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The dorsal funiculus in the spinal cord relays somatosensory information to the brain. It is made of T-shaped bifurcation of dorsal root ganglion (DRG) sensory axons. Our previous study has shown that Slit signaling is required for proper guidance during bifurcation, but loss of Slit does not affect all DRG axons. Here, we examined the role of the extracellular molecule Netrin-1 (Ntn1). Using wholemount staining with tissue clearing, we showed that mice lacking Ntn1 had axons escaping from the dorsal funiculus at the time of bifurcation. Genetic labeling confirmed that these misprojecting axons come from DRG neurons. Single axon analysis showed that loss of Ntn1 did not affect bifurcation but rather altered turning angles. To distinguish their guidance functions, we examined mice with triple deletion of Ntn1, Slit1, and Slit2 and found a completely disorganized dorsal funiculus. Comparing mice with different genotypes using immunolabeling and single axon tracing revealed additive guidance errors, demonstrating the independent roles of Ntn1 and Slit. Moreover, the same defects were observed in embryos lacking their cognate receptors. These in vivo studies thus demonstrate the presence of multi-factorial guidance mechanisms that ensure proper formation of a common branched axonal structure during spinal cord development., Competing Interests: BC, KN, AY, LG, AJ, MT, LM No competing interests declared, (© 2024, Curran et al.)

Axon remodeling through sprouting and pruning contributes to the refinement of developing neural circuits. A prominent example is the pruning of developing sensory axons deprived of neurotrophic support, which is mediated by a caspase-dependent (apoptotic) degeneration process. Distal sensory axons possess a latent apoptotic pathway, but a cell body-derived signal that travels anterogradely down the axon is required for pathway activation. The signaling mechanisms that underlie this anterograde process are poorly understood. Here, we show that the tumor suppressor P53 is required for anterograde signaling. Interestingly loss of P53 blocks axonal but not somatic (i.e., cell body) caspase activation. Unexpectedly, P53 does not appear to have an acute transcriptional role in this process and instead appears to act in the cytoplasm to directly activate the mitochondrial apoptotic pathway in axons. Our data support the operation of a cytoplasmic role for P53 in the anterograde death of developing sensory axons., Competing Interests: Declaration of interests M.T.-L. is a director of Denali Therapeutics and Regeneron Pharmaceuticals. Other authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Axon guidance molecules and neuronal activity have been implicated in the establishment and refinement of neural circuits during development. It is unclear, however, whether these guidance molecule- and activity-dependent mechanisms interact with one another to shape neural circuit formation. The formation of corticospinal (CS) circuits, which are essential for voluntary movements, involves both guidance molecule- and activity-dependent components during development. We previously showed that semaphorin6D (Sema6D)-plexinA1 (PlexA1) signaling eliminates ipsilateral projections of CS neurons in the spinal cord, while other studies demonstrate that CS projections to the spinal cord are eliminated in an activity-dependent manner. Here we show that inhibition of cortical neurons during postnatal development causes defects in elimination of ipsilateral CS projections in mice. We further show that mice that lack the activity-dependent Bax/Bak pathway or caspase-9 similarly exhibit defects in elimination of ipsilateral CS projections, suggesting that the activity-dependent Bax/Bak-caspase-9 pathway is essential for the removal of ipsilateral CS projections. Interestingly, either inhibition of neuronal activity in the cortex or deletion of Bax/Bak in mice causes a reduction in PlexA1 protein expression in corticospinal neurons. Finally, intracortical microstimulation induces activation of only contralateral forelimb muscles in control mice, whereas it induces activation of both contralateral and ipsilateral muscles in mice with cortical inhibition, suggesting that the ipsilaterally projecting CS axons that have been maintained in mice with cortical inhibition form functional connections. Together, these results provide evidence of a potential link between the repellent signaling of Sema6D-PlexA1 and neuronal activity to regulate axon elimination. SIGNIFICANCE STATEMENT Both axon guidance molecules and neuronal activity regulate axon elimination to refine neuronal circuits during development. However, the degree to which these mechanisms operate independently or cooperatively to guide network generation is unclear. Here, we show that neuronal activity-driven Bax/Bak-caspase signaling induces expression of the PlexA1 receptor for the repellent Sema6D molecule in corticospinal neurons (CSNs). This cascade eliminates ipsilateral projections of CSNs in the spinal cord during early postnatal development. The absence of PlexA1 , neuronal activity, Bax and Bak , or caspase-9 leads to the maintenance of ipsilateral projections of CSNs, which can form functional connections with spinal neurons. Together, these studies reveal how the Sema6D-PlexA1 signaling and neuronal activity may play a cooperative role in refining CS axonal projections., (Copyright © 2020 the authors.)

The projection targets of a neuronal population are a key feature of its anatomical characteristics. Historically, tissue sectioning, confocal microscopy, and manual scoring of specific regions of interest have been used to generate coarse summaries of mesoscale projectomes. We present here TrailMap, a three-dimensional (3D) convolutional network for extracting axonal projections from intact cleared mouse brains imaged by light-sheet microscopy. TrailMap allows region-based quantification of total axon content in large and complex 3D structures after registration to a standard reference atlas. The identification of axonal structures as thin as one voxel benefits from data augmentation but also requires a loss function that tolerates errors in annotation. A network trained with volumes of serotonergic axons in all major brain regions can be generalized to map and quantify axons from thalamocortical, deep cerebellar, and cortical projection neurons, validating transfer learning as a tool to adapt the model to novel categories of axonal morphology. Speed of training, ease of use, and accuracy improve over existing tools without a need for specialized computing hardware. Given the recent emphasis on genetically and functionally defining cell types in neural circuit analysis, TrailMap will facilitate automated extraction and quantification of axons from these specific cell types at the scale of the entire mouse brain, an essential component of deciphering their connectivity., Competing Interests: The authors declare no competing interest.

