INTRODUCTION

The stratified epidermis of the skin is a continuouslyregenerating tissue in which homeostasis is achieved througha balance between keratinocyte proliferation in the basal celllayer and loss of differentiated cells from the outer skinsurface. Keratinocytes of the basal layer are anchored tothe basement membrane, a specialized extracellular matrix(ECM) rich in laminins and collagens that separates the

epidermis from the underlying dermis (reviewed by Burgesonand Christiano, 1997). As non-proliferative keratinocytes inthe basal layer undergo terminal differentiation, they detachfrom the basement membrane, migrate upwards through thesuprabasal layers of the epidermis, and are eventuallysloughed from the outer skin layer as dead squames. Loss ofcells from the skin surface is balanced by the proliferation ofa stem cell population in the basal layer (Watt, 1989; Jones,1997).

3051Journal of Cell Science 113, 3051-3062 (2000)Printed in Great Britain © The Company of Biologists Limited 2000JCS1607

Continuous regeneration and homeostasis of the stratifiedepidermis requires coordinated regulation of cellproliferation, cell differentiation, and cell survival.Integrin-mediated cell adhesion to the extracellular matrixhas important roles in regulating each of these processes.Integrins α3β1 and α6β4 are both receptors on epidermalkeratinocytes for the basement membrane protein laminin-5, the major ligand for epidermal adhesion in mature skin.Ablation in mice of either α3β1 or α6β4, through nullmutation of the gene encoding the α3, α6, or β4 integrinsubunit, results in epidermal blistering of varying severity.Our previous studies showed that, despite blistering,differentiation and stratification of the epidermis appearedessentially normal in mice that lacked either α3β1 or α6β4.However, these studies did not definitively address thespecific developmental importance of each integrin, sincethey may have overlapping and/or compensatory functions.Given the individual importance of α3β1 or α6β4 inmaintaining the dermo-epidermal junction in matureskin, we sought to determine the importance of theseintegrins for embryonic skin development and epidermalmorphogenesis. In the current study, we analyzed skindevelopment in mutant embryos that completely lack both

integrins α3β1 and α6β4. Although α3β1/α6β4-deficientembryos displayed epidermal blistering by stage E15.5 ofdevelopment, they also retained regions of extensiveepidermal adhesion to the basement membrane throughstage E16.5, indicating alternative adhesion mechanisms.Apoptosis was induced in detached epidermis ofα3β1/α6β4-deficient embryos, exemplifying vividly theimportance of epithelial attachment to the basementmembrane for cell survival. However, apoptotic cells werecompletely absent from attached epidermis of α3β1/α6β4-deficient embryos, showing that epithelial adhesion thatoccurred independently of α3β1 and α6β4 also protectedcells from apoptosis. Remarkably, in the absence of theknown laminin-5 binding integrins (α3β1, α6β4, and α6β1),keratinocytes retained the capacity to proliferate inthe epidermis, and epidermal stratification and skinmorphogenesis appeared normal prior to blister formation.These findings show that while α3β1 and α6β4 are bothrequired for integrity of the dermo-epidermal junction,neither one is essential for epidermal morphogenesisduring skin development.

Key words: Integrin, Epidermis, Extracellular matrix

SUMMARY

α3β1 and α6β4 integrin receptors for laminin-5 are not essential for epidermal

morphogenesis and homeostasis during skin development

C. Michael DiPersio 1,2,*, Ronald van der Neut 3,‡, Elisabeth Georges-Labouesse 4, Jordan A. Kreidberg 5,Arnoud Sonnenberg 3 and Richard O. Hynes 1

1Howard Hughes Medical Institute, Center for Cancer Research and Department of Biology, Massachusetts Institute ofTechnology, Cambridge, Massachusetts 02139, USA2Center for Cell Biology and Cancer Research, Albany Medical College, Albany, New York 122083Division of Cell Biology, Netherlands Cancer Insititute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands4Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, BP 163, 67404 Illkirch, C. U. de Strasbourg,France5Department of Medicine, Children’s Hospital and Department of Pediatrics, Harvard Medical School, Boston, Massachusetts02115, USA*Author for correspondence at present address: Center for Cell Biology and Cancer Research, Albany Medical College, Mail Code 165, 47 New Scotland Avenue,Albany, NY 12208-3479, USA (e-mail: dipersm@mail.amc.edu)‡Present address: Department of Pathology, Academic Medical Centre, Meibergdreef 9, 1105 AZ, Amsterdam, The Netherlands

Accepted 9 June; published on WWW 9 August 2000

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Integrins are transmembrane, heterodimeric receptorsconsisting of an α and a β subunit that mediate cell adhesionto the ECM (Hynes, 1992). Integrin-mediated cell adhesionto the ECM is important in the regulation of keratinocytedifferentiation and epidermal stratification (Adams and Watt,1990). In normal epidermis, integrin expression is confinedto the basal keratinocytes in contact with the basementmembrane. Laminin-5 is the major adhesive ligand forintegrins in the mature, cutaneous basement membrane(Carter et al., 1991; Rousselle et al., 1991). Like all laminins,laminin-5 consists of three disulfide linked polypeptidechains; the specific chain composition of laminin-5 is α3, β3,γ2 (Burgeson et al., 1994). The importance of laminin-5-based adhesion at the dermo-epidermal junction isexemplified in the human blistering disease JunctionalEpidermolysis Bullosa (JEB), variants of which have beenassociated with mutations in the genes encoding each of theα3, β3, and γ2chains (Pulkkinen et al., 1994a,b; Kivirikko etal., 1995).

Adhesion of basal keratinocytes to laminin-5 is dependenton two integrins, α6β4 and α3β1, which are recruited todistinct adhesion sites within keratinocytes and regulatedifferent aspects of cell adhesion. α6β4 is a component ofhemidesmosomes, adhesion structures linking the ECM tokeratin intermediate filaments (Stepp et al., 1990; Sonnenberget al., 1991; Jones et al., 1991; Borradori and Sonnenberg,1999). In contrast, α3β1 links the ECM to the actincytoskeleton and is required for keratinocyte spreading andmigration on laminin-5 (Carter et al., 1990; Xia et al., 1996).These distinctions are reflected in the different skin phenotypesof mice homozygous for null mutations in the genes encodingα3, α6, or β4 integrin subunits. For example, α3β1 wasrequired for proper organization of the actin cytoskeleton(Hodivala-Dilke et al., 1998) and the epidermal basementmembrane (DiPersio et al., 1997), while α6β4 was required forhemidesmosome formation and stable adhesion at the dermo-epidermal junction (Georges-Labouesse et al., 1996; van derNeut et al., 1996; Dowling et al., 1996).

