Analysis of zebrafish development using explant culture assays

Abstract

Two fundamental questions of developmental biology are when cells become committed to a certain lineage and what cell interactions are involved in establishing this commitment. These questions can be answered using explant or transplant assays. We have developed explant assays to study zebrafish development. These assays involve isolating by microdissection small regions of the embryo at specific times during development, and determining their fate after culture in isolation (lineage commitment assays) or after exposure to a putative inducing tissue (induction assays). In our laboratory, we have used these assays to address questions of neural development, including those of commitment to anterior and posterior neural lineages, and the signals involved in making these decisions. This chapter contains detailed guidelines for designing explant assays. These include suggestions for the isolation and successful culture of explants, and descriptions of the methods used to assay the final fate of explants after culture. Step-by-step protocols are given for the isolation of specific explants that can be used in specification and induction assays. The application of this technique is illustrated with descriptions of experiments. Explant assays will continue to generate key information concerning the establishment of lineage commitment of many embryonic tissues and will provide extremely valuable for analysis of new genes identified molecularly and in mutant screens.