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Research Techniques Made Simple: Zebrafish Models for Human Dermatologic Disease

  • William Tyler Frantz
    Affiliations
    Program in Molecular Medicine, University of Massachusetts Chan Medical School, Worcester, Massachusetts, USA

    Department of Molecular, Cellular and Cancer Biology, University of Massachusetts Chan Medical School, Worcester, Massachusetts, USA
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  • Craig J. Ceol
    Correspondence
    Correspondence: Craig J. Ceol, Program in Molecular Medicine, University of Massachusetts Chan Medical School, 368 Plantation Street, ASC-1041, Worcester, Massachusetts 01605, USA.
    Affiliations
    Program in Molecular Medicine, University of Massachusetts Chan Medical School, Worcester, Massachusetts, USA

    Department of Molecular, Cellular and Cancer Biology, University of Massachusetts Chan Medical School, Worcester, Massachusetts, USA
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      Skin diseases affect nearly one third of the world’s population. Disease types range from oncologic to inflammatory, and outcomes can be as severe as death and disfigurement. Although many skin diseases have been modeled in murine models, the advantages of zebrafish models have led to recent increasing use in modeling human disease. Their rapid development, comparable skin architecture, tractable genetics, unparalleled optical properties, and straightforward drug screens make them an excellent model to study skin disease. In this review, we discuss the attributes of the zebrafish model system as well as current zebrafish models for dermatologic diseases, including melanoma, squamous cell carcinoma, vitiligo, epidermal bullosa, psoriasis, and wounding.

      Abbreviations:

      BCC (basal cell carcinoma), EB (epidermolysis bullosa), ENU (ethylnitrosourea), H2O2 (hydrogen peroxide), K (keratin), KC (keratinocyte), OSCC (oral squamous cell carcinoma), MMP (matrix metalloprotein), SCC (squamous cell carcinoma), WT (wild-type)

      Summary Points

      Advantages

      • Conserved disease-related genetics;
      • Conserved epidermal/dermal junction;
      • Rapid development and organogenesis;
      • Easy breeding with large numbers of offspring;
      • Unparalleled optical properties;
      • Amenable to high-throughput drug screening

      Limitations

      • Lack of some properties of mammalian skin structure such as the hair follicle, sebaceous glands, and a stratum corneum;
      • Lack of antibodies recognizing zebrafish proteins;
      • Unclear cytokine orthologs;
      • Multiple orthologs for many mammalian genes

      Introduction

      Originally used to study development, over the past two decades, zebrafish have become an important model organism for studying human skin disease. Zebrafish have highly homologous skin architecture (
      • Li Q.
      • Frank M.
      • Thisse C.I.
      • Thisse B.V.
      • Uitto J.
      Zebrafish: a model system to study heritable skin diseases.
      ). Similar to that in mammals, the zebrafish skin is organized into two layers: an epidermis, made up of multiple layers of keratinocytes (KCs), and dermis, which contains admixed fibroblasts, KCs, pigment cells, and immune cells. These two layers are separated by a basement membrane. These structures develop rapidly, with KCs appearing as early as 1 day after fertilization and an epidermal/dermal boundary by day 6 after fertilization. At approximately 25 days after fertilization, basal p63-expressing zebrafish stem cells proliferate, forming a new layer of suprabasal transient amplifying cells (
      • Guzman A.
      • Ramos-Balderas J.L.
      • Carrillo-Rosas S.
      • Maldonado E.
      A stem cell proliferation burst forms new layers of P63 expressing suprabasal cells during zebrafish postembryonic epidermal development.
      ). Similar to that in human skin, a subset of zebrafish epidermal stem cells engage in asymmetric proliferation and change shape during epidermal thickening (
      • Rangel-Huerta E.
      • Guzman A.
      • Maldonado E.
      The dynamics of epidermal stratification during post-larval development in zebrafish.
      ). In the adult zebrafish, this multilayered stratified squamous epithelium is separated from the dermis by the basement membrane zone (
      • Sonawane M.
      • Carpio Y.
      • Geisler R.
      • Schwarz H.
      • Maischein H.M.
      • Nuesslein-Volhard C.
      Zebrafish penner/lethal giant larvae 2 functions in hemidesmosome formation, maintenance of cellular morphology and growth regulation in the developing basal epidermis.
      ).
      Zebrafish melanocytes, similar to their mammalian counterparts, are neural crest‒derived cells responsible for pigment production (
      • Sauka-Spengler T.
      • Bronner-Fraser M.
      A gene regulatory network orchestrates neural crest formation.
      ). In adult zebrafish, melanocytes can be found in two locations: in the epidermal layer found on top of the scales and in highly organized stripes found in the hypodermis (
      • Hirata M.
      • Nakamura K.
      • Kanemaru T.
      • Shibata Y.
      • Kondo S.
      Pigment cell organization in the hypodermis of zebrafish.
      ). Zebrafish have a few other differences in skin architecture. Zebrafish skin does not form a stratum corneum (
      • Le Guellec D.
      • Morvan-Dubois G.
      • Sire J.Y.
      Skin development in bony fish with particular emphasis on collagen deposition in the dermis of the zebrafish (Danio rerio).
      ). As aquatic animals, zebrafish have no need for the water-impermeable barrier functions of the mammalian stratum corneum. Zebrafish also lack hair follicles; instead, zebrafish are covered in rows of overlapping scales, with the base of each serving an analogous function to a hair follicle (
      • Rakers S.
      • Gebert M.
      • Uppalapati S.
      • Meyer W.
      • Maderson P.
      • Sell A.F.
      • et al.
      ‘Fish matters’: the relevance of fish skin biology to investigative dermatology.
      ). Sebaceous glands are also absent. Despite these differences, the overall architecture and cellularity of the zebrafish skin are similar to those of mammalian skin and are well-suited to investigating human skin disease.
      Annotation of the first zebrafish reference genome revealed that 71.4% of all human protein-coding genes and 82% of disease-causing genes have clear zebrafish orthologs (
      • Howe K.
      • Clark M.D.
      • Torroja C.F.
      • Torrance J.
      • Berthelot C.
      • Muffato M.
      • et al.
      The zebrafish reference genome sequence and its relationship to the human genome [published correction appears in Nature 2014;505:248].
      ). These include orthologs for numerous skin genes implicated in human disease, including epidermal markers BPAG1, PLEC1, and keratin (K) gene K1 and K5 and numerous integrins and collagen encoding genes (
      • Li Q.
      • Frank M.
      • Thisse C.I.
      • Thisse B.V.
      • Uitto J.
      Zebrafish: a model system to study heritable skin diseases.
      ). Immune cell markers are also well-persevered. For example, zebrafish macrophages express an ortholog of MPEG1, neutrophils express an ortholog of LYZ, and zebrafish T cells express orthologs of CD4 or CD8 (
      • Kitaguchi T.
      • Kawakami K.
      • Kawahara A.
      Transcriptional regulation of a myeloid-lineage specific gene lysozyme C during zebrafish myelopoiesis.
      ;
      • Martins R.R.
      • Ellis P.S.
      • MacDonald R.B.
      • Richardson R.J.
      • Henriques C.M.
      Resident immunity in tissue repair and maintenance: the zebrafish model coming of age.
      ;
      • Moore F.E.
      • Garcia E.G.
      • Lobbardi R.
      • Jain E.
      • Tang Q.
      • Moore J.C.
      • et al.
      Single-cell transcriptional analysis of normal, aberrant, and malignant hematopoiesis in zebrafish.
      ). In addition to the conservation of skin cell‒specific genes, signaling pathways critical to skin development and function, such as Shh, TP63-associated, and Notch pathways, are well-conserved (
      • Rangel-Huerta E.
      • Guzman A.
      • Maldonado E.
      The dynamics of epidermal stratification during post-larval development in zebrafish.
      ).
      Although zebrafish were classically used as a forward genetic system through ethylnitrosourea (ENU) mutagenesis, the development of genetic tools such as morpholinos and recent advances in efficient genetic engineering approaches such as CRISPR/Cas9 allow precise manipulation of disease-causing genes and rapid development of novel disease models (
      • Haffter P.
      • Granato M.
      • Brand M.
      • Mullins M.C.
      • Hammerschmidt M.
      • Kane D.A.
      • et al.
      The identification of genes with unique and essential functions in the development of the zebrafish, Danio rerio.
      ;
      • Hwang W.Y.
      • Fu Y.
      • Reyon D.
      • Maeder M.L.
      • Tsai S.Q.
      • Sander J.D.
      • et al.
      Efficient genome editing in zebrafish using a CRISPR-Cas system.
      ;
      • Nasevicius A.
      • Ekker S.C.
      Effective targeted gene ‘knockdown’ in zebrafish.
      ). These tools, combined with well-conserved genetics, allow for the straightforward creation of new tissue-specific reporters and disease-modeling mutants.
      In addition to their conserved skin architecture and tractable genetics, zebrafish also have desirable optical properties. Zebrafish embryos and larvae are transparent, enabling unparalleled visualization of organ development. Although less transparent than embryos, adult zebrafish still exhibit desirable optical properties for visualizing external structures such as the skin. Furthermore, zebrafish’s easy breeding and large numbers of embryos, rapid ex utero development, and water-borne drug administration permit high-throughput drug screening.

