Correspondence to: Elaine Fuchs, Howard Hughes Medical Institute, Dept. of Molecular Genetics and Cell Biology, The University of Chicago, 5841 S. Maryland Ave., Rm. N314, Chicago, IL 60637. Tel:(773) 702-1347 Fax:(773) 702-0141 E-mail:lain{at}midway.uchicago.edu.
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Abstract |
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The major epidermal integrins are 3ß1 and hemidesmosome-specific
6ß4; both share laminin 5 as ligand. Keratinocyte culture studies implicate both integrins in adhesion, proliferation, and stem cell maintenance and suggest unique roles for
ß1 integrins in migration and terminal differentiation. In mice, however, whereas ablation of
6 or ß4 results in loss of hemidesmosomes, epidermal polarity, and basement membrane (BM) attachment, ablation of
3 only generates microblistering due to localized internal shearing of BM. Using conditional knockout technology to ablate ß1 in skin epithelium, we have uncovered biological roles for
ß1 integrins not predicted from either the
3 knockout or from in vitro studies. In contrast to
3 null mice, ß1 mutant mice exhibit severe skin blistering and hair defects, accompanied by massive failure of BM assembly/organization, hemidesmosome instability, and a failure of hair follicle keratinocytes to remodel BM and invaginate into the dermis. Although epidermal proliferation is impaired, a spatial and temporal program of terminal differentiation is executed. These results indicate that ß1's minor partners in skin are important, and together,
ß1 integrins are required not only for extracellular matrix assembly but also for BM formation. This, in turn, is required for hemidesmosome stability, epidermal proliferation, and hair follicle morphogenesis. However, ß1 downregulation does not provide the trigger to terminally differentiate.
Key Words: integrins, epidermis, conditional, knockout, proliferation, skin
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Introduction |
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Integrins are heterodimeric transmembrane receptors composed of an regulatory subunit and a ß signal transducing subunit (for review see
Integrin heterodimers containing the ß1 subunit are broadly expressed in many cell types, and ß1 is promiscuous, associating with many partners. The
subunit imparts ligand specificity, enabling the heterodimer to bind to specific ECM or basement membrane (BM) components. Cultured fibroblast studies with the fibronectin receptor,
5ß1, suggest that upon ligand engagement, ß1's short (50 amino acid) cytoplasmic domain binds to proteins that in turn associate with and reorganize actin filaments to form focal adhesions. Upon further activation of Rho GTPases, changes in the actin cytoskeleton lead to integrin clustering, which facilitates the polymerization and assembly of ECM on the cell surface and enables stable substratum attachment (
6ß4 associates with transmembrane collagen XVII (BPAG2) and with two intermediate filament linker proteins, plectin and BPAG1 (for review see
In the epidermis and its appendages, basal keratinocytes utilize integrins to adhere to their underlying BM, rich in ECM (for review see 3ß1 and
6ß4, both of which bind laminin 5, the major ECM component of the BM (
2ß1 (collagen/laminin),
5ß1 (fibronectin), and the wound healinginduced integrin
vß5. Whereas
6ß4 and hemidesmosomes are restricted to the basal surface of epidermis,
ß1 heterodimers are not polarized (
The functions of 6ß4 in mice have been explored through gene targeting (
6 or ß4 is missing, the partner is unstable, leading to a loss of the heterodimer. Hemidesmosomes are absent, and epidermal adhesion to the underlying BM is seriously impaired. Upon mild mechanical stress, the epidermis peels from its underlying substratum, a condition in humans known as junctional epidermolysis bullosa, also caused by mutations in laminin 5 (for review see
In contrast, mice deficient in 3 integrin exhibit a mild skin phenotype, with an epidermis that is normal in morphology, thickness, proliferation, and terminal differentiation, and that displays no overt signs of hair follicle defects (
3ß1, laminin 5 assembly may be perturbed.
