ENU-3 is a novel motor axon outgrowth and guidance protein in C. elegans

During the development of the nervous system, the migration of many cells and axons is guided by extracellular molecules. These molecules bind to receptors at the tips of the growth cones of migrating axons and trigger intracellular signaling to steer the axons along the correct trajectories. We have identified a novel mutant, enu-3 (enhancer of Unc), that enhances the motor neuron axon outgrowth defects observed in strains of Caenorhabditis elegans that lack either the UNC-5 receptor or its ligand UNC-6/Netrin. Specifically, the double-mutant strains have enhanced axonal outgrowth defects mainly in DB4, DB5 and DB6 motor neurons. enu-3 single mutants have weak motor neuron axon migration defects. Both outgrowth defects of double mutants and axon migration defects of enu-3 mutants were rescued by expression of the H04D03.1 gene product. ENU-3/H04D03.1 encodes a novel predicted putative trans-membrane protein of 204 amino acids. It is a member of a family of highly homologous proteins of previously unknown function in the C. elegans genome. ENU-3 is expressed in the PVT interneuron and is weakly expressed in many cell bodies along the ventral cord, including those of the DA and DB motor neurons. We conclude that ENU-3 is a novel C. elegans protein that affects both motor axon outgrowth and guidance.     

Trio mediates netrin-1-induced Rac1 activation in axon outgrowth and guidance

The chemotropic guidance cue netrin-1 promotes neurite outgrowth through its receptor Deleted in Colorectal Cancer (DCC) via activation of Rac1. The guanine nucleotide exchange factor (GEF) linking netrin-1/DCC to Rac1 activation has not yet been identified. Here, we show that the RhoGEF Trio mediates Rac1 activation in netrin-1 signaling. We found that Trio interacts with the netrin-1 receptor DCC in mouse embryonic brains and that netrin-1-induced Rac1 activation in brain is impaired in the absence of Trio. Trio(-/-) cortical neurons fail to extend neurites in response to netrin-1, while they are able to respond to glutamate. Accordingly, netrin-1-induced commissural axon outgrowth is reduced in Trio(-/-) spinal cord explants, and the guidance of commissural axons toward the floor plate is affected by the absence of Trio. The anterior commissure is absent in Trio-null embryos, and netrin-1/DCC-dependent axonal projections that form the internal capsule and the corpus callosum are defective in the mutants. Taken together, these findings establish Trio as a GEF that mediates netrin-1 signaling in axon outgrowth and guidance through its ability to activate Rac1.     

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.     

Role of myosin II in axon outgrowth.

The initial stages of nerve outgrowth carried out by growth cones occur in three fundamental cyclic steps. Each of these steps appears to require myosin II activity to variable degrees. The steps include the following: (a) exploration, involving extensions and retractions that are driven and controlled by the interaction of actin retrograde flow and polymerization; (b) adhesion of new extensions to the substrate, which has been shown to be mediated by complex interactions between extracellular matrix proteins, cell adhesion proteins, and the actin cytoskeleton; and (c) traction force generated during forward advance of the growth cone, resulting in the production of tension on the neurite.     

Promoting and directing axon outgrowth

Establishment of appropriate neuronal connections during development and regeneration requires the extension of processes that must then grow in the correct direction, find and recognize their targets, and make synapses with them. During development, embryonic neurons gradually establish central and peripheral connections in an evolving cellular environment in which neurotrophic factors are provided by supporting and target cells that promote neuronal survival, differentiation, and process outgrowth. Some cells also release neurotropic factors that direct the outgrowth of neuronal processes toward their targets. Following development the neurotrophic requirements of some adult neurons change so that, although they respond to neurotrophic factors, they no longer require exogenous neurotrophins to survive or to extend processes. Within the central nervous system (CNS), the ability of neurons to extend processes is eventually lost because of a change in their cellular environment from outgrowth permissive to inhibitory. Thus, neuronal connections that are lost in the adult CNS are rarely reestablished. In contrast, the environment of the adult peripheral nervous system fosters process outgrowth and synapse formation. This article discusses the neurotrophic requirements of embryonic and adult neurons, as well as the importance of neurotropic factors in directing the outgrowth of regenerating adult axons.     

The Beat generation: a multigene family encoding IgSF proteins related to the Beat axon guidance molecule in Drosophila.

A previous genetic screen led to the identification of the beaten path (beat Ia) gene in Drosophila. Beat Ia contains two immunoglobulin (Ig) domains and appears to function as an anti-adhesive factor secreted by specific growth cones to promote axon defasciculation. We identify a family of 14 beat-like genes in Drosophila. In contrast to beat Ia, four novel Beat-family genes encode membrane-bound proteins. Moreover, mutations in each gene lead to much more subtle guidance phenotypes than observed in beat Ia. Genetic interactions between beat Ic and beat Ia reveal complementary functions. Our data suggest a model whereby Beat Ic (and perhaps other membrane-bound family members) functions in a pro-adhesive fashion to regulate fasciculation, while Beat Ia (the original secreted Beat) functions in an anti-adhesive fashion to regulate defasciculation.     