      Current zebrafish disease models

      In the last two decades, zebrafish have emerged as an invaluable model for studying human disease. Early forward genetic screens identified large classes of mutants that serve as models for hematopoietic, pigmentation, and other disease types (
      • Haffter P.
      • Granato M.
      • Brand M.
      • Mullins M.C.
      • Hammerschmidt M.
      • Kane D.A.
      • et al.
      The identification of genes with unique and essential functions in the development of the zebrafish, Danio rerio.
      ,
      • Haffter P.
      • Odenthal J.
      • Mullins M.C.
      • Lin S.
      • Farrell M.J.
      • Vogelsang E.
      • et al.
      Mutations affecting pigmentation and shape of the adult zebrafish.
      ). Shortly after, the first cancer models emerged, including models for acute lymphoblastic leukemia and melanoma (
      • Langenau D.M.
      • Traver D.
      • Ferrando A.A.
      • Kutok J.L.
      • Aster J.C.
      • Kanki J.P.
      • et al.
      Myc-induced T cell leukemia in transgenic zebrafish.
      ;
      • Patton E.E.
      • Widlund H.R.
      • Kutok J.L.
      • Kopani K.R.
      • Amatruda J.F.
      • Murphey R.D.
      • et al.
      BRAF mutations are sufficient to promote nevi formation and cooperate with p53 in the genesis of melanoma.
      ). In the last 20 years, zebrafish have also been increasingly utilized to model nononcological skin diseases such as psoriasis, vitiligo, epidermal bullosa, and chronic wounding (Table 1).
      Table 1Major Models of Human Dermatologic Disease in Zebrafish
      CategoryDiseaseModelNotable FeaturesCitation
      OncologicMelanomamitfa:BRAFV600E;p53(lf)Invasive melanomas(
      • Patton E.E.
      • Widlund H.R.
      • Kutok J.L.
      • Kopani K.R.
      • Amatruda J.F.
      • Murphey R.D.
      • et al.
      BRAF mutations are sufficient to promote nevi formation and cooperate with p53 in the genesis of melanoma.
      )
      Melanomamitfa:NRASQ61K;p53(lf)Invasive melanomas, rapid onset(
      • Dovey M.
      • White R.M.
      • Zon L.I.
      Oncogenic NRAS cooperates with p53 loss to generate melanoma in zebrafish.
      )
      Squamous cell carcinomaENU mutagenesisEpidermal papillomas with hyperproliferation(
      • Beckwith L.G.
      • Moore J.L.
      • Tsao-Wu G.S.
      • Harshbarger J.C.
      • Cheng K.C.
      Ethylnitrosourea induces neoplasia in zebrafish (Danio rerio).
      )
      CongenitalAlbinismtyr(lf)

      oca2(lf)
      Pigmentless melanocytes(
      • Braasch I.
      • Schartl M.
      • Volff J.N.
      Evolution of pigment synthesis pathways by gene and genome duplication in fish.
      ;
      • Haffter P.
      • Odenthal J.
      • Mullins M.C.
      • Lin S.
      • Farrell M.J.
      • Vogelsang E.
      • et al.
      Mutations affecting pigmentation and shape of the adult zebrafish.
      )
      Waardenburg syndromemitfa(lf)

      sox10(lf)
      No melanocytes(
      • Dutton K.A.
      • Pauliny A.
      • Lopes S.S.
      • Elworthy S.
      • Carney T.J.
      • Rauch J.
      • et al.
      Zebrafish colourless encodes sox10 and specifies non-ectomesenchymal neural crest fates.
      ;
      • Lister J.A.
      • Robertson C.P.
      • Lepage T.
      • Johnson S.L.
      • Raible D.W.
      nacre encodes a zebrafish microphthalmia-related protein that regulates neural-crest-derived pigment cell fate.
      )
      Piebaldismkita(lf)Impaired melanocyte function and number(
      • Parichy D.M.
      • Rawls J.F.
      • Pratt S.J.
      • Whitfield T.T.
      • Johnson S.L.
      Zebrafish sparse corresponds to an orthologue of c-kit and is required for the morphogenesis of a subpopulation of melanocytes, but is not essential for hematopoiesis or primordial germ cell development.
      )
      Noonan syndromeMosaic

      NRASI24N

      NRASG60E
      Developmental craniofacial defects(
      • Runtuwene V.
      • Van Eekelen M.
      • Overvoorde J.
      • Rehmann H.
      • Yntema H.G.
      • Nillesen W.M.
      • et al.
      Noonan syndrome gain-of-function mutations in NRAS cause zebrafish gastrulation defects.
      )
      LEOPARD syndromeMosaic

      shp2A663T
      Hyperpigmentation(
      • Stewart R.A.
      • Sanda T.
      • Widlund H.R.
      • Zhu S.
      • Swanson K.D.
      • Hurley A.D.
      • et al.
      Phosphatase-dependent and -independent functions of Shp2 in neural crest cells underlie Leopard syndrome pathogenesis.
      )
      Phakomatosis pigmentovascularisMosaic

