Zebrafish as a Model System to Study Skin Biology and Pathology

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Research Techniques Made Simple: Zebrafish as a Model System to Study Skin Biology and Pathology Qiaoli Li and Jouni Uitto Department of Dermatology and.

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Zebrafish as a Model System to Study Skin Biology and Pathology Qiaoli Li, Jouni Uitto Journal of Investigative Dermatology Volume 134, Issue 6, Pages 1-6 (June 2014) DOI: 10.1038/jid.2014.182 Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Cutaneous biology of the developing zebrafish. (a) The growth of zebrafish from an embryo 1 day postfertilization (dpf), which is surrounded by a transparent chorion (c) and displays a prominent yolk sac (ys), to an adult fish. At 6 dpf, pigmentation becomes apparent on the skin. (b) Transmission electron microscopy reveals an epidermis (e) at 1 and 6 dpf consisting of two cell layers; at 6 dpf the epidermis is separated from the underlying collagenous stroma (cs) and dermis (d) by a clearly demarcated basement membrane (open arrowheads). In adult fish, there is a multilayered epidermis, and higher magnification of the basement membrane zone reveals the presence of hemidesmosomes (arrows in the inset). The spicule-like extensions of the surface of the skin (arrows) correspond to microridges. (c) Scanning electron microscopy reveals well-demarcated keratinocytes with distinct cell—cell borders (small arrows). In the middle of the keratinocyte surface, there are developing microridges that at 6 dpf become well organized (open arrowheads). In an adult fish, the epidermis is covered by scales. Reprinted with permission from Li et al. (2011). Journal of Investigative Dermatology 2014 134, 1-6DOI: (10.1038/jid.2014.182) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Principles of forward genetics for identification of mutated genes in zebrafish by ethylnitrosourea (ENU) mutagenesis. (a) ENU treatment of male fish results in random mutagenesis (m). Mating of mutant males with normal females results in a progeny of mutant fish with a readily observable phenotype in the case of a dominant mutation in F1 and F2 generations. In the case of a recessive mutation, random matings result in a population with the phenotype (gray fish). (b) Identification of the recessive mutations resulting in a phenotype (gray fish). The specific mutant genes will be identified by linkage analysis and the mutations can be detected by genome sequencing. SNP, single-nucleotide polymorphism; SSLP, simple sequence-length polymorphism. Modified with permission from Dahm et al., 2005. Copyright Wiley–VCH Verlag GmbH & Co. KGaA. Journal of Investigative Dermatology 2014 134, 1-6DOI: (10.1038/jid.2014.182) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Morpholino-mediated knockdown of zebrafish genes. (a) A morpholino (MO1) corresponding to the col17a1b gene was used to target the 5 untranslated region of the corresponding mRNA to prevent translation. To determine the efficacy of morpholino in downregulating the translation, an expression construct consisting of the SP6 promoter, 5 untranslated region of the col17a1b gene, and downstream enhanced green fluorescent protein (EGFP) reporter gene was generated. Microinjection of mRNA transcribed in vitro from the pCS2/EGFP vector to 1–4 cell-stage embryos shows green fluorescence at 8.5 hpf (lower left panel). Coinjection of this mRNA together with the MO1 morpholino completely abolished the fluorescence, indicating inhibition of the translation. (b) A morpholino (MO2) corresponding to the zebrafish abcc6b gene was placed on the exon 18/intron 18 splice junction. Efficiency of the morpholino in preventing splicing of the abcc6b pre-mRNA into mature mRNA was monitored by reverse transcription (RT)-PCR using primers placed on exon 18 (forward) and exon 19 (reverse). PCR of the genomic sequence resulted in a 421-bp fragment, whereas fully spliced cDNA yields a 299-bp fragment devoid of intron 18 (122 bp). RT-PCR of morpholino (MO2)-treated zebrafish embryo reveals the presence of the 421-bp mRNA sequence only, indicating complete inhibition of the removal of intron 18 by splicing. Because the intron 18 sequence is out of frame, this results in a complete absence of the abcc6b protein product. Reprinted with permission from Li et al. (2011). Journal of Investigative Dermatology 2014 134, 1-6DOI: (10.1038/jid.2014.182) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Journal of Investigative Dermatology 2014 134, 1-6DOI: (10. 1038/jid Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Journal of Investigative Dermatology 2014 134, 1-6DOI: (10. 1038/jid Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions