1. Institute for Genetics, University of Cologne, Weyertal 121, D-50931 Cologne, Germany
2. EMBL Mouse Biology Programme, via Ramarini 32, I-00016 Monterotondo, Italy
3. Unité de Biologie Moléculaire de l'Expression Génique, URA 1773 CNRS, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris Cedex 15, France
4. Department of Dermatology and Center for Molecular Medicine, University of Cologne, Joseph-Stelzmann Strasse 9, D-50924 Cologne, Germany
5. Department of Dermatology, University of Würzburg, Josef-Schneider Strasse 2, 97080 Würzburg, Germany
6. Institute for Cell Biology, ETH Hönggerberg, HPM D25, CH-8093 Zürich, Switzerland
7. Present addresses: Center for Blood Research, Harvard Medical School, 200 Longwood Avenue, Boston, Massachusetts 02115, USA (M.S.S and K.R); German Research Centre for Biotechnology, Mascheroder Weg 1, 38124 Braunschweig, Germany (M.H.).

Correspondence to: Manolis Pasparakis^1,2 Correspondence and requests for materials should be addressed to M.P. (e-mail: Email: pasparakis@embl-monterotondo.it).

Mice lacking IKK2 die in utero as a result of tumour necrosis factor (TNF)-induced hepatocyte apoptosis^{3, 4, 5}. To study the function of IKK2 in different tissues we generated mice carrying a loxP-site-flanked (floxed (FL)) Ikk2 allele (Fig. 1a). Ikk2^FL/FL mice develop normally and express normal levels of IKK2 (Fig. 1b). Excision of the floxed Ikk2 sequences by crossing to a Cre-deleter transgenic strain²⁰ produced mice carrying deleted (D) Ikk2 alleles. As expected, Ikk2^D/D mice die in utero and exhibit liver degeneration (data not shown). Analysis of mouse embryonic fibroblasts (MEFs) revealed that Ikk2^D/D cells lack expression of IKK2 (Fig. 1b) and show impaired activation of NF-B on stimulation with TNF, interleukin (IL)-1 or bacterial lipopolysaccharide (LPS) (ref. 7 and data not shown). To ablate IKK2 specifically in the epidermis we used a transgenic mouse line expressing Cre under the control of the human keratin 14 (K14) promoter. LacZ staining of tissue sections from a K14-Cre mouse carrying a ROSA26^FL Cre-reporter allele²¹ (Fig. 1c and data not shown) and Southern blot analysis of DNA isolated from various tissues of a K14-Cre/Ikk2^FL/FL mouse (Fig. 1d) showed Cre recombination specifically in the epidermis, hair follicles and the epithelium of the tongue. Western analysis of epidermal extracts prepared from newborn pups failed to detect expression of IKK2 in K14-Cre/Ikk2^FL/FL mice, demonstrating that IKK2 protein is absent from the epidermis at birth (Fig. 1e).

Figure 1: Epidermis-specific knockout of IKK2.

a, Diagram showing the wild-type Ikk2 genomic locus (WT), the neomycin-resistance-containing (neo), the loxP-flanked (FL) and the deleted (D) IKK2 alleles. Filled boxes indicate exons (E6–E9). Restriction enzyme sites and the location of the probe used for Southern blot analysis are depicted. StuI fragments are in kilobases. B, BamHI; E, EcoRI; Bg, BglII; S, StuI. LoxP sites are indicated by arrowheads. b, Western blot analysis of IKK2 and IKK1 expression in wild-type and Ikk2^D/D MEFs and in splenocytes from wild-type and Ikk2^FL/FL mice. c, Analysis of -galactosidase expression in the skin of a K14-Cre, ROSA26^FL/+ mouse shows Cre recombination specifically in the epidermis and in hair follicles. Scale bar, 200 m. d, Tissue specificity of IKK2 inactivation. DNA isolated from various tissues of a K14-Cre/Ikk2^FL/FL mouse was subjected to Southern blot analysis after digestion with StuI. Deletion is detected in DNA from epidermis, total skin and tongue. e, Western blot analysis of IKK2 and NEMO expression in the epidermis of newborn mice with the indicated genotypes. f, K14-Cre/Ikk2^FL/FL and control mice at P8.

