Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy
April 1st, 2009
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.
This procedure begins with injection of a single cell zebra fish embryo positioned in an injection mold such that the cell is oriented facing away from the injection apparatus. The embryos are then injected with MRNA with the needle moving through the yolk and into the cell. The next day.
A plastic mounting slide is filled with aros. An anesthetized embryo is then placed inverted in a hole in the aros in imaged by confocal microscopy. Hi, I'm Grabham from the lab of Dr.Hazel Sieve at the Whitehead Institute for Biomedical Research and the biology department at MIT.
Today we will show you a procedure for injecting zebrafish embryos at the single cell stage and the subsequent live imaging of the brain by confocal microscopy. In our lab, we use this procedure to study vertebrate brain morphogenesis at single cell resolution. So let's get started.
The mRNA used in this procedure is transcribed from a plasmid encoding C-A-X-E-G-F-P-R-N-A-A membrane bound GFP First linearize the plasmid by digestion with not one, then transcribe membrane GFP mRNA from the linearized plasmid using the M message and machine kit after the mRNA has been transcribed, quantifying dilute the resulting mRNA and water to one microgram per one microliter, Eloqua, and store at negative 80 degrees Celsius until needed. On the day before injection set up mating cages, separating the males from the females on the day of injection. Use a setter instrument micropipet polar to pull capillary needles for injection.
Prepare an injection mold of 1%AGA rose with lanes the width of embryos at the one cell stage on the day of injection, thaw an aliquot of membrane GFP mRNA at a concentration of one microgram per microliter on ice. Dilute the mRNA one to five in water to a final concentration of 200 nanograms per microliter and keep on ice. Load the capillary needle with one microliter of prepared membrane, GFP mRNA at 200 nanograms per microliter.
Here we add 0.05%phenol red to the mRNA to make it visible for injections. Place the loaded needle into a micro manipulator attached to a gas powered micro injector. Adjust the injection volume to one nanoliter.
Next, pull the dividers on the wild type fish set up in mating cages from the previous day. Collect the embryos as soon as they are laid. Fill the AGA rose injection mold with embryo medium and orient the embryos in the mold with a single cell on the side away from the micro manipulator.
Inject through the corion and yolks such that the mRNA is deposited directly into the single cell. Inject approximately 50 embryos per experiment. However, if the cell has divided, do not inject after the embryos have been injected, incubate them at 28 degrees Celsius overnight in embryo medium.
After the overnight incubation, the injected embryos are ready for mounting and imaging. To begin this procedure, remove the corion from the embryos with forceps under a stereo microscope one hour before the time point of interest, prepare a slide for mounting up to four embryos. Then fill the slide with 0.7%aros just to the top of the slide and wait for about 20 minutes or until the aros solidifies.
Once the aros has solidified, make a small hole in the aeros using a 200 microliter pipette tip to form cylindrical holes in which to mount each embryo. Remove the aros plugs with forceps before mounting. Place the embryos on the aeros filled slide under a dissecting microscope.
Add 50 microliters of trica to anesthetize. The embryos. Use forceps to orient the embryos inverted in the cylindrical holes in the aros with the brain or region of interest against the underlying cover slip cover.
The embryos in the AGA rose with another cover slip and secure with silicone vacuum grease. The embryos are now ready for imaging. Image the embryo using an inverted fluorescent laser scanning confocal microscope as shown here, or spinning disc confocal microscope image at 63 x or higher to collect high resolution images of single cells within the neuro epithelium.
Images are exported as TIF files from the LSM software and analyzed using Photoshop. This is a representative confocal image of a 24 hour zebrafish embryo, neuro epithelium between the midbrain and hindbrain ventricles. M indicates the midbrain, ventricle, and H indicates the hindbrain ventricle.
Each cell is labeled with membrane bound GFPE. We've just demonstrated a technique that allows you to analyze the developing zebrafish brain as single cell resolution. This technique has enabled us to study a novel type of cell shape change called BA basal constriction.
It could also be used to analyze other types of phenomena and to significantly expand our understanding of vertebrae organogenesis, as well as its underlying cell biology. So that's it. Thanks for watching and good luck with your experiments.
