Regulate Axon Guidance Research Articles

Functional analysis of the Caenorhabditis elegans UNC-73B PH domain demonstrates a role in activation of the Rac GTPase in vitro and axon guidance in vivo.

The Caenorhabditis elegans UNC-73B protein regulates axon guidance through its ability to act as a guanine nucleotide exchange factor (GEF) for the CeRAC/MIG-2 GTPases. Like other GEFs for Rho family GTPases, UNC-73B has a Dbl homology (DH) catalytic domain, followed by a C-terminal pleckstrin homology (PH) domain. We have explored whether the PH domain cooperates with the adjacent DH domain to promote UNC-73B GEF activity and axonal pathfinding. We show that the UNC-73B PH domain binds preferentially to monophosphorylated phosphatidylinositides in vitro. Replacement of residues Lys1420 and Arg1422 with Glu residues within the PH domain impaired this phospholipid binding but did not affect the in vitro catalytic activity of the DH domain. In contrast, a mutant UNC-73B protein with a Trp1502-to-Ala substitution in the PH domain still interacted with phosphorylated phosphatidylinositides but had lost its GEF activity. UNC-73B minigenes containing these mutations were microinjected into C. elegans and transferred to unc-73(e936) mutant worms. Unlike the wild-type protein, neither PH domain mutant was able to rescue the unc-73 axon guidance defect. These results suggest that the UNC-73B PH domain plays distinct roles in targeting and promoting GEF activity towards the Rac GTPase, both of which are important for the directed movements of motorneurons in vivo.

Two divergent slit1 genes in zebrafish.

Members of the Slit family regulate axon guidance and cell migration. To date, three vertebrate slit1 genes have been identified in mammals and orthologs of two, slit2 and slit3, have been identified in zebrafish. Here, we describe the cloning of full-length cDNAs for two zebrafish slit orthologs, slit1a and slit1b. Both predicted proteins contain the conserved motifs that characterize other vertebrate Slits. slit1a and slit1b are both expressed in the midline, hypochord, telencephalon, and hindbrain. Apart from these shared expression domains, however, their expression patterns largely differ. Whereas slit1a is expressed broadly in the central nervous system (CNS) and in the somites, pectoral fin buds, tail bud, and caudal fin folds, slit1b is expressed in the olfactory system throughout embryonic and larval development, and in the retina during larval stages. Their expression patterns, particularly that of slit1a, suggest that Slit proteins may have roles in tissue morphogenesis in addition to their established roles in axon guidance and cell migration.

The ligand-binding face of the semaphorins revealed by the high-resolution crystal structure of SEMA4D.

Semaphorins, proteins characterized by an extracellular sema domain, regulate axon guidance, immune function and angiogenesis. The crystal structure of SEMA4D (residues 1-657) shows the sema topology to be a seven-bladed beta-propeller, revealing an unexpected homology with integrins. The sema beta-propeller contains a distinctive 77-residue insertion between beta-strands C and D of blade 5. Blade 7 is followed by a domain common to plexins, semaphorins and integrins (PSI domain), which forms a compact cysteine knot abutting the side of the propeller, and an Ig-like domain. The top face of the beta-propeller presents prominent loops characteristic of semaphorins. In addition to limited contact between the Ig-like domains, the homodimer is stabilized through extensive interactions between the top faces in a sector of the beta-propeller used for heterodimerization in integrins. This face of the propeller also mediates ligand binding in integrins, and functional data for semaphorin-receptor interactions map to the equivalent surface.

Do filopodia enable the growth cone to find its way?

Ena/VASP family proteins are required for accurate axon pathfinding in developing Drosophila, Caenorhabditis elegans, and mouse nervous systems. Recent papers by Bear et al. and Svitkina et al. provide evidence that Ena/VASP proteins regulate actin filament elongation in the lamellipodia and filopodia of nonneuronal cells. Neuronal growth cones use filopodia as their "sensory organs" that steer the growth cone by reading guidance cues in the environment. Together, these studies suggest that Ena/VASP proteins regulate axon guidance by controlling the formation or rearrangement of growth cone filopodia.

