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

Francesco J. A. Conti1, Robert J. Rudling1, Alistair Robson, Consultant Dermapathologist2 and Kairbaan M. Hodivala-Dilke1,*

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

* Author for correspondence (e-mail: kairbaan.hodivala-dilke{at}cancer.org.uk)

Accepted 10 March 2003


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{alpha}3ß1-integrin is abundantly expressed in the epidermis, and in mice, ablation of the {alpha}3 gene results in embryonic defects and perinatal lethality. To determine the role of {alpha}3-integrin in adult skin development, we grafted skin from newborn {alpha}3-integrin-deficient mice on to ICRF nu/nu recipients. We report that adult {alpha}3-integrin-deficient skin has severe abnormalities restricted to hair follicle morphology, which include stunted hair follicle growth, increased hair follicle fragility, aberrant pigment accumulation and formation of hair follicle clusters. These abnormalities are caused by a combination of defects in: (1) keratinocyte cytoskeletal organisation, (2) outer root sheath architecture and (3) integrity of the lamina densa. Our results indicate that {alpha}3ß1 is not essential for adult interfollicular epidermal differentiation, but it is required to direct several processes important in hair follicle maintenance and morphogenesis.

Key words: {alpha}3-integrin, F-actin, Hair follicle abnormalities


    Introduction
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 Introduction
 Materials and Methods
 Results
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 References
 
The epidermis is composed of multiple layers of epithelial cells known as keratinocytes. Keratinocyte proliferation is normally confined to the basal layer, and cells differentiate as they migrate upwards through suprabasal spinous, granular and cornified layers. Each layer is characterised by the expression of different keratins and cytoskeletal proteins. For instance, basal keratinocytes express keratin 14 and 5, spinous and granular layers express keratins 1 and 10 (Fuchs and Green, 1980Go) and the upper cornified layer expresses filaggrin and loricrin (Dale et al., 1978Go).

Hair follicles consist of an outermost layer called the outer root sheath (ORS), which is contiguous with the basal epidermal layer and sebaceous gland, a lining of inner root sheath keratinocytes and an innermost hair shaft. The outer root sheath also contains the bulge region, which is at least one of the sites at which epidermal stem cells reside. The base of the hair follicle, or bulb, contains both specialised keratinocytes (matrix) and mesenchymally derived dermal papillae cells. In postnatal life the upper portion of the hair follicle (including the bulge), sebaceous glands and dermal papilla are permanent, but the remainder of the hair follicle undergoes cycles of growth (anagen), regression (catagen) and rest (telogen) (Hardy, 1992Go; Fuchs et al., 2001Go). These cycles are dependent on inductive signals between the bulge and the base of the hair follicle for new hair development (Cotsarelis et al., 1990Go; Fuchs et al., 2001Go).

Hair follicle and interfollicular epidermal (IFE) keratinocytes adhere to the basement membrane, which contains collagen type IV (Coll IV), laminin 5 (Lm-5), entactin (ent) and fibronectin (Timpl, 1989Go; Carter et al., 1991Go; Mosher et al., 1992Go) through cell-matrix adhesion molecules known as integrins. Integrins are heterodimeric transmembrane glycoproteins consisting of an {alpha} and ß subunit, and the heterodimer composition confers ligand specificity (Hynes, 1992Go). Basal epidermal keratinocytes express several integrins, including {alpha}2ß1-, {alpha}3ß1-, {alpha}5ß1-, {alpha}9ß1-, {alpha}vß5- and {alpha}6ß4-integrins (Watt, 2002Go; Palmer et al., 1993Go); in particular, {alpha}3ß1- and {alpha}6ß4-integrins are abundantly expressed by epidermal keratinocytes and are predominantly receptors for Lm-5. {alpha}6ß4 localises to hemidesmosomes, which are specialised adhesion structures that connect Lm-5 to the keratin cytoskeleton (Carter et al., 1990Go). Hemidesmosomes play an important role in maintaining structural integrity at the dermal-epidermal junction (DEJ), and genetic ablation of the {alpha}6- or ß4-integrin subunits results in severe skin blistering owing to a failure of epidermal keratinocytes to adhere to the basement membrane (Georges-Labouesse et al., 1996Go; van der Neut et al., 1996Go).

Conversely, {alpha}3ß1-integrin is localised in vivo between hemidesmosomes and links the basement membrane to the actin cytoskeleton. Mice in which the {alpha}3-integrin gene has been ablated die perinatally and present several developmental abnormalities in kidneys, lungs (Kreidberg et al., 1996Go) and brain (Anton et al., 1999Go). Newborn {alpha}3-integrin-deficient mice also display epidermal-dermal microblisters on footpads, which are reminiscent of a human blistering disease called junctional epidermolysis bullosa. Analysis of the basement membrane of these mice indicates that deposition of Lm-5 is altered, and it was suggested that this may be the cause of blistering (DiPersio et al., 1997Go). These data indicate that in newborn skin, {alpha}3ß1-integrin plays important functions in regulating the maintenance of the epidermal basement membrane. However, these studies could not address the role of {alpha}3-integrin in the development of adult epidermis and its appendages.