Mice that are homozygous for null mutations in the genesencoding laminin-5 (Kuster et al., 1997; Ryan et al., 1999), orits integrin receptors α3β1 or α6β4 (van der Neut et al., 1996;Georges-Labouesse et al., 1996; Dowling et al., 1996;Kreidberg et al., 1996; DiPersio et al., 1997), die shortly afterbirth with detachment of the epidermis from the dermis.Nevertheless, there are intriguing discrepancies in themechanisms and timing of blister formation between thedifferent mutants that raise interesting questions regardingcompensatory adhesion mechanisms. For example, laminin-5-deficient mice and α6β4-deficient mice all displayed extensiveepidermal detachment (van der Neut et al., 1996; Georges-Labouesse et al., 1996; Dowling et al., 1996; Kuster et al.,1997; Ryan et al., 1999). However, laminin-5-deficientneonatal mice (α3 chain null mutation) survived for up to threedays, and blister formation was delayed until after birth (Ryanet al., 1999). In contrast, α6β4-deficient mice died within hoursof birth and developed blisters during embryonic development(van der Neut et al., 1996, and this report). This discrepancyindicates a compensatory adhesion mechanism that is presentin laminin-5-deficient skin (i.e. see Ryan et al., 1999) but notin α6β4-deficient skin. In α3β1-deficient mice, which developrelatively small blisters caused by splitting of the basement

membrane, epidermal attachment to laminin-5 is maintainedthrough α6β4 (DiPersio et al., 1997).

Epidermal differentiation and stratification appeared largelynormal prior to blister formation in mice lacking α3β1, α6β4,or laminin-5, and only subtle defects in keratinocytedifferentiation were reported in α6β4-deficient and laminin-5-deficient mice (Dowling et al., 1996; Ryan et al., 1999).However, as mentioned above, compensatory adhesionmechanisms in these mutants may mask importantdevelopmental roles for laminin-5-binding integrins. Indeed, inaddition to overlapping laminin-5-binding functions (Carter etal., 1991; Xia et al., 1996), α3β1 and α6β4 may haveoverlapping signaling functions that regulate proliferation ofcultured epithelial cells (Gonzales et al., 1999). However,overlapping or compensatory function between these integrinshas not been studied directly in the context of developing skin.To address this important question, we produced embryos thatare deficient in all known laminin-5 receptors by interbreedingmice that harbor null mutations in the genes encoding the α3,β4 or α6 integrin subunits. Embryos that were deficient forboth α3β1 and α6β4 formed epidermal blisters in utero thatwere similar in severity to those seen in embryos lacking onlyα6β4, showing that α6β4 is required to maintain full adhesionat the dermo-epidermal junction during skin development.Apoptosis of basal keratinocytes was detected in detachedepidermis of α6β4-deficient or α3β1/α6β4-deficient embryos,but not in attached epidermis, demonstrating that adhesion-dependent cell survival did not require α3β1 or α6β4 . Despiteeventual epidermal detachment, skin development appearedto progress normally in α3β1/α6β4-deficient mutants. Inaddition, assessment of keratinocyte proliferation andepidermal stratification revealed no obvious alterations in skinhomeostasis in mutant embryos that lacked α3β1, α6β4, orboth integrins. Similar phenotypes were seen in α3/α6-deficient mice, confirming that the α6β1 laminin-5 receptordid not compensate in α3/β4-deficient embryos. This studyestablishes that keratinocyte adhesion to laminin-5 throughα3β1 and α6β4 is not essential for skin morphogenesis duringembryonic development, and that other adhesion mechanismsare sufficient to support epidermal development.

MATERIALS AND METHODS

AntibodiesRabbit polyclonal antisera against entactin and laminin-5 (α3 or γ2chain) were kindly provided by Dr A. Chung (University ofPittsburgh, Pittsburgh, PA) and Dr G. Meneguzzi (Faculté deMédecine, Nice, France), respectively. Rabbit antiserum against Ki67was purchased from Novacastra (United Kingdom). Rabbit antiserumagainst the α6 integrin subunit was raised against a GST-fusionprotein containing a fragment of the α6 extracellular domain, asdescribed (Sterk et al., 2000).

Generation of mice and genotyping by PCRThe generation of individual null mutations in the genes encoding α3,β4, or α6 integrin subunits was described previously (Kreidberg et al.,1996; van der Neut et al., 1996; Georges-Labouesse et al., 1996). Miceheterozygous for the α3 integrin null mutation (α3+/−) were crossedwith mice heterozygous for the β4 null mutation (β4+/−) to generatemice that were heterozygous for both mutations (α3+/−;β4+/−).Double heterozygotes were then mated to generate embryos that werehomozygous for null mutations in the genes encoding both the α3

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3053Skin morphogenesis in α3β1/α6β4-null mice

subunit and the β4 subunit (α3−/−;β4−/−). Embryos were collectedby Caesarean section, and genotypes for each gene were determinedseparately by PCR amplification of DNA from yolk sac or tissuesamples. PCR conditions and primers have been describedpreviously for α3 (DiPersio et al., 1997) and β4 (van der Neut et al.,1996). Embryos that were homozygous for null mutations in thegenes encoding both the α3 and α6 subunits (α3−/−;α6−/−) weregenerated and identified as described previously (De Arcangelis etal., 1999).

