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Cell Biology: Master Regulators of Sealing and Healing

2005; Elsevier BV; Volume: 15; Issue: 11 Linguagem: Inglês

10.1016/j.cub.2005.05.034

1879-0445

Brian Stramer, Paul Martin,

Cellular Mechanics and Interactions

The protective layer of the epidermis in Drosophila (cuticle) and mice (stratum corneum) are structurally unrelated. Yet new evidence suggests a conserved transcription factor, Grainyhead, controls both their development and the means by which both structures repair themselves. The protective layer of the epidermis in Drosophila (cuticle) and mice (stratum corneum) are structurally unrelated. Yet new evidence suggests a conserved transcription factor, Grainyhead, controls both their development and the means by which both structures repair themselves. Amazingly, while the eyes and hearts of Drosophila and mammals are constructed in entirely different ways and are morphologically quite distinct, their development appears to be under the control of similar master-regulatory transcription factors: Small eye/Pax6 for eyes [1Gehring W.J. The master control gene for morphogenesis and evolution of the eye.Genes Cells. 1996; 1: 11-15Crossref PubMed Scopus (202) Google Scholar], and Tinman/Nkx2-5 for heart [2Bodmer R. Venkatesh T.V. Heart development in Drosophila and vertebrates: conservation of molecular mechanisms.Dev. Genet. 1998; 22: 181-186Crossref PubMed Scopus (148) Google Scholar]. From two new papers [3Mace K.A. Pearson J.C. McGinnis W. An epidermal barrier wound repair pathway in Drosophila is mediated by grainy head.Science. 2005; 308: 381-385Crossref PubMed Scopus (176) Google Scholar, 4Ting S.B. Caddy J. Hislop N. Wilanowski T. Auden A. Zhao L.L. Ellis S. Kaur P. Uchida Y. Holleran W.M. et al.A homolog of Drosophila grainy head is essential for epidermal integrity in mice.Science. 2005; 308: 411-413Crossref PubMed Scopus (216) Google Scholar], it appears that this theme of signalling conservation controlling analogous structures and processes in these two very different phyla may also be true for epidermal barrier formation and even for wound healing. The new studies [3Mace K.A. Pearson J.C. McGinnis W. An epidermal barrier wound repair pathway in Drosophila is mediated by grainy head.Science. 2005; 308: 381-385Crossref PubMed Scopus (176) Google Scholar, 4Ting S.B. Caddy J. Hislop N. Wilanowski T. Auden A. Zhao L.L. Ellis S. Kaur P. Uchida Y. Holleran W.M. et al.A homolog of Drosophila grainy head is essential for epidermal integrity in mice.Science. 2005; 308: 411-413Crossref PubMed Scopus (216) Google Scholar] have shown that the CP2 transcription factor Grainyhead is required for assembly of the complex chitin coat of the fly as well as the outer stratum corneum of the epidermis in mice. Furthermore, in both flies and mice, Grainyhead is rapidly upregulated in the epithelium if either of these protective outer layers are damaged, and it appears to be necessary for successful healing. The embryonic epidermis of Drosophila is composed of a simple squamous epithelium covered by a protective barrier called the cuticle, which is made of a mesh of crosslinked proteins and chitins [5Wright T.R. The Wilhelmine E. Key 1992 Invitational lecture. Phenotypic analysis of the Dopa decarboxylase gene cluster mutants in Drosophila melanogaster.J. Hered. 1996; 87: 175-190Crossref PubMed Scopus (50) Google Scholar]. Two enzymes, DOPA decarboxylase and tyrosine hydroxylase, are required to catalyze the production of the quinones necessary to cross-link the cuticle proteins creating a final rigid protective barrier [5Wright T.R. The Wilhelmine E. Key 1992 Invitational lecture. Phenotypic analysis of the Dopa decarboxylase gene cluster mutants in Drosophila melanogaster.J. Hered. 1996; 87: 175-190Crossref PubMed Scopus (50) Google Scholar]. Mace et al. [3Mace K.A. Pearson J.C. McGinnis W. An epidermal barrier wound repair pathway in Drosophila is mediated by grainy head.Science. 