Labeling and Imaging Cells in the Zebrafish Hindbrain

Summary

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.

Abstract

Key to understanding the morphogenetic processes that shape the early vertebrate embryo is the ability to image cells at high resolution. In zebrafish embryos, injection of plasmid DNA results in mosaic expression, allowing for the visualization of single cells or small clusters of cells ¹ . We describe how injection of plasmid DNA encoding membrane-targeted Green Fluorescent Protein (mGFP) under the control of a ubiquitous promoter can be used for imaging cells undergoing neurulation. Central to this protocol is the methodology for imaging labeled cells at high resolution in sections and also in real time. This protocol entails the injection of mGFP DNA into young zebrafish embryos. Embryos are then processed for vibratome sectioning, antibody labeling and imaging with a confocal microscope. Alternatively, live embryos expressing mGFP can be imaged using time-lapse confocal microscopy. We have previously used this straightforward approach to analyze the cellular behaviors that drive neural tube formation in the hindbrain region of zebrafish embryos ². The fixed preparations allowed for unprecedented visualization of cell shapes and organization in the neural tube while live imaging complemented this approach enabling a better understanding of the cellular dynamics that take place during neurulation.

Protocol

1.Microinjection Dilute plasmid encoding membrane-targeted Green Fluorescent Protein (mGFP, courtesy of Richard Harland) to a concentration of 40 ng/ml. DNA is prepared by linearizing plasmid (mGFP/PCS2+, courtesy of Richard Harland) purified using the Qiagen Maxi Prep Kit. Addition of phenol red (diluted 1/10 total volume) to the solution is preferred to visualize the solution. Keep DNA solution on ice. Under a stereomicroscope, calibrate the injection needle (90mm glass capillaries from…

Discussion

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.

Materials

Solutions

• Hank’s Stock #1: 8.0 g NaCl , 0.4 g KCl in 100 ml dd H₂O
• Hank’s Stock #2: 0.358 g Na₂HPO₄ Anhydrous, 0.60 g KH₂PO₄ in 100 ml ddH₂O
• Hank’s Stock #4: 0.72 g CaCl₂ in 50 ml ddH₂O
• Hank’s Stock #5: 1.23 g MgSO₄x7H₂O in 50 ml dd H₂O
• 1 X Phosphate Buffer: 0.8% NaCl, 0.02% KCl, 0.02 M PO4, pH 7.3
• Blocking Solution: 2% normal goat serum, 1% bovine serum albumin (BSA), 1% dimethysulfoxide (DMSO)