Selective synaptic and axonal degeneration are critical aspects of both brain development and neurodegenerative disease. Inhibition of caspase signaling in neurons is a potential therapeutic strategy for neurodegenerative disease, but no neuron-specific modulators of caspase signaling have been described. Using a mass spectrometry approach, we discovered that RUFY3, a neuronally enriched protein, is essential for caspase-mediated degeneration of TRKA+ sensory axons in vitro and in vivo. Deletion of Rufy3 protects axons from degeneration, even in the presence of activated CASP3 that is competent to cleave endogenous substrates. Dephosphorylation of RUFY3 at residue S34 appears required for axon degeneration, providing a potential mechanism for neurons to locally control caspase-driven degeneration. Neuronally enriched RUFY3 thus provides an entry point for understanding non-apoptotic functions of CASP3 and a potential target to modulate caspase signaling specifically in neurons for neurodegenerative disease., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The molecular trigger of CNS myelination is unknown. By targeting Pten in cerebellar granule cells and activating the AKT1-mTOR pathway, we increased the caliber of normally unmyelinated axons and the expression of numerous genes encoding regulatory proteins. This led to the expansion of genetically wild-type oligodendrocyte progenitor cells, oligodendrocyte differentiation and de novo myelination of parallel fibers. Thus, a neuronal program dependent on the phosphoinositide PI(3,4,5)P 3 is sufficient to trigger all steps of myelination.

Axon degeneration is a tightly regulated, self-destructive program that is a critical feature of many neurodegenerative diseases, but the molecular mechanisms regulating this program remain poorly understood. Here, we identify S-phase kinase-associated protein 1A (Skp1a), a core component of a Skp/Cullin/F-box (SCF)-type E3 ubiquitin ligase complex, as a critical regulator of axon degeneration after injury in mammalian neurons. Depletion of Skp1a prolongs survival of injured axons in vitro and in the optic nerve in vivo. We demonstrate that Skp1a regulates the protein level of the nicotinamide adenine dinucleotide (NAD) + synthesizing enzyme nicotinamide mononucleotide adenylyltransferase 2 (Nmnat2) in axons. Loss of axonal Nmnat2 contributes to a local ATP deficit that triggers axon degeneration. Knockdown of Skp1a elevates basal levels of axonal Nmnat2, thereby delaying axon degeneration through prolonged maintenance of axonal ATP. Consistent with Skp1a functioning through regulation of Nmnat2, Skp1a knockdown fails to protect axons from Nmnat2 knockdown. These results illuminate the molecular mechanism underlying Skp1a-dependent axonal destruction., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Actin, spectrin, and associated molecules form a periodic, submembrane cytoskeleton in the axons of neurons. For a better understanding of this membrane-associated periodic skeleton (MPS), it is important to address how prevalent this structure is in different neuronal types, different subcellular compartments, and across different animal species. Here, we investigated the organization of spectrin in a variety of neuronal- and glial-cell types. We observed the presence of MPS in all of the tested neuronal types cultured from mouse central and peripheral nervous systems, including excitatory and inhibitory neurons from several brain regions, as well as sensory and motor neurons. Quantitative analyses show that MPS is preferentially formed in axons in all neuronal types tested here: Spectrin shows a long-range, periodic distribution throughout all axons but appears periodic only in a small fraction of dendrites, typically in the form of isolated patches in subregions of these dendrites. As in dendrites, we also observed patches of periodic spectrin structures in a small fraction of glial-cell processes in four types of glial cells cultured from rodent tissues. Interestingly, despite its strong presence in the axonal shaft, MPS is disrupted in most presynaptic boutons but is present in an appreciable fraction of dendritic spine necks, including some projecting from dendrites where such a periodic structure is not observed in the shaft. Finally, we found that spectrin is capable of adopting a similar periodic organization in neurons of a variety of animal species, including Caenorhabditis elegans, Drosophila, Gallus gallus, Mus musculus, and Homo sapiens.

During development, sensory axons compete for limiting neurotrophic support, and local neurotrophin insufficiency triggers caspase-dependent axon degeneration. The signaling driving axon degeneration upon local deprivation is proposed to reside within axons. Our results instead support a model in which, despite the apoptotic machinery being present in axons, the cell body is an active participant in gating axonal caspase activation and axon degeneration. Loss of trophic support in axons initiates retrograde activation of a somatic pro-apoptotic pathway, which, in turn, is required for distal axon degeneration via an anterograde pro-degenerative factor. At a molecular level, the cell body is the convergence point of two signaling pathways whose integrated action drives upregulation of pro-apoptotic Puma, which, unexpectedly, is confined to the cell body. Puma then overcomes inhibition by pro-survival Bcl-xL and Bcl-w and initiates the anterograde pro-degenerative program, highlighting the role of the cell body as an arbiter of large-scale axon removal., (Copyright © 2016 Elsevier Inc. All rights reserved.)

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., (© 2015 Hernandez-Enriquez et al.; Published by Cold Spring Harbor Laboratory Press.)

Axon pathfinding is orchestrated by numerous guidance cues, including Slits and their Robo receptors, but it remains unclear how information from multiple cues is integrated or filtered. Robo3, a Robo family member, allows commissural axons to reach and cross the spinal cord midline by antagonizing Robo1/2-mediated repulsion from midline-expressed Slits and potentiating deleted in colorectal cancer (DCC)-mediated midline attraction to Netrin-1, but without binding either Slits or Netrins. We identified a secreted Robo3 ligand, neural epidermal growth factor-like-like 2 (NELL2), which repels mouse commissural axons through Robo3 and helps steer them to the midline. These findings identify NELL2 as an axon guidance cue and establish Robo3 as a multifunctional regulator of pathfinding that simultaneously mediates NELL2 repulsion, inhibits Slit repulsion, and facilitates Netrin attraction to achieve a common guidance purpose., (Copyright © 2015, American Association for the Advancement of Science.)

