Migration and function of glia in the developing Drosophila eye.

Although glial cells have been implicated widely in the formation of axon tracts in both insects and vertebrates, their specific function appears to be context-dependent, ranging from providing essential guidance cues to playing a merely facilitory role. Here we examine the role of the retinal basal glia (RBG) in photoreceptor axon guidance in Drosophila. The RBG originate in the optic stalk and have been thought to migrate into the eye disc along photoreceptor axons, thus precluding any role in axon guidance. Here we show the following. (1) The RBG can, in fact, migrate into the eye disc even in the absence of photoreceptor axons in the optic stalk; they also migrate to ectopic patches of differentiating photoreceptors without axons providing a continuous physical substratum. This suggests that glial cells are attracted into the eye disc not through haptotaxis along established axons, but through another mechanism, possibly chemotaxis. (2) If no glial cells are present in the eye disc, photoreceptor axons are able to grow and direct their growth posteriorly as in wild type, but are unable to enter the optic stalk. This indicates that the RBG have a crucial role in axon guidance, but not in axonal outgrowth per se. (3) A few glia close to the entry of the optic stalk suffice to guide the axons into the stalk, suggesting that glia instruct axons by local interaction.     

Sonic hedgehog is indirectly required for intraretinal axon pathfinding by regulating chemokine expression in the optic stalk

Successful axon pathfinding requires both correct patterning of tissues, which will later harbor axonal tracts, and precise localization of axon guidance cues along these tracts at the time of axon outgrowth. Retinal ganglion cell (RGC) axons grow towards the optic disc in the central retina, where they turn to exit the eye through the optic nerve. Normal patterning of the optic disc and stalk and the expression of guidance cues at this choice point are necessary for the exit of RGC axons out of the eye. Sonic hedgehog (Shh) has been implicated in both patterning of ocular tissue and direct guidance of RGC axons. Here, we examine the precise spatial and temporal requirement for Hedgehog (Hh) signaling for intraretinal axon pathfinding and show that Shh acts to pattern the optic stalk in zebrafish but does not guide RGC axons inside the eye directly. We further reveal an interaction between the Hh and chemokine pathways for axon guidance and show that cxcl12a functions downstream of Shh and depends on Shh for its expression at the optic disc. Together, our results support a model in which Shh acts in RGC axon pathfinding indirectly by regulating axon guidance cues at the optic disc through patterning of the optic stalk.     

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.     

Robo2 is required for Slit-mediated intraretinal axon guidance

The developing optic pathway has proven one of the most informative model systems for studying mechanisms of axon guidance. The first step in this process is the directed extension of retinal ganglion cell (RGC) axons within the optic fibre layer (OFL) of the retina towards their exit point from the eye, the optic disc. Previously, we have shown that the inhibitory guidance molecules, Slit1 and Slit2, regulate two distinct aspects of intraretinal axon guidance in a region-specific manner. Using knockout mice, we have found that both of these guidance activities are mediated via Robo2. Of the four vertebrate Robos, only Robo1 and Robo2 are expressed by RGCs. In mice lacking robo1 intraretinal axon guidance occurs normally. However, in mice lacking robo2 RGC axons make qualitatively and quantitatively identical intraretinal pathfinding errors to those reported previously in Slit mutants. This demonstrates clearly that, as in other regions of the optic pathway, Robo2 is the major receptor required for intraretinal axon guidance. Furthermore, the results suggest strongly that redundancy with other guidance signals rather than different receptor utilisation is the most likely explanation for the regional specificity of Slit function during intraretinal axon pathfinding.     

