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First published online 20 May 2003
doi: 10.1242/jcs.00475

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3ß1-integrin regulates hair follicle but not interfollicular morphogenesis in adult epidermis

¹ Cancer Research UK, Cell Adhesion and Disease Laboratory, Richard Dimbleby Department of Cancer Research, St Thomas' Hospital, London SE1 7EH, UK
² 2nd Floor, South Wing, Block 7, St John's Institute of Dermatology, St Thomas' Hospital, London SE1 7EH, UK

View larger version (101K): [in a new window]   Fig. 1. 3ß1-integrin deficiency does not prevent skin development. 14-day-old WT (A) and 3-integrin-deficient (B) skin grafts developed fully and were covered by dense pelage. H&E-stained sections of WT (C) and 3-integrin-deficient (D) 14-day-old skin grafts. At this stage skin morphology was unaffected by 3-integrin deficiency. Immunofluorescence staining of cryosections using an anti-3-integrin antibody showed that 3-integrin was distributed normally in WT skin grafts (E) and was not detectable in 3-integrin-deficient skin grafts (F). Four skin grafts per genotype and over 160 hair follicles per genotype were analysed. (e) Epidermis; (d) dermis. Bar represents 4 mm in A and B and 100 µm in C-F.

View larger version (87K): [in a new window]   Fig. 2. 3-integrin deficiency in adult skin causes abnormalities in hair follicle morphology. H&E-stained sections of WT (A and C) and 3-integrin-deficient (B and D-G) adult skin grafts. Interfollicular epidermis (A and B) appeared normal in both WT and 3-integrin-deficient skin grafts. WT hair follicles appeared normal (C), but 3-integrin-deficient hair follicles at the same age were generally more stunted. In stark contrast with WT skin, longitudinal sections through 3-integrin-deficient skin revealed several abnormalities, including hair follicle clusters containing multiple hair shafts (E), aberrant pigment deposition (F) and uneven spacing of hair follicles (G). 11-15 skin grafts for each genotype were analysed. (H) Quantification of the percentage of clusters±s.e.m.; n=11-15 for each genotype; P*<0.005. (I) Quantification of the percentage of hair follicles with aberrant pigment deposition±s.e.m.; n=11-15 for each genotype; P*<0.005. Over 500 follicles per genotype were analysed. Arrows, pigment deposits; arrowheads, clustered hair follicles; brackets, unevenly spaced hair follicles. Bar represents 50 µm in A, B and D; 100 µm in C, E, F and G.

View larger version (63K): [in a new window]   Fig. 3. Abnormal hair follicle differentiation in adult 3-integrin-deficient skin. Frozen sections of WT (A,C,E,G,I,K) and 3-integrin deficient (B,D,F,H,J,L) adult skin grafts were used in immunofluorescence analysis with antibodies against keratin 14 (A-D), keratin 1 (E and F), loricrin (G and H), keratin 6 (I and J) and Hb2 (K and L). In both WT and 3-integrin deficient skin expression patterns of keratins 14, 1 and 6 and loricrin were normal. However, hair-specific keratin Hb2 was frequently absent in 3-integrin-deficient hair follicles. b, basal epidermal layer; sb, suprabasal layers. 10 skin grafts for each genotype were analysed and the experiment was repeated three times. Over 400 follicles per genotype were analysed. Arrows, normal hair follicles; arrowheads, hair follicle clusters. The dashed line indicates skin surface. Bars represent 50 µm in A-H, 100 µm in I-L.

View larger version (119K): [in a new window]   Fig. 4. Reduced proliferation in adult 3-integrin-deficient skin grafts. Longitudinal sections through WT (A,C) and 3-integrin-deficient (B,D,E) adult skin grafts were examined for proliferation by immunohistochemistry with an anti-Ki67 antibody. Keratinocyte proliferation in interfollicular epidermis (A,B) was normal in the 3-integrin-deficient skin grafts, but the number of Ki67-positive cells was significantly reduced in 3-integrin-deficient hair follicles (D) compared with WT controls (C). Clusters of 3-integrin-deficient hair follicles (E) had very small numbers of Ki67-positive cells. (F) Quantification of the percentage of keratinocytes that are Ki67 positive±s.e.m.; eight skin grafts for each genotype were analysed and the experiment was repeated three times. Over 320 follicles per genotype were analysed.; P*<0.0005. Arrows, examples of Ki67-positive nuclei. Arrowheads, base of mutant hair follicles with less Ki67-positive nuclei. Bar represents 50 µm in A, B and E; 100 µm in C and D.

