IntroductionThe epidermis is composed of multiple layers of epithelial cellsknown as keratinocytes. Keratinocyte proliferation is normallyconfined to the basal layer, and cells differentiate as theymigrate upwards through suprabasal spinous, granular andcornified layers. Each layer is characterised by the expressionof different keratins and cytoskeletal proteins. For instance,basal keratinocytes express keratin 14 and 5, spinous andgranular layers express keratins 1 and 10 (Fuchs and Green,1980) and the upper cornified layer expresses filaggrin andloricrin (Dale et al., 1978).

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

Hair follicle and interfollicular epidermal (IFE)keratinocytes adhere to the basement membrane, whichcontains collagen type IV (Coll IV), laminin 5 (Lm-5), entactin(ent) and fibronectin (Timpl, 1989; Carter et al., 1991; Mosheret al., 1992) through cell-matrix adhesion molecules known

as integrins. Integrins are heterodimeric transmembraneglycoproteins consisting of an α and β subunit, and theheterodimer composition confers ligand specificity (Hynes,1992). Basal epidermal keratinocytes express several integrins,including α2β1-, α3β1-, α5β1-, α9β1-, αvβ5- and α6β4-integrins (Watt, 2002; Palmer et al., 1993); in particular, α3β1-and α6β4-integrins are abundantly expressed by epidermalkeratinocytes and are predominantly receptors for Lm-5. α6β4localises to hemidesmosomes, which are specialised adhesionstructures that connect Lm-5 to the keratin cytoskeleton (Carteret al., 1990). Hemidesmosomes play an important role inmaintaining structural integrity at the dermal-epidermaljunction (DEJ), and genetic ablation of the α6- or β4-integrinsubunits results in severe skin blistering owing to a failure ofepidermal keratinocytes to adhere to the basement membrane(Georges-Labouesse et al., 1996; van der Neut et al., 1996).

Conversely, α3β1-integrin is localised in vivo betweenhemidesmosomes and links the basement membrane to the actincytoskeleton. Mice in which the α3-integrin gene has beenablated die perinatally and present several developmentalabnormalities in kidneys, lungs (Kreidberg et al., 1996) andbrain (Anton et al., 1999). Newborn α3-integrin-deficient micealso display epidermal-dermal microblisters on footpads, whichare reminiscent of a human blistering disease called junctionalepidermolysis bullosa. Analysis of the basement membrane ofthese mice indicates that deposition of Lm-5 is altered, and itwas suggested that this may be the cause of blistering (DiPersioet al., 1997). These data indicate that in newborn skin, α3β1-integrin plays important functions in regulating the maintenanceof the epidermal basement membrane. However, these studiescould not address the role of α3-integrin in the development ofadult epidermis and its appendages.

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α3β1-integrin is abundantly expressed in the epidermis,and in mice, ablation of the α3 gene results in embryonicdefects and perinatal lethality. To determine the role of α3-integrin in adult skin development, we grafted skin fromnewborn α3-integrin-deficient mice on to ICRF nu/nurecipients. We report that adult α3-integrin-deficientskin has severe abnormalities restricted to hair folliclemorphology, which include stunted hair follicle growth,increased hair follicle fragility, aberrant pigmentaccumulation and formation of hair follicle clusters. These

abnormalities are caused by a combination of defects in:(1) keratinocyte cytoskeletal organisation, (2) outer rootsheath architecture and (3) integrity of the lamina densa.Our results indicate that α3β1 is not essential for adultinterfollicular epidermal differentiation, but it is requiredto direct several processes important in hair folliclemaintenance and morphogenesis.

Key words: α3-integrin, F-actin, Hair follicle abnormalities

Summary

α3β1-integrin regulates hair follicle but notinterfollicular morphogenesis in adult epidermisFrancesco J. A. Conti 1, Robert J. Rudling 1, Alistair Robson 2 and Kairbaan M. Hodivala-Dilke 1,*1Cancer Research UK, Cell Adhesion and Disease Laboratory, Richard Dimbleby Department of Cancer Research, St Thomas’ Hospital, LondonSE1 7EH, UK2Consultant Dermapathologist, 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@cancer.org.uk)

Accepted 10 March 2003Journal of Cell Science 116, 2737-2747 © 2003 The Company of Biologists Ltddoi:10.1242/jcs.00475

Research Article

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Since β1-integrin-deficient mice have an embryonic lethalphenotype (Stephens et al., 1995), conditional mutant modelswere required to investigate the roles of β1-integrins inpostnatal skin development. Mice in which ablation of theβ1 gene is restricted to the basal layer of epidermalkeratinocytes present skin blistering at the DEJ, impaireddowngrowth of hair follicles, probably because of poorbasement membrane organisation (Raghavan et al., 2000), andeventual hair loss (Brakebusch et al., 2000). It has beenpostulated that because β1-integrin-deficient hair follicles arenot regenerated this may reflect an important role of β1-integrins in regulating the epidermal stem cell compartment.However, this severe phenotype reflects the cumulativedeficiency of all the β1-integrins expressed in keratinocytes,that is, α2-, α3-, α5- and α9β1-integrins, and does not addressthe specific roles of individual α-subunits in postnatalepidermal morphogenesis.

