Celsr3 and Fzd3 in axon guidance

The assembly of functional neuronal circuits depends on the correct wiring of axons and dendrites. To reach their targets, axons are guided by a variety of extracellular guidance cues, including Netrins, Ephrins, Semaphorins and Slits. Corresponding receptors in the growth cone, the dynamic structure at the tip of the growing axon, sense and integrate these positional signals, and activate downstream effectors to regulate cytoskeletal organization. In addition to the four canonical families of axon guidance cues mentioned above, some proteins that regulate planar cell polarity were recently found to be critical for axon guidance. The seven-transmembrane domain receptors Celsr3 and Fzd3, in particular, control the development of most longitudinal tracts in the central nervous system, and axon navigation in the peripheral, sympathetic and enteric nervous systems. Despite their unequivocally important role, however, underlying molecular mechanisms remain elusive. We do not know which extracellular ligands they recognize, whether they have co-receptors in the growth cone, and what their downstream effectors are. Here, we review some recent advances and discuss future trends in this emerging field.     

Analysis of zebrafish sidetracked mutants reveals a novel role for Plexin A3 in intraspinal motor axon guidance

One of the earliest guidance decisions for spinal cord motoneurons occurs when pools of motoneurons orient their growth cones towards a common, segmental exit point. In contrast to later events, remarkably little is known about the molecular mechanisms underlying intraspinal motor axon guidance. In zebrafish sidetracked (set) mutants, motor axons exit from the spinal cord at ectopic positions. By single-cell labeling and time-lapse analysis we show that motoneurons with cell bodies adjacent to the segmental exit point properly exit from the spinal cord, whereas those farther away display pathfinding errors. Misguided growth cones either orient away from the endogenous exit point, extend towards the endogenous exit point but bypass it or exit at non-segmental, ectopic locations. Furthermore, we show that sidetracked acts cell autonomously in motoneurons. Positional cloning reveals that sidetracked encodes Plexin A3, a semaphorin guidance receptor for repulsive guidance. Finally, we show that sidetracked (plexin A3) plays an additional role in motor axonal morphogenesis. Together, our data genetically identify the first guidance receptor required for intraspinal migration of pioneering motor axons and implicate the well-described semaphorin/plexin signaling pathway in this poorly understood process. We propose that axonal repulsion via Plexin A3 is a major driving force for intraspinal motor growth cone guidance.     

Metalloproteases: carving out a role in axon guidance

Two families of metalloproteases, the matrix metalloproteases (MMPs) and the A Disintegrin and Metalloproteases (ADAMs), have recently been implicated in the formation of neural connections in the developing central nervous system. Invertebrate and vertebrate axons fail to extend and/or make pathfinding errors when metalloprotease function is inhibited or absent. Culture studies suggest that this requirement for metalloprotease activity results from their ability to cleave ligands, or their receptors, so as to activate or inhibit specific axon extension or guidance signaling pathways.     

A role for S1P signalling in axon guidance in the Xenopus visual system

Sphingosine 1-phosphate (S1P), a lysophospholipid, plays an important chemotactic role in the migration of lymphocytes and germ cells, and is known to regulate aspects of central nervous system development such as neurogenesis and neuronal migration. Its role in axon guidance, however, has not been examined. We show that sphingosine kinase 1, an enzyme that generates S1P, is expressed in areas surrounding the Xenopus retinal axon pathway, and that gain or loss of S1P function in vivo causes errors in axon navigation. Chemotropic assays reveal that S1P elicits fast repulsive responses in retinal growth cones. These responses require heparan sulfate, are sensitive to inhibitors of proteasomal degradation, and involve RhoA and LIM kinase activation. Together, the data identify downstream components that mediate S1P-induced growth cone responses and implicate S1P signalling in axon guidance.     

