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Mary Whitman, Elaine Nguyen, Elizabeth Engle; Loss of CXCR4 signaling in motor neurons leads to misrouting of the oculomotor nerve and oculomotor synkinesis. Invest. Ophthalmol. Vis. Sci. 2018;59(9):1580.
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© ARVO (1962-2015); The Authors (2016-present)
Proper control of eye movements is critical to vision. Relatively little is known about the molecular mechanisms that regulate development and axon guidance in the oculomotor system. We have developed an ex vivo slice assay to allow live imaging of the growing oculomotor nerve and identification of axon guidance cues that affect oculomotor nerve. CXCR4 signaling has been implicated in oculomotor nerve growth, so we inhibited CXCR4 as a proof of concept for the slice assay.
Embryos from IslMN-GFP mice, which express a farnesylated GFP that localizes to the membrane of motor neurons and axons, are taken at E10.5, embedded in agarose, and sliced on a vibratome. The slices are grown on cell culture inserts on a microscope stage incubator. Fluorescent images are taken every 30 minutes for 2 days. To assess for CXCR4 function, AMD3100 is added to the culture media at a final concentration of 1ug/ml. Cxcr4cko/cko mice were purchased from Jackson Laboratory and crossed to Isl-cre and ISLMN:GFP reporter mice. Whole embryos were immunostained for GFP and a muscle marker (anti-smooth muscle actin) and cleared using either a BABB or modified iDisco+ protocol, depending on age, and imaged on a confocal.
When AMD3100 (specific inhibitor of CXCR4) is added to the slice cultures, oculomotor axons grow dorsally (away from the eye), rather than ventrally (towards the eye). Axons that have already left the neuroepithelium continue towards the eye. There is complete misrouting of the oculomotor nerve in in Cxcr4cko/cko::ISLMN:GFP:Isl-Cre embryos, recapitulating the slice culture results. In the absence of innervation from oculomotor nerve, motor axons from the trigeminal nerve, which normally innervate the muscles of mastication, aberrantly innervate extraocular muscles in the orbit. This represents the first mouse model of trigeminal-oculomotor synkinesis.
CXCR4 signaling is critical for initial pathfinding decisions of oculomotor axons and their proper exit from the neuroepithelium. Failure of the oculomotor nerve to innervate its extraocular muscle targets leads to aberrant innervation by other motor neurons, indicating that muscles that lack innervation may secrete cues that attract motor axons. Oculomotor synkinesis is seen in human disorders in which there is loss of innervation of extraocular muscles, including Marcus Gunn Jaw Winking, CFEOM, and Duane syndrome.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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