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.     

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.     

Two homeobox genes define the domain of EphA3 expression in the developing chick retina

Graded expression of the Eph receptor EphA3 in the retina and its two ligands, ephrin A2 and ephrin A5 in the optic tectum, the primary target of retinal axons, have been implicated in the formation of the retinotectal projection map. Two homeobox containing genes, SOHo1 and GH6, are expressed in a nasal-high, temporal-low pattern during early retinal development, and thus in opposing gradients to EphA3. Retroviral misexpression of SOHo1 or GH6 completely and specifically repressed EphA3 expression in the neural retina, but not in other parts of the central nervous system, such as the optic tectum. Under these conditions, some temporal ganglion cell axons overshot their expected termination zones in the rostral optic tectum, terminating aberrantly at more posterior locations. However, the majority of ganglion cell axons mapped to the appropriate rostrocaudal locations, although they formed somewhat more diffuse termination zones. These findings indicate that other mechanisms, in addition to differential EphA3 expression in the neural retina, are required for retinal ganglion axons to map to the appropriate rostrocaudal locations in the optic tectum. They further suggest that the control of topographic specificity along the retinal nasal-temporal axis is split into several independent pathways already at a very early time in development.     

nev (cyfip2) is required for retinal lamination and axon guidance in the zebrafish retinotectal system

In the zebrafish retinotectal system, retinal ganglion cells (RGCs) project topographically along anterior-posterior (A-P) and dorsal-ventral (D-V) axes to innervate their primary target, the optic tectum. In the nevermind (nev) mutant, D-V positional information is not maintained by dorsonasal retinal axons as they project through the optic tract to the tectum. Here we present a detailed phenotypic analysis of the retinotectal projection in nev and show that dorsonasal axons do eventually find their correct location on the tectum, albeit after taking a circuitous path. Interestingly, nev seems to be specifically required for retinal axons but not for several non-retinal axon tracts. In addition, we find that nev is required both cell autonomously and cell nonautonomously for proper lamination of the retina. We show that nev encodes Cyfip2 (Cytoplasmic FMRP interacting protein 2) and is thus the first known mutation in a vertebrate Cyfip family member. Finally, we show that CYFIP2 acts cell autonomously in the D-V sorting of dorsonasal RGC axons in the optic tract. CYFIP2 is a highly conserved protein that lacks known domains or structural motifs but has been shown to interact with Rac and the fragile-X mental retardation protein, suggesting intriguing links to cytoskeletal dynamics and RNA regulation.     

Target-Dependent Regulation of Retinal Nicotinic Acetylcholine Receptor and Tubulin RNAs During Optic Nerve Regeneration in Goldfish

A fundamental issue in central nervous system development regards the effect of target tissue on the differentiation of innervating neurons. We address this issue by characterizing the role the retinal ganglion cell target, i.e., the optic tectum, plays in regulating expression of tubulin and nicotinic acetylcholine receptor genes in regenerating retinal ganglion cells. Tubulins are involved in axonal growth, whereas nicotinic acetylcholine receptors mediate communication across synapses. Retinal ganglion cell axons were induced to regenerate by crushing the optic nerve. Following crush, there was a rapid increase in alpha-tubulin RNAs (3 days), which preceded the increase in nicotinic acetylcholine receptor RNAs (10-15 days). Both classes of RNAs approached control levels by the time retinotectal synapses and functional recovery were restored (4-6 weeks). If the optic nerve was repeatedly crushed or its target ablated, tubulin RNAs remained elevated, and the increase in receptor RNAs that would otherwise be seen 2 weeks after a single nerve crush did not occur. The interaction of retinal ganglion cell axons with their targets in the optic tectum appears, then, to exert a suppressive effect on the RNA encoding a cytoskeletal protein, tubulin, and an inductive effect on RNAs encoding nicotinic acetylcholine receptors involved in synaptic communication.     

