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At least two-thirds of spinal cord injury cases are anatomically incomplete, without complete spinal cord transection, although the initial injuries cause complete loss of sensory and motor functions. The malleability of neural circuits and networks allows varied extend of functional restoration in some individuals after successful rehabilitative training. However, in most cases, the efficiency and extent are both limited and uncertain, largely due to the many obstacles of repair. The restoration of function after anatomically incomplete injury is in part made possible by the growth of new axons or new axon branches through the spared spinal cord tissue and the new synaptic connections they make, either along the areas they grow through or in the areas they terminate. This review will discuss new progress on the understanding of the role of axon guidance molecules, particularly the Wnt family proteins, in spinal cord injury and how the knowledge and tools of axon guidance can be applied to increase the potential of recovery. These strategies, combined with others, such as neuroprotection and rehabilitation, may bring new promises. The recovery strategies for anatomically incomplete spinal cord injuries are relevant and may be applicable to traumatic brain injury and stroke.

Directional and positional information is essential for the diverse neuronal morphology and connectivity during development. The direction of axon growth is critical for building the correct networks among neurons, sometimes from far away. Neuronal synapses are asymmetric cell-cell junctions with distinct presynaptic and postsynaptic structures to convey neural activity in a directional fashion. Recent studies show that some of the key asymmetry is mediated by highly conversed cell polarity signaling pathways. These pathways, planar cell polarity and apical-basal polarity, are not required for the global axon-dendrite polarity. Therefore, the apparent distinct types of morphological asymmetry in the nervous system, growth cone turning and synaptic junctions, are mediated by similar cell polarity signaling mechanisms widely used in cellular and tissue morphogenesis.

Significance In addition to responding to directional cues, axons join one another as they extend to form highly organized projections. We show here that growth cones communicate with each other, and this communication is mediated by planar cell polarity signaling components, which are known to mediate cell–cell interactions in tissue polarization. This interaction is, in part, mediated by an intercellular interaction between Frizzled3 and Vangl2. Leucine-rich repeat kinase 2 (LRRK2), encoded by a Parkinson’s disease gene, regulates Frizzled3 phosphorylation and the intercellular interaction between Frizzled3 and Vangl2. Both loss-of-function and gain-of-function LRRK2 mutants show axon guidance defects, including those of the dopaminergic neurons, suggesting that this Parkinson’s disease gene plays important roles in growth cone–growth cone interactions during axon development., Axon–axon interactions are essential for axon guidance during nervous system wiring. However, it is unknown whether and how the growth cones communicate with each other while sensing and responding to guidance cues. We found that the Parkinson’s disease gene, leucine-rich repeat kinase 2 (LRRK2), has an unexpected role in growth cone–growth cone communication. The LRRK2 protein acts as a scaffold and induces Frizzled3 hyperphosphorylation indirectly by recruiting other kinases and also directly phosphorylates Frizzled3 on threonine 598 (T598). In LRRK1 or LRRK2 single knockout, LRRK1/2 double knockout, and LRRK2 G2019S knockin, the postcrossing spinal cord commissural axons are disorganized and showed anterior–posterior guidance errors after midline crossing. Growth cones from either LRRK2 knockout or G2019S knockin mice showed altered interactions, suggesting impaired communication. Intercellular interaction between Frizzled3 and Vangl2 is essential for planar cell polarity signaling. We show here that this interaction is regulated by phosphorylation of Frizzled3 at T598 and can be regulated by LRRK2 in a kinase activity-dependent way. In the LRRK1/2 double knockout or LRRK2 G2019S knockin, the dopaminergic axon bundle in the midbrain was significantly widened and appeared disorganized, showing aberrant posterior-directed growth. Our findings demonstrate that LRRK2 regulates growth cone–growth cone communication in axon guidance and that both loss-of-function mutation and a gain-of-function mutation (G2019S) cause axon guidance defects in development.

