Roundabout signalling, cell contact and trophic support confine longitudinal glia and axons in the Drosophila CNS

Contrary to our knowledge of the genetic control of midline crossing, the mechanisms that generate and maintain the longitudinal axon pathways of the Drosophila CNS are largely unknown. The longitudinal pathways are formed by ipsilateral pioneer axons and the longitudinal glia. The longitudinal glia dictate these axonal trajectories and provide trophic support to later projecting follower neurons. Follower interneuron axons cross the midline once and join these pathways to form the longitudinal connectives. Once on the contralateral side, longitudinal axons are repelled from recrossing the midline by the midline repulsive signal Slit and its axonal receptor Roundabout. We show that longitudinal glia also transiently express roundabout, which halts their ventral migration short of the midline. Once in contact with axons, glia cease to express roundabout and become dependent on neurons for their survival. Trophic support and cell-cell contact restrict glial movement and axonal trajectories. The significance of this relationship is revealed when neuron-glia interactions are disrupted by neuronal ablation or mutation in the glial cells missing gene, which eliminates glia, when axons and glia cross the midline despite continued midline repellent signalling.     

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.     

Axon repulsion from the midline of the Drosophila CNS requires slit function

Guidance of axons towards or away from the midline of the central nervous system during Drosophila embryogenesis reflects a balance of attractive and repulsive cues originating from the midline. Here we demonstrate that Slit, a protein secreted by the midline glial cells provides a repulsive cue for the growth cones of axons and muscle cells. Embryos lacking slit function show a medial collapse of lateral axon tracts and ectopic midline crossing of ventral muscles. Transgene expression of slit in the midline restores axon patterning. Ectopic expression of slit inhibits formation of axon tracts at locations of high Slit production and misdirects axon tracts towards the midline. slit interacts genetically with roundabout, which encodes a putative receptor for growth cone repulsion.     

Son of sevenless directly links the Robo receptor to rac activation to control axon repulsion at the midline

Son of sevenless (Sos) is a dual specificity guanine nucleotide exchange factor (GEF) that regulates both Ras and Rho family GTPases and thus is uniquely poised to integrate signals that affect both gene expression and cytoskeletal reorganization. Here, using genetics, biochemistry, and cell biology, we demonstrate that Sos is recruited to the plasma membrane, where it forms a ternary complex with the Roundabout receptor and the SH3-SH2 adaptor protein Dreadlocks (Dock) to regulate Rac-dependent cytoskeletal rearrangement in response to the Slit ligand. Intriguingly, the Ras and Rac-GEF activities of Sos can be uncoupled during Robo-mediated axon repulsion; Sos axon guidance function depends on its Rac-GEF activity, but not its Ras-GEF activity. These results provide in vivo evidence that the Ras and RhoGEF domains of Sos are separable signaling modules and support a model in which Robo recruits Sos to the membrane via Dock to activate Rac during midline repulsion.     

Hedgehog regulated Slit expression determines commissure and glial cell position in the zebrafish forebrain

Three major axon pathways cross the midline of the vertebrate forebrain early in embryonic development: the postoptic commissure (POC), the anterior commissure (AC) and the optic nerve. We show that a small population of Gfap+ astroglia spans the midline of the zebrafish forebrain in the position of, and prior to, commissural and retinal axon crossing. These glial ;bridges' form in regions devoid of the guidance molecules slit2 and slit3, although a subset of these glial cells express slit1a. We show that Hh signaling is required for commissure formation, glial bridge formation, and the restricted expression of the guidance molecules slit1a, slit2, slit3 and sema3d, but that Hh does not appear to play a direct role in commissural and retinal axon guidance. Reducing Slit2 and/or Slit3 function expanded the glial bridges and caused defasciculation of the POC, consistent with a ;channeling' role for these repellent molecules. By contrast, reducing Slit1a function led to reduced midline axon crossing, suggesting a distinct role for Slit1a in midline axon guidance. Blocking Slit2 and Slit3, but not Slit1a, function in the Hh pathway mutant yot (gli2DR) dramatically rescued POC axon crossing and glial bridge formation at the midline, indicating that expanded Slit2 and Slit3 repellent function is largely responsible for the lack of midline crossing in these mutants. This analysis shows that Hh signaling helps to pattern the expression of Slit guidance molecules that then help to regulate glial cell position and axon guidance across the midline of the forebrain.     

