Competitive interactions between retinal ganglion axons for tectal targets explain plasticity of retinotectal projection in the servomechanism model of retinotectal mapping

The mechanism of topographic mapping of retinal ganglion cells to the midbrain was previously elucidated by the servomechanism model, which is based on the fact that cells expressing Eph-receptors respond specifically to surface expressing membrane-bound ephrin-ligands at a critical level. The retina has increased nasal-to-temporal gradient of Eph receptor-density, and the optic tectum/superior colliculus has increased rostral-to-caudal gradient of membrane-bound ephrin-ligand. An axon from the retina has an identification tag of a certain level of Eph-receptor density depending on its retinal position, and adheres to the site on the tectum/superior colliculus expressing ephrin-ligands at a critical ligand-density level. The servomechanism model rigidly defines positions of axon terminals on the midbrain. However, optic nerve regeneration experiments combined with halved retina or tectum show a plastic or flexible mapping (expansion, compression and transposition of tectal projections). To reconcile the discrepancy between the rigid model and the plastic behavior, competition between retinal axon terminals for a target site was introduced to the servomechanism. The servomechanism/competition model succeeded in computer simulations of the plastic mapping of retinal axons on the tectum. Recent experiments of upregulated ligand-density on the tectum during nerve regeneration and the role of axonal competition are discussed.     

Activity-dependent mapping in the retinotectal projection

It is now 15 years since the discovery that N-methyl-d-aspartate receptor activity is required to maintain the refined topographic organization of retinotectal projections. Recent studies have identified additional components of the signaling pathways required for activity-dependent map formation and maintenance. Nitric oxide and brain-derived neurotrophic factor, candidate retrograde messengers, and serotonin and acetylcholine, modulators of neuronal excitability, all affect mapping. These studies indicate that the mapping process intersects with other processes fundamental to visual system development and function, such as process outgrowth, synaptic turnover and neuromodulation.     

Establishment of rostrocaudal polarity in tectal primordium: engrailed expression and subsequent tectal polarity.

In the E4 (embryonic day 4) chick tectal primordium, engrailed expression is strong at the caudal end and gradually weakens toward the rostral end. We used quail-chick chimeric tecta to investigate how the caudorostral gradient of engrailed expression is established and whether it is correlated with the subsequent rostrocaudal polarity of tectal development. To examine the positional value of the tectal primordium, we produced ectopic tecta in the diencephalon by transplanting a part of the mesencephalic alar plate heterotopically. In the ectopic tectum, the gradient of the engrailed expression reversed and the strength of the expression was dependent on the distance from the mes-diencephalon junction; the nearer the ectopic tectum was to the junction, the weaker the expression was. Consequently, the pattern of the engrailed expression in the host and ectopic tecta was nearly a mirror image, suggesting the existence of a repressive influence around the mes-diencephalon junction on the engrailed expression. We examined cytoarchitectonic development in the ectopic tecta, which normally proceeds in a gradient along the rostrocaudal axis; the rostral shows more advanced lamination than the caudal. In contrast, the caudal part of the ectopic tecta (near to the mes-diencephalon junction) showed more advanced lamination than the rostral. In both the host and ectopic tecta, advanced lamination was observed where the engrailed expression was repressed, and vice versa. Next we studied the correlation between engrailed expression and retinotectal projection from a view of plasticity and rigidity of rostrocaudal polarity in the tectum. We produced ectopic tecta by anisochronal transplantations between E3 host and E2 donor, and showed that there is little repressive influence at E3 around the mes-diencephalon junction. We then made chimeric double-rostral tectum (caudal half of it was replaced by rostral half of the donor tectum) or double-caudal tectum at E3. The transplants kept their original staining pattern in hosts. Consequently, the chimeric tecta showed wholly negative or positive staining of engrailed protein on the grafted side. In such tecta retinotectal projection pattern was disturbed as if the transplants retained their original position-specific characters. We propose from these heterotopic and anisochronal experiments that the engrailed expression can be a marker for subsequent rostrocaudal polarity in the tectum, both as regards cytoarchitectonic development and retinotectal projection.     