Since ß1-integrin-deficient mice have an embryonic lethal phenotype (Stephens et al., 1995Go), conditional mutant models were required to investigate the roles of ß1-integrins in postnatal skin development. Mice in which ablation of the ß1 gene is restricted to the basal layer of epidermal keratinocytes present skin blistering at the DEJ, impaired downgrowth of hair follicles, probably because of poor basement membrane organisation (Raghavan et al., 2000Go), and eventual hair loss (Brakebusch et al., 2000Go). It has been postulated that because ß1-integrin-deficient hair follicles are not regenerated this may reflect an important role of ß1-integrins in regulating the epidermal stem cell compartment. However, this severe phenotype reflects the cumulative deficiency of all the ß1-integrins expressed in keratinocytes, that is, {alpha}2-, {alpha}3-, {alpha}5- and {alpha}9ß1-integrins, and does not address the specific roles of individual {alpha}-subunits in postnatal epidermal morphogenesis.

The expression levels of {alpha}3ß1-integrin change during development and in many pathological conditions (Kreidberg et al., 2000). However, it is unknown whether these changes are causal or consequential, and it is therefore important to understand the roles that {alpha}3ß1-integrin plays during normal tissue development. Therefore, to investigate the precise roles of {alpha}3ß1-integrin in postnatal follicular and interfollicular epidermal development, we developed a method to graft full-thickness newborn mouse skin, comprising both dermis and epidermis, onto adult ICRF nu/nu athymic mice. We report that {alpha}3-deficient skin develops fully, but that maintenance of hair follicle morphology is significantly compromised after the first hair cycle. The abnormalities include stunted hair follicle growth, defective differentiation of hair follicle keratinocytes and severe aberrations in filamentous actin (F-actin) organisation and the outer root sheath. The morphology and differentiation of interfollicular epidermis was not affected by {alpha}3 deficiency, implying that loss of a single integrin subunit can have distinct effects in the interfollicular and follicular skin compartments. Our results indicate novel roles for {alpha}3-integrin in hair follicle morphogenesis that could not have been predicted based on prior in vitro and in vivo studies and show for the first time that a single {alpha}-integrin subunit can regulate adult hair follicle development.


    Materials and Methods
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 Materials and Methods
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Antibodies
Rabbit antisera to the {alpha}3- (clone 8-4) and ß1- (clone 210-H) integrin subunits were prepared as described previously (Marcantonio and Hynes, 1988Go; Hynes et al., 1989Go; DiPersio et al., 1995Go). The antilaminin 5 polyclonal antibody was a generous gift from Peter Marinkovich (Stanford University School of Medicine, Stanford, CA). The polyclonal antibody against collagen type IV was purchased from Chemicon International (Temecula, CA). Antibodies to keratins 1, 6 and 14, and to the epidermal differentiation marker loricrin, were purchased from Covance (Richmond, CA). The monoclonal antibody against entactin was purchased from Upstate Biotechnology (Lake Placid, NY). The polyclonal antibody against the hair-specific keratin 2 (Hb2) was a kind gift from Lutz Langbein (German Cancer Research Centre, Heidelberg, Germany). The monoclonal antibody to the proliferation antigen Ki67 was purchased from Novocastra Laboratories (Newcastle upon Tyne, UK). Filamentous actin (F-actin) was identified with rhodamine-conjugated phalloidin, purchased from Sigma-Aldrich (Dorset, UK). Biotin-conjugated goat anti-rabbit and all the FITC-conjugated secondary antibodies were purchased from Biosource International (Camarillo, CA).

Skin grafting
Six-week-old male ICRF nu/nu mice (ICRF nu/nu) were anaesthetised with an intraperitoneal injection of Avertin, and a full thickness skin disc (8 mm in diameter) was excised to expose the underlying fascia. Litters of newborn mice including {alpha}3-integrin-deficient (Kriedberg et al., 1996) (also available from Jackson laboratories as the Itgatm1Jak mouse) and wild-type (WT) littermates, outbred between C57Bl6/J and 129Sv/ter, were sacrificed by decapitation, and the tails were collected for genotyping (DiPersio et al., 1997Go). Transpore tape (Southern Syringe Service, London, UK) was used to support the pup skin while removing it from the carcass. Using an 8mm biopsy punch (Stiefel Labs, Buckinghamshire, UK) a disc of donor skin was cut and applied onto the recipient fascia. Steri-strips and circular plasters (Southern Syringe Service, London, UK) were applied to secure the grafts in place. Grafts were then dressed with gauze bandage, and the recipient mice were administered analgesic. Dressings were removed after seven days and grafts processed as described below. In total, over 600 skin grafts were prepared for this study. All mice were killed by CO2 inhalation at either 14 days or 45 days post grafting.

Immunohistochemistry and apoptosis detection
Skin grafts were shaved, excised and bisected along the anterior-posterior axis. Half the graft was then embedded in OCT compound and snap-frozen in isopentane chilled in liquid nitrogen. The other half was fixed in 4% formaldehyde solution in PBS for 24 hours and then embedded in paraffin. All sections were cut longitudinally through the skin. This was established for each sample, firstly by orientating the skin sample perpendicularly to the plane of the cut, further by always observing longitudinal sections through hair follicles in each section and finally by verifying that the thickness of the interfollicular epidermis was no more than two to three layers of keratinocytes. No tangential sections were used in the analysis. For morphological analysis, paraffin-embedded skin sections were dewaxed in xylene, rehydrated in decreasing concentrations of ethanol and finally stained with hematoxilin and eosin.

