Disturbed Epidermal Structure in Mice with Temporally Controlled Fatp4 Deficiency
2005; Elsevier BV; Volume: 125; Issue: 6 Linguagem: Inglês
10.1111/j.0022-202x.2005.23972.x
ISSN1523-1747
AutoresThomas Herrmann, Hermann-Josef Gröne, Lutz Langbein, Iris Kaiser, Isabella Gosch, Ute Bennemann, Daniel Metzger, Pierre Chambon, A. Francis Stewart, Wolfgang Stremmel,
Tópico(s)Amino Acid Enzymes and Metabolism
ResumoSo far, little is known about the physiological role of fatty acid transport protein 4 (Fatp4, Slc27a4). Mice with a targeted disruption of the Fatp4 gene display features of a human neonatally lethal restrictive dermopathy with a hyperproliferative hyperkeratosis, a disturbed epidermal barrier, a flat dermal–epidermal junction, a reduced number of pilo-sebaceous structures, and a compact dermis, demonstrating that Fatp4 is necessary for the formation of the epidermal barrier. Because Fatp4 is widely expressed, it is unclear whether intrinsic Fatp4 deficiency in the epidermis alone can cause changes in the epidermal structure or whether the abnormalities observed are secondary to the loss of Fatp4 in other organs. To evaluate the functional role of Fatp4 in the skin, we generated a mouse line with Fatp4 deficiency inducible in the epidermis. Mice with epidermal keratinocyte-specific Fatp4 deficiency developed a hyperproliferative hyperkeratosis with a disturbed epidermal barrier. These changes resemble the histological abnormalities in the epidermis of newborn mice with total Fatp4 deficiency. We conclude that Fatp4 in epidermal keratinocytes is essential for the maintenance of a normal epidermal structure. So far, little is known about the physiological role of fatty acid transport protein 4 (Fatp4, Slc27a4). Mice with a targeted disruption of the Fatp4 gene display features of a human neonatally lethal restrictive dermopathy with a hyperproliferative hyperkeratosis, a disturbed epidermal barrier, a flat dermal–epidermal junction, a reduced number of pilo-sebaceous structures, and a compact dermis, demonstrating that Fatp4 is necessary for the formation of the epidermal barrier. Because Fatp4 is widely expressed, it is unclear whether intrinsic Fatp4 deficiency in the epidermis alone can cause changes in the epidermal structure or whether the abnormalities observed are secondary to the loss of Fatp4 in other organs. To evaluate the functional role of Fatp4 in the skin, we generated a mouse line with Fatp4 deficiency inducible in the epidermis. Mice with epidermal keratinocyte-specific Fatp4 deficiency developed a hyperproliferative hyperkeratosis with a disturbed epidermal barrier. These changes resemble the histological abnormalities in the epidermis of newborn mice with total Fatp4 deficiency. We conclude that Fatp4 in epidermal keratinocytes is essential for the maintenance of a normal epidermal structure. after the first tamoxifen treatment 5-bromo-4-chloro-3-indolyl-β, D-galactopyranoside Recently, we generated mice with a targeted disruption of the Fatp4 gene within intron 2 to analyze the functional significance of Fatp4 (Herrmann et al., 2003Herrmann T. van der Hoeven F. Gröne H.-J. et al.Mice with targeted disruption of the fatty acid transport protein 4 (Fatp 4, Slc27a4) gene show features of lethal restrictive dermopathy.J Cell Biol. 2003; 161: 1105-1115Crossref PubMed Scopus (150) Google Scholar). The Fatp4 null mice displayed features of a human neonatally lethal restrictive dermopathy (Holbrook et al., 1987Holbrook K.A. Dale B.A. Witt D.R. Hayden M.R. Toriello H.V. Arrested epidermal morphogenesis in three newborn infants with a fatal genetic disorder (restrictive dermopathy).J Invest Dermatol. 1987; 88: 330-339Abstract Full Text PDF PubMed Google Scholar; Dean et al., 1993Dean J.C.S. Gray E.S. Stewart K.N. Brown T. Lloyd D.J. Smith N.C. Pope F.M. Restrictive dermopathy: A disorder of skin differentiation with abnormal integrin expression.Clin Genet. 1993; 44: 287-291Crossref PubMed Scopus (15) Google Scholar). Their skin was characterized by a hyperproliferative hyperkeratosis with a disturbed epidermal barrier, a flat dermal–epidermal junction, a reduced number of pilo-sebaceous structures, and a compact dermis. The rigid skin consistency resulted in an altered body shape with facial dysmorphia, generalized joint flexion contractures, and impaired movement, including suckling and breathing deficiencies. The morphological alterations were accompanied by a severely compromised epidermal barrier function. Lipid analysis demonstrated a disturbed fatty acid composition of epidermal ceramides with a reduced proportion of very long chain fatty acid residues within the ceramide fraction of Fatp4-deficient epidermis. These findings revealed a previously unknown, essential function of Fatp4 in the formation of the epidermal barrier. Another mouse line carrying a naturally occurring mutation in exon 3 of the Fatp4 gene leading to Fatp4 deficiency (Moulson et al., 2003Moulson C.L. Martin D.R. Lugus J.J. Schaffer J.E. Lind A.C. Miner J.H. Cloning of wrinkle-free, a previously uncharacterized mouse mutation, reveals crucial roles for fatty acid transport protein 4 in skin and hair development.Proc Natl Acad Sci USA. 2003; 100: 5274-5279Crossref PubMed Scopus (87) Google Scholar) displayed the same phenotype, confirming our findings. But another