Phenotypic abnormalities in mice with targeted inactivation of the Ilk gene in the epidermis. (A) Cumulative survival curve (Kaplan-Meier survival plot) of Ilk^f/f;K14Cre mice (- - - -) and control Ilk^f/+;K14Cre littermates (——, n = 36). (B) Ilk^f/f mice were bred with Ilk^f/;K14Cre transgenic mice, and their progeny was genotyped for presence the Cre transgene and the mutant, floxed Ilk allele at 4 d of age, using PCR. (C) Visible abnormalities in Ilk^f/f;K14Cre mice. The photograph shows the appearance of the animals genotyped in B at 4 d of age. The mouse with ILK-deficient epidermis shows growth retardation and its skin lacks pigmentation, apparent in the ILK-expressing littermate. (D and E) Presence of dry, scaly and fragile skin in the absence of epidermal ILK expression. (F–H) Histological examination of ILK-deficient epidermis reveals abnormally low hair follicle abundance (G) and presence of blisters, where the epidermis has detached from the dermis (arrow in H). Bar, 100 μm.

Defects in adhesion and proliferation of cultured ILK-deficient keratinocytes. (A) Primary keratinocytes isolated from 3-d-old Ilk^f/+;K14-Cre mice or Ilk^f/f;K14-Cre littermates were plated at a density of 100,000 cells/well in 12-well culture dishes at t = 0. The number of cells/well and [³H]dThd incorporated into DNA (expressed as a percentage of [³H]dThd incorporated in Ilk^f/+;K14-Cre keratinocytes at t = 0), were determined at the indicated intervals after initial seeding. The results represent the average + SEM of three experiments done in triplicate. * p < 0.05 (ANOVA). (B) Phase-contrast micrographs of live cultured keratinocytes from Ilk^f/+;K14Cre mice or Ilk^f/f;K14Cre littermates, 3 d after isolation and plating. Bar, 30 μm.

Inactivation of ILK in keratinocytes prevents directional cell migration and Rac1-GTP formation. (A) Four days after infection with AdCre (AdCre) or an empty adenovirus (No AdCre), confluent monolayers of Ilk^f/f keratinocytes were scrape-wounded and cultured in serum-free growth medium and 2.5% BSA. Photographs show representative images all along the wound edges at time = 0, and after allowing the cells to migrate into the denuded areas for 16 h. Bar, 200 μm. (B) Ilk^f/f keratinocytes were infected with AdCre or with an empty adenovirus (No AdCre), followed 20 h later by transfection with a vector encoding RFP-wGBD. The cells were cultured for an additional 24-h interval and photographed as they generated extensions, which resulted in directional migration in the control population (No AdCre), but not in the AdCre-infected, ILK-deficient cells. Bar, 50 μm. (C) Ilk^f/f keratinocytes were infected with AdCre or an empty adenovirus (No AdCre) and cultured for 4 d in normal growth medium. The confluent monolayers were cultured in serum-free medium containing 0.25% BSA for 16 h, at which time multiple scrape wounds were produced, and cell lysates were obtained at the indicated intervals after wounding. Active, GTP-bound Cdc42 (Cdc42-GTP) and Rac1 (Rac1-GTP) were isolated in GST-PAK pull down assays. The levels of indicated total cellular proteins were also assessed by immunoblot from samples of lysates containing each 20 μg protein. The graph represents the average + SD from densitometric analysis of Rac1GTP levels normalized to abundance of total Rac1 corresponding to the same time point (n = 4, * p < 0.005).

Expression of ILK in the skin. Frozen sections of 4-d-old mouse skin were used in immunofluorescence analysis with an anti-ILK antibody, and treated with Hoechst 33258 to visualize the nuclei, as indicated in individual micrographs. Expression of ILK is evident in all layers of the interfollicular epidermis and in the dermis (a), with predominance in the basolateral regions of basal keratinocytes. The line traced in panel b is just underneath the dermal/epidermal junction. ILK is also expressed all along the anagen hair follicles (c and d). Bar, 50 μm.

