Forelimb motor recovery by modulating extrinsic and intrinsic signaling as well as neuronal activity after the cervical spinal cord injury.

Singular strategies for promoting axon regeneration and motor recovery after spinal cord injury (SCI) have been attempted with limited success. Here, we propose the combinatorial approach of deleting extrinsic and intrinsic factors paired with neural stimulation, will enhance adaptive axonal growth and motor recovery after SCI. We previously showed the deletion of RhoA and Pten in corticospinal neurons inhibits axon dieback and promotes axon sprouting after lumbar SCI. Here, we examined the effects of RhoA;Pten deletion coupled with neural stimulation after cervical SCI. This combinatorial approach promoted more boutons on injured corticospinal neurons in the spinal cord compared to sole RhoA;Pten deletion. Although RhoA;Pten deletion does not promote motor recovery in the forelimb after SCI, stimulating corticospinal neurons in those mice results in partial motor recovery. These results demonstrate that a combinatorial approach that pairs genetic modifications with neuronal stimulation can promote axon sprouting and motor recovery following SCI.

Layer Va neurons, as major presynaptic partners of corticospinal neurons, play critical roles in skilled movements.

Corticospinal neurons (CSNs) are located in the cortex and projecting into the spinal cord. The activation of CSNs, which is associated with skilled motor behaviors, induces the activation of interneurons in the spinal cord. Eventually, motor neuron activation is induced by corticospinal circuits to coordinate muscle activation. Therefore, elucidating how the activation of CSNs in the brain is regulated is necessary for understanding the roles of CSNs in skilled motor behaviors. However, the presynaptic partners of CSNs in the brain remain to be identified. Here, we performed transsynaptic rabies virus-mediated brain-wide mapping to identify presynaptic partners of CSNs (pre-CSNs). We found that pre-CSNs are located in all cortical layers, but major pre-CSNs are located in layer Va. A small population of pre-CSNs are also located outside the cortex, such as in the thalamus. Inactivation of layer Va neurons in Tlx3-Cre mice results in deficits in skilled reaching and grasping behaviors, suggesting that, similar to CSNs, layer Va neurons are critical for skilled movements. Finally, we examined whether the connectivity of CSNs is altered after spinal cord injury (SCI). We found that unlike connections between CNSs and postsynaptic neurons, connections between pre-CSNs and CSNs do not change after SCI.

Anatomical and functional analysis of the corticospinal tract in an FRDA mouse model.

Friedreich's ataxia (FRDA) is one of the most common hereditary ataxias. It is caused by a GAA repeat in the first intron of the FXN gene, which encodes an essential mitochondrial protein. Patients suffer from progressive motor dysfunction due to the degeneration of mechanoreceptive and proprioceptive neurons in dorsal root ganglia (DRG) and cerebellar dentate nucleus neurons, especially at early disease stages. Postmortem analyses of FRDA patients also indicate pathological changes in motor cortex including in the projection neurons that give rise to the cortical spinal tract (CST). Yet, it remains poorly understood how early in the disease cortical spinal neurons (CSNs) show these alterations, or whether CSN/CST pathology resembles the abnormalities observed in other tissues affected by FXN loss. To address these questions, we examined CSN driven motor behaviors and pathology in the YG8JR FRDA mouse model. We find that FRDA mice show impaired motor skills, exhibit significant reductions in CSN functional output, and, among other pathological changes, show abnormal mitochondrial distributions in CSN neurons and CST axonal tracts. Moreover, some of these alterations were observed as early as two months of age, suggesting that CSN/CST pathology may be an earlier event in FRDA disease than previously appreciated. These studies warrant a detailed mechanistic understanding of how FXN loss impacts CSN health and functionality.

The Dorsal Column Nuclei Scale Mechanical Sensitivity in Naive and Neuropathic Pain States.