Calcium is a key regulator of axon degeneration caused by trauma and disease, but its specific spatial and temporal dynamics in injured axons remain unclear. To clarify the function of calcium in axon degeneration, we observed calcium dynamics in single injured neurons in live zebrafish larvae and tested the temporal requirement for calcium in zebrafish neurons and cultured mouse DRG neurons. Using laser axotomy to induce Wallerian degeneration (WD) in zebrafish peripheral sensory axons, we monitored calcium dynamics from injury to fragmentation, revealing two stereotyped phases of axonal calcium influx. First, axotomy triggered a transient local calcium wave originating at the injury site. This initial calcium wave only disrupted mitochondria near the injury site and was not altered by expression of the protective WD slow (WldS) protein. Inducing multiple waves with additional axotomies did not change the kinetics of degeneration. In contrast, a second phase of calcium influx occurring minutes before fragmentation spread as a wave throughout the axon, entered mitochondria, and was abolished by WldS expression. In live zebrafish, chelating calcium after the first wave, but before the second wave, delayed the progress of fragmentation. In cultured DRG neurons, chelating calcium early in the process of WD did not alter degeneration, but chelating calcium late in WD delayed fragmentation. We propose that a terminal calcium wave is a key instructive component of the axon degeneration program., Significance Statement: Axon degeneration resulting from trauma or neurodegenerative disease can cause devastating deficits in neural function. Understanding the molecular and cellular events that execute axon degeneration is essential for developing treatments to address these conditions. Calcium is known to contribute to axon degeneration, but its temporal requirements in this process have been unclear. Live calcium imaging in severed zebrafish neurons and temporally controlled pharmacological treatments in both zebrafish and cultured mouse sensory neurons revealed that axonal calcium influx late in the degeneration process regulates axon fragmentation. These findings suggest that temporal considerations will be crucial for developing treatments for diseases associated with axon degeneration., (Copyright © 2015 the authors 0270-6474/15/3515026-13$15.00/0.)

Axonal death disrupts functional connectivity of neural circuits and is a critical feature of many neurodegenerative disorders. Pathological axon degeneration often occurs independently of known programmed death pathways, but the underlying molecular mechanisms remain largely unknown. Using traumatic injury as a model, we systematically investigate mitogen-activated protein kinase (MAPK) families and delineate a MAPK cascade that represents the early degenerative response to axonal injury. The adaptor protein Sarm1 is required for activation of this MAPK cascade, and this Sarm1-MAPK pathway disrupts axonal energy homeostasis, leading to ATP depletion before physical breakdown of damaged axons. The protective cytoNmnat1/Wld(s) protein inhibits activation of this MAPK cascade. Further, MKK4, a key component in the Sarm1-MAPK pathway, is antagonized by AKT signaling, which modulates the degenerative response by limiting activation of downstream JNK signaling. Our results reveal a regulatory mechanism that integrates distinct signals to instruct pathological axon degeneration., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The myenteric plexus of the enteric nervous system controls the movement of smooth muscles in the gastrointestinal system. They extend their axons between two peripheral smooth muscle layers to form a tubular meshwork arborizing the gut wall. How a tubular axonal meshwork becomes established without invading centrally toward the gut epithelium has not been addressed. We provide evidence here that sonic hedgehog (Shh) secreted from the gut epithelium prevents central projections of enteric axons, thereby forcing their peripheral tubular distribution. Exclusion of enteric central projections by Shh requires its binding partner growth arrest specific gene 1 (Gas1) and its signaling component smoothened (Smo) in enteric neurons. Using enteric neurons differentiated from neurospheres in vitro, we show that enteric axon growth is not inhibited by Shh. Rather, when Shh is presented as a point source, enteric axons turn away from it in a Gas1-dependent manner. Of the Gαi proteins that can couple with Smo, G protein α Z (Gnaz) is found in enteric axons. Knockdown and dominant negative inhibition of Gnaz dampen the axon-repulsive response to Shh, and Gnaz mutant intestines contain centrally projected enteric axons. Together, our data uncover a previously unsuspected mechanism underlying development of centrifugal tubular organization and identify a previously unidentified effector of Shh in axon guidance.

Development of neuronal circuits is controlled by evolutionarily conserved axon guidance molecules, including Slits, the repulsive ligands for roundabout (Robo) receptors, and Netrin-1, which mediates attraction through the DCC receptor. We discovered that the Robo3 receptor fundamentally changed its mechanism of action during mammalian evolution. Unlike other Robo receptors, mammalian Robo3 is not a high-affinity receptor for Slits because of specific substitutions in the first immunoglobulin domain. Instead, Netrin-1 selectively triggers phosphorylation of mammalian Robo3 via Src kinases. Robo3 does not bind Netrin-1 directly but interacts with DCC. Netrin-1 fails to attract pontine neurons lacking Robo3, and attraction can be restored in Robo3(-/-) mice by expression of mammalian, but not nonmammalian, Robo3. We propose that Robo3 evolution was key to sculpting the mammalian brain by converting a receptor for Slit repulsion into one that both silences Slit repulsion and potentiates Netrin attraction., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Netrins are secreted proteins that regulate axon guidance and neuronal migration. Deleted in colorectal cancer (DCC) is a well-established netrin-1 receptor mediating attractive responses. We provide evidence that its close relative neogenin is also a functional netrin-1 receptor that acts with DCC to mediate guidance in vivo. We determined the structures of a functional netrin-1 region, alone and in complexes with neogenin or DCC. Netrin-1 has a rigid elongated structure containing two receptor-binding sites at opposite ends through which it brings together receptor molecules. The ligand/receptor complexes reveal two distinct architectures: a 2:2 heterotetramer and a continuous ligand/receptor assembly. The differences result from different lengths of the linker connecting receptor domains fibronectin type III domain 4 (FN4) and FN5, which differs among DCC and neogenin splice variants, providing a basis for diverse signaling outcomes., (Copyright © 2014, American Association for the Advancement of Science.)

During embryonic development, axons can gain and lose sensitivity to guidance cues, and this flexibility is essential for the correct wiring of the nervous system. Yet, the underlying molecular mechanisms are largely unknown. Here we show that receptor cleavage by ADAM (A Disintegrin And Metalloprotease) metalloproteases promotes murine sensory axons loss of responsiveness to the chemorepellant Sema3A. Genetic ablation of ADAM10 and ADAM17 disrupts the developmental downregulation of Neuropilin-1 (Nrp1), the receptor for Sema3A, in sensory axons. Moreover, this is correlated with gain of repulsive response to Sema3A. Overexpression of Nrp1 in neurons reverses axonal desensitization to Sema3A, but this is hampered in a mutant Nrp1 with high susceptibility to cleavage. Lastly, we detect guidance errors of proprioceptive axons in ADAM knockouts that are consistent with enhanced response to Sema3A. Our results provide the first evidence for involvement of ADAMs in regulating developmental switch in responsiveness to axonal guidance cues.