      GNA11R183C

      GNA11Q209L
      Dermal melanocytosis(
      • Thomas A.C.
      • Zeng Z.
      • Rivière J.B.
      • O'Shaughnessy R.
      • Al-Olabi L.
      • St-Onge J.
      • et al.
      Mosaic activating mutations in GNA11 and GNAQ are associated with phakomatosis pigmentovascularis and extensive dermal melanocytosis.
      )
      Epidermolysis bullosapenner/lgl2(lf)Hemidesmosome breakdown, blistering, and overproliferation(
      • Sonawane M.
      • Carpio Y.
      • Geisler R.
      • Schwarz H.
      • Maischein H.M.
      • Nuesslein-Volhard C.
      Zebrafish penner/lethal giant larvae 2 functions in hemidesmosome formation, maintenance of cellular morphology and growth regulation in the developing basal epidermis.
      )
      Epidermolysis bullosacol17a1a knockdownBlistering(
      • Kim S.H.
      • Choi H.Y.
      • So J.H.
      • Kim C.H.
      • Ho S.Y.
      • Frank M.
      • et al.
      Zebrafish type XVII collagen: gene structures, expression profiles, and morpholino "knock-down" phenotypes.
      )
      InflammatoryPsoriasism14Overproliferation of keratinocytes leading to aggregates(
      • Webb A.E.
      • Driever W.
      • Kimelman D.
      psoriasis regulates epidermal development in zebrafish.
      )
      Psoriasisspint1a(lf)Skin inflammation and overproliferation of keratinocytes(
      • Carney T.J.
      • von der Hardt S.
      • Sonntag C.
      • Amsterdam A.
      • Topczewski J.
      • Hopkins N.
      • et al.
      Inactivation of serine protease Matriptase1a by its inhibitor Hai1 is required for epithelial integrity of the zebrafish epidermis.
      ;
      • Mathias J.R.
      • Dodd M.E.
      • Walters K.B.
      • Rhodes J.
      • Kanki J.P.
      • Look A.T.
      • et al.
      Live imaging of chronic inflammation caused by mutation of zebrafish Hai1.
      )
      Vitiligonicastrin(lf)Skin inflammation and hypopigmentation(
      • Hsu C.H.
      • Liou G.G.
      • Jiang Y.J.
      Nicastrin deficiency induces tyrosinase-dependent depigmentation and skin inflammation.
      )
      WoundingWounding and regenerationHyPerROS detection after tail fin amputation(
      • Niethammer P.
      • Grabher C.
      • Look A.T.
      • Mitchison T.J.
      A tissue-scale gradient of hydrogen peroxide mediates rapid wound detection in zebrafish.
      )
      Wounding and regenerationSkinbowMulticolor CreER labeling of keratinocytes(
      • Chen C.H.
      • Puliafito A.
      • Cox B.D.
      • Primo L.
      • Fang Y.
      • Di Talia S.
      • et al.
      Multicolor cell barcoding technology for long-term surveillance of epithelial regeneration in zebrafish.
      )
      Wounding and regenerationLaser and mechanical destruction of zebrafish skin(
      • Richardson R.
      • Metzger M.
      • Knyphausen P.
      • Ramezani T.
      • Slanchev K.
      • Kraus C.
      • et al.
      Re-epithelialization of cutaneous wounds in adult zebrafish combines mechanisms of wound closure in embryonic and adult mammals.
      ,
      • Richardson R.
      • Slanchev K.
      • Kraus C.
      • Knyphausen P.
      • Eming S.
      • Hammerschmidt M.
      Adult zebrafish as a model system for cutaneous wound-healing research.
      )

      Oncologic

      Melanoma, a lethal malignancy of melanocytes, is responsible for most skin cancer deaths and kills nearly 60,000 patients worldwide each year (
      • Sung H.
      • Ferlay J.
      • Siegel R.L.
      • Laversanne M.
      • Soerjomataram I.
      • Jemal A.
      • et al.
      Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries.
      ). Whereas most cutaneous melanomas are diagnosed and excised at early stages, melanomas that progress to metastatic disease have poor outcomes. New small molecule and immunotherapies have improved patient outcomes, but a substantial fraction of patients still succumb to melanoma, underlining a need for greater insights into melanomagenesis (
      • Larkin J.
      • Chiarion-Sileni V.
      • Gonzalez R.
      • Grob J.J.
      • Rutkowski P.
      • Lao C.D.
      • et al.
      Five-year survival with combined nivolumab and ipilimumab in advanced melanoma.
      ). To develop the first zebrafish model of human melanoma,
      • Patton E.E.
      • Widlund H.R.
      • Kutok J.L.
      • Kopani K.R.
      • Amatruda J.F.
      • Murphey R.D.
      • et al.
      BRAF mutations are sufficient to promote nevi formation and cooperate with p53 in the genesis of melanoma.
      expressed the human oncogene BRAFV600E in melanocytes in the tumor suppressor p53 loss-of-function, p53(lf), mutants. These Tg(mitfa:BRAFV600E);p53(lf) zebrafish develop tumors that histopathologically mirror human melanomas. Since this landmark discovery, other human oncogenes, such as NRASQ61K, have been used to engineer zebrafish models for cutaneous melanoma (
      • Dovey M.
      • White R.M.
      • Zon L.I.
      Oncogenic NRAS cooperates with p53 loss to generate melanoma in zebrafish.
      ). Comparisons between genetic signatures in human melanomas and zebrafish melanoma models have also discovered critical melanoma-modifying genes important for disease progression (
      • Ceol C.J.
      • Houvras Y.
      • Jane-Valbuena J.
      • Bilodeau S.
      • Orlando D.A.
      • Battisti V.
      • et al.
      The histone methyltransferase SETDB1 is recurrently amplified in melanoma and accelerates its onset.
      ;
      • Venkatesan A.M.
      • Vyas R.
      • Gramann A.K.
      • Dresser K.
      • Gujja S.
      • Bhatnagar S.
      • et al.
      Ligand-activated BMP signaling inhibits cell differentiation and death to promote melanoma.
      ). These studies and the expansive use of zebrafish to model human melanoma have been extensively reviewed (
      • Frantz W.T.
      • Ceol C.J.
      From tank to treatment: modeling melanoma in zebrafish.
      ;
      • White R.
      • Rose K.
      • Zon L.
      Zebrafish cancer: the state of the art and the path forward.
      ).
      In contrast to melanoma, which has been the subject of extensive zebrafish-modeling studies, there are currently no models of basal cell carcinoma (BCC) and no transgenic models of squamous cell carcinoma (SCC). However, BCCs and SCCs are still the most common malignancies, and zebrafish can contribute to understanding their pathogenesis and treatment. Original efforts to model epidermal neoplasms exposed zebrafish to the mutagenic alkylating agent ENU. Twelve months after exposure, 100% of ENU-treated adult fish developed epidermal papillomas, with histological features consistent with those of epidermal hyperplasia (
      • Beckwith L.G.
      • Moore J.L.
      • Tsao-Wu G.S.
      • Harshbarger J.C.
      • Cheng K.C.
      Ethylnitrosourea induces neoplasia in zebrafish (Danio rerio).
      ). More recently, zebrafish embryonic xenograft models have been used to dissect the role of metastasis-associated gene expression signatures, such as AXL expression, on SCC progression. In 2014,
      • Cichoń M.A.
      • Szentpetery Z.
      • Caley M.P.
      • Papadakis E.S.
      • Mackenzie I.C.
      • Brennan C.H.
      • et al.
      The receptor tyrosine kinase Axl regulates cell-cell adhesion and stemness in cutaneous squamous cell carcinoma.
      injected AXL-knockdown SCC cells into zebrafish embryos and monitored for in vivo tumor formation. Interestingly, cells with AXL knockdown failed to form tumors, showing a role for AXL-mediated SCC progression (
      • Cichoń M.A.
      • Szentpetery Z.
      • Caley M.P.
      • Papadakis E.S.
      • Mackenzie I.C.
      • Brennan C.H.
      • et al.
      The receptor tyrosine kinase Axl regulates cell-cell adhesion and stemness in cutaneous squamous cell carcinoma.
      ). Similarly, an embryo xenograft model has also been used to model oral SCC (OSCC). To determine whether the overexpression of matrix metalloprotein 9 (MMP9) seen in human OSCCs has mechanistic importance for disease spread,
      • Wen J.
      • Yin P.
      • Li L.
      • Kang G.
      • Ning G.
      • Cao Y.
      • et al.
      Knockdown of matrix metallopeptidase 9 inhibits metastasis of oral squamous cell carcinoma cells in a zebrafish xenograft model.
      implanted OSCC cells with short hairpin RNA–mediated MMP9 knockdown into zebrafish embryos and tracked migration distance. Cells with MMP9 knockdown migrated less than wild-type (WT) controls, indicative of a role for cell-intrinsic MMP9 expression in OSCC metastasis.
      The embryonic xenograft model has also been used to investigate novel treatments of SCC. In human SCC, relapsed tumors express higher levels of the tyrosine kinase receptor DDR2 than primary tumors, indicative of a potential role in disease progression.
      • von Mässenhausen A.
      • Sanders C.
      • Brägelmann J.
      • Konantz M.
      • Queisser A.
      • Vogel W.
      • et al.
      Targeting DDR2 in head and neck squamous cell carcinoma with dasatinib.
      , investigated the role of DDR2 in SCC disease progression by transplanting DDR2-overexpressing SCC cell lines into zebrafish embryos and monitoring for migration. They observed enhanced migration in DDR2-overexpressing SCC lines compared with that in WT SCC cells, suggesting an increased metastatic potential. Interestingly, treatment with the Food and Drug Administration–approved RTK inhibitor dasatinib decreased migration in DDR2-overexpressing cells, indicating a potential therapeutic role in treating relapsed DDR2-expressing SCCs (
      • von Mässenhausen A.
      • Sanders C.
      • Brägelmann J.
      • Konantz M.
      • Queisser A.
      • Vogel W.
      • et al.
      Targeting DDR2 in head and neck squamous cell carcinoma with dasatinib.
      ).

