidermal side.
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 | Figure 8 Distortion of the basement membrane at the dermal–epidermal junction. Electron microscopy of back skin sections of 4-week-old control (A) and mutant (B and C) mice. The lamina densa is marked by short and hemidesmosomes by long arrowheads. Loss of 1 integrin expression on keratinocytes resulted in a discontinuous lamina densa [gaps marked by white arrowheads in (B)] and in a reduction of hemidesmosomes [region without hemidesmosomes and basement membrane marked by white arrowheads in (C)]. (A and B) Bar, 0.25 m; (C) bar, 0.42 m.
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Figure 9 Increased deposition of extracellular matrix components in mutant skin. Back skin sections of 6-week-old control (A, C and E) and mutant mice (B, D and F) were stained for tenascinC (TNC) (A, B), perlecan (C, D), and fibronectin (E, F). All three proteins showed increased staining in the mutant dermis (bar, 100 m).
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 | Figure 10 Infiltration of inflammatory cells in the mutant skin and RNase protection assay. Back skin sections of 16-day-old control (A and C) and mutant (B and D) mice were stained for F4/80 (macrophage/monocyte specific antigen; A, B) and Gr-1 (granulocyte specific antigen; C, D). In mutant skin, an infiltration of macrophages and granulocytes was observed. Some deformed hair follicles were surrounded by macrophages (arrows in B). (Staining of the sebaceous glands in (C) (arrow) and (D) is background staining; bar, 100 m.) (E) RNA isolated from skin of 4-week-old mutant and normal mice was analyzed by RNase protection (for details see Materials and methods). Mutant skin (fl/fl cre) showed a strong upregulation of IL-1 mRNA compared with littermate controls (control) (1000 c.p.m.: radiolabeled RNA probe; tRNA: negative control). Gel separated RNA samples were stained with ethidium bromide (EtBr) to control RNA amounts used in the assay.
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Figure 11 Defects in the esophagus of mutant mice. Esophagus sections of 2- (A–F) and 6-week-old (G and H) control (A, C, E and G) and mutant mice (B, D, F and H) were stained for lacZ activity (A and B), Ki67 (C and D), 4 integrin (E and F), and with hematoxylin–eosin (G and H). Basal cells in the mutant esophagus showed strong lacZ activity (B), reduced Ki67-positive proliferating cells (D) and a reduced staining for 4 integrin (F). In 6-week-old mice, mutant basal cells (H) displayed a different morphology than in normal mice (G). In addition, blisters were observed (H, arrow).
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