In the developing brain, initial neuronal projections are formed through extensive growth and branching of developing axons, but many branches are later pruned to sculpt the mature pattern of connections. Despite its widespread occurrence, the mechanisms controlling pruning remain incompletely characterized. Based on pharmacological and biochemical analysis in vitro and initial genetic analysis in vivo, prior studies implicated a pathway involving binding of the Amyloid Precursor Protein (APP) to Death Receptor 6 (DR6) and activation of a downstream caspase cascade in axonal pruning. Here, we further test their involvement in pruning in vivo and their mechanism of action through extensive genetic and biochemical analysis. Genetic deletion of DR6 was previously shown to impair pruning of retinal axons in vivo. We show that genetic deletion of APP similarly impairs pruning of retinal axons in vivo and provide evidence that APP and DR6 act cell autonomously and in the same pathway to control pruning. Prior analysis had suggested that β-secretase cleavage of APP and binding of an N-terminal fragment of APP to DR6 is required for their actions, but further genetic and biochemical analysis reveals that β-secretase activity is not required and that high-affinity binding to DR6 requires a more C-terminal portion of the APP ectodomain. These results provide direct support for the model that APP and DR6 function cell autonomously and in the same pathway to control pruning in vivo and raise the possibility of alternate mechanisms for how APP and DR6 control pruning.

Background: Guidance molecules are normally presented to cells in an overlapping fashion; however, little is known about how their signals are integrated to control the formation of neural circuits. In the thalamocortical system, the topographical sorting of distinct axonal subpopulations relies on the emergent cooperation between Slit1 and Netrin-1 guidance cues presented by intermediate cellular targets. However, the mechanism by which both cues interact to drive distinct axonal responses remains unknown., Results: Here, we show that the attractive response to the guidance cue Netrin-1 is controlled by Slit/Robo1 signaling and by FLRT3, a novel coreceptor for Robo1. While thalamic axons lacking FLRT3 are insensitive to Netrin-1, thalamic axons containing FLRT3 can modulate their Netrin-1 responsiveness in a context-dependent manner. In the presence of Slit1, both Robo1 and FLRT3 receptors are required to induce Netrin-1 attraction by the upregulation of surface DCC through the activation of protein kinase A. Finally, the absence of FLRT3 produces defects in axon guidance in vivo., Conclusions: These results highlight a novel mechanism by which interactions between limited numbers of axon guidance cues can multiply the responses in developing axons, as required for proper axonal tract formation in the mammalian brain., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The cell intrinsic factors that determine whether a neuron regenerates or undergoes apoptosis in response to axonal injury are not well defined. Here we show that the mixed-lineage dual leucine zipper kinase (DLK) is an essential upstream mediator of both of these divergent outcomes in the same cell type. Optic nerve crush injury leads to rapid elevation of DLK protein, first in the axons of retinal ganglion cells (RGCs) and then in their cell bodies. DLK is required for the majority of gene expression changes in RGCs initiated by injury, including induction of both proapoptotic and regeneration-associated genes. Deletion of DLK in retina results in robust and sustained protection of RGCs from degeneration after optic nerve injury. Despite this improved survival, the number of axons that regrow beyond the injury site is substantially reduced, even when the tumor suppressor phosphatase and tensin homolog (PTEN) is deleted to enhance intrinsic growth potential. These findings demonstrate that these seemingly contradictory responses to injury are mechanistically coupled through a DLK-based damage detection mechanism.

Axon degeneration initiated by trophic factor withdrawal shares many features with programmed cell death, but many prior studies discounted a role for caspases in this process, particularly Caspase-3. Recently, Caspase-6 was implicated based on pharmacological and knockdown evidence, and we report here that genetic deletion of Caspase-6 indeed provides partial protection from degeneration. However, we find at a biochemical level that Caspase-6 is activated effectively only by Caspase-3 but not other "upstream" caspases, prompting us to revisit the role of Caspase-3. In vitro, we show that genetic deletion of Caspase-3 is fully protective against sensory axon degeneration initiated by trophic factor withdrawal, but not injury-induced Wallerian degeneration, and we define a biochemical cascade from prosurvival Bcl2 family regulators to Caspase-9, then Caspase-3, and then Caspase-6. Only low levels of active Caspase-3 appear to be required, helping explain why its critical role has been obscured in prior studies. In vivo, Caspase-3 and Caspase-6-knockout mice show a delay in developmental pruning of retinocollicular axons, thereby implicating both Caspase-3 and Caspase-6 in axon degeneration that occurs as a part of normal development.

Axons must switch responsiveness to guidance cues during development for correct pathfinding. Sonic Hedgehog (Shh) attracts spinal cord commissural axons ventrally toward the floorplate. We show that after crossing the floorplate, commissural axons switch their response to Shh from attraction to repulsion, so that they are repelled anteriorly by a posterior-high/anterior-low Shh gradient along the longitudinal axis. This switch is recapitulated in vitro with dissociated commissural neurons as they age, indicating that the switch is intrinsic and time dependent. 14-3-3 protein inhibition converted Shh-mediated repulsion of aged dissociated neurons to attraction and prevented the correct anterior turn of postcrossing commissural axons in vivo, an effect mediated through PKA. Conversely, overexpression of 14-3-3 proteins was sufficient to drive the switch from Shh-mediated attraction to repulsion both in vitro and in vivo. Therefore, we identify a 14-3-3 protein-dependent mechanism for a cell-intrinsic temporal switch in the polarity of axon turning responses., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Axonal and synaptic degeneration is a hallmark of peripheral neuropathy, brain injury, and neurodegenerative disease. Axonal degeneration has been proposed to be mediated by an active autodestruction program, akin to apoptotic cell death; however, loss-of-function mutations capable of potently blocking axon self-destruction have not been described. Here, we show that loss of the Drosophila Toll receptor adaptor dSarm (sterile α/Armadillo/Toll-Interleukin receptor homology domain protein) cell-autonomously suppresses Wallerian degeneration for weeks after axotomy. Severed mouse Sarm1 null axons exhibit remarkable long-term survival both in vivo and in vitro, indicating that Sarm1 prodegenerative signaling is conserved in mammals. Our results provide direct evidence that axons actively promote their own destruction after injury and identify dSarm/Sarm1 as a member of an ancient axon death signaling pathway.