Description, The glutamatergic synapses are assembled and maintained by conserved planar cell polarity proteins., Whether there exists a common signaling mechanism that assembles all glutamatergic synapses is unknown. We show here that knocking out Prickle1 and Prickle2 reduced the formation of the PSD-95–positive glutamatergic synapses in the hippocampus and medial prefrontal cortex in postnatal development by 70–80%. Prickle1 and Prickle2 double knockout in adulthood lead to the disassembly of 70 to 80% of the postsynaptic-density(PSD)-95–positive glutamatergic synapses. PSD-95–positive glutamatergic synapses in the hippocampus of Prickle2E8Q/E8Q mice were reduced by 50% at postnatal day 14. Prickle2 promotes synapse formation by antagonizing Vangl2 and stabilizing the intercellular complex of the planar cell polarity (PCP) components, whereas Prickle2 E8Q fails to do so. Coculture experiments show that the asymmetric PCP complexes can determine the presynaptic and postsynaptic polarity. In summary, the PCP components regulate the assembly and maintenance of a large number of glutamatergic synapses and specify the direction of synaptic transmission.

Summary Normal synapse formation is fundamental to brain function. We show here that an apical-basal polarity (A-BP) protein, Lgl1, is present in the postsynaptic density and negatively regulates glutamatergic synapse numbers by antagonizing the atypical protein kinase Cs (aPKCs). A planar cell polarity protein, Vangl2, which inhibits synapse formation, was decreased in synaptosome fractions of cultured cortical neurons from Lgl1 knockout embryos. Conditional knockout of Lgl1 in pyramidal neurons led to reduction of AMPA/NMDA ratio and impaired plasticity. Lgl1 is frequently deleted in Smith-Magenis syndrome (SMS). Lgl1 conditional knockout led to increased locomotion, impaired novel object recognition and social interaction. Lgl1+/− animals also showed increased synapse numbers, defects in open field and social interaction, as well as stereotyped repetitive behavior. Social interaction in Lgl1+/− could be rescued by NMDA antagonists. Our findings reveal a role of apical-basal polarity proteins in glutamatergic synapse development and function and also suggest a potential treatment for SMS patients with Lgl1 deletion., Graphical Abstract, Highlights • Lgl1 regulates glutamatergic synapse numbers by inhibiting aPKC • Lgl1 cKO leads to reduced AMPA/NMDA ratio in development and adulthood • Lgl1 conditional knockout impairs novel object cognition and social interaction • Social interaction deficits can be rescued by NMDA receptor blockade, Biological Sciences; Cell Biology; Cellular Neuroscience; Neuroscience

Amyloid beta oligomers cause glutamatergic synapse degeneration by targeting the planar cell polarity components in the synapses., The signaling pathway directly controlling the maintenance of adult glutamatergic synapses has not been well understood. Planar cell polarity (PCP) signaling components were recently shown to play essential roles in the formation of glutamatergic synapses. Here, we show that they are localized in the adult synapses and are essential for their maintenance. Synapse loss at early stages of Alzheimer’s disease is thought to be induced by β-amyloid (Aβ) pathology. We found that oligomeric Aβ binds to Celsr3 and assists Vangl2 in disassembling synapses. Moreover, a Wnt receptor and regulator of PCP signaling, Ryk, is also required for Aβ-induced synapse loss. In the 5XFAD mouse model of Alzheimer’s disease, Ryk conditional knockout or a function-blocking monoclonal Ryk antibody protected synapses and preserved cognitive function. We propose that tipping of the fine balance of Wnt/PCP signaling components in glutamatergic synapses may cause synapse degeneration in neurodegenerative disorders with Aβ pathology.

Epilepsy and autism spectrum disorders (ASD) frequently show comorbidity, suggesting shared or overlapping neurobiological basis underlying these conditions. R104Q is the first mutation in the PRICKLE 1(PK1) gene that was discovered in human patients with progressive myoclonus epilepsy (PME). Subsequently, a number of mutations in the PK1 gene were shown to be associated with either epilepsy, autism, or both, as well as other developmental disorders. Using CRISPR-Cas9-mediated gene editing, we generated a PK1R104Q mouse line. The mutant mice showed reduced density of excitatory synapses in hippocampus and impaired interaction between PK1 and the repressor element 1(RE-1) silencing transcription factor (REST). They also displayed reduced seizure threshold, impaired social interaction, and cognitive functions. Taken together, the PK1R104Q mice display characteristic behavioral features similar to the key symptoms of epilepsy and ASD, providing a useful model for studying the molecular and neural circuit mechanisms underlying the comorbidity of epilepsy and ASD.