The loss of 3 still leaves ß1 with several partners thought to be minor in normal skin, but in fact, fibronectin receptor (
5ß1) and collagen IV receptor (
2ß1) activities are increased in cultured
3-deficient keratinocytes (
ß1 integrins in skin extends beyond merely a role for
3ß1 in laminin 5 assembly and/or BM integrity. Furthermore, there are considerable differences between the relatively mild skin defects in the
3 knockout mouse and the many different putative functions ascribed to ß1 from cell culture studies. This begs the question of what might happen when ß1 is missing, leaving
2,
3, and
5 without partners. The ß1 knockout is lethal in the early embryo, making it impossible to assess its role in skin (
Using conditional knockout technology, we now explore the in vivo function of ß1 integrin in epidermis and its appendages. The phenotype of ß1 null epidermis is markedly distinct from that seen in other knockouts of epidermally expressed integrins and could not have been predicted based upon prior in vivo or in vitro studies conducted on the role of ß1 integrins in skin. Most notable are a near complete loss of BM, a reduction in hemidesmosomes, a severely impaired proliferative compartment in the epidermis, and failure of developing hair follicles to invaginate into the underlying dermis. Surprisingly, however, the spatial and temporal program of terminal differentiation is preserved. Taken together, our results implicate
ß1 integrins in controlling proliferative potential in the epidermis by virtue of their ability to organize and assemble a BM at the DEJ. In this regard, they also are necessary for maintaining hemidesmosomes, but they are not required for maintaining the gene expression program that defines a keratinocyte, nor is their downregulation a trigger to induce terminal differentiation. Finally, our data suggest that
ß1 integrins play a major role in the BM remodeling and keratinocyte migration that is essential for hair follicle morphogenesis.
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Materials and Methods |
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Engineering ß1 Conditional Knockout Mice
The ß1 integrin gene was isolated from mouse RW4 genomic DNA, and a 5-kb BamHI restriction endonuclease fragment was subcloned and used for preparation of the targeting vector. Electroporations of DNAs into RW4 Agouti ES cells were carried out at 270 V, 500 mF in a GenePulser (Bio-Rad Laboratories). ES cells harboring the desired recombinations were injected into mouse C57BL blastocysts, which were then transferred to CD1 mothers. After breeding, heterozygous and homozygous mice were identified by PCR analysis of toe skin DNAs.
Histology and Immunofluorescence
For routine histology, tissues were fixed in Bouin's fixative, processed, and embedded in paraffin. Sections (5 µm) were stained with hematoxylin and eosin and examined and photographed using an Axiophot microscope (Carl Zeiss, Inc.). For immunofluorescence, frozen sections of tissues or cells on the glass coverslips were fixed in 4% paraformaldehyde in PBS for 10 min and were subjected to indirect immunostaining (
Unless otherwise indicated, primary antibodies were polyclonal and raised in rabbits. Antibodies and dilutions used were: rat monoclonal ß1 (1:100), 3 (1:100), rat monoclonal
4 (1:50), rat monoclonal
6 (1:100) (Chemicon); K1 (1:200), loricrin (1:250), filaggrin (1:2000) (BabCo); laminin (1:200), mouse monoclonal Ki67 (1:100) (Sigma-Aldrich); K17 (1:1000; gift of P. Coulombe, Johns Hopkins University School of Medicine, Baltimore, MD); guinea pig polyclonal K5 (1:300); and Lef1 (1:250). Fluorescence-conjugated secondary antibodies were from Jackson ImmunoResearch Laboratories. DAPI was used to stain nuclei.
Ultrastructural Analyses
Skin tissues were processed for conventional electron microscopy by fixing in 2% glutaraldehyde in 0.05 M cacodylate buffer, 2 mM CaCl2, pH 7.4, followed by a second fixation with 1% OsO4 in water for 60 min on ice. Ultrathin sections on copper grids were treated with uranyl acetate and lead citrate and were examined with a Phillips CM120 electron microscope.
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Results and Discussion |
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Generation of Mutant Mice Conditionally Targeted for ß1 Gene Inactivation in Skin Epithelium
To conditionally inactivate the ß1 gene in skin, we engineered the targeting vector to contain loxP sequences flanking the third exon (referred to as floxed), which once removed would produce an early frame shift, translation termination, and quantitative loss of ß1 protein. A restriction map of the WT allele, the targeting vector, and the mutated allele is shown in Fig 1 A. This vector was used to generate a single homologous recombination event in three independently derived RW4 ES clones, and this was confirmed by Southern analysis (Fig 1 B, 5' probe).
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To delete the floxed neo gene, two ES clones were transiently transfected with a Cre recombinase gene under the control of the cytomegalovirus (CMV) promoter. By Southern analysis, the desired clones lacked the PGK-neo gene but still harbored the floxed exon 3 (Fig 1 B, exon3 probe). ES technology was then employed to produce germline homozygous mice, and PCR analyses were used to verify that the targeting of both ß1 alleles had been successful (Fig 1 C, before K14-Cre).
