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

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.


Assembly of injection slides and imaging slides
Injection slides: Imaging slide:

Preparation of DNA constructs
To express a transgene in a tissue-specific manner, the gene of interest is cloned behind a cell-specific promoter. We use a cis-regulatory element of the zebrafish neurogenin1 gene (-3.1ngn1) 2 or the sox10 gene (-4.9sox10) 3 to drive expression in sensory neurons or neural crest cells, respectively. To visualize cell and membrane dynamics, we express cytoplasmic or membrane-localized fluorophores. To image F-actin distribution, we express DNA encoding the calponin homology domain of utrophin fused to mCherry (UtrCH-mCherry). The UtrCH-mCherry biosensor probe allows visualization of F-actin without interfering with actin dynamics 1 . We generate these DNA constructs using the Invitrogen 3. Injection of DNA constructs 4. Mounting embryos for imaging

4D imaging of cell behaviors on Olympus FV1000 confocal with IX81 microscope
The details of image acquisition will depend on the specifics of your microscope system. Here we describe the time-lapse acquisition process for the Olympus FV1000 confocal microscope. See previous JoVE protocol for time-lapse acquisition with the Zeiss LSM 510 confocal system 6 .

Representative Results
The figures and movies depict examples of sensory neurons and neural crest cells labeled with membrane-targeted fluorophores or the actin biosensor probe.

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 levels of expression. Labeled cells that appear very bright usually also show obvious signs of toxicity, such as abnormal cell morphology, mislocalization of tagged molecules, and cell death. A typical DNA injection yields 5-10% of embryos that are suitable for imaging.
The F-actin biosensor can function in a dominant negative fashion when expressed at high levels. If promoter-driven expression is problematic, the biosensor can be expressed ubiquitously by injecting mRNA. The advantage of mRNA injection is that the expression levels can be controlled by adjusting the concentration of mRNA. Individual cells in these embryos are labeled by co-injection of a DNA construct containing a cell-specific promoter driving expression of a membrane-localized fluorophore. We have used this approach to image F-actin in neural crest cells 7 .
We often carry out the single-cell labeling technique in transgenic expression lines. For example, injecting a -3.1ngn1:mcherry plasmid into embryos of the Tg(-3.1ngn1:gfp) line will label individual sensory neurons red in a background of green neurons. This strategy allows examination of the behavior of an individual cell within the context of the entire cell population.
We focus here on imaging neural and neural crest cell behaviors and actin distribution. However, the general method of tissue specific mosaic cell labeling by DNA plasmid injection can be expanded for use with fluorescent fusion proteins as well as other genetically encoded biosensor probes. Combining these techniques can allow investigators to visualize subcellular localization of specific molecules as well as signaling mechanisms in vivo.