Alterations in the epidermis and hair follicles of Ilk^f/f;K14Cre mice. (A) Hair follicle development was histologically assessed in epidermal tissue from 4-d-old Ilk^f/+;K14Cre mice or Ilk^f/f;K14Cre littermates, as indicated, and the fraction of follicles at a given stage of development was scored according to the criteria outlined by Paus et al. (1999). The results are expressed as the mean + SD of the stages of 600 follicles assessed per mouse (n = 4). (B) Reduced expression of Ki67 in ILK-deficient hair follicles. The presence of the proliferation marker Ki67 was assessed by immunofluorescence microscopy in epidermal sections of Ilk^f/+;K14Cre or Ilk^f/f;K14Cre mice using an anti-Ki67 antibody. Nuclei were visualized with Hoechst 33258. Nuclei not expressing Ki67 appear dark blue. Quantification of the relative abundance of Ki67-positive nuclei in ILK-expressing and ILK-deficient hair follicles at stages 2–5 is shown under the corresponding tissue micrographs (average + SD), evaluated from 80 follicles in each of five mice. Virtually all ILK-expressing follicles have cells positive for Ki67 staining, in contrast to ILK-deficient follicles (* p < 0.001 relative to stage-matched ILK-expressing follicles). Bar, 200 μm. (C) Histological abnormalities and presence of epidermal blisters in ILK-deficient epidermis. Epidermal tissue sections from mice of the indicated genotype were prepared and stained with hematoxylin and eosin. The arrows in micrographs b and c indicate intraepidermal blisters in the upper suprabasal layers and abnormal underlying epidermis. Bar, (a and b) 100 μm, (c) or 10 μm. (D) Proliferation and expression of basal cell markers in ILK-deficient epidermis. Sections of Ilk^f/+;K14Cre or Ilk^f/f;K14Cre epidermis from 4-d-old mice were processed for indirect immunofluorescence, using the antibodies indicated in individual panels. Expression of Ki67 is restricted to basal keratinocytes, and positive cells are indicated with arrowheads. Nuclei were visualized with Hoechst 33258. Bar, 50 μm. (E) Expression of differentiation markers in ILK-deficient epidermis. Sections of Ilk^f/+;K14Cre or Ilk^f/f;K14Cre epidermis from 4-d-old mice were processed for indirect immunofluorescence, using the antibodies indicated in individual panels. Nuclei were visualized with Hoechst 33258. Bar, 100 μm. (F) Integrin expression and F-actin organization in ILK-deficient epidermis. Sections of Ilk^f/+;K14Cre or Ilk^f/f;K14Cre epidermis from 4-d-old mice were either processed for indirect immunofluorescence, using antibodies against integrin α6 or β1, or were stained with rhodamine-conjugated phalloidin, to visualize the actin cytoskeleton by confocal microscopy, as indicated. Nuclei were detected with Hoechst 33258. Bar, 50 μm

Abnormalities in cell spreading and actin cytoskeleton in ILK-deficient keratinocytes. (A) Epidermal keratinocytes from Ilk^f/f mice were isolated and plated and 24 h later were infected with Cre-encoding adenoviruses (Ad-Cre) at an moi of 75. Twenty-four hours after AdCre infection, genomic DNA from infected and noninfected cultures was isolated and genotyped by PCR to assess excision of the loxP-containing Ilk alleles. Amplicon sizes corresponding to nonexcised floxed ILK, excised ILK, and Cre DNA are 2.1 kb, 230 base pairs, and 391 base pairs, respectively. (B) Immunoblot analysis of ILK protein levels in keratinocytes treated with AdCre viruses. Cell extracts were isolated at the indicated times after infection with AdCre, resolved by denaturing gel electrophoresis, and transferred to membranes. The membranes were probed with antibodies against ILK or β-actin, as indicated. (C) Four days after infection with adenovirus encoding Cre (AdCre) or β-gal (No AdCre), Ilk^f/f keratinocytes were briefly trypsinized and seeded on culture dishes coated with a laminin 332 matrix. Phase-contrast micrographs of live cells were obtained 45 min later (a and c). Parallel cultures were processed for fluorescence microscopy 60 min after plating and treated with rhodamine-labeled phalloidin to visualize the actin cytoskeleton (b and d–f). Bar, 100 μm (a and c) or 15 μm (b and d–f). (D) Ilk^f/f keratinocytes were infected with adenovirus encoding the indicated protein, 72 h later they were trypsinized, and 50,000 cells for each condition were seeded on collagen or a laminin 332 substrate. The percentage of cells that attached, or attached and spread, was determined 45 min after plating. Images of cells labeled with rhodamine-labeled phalloidin were acquired and analyzed with Openlab to measure cell surface. * p < 0.01 relative to uninfected cells (ANOVA).

Rescue of defects in ILK-deficient keratinocyte spreading and forward cell movement by Rac1 G12V and RhoG. (A) Ilk^f/f keratinocytes were infected with empty adenovirus (No AdCre), infected with AdCre (AdCre), or infected with AdCre followed 4 d later by infection with an adenovirus encoding both human ILK and GFP (AdCre + hILK). All cells were cultured in serum-free medium for 16 h. The cells were detached from the culture dish and seeded sparsely onto a laminin 332 matrix–coated surface in serum-free medium and observed by time-lapse videomicroscopy. Dashed borders indicate the outer edge of spread and migrating cells, and arrows denote the position of transient protrusions in an ILK-deficient cell. The inset is a direct fluorescence image of the cell indicated in the (AdCre + hILK) panels, demonstrating expression of GFP. Bar, 25 μm. (B) Ilk^f/f keratinocytes were infected with AdCre, followed 20 h later by transfection with vectors encoding the indicated proteins. The cells were cultured for additional 24 h, trypsinized, and reseeded on a surface coated with laminin 332-matrix. Forty-five minutes after seeding, images of live cells were obtained by videomicroscopy. Bar, 25 μm.