Tactile perception relies on reliable transmission and modulation of low-threshold information as it travels from the periphery to the brain. During pathological conditions, tactile stimuli can aberrantly engage nociceptive pathways leading to the perception of touch as pain, known as mechanical allodynia. Two main drivers of peripheral tactile information, low-threshold mechanoreceptors (LTMRs) and postsynaptic dorsal column neurons (PSDCs), terminate in the brainstem dorsal column nuclei (DCN). Activity within the DRG, spinal cord, and DCN have all been implicated in mediating allodynia, yet the DCN remains understudied at the cellular, circuit, and functional levels compared to the other two. Here, we show that the gracile nucleus (Gr) of the DCN mediates tactile sensitivity for low-threshold stimuli and contributes to mechanical allodynia during neuropathic pain in mice. We found that the Gr contains local inhibitory interneurons in addition to thalamus-projecting neurons, which are differentially innervated by primary afferents and spinal inputs. Functional manipulations of these distinct Gr neuronal populations resulted in bidirectional changes to tactile sensitivity, but did not affect noxious mechanical or thermal sensitivity. During neuropathic pain, silencing Gr projection neurons or activating Gr inhibitory neurons was able to reduce tactile hypersensitivity, and enhancing inhibition was able to ameliorate paw withdrawal signatures of neuropathic pain, like shaking. Collectively, these results suggest that the Gr plays a specific role in mediating hypersensitivity to low-threshold, innocuous mechanical stimuli during neuropathic pain, and that Gr activity contributes to affective, pain-associated phenotypes of mechanical allodynia. Therefore, these brainstem circuits work in tandem with traditional spinal circuits underlying allodynia, resulting in enhanced signaling of tactile stimuli in the brain during neuropathic pain.

Axon Fasciculation, Mediated by Transmembrane Semaphorins, Is Critical for the Establishment of Segmental Specificity of Corticospinal Circuits.

Axon fasciculation is thought to be a critical step in neural circuit formation and function. Recent studies have revealed various molecular mechanisms that underlie axon fasciculation; however, the impacts of axon fasciculation, and its corollary, defasciculation, on neural circuit wiring remain unclear. Corticospinal (CS) neurons in the sensorimotor cortex project axons to the spinal cord to control skilled movements. In rodents, the axons remain tightly fasciculated in the brain and traverse the dorsal funiculus of the spinal cord. Here we show that plexinA1 (PlexA1) and plexinA3 (PlexA3) receptors are expressed by CS neurons, whereas their ligands, semaphorin-5A (Sema5A) and semaphorin-5B (Sema5B) are expressed in the medulla at the decussation site of CS axons to inhibit premature defasciculation of these axons. In the absence of Sema5A/5B-PlexA1/A3 signaling, some CS axons are prematurely defasciculated in the medulla of the brainstem, and those defasciculated CS axons aberrantly transverse in the spinal gray matter instead of the spinal dorsal funiculus. In the absence of Sema5A/Sema5B-PlexA1/A3 signaling, CS axons, which would normally innervate the lumbar spinal cord, are unbundled in the spinal gray matter, and prematurely innervate the cervical gray matter with reduced innervation of the lumbar gray matter. In both Sema5A/5B and PlexA1/A3 mutant mice (both sexes), stimulation of the hindlimb motor cortex aberrantly evokes robust forelimb muscle activation. Finally, Sema5A/5B and PlexA1/A3 mutant mice show deficits in skilled movements. These results suggest that proper fasciculation of CS axons is required for appropriate neural circuit wiring and ultimately affect the ability to perform skilled movements.SIGNIFICANCE STATEMENT Axon fasciculation is believed to be essential for neural circuit formation and function. However, whether and how defects in axon fasciculation affect the formation and function of neural circuits remain unclear. Here we examine whether the transmembrane proteins semaphorin-5A (Sema5A) and semaphorin-5B (Sema5B), and their receptors, plexinA1 (PlexA1) and plexinA3 (PlexA3) play roles in the development of corticospinal circuits. We find that Sema5A/Sema5B and PlexA1/A3 are required for proper axon fasciculation of corticospinal neurons. Furthermore, Sema5A/5B and PlexA1/A3 mutant mice show marked deficits in skilled motor behaviors. Therefore, these results strongly suggest that proper corticospinal axon fasciculation is required for the appropriate formation and functioning of corticospinal circuits in mice.

Motor neurons use push-pull signals to direct vascular remodeling critical for their connectivity.

The nervous system requires metabolites and oxygen supplied by the neurovascular network, but this necessitates close apposition of neurons and endothelial cells. We find motor neurons attract vessels with long-range VEGF signaling, but endothelial cells in the axonal pathway are an obstacle for establishing connections with muscles. It is unclear how this paradoxical interference from heterotypic neurovascular contacts is averted. Through a mouse mutagenesis screen, we show that Plexin-D1 receptor is required in endothelial cells for development of neuromuscular connectivity. Motor neurons release Sema3C to elicit short-range repulsion via Plexin-D1, thus displacing endothelial cells that obstruct axon growth. When this signaling pathway is disrupted, epaxial motor neurons are blocked from reaching their muscle targets and concomitantly vascular patterning in the spinal cord is altered. Thus, an integrative system of opposing push-pull cues ensures detrimental axon-endothelial encounters are avoided while enabling vascularization within the nervous system and along peripheral nerves.

