In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain.
1. Preparing the mRNA for Injection
2. Injecting the mRNA
3. Mounting and Imaging Embryos
4. Representative Results/Outcome
Shown in Figure 1 is a representative confocal image of a 24 hpf zebrafish neuroepithelium between the midbrain and hindbrain ventricles. Each cell is labeled with membrane-bound GFP.
Figure 1. Live confocal imaging of a memGFP-injected 24 hpf zebrafish embryo.
(A) Neuroepithelium of a 24 hpf embryo with each cell outlined by GFP. Region imaged separates the midbrain and hindbrain ventricles (M, H respectively), forming the midbrain-hindbrain boundary.
In this video, we demonstrate a method for mRNA injection into single cell zebrafish embryos. Here, we use mRNA encoding a membrane-targeted GFP to label each cell. We then demonstrate how to mount and image the developing brain at single cell resolution. This technique has allowed us to study a novel type of cell shape change, basal constriction, required for formation of the midbrain-hindbrain boundary (Gutzman et al, 2008). Similar analysis of other phenomena has the potential to significantly expand our understanding of vertebrate morphogenesis and the underlying cell biology.
Many thanks to Dr. Jennifer Gutzman who first developed this technique in the Sive lab. Thanks also to J. B. Green at the Dana-Farber Cancer Institute Boston, MA who kindly provided the plasmid encoding CAAX-GFP mRNA. The confocal imaging was conducted at the W. M. Keck Foundation Biological Imaging Facility at the Whitehead. HS is supported by NIHMH70926. EG is supported by an NSF pre-doctoral fellowship.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
SeaKem GTG agarose | Reagent | Lonza | 50027 | |
3-aminobenzoic acid ethyl ester (Tricaine) | Reagent | Sigma | A-5040 | |
Capillary tubing | Tool | Frederick Haer and Co. | 30-30-1 | Borosil 1.0mmOD x 0.5mm ID/fiber 100mm |
Silicone vacuum grease | Reagent | VWR | W0S717 | |
Forceps | Tool | Fine Science Tools | 11232-20 | Dumont #5 Bio Inox |
1-200 μl Pipette tips | Tool | Tip One, US Scientific | 111-0806 | These are the correct size for 24 hpf embryos. |
mMessage mMachine transcription kit | Reagent | Ambion | AM1340 | SP6 RNA polymerase |
Zeiss LSM 510 scanning confocal | Microscope | Zeiss | ||
Micromanipulator | Tool | Narishige | ||
Microinjector | Tool | Medical Systems Research Products Harvard Apparatus | PLI-100 | |
Micropipette puller | Tool | Sutter Instruments |