Preparation of paraffin sections and immunofluorescenceEmbryos were fixed in 4% formaldehyde overnight before embeddingin paraffin. Tissue sections (4 µm) were dewaxed in xylene andrehydrated, then either stained with hematoxylin and eosin or preparedfor immunofluorescence. For staining with anti-entactin or anti-laminin, sections were pre-treated with 0.1% trypsin in PBS for 30minutes. Trypsin was inactivated with 50 µg/ml soybean trypsininhibitor for 10 minutes and sections were washed with PBS. Forstaining with anti-Ki67 or anti-α6 integrin, sections were microwavedat 90°C for 2× 10 minutes in 0.01 M citrate buffer (pH 6.0), thencooled to room temperature and washed in PBS. All sections wereblocked in 10% normal goat serum, 0.05% Tween-20 in PBS for atleast 30 minutes, then incubated overnight at 37°C with anti-entactin(1:500 dilution), anti-Ki67 (1:500 dilution), anti-α6 (1:100 dilution),or a mixture of antisera against the α3 and γ2 chains of laminin-5(1:800 dilution for each). Sections were then washed in PBS/0.05%Tween-20, blocked for 30 minutes, and incubated with FITC-conjugated goat anti-rabbit secondary antibody (Pierce, Rockford, IL)at a 1:50 dilution for 2 hours at 37°C. Representative fields werephotographed at ×40 magnification using an Olympus BX60microscope and Spot digital camera (Diagnostic Instruments, Inc.).

Analysis of apoptosis in skinApoptosis was evaluated in paraffin-embedded skin sections byTUNEL assay using an Apoptosis Detection System (Promega,Madison, WI), which measures the fragmented DNA of apoptoticnuclei by incorporating fluorescein-12-dUTP at the 3′-OH DNA endsusing terminal deoxynucleotidyl transferase (TdT). Prior to TUNELassay, paraffin sections were dewaxed in xylene, rehydrated, and fixedin 4% methanol-free formaldehyde/PBS for 15 minutes. Fixedsections were then treated with 20 µg/ml proteinase K for 12 minutes,washed in PBS, and fixed again in 4% methanol-freeformaldehyde/PBS for 5 minutes. Pretreated sections were assayed forapoptosis according to the manufacturer’s instructions, and werecounter-stained with propidium iodide to stain both apoptotic andnon-apoptotic nuclei. Representative fields were photographed asdescribed above.

RESULTS

Despite extensive blistering, epidermalmorphogenesis progresses normally in mice lackingboth integrins α3β1 and α6β4Mice heterozygous for the integrin α3-null mutation (α3+/−mice) were crossed with mice heterozygous for the β4-nullmutation (β4+/− mice) to produce double heterozygotes(α3+/−; β4+/−), which were normal and fertile. Afterintercrossing the α3+/−;β4+/− mice, we did not recoverneonates that were homozygous for both mutations(α3−/−;β4−/−) from 55 newborn mice distributed over fivelitters (3 or 4 double mutants are expected). Since it wasnecessary to collect and screen a considerable number ofanimals for our study (only 1/16 are α3−/−;β4−/−), we focusedour efforts on collecting embryos from E15.5-E16.5, at which

stage laminin-5 is prominent in the developing epidermis.Embryos homozygous for the α3-null mutation (α3−/−) or theβ4-null mutation (β4−/−) proceeded through development inthe expected Mendelian ratios (Kreidberg et al., 1996; van derNeut et al., 1996). Embryos homozygous for both mutations(α3−/−;β4−/−) were detected at stages E12.5, E14.5, and E16.5at a combined frequency (3.7%) that was slightly lower thanthe expected Mendelian ratio (6.25%). At E16.5 of mousedevelopment, three to four days prior to birth, most of theepidermis in normal skin is well stratified (Fig. 1A), and thebasal keratinocytes are attached to a basement membrane thatis rich in laminin-5 (Aberdam et al., 1994; DiPersio et al.,1997). By E16.5, skin sections from α3+/+;β4−/− embryosshowed extensive epidermal detachment (Fig. 1B), asdescribed previously for β4−/− neonates (van der Neut et al.,1996). A comparable extent of epidermal detachment was seenin α3−/−;β4−/− embryos (Fig. 1C), suggesting that the absenceof α3β1 did not augment epidermal detachment caused by theabsence of α6β4. We previously showed that small blisterswere present in α3−/− mice after birth, but were not detectedin embryos (DiPersio et al., 1997). Similarly, the skin ofα3−/−;β4+/− embryos looked like wild-type skin anddisplayed no blistering (Fig. 1D), showing that a singlefunctional copy of the β4 gene produces sufficient α6β4 tomaintain complete dermo-epidermal adhesion in the absenceof α3β1. Importantly, despite extensive blister formation inα3−/−;β4−/− skin, the gross development and stratification ofthe epidermis appeared normal.

The laminin receptor α6β1 does not compensate forepidermal adhesion in mice lacking integrins α3β1and α6β4Despite the epidermal detachment shown above, α3−/−;β4−/−embryos at stage E16.5 did maintain long stretches of dermo-epidermal attachment, demonstrating that some adhesion ismaintained at this stage independently of integrins α3β1 andα6β4. Since the latter two integrins are the only knownreceptors for laminin-5 in normal skin, this observationsuggested an additional mechanism of adhesion that does notrequire cell binding to laminin-5. Alternatively, integrin α6β1,a potential laminin-5 receptor (Delwel et al., 1994), may beupregulated in α3−/−;β4−/− embryos. Although keratinocytesexpress both α6 and β1 integrin subunits, they do not normallyexpress α6β1 on the cell surface, presumably due to the higheraffinity of the α6 subunit for the β4 subunit (Hemler et al.,1989). In addition, β1 may be associated with other α subunitsand unavailable for interactions with α6, since α6β1 was notdetected in β4-null skin (van der Neut et al., 1996; Dowling etal., 1996). Importantly, immunoblot analysis showed that theabsence of α3 synthesis in α3-null keratinocytes results in anincrease in the intracellular pool of β1 subunit (Hodivala-Dilkeet al., 1998, and data not shown). Presumably, the absence ofβ4 synthesis in β4-null skin results in a similar surplus ofintracellular α6. It therefore seemed possible that the combinedsurplus of β1 subunit and α6 subunit in α3−/−;β4−/− skinwould result in the expression of α6β1.

To assay α6 integrin expression in epidermis of α3−/−;β4−/− embryos, skin sections were stained withantibodies against the α6 integrin subunit. Although it wasdifficult to obtain consistent staining of paraffin sections usingseveral different antibodies against the α6 subunit, a polyclonal

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serum against the α6 extracellular domain produced amoderate, albeit patchy signal for α6 expression in the basalkeratinocytes of normal E16.5 skin. In some regions, α6 waslocalized to the basal aspects of the basal keratinocytes,reflecting the recruitment of α6β4 into hemidesmosomes (Fig.2A). We were unable to detect a signal for α6 in basalkeratinocytes of β4−/− skin (Fig. 2B), consistent with previousreports (van der Neut et al., 1996; Dowling et al., 1996). Incontrast, patches of basal keratinocytesin α3−/−;β4−/− epidermis showed apericellular staining pattern for α6 inboth non-blistered and blisteredregions (Fig. 2C and D, respectively),indicating expression of α6β1 in thesecells. Thus, it was conceivable thatupregulation of α6β1 in α3−/−;β4−/−skin maintained residual epidermalattachment to laminin-5 or otherlaminins.