The homozygous floxed animals appeared normal, indicating that the genomic manipulations had not interfered with ß1 function. We then bred the mice to generate newborn animals that were both transgenic for K14-Cre and homozygous for the floxed ß1 exon. PCR confirmed the successful conditional removal of exon 3 in skin and the presence of the K14-Cre transgene (Fig 1 C, after K14-Cre). Previously, we documented the near absolute efficiency and specificity of K14-Cremediated recombination in skin epithelial stem cells and their progeny (-E-catenin) expressed in all transcriptionally active epithelial cells of the skin (
Conditional ß1 Null Mice Display Extremely Severe Skin Blistering
The phenotype of newborn conditional ß1 null (KO) mice was unmistakable. These animals displayed thin and fragile skin, leading to separation at the DEJ upon mechanical trauma (Fig 2 A). However, in contrast to 6 or ß4 null mice, which exhibited extensive epidermal denuding (
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ß1 null animals usually died within a few hours after birth, most likely due to a loss of the epidermal barrier required to prevent dehydration and death (
Conditional ß1 Null Skin Displays Separations at the DEJ, Changes in Epidermal Morphology, and Impairment of Hair Follicle Downgrowth
Sections of newborn backskin (bs) of conditional ß1 null animals revealed gross abnormalities in the epithelium. In WT bs, epidermis consists of four morphologically distinct stages of differentiation, with a mitotically active basal layer (BL) of columnar keratinocytes, three to four spinous (SP) layers, and one to two granular (GR) layers of transcriptionally active but terminally differentiating cells, and a stratum corneum (SC) of dead, enucleated squames (Fig 2 B). In contrast, ß1 null epidermis was thinner, consisting of a flattened BL and only one or two layers of suprabasal layers before the SC (Fig 2C, Fig D, and Fig D'). Due in part to the altered morphology of the BL, it was often difficult to discern the boundary between ß1 null epidermis and dermis.
Many areas of ß1 null skin showed extensive separations at the DEJ (Fig 2 C, double arrow). In severely affected areas, sections of thin, flat epidermis detached entirely from dermis (Fig 2 D'). In contrast, no morphological abnormalities were reported in the epidermis of 3 null skin (
In animals where ß1 null bs was very thin (e.g., Fig 2 D'), thickness differences were not as pronounced between WT and ß1 null paw skin (Fig 2E and Fig F; dorsal surface shown). However, morphological perturbations were still prevalent and included an increase in the density of cells within the SP layers (brackets). As the surface area of ß1 null pawskin appeared to be normal and because mitotic indexes were not detected suprabasally, we surmise that the suprabasal ß1 null cells may not be as metabolically active as their WT counterparts, leading to a reduction in cell size and increase in cell density.
On the dorsal (haired) side of ß1 null paw, follicles did not extend into the dermis. Instead, epithelial masses were seen within the epidermis at comparable spacing to hair follicles and with morphology similar to the early stages of this process (Fig 2 F, single arrows and asterisk). Later, we provide further evidence to support this notion. Mutant bs also exhibited a paucity of developing hair follicles relative to WT littermate skin (Fig 2, compare frame B with C, D, and D'). A few mature hair follicles were detected, and these most likely represented guard hairs, which develop first during embryonic development (not shown). Although these severely affected mice did not survive to develop a hair coat, a near quantitative absence of hair coat would have been predicted based upon this morphology. This could be seen in juvenile animals expressing lower Cre, where their mosaic ß1 null skin resulted in alternating stripes of bald skin (Fig 2 A').
Loss of BM at the DEJ and Abnormal Distribution of Integrins in Conditional ß1 Null Epidermis
The gross morphological abnormalities at the DEJ of ß1 null epidermis were also reflected at the immunofluorescence level (Fig 3). As expected from the activity of our high-expressing K14-Cre recombinase mice (see above), antibodies against ß1 showed no staining in the newborn epidermis of mutant skin but exhibited normal staining in the underlying dermis (Fig 3 A and A', WT; and Fig 3 B, B', and B'', KO).