genetically engineered mouse line with a targeted disruption of the Fatp4 gene (Gimeno et al., 2003Gimeno R.E. Hirsch D.J. Punreddy S. et al.Targeted deletion of fatty acid transport protein-4 results in early embryonic lethality.J Biol Chem. 2003; 278: 49512-49516Crossref PubMed Scopus (100) Google Scholar) where exons 2 and 3 were replaced by a selectable marker cassette exhibited a far more severe phenotype with early embryonic lethality before day 9.5 of gestation. The reason for the differences between the two mouse lines with disruption of the Fatp4 gene within intron 2 and exon 3, respectively, and the mouse line with disruption of the Fatp4 gene at the 5′ side of exon 2 is unclear. Immunofluorescence microscopy localized Fatp4 to the stratum granulosum and the stratum spinosum of the epidermis (Herrmann et al., 2003Herrmann T. van der Hoeven F. Gröne H.-J. et al.Mice with targeted disruption of the fatty acid transport protein 4 (Fatp 4, Slc27a4) gene show features of lethal restrictive dermopathy.J Cell Biol. 2003; 161: 1105-1115Crossref PubMed Scopus (150) Google Scholar). But it remained unclear whether the lack of Fatp4 in the epidermis was the primary cause of the dominant skin phenotype of mice with generalized Fatp4 deficiency or whether the alterations in the skin architecture were secondary phenomena because of the lack of Fatp4 elsewhere. To answer this question, we used a variation of Cre/lox conditional mutagenesis based on ligand inducible recombination (Logie and Stewart, 1995Logie C. Stewart A.F. Ligand-regulated site-specific recombination.Proc Natl Acad Sci USA. 1995; 92: 5940-5944Crossref PubMed Scopus (140) Google Scholar; Metzger et al., 1995Metzger D. Clifford J. Chiba H. Chambon P. Conditional site-specific recombination in mammalian cells using a ligand-dependent chimeric Cre recombinase.Proc Natl Acad Sci USA. 1995; 92: 6991-6995Crossref PubMed Scopus (393) Google Scholar). In addition to placing loxP sites strategically within the Fatp4 gene, a Cre recombinase—mutant estrogen receptor fusion protein (ERT2) was used to render conditional mutagenesis of Fatp4 dependent upon the administration of tamoxifen (Feil et al., 1997Feil R. Wagner J. Metzger D. Chambon P. Regulation of Cre recombinase activity by mutated estrogen receptor ligand-binding domains.Biochem Biophys Res Commun. 1997; 237: 752-757Crossref PubMed Scopus (688) Google Scholar; Brocard et al., 1998Brocard J. Feil R. Chambon P. Metzger D. A chimeric Cre recombinase inducible by synthetic, but not by natural ligands of the glucocorticoid receptor.Nucleic Acids Res. 1998; 26: 4086-4090Crossref PubMed Scopus (73) Google Scholar; Metzger and Chambon, 2001Metzger D. Chambon P. Site- and time-specific gene targeting in the mouse.Methods. 2001; 24: 71-80Crossref PubMed Scopus (282) Google Scholar). Because Cre-ERT2 expression was directed by the K14 promoter (Li et al., 2000Li M. Indra A.K. Warot X. et al.Skin abnormalities generated by temporally controlled RXR-alpha mutations in mouse epidermis.Nature. 2000; 407: 633-636Crossref PubMed Scopus (263) Google Scholar), conditional mutagenesis of Fatp4 was confined to epidermal keratinocytes after tamoxifen administration. Thereby, we show that Fatp4 in the epidermis itself is essential for the maintenance of a normal epidermal structure. The strategy used for the generation of Fatp4 mutant mice with epidermis-specific Fatp4 inactivation is shown in Figure 1a. To generate mice with a floxed Fatp4 allele (Fatp4flox/wt), Fatp4neoflox/wt mice were crossed with transgenic mice expressing FLPe recombinase (Rodriguez et al., 2000Rodriguez C.I. Buchholz F. Galloway J. et al.High-efficiency deleted mice show that FLPe is an alternative to Cre-loxP.Nat Genet. 2000; 25: 139-140Crossref PubMed Scopus (850) Google Scholar). Fatp4flox/wt mice were crossed with K14-Cre-ERT2 transgenic mice (Li et al., 2000Li M. Indra A.K. Warot X. et al.Skin abnormalities generated by temporally controlled RXR-alpha mutations in mouse epidermis.Nature. 2000; 407: 633-636Crossref PubMed Scopus (263) Google Scholar) to generate Fatp4flox/wt K14-Cre-ERT2(tg/0) double-mutant mice carrying a floxed Fatp4 allele and the K14-Cre-ERT2 transgene. To generate mice with an Fatp4 null allele (Fatp4neoΔex3/wt), Fatp4neoflox/wt mice were crossed with PGK-Cre transgenic mice (Schwenk et al., 1995Schwenk 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. 1995; 23: 5080-5081Crossref PubMed Scopus (935) Google Scholar). Fatp4flox/wt K14-Cre-ERT2(tg/0) mice were crossed with Fatp4neoΔex3/wt K14-Cre-ERT2(0/0) mice to generate Fatp4neoΔex3/flox K14-Cre-ERT2(tg/0) mice, which were injected with tamoxifen to generate mice with epidermis-specific inactivation of Fatp4, now termed esi-Fatp4. PCR analysis was used to identify the different genotypes and to detect the Cre-mediated recombination of the allele Fatp4flox to the allele Fatp4Δex3 (Figure 1b). Mice with epidermis-specific Fatp4 deficiency (esi-Fatp4 mice) did not show any gross abnormality in comparison with control littermates neither 7 nor 13 wk after the first tamoxifen treatment (AFT). Their behavior and gross appearance were also normal. Only histological examination revealed distinct changes. The stratum corneum of esi-Fatp4 mice was considerably thicker than that of control littermates (Figure 2). The