Molecular gradients along the proximal-distal axis of embryonic insect legs: possible guidance cues of pioneer axon growth.

It has been proposed that gradients of environmental cues direct the proximal growth of pioneer axons in embryonic insect legs. Hybridoma techniques have been used to produce 3 monoclonal antibodies (mAbs) that bind to components associated with the basal lamina/extracellular matrix that are non-uniformly distributed along the proximal-distal axis of cockroach legs at the time of pioneer axon growth. Two of these mAbs, PROD-1 and PROD-2, label the proximal parts of the leg more intensely than the distal ends. The other mAb, DIP-1, has the reverse pattern of binding with the distal parts of the leg labeled more intensely. The graded distribution of these antigens only occurs just prior to and during the growth period of the Ti1 pioneer axons. Western blot analyses and immunoprecipitations have identified the protein antigens recognized by these mAbs. The spatial and temporal distributions of these molecules in the legs and the CNS make them good candidates for environmental guidance cues of pioneer axon growth.     

Error correction during guidance of pioneer axons in the leg of the cockroach embryo

Axons of the Til and Fe2 pioneer neurons in the legs of insect embryos possess separate and highly stereotyped proximal projections towards the CNS. However, quantitative analyses of deviations from the standard paths during the period of axon growth indicate that transient errors occur unexpectedly often. The distribution of legs with axons following deviant paths among the embryos analyzed is used to determine whether these errors are caused by random developmental noise or by non-random genetic or environmental factors. During the formation of the Til pathway all the errors are characterized by defasciculation of the 2 axons, occur with an average incidence of 7% and are statistically shown to be randomly caused. In comparison, during the formation of the Fe2 pathway the errors are characterized by both defasciculation and elongation in an inappropriate distal direction, occur with an incidence of 16%, and as revealed by statistical analyses, are caused by a non-random factor. Therefore, during pathfinding by these 2 pairs of axons there is a need for error-correcting mechanisms to insure the stereotypy of the final projections. These error-correcting mechanisms are suggested to have properties similar to those producing canalization as proposed by Waddington.     

A role for proteoglycans in the guidance of a subset of pioneer axons in cultured embryos of the cockroach.

Several molecules involved in the development of the nervous system have specific binding sites for the glycosaminoglycan (GAG) side chains of proteoglycans. Exogenous GAGs should bind to these sites, competitively inhibit interactions with proteoglycans, and perturb development. GAGs added to the culture medium perturb the in situ growth of pioneer axons in cultured cockroach embryos by producing axon defasciculation and growth in incorrect directions. The specificity of this phenomenon is evident from the following observations: Of all the GAGs tested only heparin and heparan sulfate produced perturbation; of the six axon tracts being pioneered during the culture period only two of them are perturbed by the GAGs; and similar perturbations are produced when embryos are cultured in the presence of heparinase II and heparitinase.     

The unc-5, unc-6, and unc-40 genes guide circumferential migrations of pioneer axons and mesodermal cells on the epidermis in C. elegans

Three known genes guide circumferential migrations of pioneer axons and mesodermal cells on the nematode body wall. unc-5 affects dorsal migrations, unc-40 primarily affects ventral migrations, and unc-6 affects migrations in both directions. Circumferential movements still occur, but are misdirected whereas longitudinal movements are normal in these mutants. Pioneer growth cones migrating directly on the epidermis are affected; growth cones migrating along established axon fascicles are normal. Thus these genes affect cell guidance and not cell motility per se. We propose that two opposite, adhesive gradients guide circumferential migrations on the epidermis. unc-5, unc-6, and unc-40 may encode these adhesion molecules or their cellular receptors. Neurons have access to the basal lamina and the basolateral surfaces of the epidermis, but mesodermal cells contact only the basal lamina. These genes probably identify molecular cues on the basal lamina that guide mesodermal migrations. The same basal lamina cues, or perhaps related molecules on the epidermal cell surfaces, guide pioneer neurons.     

