In order to investigate the role of extracellular matrix receptors of the integrin family in establishing the spatial organization of epidermal keratinocytes, we used immunofluorescence microscopy to examine the expression of a range of integrin subunits during development of human palm and sole skin. All of the integrins expressed during development were also present in mature epidermis and were largely confined to the basal layer of keratinocytes in a pericellular distribution. The α3 and β1 subunits were expressed prior to initiation of stratification and did not change in abundance or distribution during subsequent development, α4 and β3 were not detected at any time in the epidermis. Every other subunit examined showed spatial or temporal changes in expression. Staining for α3 was strong before stratification and until mid-development, but was greatly decreased in neonatal epidermis. α2 was first detected in small patches of basal cells prior to stratification, and thereafter was found in the entire basal layer, with greater staining in developing sweat glands, α5 was not expressed until mid-development, and then primarily in developing sweat glands, with faint expression in neonatal epidermis, αv was detected following stratification, in developing sweat glands, and occasionally in neonatal epidermis, α6 and β4 were peribasally expressed before stratification, but there-after became concentrated at the basal cell surface in contact with the basement membrane, co-localizing with hemidesmosomes as determined by staining with bullous pemphigoid antiserum. We also examined the distri-bution of three known ligands for keratinocyte integrins: laminin and collagen type IV were present in the basement membrane zone at all stages of development, whereas fibronectin was only evident there until about 13 weeks estimated gestational age. Finally, we found that the changes in integrin expression that occur on initiation of stratification in vivo could be reproduced in organ cultures of developing skin; such cultures there-fore provide a useful experimental model for further studies of the role of integrins in epidermal stratification.

The epidermis is a stratified epithelium: proliferation is confined to the basal layer of keratinocytes that adhere to the basement membrane and cells undergo terminal differentiation as they move through the upper layers (reviewed by Matoltsy, 1986). There is growing evidence that extracellular matrix (ECM) receptors of the integrin family play an important role in maintaining the spatial organization of the different keratinocyte layers.

Integrins are cell surface glycoproteins that consist of one α and one βsubunit; ligand specificity is largely determined by heterodimer composition, although the same integrin in different cell types can have different specificities (Elices and Hemler, 1989; Languino et al. 1989; and Hemler, 1990). The major integrins expressed by keratinocytes in culture are α2β, α1α3β1 (variously identified as receptors for collagen and laminin), and (ligand unknown; but see De Luca et al. 1990); they also express α5β1, which is a fibronectin receptor (Adams and Watt, 1990; Adams and Watt, 1991; Carter et al. 1990 b; De Luca et al. 1990; and Staquet et al.1990). Expression of each integrin both in culture and in the epidermis is confined to the basal layer (Fradet et al. 1984; Sonnenberg et al. 1986; Wayner et al. 1988; De Strooper et al. 1989; Peltonen et al. 1989; De Luca et al. 1990; Nicholson and Watt, 1991; and Adams and Watt, During terminal differentiation keratinocytes lose adhesiveness to fibronectin, laminin and collagen type IV, the adhesive changes precede loss of the ft integrins from the cell surface (Adams and Watt, 1990). There is evidence that a decrease in the ligand binding ability of the α5β1 integrin occurs in basal cells on commitment to terminal differentiation; such downregulation of integrin function would provide one mechanism for the selective migration of committed keratinocytes out of the basal layer (Adams and Watt, 1990). Loss or reduction of cell-substratum adhesion is one signal for keratinocyte terminal differentiation (Green, 1977 and Watt et al. 1988) and integrin-mediated interaction with the extracellular matrix may play a role in regulating the initiation of terminal differentiation (Adams and Watt, 1989).

In view of the evidence that integrins are involved in maintaining the spatial organization of keratinocytes, we decided to investigate the role of integrins in establishing that organization during human epidermal development. By about 4 weeks estimated gestational age (EGA), the ectoderm has given rise to a layer of basal keratinocytes covered by a layer of periderm cells (Holbrook, 1983). At around 9–10 weeks EGA, in a process termed stratification, the basal layer forms an intermediate keratinocyte layer (i.e. between the basal and periderm layers) through proliferation and upward cell migration. Additional suprabasal layers are added periodically until 20–24 weeks, when the periderm is sloughed off and all the epidermal layers found in mature skin are present.

In the experiments described in this report, we have used immunofluorescence microscopy to document the integrins expressed in mature epidermis and at different stages of epidermal development. We have attempted to correlate the changes observed with changes in expression of three integrin ligands, fibronectin, collagen type IV and laminin, and of bullous pemphigoid antigen (BPA), in the basement membrane zone (BMZ). Finally, we have shown that the changes in integrin expression that occur at the onset of stratification in vivo can be reproduced in an organ culture model (Fisher and Holbrook, 1987), a finding that offers significant potential for further studies of the role of integrins in assembly of the epidermis.

Tissue

Fetal tissue was obtained with permission of the ICRF Ethical Committee from the MRC Tissue Bank, London. EGA of each fetus was determined by foot or hand length measurement Skin samples were taken within six hours of collection from morphologically normal human fetuses obtained at elective terminations of pregnancy. A total of 16 samples in the embryonic period (7.8 to 9.5 weeks EGA), 8 in the early fetal period (9 5 to 11 weeks), and 6 in the later fetal period (to 16.6 weeks) was analyzed. Foreskins were obtained from routine circumcisions of neonates

Organ cultures

Organ cultures were prepared by placing four 20 mm2 pieces of skin, trimmed of excess connective tissue, on 0.45μm Milhpore HA filters supported by stainless steel grids over the central wells of Falcon 3037 dishes, so that the tissue was at the air-medium interface (Fisher and Holbrook, 1987) Medium consisted of DME (Gibco, Paisley, Scotland) supplemented with 10% fetal calf serum (Imperial Labs, Andover, England) and 300μgml-1 ascorbate (Sigma Chemical Co., Poole, England) Cultures were incubated at 37°C, in a humidified atmosphere of 5 % CO2 in air for 15–24 h, then processed as for fresh tissue. A total of 7 cultures was studied

Histology

Samples of skin were coated in OCT (Tissue-Tek) and frozen in an isopentane bath cooled in liquid nitrogen, or fixed overnight in Methacam (methanol/acetic acid/chloroform 6 31 v/v) for histological examination. Frozen sections were cut at 6jim and stored at –70°C until use. Staining was performed as follows sections were fixed for 30 min in 4% formaldehyde diluted in phosphate-buffered saline with magnesium and calcium added to ImM (PBSABC), followed by several washes in 0.1M glycine and a 30μ60 min block in PBSABC containing 0.1% fraction V BSA (Sigma) and 0.02 % Triton X-100 (Sigma) to minimize background staining Antibody dilutions and washes were performed in the same solution as used for blocking Primary antibody was applied for one hour at room temperature, followed by three 20 min washes. Biotinylated species-specific secondary antibody was applied for 45 min, followed by three 15 min washes. Fluorochrome-conjugated streptavidin was applied for 15 min, followed by three 20 min washes. For dual label immunofluorescence, anti-BPA was added after labelling of α6, and detected with a different fluorochrome. Slides were mounted in 90% glycerol, 50mM Tris pH8.9, with 2.5% 1,4-diazabicyclo(2,2,2)octane (Sigma) added to retard photob-leaching Slides were viewed on a Zeiss photomicroscope equipped for epifluorescence.