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In normal skin, the BM at the DEJ stains as a continuous line with a variety of antibodies, including laminins, collagen IV, and fibronectin (Fig 3; examples shown are laminin 5 in A and a panel laminin in A'). In ß1 null skin, however, all of these antibodies showed highly discontinuous staining patterns, often with long stretches of little or no laminin 5 staining at the DEJ (Fig 3B and Fig B'). Remarkably, as judged by immunofluorescence, the majority of BM proteins resided in the upper dermis rather than the BM (Fig 3 B). In the few regions where anti-ECM labeling was detected near or at the DEJ, blistering and splits were often seen, with staining on both sides of the split (Fig 3 B'', double arrow).
Consistent with the well established staining patterns for epidermal integrin antibodies, ß1 and its partners localized at the perimeter of WT basal cells (Fig 3A, A', and C; shown are ß1 and 3). As expected, whereas
3 was restricted to the epidermal BL, ß1 was present in dermal fibroblasts (Fig 3A and A'). In addition, antibodies against ß4 and
6 stained only the base of WT basal epidermal cells, i.e., at the DEJ (Fig 3C and Fig E, respectively).
Remarkably, the patterns of all integrins were markedly perturbed in ß1 mutant skin. It was already known from in vitro studies (for example see partners are unstable, and hence we both anticipated and observed an absence of staining with antibodies against
3 (Fig 3 D). However, given that
6ß4 is localized to hemidesmosomes whereas
ß1 integrins are not, we were very surprised to see that in many regions of ß1 null epidermis, anti-ß4 and anti-
6 staining was weak and/or discontinuous (Fig 3D and Fig F, arrowheads). The loss of
6ß4 was seen irrespective of whether separations were visible at the DEJ, and in most severely affected ß1 null newborn mice analyzed, only a few skin regions could be found where antibody staining appeared normal (see left sides of Fig 3D and Fig F). Thus far, we have not found evidence for induction of potential compensatory integrins when ß1 is ablated in the skin.
Perturbations in laminin 5, 6, and ß4 antibody stainings were not seen in
3 KO skin (
6ß4 staining were still localized to the DEJ, even though ß1 was absent (for example, see area at right of Fig 3 G). Taken together, these findings suggest that at early times after the ß1 integrin gene is mutated, the BM and hemidesmosomes remain intact, but soon afterwards, BM assembly is compromised, leading to a loss of ECM and hemidesmosomes at the DEJ.
Previous in vitro studies have indicated a role for 5ß1 integrins in the assembly of fibronectin (
3ß1 in laminin 5 assembly has been postulated based upon the occasional areas of BM perturbations and microblistering seen in the
3 knockout mouse (
ß1 integrins, not only in ECM assembly/organization but also in assembly of the BM. Thus, in striking contrast to
3 null skin, where most of the laminin 5 still resided at an intact BM (
3 knockouts lead us to wonder whether one of the minor
ß1 integrins also participates in laminin 5 assembly or whether properly assembled collagen and fibronectin provide a BM scaffold to support laminin 5 and retain it at the DEJ.
Marked Ultrastructural Differences Between ß1 Integrin Null and WT Epidermis
To gain further insights into the perturbations we have described thus far, we performed ultrastructural analyses on skin of WT littermate and conditional ß1 null mice (Fig 4). WT basal cells exhibit a classical columnar morphology, with nuclei oriented perpendicularly to the BM (Fig 4 A). At the mesenchymalepithelial junction of WT skin is a BM, composed of a lamina lucida at the base of the epidermis, and a parallel lamina densa (LD) of ECM beneath it (Fig 4A and Fig C). Contiguous with the lamina lucida are numerous electron dense hemidesmosomal plaques. Tiny anchoring filaments, in part composed of collagen XVII, extend from the base of each hemidesmosome to the LD (
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In ß1 null epidermis of a mouse that survived to four days, most basal cells were flat, and the nucleus was oriented parallel to the BM (Fig 4 B). In regions where ß1 null epidermis was attached to its underlying BM, hemidesmosomes were present (Fig 4B and Fig D). Although their morphology was indistinguishable from control skin, these structures were markedly reduced in number, and often long stretches along the base of each basal cell lacked discernible hemidesmosomes. Another marked difference between WT and KO skin was the discontinuity of the electron dense LD, which existed beneath the hemidesmosomes but not in most areas between hemidesmosomes. In many areas, neither hemidesmosomes nor LD were detected. In these regions, what appeared to be disorganized remnants of ECM dangled from the underlying surface of the basal epidermal layer (Fig 4 D, double arrows). These long stretches of disorganized BM were typical of conditional ß1 null animals that survived for several days (examples shown), but in more severely affected animals, even traces of ECM material at the DEJ were rare (not shown).
