Dysregulation of Neuropilin-2 Expression in Inhibitory Neurons Impairs Hippocampal Circuit Development Leading to Autism-Epilepsy Phenotype.

Dysregulation of development, migration, and function of interneurons, collectively termed interneuronopathies, have been proposed as a shared mechanism for autism spectrum disorders (ASDs) and childhood epilepsy. Neuropilin-2 (Nrp2), a candidate ASD gene, is a critical regulator of interneuron migration from the median ganglionic eminence (MGE) to the pallium, including the hippocampus. While clinical studies have identified Nrp2 polymorphisms in patients with ASD, whether dysregulation of Nrp2-dependent interneuron migration contributes to pathogenesis of ASD and epilepsy has not been tested. We tested the hypothesis that the lack of Nrp2 in MGE-derived interneuron precursors disrupts the excitation/inhibition balance in hippocampal circuits, thus predisposing the network to seizures and behavioral patterns associated with ASD. Embryonic deletion of Nrp2 during the developmental period for migration of MGE derived interneuron precursors (iCKO) significantly reduced parvalbumin, neuropeptide Y, and somatostatin positive neurons in the hippocampal CA1. Consequently, when compared to controls, the frequency of inhibitory synaptic currents in CA1 pyramidal cells was reduced while frequency of excitatory synaptic currents was increased in iCKO mice. Although passive and active membrane properties of CA1 pyramidal cells were unchanged, iCKO mice showed enhanced susceptibility to chemically evoked seizures. Moreover, iCKO mice exhibited selective behavioral deficits in both preference for social novelty and goal-directed learning, which are consistent with ASD-like phenotype. Together, our findings show that disruption of developmental Nrp2 regulation of interneuron circuit establishment, produces ASD-like behaviors and enhanced risk for epilepsy. These results support the developmental interneuronopathy hypothesis of ASD epilepsy comorbidity.

Balancing act of small GTPases downstream of plexin-A4 signaling motifs promotes dendrite elaboration in mammalian cortical neurons.

The precise development of neuronal morphologies is crucial to the establishment of synaptic circuits and, ultimately, proper brain function. Signaling by the axon guidance cue semaphorin 3A (Sema3A) and its receptor complex of neuropilin-1 and plexin-A4 has multifunctional outcomes in neuronal morphogenesis. Downstream activation of the RhoGEF FARP2 through interaction with the lysine-arginine-lysine motif of plexin-A4 and consequent activation of the small GTPase Rac1 promotes dendrite arborization, but this pathway is dispensable for axon repulsion. Here, we investigated the interplay of small GTPase signaling mechanisms underlying Sema3A-mediated dendritic elaboration in mouse layer V cortical neurons in vitro and in vivo. Sema3A promoted the binding of the small GTPase Rnd1 to the amino acid motif lysine-valine-serine (LVS) in the cytoplasmic domain of plexin-A4. Rnd1 inhibited the activity of the small GTPase RhoA and the kinase ROCK, thus supporting the activity of the GTPase Rac1, which permitted the growth and branching of dendrites. Overexpression of a dominant-negative RhoA, a constitutively active Rac1, or the pharmacological inhibition of ROCK activity rescued defects in dendritic elaboration in neurons expressing a plexin-A4 mutant lacking the LVS motif. Our findings provide insights into the previously unappreciated balancing act between Rho and Rac signaling downstream of specific motifs in plexin-A4 to mediate Sema3A-dependent dendritic elaboration in mammalian cortical neuron development.

Semaphorin signaling restricts neuronal regeneration in C. elegans.

Extracellular signaling proteins serve as neuronal growth cone guidance molecules during development and are well positioned to be involved in neuronal regeneration and recovery from injury. Semaphorins and their receptors, the plexins, are a family of conserved proteins involved in development that, in the nervous system, are axonal guidance cues mediating axon pathfinding and synapse formation. The Caenorhabditis elegans genome encodes for three semaphorins and two plexin receptors: the transmembrane semaphorins, SMP-1 and SMP-2, signal through their receptor, PLX-1, while the secreted semaphorin, MAB-20, signals through PLX-2. Here, we evaluate the locomotion behavior of knockout animals missing each of the semaphorins and plexins and the neuronal morphology of plexin knockout animals; we described the cellular expression pattern of the promoters of all plexins in the nervous system of C. elegans; and we evaluated their effect on the regrowth and reconnection of motoneuron neurites and the recovery of locomotion behavior following precise laser microsurgery. Regrowth and reconnection were more prevalent in the absence of each plexin, while recovery of locomotion surpassed regeneration in all genotypes.