Reelin is expressed in the accessory olfactory system, but is not a guidance cue for vomeronasal axons

Reelin is an extracellular matrix protein that regulates neuronal migration in the developing cerebral cortex, and axon outgrowth in the hippocampus. In the developing vomeronasal system, Reelin mRNA is expressed in perineural cells near the vomeronasal nerve, as well as in the vomeronasal organ, olfactory epithelium and olfactory and accessory olfactory bulbs, suggesting that it might regulate axon guidance or fasiculation. We tested that hypothesis by crossing reeler mice with VN12-IRES-tau-lacZ mice to investigate the role of reelin. The vomeronasal nerves are indistinguishable in normal and reeler mutant mice, strongly suggesting that Reelin does not provide a guidance cue for vomeronasal axons.

Target-Derived GFRα1 as an Attractive Guidance Signal for Developing Sensory and Sympathetic Axons via Activation of Cdk5

Immobilized and diffusible molecular cues regulate axon guidance during development. GFRα1, a GPI-anchored receptor for GDNF, is expressed as both membrane bound and secreted forms by accessory nerve cells and peripheral targets of developing sensory and sympathetic neurons during the period of target innervation. A relative deficit of GFRα1 in developing axons allows exogenous GFRα1 to capture GDNF and present it for recognition by axonal c-Ret receptors. Exogenous GFRα1 potentiates neurite outgrowth and acts as a long-range directional cue by creating positional information for c-Ret-expressing axons in the presence of a uniform concentration of GDNF. Soluble GFRα1 prolongs GDNF-mediated activation of cyclin-dependent kinase 5 (Cdk5), an event required for GFRα1-induced neurite outgrowth and axon guidance. Together with GDNF, target-derived GFRα1 can function in a non-cell-autonomous fashion as a chemoattractant cue with outgrowth promoting activity for peripheral neurons.

B plexins activate Rho through PDZ-RhoGEF

Plexins are receptors for the repulsive axon guidance molecules semaphorins. Previously, we have shown that plexin-B1 binds activated Rac, but that clustering of plexin-B1 causes Rho activation, resulting in stress fiber formation. Using the yeast two-hybrid system, we found that the C-terminus of B plexins interacted directly with Rho-specific exchange factors, via their PDZ domain. Mutation of the carboxy-terminal amino acids of plexin-B1 or coexpression of a dominant negative PDZ-RhoGEF abrogated the ability of plexin-B1 to cause stress fiber formation. Our results demonstrate a role for PDZ-RhoGEF in B plexin-mediated activation of Rho/Rho kinase signaling, implicated in the regulation of axon guidance and cell migration.

Expression of Vema in the developing mouse spinal cord and optic chiasm.

A critical phase of nervous system development is the formation of connections between axons and their synaptic targets. Intermediate targets play important roles in axon pathfinding by supplying growing axons with long- and short- range guidance cues at decision points along their trajectory. We recently identified Vema as a novel membrane-associated protein that is expressed at the ventral midline of the developing vertebrate central nervous system (CNS). We report that Vema is expressed in the floor plate, an intermediate target for pathfinding commissural axons located at the ventral midline of the developing mouse spinal cord. Interestingly, Vema expression overlaps with the position of an unique population of neurons situated at the midline of the ventral diencephalon and that function as intermediate targets for pathfinding retinal ganglion cell axons. The distribution of Vema in the developing spinal cord and optic chiasm resembles the expression patterns of a variety of molecules known to play important roles in axon guidance, including Robo2, Neuropilin2, and SSEA. The expression of Vema at two key choice points for pathfinding axons suggests an important role for this protein in regulating axon guidance at the midline of the developing mouse central nervous system.

NGF Enhances Sensory Axon Growth Induced by Laminin but Not by the L1 Cell Adhesion Molecule

Neurotrophins and cell adhesion molecules regulate axon guidance, but their potential coordinate interactions are not well defined. In particular, it has been difficult to define the role of signaling from different surface molecules in neurotrophin-induced axon growth because of the strong dependence of embryonic neurons on this class of molecules for survival. We have addressed this issue using Bax deficient neurons, which do not require neurotrophins for survival. The L1 neural cell adhesion molecule and laminin each supported NGF-independent axon growth of cultured sensory neurons from dorsal root ganglia of embryonic Bax−/− mice. However, nerve growth factor (NGF) stimulated additional axon growth of sensory neurons on laminin but not on L1 substrates. Inhibition of the small GTPase RhoA by the dominant-negative mutant RhoA(T19N) restored NGF responsiveness of axon growth on L1 to Bax−/− neurons. Constitutively activated RhoA(Q63L) did not affect axon growth on L1 but inhibited NGF-stimulated axon growth on laminin. Consistent with the concept that RhoA was downregulated by NGF in neurons on laminin but not L1, the RhoA inhibitor C2IN-C3 toxin stimulated axon growth on L1 in wild-type DRG neurons in NGF. These results demonstrate a novel substrate-dependent regulation of NGF-induced growth of embryonic sensory axons mediated by RhoA GTPase.