To assess skin development inembryos that lack α3β1 and α6β4, andalso lack the potential to upregulateα6β1, we examined E15.5 embryosfrom intercrosses of α3+/−;α6+/− mice(De Arcangelis et al., 1999). Dermo-epidermal adhesion appears to switchto a laminin-5-based adhesion systemaround stage E15.5 to E16.5 ofdevelopment (Aberdam et al., 1994;DiPersio et al., 1997). α3−/− micedisplayed normal epidermal attachmentat these stages (Fig. 3A; DiPersio et al.,1997), showing that α3β1 was notrequired for epidermal attachment. Incontrast, β4−/− E15.5 embryosdisplayed extensive blistering (Fig.

3B), showing that α6β4 was required for full epidermalattachment by this stage. Some blisters in the skin of β4−/−embryos were filled with blood (Fig. 3B), indicating that theyformed in utero. Nevertheless, some epidermal attachment wasmaintained in β4−/− embryos at E15.5-E16.5 (Fig. 1B, and notshown). α3−/−;α6−/− E15.5 embryos displayed blistering thatwas comparable in severity to β4−/− or α3−/−;β4−/− embryos(compare Fig. 3D with Fig. 3B,C), and they also maintained

C. M. DiPersio and others

Fig. 1.Comparison of thedermo-epidermal junction inwild-type and mutant E16.5embryos. Paraffin sections wereprepared from wild-typeembryos (A) or embryoshomozygous for null mutationsin the α3 and/or β4 genes (B-D)and stained with hematoxylinand eosin. The genotypes areindicated for each embryo.Arrowheads point to the dermo-epidermal junction. Double-headed arrows indicate a split atthis junction in B and C. Theblister in C contains blood cells.Bar, 50 µm.

Fig. 2. Expression of the α6 integrin subunit in wild-type and mutant E16.5 embryos. Sectionsfrom wild-type embryos (A), β4−/− embryos (B), or α3−/−;β4−/− embryos (C,D) were stainedby immunofluorescence with a polyclonal antibody against the α6 subunit. Homozygousity fornull mutations is indicated for each embryo; wt, wild type. Arrows point to basal keratinocytesof the epidermis. Double-headed arrow indicates a blister in D. Bar, 50 µm.

3055Skin morphogenesis in α3β1/α6β4-null mice

extensive regions of epidermal attachment (Fig. 3D,arrowhead). These results suggest that α6β1 did notcompensate for epidermal adhesion in α3−/−;β4−/− embryosand confirm that some epidermal attachment was maintainedindependently of known integrin receptors for laminin-5.

Embryos deficient either for α6β4 only or for α3β1and α6β4 display similar mechanisms of epidermalblister formationSkin blisters that form in α3β1-deficient mice appear to resultfrom a split within the basement membrane, since theycontained laminin-5, entactin, and other basement membraneproteins on both the epidermal and dermal sides of the blisters(DiPersio et al., 1997). In contrast, blisters of α6β4-deficientmice result from detachment of the epidermis from thebasement membrane, and laminin-5 was detected only at thedermal sides of blisters in α6-null or β4-null mice (van derNeut et al., 1996; Dowling et al., 1996; Georges-Labouesse etal., 1996). To compare basement membrane distribution withinblisters of the doubly deficient mutants, we stained skinsections with antibodies against entactin. Entactin serves as auseful marker of basement membrane distribution during skindevelopment (DiPersio et al., 1997), and anti-entactin stronglystained the basement membrane of normal skin at stages E16.5and E15.5 (Fig. 4A and E, respectively). Entactin stainingrevealed very limited regions of disorganized basementmembrane in α3-null skin (Fig. 4B, arrow). Basementmembrane disorganization in α3-null skin is not detected easilyuntil later than E17.5 of development (DiPersio et al., 1997),and ECM disorganization was not extensive in α3-null skin atE16.5 (Fig. 4B) or E15.5 (Fig. 4F). As expected, entactin wasdetected only at the dermal sides of blisters in β4−/− embryosat both E16.5 and E15.5 (Fig. 4C and G, respectively).Similarly, entactin was detected only at the dermal sides ofblisters in both α3−/−;β4−/− embryos (Fig. 4D) andα3−/−;α6−/− embryos (Fig. 4H). Therefore, epidermal blistersin skin lacking both α3β1 and α6β4 are similar to those in skinlacking only α6β4, which result from the loss of epidermalanchorage through hemidesmosomes (van der Neut et al.,

1996; Dowling et al., 1996; Georges-Labouesse et al., 1996).This blistering mechanism is clearly dominant over that ofbasement membrane rupture seen in α3β1-deficient mice,which does not manifest itself until after birth (DiPersio et al.,1997). Laminin-5 begins to appear in the basement membraneof stratified skin by E15.5 (Fig. 4I, arrowheads; DiPersio et al.,1997) and is present in the cutaneous basement membranes ofα3β1-deficient and α6β4-deficient mice (van der Neut et al.,1996; Dowling et al., 1996; Georges-Labouesse et al., 1996;DiPersio et al., 1997). Similarly, we were able to detectlaminin-5 in the basement membrane of α3−/−;α6−/− E15.5embryos (Fig. 4J, arrowheads), indicating that none of theknown laminin-5 receptors (α3β1, α6β1, and α6β4) arerequired for deposition of laminin-5 during skin development.

