Reverse Genetic Approach to Identify Regulators of Pigmentation using Zebrafish
Abstract
Melanocytes are specialized neural crest-derived cells present in the epidermal skin. These cells synthesize melanin pigment that protects the genome from harmful ultraviolet radiations. Perturbations in melanocyte functioning lead to pigmentary disorders such as piebaldism, albinism, vitiligo, melasma, and melanoma. Zebrafish is an excellent model system to understand melanocyte functions. The presence of conspicuous pigmented melanocytes, ease of genetic manipulation, and availability of transgenic fluorescent lines facilitate the study of pigmentation. This study employs the use of wild-type and transgenic zebrafish lines that drive green fluorescent protein (GFP) expression under mitfa and tyrp1 promoters that mark various stages of melanocytes. Morpholino-based silencing of candidate genes is achieved to evaluate the phenotypic outcome on larval pigmentation and is applicable to screen for regulators of pigmentation. This protocol demonstrates the method from microinjection to imaging and fluorescence-activated cell sorting (FACS)-based dissection of phenotypes using two candidate genes, carbonic anhydrase 14 (Ca14) and a histone variant (H2afv), to comprehensively assess the pigmentation outcome. Further, this protocol demonstrates segregating candidate genes into melanocyte specifiers and differentiators that selectively alter melanocyte numbers and melanin content per cell, respectively.
Additional Information
© 2022 JoVE Journal of Visualized Experiments. Date Published: March 1, 2022. We acknowledge the funding support from the Council for Scientific and Industrial Research vide project MLP2008 and the Department of Science and Technology for the project GAP165 for supporting the work presented in this manuscript. We thank Jeyashri Rengaraju and Chetan Mishra for their help with experiments. All authors declare no conflict of interest.Attached Files
Supplemental Material - jove-materials-62955-reverse-genetic-approach-to-identify-regulators-pigmentation-using.pdf
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Additional details
- Eprint ID
- 114120
- DOI
- 10.3791/62955
- Resolver ID
- CaltechAUTHORS:20220329-597130944
- MLP2008
- Council for Scientific and Industrial Research (CSIR)
- GAP165
- Department of Science and Technology (South Africa)
- Created
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2022-03-29Created from EPrint's datestamp field
- Updated
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2022-04-04Created from EPrint's last_modified field