Ephrin‐B2 elicits differential growth cone collapse and axon retraction in retinal ganglion cells from distinct retinal regions

The circuit for binocular vision and stereopsis is established at the optic chiasm, where retinal ganglion cell (RGC) axons diverge into the ipsilateral and contralateral optic tracts. In the mouse retina, ventrotemporal (VT) RGCs express the guidance receptor EphB1, which interacts with the repulsive guidance cue ephrin‐B2 on radial glia at the optic chiasm to direct VT RGC axons ipsilaterally. RGCs in the ventral retina also express EphB2, which interacts with ephrin‐B2, whereas dorsal RGCs express low levels of EphB receptors. To investigate how growth cones of RGCs from different retinal regions respond upon initial contact with ephrin‐B2, we utilized time‐lapse imaging to characterize the effects of ephrin‐B2 on growth cone collapse and axon retraction in real time. We demonstrate that bath application of ephrin‐B2 induces rapid and sustained growth cone collapse and axon retraction in VT RGC axons, whereas contralaterally‐projecting dorsotemporal RGCs display moderate growth cone collapse and little axon retraction. Dose response curves reveal that contralaterally‐projecting ventronasal axons are less sensitive to ephrin‐B2 treatment compared to VT axons. Additionally, we uncovered a specific role for Rho kinase signaling in the retraction of VT RGC axons but not in growth cone collapse. The detailed characterization of growth cone behavior in this study comprises an assay for the study of Eph signaling in RGCs, and provides insight into the phenomena of growth cone collapse and axon retraction in general. © 2010 Wiley Periodicals, Inc. Develop Neurobiol 70: 781–794, 2010     

Distinct roles for Robo2 in the regulation of axon and dendrite growth by retinal ganglion cells

Guidance factors act on the tip of a growing axon to direct it to its target. What role these molecules play, however, in the control of the dendrites that extend from that axon’s cell body is poorly understood. Slits, through their Robo receptors, guide many types of axons, including those of retinal ganglion cells (RGCs). Here we assess and contrast the role of Slit/Robo signalling in the growth and guidance of the axon and dendrites extended by RGCs in Xenopus laevis. As Xenopus RGCs extend dendrites, they express robo2 and robo3, while slit1 and slit2 are expressed in RGCs and in the adjacent inner nuclear layer. Interestingly, our functional data with antisense knockdown and dominant negative forms of Robo2 (dnRobo2) and Robo3 (dnRobo3) indicate that Slit/Robo signalling has no role in RGC dendrite guidance, and instead is necessary to stimulate dendrite branching, primarily via Robo2. Our in vitro culture data argue that Slits are the ligands involved. In contrast, both dnRobo2 and dnRobo3 inhibited the extension of axons and caused the misrouting of some axons. Based on these data, we propose that Robo signalling can have distinct functions in the axon and dendrites of the same cell, and that the specific combinations of Robo receptors could underlie these differences. Slit acts via Robo2 in dendrites as a branching/growth factor but not in guidance, while Robo2 and Robo3 function in concert in axons to mediate axonal interactions and respond to Slits as guidance factors. These data underscore the likelihood that a limited number of extrinsic factors regulate the distinct morphologies of axons and dendrites.     

Positional information in neural map development: lessons from the olfactory system

Positional information is fundamental in development. Although molecular gradients are thought to represent positional information in various systems, the molecular logic used to interpret these gradients remains controversial. In the nervous system, sensory maps are formed in the brain based on gradients of axon guidance molecules. However, it remains unclear how axons find their targets based on relative, not absolute, expression levels of axon guidance receptors. No model solely based on axon-target interactions explains this point. Recent studies in the olfactory system suggested that the neural map formation requires axon-axon interactions, which is known as axon sorting. This review discusses how axon-axon and axon-target interactions interpret molecular gradients and determine the axonal projection sites in neural map formation.     

