Different Consequences of β1 Integrin Deletion in Neonatal and Adult Mouse Epidermis Reveal a Context-Dependent Role of Integrins in Regulating Proliferation, Differentiation, and Intercellular Communication
2005; Elsevier BV; Volume: 125; Issue: 6 Linguagem: Inglês
10.1111/j.0022-202x.2005.23956.x
ISSN1523-1747
AutoresTeresa López‐Rovira, Violeta Silva-Vargas, Fiona M. Watt,
Tópico(s)Dermatology and Skin Diseases
ResumoThere are conflicting reports of the consequences of deleting β1 integrins from the epidermis of transgenic mice. Epidermal thinning with normal differentiation and lack of inflammation has been observed; conversely, epidermal thickening, abnormal differentiation, and dermal fibrosis can occur. β1 integrin deletion results in decreased epidermal proliferation, yet on wounding the proliferative defect is overcome. To distinguish primary from secondary consequences of β1 integrin loss, we compared epidermal β1 deletion at E14.5 via K5Cre and 4-hydroxy-tamoxifen induced deletion in adulthood via K14CreER. As reported previously, there was dermo-epidermal splitting, inflammation, reduced proliferation, and hair follicle and sebaceous gland loss in 30-d-old K5Cre β1-null mice. These changes were not observed 30 d after β1 integrin deletion in adult epidermis, however, and there were no changes in the hair follicle stem cell compartment. Deletion in adult epidermis revealed a previously unreported correlation between the level of β1 integrins and proliferation in the interfollicular epidermis that was remarkably consistent with human epidermis. In addition, the number of melanocytes in interfollicular epidermis was greatly increased. Our results highlight the context-dependent effects of β1 integrin deletion and suggest that inflammation may be responsible for some of the K5Cre β1-null phenotype. There are conflicting reports of the consequences of deleting β1 integrins from the epidermis of transgenic mice. Epidermal thinning with normal differentiation and lack of inflammation has been observed; conversely, epidermal thickening, abnormal differentiation, and dermal fibrosis can occur. β1 integrin deletion results in decreased epidermal proliferation, yet on wounding the proliferative defect is overcome. To distinguish primary from secondary consequences of β1 integrin loss, we compared epidermal β1 deletion at E14.5 via K5Cre and 4-hydroxy-tamoxifen induced deletion in adulthood via K14CreER. As reported previously, there was dermo-epidermal splitting, inflammation, reduced proliferation, and hair follicle and sebaceous gland loss in 30-d-old K5Cre β1-null mice. These changes were not observed 30 d after β1 integrin deletion in adult epidermis, however, and there were no changes in the hair follicle stem cell compartment. Deletion in adult epidermis revealed a previously unreported correlation between the level of β1 integrins and proliferation in the interfollicular epidermis that was remarkably consistent with human epidermis. In addition, the number of melanocytes in interfollicular epidermis was greatly increased. Our results highlight the context-dependent effects of β1 integrin deletion and suggest that inflammation may be responsible for some of the K5Cre β1-null phenotype. 5-bromo-2′-deoxyuridine CCAAT displacement protein label retaining cells 4-hydroxy-tamoxifen Extracellular matrix receptors of the integrin family anchor the epidermis to the underlying basement membrane (Watt, 2002Watt F.M. Role of integrins in regulating epidermal adhesion, growth and differentiation.EMBO J. 2002; 21: 3919-3926Crossref PubMed Scopus (510) Google Scholar; Danen and Sonnenberg, 2003Danen E.H. Sonnenberg A. Integrins in regulation of tissue development and function.J Pathol. 