Growth Of Ganglion Cell Axons Research Articles

Asymmetric charge balanced waveforms direct retinal ganglion cell axon growth

Failure to direct axon regeneration to appropriate targets is a major barrier to restoring function after nerve injury. Development of strategies that can direct targeted regeneration of neurons such as retinal ganglion cells (RGCs) are needed to delay or reverse blindness in diseases like glaucoma. Here, we demonstrate that a new class of asymmetric, charge balanced (ACB) waveforms are effective at directing RGC axon growth, in vitro, without compromising cell viability. Unlike previously proposed direct current (DC) stimulation approaches, charge neutrality of ACB waveforms ensures the safety of stimulation while asymmetry ensures its efficacy. Furthermore, we demonstrate the relative influence of pulse amplitude and pulse width on the overall effectiveness of stimulation. This work can serve as a practical guideline for the potential deployment of electrical stimulation as a treatment strategy for nerve injury.

SCHEEPDOG: Programming Electric Cues to Dynamically Herd Large-Scale Cell Migration.

Directed cell migration is critical across biological processes spanning healing to cancer invasion, yet no existing tools allow real-time interactive guidance over such migration. We present a new bioreactor that harnesses electrotaxis-directed cell migration along electric field gradients-by integrating four independent electrodes under computer control to dynamically program electric field patterns, and hence steer cell migration. Using this platform, we programmed and characterized multiple precise, two-dimensional collective migration maneuvers in renal epithelia and primary skin keratinocyte ensembles. First, we demonstrated on-demand, 90-degree collective turning. Next, we developed a universal electrical stimulation scheme capable of programming arbitrary 2D migration maneuvers such as precise angular turns and migration in a complete circle. Our stimulation scheme proves that cells effectively time-average electric field cues, helping to elucidate the transduction timescales in electrotaxis. Together, this work represents an enabling platform for controlling cell migration with broad utility across many cell types.

Retinal Strip Culture for Studying Ganglion Cell Axon Growth.

Retinal ganglion cell (RGC) axons extend along the inner limiting membrane, which forms the extracellular matrix (ECM) containing laminin, collagen, and proteoglycans. RGC axons express integrin, which is activated by binding to ECM proteins to regulate cytoskeleton. To study the growth of RGC axons in vitro, maintaining the natural environment for them is absolutely necessary. For this purpose, culturing a strip of embryonic chick retina in Matrigel® is a suitable method. This article describes detailed techniques of the retinal strip culture.

Regulation of axonal EphA4 forward signaling is involved in the effect of EphA3 on chicken retinal ganglion cell axon growth during retinotectal mapping

The Eph and ephrins are involved in the genesis of topographic ordered connections at the visual system. Previously we demonstrated that tectal EphA3 stimulates axon growth of nasal retinal ganglion cells (RGCs) toward the caudal tectum preventing them from branching in the rostral tectum. Now we investigated whether tectal EphA3 plays this role by modulating the axonal EphA4 forward signaling or throughout axonal ephrin-As reverse signaling. For this purpose we used cultures of nasal retinal explants and dissociated retinal neurons from chicken embryos. We treated them with clustered EphA3-Fc, Fc (control), PI-PLC (sheds ephrin-As) or KYL (inhibits ephrin-As-mediated EphA4 activation). We achieved in vitro and in vivo electroporations of chicken embryo retinas with wild type EphA4, Ki-EphA4 (kinase inactive dominant negative EphA4) or EGFP in pMES expression vector. We performed immunocytochemistry, immunoprecipitation and Western blot against Eph/ephrin-As system. Our results showed that: 1) shedding of ephrin-As and the inhibition of ephrin-A-mediated EphA4 activity increase axon length and decrease axonal interstitial filopodia density of nasal RGCs; and 2) a dominant negative form of EphA4 increases axon growth in vitro and induces nasal RGC axons to grow passing throughout their target area in the caudal tectum meanwhile overexpression of EphA4 produces the opposite effects. All together, these results demonstrate that ephrin-A-mediated EphA4 forward signaling decreases the level of axon growth and increases the density of axonal interstitial filopodia of nasal RGCs. Besides, our results showed that: 3) EphA3 ectodomain increases axon growth and decreases the density of axonal interstitial filopodia and branching in vitro and in vivo and 4) EphA3 ectodomain diminishes the ephrin-A2/EphA4 colocalization, and the EphA4 and ephexin1 phosphorylation. All together, these results show that the EphA3 ectodomain produces the opposite effects than the EphA4 forward signaling, by decreasing this signaling pathway throughout competing with EphA4 for ephrin-As binding. Furthermore, it is proposed that tectal EphA3 participates in the establishment of retinotectal mapping throughout this mechanism and that EphAs can regulate axon growth and branching by modulating other EphA receptors forward signaling.

