Oculomotor axons use external and autocrine Slit signals for fasciculation, navigation, branching, and connectivity in mouse embryos

Eye alignment and movements are controlled by three cranial nerves: the oculomotor (nIII), trochlear (nIV), and abducens nerves (nVI). Developmental errors of these nerves can lead to eye movement disorders. However, the molecular and cellular mechanisms that guide motor axons to the eye remain poorly understood. Oculomotor nerve guidance was examined in mutant mouse embryos for the secreted Slit proteins and Robo receptors. Methods included mapping Slit and Robo expression using Slit transgenic markers and Robo antibodies, imaging of oculomotor nerves and muscles using antibody labeling, and culture of oculomotor axons with Slit proteins. Robo1 and 2 were expressed in motor axons, while Slits were expressed surrounding the motor pathway, in mesenchymal tissues around the eye muscles, and by motor axons. Robo1/2 or Slit1/2 mutant embryos had similar guidance defects at several steps. The initial nerves projecting out to the eye were defasciculated. Upon reaching the eye, oculomotor axons normally stop to form a plexus in contact with muscle precursor cells, but in Robo and Slit mutants, the motor axons spread abnormally to overshoot. Later, abnormal nerve branches resulted in reduced muscle innervation. Importantly, motor neuron-specific deletion of Slit2 caused a subset of these errors. Several steps along the navigational pathway of oculomotor nerves are controlled by Slit/Robo signals, including fasciculation, correct navigation to an intermediate target, and innervation of extraocular muscle. Furthermore, important aspects of motor axon guidance are controlled by autocrine Slit2 signals produced by motor axons themselves.