1. Research Techniques Made Simple: Zebrafish as a Model System to Study Skin Biology and Pathology Qiaoli Li and Jouni Uitto Department of Dermatology and Cutaneous Biology, Jefferson Medical College, and Jefferson Institute of Molecular Medicine, Thomas Jefferson University, Philadelphia, PA, USA

2. Zebrafish and Embryos Vertebrate Has nearly the same complement of genes as mammals Develop externally Transparent Easy breeding Develop quickly

3. Zebrafish Skin Development Reprinted with permission from Li et al. (2011) J Invest Dermatol 131:565-71

4. Genetic Manipulation of Zebrafish Genome Forward genetics Ethylnitrosourea (ENU) or random mutagenesis Reverse genetics Morpholino knockdown of specific gene expression

5. ENU Mutagenesis Modified from (Dahm, 2005) Encyclopedia of Molecular Cell Biology and Molecular Medicine (Meyers RA, ed) Vol. 15: Wiley- VCH, 593-626, with permission. Dahm, 2005

6. Modulation of Gene Expression by Morpholino 25 nt oligo “Knockdown”

7. Efficiency of Morpholino Knockdown EGFP SP6 promoter pCS2/EGFP vector MO1 ATG 5’UTR target gene col17a1b/EGFP RNA + col17a1b MO1col17a1b/EGFP RNA Splice morpholino 8.5hpf CMO2 500bp 300bp 421bp 299bp forward abcc6b MO2 reverse EXON 18 EXON 19 ATG morpholino 8.5hpf Reprinted with permission from Li et al. (2011) J Invest Dermatol 131:565-71

8. Application of Zebrafish for Skin Research Wound healing and skin repair Pigmentation Epidermal adhesion Skin diseases Skin immunity Drug screening

9. Advantages A freshwater vertebrate with a complement of genes very similar to those of mammals Rapid development of transparent embryos, allowing visual inspection of internal organs Easy breeding, with a large number of embryos and larvae Easy manipulation of gene expression with phenotypic consequences A facile and cost-efficient system for large-scale screening

10. Limitations Lack of functional stratum corneum and expression of genes necessary for terminal differentiation of epidermis The zebrafish genome database is not complete and contains misjoins and misassemblies Paucity of antibodies recognizing zebrafish protein sequences