Navigating Intermediate Targets: The Nervous System Midline

In a bilaterally symmetric animal, the midline plays a key role in directing axon growth during wiring of the nervous system. Midline cells provide a variety of guidance cues for growing axons, to which different types of axons respond in different ways and at different times. For some axons, the midline is an intermediate target. They first seek it out, but then move on towards their final targets on the opposite side. For others, the midline is a repulsive barrier that keeps them on their own side of the midline. And for many of these axons the midline provides signals that guide them along specific lateral pathways or up and down the longitudinal axis.The complex guidance decisions at the midline have made it a particularly fascinating model for investigating the mechanisms and logic of axon pathfinding. Much of this work has focused on the ventral midline of the vertebrate spinal cord and its Drosophila analog, the ventral nerve cord. This work has sought to explain why some axons cross the midline, whereas others do not; why these axons respond differently to midline cues before and after crossing; and how the midline directs axon traffic along the lateral pathways. Striking similarities, as well as intriguing differences, have been documented in the way these guidance decisions are regulated in vertebrates and in Drosophila. Here, we briefly introduce the two systems, then review our current understanding of each of the key guidance decisions, and finally discuss some of the general principles and open questions that have emerged from these studies.     

Semaphorin 3d guides laterality of retinal ganglion cell projections in zebrafish

The optic chiasm is an important choice point at which retinal ganglion cell (RGC) axons either cross the midline to innervate the contralateral brain or turn back to innervate the ipsilateral brain. Guidance cues that regulate this decision, particularly those directing the midline crossing of contralateral axons, are still not well understood. Here we show that Sema3d, a secreted semaphorin expressed at the midline, guides the crossing of RGC axons in zebrafish. Both Sema3d knockdown and ubiquitous overexpression induced aberrant ipsilateral projections, suggesting that Sema3d normally guides axons into the contralateral optic tract. Live imaging in vivo showed that RGC growth cones responded to ubiquitous Sema3d overexpression by pausing for extended periods and increasing their exploratory behavior at the midline, suggesting that Sema3d overexpression causes the midline environment to become less favorable for RGC axon extension. Interestingly, Sema3d overexpression did not affect growth cone behaviors before the midline, suggesting that RGC axons normally respond to Sema3d only upon reaching the midline. After Sema3d knockdown, growth cones grew across the midline but then paused or repeatedly retracted, impairing their ability to leave the midline region. Our results indicate that a proper balance of Sema3d is needed at the midline for the progression of RGC axons from the chiasm midline into the contralateral optic tract.     

Wnt/calcium signaling mediates axon growth and guidance in the developing corpus callosum

It has been shown in vivo that Wnt5a gradients surround the corpus callosum and guide callosal axons after the midline (postcrossing) by Wnt5a-induced repulsion via Ryk receptors. In dissociated cortical cultures we showed that Wnt5a simultaneously promotes axon outgrowth and repulsion by calcium signaling. Here to test the role of Wnt5a/calcium signaling in a complex in vivo environment we used sensorimotor cortical slices containing the developing corpus callosum. Plasmids encoding the cytoplasmic marker DsRed and the genetically encoded calcium indicator GCaMP2 were electroporated into one cortical hemisphere. Postcrossing callosal axons grew 50% faster than pre-crossing axons and higher frequencies of calcium transients in axons and growth cones correlated well with outgrowth. Application of pharmacological inhibitors to the slices showed that signaling pathways involving calcium release through IP3 receptors and calcium entry through TRP channels regulate post-crossing axon outgrowth and guidance. Co-electroporation of Ryk siRNA and DsRed revealed that knock down of the Ryk receptor reduced outgrowth rates of postcrossing but not precrossing axons by 50% and caused axon misrouting. Guidance errors in axons with Ryk knockdown resulted from reduced calcium activity. In the corpus callosum CaMKII inhibition reduced the outgrowth rate of postcrossing (but not precrossing) axons and caused severe guidance errors which resulted from reduced CaMKII-dependent repulsion downstream of Wnt/calcium. We show for the first time that Wnt/Ryk calcium signaling mechanisms regulating axon outgrowth and repulsion in cortical cultures are also essential for the proper growth and guidance of postcrossing callosal axons which involve axon repulsion through CaMKII.     