Tangential neuronal migration controls axon guidance: a role for neuregulin-1 in thalamocortical axon navigation

Neuronal migration and axon guidance constitute fundamental processes in brain development that are generally studied independently. Although both share common mechanisms of cell biology and biochemistry, little is known about their coordinated integration in the formation of neural circuits. Here we show that the development of the thalamocortical projection, one of the most prominent tracts in the mammalian brain, depends on the early tangential migration of a population of neurons derived from the ventral telencephalon. This tangential migration contributes to the establishment of a permissive corridor that is essential for thalamocortical axon pathfinding. Our results also demonstrate that in this process two different products of the Neuregulin-1 gene, CRD-NRG1 and Ig-NRG1, mediate the guidance of thalamocortical axons. These results show that neuronal tangential migration constitutes a novel mechanism to control the timely arrangement of guidance cues required for axonal tract formation in the mammalian brain.     

A role for axon guidance receptors and ligands in blood vessel development and tumor angiogenesis

Nerves and blood vessels resemble each other in their ability to form branching networks. They are in close proximity suggesting possible molecular interactions. The patterning of nerves and blood vessels are not random but are regulated by attractive and repulsive cues. Four major neuronal guidance factors that are sensed by growth cones have been identified, Semaphorin, Ephrin, Slit and Netrin, and their cognate receptors, neuropilin, Eph, roundabouts (Robo) and uncoordinated-5 (UNC5). Unexpectedly, these ligand/receptor pairs also regulate developmental and tumor angiogenesis. Together, there is strong evidence that development of the nervous and vascular systems are regulated by common cues.     

Emerging evidence for cell-autonomous axon guidance

Current models of axon guidance within the central nervous system (CNS) involve the presentation of environmental cues to navigating growth cones. The surrounding and target tissues present a variety of ligands that either restrict or promote growth, thus providing pathfinding instructions to developing axons. Recent findings show that RGMb, a GPI anchored extracellular protein present on retinal ganglion cells, down-regulates Wnt3a signaling by lowering LRP5 levels at the membrane surface. When RGMb is phosphorylated by the extracellular tyrosine kinase VLK, phosphorylated RGMb (p-RGMb) is internalized and carries LRP5 towards intracellular compartments. In the eye, a dorsal-high ventral-low gradient of VLK generates a dorsal-low ventral-high gradient of LRP5 that modulates Wnt3a signaling. These molecules, which are all expressed by individual RGCs, generate Wnt-signal gradients along the dorso-ventral axis of the retina, resulting in differential axon growth which in turn regulates proper retino-tectal/collicular map formation. This pathway represents a regulatory mechanism whereby extracellular phosphorylation generates what may be the first example of a unique self-guiding mechanism that affects neuronal-target connections independent of paracrine signals from the surrounding target tissue.     

The morphogen sonic hedgehog is an axonal chemoattractant that collaborates with netrin-1 in midline axon guidance

Developing axons are guided to their targets by attractive and repulsive guidance cues. In the embryonic spinal cord, the floor plate chemoattractant Netrin-1 is required to guide commissural neuron axons to the midline. However, genetic evidence suggests that other chemoattractant(s) are also involved. We show that the morphogen Sonic hedgehog (Shh) can mimic the additional chemoattractant activity of the floor plate in vitro and can act directly as a chemoattractant on isolated axons. Cyclopamine-mediated inhibition of the Shh signaling mediator Smoothened (Smo) or conditional inactivation of Smo in commissural neurons indicate that Smo activity is important for the additional chemoattractant activity of the floor plate in vitro and for the normal projection of commissural axons to the floor plate in vivo. These results provide evidence that Shh, acting via Smo, is a midline-derived chemoattractant for commissural axons and show that a morphogen can also act as an axonal chemoattractant.     

Epithelial axon guidance in Drosophila

Evidence is offered that the axons of developing sensory neurons in the wing of Drosophila are guided (given both location and polarity information) by the epithelium over which they grow. This guidance is effective in the absence of such potential additional cues as guidepost neurons and physical channels.     

