Key to understanding the morphogenetic processes that shape the early embryo is the ability to image cells at high resolution. We describe here a technique for labeling single cells or small clusters of cells in whole zebrafish embryos with membrane-targeted Green Fluorescent Protein.
1.Microinjection
2. Imaging mGFP-labeled vibratome sections
3. Live imaging
4. Results
We describe here a straightforward approach to image single cells in the zebrafish neural tube using mosaic expression. Despite the optical transparency of the zebrafish embryo, we have found that visualization of neural cells is greatly enhanced in cross sections obtained using a vibratome. These sections are thick (50 mm) allowing for imaging of cellular extension from a given cell in different focal planes using a confocal microscope. Results using this methodology have been published elsewhere 2, but representative images of cells in the neural tube of a WT embryo (Figure 1) and an N-cadherin (N-cad) mutant (Figure 2) are provided. The latter reveals that cells in lateral regions of the neural plate in N-cad mutants fail to orient properly towards the midline (Figure 2). In contrast to this mosaic labeling, immunostaining the neuroepithelium with a general cell surface marker such as beta-catenin does not allow the morphology of individual cells to be visualized (Figure 3).
Time lapse imaging of live embryos complemented the mosaic labeling of fixed specimens, enabling a better understanding of the cellular dynamics that take place during neurulation. Movie 1 revealed that WT cells actively migrate towards the midline by extending medially-oriented membrane protrusions.
Figure 1. mGFP expression in the neural tube of a WT zebrafish embryo.
Figure 2. mGFP expression in the neural tube of a N-cadherin zebrafish embryo.
Figure 3. b-catenin immuno staining of the neuroepithelium.
In conclusion, the labeling techniques described here allow for single cell analysis of morphogenetic processes in the zebrafish embryo. The primary emphasis of this protocol is on methods for imaging labeled cells in the neural tube using mGFP under the control of a ubiquitous promoter. For additional applications of this transient expression assay readers should refer to a recent paper by Andersen et al.3.
The authors have nothing to disclose.
This work was supported by an NIH grant awarded to R. Brewster (1R01GM085290-01A1 ).
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