Role of cell adhesion molecule DM-GRASP in growth and orientation of retinal ganglion cell axons

The cell adhesion molecule (CAM) DM-GRASP was investigated with respect to a role for axonal growth and navigation in the developing visual system. Expression analysis reveals that DM-GRASP's presence is highly spatiotemporally regulated in the chick embryo retina. It is restricted to the optic fiber layer (OFL) and shows an expression maximum in a phase when the highest number of retinal ganglion cell (RGC) axons extend. In the developing retina, axons grow between the DM-GRASP-displaying OFL and the Laminin-rich basal lamina. We show that DM-GRASP enhances RGC axon extension and growth cone size on Laminin substrate in vitro. Preference assays reveal that DM-GRASP-containing lanes guide RGC axons, partially depending on NgCAM in the axonal membrane. Inhibition of DM-GRASP in organ-cultured eyes perturbs orientation of RGC axons at the optic fissure. Instead of leaving the retina, RGC axons cross the optic fissure and grow onto the opposite side of the retina. RGC axon extension per se and navigation from the peripheral retina towards the optic fissure, however, is not affected. Our results demonstrate a role of DM-GRASP for axonal pathfinding in an early phase of the formation of the higher vertebrate central nervous system.     

Fate of oligodendrocytes during retinal axon degeneration and regeneration in the goldfish visual pathway

Retinal axons in goldfish regenerate after optic nerve lesion, restore synaptic connections, and become myelinated by oligodendrocytes. The fate of oligodendrocytes during these events is not known and may require generation of new oligodendrocytes or dedifferentiation and redifferentiation of the existing ones. To determine the reaction of oligodendrocytes to optic nerve lesion, we used the terminal transferase technique to detect apoptosis, bromodeoxyuridine incorporation to reveal mitosis, antibodies to identify myelin and oligodendrocytes, and Lucifer yellow injections to reveal cell morphology. Along with the reappearance of the myelin molecules 36K protein, galactocerebroside, and myelin basic protein, myelinating oligodendrocytes (identified by Lucifer yellow injections) reappear 21 days postlesion. Prior to this time, the dye-filled cells had few processes oriented along the regenerating axons. They resembled oligodendrocytes seen both in vitro and in vivo which express the L1-related E587 antigen and synthesize the 36K myelin protein in coculture with axons. No signs of oligodendrocyte apoptosis were detected after lesion and only few of the oligodendrocytes present had recently arisen. 36K/E587 double-labeled oligodendrocytes which were most likely dedifferentiating oligodendrocytes were identified in 8-day postlesion nerves among E587-positive elongate cells whose numbers increased until 14 days postlesion. These findings suggest that oligodendrocytes dedifferentiate-like Schwann cells-from cells which express myelin molecules to elongate cells which express the L1/E587 antigen. They redifferentiate to myelinate axons from roughly 3 weeks onward. These findings suggest an adaptive plasticity of goldfish oligodendrocytes beneficial to the repair of the visual pathway.     

Neurolin, a cell surface glycoprotein on growing retinal axons in the goldfish visual system, is reexpressed during retinal axonal regeneration.

The mAb E 21 recognizes a cell surface glycoprotein selectively associated with fish retinal ganglion cell axons that are in a state of growth. All retinal axons and ganglion cells in goldfish embryos stained for E 21. In adult fish, however, E 21 immunoreactivity exhibited a patterned distribution in ganglion cells in the marginal growth zone of the continuously enlarging fish retina and the new axons emerging from these cells in the retina, optic nerve, and optic tract. The E 21 antigen was absent from older axons, except the terminal arbor layer in the tectum, the Stratum fibrosum et griseum superficiale where it was uniformly distributed. Upon optic nerve transection, the previously unlabeled axons reacquired E 21 positivity as they regenerated throughout their path to the tectum. Several months after ONS, however, E 21 staining disappeared from the regenerated axons over most of their lengths but reappeared as in normal fish in the terminal arbor layer. The immunoaffinity-purified E 21 antigen, called Neurolin, has an apparent molecular mass of 86 kD and contains the HNK1/L2 carbohydrate moiety, like several members of the class of cell adhesion molecules of the Ig superfamily. The NH2-terminal amino acid sequence has homologies to the cell adhesion molecule DM-Grasp recently described in the chicken. Thus, retinal ganglion cell axons express Neurolin during their development and are able to reexpress this candidate cell adhesion molecule during axonal regeneration, suggesting that Neurolin is functionally important for fish retinal axon growth.     