High resolution image and legend (36K)

K14-Cre/Ikk2^FL/FL pups are born at the expected mendelian ratios and are macroscopically indistinguishable from their littermates until postnatal day 4–5 (P4–P5), when their skin starts becoming hard and inflexible. This phenotype progresses rapidly and by P7–P8 the mice exhibit a highly rigid, shell-like skin with widespread scaling (Fig. 1f). At this stage the mice become 'runted' and they subsequently die between P7 and P9. The cause of death is not known at present; however, it does not seem to be the result of impaired skin barrier function as no differences were detected in toluidine blue penetration into the skin between mutant and control mice at P8 (data not shown). Ikk2^FL/FL mice lacking the K14-Cre transgene and Ikk2^FL/+ and Ikk2^+/+ mice with or without K14-Cre did not show any pathological skin phenotype and are collectively referred to as controls. Histological analysis of skin sections from K14-Cre/Ikk2^FL/FL mice at P7 revealed a markedly thickened epidermis with loss of the granular layer, pronounced hyperkeratosis, focal parakeratosis, subcorneal pustule formation and increased cellularity and dilated blood vessels in the dermis (Fig. 2a).

Figure 2: Histology of the skin in mice with epidermis-specific IKK2 deficiency.

a, b, Skin sections from K14-Cre/Ikk2^FL/FL (mutant, mt) and control (ct) mice were stained with haematoxylin/eosin (a) or immunostained for the indicated epidermal differentiation markers (green staining in b). Red counterstaining in b shows tissue structure; blue staining shows nuclei. c, Analysis of cell proliferation by staining for Ki67 (green); red staining shows nuclei. d, TUNEL staining for the detection of apoptotic cells (green); red staining shows nuclei. A white arrow points to an intraepidermal pustule with surrounding apoptotic keratinocytes. Dotted lines indicate the border between epidermis and dermis. Scale bars: a, 150 m; b, 100 m; c, d, 200 m.

High resolution image and legend (91K)

We undertook detailed immunohistological analysis of skin sections from K14-Cre/Ikk2^FL/FL and control mice at different time points after birth, using antibodies against various epidermal differentiation markers. At P0 and P1 the skin of mutant mice did not show any differences compared to controls (Fig. 2b, and data not shown), suggesting that, in contrast to IKK1, IKK2 is not essential for epidermal differentiation. At P3 the epidermis of K14-Cre/Ikk2^FL/FL mice showed upregulation of keratin 6 (K6) expression, a marker for inflamed and hyperproliferative epidermis, and suprabasal expression of K14, which is normally confined to the basal epidermal layer (Fig. 2b). K6 and K14 were expressed in all viable layers of the mutant epidermis at P7 (Fig. 2b). In addition, expression of the suprabasal keratin 10 (K10) and of the keratinocyte terminal differentiation markers loricrin (Fig. 2b) and filaggrin (not shown) was markedly reduced in K14-Cre/Ikk2^FL/FL animals at this stage. Staining for Ki67 revealed increased proliferation of keratinocytes in mutant epidermis at P4 and P7, but not P0 and P1 (Fig. 2c, and data not shown). TdT-mediated dUTP nick end labelling (TUNEL) staining showed no differences between K14-Cre/Ikk2^FL/FL and control skin at P2 (Fig. 2d); however, increased numbers of apoptotic keratinocytes in proximity to aggregates of inflammatory cells were observed in mutant epidermis at P8 (Fig. 2d).

To determine whether the cutaneous phenotype observed in K14-Cre/Ikk2^FL/FL mice is caused by a cell-autonomous defect in proliferation and/or differentiation of IKK2-deficient keratinocytes, we analysed ex vivo keratinocyte cultures from mutant and control mice. Keratinocytes isolated from newborn K14-Cre/Ikk2^FL/FL mice lack expression of IKK2 (Fig. 3a) and, similarly to Ikk2-knockout MEFs^{3, 4, 5, 7}, show impaired NF-B activation in response to stimulation by TNF or IL-1 (Fig. 3b, c). IKK2-deficient keratinocytes were not impaired in their capability to terminally differentiate in suspension culture, as demonstrated by similar expression of K10, involucrin, loricrin and filaggrin compared to control keratinocytes (Fig. 3e–f, see also Supplementary Information Table 1). Growth curves revealed that primary keratinocytes lacking IKK2 exhibit decreased proliferation compared with control cells (Fig. 3d). These findings show that, in contrast to IKK1, IKK2 deficiency does not lead to impaired differentiation and hyperproliferation of keratinocytes. Previous in vivo and in vitro experiments showed hyperproliferation and epidermal hyperplasia after NF-B inhibition and growth arrest after NF-B activation in epidermal keratinocytes, and suggested a role for NF-B in the regulation of keratinocyte growth^{12, 13, 14}. In these studies NF-B activity in keratinocytes was modulated downstream of the IKK complex. Our experiments demonstrate that the proposed growth regulatory function of NF-B in epidermal keratinocytes can be mediated through an IKK2-independent pathway and suggest that the skin phenotype of K14-Cre/Ikk2^FL/FL mice is not caused by cell-autonomous hyperproliferation of IKK2-deficient keratinocytes. TUNEL staining revealed that keratinocytes lacking IKK2 do not undergo increased spontaneous apoptosis in culture and show only mildly increased apoptosis on TNF treatment (1.9–2.8% compared with 0.1–0.2% in the controls). In agreement with the presence of only a few apoptotic keratinocytes in the epidermis of K14-Cre/Ikk2^FL/FL mice, these results suggest that TNF-induced killing of IKK2 knockout keratinocytes does not have an important role in the pathogenesis of the skin phenotype in our mice.

