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 cells.
Genetics Training Program, University of Wisconsin-Madison, USA.
Erica
E
Andersen
Namrata
N
Asuri
Matthew
M
Clay
Mary
M
Halloran
D000199:Actins / Q000032:analysis*; D000818:Animals; D015374:Biosensing Techniques; D002465:Cell Movement / Q000502:physiology*; D003599:Cytoskeleton / Q000737:chemistry*; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics; D018613:Microscopy, Confocal / Q000379:methods*; D009432:Neural Crest / Q000166:cytology*; D009474:Neurons / Q000166:cytology*; D010957:Plasmids / Q000235:genetics; D015027:Zebrafish / Q000196:embryology*
D000199:Actins; D049452:Green Fluorescent Proteins