Novel axon projection after stress and degeneration in the Dscam mutant retina.

The Down syndrome cell adhesion molecule gene (Dscam) is required for normal dendrite patterning and promotes developmental cell death in the mouse retina. Loss-of-function studies indicate that Dscam is required for refinement of retinal ganglion cell (RGC) axons in the lateral geniculate nucleus, and in this study we report and describe a requirement for Dscam in the maintenance of RGC axon projections within the retina. Mouse Dscam loss of function phenotypes related to retinal ganglion cell axon outgrowth and targeting have not been previously reported, despite the abundance of axon phenotypes reported in Drosophila Dscam1 loss and gain of function models. Analysis of the Dscam deficient retina was performed by immunohistochemistry and Western blot analysis during postnatal development of the retina. Conditional targeting of Dscam and Jun was performed to identify factors underlying axon-remodeling phenotypes. A subset of RGC axons were observed to project and branch extensively within the Dscam mutant retina after eye opening. Axon remodeling was preceded by histological signs of RGC stress. These included neurofilament accumulation, axon swelling, axon blebbing and activation of JUN, JNK and AKT. Novel and extensive projection of RGC axons within the retina was observed after upregulation of these markers, and novel axon projections were maintained to at least one year of age. Further analysis of retinas in which Dscam was conditionally targeted with Brn3b or Pax6α Cre indicated that axon stress and remodeling could occur in the absence of hydrocephalus, which frequently occurs in Dscam mutant mice. Analysis of mice mutant for the cell death gene Bax, which executes much of Dscam dependent cell death, identified a similar axon misprojection phenotype. Deleting Jun and Dscam resulted in increased axon remodeling compared to Dscam or Bax mutants. Retinal ganglion cells have a very limited capacity to regenerate after damage in the adult retina, compared to the extensive projections made in the embryo. In this study we find that DSCAM and JUN limit ectopic growth of RGC axons, thereby identifying these proteins as targets for promoting axon regeneration and reconnection.

Inhibition of Retinal Ganglion Cell Axonal Outgrowth Through the Amino-Nogo-A Signaling Pathway

Nogo-A is a myelin-derived inhibitor playing a pivotal role in the prevention of axonal regeneration. A functional domain of Nogo-A, Amino-Nogo, exerts an inhibitory effect on axonal regeneration, although the mechanism is unclear. The present study investigated the role of the Amino-Nogo-integrin signaling pathway in primary retinal ganglion cells (RGCs) with respect to axonal outgrowth, which is required for axonal regeneration. Immunohistochemistry showed that integrin αv, integrin α5 and FAK were widely expressed in the visual system. Thy-1 and GAP-43 immunofluorescence showed that axonal outgrowth of RGCs was promoted by Nogo-A siRNA and a peptide antagonist of the Nogo-66 functional domain of Nogo-A (Nep1-40), and inhibited by a recombinant rat Nogo-A-Fc chimeric protein (Δ20). Western blotting revealed increased integrin αv and p-FAK expression in Nogo-A siRNA group, decreased integrin αv expression in Δ20 group and decreased p-FAK expression in Nep1-40 group. Integrin α5 expression was not changed in any group. RhoA G-LISA showed that RhoA activation was inhibited by Nogo-A siRNA and Δ20, but increased by Nep1-40 treatment. These results suggest that Amino-Nogo inhibits RGC axonal outgrowth primarily through the integrin αv signaling pathway.