The expression levels of α3β1-integrin change duringdevelopment and in many pathological conditions (Kreidberget al., 2000). However, it is unknown whether these changesare causal or consequential, and it is therefore important tounderstand the roles that α3β1-integrin plays during normaltissue development. Therefore, to investigate the precise rolesof α3β1-integrin in postnatal follicular and interfollicularepidermal development, we developed a method to graft full-thickness newborn mouse skin, comprising both dermis andepidermis, onto adult ICRF nu/nu athymic mice. We report thatα3-deficient skin develops fully, but that maintenance of hairfollicle morphology is significantly compromised after the firsthair cycle. The abnormalities include stunted hair folliclegrowth, defective differentiation of hair follicle keratinocytesand severe aberrations in filamentous actin (F-actin)organisation and the outer root sheath. The morphology anddifferentiation of interfollicular epidermis was not affected byα3 deficiency, implying that loss of a single integrin subunitcan have distinct effects in the interfollicular and follicular skincompartments. Our results indicate novel roles for α3-integrinin hair follicle morphogenesis that could not have beenpredicted based on prior in vitro and in vivo studies and showfor the first time that a single α-integrin subunit can regulateadult hair follicle development.

Materials and MethodsAntibodiesRabbit antisera to the α3- (clone 8-4) and β1- (clone 210-H) integrinsubunits were prepared as described previously (Marcantonio andHynes, 1988; Hynes et al., 1989; DiPersio et al., 1995). The anti-laminin 5 polyclonal antibody was a generous gift from PeterMarinkovich (Stanford University School of Medicine, Stanford, CA).The polyclonal antibody against collagen type IV was purchased fromChemicon International (Temecula, CA). Antibodies to keratins 1, 6and 14, and to the epidermal differentiation marker loricrin, werepurchased from Covance (Richmond, CA). The monoclonal antibodyagainst entactin was purchased from Upstate Biotechnology (LakePlacid, NY). The polyclonal antibody against the hair-specific keratin2 (Hb2) was a kind gift from Lutz Langbein (German CancerResearch Centre, Heidelberg, Germany). The monoclonal antibody tothe proliferation antigen Ki67 was purchased from NovocastraLaboratories (Newcastle upon Tyne, UK). Filamentous actin (F-actin)was identified with rhodamine-conjugated phalloidin, purchased fromSigma-Aldrich (Dorset, UK). Biotin-conjugated goat anti-rabbit and

all the FITC-conjugated secondary antibodies were purchased fromBiosource International (Camarillo, CA).

Skin graftingSix-week-old male ICRF nu/nu mice (ICRF nu/nu) were anaesthetisedwith an intraperitoneal injection of Avertin, and a full thickness skindisc (8 mm in diameter) was excised to expose the underlying fascia.Litters of newborn mice including α3-integrin-deficient (Kriedberg etal., 1996) (also available from Jackson laboratories as the Itgatm1Jak

mouse) and wild-type (WT) littermates, outbred between C57Bl6/Jand 129Sv/ter, were sacrificed by decapitation, and the tails werecollected for genotyping (DiPersio et al., 1997). Transpore tape(Southern Syringe Service, London, UK) was used to support the pupskin while removing it from the carcass. Using an 8mm biopsy punch(Stiefel Labs, Buckinghamshire, UK) a disc of donor skin was cut andapplied onto the recipient fascia. Steri-strips and circular plasters(Southern Syringe Service, London, UK) were applied to secure thegrafts in place. Grafts were then dressed with gauze bandage, and therecipient mice were administered analgesic. Dressings were removedafter seven days and grafts processed as described below. In total, over600 skin grafts were prepared for this study. All mice were killed byCO2 inhalation at either 14 days or 45 days post grafting.

Immunohistochemistry and apoptosis detectionSkin grafts were shaved, excised and bisected along the anterior-posterior axis. Half the graft was then embedded in OCT compoundand snap-frozen in isopentane chilled in liquid nitrogen. The other halfwas fixed in 4% formaldehyde solution in PBS for 24 hours and thenembedded in paraffin. All sections were cut longitudinally through theskin. This was established for each sample, firstly by orientating theskin sample perpendicularly to the plane of the cut, further by alwaysobserving longitudinal sections through hair follicles in each sectionand finally by verifying that the thickness of the interfollicularepidermis was no more than two to three layers of keratinocytes. Notangential sections were used in the analysis. For morphologicalanalysis, paraffin-embedded skin sections were dewaxed in xylene,rehydrated in decreasing concentrations of ethanol and finally stainedwith hematoxilin and eosin.

To examine apoptosis, paraffin-embedded sections were dewaxedand rehydrated as described above and apoptosis-related DNAfragmentation was detected using the TUNEL detection kit (Intergencompany, Oxford, UK).

Immunohistochemistry of 6 µm cryosections was carried out asfollows: sections were fixed in 4% formaldehyde in PBS for 20minutes at room temperature and blocked in 0.1% BSA, 0.2% TritonX-100, 0.1% glycine in PBS for 1 hour prior to a 40 minute incubationwith primary antibodies. For detection of entactin, keratin-1, keratin-6, keratin-14 and Hb2, sections were incubated with a primaryantibody diluted in blocking solution for 40 minutes at roomtemperature, washed in PBS and incubated with FITC-conjugatedsecondary antibody diluted in blocking solution for 40 minutes.Alternatively, antibodies to loricrin, collagen type IV and α3-integrinswere followed by incubation with a biotin-conjugated secondaryantibody, washed in PBS and then incubated with FITC-conjugatedstreptavidin for 40 minutes. Finally, sections were washed in distilledwater and mounted in Gelvatol supplemented with DABCO anti-fading agent. Representative fields were photographed using aHamamatsu Digital Camera (Improvision, London, UK) on a ZeissAxioplan microscope (Zeiss, HERTS, UK), with the exception of F-actin images, which were acquired using a Zeiss LSM-10 confocalmicroscope.

