Whole-Mount In Situ Hybridization for Detection of Migrating Zebrafish Endodermal Cells.

Abstract

One of the most important events in early vertebrate development is the formation and positioning of the endoderm, the embryonic progenitor cell population that gives rise to the internal organs. Recent years have seen renewed interest in the mechanisms underlying the specification and migration of endodermal progenitor cells. The zebrafish is a well-established, accessible, and powerful model to study this cell population. Zebrafish endodermal cells are specified around 4h after fertilization and subsequently migrate as evenly spaced single cells in a stereotypical manner in the next 6h. Given the large numbers of fertilized eggs that can be obtained from a single breeding pair and the ease of chemical and genetic perturbations, the zebrafish is an excellent model to study mechanisms underlying endoderm specification and migration. An easy approach to visualizing and quantitating endodermal cells and their migratory routes is by whole-mount in situ hybridization (WISH) on fixed embryos, collected in time series. This chapter provides basic information on the organization and staging of the embryos, with an emphasis on the migrating endodermal cell population. In addition, optimized protocols for the isolation and fixation of staged embryos are provided as well as detailed probe synthesis and WISH protocols, specific for migrating endoderm. Finally, details are provided on how to approach these experiments quantitatively, and some common pitfalls are discussed.