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

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Method Article

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

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DOI:

10.3791/59911

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July 16th, 2019

Mary C. Whitman¹^,²^,³ , Jessica L. Bell¹^,³ , Elaine H. Nguyen¹^,³ , Elizabeth C. Engle¹^,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

Summary

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.

Abstract

Accurate eye movements are crucial for vision, but the development of the ocular motor system, especially the molecular pathways controlling axon guidance, has not been fully elucidated. This is partly due to technical limitations of traditional axon guidance assays. To identify additional axon guidance cues influencing the oculomotor nerve, an ex vivo slice assay to image the oculomotor nerve in real-time as it grows towards the eye was developed. E10.5 Isl^MN-GFP embryos are used to generate ex vivo slices by embedding them in agarose, slicing on a vibratome, then growing them in a microscope stage-top incubator with time-lapse photomicroscopy for 24-72 h. Control slices recapitulate the in vivo timing of outgrowth of axons from the nucleus to the orbit. Small molecule inhibitors or recombinant proteins can be added to the culture media to assess the role of different axon guidance pathways. This method has the advantages of maintaining more of the local microenvironment through which axons traverse, not axotomizing the growing axons, and assessing the axons at multiple points along their trajectory. It can also identify effects on specific subsets of axons. For example, inhibition of CXCR4 causes axons still within the midbrain to grow dorsally rather than ventrally, but axons that have already exited ventrally are not affected.

Introduction

The ocular motor system provides an elegant system for investigating axon guidance mechanisms. It is relatively uncomplicated, consisting of three cranial nerves innervating six extraocular muscles (EOMs) which move the eye, and the levator palpebrae superioris (LPS) which lifts the eyelid. The oculomotor nerve innervates the LPS and four EOMs - the inferior oblique and the medial, inferior, and superior rectus muscles. The other two nerves, the trochlear and abducens, each only innervate one muscle, the superior oblique and lateral rectus muscle, respectively. Eye movements provide an easy readout, showing if innervation was appropriate, missing, or aberrant. Additio....