Antibodies

Antibodies used were as follows and were the generous gifts of the investigators indicated. TS2/7 against α1 (M. Hemler, Dana Farber Cancer Research Institute, Boston, MA; Hemler et al 1984); 5E8 (R. Bankert, Roswell Park Memorial Institute, Buffalo, NY; Zylstra etal. 1986), P1E5 (E Wayner, Oncogen, Seattle, WA, Carter et al. 1990 b), and P1E6 (Tehos Pharmaceuticals, San Diego, CA, Carter et al. 1990 b) against α2 JI43 against (A. Albino, Sloan-Kettenng Memorial Institute, New York, NY; Kantor et al 1987); B-5G10 against α4 (M Hemler; Hemler et al. 1987), MAb 16 against α5 (K. Yamada, NCI, Bethesda, MD; Akiyama etal. 1989), GoH3 against α6 (A. Sonnenberg, University of Amsterdam, The Netherlands; Sonnenberg et al. 1986), VNR147 (Telios Pharmaceuticals, San Diego, CA) and 13C2 (M. Horton, ICRF, London, Horton et al. 1985) against αv; MAb 13 against β 1 (K. Yamada, NCI, Bethesda, MD; Akiyama et al 1989); Y2/51 against β 4 (D. Mason, John Radcliffe Hospital, Oxford, von dem Borne et al 1989), and 439-9B against β 4 (S Kennel, Oak Ridge National Laboratory, Oak Ridge, TN, Kennel et al. 1989). Rabbit anti-human fibronectin was purchased from Calbiochem (Novabiochem (UK), Nottingham, England). Polyclonal antibodies against mouse laminin and human collagen type IV were kindly provided by M. J. Warburton (Warburton et al. 1982). Human antiserum to bullous pemphigoid antigen was kindly provided by B. Bernard, Centre Internationale de Recherches Dermatol-ogtques, Sophia Antipohs, France. Biotinylated species-specific antibodies and FITC- and Texas Red-conjugated streptavidin were from Amersham International, UK. The biotin-streptavidin system was used to maximize sensitivity in order to detect weakly expressed integnns

Neonatal epidermis

The distribution of integrin subunits in neonatal foreskin is summarized in Table 1. Expression of all integnns was largely confined to the basal layer, although faint staining of the first 1-2 suprabasal layers was sometimes seen (e.g. Fig. 1G). All basal cells expressed integrins and each of the subunits was expressed on all surfaces of the basal cells (i.e. pericellular staining). Antibodies to αv, β 1, and subunits known from immunoprecipitation studies of cultured keratinocytes to associate with β 1(i.e. α2,α3,α5,;Adams and Watt, 1990 and 1991) stained all basal cell surfaces uniformly. In contrast, α6 and β 4 showed strongest expression at the basal surface of the basal cells, in contact with the basement membrane (compare Figs 1G, 2B, 3E, 4C with Fig. 5D).

Table 1.

Summary of integrin expression in neonatal skin and in palm and sole skin during development

Summary of integrin expression in neonatal skin and in palm and sole skin during development
Summary of integrin expression in neonatal skin and in palm and sole skin during development
Fig. 1.

β 1 expression. (A,B) 8.0 weeks; (C,D) 9.9 weeks; (E,F) 13.6 weeks; and (G,H) neonatal skin, with corresponding phase micrographs (B-H). α 3 expression was identical to β 1 at all stages. Scale bar=50 μ m.

Fig. 1.

β 1 expression. (A,B) 8.0 weeks; (C,D) 9.9 weeks; (E,F) 13.6 weeks; and (G,H) neonatal skin, with corresponding phase micrographs (B-H). α 3 expression was identical to β 1 at all stages. Scale bar=50 μ m.

Fig. 5.

α6 expression. (A) 8 6 weeks; (B) 9.0 weeks, (C) 13.6 weeks; and (D) neonatal skin Note that the sample in B had just stratified, β4 expression was similar to α6 at all stages. Scale bar=50 μm.

Fig. 5.

α6 expression. (A) 8 6 weeks; (B) 9.0 weeks, (C) 13.6 weeks; and (D) neonatal skin Note that the sample in B had just stratified, β4 expression was similar to α6 at all stages. Scale bar=50 μm.

Staining for the β 1,α2,α3,α6 and β 4 subunits (Figs 1G, 3E, 5D, and results not shown) was intense; in contrast, staining for α1α5 and αv (Figs 2B, 4C, and 6C) was weak. α4 and β 3were never detected in the epidermis, although blood vessel walls were stained (results not shown).

Fig. 2.

α1 expression. (A) 9.0 weeks and (B) neonatal skin. Note that the sample in A had just stratified Scale bar=50 μm.

Fig. 2.

α1 expression. (A) 9.0 weeks and (B) neonatal skin. Note that the sample in A had just stratified Scale bar=50 μm.

Epidermal development

Having established the distribution of integrins in mature epidermis, we examined expression of the same integrins at three different stages of epidermal development: prior to stratification (embryonic period; up to 9.5 weeks EGA); immediately after stratification (early fetal, 9.5 to 11 weeks EGA, although stratification was sometimes observed earlier); and mid-development (11 to 17 weeks EGA). The results are summarized in Table 1.

Palm and sole skin were chosen for analysis because, particularly at the earliest stages of development, the morphology and integrity of the tissue was better than that from other body sites In some later specimens, arm or leg skin was also examined; the results obtained were essentially the same as for the palm and sole, except that development of arm and leg epidermis was relatively less advanced than palm and sole at the same EGA (Holbrook and Odland, 1980). Palm and sole skin lack hair follicles but contain abundant sweat glands, which start to develop at about 12 weeks EGA (Holbrook, 1983).

Periderm

The periderm is a single cell layer that covers the epidermis until about 20 – 22 weeks EGA. At all stages of development, the periderm did not express any of the integrins examined, with the possible exception of αv, which showed occasional, speckled staining of individual cells (results not shown). In older specimens, the periderm was usually stained, but this was non-specific because controls using second antibody alone also stained the periderm.

Prestratification (embryonic period)

Embryonic epidermis, i.e. prior to initiation of stratification at about 9.5 weeks EGA, consists of only one layer of keratinocytes. At this early stage in development, the cells already expressed several of the integrin subunits that were present in mature epidermis, ft, β1α1,α3α6,and β 4(Figs 1A, 5A, Table 1, and results not shown). These integrins had a uniform pericellular distribution; for α6 and β 4, this was in contrast to the concentration at the BMZ noted in neonatal skin (compare Fig. 5A,D). Staining for α1 was more intense in embryonic epidermis than in foreskin (results not shown).

Two integnns detected in mature epidermis were not observed at this stage of development: α5 and αv. α5 was never detected prior to stratification (Fig. 4A) and α v was detected in only one out of 16 specimens. The staining pattern using two anti- αv monoclonal antibodies was identical. The α4 and β 3subunits, which were not present in mature epidermis, were also absent in embryonic epidermis.

Fig. 4.

α5 expression. (A) 8.0 weeks; (B) 15 3 weeks, arrow indicates elevated expression in developing sweat gland, and (C) neonatal skin.