Proteoglycans in retina.

In this article, we summarize the roles of proteoglycans in retinal tissue. Chondroitin sulfate and heparan sulfate proteoglycans are the major constituents in proteoglycans expressed in retinal tissue. Soluble heparan sulfate proteoglycans are found in the extracellular matrices of the basement membrane, such as the inner limiting membrane and Bruch's membrane, whereas heparan sulfate proteoglycans with their membrane-binding domain are localized primarily in the neurites of retinal neuronal cells, indicating their role as receptors for cytokines. The distribution of chondroitin sulfate proteoglycans is classified into two regions: nerve fiber-rich layers such as the optic nerve, inner plexiform layer and outer plexiform layer, and the interphotoreceptor matrix (IPM). The expression in the nerve fiber-rich layers of several chondroitin sulfate proteoglycans, such as neurocan and phosphacan, is restricted in the nervous tissues, and is upregulated as retinal development proceeds, then decreases after maturation of the retina. In vitro data suggest that these proteoglycans regulate axon guidance and synapse formation during the development of nervous tissue. In contrast, in adult vertebrate retina, the IPM is a rich source of chondroitin sulfate proteoglycans. Histologic data from animals with experimental retinitis pigmentosa, and the existence of the hyaluronan-binding domain in their core proteins, indicate that these proteoglycans contribute to the structural link between the neural retina and retinal pigment epithelium via the interaction with hyaluronan, which is also abundant in the IPM. Furthermore, several chondroitin sulfate proteoglycans in the nerve fiber-rich layers contain the hyaluronan-binding domain, so it is likely that the interaction of proteoglycans with hyaluronan plays an important role in neural network formation in the central nervous system.

Ectopic Expression in the Giant Fiber System ofDrosophila Reveals Distinct Roles for Roundabout (Robo), Robo2, and Robo3 in Dendritic Guidance and Synaptic Connectivity

The Roundabout (Robo) receptors have been intensively studied for their role in regulating axon guidance in the embryonic nervous system, whereas a role in dendritic guidance has not been explored. In the adult giant fiber system of Drosophila, we have revealed that ectopic Robo expression can regulate the growth and guidance of specific motor neuron dendrites, whereas Robo2 and Robo3 have no effect. We also show that the effect of Robo on dendritic guidance can be suppressed by Commissureless coexpression. Although we confirmed a role for all three Robo receptors in giant fiber axon guidance, the strong axon guidance alterations caused by overexpression of Robo2 or Robo3 have no effect on synaptic connectivity. In contrast, Robo overexpression in the giant fiber seems to directly interfere with synaptic function. We conclude that axon guidance, dendritic guidance, and synaptogenesis are separable processes and that the different Robo family members affect them distinctly.

Ectopic expression in the giant fiber system of Drosophila reveals distinct roles for roundabout (Robo), Robo2, and Robo3 in dendritic guidance and synaptic connectivity.

The Roundabout (Robo) receptors have been intensively studied for their role in regulating axon guidance in the embryonic nervous system, whereas a role in dendritic guidance has not been explored. In the adult giant fiber system of Drosophila, we have revealed that ectopic Robo expression can regulate the growth and guidance of specific motor neuron dendrites, whereas Robo2 and Robo3 have no effect. We also show that the effect of Robo on dendritic guidance can be suppressed by Commissureless coexpression. Although we confirmed a role for all three Robo receptors in giant fiber axon guidance, the strong axon guidance alterations caused by overexpression of Robo2 or Robo3 have no effect on synaptic connectivity. In contrast, Robo overexpression in the giant fiber seems to directly interfere with synaptic function. We conclude that axon guidance, dendritic guidance, and synaptogenesis are separable processes and that the different Robo family members affect them distinctly.

