Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos

Summary

This protocol describes imaging of individual neurons or neural crest cells in living zebrafish embryos. This method is used to examine cellular behaviors and actin localization using fluorescence confocal time-lapse microscopy.

Abstract

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily accessible at all stages of development. Moreover, their optical clarity allows high resolution imaging of cell and molecular dynamics in the natural environment of the intact embryo. We are using a live imaging approach to analyze cell behaviors during neural crest cell migration and the outgrowth and guidance of neuronal axons.

Live imaging is particularly useful for understanding mechanisms that regulate cell motility processes. To visualize details of cell motility, such as protrusive activity and molecular dynamics, it is advantageous to label individual cells. In zebrafish, plasmid DNA injection yields a transient mosaic expression pattern and offers distinct benefits over other cell labeling methods. For example, transgenic lines often label entire cell populations and thus may obscure visualization of the fine protrusions (or changes in molecular distribution) in a single cell. In addition, injection of DNA at the one-cell stage is less invasive and more precise than dye injections at later stages.

Here we describe a method for labeling individual developing neurons or neural crest cells and imaging their behavior in vivo. We inject plasmid DNA into 1-cell stage embryos, which results in mosaic transgene expression. The vectors contain cell-specific promoters that drive expression of a gene of interest in a subset of sensory neurons or neural crest cells. We provide examples of cells labeled with membrane targeted GFP or with a biosensor probe that allows visualization of F-actin in living cells¹.

Erica Andersen, Namrata Asuri, and Matthew Clay contributed equally to this work.

Protocol

1. Assembly of injection slides and imaging slides

Injection slides:

1. Prepare Sylgard silicone elastomer according to manufacturer's instructions.
2. Use the Sylgard to bond three standard glass microscope slides together by stacking one slide on top of the intersection of the other two slides, which are arranged side-by-side at the long edges. The top slide creates a right-angled corner or "wall" between the top and bottom slides.
3. Let Sylgard set overnight. These slides are reusable.

Imaging slide:

1. We use stainless steel rectangles cut to the same size of a stand....

Discussion

The optimal concentration of injected DNA will vary depending on size and strength of the promoter construct and should be determined empirically. Injection of too much DNA can lead to unhealthy embryos with extensive cell death, while too little will result in a very small proportion of injected embryos expressing the transgene. The DNA expression level correlates with the strength of the fluorophore signal, which varies from cell to cell. While sorting embryos under epifluorescence, exclude those with extremely high le.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Tricaine (Ethyl 3-aminobenzoate methanesulfonate)	Sigma-Aldrich	A5040-250G
Sylgard Silicone Elastomer Kit	Dow Corning	184
QIAfilter Plasmid Midi Kit	Qiagen	12243
Low melting point agarose	Invitrogen	15517-014
Picospritzer	Parker Hannifin Corporation	051-0302-900