Electron microscopySkin grafts were harvested as described above, and prepared for

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2739α3-integrin regulates adult hair morphogenesis

electron microscopy as follows: small pieces of tissue were fixed with2.5% gluteraldehyde in 0.1 M Sörensens phosphate buffer for 1 hourat room temperature, washed in Sörensens buffer, postfixed in 1%osmium tetroxide in 0.5 M Sörensens buffer for 30 minutes at roomtemperature, washed in Sörensens buffer and then finally washed indistilled water. Samples were then dehydrated in ethanol andpropylene oxide, infiltrated with araldite/propylene oxide 1:1overnight at room temperature, infiltrated with araldite, embedded inresin and polymerised at 60°C overnight. Ultrathin (80 nm) sectionswere cut, mounted on grids, stained with uranyl acetate in lead cytrateand washed in distilled water. Sections were viewed using a JEOL1010 Transmission Electron Microscope.

Results α3β1-integrin deficiency does not prevent postnatal skindevelopmentPrevious studies on the consequences of α3-integrin deficiencyin mouse development have been limited to the analysis ofnewborn mice (Kreidberg et al., 1996; DiPersio et al., 1997;Hodivala-Dilke et al., 1998; Kreidberg, 2000). To examine theeffect of α3-integrin ablation in adult skin and especially inhair follicle morphogenesis, we performed full-thickness skingrafts from newborn α3-integrin-deficient and WT mice ontoadult ICRF nu/nu athymic mice. Grafting efficiency was notaffected by α3 deficiency and was 95% for both genotypes.Hair growth was apparent at 12 days post grafting, and by 14days both WT and α3-integrin deficient skin grafts werecovered by dense pelage. At this stage the density andappearance of the hair was comparable in control and α3-integrin-deficient skin grafts (Fig. 1A,B).

Morphological analysis of the effect of α3-integrindeficiency in skin was carried out by examining hematoxilin-and eosin (H&E)-stained sections from WT and α3-integrin-deficient skin grafts. α3β1 is a major β1-integrin inkeratinocytes, and β1-integrins have been proposed to beimportant in regulating keratinocyte differentiation (Adamsand Watt, 1989). However, at 14 days the morphology andthickness of α3-integrin-deficient interfollicular epidermisappeared normal when compared to WT skin grafts (Fig.1C,D), and both genotypes had evenly spaced, anagen-phasehair follicles. Immunohistochemistry with antibodies directedagainst α3-integrin showed that in WT skin grafts, α3-integrinwas expressed in the basal layer of the interfollicular epidermisand in the ORS of hair follicles (Fig. 1E), but was undetectablein α3-integrin-deficient skin grafts (Fig. 1F). These dataindicate that α3β1, despite being a major Lm5 receptor in skin,is not required for the initial postnatal development of mouseskin and hair.

Abnormal hair follicle morphogenesis in adult α3-integrin-deficient skinTo investigate the effect of α3 deficiency on adult skindevelopment, we analysed α3-integrin-deficient and WT skingrafts 45 days post grafting. Morphological analysis of H&E-stained sections revealed that interfollicular epidermisappeared normal in both WT and α3-integrin-deficient skingrafts and was two to three layers thick (Fig. 2A,B),establishing that α3β1 is not important for the regulation ofadult interfollicular epidermal morphology. However, severalhair follicle abnormalities were observed in adult α3-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 α3-integrin-deficient hair folliclesappeared stunted and abnormal (Fig. 2D). Quantificationrevealed that in adult α3-integrin-deficient skin the averagenumber of hair follicles in the catagen or telogen phases of thehair cycle was increased when compared with age-matchedWT skin grafts (50%, WT verses 86%, α3-integrin deficient).This suggested that α3-integrin deficiency affects hair cycleprogression.

In addition, other abnormalities were observed in α3-integrin-deficient skin grafts. The most striking involved the aggregationof multiple hair follicles in clusters (Fig. 2E,H) often withmisplaced sebaceous glands (data not shown). It is important to

Fig. 1.α3β1-integrin deficiency does not prevent skin development.14-day-old WT (A) and α3-integrin-deficient (B) skin graftsdeveloped fully and were covered by dense pelage. H&E-stainedsections of WT (C) and α3-integrin-deficient (D) 14-day-old skingrafts. At this stage skin morphology was unaffected by α3-integrindeficiency. Immunofluorescence staining of cryosections using ananti-α3-integrin antibody showed that α3-integrin was distributednormally in WT skin grafts (E) and was not detectable in α3-integrin-deficient skin grafts (F). Four skin grafts per genotype andover 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.

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note that these clustered hair follicles were observed inlongitudinally cut sections through the skin and were not causedby oblique sectioning (see Materials and Methods). Indeed,clusters could be detected directly beside normal longitudinallycut hair follicles (Fig. 2D). The majority of WT hair follicles

contained a single hair shaft, and follicles containing a maximumof two hair shafts could only be found occasionally. In contrast,in α3-integrin-deficient skin, the number of hair shafts percluster unit varied and the most severely abnormal clustersincluded up to 11 hair shafts. The percentage of abnormal hairfollicles was quantified (as the percentage of all hair follicles thatformed clustered units) and showed that, although there was nochange in the total number of hair follicles between genotypes(data not shown), adult α3-integrin-deficient skin grafts had asignificantly higher number of abnormal hair follicles whencompared with WT controls (Fig. 2H).

Another frequently observed abnormality in adult α3-integrin-deficient skin grafts was the presence of pigmentdeposits, which were found in different locations: inside thehair follicle bulb, detached from hair follicles deeper in thedermis or at the base of club hairs (Fig. 2F). Histochemicalanalysis using Masson fontana staining showed that thesepigment deposits contained melanin (data not shown). Wecounted the number of deposits in WT and α3-integrin-deficient skin grafts, and although they were never be found in14 day (data not shown) or in adult WT skin grafts, they wereabundant in adult α3-integrin-deficient follicles (Fig. 2I).Pigment deposits are often indicative of deteriorating hairfollicles and may reflect an increase in hair follicle fragility inα3-integrin-deficient skin.