2003; 201: 632-641Crossref PubMed Scopus (91) Google Scholar). The most abundant epidermal integrins are the collagen receptor, α2β1, and two laminin receptors, α3β1 and α6β4 (Watt, 2002Watt F.M. Role of integrins in regulating epidermal adhesion, growth and differentiation.EMBO J. 2002; 21: 3919-3926Crossref PubMed Scopus (510) Google Scholar). In addition to their role in anchorage, functions for integrins in regulating exit from the stem cell compartment and the onset of terminal differentiation have been proposed (Watt, 2002Watt F.M. Role of integrins in regulating epidermal adhesion, growth and differentiation.EMBO J. 2002; 21: 3919-3926Crossref PubMed Scopus (510) Google Scholar), and aberrant integrin expression is linked to skin inflammation and cancer (Owens and Watt, 2001Owens D.M. Watt F.M. Influence of β1 integrins on epidermal squamous cell carcinoma formation in a transgenic mouse model: α3β1, but not α2β1, suppresses malignant conversion.Cancer Res. 2001; 61: 5248-5254PubMed Google Scholar,Owens and Watt, 2003Owens D.M. Watt F.M. Contribution of stem cells and differentiated cells to epidermal tumours.Nat Rev Cancer. 2003; 3: 444-451Crossref PubMed Scopus (259) Google Scholar; Watt, 2002Watt F.M. Role of integrins in regulating epidermal adhesion, growth and differentiation.EMBO J. 2002; 21: 3919-3926Crossref PubMed Scopus (510) Google Scholar; Hobbs and Watt, 2003Hobbs R.M. Watt F.M. Regulation of interleukin-1α expression by integrins and epidermal growth factor receptor in keratinocytes from a mouse model of inflammatory skin disease.J Biol Chem. 2003; 278: 19798-19807Crossref PubMed Scopus (36) Google Scholar; Owens et al., 2003Owens D.M. Romero M.R. Gardner C. Watt F.M. Suprabasal α6β4 integrin expression in epidermis results in enhanced tumourigenesis and disruption of TGFβ signalling.J Cell Sci. 2003; 116: 3783-3791Crossref PubMed Scopus (74) Google Scholar; Hobbs et al., 2004Hobbs R.M. Silva-Vargas V. Groves R. Watt F.M. Expression of activated MEK1 in differentiating epidermal cells is sufficient to generate hyperproliferative and inflammatory skin lesions.J Invest Dermatol. 2004; 123: 503-515Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). The α6β4 integrin is a component of hemidesmosomes and is required for epidermal attachment (Danen and Sonnenberg, 2003Danen E.H. Sonnenberg A. Integrins in regulation of tissue development and function.J Pathol. 2003; 201: 632-641Crossref PubMed Scopus (91) Google Scholar). α6β4 deletion in mice or mutation in humans with junctional epidermolysis bullosa causes the epidermis to detach from the underlying basement membrane. Overexpression of this integrin correlates with poor prognosis in human and mouse squamous cell carcinomas and one mechanism involves disruption of TGF-β responsiveness (Owens and Watt, 2003Owens D.M. Watt F.M. Contribution of stem cells and differentiated cells to epidermal tumours.Nat Rev Cancer. 2003; 3: 444-451Crossref PubMed Scopus (259) Google Scholar; Owens et al., 2003Owens D.M. Romero M.R. Gardner C. Watt F.M. Suprabasal α6β4 integrin expression in epidermis results in enhanced tumourigenesis and disruption of TGFβ signalling.J Cell Sci. 2003; 116: 3783-3791Crossref PubMed Scopus (74) Google Scholar). But there is no evidence that α6β4 regulates epidermal differentiation per se (DiPersio et al., 2000DiPersio C.M. van der Neut R. Georges-Labouesse E. Kreidberg J.A. Sonnenberg A. Hynes R.O. α3β1 and α6β4 integrin receptors for laminin-5 are not essential for epidermal morphogenesis and homeostasis during skin development.J Cell Sci. 2000; 113: 3051-3062Crossref PubMed Google Scholar). In contrast to the importance of α6β4 in epidermal anchorage, α2β1 is completely dispensable (Chen et al., 2002Chen J. Diacovo T.G. Grenache D.G. Santoro S.A. Zutter M.M. The α2 integrin subunit-deficient mouse: A multifaceted phenotype including defects of branching morphogenesis and hemostasis.Am J Pathol. 