To examine apoptosis, paraffin-embedded sections were dewaxed and rehydrated as described above and apoptosis-related DNA fragmentation was detected using the TUNEL detection kit (Intergen company, Oxford, UK).

Immunohistochemistry of 6 µm cryosections was carried out as follows: sections were fixed in 4% formaldehyde in PBS for 20 minutes at room temperature and blocked in 0.1% BSA, 0.2% Triton X-100, 0.1% glycine in PBS for 1 hour prior to a 40 minute incubation with primary antibodies. For detection of entactin, keratin-1, keratin-6, keratin-14 and Hb2, sections were incubated with a primary antibody diluted in blocking solution for 40 minutes at room temperature, washed in PBS and incubated with FITC-conjugated secondary antibody diluted in blocking solution for 40 minutes. Alternatively, antibodies to loricrin, collagen type IV and {alpha}3-integrins were followed by incubation with a biotin-conjugated secondary antibody, washed in PBS and then incubated with FITC-conjugated streptavidin for 40 minutes. Finally, sections were washed in distilled water and mounted in Gelvatol supplemented with DABCO antifading agent. Representative fields were photographed using a Hamamatsu Digital Camera (Improvision, London, UK) on a Zeiss Axioplan microscope (Zeiss, HERTS, UK), with the exception of F-actin images, which were acquired using a Zeiss LSM-10 confocal microscope.

Electron microscopy
Skin grafts were harvested as described above, and prepared for electron microscopy as follows: small pieces of tissue were fixed with 2.5% gluteraldehyde in 0.1 M Sörensens phosphate buffer for 1 hour at room temperature, washed in Sörensens buffer, postfixed in 1% osmium tetroxide in 0.5 M Sörensens buffer for 30 minutes at room temperature, washed in Sörensens buffer and then finally washed in distilled water. Samples were then dehydrated in ethanol and propylene oxide, infiltrated with araldite/propylene oxide 1:1 overnight at room temperature, infiltrated with araldite, embedded in resin and polymerised at 60°C overnight. Ultrathin (80 nm) sections were cut, mounted on grids, stained with uranyl acetate in lead cytrate and washed in distilled water. Sections were viewed using a JEOL 1010 Transmission Electron Microscope.


    Results
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 Materials and Methods
 Results
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 References
 
{alpha}3ß1-integrin deficiency does not prevent postnatal skin development
Previous studies on the consequences of {alpha}3-integrin deficiency in mouse development have been limited to the analysis of newborn mice (Kreidberg et al., 1996Go; DiPersio et al., 1997Go; Hodivala-Dilke et al., 1998Go; Kreidberg, 2000Go). To examine the effect of {alpha}3-integrin ablation in adult skin and especially in hair follicle morphogenesis, we performed full-thickness skin grafts from newborn {alpha}3-integrin-deficient and WT mice onto adult ICRF nu/nu athymic mice. Grafting efficiency was not affected by {alpha}3 deficiency and was 95% for both genotypes. Hair growth was apparent at 12 days post grafting, and by 14 days both WT and {alpha}3-integrin deficient skin grafts were covered by dense pelage. At this stage the density and appearance of the hair was comparable in control and {alpha}3-integrin-deficient skin grafts (Fig. 1A,B).



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Fig. 1. {alpha}3ß1-integrin deficiency does not prevent skin development. 14-day-old WT (A) and {alpha}3-integrin-deficient (B) skin grafts developed fully and were covered by dense pelage. H&E-stained sections of WT (C) and {alpha}3-integrin-deficient (D) 14-day-old skin grafts. At this stage skin morphology was unaffected by {alpha}3-integrin deficiency. Immunofluorescence staining of cryosections using an anti-{alpha}3-integrin antibody showed that {alpha}3-integrin was distributed normally in WT skin grafts (E) and was not detectable in {alpha}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.

 

Morphological analysis of the effect of {alpha}3-integrin deficiency in skin was carried out by examining hematoxilin- and eosin (H&E)-stained sections from WT and {alpha}3-integrin-deficient skin grafts. {alpha}3ß1 is a major ß1-integrin in keratinocytes, and ß1-integrins have been proposed to be important in regulating keratinocyte differentiation (Adams and Watt, 1989Go). However, at 14 days the morphology and thickness of {alpha}3-integrin-deficient interfollicular epidermis appeared normal when compared to WT skin grafts (Fig. 1C,D), and both genotypes had evenly spaced, anagen-phase hair follicles. Immunohistochemistry with antibodies directed against {alpha}3-integrin showed that in WT skin grafts, {alpha}3-integrin was expressed in the basal layer of the interfollicular epidermis and in the ORS of hair follicles (Fig. 1E), but was undetectable in {alpha}3-integrin-deficient skin grafts (Fig. 1F). These data indicate that {alpha}3ß1, despite being a major Lm5 receptor in skin, is not required for the initial postnatal development of mouse skin and hair.

