Inaccuracies in initial growth and arborization of chick retinotectal axons followed by course corrections and axon remodeling to develop topographic order

The retinotectal projection is organized in a precise retinotopic manner. We find, though, that during development the growth and arborization of temporal retinal axons within the optic tectum of chick embryos is initially imprecise. Axonal targeting errors occur along the rostral-caudal and medial-lateral tectal axes, and arbors are formed at topographically inappropriate positions. Subsequent course corrections along both tectal axes and large-scale axonal remodeling lead to the retinotopic ordering of terminal arborizations characteristic of the mature projection. The trajectories and branching patterns of temporal retinal axons labeled with Dil or DiO were determined in whole mounts of retina and tectum from chicks ranging in age from embryonic day 9 to posthatching. Within the retina, labeled retinofugal axons travel in a compact bundle but do not maintain strict neighbor relations, as they course to the optic fissure. The axons enter the contralateral tectum at its rostral edge and grow caudally. Many extend well past their appropriate terminal zone within rostral tectum; a proportion of these later reverse their direction of growth. Many axons grow onto the tectum at incorrect positions along the medial-lateral tectal axis. Some correct this error in a directed manner by altering their trajectory or extending collateral branches at right angles. About 80% of the positional changes of this type are made in the direction appropriate to correct axon position, and thus are likely a response to tectal positional cues. After maturation of retinotopic order, about half of the axons that project to a mature terminal zone have made abrupt course corrections along one or both tectal axes, indicating that initially mistargeted axons can establish appropriately positioned arbors and survive. The development of temporal axons within the tectum is characterized by 3 phases: elongation, branch and arbor formation, and remodeling. After considerable rostrocaudal elongation, an axon typically develops numerous side branches and arbors, many at inappropriate locations. Most arbors are formed by side branches that develop as interstitial collaterals; few axons grow directly to their appropriate terminal zone and arborize. Aberrant arbors, and axons and axon segments that fail to form arbors in the appropriate terminal zone, are rapidly eliminated over about a 2 d period. Axon degeneration appears to play a role in this remodeling process.     

Evidence for shifting connections during development of the chick retinotectal projection

The pattern in which optic axons invade the tectum and begin synaptogenesis was studied in the chick. The anterogradely transported marker, horseradish peroxidase, was injected into one eye of embryos between 5 and 16 days of development (E5 to E16). This labeled the optic axons in the brain. The first retinal axons arrived in the most superficial lamina of the tectum on E6. They entered the tectum at the rostroventral margin. During the next 6 days of development the axons grew over the tectal surface. First they filled the rostral tectum, the oldest portion of the tectum, and then they spread to the caudal pole. Shortly after the first axons entered the tectum on E6, labeled retinal axons were found penetrating from the surface into deeper tectal layers. In any given area of the tectum, optic axons were seen penetrating deeper layers shortly after arriving in that area. Electron microscopic examination showed that at least some of the labeled axons in rostral tectum formed synapses with tectal cells by E7. These results show two things which contrast with results from previous studies. First, there is no delay between the time the retinal axons enter the tectum and the time they penetrate into synaptic layers of the tectum. Second, the first retinotectal connections are formed in rostral tectum and not central tectum. Retrograde tracing showed the first optic axons that arrived in the tectum were from ganglion cells in central retina. Previous studies have shown that the ganglion cells of central retina project to the central tectum in the mature chick. This opens the possibility that the optic axons from central retina, which connect to rostral tectum in the young embryo, shift their connections to central tectum during subsequent development. As they enter the tectum the growth cones of retinal axons appear to be associated with the external limiting membrane. During the time that connections would begin to shift in the tectum a second population of axons appears at the bottom of stratum opticum, some with characteristics of growth cones. This late-appearing population may represent axons shifting their connections. These results have implications for theories on how the retinotopic pattern of retinotectal connections develops.     