A third abnormality observed in adult α3-integrin-deficientskin grafts was an aberrant distribution of hair follicles, whichwere often grouped together, with large areas of interfollicularepidermis between groups (Fig. 2G). Frequently, differentabnormalities coexisted in the same skin graft. Although thegross appearance of adult WT and α3-integrin-deficient skinwas comparable, and there was no significant loss of hair atthis or older time points (3 and 6 months post grafting), theresults establish that the maintenance of hair folliclemorphology is compromised in the absence of α3-integrin.

Hair follicle differentiation is abnormal in α3-integrin-deficient skin.Because β1-integrins have been implicated in controllingkeratinocyte differentiation, and adult α3-integrin deficienthair follicles were morphologically abnormal butinterfollicular epidermis in the same samples appeared normal,we wished to examine keratinocyte differentiation in thesetwo epidermal compartments. Immunohistochemical analysisshowed that β1 expression levels were reduced, and thedistribution of α6-, β4-, α5- and αv-integrin subunits wereunaltered in α3-integrin-deficient skin (data not shown).K14 (a basal keratinocyte marker) was confined to basalkeratinocytes in the interfollicular epidermis and was alsoexpressed in the hair follicle ORS, with no difference inexpression between WT and α3-integrin-deficient skin grafts(Fig. 3A-D). Markers of epidermal differentiation include K1,loricrin and filaggrin. In both WT and α3-integrin-deficientskin grafts, K1 was expressed in the suprabasal epidermallayers (Fig. 3E,F), and loricrin and filaggrin were expressed inthe cornified layers of the epidermis (Fig. 3G,H and data notshown). K6 is normally confined to hair follicles and itsexpression in α3-integrin-deficient skin was similar to thatfound in WT hair follicles (Fig. 3I,J). However, in contrast,hair-specific keratin Hb2 (Langbein et al., 2001) was expressed

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Fig. 2.α3-integrin deficiency in adult skin causes abnormalities inhair follicle morphology. H&E-stained sections of WT (A and C) andα3-integrin-deficient (B and D-G) adult skin grafts. Interfollicularepidermis (A and B) appeared normal in both WT and α3-integrin-deficient skin grafts. WT hair follicles appeared normal (C), but α3-integrin-deficient hair follicles at the same age were generally morestunted. In stark contrast with WT skin, longitudinal sections throughα3-integrin-deficient skin revealed several abnormalities, includinghair follicle clusters containing multiple hair shafts (E), aberrantpigment deposition (F) and uneven spacing of hair follicles (G). 11-15 skin grafts for each genotype were analysed. (H) Quantification ofthe percentage of clusters±s.e.m.; n=11-15 for each genotype;P*<0.005. (I) Quantification of the percentage of hair follicles withaberrant 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.

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in the cuticle of WT hair follicles (Fig. 3K), but never foundin hair follicle clusters (Fig. 3L). These findings indicate thatα3-integrin deficiency does not affect differentiation of theinterfollicular epidermis but does affect differentiation of hairfollicle keratinocytes.

Reduced proliferation and apoptosis in adult α3-integrin-deficient skin graftsHair follicle keratinocytes undergo coordinated cycles ofproliferation and apoptosis that control hair follicle structureand morphology. Because both these processes are thoughtto be affected by integrin expression, we wished to test ifalterations in proliferation and/or apoptosis correlate with theformation of abnormal, poorly differentiated hair follicles inα3-integrin-deficient skin.

We analysed the expression of the proliferation marker Ki67in α3-integrin-deficient and WT skin grafts. Ki67-positive cellswere found in the interfollicular epidermis, hair matrix, ORS andoccasionally in sebaceous glands. No significant difference inthe number of cells expressing Ki67 was found in theinterfollicular epidermis of α3-integrin-deficient skin graftswhen compared with WT control grafts (Fig. 4A,B). However,in α3-integrin-deficient hair follicles, the number of Ki67positive cells was significantly lower than in WT hair follicles(Fig. 4C and D). Furthermore, hair follicle clusters observed inα3-integrin deficient skin grafts had few or no Ki67-positivecells (Fig. 4E), and quantitation of the percentage of hair folliclekeratinocytes that were positive for Ki67 showed that it wassignificantly lower in α3-integrin-deficient samples than in WTcontrols (Fig. 4F). No significant difference in the number ofproliferating keratinocytes was detected in 14-day-old skingrafts (data not shown). The proliferation studies were confirmedby BrdU incorporation experiments (data not shown).

Apoptosis has been reported to occur normally in the IRS,bulge and bulb of regressing hair follicles (Lindner et al.,1997). We analysed apoptosis in WT and α3-integrin-deficientskin by using TUNEL staining for the presence of positivenuclei. No apoptotic cells were detected in the interfollicularepidermis of WT (Fig. 5A) or α3-integrin-deficient (Fig. 5B)grafts. In contrast, in WT hair follicles apoptotic profiles werenormal (Fig. 5C), but were surprisingly low in α3-integrin-deficient follicles (Fig. 5D). In addition, very few apoptoticnuclei could be detected in α3-integrin-deficient hair follicleclusters (Fig. 5E). Calculation of the percentage of TUNEL-positive cells confirmed that α3-integrin-deficient hair follicleshad significantly fewer apoptotic nuclei when compared withWT controls (Fig. 5F).

These results may reflect the difference in hair morphologyin WT and α3-integrin-deficient skin and suggest that α3-integrin is important in maintaining normal proliferative andapoptotic profiles in adult hair follicles.