2005; 308: 381-385Crossref PubMed Scopus (176) Google Scholar] showed that these enzymes are rapidly switched on in epidermal cells surrounding wounds in the epithelial surface of Drosophila embryos. So not only are these enzymes necessary for cuticle formation during development, but they may similarly be required for epidermal repair. Previous studies in flies showed that wound healing recapitulates morphogenesis at the level of the cytoskeletal machinery needed to close epidermal holes — actin pursestrings and dynamic filopodial zippers [6Wood W. Jacinto A. Grose R. Woolner S. Gale J. Wilson C. Martin P. Wound healing recapitulates morphogenesis in Drosophila embryos.Nat. Cell Biol. 2002; 4: 907-912Crossref PubMed Scopus (294) Google Scholar, 7Martin P. Parkhurst S.M. Parallels between tissue repair and embryo morphogenesis.Development. 2004; 131: 3021-3034Crossref PubMed Scopus (413) Google Scholar] (Figure 1) — but the new studies [3Mace K.A. Pearson J.C. McGinnis W. An epidermal barrier wound repair pathway in Drosophila is mediated by grainy head.Science. 2005; 308: 381-385Crossref PubMed Scopus (176) Google Scholar, 4Ting S.B. Caddy J. Hislop N. Wilanowski T. Auden A. Zhao L.L. Ellis S. Kaur P. Uchida Y. Holleran W.M. et al.A homolog of Drosophila grainy head is essential for epidermal integrity in mice.Science. 2005; 308: 411-413Crossref PubMed Scopus (216) Google Scholar] now provide evidence for a novel transcriptional regulatory process which is used during development and reused to repair wounds. The enhancer elements that mediate transcriptional upregulation of the DOPA decarboxylase and tyrosine hydroxylase genes during the wound response have consensus binding sites for Grainyhead, which is localized to the epidermis of Drosophila embryos and is required for developmental DOPA decarboxylase expression; not surprisingly, Grainyhead mutant flies have a defective cuticle [8Bray S.J. Kafatos F.C. Developmental function of Elf-1: an essential transcription factor during embryogenesis in Drosophila.Genes Dev. 1991; 5: 1672-1683Crossref PubMed Scopus (145) Google Scholar]. DOPA decarboxylase reporter constructs lacking the Grainyhead binding sites fail to be activated around wounds. Furthermore, Grainyhead mutant fly embryos only weakly induce DOPA decarboxylase expression at wound sites, confirming that Grainyhead signalling is key to wound expression of DOPA decarboxylase. The epidermis of mice and humans is far more complex than that of Drosophila embryos; in mammals this layer of the skin consists of a stratified squamous epithelium protected by a structure somewhat analogous to the cuticle in flies [9Madison K.C. Barrier function of the skin: “la raison d’etre” of the epidermis.J. Invest. Dermatol. 2003; 121: 231-241Crossref PubMed Scopus (736) Google Scholar]. This stratum corneum, much like the Drosophila cuticle, is composed of structural proteins and lipids, covalently cross-linked by the enzyme transglutaminase 1, which fulfils the same role as DOPA decarboxylase and tyrosine hydroxylase for crosslinking the fly cuticle proteins [9Madison K.C. Barrier function of the skin: “la raison d’etre” of the epidermis.J. Invest. Dermatol. 2003; 121: 231-241Crossref PubMed Scopus (736) Google Scholar]. Ting et al. [4Ting S.B. Caddy J. Hislop N. Wilanowski T. Auden A. Zhao L.L. Ellis S. Kaur P. Uchida Y. Holleran W.M. et al.A homolog of Drosophila grainy head is essential for epidermal integrity in mice.Science. 2005; 308: 411-413Crossref PubMed Scopus (216) Google Scholar] showed that Grainy head-like 3 (Grhl3), the mouse homolog of Grainyhead, is expressed in the developing embryonic epithelium from embryonic day 12.5 onward, and that Grhl3 null mice have defects in skin permeability. Grainyhead mutant embryos dipped in dye exhibit a leaky skin at stages when their wild-type siblings are fully impervious to the same dyes. The reason for this leakiness turns out to be that, just as Grainyhead is needed for cuticle formation in flies, Grhl3 is needed for formation of the stratum corneum in mammalian skin. Ting et al. [4Ting S.B. Caddy J. Hislop N. Wilanowski T. Auden A. Zhao L.L. Ellis S. Kaur P. Uchida Y. Holleran W.M. et al.A homolog of Drosophila grainy head is essential for epidermal integrity in mice.Science. 