EphA3 Expressed in the Chicken Tectum Stimulates Nasal Retinal Ganglion Cell Axon Growth and Is Required for Retinotectal Topographic Map Formation

BackgroundRetinotopic projection onto the tectum/colliculus constitutes the most studied model of topographic mapping and Eph receptors and their ligands, the ephrins, are the best characterized molecular system involved in this process. Ephrin-As, expressed in an increasing rostro-caudal gradient in the tectum/colliculus, repel temporal retinal ganglion cell (RGC) axons from the caudal tectum and inhibit their branching posterior to their termination zones. However, there are conflicting data regarding the nature of the second force that guides nasal axons to invade and branch only in the caudal tectum/colliculus. The predominant model postulates that this second force is produced by a decreasing rostro-caudal gradient of EphA7 which repels nasal optic fibers and prevents their branching in the rostral tectum/colliculus. However, as optic fibers invade the tectum/colliculus growing throughout this gradient, this model cannot explain how the axons grow throughout this repellent molecule.Methodology/Principal FindingsBy using chicken retinal cultures we showed that EphA3 ectodomain stimulates nasal RGC axon growth in a concentration dependent way. Moreover, we showed that nasal axons choose growing on EphA3-expressing cells and that EphA3 diminishes the density of interstitial filopodia in nasal RGC axons. Accordingly, in vivo EphA3 ectodomain misexpression directs nasal optic fibers toward the caudal tectum preventing their branching in the rostral tectum.ConclusionsWe demonstrated in vitro and in vivo that EphA3 ectodomain (which is expressed in a decreasing rostro-caudal gradient in the tectum) is necessary for topographic mapping by stimulating the nasal axon growth toward the caudal tectum and inhibiting their branching in the rostral tectum. Furthermore, the ability of EphA3 of stimulating axon growth allows understanding how optic fibers invade the tectum growing throughout this molecular gradient. Therefore, opposing tectal gradients of repellent ephrin-As and of axon growth stimulating EphA3 complement each other to map optic fibers along the rostro-caudal tectal axis.

Dynamic responses of Xenopus retinal ganglion cell axon growth cones to netrin‐1 as they innervate their in vivo target

Netrin-1 influences retinal ganglion cell (RGC) axon pathfinding and also participates in the branching and synaptic differentiation of mature RGC axons at their target. To investigate whether netrin also serves as an early target recognition signal in the brain, we examined the dynamic behavior of Xenopus RGC axons soon after they innervate the optic tectum. Time-lapse confocal microscopy imaging of RGC axons expressing enhanced yellow fluorescent protein demonstrated that netrin-1 is involved in early axon branching, as recombinant netrin-1 halted further advancement of growth cones into the tectum and induced back branching. RGC growth cones exhibited differential responses to netrin-1 that depended on the degree of differentiation of the axon and the developmental stage of the tadpole. Netrin-1 decreased the total number of branches on newly arrived RGC growth cones at the target, but increased the dynamic branching of more mature arbors at the later developmental stage. To further explore the response of axonal growth cones to netrin, Xenopus RGC axons were followed in culture by time-lapse imaging. Exposure to netrin-1 rapidly increased the forward advancement of the axon and decreased the size and expanse of the growth cone, while also inducing back branching. Taken together, the differential in vivo and in vitro responses to netrin-1 suggest that netrin alone is not sufficient to induce the cessation of growth cone advancement in the absence of a target but can independently modulate axon branching. Collectively, our findings reveal a novel role for netrin on RGC axon branch initiation as growth cones innervate their target.