Limited functional recovery can be achieved through rehabilitation after incomplete spinal cord injury. Eliminating the function of a repulsive Wnt receptor, Ryk, in mice and rats by either conditional knockout in the motor cortex or monoclonal antibody infusion resulted in increased corticospinal axon collateral branches with presynaptic puncta in the spinal cord and enhanced recovery of forelimb reaching and grasping function following a cervical dorsal column lesion. Using optical stimulation, we observed that motor cortical output maps underwent massive changes after injury and that hindlimb cortical areas were recruited to control the forelimb over time. Furthermore, a greater cortical area was dedicated to controlling the forelimb in Ryk conditional knockout mice than in controls (wild-type or heterozygotes). In the absence of weekly task-specific training, recruitment of ectopic cortical areas was greatly reduced and there was no significant functional recovery even in Ryk conditional knockout mice. Our study provides evidence that maximal circuit reorganization and functional recovery can be achieved by combining molecular manipulation and targeted rehabilitation.

GABAergic interneurons have many important functions in cortical circuitry, a reflection of their diversity as a cell population. However, the developmental origins of this diversity are poorly understood. Here, we identify a rostral-caudal gradient of Wnt-responsiveness that delineates the specification of the two main interneuron subclasses. Caudallysituated medial ganglionic eminence (MGE) progenitors receive high levels of Wnt signaling and give rise to somatostatin (SST)-expressing cortical interneurons. Parvalbumin (PV)-expressing basket cells, by contrast, originate mostly from the rostral MGE where Wnt signaling is attenuated. Interestingly, rather than canonical signaling through beta-catenin, Wnt signaling transmitted via the non-canonical receptor, Ryk regulates interneuron cell-fate specification in vivo and in vitro. Indeed, gain-of-function of Ryk intracellular domain signaling regulates SST and PV fate in a dose-dependent manner, suggesting Ryk signaling acts in a graded fashion. These data reveal a complex and important role for non-canonical Wnt-Ryk signaling in establishing the correct ratios of mature cortical interneuron subtypes.

Commissural axons switch on responsiveness to Wnt attraction during midline crossing and turn anteriorly only after exiting the floor plate. We report here that Sonic Hedgehog (Shh)-Smoothened signaling downregulates Shisa2, which inhibits the glycosylation and cell surface presentation of Frizzled3 in rodent commissural axon growth cones. Constitutive Shisa2 expression causes randomized turning of post-crossing commissural axons along the anterior–posterior (A–P) axis. Loss of Shisa2 led to precocious anterior turning of commissural axons before or during midline crossing. Post-crossing commissural axon turning is completely randomized along the A–P axis when Wntless, which is essential for Wnt secretion, is conditionally knocked out in the floor plate. This regulatory link between Shh and planar cell polarity (PCP) signaling may also occur in other developmental processes.

Functional synapse formation is critical for the wiring of neural circuits in the developing brain. The cell adhesion molecule N-cadherin plays important roles in target recognition and synaptogenesis. However, the molecular mechanisms that regulate the localization of N-cadherin and the subsequent effects remain poorly understood. Here, we show that protein kinase D1 (PKD1) directly binds to N-cadherin at amino acid residues 836–871 and phosphorylates it at Ser 869, 871, and 872, thereby increasing the surface localization of N-cadherin and promoting functional synapse formation in primary cultured hippocampal neurons obtained from embryonic day 18 rat embryos of either sex. Intriguingly, neuronal activity enhances the interactions between N-cadherin and PKD1, which are critical for the activity-dependent growth of dendritic spines. Accordingly, either disruption the binding between N-cadherin and PKD1 or preventing the phosphorylation of N-cadherin by PKD1 in the hippocampal CA1 region of male rat leads to the reduction in synapse number and impairment of LTP. Together, this study demonstrates a novel mechanism of PKD1 regulating the surface localization of N-cadherin and suggests that the PKD1-N-cadherin interaction is critical for synapse formation and function.SIGNIFICANCE STATEMENTDefects in synapse formation and function lead to various neurological diseases, although the mechanisms underlying the regulation of synapse development are far from clear. Our results suggest that protein kinase D1 (PKD1) functions upstream of N-cadherin, a classical synaptic adhesion molecule, to promote functional synapse formation. Notably, we identified a crucial binding fragment to PKD1 at C terminus of N-cadherin, and this fragment also contains PKD1 phosphorylation sites. Through this interaction, PKD1 enhances the stability of N-cadherin on cell membrane and promotes synapse morphogenesis and synaptic plasticity in an activity-dependent manner. Our study reveals the role of PKD1 and the potential downstream mechanism in synapse development, and contributes to the research for neurodevelopment and the therapy for neurological diseases.

