Ipsilateral Retinal Ganglion Cell Research Articles

Ten-m4 plays a unique role in the establishment of binocular visual circuits.

The patterning of binocular vision requires distinct molecular pathways for inputs arising from each side of the nervous system. Recent studies have demonstrated important roles for members of the Ten-m/Odz/teneurin family in the development of ipsilateral retinal projections. Here, we further highlight the significance of this gene family in visual development by identifying a role for Ten-m4 during the formation of the ipsilateral projection in the mouse. Ten-m4 was found to be expressed in the retina, dorsal lateral geniculate nucleus (dLGN), superior colliculus (SC), and primary visual cortex (V1) during development. Anterograde and retrograde tracing experiments in Ten-m4 knockout (KO) mice revealed a specific increase in ipsilateral retinal ganglion cells projecting to dLGN and SC. This increase was most prominent in regions corresponding to temporal retina. Consistent with this, EphB1 expression in the retina around the time of decussation was enhanced in this temporal region for KO mice, suggesting that the increased size of the ipsilateral population arises due to an increased number of retinal ganglion cells remaining ipsilaterally at the optic chiasm due to EphB1-mediated repulsion. The ectopic ipsilaterally targeted retinal ganglion cell projection observed in Ten-m4 KOs was associated with changes in response to ethologically-relevant visual stimuli. Together, these data demonstrate a requirement for Ten-m4 in the establishment of ipsilateral projections from the retina, which likely acts in combination with other Ten-m members (Ten-m2 and Ten-m3) to promote the formation of functional binocular circuits. This article is protected by copyright. All rights reserved.

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.

Bilateral visual projections exist in non-teleost bony fish and predate the emergence of tetrapods.

In most vertebrates, camera-style eyes contain retinal ganglion cell neurons that project to visual centers on both sides of the brain. However, in fish, ganglion cells were thought to innervate only the contralateral side, suggesting that bilateral visual projections appeared in tetrapods. Here we show that bilateral visual projections exist in non-teleost fishes and that the appearance of ipsilateral projections does not correlate with terrestrial transition or predatory behavior. We also report that the developmental program that specifies visual system laterality differs between fishes and mammals, as the Zic2 transcription factor, which specifies ipsilateral retinal ganglion cells in tetrapods, appears to be absent from fish ganglion cells. However, overexpression of human ZIC2 induces ipsilateral visual projections in zebrafish. Therefore, the existence of bilateral visual projections likely preceded the emergence of binocular vision in tetrapods.

Neurogenesis and Specification of Retinal Ganglion Cells.

Across all species, retinal ganglion cells (RGCs) are the first retinal neurons generated during development, followed by the other retinal cell types. How are retinal progenitor cells (RPCs) able to produce these cell types in a specific and timely order? Here, we will review the different models of retinal neurogenesis proposed over the last decades as well as the extrinsic and intrinsic factors controlling it. We will then focus on the molecular mechanisms, especially the cascade of transcription factors that regulate, more specifically, RGC fate. We will also comment on the recent discovery that the ciliary marginal zone is a new stem cell niche in mice contributing to retinal neurogenesis, especially to the generation of ipsilateral RGCs. Furthermore, RGCs are composed of many different subtypes that are anatomically, physiologically, functionally, and molecularly defined. We will summarize the different classifications of RGC subtypes and will recapitulate the specification of some of them and describe how a genetic disease such as albinism affects neurogenesis, resulting in profound visual deficits.

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.

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.

Single-cell transcriptional logic of cell-fate specification and axon guidance in early-born retinal neurons.

Retinal ganglion cells (RGCs), cone photoreceptors (cones), horizontal cells and amacrine cells are the first classes of neurons produced in the retina. However, an important question is how this diversity of cell states is transcriptionally produced. Here, we profiled 6067 single retinal cells to provide a comprehensive transcriptomic atlas showing the diversity of the early developing mouse retina. RNA velocities unveiled the dynamics of cell cycle coordination of early retinogenesis and define the transcriptional sequences at work during the hierarchical production of early cell-fate specification. We show that RGC maturation follows six waves of gene expression, with older-generated RGCs transcribing increasing amounts of guidance cues for young peripheral RGC axons that express the matching receptors. Spatial transcriptionally deduced features in subpopulations of RGCs allowed us to define novel molecular markers that are spatially restricted. Finally, the isolation of such a spatially restricted population, ipsilateral RGCs, allowed us to identify their molecular identity at the time they execute axon guidance decisions. Together, these data represent a valuable resource shedding light on transcription factor sequences and guidance cue dynamics during mouse retinal development.