stratum spinosum was characterized by an increased number of cell layers. In comparison with newborn mice with generalized Fatp4 deficiency, however, the alterations were less pronounced. The flat dermal–epidermal junction, the reduced number of pilo-sebaceous structures, and the condensed dermis with compact collagen fibers present in newborn mice with generalized Fatp4 deficiency could not be observed in adult esi-Fatp4 mice, neither 7 nor 13 wk AFT. In addition, no ultrastructural differences between control and transgenic mice could be detected, including lamellar membranes as well as the number and shape of lamellar bodies (data not shown). All other organs, including the brain, intestine, liver, spleen, kidneys, myocardium, lung, thymus, and skeletal muscle (not shown), examined by light microscopy appeared normal in esi-Fatp4 mice both 7 and 13 wk AFT. Immunohistochemical analysis revealed an increased number of Ki-67-positive nuclei in the basal layer of esi-Fatp4 mouse epidermis in comparison with control mouse epidermis (7 wk AFT: 45%vs 9%; 13 wk AFT: 42%vs 19%). Whereas in control mouse skin all positive nuclei were located in the basal layer of the epidermis or in the outer root sheath or matrix of hair follicles (data not shown), in esi-Fatp4 mouse skin some Ki-67-positive cells were observed in the first suprabasal layer (Figure 2f). Thus, the observed hyperkeratosis could be classified as a hyperproliferative hyperkeratosis. The immunofluorescence studies were performed on foot sole and on snout epidermis (data not shown). For Fatp4 immunofluorescence microscopy, we used an antiserum that we generated against a specific peptide (Herrmann et al., 2003Herrmann T. van der Hoeven F. Gröne H.-J. et al.Mice with targeted disruption of the fatty acid transport protein 4 (Fatp 4, Slc27a4) gene show features of lethal restrictive dermopathy.J Cell Biol. 2003; 161: 1105-1115Crossref PubMed Scopus (150) Google Scholar). By using staining protocols without detergent addition in any of the fixation and incubation steps (see Materials and Methods), a decoration along the cell margins of the stratum granulosum was seen in Fatp4neoΔex3/flox K14-Cre-ERT2(0/0) mice (henceforth termed control mice; Figure 3a, white open arrow), and a very faint but nonspecific cytoplasmic staining (cf.Herrmann et al., 2003Herrmann T. van der Hoeven F. Gröne H.-J. et al.Mice with targeted disruption of the fatty acid transport protein 4 (Fatp 4, Slc27a4) gene show features of lethal restrictive dermopathy.J Cell Biol. 2003; 161: 1105-1115Crossref PubMed Scopus (150) Google Scholar) was seen in the keratinocytes of the lower stratum basale. This specific Fatp4 staining was not detectable in tamoxifen-treated Fatp4neoΔex3/flox K14-Cre-ERT2(tg/0) mice (esi-Fatp4 mice; Figure 3a, yellow open arrow). Special aspects could be observed when investigating skin appendages. Besides the interfollicular epidermis, in control snout skin, Fatp4 was most prominent at the cellular margins of trichocytes of hair follicle cortex and keratinocytes of the inner root sheath and less pronounced in those of the outer root sheath (Figure 4a, b). Furthermore, the epithelial cells of the sebaceous glands were also positive for Fatp4 (Figure 4c). In mutant animals, as already mentioned, Fatp4 staining of the interfollicular epidermis was no longer detectable and the staining in the sebaceous glands and the outer root sheath was also lost (Figure 4d). Remarkably, the staining of the hair follicle cortex, the inner root sheath, and the companion layer was not affected (Figure 4d, inset and E). No specific Fatp4 staining of dermal components could be detected.Figure 4Appendages in snout skin of Fatp4 mutant and control mice, as shown by immunofluorescence microscopy, and disturbed epidermal barrier in esi-Fatp4 mice. (A–C) In control mice, Fatp4 – besides the interfollicular epidermis – can clearly be detected in skin appendages, in the hair follicle, in particular, in the outer root sheath and most prominently in the inner root sheath and in the hair cortex (cross-section, inset in (A); longitudinal section in (B)). Note the positive large whisker and small pelage follicles (A). The sebaceous glands are also clearly positive for Fatp4 (C). (D–E) In mutant mice (7 wk AFT), Fatp4 staining is missed in the interfollicular skin (D, ep*), the sebaceous glands (D, gl*), and the hair follicle outer root sheath (open triangles in the inset in (D, E)). Note that the inner root sheath and the hair cortex remain positive for Fatp4 in esi-Fatp4 mice. Remarkably, the hair follicle companion layer also remains Fatp4 positive (triangle in E). (F) Access of 5-bromo-4-chloro-3-indolyl-β, D-galactopyranoside (X-gal) to ear skin. Control epidermis is impermeable to X-gal. In contrast, X-gal permeates Fatp4 mutant skin, where it is cleaved by endogenous β-galactosidase activity to produce a colored precipitate. (G) Diffusion of Lucifer yellow in ear skin. The fluorescent dye Lucifer yellow does not pass the upper layers of the stratum corneum in control mice. In contrast, in Fatp4 mutant mice it permeates the entire epidermis. Scale bar, 50 μm (A–E, G).View Large Image Figure ViewerDownload (PPT) As keratin K14 is expressed neither in the hair fiber nor in the inner root sheath (Langbein et al., 1999Langbein L. Rogers