In ß1 null animals that still showed some skin areas with traces of BM, ECM material, and hemidesmosomes at the DEJ, it was evident that where the DEJ separated, splits occurred within the disorganized BM (Fig 4 E). This left fragments of ECM at both the base of the epidermis and the upper surface of the dermis. In addition, the hemidesmosomes often contained an intact LD beneath them, even though elsewhere LD was not detected (Fig 4 E'). We surmise that through the ability of hemidesmosomes to anchor to and maintain organized BM through their collagen XVII anchoring fibers, these areas of LD are the last to survive in the ß1 null epidermis.
The dramatic reduction in hemidesmosomes, not seen in the 3 knockout but a prominent feature of the ß1 null epidermis, suggests strongly that
6ß4 relies upon
ß1 integrins for BM assembly, and without a BM, they are unable to assemble into stable hemidesmosomes. Although the progressive loss of hemidesmosomes and
6ß4 antibody staining would seem to favor this hypothesis, we cannot exclude the possibility that hemidesmosome assembly and/or stability might be governed by an
ß1-regulated intracellular signaling pathway. In support of this view is the finding that in vitro hemidesmosomes can be formed upon adhesion of
6ß4-expressing cells to fibronectin, the receptor for
5ß1 (
ß1 and
ß4 integrins.
Terminal Differentiation Is Spatially and Temporally Maintained in ß1 Null Epidermis, but Proliferation Is Impaired
Epidermal cells downregulate integrin expression as they detach from the BM, terminally differentiate, and move outward towards the skin surface. Based upon a number of gene transfection and keratinocyte suspension studies, investigators have postulated that downregulation of ß1 integrin expression may be a trigger for inducing terminal differentiation (
In normal epidermis, an early hallmark of terminal differentiation is the switch from expression of keratins 5 and 14 to keratins 1 and 10 (Fig 5 A;
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Expression of late stage markers of epidermal differentiation, including loricrin and filaggrin, were also faithful in their gene expression patterns (Fig 5, CF), despite the fact that some differences in epidermal morphology had been noted in the upper skin layers (Fig 2). Finally, cornified envelopes, an end-stage product of terminal differentiation, appeared morphologically normal when isolated from ß1 null skin and examined under the light microscope (data not shown; for procedures see partners, basal epidermal cells still maintained their basal-like properties and refrained from premature execution of the program of terminal differentiation. This result argues against the long-standing hypothesis that downregulation of ß1 integrin is a molecular trigger of terminal differentiation (
Another hypothesis regarding the possible function of ß1 integrins is that they control proliferation in epidermal cells (for review see
6ß4 in regulating epidermal proliferation (
In the most severely affected conditional ß1 null mice, few mitoses were detected in the basal epidermal layer. In contrast, 5% of basal cells in littermate skin displayed mitotic figures. Antibodies against Ki67, a proliferating nuclear antigen present throughout the cell cycle, permitted more rigorous examination of proliferation within the newborn epidermis. The majority of WT basal cells were Ki67 positive, and labeled cells were especially abundant in hair follicles (Fig 5 G). In contrast, very few Ki67-positive cells were found in ß1 null newborn epidermis. Fig 5 H illustrates a region where at least some basal cells were labeled, but most stretches of epidermis were entirely negative. Taken together, these findings demonstrate that although terminal differentiation is spatially and temporally defined in ß1 null epidermis, the proliferative potential is markedly reduced.
