Fig 1.
Keratinocyte-restricted deletion of α-pv results in progressive and persistent hair loss.
(A) Tail-skin of 8-week-old (would-mount) and back-skin of 2-week-old (paraffin section) control and α-pvΔK mice stained for α-pv and α6 integrin. Scale bars are 40 μm and 20 μm, respectively. E: epidermis, D: dermis, ORS: outer-root-sheath and SG: sebaceous gland. Note the expression of α-pv in basal keratinocytes of interfollicular epidermis and ORS, and the strong reduction in the α-pv signal in α-pvΔK mice when compared to control mice. (B) Protein levels of α-pv and β-pv in keratinocyte lysates of control and α-pvΔK mice and in fibroblast (fibro) lysates isolated from α-pv-/- mice [11]. (C) Gross morphology analysis of control and α-pvΔK mice at indicated time points.
Fig 2.
Keratinocyte-restricted deletion of α-pv results in epidermis thickening and subepidermal blistering and impaired HF development.
(A-C) Hematoxylin-eosin staining of back skin section of control and α-pvΔK mice. Black arrows indicate areas of epidermal detachments at the DEJ, white arrow indicate subepidermal blister filled with repair tissue, white arrowheads indicate abnormal melanin-deposits. Triangle: fully developed HFs, square: short and prematurely growth arrested HFs and asterisk: epidermis hyperthickening. E: epidermis; D: dermis; SC: subcutis; PC: panniculus carnosum. Scale bar: 200 μm. (D) Histogram of HF length distributions at distinct stages of the HF cycle. At least 3 mice per genotype and time point were analyzed, and a minimum of 100 HFs per time point are presented in the histograms. Images were processed with the Image-J software.
Fig 3.
Deletion of α-pv results in the accumulation of ectopically proliferating cells in suprabasal layers of the epidermis and in the ORS.
(A) Ki67 staining of control and α-pvΔK skin sections. Arrowheads indicate proliferating suprabasal cells and arrow point to proliferative cells in the ORS of α-pv-null HFs. Asterisks indicate a region in the α-pvΔK HF-bulb devoid of proliferative cells. Scale bar: 50 μm. (B) Double-fluorescent labeling for phospho-histone 3 and α6-integrin of control and α-pvΔK skin sections. Nuclei were visualized with DAPI. Arrowheads indicate proliferating suprabasal cells. Scale bar: 20 μm. E: epidermis, D: dermis and HF: hair follicles (C) Quantification of Ki67-positive cells in basal and suprabasal layers of control and α-pvΔK epidermis. Values represent means+SD.
Fig 4.
Impaired integrin distribution and distorted BM organization in the skin of α-pvΔK mice.
Double-fluorescent labeling for (A) LN332 and β4-integrin, and (B) LN332 and β1-integrin of control and α-pvΔK skin sections. Nuclei were visualized with DAPI. Arrowhead indicates β1 integrin expressing suprabasal cells. Scale bar: 20 μm. (C) Ultrastructure of the skin of control and α-pvΔK mice. α-pvΔK mice are characterized by displacement of the basement membrane into the collagen-fibrils of the dermis (cross) and the widening of intercellular spaces (red asterisks), associated with the occurrence of microvilli-like cell protrusions. Double black arrows: desmosomes, single black arrow: lamina densa, red arrows: desmosomes and black asterisks: dermis. (D) Double-fluorescent labeling for LN332 and Gr1, and LN332 and Mac-1 of control and α-pvΔK skin sections. Nuclei were visualized with DAPI. Arrows indicate Gr1-positive granulocytes and Mac1-positive macrophages in the dermis and adjacent to HFs in 2-week-old α-pvΔK mice. Scale bar: 20 μm. E: epidermis, D: dermis, HF: hair follicle and RT: repair tissue.
Fig 5.
Impaired keratinocyte polarity and differentiation in α-pvΔK mice.
Double-fluorescent labeling for (A) F-actin and α6-integrin, (B) E-cadherin and LN332, and (C) β-catenin and LN332 of control and α-pvΔK skin sections. Nuclei were visualized with DAPI. Arrowheads indicate basal stain of F-actin, E-cadherin and β-catenin in α-pv-null basal keratinocytes. Scale bar: 10, 20 and 20 μm respectively. (D) Double-fluorescent labeling for keratin-5 and α6-integrin, keratin-10 and α6-integrin, and loricrin and α6-integrin of control and α-pv-null skin sections. Nuclei were visualized with DAPI. Dotted line: basement membrane. Scale bar: 20 μm.
Fig 6.
Adhesion and migration of α-pv-deficient keratinocytes.
(A) Quantification of adhesion of control and α-pv-null keratinocytes to PLL, LN332, FN and Col-I. Values represent means of the percentage of adhering cells + SD. At least three independent adhesion assays were performed. (B) Double-fluorescent labeling for α-pv and F-actin of control and α-pv-null keratinocytes cultured on fibronectin. Nuclei were visualized with DAPI. (C) Double-fluorescent labeling for α-pv and paxillin of control and α-pv-null keratinocytes cultured on fibronectin. Nuclei were visualized with DAPI. (D) Scratch wounding of a confluent monolayer of control and α-pv-null keratinocytes. (E) Quantification of migration parameters as indicated. Values represent means+SD. At least three independent scratch assay experiments were performed and more than 20 individual cells were tracked in randomly chosen regions in each experiment.