Capsaicin, the ingredient responsible for the pungent taste of hot chili peppers, is widely used in the study and management of pain. Recently, its neuroprotective effect has been described in multiple studies. Herein, we investigated the underlying mechanisms for the neuroprotective effect of capsaicin. Direct injection of capsaicin (1 or 3 nmol) into the peri-infarct area reduced the infarct volume and improved neurological behavioral scoring and motor coordination function in the middle cerebral artery occlusion (MCAO)/reperfusion model in rats. The time window of the protective effect of capsaicin was within 1 h after reperfusion, when excitotoxicity is the main reason of cell death. In cultured cortical neurons, administration of capsaicin attenuated glutamate-induced excitotoxic injury. With respect to the mechanisms of the neuroprotective effect of capsaicin, reduced calcium influx after glutamate stimulation was observed following capsaicin pretreatment in cortical neurons. Trpv1 knockout abolished the inhibitory effect of capsaicin on glutamate-induced calcium influx and subsequent neuronal death. Reduced expression of GluN1 and GluN2B, subunits of NMDA receptor, was examined after capsaicin treatment in cortical neurons. In summary, our studies reveal that the neuroprotective effect of capsaicin in cortical neurons is TRPV1-dependent and down-regulation of the expression and function of NMDA receptors contributes to the protection afforded by capsaicin.

Many studies in the past decade have revealed the role and mechanisms of Wnt signaling in axon guidance during development and the reinduction of Wnt signaling in adult central nervous system axons upon traumatic injury, which has profound influences on axon regeneration. With 19 Wnts and 14 known receptors (10 Frizzleds (Fzds), Ryk, Ror1/2 and PTK7), the Wnt family signaling proteins contribute significantly to the wiring specificity of the complex brain and spinal cord circuitry. Subsequent investigation into the signaling mechanisms showed that conserved cell polarity pathways mediate growth cone steering. These cell polarity pathways may unveil general principles of growth cone guidance. The reappeared Wnt signaling system after spinal cord injury limits the regrowth of both descending and ascending motor and sensory axons. Therefore, the knowledge of Wnt signaling mechanisms learned from axon development can be applied to axon repair in adulthood.

The signaling mechanisms that choreograph the assembly of the highly asymmetric pre- and postsynaptic structures are still poorly defined. Using synaptosome fractionation, immunostaining, and coimmunoprecipitation, we found that Celsr3 and Vangl2, core components of the planar cell polarity (PCP) pathway, are localized at developing glutamatergic synapses and interact with key synaptic proteins. Pyramidal neurons from the hippocampus of Celsr3 knockout mice exhibit loss of ∼50% of glutamatergic synapses, but not inhibitory synapses, in culture. Wnts are known regulators of synapse formation, and our data reveal that Wnt5a inhibits glutamatergic synapses formed via Celsr3. To avoid affecting earlier developmental processes, such as axon guidance, we conditionally knocked out Celsr3 in the hippocampus 1 week after birth. The CA1 neurons that lost Celsr3 also showed a loss of ∼50% of glutamatergic synapses in vivo without affecting the inhibitory synapses assessed by miniature excitatory postsynaptic current (mEPSC) and electron microscopy. These animals displayed deficits in hippocampus-dependent behaviors in adulthood, including spatial learning and memory and fear conditioning. In contrast to Celsr3 conditional knockouts, we found that the conditional knockout of Vangl2 in the hippocampus 1 week after birth led to a large increase in synaptic density, as evaluated by mEPSC frequency and spine density. PCP signaling is mediated by multiple core components with antagonizing functions. Our results document the opposing roles of Celsr3 and Vangl2 in glutamatergic synapse formation.