Upregulation of IGF-I in the goldfish retinal ganglion cells during the early stage of optic nerve regeneration

Goldfish retinal ganglion cells (RGCs) can regrow their axons after optic nerve injury. However, the reason why goldfish RGCs can regenerate after nerve injury is largely unknown at the molecular level. To investigate regenerative properties of goldfish RGCs, we divided the RGC regeneration process into two components: (1) RGC survival, and (2) axonal elongation processes. To characterize the RGC survival signaling pathway after optic nerve injury, we investigated cell survival/death signals such as Bcl-2 family members in the goldfish retina. Amounts of phospho-Akt (p-Akt) and phospho-Bad (p-Bad) in the goldfish retina rapidly increased four- to five-fold at the protein level by 3-5 days after nerve injury. Subsequently, Bcl-2 levels increased 1.7-fold, accompanied by a slight reduction in caspase-3 activity 10-20 days after injury. Furthermore, level of insulin-like growth factor-I (IGF-I), which activates the phosphatidyl inositol-3-kinase (PI3K)/Akt system, increased 2-3 days earlier than that of p-Akt in the goldfish retina. The cellular localization of these molecular changes was limited to RGCs. IGF-I treatment significantly induced phosphorylation of Akt, and strikingly induced neurite outgrowth in the goldfish retina in vitro. On the contrary, addition of the PI3K inhibitor wortmannin, and IGF-I antibody inhibited Akt phosphorylation and neurite outgrowth in an explant culture. Thus, we demonstrated, for the first time, the signal cascade for early upregulation of IGF-I, leading to RGC survival and axonal regeneration in adult goldfish retinas through PI3K/Akt system after optic nerve injury. The present data strongly indicate that IGF-I is one of the most important molecules for controlling regeneration of RGCs after optic nerve injury.     

NF-Protocadherin Regulates Retinal Ganglion Cell Axon Behaviour in the Developing Visual System

Cell adhesion molecules play a central role in mediating axonal tract development within the nascent nervous system. NF-protocadherin (NFPC), a member of the non-clustered protocadherin family, has been shown to regulate retinal ganglion cell (RGC) axon and dendrite initiation, as well as influencing axonal navigation within the mid-optic tract. However, whether NFPC mediates RGC axonal behaviour at other positions within the optic pathway remains unclear. Here we report that NFPC plays an important role in RGC axonogenesis, but not in intraretinal guidance. Moreover, axons with reduced NFPC levels exhibit insensitivity to Netrin-1, an attractive guidance cue expressed at the optic nerve head. Netrin-1 induces rapid turnover of NFPC localized to RGC growth cones, suggesting that the regulation of NFPC protein levels may underlie Netrin-1-mediated entry of RGC axons into the optic nerve head. At the tectum, we further reveal a function for NFPC in controlling RGC axonal entry into the final target area. Collectively, our results expand our understanding of the role of NFPC in RGC guidance and illustrate that this adhesion molecule contributes to axon behaviour at multiple points in the optic pathway.     