SNARE proteins play a role in motor axon guidance in vertebrates and invertebrates

Axonal growth and guidance rely on correct growth cone responses to guidance cues, both in the central nervous system (CNS) and in the periphery. Unlike the signaling cascades that link axonal growth to cytoskeletal dynamics, little is known about the cross‐talk mechanisms between guidance and membrane dynamics and turnover in the axon. Our studies have shown that Netrin‐1/deleted in colorectal cancer signaling triggers exocytosis through the SNARE Syntaxin‐1 (STX‐1) during the formation of commissural pathways. However, limited in vivo evidence is available about the role of SNARE proteins in motor axonal guidance. Here we show that loss‐of‐function of SNARE complex members results in motor axon guidance defects in fly and chick embryos. Knock‐down of Syntaxin‐1, VAMP‐2, and SNAP‐25 leads to abnormalities in the motor axon routes out of the CNS. Our data point to an evolutionarily conserved role of the SNARE complex proteins in motor axon guidance, thereby pinpointing an important function of SNARE proteins in axonal navigation in vivo . © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 963–974, 2017     

Lazarillo, a neuronal lipocalin in grasshoppers with a role in axon guidance

In this report we present a review on the grasshopper lipocalin Lazarillo with special emphasis on how its molecular properties could account for its known function: the guidance of pioneer neurons during nervous system development. The expression and function of Lazarillo in a subset of developing neurons, its heavy glycosylation and its glycosylphosphatidylinositol linkage to the plasma membrane, make Lazarillo a unique member of the lipocalin family. We have built a model of the tertiary structure of Lazarillo in which we have studied the exposed surfaces in search for clues about ligand and protein interactions with Lazarillo. Our hypotheses about how this lipocalin can exert its function are discussed.     

Repellent cues in axon guidance.

There is increasing evidence that axons are guided by repulsion in several regions of the developing nervous system, although this has yet to be confirmed directly in vivo. As more candidate repulsion molecules are identified, it is becoming clear that collapse of the growth cone in vitro may be mediated by more than one intracellular mechanism. The present emphasis on molecular cloning of the ligands and their receptors should enable a proper definition of their function during development.     

Retinal axon guidance: novel mechanisms for steering

Axons from the retina traverse different molecular territories as they navigate to the tectum. A single territory might span only a few cell diameters and harbour multiple guidance cues, many of which are beginning to be characterized. Also present in the pathway are 'modulators' that influence a growth cone's response to a coincident signal but do not guide growth directly. An emerging principle is that the growth cone, itself, changes molecularly as it journeys through the visual pathway. Growing retinal axons contain mRNAs, ubiquitinating and apoptotic enzymes, translation and degradation machinery. Guidance cues can trigger rapid and local synthesis, degradation and endocytosis of proteins, providing a fast and flexible way for growth cones to respond to cues in their microenvironment and to alter their responsiveness. The data raise the idea that the localized synthesis and downregulation of proteins might help to steer retinal axon growth and, further, might contribute to the changing character of a growth cone as it ages.     

Novel mutations affecting axon guidance in zebrafish and a role for plexin signalling in the guidance of trigeminal and facial nerve axons

In zebrafish embryos, the axons of the posterior trigeminal (Vp) and facial (VII) motoneurons project stereotypically to a small number of target muscles derived from the first and second branchial arches (BA1, BA2). Use of the Islet1 (Isl1)-GFP transgenic line enabled precise real-time observations of the growth cone behaviour of the Vp and VII motoneurons within BA1 and BA2. Screening for N-ethyl-N-nitrosourea-induced mutants identified seven distinct mutations affecting different steps in the axonal pathfinding of these motoneurons. The class 1 mutations caused severe defasciculation and abnormal pathfinding in both Vp and VII motor axons before they reached their target muscles in BA1. The class 2 mutations caused impaired axonal outgrowth of the Vp motoneurons at the BA1-BA2 boundary. The class 3 mutation caused impaired axonal outgrowth of the Vp motoneurons within the target muscles derived from BA1 and BA2. The class 4 mutation caused retraction of the Vp motor axons in BA1 and abnormal invasion of the VII motor axons in BA1 beyond the BA1-BA2 boundary. Time-lapse observations of the class 1 mutant, vermicelli (vmc), which has a defect in the plexin A3 (plxna3) gene, revealed that Plxna3 acts with its ligand Sema3a1 for fasciculation and correct target selection of the Vp and VII motor axons after separation from the common pathways shared with the sensory axons in BA1 and BA2, and for the proper exit and outgrowth of the axons of the primary motoneurons from the spinal cord.     