Focus on Molecules: Math5 and retinal ganglion cells

The LIM-homeodomain transcription factor Islet-1 (Isl1) has been widely used as a marker of different subtypes of neurons in the developing and mature retina of vertebrates. During retinal neurogenesis, early Isl1 expression is detected in the nuclei of neuroblasts that give rise to ganglion, amacrine, bipolar, and horizontal cells. In the mature retina, Isl1 expression is restricted to the nuclei of ganglion cells, cholinergic amacrine cells, ON-bipolar cells, and subpopulations of horizontal cells. Recent studies have explored the functional mechanisms of Isl1 during specification and differentiation of these retinal cell types. Thus, conditional inactivation of Isl1 in the developing mouse retina disrupts retinal function, and also results in optic nerve hypoplasia, marked reductions in mature ganglion, amacrine, and bipolar cells, and a substantial increase in horizontal cells. Furthermore, conditional knockout shows delayed ganglion cell axon growth, ganglion cell axon guidance error, and ganglion cell nerve fiber defasciculation. These data together suggest a possible role for Isl1 in the early differentiation and maintenance of different vertebrate retinal cell types. This review examines whether the expression pattern of Isl1 during vertebrate retinal development is conserved across vertebrate species, and discusses current understanding of the developmental functions of Isl1 in retinogenesis.

Off-target effects of epidermal growth factor receptor antagonists mediate retinal ganglion cell disinhibited axon growth

Inhibition of central nervous system axon growth is reportedly mediated in part by calcium-dependent phosphorylation of axonal epidermal growth factor receptor, with local administration of the epidermal growth factor receptor kinase inhibitors AG1478 and PD168393 to an optic nerve lesion site promoting adult retinal ganglion cell axon regeneration. Here, we show that epidermal growth factor receptor was neither constitutively expressed, nor activated in optic nerve axons in our non-regenerating and regenerating optic nerve injury models, a finding that is inconsistent with phosphorylated epidermal growth factor receptor-dependent intra-axonal signalling of central nervous system myelin-related axon growth inhibitory ligands. However, epidermal growth factor receptor was localized and activated within most glia in the retina and optic nerve post-injury, and thus an indirect glial-dependent mechanism for stimulated retinal ganglion cell axon growth by epidermal growth factor receptor inhibitors seemed plausible. Using primary retinal cultures with added central nervous system myelin extracts, we confirmed previous reports that AG1478/PD168393 blocks epidermal growth factor receptor activation and promotes disinhibited neurite outgrowth. Paradoxically, neurites did not grow in central nervous system myelin extract-containing cultures after short interfering ribonucleic acid-mediated knockdown of epidermal growth factor receptor. However, addition of AG1478 restored neurite outgrowth to short interfering ribonucleic acid-treated cultures, implying that epidermal growth factor receptor does not mediate AG1478-dependent effects. TrkA-/B-/C-Fc fusion proteins and the kinase blocker K252a abrogated the neuritogenic activity in these cultures, correlating with the presence of the neurotrophins brain derived neurotrophic factor, nerve growth factor and neurotrophin-3 in the supernatant and increased intracellular cyclic adenosine monophosphate activity. Neurotrophins released by AG1478 stimulated disinhibited retinal ganglion cell axon growth in central nervous system myelin-treated cultures by the induction of regulated intramembraneous proteolysis of p75(NTR) and Rho inactivation. Retinal astrocytes/Müller cells and retinal ganglion cells were the source of neurotrophins, with neurite outgrowth halved in the presence of glial inhibitors. We attribute AG1478-stimulated neuritogenesis to the induced release of neurotrophins together with raised cyclic adenosine monophosphate levels in treated cultures, leading to axon growth and disinhibition by neurotrophin-induced regulated intramembraneous proteolysis of p75(NTR). These off-target effects of epidermal growth factor receptor kinase inhibition suggest a novel therapeutic approach for designing treatments to promote central nervous system axon regeneration.

Abnormal Axons in the Albino Optic Tract

There have been suggestions that the misrouting of axons at the optic chiasm leading to the abnormal ratio of crossed to uncrossed axons in the optic tract of albinos is a consequence of abnormal timing of ganglion cell axon outgrowth. The sequence of genesis of ganglion cell classes and their axon outgrowth is correlated with the deep-to-superficial distribution of their axons by size in the mammalian optic tract. Optic tract axon order in albino and normally pigmented ferrets was, therefore, examined to determine whether an abnormal pattern of retinal ganglion cell genesis and axon outgrowth is evident in albinos. Light and electron microscopy were used to study axon diameters and myelin thickness of axons in the optic tracts of adult albino and pigmented ferrets. In the optic tract, large-diameter axons are confined superficially in the normally pigmented ferret but are present throughout its depth in albinos. The abnormally located large axons and neighboring small-diameter axons in the albino have an abnormal axon diameter/myelin thickness ratio; large-diameter axons are poorly myelinated, and small-diameter axons exhibit an abnormally thick myelin sheath. These deep abnormal axons originate from the contralateral retina. In addition to the known disruptions of normal organization in the visual system, albinos have an abnormal axon diameter distribution and a population of morphologically abnormal axons in the optic tract. These abnormal axons may represent the population of aberrantly crossed axons found in all albino mammals.