2002; 161: 337-344Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar) and deletion of α3β1 results only in small epidermal blisters on the palms and soles of mice (DiPersio et al., 1997DiPersio C.M. Hodivala-Dilke K.M. Jaenisch R. Kreidberg J.A. Hynes R.O. α3β1 Integrin is required for normal development of the epidermal basement membrane.J Cell Biol. 1997; 137: 729-742Crossref PubMed Scopus (331) Google Scholar). When all β1 integrins are deleted, via keratin (K) 5 or K14Cre-mediated recombination, there is detachment of the epidermis from the underlying dermis (Brakebusch et al., 2000Brakebusch C. Grose R. Quondamatteo F. et al.Skin and hair follicle integrity is crucially dependent on β1 integrin expression on keratinocytes.EMBO J. 2000; 19: 3990-4003Crossref PubMed Scopus (307) Google Scholar; Raghavan et al., 2000Raghavan S. Bauer C. Mundschau G. Li Q. Fuchs E. Conditional ablation of β1 integrin in skin. Severe defects in epidermal proliferation, basement membrane formation, and hair follicle invagination.J Cell Biol. 2000; 150: 1149-1160Crossref PubMed Scopus (308) Google Scholar). The phenotype, however, is more complex to interpret than that of α6β4 deletion. Hair follicles either fail to form (Raghavan et al., 2000Raghavan S. Bauer C. Mundschau G. Li Q. Fuchs E. Conditional ablation of β1 integrin in skin. Severe defects in epidermal proliferation, basement membrane formation, and hair follicle invagination.J Cell Biol. 2000; 150: 1149-1160Crossref PubMed Scopus (308) Google Scholar) or form and then regress (Brakebusch et al., 2000Brakebusch C. Grose R. Quondamatteo F. et al.Skin and hair follicle integrity is crucially dependent on β1 integrin expression on keratinocytes.EMBO J. 2000; 19: 3990-4003Crossref PubMed Scopus (307) Google Scholar). Mice that die shortly after birth (K14Cre deletion) have reduced epidermal proliferation and thinning of the epidermis but differentiation is normal (Raghavan et al., 2000Raghavan S. Bauer C. Mundschau G. Li Q. Fuchs E. Conditional ablation of β1 integrin in skin. Severe defects in epidermal proliferation, basement membrane formation, and hair follicle invagination.J Cell Biol. 2000; 150: 1149-1160Crossref PubMed Scopus (308) Google Scholar). In mice that survive for up to 6 wk after birth (K5Cre deletion) proliferation is also reduced, but there is epidermal thickening, abnormal differentiation, dermal fibrosis, and inflammation (Brakebusch et al., 2000Brakebusch C. Grose R. Quondamatteo F. et al.Skin and hair follicle integrity is crucially dependent on β1 integrin expression on keratinocytes.EMBO J. 2000; 19: 3990-4003Crossref PubMed Scopus (307) Google Scholar). Epidermal wound healing in these mice is delayed, but nevertheless proliferation is increased to the same extent as in wounded wild-type epidermis (Grose et al., 2002Grose R. Hutter C. Bloch W. et al.A crucial role of β1 integrins for keratinocyte migration in vitro and during cutaneous wound repair.Development. 2002; 129: 2303-2315PubMed Google Scholar). The K5 and K14 promoters used to drive transgene expression are largely faithful to the patterns of expression of the endogenous keratins and the transgene promoters are of comparable strength with one another (Byrne et al., 1994Byrne C. Tainsky M. Fuchs E. Programming gene expression in developing epidermis.Development. 1994; 120: 2369-2383Crossref PubMed Google Scholar; Ramirez et al., 1994Ramirez A. Bravo A. Jorcano J.L. Vidal M. Sequences 5' of the bovine keratin 5 gene direct tissue- and cell-type-specific expression of a lacZ gene in the adult and during development.Differentiation. 