Figure 3: Analysis of NF-B activation, proliferation and differentiation in cultured keratinocytes.

a, Western blot analysis showing expression of IKK2, IKK1, NEMO and actin in keratinocytes isolated from mice with the indicated genotypes. b, IB kinase activity (KA), IB degradation and NF-B activation in primary keratinocytes isolated from control (ct) and K14-Cre/Ikk2^FL/FL (mt) mice. Cells were treated with TNF (20 ng ml^-1) for the indicated times. The kinase activity of IKK complexes immunoprecipitated with anti-NEMO antibody was measured. NEMO blot is shown as loading control. IB degradation was assayed by western analysis of cytoplasmic extracts and NF-B DNA-binding activity was measured by gel mobility shift analysis of nuclear extracts. c, Immunostaining for p65/RelA in unstimulated or IL-1-treated (20 ng ml^-1 for 20 min) mutant and control keratinocytes. d, Growth curves of primary keratinocytes in culture. A total of 30,000 cells isolated from mutant (red circles) and control mice (blue squares) were seeded per well, cultured for 17 days and collected at different time points. The graph shows mean values s.d. from triplicate wells. e, f, Expression of differentiation markers in IKK2-deficient primary keratinocytes before (start) or after 24 h in suspension culture (susp). Fluorescence images of keratinocytes expressing keratin 10 (red in e) and involucrin (red in f) are shown. Blue signal shows nuclei stained with 4,6-diamidino-2-phenylindole (DAPI). Scale bars: c, e, f, 200 m.

High resolution image and legend (52K)

The progressive disturbance of epidermal differentiation and proliferation in the skin of K14-Cre/Ikk2^FL/FL mice seems to be associated with an inflammatory response, suggested by the presence of subcorneal pustules (Fig. 4a) and increased cellularity in the dermis at P7. Immunohistological analysis revealed the presence of increased numbers of macrophages, granulocytes and CD4-positive T cells in the dermis of mutant mice at P7 (Fig. 4a). Examination of inflammatory cytokine gene expression showed increased levels of IL-1 messenger RNA in the skin of K14-Cre/Ikk2^FL/FL mice as early as P2, but not at P1 (Fig. 4b and data not shown). Curiously, IKK2-deficient epidermal keratinocytes are identified as the major source of these elevated IL-1 levels, as demonstrated by immunostaining with an antibody against IL-1 (Fig. 4d) and by analysis of IL-1 mRNA in epidermal sheets (data not shown). However, the increased expression of IL-1 does not seem to be induced by a cell-autonomous mechanism triggered by the lack of IKK2, as cultured IKK2-deficient keratinocytes do not show increased expression of IL-1 (Fig. 4c and data not shown). Immunohistology with anti-TNF antibodies revealed increased expression of TNF by cells in the dermis—most probably macrophages and granulocytes—of K14-Cre/Ikk2^FL/FL mice at P4 and P7, but not at P1 and P2 (Fig. 4d and data not shown). To identify the signals that attract the inflammatory cells into the skin we performed in situ hybridization analysis for various chemokine mRNAs. These experiments showed expression of MCP-1 (Fig. 4e), LIX, C10, IP-10 and Mig (data not shown) in the dermis of K14-Cre/Ikk2^FL/FL mice at P4 and P7, but not in the controls. MIP-2, one of the murine homologues of human IL-8, was expressed mainly in pustules of inflammatory cells in the epidermis of K14-Cre/Ikk2^FL/FL mice at P7 (Fig. 4e). These results suggest that an immune response orchestrated by the expression of inflammatory cytokines and multiple chemokines in the dermis, and involving T lymphocytes, macrophages and granulocytes, is implicated in the pathogenesis of the skin disease that develops in these mice.