HoxD transcription factors define monosynaptic sensory-motor specificity in the developing spinal cord.

The specificity of monosynaptic connections between proprioceptive sensory neurons and their recipient spinal motor neurons depends on multiple factors, including motor neuron positioning and dendrite morphology, axon projection patterns of proprioceptive sensory neurons in the spinal cord, and the ligand-receptor molecules involved in cell-to-cell recognition. However, with few exceptions, the transcription factors engaged in this process are poorly characterized. Here, we show that members of the HoxD family of transcription factors play a crucial role in the specificity of monosynaptic sensory-motor connections. Mice lacking Hoxd9, Hoxd10 and Hoxd11 exhibit defects in locomotion but have no obvious defects in motor neuron positioning or dendrite morphology through the medio-lateral and rostro-caudal axes. However, we found that quadriceps motor neurons in these mice show aberrant axon development and receive inappropriate inputs from proprioceptive sensory axons innervating the obturator muscle. These genetic studies demonstrate that the HoxD transcription factors play an integral role in the synaptic specificity of monosynaptic sensory-motor connections in the developing spinal cord.

Molecular mechanisms underlying monosynaptic sensory-motor circuit development in the spinal cord.

Motor behaviors are precisely controlled by the integration of sensory and motor systems in the central nervous system (CNS). Proprioceptive sensory neurons, key components of the sensory system, are located in the dorsal root ganglia and project axons both centrally to the spinal cord and peripherally to muscles and tendons, communicating peripheral information about the body to the CNS. Changes in muscle length detected by muscle spindles, and tension variations in tendons conveyed by Golgi tendon organs, are communicated to the CNS through group Ia /II, and Ib proprioceptive sensory afferents, respectively. Group Ib proprioceptive sensory neurons connect with motor neurons indirectly through spinal interneurons, whereas group Ia/II axons form both direct (monosynaptic) and indirect connections with motor neurons. Although monosynaptic sensory-motor circuits between spindle proprioceptive sensory neurons and motor neurons have been extensively studied since 1950s, the molecular mechanisms underlying their formation and upkeep have only recently begun to be understood. We will discuss our current understanding of the molecular foundation of monosynaptic circuit development and maintenance involving proprioceptive sensory neurons and motor neurons in the mammalian spinal cord. Developmental Dynamics 247:581-587, 2018. © 2017 Wiley Periodicals, Inc.

Requirement for Dicer in Maintenance of Monosynaptic Sensory-Motor Circuits in the Spinal Cord.

In contrast to our knowledge of mechanisms governing circuit formation, our understanding of how neural circuits are maintained is limited. Here, we show that Dicer, an RNaseIII protein required for processing microRNAs (miRNAs), is essential for maintenance of the spinal monosynaptic stretch reflex circuit in which group Ia proprioceptive sensory neurons form direct connections with motor neurons. In postnatal mice lacking Dicer in proprioceptor sensory neurons, there are no obvious defects in specificity or formation of monosynaptic sensory-motor connections. However, these circuits degrade through synapse loss and retraction of proprioceptive axonal projections from the ventral spinal cord. Peripheral terminals are also impaired without retracting from muscle targets. Interestingly, despite these central and peripheral axonal defects, proprioceptive neurons survive in the absence of Dicer-processed miRNAs. These findings reveal that Dicer, through its production of mature miRNAs, plays a key role in the maintenance of monosynaptic sensory-motor circuits.

Semaphorin 3A is a retrograde cell death signal in developing sympathetic neurons.