Fig. 4.

α5 expression. (A) 8.0 weeks; (B) 15 3 weeks, arrow indicates elevated expression in developing sweat gland, and (C) neonatal skin.

α2 was not expressed in the youngest specimens examined (n=3 at 7.8 weeks EGA; see Fig. 3A). However, α2 showed patchy expression in about half of the other prestratification specimens. When present, α2 expression was pericellular and confined to stretches of faintly positive cells bordered by negative areas (Fig. 3B). There was no apparent difference in epider-trial mal morphology between the positive and negative areas. The same staining pattern was observed with three different anti-α2 monoclonal antibodies.

Fig. 3.

α2 expression. (A) 7.8 weeks; (B) 9 0 weeks; arrowhead indicates start of negative area to right; (C) 10.7 weeks; (D) 15.3 weeks; note intense sweat duct staining; and (E) neonatal skin. Scale bar=50 μm.

Fig. 3.

α2 expression. (A) 7.8 weeks; (B) 9 0 weeks; arrowhead indicates start of negative area to right; (C) 10.7 weeks; (D) 15.3 weeks; note intense sweat duct staining; and (E) neonatal skin. Scale bar=50 μm.

Onset of stratification

The fetal stage of development starts at stratification, when an intermediate layer of keratinocytes forms between the basal and periderm layers through proliferation and upward migration of basal keratinocytes at about 9.5 weeks EGA.

The bi, β 1α1and α3 integrin subunits continued to be uniformly and strongly expressed in a pericellular distribution in the basal layer of newly stratified epidermis (Figs 1C, 2A, and results not shown). The α4, α5 and β 3subunits were not detected, whereas was faintly expressed in the basal layer in 5 out of 8 specimens examined (Fig. 6A). and fa were expressed in the basal layer of keratinocytes, but in contrast to the prestratification distribution (Fig. 5A), there was a concentration of staining at the basal surface of the basal cells (Fig. 5B), as observed in neonatal epidermis (Fig. 5D).

Fig. 6.

αv expression. (A) 9 0 weeks; (B) 15 3 weeks; the periderm staining is non-specific; arrow indicates sweat duct; and (C) neonatal skin, arrows indicate basal cell layer. Note that the sample in A had just stratified. Scale bar=50μm.

Fig. 6.

αv expression. (A) 9 0 weeks; (B) 15 3 weeks; the periderm staining is non-specific; arrow indicates sweat duct; and (C) neonatal skin, arrows indicate basal cell layer. Note that the sample in A had just stratified. Scale bar=50μm.

Whereas prior to stratification α2-positive cells were found in patches (Fig. 3B), after stratification α2 was expressed by all basal keratinocytes, in 7 out of 8 specimens examined (Fig. 3C). In the other specimen, no positive staining was observed, even though the specimen showed strong expression of the other integrins.

Further development

Between 12 and 17 weeks of gestation, more layers of keratinocytes are formed, reaching a total of 5 –6 by 17 weeks. In the oldest specimen examined, cornified layers were beginning to form. As observed at the earlier stages of development, integrin expression was largely confined to the basal layer. The β 1and α3 subunits continued to show strong pericellular expression (Fig. IE and results not shown), α1 was also expressed, but starting from about 15 weeks of gestation onwards the intensity of staining started to decrease to the low level observed in neonatal skin (Fig. 2B). α6 and β4 staining was concentrated at the BMZ, but there was also clear pericellular staining of the basal cells and faint staining of the first suprabasal layers (Fig. 5C).

Three of the integrin subunits, α2, α5 and αv appeared to be expressed at a higher level in developing sweat glands than in the regions between the sweat glands. α2 was expressed in all specimens of epidermis (Fig. 3D), but α5 was detected in about half of the specimens and then at a very low level (Fig. 4B), as previously reported (Peltonen et al. 1989; but see Wayner et al. 1988). Staining with αvwas also faint, comparable to α5, and was only detected in about half of the samples (Fig. 6B).

Basement membrane components

The expression of fibronectin, collagen type IV and laminin, representing ligands for some of the integrins studied, was examined. Fibronectin was detected at high levels in the dermis throughout development. Staining formed a bright line at the BMZ early in development (Fig. 7A), until about 13 weeks EGA, after which staining was uniform throughout the dermis (see Fig. 7D,G; see also Fine et al 1984). Collagen type IV and laminin staining were concentrated in the BMZ and around blood vessels throughout development, with no apparent changes in levels of expression during development (Fig. 7), as reported previously (Fine et al. 1984). Just after stratification, collagen type IV and laminin were also observed along the lateral and apical surfaces of the basal cells (Fig. 7B,C).

Fig. 7.

ECM protein localization. (A-C) 10 7 weeks; (D-F) 13 6 weeks, and (G-I) neonatal skin. (A,D,G) fibronectin; (B,E,H) collagen type IV; and (C,F,I) laminin. Scale bar=50 μm

Fig. 7.

ECM protein localization. (A-C) 10 7 weeks; (D-F) 13 6 weeks, and (G-I) neonatal skin. (A,D,G) fibronectin; (B,E,H) collagen type IV; and (C,F,I) laminin. Scale bar=50 μm

In vitro expression

Staining with antibodies to integrin subunits showed that major changes in integrin expression occur at the onset of stratification. It has previously been reported that embryonic skin maintained in organ culture can undergo near-normal development, although at an accelerated rate (Fisher and Holbrook, 1987). We therefore investigated whether such cultures would provide an experimental model for analyzing the significance of changes in integrin expression observed in vivo.

Prestratification specimens of palm or sole tissue were dissected into several pieces. One piece was frozen immediately and the others were cultured for 15-24h, then frozen. The histological appearance of cultured epidermis is shown in Fig, 8B, D and F. In every case, stratification had taken place in culture, and the cell layers were almost indistinguishable from layers formed after stratification in vivo (compare with Fig. ID).

Cultures were stained with antibodies to α2 and α6, two subunits that underwent marked changes on stratification (see Figs 3 and 5), and with antibodies to β 1and α3(Fig. 8A and results not shown). α2 expression was absent or patchy prior to stratification but, after culture, we detected uniform expression in the basal layer (Fig. 8C). α6, showed uniform pericellular distribution prior to stratification but, after culture, there was a concentration of stain at the BMZ (Fig. 8E). β 1 (Fig. 8A) and α 3(results not shown) continued to be expressed in the basal layer following stratification. Thus the patterns of integrin staining in vitro were identical to those seen in vivo.

Fig. 8.

β 1, α2α6 and BPA expression in vitro. (A) β 1 8.3 week specimen after 22-hour culture, with (B) corresponding phase micrograph; (C) α2 8.3 week specimen after 22-hour culture, with (D) corresponding phase micrograph; (E) α6, 8.6 week specimen after 15-hour culture, with (F) corresponding phase micrograph; (G,H) dual labeling of 8.6 week specimen after 24-hour culture: (G) α6 (H) BPA on same field. On single-labeled specimens, there was fluorescence only with the appropriate filter. Scale bar=50 μm for A-F, scale bar=12.5 μm for G and H.

Fig. 8.