1994; 58: 53-64PubMed Google Scholar,Ramirez et al., 2004Ramirez A. Page A. Gandarillas A. et al.A keratin K5Cre transgenic line appropriate for tissue-specific or generalized Cre-mediated recombination.Genesis. 2004; 39: 52-57Crossref PubMed Scopus (156) Google Scholar; Wang et al., 1997Wang X. Zinkel S. Polonsky K. Fuchs E. Transgenic studies with a keratin promoter-driven growth hormone transgene: Prospects for gene therapy.Proc Natl Acad Sci USA. 1997; 94: 219-226Crossref PubMed Scopus (145) Google Scholar). Nevertheless, it has been suggested that the different phenotypes of mice with epidermal deletion of β1 integrins reflect the impact of more or less efficient Cre-mediated recombination (Raghavan et al., 2000Raghavan S. Bauer C. Mundschau G. Li Q. Fuchs E. Conditional ablation of β1 integrin in skin. Severe defects in epidermal proliferation, basement membrane formation, and hair follicle invagination.J Cell Biol. 2000; 150: 1149-1160Crossref PubMed Scopus (308) Google Scholar). It has also been proposed that skin inflammation is triggered by hair follicle degeneration (Brakebusch et al., 2000Brakebusch C. Grose R. Quondamatteo F. et al.Skin and hair follicle integrity is crucially dependent on β1 integrin expression on keratinocytes.EMBO J. 2000; 19: 3990-4003Crossref PubMed Scopus (307) Google Scholar) and that elevated cytokine production in wounded skin may overcome the defect in proliferation (Grose et al., 2002Grose R. Hutter C. Bloch W. et al.A crucial role of β1 integrins for keratinocyte migration in vitro and during cutaneous wound repair.Development. 2002; 129: 2303-2315PubMed Google Scholar). In addition, in cultured keratinocytes there can be compensation for β1 integrin loss by upregulation of αv integrins (Raghavan et al., 2003Raghavan S. Vaezi A. Fuchs E. A role for αβ1 integrins in focal adhesion function and polarized cytoskeletal dynamics.Dev Cell. 2003; 5: 415-427Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). As the K5 and K14 promoters are upregulated by E14.5 of gestation (Byrne et al., 1994Byrne C. Tainsky M. Fuchs E. Programming gene expression in developing epidermis.Development. 1994; 120: 2369-2383Crossref PubMed Google Scholar; Brakebusch et al., 2000Brakebusch C. Grose R. Quondamatteo F. et al.Skin and hair follicle integrity is crucially dependent on β1 integrin expression on keratinocytes.EMBO J. 2000; 19: 3990-4003Crossref PubMed Scopus (307) Google Scholar; Raghavan et al., 2000Raghavan S. Bauer C. Mundschau G. Li Q. Fuchs E. Conditional ablation of β1 integrin in skin. Severe defects in epidermal proliferation, basement membrane formation, and hair follicle invagination.J Cell Biol. 2000; 150: 1149-1160Crossref PubMed Scopus (308) Google Scholar) it is also to be expected that effects of β1 deletion in embryonic epidermis will influence the postnatal phenotype. In order to distinguish primary and secondary consequences of β1 deletion, we have compared the K5Cre β1-null phenotype (Brakebusch et al., 2000Brakebusch C. Grose R. Quondamatteo F. et al.Skin and hair follicle integrity is crucially dependent on β1 integrin expression on keratinocytes.EMBO J. 2000; 19: 3990-4003Crossref PubMed Scopus (307) Google Scholar) with the phenotype of mice in which β1 deletion is induced in adulthood via Tamoxifen activatable K14Cre (K14CreER) (Vasioukhin et al., 1999Vasioukhin V. Degenstein L. Wise B. Fuchs E. The magical touch: Genome targeting in epidermal stem cells induced by tamoxifen application to mouse skin.Proc Natl Acad Sci USA. 1999; 96: 8551-8556Crossref PubMed Scopus (444) Google Scholar; Hong et al., 2004Hong K.U. Reynolds S.D. Watkins S. Fuchs E. Stripp B.R. Basal cells are a multipotent progenitor capable of renewing the bronchial epithelium.Am J Pathol. 