Ex Vivo Oculomotor Slice Culture from Embryonic GFP-Expressing Mice for Time-Lapse Imaging of Oculomotor Nerve Outgrowth

Mary  C. Whitman; Jessica  L. Bell; Elaine  H. Nguyen; Elizabeth  C. Engle

doi:10.3791/59911

Ex Vivo Oculomotor Slice Culture from Embryonic GFP-Expressing Mice for Time-Lapse Imaging of Ocu...

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Neuroscience

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JoVE Journal Neuroscience

Ex Vivo Oculomotor Slice Culture from Embryonic GFP-Expressing Mice for Time-Lapse Imaging of Oculomotor Nerve Outgrowth

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06:04 min

July 16, 2019

DOI: 10.3791/59911-v

Mary C. Whitman^1,2,3, Jessica L. Bell^1,3, Elaine H. Nguyen^1,3, Elizabeth C. Engle^1,2,3,4,5,6

¹Department of Ophthalmology,Boston Children's Hospital, ²Department of Ophthalmology,Harvard Medical School, ³F.M. Kirby Neurobiology Center,Boston Children's Hospital, ⁴Department of Neurology,Boston Children's Hospital, ⁵Department of Neurology,Harvard Medical School, ⁶Howard Hughes Medical Institute

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

What are the advantages of using an ex vivo slice assay?

This approach preserves the local environment of developing axons, allowing for real-time imaging and analysis of axon outgrowth without cutting the axons.

How is the biological model implemented in this study?

E10.5 Isl MN:GFP embryos are used, which are embedded in agarose and sectioned to create slices for culture and imaging.

What types of data are obtained from this method?

The method provides imaging data to visualize axon outgrowth and allows for the assessment of molecular interactions through the addition of inhibitors.

How can this method be adapted for other studies?

The ex vivo slice culture technique can be modified to study axon guidance mechanisms in various types of nerves beyond the oculomotor nerve.

What are the key limitations of this technique?

Practicing the method is crucial for mastery, and proper orientation of the slices is necessary for interpretable results. Additionally, care must be taken when handling embryos and reagents.

An ex vivo slice assay allows oculomotor nerve outgrowth to be imaged in real time. Slices are generated by embedding E10.5 Isl^MN: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.

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