Contralateral Retinal Ganglion Cell Research Articles

Development of the Binocular Circuit.

Seeing in three dimensions is a major property of the visual system in mammals. The circuit underlying this property begins in the retina, from which retinal ganglion cells (RGCs) extend to the same or opposite side of the brain. RGC axons decussate to form the optic chiasm, then grow to targets in the thalamus and midbrain, where they synapse with neurons that project to the visual cortex. Here we review the cellular and molecular mechanisms of RGC axonal growth cone guidance across or away from the midline via receptors to cues in the midline environment. We present new views on the specification of ipsi- and contralateral RGC subpopulations and factors implementing their organization in the optic tract and termination in subregions of their targets. Lastly, we describe the functional and behavioral aspects of binocular vision, focusing on the mouse, and discuss recent discoveries in the evolution of the binocular circuit.

Pou3f1 orchestrates a gene regulatory network controlling contralateral retinogeniculate projections

The balance of contralateral and ipsilateral retinogeniculate projections is critical for binocular vision, but the transcriptional programs regulating this process remain ill defined. Here we show that the Pou class homeobox protein POU3F1 is expressed in nascent mouse contralateral retinal ganglion cells (cRGCs) but not ipsilateral RGCs (iRGCs). Upon Pou3f1 inactivation, the proportion of cRGCs is reduced in favor of iRGCs, leading to abnormal projection ratios at the optic chiasm. Conversely, misexpression of Pou3f1 in progenitors increases the production of cRGCs. Using CUT&RUN and RNA sequencing in gain- and loss-of-function assays, we demonstrate that POU3F1 regulates expression of several key members of the cRGC gene regulatory network. Finally, we report that POU3F1 is sufficient to induce RGC-like cell production, even in late-stage retinal progenitors of Atoh7 knockout mice. This work uncovers POU3F1 as a regulator of the cRGC transcriptional program, opening possibilities for optic nerve regenerative therapies.

BDNF signaling in correlation-dependent structural plasticity in the developing visual system.

During development, patterned neural activity instructs topographic map refinement. Axons with similar patterns of neural activity converge onto target neurons and stabilize their synapses with these postsynaptic partners, restricting exploratory branch elaboration (Hebbian structural plasticity). On the other hand, non-correlated firing in inputs leads to synapse weakening and increased exploratory growth of axons (Stentian structural plasticity). We used visual stimulation to control the correlation structure of neural activity in a few ipsilaterally projecting (ipsi) retinal ganglion cell (RGC) axons with respect to the majority contralateral eye inputs in the optic tectum of albino Xenopus laevis tadpoles. Multiphoton live imaging of ipsi axons, combined with specific targeted disruptions of brain-derived neurotrophic factor (BDNF) signaling, revealed that both presynaptic p75NTR and TrkB are required for Stentian axonal branch addition, whereas presumptive postsynaptic BDNF signaling is necessary for Hebbian axon stabilization. Additionally, we found that BDNF signaling mediates local suppression of branch elimination in response to correlated firing of inputs. Daily in vivo imaging of contralateral RGC axons demonstrated that p75NTR knockdown reduces axon branch elongation and arbor spanning field volume.

Characterizing a novel retinal organoid model of Oculocutaneous albinism and optic nerve misrouting