Proteoglycans as cues for axonal guidance in formation of retinotectal or retinocollicular projections

Understanding the formation of neuronal circuits has long been one of the basic problems in developmental neurobiology. Projections from the retina to their higher center, the optic tectum in nonmammalian vertebrates and the superior colliculus in mammals, are most amenable to experimental approaches; thus, much information has been accumulated about the mechanisms of axonal guidance. The retinal axons navigate along the appropriate pathway with the help of a series of guidance cues. Although much of the work has focused on proteinaceous factors, proteoglycans have been identified as playing important roles in retinal axon guidance. Chondroitin sulfate proteoglycans and heparan sulfate proteoglycans are involved in essential decisions of axon steering along the pathway. However, it has not been determined whether diversity of the carbohydrate chains results in differential effects and how their diversity is recognized by growth cones, which represent an important area of future research.     

Hierarchical guidance cues in the developing nervous system of C. elegans

During embryogenesis, the basic axon scaffold of the nervous system is formed by special axons that pioneer pathways between groups of cells. To find their way, the pioneer growth cones detect specific cues in their extracellular environment. One of these guidance cues is netrin. Observations and experimental manipulations in vertebrates and nematodes have shown that netrin is a bifunctional guidance cue that can simultaneously attract and repel axons. During the formation of this basic axon scaffold in Caenorhabditis elegans, the netrin UNC-6 is expressed by neuroglia and pioneer neurons, providing hierarchical guidance cues throughout the animal. Each cue has a characteristic role depending on the cell type, its position and the developmental stage. These roles include activities as global, decussation and labeled-pathway cues. This hierarchical model of UNC-6 netrin-mediated guidance suggests a method by which guidance cues can direct formation of basic axon scaffolds in developing nervous systems.     

Glypican Is a Modulator of Netrin-Mediated Axon Guidance

Netrin is a key axon guidance cue that orients axon growth during neural circuit formation. However, the mechanisms regulating netrin and its receptors in the extracellular milieu are largely unknown. Here we demonstrate that in Caenorhabditis elegans, LON-2/glypican, a heparan sulfate proteoglycan, modulates UNC-6/netrin signaling and may do this through interactions with the UNC-40/DCC receptor. We show that developing axons misorient in the absence of LON-2/glypican when the SLT-1/slit guidance pathway is compromised and that LON-2/glypican functions in both the attractive and repulsive UNC-6/netrin pathways. We find that the core LON-2/glypican protein, lacking its heparan sulfate chains, and secreted forms of LON-2/glypican are functional in axon guidance. We also find that LON-2/glypican functions from the epidermal substrate cells to guide axons, and we provide evidence that LON-2/glypican associates with UNC-40/DCC receptor–expressing cells. We propose that LON-2/glypican acts as a modulator of UNC-40/DCC-mediated guidance to fine-tune axonal responses to UNC-6/netrin signals during migration.     

Semaphorin 5A is a bifunctional axon guidance cue regulated by heparan and chondroitin sulfate proteoglycans

The response of neuronal growth cones to axon guidance cues depends on the developmental context in which these cues are encountered. We show here that the transmembrane protein semaphorin 5A (Sema5A) is a bifunctional guidance cue exerting both attractive and inhibitory effects on developing axons of the fasciculus retroflexus, a diencephalon fiber tract associated with limbic function. The thrombospondin repeats of Sema5A physically interact with the glycosaminoglycan portion of both chondroitin sulfate proteoglycans (CSPGs) and heparan sulfate proteoglycans (HSPGs). CSPGs function as precisely localized extrinsic cues that convert Sema5A from an attractive to an inhibitory guidance cue. Therefore, glycosaminoglycan bound guidance cues provide a molecular mechanism for CSPG-mediated inhibition of axonal extension. Further, axonal HSPGs are required for Sema5A-mediated attraction, suggesting that HSPGs are components of functional Sema5A receptors. Thus, neuronal responses to Sema5A are proteoglycan dependent and interpreted according to the biological context in which this membrane bound guidance cue is presented.     