GAP-43 promoter elements in transgenic zebrafish reveal a difference in signals for axon growth during CNS development and regeneration

A pivotal event in neural development is the point at which differentiating neurons become competent to extend long axons. Initiation of axon growth is equally critical for regeneration. Yet we have a limited understanding of the signaling pathways that regulate the capacity for axon growth during either development or regeneration. Expression of a number of genes encoding growth associated proteins (GAPs) accompanies both developmental and regenerative axon growth and has led to the suggestion that the same signaling pathways regulate both modes of axon growth. We have tested this possibility by asking whether a promoter fragment from a well characterized GAP gene, GAP-43, is sufficient to activate expression in both developing and regenerating neurons. We generated stable lines of transgenic zebrafish that express green fluorescent protein (GFP) under regulation of a 1 kb fragment of the rat GAP-43 gene, a fragment that contains a number of evolutionarily conserved elements. Analysis of GFP expression in these lines confirms that the rat 1 kb region can direct growth-associated expression of the transgene in differentiating neurons that extend long axons. Furthermore, this region supports developmental down-regulation of transgene expression which, like the endogenous gene, coincides with neuronal maturation. Strikingly, these same sequences are insufficient for directing expression in regenerating neurons. This finding suggests that signaling pathways regulating axon growth during development and regeneration are not the same. While these results do not exclude the possibility that pathways involved in developmental axon growth are also active in regenerative growth, they do indicate that signaling pathway(s) controlling activation of the GAP-43 gene after CNS injury differ in at least one key component from the signals controlling essential features of developmental axon growth.     

DCC plays a role in navigation of forebrain axons across the ventral midbrain commissure in embryonic xenopus

In the developing vertebrate brain, growing axons establish a scaffold of axon tracts connected across the midline via commissures. We have previously identified a population of telencephalic neurons that express NOC-2, a novel glycoform of the neural cell adhesion molecule N-CAM that is involved in axon guidance in the forebrain. These axons arise from the presumptive telencephalic nucleus, course caudally along the principal longitudinal tract of the forebrain, cross the ventral midline in the midbrain, and then project to the contralateral side of the brain. In the present study we have investigated mechanisms controlling the growth of these axons across the ventral midline of the midbrain. The axon guidance receptor DCC is expressed by the NOC-2 population of axons both within the longitudinal tract and within the ventral midbrain commissure. Disruption of DCC-dependent interactions, both in vitro and in vivo, inhibited the NOC-2 axons from crossing the ventral midbrain. Instead, these axons grew along aberrant trajectories away from the midline, suggesting that DCC-dependent interactions are important for overcoming inhibitory mechanisms within the midbrain of the embryonic vertebrate brain. Thus, coordinated responsiveness of forebrain axons to both chemostimulatory and chemorepulsive cues appears to determine whether they cross the ventral midline in the midbrain.     

Abelson tyrosine kinase is required to transduce midline repulsive cues

Tyrosine phosphorylation-dependent signaling cascades play key roles in determining the formation of an axon pathway. The cytoplasmic Abelson tyrosine kinase participate in several signaling pathways that orchestrate both growth cone advance and steering in response to guidance cues. Here, a genetic approach is used to evaluate the role for Abelson in growth cones during a decision to cross or not to cross the Drosophila embryonic midline. Our data indicate that both loss- and gain-of-function conditions for Abl cause neurons within the pCC/MP2 pathway to project across the midline incorrectly. The frequency of abnormal crossovers is enhanced by mutations in the genes encoding the midline repellent, Slit, or its receptor, Roundabout. In comm mutants, where repulsive signals remain elevated, increasing or decreasing Abl activity partially rescues commissure formation. Thus, both too much and too little Abl activity causes axons to cross the midline inappropriately, indicating that Abl plays a critical role in transducing midline repulsive cues. How Abl functions in this role is not yet clear, but we suggest that Abl may help regulate cytoskeletal dynamics underlying a growth cone's response to midline cues.     

GAP-43 mediates retinal axon interaction with lateral diencephalon cells during optic tract formation

GAP-43 is an abundant intracellular growth cone protein that can serve as a PKC substrate and regulate calmodulin availability. In mice with targeted disruption of the GAP-43 gene, retinal ganglion cell (RGC) axons fail to progress normally from the optic chiasm into the optic tracts. The underlying cause is unknown but, in principle, can result from either the disruption of guidance mechanisms that mediate axon exit from the midline chiasm region or defects in growth cone signaling required for entry into the lateral diencephalic wall to form the optic tracts. Results here show that, compared to wild-type RGC axons, GAP-43-deficient axons exhibit reduced growth in the presence of lateral diencephalon cell membranes. Reduced growth is not observed when GAP-43-deficient axons are cultured with optic chiasm, cortical, or dorsal midbrain cells. Lateral diencephalon cell conditioned medium inhibits growth of both wild-type and GAP-43-deficient axons to a similar extent and does not affect GAP-43-deficient axons more so. Removal or transplant replacement of the lateral diencephalon optic tract entry zone in GAP-43-deficient embryo preparations results in robust RGC axon exit from the chiasm. Together these data show that RGC axon exit from the midline region does not require GAP-43 function. Instead, GAP-43 appears to mediate RGC axon interaction with guidance cues in the lateral diencephalic wall, suggesting possible involvement of PKC and calmodulin signaling during optic tract formation.     