The cell adhesion molecule NrCAM is crucial for growth cone behaviour and pathfinding of retinal ganglion cell axons

We investigated the role of the cell adhesion molecule NrCAM for axonal growth and pathfinding in the developing retina. Analysis of the distribution pattern of NrCAM in chick embryo retina sections and flat-mounts shows its presence during extension of retinal ganglion cell (RGC) axons; NrCAM is selectively present on RGC axons and is absent from the soma. Single cell cultures show an enrichment of NrCAM in the distal axon and growth cone. When offered as a substrate in addition to Laminin, NrCAM promotes RGC axon extension and the formation of growth cone protrusions. In substrate stripe assays, mimicking the NrCAM-displaying optic fibre layer and the Laminin-rich basal lamina, RGC axons preferentially grow on NrCAM lanes. The three-dimensional analysis of RGC growth cones in retina flat-mounts reveals that they are enlarged and form more protrusions extending away from the correct pathway under conditions of NrCAM-inhibition. Time-lapse analyses show that these growth cones pause longer to explore their environment, proceed for shorter time spans, and retract more often than under control conditions; in addition, they often deviate from the correct pathway towards the optic fissure. Inhibition of NrCAM in organ-cultured intact eyes causes RGC axons to misroute at the optic fissure; instead of diving into the optic nerve head, these axons cross onto the opposite side of the retina. Our results demonstrate a crucial role for NrCAM in the navigation of RGC axons in the developing retina towards the optic fissure, and also for pathfinding into the optic nerve.     

Mutations affecting retinotectal axonal pathfinding in Medaka, Oryzias latipes

We screened for mutations affecting retinotectal axonal projection in Medaka, Oryzias latipes. In wild-type Medaka embryos, all the axons of retinal ganglion cells (RGCs) project to the contralateral tectum, such that the topological relationship of the retinal field is maintained. We labeled RGC axons using DiI/DiO at the nasodorsal and temporoventral positions of the retina, and screened for mutations affecting the pattern of stereotypic projections to the tectum. By screening 184 mutagenized haploid genomes, seven mutations in five genes causing defects in axonal pathfinding were identified, whereas mutations affecting the topographic projection of RGC axons were not found. The mutants were grouped into two classes according to their phenotypes. In mutants of Class I, a subpopulation of the RGC axons branched out either immediately after leaving the eye or after reaching the midline, and this axonal subpopulation projected to the ipsilateral tectum. In mutants of Class II, subpopulations of RGC axons branched out after crossing the midline and projected aberrantly. These mutants will provide clues to understanding the functions of genes essential for axonal pathfinding, which may be conserved or partly divergent among vertebrates.     

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.     

Selective fasciculation of nerve fibres in culture

Small aggregates of embryonic rat retina and perinatal rat sympathetic ganglia were put into culture and allowed to form axonal outgrowths. Neuritic outgrowths from adjacent sympathetic explants grew freely into one another and appeared to form common bundles; neurites from adjacent retinal explants showed a similar pattern of interaction. In contrast, when neurites from retinal and sympathetic explants confronted one another they showed a marked avoidance reaction. This response included the partial retraction of some axons, changes in the direction of their growth and, eventually, the formation of discrete bundles of a single kind of axon. In a second kind of experiment, single-cell preparations from retina and sympathetic ganglia were mixed and allowed to form aggregates. These were put into culture and the distribution of sympathetic fibres within the resulting outgrowth was detected by incubation with radioactive norepinephrine followed by radioautography. It was found that the sympathetic axons segregated from the retinal axons as they grew and formed separate bundles of predominantly one kind of fibre. It is concluded that selective fasciculation of nerve axons can occur in culture and we discuss some possible contributory mechanisms.     

Distribution of GAP-43, β-III tubulin and F-actin in developing and regenerating axons and their growth cones in vitro, following neurotrophin treatment