Adhesion-dependent survival of basal epidermalkeratinocytes does not require integrins α3β1 andα6β4Integrin-mediated adhesion of epithelial cells to theirunderlying basement membranes promotes cell survival byinhibiting apoptosis (Frisch and Francis, 1994; Boudreau et al.,1995; Pullan et al., 1996). To determine whether α3β1- and/orα6β4-mediated adhesion is required to maintain survival ofepidermal keratinocytes, we performed TUNEL assays onsections of developing skin from normal and mutant embryos.At E16.5, we did not detect apoptotic cells in epidermis ofeither wild type (Fig. 5A) or α3−/− embryos (Fig. 5B),indicating that the absence of α3β1 did not promote apoptosis.In β4−/− skin, apoptotic basal keratinocytes were detectedfrequently in epidermis that had detached from the basementmembrane (Fig. 5C, arrows), but were not detected in non-blistered epidermis (not shown), indicating that loss ofepidermal attachment to the basement membrane inducedapoptosis. Apoptotic keratinocytes were similarly abundant inblistered epidermis of α3−/−;β4−/− skin (Fig. 5D, arrows);however, we found no evidence of apoptosis in attachedepidermis of the α3−/−;β4−/− mutants (Fig. 5E). These resultsdemonstrate that while epidermal attachment to the basementmembrane protects keratinocytes from apoptosis, survival of

Fig. 3. Integrin α6β1 does notcompensate for epidermalattachment or morphogenesis inskin lacking both α3β1 andα6β4. Paraffin sections wereprepared from embryos that werehomozygous for the α3-nullmutation (A), the β4-nullmutation (B), the α3-null andβ4-null mutations (C), or the α3-null and α6-null mutations (D)and stained with hematoxylinand eosin. Embryos were fromE15.5 (A,B,D) or E16.5 (C).Arrowheads point to regions ofintact dermo-epidermal adhesionin A and D. Double-headedarrows indicate a split at thisjunction in B-D. The blister in Bis blood-filled. Bar, 50 µm.

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attached cells does not require the laminin receptors α3β1 andα6β4.

It is worth noting that the frequent presence of apoptoticcells in blisters of β4−/− or α3−/−;β4−/− embryos indicatesthat at least some of the blisters formed in utero, rather than asa result of embryo harvest and tissue preparation. This isconsistent with the detection of blood filled blisters in mutantembryos (see Figs 1C and 3B). Interestingly, in contrast withstage E16.5 embryos, we did not detect apoptotic cells overblistered regions of either β4−/− or α3−/−;α6−/− embryos atstage E15.5 (Fig. 5F,G). Our interpretation of this difference isthat blisters at E15.5 are too new to contain apoptotic cells.Indeed, we may expect that blisters of E15.5 embryos arerecently formed, since deposition of laminin-5 into thebasement membrane and, presumably, laminin-5 basedadhesion are established near this stage of development(Aberdam et al., 1994; DiPersio et al., 1997). The observation

that embryonic blistering precedes apoptosis is consistent withthe notion that blistering is the cause of apoptosis, rather thana result of increased apoptosis.

Keratinocytes lacking α3β1 and α6β4 retain thecapacity to proliferate in the developing epidermisStratification and maturation of the epidermis in the developingmouse embryo occurs over a period of about 5-6 days,beginning at about E12 (summarized in Fig. 7A; for a review,see Turksen and Troy, 1998). At E8-E12, the epidermisconsists of a single layer of proliferating cells, the stratumgerminatum, that is in contact with a basement membrane andis overlaid by an outer cell layer, the periderm. At E12-E14,the stratum germinatum gives rise to the stratum intermedium,a suprabasal layer that lies between the basal layer and theperiderm. Cells in the stratum intermedium retain the ability toproliferate at these early stages. At around E15, further

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Fig. 4.The basementmembrane remains at thedermal sides of blisters in allmutants that lack α6β4.Sections from normalembryos (A,E,I) or embryoshomozygous for nullmutations in the α3, β4,and/or α6 genes (B-D,F-H,J)were stained byimmunofluorescence withantisera against the basementmembrane proteins entactin(ent., A-H) or laminin-5(LM-5, I,J). Embryos werefrom E16.5 (A-D) or E15.5(E-J), as indicated.Homozygous null mutationsare indicated for each embryo;normal embryos are eitherwild-type or heterozygous formutations. Arrowheads pointto the basement membrane.Double-headed arrowsindicate blisters in C, D, G, H,and J. The arrow in B pointsto a region of slightlydisorganized basementmembrane. Bar, 50 µm.

3057Skin morphogenesis in α3β1/α6β4-null mice

stratification of the stratum intermedium marks the beginningof differentiation into mature epidermis. By E17-E18, just priorto birth, the mature epidermis has shed the periderm and thestratum intermedium has differentiated into the granular andcornified layers. By the time the epidermis has matured,proliferating keratinocytes are restricted mainly to the basaland immediately suprabasal cell layers (Fuchs, 1993; Murgiaet al., 1998).

As shown in the preceding figures, histological analysisrevealed that the degree of epidermalstratification was comparable in normal andα3β1/α6β4-deficient embryos, at least untilE16.5. Nevertheless, since maturation of theepidermis between E15 and E17/E18correlates well with accumulation of laminin-5 in the cutaneous basement membrane(Aberdam et al., 1994; DiPersio et al., 1997),we were interested in determining whetherthis correlation reflects a role for laminin-5-binding integrins in regulating epidermal cellproliferation. Indeed, α3β1 and α6β4 haveeach been implicated in signal transductionevents that regulate proliferation ofkeratinocytes and other epithelial cells in vitroand in vivo (Mainiero et al., 1997; Murgia etal., 1998; Gonzales et al., 1999). To identifyproliferating cells in epidermis that lackedα3β1 and/or α6β4 integrins, we stained skinsections by immunofluorescence with anantibody against the nuclear antigen Ki67, awidely used marker for cell proliferation. AtE14.5 (not shown) and E15.5 (Fig. 6A,C,E,G),each normal or mutant embryo examinedcontained proliferating keratinocytes in bothbasal and suprabasal epidermal layers. ByE16.5, proliferating keratinocytes in normalepidermis were, for the most part, restricted tothe basal and immediately suprabasal layers(Fig. 6B). Similarly, in E16.5 epidermis ofα3−/− embryos (Fig. 6D), and in non-blistered(Fig. 6F) or blistered (not shown) epidermis ofβ4−/− embryos, Ki67-positive cells weredetected mainly in the basal layers. Finally,the distribution of Ki67-positive cells in E16.5epidermis of α3−/−;β4−/− embryos (Fig. 6H)was similar to that seen in normal epidermis

(Fig. 6B). Dowling et al. (1996) previously reported anincrease in mitotically active suprabasal cells in β4-nullneonatal skin, identified by ultrastructural morphology.Although we occasionally detected Ki67-positive suprabasalcells at stage E16.5, we did not observe any consistentdifferences between normal and mutant embryos. However, itis possible that the number of proliferating suprabasal cells inβ4−/− mutants increases at later developmental stages.