View larger version (56K): [in a new window]   Fig. 5. Reduced apoptosis in adult 3-integrin-deficient skin grafts. Longitudinal sections through WT (A,C) and 3-integrin deficient (B,D,E) adult skin grafts were examined for apoptosis by TUNEL detection. Sections were double labelled with propidium iodide (PI) to detect cell nuclei (A', B', C', D' and E', respectively). Apoptotic cells were very rarely detected in either WT (A and A') or 3-integrin-deficient (B and B') interfollicular epidermis. In hair follicles the numbers of TUNEL-positive cells were significantly reduced in 3-integrin-deficient skin grafts (D) compared with WT controls (C). Clusters of hair follicles in the 3-integrin deficient samples had very few numbers of TUNEL-positive cells (E and E'). Eight skin grafts for each genotype were analysed and the experiment was repeated three times. Over 320 follicles per genotype were analysed. (F) Quantitation of the percentage of hair follicle keratinocytes with TUNEL-positive signals ± s.e.m.; n=5-6 per genotype; P*<0.005. Bar, 50 µm (A,B,E); 100 µm (C,D).

View larger version (128K): [in a new window]   Fig. 6. Laminin 5 and entactin are disorganised in adult 3-integrin-deficient interfollicular but not follicular epidermis. WT (A,D,G) and 3-integrin-deficient (B,C,E,F,H,I) adult skin grafts were analysed by immunofluorescence with antibodies to Lm-5 (A-C), entactin (D-F) and Coll IV (G-I). In WT skin grafts, Lm-5, entactin and Coll IV were localised in the basement membrane zone of the interfollicular and follicular epidermis (A,D,G, respectively). In contrast, in 3-integrin-deficient skin Lm-5 and entactin appeared disorganised at the dermal-epidermal junction of the interfollicular epidermis but not in the basement membrane of normal hair follicles (B and E, respectively) or hair follicle clusters (C and F, respectively). The distribution of Coll IV was not affected in 3-integrin-deficient skin samples in either the interfollicular or follicular compartments (H,I). 11-15 skin grafts for each genotype were analysed and the experiment was repeated three times. Over 500 follicles per genotype were analysed. Electron micrographs of interfollicular epidermis of adult WT (J) and 3-integrin-deficient (K,L) skin grafts. The epidermis appeared to stratify normally, and hemidesmosomes were evident in both WT and 3-integrin-deficient samples. Note that the lamina densa is disrupted between hemidesmosomes. For electron microscopy, four skin grafts per genotype were analysed. Arrows, basement membrane zone; arrowheads, disorganised basement membrane; b, basal layer; s, spinous layer; c, cornified layer; HD, hemidesmosomes; LD, lamina densa; empty arrowheads, interrupted lamina densa. Bar represents 50 µm in A-I; 1 µm in J and K, 200 nm in L.

View larger version (179K): [in a new window]   Fig. 7. Severe disorganisation and fragility of outer and inner root sheath cells in 3-integrin-deficient hair follicles. Electron micrographs of WT (A,C,E) and 3-integrin deficient (B,D,F,G) hair follicles. (A,B) Distal area of hair follicle ORS, where hemidesmosomes are present. In 3-integrin-deficient samples (B) the ORS cells are ruffled, extending finger-like projections, and appear to be retracting from the lamina densa compared with WT controls (A). (C,D) ORS of proximal hair follicle, where hemidesmosomes are not present. In 3-integrin-deficient samples multiple layers of lamina densa were produced (D) in contrast to a single layer of lamina densa in WT controls (C). An abnormally dense accumulation of dermal fibroblasts was also evident in 3-integrin-deficient samples (D,F). (E,F) Low power micrographs of the hair follicle ORS and IRS. Note the loss of organisation of the 3-integrin-deficient ORS, with increased cellular fragility and loss of cell-cell junctions in the inner and outer layers. (G) High-power micrograph of 3-integrin-deficient inner and outer root sheath showing disruption of cell-cell contact. Four skin grafts per genotype were analysed. ORS, outer root sheath; IRS, inner root sheath; HD, hemidesmosomes; LD, lamina densa; P, cell processes or projections, DF, dermal fibroblasts. Bracket and MLLD, multi-layered lamina densa. Asterisk, cellular space. Bar represents 5 µm in A and B, 2 µm in C, 1 µm in D, E, F and G.

View larger version (141K): [in a new window]   Fig. 8. 3-integrin deficient hair follicles have both disorganised and prominent F-actin bundles. Cryosections of WT (A,C) and 3-integrin deficient (B,D,E,F) adult skin grafts were analysed by immunofluorescence with rhodamine-conjugated phalloidin. In WT and 3-integrin deficient interfollicular epidermis F-actin was distributed subcortically in all keratinocyte layers (A,B). A similar pattern of F-actin was observed in WT hair follicles (C). In contrast, in many 3-integrin deficient hair follicles F-actin appeared disorganised (D,E) or formed heavy bundles especially at the basal face of the cells (F). Inserts in C, D and F show a high magnification of the selected areas. 11-15 skin grafts per genotype were analysed and the experiment was repeated three times. Over 500 follicles per genotype were analysed. Bar, 10 µm (A,B); 20 µm in (C,D,E).

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