During development of the peripheral nervous system, excess neurons are generated, most of which will be lost by programmed cell death due to a limited supply of neurotrophic factors from their targets. Other environmental factors, such as 'competition factors' produced by neurons themselves, and axon guidance molecules have also been implicated in developmental cell death. Semaphorin 3A (Sema3A), in addition to its function as a chemorepulsive guidance cue, can also induce death of sensory neurons in vitro The extent to which Sema3A regulates developmental cell death in vivo, however, is debated. We show that in compartmentalized cultures of rat sympathetic neurons, a Sema3A-initiated apoptosis signal is retrogradely transported from axon terminals to cell bodies to induce cell death. Sema3A-mediated apoptosis utilizes the extrinsic pathway and requires both neuropilin 1 and plexin A3. Sema3A is not retrogradely transported in older, survival factor-independent sympathetic neurons, and is much less effective at inducing apoptosis in these neurons. Importantly, deletion of either neuropilin 1 or plexin A3 significantly reduces developmental cell death in the superior cervical ganglia. Taken together, a Sema3A-initiated apoptotic signaling complex regulates the apoptosis of sympathetic neurons during the period of naturally occurring cell death.

Synapse Formation in Monosynaptic Sensory-Motor Connections Is Regulated by Presynaptic Rho GTPase Cdc42.

UNLABELLED: Spinal reflex circuit development requires the precise regulation of axon trajectories, synaptic specificity, and synapse formation. Of these three crucial steps, the molecular mechanisms underlying synapse formation between group Ia proprioceptive sensory neurons and motor neurons is the least understood. Here, we show that the Rho GTPase Cdc42 controls synapse formation in monosynaptic sensory-motor connections in presynaptic, but not postsynaptic, neurons. In mice lacking Cdc42 in presynaptic sensory neurons, proprioceptive sensory axons appropriately reach the ventral spinal cord, but significantly fewer synapses are formed with motor neurons compared with wild-type mice. Concordantly, electrophysiological analyses show diminished EPSP amplitudes in monosynaptic sensory-motor circuits in these mutants. Temporally targeted deletion of Cdc42 in sensory neurons after sensory-motor circuit establishment reveals that Cdc42 does not affect synaptic transmission. Furthermore, addition of the synaptic organizers, neuroligins, induces presynaptic differentiation of wild-type, but not Cdc42-deficient, proprioceptive sensory neurons in vitro Together, our findings demonstrate that Cdc42 in presynaptic neurons is required for synapse formation in monosynaptic sensory-motor circuits. SIGNIFICANCE STATEMENT: Group Ia proprioceptive sensory neurons form direct synapses with motor neurons, but the molecular mechanisms underlying synapse formation in these monosynaptic sensory-motor connections are unknown. We show that deleting Cdc42 in sensory neurons does not affect proprioceptive sensory axon targeting because axons reach the ventral spinal cord appropriately, but these neurons form significantly fewer presynaptic terminals on motor neurons. Electrophysiological analysis further shows that EPSPs are decreased in these mice. Finally, we demonstrate that Cdc42 is involved in neuroligin-dependent presynaptic differentiation of proprioceptive sensory neurons in vitro These data suggest that Cdc42 in presynaptic sensory neurons is essential for proper synapse formation in the development of monosynaptic sensory-motor circuits.

Expression of the immunoglobulin superfamily cell adhesion molecules in the developing spinal cord and dorsal root ganglion.

Cell adhesion molecules belonging to the immunoglobulin superfamily (IgSF) control synaptic specificity through hetero- or homophilic interactions in different regions of the nervous system. In the developing spinal cord, monosynaptic connections of exquisite specificity form between proprioceptive sensory neurons and motor neurons, however, it is not known whether IgSF molecules participate in regulating this process. To determine whether IgSF molecules influence the establishment of synaptic specificity in sensory-motor circuits, we examined the expression of 157 IgSF genes in the developing dorsal root ganglion (DRG) and spinal cord by in situ hybridization assays. We find that many IgSF genes are expressed by sensory and motor neurons in the mouse developing DRG and spinal cord. For instance, Alcam, Mcam, and Ocam are expressed by a subset of motor neurons in the ventral spinal cord. Further analyses show that Ocam is expressed by obturator but not quadriceps motor neurons, suggesting that Ocam may regulate sensory-motor specificity in these sensory-motor reflex arcs. Electrophysiological analysis shows no obvious defects in synaptic specificity of monosynaptic sensory-motor connections involving obturator and quadriceps motor neurons in Ocam mutant mice. Since a subset of Ocam+ motor neurons also express Alcam, Alcam or other functionally redundant IgSF molecules may compensate for Ocam in controlling sensory-motor specificity. Taken together, these results reveal that IgSF molecules are broadly expressed by sensory and motor neurons during development, and that Ocam and other IgSF molecules may have redundant functions in controlling the specificity of sensory-motor circuits.