Although we cannot rule out a participatory role for 6ß4 in this process, the marked inhibition of proliferation seen in ß1 null epidermis was not seen in ß4 null skin (
As a final evaluation of the early consequences of ß1 ablation, we examined skin from a 4-d-surviving animal that expressed lower Cre levels and thus was still undergoing homologous recombination in some basal epidermal cells (same skin as that analyzed in Fig 3G and Fig H). In this skin, many areas could be found where ß1 expression had been ablated, but laminin 5 and hemidesmosomal markers still localized to the DEJ. Since rupturing at the BM was the earliest sign that ß1 had been ablated, we focused on one of these areas (Fig 5; serial sections shown in IK). Ki67-positive cells were fewer in number but still found within these areas (Fig 5 J). However, no basal cell was found that was positive for K1 or any other terminal differentiation marker that we examined, and no basal cell was found that was negative for K5 or K14 (Fig 5 K). Thus, despite the clear loss of ß1, in a skin area where this was a very recent event, some cell proliferation still existed and terminal differentiation was not induced in the BL. Therefore, whether we examined skin at early or late times after ß1 ablation, our results did not support the notion that downregulation of ß1 is the trigger for terminal differentiation in vivo. Rather, the results suggest that keratinocyte culture may not always be reliable as a model system for studying the roles of integrins in controlling the balance between epidermal proliferation and terminal differentiation.
Hair Follicle Invagination and Differentiation Is Impaired in Conditional ß1 Null Skin
The striking perturbations of the hair coat of ß1-floxed animals expressing low Cre recombinase levels and the paucity of hair follicles in the dermis of conditional ß1 null skin led us to suspect that the epithelial masses represented hair germs that failed to invaginate into the underlying dermis. We therefore examined the ß1 null skin for markers of hair follicle differentiation.
In WT skin, the leading front of developing hair follicles express nuclear Lef1, a DNA binding protein that collaborates with stabilized ß-catenin to activate downstream target genes (Fig 6 A; see also 6, even though regions flanking these masses showed little or no labeling (Fig 6 F; compare with WT in Fig 6 E). Currently, we have no molecular understanding for why these cells express
6ß4; however, it may explain why these regions display more Ki67-positive cells and a more organized BM than other areas of ß1 null epithelium.
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Little is known about the molecular mechanisms underlying the ability of developing hair follicles to locally remodel their ECM and invaginate into underlying mesenchyme. Our findings suggest a critical role for ß1 integrins in this process, a feature hitherto unrecognized. This is in agreement with
ß1 integrins to remodel its BM and grow downward remains an intriguing issue that awaits further exploration.
In summary, the conditional ablation of the ß1 integrin gene in mouse epidermis has provided major new insights into the functions of ß1 integrins and into the differential roles of ß1 and ß4 integrins in the skin. Our findings argue against an essential role for
ß1 integrins in regulating the spatial and temporal program of epidermal terminal differentiation, a function predicted from keratinocyte culture studies conducted with mutant ß1 transgenes (
ß1 integrins to assemble BM. This insight was not obtained from the
3 knockout, where BM was largely intact and proliferation unaffected, although some clues to potential roles for
ß1 integrins in BM assembly have emerged from studies on other tissues (
ß1 integrins in hemidesmosome assembly/stabilization and in hair follicle invagination into the underlying dermis. The challenge that faces us now will be to dissect the molecular pathways used by
ß1 integrins in orchestrating these events.
Note Added in Proof. A related paper was recently published by Fässler and colleagues (Brakebusch, C., R. Grose, F. Quondamatteo, A. Ramirez, J.L. Jorcano, A. Pirro, M. Svensson, R. Herken, T. Sasaki, R. Timpl, S. Werner, and R. Fässler. 2000. EMBO (Eur. Mol. Biol. Organ.) J. 19:39904003).
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Footnotes |
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1 Abbreviations used in this paper: BL, basal layer; BM, basement membrane; bs, backskin; DEJ, dermalepidermal junction; ECM, extracellular matrix; ES, embryonic stem; GR, granular; LD, lamina densa; SC, stratum corneum; SP, spinous; WT, wild-type.
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Acknowledgements |
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A special thank you goes to Ms. Linda Degenstein for her expert care in handling, caring, observing, and photographing these mice and to Dr. Valera Vasioukhin, Ramanuj DasGupta, Brad Merrill, Satrajit Sinha, and Colin Jamora for their generous and thoughtful suggestions, discussions, and assistance regarding various aspects of this work, including figure preparation (R. DasGupta). We thank Dr. Pierre Coulombe and Dr. Robert Burgeson for their generous gifts of antibodies.
S. Raghavan is the recipient of a Human Frontiers Postdoctoral Fellowship, and E. Fuchs is an Investigator of the Howard Hughes Medical Institute. The work was supported by the Howard Hughes Medical Institute and by a grant from the National Institutes of Health (R01AR27883).
Submitted: 19 July 2000
Revised: 31 July 2000
Accepted: 31 July 2000
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References |
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