Retina development in zebrafish requires the heparan sulfate proteoglycan agrin

Recent studies from our laboratory have begun to elucidate the role of agrin in zebrafish development. One agrin morphant phenotype that results from agrin knockdown is microphthalmia (reduced eye size). To begin to understand the mechanisms underlying the role of agrin in eye development, we have analyzed retina development in agrin morphants. Retinal differentiation is impaired in agrin morphants, with retinal lamination being disrupted following agrin morpholino treatment. Pax 6.1 and Mbx1 gene expression, markers of eye development, are markedly reduced in agrin morphants. Formation of the optic fiber layer of the zebrafish retina is also impaired, exhibited as both reduced size of the optic fiber layer, and disruption of retinal ganglion cell axon growth to the optic tectum. The retinotectal topographic projection to the optic tectum is perturbed in agrin morphants in association with a marked loss of heparan sulfate expression in the retinotectal pathway, with this phenotype resembling retinotectal phenotypes observed in mutant zebrafish lacking enzymes for heparan sulfate synthesis. Treatment of agrin morphants with a fibroblast growth factor (Fgf) receptor inhibitor, rescue of the retinal lamination phenotype by transplantation of Fgf8-coated beads, and disruption of both the expression of Fgf-dependent genes and activation of ERK in agrin morphants provides evidence that agrin modulation of Fgf function contributes to retina development. Collectively, these agrin morphant phenotypes provide support for a crucial role of agrin in retina development and formation of an ordered retinotectal topographic map in the optic tectum of zebrafish.

Pbx homeodomain proteins pattern both the zebrafish retina and tectum

BackgroundPbx genes encode TALE class homeodomain transcription factors that pattern the developing neural tube, pancreas, and blood. Within the hindbrain, Pbx cooperates with Hox proteins to regulate rhombomere segment identity. Pbx cooperates with Eng to regulate midbrain-hindbrain boundary maintenance, and with MyoD to control fast muscle cell differentiation. Although previous results have demonstrated that Pbx is required for proper eye size, functions in regulating retinal cell identity and patterning have not yet been examined.ResultsAnalysis of retinal ganglion cell axon pathfinding and outgrowth in pbx2/4 null embryos demonstrated a key role for pbx genes in regulating neural cell behavior. To identify Pbx-dependent genes involved in regulating retino-tectal pathfinding, we conducted a microarray screen for Pbx-dependent transcripts in zebrafish, and detected genes that are specifically expressed in the eye and tectum. A subset of Pbx-dependent retinal transcripts delineate specific domains in the dorso-temporal lobe of the developing retina. Furthermore, we determined that some Pbx-dependent transcripts also require Meis1 and Gdf6a function. Since gdf6a expression is also dependent on Pbx, we propose a model in which Pbx proteins regulate expression of the growth factor gdf6a, which in turn regulates patterning of the dorso-temporal lobe of the retina. This, in concert with aberrant tectal patterning in pbx2/4 null embryos, may lead to the observed defects in RGC outgrowth.ConclusionThese data define a novel role for Pbx in patterning the vertebrate retina and tectum in a manner required for proper retinal ganglion cell axon outgrowth.

Targeted disruption of Aldh1a1 (Raldh1) provides evidence for a complex mechanism of retinoic acid synthesis in the developing retina.