A GFP-based genetic screen reveals mutations that disrupt the architecture of the zebrafish retinotectal projection

The retinotectal projection is a premier model system for the investigation of molecular mechanisms that underlie axon pathfinding and map formation. Other important features, such as the laminar targeting of retinal axons, the control of axon fasciculation and the intrinsic organization of the tectal neuropil, have been less accessible to investigation. In order to visualize these processes in vivo, we generated a transgenic zebrafish line expressing membrane-targeted GFP under control of the brn3c promoter/enhancer. The GFP reporter labels a distinct subset of retinal ganglion cells (RGCs), which project mainly into one of the four retinorecipient layers of the tectum and into a small subset of the extratectal arborization fields. In this transgenic line, we carried out an ENU-mutagenesis screen by scoring live zebrafish larvae for anatomical phenotypes. Thirteen recessive mutations in 12 genes were discovered. In one mutant, ddl, the majority of RGCs fail to differentiate. Three of the mutations, vrt, late and tard, delay the orderly ingrowth of retinal axons into the tectum. Two alleles of drg disrupt the layer-specific targeting of retinal axons. Three genes, fuzz, beyo and brek, are required for confinement of the tectal neuropil. Fasciculation within the optic tract and adhesion within the tectal neuropil are regulated by vrt, coma, bluk, clew and blin. The mutated genes are predicted to encode molecules essential for building the intricate neural architecture of the visual system.     

Acute blockade of nitric oxide synthesis induces disorganization and amplifies lesion-induced plasticity in the rat retinotectal projection

In the rat visual system, the uncrossed retinotectal projection undergoes a topographical refinement within the first two postnatal weeks. We have studied the role of nitric oxide (NO), a retrograde messenger which couples pre- and postsynaptic activation, in the development of the uncrossed retinotectal projection and in the plasticity of this pathway as a result of a restricted retinal lesion in the opposite eye. During development, maximal nitric oxide synthase (NOS) activity was observed in homogenates of tectal tissue at postnatal day 5 (PND 5), followed by a two-step decrease at the end of the topographical fine tuning period (PND 21) and the adult stage (PND 42). We also tested the effects of an acute in vivo blockade of NOS during the development of both animals that had not been operated on, and lesioned animals. Animals ranging from PND 4 to PND 42 were treated either with the NOS inhibitor, L-nitro-arginine (Narg 50 mg/kg ip.) or vehicle (NaCl 0.9%) during 4 days (from PND 4-7 or PND 9-12) or 8 days (from PND 20-27 or PND 34-41). Reduction of NOS activity induced sprouting of the ipsilateral pathway up to the second postnatal week in the animals that had not been operated on. Rats that had been operated on, however, showed an amplification of the lesion-induced plasticity up to the fourth postnatal week under NOS blockade. The data suggest that NO plays a role in the stabilization of retinotectal synapses during the critical period of topographic refinement, and indicate that an acute blockade of retrograde signals enables plastic rearrangements in the visual system within this time window.     

Invariants in the ipsilateral retinotectal visual projection of frogs

We determined whether the sensitivity of the ipsilateral type II units of Rana esculenta to prey (W/H-oriented bars) and non-prey (A/V-oriented bars)-like targets remains invariant under various experimental conditions. We show that the shape of the 'discrimination' curve is largely unaffected by the level of general illumination and by the background texture. An increase in the stimulus velocity and in the width of the bars moderately affects the salient points (negative peak and preference reversal) of the curve, but does not alter the overall configurational preference of these units. As for retinal ganglion cells: (i) this curve expresses better a 'contrast' between two vertically oriented edges of different dimensions than a 'contrast' between two edges of equal dimension but of different orientation; and (ii) the experimentally induced variations can be explained on the basis of the spatial and temporal properties of the neuronal elements forming the antagonistic center-surround arrangement of the receptive field.     

