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In Bilateria, many axons cross the midline of the central nervous system, forming well-defined commissures. Whereas in mammals the functions of commissures in the forebrain and in the visual system are well established, functions at other axial levels are less clearly understood. Here, we have dissected the function of several hindbrain commissures using genetic methods. By taking advantage of multiple Cre transgenic lines, we have induced site-specific deletions of the Robo3 receptor. These lines developed with the disruption of specific commissures in the sensory, motor, and sensorimotor systems, resulting in severe and permanent functional deficits. We show that mice with severely reduced commissures in rhombomeres 5 and 3 have abnormal lateral eye movements and auditory brainstem responses, respectively, whereas mice with a primarily uncrossed climbing fiber/Purkinje cell projection are strongly ataxic. Surprisingly, although rerouted axons remain ipsilateral, they still project to their appropriate neuronal targets. Moreover, some Cre;Robo3 lines represent potential models that can be used to study human syndromes, including horizontal gaze palsy with progressive scoliosis (HGPPS). To our knowledge, this study is one of the first to link defects in commissural axon guidance with specific cellular and behavioral phenotypes.

In Bilateria, many axons cross the midline of the central nervous system, forming well-defined commissures. Whereas in mammals the functions of commissures in the forebrain and in the visual system are well established, functions at other axial levels are less clearly understood. Here, we have dissected the function of several hindbrain commissures using genetic methods. By taking advantage of multiple Cre transgenic lines, we have induced site-specific deletions of the Robo3 receptor. These lines developed with the disruption of specific commissures in the sensory, motor, and sensorimotor systems, resulting in severe and permanent functional deficits. We show that mice with severely reduced commissures in rhombomeres 5 and 3 have abnormal lateral eye movements and auditory brainstem responses, respectively, whereas mice with a primarily uncrossed climbing fiber/Purkinje cell projection are strongly ataxic. Surprisingly, although rerouted axons remain ipsilateral, they still project to their appropriate neuronal targets. Moreover, some Cre; Robo3 lines represent potential models that can be used to study human syndromes, including horizontal gaze palsy with progressive scoliosis (HGPPS). To our knowledge, this study is one of the first to link defects in commissural axon guidance with specific cellular and behavioral phenotypes.

Neuropilin-1 and neuropilin-2 bind differentially to different class 3 semaphorins and are thought to provide the ligand-binding moieties in receptor complexes mediating repulsive responses to these semaphorins. Here, we have studied the function of neuropilin-2 through analysis of a neuropilin-2 mutant mouse, which is viable and fertile. Repulsive responses of sympathetic and hippocampal neurons to Sema3F but not to Sema3A are abolished in the mutant. Marked defects are observed in the development of several cranial nerves, in the initial central projections of spinal sensory axons, and in the anterior commissure, habenulo-interpeduncular tract, and the projections of hippocampal mossyfiber axons in the infrapyramidal bundle. Our results show that neuropilin-2 is an essential component of the Sema3F receptor and identify key roles for neuropilin-2 in axon guidance in the PNS and CNS.

Commissural axons cross the nervous system midline and then turn to grow alongside it, neither recrossing nor projecting back into ventral regions. In Drosophila, the midline repellent Slit prevents recrossing: axons cross once because they are initially unresponsive to Slit, becoming responsive only upon crossing. We show that commissural axons in mammals similarly acquire responsiveness to a midline repellent activity upon crossing. Remarkably, they also become responsive to a repellent activity from ventral spinal cord, helping explain why they never reenter that region. Several Slit and Semaphorin proteins, expressed in midline and/or ventral tissues, mimic these repellent activities, and midline guidance defects are observed in mice lacking neuropilin-2, a Semaphorin receptor. Thus, Slit and Semaphorin repellents from midline and nonmidline tissues may help prevent crossing axons from reentering gray matter, squeezing them into surrounding fiber tracts.