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Overview
This video demonstrates a method for analyzing the developing vertebrate brain in live zebrafish embryos at single-cell resolution using confocal microscopy. The process includes injecting the zebrafish embryo and subsequently mounting and imaging the developing brain.
Key Study Components
Area of Science
- Neuroscience
- Developmental Biology
- Imaging Techniques
Background
- Zebrafish are a model organism for studying vertebrate development.
- Confocal microscopy allows for high-resolution imaging of live tissues.
- Single-cell analysis provides insights into cellular development and differentiation.
- This method can be applied to various studies in developmental biology.
Purpose of Study
- To visualize the developing brain in zebrafish embryos.
- To demonstrate a technique for single-cell resolution imaging.
- To enhance understanding of vertebrate brain development.
Methods Used
- Injection of mRNA into single-cell zebrafish embryos.
- Use of a plastic mounting slide for imaging.
- Confocal microscopy for high-resolution imaging.
- Positioning embryos in an injection mold for optimal orientation.
Main Results
- Successful injection of mRNA into zebrafish embryos.
- High-resolution images of the developing brain obtained.
- Demonstration of the feasibility of live imaging techniques.
- Insights into cellular behavior during early development.
Conclusions
- The method allows for detailed observation of brain development in zebrafish.
- Single-cell resolution imaging is achievable with confocal microscopy.
- This technique can be utilized for further studies in developmental neuroscience.
Frequently Asked Questions
Executive Industry Relevance
Live imaging of the zebrafish embryonic brain enables high-resolution visualization of cellular dynamics during neural tube morphogenesis, providing a disease-relevant system for studying vertebrate brain development. This approach supports target validation and mechanistic de-risking by allowing direct observation of cell shape changes and rearrangements critical to organogenesis. The method enhances predictive confidence in preclinical models by linking subcellular behaviors to structural outcomes in a transparent, genetically tractable system.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of therapeutic hypotheses through direct visualization of membrane-bound protein dynamics in developing neuroepithelium.
- Operational Value: Supports biological de-risking by linking cytoskeletal mechanisms to tissue-level morphogenetic events.
Screening & Assay Development
- Scientific Value: Prepares validated biological systems for downstream compound screening by establishing baseline morphogenetic benchmarks.
- Operational Value: Promotes assay standardization through reproducible embryo mounting and inversion protocols.
Translational & Preclinical Research
- Scientific Value: Offers disease-relevant system alignment by capturing conserved mechanisms of vertebrate brain folding applicable to human neural tube defects.
- Operational Value: Facilitates translational biomarker alignment through quantifiable readouts of cell shape changes such as basal constriction.
Pipeline & Workflow Integration
The method integrates into early discovery workflows by providing single-cell resolution data that informs target confidence prior to lead identification efforts.
- Discovery Biology: Supports hypothesis testing and pathway clarification by enabling real-time observation of cell behaviors in intact tissues.
- Screening: Enhances assay readiness through standardized production of fluorescently labeled embryos suitable for time-lapse analysis.
- Analytics: Generates quantitative dependent variable measurements via fluorescence intensity and spatial tracking of GFP-labeled membranes.
- Translational Research: Connects to preclinical continuity by modeling conserved morphogenetic processes across vertebrate species.
- Enterprise Reuse: Establishes a reusable platform for neurodevelopmental screening due to embryo transparency and external development.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence by reducing mechanistic ambiguity in brain morphogenesis pathways.
- Operational Value: Improves reproducibility through standardized injection volumes, mounting orientation, and imaging parameters.
- Strategic Value: Supports better go/no-go decisions by enabling early detection of phenotypic anomalies in developing tissues.
- Portfolio Impact: Informs risk-adjusted prioritization by linking cellular phenotypes to structural outcomes in a high-fidelity model.
Implementation Considerations
- Requires expertise in microinjection techniques and embryonic staging.
- Dependent on confocal microscopy infrastructure with 63x or higher objective capabilities.
- Necessitates standardization across teams for embryo handling, anesthesia timing, and mounting consistency.
- Involves adaptation considerations when extending to other brain regions or developmental stages beyond 18–24 hpf.
- Limited by imaging depth of 80–100 μm, restricting analysis of deeper brain structures.