2004; 164: 577-588Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar). We made use of a recently described method for whole-mount labeling (Braun et al., 2003Braun K.M. Niemann C. Jensen U.B. Sundberg J.P. Silva-Vargas V. Watt F.M. Manipulation of stem cell proliferation and lineage commitment: Visualisation of label-retaining cells in wholemounts of mouse epidermis.Development. 2003; 130: 5241-5255Crossref PubMed Scopus (353) Google Scholar), which facilitates examination of proliferation and differentiation in large areas of the epidermis without the need to prepare conventional histological sections. As previously reported (Brakebusch et al., 2000Brakebusch C. Grose R. Quondamatteo F. et al.Skin and hair follicle integrity is crucially dependent on β1 integrin expression on keratinocytes.EMBO J. 2000; 19: 3990-4003Crossref PubMed Scopus (307) Google Scholar), mice with K5Cre-mediated deletion of the β1 integrin subunit could be distinguished at 2 d after birth because their skin was pink, not black (data not shown). By 5 d the mice were smaller than wild-type littermates. They did not develop a full coat of hair and by 4 wk they walked with an odd gait as a result of tight and blistered skin. The mice had to be killed at 4 wk after birth. It has previously been reported that when the β1 integrin subunit is deleted from the epidermis via K5Cre there is a decrease in epidermal proliferation, together with hair loss, epidermal blistering and dermal fibrosis (Brakebusch et al., 2000Brakebusch C. Grose R. Quondamatteo F. et al.Skin and hair follicle integrity is crucially dependent on β1 integrin expression on keratinocytes.EMBO J. 2000; 19: 3990-4003Crossref PubMed Scopus (307) Google Scholar). Hematoxylin and eosin (H & E)-stained sections of back skin showed that the hair follicles were abnormal at 1 wk (Figure 1a, b). By 2 wk the interfollicular epidermis (IFE) was considerably thickened compared with wild-type (Figure 1c). At 3 and 4 wk there was an inflammatory infiltrate in the dermis, marked degeneration of the hair follicles and splits at the dermal–epidermal junction (Figure 1d, e). All of these findings are in good agreement with previous observations (Brakebusch et al., 2000Brakebusch C. Grose R. Quondamatteo F. et al.Skin and hair follicle integrity is crucially dependent on β1 integrin expression on keratinocytes.EMBO J. 2000; 19: 3990-4003Crossref PubMed Scopus (307) Google Scholar). We next examined how these changes looked in whole mounts of tail epidermis. At least 20 K5Cre × floxed β1 mice were examined, with a minimum of five at each time point. In the wild-type whole mounts there was the characteristic arrangement of the hair follicles in groups of three, with prominent sebaceous glands (Figure 1f). As described previously (Braun et al., 2003Braun K.M. Niemann C. Jensen U.B. Sundberg J.P. Silva-Vargas V. Watt F.M. Manipulation of stem cell proliferation and lineage commitment: Visualisation of label-retaining cells in wholemounts of mouse epidermis.Development. 2003; 130: 5241-5255Crossref PubMed Scopus (353) Google Scholar), there was relatively uniform expression of β1 integrins (Figure 1f). Proliferating cells, visualized by Ki67 immunoreactivity, were found throughout the IFE basal layer and the periphery of the sebaceous glands (Figure 1f). The permanent portion of the follicles, below the sebaceous glands and above the follicle bulb, is known as the bulge (Figure 1f). The bulge region was relatively devoid of proliferating cells, whereas the bulb of anagen follicles was highly proliferative (Figure 1f). Antibody staining confirmed the loss of β1 integrins in whole mounts of tail epidermis from K5Cre β1-null mice (Figure 1g–j). As reported previously (Brakebusch et al., 2000Brakebusch C. Grose R. Quondamatteo F. et al.Skin and hair follicle integrity is crucially dependent on β1 integrin expression on