Abnormal hair follicle morphogenesis in adult {alpha}3-integrin-deficient skin
To investigate the effect of {alpha}3 deficiency on adult skin development, we analysed {alpha}3-integrin-deficient and WT skin grafts 45 days post grafting. Morphological analysis of H&E-stained sections revealed that interfollicular epidermis appeared normal in both WT and {alpha}3-integrin-deficient skin grafts and was two to three layers thick (Fig. 2A,B), establishing that {alpha}3ß1 is not important for the regulation of adult interfollicular epidermal morphology. However, several hair follicle abnormalities were observed in adult {alpha}3-integrin-deficient skin grafts. WT hair follicles were distributed evenly, and a significant proportion was in the anagen growth phase (Fig. 2C). In contrast, most {alpha}3-integrin-deficient hair follicles appeared stunted and abnormal (Fig. 2D). Quantification revealed that in adult {alpha}3-integrin-deficient skin the average number of hair follicles in the catagen or telogen phases of the hair cycle was increased when compared with age-matched WT skin grafts (50%, WT verses 86%, {alpha}3-integrin deficient). This suggested that {alpha}3-integrin deficiency affects hair cycle progression.



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Fig. 2. {alpha}3-integrin deficiency in adult skin causes abnormalities in hair follicle morphology. H&E-stained sections of WT (A and C) and {alpha}3-integrin-deficient (B and D-G) adult skin grafts. Interfollicular epidermis (A and B) appeared normal in both WT and {alpha}3-integrin-deficient skin grafts. WT hair follicles appeared normal (C), but {alpha}3-integrin-deficient hair follicles at the same age were generally more stunted. In stark contrast with WT skin, longitudinal sections through {alpha}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.

 

In addition, other abnormalities were observed in {alpha}3-integrin-deficient skin grafts. The most striking involved the aggregation of multiple hair follicles in clusters (Fig. 2E,H) often with misplaced sebaceous glands (data not shown). It is important to note that these clustered hair follicles were observed in longitudinally cut sections through the skin and were not caused by oblique sectioning (see Materials and Methods). Indeed, clusters could be detected directly beside normal longitudinally cut hair follicles (Fig. 2D). The majority of WT hair follicles contained a single hair shaft, and follicles containing a maximum of two hair shafts could only be found occasionally. In contrast, in {alpha}3-integrin-deficient skin, the number of hair shafts per cluster unit varied and the most severely abnormal clusters included up to 11 hair shafts. The percentage of abnormal hair follicles was quantified (as the percentage of all hair follicles that formed clustered units) and showed that, although there was no change in the total number of hair follicles between genotypes (data not shown), adult {alpha}3-integrin-deficient skin grafts had a significantly higher number of abnormal hair follicles when compared with WT controls (Fig. 2H).

Another frequently observed abnormality in adult {alpha}3-integrin-deficient skin grafts was the presence of pigment deposits, which were found in different locations: inside the hair follicle bulb, detached from hair follicles deeper in the dermis or at the base of club hairs (Fig. 2F). Histochemical analysis using Masson fontana staining showed that these pigment deposits contained melanin (data not shown). We counted the number of deposits in WT and {alpha}3-integrin-deficient skin grafts, and although they were never be found in 14 day (data not shown) or in adult WT skin grafts, they were abundant in adult {alpha}3-integrin-deficient follicles (Fig. 2I). Pigment deposits are often indicative of deteriorating hair follicles and may reflect an increase in hair follicle fragility in {alpha}3-integrin-deficient skin.

A third abnormality observed in adult {alpha}3-integrin-deficient skin grafts was an aberrant distribution of hair follicles, which were often grouped together, with large areas of interfollicular epidermis between groups (Fig. 2G). Frequently, different abnormalities coexisted in the same skin graft. Although the gross appearance of adult WT and {alpha}3-integrin-deficient skin was comparable, and there was no significant loss of hair at this or older time points (3 and 6 months post grafting), the results establish that the maintenance of hair follicle morphology is compromised in the absence of {alpha}3-integrin.

Hair follicle differentiation is abnormal in {alpha}3-integrin-deficient skin.
Because ß1-integrins have been implicated in controlling keratinocyte differentiation, and adult {alpha}3-integrin deficient hair follicles were morphologically abnormal but interfollicular epidermis in the same samples appeared normal, we wished to examine keratinocyte differentiation in these two epidermal compartments. Immunohistochemical analysis showed that ß1 expression levels were reduced, and the distribution of {alpha}6-, ß4-, {alpha}5- and {alpha}v-integrin subunits were unaltered in {alpha}3-integrin-deficient skin (data not shown). K14 (a basal keratinocyte marker) was confined to basal keratinocytes in the interfollicular epidermis and was also expressed in the hair follicle ORS, with no difference in expression between WT and {alpha}3-integrin-deficient skin grafts (Fig. 3A-D). Markers of epidermal differentiation include K1, loricrin and filaggrin. In both WT and {alpha}3-integrin-deficient skin grafts, K1 was expressed in the suprabasal epidermal layers (Fig. 3E,F), and loricrin and filaggrin were expressed in the cornified layers of the epidermis (Fig. 3G,H and data not shown). K6 is normally confined to hair follicles and its expression in {alpha}3-integrin-deficient skin was similar to that found in WT hair follicles (Fig. 3I,J). However, in contrast, hair-specific keratin Hb2 (Langbein et al., 2001Go) was expressed in the cuticle of WT hair follicles (Fig. 3K), but never found in hair follicle clusters (Fig. 3L). These findings indicate that {alpha}3-integrin deficiency does not affect differentiation of the interfollicular epidermis but does affect differentiation of hair follicle keratinocytes.



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Fig. 3. Abnormal hair follicle differentiation in adult {alpha}3-integrin-deficient skin. Frozen sections of WT (A,C,E,G,I,K) and {alpha}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 {alpha}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 {alpha}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.