The Sixth Biennial Meeting on Axon Guidance, Synaptogenesis & Neural Plasticity took place between 10 and 14 September 2008, at Cold Spring Harbor, New York, USA, and was organized by A. Ghosh, C. Holt & A. Kolodkin. ![][1] See Glossary for abbreviations used in this article In September 2008, more than 400 neuroscientists gathered in Cold Spring Harbor (NY, USA) to discuss how axons in the nervous system navigate to their synaptic targets to create highly organized connections with intriguing properties of plasticity and adaptability. Traditionally, this meeting not only features research on axon guidance, but also includes a consideration of additional processes in neural development that are relevant to circuit formation, including cell positioning and migration, synapse formation and plasticity, dendrite development and axon regeneration. This year was no exception. Coincidentally, many of the molecules that were initially identified as axon‐guidance molecules are now also known to have a role in many of these other events. A rich diversity of work was presented from the identification of guidance cues, receptors and signal‐transduction pathways to the function of these cues in pathfinding and target selection. In addition, the cell‐biological mechanisms by which these cues act were discussed, as well as the transcriptional and translational mechanisms that control their action. Functional imaging of developing and adult circuits was also presented. Of course, the meeting did not solve all problems in axon guidance; however, it clearly reflected new directions in axon‐guidance studies. The field is becoming a more interdisciplinary and multilevel forum that addresses a central problem of developmental neuroscience: the formation of functional neural circuits. This meeting report highlights some of the exciting advances that were presented at the meeting. Many classes of axon‐guidance molecules have been discovered during the past two decades (Dickson, 2002), and only a few new ones were … [1]: /embed/graphic-1.gif

The Wnt family of secreted proteins plays a crucial role in nervous system wiring. Wnts regulate neuronal positioning, polarization, axon and dendrite development, and synaptogenesis. These diverse roles of Wnt proteins are due not only to the large numbers of Wnt ligands and receptors but also to their ability to signal through distinct signaling pathways in different cell types and developmental contexts. Studies on Wnts have shed new light on novel molecular mechanisms that control the development of complex neuronal connections. This review discusses recent advances on how Wnt signaling influences different aspects of neuronal circuit assembly through changes in gene expression and/or cytoskeletal modulation.

Wnt proteins are conserved axon guidance cues that control growth cone navigation. However, the intracellular signaling mechanisms that mediate growth cone turning in response to Wnts are unknown. We previously showed that Wnt-Frizzled signaling directs spinal cord commissural axons to turn anteriorly after midline crossing through an attractive mechanism. Here we show that atypical protein kinase C (aPKC), is required for Wnt-mediated attraction of commissural axons and proper anterior-posterior (A-P) pathfinding. A PKCzeta pseudosubstrate, a specific blocker of aPKC activity, and expression of a kinase-defective PKCzeta mutant in commissural neurons resulted in A-P randomization in "open-book" explants. Upstream of PKCzeta, heterotrimeric G-proteins and phosphatidylinositol-3-kinases (PI3Ks), are also required for A-P guidance, because pertussis toxin, wortmannin, and expression of a p110gamma kinase-defective construct all resulted in A-P randomization. Overexpression of p110gamma, the catalytic subunit of PI3Kgamma, caused precocious anterior turning of commissural axons before midline crossing in open-book explants and caused dissociated precrossing commissural axons, which are normally insensitive to Wnt attraction, to turn toward Wnt4-expressing cells. Therefore, we propose that atypical PKC signaling is required for Wnt-mediated A-P axon guidance and that PI3K can act as a switch to activate Wnt responsiveness during midline crossing.

Studies show that limited functional recovery can be achieved by plasticity and adaptation of the remaining circuitry in partial injuries in the central nervous system, although the new circuits that arise in these contexts have not been clearly identified or characterized. We show here that synaptic contacts from dorsal root ganglions to a small number of dorsal column neurons, a caudal extension of nucleus gracilis, whose connections to the thalamus are spared in a precise cervical level 1 lesion, underwent remodeling over time. These connections support proprioceptive functional recovery in a conditioning lesion paradigm, as silencing or eliminating the remodelled circuit completely abolishes the recovered proprioceptive function of the hindlimb. Furthermore, we show that blocking repulsive Wnt signalling increases axon plasticity and synaptic connections that drive greater functional recovery.