Investigations on the development and topographic order of retinotectal axons: anterograde and retrograde staining of axons and perikarya with rhodamine in vivo

Rhodamine-B-isothiocyanate (RITC) is shown to be a convenient and advantageous fluorescence tracer both for anterograde staining of retinal ganglion cell axons on the tectum and for retrograde staining of ganglion cell bodies in the retina of chick embryos. After intravitreal injection the dye is taken up by ganglion cells of the retina from the extracellular space and is transported anterogradely at about 10 mm/day up to the axonal growth cones on the tectum. RITC can be taken up by growing axons on the tectum and it is transported retrogradely at about 5 mm/day to the cell bodies in the retina. Local staining can be achieved if RITC is applied in its crystalline form. RITC is nontoxic for the cells and their axons, is resistant to histological fixation procedures, and allows quick observation in vivo and on dissection stained tissue. Local application of RITC to distinct retinal areas allows examination of the position of the corresponding stained fibers along the retinotectal pathway. Fibers which arise from the central temporal retina occupy deeper layers, whereas fibers from the peripheral temporal retina occupy more superficial layers in the optic tract and in the stratum opticum on the anterior tectum. The growth cones of early retinal fibers growing directly on the tectal surface show a different morphology to later growth cones growing on top of the stratum opticum on the tectum.     

Pretarget sorting of retinocollicular axons in the mouse

The map of the retina onto the optic tectum is a highly conserved feature of the vertebrate visual system; the mechanism by which this mapping is accomplished during development is a long-standing problem of neurobiology. The early suggestion by Roger Sperry that the map is formed through interactions between retinal ganglion cell axons and target cells within the tectum has gained significant experimental support and widespread acceptance. Nonetheless, reports in a variety of species indicate that some aspects of retinotopic order exist within the optic tract, leading to the suggestion that this "preordering" of retinal axons may play a role in the formation of the mature tectal map. A satisfactory account of pretarget order must provide the mechanism by which such axon order develops. Insofar as this mechanism must ultimately be determined genetically, the mouse suggests itself as the natural species in which to pursue these studies. Quantitative and repeatable methods are required to assess the contribution of candidate genes in mouse models. For these reasons, we have undertaken a quantitative study of the degree of retinotopic order within the optic tract and nerve of wild-type mice both before and after the development of the retinotectal map. Our methods are based on tract tracing using lipophilic dyes, and our results indicate that there is a reestablishment of dorsoventral but not nasotemporal retinal order when the axons pass through the chiasm and that this order is maintained throughout the subsequent tract. Furthermore, this dorsoventral retinotopic order is well established by the day after birth, long before the final target zone is discernible within the tectum. We conclude that pretarget sorting of axons according to origin along the dorsoventral axis of the retina is both spatially and chronologically appropriate to contribute to the formation of the retinotectal map, and we suggest that these methods be used to search for the molecular basis of such order by using available mouse genetic models.     

EphrinA6 on chick retinal axons is a key component for p75(NTR)-dependent axon repulsion and TrkB-dependent axon branching

A characteristic of the ephrin/Eph family is their capacity for bi-directional signalling. This means that an ephrin, for example, can function either as a ligand for an Eph 'receptor', or as a receptor for an Eph 'ligand'. A system in which this phenomenon is well studied is the retinotectal projection in which the guidance of retinal ganglion cell (RGC) axons to their target area in the tectum is controlled by both Ephs and ephrins expressed in gradients in both the retina and tectum. Here we have analysed the receptor function of ephrinAs on RGC axons in further detail by focussing on ephrinA6, which is the most strongly expressed ephrinA in the chick retina. EphrinAs are GPI-anchored proteins and therefore require the interaction with transmembrane proteins to exert this receptor function. Previous work has shown that ephrinAs interact on RGC axons in cis with the neurotrophin receptors p75(NTR) and TrkB. P75(NTR) then was shown to be necessary for the repulsion of ephrinA-expressing RGC axons from an EphA substrate and for the downregulation of axon branching. In turn, an interaction of ephrinAs with TrkB as well as an increase in axonal ephrinA expression augments the axon branch-promoting activity of TrkB. We now show that ephrinA6 is the necessary ephrinA component of the repulsive ephrinA/p75(NTR) receptor complex on chick RGC axons as axons lacking ephrinA6 no longer avoid an EphA matrix in stripe assay experiments. We also demonstrate that the branch-promoting activity of TrkB is dependent on ephrinA6 as a knockdown of ephrinA6 renders RGC axons insensitive to BDNF, the high affinity ligand for TrkB. In sum our data further strengthen the hypothesis that a fine-tuned interplay of ephrinAs with p75(NTR) and TrkB is important for the guidance and branching of RGC axons.     