Laminin 5 and entactin are disorganised in adult α3-integrin-deficient interfollicular but not follicularepidermisSince keratinocyte interaction with the underlying basementmembrane is important in regulating epidermal morphology,we examined the distribution of basement membrane proteinsin adult α3-integrin-deficient and WT skin grafts. Adultα3-integrin-deficient and WT skin were analysed by

Fig. 3.Abnormal hair follicle differentiation in adult α3-integrin-deficient skin. Frozen sections of WT (A,C,E,G,I,K) and α3-integrindeficient (B,D,F,H,J,L) adult skin grafts were used inimmunofluorescence analysis with antibodies against keratin 14 (A-D), keratin 1 (E and F), loricrin (G and H), keratin 6 (I and J) andHb2 (K and L). In both WT and α3-integrin deficient skin expressionpatterns of keratins 14, 1 and 6 and loricrin were normal. However,hair-specific keratin Hb2 was frequently absent in α3-integrin-deficient hair follicles. b, basal epidermal layer; sb, suprabasal layers.10 skin grafts for each genotype were analysed and the experimentwas repeated three times. Over 400 follicles per genotype wereanalysed. Arrows, normal hair follicles; arrowheads, hair follicleclusters. The dashed line indicates skin surface. Bars represent50µm in A-H, 100 µm in I-L.

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immunofluorescence with antibodies to different basementmembrane proteins, including laminin 5 (Lm-5), entactin (ent),collagen type IV (Coll IV) and the extracellular matrix proteinsfibronectin (Fn) and collagen type I (data not shown). In WTskin grafts Lm-5, entactin and Coll IV were expressed in thebasement membrane zones of interfollicular epidermis andhair follicles (Fig. 6A,D,G). In contrast, in the interfollicularepidermis of α3-integrin-deficient skin grafts, Lm-5 andentactin were often deposited in multiple layers below theplane of the basement membrane but appeared to be intactaround hair follicles (Fig. 6B,E) and hair follicle clusters (Fig.6C,F). In some conditions, such as psoriasis, disruption ofthe basement membrane may be associated with mast cellinfiltration. However, we did not observe any changes indermal cell infiltrate in the mutant skin grafts (data not shown).In WT and α3-integrin-deficient skin grafts Fn was expressedat the dermal-epidermal junction and throughout the dermis,with no significant difference between genotypes. Sometimes,in areas of α3-integrin-deficient interfollicular epidermiswhere Lm-5 expression was disrupted, a dense accumulation

of Fn was observed. In addition, the distribution of Fn appearedless organised around α3-integrin-deficient hair folliclescompared with WT controls (data not shown). In contrast, Coll

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Fig. 4.Reduced proliferation in adult α3-integrin-deficient skingrafts. Longitudinal sections through WT (A,C) and α3-integrin-deficient (B,D,E) adult skin grafts were examined for proliferation byimmunohistochemistry with an anti-Ki67 antibody. Keratinocyteproliferation in interfollicular epidermis (A,B) was normal in the α3-integrin-deficient skin grafts, but the number of Ki67-positive cellswas significantly reduced in α3-integrin-deficient hair follicles (D)compared with WT controls (C). Clusters of α3-integrin-deficienthair follicles (E) had very small numbers of Ki67-positive cells.(F) Quantification of the percentage of keratinocytes that are Ki67positive±s.e.m.; eight skin grafts for each genotype were analysedand the experiment was repeated three times. Over 320 follicles pergenotype were analysed.; P*<0.0005. Arrows, examples of Ki67-positive nuclei. Arrowheads, base of mutant hair follicles with lessKi67-positive nuclei. Bar represents 50 µm in A, B and E; 100 µm inC and D.

Fig. 5.Reduced apoptosis in adult α3-integrin-deficient skin grafts.Longitudinal sections through WT (A,C) and α3-integrin deficient(B,D,E) adult skin grafts were examined for apoptosis by TUNELdetection. Sections were double labelled with propidium iodide (PI)to detect cell nuclei (A′, B′, C′, D′ and E′, respectively). Apoptoticcells were very rarely detected in either WT (A and A′) or α3-integrin-deficient (B and B′) interfollicular epidermis. In hairfollicles the numbers of TUNEL-positive cells were significantlyreduced in α3-integrin-deficient skin grafts (D) compared with WTcontrols (C). Clusters of hair follicles in the α3-integrin deficientsamples had very few numbers of TUNEL-positive cells (E and E′).Eight skin grafts for each genotype were analysed and theexperiment was repeated three times. Over 320 follicles per genotypewere analysed. (F) Quantitation of the percentage of hair folliclekeratinocytes with TUNEL-positive signals ± s.e.m.; n=5-6 pergenotype; P*<0.005. Bar, 50 µm (A,B,E); 100 µm (C,D).

2743α3-integrin regulates adult hair morphogenesis

IV (Fig. 6G,H,I) and Coll I (data not shown) were distributednormally in α3-integrin-deficient skin grafts.

The lamina densa is disrupted in α3-integrin-deficientinterfollicular epidermisSince the morphology and differentiation of interfollicularepidermis in α3-integrin-deficient skin grafts appeared normalby histological and immunohistochemical analyses, but Lm-5and entactin distribution was disrupted, we went on to studyadult α3-integrin-deficient skin at the ultrastructural levelin order to examine better the keratinocyte structure andbasement membrane integrity. The morphology of the basal,spinous and cornified layers was comparable in theinterfollicular epidermis of WT and α3-integrin-deficientsamples (Fig. 6J,K), and there was no significant difference inthe number of hemidesmosomes expressed by α3-integrin-deficient grafts when compared with WT skin. However, inα3-integrin-deficient interfollicular epidermis the laminadensa often appeared discontinuous in betweenhemidesmosomes (Fig. 6L), confirming that the integrity of

the interfollicular basal lamina was compromised in adult α3-integrin-deficient skin.