Since our original papers reporting a role for Wnt signaling in regulating regeneration after spinal cord injury, there have been several publications addressing the role of Wnt signaling in spinal cord injury. Although there may be some slight differences in some details of Wnt expression, such as the baseline levels of expression of some of the Wnt’s before injury, which may be due to different sensitivity of methods used, there is overall an agreement that Wnt expression levels change drastically after injury, particularly being upregulated. We also showed that the reinduced Wnt system has strong effects on axon regeneration. Although evidence is accumulating, genetic studies have not yet been reported. A genetic model to test Wnt signaling in axon regeneration will not only help establish the role of Wnt’s in spinal cord injury, it will also provide an important tool to probe what types of therapeutic benefit can be achieved by manipulating Wnt signaling.

Molecular and cellular mechanisms underlying the peripheral conditioning lesion remain unsolved. We show here that injection of a chemical demyelinating agent, ethidium bromide, into the sciatic nerve induces a similar set of regeneration-associated genes and promotes a 2.7-fold greater extent of sensory axon regeneration in the spinal cord than sciatic nerve crush. We found that more severe peripheral demyelination correlates with more severe functional and electrophysiological deficits, but more robust central regeneration. Ethidium bromide injection does not activate macrophages at the demyelinated sciatic nerve site, as observed after nerve crush, but briefly activates macrophages in the dorsal root ganglion. This study provides a new method for investigating the underlying mechanisms of the conditioning response and suggests that loss of the peripheral myelin may be a major signal to change the intrinsic growth state of adult sensory neurons and promote regeneration.

Conditioning lesion of the peripheral branch of dorsal column axons is a well-known paradigm enabling the central branch to regenerate after injury to the spinal cord. However, only a small number of regenerating axons enter grafted substrates, and they do not grow beyond the lesion. We found that conditioning lesion induces, in addition to growth-stimulating genes, related to receptor tyrosine kinase (Ryk), a potent repulsive receptor for Wnts. Wnts are expressed around the site of spinal cord injury, and we found that grafted bone marrow stromal cells secreting the Wnt inhibitors secreted frizzled-related protein 2 or Wnt inhibitory factor 1 enhanced regeneration of the central branch after peripheral conditioning lesion. Furthermore, we found that Wnt4-expressing grafts caused dramatic long-range retraction of the injured central branch of conditioned dorsal root ganglion neurons. Macrophages accumulate along the path of receding axons but not around Wnt4-expressing cells, suggesting that the retraction of dorsal column axons is not a secondary effect of increased macrophages attracted by Wnt4. Therefore, Wnt-Ryk signaling is an inhibitory force co-induced with growth-stimulating factors after conditioning lesion. Overcoming Wnt inhibition may further enhance therapies being designed on the basis of the conditioning-lesion paradigm.

Wnt signaling is essential for axon wiring throughout the development of the nervous system in vertebrates and invertebrates. In rodents, Wnts are expressed in gradients that span the entire anterior–posterior (A–P) axis in the spinal cord and the medial–lateral axis in the superior colliculus. In the brainstem, Wnts are expressed in more complex gradients along the A–P axis. These gradients provide directional information for axon pathfinding and positional information for topographic mapping and are detected by cell polarity signaling pathways in the growth cone. The gradient expression of Wnts and the coordinated expression of Wnt signaling systems are regulated by mechanisms which are currently unknown. Injury to the adult spinal cord results in the re-induction of Wnts in multiple cell types around the lesion site and their signaling system in injured axons. The re-induced Wnts form gradients around the lesion site, with the lesion site being the peak. The re-inducedWnts may be responsible for the well-known retraction of descending motor axons through the receptor Ryk (related receptor tyrosine kinases). Wnt signaling is an appealing new therapeutic target for CNS repair. The mechanisms regulating the re-induction are unknown but will be informative for therapeutic design.

The peripheral growth cones of statoacoustic ganglion (SAG) neurons are presumed to sense molecular cues to navigate to their sensory targets during development. Based on previously reported expression data for Frizzled receptors, Wnt ligands, and Wnt inhibitors, we hypothesized that some members of the Wnt morphogen family may function as repulsive cues for SAG neurites. The responses of SAG neurons to mammalian Wnts -1, -4, -5a, -6, and -7b, and the Wnt inhibitors sFRP -1, -2, and -3, were tested in vitro by growing SAG explants from embryonic day 4 (E4) chicken embryos for two days in 3D collagen gels. Average neurite length and density were quantified to determine effects on neurite outgrowth. SAG neurites were strongly repelled by human Sema3E, demonstrating SAG neurons are responsive under these assay conditions. In contrast, SAG neurons showed no changes in neurite outgrowth when cultured in the presence of Wnts and Wnt inhibitors. As an alternative approach, Wnt4 and Wnt5a were also tested in vivo by injecting retroviruses encoding these genes into the chicken otocyst on E3. On E6, no differences were evident in the peripheral projections of SAG axons terminating in infected sensory organs as compared to uninfected organs on the contralateral side of the same embryo. For all Wnt sources, bioactivity was confirmed on E6 chick spinal cord explants by observing enhanced axon outgrowth, as reported previously in the mouse. These results suggest that the tested Wnts do not play a role in guiding peripheral axons in the chicken inner ear.