keratinocytes.EMBO J. 2000; 19: 3990-4003Crossref PubMed Scopus (307) Google Scholar), however, the deletion was not uniform, and at 1 and 2 wk significant β1 immunoreactivity remained in some follicles (Figure 1g, h). By 4 wk the previously reported decrease in proliferation was clear in all regions, but was particularly striking at the bulb (Figure 1j). From 3 wk onward the sebaceous glands were either absent or greatly reduced in size (Figure 1i, j). In normal epidermis, the major β1 integrins are α2β1 and α3β1, whereas αv forms a heterodimer with the β5 subunit (Watt, 2002Watt F.M. Role of integrins in regulating epidermal adhesion, growth and differentiation.EMBO J. 2002; 21: 3919-3926Crossref PubMed Scopus (510) Google Scholar). In wild-type epidermis the α2 and αv integrin subunits were detected in the basal layer of the interfollicular epidermis (Figure 2a, c). In 4-wk-old K5Cre β1-null epidermis there was very little detectable α2 integrin (Figure 2b). In contrast, the αv subunit was upregulated in the suprabasal layers (Figure 2d), consistent with earlier observations of suprabasal integrin expression in thickened IFE (Watt, 2002Watt F.M. Role of integrins in regulating epidermal adhesion, growth and differentiation.EMBO J. 2002; 21: 3919-3926Crossref PubMed Scopus (510) Google Scholar) and with the upregulation of αv integrins in some cultures of β1-null keratinocytes (Raghavan et al., 2003Raghavan S. Vaezi A. Fuchs E. A role for αβ1 integrins in focal adhesion function and polarized cytoskeletal dynamics.Dev Cell. 2003; 5: 415-427Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). As previously reported, deletion of β1 integrins led to abnormal deposition of type IV collagen (ligand for α2β1) (Figure 2e, f) and laminin 5 (ligand for α3β1) (Figure 2g, h) in the dermis. It was previously reported (Raghavan et al., 2000Raghavan S. Bauer C. Mundschau G. Li Q. Fuchs E. Conditional ablation of β1 integrin in skin. Severe defects in epidermal proliferation, basement membrane formation, and hair follicle invagination.J Cell Biol. 2000; 150: 1149-1160Crossref PubMed Scopus (308) Google Scholar) that deletion of β1 integrins via K14Cre-mediated recombination does not alter the program of terminal differentiation within the IFE. But the number of differentiated cell layers is increased in K5Cre β1-null epidermis (Brakebusch et al., 2000Brakebusch C. Grose R. Quondamatteo F. et al.Skin and hair follicle integrity is crucially dependent on β1 integrin expression on keratinocytes.EMBO J. 2000; 19: 3990-4003Crossref PubMed Scopus (307) Google Scholar). We re-evaluated the impact of β1 deletion on IFE differentiation using a combination of whole-mount labeling and conventional histology of 30-d-old tail epidermis and a wider range of terminal differentiation markers than examined previously (Figure 3). Whole mounts predominantly reveal staining in the epidermal basal layer because the permeabilization conditions do not allow access of antibodies to all the suprabasal layers (Jensen et al., 1999Jensen U.B. Lowell S. Watt F.M. The spatial relationship between stem cells and their progeny in the basal layer of human epidermis: A new view based on whole-mount labelling and lineage analysis.Development. 1999; 126: 2409-2418PubMed Google Scholar; Braun et al., 2003Braun K.M. Niemann C. Jensen U.B. Sundberg J.P. Silva-Vargas V. Watt F.M. Manipulation of stem cell proliferation and lineage commitment: Visualisation of label-retaining cells in wholemounts of mouse epidermis.Development. 