      Congenital skin disease

      Zebrafish are also a practical platform for studying congenital skin diseases. Zebrafish are particularly well-suited to investigating inherited hypopigmentation and hyperpigmentation disorders. Hypopigmentation diseases, which include albinism, piebaldism, and Waardenburg syndrome, are caused by varying levels of impairment in genes governing melanocyte function. The mechanisms governing melanocyte development are well-conserved in zebrafish, and mutations in orthologs of pigment production genes display hypopigmentation phenotypes. Waardenburg syndrome, an auditory pigment disorder, is caused by mutations in genes essential for neural crest formation and melanocyte development. In zebrafish, such as in humans, loss of sox10 or mitfa results in pigmentation defects and melanocyte deficiency (
      • Dutton K.A.
      • Pauliny A.
      • Lopes S.S.
      • Elworthy S.
      • Carney T.J.
      • Rauch J.
      • et al.
      Zebrafish colourless encodes sox10 and specifies non-ectomesenchymal neural crest fates.
      ;
      • Lister J.A.
      • Robertson C.P.
      • Lepage T.
      • Johnson S.L.
      • Raible D.W.
      nacre encodes a zebrafish microphthalmia-related protein that regulates neural-crest-derived pigment cell fate.
      ). The receptor tyrosine kinase c-KIT, which is partially lost in piebaldism, is also conserved in zebrafish (
      • Parichy D.M.
      • Rawls J.F.
      • Pratt S.J.
      • Whitfield T.T.
      • Johnson S.L.
      Zebrafish sparse corresponds to an orthologue of c-kit and is required for the morphogenesis of a subpopulation of melanocytes, but is not essential for hematopoiesis or primordial germ cell development.
      ). Loss of the ortholog kita results in defects of melanocyte survival and migration, the hypothesized causes of the patterning seen in human disease (
      • Rawls J.
      • Johnson S.L.
      Temporal and molecular separation of the kit receptor tyrosine kinase’s roles in zebrafish melanocyte migration and survival.
      ). Albinism, typified by hair and skin lacking pigmentation, is caused by mutations in pigment synthesis and trafficking genes such as TYR and the aptly named OCA2 (
      • Braasch I.
      • Schartl M.
      • Volff J.N.
      Evolution of pigment synthesis pathways by gene and genome duplication in fish.
      ;
      • Haffter P.
      • Granato M.
      • Brand M.
      • Mullins M.C.
      • Hammerschmidt M.
      • Kane D.A.
      • et al.
      The identification of genes with unique and essential functions in the development of the zebrafish, Danio rerio.
      ). Mutations in zebrafish orthologs of these genes lead to depigmented zebrafish, mirroring the genetic/phenotypic relationship of human disease.
      Zebrafish have also been used to model congenital hyperpigmentation diseases and elicit mechanisms underpinning their biology. In addition to the nevus-like hyperpigmentations seen in
      • Patton E.E.
      • Widlund H.R.
      • Kutok J.L.
      • Kopani K.R.
      • Amatruda J.F.
      • Murphey R.D.
      • et al.
      BRAF mutations are sufficient to promote nevi formation and cooperate with p53 in the genesis of melanoma.
      seminal work, numerous other studies have used zebrafish to study hyperpigmentation disorders. In 2016,
      • Thomas A.C.
      • Zeng Z.
      • Rivière J.B.
      • O'Shaughnessy R.
      • Al-Olabi L.
      • St-Onge J.
      • et al.
      Mosaic activating mutations in GNA11 and GNAQ are associated with phakomatosis pigmentovascularis and extensive dermal melanocytosis.
      found that mosaic expression of ocular melanoma drivers GNAQ and GNA11 drives the congenital pigmentation found in phakomatosis pigmentovascularis.
      Zebrafish models have also contributed to understanding two other MAPK-signaling related diseases: the RASopathies Noonan syndrome and Noonan syndrome with multiple letingies (formerly LEOPARD syndrome). In 2011,
      • Runtuwene V.
      • Van Eekelen M.
      • Overvoorde J.
      • Rehmann H.
      • Yntema H.G.
      • Nillesen W.M.
      • et al.
      Noonan syndrome gain-of-function mutations in NRAS cause zebrafish gastrulation defects.
      sequenced a patient with Noonan syndrome features and identified a novel potential activating mutation in NRAS. Expression of this mutant NRAS in zebrafish embryos led to craniofacial features phenocopying those found in Noonan syndrome, supporting the role of activating NRAS mutations in disease pathogenesis (
      • Runtuwene V.
      • Van Eekelen M.
      • Overvoorde J.
      • Rehmann H.
      • Yntema H.G.
      • Nillesen W.M.
      • et al.
      Noonan syndrome gain-of-function mutations in NRAS cause zebrafish gastrulation defects.
      ). Similarly,
      • Stewart R.A.
      • Sanda T.
      • Widlund H.R.
      • Zhu S.
      • Swanson K.D.
      • Hurley A.D.
      • et al.
      Phosphatase-dependent and -independent functions of Shp2 in neural crest cells underlie Leopard syndrome pathogenesis.
      compared cohorts of patients with Noonan and LEOPARD syndrome and identified PTPN11, which encodes an SHP2 tyrosine phosphatase ortholog that acts upstream of NRAS, as being differentially activated, suggestive of a possible causative role in LEOPARD pathogenesis. When ptpn11 was inhibited with morpholinos, zebrafish exhibited phenotypes supporting a role for SHP2 in regulating the numerous hyperpigmented café-au-lait spots seen on patients with LEOPARD syndrome (
      • Stewart R.A.
      • Sanda T.
      • Widlund H.R.
      • Zhu S.
      • Swanson K.D.
      • Hurley A.D.
      • et al.
      Phosphatase-dependent and -independent functions of Shp2 in neural crest cells underlie Leopard syndrome pathogenesis.
      ).
      The advantages of these conserved genetics in modeling human disease extends beyond inherited pigment disorders and to other skin disorders. Epidermolysis bullosa (EB) is a group of rare inherited skin disorders characterized by fragile blistering skin. These blisters are caused by deficiencies in structural proteins that make up dermoepidermal complexes and govern epidermal adhesion. Owing to their conserved epidermal and dermal structures, zebrafish are an appropriate model for EB. The first zebrafish model for basal epidermal structural weaknesses was the pen/lgl2 mutant (
      • Sonawane M.
      • Carpio Y.
      • Geisler R.
      • Schwarz H.
      • Maischein H.M.
      • Nuesslein-Volhard C.
      Zebrafish penner/lethal giant larvae 2 functions in hemidesmosome formation, maintenance of cellular morphology and growth regulation in the developing basal epidermis.
      ). These loss-of-function mutants fail to form basal hemidesmosomes, resulting in skin detachment from the basement membrane and epidermal hyperproliferation, reminiscent of human EB (
      • Sonawane M.
      • Martin-Maischein H.
      • Schwarz H.
      • Nüsslein-Volhard C.
      Lgl2 and E-cadherin act antagonistically to regulate hemidesmosome formation during epidermal development in zebrafish.
      ,
      • Sonawane M.
      • Carpio Y.
      • Geisler R.
      • Schwarz H.
      • Maischein H.M.
      • Nuesslein-Volhard C.
      Zebrafish penner/lethal giant larvae 2 functions in hemidesmosome formation, maintenance of cellular morphology and growth regulation in the developing basal epidermis.
      ). Although the epidermal hyperproliferation aspect of this mutant has been used to model psoriasis, the destruction of the epidermal/dermal adhesion makes this model most analogous to EB. Subsequent studies have utilized reverse genetics to target the genes implicated in human EB. In 2010,
      • Kim S.H.
      • Choi H.Y.
      • So J.H.
      • Kim C.H.
      • Ho S.Y.
      • Frank M.
      • et al.
      Zebrafish type XVII collagen: gene structures, expression profiles, and morpholino "knock-down" phenotypes.
      knocked down an ortholog for the gene encoding for the hemidesmosome protein type XVII collagen, col17a1a. The knockdown led to perturbations in the basement membrane zone and a blistering phenotype, both characteristics of human EB (
      • Kim S.H.
      • Choi H.Y.
      • So J.H.
      • Kim C.H.
      • Ho S.Y.
      • Frank M.
      • et al.
      Zebrafish type XVII collagen: gene structures, expression profiles, and morpholino "knock-down" phenotypes.
      ).
      Many other hemidesmosome and basement membrane proteins implicated in EB are conserved and expressed in the zebrafish skin, such as the integrin itga6, yet the role of these proteins in EB has yet to be studied in zebrafish. With a firm beginning to model EB in zebrafish, future studies can combine new genetic knockout models with small-molecule epistatic experiments to elucidate the mechanisms of EB pathogenesis.