Neuropilin 2/Plexin-A3 Receptors Regulate the Functional Connectivity and the Excitability in the Layers 4 and 5 of the Cerebral Cortex.

The functions of cortical networks are progressively established during development by series of events shaping the neuronal connectivity. Synaptic elimination, which consists of removing the supernumerary connections generated during the earlier stages of cortical development, is one of the latest stages in neuronal network maturation. The semaphorin 3F coreceptors neuropilin 2 (Nrp2) and plexin-A3 (PlxnA3) may play an important role in the functional maturation of the cerebral cortex by regulating the excess dendritic spines on cortical excitatory neurons. Yet, the identity of the connections eliminated under the control of Nrp2/PlxnA3 signaling is debated, and the importance of this synaptic refinement for cortical functions remains poorly understood. Here, we show that Nrp2/PlxnA3 controls the spine densities in layer 4 (L4) and on the apical dendrite of L5 neurons of the sensory and motor cortices. Using a combination of neuroanatomical, ex vivo electrophysiology, and in vivo functional imaging techniques in Nrp2 and PlxnA3 KO mice of both sexes, we disprove the hypothesis that Nrp2/PlxnA3 signaling is required to maintain the ectopic thalamocortical connections observed during embryonic development. We also show that the absence of Nrp2/PlxnA3 signaling leads to the hyperexcitability and excessive synchronization of the neuronal activity in L5 and L4 neuronal networks, suggesting that this system could participate in the refinement of the recurrent corticocortical connectivity in those layers. Altogether, our results argue for a role of semaphorin-Nrp2/PlxnA3 signaling in the proper maturation and functional connectivity of the cerebral cortex, likely by controlling the refinement of recurrent corticocortical connections.SIGNIFICANCE STATEMENT The function of a neuronal circuit is mainly determined by the connections that neurons establish with one another during development. Understanding the mechanisms underlying the establishment of the functional connectivity is fundamental to comprehend how network functions are implemented, and to design treatments aiming at restoring damaged neuronal circuits. Here, we show that the cell surface receptors for the family of semaphorin guidance cues neuropilin 2 (Nrp2) and plexin-A3 (PlxnA3) play an important role in shaping the functional connectivity of the cerebral cortex likely by trimming the recurrent connections in layers 4 and 5. By removing the supernumerary inputs generated during early development, Nrp2/PlxnA3 signaling reduces the neuronal excitability and participates in the maturation of the cortical network functions.

Neuronal Microsurgery with an Yb-Doped Fiber Femtosecond Laser.

Laser microsurgery allows the user to ablate cell bodies or disconnect nerve fibers by using a laser microbeam focused through a microscope. This technique was pioneered in C. elegans where it led to exciting discoveries in the fields of development and neurobiology. All neurons studied so far in C. elegans can regenerate and regrow axons and dendrites after injury, allowing studies of the molecular and cellular basis of neuroregeneration. In this chapter, we describe how to assemble and operate a platform for Yb-doped fiber laser microsurgery. The novel laser setup described here is a more robust, lower cost, and user-friendly alternative to other femtosecond-pulsed laser systems.

Semaphorin3A/PlexinA3 association with the Scribble scaffold for cGMP increase is required for apical dendrite development.

The development of the apical dendrite from the leading process of the bipolar pyramidal neuron might be directed by spatially organized extrinsic cues acting on localized intrinsic determinants. The extracellular cues regulating apical dendrite polarization remain elusive. We show that leading process and apical dendrite development are directed by class III Semaphorins and mediated by a localized cGMP-synthesizing complex. The scaffolding protein Scribble that associates with the cGMP-synthesizing enzyme soluble guanylate cyclase (sGC) also associates with the Semaphorin3A (Sema3A) co-receptor PlexinA3. Deletion or knockdown of PlexinA3 and Sema3A or disruption of PlexinA3-Scribble association prevents Sema3A-mediated cGMP increase and causes defects in apical dendrite development. These manipulations also impair bipolar polarity and leading process establishment. Local cGMP elevation or sGC expression rescues the effects of PlexinA3 knockdown or PlexinA3-Scribble complex disruption. During neuronal polarization, leading process and apical dendrite development are directed by a scaffold that links Semaphorin cue to cGMP increase.

