The early development of retinal ganglion cells with uncrossed axons in the mouse: retinal position and axonal course

The carbocyanine dye, DiI, has been used to study the retinal origin of the uncrossed retinofugal component of the mouse and to show the course taken by these fibres through the optic nerve and chiasm during development. Optic axons first arrive at the chiasm at embryonic day 13 (E13) but do not cross the midline until E14. After this stage, fibres taking an uncrossed course can be selectively labelled by unilateral tract implants of DiI. The earliest ipsilaterally projecting ganglion cells are located in the dorsal central retina. The first sign of the adult pattern of distribution of ganglion cells with uncrossed axons located mainly in the ventrotemporal retina is seen on embryonic day 16.5, thus showing that the adult line of decussation forms early in development. A small number of labelled cells continue to be found in nasal and dorsal retina at all later stages. At early stages (E14-15), retrogradely labelled uncrossed fibres are found in virtually all fascicles of the developing nerve, intermingling with crossed axons throughout the length of the nerve. At later stages of development (E16-17), although uncrossed fibres pass predominantly within the temporal part of the stalk, they remain intermingled with crossed axons. A significant number of uncrossed axons also lie within the nasal part of the optic stalk. The position of uncrossed fibres throughout the nerve in the later developmental stages is comparable to that seen in the adult rodent (Baker and Jeffery, 1989). The distribution of uncrossed axons thus indicates that positional cues are not sufficient to account for the choice made by axons when they reach the optic chiasm.     

Retinal axon pathfinding in the optic chiasm: divergence of crossed and uncrossed fibers

In the developing mammalian visual system, retinal fibers grow through the optic chiasm, where one population crosses to the opposite side of the brain and the other does not. Evidence from labeling growing retinal axons with the carbocyanine dye Dil in mouse embryos indicates that the two subpopulations diverge at a zone along the midline of the optic chiasm. At the border of this zone, crossed fibers grow directly across, whereas uncrossed fibers turn back, developing highly complex terminations with bifurcating and wide-ranging growth cones. When one eye is removed at early stages, uncrossed fibers from the remaining eye stall at the chiasm midline. These results suggest that crossed and uncrossed retinal fibers respond differently to cues along the midline of the chiasm and that the uncrossed fibers from one eye grow along crossed fibers from the other eye, both guidance mechanisms contributing to the establishment of the bilateral pattern of visual projections in mammalian brain.     

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.     

VEGF signaling through neuropilin 1 guides commissural axon crossing at the optic chiasm

During development, the axons of retinal ganglion cell (RGC) neurons must decide whether to cross or avoid the midline at the optic chiasm to project to targets on both sides of the brain. By combining genetic analyses with in vitro assays, we show that neuropilin 1 (NRP1) promotes contralateral RGC projection in mammals. Unexpectedly, the NRP1 ligand involved is not an axon guidance cue of the class 3 semaphorin family, but VEGF164, the neuropilin-binding isoform of the classical vascular growth factor VEGF-A. VEGF164 is expressed at the chiasm midline and is required for normal contralateral growth in vivo. In outgrowth and growth cone turning assays, VEGF164 acts directly on NRP1-expressing contralateral RGCs to provide growth-promoting and chemoattractive signals. These findings have identified a permissive midline signal for axons at the chiasm midline and provide in vivo evidence that VEGF-A is an essential axon guidance cue.     

Retinal axon misrouting at the optic chiasm in mice with neural tube closure defects

In a new mouse mutant, circletail (Crc), failure of neural tube closure (embryonic day [E] 8-9) is associated with errors in retinal axon projection at the optic chiasm (E12-18), such that many axons normally projecting contralaterally instead grow to ipsilateral targets. Although the architecture of the chiasmatic region is altered, neurons and glia containing putative cues for axon guidance are present. The aberrant ipsilateral-projecting cells originate from a nonrandom expansion of the wild-type uncrossed retinal region. These axon pathway defects are found in two other mutants with cephalic neural tube defects (NTD), loop-tail (Lp) and Pax3 (splotch; Sp(2H)). Crc is phenotypically similar to Lp, exhibiting an open neural tube from midbrain to tail (craniorachischisis), while splotch has spina bifida with or without a cranial NTD. The retinal axon abnormalities occur only in the presence of NTD and not in homozygous mutants lacking cranial NTD. Thus, failure of neural tube closure is associated with failure of many retinal axons to cross the ventral midline. This study therefore reveals an unexpected connection between closure of the neural tube at the dorsal midline and development of ventral axon tracts. genesis 27:32-47, 2000.     

Chondroitin sulfate disrupts axon pathfinding in the optic tract and alters growth cone dynamics

Little is known about the cues that guide retinal axons across the diencephalon en route to their midbrain target, the optic tectum. Here we show that chondroitin sulfate proteoglycans are differentially expressed within the diencephalon at a time when retinal axons are growing within the optic tract. Using exposed brain preparations, we show that the addition of exogenous chondroitin sulfate results in retinal pathfinding errors. Retinal axons disperse widely from their normal trajectory within the optic tract and extend aberrantly into inappropriate regions of the forebrain. Time-lapse analysis of retinal growth cone dynamics in vivo shows that addition of exogenous chondroitin sulfate causes intermittent stalling and increases growth cone complexity. These results suggest that chondroitin sulfate may modulate the guidance of retinal axons as they grow through the diencephalon towards the optic tectum.     

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.     

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.     

Perturbations of MicroRNA Function in Mouse Dicer Mutants Produce Retinal Defects and Lead to Aberrant Axon Pathfinding at the Optic Chiasm

Background

During development axons encounter a variety of choice points where they have to make appropriate pathfinding decisions. The optic chiasm is a major decision point for retinal ganglion cell (RGC) axons en route to their target in order to ensure the correct wiring of the visual system. MicroRNAs (miRNAs) belong to the class of small non-coding RNA molecules and have been identified as important regulators of a variety of processes during embryonic development. However, their involvement in axon guidance decisions is less clear.