      Immune-mediated and inflammatory disease

      Zebrafish have also been used to model inflammatory diseases. Vitiligo, a disease characterized by autoimmune-mediated destruction of the epidermal melanocytes, has been the subject of recent modeling attempts in zebrafish. In 2018,
      • Zhou J.
      • An X.
      • Dong J.
      • Wang Y.
      • Zhong H.
      • Duan L.
      • et al.
      IL-17 induces cellular stress microenvironment of melanocytes to promote autophagic cell apoptosis in vitiligo.
      investigated the role of stress-induced IL17 and IL1β expression in precipitating the melanocyte destruction that occurs in vitiligo. IL-17 treatment of developing zebrafish larvae resulted in mitochondrial dysfunction and inhibition of melanogenesis, presumptively through KC-derived il1b expression (
      • Zhou J.
      • An X.
      • Dong J.
      • Wang Y.
      • Zhong H.
      • Duan L.
      • et al.
      IL-17 induces cellular stress microenvironment of melanocytes to promote autophagic cell apoptosis in vitiligo.
      ). Another study developed a knockout for the gamma secretase complex subunit nicastrin (
      • Hsu C.H.
      • Liou G.G.
      • Jiang Y.J.
      Nicastrin deficiency induces tyrosinase-dependent depigmentation and skin inflammation.
      ;
      • Shah S.
      • Lee S.F.
      • Tabuchi K.
      • Hao Y.H.
      • Yu C.
      • LaPlant Q.
      • et al.
      Nicastrin functions as a gamma-secretase-substrate receptor.
      ). It has been established that gamma secretase facilitates NOTCH signaling, and NOTCH signaling is required for proper pigmentation (
      • Osawa M.
      • Fisher D.E.
      Notch and melanocytes: diverse outcomes from a single signal.
      ). Similar to that in human vitiligo, these nicastrin-deficient animals exhibit skin inflammation. However, unlike in vitiligo where the immune cells likely cause melanocyte death, in these mutants, nicastrin deficiency leads to necrotic melanocytes, which likely recruit immune cells (
      • Hsu C.H.
      • Liou G.G.
      • Jiang Y.J.
      Nicastrin deficiency induces tyrosinase-dependent depigmentation and skin inflammation.
      ). Although these studies provide interesting insights into the role of KCs, cytokines, immune cells, and melanocytes in skin inflammation, a vitiligo model comparable with murine models remains elusive (
      • Harris J.E.
      • Harris T.H.
      • Weninger W.
      • Wherry E.J.
      • Hunter C.A.
      • Turka L.A.
      A mouse model of vitiligo with focused epidermal depigmentation requires IFN-γ for autoreactive CD8+ T-cell accumulation in the skin.
      ).
      Similar to vitiligo, psoriasis is a complex immune-mediated disease. Instead of melanocyte destruction, psoriasis is characterized by an overproliferation of KCs. KC proliferation results in scaling erythematous plaques and occurs in roughly 3% of people worldwide (
      • Lebwohl M.
      Psoriasis.
      ). In 2008,
      • Webb A.E.
      • Driever W.
      • Kimelman D.
      psoriasis regulates epidermal development in zebrafish.
      established one of the first zebrafish psoriasis models when they described atp1b1a mutants, which were obtained from an ethylmethanesulfonate screen. These mutants, with a loss of atp1b1a function, display widespread overproliferation of the epidermis and a defect in KC differentiation, resulting in aggregates of epidermal cells, all key hallmarks of human psoriasis (
      • Hatzold J.
      • Beleggia F.
      • Herzig H.
      • Altmüller J.
      • Nürnberg P.
      • Bloch W.
      • et al.
      Tumor suppression in basal keratinocytes via dual non-cell-autonomous functions of a Na,K-ATPase beta subunit.
      ;
      • Webb A.E.
      • Driever W.
      • Kimelman D.
      psoriasis regulates epidermal development in zebrafish.
      ). Other studies have sought to model the immunologic component to psoriatic disease. A series of studies have developed models where the loss of spint1a, also known as hai1a, results in neutrophil infiltration of the skin and loss of KC–KC contact (
      • Carney T.J.
      • von der Hardt S.
      • Sonntag C.
      • Amsterdam A.
      • Topczewski J.
      • Hopkins N.
      • et al.
      Inactivation of serine protease Matriptase1a by its inhibitor Hai1 is required for epithelial integrity of the zebrafish epidermis.
      ;
      • Mathias J.R.
      • Dodd M.E.
      • Walters K.B.
      • Rhodes J.
      • Kanki J.P.
      • Look A.T.
      • et al.
      Live imaging of chronic inflammation caused by mutation of zebrafish Hai1.
      ). The neutrophil infiltration causes chronic inflammation and eventual KC proliferation, both characteristics of human psoriasis (
      • Carney T.J.
      • von der Hardt S.
      • Sonntag C.
      • Amsterdam A.
      • Topczewski J.
      • Hopkins N.
      • et al.
      Inactivation of serine protease Matriptase1a by its inhibitor Hai1 is required for epithelial integrity of the zebrafish epidermis.
      ;
      • Mathias J.R.
      • Dodd M.E.
      • Walters K.B.
      • Rhodes J.
      • Kanki J.P.
      • Look A.T.
      • et al.
      Live imaging of chronic inflammation caused by mutation of zebrafish Hai1.
      ). These studies establish the models for understanding and visualizing proinflammatory and proproliferative events in real time, which will be invaluable for a better understanding of the pathogenesis of human psoriasis.