Matrix Metalloproteinase 14 in the Zebrafish: An Eye on Retinal and Retinotectal Development

Background

Matrix metalloproteinases (MMPs) are members of the metzincin superfamily of proteinases that cleave structural elements of the extracellular matrix and many molecules involved in signal transduction. Although there is evidence that MMPs promote the proper development of retinotectal projections, the nature and working mechanisms of specific MMPs in retinal development remain to be elucidated. Here, we report a role for zebrafish Mmp14a, one of the two zebrafish paralogs of human MMP14, in retinal neurogenesis and retinotectal development.

Results

Whole mount in situ hybridization and immunohistochemical stainings for Mmp14a in developing zebrafish embryos reveal expression in the optic tectum, in the optic nerve and in defined retinal cell populations, including retinal ganglion cells (RGCs). Furthermore, Mmp14a loss-of-function results in perturbed retinoblast cell cycle kinetics and consequently, in a delayed retinal neurogenesis, differentiation and lamination. These Mmp14a-dependent retinal defects lead to microphthalmia and a significantly reduced innervation of the optic tectum (OT) by RGC axons. Mmp14b, on the contrary, does not appear to alter retinal neurogenesis or OT innervation. As mammalian MMP14 is known to act as an efficient MMP2-activator, we also explored and found a functional link and a possible co-involvement of Mmp2 and Mmp14a in zebrafish retinotectal development.

Conclusion

Both the Mmp14a expression in the developing visual system and the Mmp14a loss-of-function phenotype illustrate a critical role for Mmp14a activity in retinal and retinotectal development.     

Pathways of Retinotectal Projection in Developing Xenopus Tadpoles Revealed by Selectively Labeling of Retinal Axons with Horseradish Peroxidase (HRP)

Anatomical mapping was made of the retinal central pathways from the chiasm to the targets within the tectum in the developing Xenopus tadpoles, after labeling a specific regional population of retinal axons with horseradish peroxidase (HRP). In the tadpoles at stage 50, pathway sorting of retinal axons within the optic tract was clear for the dorsoventral axis of the retina, but not for the nasotemporal axis. Most nasal retinal axons and some dorsal and ventral retinal axons invaded the tectum directly at the diencephalotectal junction, and arrived at their correct sites of innervation after running through ectopic parts of the tectum. These findings indicate that the pathway orientation before targets is not a prerequisite factor for establishment of the orderly map of the retinotectal projection. Rather, a direct interaction between ingrowing retinal axons and tectal cells seems to be a predominant factor for specification of retinal central connections.     

Trying to understand axonal regeneration in the CNS of fish.

In contrast to the situation in mammals and birds, neurons in the central nervous system (CNS) of fish--such as the retinal ganglion cells--are capable of regenerating their axons and restoring vision. Special properties of the glial cells and the neurons of the fish visual pathway appear to contribute to the success of axonal regeneration. The fish oligodendrocytes lack the axon growth inhibiting molecules that interfere with axonal extension in mammals. Instead, fish optic nerve oligodendrocytes support--at least in vitro--axonal elongation of fish as well as that of rat retinal axons. Moreover, the fish retinal ganglion cells re-express upon injury a set of growth-associated cell surface molecules and equip the regenerating axons throughout their path and up into their target, the tectum opticum with these molecules. This may indicate that the injured fish ganglion cells reactivate the cellular machinery necessary for axonal regrowth and pathfinding. Furthermore, the target itself provides positional marker molecules even in adult fish. These marker molecules are required to guide the regenerating axons back to their retinotopic home territory within the tectum.     

Trying to understand axonal regeneration in the CNS of fish

In contrast to the situation in mammals and birds, neurons in the central nervous system (CNS) of fish—such as the retinal ganglion cells—are capable of regenerating their axons and restoring vision. Special properties of the glial cells and the neurons of the fish visual pathway appear to contribute to the success of axonal regeneration. The fish oligodendrocytes lack the axon growth inhibiting molecules that interfere with axonal extension in mammals. Instead, fish optic nerve oligodendrocytes support—at least in vitro—axonal elongation of fish as well as that of rat retinal axons. Moreover, the fish retinal ganglion cells re-express upon injury a set of growth associated cell surface molecules and equip the regenerating axons throughout their path and up into their target, the tectum opticum with these molecules. This may indicate that the injured fish ganglion cells reactivate the cellular machinery necessary for axonal regrowth and pathfinding. Furthermore, the target itself provides positional marker molecules even in adult fish. These marker molecules are required to guide the regenerating axons back to their retinotopic home territory within the tectum. © 1992 John Wiley & Sons, Inc.     