Acute blockade of nitric oxide synthesis induces disorganization and amplifies lesion‐induced plasticity in the rat retinotectal projection

In the rat visual system, the uncrossed retinotectal projection undergoes a topographical refinement within the first two postnatal weeks. We have studied the role of nitric oxide (NO), a retrograde messenger which couples pre‐ and postsynaptic activation, in the development of the uncrossed retinotectal projection and in the plasticity of this pathway as a result of a restricted retinal lesion in the opposite eye. During development, maximal nitric oxide synthase (NOS) activity was observed in homogenates of tectal tissue at postnatal day 5 (PND 5), followed by a two‐step decrease at the end of the topographical fine tuning period (PND 21) and the adult stage (PND 42). We also tested the effects of an acute in vivo blockade of NOS during the development of both animals that had not been operated on, and lesioned animals. Animals ranging from PND 4 to PND 42 were treated either with the NOS inhibitor, L‐nitro‐arginine (Narg 50 mg/kg ip.) or vehicle (NaCl 0.9%) during 4 days (from PND 4–7 or PND 9–12) or 8 days (from PND 20–27 or PND 34–41). Reduction of NOS activity induced sprouting of the ipsilateral pathway up to the second postnatal week in the animals that had not been operated on. Rats that had been operated on, however, showed an amplification of the lesion‐induced plasticity up to the fourth postnatal week under NOS blockade. The data suggest that NO plays a role in the stabilization of retinotectal synapses during the critical period of topographic refinement, and indicate that an acute blockade of retrograde signals enables plastic rearrangements in the visual system within this time window. © 2000 John Wiley & Sons, Inc. J Neurobiol 44: 371–381, 2000     

Fibre organization and reorganization in the retinotectal projection of Xenopus

This study concerns the retinotopic organization of the ganglion cell fibres in the visual system of the frog Xenopus laevis. HRP was used to trace the pathways taken by fibres from discrete retinal positions as they pass from the retina, along the optic nerve and into the chiasma. The ganglion cell fibres in the retina are arranged in fascicles which correspond with their circumferential positions of origin. Within the fascicles the fibres show little age-related layering and do not have a strict radial organization. As the fascicles of fibres pass into the optic nerve head there is some exchange of position resulting in some loss of the retinal circumferential organization. The poor radial organization of the fibres in the retinal fascicles persists as the fibres pass through the intraocular part of the nerve. At a position just behind the eye there is a major fibre reorganization in which fibres arising from cells of increasingly peripheral retinal locations are found to have passed into increasingly peripheral positions in the nerve. Thus, fibres from peripheral-most retina are located at the nerve perimeter, whilst fibres from central retina are located in the nerve core. It is at this point that the radial, chronotopic, ordering of the ganglion cell axons, found throughout the rest of the optic pathway, is established. This annular organization persists along the length of the nerve until a position just before the nerve enters the brain. Here, fibres from each annulus move to form layers as they pass into the optic chiasma. This change in the radial organization appears to be related to the pathway followed by all newly growing fibres, in the most superficial part of the optic tract, adjacent to the pia. Just behind the eye, where fibres become radially ordered, the circumferential organization of the projection is largely lost. Fibres from every circumferential retinal position, which are of similar radial position, are distributed within the same annulus of the nerve. At the nerve-chiasma junction where each annulus forms a single layer as it enters the optic tract, there is a further mixing of fibres from all circumferential positions. However, as the fibres pass through the chiasma some active pathway selection occurs, generating the circumferential organization of the fibres in the optic tract. Additional observations of the organization of fibres in the optic nerve of Rana pipiens confirm previous reports of a dual representation of fibres within the nerve. The difference in the organization of fibres in the optic nerve of Xenopus and Rana pipiens is discussed.     