      Wound repair

      Wound repair is critical to recovery from wounds induced by trauma, diabetes, infection, and numerous other diseases. Repair requires complex cell‒cell signaling events to restore proper tissue function and homeostasis. A rapid cascade of injury detection, wound closure, acute scarring, and subacute wound healing occurs in a finely orchestrated fashion. These processes require coordination among KCs, fibroblasts, and resident immune cells. Zebrafish are a highly regenerative species, capable of regenerating their fins, melanocytes, skin, axons, and even hearts (
      • Gemberling M.
      • Bailey T.J.
      • Hyde D.R.
      • Poss K.D.
      The zebrafish as a model for complex tissue regeneration.
      ;
      • Iyengar S.
      • Kasheta M.
      • Ceol C.J.
      Poised regeneration of zebrafish melanocytes involves direct differentiation and concurrent replenishment of tissue-resident progenitor cells.
      ;
      • Richardson R.
      • Slanchev K.
      • Kraus C.
      • Knyphausen P.
      • Eming S.
      • Hammerschmidt M.
      Adult zebrafish as a model system for cutaneous wound-healing research.
      ). Owing to their regenerative capacity, optical transparency, and conserved skin architecture, zebrafish models have yielded unique insights into the mechanisms governing these processes.
      The first zebrafish wounding models utilized caudal fin amputation, where the distal two third of the fin is clipped and regenerates over the following weeks. In 2009,
      • Niethammer P.
      • Grabher C.
      • Look A.T.
      • Mitchison T.J.
      A tissue-scale gradient of hydrogen peroxide mediates rapid wound detection in zebrafish.
      utilized a genetically encoded radiometric sensor for hydrogen peroxide (H2O2) to visualize dynamic ROS gradients created after fin amputation. They found that a Nox/Duox-mediated burst of H2O2 production was required for leukocyte recruitment to the wound site (
      • Niethammer P.
      • Grabher C.
      • Look A.T.
      • Mitchison T.J.
      A tissue-scale gradient of hydrogen peroxide mediates rapid wound detection in zebrafish.
      ). Additional studies confirmed that ROS production was critical for compensatory proliferation and proper fin regeneration (
      • Gauron C.
      • Rampon C.
      • Bouzaffour M.
      • Ipendey E.
      • Teillon J.
      • Volovitch M.
      • et al.
      Sustained production of ROS triggers compensatory proliferation and is required for regeneration to proceed.
      ). Similar studies have elucidated the roles of another innate immune cell, the macrophage. In 2014,
      • Petrie T.A.
      • Strand N.S.
      • Yang C.T.
      • Rabinowitz J.S.
      • Moon R.T.
      Macrophages modulate adult zebrafish tail fin regeneration [published correction appears in Development 2015;142:406].
      found that macrophages are recruited to the wound site, and knockout of the macrophage lineage through inducible macrophage-specific cell killing resulted in impaired wound healing and tail fin regeneration. Tetracycline inducible tools (TetON) have also been adapted to study zebrafish tailfin regeneration. In 2014,
      • Wehner D.
      • Cizelsky W.
      • Vasudevaro M.D.
      • Ozhan G.
      • Haase C.
      • Kagermeier-Schenk B.
      • et al.
      Wnt/β-catenin signaling defines organizing centers that orchestrate growth and differentiation of the regenerating zebrafish caudal fin [published correction appears in Cell Rep 2014;6:777–8].
      used a TetON system to show that WNT signaling is critical to regeneration.
      Recent studies have also sought to expand the modeling of skin wounding and healing beyond the tail fin amputation model. In 2013,
      • Richardson R.
      • Slanchev K.
      • Kraus C.
      • Knyphausen P.
      • Eming S.
      • Hammerschmidt M.
      Adult zebrafish as a model system for cutaneous wound-healing research.
      developed the first dermal regeneration model when they created full-thickness wounds with a dermatology laser. They later expanded on this model by utilizing depilation of the zebrafish scales to induce partial-thickness wounds of the zebrafish skin (
      • Richardson R.
      • Metzger M.
      • Knyphausen P.
      • Ramezani T.
      • Slanchev K.
      • Kraus C.
      • et al.
      Re-epithelialization of cutaneous wounds in adult zebrafish combines mechanisms of wound closure in embryonic and adult mammals.
      ). In these studies,
      • Richardson R.
      • Slanchev K.
      • Kraus C.
      • Knyphausen P.
      • Eming S.
      • Hammerschmidt M.
      Adult zebrafish as a model system for cutaneous wound-healing research.
      found that zebrafish engage in re-epithelization of the wound without scar formation or a lag phase. Furthermore, the surrounding KCs stretch and migrate to seal the wound, before engaging in proliferation to reconstitute the epithelial sheet. Further investigation by
      • Morris J.L.
      • Cross S.J.
      • Lu Y.
      • Kadler K.E.
      • Lu Y.
      • Dallas S.L.
      • et al.
      Live imaging of collagen deposition during skin development and repair in a collagen I - GFP fusion transgenic zebrafish line.
      developed a GFP-tagged collagen I fusion line to reveal the role of collagen deposition during wound healing.
      • Chen C.H.
      • Puliafito A.
      • Cox B.D.
      • Primo L.
      • Fang Y.
      • Di Talia S.
      • et al.
      Multicolor cell barcoding technology for long-term surveillance of epithelial regeneration in zebrafish.
      engineered a CreER-based multicolor-labeling Skinbow system to track individual epithelial cells during regeneration. This powerful system makes intravital imaging of individual migrating and proliferating KCs possible during regeneration, yielding unparalleled resolution to the skin wounding and regenerative process.
      Inflammatory wound responses are also important to understanding the interplay between melanocytes, melanoma, and immune cells. In 2010,
      • Feng Y.
      • Santoriello C.
      • Mione M.
      • Hurlstone A.
      • Martin P.
      Live imaging of innate immune cell sensing of transformed cells in zebrafish larvae: parallels between tumor initiation and wound inflammation [published correction appears in PLoS Biol 2016;14:e1002377].
      showed that H2O2 mediates leukocyte recruitment to transformed oncogenic cells. Further investigation of immune recruitment with transgenic collagen reporter zebrafish revealed that leukocytes migrate through the basement membrane to early melanomas (
      • Van Den Berg M.C.W.
      • Maccarthy-Morrogh L.
      • Carter D.
      • Morris J.
      • Ribeiro Bravo I.
      • Feng Y.
      • et al.
      Proteolytic and opportunistic breaching of the basement membrane zone by immune cells during tumor initiation.
      ). Immune cell‒mediated inflammation is also capable of recruiting untransformed melanocytes to sites of tissue damage, resulting in hyperpigmentation of healing wounds, reinforcing the importance of inflammation in immune cell‒melanocyte interactions (
      • Lévesque M.
      • Feng Y.
      • Jones R.A.
      • Martin P.
      Inflammation drives wound hyperpigmentation in zebrafish by recruiting pigment cells to sites of tissue damage.
      ).

      Expanded models and improvements on the horizon

      Further understanding of how the immune system functions in the skin will yield crucial insights into the pathogenesis and treatment of diverse types of skin disease. Much as in human skin, the zebrafish skin contains KCs, fibroblasts, resident immune cells, melanocytes, vasculature, and adipocytes. Human skin disease involves any number of these cells, and zebrafish offer an opportunity to utilize in vivo imaging to understand complex cell type‒specific contributions to the pathogenesis and treatment of these diseases. In oncologic disease, inflammatory disease, and wound recovery, the immune system plays a critical role in disease progression, modulation, and recovery. Zebrafish have an innate immune system with macrophages, neutrophils, eosinophils, and mast cells. They also have an adaptive immune system with B cells, T cells, and regulatory T cells (
      • Martins R.R.
      • Ellis P.S.
      • MacDonald R.B.
      • Richardson R.J.
      • Henriques C.M.
      Resident immunity in tissue repair and maintenance: the zebrafish model coming of age.
      ). However, it is not yet clear whether all of the immune cell types found in humans have clear orthologs in zebrafish and vice versa. Deeper knowledge of the similarities and differences between the human and zebrafish immune systems will enable apt comparisons and invaluable insights into the pathogenesis of human diseases.