Methodology/Principal Findings

We report here that the early loss of Dicer, an essential protein for the maturation of miRNAs, in all cells of the forming retina and optic chiasm leads to severe phenotypes of RGC axon pathfinding at the midline. Using a conditional deletion approach in mice, we find in homozygous Dicer mutants a marked increase of ipsilateral projections, RGC axons extending outside the optic chiasm, the formation of a secondary optic tract and a substantial number of RGC axons projecting aberrantly into the contralateral eye. In addition, the mutant mice display a microphthalmia phenotype.

Conclusions

Our work demonstrates an important role of Dicer controlling the extension of RGC axons to the brain proper. It indicates that miRNAs are essential regulatory elements for mechanisms that ensure correct axon guidance decisions at the midline and thus have a central function in the establishment of circuitry during the development of the nervous system.     

Ephrin-B2 and EphB1 mediate retinal axon divergence at the optic chiasm

In animals with binocular vision, retinal ganglion cell (RGC) axons either cross or avoid the midline at the optic chiasm. Here, we show that ephrin-Bs in the chiasm region direct the divergence of retinal axons through the selective repulsion of a subset of RGCs that express EphB1. Ephrin-B2 is expressed at the mouse chiasm midline as the ipsilateral projection is generated and is selectively inhibitory to axons from ventrotemporal (VT) retina, where ipsilaterally projecting RGCs reside. Moreover, blocking ephrin-B2 function in vitro rescues the inhibitory effect of chiasm cells and eliminates the ipsilateral projection in the semiintact mouse visual system. A receptor for ephrin-B2, EphB1, is found exclusively in regions of retina that give rise to the ipsilateral projection. EphB1 null mice exhibit a dramatically reduced ipsilateral projection, suggesting that this receptor contributes to the formation of the ipsilateral retinal projection, most likely through its repulsive interaction with ephrin-B2.     

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.     

Matrix metalloproteinases are required for retinal ganglion cell axon guidance at select decision points

Axons receive guidance information from extrinsic cues in their environment in order to reach their targets. In the frog Xenopus laevis, retinal ganglion cell (RGC) axons make three key guidance decisions en route through the brain. First, they cross to the contralateral side of the brain at the optic chiasm. Second, they turn caudally in the mid-diencephalon. Finally, they must recognize the optic tectum as their target. The matrix metalloproteinase (MMP) and a disintegrin and metalloproteinase (ADAM) families are zinc (Zn)-dependent proteolytic enzymes. The latter functions in axon guidance, but a similar role has not yet been identified for the MMP family. Our previous work implicated metalloproteinases in the guidance decisions made by Xenopus RGC axons. To test specifically the importance of MMPs, we used two different in vivo exposed brain preparations in which RGC axons were exposed to an MMP-specific pharmacological inhibitor (SB-3CT), either as they reached the optic chiasm or as they extended through the diencephalon en route to the optic tectum. Interestingly, SB-3CT affected only two of the guidance decisions, with misrouting defects at the optic chiasm and tectum. Only at higher concentrations was RGC axon extension also impaired. These data implicate MMPs in the guidance of vertebrate axons, and suggest that different metalloproteinases function to regulate axon behaviour at distinct choice points: an MMP is important in guidance at the optic chiasm and the target, while either a different MMP or an ADAM is required for axons to make the turn in the mid-diencephalon.     

Optic nerve regeneration after intravitreal peripheral nerve implants: trajectories of axons regrowing through the optic chiasm into the optic tracts

We have studied axon regeneration through the optic chiasm of adult rats 30 days after prechiasmatic intracranial optic nerve crush and serial intravitreal sciatic nerve grafting on day 0 and 14 post-lesion. The experiments comprised three groups of treated rats and three groups of controls. All treated animals received intravitreal grafts either into the left eye after both left sided (unilateral) and bilateral optic nerve transection, or into both eyes after bilateral optic nerve transection. Control eyes were all sham grafted on day 0 and 14 post-lesion, and the optic nerves either unlesioned, or crushed unilaterally or bilaterally. No regeneration through the chiasm was seen in any of the lesioned control optic nerves. In all experimental groups, large numbers of axons regenerated across the optic nerve lesions ipsilateral to the grafted eyes, traversed the short distal segment of the optic nerve and invaded the chiasm without deflection. Regeneration was correlated with the absence of the mesodermal components in the scar. In all cases, axon regrowth through the chiasm appeared to establish a major crossed and a minor uncrossed projection into both optic tracts, with some aberrant growth into the contralateral optic nerve. Axons preferentially regenerated within the degenerating trajectories from their own eye, through fragmented myelin and axonal debris, and reactive astrocytes, oligodendrocytes, microglia and macrophages. In bilaterally lesioned animals, no regeneration was detected in the optic nerve of the unimplanted eye. Although astrocytes became reactive and their processes proliferated, the architecture of their intrafascicular processes was little perturbed after optic nerve transection within either the distal optic nerve segment or the chiasm. The re-establishment of a comparatively normal pattern of passage through the chiasm by regenerating axons in the adult might therefore be organised by this relatively immutable scaffold of astrocyte processes. Binocular interactions between regenerating axons from both nerves (after bilateral optic nerve transection and intravitreal grafting), and between regenerating axons and the intact transchiasmatic projections from the unlesioned eye (after unilateral optic nerve lesions and after ipsilateral grafting) may not be important in establishing the divergent trajectories, since regenerating axons behave similarly in the presence and absence of an intact projection from the other eye.     

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.