Imaging brain development and organogenesis in zebrafish using immobilized embryonic explants

Abstract

Owing to its optical clarity and rapid rate of development, the zebrafish embryo is an ideal model system for studying the cellular mechanics of organogenesis. Unfortunately, extended time-lapse recordings of zebrafish embryos are often disrupted by the extension and straightening of the embryonic axis, as well as movement artifacts associated with developing musculature. In addition, the embryo's massive yolk cell often prevents optical access to tissues of interest. To circumvent these imaging problems, we have developed a procedure to deflate and mechanically remove the yolk cell. A "paralyzing" agent, AMP-PNP (a membrane-impermeant nonhydrolyzable analog of ATP), is first injected into the embryo's contractile yolk cell. The yolk cell is then removed using sharpened tungsten needles. Deyolked embryos, or organ rudiments explanted from them, are then immobilized on a microscope coverslip using a thin plasma clot. This plasma clot immobilization allows novel mountings of the explants so that ventral, lateral, and even cross-sectional fields of views are possible using high numerical aperture objectives. We show that isolated head rudiments undergo normal morphogenesis and gene expression for at least 1 day after being explanted into organotypic culture. These procedures can be used to study the cellular mechanics of organogenesis in "deyolked" embryos, as well as in tissues explanted from green fluorescent protein transgenic animals.