Slit Branches Out

The secreted proteins of the Slit family have been characterized as chemorepellents that regulate axon guidance and neuronal migration. However, they also stimulate branching of dorsal root ganglion axons. Whitford et al . now show that Slit stimulates branching of cortical neuronal dendrites as well. Slit and its receptor Robo are expressed during the time when the migrating patterns of neurons and axons are elaborating the layers of the cerebral cortex. As expected, exogenously added and endogenously expressed Slit repelled axons of cultured cortical neurons. However, Slit stimulated the total number of neuronal dendrites and dendritic branch points, as well as dendritic length. Expression of a dominant negative Robo receptor inhibited the stimulatory effect of Slit on dendrites. Hence, branch formation may be a general property of Slit, making these secreted proteins bifunctional chemotropic cues that differentially regulate axonal and dendritic patterning in the developing mammalian brain. K. L. Whitford, V. Marillat, E. Stein, C. S. Goodman, M. Tessier-Lavigne, A. Chedotal, A. Ghosh, Regulation of cortical dendrite development by Slit-Robo interactions. Neuron 33 , 47-61 (2001). [Online Journal]

Regulation of Cortical Dendrite Development by Slit-Robo Interactions

Slit proteins have previously been shown to regulate axon guidance, branching, and neural migration. Here we report that, in addition to acting as a chemorepellant for cortical axons, Slit1 regulates dendritic development. Slit1 is expressed in the developing cortex, and exposure to Slit1 leads to increased dendritic growth and branching. Conversely, inhibition of Slit-Robo interactions by Robo-Fc fusion proteins or by a dominant-negative Robo attenuates dendritic branching. Stimulation of neurons transfected with a Met-Robo chimeric receptor with Hepatocyte growth factor leads to a robust induction of dendritic growth and branching, suggesting that Robo-mediated signaling is sufficient to induce dendritic remodeling. These experiments indicate that Slit-Robo interactions may exert a significant influence over the specification of cortical neuron morphology by regulating both axon guidance and dendritic patterning.

Regulation of CNS and motor axon guidance in Drosophila by the receptor tyrosine phosphatase DPTP52F.

Receptor-linked protein tyrosine phosphatases (RPTPs) regulate axon guidance and synaptogenesis in Drosophila embryos and larvae. We describe DPTP52F, the sixth RPTP to be discovered in Drosophila. Our genomic analysis indicates that there are likely to be no additional RPTPs encoded in the fly genome. Five of the six Drosophila RPTPs have C. elegans counterparts, and three of the six are also orthologous to human RPTP subfamilies. DPTP52F, however, has no clear orthologs in other organisms. The DPTP52F extracellular domain contains five fibronectin type III repeats and it has a single phosphatase domain. DPTP52F is selectively expressed in the CNS of late embryos, as are DPTP10D, DLAR, DPTP69D and DPTP99A. To define developmental roles of DPTP52F, we used RNA interference (RNAi)-induced phenotypes as a guide to identify Ptp52F alleles among a collection of EMS-induced lethal mutations. Ptp52F single mutant embryos have axon guidance phenotypes that affect CNS longitudinal tracts. This phenotype is suppressed in Dlar Ptp52F double mutants, indicating that DPTP52F and DLAR interact competitively in regulating CNS axon guidance decisions. Ptp52F single mutations also cause motor axon phenotypes that selectively affect the SNa nerve. DPTP52F, DPTP10D and DPTP69D have partially redundant roles in regulation of guidance decisions made by axons within the ISN and ISNb motor nerves.

Dynamic regulation of axon guidance.

To reach their proper targets, axons rely upon the actions of highly conserved families of attractive and repulsive guidance molecules, including the netrins, Slits, semaphorins and ephrins. These guidance systems are used to generate an astonishingly varied set of neuronal circuits. Here we consider the mechanisms by which a few guidance systems can be used to generate diverse outcomes. Recent studies have revealed extensive transcriptional and post-transcriptional regulation of guidance cues and their receptors, as well as combinatorial mechanisms that integrate information from different families of guidance cues.