Topographic projections of the retina and optic tectum upon the ventral lateral geniculate nucleus in the chick

The topographic projections of the retina upon the optic tectum and ventral lateral geniculate nucleus (GLv) of the chick were investigated by making small intraretinal injections of ³H-proline. The retinotectal projection pattern was similar to that described for the pigeon. The retinal projection to the GLv was also topographic and was restricted to the outermost lamina of the nucleus. The anteroposterior retinal axis was reversed in the GLv relative to its orientation in the tectum but the superoinferior axis was oriented identically in both. Furthermore, the posterior retina had an enlarged area of projection in the GLv similar to the enlarged area of retinotectal projection for the “red field” found in pigeons. The tectogeniculate projection was topographic and was confined to the outermost geniculate lamina. The secondorder retionotopic map made by the tectogeniculate projections was in register with the retinogeniculate projection. Although the retinal and tectal projection areas were coextensive in the outermost geniculate lamina, the grain density distributions peaked at different points along a radial path through the geniculate laminae. Injections of HRP into the optic tectum led to very light retrograde labeling of a small population of GLv cells topographically corresponding to the tectogeniculate projection zone of the injection site. The data suggest that the chick GLv is comparable to the GLv of other non-primate mammals.     

Development of the transient ipsilateral retinotectal projection in the chick embryo: A numerical flourescence-microscopic analysis

The ipsilateral retinotectal projection in the developing chick was examined by using rhodamine-B-isothiocyanate (RITC)as an anterograde and retrograde vital marker for the retinal ganglion cells and their axons. Staining of the entire retina following intravitreal RITC injection between incubation days 3 and 16 revealed a small number of anterogradely labeled fibers in the optic tract and the anterior half of the optic tectum ipsilateral to the injection site. The total number of ipsilaterally projecting fibers was estimated to be about 2,000 on developmental day 9. The ipsilateral projection totally disappeared after day 15. The arrangement of fibers within the ipsilateral projection was examined by local anterograde RITC staining of localized retinal regions between days 9 and 10. The projection was retinotopically organized along the dorsoventral axis such that fibers of dorsal retinal origin projected on the ventral tectal half, whereas fibers of ventral retinal orgin projected on the dorsal tectal half. The localization of ipsilaterally projecting ganglion cell bodies was examined by retrograde RITC staining during days 9 and 15. Ganglion cells of all four quadrants of the central retina contributed to the production of the ipsilateral projection. The ipsilaterally growing retinotectal fibers did not represent collaterals of contralaterally projecting retinotectal axons. We assume that the tendency of early growing retinotectal axons to grow straight, as well as the ability of axonal growth cones to “sample” the environment, lead to a crossing of axons to the contralateral side. Ipsilateral projections would therefore represent “pathfinding errors.” Explanations for the elimination of the ipsilateral retinotectal projection are discussed.     

Retinotectal ligands for the receptor tyrosine phosphatase CRYPalpha.

The cell adhesion molecule-like tyrosine phosphatase CRYPalpha is localized on retinal axons and their growth cones. We present evidence that two isoforms of this type IIa phosphatase, CRYPalpha1 and CRYPalpha2, have extracellular ligands along the developing retinotectal pathway. Using alkaline phosphatase fusion proteins containing the CRYPalpha1 ectodomain, we detect a prominent ligand on basement membranes of the early retina, optic stalk, and chiasm. A second ligand is observed in the endfeet region of radial processes in the developing stratum opticum, the site of initial retinal axon invasion. This latter ligand binds CRYPalpha2 preferentially. Further ligand interactions are detected for both CRYPalpha protein isoforms in retinorecipient tectal laminae and on retinal fibers themselves. CRYPalpha thus has cell- and matrix-associated ligands along the entire retinotectal projection. Moreover, these ligands appear to be heterotypic and interact with CRYPalpha through both its immunoglobulin and fibronectin type III regions. The anteroposterior levels of the ligands are relatively uniform within the retina and tectum, suggesting that the CRYPalpha protein within retinal axons does not directly recognise topographically graded guidance cues. We propose that CRYPalpha may have a permissive role in promoting retinal axon growth across the eye and tectum and that its functions are modulated temporally and spatially by isoform-specific interactions with cell- and matrix-associated ligands.     