β 1, α2α6 and BPA expression in vitro. (A) β 1 8.3 week specimen after 22-hour culture, with (B) corresponding phase micrograph; (C) α2 8.3 week specimen after 22-hour culture, with (D) corresponding phase micrograph; (E) α6, 8.6 week specimen after 15-hour culture, with (F) corresponding phase micrograph; (G,H) dual labeling of 8.6 week specimen after 24-hour culture: (G) α6 (H) BPA on same field. On single-labeled specimens, there was fluorescence only with the appropriate filter. Scale bar=50 μm for A-F, scale bar=12.5 μm for G and H.

α6,and β 4have recently been reported to localize to hemidesmosomes at the BMZ (Kurpakus et al. 1990 and Stepp etal. 1990). Hemidesmosomes are first formed at the onset of stratification in the epidermis (Fine et al. 1984 and Lane et al. 1985). We therefore examined whether the redistribution of α6 to the BMZ correlated with the appearance of hemidesmosomes, as determined by staining for bullous pemphigoid antigen (BPA), a hemidesmosome protein (Robledo et al. 1990). Staining for BPA was rarely present at the BMZ before stratification, but appeared discontinuously at the BMZ following culture, consistent with the appearance of BPA following stratification in vivo (Fig. 8H and results not shown). Dual labeling of stratified epidermis with BPA and α6 antibodies showed that BPA co-localized with α6 at the BMZ (Fig. 8G and H), but, in contrast to BPA, α6staining was uniform along the BMZ and pericellular, as described above.

Integrin-mediated interactions between cells and extracellular matrix molecules play an important role in differentiation and morphogenesis (see, for example: Menko and Boettiger, 1987; Patel and Lodish, 1987; Korhonen et al. 1990; and Sorokin et al. 1990). In the epidermis, integrins are involved not only in maintaining the spatial organization of keratinocytes but also in regulating the initiation of terminal differentiation (Adams and Watt, 1989, 1990). In this report, we have begun to investigate the role of integnns in the development of the epidermis from a single layer of keratinocytes to the mature stratified tissue.

We have examined the pattern of integrin expression in human epidermis at different stages of gestation, and have compared it with the distribution of receptor ligands within the basement membrane. We have also determined that the changes observed during development in vivo can be reproduced in organ culture, thus providing the opportunity to analyze factors that regulate integrin expression. Wherever possible, we have used more than one antibody to each integrin subunit, and we have used a biotin-streptavidin detection system to maximize sensitivity. The different stages of epidermal development that we examined and the major changes in integrin expression we observed are summarized in Fig. 9.

Fig. 9.

Diagram illustrating the stages of epidermal development examined and the main changes in integrin expression at each stage. The different layers of neonatal epidermis are indicated Intermed=intermediate, kerats=keratinocytes.

Fig. 9.

Diagram illustrating the stages of epidermal development examined and the main changes in integrin expression at each stage. The different layers of neonatal epidermis are indicated Intermed=intermediate, kerats=keratinocytes.

The staining that we found in neonatal (i.e. mature) epidermis largely confirms the observations of others (Kajiji et al. 1987; De Strooper et al. 1989; Carter et al. 1990b; Nazzaro et al. 1990; and Peltonen et al. 1989). Integrin expression was mainly confined to the basal layer and there was no evidence of basal cell heterogeneity. All of the integrins showed a pericellular distribution, although α6 and β4were concentrated at the BMZ. The strongest staining detected was for integrin subunits α2α3, β 1α6 and β 4 We also detected very weak staining for α1α5,and αv, These results are in agreement with data on the relative abundance of different integrins obtained from immunoprecipitation of metabolically labeled cultured keratinocytes (De Luca et al. 1990; Larjava et al. 1990; and Adams and Watt, 1991). It is not possible to determine which integrin subunits form heterodimers on the basis of immunofluorescence staining, but the immunoprecipitation studies show that α2α3 and α5 are complexed with β 1α6; and β 4 form a heterodimer and αv is probably expressed in association with α5 (De Luca et al. 1990; Staquet et al. 1990; and Adams and Watt, 1991).

The integrin subunits expressed in developing epidermis were the same as those expressed in the neonatal, mature tissue. As in mature epidermis, the integrins were largely confined to the basal layer. However, with the exception of α3 β 1,all of the integrins showed temporal or spatial variation in expression. As has been reported previously, collagen type IV and laminin were present in the BMZ throughout development (Fine et al. 1984); just after stratification they were also found in a pericellular distribution in the basal layer. Fibronectin was concentrated at the BMZ until about 13 weeks EGA (in agreement with observations by Fine et al. 1984).

α1 β 1

This integrin has been shown to bind to collagen types I-IV (Kramer and Marks, 1989 and Belkin et al. 1990), and to the El region of laminin (Ignatius and Reichardt, 1988 and Hall et al. 1990). α1 β 1 was expressed prior to epidermal stratification and staining was relatively strong until about 15 weeks EGA. Later in development and in neonatal epidermis we observed only weak staining; others have reported it to be absent in mature epidermis (Hemler et al. 1984, De Luca et al. 1990; Nazzaro et al. 1990; but see Belkin et al 1990). Using immunoprecipitation, α1 β 1is not detected consistently in cultured keratinocytes, but it is expressed by ndk, a strain of epidermal cells with a complete block in terminal differentiation (Adams and Watt, 1991). The ligand for the keratinocyte α1 β 1 has yet to be determined, but until about 15 weeks EGA, α1 β 1α2 β 1and α3 β 1 are all highly expressed; it is therefore likely that the basal keratinocytes are simultaneously expressing two laminin or collagen receptors. An intriguing possibility is that different receptors for the same ECM ligand are coupled to different second messenger pathways or cytoskeletal components.

There are a number of possible explanations for the decrease in α1 β 1 staining during epidermal development. A formal possibility, which could also apply for the other subunits, is that α1 is present at the same level, but the epitope recognized by the antibody becomes masked; this can only be tested by extraction and biochemical analysis. A second possibility is that decreased α1 β 1expression reflects changes in basement membrane composition. Laminin and collagen type IV have recently been shown to be products of multigene families and different isoforms are present in basement membranes of different tissues (Sanes et al. 1990). The antibodies that we used might not detect such changes. Furthermore, the relative abundance of other collagen types (in particular, types III and V) at the dermoepi-dermal junction changes during skin development (Smith et al. 1986), although there is, so far, no evidence for collagen type-specificity in integrin binding. It is intriguing that the reduction in α1 staining was concurrent with the appearance of α5 and decreased fibronectin staining at the BMZ; at present, we have no evidence that these correlations are significant. Finally, it is interesting that in another tissue, the aorta, the level of α1 β 1also decreases with increasing gestational age (Belkin et al. 1990); down-regulation of may turn out to be a widespread phenomenon during development.

α2 β 1

This has been reported to be a receptor for collagen types I-IV (Kramer et al. 1989, Staatz et al. 1989; Santoro et al. 1988; and Wayner et al. 1988), laminin (Languino et al 1989), and possibly fibronectin (Kirchhofer et al. 1990). In cultured keratinocytes, it is a collagen (Wayner et al. 1988; Carter et al. 19906; Staquet et al. 1990; and Adams and Watt, 1991) and laminin receptor (Carter et al. 19906). There is some evidence that α2 β 1may also play a role in cell-cell adhesion (Larjava et al. 1990 and Carter et al. 1990b); the nature of the intercellular ligand(s) is unknown, but it is interesting that at least for a short period of development (Fig. 7B and C) and also in culture (Nicholson and Watt, unpublished data), pericellular staining for laminin and collagen type IV is observed.