Severe fragility of α3-deficient hair follicle keratinocytesand lamina densa disorganisationTo better understand the cause of α3-integrin-deficient hairfollicle defects we examined the ultrastructure of hair folliclesin adult WT and α3-integrin-deficient skin grafts. Follicularkeratinocytes normally assemble hemidesmosomes in the regionsof the follicle proximal to the epidermis. In WT skin grafts, thelamina densa was intact regardless of whether hemidesmosomeswere present or not (Fig. 7A,C). In contrast, we observed thatmaintenance of the basal lamina in α3-integrin-deficient hairfollicles differed depending on whether keratinocytes assembledhemidesmosomes or not. Where hemidesmosomes were present,the lamina densa was disorganised and keratinocytes appeared toretract from it, adhering poorly with finger-like projections,giving the cells a ruffled appearance (Fig. 7B). In the areaslacking hemidesmosomes the lamina densa appeared to bedeposited in multiple layers (Fig. 7D). In addition, dermal

Fig. 6.Laminin 5 and entactin are disorganised inadult α3-integrin-deficient interfollicular but notfollicular epidermis. WT (A,D,G) and α3-integrin-deficient (B,C,E,F,H,I) adult skin grafts wereanalysed by immunofluorescence with antibodies toLm-5 (A-C), entactin (D-F) and Coll IV (G-I). In WTskin grafts, Lm-5, entactin and Coll IV werelocalised in the basement membrane zone of theinterfollicular and follicular epidermis (A,D,G,respectively). In contrast, in α3-integrin-deficientskin Lm-5 and entactin appeared disorganised at thedermal-epidermal junction of the interfollicularepidermis but not in the basement membrane ofnormal hair follicles (B and E, respectively) or hairfollicle clusters (C and F, respectively). Thedistribution of Coll IV was not affected in α3-integrin-deficient skin samples in either theinterfollicular or follicular compartments (H,I). 11-15skin grafts for each genotype were analysed and theexperiment was repeated three times. Over 500follicles per genotype were analysed. Electronmicrographs of interfollicular epidermis of adult WT(J) and α3-integrin-deficient (K,L) skin grafts. Theepidermis appeared to stratify normally, andhemidesmosomes were evident in both WT and α3-integrin-deficient samples. Note that the lamina densais disrupted between hemidesmosomes. For electronmicroscopy, four skin grafts per genotype wereanalysed. Arrows, basement membrane zone;arrowheads, disorganised basement membrane;b, basal layer; s, spinous layer; c, cornified layer;HD, hemidesmosomes; LD, lamina densa; emptyarrowheads, interrupted lamina densa. Bar represents50 µm in A-I; 1 µm in J and K, 200 nm in L.

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fibroblasts accumulated in dense layers close to the lamina densain α3-integrin-deficient hair follicles (Fig. 7D,F), and this was notobserved in WT samples. Thus, although by immunofluorescencedetection of basement membrane molecules no defects wereapparent in the basement membrane of α3-integrin-deficient hairfollicles, ultrastructural analysis revealed severe abnormalities inthe lamina densa organisation.

The presence of pigment deposits and stunted hair folliclesin α3-integrin-deficient skin strongly suggest that α3-integrindeficiency causes increased hair follicle fragility. At theultrastructural level WT hair follicle keratinocytes in the outerand inner root sheaths were well organised and had goodintercellular contacts (Fig. 7E). In contrast, cells in the ORS ofα3-integrin-deficient hair follicles were severely ruptured,indicating an increase in cellular fragility, and keratinocytes ofboth the ORS and IRS presented large intra- and intercellularspaces, in which the IRS cells extended numerous filopodialprocesses (Fig. 7F,G). Thus, the severe ultrastructuralabnormalities in the hair follicle keratinocytes most probablycontributes significantly to the stunted and fragile nature of α3-integrin-deficient hair follicles.

F-actin is both disorganised and arranged in prominentbundles in α3-integrin-deficient hair folliclesSince the assembly of actin filaments profoundly affects the

structural organisation of cells within tissues and theiradhesive behaviour, we wondered whether changes in F-actindistribution correlated with the morphological abnormalitiesand cellular fragility observed in adult α3-integrin-deficienthair follicles. Cryosections of adult WT and α3-integrin-deficient skin were stained with rhodamine-conjugatedphalloidin. In both WT and α3-integrin-deficientinterfollicular epidermis, F-actin was distributed in apericellular fashion in all keratinocyte layers with nosignificant difference between genotypes (Fig. 8A,B).However, comparison of F-actin distribution in WT and α3-integrin-deficient hair follicles gave a strikingly differentresult. F-actin was distributed subcortically in WT hair folliclekeratinocytes (Fig. 8C). In contrast, in α3-integrin-deficienthair follicles it appeared either severely disorganised (Fig.8D,E) or formed prominent actin bundles, especially at thebase cells, within ORS and IRS keratinocytes (Fig. 8F). Thesedata establish that in adult skin, α3-integrin deficiencyspecifically affects the organisation of filamentous actin infollicular but not interfollicular epidermal keratinocytes andthat this correlates with hair follicle fragility in α3-integrin-deficient skin.