Albinism refers to a group of genetic disorders typically characterized by a loss/reduction of melanin in the hair, skin and eyes of affected patients. Apart from pigment changes, all albinism patients present with foveal hypoplasia and optic nerve misrouting, and have blurred vision. The molecular mechanisms that link this lack of pigment with neural retinal development are poorly understood, with foveal and optic tract development being difficult to model. To advance our knowledge, we developed a novel retinal organoid model of albinism, and characterized the development and outgrowth of retinal ganglion cells affected during albinism as a model for future studies. Human oculocutaneous albinism 1 (OCA1) patient-derived stem cells were differentiated alongside controls into retinal organoids, as published previously1,2 . Early retinal ganglion cells develop in the first 4 weeks of differentiation, at which point whole organoids could be plated to allow for optic nerve-like outgrowth. Whole organoids were also fixed and analysed with immunohistochemistry (IHC) to visualize contralateral and ipsilateral ganglion cells present in the organoid. IHC analysis showed differences in the number of ipsilateral and contralateral retinal ganglion cells between the healthy control and albinism organoids, in line with in vivo observations. Further, optic nerve-like outgrowth could be achieved with both models, allowing for future research into optic nerve misrouting in albinism. We generated a novel retinal organoid model of oculocutaneous albinism, and characterized the retinal ganglion cell development and outgrowth. This will allow us in the future to study a different and sometimes overlooked aspect of albinism; optic nerve misrouting.

CyclinD2-mediated regulation of neurogenic output from the retinal ciliary margin is perturbed in albinism

In albinism, aberrations in the ipsi-/contralateral retinal ganglion cell (RGC) ratio compromise the functional integrity of the binocular circuit. Here, we focus on the mouse ciliary margin zone (CMZ), a neurogenic niche at the embryonic peripheral retina, to investigate developmental processes regulating RGC neurogenesis and identity acquisition. We found that the mouse ventral CMZ generates predominantly ipsilaterally projecting RGCs, but this output is altered in the albino visual system because of CyclinD2 downregulation and disturbed timing of the cell cycle. Consequently, albino as well as CyclinD2-deficient pigmented mice exhibit diminished ipsilateral retinogeniculate projection and poor depth perception. In albino mice, pharmacological stimulation of calcium channels, known to upregulate CyclinD2 in other cell types, augmented CyclinD2-dependent neurogenesis of ipsilateral RGCs and improved stereopsis. Together, these results implicate CMZ neurogenesis and its regulators as critical for the formation and function of the mammalian binocular circuit.

Multiomic Analysis of Neurons with Divergent Projection Patterns Identifies Novel Regulators of Axon Pathfinding.

Axon pathfinding is a key step in neural circuits formation. However, the transcriptional mechanisms regulating its progression remain poorly understood. The binary decision of crossing or avoiding the midline taken by some neuronal axons during development represents a robust model to investigate the mechanisms that control the selection of axonal trajectories. Here, to identify novel regulators of axon guidance, this work compares the transcriptome and chromatin occupancy profiles of two neuronal subpopulations, ipsilateral (iRGC) and contralateral retinal ganglion cells (cRGC), with similar functions but divergent axon trajectories. These analyses retrieved a number of genes encoding for proteins not previously implicated in axon pathfinding. In vivo functional experiments confirm the implication of some of these candidates in axonal navigation. Among the candidate genes, γ‐synuclein is identified as essential for inducing midline crossing. Footprint and luciferase assays demonstrate that this small‐sized protein is regulated by the transcription factor (TF) Pou4f1 in cRGCs. It is also shown that Lhx2/9 are specifically expressed in iRGCs and control a program that partially overlaps with that regulated by Zic2, previously described as essential for iRGC specification. Overall, the analyses identify dozens of new molecules potentially involved in axon guidance and reveal the regulatory logic behind the selection of axonal trajectories.

Wiring the Binocular Visual Pathways.

Retinal ganglion cells (RGCs) extend axons out of the retina to transmit visual information to the brain. These connections are established during development through the navigation of RGC axons along a relatively long, stereotypical pathway. RGC axons exit the eye at the optic disc and extend along the optic nerves to the ventral midline of the brain, where the two nerves meet to form the optic chiasm. In animals with binocular vision, the axons face a choice at the optic chiasm—to cross the midline and project to targets on the contralateral side of the brain, or avoid crossing the midline and project to ipsilateral brain targets. Ipsilaterally and contralaterally projecting RGCs originate in disparate regions of the retina that relate to the extent of binocular overlap in the visual field. In humans virtually all RGC axons originating in temporal retina project ipsilaterally, whereas in mice, ipsilaterally projecting RGCs are confined to the peripheral ventrotemporal retina. This review will discuss recent advances in our understanding of the mechanisms regulating specification of ipsilateral versus contralateral RGCs, and the differential guidance of their axons at the optic chiasm. Recent insights into the establishment of congruent topographic maps in both brain hemispheres also will be discussed.