α2 β 1was not expressed in the earliest specimens examined; in older prestratification specimens, it was sometimes present, but then only in a patchy distribution. From stratification onwards, α2 was strongly stained in all cells of the basal layer, with particularly high expression in developing sweat ducts. α2 was the only integrin subunit to show heterogeneous expression among basal cells and at present we can only speculate as to its significance: it could represent local variation in the composition of the basement membrane; or it might be a marker of areas that are about to stratify. Zutter and Santoro (1990) have noted that α2 β 1is highly expressed in proliferating populations of epithelial cells; another explanation for the patchy distribution prior to stratification is that it reflects proliferative heterogeneity within the basal layer. The organ cultures that we have employed will be useful for testing these ideas.

α3 β 1

This has been reported to be a collagen, laminin and fibronectin receptor (Wayner and Carter, 1987; Takada et al. 1988; and Gehlsen et al. 1989). The laminin binding site has been localized to the globular domain of the long arm (Gehlsen et al. 1989). In cultured keratinocytes, it acts as a laminin receptor (Carter et al. 1990b; Staquet et al. 1990; and Adams and Watt, 1991). It is also found at cell-cell contacts in a variety of cells (Kaufmann et al. 1989) and may play a role in keratinocyte-keratinocyte adhesion (Carter et al. 1990b and Larjava et al. 1990). α3 β 1was the only integrin that showed no change in abundance or distribution in the epidermis; it was present in the basal layer prior to stratification and throughout development. Since it can bind to a range of ligands, it may fulfil multiple functions in the developing tissue.

α5 β 1

In keratinocytes and other cell types, the only reported ligand for α5 β 1 is the central cell binding domain of fibronectin (Hynes, 1987; Wayner et al 1989; and Adams and Watt, 1990). Some cell types adhere to the IIICS site of fibronectin via α4 β 1 (Wayner et al. 1989). However, keratinocytes do not adhere to IIICS (Adams and Watt, 1990) and did not express α4 in mature epidermis or at any stage of development.

α5 was the last integrin subunit to be expressed during development. Expression was always low, although higher in the cells of developing sweat ducts than in the basal layer, as previously reported by Peltonen et al. (1989). The low level of α5 β 1expression is consistent with the low level of fibronectin in mature basement membrane (Stenman and Vaheri, 1978 and Fleisch-majer and Timpl, 1984). It is, however, surprising that α5 β 1 only appeared in the epidermis after fibronectin was no longer concentrated at the basement membrane zone. Our finding is similar to the observation of Korhonen et al (1990) that α5 β 1does not consistently codistribute with fibronectin in developing kidney.

Fibronectin binding is upregulated when keratinocytes are placed in culture (Toda et al. 1987), yet although cultured keratinocytes adhere better to fibronectin than laminin or collagen type IV (Clarke et al. 1985 and Adams and Watt, 1990), immunoprecipitation of metabolically labeled cell extracts shows that α5 β 1 is less abundant than α2 β 1and α3 β 1 (Adams and Watt, 1991). Further evidence that there need not be a simple correlation between α5 β 1expression and function comes from the observation that during keratino-cyte terminal differentiation, the ability of α5 β 1 to bind fibronectin decreases prior to a decrease in the level of α5 β 1 on the cell surface (Adams and Watt, 1990).

In contrast to our findings and those of Peltonen et al (1989), Wayner etal. (1988) found that α5 was present in all the epidermal layers of midgestation skin. One explanation for the discrepancy may be that the antibodies used recognize different epitopes; De Strooper et al. (1990) have recently reported that a monoclonal antibody to ag stains suprabasal but not basal keratinocytes in mature epidermis. At least in culture, the immunofluorescence localization of ag to the basal layer is supported by the finding that the level of the protein detected by immunoprecipitation (Adams and Watt, 1990) and of the α5 mRNA (Nicholson and Watt, 1991) decrease during terminal differentiation.

αv

This subunit can form heterodimers with β 1, α3, or β 1 and can participate in binding to vitronectin, fibronectin, fibrinogen, von Willebrand factor, osteonectin, or thrombospondin (Cheresh et al. 1989; Bodary and McLean, 1990; Hemler, 1990; and Ramaswamy and Hemler, 1990). In cultured keratinocytes, αv does not form a complex with β 1 and appears to form an αv β 5 heterodimer; it mediates adhesion to vitronectin, not fibronectin (Adams and Watt, 1991). The β 3 subunit is not expressed in keratinocyte cultures or at any stage of epidermal development (results not shown) We were unable to look for β 5 expression in skin and therefore cannot establish whether αv formed a complex with β 1 or α5.

αv was expressed at stratification. Staining was always very weak although, like β 2 and α5, it appeared to be elevated in developing sweat glands. We detected av less consistently than the other integrins, and this may be because it was expressed at a level that was at the limit of sensitivity for the staining protocol. Vitronectin has been reported to be absent in basement membrane (Reilly and Nash, 1988); however, if αv were complexed with β 1, it could be binding to fibronectin (Vogel et al. 1990).

α6 β 4

β 4 has only been reported complexed to α6 but α6 can form heterodimers with β 1 or β 4 (Sonnenberg et al.1990a). In cultured keratinocytes, is only present as α6 β 4 (Carter et al. 1990b and Adams and Watt, 1991) and since the staining patterns of α6 and β 4 were similar in the epidermis throughout development, it is likely that α6, is associated with β 4in the tissue.

A ligand for α6 β 4 has not been conclusively identified. A polyclonal antibody to β 4 blocks adhesion of keratinocytes to laminin (De Luca et al. 1990), but Hall et al. (1990) and Sonnenberg et al. (1990b) report that α6 β 4 is not a laminin receptor, α6 β 4 has been reported to be a component of hemidesmosomes (Kurpakus et al. 1990 and Stepp et al. 1990) and to co-localize with BPA in cultured keratinocytes (Carter et al. 1990a); our results, as outlined below, are consistent with this conclusion.

Like α3 β 1, α6 β 4 was expressed prior to stratification and throughout development. Before stratification, it showed a uniform pericellular distribution but, with the onset of stratification, it became concentrated at the basal surface of the basal cells. Hemidesmosomes are reported to appear at stratification and, consistent with earlier reports (Fine et al. 1984 and Lane et al. 1985), we found bullous pemphigoid antigen (BPA), a 230 × 103Mr hemidesmosome protein (Robledo et al. 1990), to be expressed at stratification. Dual labelling of newly stratified epidermis in organ culture showed co-localization of BPA with the α6 subunit. However, α6 β 4 cannot be restricted to hemidesmosomes, because it was also expressed on the apical and lateral surfaces of basal cells and is abundant in cultured keratinocytes that form few hemidesmosomes (Bohnert et al. 1986).