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.

Retinal ganglion cell axon sorting at the optic chiasm requires dystroglycan

In the developing visual system, retinal ganglion cell (RGC) axons project from the retina to several distal retinorecipient regions in the brain. Several molecules have been implicated in guiding RGC axons in vivo, but the role of extracellular matrix molecules in this process remains poorly understood. Dystroglycan is a laminin-binding transmembrane protein important for formation and maintenance of the extracellular matrix and basement membranes and has previously been implicated in axon guidance in the developing spinal cord. Using two genetic models of functional dystroglycan loss, we show that dystroglycan is necessary for correct sorting of contralateral and ipsilateral RGC axons at the optic chiasm. Mis-sorted axons still target retinorecipient brain regions and persist in adult mice, even after axon pruning is complete. Our results highlight the importance of the extracellular matrix for axon sorting at an intermediate choice point in the developing visual circuit.

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.

Activation of Wnt signaling reduces ipsilaterally projecting retinal ganglion cells in pigmented retina.

In mammalian albinism, disrupted melanogenesis in the retinal pigment epithelium (RPE) is associated with fewer retinal ganglion cells (RGCs) projecting ipsilaterally to the brain, resulting in numerous abnormalities in the retina and visual pathway, especially binocular vision. To further understand the molecular link between disrupted RPE and a reduced ipsilateral RGC projection in albinism, we compared gene expression in the embryonic albino and pigmented mouse RPE. We found that the Wnt pathway, which directs peripheral retinal differentiation and, generally, cell proliferation, is dysregulated in the albino RPE. Wnt2b expression is expanded in the albino RPE compared with the pigmented RPE, and the expanded region adjoins the site of ipsilateral RGC neurogenesis and settling. Pharmacological activation of Wnt signaling in pigmented mice by lithium (Li+) treatment in vivo reduces the number of Zic2-positive RGCs, which are normally fated to project ipsilaterally, to numbers observed in the albino retina. These results implicate Wnt signaling from the RPE to neural retina as a potential factor in the regulation of ipsilateral RGC production, and thus the albino phenotype.

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.

The Ciliary Margin Zone of the Mammalian Retina Generates Retinal Ganglion Cells

The retina of lower vertebrates grows continuously by integrating new neurons generated from progenitors in the ciliary margin zone (CMZ). Whether the mammalian CMZ provides the neural retina with retinal cells is controversial. Live imaging of embryonic retina expressing eGFP in the CMZ shows that cells migrate laterally from the CMZ to the neural retina where differentiated retinal ganglion cells (RGCs) reside. Because Cyclin D2, a cell-cycle regulator, is enriched in ventral CMZ, we analyzed Cyclin D2-/- mice to test whether the CMZ is a source of retinal cells. Neurogenesis is diminished in Cyclin D2 mutants, leading to a reduction of RGCs in the ventral retina. In line with these findings, in the albino retina, the decreased production of ipsilateral RGCs is correlated with fewer Cyclin D2+ cells. Together, these results implicate the mammalian CMZ as a neurogenic site that produces RGCs and whose proper generation depends on Cyclin D2 activity.

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.

Retinal pigment epithelial integrity is compromised in the developing albino mouse retina

In the developing murine eye, melanin synthesis in the retinal pigment epithelium (RPE) coincides with neurogenesis of retinal ganglion cells (RGCs). Disruption of pigmentation in the albino RPE is associated with delayed neurogenesis in the ventrotemporal retina, the source of ipsilateral RGCs, and a reduced ipsilateral RGC projection. To begin to unravel how melanogenesis and the RPE regulate RGC neurogenesis and cell subpopulation specification, we compared the features of albino and pigmented mouse RPE cells during the period of RGC neurogenesis (embryonic day, E, 12.5 to 18.5) when the RPE is closely apposed to developing RGC precursors. At E12.5 and E15.5, although albino and pigmented RPE cells express RPE markers Otx2 and Mitf similarly, albino RPE cells are irregularly shaped and have fewer melanosomes compared with pigmented RPE cells. The adherens junction protein P-cadherin appears loosely distributed within the albino RPE cells rather than tightly localized on the cell membrane, as in pigmented RPE. Connexin 43 (gap junction protein) is expressed in pigmented and albino RPE cells at E13.5 but at E15.5 albino RPE cells have fewer small connexin 43 puncta, and a larger fraction of phosphorylated connexin 43 at serine 368. These results suggest that the lack of pigment in the RPE results in impaired RPE cell integrity and communication via gap junctions between RPE and neural retina during RGC neurogenesis. Our findings should pave the way for further investigation of the role of RPE in regulating RGC development toward achieving proper RGC axon decussation. J. Comp. Neurol. 524:3696-3716, 2016. © 2016 Wiley Periodicals, Inc.

