Ex Vivo Oculomotor Slice Culture from Embryonic GFP-Expressing Mice for Time-Lapse Imaging of Oculomotor Nerve Outgrowth
July 16th, 2019
An ex vivo slice assay allows oculomotor nerve outgrowth to be imaged in real time. Slices are generated by embedding E10.5 IslMN:GFP embryos in agarose, slicing on a vibratome, and growing in a stage-top incubator. The role of axon guidance pathways is assessed by adding inhibitors to the culture media.
This protocol allows us to identify axon guidance pathways active in the oculomotor nerve and to assess their roles at different points along the nerve trajectory in real time. This technique preserves the local environments through which axons travel and their final targets. The growing axons are not cut, so initial axon outgrowth, rather than regeneration, can be assessed.
This method provides insights into axon guidance in the ocular motor system but could be adapted to the study of axon guidance of other nerves. This technique takes practice to master, and working quickly is extremely important. When trying it for the first time, use just a few embryos, rather than a whole litter.
Before beginning the procedure, first confirm the pregnancy by ultrasound on embryonic day 10.5 from a timed mating. Harvest the embryos, spray the abdomen of the pregnant mouse with 70%ethanol, and use scissors to open the abdominal cavity to extract the uterus. Wash the uterus in a Petri dish of ice-cold HBSS before placing the organ in a second Petri dish of fresh ice-cold HBSS.
Under a dissecting scope, remove the embryos from the uterine horn and their individual amniotic sacs, placing each embryo on the underside of the lid of a 12-well plate, on ice, as they are harvested. Use filter paper to remove any liquid surrounding each embryo, without touching the embryos themselves, and submerge the embryos in liquid 4%low-melting agarose. Place the plate lid on ice to solidify the agarose.
When the agarose has hardened, flip over the embryos and cover the other side of each sample with additional agarose. When the second volume of agarose has solidified, use a fluorescence dissecting microscope to trim the agarose around each embryo so that each embryo will be oriented properly on the vibratome stage. The oculomotor nuclei and early axon outgrowths should be fluorescent.
Align each embryo so that the nucleus, outgrowing axons, and eye form a line, and use a razor blade to trim the agarose parallel to this line. Next, fill the vibratome chamber with ice-cold slice buffer, and superglue the first embryo to the vibratome stage so that the blade will be parallel with the oculomotor nucleus and eyes. When the superglue is dry, submerge the vibratome stage so that the embryo is oriented facing away from blade, and use a new vibratome blade to obtain 400-to 450-micrometer slices.
Use a sterile transfer pipette to transfer each slice into cold slicing buffer as it is acquired, and use the fluorescence dissecting microscope to select the slice containing the oculomotor nuclei and eyes. Using a sterile transfer pipette, transfer the slice onto a cell culture insert in a six-well plate containing 1.5 milliliters of culture medium per well, and place the plate in a 37-degree Celsius incubator. Remove the residual agarose from the vibratome stage, and superglue the next embryo onto the stage, continuing to collect slices until all of the embryos have been sectioned and plated.
Then add the appropriate concentration of the inhibitor or recombinant molecule of interest, diluted in an appropriate solvent, to the medium of each well. Create a dose-response curve. Image the slices every 30 minutes by phase contrast and fluorescence microscopy for up to 72 hours.
During normal in utero development, the first green fluorescent protein-positive oculomotor axons reach the orbit by embryonic day 11.5 before branching to their final targets, as observed in this representative slice culture. The orientation of the slice on the vibratome stage is crucial, as the slices are not interpretable if they're not oriented properly. For example, if the embryo is tilted to its side, only one oculomotor nucleus will be observed within the slice.
If the embedded embryo is tilted too far towards its back, the eyes will not be present within the slice, and instead the upper limb and hindbrain or spinal cord may be included. It is also important to take care that during the placement of the slice onto the culture membrane, that the tissue does not become folded. Take care when dissolving the inhibitors and growth factors in organic solvents.
For example, in this experiment, the slice died after the addition of ethanol to the medium. Remember to keep everything on ice and to minimize the time between the extraction of the embryos and placing the slices in the incubator. Using this technique, we have begun to identify additional axon guidance mechanisms at work in the ocular motor system.
Remember to use caution when working with razor blades and that some inhibitors may be hazardous and should be handled carefully according to their safety profiles.
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Overview
This study presents an ex vivo slice assay that enables the imaging of oculomotor nerve outgrowth in real time. Using embryos embedded in agarose, the research investigates the role of axon guidance pathways during this developmental process.