Conversations with Ray Guillery on albinism: linking Siamese cat visual pathway connectivity to mouse retinal development.

In albinism of all species, perturbed melanin biosynthesis in the eye leads to foveal hypoplasia, retinal ganglion cell misrouting, and, consequently, altered binocular vision. Here, written before he died, Ray Guillery chronicles his discovery of the aberrant circuitry from eye to brain in the Siamese cat. Ray's characterization of visual pathway anomalies in this temperature sensitive mutation of tyrosinase and thus melanin synthesis in domestic cats opened the exploration of albinism and simultaneously, a genetic approach to the organization of neural circuitry. I follow this account with a remembrance of Ray's influence on my work. Beginning with my postdoc research with Ray on the cat visual pathway, through my own work on the mechanisms of retinal axon guidance in the developing mouse, Ray and I had a continuous and rich dialogue about the albino visual pathway. I will present the questions Ray posed and clues we have to date on the still-elusive link between eye pigment and the proper balance of ipsilateral and contralateral retinal ganglion cell projections to the brain.

Nell2 regulates the contralateral-versus-ipsilateral visual projection as a domain-specific positional cue.

In mammals with binocular vision, retinal ganglion cell (RGC) axons from each eye project to eye-specific domains in the contralateral and ipsilateral dorsal lateral geniculate nucleus (dLGN), underpinning disparity-based stereopsis. Although domain-specific axon guidance cues that discriminate contralateral and ipsilateral RGC axons have long been postulated as a key mechanism for development of the eye-specific retinogeniculate projection, the molecular nature of such cues has remained elusive. Here, we show that the extracellular glycoprotein Nell2 (neural epidermal growth factor-like-like 2) is expressed in the dorsomedial region of the dLGN, which ipsilateral RGC axons terminate in and contralateral axons avoid. In Nell2 mutant mice, contralateral RGC axons abnormally invaded the ipsilateral domain of the dLGN, and ipsilateral axons terminated in partially fragmented patches, forming a mosaic pattern of contralateral and ipsilateral axon-termination zones. In vitro, Nell2 exerted inhibitory effects on contralateral, but not ipsilateral, RGC axons. These results provide evidence that Nell2 acts as a domain-specific positional label in the dLGN that discriminates contralateral and ipsilateral RGC axons, and that it plays essential roles in the establishment of the eye-specific retinogeniculate projection.

Spatiotemporal distribution of glia in and around the developing mouse optic tract.

In the developing mouse optic tract, retinal ganglion cell (RGC) axon position is organized by topography and laterality (i.e., eye-specific or ipsi- and contralateral segregation). Our lab previously showed that ipsilaterally projecting RGCs are segregated to the lateral aspect of the developing optic tract and found that ipsilateral axons self-fasciculate to a greater extent than contralaterally projecting RGC axons in vitro. However, the full complement of axon-intrinsic and -extrinsic factors mediating eye-specific segregation in the tract remain poorly understood. Glia, which are known to express several guidance cues in the visual system and regulate the navigation of ipsilateral and contralateral RGC axons at the optic chiasm, are natural candidates for contributing to eye-specific pre-target axon organization. Here, we investigate the spatiotemporal expression patterns of both putative astrocytes (Aldh1l1+ cells) and microglia (Iba1+ cells) in the embryonic and neonatal optic tract. We quantified the localization of ipsilateral RGC axons to the lateral two-thirds of the optic tract and analyzed glia position and distribution relative to eye-specific axon organization. While our results indicate that glial segregation patterns do not strictly align with eye-specific RGC axon segregation in the tract, we identify distinct spatiotemporal organization of both Aldh1l1+ cells and microglia in and around the developing optic tract. These findings inform future research into molecular mechanisms of glial involvement in RGC axon growth and organization in the developing retinogeniculate pathway.