M.A. Winter H. Praetzel S. Beckhaus U. Rackwitz H.R. Schweizer J. The catalog of human hair keratins: I. Expression of the nine type I members in the hair follicle.J Biol Chem. 1999; 274: 19874-19893Crossref PubMed Scopus (203) Google Scholar, Langbein et al., 2001Langbein L. Rogers M.A. Winter H. Praetzel S. Schweizer J. The catalog of human hair keratins: II. Expression of the six type II members in the hair follicle and the combined catalog of human type I and type II keratins.J Biol Chem. 2001; 276: 35123-35132Crossref PubMed Scopus (226) Google ScholarLangbein et al., 2002bLangbein L. Rogers M.A. Praetzel S. Aoki N. Winter H. Schweizer J. A novel epithelial keratin, hK6irs1, is expressed differentially in all layers of the inner root sheath, including specialized Huxley cells (“Flügelzellen”) of the human hair follicle.J Invest Dermatol. 2002; 118: 789-800Crossref PubMed Scopus (73) Google Scholar,Langbein et al., 2003Langbein L. Rogers M.A. Praetzel S. Winter H. Schweizer J. K6irs1, K6irs 2, K6irs 3, and K6irs 4 represent the inner-root-sheath (IRS)-specific type II epithelial keratins of the human hair follicle.J Invest Dermatol. 2003; 120: 512-522Crossref PubMed Scopus (97) Google Scholar; for a review seeLangbein and Schweizer, 2005Langbein L. Schweizer J. The keratins of the human hair follicle.Int Rev Cytol. 2005; 243: 1-78Crossref PubMed Scopus (188) Google Scholar), the maintenance of Fatp4 staining after the tamoxifen-induced, K14 promoter-controlled inactivation of Fatp4 in these K14-negative tissue compartments is reasonable. Otherwise, in the K14-positive sebaceous glands (Hughes et al., 1996Hughes B.R. Morris C. Cunliffe W.J. Leigh I.M. Keratin expression in pilosebaceous epithelia in truncal skin of acne patients.Br J Dermatol. 1996; 134: 247-256Crossref PubMed Scopus (73) Google Scholar) and the outer root sheath (Coulombe et al., 1989Coulombe P.A. Kopan R. Fuchs E. Expression of keratin K14 in the epidermis and hair follicle: Insights into complex programs of differentiation.J Cell Biol. 1989; 109: 2295-2312Crossref PubMed Scopus (148) Google Scholar), Fatp4 staining was, like in the interfollicular epidermis, lost. These results can clearly be assessed as an “internal proof” of the specificity and efficiency of the knockout technique used in this work. The skin of esi-Fatp4 mice was hyperplastic, hyperkeratotic, and parakeratotic. Therefore, we investigated the differentiation of keratinocytes, particularly the occurrence and distribution of specific keratins and other proteins of the cornified envelope, by using standard protocols including detergent permeabilization (see Materials and Methods). Keratin K14 (Figure 3b) was markedly restricted to the stratum basale of control skin. The skin of esi-Fatp4 mice showed a comparable immunostaining pattern but with K14 labelling extended to the first or more suprabasal layers (Figure 3b, yellow open arrow), indicative of increased cell proliferation in the Fatp4 mutant skin. This proliferative characteristic of the esi-Fatp4 epidermis was also demonstrable by the intense reaction for keratin K6 (Figure 3c): whereas in the foot sole epidermis of control mice typically only the keratinocytes of the basal layer and few suprabasal cells within the secondary ridges were positive for K6 (Figure 3c, white bracket), in esi-Fatp4 mouse cells, this keratin was labelled in the basal and also in the suprabasal layers, in the stratum spinosum, and particularly in the stratum granulosum (Figure 3c, yellow bracket). Strong keratin K10 reactivity was observed in all living suprabasal cell layers (Figure 3d, white bracket, ss+sg), but appeared much broader in the hyperkeratotic foot sole of esi-Fatp4 mice (Figure 3d, yellow bracket, ss+sg). Neither the stratum basale nor the stratum corneum reacted in either kind of mice. The immunostaining of keratin K2e was restricted to the keratinocytes of the stratum granulosum in control mice (Figure 3e; white bracket, sg), whereas it was greatly widened, extending to the cells of the upper stratum spinosum, in esi-Fatp4 mice (Figure 3e; yellow bracket, ss+sg). The stage of cornification of the skin keratinocytes was investigated by the immunolocalization of loricrin (Figure 3f), filaggrin (Figure 3g), and transglutaminase I (Figure 3h): all of these proteins were seen in their typical localizations in both esi-Fatp4 and control animals, the only consistent difference being that more cell layers showed immunoreactivity in the skin of esi-Fatp4 mice, indicative of the hyperkeratinization (Figure 3f–g, yellow brackets). Immunohistochemical staining of the tight junction proteins claudin-1, occludin, and protein ZO-1 virtually reproduced their typical wild-type localization (cf.Morita et al., 1999Morita K. Furuse M. Fujimoto K. Tsukita S. Claudin multigene family encoding four-transmembrane domain protein components of tight junction strands.Proc Natl Acad Sci USA. 1999; 96: 511-516Crossref PubMed Scopus (937) Google Scholar; Brandner et al., 2002Brandner J.M. Kief S. Grund C. et al.Organization and formation of the tight junction system in human epidermis and cultured keratinocytes.Eur J Cell Biol. 2002; 81: 253-263Crossref PubMed Scopus (224) Google Scholar; Langbein et al., 2002aLangbein L. Grund C. Kuhn C. et al.Tight junctions and compositionally related