Developing axons must control their growth rate to follow the appropriate pathways and establish specific connections. However, the regulatory mechanisms involved remain elusive. By combining live imaging with transplantation studies in mice, we found that spontaneous calcium activity in the thalamocortical system and the growth rate of thalamocortical axons were developmentally and intrinsically regulated. Indeed, the spontaneous activity of thalamic neurons governed axon growth and extension through the cortex in vivo. This activity-dependent modulation of growth was mediated by transcriptional regulation of Robo1 through an NF-κB binding site. Disruption of either the Robo1 or Slit1 genes accelerated the progression of thalamocortical axons in vivo, and interfering with Robo1 signaling restored normal axon growth in electrically silent neurons. Thus, modifications to spontaneous calcium activity encode a switch in the axon outgrowth program that allows the establishment of specific neuronal connections through the transcriptional regulation of Slit1 and Robo1 signaling.

Axons travel to their targets in bundles or fascicles, but the molecules regulating fasciculation remain incompletely characterized. We found that Slit2 and its Robo receptors are expressed by motor axons, and that inactivation of Slit2 or Robo1 and Robo2 in mice caused axons to defasciculate prematurely at muscle targets. In vitro, Slit2 secreted by motoneurons regulated fasciculation through Robo1 and Robo2. These results support the idea that Slit2 promotes axon fasciculation via an autocrine and/or juxtaparacrine mechanism.

How guidance cues are integrated during the formation of complex axonal tracts remains largely unknown. Thalamocortical axons (TCAs), which convey sensory and motor information to the neocortex, have a rostrocaudal topographic organization initially established within the ventral telencephalon [1-3]. Here, we show that this topography is set in a small hub, the corridor, which contains matching rostrocaudal gradients of Slit1 and Netrin 1. Using in vitro and in vivo experiments, we show that Slit1 is a rostral repellent that positions intermediate axons. For rostral axons, although Slit1 is also repulsive and Netrin 1 has no chemotactic activity, the two factors combined generate attraction. These results show that Slit1 has a dual context-dependent role in TCA pathfinding and furthermore reveal that a combination of cues produces an emergent activity that neither of them has alone. Our study thus provides a novel framework to explain how a limited set of guidance cues can generate a vast diversity of axonal responses necessary for proper wiring of the nervous system., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Draxin, a recently identified axon guidance protein, is essential for the formation of forebrain commissures, and can mediate repulsion of netrin-stimulated spinal commissural axons. Here, we report that draxin binds multiple netrin receptors: DCC (deleted in colorectal cancer), Neogenin, UNC5s (H1, H2, H3), and DSCAM (Down's syndrome cell adhesion molecule). Since draxin and Dcc knockouts showed similar phenotype in forebrain commissures formation, we show here the functional importance of draxin/DCC interaction. Draxin interacts with subnanomolar affinity to the netrin receptor DCC, in a region of DCC distinct from its netrin-binding domain. In vitro, neurite outgrowth from cortical and olfactory bulb explants of Dcc knock-out mice is significantly less inhibited by draxin, when compared with neurites from explants of wild-type mice. Furthermore, in comparison with wild-type mice, the growth cone collapse in response to draxin is largely abolished in Dcc-deficient cortical neurons. In vivo, double heteros of draxin/Dcc mice show markedly higher frequency of complete agenesis of corpus callosum than either of the single hetero. These results identify DCC as a convergent receptor for netrin and draxin in axon growth and guidance.

Growing axons are guided to their targets by attractive and repulsive cues. In the developing spinal cord, Netrin-1 and Shh guide commissural axons toward the midline. However, the combined inhibition of their activity in commissural axon turning assays does not completely abrogate turning toward floor plate tissue, suggesting that additional guidance cues are present. Here we show that the prototypic angiogenic factor VEGF is secreted by the floor plate and is a chemoattractant for commissural axons in vitro and in vivo. Inactivation of Vegf in the floor plate or of its receptor Flk1 in commissural neurons causes axon guidance defects, whereas Flk1 blockade inhibits turning of axons to VEGF in vitro. Similar to Shh and Netrin-1, VEGF-mediated commissural axon guidance requires the activity of Src family kinases. Our results identify VEGF and Flk1 as a novel ligand/receptor pair controlling commissural axon guidance., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Axon growth potential is highest in young neurons but diminishes with age, thus becoming a significant obstacle to axonal regeneration after injury in maturity. The mechanism for the decline is incompletely understood, and no effective clinical treatment is available to rekindle innate growth capability. Here, we show that Smad1-dependent bone morphogenetic protein (BMP) signaling is developmentally regulated and governs axonal growth in dorsal root ganglion (DRG) neurons. Down-regulation of the pathway contributes to the age-related decline of the axon growth potential. Reactivating Smad1 selectively in adult DRG neurons results in sensory axon regeneration in a mouse model of spinal cord injury (SCI). Smad1 signaling can be effectively manipulated by an adeno-associated virus (AAV) vector encoding BMP4 delivered by a clinically applicable and minimally invasive technique, an approach devoid of unwanted abnormalities in mechanosensation or pain perception. Importantly, transected axons are able to regenerate even when the AAV treatment is delivered after SCI, thus mimicking a clinically relevant scenario. Together, our results identify a therapeutic target to promote axonal regeneration after SCI.

Commissural neurons project axons across the floor plate at the spinal cord ventral midline. After crossing, commissural axons turn rostrally, sort into distinct positions within the ventrolateral funiculus, and never reenter the floor plate. Robo1 and Robo2 are receptors for the midline repellents Slit1-Slit3, and upregulation of Robos in post-crossing axons allows expulsion from the floor plate and prevents recrossing. Before crossing, Robo-mediated repulsion is attenuated by the divergent family member Robo3/Rig-1. To define the relative contributions of Robo family members to commissural axon guidance in mice, we studied commissural axon trajectories in combination mutants between Robo1, Robo2, and Robo3. Our results suggest the existence of another receptor contributing to Slit repulsion because the failure of midline crossing in Robo3 mutants is rescued largely but not entirely by loss of both Robo1 and Robo2 and because axon guidance defects in mice lacking both Robo1 and Robo2 are less severe than in mice lacking all Slits. Analysis of post-crossing axon trajectories indicates that Robo1 and Robo2 collaborate to prevent axons from reentering the gray matter and projecting dorsally alongside contralateral pre-crossing axons. We also discovered a previously unappreciated division of labor between Robo1 and Robo2 in post-crossing axons. Robo2 is required for axons to project away from the floor plate into the lateral funiculus. In contrast, Robo1 prevents axonal stalling after crossing. Our results reveal specialized and complementary actions of Robo1 and Robo2 in commissural axon guidance and suggest the existence of an as yet unidentified Slit receptor.