Figure 4: Inflammation in the skin of K14-Cre/Ikk2^FL/FL mice.

a, Sections were stained with Giemsa or immunostained with antibodies recognizing T cells (CD3, CD4), granulocytes (Gr-1) and macrophages (F4/80) (green signal). Counterstaining and nuclear staining is as in Fig. 2b. Arrows point to intraepidermal pustules. b, RNase protection assay showing IL-1 expression in the skin of mice 2 or 4 days after birth. Left lane shows positive controls; tRNA is used as negative control. c, RT-PCR analysis of mouse IL-1 expression in skin samples from mutant and control mice at P4 and in cultured IKK2-deficient (mt) and control (ct) keratinocytes (cells). d, Immunostaining using antibodies specific for either IL-1 or TNF (green) in skin sections from mice at P3, and P7–P8; red staining shows nuclei. e, In situ hybridization for MCP1 and MIP-2 mRNA (black) in skin sections from mice at P4 (MCP-1) or P7 (MIP-2). mt, mutant; ct, control mice. Magnification 250. Dotted lines indicate the border between epidermis and dermis. Scale bars, a, d, 200 m.

High resolution image and legend (163K)

To investigate whether T lymphocytes are essential for the development of the skin disease we crossed the K14-Cre/Ikk2^FL/FL mice with T-cell antigen receptor- (TCR) knockout mice, which lack mature T cells²². K14-Cre/Ikk2^FL/FL/TCR^-/- mice develop skin disease with similar kinetics and histological characteristics as the K14-Cre/Ikk2^FL/FL mice (Fig. 5b), demonstrating that T cells are not required for the development of the inflammatory skin lesions. This finding, together with the early age of disease onset, suggests that the inflammation of the skin in K14-Cre/Ikk2^FL/FL mice is not triggered by an antigen-specific immune response but rather by a reaction driven from the innate immune system.

Figure 5: Requirement of TNFRI for the development of the skin phenotype in K14-Cre/Ikk2^FL/FL mice.

a, K14-Cre/Ikk2^FL/FL/TnfrI^-/- mice do not develop inflammatory skin disease. b, Analysis of skin samples from K14-Cre/Ikk2^FL/FL/TnfrI^-/- and K14-Cre/Ikk2^FL/FL/TCR^-/- mice. Sections were stained with haematoxylin/eosin (H/E) or Giemsa, or with antibodies against the indicated differentiation, proliferation or immune cell markers (green signal; see Fig. 4 for abbreviations). Nuclei are in red. Top scale bar, 100 m; bottom scale bar, 200 m. Dotted lines indicate the border between epidermis and dermis.

High resolution image and legend (152K)

To investigate the role of TNF signalling in the pathogenesis of the skin disease we crossed the K14-Cre/Ikk2^FL/FL mice with TNF-receptor I (TNFRI)-deficient mice²³. Surprisingly, K14-Cre/Ikk2^FL/FL mice lacking TNFRI do not develop inflammatory skin disease (Fig. 5a). Immunohistological analysis of skin sections from K14-Cre/Ikk2^FL/FL/TnfrI^-/- mice revealed a normal pattern of keratinocyte differentiation and proliferation in the epidermis and the absence of inflammatory cells from the dermis (Fig. 5b). These results demonstrate that TNFRI signalling is critical for the inflammatory response that causes the skin disease in K14-Cre/Ikk2^FL/FL mice. As TNFRI mediates its effects largely through activation of NF-B-dependent gene transcription, the presence of an intact NF-B signalling pathway in all other cell types except epidermal keratinocytes in K14-Cre/Ikk2^FL/FL mice may be critical for disease pathogenesis. Finally, the normal development of the epidermis in K14-Cre/Ikk2^FL/FL/TnfrI^-/- mice confirms that IKK2 deficiency does not interfere directly with keratinocyte differentiation and proliferation, and suggests that the observed hyperplasia and the disturbed expression of keratinocyte differentiation markers in the mutant epidermis after P3 are secondary to the inflammation of the skin.