      Xenografts and drug screens

      Zebrafish xenografts are a key tool in understanding skin cancer disease progression and treatment (Figure 1) (
      • Cichoń M.A.
      • Szentpetery Z.
      • Caley M.P.
      • Papadakis E.S.
      • Mackenzie I.C.
      • Brennan C.H.
      • et al.
      The receptor tyrosine kinase Axl regulates cell-cell adhesion and stemness in cutaneous squamous cell carcinoma.
      ;
      • Haldi M.
      • Ton C.
      • Seng W.L.
      • McGrath P.
      Human melanoma cells transplanted into zebrafish proliferate, migrate, produce melanin, form masses and stimulate angiogenesis in zebrafish.
      ;
      • Lee L.M.
      • Seftor E.A.
      • Bonde G.
      • Cornell R.A.
      • Hendrix M.J.
      The fate of human malignant melanoma cells transplanted into zebrafish embryos: assessment of migration and cell division in the absence of tumor formation.
      ;
      • Stoletov K.
      • Montel V.
      • Lester R.D.
      • Gonias S.L.
      • Klemke R.
      High-resolution imaging of the dynamic tumor cell vascular interface in transparent zebrafish.
      ;
      • Topczewska J.M.
      • Postovit L.M.
      • Margaryan N.V.
      • Sam A.
      • Hess A.R.
      • Wheaton W.W.
      • et al.
      Embryonic and tumorigenic pathways converge via nodal signaling: role in melanoma aggressiveness.
      ;
      • von Mässenhausen A.
      • Sanders C.
      • Brägelmann J.
      • Konantz M.
      • Queisser A.
      • Vogel W.
      • et al.
      Targeting DDR2 in head and neck squamous cell carcinoma with dasatinib.
      ). Their transparency, available transgenic reporters, and the absence of an adaptive immune system make zebrafish embryos a convenient system for performing xenografts (
      • Lieschke G.J.
      • Trede N.S.
      Fish immunology.
      ). Furthermore, their small size, high fecundity, and high fertility facilitate high-throughput drug screening (
      • Precazzini F.
      • Pancher M.
      • Gatto P.
      • Tushe A.
      • Adami V.
      • Anelli V.
      • et al.
      Automated in vivo screen in zebrafish identifies clotrimazole as targeting a metabolic vulnerability in a melanoma model.
      ). In addition to their utility in xenograft drug screens, drug screens on zebrafish larvae (Figure 1) with cell-specific reporters have been used to discover novel functions for oncogenes such as PRL3 or identify small-molecule regulators of the neural crest such as capheic acid phenethyl ester (
      • Ciarlo C.
      • Kaufman C.K.
      • Kinikoglu B.
      • Michael J.
      • Yang S.
      • D Amato C.
      • et al.
      A chemical screen in zebrafish embryonic cells establishes that Akt activation is required for neural crest development.
      ;
      • Johansson J.A.
      • Marie K.L.
      • Lu Y.
      • Brombin A.
      • Santoriello C.
      • Zeng Z.
      • et al.
      PRL3-DDX21 transcriptional control of endolysosomal genes restricts melanocyte stem cell differentiation.
      ).
      Figure thumbnail gr1
      Figure 1A selection of techniques used in zebrafish to investigate dermatologic processes and pathologies. Clockwise from top left. Transgenesis: Injection of BRAFV600E into a p53(lf)-mutant embryo causes melanoma (
      • Patton E.E.
      • Widlund H.R.
      • Kutok J.L.
      • Kopani K.R.
      • Amatruda J.F.
      • Murphey R.D.
      • et al.
      BRAF mutations are sufficient to promote nevi formation and cooperate with p53 in the genesis of melanoma.
      ). Gene editing: Knockout of the albino locus with CRISPR‒Cas9 produces hypopigmented mutants (
      • Irion U.
      • Krauss J.
      • Nüsslein-Volhard C.
      Precise and efficient genome editing in zebrafish using the CRISPR/Cas9 system.
      ). Skin and tissue injury: Zebrafish caudal fins regenerate after amputation, allowing insights into skin and tissue regeneration (
      • Grotek B.
      • Wehner D.
      • Weidinger G.
      Notch signaling coordinates cellular proliferation with differentiation during zebrafish fin regeneration.
      ). Live-cell imaging: Tracking of fluorescently labeled macrophages during melanocyte death in vivo allows for insight into inflammatory and immune processes (
      • Hsu C.H.
      • Liou G.G.
      • Jiang Y.J.
      Nicastrin deficiency induces tyrosinase-dependent depigmentation and skin inflammation.
      ). Drug screening: Screening of zebrafish embryos developmental phenotypes for potential therapeutic small molecules (
      • Peterson R.T.
      • Link B.A.
      • Dowling J.E.
      • Schreiber S.L.
      Small molecule developmental screens reveal the logic and timing of vertebrate development.
      ). Transplantation: Transplantation of fluorescently tagged melanoma cells into adult zebrafish to assay for metastasis (
      • Yan C.
      • Brunson D.C.
      • Tang Q.
      • Do D.
      • Iftimia N.A.
      • Moore J.C.
      • et al.
      Visualizing engrafted human cancer and therapy responses in immunodeficient zebrafish.
      ). KO, knockout; sgRNA, single guide RNA; WT, wild-type.
      New adult zebrafish xenograft models have been developed to allow long-term engraftment and study of tumor biology in a stable adult milieu (
      • Yan C.
      • Brunson D.C.
      • Tang Q.
      • Do D.
      • Iftimia N.A.
      • Moore J.C.
      • et al.
      Visualizing engrafted human cancer and therapy responses in immunodeficient zebrafish.
      ). These tools allow for creating new models for rare subtypes of melanoma and testing novel therapeutics in an expedited fashion. These screens can be integrated with murine studies to further elucidate the conserved mechanisms of disease and therapy (
      • Patton E.E.
      • Mueller K.L.
      • Adams D.J.
      • Anandasabapathy N.
      • Aplin A.E.
      • Bertolotto C.
      • et al.
      Melanoma models for the next generation of therapies.
      ). Together, these models present a comprehensive arsenal for using zebrafish to understand the mechanisms governing the initiation, progression, and treatment of skin cancer.

      Efficient genetic tools for the generation of new models

      Although zebrafish have been utilized as a powerful model for forward genetic screens, recent development of efficient genetic editing techniques in the zebrafish has enabled easier loss-of-function studies (Figure 1). The CRISPR‒Cas9 system efficiently creates somatic and germline mutant zebrafish (
      • Hwang W.Y.
      • Fu Y.
      • Reyon D.
      • Maeder M.L.
      • Kaini P.
      • Sander J.D.
      • et al.
      Heritable and precise zebrafish genome editing using a CRISPR-Cas system.
      ,
      • Hwang W.Y.
      • Fu Y.
      • Reyon D.
      • Maeder M.L.
      • Tsai S.Q.
      • Sander J.D.
      • et al.
      Efficient genome editing in zebrafish using a CRISPR-Cas system.
      ). These tools have since been applied to create novel disease models. In 2015,
      • Ablain J.
      • Durand E.M.
      • Yang S.
      • Zhou Y.
      • Zon L.I.
      A CRISPR/Cas9 vector system for tissue-specific gene disruption in zebrafish.
      utilized CAS9 expressed behind a cell-specific promoter to show the ability to disrupt cell-specific gene expression in zebrafish using CRISPR‒Cas9. These tools have recently been utilized to elucidate the novel roles for HEXIM1 and SPRED1 as modulators of melanoma (
      • Ablain J.
      • Xu M.
      • Rothschild H.
      • Jordan R.C.
      • Mito J.K.
      • Daniels B.H.
      • et al.
      Human tumor genomics and zebrafish modeling identify SPRED1 loss as a driver of mucosal melanoma.
      ;
      • Tan J.L.
      • Fogley R.D.
      • Flynn R.A.
      • Ablain J.
      • Yang S.
      • Saint-André V.
      • et al.
      Stress from nucleotide depletion activates the transcriptional regulator HEXIM1 to suppress melanoma.
      ). Application of these new reverse genetic tools to generate models for other skin diseases such as SCC, EB, vitiligo, and psoriasis will create opportunities to better understand their pathogenesis.