Persistence of graded EphA/Ephrin-A expression in the adult frog visual system

Many studies have demonstrated the involvement of the EphA family of receptor tyrosine kinases and their ligands, ephrin-A2 and -A5, in the development of the temporonasal axis of the retinotectal/collicular map, but the role of these molecules in optic nerve regeneration has not been well studied. Noting that the characteristic gradients of the EphA/ephrin-A family that are expressed topographically in the retina and tectum of embryonic chicks and mice tend to disappear after birth, we took as our starting point an analysis of EphA and ephrin-A expression in leopard frogs (Rana pipiens and utricularia), species capable of regenerating the retinotectal map as adults. For the EphA family to be involved in the regeneration, one would expect these topographic gradients to persist in the adult or, if downregulated after metamorphosis, to be reexpressed after optic nerve injury. Using EphA3 receptor and ephrin-A5 ligand alkaline phosphatase in situ affinity probes (RAP and LAP, respectively) in whole-mount applications, we report that reciprocally complementary gradients of RAP and LAP binding persist in the optic tract and optic tectum of postmetamorphic frogs, including mature adults. EphA expression in temporal retinal axons in the optic tract was significantly reduced after nerve section but returned during regeneration. However, ephrin-A expression in the tectal parenchyma was not significantly elevated by either eye removal, with degeneration of optic axons, or during regeneration of the retinotectal projection. Thus, the present study has demonstrated a persisting expression of EphA/ephrin-A family members in the retinal axons and tectal parenchyma that may help guide regenerating fibers, but we can offer no evidence for an upregulation of ephrin-A expression in conjunction with optic nerve injury.     

Plasticity in the chick ventral lateral geniculate nucleus

The chick ventral lateral geniculate nucleus (GLv) receives topographically corresponding projections from the retina and optic tectum. Tectal lesions produced on the day of hatching removed the tectogeniculate input to the GLv region corresponding to the tectal lesion and also severed some retinotectal axons. Following a survival period of 3 to 10 weeks, a patch of augmented retinogeniculate projection was noted in the GLv segment that corresponds topographically to the damaged area of the tectum. Changing the site of the tectal lesion led to changes in the locus of heavy retinal projection to the GLv predictable from topographic maps. Nuclei which received retinal but not tectal projections did not appear to have regions of augmented retinal termination nor did nuclei which received tectal but not retinal innervation. It is unlikely that the increased retinogeniculate termination is due to rerouting of growing retinotectal axons since the chick retinofugal pathway is well established by the time of hatching. Furthermore, there was no evidence of a projection from the ipsilateral eye to the affected GLv. On the basis of these light microscopic studies, it would appear that retinogeniculate terminals have sprouted in the GLv and that competition for terminal space, conservation of terminal space, proximity, and perhaps other factors are necessary for the augmented projection to occur.     