Distinct timing of neurogenesis of ipsilateral and contralateral retinal ganglion cells.

In higher vertebrates, the circuit formed by retinal ganglion cells (RGCs) projecting ipsilaterally (iRGCs) or contralaterally (cRGCs) to the brain permits binocular vision and depth perception. iRGCs and cRGCs differ in their position within the retina and in expression of transcription, guidance and activity-related factors. To parse whether these two populations also differ in the timing of their genesis, a feature of distinct neural subtypes and associated projections, we used newer birthdating methods and cell subtype specific markers to determine birthdate and cell cycle exit more precisely than previously. In the ventrotemporal (VT) retina, i- and cRGCs intermingle and neurogenesis in this zone lags behind RGC production in the rest of the retina where only cRGCs are positioned. In addition, within the VT retina, i- and cRGC populations are born at distinct times: neurogenesis of iRGCs surges at E13, and cRGCs arise as early as E14, not later in embryogenesis as reported. Moreover, in the ventral ciliary margin zone (CMZ), which contains progenitors that give rise to some iRGCs in ventral neural retina (Marcucci et al., 2016), cell cycle exit is slower than in other retinal regions in which progenitors give rise only to cRGCs. Further, when the cell cycle regulator Cyclin D2 is missing, cell cycle length in the CMZ is further reduced, mirroring the reduction of both i- and cRGCs in the Cyclin D2 mutant. These results strengthen the view that differential regulation of cell cycle dynamics at the progenitor level is associated with specific RGC fates and laterality of axonal projection.

Eye-specific segregation and differential fasciculation of developing retinal ganglion cell axons in the mouse visual pathway.

Prior to forming and refining synaptic connections, axons of projection neurons navigate long distances to their targets. While much is known about guidance cues for axon navigation through intermediate choice points, whether and how axons are organized within tracts is less clear. Here we analyze the organization of retinal ganglion cell (RGC) axons in the developing mouse retinogeniculate pathway. RGC axons are organized by both eye-specificity and topography in the optic nerve and tract: ipsilateral RGC axons are segregated from contralateral axons and are offset laterally in the tract relative to contralateral axon topographic position. To identify potential cell-autonomous factors contributing to the segregation of ipsilateral and contralateral RGC axons in the visual pathway, we assessed their fasciculation behavior in a retinal explant assay. Ipsilateral RGC neurites self-fasciculate more than contralateral neurites in vitro and maintain this difference in the presence of extrinsic chiasm cues. To further probe the role of axon self-association in circuit formation in vivo, we examined RGC axon organization and fasciculation in an EphB1-/- mutant, in which a subset of ipsilateral RGC axons aberrantly crosses the midline but targets the ipsilateral zone in the dorsal lateral geniculate nucleus on the opposite side. Aberrantly crossing axons retain their association with ipsilateral axons in the contralateral tract, indicating that cohort-specific axon affinity is maintained independently of guidance signals present at the midline. Our results provide a comprehensive assessment of RGC axon organization in the retinogeniculate pathway and suggest that axon self-association contributes to pre-target axon organization.

Sonic Hedgehog Is a Remotely Produced Cue that Controls Axon Guidance Trans-axonally at a Midline Choice Point

At the optic chiasm choice point, ipsilateral retinal ganglion cells (RGCs) are repelled away from the midline by guidance cues, including Ephrin-B2 and Sonic Hedgehog (Shh). Although guidance cues arenormally produced by cells residing at the choice point, the mRNA for Shh is not found at the optic chiasm. Here we show that Shh protein is instead produced by contralateral RGCs at the retina, transported anterogradely along the axon, and accumulates at the optic chiasm to repel ipsilateral RGCs. Invitro, contralateral RGC axons, which secrete Shh, repel ipsilateral RGCs in a Boc- and Smo-dependent manner. Finally, knockdown of Shh in the contralateral retina causes a decrease in the proportion of ipsilateral RGCs in a non-cell-autonomous manner. These findings reveal a role for axon-axon interactions in ipsilateral RGC guidance, and they establish that remotely produced cues can act at axon guidance midline choice points.