Robo-Slit interactions regulate longitudinal axon pathfinding in the embryonic vertebrate brain

The early network of axons in the embryonic brain provides connectivity between functionally distinct regions of the nervous system. While many of the molecular interactions driving commissural pathway formation have been deciphered, the mechanisms underlying the development of longitudinal tracts remain unclear. We have identified here a role for the Roundabout (Robo) family of axon guidance receptors in the positioning of longitudinally projecting axons along the dorsoventral axis in the embryonic zebrafish forebrain. Using a loss-of-function approach, we established that Robo family members exhibit complementary functions in the tract of the postoptic commissure (TPOC), the major longitudinal tract in the forebrain. Robo2 acted initially to split the TPOC into discrete fascicles upon entering a broad domain of Slit1a expression in the ventrocaudal diencephalon. In contrast, Robo1 and Robo3 restricted the extent of defasciculation of the TPOC. In this way, the complementary roles of Robo family members balance levels of fasciculation and defasciculation along this trajectory. These results demonstrate a key role for Robo-Slit signaling in vertebrate longitudinal axon guidance and highlight the importance of context-specific guidance cues during navigation within complex pathways.     

Chemoattractant axon guidance cues regulate de novo axon trajectories in the embryonic forebrain of zebrafish

The development of axon tracts in the early vertebrate brain is controlled by combinations of soluble, membrane-bound and extracellular matrix molecules. How these multiple and sometimes conflicting guidance cues are integrated in order to establish stereotypical pathways remains to be determined. We show here that when interactions between the chemoattractive signal Netrin1a and its receptor Dcc are suppressed using a loss-of-function approach, a novel axon trajectory emerges in the dorsal diencephalon. Axons arising from a subpopulation of telencephalic neurons failed to project rostrally into the anterior commissure in the absence of either Netrin1a or Dcc. Instead these axons inappropriately exited the telencephalon and ectopically coursed caudally into virgin neuroepithelium. This response was highly specific since loss-of-function of Netrin1b, a paralogue of Netrin1a, generated a distinct phenotype in the rostral brain. These results show that a subpopulation of telencephalic neurons, when freed from long-range chemoattraction mediated by Netrin1a-Dcc interactions, follow alternative instructive cues that lead to creation of an ectopic axon bundle in the diencephalon. This work provides insight into how integration of multiple guidance signals defines the initial scaffold of axon tracts in the embryonic vertebrate forebrain.     

Neogenin interacts with RGMa and netrin-1 to guide axons within the embryonic vertebrate forebrain

In the embryonic forebrain, pioneer axons establish a simple topography of dorsoventral and longitudinal tracts. The cues used by these axons during the initial formation of the axon scaffold remain largely unknown. We have investigated the axon guidance role of Neogenin, a member of the immunoglobulin (Ig) superfamily that binds to the chemoattractive ligand Netrin-1, as well as to the chemorepulsive ligand repulsive guidance molecule (RGMa). Here, we show strong expression of Neogenin and both of its putative ligands in the developing Xenopus forebrain. Neogenin loss-of-function mutants revealed that this receptor was essential for axon guidance in an early forming dorsoventral brain pathway. Similar mutant phenotypes were also observed following loss of either RGMa or Netrin-1. Simultaneous partial knock downs of these molecules revealed dosage-sensitive interactions and confirmed that these receptors and ligands were acting in the same pathway. The results provide the first evidence that Neogenin acts as an axon guidance molecule in vivo and support a model whereby Neogenin-expressing axons respond to a combination of attractive and repulsive cues as they navigate their ventral trajectory.     

New views on retinal axon development: a navigation guide

The eye is a peripheral outpost of the central nervous system (CNS) where the retinal ganglion cells (RGCs) reside. RGC axons navigate to their targets in a remarkably stereotyped and error-free manner and it is this process of directed growth that underlies the complex organization of the adult brain. The RGCs are the only retinal neurons to project into the brain and their peripheral location makes them an unusually accessible population of projection neurons for experiments involving in vivo gene transfer, anatomical tracing, transplantation and in vitro culture. In this paper, we review recent findings that have contributed to our understanding of some of the guidance decisions that axons make in the developing visual system. We look at two choice points in the pathway, the optic nerve head (onh) and the midline chiasm, and discuss evidence that supports the idea that key molecules in guiding axon growth at these junctures are netrin-1 (onh) and ephrin-B (chiasm). In the optic tectum where RGC axon terminals are arrayed in topographic order, we present experimental evidence to suggest that in the dorso-ventral dimension, the B-type ephrins and Eph receptors are of prime importance, possibly through attractive interactions. This complements the anterior-posterior topographic mapping known to be mediated through A-type ephrin/Eph repulsive interactions. An emerging theme is that guidance molecules such as ephrin-B and netrin-1 have complex patterns of restricted expression in the pathway and play multiple and changing roles in axon guidance.     

