Preparation of transgenic zebrafish embryos for imaging the developing retina.

The zebrafish retina is an ideal model system for addressing neural fate specification in vivo. As in all vertebrate species studied, the retina is composed of seven major cell types distributed in a laminated histogenetic arrangement. The major connections and final positioning of cell types are well known, allowing lineage tracing and identification of final cell outcome by location, morphology, and subsequent immunostaining. The retina is conveniently located on the outside of the fish, allowing the embryos to be mounted such that the eye is close to the coverslip. This enables the entire structure to be imaged in four dimensions (4D) within the given focusing depth constraints. When preparing cells for lineage tracing, it is very important to label isolated cells mosaically, so that they stand out in a mostly unlabeled background. This can be achieved by transplanting cells from transgenic or injected embryos into uninjected embryos, as is described in this protocol. Transgenic (wild-type or mutant) embryos expressing stable green or red fluorescent protein (GFP or RFP) are used as donors, and non-transgenics or transgenics expressing a different fluorescent label are used as hosts. Mosaic labeling can also be achieved by DNA injection.