 

Reduced proliferation and apoptosis in adult {alpha}3-integrin-deficient skin grafts
Hair follicle keratinocytes undergo coordinated cycles of proliferation and apoptosis that control hair follicle structure and morphology. Because both these processes are thought to be affected by integrin expression, we wished to test if alterations in proliferation and/or apoptosis correlate with the formation of abnormal, poorly differentiated hair follicles in {alpha}3-integrin-deficient skin.

We analysed the expression of the proliferation marker Ki67 in {alpha}3-integrin-deficient and WT skin grafts. Ki67-positive cells were found in the interfollicular epidermis, hair matrix, ORS and occasionally in sebaceous glands. No significant difference in the number of cells expressing Ki67 was found in the interfollicular epidermis of {alpha}3-integrin-deficient skin grafts when compared with WT control grafts (Fig. 4A,B). However, in {alpha}3-integrin-deficient hair follicles, the number of Ki67 positive cells was significantly lower than in WT hair follicles (Fig. 4C and D). Furthermore, hair follicle clusters observed in {alpha}3-integrin deficient skin grafts had few or no Ki67-positive cells (Fig. 4E), and quantitation of the percentage of hair follicle keratinocytes that were positive for Ki67 showed that it was significantly lower in {alpha}3-integrin-deficient samples than in WT controls (Fig. 4F). No significant difference in the number of proliferating keratinocytes was detected in 14-day-old skin grafts (data not shown). The proliferation studies were confirmed by BrdU incorporation experiments (data not shown).



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Fig. 4. Reduced proliferation in adult {alpha}3-integrin-deficient skin grafts. Longitudinal sections through WT (A,C) and {alpha}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 {alpha}3-integrin-deficient skin grafts, but the number of Ki67-positive cells was significantly reduced in {alpha}3-integrin-deficient hair follicles (D) compared with WT controls (C). Clusters of {alpha}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.

 

Apoptosis has been reported to occur normally in the IRS, bulge and bulb of regressing hair follicles (Lindner et al., 1997Go). We analysed apoptosis in WT and {alpha}3-integrin-deficient skin by using TUNEL staining for the presence of positive nuclei. No apoptotic cells were detected in the interfollicular epidermis of WT (Fig. 5A) or {alpha}3-integrin-deficient (Fig. 5B) grafts. In contrast, in WT hair follicles apoptotic profiles were normal (Fig. 5C), but were surprisingly low in {alpha}3-integrin-deficient follicles (Fig. 5D). In addition, very few apoptotic nuclei could be detected in {alpha}3-integrin-deficient hair follicle clusters (Fig. 5E). Calculation of the percentage of TUNEL-positive cells confirmed that {alpha}3-integrin-deficient hair follicles had significantly fewer apoptotic nuclei when compared with WT controls (Fig. 5F).



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Fig. 5. Reduced apoptosis in adult {alpha}3-integrin-deficient skin grafts. Longitudinal sections through WT (A,C) and {alpha}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 {alpha}3-integrin-deficient (B and B') interfollicular epidermis. In hair follicles the numbers of TUNEL-positive cells were significantly reduced in {alpha}3-integrin-deficient skin grafts (D) compared with WT controls (C). Clusters of hair follicles in the {alpha}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).

 

These results may reflect the difference in hair morphology in WT and {alpha}3-integrin-deficient skin and suggest that {alpha}3-integrin is important in maintaining normal proliferative and apoptotic profiles in adult hair follicles.

Laminin 5 and entactin are disorganised in adult {alpha}3-integrin-deficient interfollicular but not follicular epidermis
Since keratinocyte interaction with the underlying basement membrane is important in regulating epidermal morphology, we examined the distribution of basement membrane proteins in adult {alpha}3-integrin-deficient and WT skin grafts. Adult {alpha}3-integrin-deficient and WT skin were analysed by immunofluorescence with antibodies to different basement membrane proteins, including laminin 5 (Lm-5), entactin (ent), collagen type IV (Coll IV) and the extracellular matrix proteins fibronectin (Fn) and collagen type I (data not shown). In WT skin grafts Lm-5, entactin and Coll IV were expressed in the basement membrane zones of interfollicular epidermis and hair follicles (Fig. 6A,D,G). In contrast, in the interfollicular epidermis of {alpha}3-integrin-deficient skin grafts, Lm-5 and entactin were often deposited in multiple layers below the plane of the basement membrane but appeared to be intact around hair follicles (Fig. 6B,E) and hair follicle clusters (Fig. 6C,F). In some conditions, such as psoriasis, disruption of the basement membrane may be associated with mast cell infiltration. However, we did not observe any changes in dermal cell infiltrate in the mutant skin grafts (data not shown). In WT and {alpha}3-integrin-deficient skin grafts Fn was expressed at the dermal-epidermal junction and throughout the dermis, with no significant difference between genotypes. Sometimes, in areas of {alpha}3-integrin-deficient interfollicular epidermis where Lm-5 expression was disrupted, a dense accumulation of Fn was observed. In addition, the distribution of Fn appeared less organised around {alpha}3-integrin-deficient hair follicles compared with WT controls (data not shown). In contrast, Coll IV (Fig. 6G,H,I) and Coll I (data not shown) were distributed normally in {alpha}3-integrin-deficient skin grafts.