Frizzled-3a and slit2 genetically interact to modulate midline axon crossing in the telencephalon

The anterior commissure forms the first axon connections between the two sides of the embryonic telencephalon. We investigated the role of the transmembrane receptor Frizzled-3a in the development of this commissure using zebrafish as an experimental model. Knock down of Frizzled-3a resulted in complete loss of the anterior commissure. This defect was accompanied by a loss of the glial bridge, expansion of the slit2 expression domain and perturbation of the midline telencephalic-diencephalic boundary. Blocking Slit2 activity following knock down of Frizzled-3a effectively rescued the anterior commissure defect which suggested that Frizzled-3a was indirectly controlling the growth of axons across the rostral midline. We have shown here that Frizzled-3a is essential for normal development of the commissural plate and that loss-of-function causes Slit2-dependent defects in axon midline crossing in the embryonic vertebrate forebrain. These data supports a model whereby Wnt signaling through Frizzled-3a attenuates expression of Slit2 in the rostral midline of the forebrain. The absence of Slit2 facilitates the formation of a midline bridge of glial cells which is used as a substrate for commissural axons. In the absence of this platform of glia, commissural axons fail to cross the rostral midline of the forebrain.     

Cross-midline interactions between mouse commissural hindbrain axons contribute to their efficient decussation

Information from both sides of the brain is integrated by axons that project across the midline of the central nervous system via numerous commissures present at all axial levels. Despite the accumulated experimental evidence, questions remain regarding the formation of commissures in the presence of strong repulsive signals in the ventral midline. Studies from invertebrates suggest that interaction at the midline between homologous axons of specific decussating neurons contributes to efficient midline crossing, but such evidence is lacking in vertebrate systems. We performed experiments to determine whether commissural axons of the caudal region of the hindbrain interact with their contralateral counterparts at the ventral midline and to evaluate the relevance of this reciprocal interaction. Double anterograde axon labeling with lipophilic tracers revealed close apposition between growth cones of contralateral pioneer decussating axons at the midline. Later, we detected fasciculation between contralateral axons that is maintained even after they have crossed the midline. Blocking axon projections unilaterally with a solid mechanical barrier decreased dramatically the midline crossing of the equivalent population from the contralateral side. Decussation was also blocked by a unilateral barrier permeable to diffusible molecules but not by an axon-permeable barrier. These results suggest that in the caudal region of the hindbrain, midline crossing is facilitated by interactions between decussating contralateral axon partners.     

A novel Eph receptor-interacting IgSF protein provides C. elegans motoneurons with midline guidepost function

BACKGROUND: The ventral midline is a prominent structure in vertebrate and invertebrate nervous systems that provides crucial topological information for guiding axons to their appropriate target destinations. Rather than being composed of specialized midline glia cells as in many other species, the embryonic midline of the nematode Caenorhabditis elegans is physically defined by motoneuron cell bodies that separate the left from the right ventral cord fascicles. Their function during development, if any, is not known. RESULTS: We show here that besides being components of the postembryonic locomotory circuit, these embryonic motoneurons (eMNs) actively provide midline guidance information for a specific subset of ventral midline axons. This information is provided in the form of a novel, cell-surface-anchored immunoglobulin superfamily (IgSF) member, WRK-1. WRK-1 acts in eMNs to prevent follower axons from inappropriately crossing the ventral midline. We describe the function of the Eph receptor vab-1 and multiple ephrin ligands at the midline, and we show by double mutant analysis and physical interaction tests that WRK-1 functionally interacts with the Eph receptor system. This interaction appears to occur exclusively in the context of axon guidance at the ventral midline but not in other cellular contexts, thereby suggesting that Eph receptor signaling is mechanistically distinct in different tissue types. CONCLUSIONS: Our studies reveal cellular and molecular components of axon midline patterning and suggest that Ephrin signaling relies on previously unknown accessory components.     

Slit1 and Slit2 cooperate to prevent premature midline crossing of retinal axons in the mouse visual system.