Vav family GEFs link activated Ephs to endocytosis and axon guidance

Ephrin signaling through Eph receptor tyrosine kinases can promote attraction or repulsion of axonal growth cones during development. However, the mechanisms that determine whether Eph signaling promotes attraction or repulsion are not known. We show here that the Rho family GEF Vav2 plays a key role in this process. We find that, during axon guidance, ephrin binding to Ephs triggers Vav-dependent endocytosis of the ligand-receptor complex, thus converting an initially adhesive interaction into a repulsive event. In the absence of Vav proteins, ephrin-Eph endocytosis is blocked, leading to defects in growth cone collapse in vitro and significant defects in the ipsilateral retinogeniculate projections in vivo. These findings suggest an important role for Vav family GEFs as regulators of ligand-receptor endocytosis and determinants of repulsive signaling during axon guidance.     

Conserved roles for Slit and Robo proteins in midline commissural axon guidance

In Drosophila, Slit at the midline activates Robo receptors on commissural axons, thereby repelling them out of the midline into distinct longitudinal tracts on the contralateral side of the central nervous system. In the vertebrate spinal cord, Robo1 and Robo2 are expressed by commissural neurons, whereas all three Slit homologs are expressed at the ventral midline. Previous analysis of Slit1;Slit2 double mutant spinal cords failed to reveal a defect in commissural axon guidance. We report here that when all six Slit alleles are removed, many commissural axons fail to leave the midline, while others recross it. In addition, Robo1 and Robo2 single mutants show guidance defects that reveal a role for these two receptors in guiding commissural axons to different positions within the ventral and lateral funiculi. These results demonstrate a key role for Slit/Robo signaling in midline commissural axon guidance in vertebrates.     

Conservation and divergence of axon guidance mechanisms.

Analysis of axon guidance mechanisms in vertebrates, Caenorhabditis elegans, and Drosophila melanogaster has led to the identification of several signaling pathways, many of which are strikingly conserved in function. Recent studies indicate that several axon guidance mechanisms are highly conserved in all animals, whereas others, though still conserved in a general sense, show strong evolutionary divergence at a detailed mechanistic level.     

Structure and axon outgrowth inhibitor binding of the Nogo-66 receptor and related proteins

The myelin-derived proteins Nogo, MAG and OMgp limit axonal regeneration after injury of the spinal cord and brain. These cell-surface proteins signal through multi-subunit neuronal receptors that contain a common ligand-binding glycosylphosphatidylinositol-anchored subunit termed the Nogo-66 receptor (NgR). By deletion analysis, we show that the binding of soluble fragments of Nogo, MAG and NgR to cell-surface NgR requires the entire leucine-rich repeat (LRR) region of NgR, but not other portions of the protein. Despite sharing extensive sequence similarity with NgR, two related proteins, NgR2 and NgR3, which we have identified, do not bind Nogo, MAG, OMgp or NgR. To investigate NgR specificity and multi-ligand binding, we determined the crystal structure of the biologically active ligand-binding soluble ectodomain of NgR. The molecule is banana shaped with elongation and curvature arising from eight LRRs flanked by an N-terminal cap and a small C-terminal subdomain. The NgR structure analysis, as well as a comparison of NgR surface residues not conserved in NgR2 and NgR3, identifies potential protein interaction sites important in the assembly of a functional signaling complex.     

Cytoskeletal mechanisms of axon outgrowth and pathfinding

The use of modern techniques involving gene transfer and functional knock-out strategies has lead to new concepts of the way in which cytoskeletal elements interact to produce the unique morphologies of neurons. This review presents these concepts and discusses their implications for neuronal development, especially with respect to the role of microtubules, microfilaments, and neurofilaments. Received: 29 July 1997 / Accepted: 27 October 1997     

Semaphorins and their receptors in olfactory axon guidance.

The mammalian olfactory system is capable of discriminating among a large variety of odor molecules and is therefore essential for the identification of food, enemies and mating partners. The assembly and maintenance of olfactory connectivity have been shown to depend on the combinatorial actions of a variety of molecular signals, including extracellular matrix, cell adhesion and odorant receptor molecules. Recent studies have identified semaphorins and their receptors as putative molecular cues involved in olfactory pathfinding, plasticity and regeneration. The semaphorins comprise a large family of secreted and transmembrane axon guidance proteins, being either repulsive or attractive in nature. Neuropilins were shown to serve as receptors for secreted class 3 semaphorins, whereas members of the plexin family are receptors for class 1 and V (viral) semaphorins. The present review will discuss a role for semaphorins and their receptors in the establishment and maintenance of olfactory connectivity.     