junctional structures in mammalian stratified epithelia and cell cultures derived therefrom.Eur J Cell Biol. 2002; 81: 419-435Crossref PubMed Scopus (169) Google Scholar) i.e. occludin exclusively in the stratum granulosum (data not shown), whereas protein ZO-1 (data not shown) and claudin-1 could also be detected at the cell margins of cells of the lower strata (Figure 3i, white bracket, sb-sg; cf. alsoMorita et al., 1999Morita K. Furuse M. Fujimoto K. Tsukita S. Claudin multigene family encoding four-transmembrane domain protein components of tight junction strands.Proc Natl Acad Sci USA. 1999; 96: 511-516Crossref PubMed Scopus (937) Google Scholar; Brandner et al., 2002Brandner J.M. Kief S. Grund C. et al.Organization and formation of the tight junction system in human epidermis and cultured keratinocytes.Eur J Cell Biol. 2002; 81: 253-263Crossref PubMed Scopus (224) Google Scholar; Langbein et al., 2002aLangbein L. Grund C. Kuhn C. et al.Tight junctions and compositionally related junctional structures in mammalian stratified epithelia and cell cultures derived therefrom.Eur J Cell Biol. 2002; 81: 419-435Crossref PubMed Scopus (169) Google Scholar). In the Fatp4 mutant animals, both proteins were often detected in the additional layers of the thickened skin. Remarkably, under these conditions, claudin-1 was no longer detectable in the stratum basale of esi-Fatp4 mice (Figure 3i, yellow bracket, ss+sg). To examine whether the observed abnormal skin structure might also be associated with an impaired skin barrier function, two different techniques were applied. The substrate 5-bromo-4-chloro-3-indolyl-β, D-galactopyranoside (X-gal) penetrated esi-Fatp4 skin, where it was cleaved to produce a colored precipitate by endogenous β-galactosidase activity, whereas the skin of control mice was impermeable (Figure 4f). In another set of experiments it was shown that the fluorescent dye Lucifer yellow permeated throughout the entire epidermis in esi-Fatp4 mice; in contrast, it did not pass through the upper layers of the stratum corneum in control mice (Figure 4g). Thus, we concluded that the abnormal epidermal structure of esi-Fatp4 mice was accompanied by a compromised epidermal barrier function. Mice with generalized Fatp4 deficiency exhibit a characteristic phenotype with distinct abnormalities in the structure and a disturbed barrier function of the epidermis (Herrmann et al., 2003Herrmann T. van der Hoeven F. Gröne H.-J. et al.Mice with targeted disruption of the fatty acid transport protein 4 (Fatp 4, Slc27a4) gene show features of lethal restrictive dermopathy.J Cell Biol. 2003; 161: 1105-1115Crossref PubMed Scopus (150) Google Scholar; Moulson et al., 2003Moulson C.L. Martin D.R. Lugus J.J. Schaffer J.E. Lind A.C. Miner J.H. Cloning of wrinkle-free, a previously uncharacterized mouse mutation, reveals crucial roles for fatty acid transport protein 4 in skin and hair development.Proc Natl Acad Sci USA. 2003; 100: 5274-5279Crossref PubMed Scopus (87) Google Scholar). How Fatp4 deficiency leads to these severe alterations is still unclear. As Fatp4 acts as an acyl-CoA synthetase for long and very long chain fatty acids (Herrmann et al., 2001Herrmann T. Buchkremer F. Gosch I. Hall A.M. Bernlohr D.A. Stremmel W. Mouse fatty acid transport protein 4 (FATP4): Characterization of the gene and functional assessment as a very long chain acyl-CoA synthetase.Gene. 2001; 270: 31-40Crossref PubMed Scopus (121) Google Scholar; Hall et al., 2005Hall A.M. Wiczer B.M. Herrmann T. Stremmel W. Bernlohr D.A. Enzymatic properties of purified murine fatty acid transport protein 4 and analysis of acyl-CoA synthetase activities in tissues from FATP4 null mice.J Biol Chem. 2005; 280: 11948-11954Crossref PubMed Scopus (111) Google Scholar) and Fatp4-/- mice exhibit a disturbed fatty acid composition of epidermal ceramides (Herrmann et al., 2003Herrmann T. van der Hoeven F. Gröne H.-J. et al.Mice with targeted disruption of the fatty acid transport protein 4 (Fatp 4, Slc27a4) gene show features of lethal restrictive dermopathy.J Cell Biol. 2003; 161: 1105-1115Crossref PubMed Scopus (150) Google Scholar), Fatp4 might play an important role in the epidermal ceramide metabolism. The mutation within the Fatp4 gene of our mouse line and the Fatp4-deficient mouse line described byMoulson et al., 2003Moulson C.L. Martin D.R. Lugus J.J. Schaffer J.E. Lind A.C. Miner J.H. Cloning of wrinkle-free, a previously uncharacterized mouse mutation, reveals crucial roles for fatty acid transport protein 4 in skin and hair development.Proc Natl Acad Sci USA. 2003; 100: 5274-5279Crossref PubMed Scopus (87) Google Scholar is located within intron 2 and exon 3, respectively. Because we generated a gene trap vector to target the Fatp4 gene with a mutant multipurpose Fatp4 allele, we had to integrate the cassette with the selectable marker after the first coding exon, i.e., after exon 2. Another mouse line with a disruption of the Fatp4 gene at the 5′ side of exon 2 exhibits a far more severe phenotype with early embryonic lethality before gestational day 9.5 (Gimeno et al., 2003Gimeno R.E. Hirsch D.J. Punreddy S. et al.Targeted deletion of fatty acid transport protein-4 results in early embryonic lethality.J Biol Chem. 2003; 278: 49512-49516Crossref PubMed Scopus (100) Google Scholar). The reason for