The tumour suppressor LKB1 regulates myelination through mitochondrial metabolism.

A prerequisite to myelination of peripheral axons by Schwann cells (SCs) is SC differentiation, and recent evidence indicates that reprogramming from a glycolytic to oxidative metabolism occurs during cellular differentiation. Whether this reprogramming is essential for SC differentiation, and the genes that regulate this critical metabolic transition are unknown. Here we show that the tumour suppressor Lkb1 is essential for this metabolic transition and myelination of peripheral axons. Hypomyelination in the Lkb1-mutant nerves and muscle atrophy lead to hindlimb dysfunction and peripheral neuropathy. Lkb1-null SCs failed to optimally activate mitochondrial oxidative metabolism during differentiation. This deficit was caused by Lkb1-regulated diminished production of the mitochondrial Krebs cycle substrate citrate, a precursor to cellular lipids. Consequently, myelin lipids were reduced in Lkb1-mutant mice. Restoring citrate partially rescued Lkb1-mutant SC defects. Thus, Lkb1-mediated metabolic shift during SC differentiation increases mitochondrial metabolism and lipogenesis, necessary for normal myelination.

Stem-loop binding protein is required for retinal cell proliferation, neurogenesis, and intraretinal axon pathfinding in zebrafish.

In the developing retina, neurogenesis and cell differentiation are coupled with cell proliferation. However, molecular mechanisms that coordinate cell proliferation and differentiation are not fully understood. In this study, we found that retinal neurogenesis is severely delayed in the zebrafish stem-loop binding protein (slbp) mutant. SLBP binds to a stem-loop structure at the 3'-end of histone mRNAs, and regulates a replication-dependent synthesis and degradation of histone proteins. Retinal cell proliferation becomes slower in the slbp1 mutant, resulting in cessation of retinal stem cell proliferation. Although retinal stem cells cease proliferation by 2 days postfertilization (dpf) in the slbp mutant, retinal progenitor cells in the central retina continue to proliferate and generate neurons until at least 5dpf. We found that this progenitor proliferation depends on Notch signaling, suggesting that Notch signaling maintains retinal progenitor proliferation when faced with reduced SLBP activity. Thus, SLBP is required for retinal stem cell maintenance. SLBP and Notch signaling are required for retinal progenitor cell proliferation and subsequent neurogenesis. We also show that SLBP1 is required for intraretinal axon pathfinding, probably through morphogenesis of the optic stalk, which expresses attractant cues. Taken together, these data indicate important roles of SLBP in retinal development.

Specificity of monosynaptic sensory-motor connections imposed by repellent Sema3E-PlexinD1 signaling.

In mammalian spinal cord, group Ia proprioceptive afferents form selective monosynaptic connections with a select group of motor pool targets. The extent to which sensory recognition of motor neurons contributes to the selectivity of sensory-motor connections remains unclear. We show here that proprioceptive sensory afferents that express PlexinD1 avoid forming monosynaptic connections with neurons in Sema3E(+) motor pools yet are able to form direct connections with neurons in Sema3E(off) motor pools. Anatomical and electrophysiological analysis of mice in which Sema3E-PlexinD1 signaling has been deregulated or inactivated genetically reveals that repellent signaling underlies aspects of the specificity of monosynaptic sensory-motor connectivity in these reflex arcs. A semaphorin-based system of motor neuron recognition and repulsion therefore contributes to the formation of specific sensory-motor connections in mammalian spinal cord.

Deletion of Sema3a or plexinA1/plexinA3 causes defects in sensory afferent projections of statoacoustic ganglion neurons.

Statoacoustic ganglion (SAG) neurons project sensory afferents to appropriate targets in the inner ear to form functional vestibular and auditory circuits. Neuropilin1 (Npn1), a receptor for class 3 semaphorins, is required to generate appropriate afferent projections in SAG neurons; however, the ligands and coreceptors involved in Npn1 functioning remain unknown. Here we show that both plexinA1 and plexinA3 are expressed by SAG neurons, and plexinA1/plexinA3 double mutant mice show defects in afferent projections of SAG neurons in the inner ear. In control mice, sensory afferents of SAG neurons terminate at the vestibular sensory patches, whereas in plexinA1/plexinA3 double mutants, they extend more dorsally in the inner ear beyond normal vestibular target areas. Moreover, we find that semaphorin3a (Sema3a) is expressed in the dorsal otocyst, and Sema3a mutant mice show defects in afferent projections of SAG neurons similar to those observed in plexinA1/plexinA3 double mutants and in mice lacking a functional Npn1 receptor. Taken together, these genetic findings demonstrate that Sema3a repellent signaling plays a role in the establishment of proper afferent projections in SAG neurons, and this signaling likely occurs through a receptor complex involving Npn1 and either plexinA1 or plexinA3.