During development, retinal ganglion cell (RGC) axons either cross or avoid the midline at the optic chiasm. In Drosophila, the Slit protein regulates midline axon crossing through repulsion. To determine the role of Slit proteins in RGC axon guidance, we disrupted Slit1 and Slit2, two of three known mouse Slit genes. Mice defective in either gene alone exhibited few RGC axon guidance defects, but in double mutant mice a large additional chiasm developed anterior to the true chiasm, many retinal axons projected into the contralateral optic nerve, and some extended ectopically-dorsal and lateral to the chiasm. Our results indicate that Slit proteins repel retinal axons in vivo and cooperate to establish a corridor through which the axons are channeled, thereby helping define the site in the ventral diencephalon where the optic chiasm forms.     

The Abelson tyrosine kinase, the Trio GEF and Enabled interact with the Netrin receptor Frazzled in Drosophila

The attractive Netrin receptor Frazzled (Fra), and the signaling molecules Abelson tyrosine kinase (Abl), the guanine nucleotide-exchange factor Trio, and the Abl substrate Enabled (Ena), all regulate axon pathfinding at the Drosophila embryonic CNS midline. We detect genetic and/or physical interactions between Fra and these effector molecules that suggest that they act in concert to guide axons across the midline. Mutations in Abl and trio dominantly enhance fra and Netrin mutant CNS phenotypes, and fra;Abl and fra;trio double mutants display a dramatic loss of axons in a majority of commissures. Conversely, heterozygosity for ena reduces the severity of the CNS phenotype in fra, Netrin and trio,Abl mutants. Consistent with an in vivo role for these molecules as effectors of Fra signaling, heterozygosity for Abl, trio or ena reduces the number of axons that inappropriately cross the midline in embryos expressing the chimeric Robo-Fra receptor. Fra interacts physically with Abl and Trio in GST-pulldown assays and in co-immunoprecipitation experiments. In addition, tyrosine phosphorylation of Trio and Fra is elevated in S2 cells when Abl levels are increased. Together, these data suggest that Abl, Trio, Ena and Fra are integrated into a complex signaling network that regulates axon guidance at the CNS midline.     

Receptor tyrosine phosphatases in axon growth and guidance

Receptor-like protein tyrosine phosphatases (RPTPs) continue to emerge as important signalling molecules in axons and their growth cones. Recent findings show that Drosophila RPTPs play key roles in guiding retinal axons and in preventing midline crossing of longitudinal axons. Vertebrate RPTPs are now implicated in controlling axon outgrowth, and preliminary evidence suggests that they too may influence axon guidance.     

Axon pathfinding proceeds normally despite disrupted growth cone decisions at CNS midline

Axons in the bilateral brain of Drosophila decide whether or not to cross the midline before following their specific subsequent pathways. In commissureless mutants, the RP3 and V motoneuron axons often fail to cross the midline but subsequently follow the mirror-image pathways and innervate corresponding muscle targets on the ipsilateral side. Conversely, in roundabout mutants, the RP2 and aCC motoneuron axons sometimes cross the midline abnormally but their subsequent pathways and synaptic targeting are the perfect mirror images of those seen in wild type. Furthermore, within a single segment of these mutants, bilateral pairs of motoneuron axons can make their midline decisions independently of each other. Thus, neither the growth cones' particular molecular experience nor the decision at the midline caused by these mutations affects their ability to respond normally to subsequently presented cues.     

Evidence for a role of srGAP3 in the positioning of commissural axons within the ventrolateral funiculus of the mouse spinal cord

Slit-Robo signaling guides commissural axons away from the floor-plate of the spinal cord and into the longitudinal axis after crossing the midline. In this study we have evaluated the role of the Slit-Robo GTPase activating protein 3 (srGAP3) in commissural axon guidance using a knockout (KO) mouse model. Co-immunoprecipitation experiments confirmed that srGAP3 interacts with the Slit receptors Robo1 and Robo2 and immunohistochemistry studies showed that srGAP3 co-localises with Robo1 in the ventral and lateral funiculus and with Robo2 in the lateral funiculus. Stalling axons have been reported in the floor-plate of Slit and Robo mutant spinal cords but our axon tracing experiments revealed no dorsal commissural axon stalling in the floor plate of the srGAP3 KO mouse. Interestingly we observed a significant thickening of the ventral funiculus and a thinning of the lateral funiculus in the srGAP3 KO spinal cord, which has also recently been reported in the Robo2 KO. However, axons in the enlarged ventral funiculus of the srGAP3 KO are Robo1 positive but do not express Robo2, indicating that the thickening of the ventral funiculus in the srGAP3 KO is not a Robo2 mediated effect. We suggest a role for srGAP3 in the lateral positioning of post crossing axons within the ventrolateral funiculus.