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.     

Calmodulin and profilin coregulate axon outgrowth in Drosophila

Coordinated regulation of actin cytoskeletal dynamics is critical to growth cone movement. The intracellular molecules calmodulin and profilin actively regulate actin-based motility and participate in the signaling pathways used to steer growth cones. Here we show that in the developing Drosophila embryo, calmodulin and profilin convey complimentary information that is necessary for appropriate growth cone advance. Reducing calmodulin activity by expression of a dominant inhibitor (KA) stalls axon extension of pioneer neurons within the CNS, while a partial loss of profilin function decreases extension of motor axons in the periphery. Yet, surprisingly, when calmodulin and profilin are simultaneously reduced, the ability of both CNS pioneer axons and motor axons to extend beyond the choice points is restored. In the CNS, at the time when growth cones must decide whether to cross or not to cross the midline, a reduction in calmodulin and/or roundabout signaling causes axons to cross the midline inappropriately. These inappropriate crossings are suppressed when profilin activity is simultaneously reduced. Interestingly, the mutual suppression of calmodulin and profilin activity requires a minimal level of profilin. In KA combinations with profilin null alleles, defects in axon extension and midline guidance are synergistically enhanced rather than suppressed. Together, our data indicate that the growth cone must coordinate the activity of both calmodulin and profilin in order to advance past selected choice points, including those dictating midline crossovers.     

Reexpression of LGI1 in glioma cells results in dysregulation of genes implicated in the canonical axon guidance pathway

The LGI1 gene suppresses invasion in glioma cells and predisposes to epilepsy. In a gene expression array comparison between parental cells and T98G cell clones forced to express LGI1, we demonstrate that the canonical axon guidance pathway is the most significantly affected. In particular, aspects of axon guidance that involve reorganization of the actin cytoskeleton, which is also involved in cell movement and invasion, were affected. Analysis of actin fiber organization using fluorescence microscopy demonstrated that different T98G cell clones expressing the exogenous LGI1 gene show high levels of stress fibers compared with controls. Since stress fiber formation is associated with loss of cell mobility, we used scratch wound assays to demonstrate that LGI1-expressing clones show a significant reduction in cell mobility. LGI1 reexpression also resulted in loss of the PDGFRA and EGFR proteins, suggesting a rapid turnover of these receptors despite increased mRNA levels for PDGFRA. LGI1 suppression of invasion is associated with loss of ERK/MAPK1 activation. LGI1 is a secreted protein, and when the culture supernatant from cells expressing FLAG- and GFP-tagged proteins were applied to parental T98G cells, ERK/MAPK1 phosphorylation and cell mobility was suppressed, demonstrating that the LGI1 protein acts as a suppressive agent for cell movement in this assay. These observations support a previous suggestion that LGI1 can reduce cellular invasion in in vitro assays and, as a secreted agent, may be developed as a means of treating metastatic cancer. In addition, this observation provides a mechanistic link for LGI1's common role in metastasis and epilepsy development.     

Differential functions of the C. elegans FGF receptor in axon outgrowth and maintenance of axon position

Wiring of the nervous system requires that axons navigate to their targets and maintain their correct positions in axon fascicles after termination of axon outgrowth. We show here that the C. elegans fibroblast growth factor receptor (FGFR), EGL-15, affects both processes in fundamentally distinct manners. FGF-dependent activation of the EGL-15 tyrosine kinase and subsequently the GTPase LET-60/ras is required within epidermal cells, the substratum for most outgrowing axon, for appropriate outgrowth of specific axon classes to their target area. In contrast, genetic elimination of the FGFR isoform EGL-15(5A), defined by the inclusion of an alternative extracellular interimmunoglobulin domain, has no consequence for axon outgrowth but leads to a failure to postembryonically maintain axon position within defined axon fascicles. An engineered, secreted form of EGL-15(5A) containing only its ectodomain is sufficient for maintenance of axon position, thus providing novel insights into receptor tyrosine kinase function and the process of maintaining axon position.     

Molecular mechanisms of axon guidance

In order to form a functional nervous system, neurones extend axons, often over long distances, to reach their targets. This process is controlled by extracellular receptors and their ligands, several families of which have been identified. These proteins may act to either repel or attract growth cones and a given receptor may transduce either type of signal, depending on the cellular context. In addition to these archetypal axon guidance molecules, it is becoming apparent that molecules previously known for their role in patterning can also direct axonal outgrowth. The growth cone receptors do not act in isolation and combine with members of the same or other families to produce a graded response or even a complete reversal in its polarity. These signals can be further combined and/or modulated by processing of the molecule both directly at the cell surface and by the network of intracellular signalling pathways which are activated. The result is a sophisticated and dynamic set of cues that enable a growth cone to successfully navigate to its destination, modulating its response to changing environmental cues along its pathway.