Reduced hippocampal inhibition and enhanced autism-epilepsy comorbidity in mice lacking neuropilin 2.

The neuropilin receptors and their secreted semaphorin ligands play key roles in brain circuit development by regulating numerous crucial neuronal processes, including the maturation of synapses and migration of GABAergic interneurons. Consistent with its developmental roles, the neuropilin 2 (Nrp2) locus contains polymorphisms in patients with autism spectrum disorder (ASD). Nrp2-deficient mice show autism-like behavioral deficits and propensity to develop seizures. In order to determine the pathophysiology in Nrp2 deficiency, we examined the hippocampal numbers of interneuron subtypes and inhibitory regulation of hippocampal CA1 pyramidal neurons in mice lacking one or both copies of Nrp2. Immunostaining for interneuron subtypes revealed that Nrp2-/- mice have a reduced number of parvalbumin, somatostatin, and neuropeptide Y cells, mainly in CA1. Whole-cell recordings identified reduced firing and hyperpolarized shift in resting membrane potential in CA1 pyramidal neurons from Nrp2+/- and Nrp2-/- mice compared to age-matched wild-type controls indicating decrease in intrinsic excitability. Simultaneously, the frequency and amplitude of spontaneous inhibitory postsynaptic currents (sIPSCs) are reduced in Nrp2-deficient mice. A convulsive dose of kainic acid evoked electrographic and behavioral seizures with significantly shorter latency, longer duration, and higher severity in Nrp2-/- compared to Nrp2+/+ animals. Finally, Nrp2+/- and Nrp2-/- but not Nrp2+/+, mice have impaired cognitive flexibility demonstrated by reward-based reversal learning, a task associated with hippocampal circuit function. Together these data demonstrate a broad reduction in interneuron subtypes and compromised inhibition in CA1 of Nrp2-/- mice, which could contribute to the heightened seizure susceptibility and behavioral deficits consistent with an ASD/epilepsy phenotype.

Modular and Distinct Plexin-A4/FARP2/Rac1 Signaling Controls Dendrite Morphogenesis.

Diverse neuronal populations with distinct cellular morphologies coordinate the complex function of the nervous system. Establishment of distinct neuronal morphologies critically depends on signaling pathways that control axonal and dendritic development. The Sema3A-Nrp1/PlxnA4 signaling pathway promotes cortical neuron basal dendrite arborization but also repels axons. However, the downstream signaling components underlying these disparate functions of Sema3A signaling are unclear. Using the novel PlxnA4KRK-AAA knock-in male and female mice, generated by CRISPR/cas9, we show here that the KRK motif in the PlxnA4 cytoplasmic domain is required for Sema3A-mediated cortical neuron dendritic elaboration but is dispensable for inhibitory axon guidance. The RhoGEF FARP2, which binds to the KRK motif, shows identical functional specificity as the KRK motif in the PlxnA4 receptor. We find that Sema3A activates the small GTPase Rac1, and that Rac1 activity is required for dendrite elaboration but not axon growth cone collapse. This work identifies a novel Sema3A-Nrp1/PlxnA4/FARP2/Rac1 signaling pathway that specifically controls dendritic morphogenesis but is dispensable for repulsive guidance events. Overall, our results demonstrate that the divergent signaling output from multifunctional receptor complexes critically depends on distinct signaling motifs, highlighting the modular nature of guidance cue receptors and its potential to regulate diverse cellular responses.SIGNIFICANCE STATEMENT The proper formation of axonal and dendritic morphologies is crucial for the precise wiring of the nervous system that ultimately leads to the generation of complex functions in an organism. The Semaphorin3A-Neuropilin1/Plexin-A4 signaling pathway has been shown to have multiple key roles in neurodevelopment, from axon repulsion to dendrite elaboration. This study demonstrates that three specific amino acids, the KRK motif within the Plexin-A4 receptor cytoplasmic domain, are required to coordinate the downstream signaling molecules to promote Sema3A-mediated cortical neuron dendritic elaboration, but not inhibitory axon guidance. Our results unravel a novel Semaphorin3A-Plexin-A4 downstream signaling pathway and shed light on how the disparate functions of axon guidance and dendritic morphogenesis are accomplished by the same extracellular ligand in vivo.

Neuropilin 2 Signaling Mediates Corticostriatal Transmission, Spine Maintenance, and Goal-Directed Learning in Mice.