Alterations in the Xenopus retinotectal projection by antibodies to Xenopus N-CAM

The patterned neural projection from the eye to the optic tectum of lower vertebrates (the retinotectal projection) has been proposed to be ordered by interactions between the optic nerve fibers and their surrounding tissues. To investigate the role of one such defined cell interaction, agarose implants containing antibodies to the neural cell adhesion molecule, N-CAM, were inserted into the tectum of the African clawed frog, Xenopus laevis. Both monoclonal and polyclonal antibodies against N-CAM reversibly and specifically distorted the pattern of the retinotectal projection, decreasing the precision of the projection as determined by electrophysiological techniques as well as decreasing the density of retinal innervation of the tectum and the branching of single axons as determined by horseradish peroxidase tracing. The anatomical effects became maximal at 4 to 6 days after implantation and returned to undetectable levels by 2 weeks, whereas the physiological effects became maximal by 8 to 10 days and a normal physiological map was reestablished within 4 weeks. The results are consistent with the hypothesis that anti-N-CAM antibodies perturb the ongoing growth and retraction of the terminal arbors of the optic nerve fibers, such that a region of the tectum becomes largely denuded of fibers. The physiological defects may then be a consequence both of the initial retraction of optic nerve terminals and of the rapid ingrowth of the perturbed and neighboring optic nerve fibers into the denuded region after the antibodies were cleared from the tectum. These results support the concept of a major role for N-CAM-mediated adhesion during map regeneration and maintenance.     

Generation of grouped discharges by tectal projection cells

In this paper we briefly summarize our recent data on the transduction properties of tecto-bulbo-spinal neurons (TBSN) in the cat. These neurons from an important link between the superior colliculus and the "premotor" structures of the brain stem and the cervical spinal cord. They are closely similar to spinal alpha-motoneurons, as concerns the soma-dendritic geometry and electrotonic parameters. In contrast, their rhythmic firing behavior is characterized by much higher sensitivity to depolarizing currents and by the capability ot generate extraspikes ("regenerative firing mode"). This results in an abrupt increase of sensitivity when a certain limit of depolarization is surpassed. Membrane parameters of TBSN which are responsible for their characteristic transduction properties are presented. We forward an hypothesis that different modes of rhythmic firing, as dependent on behavioral situation, play a role in the distribution of efferent signals among many different target areas of TBSNs.     

Compression and expansion without impulse activity in the retinotectal projection of goldfish

The capacity of regenerating optic fibers to undergo retinotopic compression and expansion in the absence of impulse activity was tested by eliminating activity with periodic intraocular injections of tetrodotoxin (TTX) during regeneration. To test for compression, the posterior half of tectum was removed and the optic nerve crushed. For expansion, the temporal half of retina was ablated and the nerve also crushed. The projection was then subsequently examined with electrophysiological mapping and autoradiographic tracing. Like electrically active fibers, silent fibers formed a retinotopically ordered projection that was compressed onto the anterior half tectum. Similarly, TTX-treated fibers from a nasal half retina formed a retinotopic projection that was expanded across the entire tectum. Except for some enlargement of receptive fields produced by the TTX, the topography was equivalent to that formed by active fibers. Thus, fibers can apparently maintain relative positions irrespective of absolute tectal position without the benefit of activity-dependent ordering. This implies the existence of an activity-independent mechanism for relative positioning that may operate across larger distances than the activity-dependent ordering responsible for fine topography and ocular dominance columns.     

Role for cell adhesion and glycosyl (HNK-1 and oligomannoside) recognition in the sharpening of the regenerating retinotectal projection in goldfish