RhoA and Cdc42 are required in pre-migratory progenitors of the medial ganglionic eminence ventricular zone for proper cortical interneuron migration.

Cortical interneurons arise from the ganglionic eminences in the ventral telencephalon and migrate tangentially to the cortex. Although RhoA and Cdc42, members of the Rho family of small GTPases, have been implicated in regulating neuronal migration, their respective roles in the tangential migration of cortical interneurons remain unknown. Here we show that loss of RhoA and Cdc42 in the ventricular zone (VZ) of the medial ganglionic eminence (MGE) using Olig2-Cre mice causes moderate or severe defects in the migration of cortical interneurons, respectively. Furthermore, RhoA- or Cdc42-deleted MGE cells exhibit impaired migration in vitro. To determine whether RhoA and Cdc42 directly regulate the motility of cortical interneurons during migration, we deleted RhoA and Cdc42 in the subventricular zone (SVZ), where more fate-restricted progenitors are located within the ganglionic eminences, using Dlx5/6-Cre-ires-EGFP (Dlx5/6-CIE) mice. Deletion of either gene within the SVZ does not cause any obvious defects in cortical interneuron migration, indicating that cell motility is not dependent upon RhoA or Cdc42. These findings provide genetic evidence that RhoA and Cdc42 are required in progenitors of the MGE in the VZ, but not the SVZ, for proper cortical interneuron migration.

RhoA is dispensable for axon guidance of sensory neurons in the mouse dorsal root ganglia.

RhoA, a member of the Rho family small GTPases, has been shown to play important roles in axon guidance. However, to date, the physiological function of RhoA in axon guidance events in vivo has not been determined genetically in animals. Here we show that RhoA mRNA is strongly expressed by sensory neurons in the developing mouse dorsal root ganglia (DRG). We have deleted RhoA in sensory neurons of the DRG using RhoA-floxed mice under the Wnt1-Cre driver in which Cre is strongly expressed in sensory neurons. Peripheral projections of sensory neurons appear normal and there are no detectable defects in the central projections of either cutaneous or proprioceptive sensory neurons in RhoA(f/f); Wnt1-Cre mice. Furthermore, a co-culture assay using DRG explants from RhoA(f/f); Wnt1-Cre embryos, and 293T cells expressing semaphorin3A (Sema3A) reveals that RhoA is not required for Sema3A-mediated axonal repulsion of sensory neurons. Expression of RhoC, a closely related family member, is increased in RhoA-deficient sensory neurons and may play a compensatory role in this context. Taken together, these genetic studies demonstrate that RhoA is dispensable for peripheral and central projections of sensory neurons in the DRG.

Ectopic myelinating oligodendrocytes in the dorsal spinal cord as a consequence of altered semaphorin 6D signaling inhibit synapse formation.

Different types of sensory neurons in the dorsal root ganglia project axons to the spinal cord to convey peripheral information to the central nervous system. Whereas most proprioceptive axons enter the spinal cord medially, cutaneous axons typically do so laterally. Because heavily myelinated proprioceptive axons project to the ventral spinal cord, proprioceptive axons and their associated oligodendrocytes avoid the superficial dorsal horn. However, it remains unclear whether their exclusion from the superficial dorsal horn is an important aspect of neural circuitry. Here we show that a mouse null mutation of Sema6d results in ectopic placement of the shafts of proprioceptive axons and their associated oligodendrocytes in the superficial dorsal horn, disrupting its synaptic organization. Anatomical and electrophysiological analyses show that proper axon positioning does not seem to be required for sensory afferent connectivity with motor neurons. Furthermore, ablation of oligodendrocytes from Sema6d mutants reveals that ectopic oligodendrocytes, but not proprioceptive axons, inhibit synapse formation in Sema6d mutants. Our findings provide new insights into the relationship between oligodendrocytes and synapse formation in vivo, which might be an important element in controlling the development of neural wiring in the central nervous system.