In Bilateria, many axons cross the midline of the central nervous system, forming well-defined commissures. Whereas in mammals the functions of commissures in the forebrain and in the visual system are well established, functions at other axial levels are less clearly understood. Here, we have dissected the function of several hindbrain commissures using genetic methods. By taking advantage of multiple Cre transgenic lines, we have induced site-specific deletions of the Robo3 receptor. These lines developed with the disruption of specific commissures in the sensory, motor, and sensorimotor systems, resulting in severe and permanent functional deficits. We show that mice with severely reduced commissures in rhombomeres 5 and 3 have abnormal lateral eye movements and auditory brainstem responses, respectively, whereas mice with a primarily uncrossed climbing fiber/Purkinje cell projection are strongly ataxic. Surprisingly, although rerouted axons remain ipsilateral, they still project to their appropriate neuronal targets. Moreover, some Cre;Robo3 lines represent potential models that can be used to study human syndromes, including horizontal gaze palsy with progressive scoliosis (HGPPS). To our knowledge, this study is one of the first to link defects in commissural axon guidance with specific cellular and behavioral phenotypes., Competing Interests: The authors have declared that no competing interests exist.

The ability of sensory systems to detect and process information from the environment relies on the elaboration of precise connections between sensory neurons in the periphery and second order neurons in the CNS. In mice, the accessory olfactory system is thought to regulate a wide variety of social and sexual behaviors. The expression of the Slit receptors Robo-1 and Robo-2 in vomeronasal sensory neurons (VSNs) suggests they may direct the stereotypic targeting of their axons to the accessory olfactory bulb (AOB). Here, we have examined the roles of Robo-1 and Robo-2 in the formation of connections by VSN axons within the AOB. While Robo-1 is not necessary for the segregation of VSN axons within the anterior and posterior regions of the AOB, Robo-2 is required for the targeting of some basal VSN axons to the posterior region of the AOB but is dispensable for the fasciculation of VSN axons. Furthermore, the specific ablation of Robo-2 expression in VSNs leads to mistargeting of a portion of basal VSN axons to the anterior region of the AOB, indicating that Robo-2 expression is required on projecting VSN axons. Together, these results identify Robo-2 as a receptor that controls the targeting of basal VSN axons to the posterior AOB.

Mature neurons have diminished intrinsic regenerative capacity. Axotomy of the peripheral branch of adult dorsal root ganglia (a "conditioning" lesion) triggers a transcription-dependent axon growth program. Here, we show that this growth program requires the function of the transcription factor Smad1. After peripheral axotomy, neuronal Smad1 is upregulated, and phosphorylated Smad1 accumulates in the nucleus. Both events precede the onset of axonal extension. Reducing Smad1 by RNA interference in vitro impairs axonal growth, and the continued presence of Smad1 is required to maintain the growth program. Furthermore, intraganglionic injection of BMP2 or 4, which activates Smad1, markedly enhances axonal growth capacity, mimicking the effect of a conditioning lesion. Thus, activation of Smad1 by axotomy is a key component of the transcriptional switch that promotes an enhanced growth state of adult sensory neurons.

Naturally occurring axonal pruning and neuronal cell death help to sculpt neuronal connections during development, but their mechanistic basis remains poorly understood. Here we report that beta-amyloid precursor protein (APP) and death receptor 6 (DR6, also known as TNFRSF21) activate a widespread caspase-dependent self-destruction program. DR6 is broadly expressed by developing neurons, and is required for normal cell body death and axonal pruning both in vivo and after trophic-factor deprivation in vitro. Unlike neuronal cell body apoptosis, which requires caspase 3, we show that axonal degeneration requires caspase 6, which is activated in a punctate pattern that parallels the pattern of axonal fragmentation. DR6 is activated locally by an inactive surface ligand(s) that is released in an active form after trophic-factor deprivation, and we identify APP as a DR6 ligand. Trophic-factor deprivation triggers the shedding of surface APP in a beta-secretase (BACE)-dependent manner. Loss- and gain-of-function studies support a model in which a cleaved amino-terminal fragment of APP (N-APP) binds DR6 and triggers degeneration. Genetic support is provided by a common neuromuscular junction phenotype in mutant mice. Our results indicate that APP and DR6 are components of a neuronal self-destruction pathway, and suggest that an extracellular fragment of APP, acting via DR6 and caspase 6, contributes to Alzheimer's disease.

A major barrier to regenerating axons after injury in the mammalian central nervous system is an unfavorable milieu. Three proteins found in myelin--Nogo, MAG, and OMgp--inhibit axon regeneration in vitro and bind to the glycosylphosphatidylinositol-anchored Nogo receptor (NgR). However, genetic deletion of NgR has only a modest disinhibitory effect, suggesting that other binding receptors for these molecules probably exist. With the use of expression cloning, we have found that paired immunoglobulin-like receptor B (PirB), which has been implicated in nervous system plasticity, is a high-affinity receptor for Nogo, MAG, and OMgp. Interfering with PirB activity, either with antibodies or genetically, partially rescues neurite inhibition by Nogo66, MAG, OMgp, and myelin in cultured neurons. Blocking both PirB and NgR activities leads to near-complete release from myelin inhibition. Our results implicate PirB in mediating regeneration block, identify PirB as a potential target for axon regeneration therapies, and provide an explanation for the similar enhancements of visual system plasticity in PirB and NgR knockout mice.