Mechanisms in the development of retinotectal projections in the chick embryo studied by surgical deflection of the retinal pathway

Retinotopic analysis of the pathways of normal and aberrant retinal axons within the tectum of developing chick embryos was performed by selective labeling of retinal axons with a fluorescent dye, rhodamine-B isothiocyanate. To produce aberrant retinal axons, the presumptive optic chiasma was surgically disorganized at the 3rd day of incubation. At the 11th and 13th days of incubation, more than half of the operated embryos exhibited several aberrant retinal axons which reached ectopic parts of the tectum. The pathways of these aberrant axons within the tectum depended on the position of their initial invasion into the tectum at the diencephalotectal junction, and not on their position of origin within the retina. The aberrant retinal axons did not show any sign of correction of their pathways toward their normal sites of innervation within the tectum. As development proceeded, elimination of the aberrant retinal axons occurred. By the 16th day of incubation, almost all operated embryos lacked aberrant retinal axons and although the total number of axons often appeared reduced, a nearly normal topography of retinotectal projections was established. These findings indicate that the initial invasion of the retinal axons into the tectum is conducted predominantly by nonspecific mechanisms and, thereafter, a selective maintenance of appropriate retinal axons occurs.     

Retina development in zebrafish requires the heparan sulfate proteoglycan agrin

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

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.     

Up-regulation of protein kinase C in regenerating optic nerve fibers of goldfish: Immunohistochemistry and kinase activity assay

Protein kinase C (PKC) activation has been associated with synaptic plasticity in many projections, and manipulating PKC in the retinotectal projection strongly affects the activity-driven sharpening of the retinotopic map. This study examined levels of PKC in the regenerating retinotectal projection via immunostaining and assay of activity. A polyclonal antibody to the conserved C2 (Ca²⁺ binding) domain of classical PKC isozymes (anti-panPKC) recognized a single band at 79–80 kD on Western blots of goldfish brain. It stained one class of retinal bipolar cells and the ganglion cells in normal retina, as shown previously. Strong staining was not present in the optic fiber layer of retina or in optic nerve, optic tract, or terminal zone in tectum, with the exception of a single fascicle of optic nerve fibers that by their location and by L1 (E587) staining were identified as those arising from newly added ganglion cells at the retinal margin. Normal tectal sections showed dark staining of a subclass of type XIV neuron with somas at the top of the periventricular layer and an apical dendrite ascending to stratum opticum. In regenerating retina, swollen ganglion cells stained darkly and stained axons were seen in the optic fiber layer. In regenerating optic nerve (2–11 weeks postcrush), all fascicles of optic fibers stained darkly for both PKC and L1(E587). At 5 weeks postcrush, PKC staining could also be seen in the medial and lateral optic tracts and stratum opticum at the front half of the tectum and very lightly over the terminal zones. PKC activity was measured in homogenized tissues dissected from a series of fish with unilateral nerve crush from 1 to 5 weeks previously. Activity levels stimulated by phorbols and Ca²⁺ were measured by phosphorylation of a specific peptide and referred to levels measured in the opposite control side. Regeneration did not increase overall PKC activity in retina or tectum, but in optic nerve there was an 80% rise after the first week. The increased activity verifies that the increased staining in nerve represented an up-regulation of functional PKC during nerve regeneration. © 1998 John Wiley & Sons, Inc. J Neurobiol 36: 315–324, 1998     