Presenilin-dependent receptor processing is required for axon guidance

The Alzheimer's disease-linked gene presenilin is required for intramembrane proteolysis of amyloid-β precursor protein, contributing to the pathogenesis of neurodegeneration that is characterized by loss of neuronal connections, but the role of Presenilin in establishing neuronal connections is less clear. Through a forward genetic screen in mice for recessive genes affecting motor neurons, we identified the Columbus allele, which disrupts motor axon projections from the spinal cord. We mapped this mutation to the Presenilin-1 gene. Motor neurons and commissural interneurons in Columbus mutants lacking Presenilin-1 acquire an inappropriate attraction to Netrin produced by the floor plate because of an accumulation of DCC receptor fragments within the membrane that are insensitive to Slit/Robo silencing. Our findings reveal that Presenilin-dependent DCC receptor processing coordinates the interplay between Netrin/DCC and Slit/Robo signaling. Thus, Presenilin is a key neural circuit builder that gates the spatiotemporal pattern of guidance signaling, thereby ensuring neural projections occur with high fidelity.     

Neurotrophic factor NT-3 displays a non-canonical cell guidance signaling function for cephalic neural crest cells

Chemotactic cell migration is triggered by extracellular concentration gradients of molecules segregated by target fields. Neural crest cells (NCCs), paradigmatic as an accurately moving cell population, undergo wide dispersion along multiple pathways, invading with precision defined sites of the embryo to differentiate into many derivatives. This report addresses the involvement of NT-3 in early colonization by cephalic NCCs invading the optic vesicle region. The results of in vitro and in vivo approaches showed that NCCs migrate directionally up an NT-3 concentration gradient. We also demonstrated the expression of NT-3 in the ocular region as well as their functional TrkB, TrkC and p75 receptors on cephalic NCCs. On whole-mount embryo, a perturbed distribution of NCCs colonizing the optic vesicle target field was shown after morpholino cancelation of cephalic NT-3 or TrkC receptor on NCCs, as well as in situ blocking of TrkC receptor of mesencephalic NCCs by specific antibody released from inserted microbeads. The present results strongly suggest that, among other complementary cell guidance factor(s), the chemotactic response of NCCs toward the ocular region NT-3 gradient is essential for spatiotemporal cell orientation, amplifying the functional scope of this neurotrophic factor as a molecular guide for the embryo cells, besides its well-known canonical functions.     

Repulsive axon guidance molecule Sema3A inhibits branching morphogenesis of fetal mouse lung

Semaphorin III/collapsin-1 (Sema3A) guides a specific subset of neuronal growth cones as a repulsive molecule. In this study, we have investigated a possible role of non-neuronal Sema3A in lung morphogenesis. Expression of mRNAs of Sema3A and neuropilin-1 (NP-1), a Sema3A receptor, was detected in fetal and adult lungs. Sema3A-immunoreactive cells were found in airway and alveolar epithelial cells of the fetal and adult lungs. Immunoreactivity for NP-1 was seen in fetal and adult alveolar epithelial cells as well as endothelial cells. Immunoreactivity of collapsin response mediator protein CRMP (CRMP-2), an intracellular protein mediating Sema3A signaling, was localized in alveolar epithelial cells, nerve tissue and airway neuroendocrine cells. The expression of CRMP-2 increased during the fetal, neonate and adult periods, and this pattern paralleled that of NP-1. In a two-day culture of lung explants from fetal mouse lung (E11.5), with exogenous Sema3A at a dose comparable to that which induces growth cone collapse of dorsal root ganglia neurons, the number of terminal buds was reduced in a dose-dependent manner when compared with control or untreated lung explants. This decrease was not accompanied with any alteration of the bromodeoxyuridine-positive DNA-synthesizing fraction. A soluble NP-1 lacking the transmembrane and intracellular region, neutralized the inhibitory effect of Sema3A. The fetal lung explants from neuropilin-1 homozygous null mice grew normally in vitro regardless of Sema3A treatment. These results provide evidence that Sema3A inhibits branching morphogenesis in lung bud organ cultures via NP-1 as a receptor or a component of a possible multimeric Sema3A receptor complex.