Monoaminergic neurons [serotonergic (5-HT) and dopaminergic (mdDA)] in the brainstem project axons along the anterior–posterior axis. Despite their important physiological functions and implication in disease, the molecular mechanisms that dictate the formation of these projections along the anterior–posterior axis remain unknown. Here we reveal a novel requirement for Wnt/planar cell polarity signaling in the anterior–posterior organization of the monoaminergic system. We find that 5-HT and mdDA axons express the core planar cell polarity components Frizzled3, Celsr3, and Vangl2. In addition, monoaminergic projections show anterior–posterior guidance defects inFrizzled3,Celsr3, andVangl2mutant mice. The only known ligands for planar cell polarity signaling are Wnt proteins. In culture, Wnt5a attracts 5-HT but repels mdDA axons, and Wnt7b attracts mdDA axons. However, mdDA axons fromFrizzled3mutant mice are unresponsive to Wnt5a and Wnt7b. Both Wnts are expressed in gradients along the anterior–posterior axis, consistent with their role as directional cues. Finally,Wnt5amutants show transient anterior–posterior guidance defects in mdDA projections. Furthermore, we observe during development that the cell bodies of migrating descending 5-HT neurons eventually reorient along the direction of their axons. InFrizzled3mutants, many 5-HT and mdDA neuron cell bodies are oriented abnormally along the direction of their aberrant axon projections. Overall, our data suggest that Wnt/planar cell polarity signaling may be a global anterior–posterior guidance mechanism that controls axonal and cellular organization beyond the spinal cord.

Pathfinding axons change responses to guidance cues at intermediate targets. During midline crossing, spinal cord commissural axons acquire responsiveness to class 3 Semaphorins and Slits, which regulate their floor plate exit and restrict their post-crossing trajectory into a longitudinal pathway. We found that Sonic Hedgehog (Shh) could activate the repulsive response of pre-crossing axons to Semaphorins. Blocking Shh function with a monoclonal antibody to Shh, 5E1, in 'open-book' explants or by expressing a dominant-negative form of Patched-1, Ptch1(Delta loop2), or a Smoothened (Smo) shRNA construct in commissural neurons resulted in severe guidance defects, including stalling and knotting inside the floor plate, recrossing, randomized anterior-posterior projection and overshooting after crossing, reminiscent of Neuropilin-2 mutant embryos. Enhancing protein kinase A activity in pre-crossing axons diminished Shh-induced Semaphorin repulsion and caused profound midline stalling and overshooting/wandering of post-crossing axons. Therefore, a morphogen, Shh, can act as a switch of axon guidance responses.

Background Wnt proteins comprise a large class of signaling molecules that regulate a variety of developmental processes, including synapse formation. Previous studies have shown Wnts to be involved in both the induction and prevention of synapses in a number of different organisms. However, it is not clear whether the influence of Wnts on synapses is a result of Wnts' behavior in different organisms or differences in the activity of different Wnt ligands. Results We used in situ hybridization to show that several Wnt ligands (Wnt3, Wnt5a, Wnt7a, and Wnt7b) and their receptors, Frizzled, are expressed in the developing hippocampus during the period of synapse formation in rodents. We used recombinant Wnt protein or Wnt conditioned media to explore the effects of Wnts on synapses in hippocampal cultures. We found that Wnt7a and Wnt7b activate canonical signaling, whereas Wnt5a activates a noncanonical pathway. The activation of the canonical pathway, either through pathway manipulations or through Wnt stimulation, increases presynaptic inputs. In contrast, exposure to Wnt5a, which activates a noncanonical signaling pathway, decreases the number of presynaptic terminals. Conclusion Our observations suggest that the pro- and antisynaptogenic effects of Wnt proteins are associated with the activation of the canonical and noncanonical Wnt signaling pathways.