The receptor tyrosine phosphatase CRYPalpha affects growth cone morphology

During development of the nervous system receptor tyrosine kinases and receptor protein tyrosine phosphatases act in a coordinate way during axon growth and guidance. In the developing avian retinotectal system, many different receptor protein tyrosine phosphatases are expressed. Most of them have unknown functions. Retinal ganglion cells express at least three different members of this receptor family on their axons and growth cones: CRYPalpha, CRYP-2 and PTPmu. CRYPalpha interacts heterophilically with at least two different ligands found in the basal membranes of the retina and the optic tectum. To analyze the role of the CRYPalpha-ligand interaction, retinal ganglion cell axons were grown on retinal basal membranes (inner limiting membrane) and the receptor-ligand interaction was blocked from both the receptor side (by receptor specific antibodies) and from the ligand side by using a receptor-alkaline phosphatase fusion protein. Both of these treatments reduced average retinal axon length and induced a dramatic change in morphology of retinal ganglion cell growth cones on basal membranes, but not on other substrates like laminin, N-cadherin, matrigel- and detergent-treated basal membranes. These results suggest that CRYPalpha and its ligand act as growth-promoting molecules during intraretinal axon growth.     

Recognition of position-specific properties of tectal cell membranes by retinal axons in vitro

In order to test the preference of growing axons for membrane-associated positional specificity a new in vitro assay was developed. In this assay, membrane fragments of two different sources are arranged as a carpet of very narrow alternating strips. Axons growing on such striped carpets are simultaneously confronted with the two substrates at the stripe borders. If there is a preference of axons for one or the other substrate they become oriented by the stripes and grow within the lanes of the preferred substrate. Such preferential growth could, in principle, be due to affinity to attractive factors on the preferred stripes or avoidance of repulsive factors on the alternate stripes. This assay system was used to investigate growth of chick retinal axons on tectal membranes. Tissue strips cut from various areas of the retina were explanted and the extending axons were confronted with stripes of cell membranes from various areas within the optic tectum. Tectal cell membranes prove to be an excellent substrate for the growth of retinal axons. Nasal and temporal axons can grow well on membranes of both posterior and anterior tectal cells. If, however, temporal axons are given a choice and encounter the border between anterior and posterior membranes they show a marked preference for growth on membranes of the anterior tectum, their natural target area. Nasal axons do not show a preference in this assay system. The transition from nasal to temporal properties within the retina is abrupt. In contrast, the transition from anterior to posterior properties of the tectal cell membranes occurs as a smooth gradient. Significantly, the positional differences of tectal membrane properties are only seen during the period of development of the retinotectal projection and are independent of tectal innervation by retinal axons. These anterior-posterior differences disappear by embryonic day 14.     

金鱼再生的视神经在顶盖投射精确化的DiI顺行标记研究

本文用微量显微注射法,在金鱼视网膜的背侧用亲脂类荧光染料DiI标记少量神经节细胞,通过顺行标记研究了视神经再生过程中视网膜顶盖投射的精确化过程。在损伤视神经后的不同时期观察了再生视神经纤维在顶盖整装片上的分布。在再生早期它们以超出正常的途径由背腹两侧进入顶盖,广泛分布。但其中大部分仍分布于顶盖腹侧的靶区。在再生晚期通过精确化,重建如正常鱼一样精确的视网膜顶盖投射。这个精确化过程表现在以下三方面:(1)再生于顶盖错误区域的再生视神经纤维的消失;(2)再生早期视神经纤维主干上生长的侧部分支的消失;(3)到达靶区的再生视神经纤维形成重迭的终末分支。由以上结果推测,顶盖中可能存在两类不同的因子:一类是普通诱向因子,存在于整个顶盖中,它在再生早期引导再生的视神经纤维长入顶盖。另一类是神经营养因子,它具区域特异性,在再生晚期引导视神经纤维到达顶盖靶区,形成精确的视网膜顶盖投射。     

Induction of target-directed optic axon outgrowth: effect of retinae transplanted to anophthalmic mice