Genetic studies have shown that retinoic acid (RA) signaling is required for mouse retina development, controlled in part by an RA-generating aldehyde dehydrogenase encoded by Aldh1a2 (Raldh2) expressed transiently in the optic vesicles. We examined the function of a related gene, Aldh1a1 (Raldh1), expressed throughout development in the dorsal retina. Raldh1(-/-) mice are viable and exhibit apparently normal retinal morphology despite a complete absence of Raldh1 protein in the dorsal neural retina. RA signaling in the optic cup, detected by using a RARE-lacZ transgene, is not significantly altered in Raldh1(-/-) embryos at embryonic day 10.5, possibly due to normal expression of Aldh1a3 (Raldh3) in dorsal retinal pigment epithelium and ventral neural retina. However, at E16.5 when Raldh3 is expressed ventrally but not dorsally, Raldh1(-/-) embryos lack RARE-lacZ expression in the dorsal retina and its retinocollicular axonal projections, whereas normal RARE-lacZ expression is detected in the ventral retina and its axonal projections. Retrograde labeling of adult Raldh1(-/-) retinal ganglion cells indicated that dorsal retinal axons project to the superior colliculus, and electroretinography revealed no defect of adult visual function, suggesting that dorsal RA signaling is unnecessary for retinal ganglion cell axonal outgrowth. We observed that RA synthesis in liver of Raldh1(-/-) mice was greatly reduced, thus showing that Raldh1 indeed participates in RA synthesis in vivo. Our findings suggest that RA signaling may be necessary only during early stages of retina development and that if RA synthesis is needed in dorsal retina, it is catalyzed by multiple enzymes, including Raldh1.

The Rho/ROCK pathway mediates neurite growth-inhibitory activity associated with the chondroitin sulfate proteoglycans of the CNS glial scar

Axons fail to regenerate in the central nervous system after injury. Chondroitin sulfate proteoglycans (CSPG) expressed in the scar significantly contribute to the nonpermissive properties of the central nervous system environment. To examine the inhibitory activity of a CSPG mixture on retina ganglion cell (RGC) axon growth, we employed both a stripe assay and a nerve fiber outgrowth assay. We show that the inhibition exerted by CSPGs in vitro can be blocked by application of either C3 transferase, a specific inhibitor of the Rho GTPase, or Y27632, a specific inhibitor of the Rho kinase. These results demonstrate that CSPG-associated inhibition of neurite outgrowth is mediated by the Rho/ROCK signaling pathway. Consistent with these results, we found that retina ganglion cell axon growth on glial scar tissue was enhanced in the presence of C3 transferase and Y27632, respectively. In addition, we show that the recently identified inhibitory CSPG Te38 is upregulated in the lesioned spinal cord.

The roles of Hedgehogs and Fibroblast Growth Factors in eye development and retinal cell rescue

Knowledge of normal eye development is crucial for the development of retinal rescue strategies. I shall focus on two signalling pathways that affect retinal development. Fibroblast growth factors function in retinal cell proliferation, retinal ganglion cell axon guidance and target recognition, craniofacial patterning and lens induction. Hedgehog proteins are required for progression of the neurogenic wave, cell proliferation, photoreceptor differentiation, retinal ganglion cell axon growth and craniofacial patterning. These signalling pathways have pleiotropic effects, can interact and have the potential to be used therapeutically. The zebrafish model organism may be well suited to studying how signalling pathways interact.

The role of bone morphogenetic proteins in the differentiation of the ventral optic cup.

The ventral region of the chick embryo optic cup undergoes a complex process of differentiation leading to the formation of four different structures: the neural retina, the retinal pigment epithelium (RPE), the optic disk/optic stalk, and the pecten oculi. Signaling molecules such as retinoic acid and sonic hedgehog have been implicated in the regulation of these phenomena. We have now investigated whether the bone morphogenetic proteins (BMPs) also regulate ventral optic cup development. Loss-of-function experiments were carried out in chick embryos in ovo, by intraocular overexpression of noggin, a protein that binds several BMPs and prevents their interactions with their cognate cell surface receptors. At optic vesicle stages of development, this treatment resulted in microphthalmia with concomitant disruption of the developing neural retina, RPE and lens. At optic cup stages, however, noggin overexpression caused colobomas, pecten agenesis, replacement of the ventral RPE by neuroepithelium-like tissue, and ectopic expression of optic stalk markers in the region of the ventral retina and RPE. This was frequently accompanied by abnormal growth of ganglion cell axons, which failed to enter the optic nerve. The data suggest that endogenous BMPs have significant effects on the development of ventral optic cup structures.