Brain derived neurotrophic factor (BDNF) when added to explant cultures of both embryonic and adult retinal ganglion cell (RGC) axons exerted a marked effect on their growth cone size and complexity and also on the intensity of GAP-43, ß-III tubulin and F-actin immunoreaction product in their axons. GAP-43 was distributed in axons, lamellipodia, and filopodia whereas ß-III tubulin was distributed along the length of developing and adult regenerating axons and also in the C-domain of their growth cones. BDNF-treated developing RGC growth cones were larger and displayed increased numbers of GAP-43 and microtubule-containing branches. Although filopodia and lamellipodia were lost from both developing and adult RGC growth cones following trkB-IgG treatment, the intensity of the immunoreaction product of all these molecules was reduced and trkB-IgGs had no effect on the axonal distribution of ß-III tubulin and GAP-43. BDNF-treated growth cones also displayed increased numbers of F-actin containing filopodia and axonal protrusions. This study demonstrates, for the first time, that trkB-IgG treatment causes the loss of F-actin in the P-domain of growth cone tips in developing and regenerating RGC axons. Although microtubules and F-actin domains normally remained distinct in cultured growth cones, ß-III tubulin and F-actin overlapped within the growth cone C-domain, and within axonal protrusions of adult RGC axons, under higher concentrations of BDNF. The collapse of RGC growth cones appeared to correlate with the loss of F-actin. In vitro, trkB signalling may therefore be involved in the maintenance and stabilisation of RGC axons, by influencing F-actin polymerisation, stabilisation and distribution.     

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. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 75: 1385–1401, 2015     

A multifunctional cell surface developmental stage-specific antigen in the cockroach embryo: involvement in pathfinding by CNS pioneer axons

mAb DSS-8 binds to a 164-kD developmental stage-specific cell surface antigen in the nervous system of the cockroach, Periplaneta americana. The antigen is localized to different subsets of cells at various stages of development. The spatial and temporal distributions of DSS-8 binding were determined and are consistent with this antigen playing multiple roles in the development of the nervous system. Direct identification of some of these functions was made by perturbation experiments in which pioneer axon growth occurs in embryos that are cultured in vitro in the presence of mAb DSS-8 or its Fab fragment. Under these conditions the pioneer axons of the median fiber tract grow but follow altered pathways. In a smaller percentage of the ganglia, the immunoreagents additionally produce defasciculation of a subset of DSS-8 labeled axons. Therefore, direct roles for the DSS-8 antigen in both the guidance of pioneer axons and selective fasciculation have been demonstrated.     

The role of laminin and the laminin/fibronectin receptor complex in the outgrowth of retinal ganglion cell axons

Chick embryo retinal ganglion cell (RGC) axons grow to the optic tectum along a stereotyped route, as if responding to cues distributed along the pathway. We showed previously that, in culture, RGCs from embryonic Day 6 retina are responsive to the neurite-promoting effects of the extracellular matrix glycoprotein laminin and that this response is lost by RGCs at a later stage of development. Here we report that, before axon outgrowth is initiated in vivo, laminin, is expressed along the optic pathway at nonbasal lamina sites that are accessible to the growth cones of RGC axons. The distribution of laminin within the pathway is consistent with its localization at the end-feet of neuroepithelial cells that line the route, and it continues to be expressed at these marginal sites during the first week of embryonic development. At later stages, concomitant with the loss of response by RGCs in culture, laminin becomes restricted to basal laminae at the retinal inner limiting membrane and pial surface of the optic pathway. Neurofilament-positive RGC axons bind a monoclonal antibody, JG22, which recognizes the laminin/fibronectin receptor complex, and continue to do so throughout embryonic development. We show that, in vitro, the JG22 antigen expressed by RGCs appears to function as a laminin receptor, by demonstrating that JG22 antibody blocks neurite outgrowth on a substrate of laminin. These findings are consistent with the possibility that laminin defines a transient performed pathway specifically recognized by early RGC growth cones as they navigate toward their central target.     

