BackgroundLongitudinal axons grow parallel to the embryonic midline to connect distant regions of the central nervous system. Previous studies suggested that repulsive midline signals guide pioneer longitudinal axons by blocking their entry into the floor plate; however, the role of midline attractants, and whether attractant signals may cooperate with repulsive signals, remains unclear. In this study we investigated the navigation of a set of pioneer longitudinal axons, the medial longitudinal fasciculus, in mouse embryos mutant for the Netrin/Deleted in Colorectal Cancer (DCC) attractants, and for Slit repellents, as well as the responses of explanted longitudinal axons in vitro.ResultsIn mutants for Netrin1 chemoattractant or DCC receptor signaling, longitudinal axons shifted away from the ventral midline, suggesting that Netrin1/DCC signals act attractively to pull axons ventrally. Analysis of mutants in the three Slit genes, including Slit1/2/3 triple mutants, suggest that concurrent repulsive Slit/Robo signals push pioneer axons away from the ventral midline. Combinations of mutations between the Netrin and Slit guidance systems provided genetic evidence that the attractive and repulsive signals balance against each other. This balance is demonstrated in vitro using explant culture, finding that the cues can act directly on longitudinal axons. The explants also reveal an unexpected synergy of Netrin1 and Slit2 that promotes outgrowth.ConclusionsThese results support a mechanism in which longitudinal trajectories are positioned by a push-pull balance between opposing Netrin and Slit signals. Our evidence suggests that longitudinal axons respond directly and simultaneously to both attractants and repellents, and that the combined signals constrain axons to grow longitudinally.
BackgroundOculomotor neurons develop initially like typical motor neurons, projecting axons out of the ventral midbrain to their ipsilateral targets, the extraocular muscles. However, in all vertebrates, after the oculomotor nerve (nIII) has reached the extraocular muscle primordia, the cell bodies that innervate the superior rectus migrate to join the contralateral nucleus. This motor neuron migration represents a unique strategy to form a contralateral motor projection. Whether migration is guided by diffusible cues remains unknown.MethodsWe examined the role of Slit chemorepellent signals in contralateral oculomotor migration by analyzing mutant mouse embryos.ResultsWe found that the ventral midbrain expresses high levels of both Slit1 and 2, and that oculomotor neurons express the repellent Slit receptors Robo1 and Robo2. Therefore, Slit signals are in a position to influence the migration of oculomotor neurons. In Slit 1/2 or Robo1/2 double mutant embryos, motor neuron cell bodies migrated into the ventral midbrain on E10.5, three days prior to normal migration. These early migrating neurons had leading projections into and across the floor plate. In contrast to the double mutants, embryos which were mutant for single Slit or Robo genes did not have premature migration or outgrowth on E10.5, demonstrating a cooperative requirement of Slit1 and 2, as well as Robo1 and 2. To test how Slit/Robo midline repulsion is modulated, we found that the normal migration did not require the receptors Robo3 and CXCR4, or the chemoattractant, Netrin 1. The signal to initiate contralateral migration is likely autonomous to the midbrain because oculomotor neurons migrate in embryos that lack either nerve outgrowth or extraocular muscles, or in cultured midbrains that lacked peripheral tissue.ConclusionOverall, our results demonstrate that a migratory subset of motor neurons respond to floor plate-derived Slit repulsion to properly control the timing of contralateral migration.
Motor neurons differentiate from progenitor cells and cluster as motor nuclei, settling next to the floor plate in the brain stem and spinal cord. Although precise positioning of motor neurons is critical for their functional input and output, the molecular mechanisms that guide motor neurons to their proper positions remain poorly understood. Here, we review recent evidence of motor neuron positioning mechanisms, highlighting situations in which motor neuron cell bodies can migrate, and experiments that show that their migration is regulated by axon guidance cues. The view that emerges is that motor neurons are actively trapped or restricted in static positions, as the cells balance a push in the dorsal direction by repulsive Slit/Robo cues and a pull in the ventral direction by attractive Netrin-1/DCC cues. These new functions of guidance cues are necessary fine-tuning to set up patterns of motor neurons at their proper positions in the neural tube during embryogenesis.
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 nerveprecursor 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.
Large controlled studies of Chronic Fatigue Syndrome/Myalgic Encephalomyelitis (CFS/ME) have shown no effective medical treatment for this disorder. There are individual patients, however, with dramatic responses to some medications. We report two patients with clear responses to rintatolimid and galantamine characterized by rapid reduction of symptoms on starting treatment and return of symptoms on withdrawal. As in cancer, CFS/ME is a heterogeneous disorder but unlike most cancers, such as melanoma, breast cancer, and B-cell lymphoma, CFS/ME has no known biological marker that can distinguish between subtypes. We suggest an approach to medical treatment of CFS/ME that could be utilized by primary caregivers that offer the possibility of more rapid and complete recovery from this debilitating disorder. Current studies indicate that prolonged symptomatic recovery from infection with Covid-19 (“long hauler syndrome” or PASC, for post-acute sequelae of Covid-19) represents a severe form of CFS/ME and thus may also be amenable to personalized medicine with specific medications.
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