Mice with targeted disruption of the fatty acid transport protein 4 (Fatp 4, Slc27a4) gene show features of lethal restrictive dermopathy - PubMed (original) (raw)

. 2003 Jun 23;161(6):1105-15.

doi: 10.1083/jcb.200207080.

Frank van der Hoeven, Hermann-Josef Grone, Adrian Francis Stewart, Lutz Langbein, Iris Kaiser, Gerhard Liebisch, Isabella Gosch, Florian Buchkremer, Wolfgang Drobnik, Gerd Schmitz, Wolfgang Stremmel

Affiliations

Mice with targeted disruption of the fatty acid transport protein 4 (Fatp 4, Slc27a4) gene show features of lethal restrictive dermopathy

Thomas Herrmann et al. J Cell Biol. 2003.

Abstract

The fatty acid transport protein family is a group of evolutionarily conserved proteins that are involved in the cellular uptake and metabolism of long and very long chain fatty acids. However, little is known about their respective physiological roles. To analyze the functional significance of fatty acid transport protein 4 (Fatp4, Slc27a4), we generated mice with a targeted disruption of the Fatp4 gene. Fatp4-null mice displayed features of a neonatally lethal restrictive dermopathy. Their skin was characterized by 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. Lipid analysis demonstrated a disturbed fatty acid composition of epidermal ceramides, in particular a decrease in the C26:0 and C26:0-OH fatty acid substitutes. These findings reveal a previously unknown, essential function of Fatp4 in the formation of the epidermal barrier.

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Figures

Figure 1.

Figure 1.

Fatp4 knockout strategy, genotype analysis, and verification of absent Fatp4 expression in Fatp4 −/− mice. (A) Fatp4 locus before (wild-type, Fatp-WT) and after (mutant allele, Fatp-K) homologous recombination with the targeting vector insert. Cre recombinase converts the allele Fatp4-K to the allele Fatp4-KO, which lacks exon 3. LoxP sites are depicted as open triangles, FRT sites as open rectangles. (B) Southern analysis of the Fatp4 locus. Fatp4 +/+, +/−, and −/− DNA was digested with ScaI, subjected to gel electrophoresis, and transferred to a nylon membrane. The filter was hybridized with a Fatp4 5′ probe, washed, and exposed to film. (C) PCR genotype analysis of tail DNA. An ethidium bromide–stained agarose gel containing amplification products from Fatp4 +/+, +/−, and −/− mice is shown. The primer combination mmF4-In2f/mmF4-pUX4r detects the mutant allele (K), the primer combination mmF4-In2f/mmF4-wtIn3r the wild-type allele (WT). (D) Northern blot analysis of Fatp4 +/+ and Fatp4 −/− RNA from total intestine using a specific Fatp4 cDNA probe and a β-actin control probe, respectively. The respective probe is noted on the right, the genotype is marked above the lanes. (E and F) Immunoblot of Fatp4 and β-actin in total intestine (E) and skin (F) homogenates. Tissue homogenates were prepared from Fatp4 +/+, Fatp4 +/−, and Fatp4 −/− mice, subjected to SDS/PAGE, and blotted. The respective antibody is noted on the right, the genotype is marked above the lanes. The Fatp4 peptide sequence recognized by the antibody is APKHLPSHPDKGFTD (aa 225–239 of the Fatp4 protein), which is encoded by a nucleotide sequence in exons 4 and 5. The data are representative of three separate experiments.

Figure 2.

Figure 2.

Gross analyses of newborn Fatp4 −/− mice and Fatp4 +/+ control littermates. (A–C) Gross morphology of Fatp4 mutant neonates. At birth, Fatp4 −/− progeny were smaller and had a markedly abnormal appearance with facial dysmorphia, a compressed torso, and a taut skin exhibiting a scaly, wrinkle-free surface and extending over and covering the proximal extremities, the joints of which were fixed in flexion contracture. (D) Alcian blue/Alizarin red S staining of wild-type and homozygous mutant skeletons revealed no gross morphological differences, neither in bone nor in cartilage. The flexion joint contractures persisted after the staining procedure.

Figure 3.

Figure 3.