Failure of axon regeneration in the mammalian CNS is attributable in part to the presence of various inhibitory molecules, including myelin-associated proteins and proteoglycans enriched in glial scars. Here, we evaluate whether axon guidance molecules also regulate regenerative growth after injury in adulthood. Wnts are a large family of axon guidance molecules that can attract ascending axons and repel descending axons along the length of the developing spinal cord. Their expression (all 19Wnts) is not detectable in normal adult spinal cord byin situhybridization. However, three of them are clearly reinduced after spinal cord injury.Wnt1andWnt5a, encoding potent repellents of the descending corticospinal tract (CST) axons, were robustly and acutely induced broadly in the spinal cord gray matter after unilateral hemisection. Ryk, the conserved repulsive Wnt receptor, was also induced in the lesion area, and Ryk immunoreactivity was found on the lesioned CST axons.Wnt4, which attracts ascending sensory axons in development, was acutely induced in areas closer to the lesion thanWnt1andWnt5a. Injection of function-blocking Ryk antibodies into the dorsal bilateral hemisectioned spinal cord either prevented the retraction of CST axons or promoted their regrowth but clearly enhanced the sprouting of CST collateral branches around and beyond the injury site. Therefore, repulsive Wnt signaling may be a cause of cortical spinal tract axon retraction and inhibits axon sprouting after injury.

Morphogen family proteins are now widely appreciated as axon guidance cues. Because their roles as morphogens are highly conserved across phylogeny, their functional conservation in axon guidance is now being rigorously examined. Recent studies suggest that morphogens are important in shaping topographic projections in chick and Drosophila visual systems, a process that occurs even later in development.

Recent studies have begun to shed light on the molecular guidance cues controlling anterior-posterior axon guidance. Two recent studies in the current issue of Developmental Cell show that Wnts play critical roles in patterning processes and directing neuronal migration in C. elegans. Together with previous findings in vertebrates and flies, these new results establish conserved function of Wnts in A-P guidance.

The Wingless-type (Wnt) family proteins are well known for their functions in embryonic patterning, cell fate determination, and cell proliferation ([Wodarz and Nusse, 1998][1]). Wnts act through both canonical and noncanonical signal transduction pathways. Canonical Wnt signaling results in the

Guidance cues along the longitudinal axis of the CNS are poorly understood. Wnt proteins attract ascending somatosensory axons to project from the spinal cord to the brain. Here we show that Wnt proteins repel corticospinal tract (CST) axons in the opposite direction. Several Wnt genes were found to be expressed in the mouse spinal cord gray matter, cupping the dorsal funiculus, in an anterior-to-posterior decreasing gradient along the cervical and thoracic cord. Wnts repelled CST axons in collagen gel assays through a conserved high-affinity receptor, Ryk, which is expressed in CST axons. Neonatal spinal cord secretes diffusible repellent(s) in an anterior-posterior graded fashion, with anterior cord being stronger, and the repulsive activity was blocked by antibodies to Ryk (anti-Ryk). Intrathecal injection of anti-Ryk blocked the posterior growth of CST axons. Therefore, Wnt proteins may have a general role in anterior-posterior guidance of multiple classes of axons.

Recent studies have identified Wnt proteins as conserved axon guidance molecules in vertebrates and invertebrates. Wnt proteins are a large family of diffusible factors that play several important roles, both in embryonic development and in adult function. The signaling mechanisms of Wnt proteins are complex and, because Wnts are newly discovered as axon guidance cues, little is known about how Wnt signaling controls the direction of growth cone navigation - a process that is crucial in development of the nervous system. This review summarizes recent work on the role of Wnts in axon guidance and discusses the possible signaling mechanisms involved in growth cone guidance. Understanding how Wnts regulate axon wiring will not only help us to understand how the nervous system is connected but also provide possible tools for axon regeneration.

Nat. Neurosci. 8, 1151–1159 (2005) In the print version of this article and the version initially published online, one author's name was spelled incorrectly. The correct spelling is Xue-Jun Song. The error has been corrected in the HTML and PDF versions of the article. This correction has been appended to the PDF version.

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 mossy fiber 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.