Tissue-specific knockout of the mouse Pig-a gene reveals important roles for GPI-anchored proteins in skin development - PubMed (original) (raw)
Tissue-specific knockout of the mouse Pig-a gene reveals important roles for GPI-anchored proteins in skin development
M Tarutani et al. Proc Natl Acad Sci U S A. 1997.
Abstract
Glycosylphosphatidylinositol (GPI)-anchored proteins are widely distributed on plasma membranes of eukaryotes. More than 50 GPI-anchored proteins have been shown to be spatiotemporally expressed in mice with a deficiency of GPI-anchor biosynthesis that causes embryonic lethality. Here, we examine the functional roles of GPI-anchored proteins in mouse skin using the Cre-loxP recombination system. We disrupted the Pig-a gene, an X-linked gene essential for GPI-anchor biosynthesis, in skin. The Cre-mediated Pig-a disruption occurred in skin at almost 100% efficiency in male mice bearing two identically orientated loxP sites within the Pig-a gene. Expression of GPI-anchored proteins was completely absent in the skin of these mice. The skin of such mutants looked wrinkled and more scaly than that of wild-type mice. Furthermore, histological examination of mutant mice showed that the epidermal horny layer was tightly packed and thickened. Electron microscopy showed that the intercellular space was narrow and there were many small vesicles embedded in the intercellular space that were not observed in equivalent wild-type mouse skin preparations. Mutant mice died within a few days after birth, suggesting that Pig-a function is essential for proper skin differentiation and maintenance.
Figures
Figure 1
Targeted insertion of loxP sites into the Pig-a gene. (A) Part of the wild-type Pig-a locus showing the positions of exons 3–6, the targeting construct, and Pig-a allele containing the introduced loxP sites are shown. Solid and open boxes indicate the coding and noncoding exons, respectively. Restriction sites for _Bam_HI (B) and _Eco_RI (E) are indicated. (B) Cre-mediated disappearance of GPI-anchored proteins on the surface of ES cells containing the loxP sites in the Pig-a gene. The plasmid pMC-Cre (13) with hygr was transfected into clone 58. The surface expression of heat-stable antigens (HSAg) was examined before and after selection with 150 μg/ml of hygromycin B for 7 days. Five of the 12 clones lost surface GPI-anchored proteins after selection. A representative clone that lost GPI-anchored proteins is shown. (C) Southern hybridization of the DNA from the germ-line-transmitted and control mice. Genomic DNA was obtained from tails and digested with _Bam_HI. The 3.5- and 2.0-kb fragments represent targeted and endogenous alleles, respectively.
Figure 2
Creation of the K5Cre transgenic lines. (A) K5 promoter activity in cultured cell lines. The construct in which the luciferase gene was placed under the control of the K5 promoter (Upper) was transfected into a keratinocyte cell line (PAM 212) and a fibroblast line (NIH 3T3). The control vector in which the luciferase gene was driven by an simian virus 40 early promoter and its enhancer was also transfected into these cell lines. The luciferase activities were measured and are indicated as arbitrary units (Lower). (B) Structure of the K5Cre transgene. A 14-kb fragment of human K5 promoter used to control the expression of Cre transgene and the nuclear localizing signal was added at 5′ of Cre gene.
Figure 3
Tissue-specific disruption of the Pig-a gene and deficient expression of GPI-anchored proteins in Cre:_Pig-a_flox male mice. (A) Tissue-specific disruption of the Pig-a gene in Cre:_Pig-a_flox male mice. To estimate the efficiency of Pig-a disruption, allele-specific PCR was performed using the three primers shown on the right. Size markers are included. (B) Deficient expression of a GPI-anchored protein, heat-stable antigen (HSAg), in keratinocytes. Epidermis from 1-day-old mice was separated from dermis by trypsin, and then individual cells were obtained. The surface expression of HSAg was examined.
Figure 4
Macro- and microscopic changes of epidermis from wild-type and skin-specific, _Pig-a_-disrupted mouse (Cre:_Pig-a_flox male mouse). (A) Macroscopic appearance of the wild-type and the skin-specific, _Pig-a_-disrupted mouse. (B) Histological examination of the epidermis from the wild-type and the skin-specific, disrupted mouse. (Hematoxylin-eosin staining, ×200.) (C) Histological examination of the horny layers by electron microscopy. The vesicles in the horny layers are indicated by white arrows.
Similar articles
- Rapid compensation for glycosylphosphatidylinositol anchor deficient keratinocytes after birth: visualization of glycosylphosphatidylinositol-anchored proteins in situ.