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Fig. 6. Laminin 5 and entactin are disorganised in adult {alpha}3-integrin-deficient interfollicular but not follicular epidermis. WT (A,D,G) and {alpha}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 {alpha}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 {alpha}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 {alpha}3-integrin-deficient (K,L) skin grafts. The epidermis appeared to stratify normally, and hemidesmosomes were evident in both WT and {alpha}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.

 

The lamina densa is disrupted in {alpha}3-integrin-deficient interfollicular epidermis
Since the morphology and differentiation of interfollicular epidermis in {alpha}3-integrin-deficient skin grafts appeared normal by histological and immunohistochemical analyses, but Lm-5 and entactin distribution was disrupted, we went on to study adult {alpha}3-integrin-deficient skin at the ultrastructural level in order to examine better the keratinocyte structure and basement membrane integrity. The morphology of the basal, spinous and cornified layers was comparable in the interfollicular epidermis of WT and {alpha}3-integrin-deficient samples (Fig. 6J,K), and there was no significant difference in the number of hemidesmosomes expressed by {alpha}3-integrin-deficient grafts when compared with WT skin. However, in {alpha}3-integrin-deficient interfollicular epidermis the lamina densa often appeared discontinuous in between hemidesmosomes (Fig. 6L), confirming that the integrity of the interfollicular basal lamina was compromised in adult {alpha}3-integrin-deficient skin.

Severe fragility of {alpha}3-deficient hair follicle keratinocytes and lamina densa disorganisation
To better understand the cause of {alpha}3-integrin-deficient hair follicle defects we examined the ultrastructure of hair follicles in adult WT and {alpha}3-integrin-deficient skin grafts. Follicular keratinocytes normally assemble hemidesmosomes in the regions of the follicle proximal to the epidermis. In WT skin grafts, the lamina densa was intact regardless of whether hemidesmosomes were present or not (Fig. 7A,C). In contrast, we observed that maintenance of the basal lamina in {alpha}3-integrin-deficient hair follicles differed depending on whether keratinocytes assembled hemidesmosomes or not. Where hemidesmosomes were present, the lamina densa was disorganised and keratinocytes appeared to retract from it, adhering poorly with finger-like projections, giving the cells a ruffled appearance (Fig. 7B). In the areas lacking hemidesmosomes the lamina densa appeared to be deposited in multiple layers (Fig. 7D). In addition, dermal fibroblasts accumulated in dense layers close to the lamina densa in {alpha}3-integrin-deficient hair follicles (Fig. 7D,F), and this was not observed in WT samples. Thus, although by immunofluorescence detection of basement membrane molecules no defects were apparent in the basement membrane of {alpha}3-integrin-deficient hair follicles, ultrastructural analysis revealed severe abnormalities in the lamina densa organisation.



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Fig. 7. Severe disorganisation and fragility of outer and inner root sheath cells in {alpha}3-integrin-deficient hair follicles. Electron micrographs of WT (A,C,E) and {alpha}3-integrin deficient (B,D,F,G) hair follicles. (A,B) Distal area of hair follicle ORS, where hemidesmosomes are present. In {alpha}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 {alpha}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 {alpha}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 {alpha}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 {alpha}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.

 

The presence of pigment deposits and stunted hair follicles in {alpha}3-integrin-deficient skin strongly suggest that {alpha}3-integrin deficiency causes increased hair follicle fragility. At the ultrastructural level WT hair follicle keratinocytes in the outer and inner root sheaths were well organised and had good intercellular contacts (Fig. 7E). In contrast, cells in the ORS of {alpha}3-integrin-deficient hair follicles were severely ruptured, indicating an increase in cellular fragility, and keratinocytes of both the ORS and IRS presented large intra- and intercellular spaces, in which the IRS cells extended numerous filopodial processes (Fig. 7F,G). Thus, the severe ultrastructural abnormalities in the hair follicle keratinocytes most probably contributes significantly to the stunted and fragile nature of {alpha}3-integrin-deficient hair follicles.

F-actin is both disorganised and arranged in prominent bundles in {alpha}3-integrin-deficient hair follicles
Since the assembly of actin filaments profoundly affects the structural organisation of cells within tissues and their adhesive behaviour, we wondered whether changes in F-actin distribution correlated with the morphological abnormalities and cellular fragility observed in adult {alpha}3-integrin-deficient hair follicles. Cryosections of adult WT and {alpha}3-integrin-deficient skin were stained with rhodamine-conjugated phalloidin. In both WT and {alpha}3-integrin-deficient interfollicular epidermis, F-actin was distributed in a pericellular fashion in all keratinocyte layers with no significant difference between genotypes (Fig. 8A,B). However, comparison of F-actin distribution in WT and {alpha}3-integrin-deficient hair follicles gave a strikingly different result. F-actin was distributed subcortically in WT hair follicle keratinocytes (Fig. 8C). In contrast, in {alpha}3-integrin-deficient hair follicles it appeared either severely disorganised (Fig. 8D,E) or formed prominent actin bundles, especially at the base cells, within ORS and IRS keratinocytes (Fig. 8F). These data establish that in adult skin, {alpha}3-integrin deficiency specifically affects the organisation of filamentous actin in follicular but not interfollicular epidermal keratinocytes and that this correlates with hair follicle fragility in {alpha}3-integrin-deficient skin.