Axon guidance and somites

The segmental arrangement of spinal nerves in higher vertebrate embryos provides a simple system in which to study the factors that influence axon pathfinding. Developing motor and sensory axons are intimately associated with surrounding tissues that direct axon guidance. We argue that two distinct guidance mechanisms, viz. contact repulsion and chemorepulsion, act simultaneously to prescribe spinal axon trajectories by ’surround-repulsion’. Motor and sensory axons grow freely within the anterior half of each mesodermal somite, because they are excluded from posterior half-somites by contact repulsion. By contrast, the dorsoventral trajectory that bipolar sensory axons of the dorsal root ganglia follow is governed by diffusible repellents originating from the notochord medially and dermamyotome laterally. Even though spinal nerve development appears to be a simple system for elucidating axon guidance mechanisms, many distinct candidate guidance molecules have been implicated and their relative contributions remain to be evaluated. Received: 28 May 1997 / Accepted: 27 June 1997     

Pioneer longitudinal axons navigate using floor plate and Slit/Robo signals

Longitudinal axons transmit all signals between the brain and spinal cord. Their axon tracts through the brain stem are established by a simple set of pioneer axons with precise trajectories parallel to the floor plate. To identify longitudinal guidance mechanisms in vivo, the overall role of floor plate tissue and the specific roles of Slit/Robo signals were tested. Ectopic induction or genetic deletion of the floor plate diverted longitudinal axons into abnormal trajectories. The expression patterns of the diffusible cues of the Slit family were altered in the floor plate experiments, suggesting their involvement in longitudinal guidance. Genetic tests of Slit1 and Slit2, and the Slit receptors Robo1 and Robo2 were carried out in mutant mice. Slit1;Slit2 double mutants had severe longitudinal errors, particularly for ventral axons, including midline crossing and wandering longitudinal trajectories. Robo1 and Robo2 were largely genetically redundant, and neither appeared to specify specific tract positions. However, combined Robo1 and Robo2 mutations strongly disrupted each pioneer tract. Thus, pioneer axons depend on long-range floor plate cues, with Slit/Robo signaling required for precise longitudinal trajectories.     

Heparan sulfate proteoglycans are ligands for receptor protein tyrosine phosphatase sigma

RPTPsigma is a cell adhesion molecule-like receptor protein tyrosine phosphatase involved in nervous system development. Its avian orthologue, known as cPTPsigma or CRYPalpha, promotes intraretinal axon growth and controls the morphology of growth cones. The molecular mechanisms underlying the functions of cPTPsigma are still to be determined, since neither its physiological ligand(s) nor its substrates have been described. Nevertheless, a major class of ligand(s) is present in the retinal basal lamina and glial endfeet, the potent native growth substrate for retinal axons. We demonstrate here that cPTPsigma is a heparin-binding protein and that its basal lamina ligands include the heparan sulfate proteoglycans (HSPGs) agrin and collagen XVIII. These molecules interact with high affinity with cPTPsigma in vitro, and this binding is totally dependent upon their heparan sulfate chains. Using molecular modelling and site-directed mutagenesis, a binding site for heparin and heparan sulfate was identified in the first immunoglobulin-like domain of cPTPsigma. HSPGs are therefore a novel class of heterotypic ligand for cPTPsigma, suggesting that cPTPsigma signaling in axons and growth cones is directly responsive to matrix-associated cues.     

C. elegans dystroglycan coordinates responsiveness of follower axons to dorsal/ventral and anterior/posterior guidance cues

Neural development in metazoans is characterized by the establishment of initial process tracts by pioneer axons and the subsequent extension of follower axons along these pioneer processes. Mechanisms governing the fidelity of follower extension along pioneered routes are largely unknown. In C. elegans, formation of the right angle‐shaped lumbar commissure connecting the lumbar and preanal ganglia is an example of pioneer/follower dynamics. We find that the dystroglycan ortholog DGN‐1 mediates the fidelity of follower lumbar commissure axon extension along the pioneer axon route. In dgn‐1 mutants, the axon of the pioneer PVQ neuron faithfully establishes the lumbar commissure, but axons of follower lumbar neurons, such as PVC, frequently bypass the lumbar commissure and extend along an oblique trajectory directly toward the preanal ganglion. In contrast, disruption of the UNC‐6/netrin guidance pathway principally perturbs PVQ ventral guidance to pioneer the lumbar commissure. Loss of DGN‐1 in unc‐6 mutants has a quantitatively similar effect on follower axon guidance regardless of PVQ axon route, indicating that DGN‐1 does not mediate follower/pioneer adhesion. Instead, DGN‐1 appears to block premature responsiveness of follower axons to a preanal ganglion‐directed guidance cue, which mediates ventral‐to‐anterior reorientation of lumbar commissure axons. Deletion analysis shows that only the most N‐terminal DGN‐1 domain is required for these activities. These studies suggest that dystroglycan modulation of growth cone responsiveness to conflicting guidance cues is important for restricting follower axon extension to the tracts laid down by pioneers. © 2011 Wiley Periodicals, Inc. Develop Neurobiol, 2012     