The striatum represents the main input structure of the basal ganglia, receiving massive excitatory input from the cortex and the thalamus. The development and maintenance of cortical input to the striatum is crucial for all striatal function including many forms of sensorimotor integration, learning, and action control. The molecular mechanisms regulating the development and maintenance of corticostriatal synaptic transmission are unclear. Here we show that the guidance cue, Semaphorin 3F and its receptor Neuropilin 2 (Nrp2), influence dendritic spine maintenance, corticostriatal short-term plasticity, and learning in adult male and female mice. We found that Nrp2 is enriched in adult layer V pyramidal neurons, corticostriatal terminals, and in developing and adult striatal spiny projection neurons (SPNs). Loss of Nrp2 increases SPN excitability and spine number, reduces short-term facilitation at corticostriatal synapses, and impairs goal-directed learning in an instrumental task. Acute deletion of Nrp2 selectively in adult layer V cortical neurons produces a similar increase in the number of dendritic spines and presynaptic modifications at the corticostriatal synapse in the Nrp2-/- mouse, but does not affect the intrinsic excitability of SPNs. Furthermore, conditional loss of Nrp2 impairs sensorimotor learning on the accelerating rotarod without affecting goal-directed instrumental learning. Collectively, our results identify Nrp2 signaling as essential for the development and maintenance of the corticostriatal pathway and may shed novel insights on neurodevelopmental disorders linked to the corticostriatal pathway and Semaphorin signaling.SIGNIFICANCE STATEMENT The corticostriatal pathway controls sensorimotor, learning, and action control behaviors and its dysregulation is linked to neurodevelopmental disorders, such as autism spectrum disorder (ASD). Here we demonstrate that Neuropilin 2 (Nrp2), a receptor for the axon guidance cue semaphorin 3F, has important and previously unappreciated functions in the development and adult maintenance of dendritic spines on striatal spiny projection neurons (SPNs), corticostriatal short-term plasticity, intrinsic physiological properties of SPNs, and learning in mice. Our findings, coupled with the association of Nrp2 with ASD in human populations, suggest that Nrp2 may play an important role in ASD pathophysiology. Overall, our work demonstrates Nrp2 to be a key regulator of corticostriatal development, maintenance, and function, and may lead to better understanding of neurodevelopmental disease mechanisms.

Sema3A Reduces Sprouting of Adult Rod Photoreceptors In Vitro.

Purpose: Rod photoreceptor terminals respond to retinal injury with retraction and sprouting. Since the guidance cue Semaphorin3A (Sema3A) is observed in the retina after injury, we asked whether Sema3A contributes to structural plasticity in rod photoreceptors. Methods: We used Western blots and alkaline phosphatase (AP)-tagged neuropilin-1 (NPN-1) to detect the expression of Sema3A in an organotypic model of porcine retinal detachment. We then examined Sema3A binding to cultured salamander rod photoreceptors using AP-tagged Sema3A. For functional analysis, we used a microspritzer to apply a gradient of Sema3A-Fc to isolated salamander rod photoreceptors over 24 hours. Results: Sema3A protein was biochemically detected in porcine retinal explants in the retina 7, 24, and 72 hours after detachment. In sections, NPN-1 receptor was bound to the inner and outer retina. For isolated rod photoreceptors, Sema3A localized to synaptic terminals and to neuritic processes after 1 week in vitro. In microspritzed rod photoreceptors, process initiation occurred away from high concentrations of Sema3A. Sema3A significantly decreased the number of processes formed by rod photoreceptors although the average length of processes was not affected. The cellular orientation of rod photoreceptors relative to the microspritzer also significantly changed over time; this effect was reduced with the Sema3A inhibitor, xanthofulvin. Conclusion: Sema3A is expressed in the retina after detachment, binds to rod photoreceptors, affects cell orientation, and reduces photoreceptor process initiation in vitro. Our results suggest that Sema3A contributes to axonal retraction in retinal injury, whereas rod neuritic sprouting and regenerative synaptogenesis may require a reduction in semaphorin signaling.

Regulation of Cortical Dendrite Morphology and Spine Organization by Secreted Semaphorins: A Primary Culture Approach.

Primary tissue culture is an invaluable technique in cell biology and has a long history in demonstrating its versatility in characterizing cellular morphology, function, and behavior. Here, we describe a modified, low density, long-term, primary neuron culture system to characterize dendritic morphology and synaptic spine organization in developing mouse cortical neurons. While this method can be applied to investigate the signaling pathways of a range of extracellular cues' effect on neuronal development, we focus on how distinct secreted semaphorins regulate dendritic elaboration and spine morphogenesis in deep layer cortical neurons.