Cell-adhesion molecules (CAMs) are thought to play crucial roles in development and plasticity in the nervous system. This study tested for a role for cell adhesion and in particular, the recognition of two glycosyl epitopes (HNK-1 and oligomannoside) in the activity-driven sharpening of the retinotopic map formed by the regenerating retinal fibers of goldfish. HNK-1 is a prominent glycosyl epitope on many CAMs and extracellular matrix (ECM) molecules, including NCAM, L1, ependymin, and integrins, which have all been implicated in synaptic plasticity. To test for a role of HNK-1 in the sharpening process, we used osmotic minipumps to infuse HNK-1 antibodies for 7–21 days into the tectal ventricle starting at 18 days after optic nerve crush. Retinotopic maps recorded at 76–86 days postcrush showed a lack of sharpening similar to that seen previously with two antibodies to ependymin, an HNK-1–positive ECM component present in cerebrospinal fluid. The multiunit receptive fields at each point averaged 26° versus 11–12° in regenerates infused with control antibodies or Ringer's alone. The HNK-1 epitope also binds to the G2 domain of laminin to mediate neuron-ECM adhesion. To test for a role for laminin, a polyclonal antibody was similarly infused and also prevented sharpening to approximately the same degree. The results support a role for the HNK-1 epitope and laminin in retinotectal sharpening. The oligomannoside epitope (recognized by monoclonal antibody L3) on the CAM L1 interacts with NCAM on the same cell to promote stronger L1 homophilic interactions between cells. Both an L1-like molecule and NCAM are prominently reexpressed in the regenerating retinotectal system of fish. Infusion of oligomannosidic glycopeptides resulted in decreased sharpening, with multiunit receptive fields that averaged 22.7°. Infusions of mannose-poor glycopeptides less prominently disrupted sharpening, with average multiunit receptive fields of 18°. Thus, oligomannosidic glycans in particular may play a role in retinotopic sharpening. Blocking glycan-mediated interactions between CAMs and ECM molecules could decrease the extent of exploratory growth of retinal axon collaterals, preventing them from finding their retinotopic sites, or could interfere with L1 or NCAM and laminin binding at the synaptic densities preventing stabilization of retinotopically appropriate synapses. Together, these results support a prominent role for cell adhesion and glycan epitopes in visual synaptic plasticity. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 659–671, 1998     

Gradual appearance of a regulated retinotectal projection pattern in Xenopus laevis

The topographic projection pattern formed by the retinal ganglion cell axons in the tectum of the lower vertebrate appears to require positional cues that guide the optic nerve fibers to their appropriate targets. One approach to understanding these positional cues or "positional information" has been to investigate changes in the pattern of the retinotectal projection after surgical manipulation of the embryonic eyebud. Analysis of these apparent changes in the patterns of positional information in the eye, termed "pattern regulation," may provide clues to both the nature of positional information and the mechanisms by which it is assigned to cells in the eyebud. Here we examine pattern regulation in the Xenopus visual system following the replacement of the temporal half of a right eyebud with the temporal half of a left eyebud. This manipulation requires that the left half-eyebud be inverted along its dorsoventral axis. Electrophysiological maps of these compound eyes in postmetamorphic frogs reveal regulated maps; the cells in the temporal half of the NrTl eye project to the tectum with a dorsoventral polarity appropriate for their position in the host eye and not appropriate for the original positions of the grafted cells in the donor eyebud. Paradoxically, the regulated patterns are not apparent in the projections of the original grafted eyebud cells during early larval development. Using fiber-tracing and electrophysiological mapping techniques, we now show that the regulated patterns appear gradually in the projections made by peripheral retinal cells added during mid-larval development. Because the regulation occurs relatively late in development and probably only in the peripheral retinal cells, simple models of epimorphic or morphallactic regulation do not appear to fit this system. Thus, new or more complex models must be invoked to explain the phenomenon of pattern regulation in the developing visual system of Xenopus.     

NMDA receptor activity stabilizes presynaptic retinotectal axons and postsynaptic optic tectal cell dendrites in vivo

To investigate the role of N-methyl-D-aspartate (NMDA) receptor activity in the stability of the presynaptic axon arbor and postsynaptic dendritic arbors in vivo, we took time-lapse confocal images of single DiI-labeled Xenopus retinotectal axons and optic tectal neurons in the presence and absence of the NMDA receptor antagonist, APV. Retinotectal axons or tectal neurons were imaged at 30-min intervals over 2 h, or twice over a 24-h period. Retinal axons in animals exposed to DL-APV (100 microM) showed an increase in rates of branch additions and a decrease in branch lifetimes over 2 h compared to untreated axons. Under the same experimental conditions, tectal neurons showed a decreased rate of branch tip additions and retractions. APV treatment over 24 h had no apparent effect on axon arbor morphology, but did decrease tectal cell dendritic arbor elaboration. These observations demonstrate that NMDA receptor activity in postsynaptic neurons stabilizes pre- and postsynaptic neuronal morphology in vivo.. However, when NMDA receptor activity is blocked, presynaptic retinal axons respond with increased arbor dynamics while postsynaptic tectal cell dendrites decrease arbor dynamics. Such differential responses of pre- and postsynaptic partners might increase the probability of coactive afferents converging onto a common target under conditions of lower NMDA receptor activity.     