In conclusion, we have obtained evidence that integrins may play an important role in establishing the spatial organization of keratinocytes during epidermal development. Several integrin subunits are expressed prior to stratification, before the earliest markers of terminal differentiation appear (Watt et al. 1989) and the profile and distribution of integrins changes during subsequent development, most notably at the onset of stratification. The fact that those changes take place in organ culture offers great potential for further analysis of the role of integrins in tissue assembly. It will now be possible to use function-blocking antibodies to individual subunits in order to look at the effect on stratification, and to study whether there are environmental factors that can regulate integrin expression and the onset of stratification.

     
  • EGA

    (estimated gestational age)

  •  
  • ECM

    (extracellular matrix)

  •  
  • BMZ

    (basement membrane zone)

  •  
  • BPA

    (bullous pemphigoid antigen)

We are grateful to the many investigators who generously provided antibodies, to the MRC Tissue Bank for fetal tissue, to Dr Chns Fisher for teaching us the technique of organ culture, and to the ICRF Histopathology Unit for cryosec-tioning.

Adams
,
J C.
and
Watt
,
F M
(
1988
).
An unusual strain of human keratinocytes which do not stratify or undergo terminal differentiation in culture
.
J. Cell Biol
107
,
1927
1938
Adams
,
J C
and
Watt
,
F M
(
1989
).
Fibronectin inhibits the terminal differentiation of human keratinocytes
Nature
340
,
307
309
.
Adams
,
J C
and
Watt
,
F M
(
1990
)
Changes in keratinocyte adhesion during terminal differentiation reduction in fibronectin binding precedes α5 β 1 integnn loss from the cell surface
Cell
63
,
425
435
Adams
,
J C
and
Watt
,
F M
(
1991
)
Expression of β 1, β 3, and β 4 integnns by human epidermal keratinocytes and non-differentiating keratinocytes (ndk)
J Cell Biol Submitted
Akiyama
,
S K
,
Yamada
,
S S
,
Chen
,
W-T
and
Yamada
,
K M.
(
1989
)
Analysis of fibronectin receptor function with monoclonal antibodies, roles in cell adhesion, migration, matnx assembly, and cytoskeletal organization
J Cell Biol
109
,
863
875
Belkin
,
V M
,
Belkin
,
A M
and
Koteuansky
,
V E.
(
1990
).
Human smooth muscle VLA-1 integrin punfication, substrate specificity, localization in aorta, and expression during development
J Cell Biol
111
,
2159
2170
Bodary
,
S C.
and
Mclean
,
J W.
(
1990
).
The integnn subunit associates with the vitronectin receptor αv, subunit to form a novel vitronectin receptor in a human embryonic kidney cell hne
J. biol Chem
265
,
5938
5941
Bohnert
,
A
,
Hornung
,
J
,
Mackenzie
,
I C
and
Fusenig
,
N E
(
1986
)
Epithelial-mesenchymal interactions control basement membrane production and differentiation in cultured and transplanted mouse keratinocytes
Cell Tissue Res
244
,
413
429
Carter
,
W G
,
Kaur
,
P
,
Gil
,
S G
,
Gahr
,
P J
and
Wayner
,
E A
(
1990a
)
Distinct functions for integnns α3 β 1 in focal adhesions and α6 β 4 /bullous pemphigoid antigen in a new stable anchonng contact (SAC) of keratinocytes relation to hemidesmosomes
J Cell Biol
111
,
3141
3154
Carter
,
W G
,
Wayner
,
E A.
,
Bouchard
,
T S
and
Kaur
,
P
(
1990b
)
The role of integrins and in cell-cell and cell-substrate adhesion of human epidermal cells
.
J Cell Biol
110
,
1387
1404
Cheresh
,
D A
,
Smith
,
J W
,
Cooper
,
H M
and
Quaranta
,
V
(
1989
)
A novel vitronectin receptor integnn (αv β x) is responsible for distinct adhesive properties of carcinoma cells
.
Cell
57
,
59
69
Clarke
,
RAF
,
Folkvord
,
J M
and
Wertz
,
R L
(
1985
)
Fibronectin, as well as other extracellular matnx proteins, mediate human keratinocyte adherence
J invest Dermatol
.
85
,
378
383
Deluca
,
M
,
Tamura
,
R. N
,
Kajui
,
S
,
Bondanza
,
S.
,
Rossino
,
P
,
Cancedda
,
R
,
Marchisio
,
P C
and
Quaranta
,
V
(
1990
).
Polanzed integnn mediates human keratinocyte adhesion to basal lamina
Proc natn Acad Set USA
87
,
6888
6892
De Strooper
,
B
,
Van Der Schueren
,
B
,
Jaspers
,
M
,
Saison
,
M.
,
Spaepen
,
M.
,
Van Leuven
,
F
,
Van Den Berghe
,
H
and
Cassiman
,
J -J
(
1989
).
Distnbution of the β 1 subgroup of the integnns in human cells and tissues
J Histochem Cytochem
37
,
299
307
De Strooper
,
B
,
Vekemans
,
S
,
Carmeliet
,
G
,
Jaspers
,
M
,
Van Der Schueren
,
B
,
Wu
,
R R
and
Cassiman
,
J-J.
(
1990
)
mAb 8D9 reacts with α5 -integnn and stains the stratum spinosum of adult skin
Cell Biol Int Rep
14
,
233
Elices
,
M. J.
and
Hemler
,
M E
(
1989
)
The human integnn VLA-2 is a collagen receptor on some cells and a collagen/lamimn receptor on others Proc natn
.
Acad Set U SA
86
,
9906
9910
Fine
,
J-D
,
Smith
,
L. T
,
Holbrook
,
K A
and
Katz
,
S I
(
1984
)
The appearance of four basement membrane zone antigens in developing human fetal skin
.
J. invest Dermatol
.
83
,
66
69
Fisher
,
C
and
Holbrook
,
K A
(
1987
)
Cell surface and cytoskeletal changes associated with epidermal stratification and differentiation in organ cultures of embryonic human skin
Devi Biol
119
,
231
241
Fleischmajer
,
R.
and
Timpl
,
R
(
1984
)
Ultrastructural localization of fibronectin to different anatomic structures of human skin
J Histochem Cytochem
32
,
315
321
Fradet
,
Y
,
Cordon-Cardo
,
C
,
Thomson
,
T
,
Daly
,
M. E
,
Whitmore
,
W F.
,
Lloyd
,
K O
,
Melamed
,
M R
and
Old
,
L J
(
1984
)
Cell surface antigens of human bladder cancer defined by mouse monoclonal antibodies
Proc natn. Acad Set USA
81
,
224
228
Gehlsen
,
K R
,
Dickerson
,
K
,
Argraves
,
W. S