Floor plate-derived neuropilin-2 functions as a secreted semaphorin sink to facilitate commissural axon midline crossing.

Commissural axon guidance depends on a myriad of cues expressed by intermediate targets. Secreted semaphorins signal through neuropilin-2/plexin-A1 receptor complexes on post-crossing commissural axons to mediate floor plate repulsion in the mouse spinal cord. Here, we show that neuropilin-2/plexin-A1 are also coexpressed on commissural axons prior to midline crossing and can mediate precrossing semaphorin-induced repulsion in vitro. How premature semaphorin-induced repulsion of precrossing axons is suppressed in vivo is not known. We discovered that a novel source of floor plate-derived, but not axon-derived, neuropilin-2 is required for precrossing axon pathfinding. Floor plate-specific deletion of neuropilin-2 significantly reduces the presence of precrossing axons in the ventral spinal cord, which can be rescued by inhibiting plexin-A1 signaling in vivo. Our results show that floor plate-derived neuropilin-2 is developmentally regulated, functioning as a molecular sink to sequester semaphorins, preventing premature repulsion of precrossing axons prior to subsequent down-regulation, and allowing for semaphorin-mediated repulsion of post-crossing axons.

Vertebrate spinal commissural neurons: a model system for studying axon guidance beyond the midline.

For bilaterally symmetric organisms, the transfer of information between the left and right side of the nervous system is mediated by commissures formed by neurons that project their axons across the body midline to the contralateral side of the central nervous system (CNS). After crossing the midline, many of these axons must travel long distances to reach their targets, including those that extend from spinal commissural neurons. Owing to the highly stereotyped trajectories of spinal commissural neurons that can be divided into several segments as these axons project to their targets, it is an ideal system for investigators to ask fundamental questions related to mechanisms of short- and long-range axon guidance, fasciculation, and choice point decisions at the midline intermediate target. In addition, studies of patterning genes of the nervous system have revealed complex transcription factor codes that function in a combinatorial fashion to specify individual classes of spinal neurons including commissural neurons. Despite these advances and the functional importance of spinal commissural neurons in mediating the transfer of external sensory information from the peripheral nervous system (PNS) to the CNS, only a handful of studies have begun to elucidate the mechanistic logic underlying their long-range pathfinding and the characterization of their synaptic targets. Using in vitro assays, in vivo labeling methodologies, in combination with both loss- and gain-of-function experiments, several studies have revealed that the molecular mechanisms of long-range spinal commissural axon pathfinding involve an interplay between classical axon guidance cues, morphogens and cell adhesion molecules. For further resources related to this article, please visit the WIREs website.

Global ablation of the mouse Rab11a gene impairs early embryogenesis and matrix metalloproteinase secretion.

Rab11a has been conceived as a prominent regulatory component of the recycling endosome, which acts as a nexus in the endo- and exocytotic networks. The precise in vivo role of Rab11a in mouse embryonic development is unknown. We globally ablated Rab11a and examined the phenotypic and molecular outcomes in Rab11a(null) blastocysts and mouse embryonic fibroblasts. Using multiple trafficking assays and complementation analyses, we determined, among multiple important membrane-associated and soluble cargos, the critical contribution of Rab11a vesicular traffic to the secretion of multiple soluble MMPs. Rab11a(null) embryos were able to properly form normal blastocysts but died at peri-implantation stages. Our data suggest that Rab11a critically controls mouse blastocyst development and soluble matrix metalloproteinase secretion.

Neural cell adhesion molecule NrCAM regulates Semaphorin 3F-induced dendritic spine remodeling.