2005; 308: 411-413Crossref PubMed Scopus (216) Google Scholar] realized that the Grhl3 mutant phenotype is rather similar to that of transglutaminase 1 knockout mice [10Kuramoto N. Takizawa T. Matsuki M. Morioka H. Robinson J.M. Yamanishi K. Development of ichthyosiform skin compensates for defective permeability barrier function in mice lacking transglutaminase 1.J. Clin. Invest. 2002; 109: 243-250Crossref PubMed Scopus (98) Google Scholar, 11Matsuki M. Yamashita F. Ishida-Yamamoto A. Yamada K. Kinoshita C. Fushiki S. Ueda E. Morishima Y. Tabata K. Yasuno H. 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]. Further analysis of the Grhl3 target by screening random sequences revealed a site identical to Drosophila’s Grainyhead, and the authors went on to show that the transglutaminase 1 gene contains consensus binding sites for Grhl3. It is interesting to note that transglutaminase 1 and DOPA decarboxylase/tyrosine hydroxylase are not actual homologs but rather are examples of a convergent evolutionary process that selected for crosslinking enzymes in creation of the outer protective layer of the epidermis; yet the signals driving assembly of these different protective layers appear to be conserved across phyla. The genome of the nematode Caenorhabditis elegans also contains a Grainyhead orthologue, Ce-GRH-1, and depletion by RNA interference (RNAi) results in larvae with a fragile epidermal cuticle, similar to the effect of loss of the respective Grainyhead homologue in mouse and flies [12Venkatesan K. McManus H.R. Mello C.C. Smith T.F. Hansen U. Functional conservation between members of an ancient duplicated transcription factor family, LSF/Grainyhead.Nucleic Acids Res. 2003; 31: 4304-4316Crossref PubMed Scopus (69) Google Scholar]. Clearly, hunting down the downstream targets of Grainyhead that may regulate morphogenetic sealing processes and/or aspects of the healing response, will be the new goals of several labs in the next few years. It is obviously premature to give Grainyhead master-regulatory status, as the new studies show its conservation in the differentiation of only a single aspect of the epidermis. But the papers [3Mace K.A. Pearson J.C. McGinnis W. An epidermal barrier wound repair pathway in Drosophila is mediated by grainy head.Science. 2005; 308: 381-385Crossref PubMed Scopus (176) Google Scholar, 4Ting S.B. Caddy J. Hislop N. Wilanowski T. Auden A. Zhao L.L. Ellis S. Kaur P. Uchida Y. Holleran W.M. et al.A homolog of Drosophila grainy head is essential for epidermal integrity in mice.Science. 2005; 308: 411-413Crossref PubMed Scopus (216) Google Scholar] leave several unanswered questions that suggest significant additional roles for Grainyhead transcription factors. Ting et al. [4Ting S.B. Caddy J. Hislop N. Wilanowski T. Auden A. Zhao L.L. Ellis S. Kaur P. Uchida Y. Holleran W.M. et al.A homolog of Drosophila grainy head is essential for epidermal integrity in mice.Science. 2005; 308: 411-413Crossref PubMed Scopus (216) Google Scholar] observed a very dramatic failure of Grhl3 mutant mice to re-epithelialise wounds at both E12.5 (about half way through gestation) and later at E16.5. If Grhl3 is really only involved in transglutaminase 1 expression and stratum corneum development, one would not expect a complete failure in re-epithelialization. Indeed, the barrier function in mice is not even developed until around E18, so why epidermal wound defects found at E12.5? Grhl3 mutant mouse embryos also show defects in neural tube closure [13Ting S.B. Wilanowski T. Auden A. Hall M. Voss A.K. Thomas T. Parekh V. Cunningham J.M. Jane S.M. Inositol- and folate-resistant neural tube defects in mice lacking the epithelial-specific factor Grhl-3.Nat. Med. 2003; 9: 1513-1519Crossref PubMed Scopus (143) Google Scholar], an epithelial fusion that shares parallels with the final sealing stages of wound repair [7Martin P. Parkhurst S.M. Parallels between tissue repair and embryo morphogenesis.Development. 