Zebrafish topped is required for ventral motor axon guidance

Zebrafish primary motor axons extend along stereotyped pathways innervating distinct regions of the developing myotome. During development, these axons make stereotyped projections to ventral and dorsal myotome regions. Caudal primary motoneurons, CaPs, pioneer axon outgrowth along ventral myotomes; whereas, middle primary motoneurons, MiPs, extend axons along dorsal myotomes. Although the development and axon outgrowth of these motoneurons has been characterized, cues that determine whether axons will grow dorsally or ventrally have not been identified. The topped mutant was previously isolated in a genetic screen designed to uncover mutations that disrupt primary motor axon guidance. CaP axons in topped mutants fail to enter the ventral myotome at the proper time, stalling at the nascent horizontal myoseptum, which demarcates dorsal from ventral axial muscle. Later developing secondary motor nerves are also delayed in entering the ventral myotome whereas all other axons examined, including dorsally projecting MiP motor axons, are unaffected in topped mutants. Genetic mosaic analysis indicates that Topped function is non-cell autonomous for motoneurons, and when wild-type cells are transplanted into topped mutant embryos, ventromedial fast muscle are the only cell type able to rescue the CaP axon defect. These data suggest that Topped functions in the ventromedial fast muscle and is essential for motor axon outgrowth into the ventral myotome.     

Syndecan regulates cell migration and axon guidance in C. elegans

During nervous system development, axons that grow out simultaneously in the same extracellular environment are often sorted to different target destinations. As there is only a restricted set of guidance cues known, regulatory mechanisms are likely to play a crucial role in controlling cell migration and axonal pathfinding. Heparan sulfate proteoglycans (HSPGs) carry long chains of differentially modified sugar residues that have been proposed to encode specific information for nervous system development. Here, we show that the cell surface proteoglycan syndecan SDN-1 functions autonomously in neurons to control the neural migration and guidance choices of outgrowing axons. Epistasis analysis suggests that heparan sulfate (HS) attached to SDN-1 can regulate guidance signaling by the Slit/Robo pathway. Furthermore, SDN-1 acts in parallel with other HSPG core proteins whose HS side chains are modified by the C5-epimerase HSE-5, and/or the 2O-sulfotransferase HST-2, depending on the cellular context. Taken together, our experiments show that distinct HS modification patterns on SDN-1 are involved in regulating axon guidance and cell migration in C. elegans.     

Axon routing at the optic chiasm after enzymatic removal of chondroitin sulfate in mouse embryos

The effects of removing chondroitin sulfate from chondroitin sulfate proteoglycan molecules on guidance of retinal ganglion cell axons at the optic chiasm were investigated in a brain slice preparation of mouse embryos of embryonic day 13 to 15. Slices were grown for 5 hours and growth of dye-labeled axons was traced through the chiasm. After continuous enzymatic digestion of the chondroitin sulfate proteoglycans with chondroitinase ABC, which removes the glycosaminoglycan chains, navigation of retinal axons was disrupted. At embryonic day 13, before the uncrossed projection forms in normal development, many axons deviated from their normal course, crossing the midline at aberrant positions and invading the ventral diencephalon. In slices from embryonic day 14 embryos, axons that would normally form the uncrossed projection at this stage failed to turn into the ipsilateral optic tract. In embryonic day 15 slices, enzyme treatment caused a reduction of the uncrossed projection that develops at this stage. Growth cones in enzyme-treated slices showed a significant increase in the size both before and after they crossed the midline. This indicates that responses of retinal axons to guidance signals at the chiasm have changed after removal of the chondroitin sulfate epitope. We concluded that the chondroitin sulfate moieties of the proteoglycans are involved in patterning the early phase of axonal growth across the midline and at a later stage controlling the axon divergence at the chiasm.     