Recent studies have demonstrated that the topography of thalamocortical (TC) axon projections is initiated before they reach the cortex, in the ventral telencephalon (VTel). However, at this point, the molecular mechanisms patterning the topography of TC projections in the VTel remains poorly understood. Here, we show that a long-range, high-rostral to low-caudal gradient of Netrin-1 in the VTel is required in vivo for the topographic sorting of TC axons to distinct cortical domains. We demonstrate that Netrin-1 is a chemoattractant for rostral thalamic axons but functions as a chemorepulsive cue for caudal thalamic axons. In accordance with this model, DCC is expressed in a high-rostromedial to low-caudolateral gradient in the dorsal thalamus (DTh), whereas three Unc5 receptors (Unc5A-C) show graded expression in the reverse orientation. Finally, we show that DCC is required for the attraction of rostromedial thalamic axons to the Netrin-1-rich, anterior part of the VTel, whereas DCC and Unc5A/C receptors are required for the repulsion of caudolateral TC axons from the same Netrin-1-rich region of the VTel. Our results demonstrate that a long-range gradient of Netrin-1 acts as a counteracting force from ephrin-A5 to control the topography of TC projections before they enter the cortex.

Alternative splicing provides a means to increase the complexity of gene function in numerous biological processes, including nervous system wiring. Navigating axons switch responses from attraction to repulsion at intermediate targets, allowing them to grow to each intermediate target and then to move on. The mechanisms underlying this switch remain poorly characterized. We previously showed that the Slit receptor Robo3 is required for spinal commissural axons to enter and cross the midline intermediate target. We report here the existence of two functionally antagonistic isoforms of Robo3 with distinct carboxy termini arising from alternative splicing. Robo3.1 is deployed on the precrossing and crossing portions of commissural axons and allows midline crossing by silencing Slit repulsion. Robo3.2 becomes expressed on the postcrossing portion and blocks midline recrossing, favoring Slit repulsion. The tight spatial regulation of opponent splice variants helps ensure high-fidelity transition of axonal responses from attraction to repulsion at the midline.

Commissural axons are attracted to the midline intermediate target by chemoattractants, but upon crossing the midline they switch off responsiveness to attractants and switch on responsiveness to midline repellents, which expel the axons from the midline and enable them to move on. Here we show that midline exit also requires the stimulation of axon outgrowth by Stem Cell Factor (SCF, also known as Steel Factor). SCF is expressed by midline floor plate cells, and its receptor Kit, a receptor tyrosine kinase, is expressed by commissural axons. In Steel and Kit mutant mice, the majority of commissural axons line up transiently at the contralateral edge of the floor plate, showing a delay in midline exit. In vitro, SCF selectively promotes outgrowth of postcrossing, but not precrossing, commissural axons. Our findings identify SCF as a guidance cue in the CNS, and provide evidence that exiting intermediate targets requires activation of outgrowth-promoting mechanisms.

We investigated the influence of the bifunctional guidance molecule netrin-1 on axonal growth in the injured adult spinal cord. In the adult, netrin-1 is expressed on mature oligodendrocytes, cells of the central canal, and the meninges. Netrin-1 protein in white matter is selectively enriched adjacent to paranodal loops of myelin in nodes of Ranvier. The repulsion-mediating netrin-1 uncoordinated-5 (UNC5) receptors are expressed by neurons of the corticospinal and rubrospinal projections, and by intrinsic neurons of the spinal cord, both before and after spinal cord injury. Neutralization of netrin-1 in myelin prepared from adult rat spinal cord using UNC5 receptor bodies increases neurite outgrowth from UNC5-expressing spinal motor neurons in vitro. Furthermore, axon regeneration is inhibited in a netrin-1-enriched zone, devoid of other myelin-associated inhibitors, within spinal cord lesion sites in vivo. We conclude that netrin-1 is a novel oligodendrocyte-associated inhibitor that can contribute to axonal growth failure after adult spinal cord injury.

Secreted Slit proteins signal through Robo receptors and negatively regulate axon guidance and cell migration, but in vertebrates, Slit proteins can also stimulate branching and elongation of sensory axons and cortical dendrites in vitro. Here, we show that this branching activity is required for developing peripheral sensory arbors in vivo, because trigeminal sensory branching above the eye is reduced in Slit2;Slit3 double or Slit1,2,3 triple mutants. A similar phenotype is observed in Robo1;Robo2 double mutants, implicating Robo receptors in mediating this activity. Interestingly, by studying the central projection of sensory neurons in the spinal cord, we discovered that Slit ligands are also required for proper guidance of sensory branches during bifurcation but through a different cellular mechanism. In Slit1;Slit2 or Robo1;Robo2 double mutant mice, sensory axons enter the spinal cord prematurely because of the loss of an inhibitory guidance function on one of the daughter branches of each afferent during bifurcation. Together, our studies reveal that Slit/Robo signaling contributes to patterning both the peripheral and central branches of sensory neurons but via distinct positive branching and negative guidance actions, respectively.

There is increasing evidence that heparan sulfate (HS) plays an essential role in various axon guidance processes. These observations, however, have not addressed whether HS is required cell autonomously as an axonal coreceptor or as an environmental factor that modulates the localization of guidance molecules in the terrain in which growing axons navigate. Here we demonstrate that netrin-1-mediated commissural axon guidance requires cell-autonomous expression of HS in commissural neurons in vivo. We used the Wnt1-Cre transgene to drive region-specific ablation of Ext1, which encodes an enzyme essential for HS synthesis, in the dorsal part of the spinal cord. Remarkably, Wnt1-Cre-mediated ablation of Ext1 causes commissural axon pathfinding defects that share similarities with those of Netrin-1-deficient and DCC (deleted in colorectal cancer)-deficient mice. Neither Ext1-deficient dorsal spinal cord explants nor wild-type explants in which HS expression was ablated could extend axons in response to netrin-1. Intracellular signaling downstream of netrin-1 and DCC was defective in Ext1-deficient commissural neurons and in DCC-transfected HEK293T cells from which HS was removed. These results demonstrate that the expression of HS by commissural neurons is essential for these neurons to transduce netrin-1 signals, thus providing evidence for a cell-autonomous role of HS in netrin-1/DCC-mediated axon guidance.