Brn3b/Brn3c double knockout mice reveal an unsuspected role for Brn3c in retinal ganglion cell axon outgrowth.

In mice, Brn3 POU domain transcription factors play essential roles in the differentiation and survival of projection neurons within the retina, inner ear, dorsal root and trigeminal ganglia. During retinal ganglion cell differentiation, Brn3b is expressed first, followed by Brn3a and Brn3c. Targeted deletion of Brn3b, but not Brn3a or Brn3c, leads to a loss of most retinal ganglion cells before birth. However, as a few retinal ganglion cells are still present in Brn3b(-/-) mice, Brn3a and Brn3c may partially compensate for the loss of Brn3b. To examine the role of Brn3c in retinal ganglion cell development, we generated Brn3b/Brn3c double knockout mice and analyzed their retinas and optic chiasms. Retinal ganglion cell axons from double knockout mice were more severely affected than were those from Brn3b-deficient mice, indicating that Brn3c was required for retinal ganglion cell differentiation and could partially compensate for the loss of Brn3b. Moreover, Brn3c had functions in retinal ganglion cell differentiation separate from those of Brn3b. Ipsilateral and misrouted projections at the optic chiasm were overproduced in Brn3b(-/-) mice but missing were entirely in optic chiasms of Brn3b/Brn3c double knockout mice, suggesting that Brn3c controlled ipsilateral axon production. Forced expression of Brn3c in Brn3b(-/-) retinal explants restored neurite outgrowth, demonstrating that Brn3c could promote axon outgrowth in the absence of Brn3b. Our results reveal a complex genetic relationship between Brn3b and Brn3c in regulating the retinal ganglion cell axon outgrowth.

Characterization of a New Brain-Derived Proteoglycan Inhibiting Retinal Ganglion Cell Axon Outgrowth

A proteoglycan was identified and isolated from physiological saline extracts of chick embryo brains by using a new monoclonal antibody (hybridoma clone mab Te38). The purified proteoglycan displayed an apparent molecular mass of 2500–3500 kDa, which became reduced to 370 and 600 kDa after digestion with chondroitinase ABC or chondroitinase AC. After additional treatment with keratanase the 600-kDa band was no longer detectable in Western blots. The specific epitope recognized by mab Te38 is an O-linked carbohydrate associated with the core protein. Tenascin-C, an extracellular matrix protein known to associate with several proteoglycans, copurified with the mab Te38 proteoglycan on the immunoaffinity column. Mab Te38 binds to the surface of nonneuronal cells; in sections from the primary visual system, expression is restricted to cells in the optic fissure, the dorsal optic nerve, and the chiasm. No retinal cells were found to express the mab Te38 epitope. The isolated molecule inhibited axon outgrowth from retinal explants when offered bound to a substrate consisting of either matrigel or collagen, chondroitinase treatment did not alter the inhibitory properties. The distribution and in vitro function of the Te38 proteoglycan indicate that it may serve a role in guidance of retinal ganglion cell axons.

Control of retinal ganglion cell axon growth: a new role for Sonic hedgehog.

Retinal ganglion cell (RGC) axons grow towards the diencephalic ventral midline during embryogenesis guided by cues whose nature is largely unknown. We provide in vitro and in vivo evidence for a novel role of Sonic hedgehog (SHH) as a negative regulator of growth cone movement. SHH suppresses both the number and the length of neurites emerging from the chick retina but not from neural tube or dorsal root ganglia explants, without interfering with their rate of proliferation and differentiation. Similarly, retroviral-mediated ectopic expression of Shh along the chick visual pathway greatly interferes the growth of RGC axons. Upon SHH addition to grown neurites, the intracellular level of cAMP decreases, suggesting that the dampening of growth cone extension mediated by SHH may involve interaction with its receptor Patched which is expressed by RGC. Based on these findings, we propose that Shh expression at the chiasm border defines a constrained pathway within the ventral midline which serves to guide the progression of RGC axons.