We calculated the number of Ki67-positive cells per linear

Fig. 5. Epidermal apoptosis is restricted toblistered regions of α6β4-deficient or α3β1/α6β4-deficient skin. Sections were prepared from E16.5(A-E) or E15.5 (F,G) embryos and assayed byTUNEL for apoptotic cells (TUNEL, left column),or counter-stained with propidium iodide to stainall nuclei (PI, right column). Sections were fromwild-type embryos (A) or mutant embryos (B-G)homozygous for null mutations, as indicated.Arrows point to basal keratinocytes; brightTUNEL staining of apoptotic keratinocytes occursonly over blistered regions of E16.5 skin (C,D).Double-headed arrows indicate blisters in C, D, F,and G. Bar, 50 µm.

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centimeter of epidermis for normal and double mutant embryos(only non-blistered regions of mutant embryos wereexamined). Comparison of E16.5 epidermal sections from twonormal embryos and from two α3−/−;β4−/− mutant embryosrevealed similar numbers of Ki67-positive cells (35±2.8cells/cm and 37±1.4 cells/cm, respectively). Analyses of aα3−/− and a β4−/− mutant revealed similar results (33 cells/cmand 27 cells/cm, respectively). Although we detected roughlytwice as many proliferating cells in sections of E15.5epidermis, only slightly fewer Ki67-positive cells weredetected in α3−/−;α6−/− epidermis (60±13 cells/cm) than innormal epidermis (70±6.4 cells/cm). In a recent study byMurgia et al. (1998), a targeted deletion of the β4 cytoplasmicdomain that resulted in expression of a functionally impairedα6β4 integrin caused a 2-fold decrease in the number of Ki67-positive cells in the epidermis of E18.5 embryos. We detectedconsiderably fewer Ki67-positive cells per unit length ofepidermis than did Murgia and coworkers, possibly due to thefact that different embryonic stages were assayed in each study.In contrast to Murgia et al. we did not detect a 2-fold decreasein keratinocyte proliferation in our mutant embryos. Onepossible explanation for this discrepancy is that α6β4 may berequired for optimal proliferation atsome point after stage E16.5.Alternatively, expression of the α6β4mutant in the study by Murgia et al.may have altered cell adhesion andintracellular signaling in a way thatinhibited proliferation, while thecomplete absence of α6β4 in our studydid not have the same inhibitory effect.In any case, our results show clearlythat α3β1 and α6β4 are not requiredin the developing epidermis forkeratinocyte proliferation per se, orto support essentially normal skinmorphogenesis.

DISCUSSION

Integrins α3β1 and α6β4 are two ofthe most abundant integrins expressedby basal keratinocytes of normalepidermis, and are the only knownreceptors on these cells for adhesion tolaminin-5. Although there has been areport that integrin α2β1 contributed tothe adhesion of colonic cancer cells tolaminin-5 (Orian-Rousseau et al.,1998), adhesion of both mouse andhuman keratinocytes to laminin-5 wascompletely dependent on α3β1 andα6β4, and α2β1 was not involved(Niessen et al., 1996; Xia et al., 1996;DiPersio et al., 1997). α3β1-mediatedand α6β4-mediated cell adhesion haveeach been shown to regulate importantsignaling functions in culturedkeratinocytes and other epithelial cells(Mainiero et al., 1995, 1997; Clarke et

al., 1995; Xia et al., 1996; Shaw et al., 1997; Gonzales et al.,1999), and it has been predicted that some of these signals maybe essential for normal stratification and differentiation of theepidermis. Previous studies of mice lacking either α3β1 orα6β4 showed that, while each was necessary for distinctaspects of dermo-epidermal adhesion, neither of these integrinswas necessary for skin morphogenesis during embryonicdevelopment. However, given the overlapping ligand-bindingspecificities of these two laminin receptors, it was possible thatone was sufficient for epidermal development when the otherwas absent. Similarly, although skin morphogenesis proceedsin mice that are completely deficient in laminin-5, the absenceof laminin-5 appears to unmask, or activate, compensatoryadhesion to other laminins (Ryan et al., 1999). In short, thepotential for overlapping and/or compensatory mechanisms ineach of the mutant mice described above has made it difficultin the past to ascertain the importance of laminin-5-bindingintegrins during skin development.

Our current study supports the previously established notionthat integrins α3β1 and α6β4 perform complementary butdistinct roles in maintaining the dermo-epidermal junction.Similarly, a recent study by van der Neut et al. (1999) suggests

C. M. DiPersio and others

Fig. 6.Epidermal cell proliferation in normal and mutant embryos. Sections from normalembryos (A, B) or embryos homozygous for null mutations in the α3, β4, and/or α6 genes (C-H)were stained by immunofluorescence with an antiserum against the proliferation marker Ki67.Homozygous null mutations are indicated for each embryo; normal embryos are either wild-typeor heterozygous for mutations. Embryos were from E15.5 (A,C,E,G) or E16.5 (B,D,F,H), asindicated. Arrowheads point to the dermo-epidermal junction directly beneath proliferating cells.Only non-blistered epidermis is shown for mutants. Bar, 50 µm.

3059Skin morphogenesis in α3β1/α6β4-null mice

that α3β1 and α6β4 act in concert through distinct mechanismsto maintain dermo-epidermal adhesion in plantar and palmar(i.e. hairless) regions of the skin. However, our current studyprovides no evidence for overlapping or compensatoryfunctions between these integrins during skin development.Furthermore, a surprising finding was that skin morphogenesisappears normal, despite blistering, in embryos that lack thelaminin-5 receptors α3β1 and α6β4, proving that integrin-mediated adhesion to laminin-5 is not essential for epidermaldevelopment.