SoxC Transcription Factors Promote Contralateral Retinal Ganglion Cell Differentiation and Axon Guidance in the Mouse Visual System

Transcription factors control cell identity by regulating diverse developmental steps such as differentiation and axon guidance. The mammalian binocular visual circuit is comprised of projections of retinal ganglion cells (RGCs) to ipsilateral and contralateral targets in the brain. A transcriptional code for ipsilateral RGC identity has been identified, but less is known about the transcriptional regulation of contralateral RGC development. Here we demonstrate that SoxC genes (Sox4, 11, and 12) act on the progenitor-to-postmitotic transition to implement contralateral, but not ipsilateral, RGC differentiation, by binding to Hes5 and thus repressing Notch signaling. When SoxC genes are deleted in postmitotic RGCs, contralateral RGC axons grow poorly on chiasm cells invitro and project ipsilaterally at the chiasm midline invivo, and Plexin-A1 and Nr-CAM expression in RGCs is downregulated. These data implicate SoxC transcription factors in the regulation of contralateral RGC differentiation and axon guidance.

Ipsilateral and Contralateral Retinal Ganglion Cells Express Distinct Genes during Decussation at the Optic Chiasm.

The increasing availability of transcriptomic technologies within the last decade has facilitated high-throughput identification of gene expression differences that define distinct cell types as well as the molecular pathways that drive their specification. The retinal projection neurons, retinal ganglion cells (RGCs), can be categorized into distinct morphological and functional subtypes and by the laterality of their projections. Here, we present a method for purifying the sparse population of ipsilaterally projecting RGCs in mouse retina from their contralaterally projecting counterparts during embryonic development through rapid retrograde labeling followed by fluorescence-activated cell sorting. Through microarray analysis, we uncovered the distinct molecular signatures that define and distinguish ipsilateral and contralateral RGCs during the critical period of axonal outgrowth and decussation, with more than 300 genes differentially expressed within these two cell populations. Among the differentially expressed genes confirmed through in vivo expression validation, several genes that mark "immaturity" are expressed within postmitotic ipsilateral RGCs. Moreover, at least one complementary pair, Igf1 and Igfbp5, is upregulated in contralateral or ipsilateral RGCs, respectively, and may represent signaling pathways that determine ipsilateral versus contralateral RGC identity. Importantly, the cell cycle regulator cyclin D2 is highly expressed in peripheral ventral retina with a dynamic expression pattern that peaks during the period of ipsilateral RGC production. Thus, the molecular signatures of ipsilateral and contralateral RGCs and the mechanisms that regulate their differentiation are more diverse than previously expected.

The retino–retinal projection: Tracing retinal ganglion cells projecting to the contralateral retina