Gao XH, Kondoh G, Tarutani M, Hara M, Inoue S, Nakanishi T, Okabe M, Yamaguchi Y, Yoshikawa K, Itami S, Takeda J. Gao XH, et al. J Invest Dermatol. 2002 Jun;118(6):998-1002. doi: 10.1046/j.1523-1747.2002.01778.x. J Invest Dermatol. 2002. PMID: 12060394 - Developmental abnormalities of glycosylphosphatidylinositol-anchor-deficient embryos revealed by Cre/loxP system.
Nozaki M, Ohishi K, Yamada N, Kinoshita T, Nagy A, Takeda J. Nozaki M, et al. Lab Invest. 1999 Mar;79(3):293-9. Lab Invest. 1999. PMID: 10092065 - Infertility in female mice with an oocyte-specific knockout of GPI-anchored proteins.
Alfieri JA, Martin AD, Takeda J, Kondoh G, Myles DG, Primakoff P. Alfieri JA, et al. J Cell Sci. 2003 Jun 1;116(Pt 11):2149-55. doi: 10.1242/jcs.00430. Epub 2003 Apr 8. J Cell Sci. 2003. PMID: 12692150 - CHO glycosylation mutants: GPI anchor.
Maeda Y, Ashida H, Kinoshita T. Maeda Y, et al. Methods Enzymol. 2006;416:182-205. doi: 10.1016/S0076-6879(06)16012-7. Methods Enzymol. 2006. PMID: 17113867 Review. - [Paroxysmal nocturnal hemoglobinuria].
Wada H, Kanamaru A. Wada H, et al. Nihon Rinsho. 1996 Sep;54(9):2507-12. Nihon Rinsho. 1996. PMID: 8890586 Review. Japanese.
Cited by
- The role of GABA in modulation of taste signaling within the taste bud.
Mikami A, Huang H, Hyodo A, Horie K, Yasumatsu K, Ninomiya Y, Mitoh Y, Iida S, Yoshida R. Mikami A, et al. Pflugers Arch. 2024 Nov;476(11):1761-1775. doi: 10.1007/s00424-024-03007-x. Epub 2024 Aug 29. Pflugers Arch. 2024. PMID: 39210062 Free PMC article. - MAPK13 controls structural remodeling and disease after epithelial injury.
Wu K, Zhang Y, Mao D, Iberg CA, Yin-Declue H, Sun K, Keeler SP, Wikfors HA, Young D, Yantis J, Austin SR, Byers DE, Brody SL, Crouch EC, Romero AG, Holtzman MJ. Wu K, et al. bioRxiv [Preprint]. 2024 Aug 10:2024.05.31.596863. doi: 10.1101/2024.05.31.596863. bioRxiv. 2024. PMID: 38895360 Free PMC article. Preprint. - AAV-based gene therapy ameliorated CNS-specific GPI defect in mouse models.
Murakami Y, Umeshita S, Imanishi K, Yoshioka Y, Ninomiya A, Sunabori T, Likhite S, Koike M, Meyer KC, Kinoshita T. Murakami Y, et al. Mol Ther Methods Clin Dev. 2023 Dec 14;32(1):101176. doi: 10.1016/j.omtm.2023.101176. eCollection 2024 Mar 14. Mol Ther Methods Clin Dev. 2023. PMID: 38225934 Free PMC article. - Luminal Rank loss decreases cell fitness leading to basal cell bipotency in parous mammary glands.
Rocha AS, Collado-Solé A, Graña-Castro O, Redondo-Pedraza J, Soria-Alcaide G, Cordero A, Santamaría PG, González-Suárez E. Rocha AS, et al. Nat Commun. 2023 Oct 9;14(1):6213. doi: 10.1038/s41467-023-41741-5. Nat Commun. 2023. PMID: 37813842 Free PMC article. - Autophagy critically controls skin inflammation and apoptosis-induced stem cell activation.
Van Hove L, Toniolo A, Ghiasloo M, Lecomte K, Boone F, Ciers M, Raaijmakers K, Vandamme N, Roels J, Maschalidi S, Ravichandran KS, Kasper M, van Loo G, Hoste E. Van Hove L, et al. Autophagy. 2023 Nov;19(11):2958-2971. doi: 10.1080/15548627.2023.2247742. Epub 2023 Sep 1. Autophagy. 2023. PMID: 37615626 Free PMC article.
References
- Udenfriend S, Kodukula K. Annu Rev Biochem. 1995;64:563–591. - PubMed
- Takeda J, Kinoshita T. Trends Biochem Sci. 1995;20:367–371. - PubMed
- Kawagoe K, Kitamura D, Okabe M, Taniuchi I, Ikawa M, Watanabe T, Kinoshita T, Takeda J. Blood. 1996;87:3600–3606. - PubMed
- Kinoshita T, Inoue N, Takeda J. Adv Immunol. 1995;60:57–103. - PubMed
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources
Molecular Biology Databases