Skin of newborn Fatp4 −/− mice and Fatp4 +/+ control littermates. (A) Neonatal dorsal skin stained with hematoxylin and eosin. In comparison with control epidermis, Fatp4 −/− epidermis exhibits an increased number of cell layers in the stratum spinosum and the basal layer, significantly smaller granula in the stratum granulosum, and a compact, thickened stratum corneum. Bars, 50 μm. (B) Neonatal snout skin stained with Masson's trichrome. In comparison with wild-type dermis, the dermis of Fatp4 −/− mice appears condensed with more compact collagen fibers. Bars, 50 μm. (C) Ultrastructural analysis of epidermis of Fatp4 −/− mice and wild-type littermates (osmium tetroxide fixation). In comparison with control epidermis, the epidermis of Fatp4 −/− mice is characterized by significantly smaller, often irregularly shaped granules (arrow) in the stratum granulosum. Bars, 5 μm. (D, top) In Fatp4 +/+ mice, immunostaining using Fatp4 antiserum shows prominent staining along the cell–cell borders (arrows) in upper layers of the stratum spinosum and in the stratum granulosum subjacent to the stratum corneum (sc), besides a very faint and sparse staining in the lower layers of the stratum spinosum. The inset shows a higher magnification. In Fatp4 −/− mice, these structures appear negative for such a staining pattern. Only a very faint staining is detectable in cells of the stratum basale (sb) of both wild-type and Fatp4 −/− mice. (Bottom) Phase-contrast images of the same sections. Blue: DAPI nuclear staining. Bars, 50 μm.

Figure 4.

Figure 4.

Differentiation of neonatal skin of newborn Fatp4 −/− and control Fatp4 +/+ mice, as shown by immunofluorescence microscopy. (A) Immunofluorescence microscopy of keratin K14 in dorsal skin. In the Fatp4 +/+ control mice, K14 is restricted to the stratum basale (sb) and in the Fatp4 −/− mice is additionally detected in the first layers of the stratum spinosum (open arrow). (B) With keratin K10 antibodies, the stratum spinosum and the stratum granulosum (ss+sg) are strongly stained in both wild-type and Fatp4 −/− mice. However, note that both layers are thicker in the mutant. (C) Immunostaining of K6 is negative in the Fatp4 +/+, but intense in the upper suprabasal layers, including the stratum corneum (sc), of the Fatp4 −/− mice. (D) Loricrin as a typical protein of the cornified envelope is detected in the stratum granulosum (sg) and seems to be a bit more prominent in the mutant mice. (E) The presence of occludin in the plasma membranes of the stratum granulosum (bracket; sb, stratum basale) is seen in a somewhat broader appearing zone in the mutant mice, indicating an intact tight junction barrier in both kinds of mice. To the right of each of the immunofluorescence micrographs are the specific phase-contrast images of the same sections. Blue: DAPI nuclear staining. Bars, 50 μm.

Figure 5.

Figure 5.

Disturbed epidermal barrier in Fatp4 −/− mice. (A) Diffusion of Lucifer yellow in neonatal mice. The fluorescent dye Lucifer yellow did not pass the upper layers of the stratum corneum in wild-type neonatal mice. In contrast, in Fatp4 −/− pups, it permeated the entire epidermis. Bars, 50 μm. (B) Access of X-gal to skin of embryonic day 18.5 mice. Fatp4 +/− epidermis is impermeable for X-gal. In contrast, X-gal permeates Fatp4 −/− skin, where it is cleaved by endogenous β-galactosidase activity to produce a colored precipitate. A Fatp4 +/− mouse is shown as a control to exclude false-positive staining due to β-galactosidase encoded by the lacZ gene of the mutant Fatp4 allele. (C) TEWL of dorsal skin, determined in three litters consisting of 10 Fatp4 +/+, 19 Fatp4 +/−, and 9 Fatp4 −/− pups in total. Error bars depict the SEM. ***, P < 0.001.

Figure 6.

Figure 6.

Lungs of newborn Fatp4 −/− mice and Fatp4 +/+ control littermates. (A) Severe atelectasis of the lung in Fatp4 mutant mice. Fatp4 −/− lungs, stained with hematoxylin/eosin, exhibit a reduced extension of the alveolae and an increase in width of the cellular alveolar septae. Bars, 100 μm. (B) Ultrastructural analysis of type II alveolar cells and alveolar surfactant in Fatp4-deficient mice (ruthenium tetroxide fixation). Lung tissue was obtained immediately after birth and prepared for EM. Extracellular forms of surfactant, which can be detected in both Fatp4 +/+ and Fatp4 −/− mice without distinction between genotypes, are marked by arrows. Bars, 5 μm.

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