this difference is still elusive. It has been speculated that the disruption of intron 2 or exon 3, respectively, might give rise to a trunkated form of Fatp4 with a rest activity leading to a less severe phenotype than observed when the Fatp4 gene is disrupted within exon 1. This truncated protein, however, would carry only the first 53 amino acids of Fatp4, whereas the length of the total deduced Fatp4 amino acid sequence is 643 amino acids. Fatp4 is widely expressed in many organs. Its physiological function in these organs has so far remained elusive. The gene is also strongly expressed in the skin, as we have shown at the RNA level by Northern blot analysis (Herrmann et al., 2001Herrmann T. Buchkremer F. Gosch I. Hall A.M. Bernlohr D.A. Stremmel W. Mouse fatty acid transport protein 4 (FATP4): Characterization of the gene and functional assessment as a very long chain acyl-CoA synthetase.Gene. 2001; 270: 31-40Crossref PubMed Scopus (121) Google Scholar) and quantitative RT-PCR (not shown), as well as at the protein level by immunofluorescence (Herrmann et al., 2003Herrmann T. van der Hoeven F. Gröne H.-J. et al.Mice with targeted disruption of the fatty acid transport protein 4 (Fatp 4, Slc27a4) gene show features of lethal restrictive dermopathy.J Cell Biol. 2003; 161: 1105-1115Crossref PubMed Scopus (150) Google Scholar). But proof has not been provided that the lack of Fatp4 in the epidermis causes the phenotype of Fatp4-deficient mice. It might be conceivable that the lack of Fatp4 in another tissue or other tissues causes secondary effects on the generation and maintenance of the epidermal structure. To find out whether Fatp4 indeed plays a pivotal role in the epidermis for the maintenance of a normal skin structure, the generation of a mouse line with keratinocyte-specific Fatp4 deficiency (Li et al., 2000Li M. Indra A.K. Warot X. et al.Skin abnormalities generated by temporally controlled RXR-alpha mutations in mouse epidermis.Nature. 2000; 407: 633-636Crossref PubMed Scopus (263) Google Scholar; Metzger and Chambon, 2001Metzger D. Chambon P. Site- and time-specific gene targeting in the mouse.Methods. 2001; 24: 71-80Crossref PubMed Scopus (282) Google Scholar) appears to be a promising approach. In this paper, we describe the generation of the first conditional Fatp4-deficient mouse line at all. This mouse line displays distinct changes in the structure of the epidermis and a compromised epidermal barrier function resembling the changes in mice with generalized Fatp4 deficiency. Thus, we show that the function of Fatp4 in keratinocytes is essential for the maintenance of a normal epidermal structure. Certain features of newborn mice with generalized Fatp4 deficiency could not be observed in our mice with epidermis-specific Fatp4 deficiency, like the flat dermal–epidermal junction, the reduced number of pilo-sebaceous structures, and the condensed dermis with compact collagen fibers. Whereas newborn mice with total Fatp4 deficiency can be recognized at first sight because of their characteristic appearance, esi-Fatp4 mice are not distinguishable from their control littermates macroscopically. Possible explanations for the milder phenotype of adult esi-Fatp4 mice in comparison with newborn mice with total Fatp4 deficiency are: (1) as described (Li et al., 2000Li M. Indra A.K. Warot X. et al.Skin abnormalities generated by temporally controlled RXR-alpha mutations in mouse epidermis.Nature. 2000; 407: 633-636Crossref PubMed Scopus (263) Google Scholar), we treated mice at the age of 10 wk with tamoxifen for 5 consecutive days, and again for 5 consecutive days, 2, 4, and 6 wk later, and examined them 7 and 13 wk AFT, respectively. Possibly, this period of time might not be sufficient for the development of all alterations observed in newborn mice with generalized Fatp4 deficiency, and further changes in the skin architecture might become visible after a longer period of observation or a longer time course of tamoxifen treatment. (2) The recombination efficiency of K14-Cre-ERT2 might be below 100%. A small rest activity of Fatp4 in the epidermis of esi-Fatp mice might lead to a milder phenotype than in mice with total Fatp4 deficiency; however, we could not detect Fatp4 protein by immunofluorescence 7 or 13 wk AFT. Furthermore,Li et al., 2000Li M. Indra A.K. Warot X. et al.Skin abnormalities generated by temporally controlled RXR-alpha mutations in mouse epidermis.Nature. 2000; 407: 633-636Crossref PubMed Scopus (263) Google Scholar already described a complete recombination of a floxed RXRα allele by K14-Cre-ERT2. (3) Fatp4 might be more important for the generation than for the maintenance of the epidermal barrier. The epidermis of mice with generalized Fatp4 deficiency exhibits an abnormal lipid composition, the most striking difference being an increase of the ceramide fraction by about 80% and a decrease of very long chain fatty acids with at least 26 C atoms within the ceramide fraction from 64% to 26% in Fatp4-deficient mice (Herrmann et al., 2003Herrmann T. van der Hoeven F. Gröne H.