How can deletion of IKK2 in epidermal keratinocytes initiate an inflammatory skin disease? A tightly regulated microenvironment of cytokines and growth factors produced by keratinocytes, dermal fibroblasts and immune cells is critical for the maintenance of physiological skin homeostasis²⁴. The NF-B signalling pathway may be essential for epidermal keratinocytes to respond to such regulatory mediators and/or to produce factors that are important for the maintenance of a well balanced interplay between the epidermis, the dermis and the immune system. Although the exact mechanism of disease initiation remains unclear, ablation of IKK2 from epidermal keratinocytes seems to interfere with this balance and triggers an inflammatory response that is marked by the induction of inflammatory cytokines such as IL-1 and TNF, resulting in the development of skin disease in K14-Cre/Ikk2^FL/FL mice. The neutralization of the skin disease in K14-Cre/Ikk2^FL/FL mice lacking TNFRI demonstrates the essential pathogenic role of TNF for the development of this phenotype. This result does not exclude a function for IL-1 in our model, but it confirms the role of TNF as a master cytokine in inflammation and highlights its significance in the pathogenesis of inflammatory skin disease. The initiation of the skin disease during the first few days after birth may indicate the involvement of environmental factors. Alternatively, mechanisms related to developmental changes of the skin at this early age might be involved in disease initiation. Our finding that interference with NF-B signalling in epidermal keratinocytes triggers skin inflammation provides new insight into the function of NF-B in the epidermis and suggests that keratinocytes can act as the initiating cell type in inflammatory skin disease.