We investigated the presence of a direct retino–retinal (R–R) projection between the two eyes via the optic chiasm of retinal ganglion cells (RGCs) in adult Long-Evans rats. We also explored the presence of collateral projections originating from these cells to the brain. In the first group of animals, right optic nerves (ONs) were orbitally transected approximately 2mm behind the globe followed by application of fluorochrome (2% Fluorogold [FG]) to the optic nerve stump to retrogradely label the R–R projection RGCs (R–RGCs) on the contralateral side. Animals were then sacrificed after 3, 5, 7, or 21 days. Contralateral retinas were fixed, whole-mounted, and imaged for R–RGCs. In a second group of animals, RGCs were retrogradely labeled with 15% rhodamine-β-isothiocynate (RITC) at the superior colliculi, where approximately 96% of rat RGCs synapse. Seven days later, the right ONs were transected and 2% FG applied to the proximal and distal ON stumps. Animals were then sacrificed after 5 days. Contralateral retinas were examined for co-labeled (RITC/FG) RGCs. Control rats underwent the same procedures excluding fluorescent tracer application. In the first group of animals, the number of R–RGCs in the contralateral eye ranged from 3 to 25 and did not depend on survival time. The second group of animals revealed evidence of co-labeled contralateral RGCs. Results suggest that a greater number of R–RGCs persist into adulthood than previously reported [M. Müller, H. Holländer, 1988]. Furthermore, the presence of co-labeled RGCs in the contralateral eye indicates that in adult rodents some R–R projections have a collateral projection to the brain, whereas previous reports had only found collateral projections in newborns.

Delayed neurogenesis leads to altered specification of ventrotemporal retinal ganglion cells in albino mice.

BackgroundProper binocular vision depends on the routing at the optic chiasm of the correct proportion of retinal ganglion cell (RGC) axons that project to the same (ipsilateral) and opposite (contralateral) side of the brain. The ipsilateral RGC projection is reduced in mammals with albinism, a congenital disorder characterized by deficient pigmentation in the skin, hair, and eyes. Compared to the pigmented embryonic mouse retina, the albino embryonic mouse retina has fewer RGCs that express the zinc-finger transcription factor, Zic2, which is transiently expressed by RGCs fated to project ipsilaterally. Here, using Zic2 as a marker of ipsilateral RGCs, Islet2 as a marker of contralateral RGCs, and birthdating, we investigate spatiotemporal dynamics of RGC production as they relate to the phenotype of diminished ipsilateral RGC number in the albino retina.ResultsAt embryonic day (E)15.5, fewer Zic2-positive (Zic2+) RGCs are found in the albino ventrotemporal (VT) retina compared with the pigmented VT retina, as we previously reported. However, the reduction in Zic2+ RGCs in the albino is not accompanied by a compensatory increase in Zic2-negative (Zic2−) RGCs, resulting in fewer RGCs in the VT retina at this time point. At E17.5, however, the number of RGCs in the VT region is similar in pigmented and albino retinae, implicating a shift in the timing of RGC production in the albino. Short-term birthdating assays reveal a delay in RGC production in the albino VT retina between E13 and E15. Specifically, fewer Zic2+ RGCs are born at E13 and more Zic2− RGCs are born at E15. Consistent with an increase in the production of Zic2− RGCs born at later ages, more RGCs at E17.5 express the contralateral marker, Islet2, in the albino VT retina compared with the pigmented retina.ConclusionsA delay in neurogenesis in the albino retina is linked to the alteration of RGC subtype specification and consequently leads to the reduced ipsilateral projection that characterizes albinism.

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.

VEGF Signaling through Neuropilin 1 Guides Commissural Axon Crossing at the Optic Chiasm

SummaryDuring 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.

Zic2 regulates the expression of Sert to modulate eye-specific refinement at the visual targets

The development of the nervous system is a time-ordered and multi-stepped process that requires neural specification, axonal navigation and arbor refinement at the target tissues. Previous studies have demonstrated that the transcription factor Zic2 is necessary and sufficient for the specification of retinal ganglion cells (RGCs) that project ipsilaterally at the optic chiasm midline. Here, we report that, in addition, Zic2 controls the refinement of eye-specific inputs in the visual targets by regulating directly the expression of the serotonin transporter (Sert), which is involved in the modulation of activity-dependent mechanisms during the wiring of sensory circuits. In agreement with these findings, RGCs that express Zic2 ectopically show defects in axonal refinement at the visual targets and respond to pharmacological blockage of Sert, whereas Zic2-negative contralateral RGCs do not. These results link, at the molecular level, early events in neural differentiation with late activity-dependent processes and propose a mechanism for the establishment of eye-specific domains at the visual targets.