The heparan sulfate proteoglycans Dally-like and Syndecan have distinct functions in axon guidance and visual-system assembly in Drosophila

Heparan sulfate proteoglycans (HSPGs), a class of glycosaminoglycan-modified proteins, control diverse patterning events via their regulation of growth-factor signaling and morphogen distribution. In C. elegans, zebrafish, and the mouse, heparan sulfate (HS) biosynthesis is required for normal axon guidance, and mutations affecting Syndecan (Sdc), a transmembrane HSPG, disrupt axon guidance in Drosophila embryos. Glypicans, a family of glycosylphosphatidylinositol (GPI)-linked HSPGs, are expressed on axons and growth cones in vertebrates, but their role in axon guidance has not been determined. We demonstrate here that the Drosophila glypican Dally-like protein (Dlp) is required for proper axon guidance and visual-system function. Mosaic studies revealed that Dlp is necessary in both the retina and the brain for different aspects of visual-system assembly. Sdc mutants also showed axon guidance and visual-system defects, some that overlap with dlp and others that are unique. dlp+ transgenes were able to rescue some sdc visual-system phenotypes, but sdc+ transgenes were ineffective in rescuing dlp abnormalities. Together, these findings suggest that in some contexts HS chains provide the biologically critical component, whereas in others the structure of the protein core is also essential.     

Function of Neurolin (DM-GRASP/SC-1) in guidance of motor axons during zebrafish development.

Neurolin (zf DM-GRASP), a transmembrane protein with five extracellular immunoglobulin domains, is expressed by secondary but not primary motoneurons during zebrafish development. The spatiotemporally restricted expression pattern suggests that Neurolin plays a role in motor axon growth and guidance. To test this hypothesis, we injected zebrafish embryos with function-blocking Neurolin antibodies. In injected embryos, secondary motor axons form a broadened bundle along the common path and ectopic branches leave the common path at right angles. Moreover, the formation of the ventral and the rostral projection of secondary motor axons is inhibited during the second day of development. Pathfinding errors, resulting in secondary motor axons growing through ectopic regions of the somites, occur along the common path and in the dorsal and rostral projection. Our data are compatible with the view that Neurolin is involved in the recognition of guidance cues and acts as a receptor on secondary motor axons. Consistent with this idea is the binding pattern of a soluble Neurolin-Fc construct showing that putative ligands are distributed along the common path, the ventral projection, and in the area where the rostral projection develops.     

Migration of zebrafish spinal motor nerves into the periphery requires multiple myotome-derived cues

In vertebrate embryos, spinal motor neurons project through segmentally reiterated nerves into the somites. Here, we report that zebrafish secondary motor neurons, which are similar to motor neurons in birds and mammals, depend on myotomal cues to navigate into the periphery. We show that the absence of myotomal adaxial cells in you-too/gli2 embryos severely impairs secondary motor axonal pathfinding, including their ability to project into the somites. Moreover, in diwanka mutant embryos, in which adaxial cells are present but fail to produce cues essential for primary motor growth cones to pioneer into the somites, secondary motor axons display similar pathfinding defects. The similarities between the axonal defects in you-too/gli2 and diwanka mutant embryos strongly suggest that pathfinding of secondary motor axons depends on myotome-derived cues, and that the diwanka gene is a likely candidate to produce or encode such a cue. Our experiments also demonstrate that diwanka plays a central role in the migration of primary and secondary motor neurons, suggesting that both neural populations share mechanisms underlying axonal pathfinding. In summary, we provide compelling evidence that myotomal cells produce multiple signals to initiate and control the migration of spinal nerve axons into the somites.     

The zebrafish unplugged gene controls motor axon pathway selection

En route to their targets, motor axons encounter choice points at which they select their future path. Experimental studies predict that at each choice point specialized cells provide local guidance to pathfinding motor axons, however, the identity of these cells and their signals is unknown. Here, we identify the zebrafish unplugged gene as a key component for choice point navigation of pioneering motor axons. We show that in unplugged mutant embryos, motor neuron growth cones reach the choice point but make inappropriate pathway decisions. Analysis of chimeric embryos demonstrates that unplugged activity is produced by a selective group of mesodermal cells located adjacent to the choice point. As the first motor growth cones approach the choice point, these mesodermal cells migrate away, suggesting that unplugged activity influences growth cones by a contact-independent mechanism. These data suggest that unplugged defines a somite-derived signal that elicits differential guidance decisions in motor growth cones.