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Fig. 8. {alpha}3-integrin deficient hair follicles have both disorganised and prominent F-actin bundles. Cryosections of WT (A,C) and {alpha}3-integrin deficient (B,D,E,F) adult skin grafts were analysed by immunofluorescence with rhodamine-conjugated phalloidin. In WT and {alpha}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 {alpha}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).

 


    Discussion
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Understanding the precise role of integrins in the morphogenesis and maintenance of epidermis is of fundamental importance. In this paper we describe the phenotype of {alpha}3-integrin-deficient adult skin and demonstrate that deficiency of {alpha}3-integrin in skin causes morphological abnormalities which are, surprisingly, restricted to adult hair follicles. These abnormalities include stunted and clustered hair follicle growth, which correlates with an increase in hair follicle fragility, reduced proliferation and apoptosis, reduced expression of hair-specific keratins and severely disorganised F-actin. Our results reveal functions for {alpha}3-integrin in regulating the morphology of hair follicles that previously were unknown.

{alpha}3ß1 is important for structural integrity of hair follicles via regulation of the cytoskeleton
Our observations show that {alpha}3-integrin-deficient skin begins to develop normally and that abnormalities in hair follicle morphology were not observed in 14-day-old grafts. At this early stage hair follicles in both genotypes appeared to be in the first anagen growth phase. Between 14 and 45 days post grafting, the WT hair follicles regressed, through catagen and telogen, and entered the second anagen phase (data not shown). However, in adult {alpha}3-integrin-deficient skin, the majority of hair follicles remained in catagen or telogen, implying that {alpha}3 deficiency may inhibit entrance into the second anagen phase. Clusters of hair follicles in the {alpha}3-integrin-deficient skin may represent multiple unsuccessful attempts of a hair follicle to enter anagen. Furthermore, the general level of reduced proliferation and apoptosis in the {alpha}3-integrin-deficient hair follicles suggests that these follicles may be in an extended resting phase. These results indicate that {alpha}3-integrin plays a crucial role in regulating hair follicle morphology specifically during the catagen phase of the hair cycle.

Since {alpha}3ß1 is one of the most highly expressed epidermal ß1-integrins, one might expect that deficiency in {alpha}3- or ß1-subunits would have overlapping or similar effects on hair follicle cycling. The epidermis-specific, ß1-integrin-deficient mice have severely compromised survival (Brakebusch et al., 2000Go; Raghavan et al., 2000Go) and hair follicles either do not invaginate properly (Raghavan et al., 2000Go) or are lost by 16 days after birth implying that ß1-integrins are essential in matrix formation (Brakebusch et al., 2000Go). In short, both {alpha}3- and ß1-integrins affect hair follicle growth, but they do so at different stages of the hair cycle. In addition, we observed that in adult {alpha}3-integrin-deficient skin grafts, stunted hair follicles often appeared to associate with pigment deposits or casts in the dermis. Such pigment deposits are regarded to reflect an increase in hair follicle fragility. Together with an increase in cell fragility in the ORS and a decrease in hair-specific keratin 2 (Hb2) expression, our data imply that {alpha}3ß1, unlike the combination of all ß1-integrins, is important in maintaining the structural integrity of hair follicles.

What could cause increased hair follicle fragility in {alpha}3-integrin-deficient adult skin? Changes in F-actin organisation within cells has been shown to alter both cell-cell and cell-matrix adhesion (Vasioukhin et al., 2000Go; Vasioukhin et al., 2001). In {alpha}3-integrin-deficient hair follicles F-actin was both severely disorganised and formed prominent actin bundles. Neither of these patterns of F-actin distribution were found in WT hair follicles. This indicates that, in adult skin, {alpha}3-integrin is important in maintaining the correct organisation of the actin cytoskeleton in hair follicles and suggests that F-actin disorganisation causes the observed cell fragility. Consistent with these observations, F-actin is organised into prominent bundles in {alpha}3-integrin-deficient newborn pad skin and keratinocytes (Hodivala-Dilke et al., 1998Go). The reason for the apparent recovery of F-actin organisation in adult interfollicular epidermis is unclear. One explanation may be that the adult skin grafts are essentially from thin, back skin, and the newborn phenotype was described for thick, pad skin that developed blisters. Blistering is a type of wound, and it has been reported previously that keratinocytes in a wound have bundles of F-actin on their basal face. Furthermore since newborn epidermis is less quiescent than adult interfollicular epidermis and no blisters were evident in the adult thin skin, this may explain why the F-actin appears normal in adult thin interfollicular epidermis. Normally, activation of Rho GTPases leads to changes in the actin cytoskeleton, thus enhancing integrin clustering and ECM organisation (Wennerberg et al., 1996Go; Nguyen et al., 2001Go). One might speculate that in the absence of {alpha}3ß1 abnormal activation of Rho results in disorganised F-actin and an increase cell fragility. Taken together, these symptoms represent a likely cause for both the stunted and clustered abnormal hair follicle growth in {alpha}3-integrin-deficient skin.

The role of {alpha}3-integrin in follicular versus interfollicular epidermal development
As {alpha}3ß1-integrin is expressed in both IFE keratinocytes and hair follicles, why does its deficiency affect follicular and not inter-follicular epidermal morphogenesis and differentiation? One difference between hair follicles and IFE is that although hemidesmosomes (and thus {alpha}6ß4) are expressed throughout the IFE, they are present only in the distal part of hair follicles (Nutbrown and Randall, 1995Go). Consequently, in adult {alpha}3-integrin-deficient skin, the lower part of the hair follicle is doubly-deficient in both {alpha}3ß1- and {alpha}6ß4-integrins. Hence, this combined loss of Lm-5 receptors, although not necessarily the only reason, might contribute to the severe loss of cytoskeletal (both F-actin and hair specific keratin 2) organisation and the extreme cellular fragility of {alpha}3-integrin-deficient hair follicles.

