Visualizing Cell Behavior in the Neural Plate

High-resolution video microscopy of neural plate explants similar to the type described above can be used to characterize the cell behaviors driving neural convergent extension. Protrusive activity of cells in a tissue is best visualized if a scattered population of the cells is labeled with a vital dye. To achieve such a labeling in the neural plate, we inject 20-30 nL of rhodamine dextran amine (RDA, Molecular Probes Inc., Eugene, OR) into the "A" or "B" dorsal tiers of 32 cell-stage embryos. Dorsal is identified by the tipping and marking method described above. By the midgastrula stage (stage 11.5), cell rearrangement associated with convergence and extension has occurred among the derivatives of the injected blastomere, so that labeled cells are scattered along the length of the neural plate. At stage 12.5, the outer epithelial layer of the neural plate is removed and discarded, exposing the labeled deep cells of the neural plate. The remaining layer of neural plate deep cells, with or without the underlying mesoderm, is removed and cultured under a restraining coverslip, oriented with the neural deep cells facing the bottom of the culture chamber. In this configuration, the neural deep cells can be visualized with time-lapse video recording, using fluorescence illumination, a low-light camera, and image processing, as described previously (39). We have used this method to study the cell behavior underlying convergence extension of the posterior neural plate (89).

Was this article helpful?

0 0

Post a comment