,
Engvall
,
E
and
Ruoslahti
,
E
(
1989
)
Subunit structure of a lamimn-binding integnn and localization of its binding site on laminin
J biol Chem
264
,
19034
19038
Hall
,
D E
,
Reichardt
,
L F
,
Crowley
,
E
,
Holley
,
B
,
Moezzi
,
H
,
Sonnenberg
,
A
and
Damsky
,
C H
(
1990
)
The α1 / β 1 and α6 / β 1 integrin heterodimers mediate cell attachment to distinct sites on laminin
J Cell Biol
110
,
2175
2184
Hemler
,
M E
(
1990
)
VLA proteins in the integnn family structures, functions and their role on leukocytes
Annu Rev Immunol
8
,
365
400
Hemler
,
M E
,
Crouse
,
C
and
Sonnenberg
,
A
(
1989
)
Association of the VLA α6, subunit with a novel protein A possible alternative to the common VLA subunit on certain cell lines
J biol Chem
264
,
6529
6535
Hemler
,
M E
,
Huang
,
C
,
Takada
,
Y
,
Schwarz
,
L
,
Strominger
,
J L
and
Clabby
,
M L
(
1987
)
Characterization of the cell surface heterodimer VLA-4 and related peptides
J biol Chem
262
,
11478
11485
Hemler
,
M E
,
Sanchez-Madrid
,
F
,
Flotte
,
T J
,
Krensky
,
A M
,
Burakoff
,
S J
,
Bhan
,
A K
,
Springer
,
T A
and
Strominger
,
J L
(
1984
)
Glycoproteins of 210,000 and 130,000 M.W on activated T cells cell distnbution and antigenic relation to components on resting cells and T cell lines
J Immunol
132
,
3011
3018
.
Holbrook
,
K A
(
1983
) Structure and function of the developing human skin
In Biochemistry and Physiology of the Skm
, vol
1
(ed
L A
Goldsmith
), pp
64
101
Oxford Oxford University Press
Holbrook
,
K A
and
Odland
,
G F
(
1980
)
Regional development of the human epidermis in the first trimester embryo and the second trimester fetus (ages related to the timing of amniocentesis and fetal biopsy)
J invest Dermatol
80
,
161
168
Horton
,
M A
,
Lewis
,
D
,
Mcnulty
,
K
,
Pringle
,
JAS
and
Chambers
,
T J
(
1985
)
Monoclonal antibodies to osteoclastomas (giant cell bone tumors): definition of osteoclast-specific cellular antigens
Cancer Res
45
,
5663
5669
Hynes
,
R O.
(
1987
)
Integrins a family of cell surface receptors
Cell
48
,
549
554
Ignatius
,
M J
and
Reichardt
,
L F
(
1988
)
Identification of a neuronal laminin receptor: an Mr 200K/120K integnn heterodimer that binds laminin in a divalent cation-dependent manner
Neuron
1
,
713
725
Kajui
,
S M
,
Davceva
,
B
and
Quaranta
,
V
(
1987
)
Six monoclonal antibodies to human pancreatic cancer antigens
Cancer Res
47
,
1367
1376
Kantor
,
R R S
,
Mattes
,
M. J
,
Lloyd
,
K O
,
Old
,
L J
and
Albino
,
A P.
(
1987
)
Biochemical analysis of two cell surface glycoprotein complexes, very common antigen 1 and very common antigen 2
J biol Chem
262
,
15 158
15 165
Kaufmann
,
R
,
Frosch
,
D
,
Westphal
,
C.
,
Weber
,
L
and
Klein
,
C. E
(
1989
)
Integnn VLA-3 ultrastructural localization at cell-cell contact sites of human cell cultures
J Cell Biol
109
,
1807
1815
Kennel
,
S J
,
Foote
,
L J
,
Falaoni
,
R
,
Sonnenberg
,
A
,
Stringer
,
C D
,
Crouse
,
C
and
Hemler
,
M E
(
1989
)
Analysis of the tumor-associated antigen TSP-180
J biol Chem
264
,
15515
15521
.
Kirchhofer
,
D
,
Languino
,
L. R
,
Ruoslahti
,
E
and
Pierschbacher
,
M D
(
1990
)
α2 β 1 integnns from different cell types show different binding specificities
J biol Chem
265
,
615
618
Korhonen
,
M
,
Ylanne
,
J
,
Laitinen
,
L
and
Virtanen
,
I
(
1990
).
The α2 6 subunits of integnns are charactenstically expressed in distinct segments of developing and adult human nephron
J Cell Biol
111
,
1245
1254
Kramer
,
R. H.
and
Marks
,
N.
(
1989
)
Identification of integnn collagen receptors on human melanoma cells
.
J. biol Chem
.
264
,
4684
4688
Kurpakus
,
M A
,
Quaranta
,
V
,
Cooper
,
H M
and
Jones
,
J. C R
(
1990
)
Integnns in the hemidesmosome
J Cell Biol
.
111
,
402a
Lane
,
A T
,
Helm
,
K F
and
Goldsmith
,
L A
(
1985
)
Identification of bullous pemphigoid, pemphigus, laminin, and anchonng fibnl antigens in human fetal skm
J invest Dermatol
84
,
27
30
Languino
,
L R
,
Gehlsen
,
K R
,
Wayner
,
E
,
Carter
,
W G
,
Engvall
,
E
and
Ruoslahti
,
E
(
1989
)
Endothelial cells use α2 β 1 integnn as a laminin receptor
J Cell Biol
109
,
2455
2462
Larjava
,
H
,
Peltonen
,
J
,
Akiyama
,
S K
,
Yamada
,
S S
,
Galnick
,
H R
,
Urrro
,
J
and
Yamada
,
K M
(
1990
)
Novel function for β 1 integnns in keratinocyte cell-cell interactions
J Cell Biol
110
,
803
815
Matoltsy
,
A G
(
1986
) Structure and function of the mammahan epidermis
In Biology of the Integument
, vol
2
vertebrates (ed
J
Bereiter-Hahn
,
A G
Matoltsy
, and
K S
Richards
), pp
255
271
Berlin
Sponger Verlag
Menko
,
A S
and
Boettiger
,
D
(
1987
)
Occupation of the extracellular matnx receptor, integnn, is a control point for myogenic differentiation
Cell
51
,
51
57
.
Nazzaro
,
V
,
Berti
,
E
,
Cerri
,
A
,
Brusasco
,
A
,
Cavalu
,
R
and
Caputo
,
R
(
1990
).
Expression of integnns in junctional and dystrophic epidermolysis bullosa
J invest Dermatol
95
,
60
64
Nicholson
,
L J
and
Watt
,
F M
(
1991
)
Decreased expression of fibronectin and the α5 β 1 integnn dunng terminal differentiation of human keratinocytes
J Cell Sci
98
,
225
232
.
Patel
,
V P
and
Lodish
,
H. F
(
1987
)
A fibronectin matnx is required for differentiation of munne erythroleukemia cells into reticulocytes
J Cell Biol
105
,
3105
3118
.
Peltonen
,
J
,
Larjava
,
H
,
Jaakkola
,
S
,
Gralnick
,
H
,
Akiyama
,
S K
,
Yamada
,
S S
,
Yamada
,
K M
and
Urrro
,
J
(
1989
)
Localization of integnn receptors for fibronectin, collagen, and laminin in human skin
J chn Invest
84
,
1916
1923
Ramaswamy
,
H
and
Hemler
,
M E
(
1990
)
Cloning, pnmary structure and properties of a novel human integnn )3 subunit