DiscussionUnderstanding the precise role of integrins in the

Journal of Cell Science 116 (13)

Fig. 7.Severe disorganisationand fragility of outer and innerroot sheath cells in α3-integrin-deficient hair follicles.Electron micrographs of WT(A,C,E) and α3-integrindeficient (B,D,F,G) hairfollicles. (A,B) Distal area ofhair follicle ORS, wherehemidesmosomes are present.In α3-integrin-deficientsamples (B) the ORS cells areruffled, extending finger-likeprojections, and appear to beretracting from the laminadensa compared with WTcontrols (A). (C,D) ORS ofproximal hair follicle, wherehemidesmosomes are notpresent. In α3-integrin-deficient samples multiplelayers of lamina densa wereproduced (D) in contrast to asingle layer of lamina densa inWT controls (C). Anabnormally denseaccumulation of dermalfibroblasts was also evident inα3-integrin-deficient samples(D,F). (E,F) Low powermicrographs of the hair follicleORS and IRS. Note the loss of organisation of the α3-integrin-deficient ORS, with increased cellular fragility and loss of cell-cell junctions inthe inner and outer layers. (G) High-power micrograph of α3-integrin-deficient inner and outer root sheath showing disruption of cell-cellcontact. 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 represents5 µm in A and B, 2 µm in C, 1 µm in D, E, F and G.

2745α3-integrin regulates adult hair morphogenesis

morphogenesis and maintenance of epidermis is of fundamentalimportance. In this paper we describe the phenotype of α3-integrin-deficient adult skin and demonstrate that deficiency ofα3-integrin in skin causes morphological abnormalities whichare, surprisingly, restricted to adult hair follicles. Theseabnormalities 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 resultsreveal functions for α3-integrin in regulating the morphologyof hair follicles that previously were unknown.

α3β1 is important for structural integrity of hair folliclesvia regulation of the cytoskeletonOur observations show that α3-integrin-deficient skin beginsto develop normally and that abnormalities in hair follicle

morphology were not observed in 14-day-old grafts. At thisearly stage hair follicles in both genotypes appeared to be inthe first anagen growth phase. Between 14 and 45 days postgrafting, the WT hair follicles regressed, through catagen andtelogen, and entered the second anagen phase (data not shown).However, in adult α3-integrin-deficient skin, the majority ofhair follicles remained in catagen or telogen, implying that α3deficiency may inhibit entrance into the second anagen phase.Clusters of hair follicles in the α3-integrin-deficient skin mayrepresent multiple unsuccessful attempts of a hair follicle toenter anagen. Furthermore, the general level of reducedproliferation and apoptosis in the α3-integrin-deficient hairfollicles suggests that these follicles may be in an extendedresting phase. These results indicate that α3-integrin plays acrucial role in regulating hair follicle morphology specificallyduring the catagen phase of the hair cycle.

Since α3β1 is one of the most highly expressed epidermalβ1-integrins, one might expect that deficiency in α3- or β1-subunits would have overlapping or similar effects on hairfollicle cycling. The epidermis-specific, β1-integrin-deficientmice have severely compromised survival (Brakebusch et al.,2000; Raghavan et al., 2000) and hair follicles either do notinvaginate properly (Raghavan et al., 2000) or are lost by 16days after birth implying that β1-integrins are essential inmatrix formation (Brakebusch et al., 2000). In short, both α3-and β1-integrins affect hair follicle growth, but they do so atdifferent stages of the hair cycle. In addition, we observed thatin adult α3-integrin-deficient skin grafts, stunted hair folliclesoften appeared to associate with pigment deposits or casts inthe dermis. Such pigment deposits are regarded to reflect anincrease in hair follicle fragility. Together with an increase incell fragility in the ORS and a decrease in hair-specific keratin2 (Hb2) expression, our data imply that α3β1, unlike thecombination of all β1-integrins, is important in maintaining thestructural integrity of hair follicles.

What could cause increased hair follicle fragility in α3-integrin-deficient adult skin? Changes in F-actin organisationwithin cells has been shown to alter both cell-cell and cell-matrix adhesion (Vasioukhin et al., 2000; Vasioukhin et al.,2001). In α3-integrin-deficient hair follicles F-actin was bothseverely disorganised and formed prominent actin bundles.Neither of these patterns of F-actin distribution were found inWT hair follicles. This indicates that, in adult skin, α3-integrinis important in maintaining the correct organisation of the actincytoskeleton in hair follicles and suggests that F-actindisorganisation causes the observed cell fragility. Consistentwith these observations, F-actin is organised into prominentbundles in α3-integrin-deficient newborn pad skin andkeratinocytes (Hodivala-Dilke et al., 1998). The reason forthe apparent recovery of F-actin organisation in adultinterfollicular epidermis is unclear. One explanation may bethat the adult skin grafts are essentially from thin, back skin,and the newborn phenotype was described for thick, pad skinthat developed blisters. Blistering is a type of wound, and ithas been reported previously that keratinocytes in a woundhave bundles of F-actin on their basal face. Furthermore sincenewborn epidermis is less quiescent than adult interfollicularepidermis and no blisters were evident in the adult thin skin,this may explain why the F-actin appears normal in adult thininterfollicular epidermis. Normally, activation of Rho GTPasesleads to changes in the actin cytoskeleton, thus enhancing

Fig. 8.α3-integrin deficient hair follicles have both disorganised andprominent F-actin bundles. Cryosections of WT (A,C) and α3-integrin deficient (B,D,E,F) adult skin grafts were analysed byimmunofluorescence with rhodamine-conjugated phalloidin. In WTand α3-integrin deficient interfollicular epidermis F-actin wasdistributed subcortically in all keratinocyte layers (A,B). A similarpattern of F-actin was observed in WT hair follicles (C). In contrast,in many α3-integrin deficient hair follicles F-actin appeareddisorganised (D,E) or formed heavy bundles especially at the basalface of the cells (F). Inserts in C, D and F show a high magnificationof the selected areas. 11-15 skin grafts per genotype were analysedand the experiment was repeated three times. Over 500 follicles pergenotype were analysed. Bar, 10 µm (A,B); 20 µm in (C,D,E).