Zebrafish neurolin‐a and ‐b,orthologs of ALCAM,are involved in retinal ganglion cell differentiation and retinal axon pathfinding

Neurolin‐a and Neurolin‐b (also called alcam and nlcam, respectively) are zebrafish orthologs of human ALCAM, an adhesion protein of the immunoglobulin superfamily with functions in axon growth and guidance. Within the developing zebrafish retina, onset and progression of Neurolin‐a expression parallels the pattern of retinal ganglion cell (RGC) differentiation. By using a morpholino‐based knockdown approach, we show that Neurolin‐a (but not Neurolin‐b) is necessary for a crucial step in RGC differentiation. Without Neurolin‐a, a large proportion of RGCs fail to develop, and RGC axons are absent or reduced in number. Subsequently, Neurolin‐a is required for RGC survival and for the differentiation of all other retinal neurons. Neurolin‐b is expressed later in well‐differentiated RGCs and is required for RGC axon pathfinding. Without Neurolin‐b, RGC axons grow in highly aberrant routes along the optic tract and/or fail to reach the optic tectum. Thus, the zebrafish Neurolin paralogs are involved in distinct steps of retinotectal development. J. Comp. Neurol. 513:38–50, 2009. © 2008 Wiley‐Liss, Inc.     

Thy-1 expression in the retinotectal system of the chick

Previous investigations into the occurrence of Thy-1 in the chick retina have not clearly defined when the antigen first appears and have not adequately described its expression during the relatively early phases of retinal ontogeny. We have investigated these issues, using improved immunohistochemical procedures and show that Thy-1 is associated with the retinal ganglion cells from the time they begin to differentiate by extending their axonal projections. In addition, we have found that its expression reflects the growth of the optic fibre layer and the elaboration of the ganglion cell dendritic processes into the inner plexiform layer. For the first time we describe the appearance and the developmental expression of Thy-1 in the chick tectum. We have found that Thy-1 is associated with retinal axons from the time of their arrival at the tectum and that its expression reflects the elaboration of the stratum opticum. Within the tectum proper Thy-1 appears first in 3 distinct layers all of which are plexiform in nature. By the time that tectal histogenesis is essentially complete the antigen is expressed by all the layers of the tectum. The implications of these findings are discussed in terms of the development of the individual tissues and with respect to the elaboration of the retinotectal pathway.     

Differential expression of the mRNAs of the axonal glycoproteins axonin-1 and NgCAM in the developing chick retina

Cell adhesion molecules expressed on the axonal membrane have been thought to be involved in the guidance of axons to their target area. In the chick, axonin-1 and NgCAM have been shown to promote, through reciprocal interactions, neurite outgrowth in vitro. We have recently shown that chick retinal ganglion cells (RGC) express both proteins as early as the axonal elongation begins. Their expression continues throughout the development of the retinotectal system synchronously with the chronotopic spread of axons. To further investigate the spatiotemporal distribution of axonin-1 and NgCAM in the retina, we have analysed the expression of their mRNAs in the present study. From stage 36 (E10) until hatching photoreceptors express axonin-1 but not NgCAM. In the inner nuclear layer groups of amacrine cells were strongly labelled with both probes but they seemed to belong to different subgroups. These patterns of expression might indicate a differential influence of the two proteins on the development of the local neural circuits of the retina.     

Rules of order in the retinotectal fascicles of goldfish

Individual fascicles of retinal axons were labeled in the goldfish tectum with horseradish peroxidase (HRP). The contralateral retina was later processed for HRP histochemistry to mark the cells that had axons in the fascicles. Labeled cells were found in a partial half anulus in ventral hemiretina, centered on the optic disk. The distance of the partial anulus from the disk depended on which tectal fascicle had been labeled; the more rostrocentral the fascicle, the smaller was the annular radius. The angular subtense of the partial anulus with respect to the disk depended on where (along its tectal course) the fascicle had been labeled; the more rostral the label site, the longer was the angular subtense. These results were interpreted in the context of retinotectal growth, and it was inferred that the axons followed two rules: (1) grow in along the edge of the tectum and (2) exit and terminate in order, axons from temporal retina first, nasal retina last. These rules would produce a retinotopic projection in peripheral tectum, but they require that some of the terminals already in place must shift as the tectum grows.     