Neuron-glial related cell adhesion molecule (NrCAM) is a regulator of axon growth and repellent guidance, and has been implicated in autism spectrum disorders. Here a novel postsynaptic role for NrCAM in Semaphorin3F (Sema3F)-induced dendritic spine remodeling was identified in pyramidal neurons of the primary visual cortex (V1). NrCAM localized to dendritic spines of star pyramidal cells in postnatal V1, where it was coexpressed with Sema3F. NrCAM deletion in mice resulted in elevated spine densities on apical dendrites of star pyramidal cells at both postnatal and adult stages, and electron microscopy revealed increased numbers of asymmetric synapses in layer 4 of V1. Whole-cell recordings in cortical slices from NrCAM-null mice revealed increased frequency of mEPSCs in star pyramidal neurons. Recombinant Sema3F-Fc protein induced spine retraction on apical dendrites of wild-type, but not NrCAM-null cortical neurons in culture, while re-expression of NrCAM rescued the spine retraction response. NrCAM formed a complex in brain with Sema3F receptor subunits Neuropilin-2 (Npn-2) and PlexinA3 (PlexA3) through an Npn-2-binding sequence (TARNER) in the extracellular Ig1 domain. A trans heterozygous genetic interaction test demonstrated that Sema3F and NrCAM pathways interacted in vivo to regulate spine density in star pyramidal neurons. These findings reveal NrCAM as a novel postnatal regulator of dendritic spine density in cortical pyramidal neurons, and an integral component of the Sema3F receptor complex. The results implicate NrCAM as a contributor to excitatory/inhibitory balance in neocortical circuits.

Distinct cytoplasmic domains in Plexin-A4 mediate diverse responses to semaphorin 3A in developing mammalian neurons.

Guidance receptor signaling is crucial for neural circuit formation and elicits diverse cellular events in specific neurons. We found that signaling from the guidance cue semaphorin 3A diverged through distinct cytoplasmic domains in its receptor Plexin-A4 to promote disparate cellular behavior in different neuronal cell types. Plexin-A4 has three main cytoplasmic domains--C1, Hinge/RBD, and C2--and interacts with family members of the Rho guanine nucleotide exchange factor FARP proteins. We show that growth cone collapse occurred in Plexin-A4-deficient dorsal root ganglion sensory neurons reconstituted with Plexin-A4 containing either the Hinge/RBD or C2 domain, whereas both of the Hinge/RBD and C1 domains were required for dendritic arborization in cortical neurons. Although knockdown studies indicated that both the collapse and arborization responses involved FARP2, mutations in the cytoplasmic region of Plexin-A4 that reduced its interaction with FARP2 strongly inhibited semaphorin 3A-induced dendritic branching but not growth cone collapse, suggesting that different degrees of interaction are required for the two responses or that developing axons have an indirect path to FARP2 activation. Thus, our study provided insights into the multifunctionality of guidance receptors, in particular showing that the semaphorin 3A signal diverges through specific functions of the modular domains of Plexin-A4.

Neuropilin2 regulates the guidance of post-crossing spinal commissural axons in a subtype-specific manner.

BACKGROUND: Spinal commissural axons represent a model system for deciphering the molecular logic that regulates the guidance of midline-crossing axons in the developing central nervous system (CNS). Whether the same or specific sets of guidance signals control the navigation of molecularly distinct subtypes of these axons remains an open and largely unexplored question. Although it is well established that post-crossing commissural axons alter their responsiveness to midline-associated guidance cues, our understanding of the repulsive mechanisms that drive the post-crossing segments of these axons away from the midline and whether the underlying guidance systems operate in a commissural axon subtype-specific manner, remains fragmentary at best. RESULTS: Here, we utilize axonally targeted transgenic reporter mice to visualize genetically distinct dorsal interneuron (dI)1 and dI4 commissural axons and show that the repulsive class 3 semaphorin (Sema3) guidance receptor Neuropilin 2 (Npn2), is selectively expressed on the dI1 population and is required for the guidance of post-crossing dI1, but not dI4, axons. Consistent with these observations, the midline-associated Npn2 ligands, Sema3F and Sema3B, promote the collapse of dI1, but not dI4, axon-associated growth cones in vitro. We also identify, for the first time, a discrete GABAergic population of ventral commissural neurons/axons in the embryonic mouse spinal cord that expresses Npn2, and show that Npn2 is required for the proper guidance of their post-crossing axons. CONCLUSIONS: Together, our findings indicate that Npn2 is selectively expressed in distinct populations of commissural neurons in both the dorsal and ventral spinal cord, and suggest that Sema3-Npn2 signaling regulates the guidance of post-crossing commissural axons in a population-specific manner.

Semaphorin 3A contributes to distal pulmonary epithelial cell differentiation and lung morphogenesis.