-J. et al.Mice with targeted disruption of the fatty acid transport protein 4 (Fatp 4, Slc27a4) gene show features of lethal restrictive dermopathy.J Cell Biol. 2003; 161: 1105-1115Crossref PubMed Scopus (150) Google Scholar). Although an analysis of the lipid composition of the epidermis of esi-Fatp4 mice has not been performed, we assume that the mechanisms that lead to these very similar phenotypes are identical because of the identical underlying genetic cause. Of note, in addition to their structural role in the epidermis, ceramides are also involved in various signalling pathways that control differentiation and proliferation (Huwiler et al., 2000Huwiler A. Kolter T. Pfeilschifter J. Sandhoff K. Physiology and pathophysiology of sphingolipid metabolism and signaling.Biochim Biophys Acta. 2000; 1485: 63-99Crossref PubMed Scopus (373) Google Scholar). The availability of a mouse model with inducible epidermis-specific Fatp4 deficiency in the skin will enable further projects for analysis of the details of Fatp4 function in the skin of adult mice. By means of this mouse line, it will be possible to determine the sequence of morphological events that lead to the observed hyperkeratosis, to characterize the metabolic changes in Fatp4-deficient epidermis further, and to detect delayed consequences of the observed morphological and functional changes. Genomic DNA was isolated from tissues by standard techniques. The epidermis was separated from the dermis after heating tail skin in distilled water to 64°C for 10 s. Fatp4 genotyping was performed by PCR with primer pairs specific for the wt, the flox, the neoΔex3, and the Δex3 alleles, respectively. The generation of Fatp4 mutant mice has been reported previously (Herrmann et al., 2003Herrmann T. van der Hoeven F. Gröne H.-J. et al.Mice with targeted disruption of the fatty acid transport protein 4 (Fatp 4, Slc27a4) gene show features of lethal restrictive dermopathy.J Cell Biol. 2003; 161: 1105-1115Crossref PubMed Scopus (150) Google Scholar). The original mouse line carried the mutant allele Fatp4neoflox (Fatp4-K;Herrmann et al., 2003Herrmann T. van der Hoeven F. Gröne H.-J. et al.Mice with targeted disruption of the fatty acid transport protein 4 (Fatp 4, Slc27a4) gene show features of lethal restrictive dermopathy.J Cell Biol. 2003; 161: 1105-1115Crossref PubMed Scopus (150) Google Scholar) harboring a gene trap cassette in intron 2 (Figure 1a). By crossing this mouse line with a transgenic mouse line expressing FLPe recombinase (Rodriguez et al., 2000Rodriguez C.I. Buchholz F. Galloway J. et al.High-efficiency deleted mice show that FLPe is an alternative to Cre-loxP.Nat Genet. 2000; 25: 139-140Crossref PubMed Scopus (850) Google Scholar), the gene trap cassette was excised, and the mutant allele Fatp4neoflox was converted into the mutant allele Fatp4flox, which carried two loxP sites within introns 2 and 3, respectively (Figure 1a). Mice homozygous for the “floxed”Fatp4 allele showed no phenotypic abnormalities, indicating that the allele Fatp4flox was functionally fully active. When mice carrying the Fatp4flox allele were crossed with PGK-Cre transgenic mice (kindly provided by Klas Kullander and Ruediger Klein) exhibiting ubiquitous Cre expression (Schwenk et al., 1995Schwenk 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. 1995; 23: 5080-5081Crossref PubMed Scopus (935) Google Scholar), the Fatp4flox allele was converted into the null allele Fatp4Δex3 (Figure 1a). Offspring homozygous for the Fatp4Δex3 or Fatp4neoflox alleles were analyzed for morphological differences and were found to be identical (data not shown). All procedures were in compliance with the guidelines of the institutional animal care and use committees, and in accordance with governmental guidelines. Institutional approval was granted for all experiments. To generate mice with tamoxifen-inducible Fatp4 inactivation in the epidermis, mice carrying the Fatp4flox allele were crossed with transgenic mice carrying the K14-Cre-ERT2 transgene that expresses the tamoxifen-inducible Cre-ERT2 recombinase specifically in keratinocytes (Li et al., 2000Li M. Indra A.K. Warot X. et al.Skin abnormalities generated by temporally controlled RXR-alpha mutations in mouse epidermis.Nature. 2000; 407: 633-636Crossref PubMed Scopus (263) Google Scholar). The resulting mice used for the experiments had a mixed genetic background C57BL/6–129SV/Ola. As described (Li et al., 2000Li M. Indra A.K. Warot X. et al.Skin abnormalities generated by temporally controlled RXR-alpha mutations in mouse epidermis.Nature. 2000; 407: 633-636Crossref PubMed Scopus (263) Google Scholar), tamoxifen (Sigma, St. Louis, Missouri; 0.1 mg in 100 μL sunflower oil) was injected intraperitoneally into female mice at the age of 10 wk for 5 consecutive days, and again for 5 consecutive days, 2, 4, and 6 wk later. Animals were sacrificed 7 or 13 wk AFT, and tissues were analyzed by histology, immunohistochemistry, and electron microscopy. Histological analysis of the heart, lung, liver, spleen, kidney, stomach, duodenum, jejunum, ileum, colon, skeletal muscle, adipose tissue, and skin from dorsal and ventral trunk, ears, eyelids, snout, feet, and tail from control and tamoxifen-treated (7 wk AFT and 13 wk AFT) mice was performed as described previously (Herrmann et al., 2003Herrmann T. van der Hoeven F. Gröne H.-J. et al.Mice with targeted disruption of the fatty acid transport protein 4 (Fatp 4, Slc27a4) gene show features of lethal restrictive dermopathy.J Cell Biol. 2003; 161: 1105-1115Crossref PubMed Scopus (150) Google Scholar). The primary antibodies used in immunohistochemistry were as follows: (1) guinea-pig antibodies against mouse Fatp4 (1:500;Herrmann et al., 2003Herrmann T. van der Hoeven F. Gröne H.