The application of the Gierer-Meinhardt equations to the development of the retinotectal projection

Model calculations are presented for the several properties of the development of the retinotectal projection in amphibians and fishes, using the Gierer-Meinhardt equations. One of these properties is the maintenance of topographic mapping between the retina and the tectum during their development despite the fact that the two tissues grow in morphologically different ways. Another is the existence of a critical period, at which the coordinates of the retina with respect to the tectum are irrevocably determined. It is assumed that the connections between the retinal and the tectal cells are made on the correspondence of positional markers which are given as a form of the distribution of a specific activator, the dynamics of which is described by the Gierer-Meinhardt equations. The monotonic distributions of the activator and the existence of the critical period are shown by a computer simulation of the proliferating retina. Several changes of the retinotectal projection after surgical operations on the retina or the tectum are also explained.Some of the results in this paper were presented at the poster session of the 6th International Biophysics Congress in Kyoto 1978     

C-kinase manipulations disrupt activity-driven retinotopic sharpening in regenerating goldfish retinotectal projection

Regenerating optic axons initially branch over a wide area in tectum to form a crude retinotopic map. The map is sharpened, and retinotopically appropriate synapses are stabilized via NMDA receptors that detect, via summation of EPSPs, the coincident activity of neighboring ganglion cells that make synapses onto common tectal cells. Sharpening shares a number of properties with long-term potentiation (LTP) in hippocampus. This study tested whether protein kinase C (PKC) activation is necessary for sharpening as it is for LTP. Intracular (IO) or intracranial (IC) injections of kinase inhibitors or activators were made every other day from 19 to 37 days postcrush (sensitive period), and the projections formed were later recorded. Retinotopic sharpening was prevented by IC injection of the following agents: (1) general kinase inhibitors sphingosine and H7 (100-200 μM in fluid above brain), (2) active but not inactive phorbols (TPA, 1 μM), and (3) calphostin C (1 μM), a specific and irreversible PKC inhibitor. The mature projection on the opposite tectum, however, when examined was not unsharpened. Lack of sharpening was reflected in multiunit fields at each tectal point that averaged 27°–30° versus 11° in Ringers and inactive phorbol control regenerates. Intraocular injections of either TPA (1 μM), or calphostin C (1 μM) also prevented sharpening (26° and 32° multiunit fields), suggesting action on PKC axonally transported to the presynaptic terminals. Calphostin C had no noticeable effect on the firing patterns of retinal ganglion cells. The endogenous activator of PKC, arachidonic acid (AA), disrupted sharpening at 20 μM or higher (IC injection, 32° multiunit fields), while a control fatty acid, elaidic acid, had no effect. Although AA at 5 μM showed no effect, and diacylglycerol at 5 μM exhibited only small effects, together they produced a large synergistic effect (32° multiunit fields). Such synergy mirrors the synergy in the activation of several isoforms of PKC. Actual concentrations in the extradural fluid around the brain were assayed via injections of ³H-AA. Levels fell about sixfold after a day and by an additional fivefold the second day before the next injection. The results confirm that activity-driven retinotopic sharpening is very sensitive to manipulations of kinases, especially PKC. © 1994 John Wiley & Sons, Inc.     

In vitro evolution of recognition specificity mediated by SH3 domains reveals target recognition rules

We have designed a repertoire of 10(7) different SH3 domains by grafting the residues that are represented in the binding surfaces of natural SH3 domains onto the scaffold of the human Abl-SH3 domain. This phage-displayed library was screened by affinity selection for SH3 domains that bind to the synthetic peptides, APTYPPPLPP and LSSRPLPTLPSP, which are peptide ligands for the human Abl or Src SH3 domains, respectively. By characterizing the isolates, we have observed that as few as two or three amino acid substitutions lead to dramatic changes in recognition specificity. We propose that the ability to shift recognition specificity with a small number of amino acid replacements is an important evolutionary characteristic of protein binding modules. Furthermore, we have used the information obtained by these in vitro evolution experiments to generate a scoring matrix that evaluates the probability that any SH3 domain binds to the peptide ligands for the Abl and Src SH3 domains. A table of predictions for the 28 SH3 domains of baker's yeast is presented.     