RATIONALE: Semaphorin 3A (Sema3A) is a neural guidance cue that also mediates cell migration, proliferation and apoptosis, and inhibits branching morphogenesis. Because we have shown that genetic deletion of neuropilin-1, which encodes an obligatory Sema3A co-receptor, influences airspace remodeling in the smoke-exposed adult lung, we sought to determine whether genetic deletion of Sema3A altered distal lung structure. METHODS: To determine whether loss of Sema3A signaling influenced distal lung morphology, we compared pulmonary histology, distal epithelial cell morphology and maturation, and the balance between lung cell proliferation and death, in lungs from mice with a targeted genetic deletion of Sema3A (Sema3A(-/-)) and wild-type (Sema3A(+/+)) littermate controls. RESULTS: Genetic deletion of Sema3A resulted in significant perinatal lethality. At E17.5, lungs from Sema3A(-/-) mice had thickened septae and reduced airspace size. Distal lung epithelial cells had increased intracellular glycogen pools and small multivesicular and lamellar bodies with atypical ultrastructure, as well as reduced expression of type I alveolar epithelial cell markers. Alveolarization was markedly attenuated in lungs from the rare Sema3A(-/-) mice that survived the immediate perinatal period. Furthermore, Sema3A deletion was linked with enhanced postnatal alveolar septal cell death. CONCLUSIONS: These data suggest that Sema3A modulates distal pulmonary epithelial cell development and alveolar septation. Defining how Sema3A influences structural plasticity of the developing lung is a critical first step for determining if this pathway can be exploited to develop innovative strategies for repair after acute or chronic lung injury.

NrCAM deletion causes topographic mistargeting of thalamocortical axons to the visual cortex and disrupts visual acuity.

NrCAM is a neural cell adhesion molecule of the L1 family that has been linked to autism spectrum disorders, a disease spectrum in which abnormal thalamocortical connectivity may contribute to visual processing defects. Here we show that NrCAM interaction with neuropilin-2 (Npn-2) is critical for semaphorin 3F (Sema3F)-induced guidance of thalamocortical axon subpopulations at the ventral telencephalon (VTe), an intermediate target for thalamic axon sorting. Genetic deletion of NrCAM or Npn-2 caused contingents of embryonic thalamic axons to misproject caudally in the VTe. The resultant thalamocortical map of NrCAM-null mutants showed striking mistargeting of motor and somatosensory thalamic axon contingents to the primary visual cortex, but retinogeniculate targeting and segregation were normal. NrCAM formed a molecular complex with Npn-2 in brain and neural cells, and was required for Sema3F-induced growth cone collapse in thalamic neuron cultures, consistent with a vital function for NrCAM in Sema3F-induced axon repulsion. NrCAM-null mice displayed reduced responses to visual evoked potentials recorded from layer IV in the binocular zone of primary visual cortex (V1), particularly when evoked from the ipsilateral eye, indicating abnormal visual acuity and ocularity. These results demonstrate that NrCAM is required for normal maturation of cortical visual acuity, and suggest that the aberrant projection of thalamic motor and somatosensory axons to the visual cortex in NrCAM-null mutant mice impairs cortical functions.

Secreted semaphorins control spine distribution and morphogenesis in the postnatal CNS.

The majority of excitatory synapses in the mammalian CNS (central nervous system) are formed on dendritic spines, and spine morphology and distribution are critical for synaptic transmission, synaptic integration and plasticity. Here, we show that a secreted semaphorin, Sema3F, is a negative regulator of spine development and synaptic structure. Mice with null mutations in genes encoding Sema3F, and its holoreceptor components neuropilin-2 (Npn-2, also known as Nrp2) and plexin A3 (PlexA3, also known as Plxna3), exhibit increased dentate gyrus (DG) granule cell (GC) and cortical layer V pyramidal neuron spine number and size, and also aberrant spine distribution. Moreover, Sema3F promotes loss of spines and excitatory synapses in dissociated neurons in vitro, and in Npn-2(-/-) brain slices cortical layer V and DG GCs exhibit increased mEPSC (miniature excitatory postsynaptic current) frequency. In contrast, a distinct Sema3A-Npn-1/PlexA4 signalling cascade controls basal dendritic arborization in layer V cortical neurons, but does not influence spine morphogenesis or distribution. These disparate effects of secreted semaphorins are reflected in the restricted dendritic localization of Npn-2 to apical dendrites and of Npn-1 (also known as Nrp1) to all dendrites of cortical pyramidal neurons. Therefore, Sema3F signalling controls spine distribution along select dendritic processes, and distinct secreted semaphorin signalling events orchestrate CNS connectivity through the differential control of spine morphogenesis, synapse formation, and the elaboration of dendritic morphology.