Patched regulation of axon guidance is by specifying neural identity in the<Emphasis Type="Italic"> Drosophila</Emphasis> nerve cord

Within an axon bundle, one or two are pioneering axons and the rest are follower axons. Pioneering axons are projected first and the follower axons are projected later but follow a pioneering axon(s) pathway. It is not clear whether the pioneering axons have a guidance role for follower axons. In this paper, we have investigated the role of Patched (Ptc) in regulating the guidance of medial tract, one of the longitudinal tracts in the nerve cord. In patched mutants the medial longitudinal tract fails to fasciculate on its own side along the nerve cord, instead it abnormally crosses the midline and fasciculates with the contralateral tract. Interestingly, the medial tracts cross the midline ignoring the axon-repellant Slit on the midline and Roundabout on growth cones. The medial tract is pioneered by neurons pCC and vMP2. Our results show that guidance defects of this tract are due to loss and mis-specification of vMP2, which results in the projection from pCC to either stall or project outward near the location of vMP2. Thus, both pioneering neurons are necessary for the proper guidance of pioneering and follower axons. We also show that the loss of Ptc activity in the neuroectoderm prior to the formation of S1 and S2 neuroblasts causes the majority of axon guidance defects. These results provide insight into how mis-specification and loss of neurons can non-autonomously contribute to defects in axon pathfinding.     

Pax genes in development and maturation of the vertebrate visual system: implications for optic nerve regeneration

Pax genes play a pivotal role in development of the vertebrate visual system. Pax6 is the master control gene for eye development: ectopic expression of Pax6 in Xenopus laevis and Drosphila melanogaster leads to the formation of differentiated eyes on the legs or wings. Pax6 is involved in formation of ganglion cells of the retina, as well as cells of the lens, iris and cornea. In addition Pax6 may play a role in axon guidance in the visual system. Pax2 regulates differentiation of the optic disk through which retinal ganglion cell axons exit the eye. Furthermore, Pax2 plays a critical role in development of the optic chiasm and in the guidance of axons along the contralateral or ipsilateral tracts of the optic nerve to visual targets in the brain. During development Pax7 is expressed in neuronal cells of one of the major visual targets in the brain, the optic tectum/superior colliculus. Neurons expressing Pax7 migrate towards the pia and concentrate in the stratum griseum superficiale (SGFS), the target site for retinal axons. Together, expression of Pax2, 6 and 7 may guide axons during formation of functional retinotectal/collicular projections. Highly regulated Pax gene expression is also observed in mature animals. Moreover, evidence suggests that Pax genes are important for regeneration of the visual system. We are currently investigating Pax gene expression in species that display a range of outcomes of optic nerve regeneration. We predict that such information will provide valuable insights for the induction of successful regeneration of the optic nerve and of other regions of the central nervous system in mammals including man.     

Novel guidance cues during neuronal pathfinding in the early scaffold of axon tracts in the rostral brain

A scaffold of axons consisting of a pair of longitudinal tracts and several commissures is established during early development of the vertebrate brain. We report here that NOC-2, a cell surface carbohydrate, is selectively expressed by a subpopulation of growing axons in this scaffold in Xenopus. NOC-2 is present on two glycoproteins, one of which is a novel glycoform of the neural cell adhesion molecule N-CAM. When the function of NOC-2 was perturbed using either soluble carbohydrates or anti-NOC-2 antibodies, axons expressing NOC-2 exhibited aberrant growth at specific points in their pathway. NOC-2 is the first-identified axon guidance molecule essential for development of the axon scaffold in the embryonic vertebrate brain.     