EMBO J
9
,
1561
1568
Reilly
,
J T
and
Nash
,
J R
(
1988
)
Vitronectin (serum spreading factor) its localization in normal and fibrotic tissue
J chn Pathol
41
,
1269
1272
Robledo
,
M A
,
Kjm
,
S. C
,
Korman
,
N J
,
Stanley
,
J R
,
Labib
,
R S
,
Futamura
,
S
and
Anhalt
,
G J
(
1990
)
Studies of the relationship of the 230-kD and 180-kD bullous pemphigoid antigens
J invest Dermatol
94
,
793
797
Sanes
,
J R
,
Engvall
,
E
,
Butkowski
,
R
and
Hunter
,
D
(
1990
)
Molecular heterogeneity of basal laminae isoforms of laminin and collagen IV at the neuromuscular junction and elsewhere
J Cell Btol
111
,
1685
1699
Santoro
,
S A
,
Rajpara
,
S M
,
Staatz
,
W D
and
Woods
,
V L
(
1988
)
Isolation and charactenzation of a platelet surface collagen binding complex related to VLA-2
Biochem biophys Res Commun
153
,
217
223
Smith
,
L T
,
Holbrook
,
K A.
and
Maori
,
J A
(
1986
).
Collagen types I, III, and V in human embryonic and fetal skin
Am J Anat
175
,
507
521
Sonnenberg
,
A
,
Daams
,
H
,
Van Der Valk
,
M A
,
Hilken
,
J
and
Hilgers
,
J
(
1986
)
Development of mouse mammary gland identification of stages in differentiation of luminal and myoepithelial cells using monoclonal antibodies and polyvalent antiserum against keratin
J Htstochem Cytochem
34
,
1037
1046
Sonnenberg
,
A
,
Linders
,
C J T
,
Daams
,
J H
and
Kennel
,
S J.
(
1990a
)
The α6 β 1 (VLA-6) and α6 β 4 protein complexes tissue distribution and biochemical properties
J Cell Set
96
,
207
217
Sonnenberg
,
A
,
Linders
,
C J. T
,
Modderman
,
P W
,
Damsky
,
C. J.
,
Aumailley
,
M
and
Timpl
,
R
(
1990b
)
Integnn recognition of different cell-binding fragments of laminin (Pl, E3, E8) and evidence that α6 β 1 but not α6 β 4 functions as a major receptor for fragment E8
J Cell Biol
110
,
2145
2155
.
Sorokin
,
L
,
Sonnenberg
,
A
,
Aumailley
,
M
,
Timpl
,
R
and
Ekblom
,
P.
(
1990
)
Recognition of the laminin E8 cell-binding site by an integnn possessing the ag subunit is essential for epithelial polarization in developing kidney tubules
.
J Cell Biol
111
,
1265
1273
Staatz
,
W D
,
Rajpara
,
S. M
,
Wayner
,
E. A
,
Carter
,
W G
and
Santoro
,
S A
(
1989
)
The membrane glycoprotein la-IIa (VLA-2) complex mediates the Mg++-dependent adhesion of platelets to collagen
J Cell Biol
108
,
1917
1924
Staquet
,
M J
,
Levarlet
,
B
,
Dezutter-Dambuyant
,
C
,
Schmitt
,
D
and
Thivolet
,
J
(
1990
)
Identification of specific human epithelial cell integnn receptors as VLA proteins
Expl Cell Res
187
,
277
283
.
Stenman
,
S
and
Vaheri
,
A
(
1978
)
Distribution of a major connnective tissue protein, fibronectin, in normal human tissues
J exp Med
147
,
1054
1064
Stepp
,
M A.
,
Spurr-Michaud
,
S
,
Tisdale
,
A
,
Elwell
,
J
and
Gipson
,
I K
(
1990
)
α6 β 4 integnn heterodimer is a component of hemidesmosomes
Proc natn Acad Sci USA
87
,
8970
8974
.
Takada
,
Y
,
Wayner
,
E A
,
Carter
,
W G
and
Hemler
,
M E
(
1988
)
Extracellular matnx receptors, ECMRII and ECMRI, for collagen and fibronectin corresjxind to VLA-2 and VLA-3 in the VLA family of heterodimers
J cell Biochem
37
,
385
393
Toda
,
K-I
,
Tuan
,
T-L
,
Brown
,
P J
and
Grinnell
,
F
(
1987
)
Fibronectin receptors of human keratinocytes and their expression dunng cell culture
J Cell Biol
105
,
3097
3104
Vogel
,
B. E
,
Tarone
,
G.
,
Giancotti
,
F. G
,
Gahjt
,
J
and
Ruoslahti
,
E
(
1990
)
A novel fibronectin receptor with an unexpected subunit composition (oyA)
J biol Chem
.
265
,
5934
5937
VON DEM
Borne
,
Aeg
,
KR
,
Modderman
,
P W
,
Admiraal
,
L G
and
Nieuwenhuis
,
J K
(
1989
)
In Leucocyte Typing IV
(ed
W
Knapp
, et al. 
), p
951
.
Oxford Oxford University Press
Warburton
,
M J.
,
Ferns
,
S A
and
Rudland
,
P S
(
1982
)
Enhanced synthesis of basement membrane proteins dunng the differentiation of rat mammary tumor epithehal cells into myoepithehal-hke cells in vitro
Expl Cell Res
137
,
373
380
.
Watt
,
F M
,
Jordan
,
P W
and
O’Neill
,
C H
(
1988
)
Cell shape controls terminal differentiation of human epidermal keratinocytes
Proc natn Acad Sci USA
85
,
5576
5580
Watt
,
F M
,
Keeble
,
S
,
Fisher
,
C
,
Hudson
,
D L
,
Codd
,
J
and
Salisbury
,
J R
(
1989
)
Onset of expression of peanut lectin-binding glycoproteins is correlated with stratification of keratinocytes dunng human epidermal development in vivo and in vitro
J Cell Sci
94
,
355
359
Wayner
,
E A
and
Carter
,
W G
(
1987
).
Identification of multiple cell adhesion receptors for collagen and fibronectin in human fibrosarcoma cells possessing unique alpha and common beta subunits
J Cell Biol
105
,
1873
1884
Wayner
,
E A
,
Carter
,
W G
,
Piotrowicz
,
R S
and
Kunicki
,
T J
(
1988
)
The function of multiple extracellular matnx receptors in mediating cell adhesion to extracellular matnx. preparation of monoclonal antibodies to the fibronectin receptor that specifically inhibit cell adhesion to fibronectin and react with platelet glycoproteins Ic-IIa
J Cell Biol
107
,
1881
1891
Wayner
,
E A
,
Garcia-Pardo
,
A
,
Humphries
,
M J.
,
Mcdonald
,
J A
and
Carter
,
W G
(
1989
)
Identification and charactenzation of the T lymphocyte adhesion receptor for an alternative cell attachment domain (CS-1) in plasma fibronectin
J Cell Biol
109
,
1321
1330
Zutter
,
M M
and
Santoro
,
S. A.
(
1990
).
Widespread histologic distnbution of the α2 β 1 integnn cell-surface collagen receptor
Am J Path
137
,
113
120
Zylstra
,
S
,
Chen
,
F A
,
Ghosh
,
S K
,
Repasky
,
E A
,
Rao
,
U
,
Takita
,
H.
and
Bankert
,
R B
(
1986
)
Membrane-associated glycoprotein (gp 160) identified on human lung tumors by a monoclonal antibody
Cancer Res
46
,
6446
6451