It was shown previously that α6β4-deficiency results inincreased recruitment of α3β1 to the basal surface ofkeratinocytes in the epidermis, suggesting that α3β1 maycompensate to some extent for the loss of α6β4-mediatedadhesion (van der Neut et al., 1996). However, we have nowshown that the extent of blistering in embryos lacking bothα3β1 and α6β4 was similar to that in embryos lacking onlyα6β4, suggesting that α3β1 does not compensate significantlydespite its re-distribution. The mechanisms of blister formationin α3β1-deficient mice and α6β4-deficient mice are different.α3β1-deficiency results in mild, perinatal blisters that appearto be caused by rupture of a disorganized basement membrane

(DiPersio et al., 1997). In contrast, α6β4-deficiency results inextensive detachment of the epidermis caused by the absenceof hemidesmosomes and lack of stable adhesion (van der Neutet al., 1996; Georges-Labouesse et al., 1996; Dowling et al.,1996). Not surprisingly, the β4-null blistering phenotype wasdominant over the α3-null blistering phenotype in α3β1/α6β4-deficient mice. Indeed, antibodies against the basementmembrane marker entactin stained only the dermal sides ofα3−/−;β4−/− or α3−/−;α6−/− blisters, indicating epidermaldetachment from the basement membrane.

Laminin-5-deficient mice retained epidermal attachmentuntil after birth (Kuster et al., 1997; Ryan et al., 1999). It hasbeen suggested that binding interactions between integrinα3β1 and other laminins that are present in skin (i.e. laminin-10 or −11) may be activated or unmasked in the absence oflaminin-5 (Ryan et al., 1999). Thus, compensatory adhesion inlaminin-5-deficient mice may conceal a role for laminin-5 inmaintaining integrity of the dermo-epidermal junction duringskin development. Importantly, α6β4-deficient mice retainedthe ability to deposit laminin-5 into the basement membrane(van der Neut et al., 1996; Georges-Labouesse et al., 1996;Dowling et al., 1996), so that alternative mechanisms that are

α3β1 / α6β4-deficient epidermis

E8-E12

E14-E15

E15-E17

A

LM-5 binding integrin

LM-5

other BM receptor

other BM ligand

embryonic basal cells(stratum germinatum)

basement membrane

stratum intermedium

basal cell layer

suprabasal cell layers

cornified layers

basal cell layer

normal epidermis B

periderm

Fig. 7. A model for epidermal morphogenesis in normal versus α3β1/α6β4-deficient embryos. (A) In normal epidermis, laminin-5 (LM-5) isnot yet present during early stages of development (E8-E12), and other basement membrane (BM) receptors mediate epidermal attachment.From E12 to E15, accumulation of laminin-5 in the basement membrane, and presumably its binding to integrins α6β4 and α3β1, coincideswith increased stratification of the epidermis (Aberdam et al., 1994; DiPersio et al., 1997). For example, α6β4 is expressed by E15.5 in basalcells and is recruited to the basal surface by interactions with laminin-5. Importantly, other basement membrane receptors may remain activeduring stratification. As the epidermis matures and stratifies further (E15-E17), laminin-5 becomes the major adhesive ligand in the basementmembrane. (B) In α3β1/α6β4-deficient (or α6β4-deficient) epidermis, adhesion through other receptors during early developmental stagessupports epidermal differentiation and stratification. However, stable adhesion to laminin-5 fails to develop at later stages, and these otheradhesion mechanisms are not sufficient to maintain anchorage of the stratified epidermis to the basement membrane, resulting in blisters.Shaded cells over the blistered region are apoptotic. LM-5, laminin-5; BM, basement membrane.

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unmasked in laminin-5-deficient mice (Ryan et al., 1999) arenot expected to be activated in α6β4-deficient mice. Indeed,we detected blisters in α6β4-deficient mice as early as E15.5,suggesting that laminin-5 has a developmental role in dermo-epidermal adhesion that was not predicted from the perinatalblistering of laminin-5-deficient mice.

Fig. 7 summarizes schematically the epidermal phenotypeof embryos lacking both α3β1 and α6β4. Despite extensiveepidermal detachment in these embryos, mechanisms ofepidermal adhesion that were independent of α3β1 and α6β4were clearly in effect, since both α3−/−;β4−/− andα3−/−;α6−/− embryos from E15.5/E16.5 retained considerabledermo-epidermal adhesion (illustrated in Fig. 7B). Thisadhesion was sufficient to support epidermal morphogenesisprior to blistering. Presumably, other integrins or non-integrinreceptors mediate keratinocyte adhesion to ligands present inthe embryonic basement membrane prior to E15, at whichpoint a switch-over to laminin-5-based adhesion begins tooccur in normal skin (illustrated in Fig. 7A). In a recent studyby De Arcangelis et al. (1999), kidney development was nearlynormal in early α3−/−;α6−/− embryos, and the epithelial andbasement membrane defects seen in kidneys of α3−/− mice atlater developmental stages (Kreidberg et al., 1996) were notexacerbated in α3−/−;α6−/− embryos. These findings alsosupport the notion that other integrin or non-integrin basementmembrane receptors are sufficient for the early stages ofdevelopmental morphogenesis of certain epithelial tissues.Indeed, both β1 integrins and non-integrin laminin receptors,such as dystroglycan, have been implicated in basementmembrane assembly during early embryogenesis (Henry andCampbell, 1998; Aumailley et al., 2000).

β1 integrins appear to be required for the synthesis anddeposition of laminin-1 and certain other basement membraneproteins during early steps of embryogenesis (Aumailley et al.,2000). In addition, both integrin and non-integrin receptors,such as dystroglycan, facilitate the polymerization of newlysecreted laminins on the cell surface, which is prerequisite forthe organized assembly of basement membranes (Henry andCampbell, 1998; Fleischmajer et al., 1998; Colognato et al.,1999). Although integrin α3β1 is involved in the organizationof laminin-5 and other basement membrane proteins that aredeposited into the dermo-epidermal region, laminin-5 wassecreted efficiently in α3β1-deficient epidermis (DiPersio etal., 1997), and was deposited into the extracellular matrix byα3β1-deficient keratinocytes in culture (DiPersio et al., 2000).Laminin-5 was also present in the basement membranes ofα6β4-deficient epidermis (van der Neut et al., 1996; Georges-Labouesse et al., 1996; Dowling et al., 1996). An interestingobservation from the current study was that laminin-5 was alsodetected by immunofluorescence in the basement membranezone of α3−/−;α6−/− embryos. Although we could notquantitative the amount of laminin-5 deposited by normal ormutant embryos, this result indicates that none of the knownlaminin-5 receptors (α3β1, α6β1, and α6β4) were required forlaminin-5 secretion per se, and suggests that laminin-5deposition can be mediated by an unidentified receptor. Asmentioned above, integrin α2β1 has been reported to bindlaminin-5 in colonic cancer cells (Orian-Rousseau et al., 1998).Although α2β1 was not involved in stable adhesion ofkeratinocytes to laminin-5 (Niessen et al., 1996; Xia et al.,1996; DiPersio et al., 1997), it seems possible that this integrin

could facilitate laminin-5 deposition by keratinocytes, at leastin the absence of α3 and α6 integrins. Alternatively, it ispossible that a non-integrin receptor for laminins, such asdystroglycan, can facilitate laminin-5 deposition (Henry andCampbell, 1998).