2004; 131: 3021-3034Crossref PubMed Scopus (413) Google Scholar]. But neural tubes do not contain a stratum corneum, suggesting that Grhl3 may have other roles in epithelial fusion events. Furthermore, overexpression of Grainyhead in Drosophila embryos causes a failure in dorsal closure [14Attardi L.D. Von Seggern D. Tjian R. Ectopic expression of wild-type or a dominant-negative mutant of transcription factor NTF-1 disrupts normal Drosophila development.Proc. Natl. Acad. Sci. USA. 1993; 90: 10563-10567Crossref PubMed Scopus (31) Google Scholar], a widely studied epithelial fusion event that has many similarities to wound repair [6Wood W. Jacinto A. Grose R. Woolner S. Gale J. Wilson C. Martin P. Wound healing recapitulates morphogenesis in Drosophila embryos.Nat. Cell Biol. 2002; 4: 907-912Crossref PubMed Scopus (294) Google Scholar, 7Martin P. Parkhurst S.M. Parallels between tissue repair and embryo morphogenesis.Development. 2004; 131: 3021-3034Crossref PubMed Scopus (413) Google Scholar]. These are not the first reports to suggest a conservation in wound repair signalling between flies and higher vertebrates. The Jun N-terminal kinase (JNK) cascade leading to activation of the transcription factor AP-1 has been widely characterized in tissue repair in both flies and higher organisms [15Galko M.J. Krasnow M.A. Cellular and genetic analysis of wound healing in Drosophila larvae.PLoS Biol. 2004; 2: E239Crossref PubMed Scopus (262) Google Scholar, 16Ramet M. Lanot R. Zachary D. Manfruelli P. JNK signaling pathway is required for efficient wound healing in Drosophila.Dev. Biol. 2002; 241: 145-156Crossref PubMed Scopus (227) Google Scholar, 17Li G. Gustafson-Brown C. Hanks S.K. Nason K. Arbeit J.M. Pogliano K. Wisdom R.M. Johnson R.S. c-Jun is essential for organization of the epidermal leading edge.Dev. Cell. 2003; 4: 865-877Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar]. Interestingly, the DOPA decarboxylase wound response enhancer has AP-1 consensus sequences, as well as Grainyhead binding sites, and the AP-1 binding sites are necessary for full function of the enhancer, suggesting that this transcription factor may cooperate with Grainyhead. Mace et al. [3Mace K.A. Pearson J.C. McGinnis W. An epidermal barrier wound repair pathway in Drosophila is mediated by grainy head.Science. 2005; 308: 381-385Crossref PubMed Scopus (176) Google Scholar], however, found that the extracellular signal-regulated kinase (ERK), and not the JNK pathway, appears to be necessary. One of the holy grails of wound healing research is to determine what are the initial signal(s) that activate all of these cascades. What is upstream of ERK, JNK, Grainyhead, AP-1, small GTPase activation and the other immediate early wound responses, and are these triggering events conserved across phyla? The activation episodes may be growth factor mediated or triggered by mechanical stretching of the wound edge cells as tensions change within the epithelial sheet; in both scenarios, these cues could be shared by analogous morphogenetic episodes like gastrulation, dorsal closure or neurulation [7Martin P. Parkhurst S.M. Parallels between tissue repair and embryo morphogenesis.Development. 2004; 131: 3021-3034Crossref PubMed Scopus (413) Google Scholar]. Alternatively, they could be cell damage related cues, for example release of intracellular ATP stores, or signals brought in by inflammatory cells recruited to the wound [18Stramer B. Wood W. Galko M.J. Redd M.J. Jacinto A. Parkhurst S.M. Martin P. Live imaging of wound inflammation in Drosophila embryos reveals key roles for small GTPases during in vivo cell migration.J. Cell Biol. 2005; 168: 567-573Crossref PubMed Scopus (223) Google Scholar], in which case they might be unique components of the repair response. Either way, adding another conserved repair response to the list lends credence to Drosophila as a clinically relevant model of repair. And no doubt, its genetic tractability will lead to new clues into how wound repair may be controlled in the future.