Temporary colonization of the site of lesion by macrophages is a prelude to the arrival of regenerated axons in injured goldfish optic nerve

Summary. In crushed goldfish optic nerve, regenerating axons cross the site of lesion within 10 days following injury. Some 30 days later, Schwann cells accumulate at the lesion, where they myelinate the new axons. In this study, we have used immunohistochemistry and electron microscopy to examine the cellular environment of the crush site prior to the establishment of Schwann cells in order to learn more about the early events that contribute to axonal regeneration. During the first week following injury, macrophages enter the site of lesion and efficiently phagocytose the debris. The infiltration of macrophages precedes the arrival of regenerating axons that abut and surround these phagocytes. Based on EM morphology and phagocytic capacity, macrophages of the type observed at the site of lesion are not present in the degenerating distal nerve segment, where debris clearance is shared between conventional microglia and astrocytes over a period of several weeks. During this period, axon bundles emerging distally from the injury zone become enwrapped by astrocyte processes, thereby re-establishing the characteristic fascicular cytoarchitecture of the optic nerve. The process of fasciculation also leads to the displacement of myelin debris to the margins of the fiber bundles, where it is trapped by the astrocytes. Our results suggest that the early robust appearance of macrophages at the lesion, and their effectiveness as phagocytes compared with the microglia distally, may contribute to the vigorous axonal regeneration across the crush, beyond which axons<197>excepting the pioneers<197>extend through newly formed debris-free channels delineated by astrocyte processes.     

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.     

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.     

Molecular mechanisms separating two axonal pathways during embryonic development of the avian optic tectum

During embryonic development of the avian optic tectum, retinal and tectobulbar axons form an orthogonal array of nerve processes. Growing axons of both tracts are transiently very closely apposed to each other. Despite this spatial proximity, axons from the two pathways do not intermix, but instead restrict their growth to defined areas, thus forming two separate plexiform layers, the stratum opticum and the stratum album centrale. In this study we present experimental evidence indicating that the following three mechanisms might play a role in segregating both axonal populations: Retinal and tectobulbar axons differ in their ability to use the extracellular matrix protein laminin as a substrate for axonal elongation; the environment in the optic tectum is generally permissive for retinal axons, but is specifically nonpermissive for tectobulbar axons, resulting in a strong fasciculation of the latter; and growth cones of temporal retinal axons are reversibly inhibited in their motility by direct contact with the tectobulbar axon's membrane.     

Multiple fates of newly synthesized neurofilament proteins: evidence for a stationary neurofilament network distributed nonuniformly along axons of retinal ganglion cell neurons

We have studied the fate of neurofilament proteins (NFPs) in mouse retinal ganglion cell (RGC) neurons from 1 to 180 d after synthesis and examined the proximal-to-distal distribution of the newly synthesized 70-, 140-, and 200-kD subunits along RGC axons relative to the distribution of neurofilaments. Improved methodology for intravitreal delivery of [3H]proline enabled us to quantitate changes in the accumulation and subsequent decline of radiolabeled NFP subunits at various postinjection intervals and, for the first time, to estimate the steady state levels of NFPs in different pools within axons. Two pools of newly synthesized triplet NFPs were distinguished based on their kinetics of disappearance from a 9-mm "axonal window" comprising the optic nerve and tract and their temporal-spatial distribution pattern along axons. The first pool disappeared exponentially between 17 and 45 d after injection with a half-life of 20 d. Its radiolabeled wavefront advanced along axons at 0.5-0.7 mm/d before reaching the distal end of the axonal window at 17 d, indicating that this loss represented the exit of neurofilament proteins composing the slowest phase of axoplasmic transport (SCa or group V) from axons. About 32% of the total pool of radiolabeled neurofilament proteins, however, remained in axons after 45 d and disappeared exponentially at a much slower rate (t 1/2 = 55 d). This second NFP pool assumed a nonuniform distribution along axons that was characterized proximally to distally by a 2.5-fold gradient of increasing radioactivity. This distribution pattern did not change between 45 and 180 d indicating that neurofilament proteins in the second pool constitute a relatively stationary structure in axons. Based on the relative radioactivities and residence time (or turnover) of each neurofilament pool in axons, we estimate that, in the steady state, more neurofilament proteins in mouse RGC axons may be stationary than are undergoing continuous slow axoplasmic transport. This conclusion was supported by biochemical analyses of total NFP content and by electron microscopic morphometric studies of neurofilament distribution along RGC axons. The 70-, 140-, and 200-kD subunits displayed a 2.5-fold proximal to distal gradient of increasing content along RGC axons. Neurofilaments were more numerous at distal axonal levels, paralleling the increased content of NFP.(ABSTRACT TRUNCATED AT 400 WORDS)     

A chondroitin sulfate proteoglycan may influence the direction of retinal ganglion cell outgrowth.