2746

integrin clustering and ECM organisation (Wennerberg et al.,1996; Nguyen et al., 2001). One might speculate that in theabsence of α3β1 abnormal activation of Rho results indisorganised F-actin and an increase cell fragility. Takentogether, these symptoms represent a likely cause for both thestunted and clustered abnormal hair follicle growth in α3-integrin-deficient skin.

The role of α3-integrin in follicular versus interfollicularepidermal developmentAs α3β1-integrin is expressed in both IFE keratinocytes andhair follicles, why does its deficiency affect follicular and notinter-follicular epidermal morphogenesis and differentiation?One difference between hair follicles and IFE is that althoughhemidesmosomes (and thus α6β4) are expressed throughoutthe IFE, they are present only in the distal part of hair follicles(Nutbrown and Randall, 1995). Consequently, in adult α3-integrin-deficient skin, the lower part of the hair follicle isdoubly-deficient in both α3β1- and α6β4-integrins. Hence, thiscombined loss of Lm-5 receptors, although not necessarily theonly reason, might contribute to the severe loss of cytoskeletal(both F-actin and hair specific keratin 2) organisation and theextreme cellular fragility of α3-integrin-deficient hair follicles.

Another difference between hair follicles and IFEs is thathair follicles undergo cycles of massive proliferation andapoptosis, corresponding to the anagen and catagen phases ofthe hair cycle, respectively. This is unlike the relatively lowrates of proliferation and apoptosis in adult quiescent IFEs.Since β1-integrins have been implicated in the control ofproliferation and apoptosis (Gonzales et al., 1999; DiPersio etal., 2000; De Arcangelis and Georges-Labouesse, 2000; Frischand Screaton, 2001), it is perhaps not surprising that α3deficiency alters the proliferative and apoptotic potential ofkeratinocytes predominantly in hair follicles. These changes,in turn, probably contribute to the increase in telogen phase andmorphological abnormalities observed in α3-integrin-deficienthair follicles.

α3-integrin is required for correct maintenance of thebasement membrane in adult skinBasement membrane disruption is associated with dermal-epidermal blistering in α3-integrin-deficient newborn pads(DiPersio et al., 1997; Hodivala-Dilke et al., 1998), and,importantly, the blistering was confined to hairless areas ofthick skin. Interestingly, in adult α3-integrin-deficient skin, thelack of basement membrane integrity in the IFE does not causeskin blistering. How could this be explained? One possibilityis that in α3-integrin-deficient skin grafts the presence of hairprovides the skin with increased mechanical resistance, therebypreventing skin blisters. In addition, because blistering is oftena skin-site-associated problem, our results may simply indicatethat α3-integrin deficiency in thick skin causes blistering,whereas in thin skin it does not. Although the follicularbasal lamina was disrupted in ultrastructural experiments,immunohistochemistry revealed that entactin and Lm-5appeared intact, indicating that the basement membrane is lessseverely disorganised in follicular than in interfollicularepidermis. One reason for this may be that cycling of hairfollicles induces a constant regeneration of the surrounding

basement membrane, thereby preventing the accumulation ofdisorganised ECM proteins around hair follicles and thusprotecting the skin from sheer stress effects.

A possible role for α3-integrin in maintenance of theepidermal stem cell compartment?Epidermal stem cells express high levels of α2β1- and α3β1-integrins (Jones and Watt, 1993; Jones et al., 1995; Bagutti etal., 1996; Moles and Watt, 1997; Jensen et al., 1999; Zhu etal., 1999; Watt, 2001). An important question is whether theseintegrins are simply markers of stem cells or whether theyare vital for the functional maintenance of the stem cellcompartment in vivo (Levy et al., 2000; Raghavan et al., 2000).Previous studies using mice either totally deficient in α3-integrin (Kreidberg et al., 1996; DiPersio et al., 1997) orepidermis-specific β1-integrin-deficient mice (Raghavan et al.,2000; Brakebusch et al., 2000) have demonstrated that theepidermis can develop in the absence of these integrins, at leastuntil birth. In the β1 mutant mice, hair follicles disappear afterthe first hair cycle, implying that β1-integrin might be involvedin the maintenance of an epithelial stem cell compartment ormight play a role in the activation of stem cells at the onset ofanagen (Brakebusch et al., 2000). However, immune cellinfiltration and hair follicle breakdown at this stage make itdifficult to state categorically whether hair follicles are notregenerated because of an intrinsic stem cell defect and/orbecause an immune response has eradicated all follicular stemcells. The approach that we have used overcame the majorityof these immune-response problems since α3-integrin-deficient skin was grafted onto immunocompromised mice.Our observations, that adult α3-deficient skin can develop fullyand form hair follicles and sebaceous glands indicate that α3β1probably is not essential for the maintenance of the stem cellcompartment.

In conclusion, we present strong evidence that adult α3-integrin-deficient skin can develop fully but, surprisingly, ithas hair follicle and not interfollicular abnormalities. Theseabnormalities are probably caused by a combination ofcytoskeletal aberrations, loss of basement membrane integrityand a resultant increase in cell fragility. Taken together our dataestablish that α3β1-integrin is important in maintaining hairfollicle architecture during the hair cycle and provides the firstevidence for the role of a single α-subunit in regulating adulthair follicle morphology.

We thank Lutz Langbein and Peter Marinkovich for kindlyproviding us with antibodies. We are grateful to Barbara Cross, ToniHill, Jessica Gruninger, Garry Saunders, Sue Watling, Colin Wren andto the staff of the Cancer Research UK Histopathology andMicroscopy Units for their expert technical assistance. We are gratefulto Richard Hynes, Ed Yoo and Erika for their support and help in theearly pilot experiments. We also wish to thank Louise Reynolds,Stephen Robinson, Catherin Niemann, Simon Broad, Ian Hart, CliveDickson and Fiona Watt for their help in this study and for criticalrevision of this manuscript.

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