      Where do we go from here?

      The recent expansion of sequencing capacity and adjunct modalities such as chromatin profiling and single-cell sequencing enables previously impractical dissection of tissue heterogeneity and signaling mechanisms during homeostasis, regeneration, and disease. Leveraging insights garnered using these technologies into the similarities and differences between zebrafish and human skin biology will enhance the utility of further zebrafish models for human skin disease. Much of the expansive technique toolbox for zebrafish has been built and tested in developmental and oncologic models, leaving great room for growth in other skin disease models. The application of these tools will lead to deeper understandings of cell–cell interactions and signaling pathways relevant to skin disease. In addition, efficient genetic engineering tools will undoubtedly be used to generate new disease models that will prove invaluable in understanding skin pathologies. Similarly, these biological insights and tools can be combined with zebrafish’s high-throughput screening attributes to enable the discovery and evaluation of novel therapeutics for human skin disease.

      Conclusion

      Although in vitro experiments and murine models have produced critical insights into the pathogenesis of and development of treatments for skin diseases, there remain significant gaps in our understanding of many of these diseases. The use of zebrafish models for human skin disease has already yielded promising results, and the evolution of new genetic tools will increase the number and quality of available models, presenting greater opportunities to model human skin disease and develop future therapeutics.

      Conflict of Interest

      The authors state no conflict of interest.

      Multiple Choice Questions

      • 1.
        Zebrafish were classically used to model which of the following:
        • A.
          Cancer progression
        • B.
          Inflammatory disorders
        • C.
          Inherited hypopigmentation disorders
        • D.
          Embryonic development
      • 2.
        Zebrafish are lacking a clear analog of which layer of the human skin
        • A.
          Dermis
        • B.
          Basement membrane
        • C.
          Stratum corneum
        • D.
          Hypodermis
      • 3.
        Zebrafish have been used to model the following skin diseases EXCEPT for:
        • A.
          Melanoma
        • B.
          Basal cell carcinoma
        • C.
          Epidermolysis bullosa
        • D.
          Psoriasis
      • 4.
        Which of the following is available for modeling cancer in zebrafish?
        • A.
          Genetic mutations in tumor suppressor genes
        • B.
          Transgenic expression of human oncogenes
        • C.
          Xenografts of tumor cells
        • D.
          All of the above
      • 5.
        Regeneration studies in zebrafish have used the following tools to wound skin EXCEPT for:
        • A.
          Chemical burns
        • B.
          Caudal fin amputation
        • C.
          Scale depilation
        • D.
          Dermatology lasers

      Acknowledgments

      This work was supported by grants from the National Cancer Institute ( T32 CA130807 ) to W.T.F. and US Department of Defense ( W81XWH2010288 ) to C.J.C.

      Supplementary Material

      Detailed Answers

      • 1.
        Zebrafish were classically used to model which of the following:
      • CORRECT ANSWER: D. Embryonic development.
      • Zebrafish were first utilized for investigating development owing to their transparent structures and rapid ex utero development. Many of the first disease models were created with forward genetic screens, which were published in a special issue of Development in 1996 (
        • Haffter P.
        • Granato M.
        • Brand M.
        • Mullins M.C.
        • Hammerschmidt M.
        • Kane D.A.
        • et al.
        The identification of genes with unique and essential functions in the development of the zebrafish, Danio rerio.
        ).
      • 2.
        Zebrafish are lacking a clear analog of which layer of the human skin
      • CORRECT ANSWER: C. Stratum corneum.
      • As aquatic animals, zebrafish never developed the water-impermeable stratum corneum found in most land animals (
        • Li Q.
        • Frank M.
        • Thisse C.I.
        • Thisse B.V.
        • Uitto J.
        Zebrafish: a model system to study heritable skin diseases.
        ).
      • 3.
        Zebrafish have been used to model the following skin diseases EXCEPT for:
      • CORRECT ANSWER: B. Basal cell carcinoma.
      • Zebrafish have been used to model melanoma (
        • Patton E.E.
        • Widlund H.R.
        • Kutok J.L.
        • Kopani K.R.
        • Amatruda J.F.
        • Murphey R.D.
        • et al.
        BRAF mutations are sufficient to promote nevi formation and cooperate with p53 in the genesis of melanoma.
        ), epidermolysis bullosa (
        • Sonawane M.
        • Carpio Y.
        • Geisler R.
        • Schwarz H.
        • Maischein H.M.
        • Nuesslein-Volhard C.
        Zebrafish penner/lethal giant larvae 2 functions in hemidesmosome formation, maintenance of cellular morphology and growth regulation in the developing basal epidermis.
        ), and psoriasis (
        • Carney T.J.
        • von der Hardt S.
        • Sonntag C.
        • Amsterdam A.
        • Topczewski J.
        • Hopkins N.
        • et al.
        Inactivation of serine protease Matriptase1a by its inhibitor Hai1 is required for epithelial integrity of the zebrafish epidermis.
        ;
        • Mathias J.R.
        • Dodd M.E.
        • Walters K.B.
        • Rhodes J.
        • Kanki J.P.
        • Look A.T.
        • et al.
        Live imaging of chronic inflammation caused by mutation of zebrafish Hai1.
        ;
        • Webb A.E.
        • Driever W.
        • Kimelman D.
        psoriasis regulates epidermal development in zebrafish.
        ) but have not yet been used to model basal cell carcinoma, possibly owing to low morbidity and readily available disease treatments.
      • 4.
        Which of the following is available for modeling cancer in zebrafish?
      • CORRECT ANSWER: D. All of the above.
      • Zebrafish have an expansive genetic toolbox that includes genetic mutations in tumor suppressor genes such as tp53 (
        • Berghmans S.
        • Murphey R.D.
        • Wienholds E.
        • Neuberg D.
        • Kutok J.L.
        • Fletcher C.D.
        • et al.
        tp53 mutant zebrafish develop malignant peripheral nerve sheath tumors.
        ), transgenic expression of human oncogenes (
        • Patton E.E.
        • Widlund H.R.
        • Kutok J.L.
        • Kopani K.R.
        • Amatruda J.F.
        • Murphey R.D.
        • et al.
        BRAF mutations are sufficient to promote nevi formation and cooperate with p53 in the genesis of melanoma.
        ), and xenografts of tumor cells (
        • Lee L.M.
        • Seftor E.A.
        • Bonde G.
        • Cornell R.A.
        • Hendrix M.J.
        The fate of human malignant melanoma cells transplanted into zebrafish embryos: assessment of migration and cell division in the absence of tumor formation.
        ).
      • 5.
        Regeneration studies in zebrafish have used the following tools to wound skin EXCEPT for:
      • CORRECT ANSWER: A. Chemical burns.
      • Investigators of regenerative mechanisms have used caudal fin amputation (
        • Becerra J.
        • Montes G.S.
        • Bexiga S.R.
        • Junqueira L.C.
        Structure of the tail fin in teleosts.
        ), scale depilation (
        • Richardson R.
        • Metzger M.
        • Knyphausen P.
        • Ramezani T.
        • Slanchev K.
        • Kraus C.
        • et al.
        Re-epithelialization of cutaneous wounds in adult zebrafish combines mechanisms of wound closure in embryonic and adult mammals.
        ), and dermatology lasers (
        • Richardson R.
        • Slanchev K.
        • Kraus C.
        • Knyphausen P.
        • Eming S.
        • Hammerschmidt M.
        Adult zebrafish as a model system for cutaneous wound-healing research.
        ) to induce wounds and study regeneration involving skin tissue in zebrafish. However, there are currently no works using chemical agents to wound the zebrafish dermis.

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