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Zebrafish is our favorite model organism because it has development programs similar to those operating during human development. This small fish breeds all year, producing hundreds of transparent embryos that develop rapidly. This facilitates the analysis of organ development in normal conditions or when genes have been mutated, which is the case for many available mutants obtained during several large-scale mutagenic screenings. We are interested in characterizing genes that participate in the development of fish pigmentation and the formation of the epidermis, particularly studying proteins involved on vesicles traffic. Some of our research is also focused on establishing novel techniques to study epidermal and germ cells during development. A typical experiment from our group would consist of setting up fish crosses and obtaining embryos to be processed at the appropriate developmental stage for in situ hybridization, antibody staining, RNA, or protein purification or live fluorescence microscopy.
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The Technique
Our collaborator, Dr. Rosa E. Navarro, showed us how she applied the freeze-crack technique to study Caenorhabditis elegans germ cells in the gonads. We recognize that by modifying her original setup we may be able to use the same principle to obtain epidermal cells during zebrafish development. Our laboratory staff assayed dozens of different setups until they found an appropriate one, which allowed us to obtain intact epidermis cells. The epidermis tissue is part of the skin, which at early stages of development is formed only by two cell monolayers; therefore it is a much simpler version of the epidermis in mammals, which is formed of several monolayers of cells. In zebrafish larvae, the cells from these two types of adjacent and connected monolayers are very different. The most external monolayer specializes in protecting the fish body, whereas the internal monolayer is dedicated to producing large amounts of collagen necessary to form the subepidermal space and make a more resistant but flexible skin. The new freeze-crack epidermis technique will allow us to determine which genes are specifically expressed in both kinds of epidermis cells during zebrafish development.
See “Freeze-crack technique to study epidermal development in zebrafish using differential interference contrast microscopy and fluorescent markers” on page 313.