Key Study Components
Area of Science
- Neuroscience
- Developmental Biology
- Axon Guidance
Background
- The oculomotor nerve is critical for eye movement and visual tracking.
- Understanding axon guidance pathways is vital for insights into developmental neurobiology.
- This technique preserves local microenvironments, allowing for real-time assessment.
- Initial axon outgrowth is evaluated instead of regeneration.
Purpose of Study
- To identify axon guidance pathways in the oculomotor nerve.
- To assess their roles at different points along the nerve trajectory.
- To provide insights applicable to other nerve types.
Methods Used
- Ex vivo slice culture method using agarose-embedded embryos for imaging.
- The biological model consists of E10.5 Isl MN:GFP embryos, allowing fluorescent labeling of developing axons.
- No multiomics workflows are mentioned in the text.
- Key steps include the careful extraction of embryos, solidification of agarose, and precise slicing on a vibratome.
- Images are captured every 30 minutes post-culture for up to 72 hours.
Main Results
- Real-time imaging reveals critical insights into axon guidance mechanisms.
- Timelines for axon outgrowth indicate that initial GFP-positive axons reach their target by E11.5.
- Orientation of slices directly influences the interpretability of results.
- The identification of additional guidance mechanisms enhances our understanding of the ocular motor system.
Conclusions
- This method enables detailed exploration of axon guidance during oculomotor nerve development.
- The results underscore the importance of methodological precision for valid interpretations.
- Insights gained may inform broader studies on nerve guidance and regeneration.
Frequently Asked Questions
Executive Industry Relevance
Understanding axon guidance mechanisms is critical for target validation in neurodevelopmental disorders and neural repair strategies. This ex vivo slice culture method enables real-time assessment of axon outgrowth, supporting mechanistic de-risking of guidance pathway targets. By preserving the native microenvironment and allowing dynamic intervention testing, it improves predictive confidence in early target engagement studies.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Interrogates therapeutic hypotheses on axon guidance pathways like CXCR4 signaling in ocular motor development.
- Operational Value: Enables functional target validation by assessing axon trajectory changes at defined developmental stages.
- Predictive Value: Supports portfolio triage by identifying guidance cues that affect specific axon subsets along their trajectory.
Screening & Assay Development
- Scientific Value: Prepares validated biological systems for dose-response screening of guidance molecules.
- Operational Value: Standardizes slice preparation and imaging for reproducible quantitative axon outgrowth measurements.
- Scalability: Supports platform reuse across nerve types with adaptable culture and inhibition protocols.
Translational & Preclinical Research
- Translational Continuity: Connects discovery-phase axon guidance insights to preclinical models of neural development.
- Mechanistic De-risking: Clarifies pathway-specific effects on axon subsets, reducing ambiguity in target mechanism.
- Disease-Relevant System: Uses embryonic GFP-expressing neurons to model early neurodevelopmental processes.
Pipeline & Workflow Integration
The method fits within the discovery continuum from target hypothesis testing to lead identification, particularly for neurodevelopmental targets requiring axon trajectory validation.
- Discovery Biology: Supports hypothesis testing of guidance cues through real-time imaging of axon outgrowth in intact tissue.
- Screening: Enables assay readiness via standardized slice culture and time-lapse imaging for compound or protein screening.
- Analytics: Generates quantitative imaging readouts to compare axon trajectory under different guidance cue conditions.
- Translational Research: Connects to preclinical continuity by modeling developmental axon guidance relevant to neural repair.
- Enterprise Reuse: Establishes a reusable platform for studying axon guidance across multiple neural systems.
Operational & Enterprise Impact
- Scientific Value: Provides predictive confidence in target mechanism by visualizing axon guidance in real time without axotomy.
- Operational Value: Ensures reproducibility through standardized embryo handling, slicing, and culture conditions.
- Strategic Value: Improves go/no-go decisions by identifying stage-specific effects of guidance inhibitors on axon subsets.
- Portfolio Impact: Enables risk-adjusted prioritization of targets based on their influence on axon trajectory in native microenvironments.
Implementation Considerations
- Requires expertise in embryonic dissection, agarose embedding, and vibratome sectioning.
- Dependent on fluorescence microscopy and stage-top incubator infrastructure for time-lapse imaging.
- Necessitates cross-team standardization for embryo handling, slice orientation, and culture timing.
- Involves adaptation considerations when applying the method to different nerve types or developmental stages.
- Includes practical limitations such as slice orientation sensitivity and embryo viability constraints.