Transient ipsilateral retinal ganglion cell projections to the brain: Extent, targeting, and disappearance

During development of the mammalian eye, the first retinal ganglion cells (RGCs) that extend to the brain are located in the dorsocentral (DC) retina. These RGCs extend to either ipsilateral or contralateral targets, but the ipsilateral projections do not survive into postnatal periods. The function and means of disappearance of the transient ipsilateral projection are not known. We have followed the course of this transient early ipsilateral cohort of RGCs, paying attention to how far they extend, whether they enter targets and if so, which ones, and the time course of their disappearance. The DC ipsilateral RGC axons were traced using DiI labeling at E13.5 and E15.5 to compare the proportion of ipsi- versus contralateral projections during the first period of growth. In utero electroporation of E12.5 retina with GFP constructs was used to label axons that could be visualized at succeeding time points into postnatal ages. Our results show that the earliest ipsilateral axons grow along the cellular border of the brain, and are segregated from the laterally positioned contralateral axons from the same retinal origin. In agreement with previous reports, although many early RGCs extend ipsilaterally, after E16 their number rapidly declines. Nonetheless, some ipsilateral axons from the DC retina enter the superior colliculus and arborize minimally, but very few enter the dorsal lateral geniculate nucleus and those that do extend only short branches. While the mechanism of selective axonal disappearance remains elusive, these data give further insight into establishment of the visual pathways.

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.

Ten-m2 Is Required for the Generation of Binocular Visual Circuits

Functional binocular vision requires that inputs arising from the two retinae are integrated and precisely organized within central visual areas. Previous studies have demonstrated an important role for one member of the Ten-m/Odz/teneurin family, Ten-m3, in the mapping of ipsilateral retinal projections. Here, we have identified a distinct role for another closely related family member, Ten-m2, in the formation of the ipsilateral projection in the mouse visual system. Ten-m2 expression was observed in the retina, dorsal lateral geniculate nucleus (dLGN), superior colliculus (SC), and primary visual cortex (V1) of the developing mouse. Anterograde and retrograde tracing experiments in Ten-m2 knock-out (KO) mice revealed a specific decrease in ipsilateral retinal ganglion cells projecting to dLGN and SC. This reduction was most prominent in regions corresponding to ventral retina. No change in the topography of ipsilateral or contralateral projections was observed. While expression of a critical ipsilateral fate determinant, Zic2, appeared unaltered, a notable reduction in one of its downstream targets, EphB1, was observed in ventral retina, suggesting that Ten-m2 may interact with this molecular pathway. Immunohistochemistry for c-fos, a neural activity marker, revealed that the area of V1 driven by ipsilateral inputs was reduced in KOs, while the ratio of ipsilateral-to-contralateral responses contributing to binocular activation during visually evoked potential recordings was also diminished. Finally, a novel two-alternative swim task revealed specific deficits associated with dorsal visual field. These data demonstrate a requirement for Ten-m2 in the establishment of ipsilateral projections, and thus the generation of binocular circuits, critical for mammalian visual function.

Restricted perinatal retinal degeneration induces retina reshaping and correlated structural rearrangement of the retinotopic map

The formation of the retinotopic map depends on the action of axon guidance molecules, activity-dependent mechanisms and axonal competition. However, little is known about the plasticity potential of the system and the effects on the remodelling of retinocollicular connections upon retinal insults. Here we create a mouse model in which retinal ganglion cells that project to anterior and posterior superior colliculus undergo cell death during topographic map formation. We show that the remaining retinal ganglion cells expand the targeted area in the superior colliculus and at the same time increase their spatial coverage in the retina in a correlated fashion. The resulting contralateral topographic map is overall maintained but less precise, while ipsilateral retinal ganglion cell axons are abnormally distributed in anterior and posterior superficial superior colliculus. These results suggest the presence of plastic mechanisms in the developing mammalian visual system to adjust retinal space and its target coverage and ensure a uniform map.