Another difference between hair follicles and IFEs is that hair follicles undergo cycles of massive proliferation and apoptosis, corresponding to the anagen and catagen phases of the hair cycle, respectively. This is unlike the relatively low rates of proliferation and apoptosis in adult quiescent IFEs. Since ß1-integrins have been implicated in the control of proliferation and apoptosis (Gonzales et al., 1999Go; DiPersio et al., 2000Go; De Arcangelis and Georges-Labouesse, 2000Go; Frisch and Screaton, 2001Go), it is perhaps not surprising that {alpha}3 deficiency alters the proliferative and apoptotic potential of keratinocytes predominantly in hair follicles. These changes, in turn, probably contribute to the increase in telogen phase and morphological abnormalities observed in {alpha}3-integrin-deficient hair follicles.

{alpha}3-integrin is required for correct maintenance of the basement membrane in adult skin
Basement membrane disruption is associated with dermal-epidermal blistering in {alpha}3-integrin-deficient newborn pads (DiPersio et al., 1997Go; Hodivala-Dilke et al., 1998Go), and, importantly, the blistering was confined to hairless areas of thick skin. Interestingly, in adult {alpha}3-integrin-deficient skin, the lack of basement membrane integrity in the IFE does not cause skin blistering. How could this be explained? One possibility is that in {alpha}3-integrin-deficient skin grafts the presence of hair provides the skin with increased mechanical resistance, thereby preventing skin blisters. In addition, because blistering is often a skin-site-associated problem, our results may simply indicate that {alpha}3-integrin deficiency in thick skin causes blistering, whereas in thin skin it does not. Although the follicular basal lamina was disrupted in ultrastructural experiments, immunohistochemistry revealed that entactin and Lm-5 appeared intact, indicating that the basement membrane is less severely disorganised in follicular than in interfollicular epidermis. One reason for this may be that cycling of hair follicles induces a constant regeneration of the surrounding basement membrane, thereby preventing the accumulation of disorganised ECM proteins around hair follicles and thus protecting the skin from sheer stress effects.

A possible role for {alpha}3-integrin in maintenance of the epidermal stem cell compartment?
Epidermal stem cells express high levels of {alpha}2ß1- and {alpha}3ß1-integrins (Jones and Watt, 1993Go; Jones et al., 1995Go; Bagutti et al., 1996Go; Moles and Watt, 1997Go; Jensen et al., 1999Go; Zhu et al., 1999Go; Watt, 2001Go). An important question is whether these integrins are simply markers of stem cells or whether they are vital for the functional maintenance of the stem cell compartment in vivo (Levy et al., 2000Go; Raghavan et al., 2000Go). Previous studies using mice either totally deficient in {alpha}3-integrin (Kreidberg et al., 1996Go; DiPersio et al., 1997Go) or epidermis-specific ß1-integrin-deficient mice (Raghavan et al., 2000Go; Brakebusch et al., 2000Go) have demonstrated that the epidermis can develop in the absence of these integrins, at least until birth. In the ß1 mutant mice, hair follicles disappear after the first hair cycle, implying that ß1-integrin might be involved in the maintenance of an epithelial stem cell compartment or might play a role in the activation of stem cells at the onset of anagen (Brakebusch et al., 2000Go). However, immune cell infiltration and hair follicle breakdown at this stage make it difficult to state categorically whether hair follicles are not regenerated because of an intrinsic stem cell defect and/or because an immune response has eradicated all follicular stem cells. The approach that we have used overcame the majority of these immune-response problems since {alpha}3-integrin-deficient skin was grafted onto immunocompromised mice. Our observations, that adult {alpha}3-deficient skin can develop fully and form hair follicles and sebaceous glands indicate that {alpha}3ß1 probably is not essential for the maintenance of the stem cell compartment.

In conclusion, we present strong evidence that adult {alpha}3-integrin-deficient skin can develop fully but, surprisingly, it has hair follicle and not interfollicular abnormalities. These abnormalities are probably caused by a combination of cytoskeletal aberrations, loss of basement membrane integrity and a resultant increase in cell fragility. Taken together our data establish that {alpha}3ß1-integrin is important in maintaining hair follicle architecture during the hair cycle and provides the first evidence for the role of a single {alpha}-subunit in regulating adult hair follicle morphology.


    Acknowledgments
 
We thank Lutz Langbein and Peter Marinkovich for kindly providing us with antibodies. We are grateful to Barbara Cross, Toni Hill, Jessica Gruninger, Garry Saunders, Sue Watling, Colin Wren and to the staff of the Cancer Research UK Histopathology and Microscopy Units for their expert technical assistance. We are grateful to Richard Hynes, Ed Yoo and Erika for their support and help in the early pilot experiments. We also wish to thank Louise Reynolds, Stephen Robinson, Catherin Niemann, Simon Broad, Ian Hart, Clive Dickson and Fiona Watt for their help in this study and for critical revision of this manuscript.


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