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.     

Embryonic lens repels retinal ganglion cell axons.

During development of the vertebrate visual system, retinal ganglion cell (RGC) axons follow a precise path toward their midbrain targets. Although much is known about the cues that direct RGC axons once they have left the optic disc, less is known about the guidance of axons at earlier stages, when RGCs first send out their axons to navigate within the developing retina. Using collagen gel coculture experiments, we find that the embryonic lens produces a powerful diffusible repulsive activity for RGC axons. We also find that this activity is localized to the lens epithelium and not the lens fiber layer, while the pigmented epithelium and vitreous humour are devoid of activity. The further observation that the lens also chemorepels primary sensory axons, but does not repel olfactory bulb axons, shows that this activity is specific for subsets of axons. Our experiments have excluded two candidate repellents for RGC axons (collapsin-1/sema III and chondroitin sulfate proteoglycans). These results implicate the lens in the earliest stages of RGC axon guidance. One function of the lens repellent may be to prevent aberrant targeting toward the lens, and it may also be involved in the directional guidance of RGC axons toward the optic disc.     

The transmembrane protein Golden goal regulates R8 photoreceptor axon-axon and axon-target interactions

During Drosophila visual system development, photoreceptor (R) axons choose their correct paths and targets in a step-wise fashion. R axons with different identities make specific pathfinding decisions at different stages during development. We show here that the transmembrane protein Golden goal (Gogo), which is dynamically expressed in all R neurons and localizes predominantly to growth cones, is required in two distinct steps of R8 photoreceptor axon pathfinding: Gogo regulates axon-axon interactions and axon-target interactions in R8 photoreceptor axons. gogo loss-of-function and gain-of-function phenotypes suggest that Gogo mediates repulsive axon-axon interaction between R8 axons to maintain their proper spacing, and it promotes axon-target recognition at the temporary layer to enable R8 axons to enter their correct target columns in the medulla. From detailed structure-function experiments, we propose that Gogo functions as a receptor that binds an unidentified ligand through its conserved extracellular domain.     

Ephrin-B regulates the Ipsilateral routing of retinal axons at the optic chiasm

In Xenopus tadpoles, all retinal ganglion cells (RGCs) send axons contralaterally across the optic chiasm. At metamorphosis, a subpopulation of EphB-expressing RGCs in the ventrotemporal retina begin to project ipsilaterally. However, when these metamorphic RGCs are grafted into embryos, they project contralaterally, suggesting that the embryonic chiasm lacks signals that guide axons ipsilaterally. Ephrin-B is expressed discretely at the chiasm of metamorphic but not premetamorphic Xenopus. When expressed prematurely in the embryonic chiasm, ephrin-B causes precocious ipsilateral projections from the EphB-expressing RGCs. Ephrin-B is also found in the chiasm of mammals, which have ipsilateral projections, but not in the chiasm of fish and birds, which do not. These results suggest that ephrin-B/EphB interactions play a key role in the sorting of axons at the vertebrate chiasm.     

Ganglion cell axon pathfinding in the retina and optic nerve

The eye is a highly specialized structure that gathers and converts light information into neuronal signals. These signals are relayed along axons of retinal ganglion cells (RGCs) to visual centers in the brain for processing. In this review, we discuss the pathfinding tasks RGC axons face during development and the molecular mechanisms known to be involved. The data at hand support the presence of multiple axon guidance mechanisms concentrically organized around the optic nerve head, each of which appears to involve both growth-promoting and growth-inhibitory guidance molecules. Together, these strategies ensure proper optic nerve formation and establish the anatomical pathway for faithful transmission of information between the retina and the brain.