References

References

1.	Karin, M. & Ben-Neriah, Y. Phosphorylation meets ubiquitination: the control of NF-B activity. Annu. Rev. Immunol. 18, 621-663 (2000) | Article | PubMed | ISI |
2.	Israel, A. The IKK complex: an integrator of all signals that activate NF-B? Trends Cell Biol. 10, 129-133 (2000) | Article | PubMed | ISI |
3.	Li, Q., Van Antwerp, D., Mercurio, F., Lee, K. F. & Verma, I. M. Severe liver degeneration in mice lacking the IB kinase 2 gene. Science 284, 321-325 (1999) | Article | PubMed | ISI |
4.	Tanaka, M. et al. Embryonic lethality, liver degeneration, and impaired NF-B activation in IKK--deficient mice. Immunity 10, 421-429 (1999) | PubMed | ISI |
5.	Li, Z. W. et al. The IKK subunit of IB kinase (IKK) is essential for nuclear factor B activation and prevention of apoptosis. J. Exp. Med. 189, 1839-1845 (1999) | Article | PubMed | ISI |
6.	Rudolph, D. et al. Severe liver degeneration and lack of NF-B activation in NEMO/IKK-deficient mice. Genes Dev. 14, 854-862 (2000) | PubMed | ISI |
7.	Schmidt-Supprian, M. et al. NEMO/IKK-deficient mice model incontinentia pigmenti. Mol. Cell 5, 981-992 (2000) | PubMed | ISI |
8.	Makris, C. et al. Female mice heterozygous for IKK/NEMO deficiencies develop a dermatopathy similar to the human X-linked disorder incontinentia pigmenti. Mol. Cell 5, 969-979 (2000) | PubMed | ISI |
9.	Takeda, K. et al. Limb and skin abnormalities in mice lacking IKK. Science 284, 313-316 (1999) | Article | PubMed | ISI |
10.	Li, Q. et al. IKK1-deficient mice exhibit abnormal development of skin and skeleton. Genes Dev. 13, 1322-1328 (1999) | PubMed | ISI |
11.	Hu, Y. et al. Abnormal morphogenesis but intact IKK activation in mice lacking the IKK subunit of IB kinase. Science 284, 316-320 (1999) | Article | PubMed | ISI |
12.	Hu, Y. et al. IKK controls formation of the epidermis independently of NF-B. Nature 410, 710-714 (2001) | Article | PubMed | ISI |
13.	Seitz, C. S., Lin, Q., Deng, H. & Khavari, P. A. Alterations in NF-B function in transgenic epithelial tissue demonstrate a growth inhibitory role for NF-B. Proc. Natl Acad. Sci. USA 95, 2307-2312 (1998) | Article | PubMed | ISI |
14.	Seitz, C. S., Deng, H., Hinata, K., Lin, Q. & Khavari, P. A. Nuclear factor B subunits induce epithelial cell growth arrest. Cancer Res. 60, 4085-4092 (2000) | PubMed | ISI |
15.	Beg, A. A., Sha, W. C., Bronson, R. T. & Baltimore, D. Constitutive NF-B activation, enhanced granulopoiesis, and neonatal lethality in IB-deficient mice. Genes Dev. 9, 2736-2746 (1995) | PubMed | ISI |
16.	Klement, J. F. et al. IB deficiency results in a sustained NF-B response and severe widespread dermatitis in mice. Mol. Cell Biol. 16, 2341-2349 (1996) | PubMed | ISI |
17.	Barton, D., HogenEsch, H. & Weih, F. Mice lacking the transcription factor RelB develop T cell-dependent skin lesions similar to human atopic dermatitis. Eur. J. Immunol. 30, 2323-2332 (2000) | Article | PubMed | ISI |
18.	The International Incontinentia Pigmenti (IP) Consortium. Genomic rearrangement in NEMO impairs NF-B activation and is a cause of incontinentia pigmenti. Nature 405, 466-472 (2000) | Article | PubMed |
19.	Fuchs, E. & Raghavan, S. Getting under the skin of epidermal morphogenesis. Nature Rev. Genet. 3, 199-209 (2002) | Article | PubMed | ISI |
20.	Schwenk, F., Baron, U. & Rajewsky, K. A cre-transgenic mouse strain for the ubiquitous deletion of loxP-flanked gene segments including deletion in germ cells. Nucleic Acids Res. 23, 5080-5081 (1995) | PubMed | ISI |
21.	Mao, X., Fujiwara, Y. & Orkin, S. H. Improved reporter strain for monitoring Cre recombinase-mediated DNA excisions in mice. Proc. Natl Acad. Sci. USA 96, 5037-5042 (1999) | Article | PubMed | ISI |
22.	Mombaerts, P. et al. Mutations in T-cell antigen receptor genes and block thymocyte development at different stages. Nature 360, 225-231 (1992) | PubMed | ISI |
23.	Pfeffer, K. et al. Mice deficient for the 55 kd tumour necrosis factor receptor are resistant to endotoxic shock, yet succumb to L. monocytogenes infection. Cell 73, 457-467 (1993) | PubMed | ISI |
24.	Szabowski, A. et al. c-Jun and JunB antagonistically control cytokine-regulated mesenchymal-epidermal interaction in skin. Cell 103, 745-755 (2000) | PubMed | ISI |
25.	Kontgen, F., Suss, G., Stewart, C., Steinmetz, M. & Bluethmann, H. Targeted disruption of the MHC class II Aa gene in C57BL/6 mice. Int. Immunol. 5, 957-964 (1993) | PubMed | ISI |
26.	Yamaoka, S. et al. Complementation cloning of NEMO, a component of the IB kinase complex essential for NF-B activation. Cell 93, 1231-1240 (1998) | PubMed | ISI |
27.	Roper, E., Weinberg, W., Watt, F. M. & Land, H. p19ARF-independent induction of p53 and cell cycle arrest by Raf in murine keratinocytes. EMBO Rep. 2, 145-150 (2001) | Article | PubMed | ISI |
28.	Romero, M. R., Carroll, J. M. & Watt, F. M. Analysis of cultured keratinocytes from a transgenic mouse model of psoriasis: effects of suprabasal integrin expression on keratinocyte adhesion, proliferation and terminal differentiation. Exp. Dermatol. 8, 53-67 (1999) | PubMed | ISI |
29.	Goebeler, M. et al. Differential and sequential expression of multiple chemokines during elicitation of allergic contact hypersensitivity. Am. J. Pathol. 158, 431-440 (2001) | PubMed | ISI |
30.	Werner, S. et al. Targeted expression of a dominant-negative FGF receptor mutant in the epidermis of transgenic mice reveals a role of FGF in keratinocyte organization and differentiation. EMBO J. 12, 2635-2643 (1993) | PubMed | ISI |

Supplementary Information

Supplementary information accompanies this paper.

Acknowledgements

We thank B. Hampel, A. Egert, A. Leinhaas, R. Pofahl, A. Arora, R. Knaup, C. Bessia and the members of the laboratory for skin histopathology of the University of Cologne for technical support. We also thank J. Peschon, G. Kollias and S. Werner for critical reading of the manuscript, G. Mahrle for discussions and F. M. Watt and T. Magin for providing antibodies. M.P. was supported by fellowships from EMBO and the Leukemia and Lymphoma Society; I.H. received grants from the German Ministry of Education and Research and from the Köln Fortune Program. This work was supported by grants from the Cologne Centre for Molecular Medicine (ZMMK) to W. Muller and K.R, from Ligue contre le Cancer (Equipe Labellisée) to A.I., and from the Körber Foundation, the European Union and the Deutsche Forschungsgemeinschaft to K.R.

Competing interests statement

The authors declare no competing financial interests.