-J. et al.Mice with targeted disruption of the fatty acid transport protein 4 (Fatp 4, Slc27a4) gene show features of lethal restrictive dermopathy.J Cell Biol. 2003; 161: 1105-1115Crossref PubMed Scopus (150) Google Scholar), K14 (1:3000), K2e (1:2000) (both from Progen Biotechnik, Heidelberg, Germany), (2) mouse antibodies against transglutaminase 1 (1:20, CellSystems, St. Katharinen, Germany), (3) rabbit antibodies against keratin K10 (1:500, Covance, Denver, Pennsylvania), filaggrin (1:500, Covance), claudin-1 (1:100, Neomarkers Labvision, Fremont, California), loricrin (1:500, Covance), periplakin (1:500, a gift from Dr D. Hohl, Baylor College, Houston, Texas), and keratin K6 (1:500, Covance), (4) rat antibodies against Ki-67 (1:200, clone TEC-3, DAKO, Glostrup, Denmark). The secondary antibodies used for indirect immunofluorescence were goat antibodies to guinea-pig, rabbit, mouse, or rat immunoglobulins, coupled to Alexa 568 (dilution of 1:200; Molecular Probes, Eugene, Oregon). Indirect immunofluorescence microscopy procedures for membrane-associated proteins and cytoskeletal components were essentially performed as described inHerrmann et al., 2003Herrmann T. van der Hoeven F. Gröne H.-J. et al.Mice with targeted disruption of the fatty acid transport protein 4 (Fatp 4, Slc27a4) gene show features of lethal restrictive dermopathy.J Cell Biol. 2003; 161: 1105-1115Crossref PubMed Scopus (150) Google Scholar (see alsoSchmidt et al., 1997Schmidt A. Langbein L. Rode M. Praetzel S. Zimbelmann R. Franke W.W. Plakophilins 1a and 1b: Widespread nuclear proteins recruited in specific epithelial cells as desmosomal plaque components.Cell Tiss Res. 1997; 29: 481-499Crossref Scopus (148) Google Scholar; Langbein et al., 2002aLangbein L. Grund C. Kuhn C. et al.Tight junctions and compositionally related junctional structures in mammalian stratified epithelia and cell cultures derived therefrom.Eur J Cell Biol. 2002; 81: 419-435Crossref PubMed Scopus (169) Google Scholar). All investigations were performed in parallel on foot sole and snout skin of mice 7 and 13 wk AFT. In brief, the standard protocol was applied by using cryostat sections of freshly frozen tissues, fixed in acetone (5 min at -20°C), permeabilized with 0.1% Triton X-100/PBS for 2 min, followed by blocking with 5% normal goat serum in PBS. Primary and secondary antibodies were applied for ca. 45 min. For Fatp4 protein demonstration, various fixative protocols were used to prevent loss of soluble proteins (see protocols 2 and 3;Schmidt et al., 1997Schmidt A. Langbein L. Rode M. Praetzel S. Zimbelmann R. Franke W.W. Plakophilins 1a and 1b: Widespread nuclear proteins recruited in specific epithelial cells as desmosomal plaque components.Cell Tiss Res. 1997; 29: 481-499Crossref Scopus (148) Google Scholar). The tissues were fixed with formaldehyde and antibodies applied for 45 min, or for a shorter time without using any detergents. Negative control immunostaining reactions were performed using non-immunized serum instead of the primary antibodies at the respective dilution or only the secondary antibody against immunoglobulins of the respective species. All of them were found to be negative. Moreover, “internal controls” exist by using the panel of different antibodies of several species indicated in the paragraph “Antibodies.” DAPI for nuclear counterstaining was added to the secondary antibodies. Visualization and documentation were done by using a photomicroscope (Axiophot II, Carl Zeiss, Germany). Electron microscopical investigations on the skin of esi-Fapt4 and control mice 7 and 13 wk AFT were conducted as described inHerrmann et al., 2003Herrmann T. van der Hoeven F. Gröne H.-J. et al.Mice with targeted disruption of the fatty acid transport protein 4 (Fatp 4, Slc27a4) gene show features of lethal restrictive dermopathy.J Cell Biol. 2003; 161: 1105-1115Crossref PubMed Scopus (150) Google Scholar. The diffusion of 1 mM Lucifer yellow in Ringer's solution (pH 7.4) at 37°C was analyzed as described (Matsuki et al., 1998Matsuki M. Yamashita F. Ishida-Yamamoto A. et al.Defective stratum corneum and early neonatal death in mice lacking the gene for transglutaminase 1 (keratinocyte transglutaminase).Proc Natl Acad Sci USA. 1998; 95: 1044-1049Crossref PubMed Scopus (230) Google Scholar). The frozen ear skin samples of animals 7 and 13 wk AFT, respectively, were sliced at a thickness of 5 μm and the sections were analyzed by fluorescence microscopy. In addition, barrier-dependent access of X-gal to untreated skin was examined as described (Hardman et al., 1998Hardman M.J. Paraskevi S. Banbury D.N. Byrne C. Patterned acquisition of skin barrier function during development.Development. 1998; 125: 1541-1552Crossref PubMed Google Scholar). We thank A. Peter, P. Hornsberger, G. Schmidt, S. Kaden, and S. Praetzel for their expert technical assistance. This work was supported by grants from the Deutsche Forschungsgemeinschaft (STR 216/11-1) and the Dietmar-Hopp-Foundation to W. Stremmel, and the Faculty of Medicine, University of Heidelberg to T. Herrmann.
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