The function of the nervous system depends on the precision of axon wiring during development. Previous studies have demonstrated that Slits, a family of secreted chemorepellent proteins, are crucial for the proper development of several major forebrain tracts. Mice deficient in Slit2 or, even more so, in both Slit1 and Slit2 have defects in multiple axonal pathways, including corticofugal, thalamocortical, and callosal connections. In the spinal cord, members of the Robo family of proteins help mediate the function of Slits, but the relative contribution of these receptors to the guidance of forebrain projections remains to be determined. In the present study, we addressed the function of Robo1 and Robo2 in the guidance of forebrain projections by analyzing Robo1-, Robo2-, and Robo1;Robo2-deficient mice. Mice deficient in Robo2 and, more dramatically, in both Robo1 and Robo2, display prominent axon guidance errors in the development of corticofugal, thalamocortical, and corticocortical callosal connections. Our results demonstrate that Robo1 and Robo2 mostly cooperate to mediate the function of Slit proteins in guiding the major forebrain projections.

During embryonic development, morphogens act as graded positional cues to dictate cell fate specification and tissue patterning. Recent findings indicate that morphogen gradients also serve to guide axonal pathfinding during development of the nervous system. These findings challenge our previous notions about morphogens and axon guidance molecules and suggest that these proteins, rather than having sharply divergent functions, act more globally to provide graded positional information that can be interpreted by responding cells either to specify cell fate or to direct axonal pathfinding. This chapter presents the roles identified for members of three prominent morphogen families-the Hedgehog, Wnt and TGF-beta/BMP families-in axon guidance, and discusses potential implications for the molecular mechanisms underlying their guidance functions.

In the spinal cord, sonic hedgehog (Shh) is secreted by the floor plate to control the generation of distinct classes of ventral neurons along the dorsoventral axis. Genetic and in vitro studies have shown that Shh also later acts as a midline-derived chemoattractant for commissural axons. However, the receptor(s) responsible for Shh attraction remain unknown. Here we show that two Robo-related proteins, Boc and Cdon, bind specifically to Shh and are therefore candidate receptors for the action of Shh as an axon guidance ligand. Boc is expressed by commissural neurons, and targeted disruption of Boc in mouse results in the misguidance of commissural axons towards the floor plate. RNA-interference-mediated knockdown of Boc impairs the ability of rat commissural axons to turn towards an ectopic source of Shh in vitro. Taken together, these data suggest that Boc is essential as a receptor for Shh in commissural axon guidance.

Gradients of diffusible long-range attractant and repellent proteins have been proposed to guide growing axons during nervous system development, but such gradients have never been visualized directly. In the embryonic spinal cord, commissural axons pioneer a circumferential trajectory to the floor plate at the ventral midline directed by secreted proteins of the netrin family. In the embryonic chick spinal cord netrin-1 mRNA is expressed by floor plate cells and netrin-2 mRNA by neural epithelial cells. Antibodies to the two netrins reveal a gradient of netrin protein directly in the path of commissural axons. The netrin-1 gradient itself extends many cell diameters dorsal to the floor plate, the site of netrin-1 expression. A similar distribution of netrin-1 protein has been detected in embryonic rat and mouse spinal cord. The detection of a gradient of netrin-1 protein supports the operation of long-range chemotropic mechanisms in the developing nervous system.

Background: The cytoplasmic C. elegans protein MIG-10 affects cell migrations and is related to mammalian proteins that bind phospholipids and Ena/VASP actin regulators. In cultured cells, mammalian MIG-10 promotes lamellipodial growth and Ena/VASP proteins induce filopodia., Results: We show here that during neuronal development, mig-10 and the C. elegans Ena/VASP homolog unc-34 cooperate to guide axons toward UNC-6 (netrin) and away from SLT-1 (Slit). The single mutants have relatively mild phenotypes, but mig-10; unc-34 double mutants arrest early in development with severe axon guidance defects. In axons that are guided toward ventral netrin, unc-34 is required for the formation of filopodia and mig-10 increases the number of filopodia. In unc-34 mutants, developing axons that lack filopodia are still guided to netrin through lamellipodial growth. In addition to its role in axon guidance, mig-10 stimulates netrin-dependent axon outgrowth in a process that requires the age-1 phosphoinositide-3 lipid kinase but not unc-34., Conclusions: mig-10 and unc-34 organize intracellular responses to both attractive and repulsive axon guidance cues. mig-10 and age-1 lipid signaling promote axon outgrowth; unc-34 and to a lesser extent mig-10 promote filopodia formation. Surprisingly, filopodia are largely dispensable for accurate axon guidance.

Inhibitory molecules associated with myelin and the glial scar limit axon regeneration in the adult central nervous system (CNS), but the underlying signaling mechanisms of regeneration inhibition are not fully understood. Here, we show that suppressing the kinase function of the epidermal growth factor receptor (EGFR) blocks the activities of both myelin inhibitors and chondroitin sulfate proteoglycans in inhibiting neurite outgrowth. In addition, regeneration inhibitors trigger the phosphorylation of EGFR in a calcium-dependent manner. Local administration of EGFR inhibitors promotes significant regeneration of injured optic nerve fibers, pointing to a promising therapeutic avenue for enhancing axon regeneration after CNS injury.

Blood vessels and nerve fibres course throughout the body in an orderly pattern, often alongside one another. Although superficially distinct, the mechanisms involved in wiring neural and vascular networks seem to share some deep similarities. The discovery of key axon guidance molecules over the past decade has shown that axons are guided to their targets by finely tuned codes of attractive and repulsive cues, and recent studies reveal that these cues also help blood vessels to navigate to their targets. Parallels have also emerged between the actions of growth factors that direct angiogenic sprouting and those that regulate axon terminal arborization.

During embryonic development, morphogens act as graded positional cues to dictate cell fate specification and tissue patterning. Recent findings indicate that morphogen gradients also serve to guide axonal pathfinding during development of the nervous system. These findings challenge our previous notions about morphogens and axon guidance molecules, and suggest that these proteins, rather than having sharply divergent functions, act more globally to provide graded positional information that can be interpreted by responding cells either to specify cell fate or to direct axonal pathfinding. This review presents the roles identified for members of three prominent morphogen families--the Hedgehog, Wnt and TGFbeta/BMP families--in axon guidance, and discusses potential implications for the molecular mechanisms underlying their guidance functions.