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.     

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.     

Optic chiasm presentation of Semaphorin6D in the context of Plexin-A1 and Nr-CAM promotes retinal axon midline crossing

At the optic chiasm, retinal ganglion cells (RGCs) project ipsi- or contralaterally to establish the circuitry for binocular vision. Ipsilateral guidance programs have been characterized, but contralateral guidance programs are not well understood. Here, we identify a tripartite molecular system for contralateral RGC projections: Semaphorin6D (Sema6D) and Nr-CAM are expressed on midline radial glia and Plexin-A1 on chiasm neurons, and Plexin-A1 and Nr-CAM are also expressed on contralateral RGCs. Sema6D is repulsive to contralateral RGCs, but Sema6D in combination with Nr-CAM and Plexin-A1 converts repulsion to growth promotion. Nr-CAM functions as a receptor for Sema6D. Sema6D, Plexin-A1, and Nr-CAM are all required for efficient RGC decussation at the optic chiasm. These findings suggest a mechanism by which a complex of Sema6D, Nr-CAM, and Plexin-A1 at the chiasm midline alters the sign of Sema6D and signals Nr-CAM/Plexin-A1 receptors on RGCs to implement the contralateral RGC projection.     

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     

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.     

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.     

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.     

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.     

Serotonin transporter-mediated molecular axis regulates regional retinal ganglion cell vulnerability and axon regeneration after nerve injury

Molecular insights into the selective vulnerability of retinal ganglion cells (RGCs) in optic neuropathies and after ocular trauma can lead to the development of novel therapeutic strategies aimed at preserving RGCs. However, little is known about what molecular contexts determine RGC susceptibility. In this study, we show the molecular mechanisms underlying the regional differential vulnerability of RGCs after optic nerve injury. We identified RGCs in the mouse peripheral ventrotemporal (VT) retina as the earliest population of RGCs susceptible to optic nerve injury. Mechanistically, the serotonin transporter (SERT) is upregulated on VT axons after injury. Utilizing SERT-deficient mice, loss of SERT attenuated VT RGC death and led to robust retinal axon regeneration. Integrin β3, a factor mediating SERT-induced functions in other systems, is also upregulated in RGCs and axons after injury, and loss of integrin β3 led to VT RGC protection and axon regeneration. Finally, RNA sequencing analyses revealed that loss of SERT significantly altered molecular signatures in the VT retina after optic nerve injury, including expression of the transmembrane protein, Gpnmb. GPNMB is rapidly downregulated in wild-type, but not SERT- or integrin β3-deficient VT RGCs after injury, and maintaining expression of GPNMB in RGCs via AAV2 viruses even after injury promoted VT RGC survival and axon regeneration. Taken together, our findings demonstrate that the SERT-integrin β3-GPNMB molecular axis mediates selective RGC vulnerability and axon regeneration after optic nerve injury.     

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.     

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.     

Autonomous and non-autonomous Shh signalling mediate the in vivo growth and guidance of mouse retinal ganglion cell axons

In non-mammalian vertebrates, the relatively homogeneous population of retinal ganglion cells (RGCs) differentiates and projects entirely to the contralateral side of the brain under the influence of sonic hedgehog (Shh). In mammals, by contrast, there are two different RGC types: the Zic2-positive ipsilateral projecting and the Isl2-positive contralateral projecting. We asked whether the axons of these two populations respond to Shh and if their response differs. We have also analysed whether midline- and RGC-derived Shh contributes to the growth of the axons in the proximal visual pathway. We show that these two RGC types are characterised by a differential expression of Shh signalling components and that they respond differently to Shh when challenged in vitro. In vivo blockade of Shh activity, however, alters the path and distribution mostly of the contralateral projecting RGC axons at the chiasm, indicating that midline-derived Shh participates in funnelling contralateral visual fibres in this region. Furthermore, interference with Shh signalling in the RGCs themselves causes abnormal growth and navigation of contralateral projecting axons in the proximal portion of the pathway, highlighting a novel cell-autonomous mechanism by which Shh can influence growth cone behaviour.