Zebrafish Course 2010 Retinotectal System

OVERVIEW: To study the development of the retinotectal system, it's important to be able to visualize and perturb its development. We will try two different methods for labeling retinal axons with lipophilic dyes, and two methods for conditional activation of gene expression. We will also discuss methods for 3D visualization of confocal data. Several of these methods are applicable to other neurobiological systems, and indeed to nonneural systems. Labeling axons. Lipophilic dyes are bright and diffuse along axonal membranes in either living or fixed tissue. We will use two methods to label retinal axons. Whole-eye labeling uses pressureinjection of lipophilic dyes to label all the retinal axons; focal labeling uses a dye-coated microneedle to label a small population of retinal axons. Conditional gene activation. The most commonly-used method for conditional gene activation employs heat-inducible heat shock protein (hsp) promoters, usually the hsp70l promoter. Brief (30-60 min) heating of embryos in a water bath or PCR machine can give temporal control of gene expression. Here we will demonstrate spatial control of hsp70l promoter-driven transgenes using local heating. We will compare the two relatively simple methods that have been developed for locally heating tissue. One employs a sharpened soldering iron to deliver heat, while the other uses a laserpointer and a drawn-out optical fiber.