Expression of receptor tyrosine phosphatases during development of the retinotectal projection of the chick

Receptor tyrosine kinases and receptor protein tyrosine phosphatases (RPTPs) appear to coordinate many aspects of neural development, including axon growth and guidance. Here, we focus on the possible roles of RPTPs in the developing avian retinotectal system. Using both in situ hybridization analysis and immunohistochemistry, we show for the first time that five RPTP genes--CRYPalpha, CRYP-2, PTPmu, PTPgamma, and PTPalpha--have different but overlapping expression patterns throughout the retina and the tectum. PTPalpha is restricted to Muller glia cells and radial glia of the tectum, indicating a possible function in controlling neuronal migration. PTPgamma expression is restricted to amacrine neurons. CRYPalpha and CRYP-2 mRNAs in contrast are expressed throughout the retinal ganglion cell layer from where axons grow out to their tectal targets. PTPmu is expressed in a subset of these ganglion cells. CRYPalpha, CRYP-2, and PTPmu proteins are also localized in growth cones of retinal ganglion cell axons and are present in defined laminae of the tectum. Thus, the spatial and temporal expression of three distinct RPTP subtypes--CRYPalpha, CRYP-2, and PTPmu--are consistent with the possibility of their involvement in axon growth and guidance of the retinotectal projection.     

Analysis of ephrin-A2 in the chick retinotectal projection using a function-blocking monoclonal antibody

Eph receptor tyrosine kinases and their ligands have been shown to be involved in processes of cell migration and axon guidance during embryonic development. Here we describe the development of a function-blocking monoclonal antibody against chick ephrin-A2, and its effect on retinal ganglion cell axons studied both in vitro and in vivo. In the stripe assay, the blocking antibody completely abolished the repulsive effect of posterior tectal membranes. In vivo, in a loss-of-function approach, hybridoma cells secreting the antiephrin-A2 antibody were applied to chick embryos from embryonic day 3 (E3) on, and the retinotectal projection was subsequently analyzed at E16. DiI tracing analyses showed that although the projection of both temporal and nasal retinal ganglion axons in the tectum was, overall, normal, occasionally diffuse and extra termination zones were observed, in addition to axons over-shooting their termination zones. These data support the idea that ephrin-A2 contributes to the establishment of the chick retinotectal projection.     

Homotopic and heterotopic transplantations of quail tectal primordia in chick embryos: Organization of the retinotectal projections in the chimeric embryos

To study the adaptative capabilities of the retinotectal system in birds, the primordium of one optic tectum from 12-somite embryos of Japanese quail was transplanted either homotopically, to replace the ablated same primordium, or heterotopically, to replace the ablated dorsal diencephalon in White Leghorn chick embryos of the same stage. The quail nucleolar marker was used to recognize the transplants. The cytoarchitecture of the tecta and the retinal projections from the eye contralateral to the graft were studied on the 17th or 18th day of incubation in the chimeric embryos by autoradiographic or horseradish peroxidase tracing methods. Morphometric analysis was applied to evaluate the percentage of the tectal surface receiving optic projections. It was observed that: (i) quail mesencephalic alar plate can develop a fully laminated optic tectum even when transplanted heterotopically; (ii) retinal ganglion cells from the chick not only recognize the tectal neurons of the quail as their specific targets in homotopic grafts, but the optic fibers deviate to innervate the heterotopically grafted tectum; (iii) in the presence of a graft, the chick retina is unable to innervate a tectal surface of similar or larger size than that of the control tectum; (iv) tectal regions devoid of optic projections, whether formed by donor or by host cells, always present an atrophic lamination; (v) the diencephalic supernumerary optic tectum competes with and prevails over the host tectum as a target for optic fiber terminals.