It has been known for some time that epithelial cell adhesionto basement membranes prevents apoptosis and promotes cellsurvival (Frisch and Francis, 1994; Boudreau et al., 1995;Pullan et al., 1996). We used TUNEL assay to show thatdetachment of the epidermis from the basement membrane ineither β4−/− or α3−/−;β4−/− embryos lead to increasedapoptosis of basal keratinocytes. Previous data by Dowling etal. (1996) suggested an increase in apoptotic cells, as identifiedby ultrastructural morphology, in both blistered and non-blistered regions of β4-null skin. In contrast, our present resultsusing TUNEL assay showed unambiguous restriction ofapoptotic cells to blistered regions of α6β4-deficient embryos.Interestingly, Murgia et al. (1998) reported that TUNEL assayrevealed no apoptosis in epidermal blisters of E18.5 embryosexpressing a mutant β4 subunit that lacks a cytoplasmicdomain and cannot integrate with the cytoskeleton; themechanism of blister formation in these embryos was distinctfrom that in α6β4-deficient embryos. Ryan et al. (1999)similarly reported that apoptotic cells were not detected byTUNEL assay in laminin-5-deficient, post-natal epidermis. Apossible explanation for the lack of apoptosis in the latter twostudies is that there was insufficient time between blisterformation and tissue preparation for apoptosis to be detectedby TUNEL assay, since DNA fragmentation is a later step inthe apoptotic program. Similarly, we were unable to detectapoptotic cells in blisters of E15.5 embryos, or in all blistersof E16.5 embryos, presumably because some blisters had onlyrecently formed and apoptotic cells had not yet accumulated.Importantly, our results show that while cell survival wasdependent on epidermal adhesion per se, it was not dependenton α3β1- or α6β4-mediated adhesion, since apoptotic cellswere absent from attached regions of α3−/−;β4−/− epidermisin E16.5 embryos. While our results do not rule out specificroles for integrins α3β1 and/or α6β4 in maintainingkeratinocyte survival in adult skin, they do show that adhesionspecific to these integrins is not required to prevent apoptosisof attached basal keratinocytes during skin development.

In a previous study by Murgia et al. (1998), mutation of theα6β4 integrin through targeted deletion of the β4 cytoplasmicdomain resulted in a 2-fold decrease in the number of Ki67-positive cells in the epidermis, suggesting that intact β4 isrequired for optimal proliferation. Despite this reduction in cellproliferation, skin morphogenesis appeared essentially normalin mutant embryos expressing the functionally deficient α6β4(Murgia et al., 1998). Gonzales et al. (1999) recently showedthat α3β1 is also involved in regulating proliferation ofcultured epithelial cells, suggesting that α3β1 and α6β4 mayhave overlapping or compensatory roles in cell proliferation.Based on their results, these authors suggested that α3β1 maycompensate for the functionally deficient α6β4 in the study ofMurgia et al. (1998), and that α6β4 may similarly compensatefor the loss of α3β1-mediated proliferation in the normallystratified epidermis of α3β1-deficient mice (DiPersio et al.,1997). However, we detected normal numbers and distributionsof Ki67-positive cells, and apparently normal epidermalstratification, in both α3−/−;β4−/− embryos and α3−/−;α6−/−

C. M. DiPersio and others

3061Skin morphogenesis in α3β1/α6β4-null mice

embryos, establishing that epidermal precursor cells retain thecapacity to proliferate and support skin morphogenesis in theabsence of both α3β1 and α6β4.

In summary, it was clear from previous studies withknockout mice that integrins α3β1 and α6β4 are eachimportant for maintaining distinct aspects of mechanicalintegrity at the dermo-epidermal junction, but that neither isotherwise necessary for essentially normal skinmorphogenesis. We have now shown that skin morphogenesisprogresses in an essentially normal fashion in embryos thatlack both α3β1 and α6β4. Although these integrins mayregulate subtle aspects of skin development and differentiationthat we did not assay in our study, epidermal morphogenesisappears to depend on other integrin or non-integrin adhesionreceptors. Nevertheless, it remains possible that α3β1 andα6β4 have important roles in differentiation and/orproliferation during post-developmental aspects of skinfunction, including homeostasis of adult skin and woundhealing. Consistent with this model, stratification anddifferentiation of the skin is already underway when laminin-5 begins to accumulate in the cutaneous basement membranearound E14-15 of mouse development. α6β4 is recruited to thebasal aspect of basal keratinocytes around this stage ofdevelopment, reflecting its binding to laminin-5 andrecruitment into hemidesmosomes. This establishment ofstable adhesion to laminin-5 may prepare the developingepidermis for adult life, when the ability of keratinocytes todynamically alter this stable adhesion will become critical forkeratinocyte migration during wound healing. To date, theperinatal lethality caused by null mutations of the genesencoding either individual chains of laminin-5 or its integrinreceptors has precluded post-developmental studies in vivo.However, future studies with viable mice that harbor inducible,skin-specific knockouts of α3β1 and/or α6β4 will helpevaluate the roles of these integrins in regulating proliferationand cell migration during wound healing and other post-developmental skin processes.

We are grateful to Denise Crowley for preparation of embryosections for histology. We also thank Drs A. Chung and G. Meneguzzifor antibodies against entactin and laminin-5, respectively. Thisresearch was supported by a grant from the National Institutes ofHealth (R01CA17007) to R.O.H, and in part by a grant from theNetherlands Organisation for Scientific Research (N.W.O. grant 902-11-043) to A.S. R.O.H. is an Investigator of the Howard HughesMedical Institute.

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C. M. DiPersio and others