The EphA4 receptor tyrosine kinase is necessary for the guidance of nasal retinal ganglion cell axons in vitro

The retinotectal projection serves as a model system for the study of topographic projections. It has been shown in the past few years that members of the Eph family are strongly involved in establishing this projection. The analysis so far has focused on a characterization of Ephrin ligands which are expressed in a gradient in both the tectum and the retina. Here we investigate the role of one of the multiple EphA receptors expressed on retinal ganglion cell axons, EphA4, which is uniformly expressed on nasal and temporal axons. We have adopted both a dominant negative approach and a method using neutralizing monoclonal antibodies in order to inactivate this receptor. The results of these in vitro experiments suggest that EphA4 is crucially involved in the repulsive guidance of nasal but not of temporal axons.     

Axons added to the regenerated visual pathway of goldfish establish a normal fiber topography along the age-axis

Throughout a goldfish's life, new generations of ganglion cells are added on the retinal margin and their axons extend centrally to occupy predictable positions in the retinotectal pathway, adjacent to their predecessors and subjacent to the pia. The stacking of successive generations of axons defines the age-axis of the pathway. This study examined whether an ordered array of predecessor axons is a prerequisite for the patterned growth of new axons. One optic nerve was crushed intraorbitally and the fish was injected with 3H-thymidine to label the proliferating cells on the retinal margin. The ring of 3H-thymidine-labeled cells separated retina that was present at the time of nerve crush (inside the ring) from new retina added afterward (outside). After a period of 14-16 months postcrush, both tectal lobes received two punctate applications of horseradish peroxidase (HRP), one in the central and the other in peripheral tectum, to retrogradely label contralateral retinal ganglion cell bodies and their axons. The pattern of HRP labeling from the control tectum confirmed earlier work: axons on the central tectum had somata in the central retina, and axons on the peripheral tectum had somata in the peripheral retina. The labeled cells and axons were both in predictable patterns. The somata that were backfilled from applications to the center of the experimental tectum lay inside the radioactive ring and had therefore regenerated their axons. The patterns of their labeled axons in the optic pathway and of their somata in the retina were typical of the regenerated condition as described in earlier studies. The somata backfilled from the periphery of the experimental tectum were outside the radioactive ring and had been added after the optic nerve crush. The patterns of their labeled axons and somata were comparable to the normal pattern. These observations indicate that new axons do not depend on an ordered array of predecessors to reestablish normal order along the age-axis of the pathway.     

The Extracellular Matrix Molecule Tenascin: Expression in the Developing Chick Retinotectal System and Substrate Properties for Retinal Ganglion Cell Neurites In vitro

To investigate the molecular mechanisms involved in the outgrowth of retinal ganglion cell axons in the tectum, the expression of the extracellular matrix molecule tenascin was analysed in the tectum and retina of chickens by immunocytochemistry and in situ hybridization. Tissue was analysed between embryonic days 4 and 12, just before and during the period when retinal ganglion cell axons innervate their target region, the optic tectum. In the tectum, tenascin immunoreactivity becomes detectable at the anterior pole at embryonic day 4, 2 days before retinal ganglion cell axons arrive, and spreads caudally with increasing age. At early stages, tenascin is predominantly accumulated in the stratum opticum, the zone of ingrowing retinal ganglion cell axons, and along their prospective pathway. In the stratum opticum, the molecule is associated with radial glial fibres, glial endfeet and retinal ganglion cell axons located in the immediate neighbourhood of radial glial fibres. At all ages investigated, tenascin mRNA is mainly restricted to cells located in the periventricular region, suggesting that the molecule is synthesized by radial glial cells. In the retina, tenascin is expressed by amacrine, displaced amacrine and horizontal cells but not by retinal ganglion cells. To investigate whether the accumulation of tenascin in the developing and prospective pathway of retinal ganglion cell axons may affect their rate of growth we assayed the substrate properties of tenascin for retinal ganglion cell neurites in vitro. When retinal ganglion cell suspensions from 6–day-old chick embryos were maintained on homogeneous mouse or chick tenascin/ polyornithine substrates, neurite length was significantly increased when compared to polyornithine substrates at coating concentrations of 10 or 20 μg/ml. Higher coating concentrations (35 or 70 μg/ml) resulted in neurite lengths comparable to control values. Together, these observations suggest that tenascin in the developing and prospective stratum opticum might serve as a preformed pathway to support growth of retinal ganglion cell axons in the tectum.