Development and reorganization of corticospinal projections in EphA4 deficient mice

The Eph family of receptor tyrosine kinases and their ligands, the ephrins, are important regulators of axon guidance and cell migration in the developing nervous system. Inactivation of the EphA4 gene results in axon guidance defects of the corticospinal tract, a major descending motor pathway that originates in the cortex and terminates at all levels of the spinal cord. In this investigation, we report that although the initial development of the corticospinal projection is normal through the cortex, internal capsule, cerebral peduncle, and medulla in the brain of EphA4 deficient animals, corticospinal axons exhibit gross abnormalities when they enter the gray matter of the spinal cord. Notably, many corticospinal axons fail to remain confined to one side of the spinal cord during development and instead, aberrantly project across the midline, terminating ipsilateral to their cells of origin. Given the possible repulsive interactions between EphA4 and one of its ligands, ephrinB3, this defect could be consistent with a loss of responsiveness by corticospinal axons to ephrinB3 that is expressed at the spinal cord midline. Furthermore, we show that EphA4 deficient animals exhibit ventral displacement of the mature corticospinal termination pattern, suggesting that developing corticospinal axons, which may also express ephrinB3, fail to be repelled from areas of high EphA4 expression in the intermediate zone of the normal spinal cord. Taken together, these results suggest that the dual expression of EphA4 on corticospinal axons and also within the surrounding gray matter is very important for the correct development and termination of the corticospinal projection within the spinal cord. J. Comp. Neurol. 436:248–262, 2001. © 2001 Wiley-Liss, Inc.

Crystal Structure of an Ephrin Ectodomain

Eph receptor tyrosine kinases and their membrane-associated ligands, the ephrins, are essential regulators of axon guidance, cell migration, segmentation, and angiogenesis. There are two classes of vertebrate ephrin ligands which have distinct binding specificities for their cognate receptors. Multimerization of the ligands is required for receptor activation, and ephrin ligands themselves signal intracellularly upon binding Eph receptors. We have determined the structure of the extracellular domain of mouse ephrin-B2. The ephrin ectodomain is an eight-stranded β barrel with topological similarity to plant nodulins and phytocyanins. Based on the structure, we have identified potential surface determinants of Eph/ephrin binding specificity and a ligand dimerization region. The high sequence similarity among ephrin ectodomains indicates that all ephrins may be modeled upon the ephrin-B2 structure presented here.

Development and reorganization of corticospinal projections in EphA4 deficient mice

The Eph family of receptor tyrosine kinases and their ligands, the ephrins, are important regulators of axon guidance and cell migration in the developing nervous system. Inactivation of the EphA4 gene results in axon guidance defects of the corticospinal tract, a major descending motor pathway that originates in the cortex and terminates at all levels of the spinal cord. In this investigation, we report that although the initial development of the corticospinal projection is normal through the cortex, internal capsule, cerebral peduncle, and medulla in the brain of EphA4 deficient animals, corticospinal axons exhibit gross abnormalities when they enter the gray matter of the spinal cord. Notably, many corticospinal axons fail to remain confined to one side of the spinal cord during development and instead, aberrantly project across the midline, terminating ipsilateral to their cells of origin. Given the possible repulsive interactions between EphA4 and one of its ligands, ephrinB3, this defect could be consistent with a loss of responsiveness by corticospinal axons to ephrinB3 that is expressed at the spinal cord midline. Furthermore, we show that EphA4 deficient animals exhibit ventral displacement of the mature corticospinal termination pattern, suggesting that developing corticospinal axons, which may also express ephrinB3, fail to be repelled from areas of high EphA4 expression in the intermediate zone of the normal spinal cord. Taken together, these results suggest that the dual expression of EphA4 on corticospinal axons and also within the surrounding gray matter is very important for the correct development and termination of the corticospinal projection within the spinal cord.

The Trio family of guanine-nucleotide-exchange factors: regulators of axon guidance.

Axon guidance requires the integration of diverse guidance signals presented by numerous extracellular cues and cell-cell interactions. The molecular mechanisms that interpret these signals involve networks of intracellular signaling proteins that coordinate a variety of responses to the environment, including remodeling and assembly of the actin cytoskeleton. Although it has been clear for some time that Rho family GTPases play a central role in the orchestration of cytoskeletal assembly, our understanding of the components that regulate these important molecules is far more primitive. Recent functional studies of the Trio family of guanine-nucleotide-exchange factors reveal that Trio proteins play a vital role in neuronal cell migration and axon guidance. Although the molecular analysis of Trio proteins is still in its infancy, accumulated evidence suggests that Trio proteins function as integrators of multiple upstream inputs and as activators of multiple downstream pathways. Future studies of these mechanisms promise to yield insights not only into neural development but also into the ongoing function and remodeling of the adult nervous system.