Loss of CXCR4/CXCL12 Signaling Causes Oculomotor Nerve Misrouting and Development of Motor Trigeminal to Oculomotor Synkinesis

Abstract

PurposeProper control of eye movements is critical to vision, but relatively little is known about the molecular mechanisms that regulate development and axon guidance in the ocular motor system or cause the abnormal innervation patterns (oculomotor synkinesis) seen in developmental disorders and after oculomotor nerve palsy. We developed an ex vivo slice assay that allows for live imaging and molecular manipulation of the growing oculomotor nerve, which we used to identify axon guidance cues that affect the oculomotor nerve.MethodsEx vivo slices were generated from E10.5 IslMN-GFP embryos and grown for 24 to 72 hours. To assess for CXCR4 function, the specific inhibitor AMD3100 was added to the culture media. Cxcr4cko/cko:Isl-Cre:ISLMN-GFP and Cxcl12KO/KO:ISLMN-GFP embryos were cleared and imaged on a confocal microscope.ResultsWhen AMD3100 was added to the slice cultures, oculomotor axons grew dorsally (away from the eye) rather than ventrally (toward the eye). Axons that had already exited the midbrain continued toward the eye. Loss of Cxcr4 or Cxcl12 in vivo caused misrouting of the oculomotor nerve dorsally and motor axons from the trigeminal motor nerve, which normally innervate the muscles of mastication, aberrantly innervated extraocular muscles in the orbit. This represents the first mouse model of trigeminal-oculomotor synkinesis.ConclusionsCXCR4/CXCL12 signaling is critical for the initial pathfinding decisions of oculomotor axons and their proper exit from the midbrain. Failure of the oculomotor nerve to innervate its extraocular muscle targets leads to aberrant innervation by other motor neurons, indicating that muscles lacking innervation may secrete cues that attract motor axons.

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

“…This ex vivo slice culture protocol provides significant advantages over traditional axon guidance assays 23 . The size of each cranial motor nucleus is not a limiting factor, and no difficult dissection is necessary.…”

“…For example, with CXCR4 inhibition, there is mild decreased overall growth of the slice (see Figure 2), consistent with the multiple functions of CXCR4. In mouse models, however, the axon phenotype seen in the slice was recapitulated 23 .…”

“…To overcome the obstacles of in vitro assays and allow rapid screening of signaling pathways, an ex vivo embryonic slice culture technique was developed 23 , adapted from a previously published protocol for peripheral nerve outgrowth 24 . Using this protocol, the developing oculomotor nerve can be imaged over time in the presence of many of the surrounding structures along its trajectory, including EOM targets.…”

MET Receptor Tyrosine Kinase Regulates Vagal Laryngeal Motor Neuron Development and Lifespan Ultrasonic Vocal Communication

“…Synkinesis also can arise developmentally as demonstrated in mouse models of strabismus where chemokine receptor deletion has been shown to result in improper ocular muscle innervation by trigeminal motor neurons (Whitman et al, 2018). To our knowledge, no studies have specifically demonstrated laryngeal synkinesis of a developmental origin.…”

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“…Subsequent non-selective reinnervation of laryngeal muscle groups can lead to synkinesis (Crumley, 1979(Crumley, , 2000Flint et al, 1991). Synkinesis also can arise developmentally as demonstrated in mouse models of strabismus where chemokine receptor deletion has been shown to result in improper ocular muscle innervation by trigeminal motor neurons (Whitman et al, 2018). To our knowledge, no studies have specifically demonstrated laryngeal synkinesis of a developmental origin.…”