In the developing retina, retinal ganglion cell (RGC) axons elongate toward the optic fissure, even though no obvious directional restrictions exist. Previous studies indicate that axon-matrix interactions are important for retinal ganglion cell axon elongation, but the factors that direct elongation are unknown. Chondroitin sulfate proteoglycan (CS-PG), a component of the extracellular matrix, repels elongating dorsal root ganglion (DRG) axons in vitro and is present in vivo in the roof plate of the spinal cord, a structure that acts as a barrier to DRG axons during development. In this study, we examined whether CS-PG may regulate the pattern of retinal ganglion cell outgrowth in the developing retina. Immunocytochemical analysis showed that CS-PG was present in the innermost layers of the developing rat retina. The expression of CS-PG moved peripherally with retinal development, always remaining at the outer edge of the front of the developing axons. CS-PG was no longer detectable with immunocytochemical techniques when RGC axon elongation in the retina is complete. Results of studies in vitro showed that CS-PG, isolated from bovine nasal cartilage and chick limb, was inhibitory to elongating RGC axons and that RGC growth cones were more sensitive to CS-PG than were DRG neurites tested at the same concentrations of CS-PG. The behavior of retinal growth cones as they encounter CS-PG was characterized using time-lapse video microscopy. Filopodia of the RGC growth cones extended to and sampled the CS-PG repeatedly. With time, the growth cones turned to avoid outgrowth on the CS-PG and grew only on laminin. While numerous studies have shown the presence of positive factors within the retina that may guide developing RGC axons, this is the first demonstration of an inhibitory or repelling molecule in the retina that may regulate axon elongation. Taken together, these data suggest that the direction of RGC outgrowth in the retina may be regulated by the proper ratio of growth-promoting molecules, such as laminin, to growth-inhibiting molecules, like CS-PG, present in the correct pattern and concentrations along the retinal ganglion cell pathway.     

Requirement for the zebrafish mid-hindbrain boundary in midbrain polarisation, mapping and confinement of the retinotectal projection.

The organizer at the midbrain-hindbrain boundary (MHB organizer) has been proposed to induce and polarize the midbrain during development. We investigate the requirement for the MHB organizer in acerebellar mutants, which lack a MHB and cerebellum, but retain a tectum, and are mutant for fgf8, a candidate inducer and polarizer. We examine the retinotectal projection in the mutants to assay polarity in the tectum. In mutant tecta, retinal ganglion cell (RGC) axons form overlapping termination fields, especially in the ventral tectum, and along both the anterior-posterior and dorsal-ventral axis of the tectum, consistent with a MHB requirement in generating midbrain polarity. However, polarity is not completely lost in the mutant tecta, in spite of the absence of the MHB. Moreover, graded expression of the ephrin family ligand Ephrin-A5b is eliminated, whereas Ephrin-A2 and Ephrin-A5a expression is leveled in acerebellar mutant tecta, showing that ephrins are differentially affected by the absence of the MHB. Some RGC axons overshoot beyond the mutant tectum, suggesting that the MHB also serves a barrier function for axonal growth. By transplanting whole eye primordia, we show that mapping defects and overshooting largely, but not exclusively, depend on tectal, but not retinal genotype, and thus demonstrate an independent function for Fgf8 in retinal development. The MHB organizer, possibly via Fgf8 itself, is thus required for midbrain polarisation and for restricting axonal growth, but other cell populations may also influence midbrain polarity.