2003; 130: 5241-5255Crossref PubMed Scopus (353) Google Scholar). Thus, when wild-type epidermis is stained with an antibody to keratin 10 there is scattered staining, reflecting keratin 10-positive basal cells in the interfollicular epidermis (Figure 3a). Keratin 10 is not expressed in the sebaceous glands and hair follicles (Figure 3a). Whole-mount labeling of wild-type epidermis reveals an area of IFE encircling each hair follicle triplet that is not labeled with antibodies to keratin 10 (Braun et al., 2003Braun K.M. Niemann C. Jensen U.B. Sundberg J.P. Silva-Vargas V. Watt F.M. Manipulation of stem cell proliferation and lineage commitment: Visualisation of label-retaining cells in wholemounts of mouse epidermis.Development. 2003; 130: 5241-5255Crossref PubMed Scopus (353) Google Scholar; Figure 3a). Tail IFE consists of zones of parakeratotic and orthokeratotic differentiation (Schweizer et al., 1987Schweizer J. Furstenberger G. Winter H. Selective suppression of two postnatally acquired 70 kD and 65 kD keratin proteins during continuous treatment of adult mouse tail epidermis with vitamin A.J Invest Dermatol. 1987; 89: 125-131Abstract Full Text PDF PubMed Google Scholar) and the keratin 10-negative regions correspond to the parakeratotic scale epidermis (see Figure 8a). The keratin 10-negative regions of interfollicular epidermis were not seen in β1-null epidermis; instead, the entire IFE had scattered keratin 10-positive basal cells (Figure 3b). Conventional sections revealed that in the thickened β1-null epidermis there were more layers of keratin 10-positive cells than in the wild-type (Figure 3c, d). But there was no significant increase in the number of keratin 10-positive basal cells (Figure 3c, d). Involucrin, like keratin 10, is normally expressed in the suprabasal layers of IFE (Figure 3g) and is also expressed in scattered basal cells that are visualized in whole mounts of wild-type epidermis (Figure 3e). In whole mounts of K5Cre β1-null epidermis, staining of involucrin was decreased overall (Figure 3f). Immunostaining of sections revealed that involucrin expression was more patchy in β1-null epidermis than in wild-type: it was reduced overall, but extended to the basal layer in places (Figure 3h). Keratin 14 staining of whole mounts revealed an increase in surface area of the β1-null basal cells in the IFE (Figure 3i, j), in agreement with the observation that K14Cre β1-null basal keratinocytes are abnormally flattened (Raghavan et al., 2000Raghavan S. Bauer C. Mundschau G. Li Q. Fuchs E. Conditional ablation of β1 integrin in skin. Severe defects in epidermal proliferation, basement membrane formation, and hair follicle invagination.J Cell Biol. 2000; 150: 1149-1160Crossref PubMed Scopus (308) Google Scholar). Conventional sections showed increased numbers of cell layers that were positive for cornifin (Figure 3m, n), loricrin (Figure 3k, l), and transglutaminase 1 (Figure 3o, p), all of which, like involucrin, are components of the epidermal cornified envelope. The transglutaminase staining (Figure 3p) was reminiscent of involucrin (Figure 3h) in being patchy and partially localized to the basal layer (Figure 3p). Keratin 6 was confined to the follicles in wild-type mice; however, there was upregulation in the IFE of transgenic mice (data not shown). We conclude that when β1 integrins are deleted from the epidermis terminal differentiation is disturbed, in contrast to an earlier report (Raghavan et al., 2000Raghavan S. Bauer C. Mundschau G. Li Q. Fuchs E. Conditional ablation of β1 integrin in skin. Severe defects in epidermal proliferation, basement membrane formation, and hair follicle invagination.J Cell Biol. 2000; 150: 1149-1160Crossref PubMed Scopus (308) Google Scholar). There are increased numbers of cell layers expressing keratin 10, cornifin, loricrin, involucrin, and transglutaminase. The terminal differentiation program is not entirely normal because of the upregulation of keratin 6, the patchy expression of involucrin and transglutaminase 1, and the altered