Abstract
Muscle function is dependent on innervation by the correct motor nerves. Motor nerves are composed of motor axons that extend through peripheral tissues as a compact bundle, but then diverge to create nerve branches to specific muscle targets. A transition point typically occurs as motor nerves grow near their targets, where the fasciculated nerve halts further growth, then later initiates branching to muscles. The motor nerve transition point is potentially an intermediate target acting as a guidepost to present specific cellular and molecular signals for navigation. Here we describe the navigation of the oculomotor nerve with respect to eye muscle precursor cells in mouse embryos. We found that the oculomotor nerve initially grew to the eye three days prior to the appearance of any eye muscles. The oculomotor axons spread to form a plexus within a mass of eye muscle precursors, then the nerve growth paused for more than two days. This plexus persisted during primary extraocular myogenesis, with a subsequent phase in which the nerve branched out to specific muscles. To test the functional significance of the nerve-precursor contact in the plexus, we genetically ablated muscle precursors early in nerve development, prior to nerve contact. Ablation of muscle precursors resulted in oculomotor nerve fibers failing to stop to form the plexus, but instead growing past the eye. In contrast, ablating the precursor pool at later stages, after the nerve has contacted the precursor cells, results in ectopic branching restricted near the eye. These results demonstrate that muscle precursors act as an intermediate target for nerve guidance, and are required for the oculomotor nerve to transition between nerve growth and distinct stages of terminal axon branching.
Acknowledgements
Several individuals contributed technical assistance in genotyping, and assistance in optimizing immunohistochemistry including Minkyung Kim, Thomas Kidd, and Hillary Price.
Author contributions
BB, GSM, KGW, and TWG formulated ideas for these experiments. Methodology: BB performed nerve tracing, antibody labeling, and analysis; several antibody labels were performed by GER, MV and KGW. LC and PJG contributed Pitx2 mutant embryos and advice on Pitx2 antibody labeling. BB and GSM drafted the manuscript, with editing by KGW and TWG. All authors reviewed and approved the manuscript.
Footnotes
Funding sources Funding was provided by RO1 EY 025205 to GSM, RO1 EY014126 to PJG, and R21 NS 107922 to TWG. Further support for core facilities at the University of Nevada was provided by NIH COBREs RR024210, GM103650, GM103554, and the Nevada INBRE 8 P20 GM103440-11, P20 GM103554. The funding bodies had no roles in the study, collection, analysis, interpretation of data, nor in writing the manuscript.
Competing interests The authors declare no competing or financial interests.
Ethics approval Animal experiments were approved by the UNR IACUC, following NIH guidelines, with the approved protocol #2015-00435.