pattern of keratin 10 expression that is visualized in the whole mounts. It has previously been reported that the hair follicles degenerate in K5Cre β1-null skin (Brakebusch et al., 2000Brakebusch C. Grose R. Quondamatteo F. et al.Skin and hair follicle integrity is crucially dependent on β1 integrin expression on keratinocytes.EMBO J. 2000; 19: 3990-4003Crossref PubMed Scopus (307) Google Scholar), and this was clear from the whole mounts, as well as conventional sections (Figure 1). No anagen (growing) follicles were observed after 2 wk; the follicles were shorter than wild-type and lacked a discrete bulb (Figure 1i, j). In addition, the sebaceous glands were largely absent from 2 wk after birth (Figure 1i, j). CCAAT displacement protein (CDP) is normally expressed in the bulb of hair follicles and is prominent at anagen (Braun et al., 2003Braun K.M. Niemann C. Jensen U.B. Sundberg J.P. Silva-Vargas V. Watt F.M. Manipulation of stem cell proliferation and lineage commitment: Visualisation of label-retaining cells in wholemounts of mouse epidermis.Development. 2003; 130: 5241-5255Crossref PubMed Scopus (353) Google Scholar) (Figure 4a). However, essentially all CDP expression was lost by day 30 in K5Cre β1-null epidermis (Figure 4b). Nile Red staining revealed differentiated sebocytes in wild-type epidermis (Figure 4c). In contrast, there was very little Nile Red staining in the β1-null epidermis (Figure 4d), consistent with loss of the sebaceous glands. The changes in the hair follicles and sebaceous glands were not accompanied by any increase in apoptosis, as detected by TUNEL labeling (Figure 4e, f). In both wild-type and β1-null epidermis scattered apoptotic keratinocytes were found at the base of the hair follicles (Figure 4e, f). To examine the effects of K5Cre-mediated deletion of β1 integrins on the hair follicle stem cell compartment, we used two protein markers of the bulge (Braun et al., 2003Braun K.M. Niemann C. Jensen U.B. Sundberg J.P. Silva-Vargas V. Watt F.M. Manipulation of stem cell proliferation and lineage commitment: Visualisation of label-retaining cells in wholemounts of mouse epidermis.Development. 2003; 130: 5241-5255Crossref PubMed Scopus (353) Google Scholar), CD34 (Trempus et al., 2003Trempus C.S. Morris R.J. Bortner C.D. Cotsarelis G. Faircloth R.S. Reece J.M. Tennant R.W. Enrichment for living murine keratinocytes from the hair follicle bulge with the cell surface marker CD34.J Invest Dermatol. 2003; 120: 501-511Crossref PubMed Scopus (472) Google Scholar; Figure 5a), and the α6β4 integrin (Tani et al., 2000Tani H. Morris R.J. Kaur P. Enrichment for murine keratinocyte stem cells based on cell surface phenotype.Proc Natl Acad Sci USA. 2000; 97: 10960-10965Crossref PubMed Scopus (334) Google Scholar; Figure 5e). In regressing β1-null follicles CD34 was still expressed, but the CD34-positive zone now extended to the bottom of the follicles, suggesting that the bulge remained even after the bulb had disappeared (Figure 5b). In wild-type epidermis the α6β4 integrin is expressed in the basal layer of the IFE, the periphery of the sebaceous glands, and along the outer root sheath of the hair follicle, but is upregulated in the bulge (Figure 5e). Strong staining was also seen along the remaining outer root sheath of the β1 null follicles (Figure 5f), the same region that was positive for CD34. Whereas in wild-type epidermis α6β4 was concentrated at the basement membrane zone (Figure 5i), in β1-null epidermis the distribution was more pericellular (Figure 5j). One characteristic of epidermal stem cells is that they tend to divide infrequently; as a result they can be visualized as DNA label retaining cells (LRC) (Bickenbach, 1981Bickenbach J.R. Identification
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