In previous work using neural transplants (Hankin and Lund, 1987) we demonstrated two basic components of optic axon outgrowth in the mammalian retinotectal system: one category of outgrowth utilizes the subpial margin of the rostral brain stem as a preferential substrate (as do normal retinotectal axons); the other type of outgrowth, from retinae embedded deep within the midbrain parenchyma, is distance-dependent and highly target-oriented, but shows little apparent substrate specificity. One explanation for this directed outgrowth is that it is in response to a diffusible factor emanating from cells in the superior colliculus. In the present study we use congenitally anophthalmic mice as recipients for retinal transplants to test whether prior optic innervation of the superior colliculus plays a role in establishing either component of outgrowth. We show that outgrowth along the subpial pathway from a graft placed on the surface of the brain stem can take place in the absence of prior innervation of the superior colliculus. The target-directed outgrowth exhibited by embedded grafts only occurs if the tectum is also innervated by a second graft placed on the surface of the brain stem. It is proposed that tectal cells produce a factor in response to optic innervation and that this directs the growth patterns of embedded grafts. This suggests that optic innervation is a necessary prerequisite for the superior colliculus to produce the proposed diffusible chemotropic signal. In normal development such a factor could function to improve the efficiency of target-finding by later growing optic axons, but it might serve a quite different role, encouraging branching and trophic maintenance of the optic pathway once it has reached the tectum.     

Cell adhesion molecules in the early developing mouse retina: Retinal neurons show preferential outgrowth in vitro on L1 but not N-CAM

Both L1 and N-CAM are present on optic axons early in the developing mouse retina and optic nerve. In in vitro assays on substrates of purified cell adhesion molecules cells derived from E13 mouse retinae showed vigorous neurite extension on L1 but not on N-CAM. Although retinal neurons on N-CAM showed only limited attachment to the substrate, they were able to form lamellipodia immediately around the cell perimeter. In contrast, similarly derived cortical cells showed extensive neurite outgrowth on both substrates. Under these culture conditions, nearly all of the L1 and N-CAM present in the cell membrane appeared to be sequestered on the lower surface of the growth cones and neurites, indicating that most of these cell adhesion molecules were involved in homophilic interactions. Our results suggest differential roles for L1 and N-CAM in intitiation and establishment of the optic pathway. © 1994 John Wiley & Sons, Inc.     

Convergence of multisensory inputs in Xenopus tadpole tectum

The integration of multisensory information takes place in the optic tectum where visual and auditory/mechanosensory inputs converge and regulate motor outputs. The circuits that integrate multisensory information are poorly understood. In an effort to identify the basic components of a multisensory integrative circuit, we determined the projections of the mechanosensory input from the periphery to the optic tectum and compared their distribution to the retinotectal inputs in Xenopus laevis tadpoles using dye‐labeling methods. The peripheral ganglia of the lateral line system project to the ipsilateral hindbrain and the axons representing mechanosensory inputs along the anterior/posterior body axis are mapped along the ventrodorsal axis in the axon tract in the dorsal column of the hindbrain. Hindbrain neurons project axons to the contralateral optic tectum. The neurons from anterior and posterior hindbrain regions project axons to the dorsal and ventral tectum, respectively. While the retinotectal axons project to a superficial lamina in the tectal neuropil, the hindbrain axons project to a deep neuropil layer. Calcium imaging showed that multimodal inputs converge on tectal neurons. The layer‐specific projections of the hindbrain and retinal axons suggest a functional segregation of sensory inputs to proximal and distal tectal cell dendrites, respectively. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2009     

Expression patterns of Ephs and ephrins throughout retinotectal development in Xenopus laevis

The Eph family of receptor tyrosine kinases and their ligands the ephrins play an essential role in the targeting of retinal ganglion cell axons to topographically correct locations in the optic tectum during visual system development. The African claw-toed frog Xenopus laevis is a popular animal model for the study of retinotectal development because of its amenability to live imaging and electrophysiology. Its visual system undergoes protracted growth continuing beyond metamorphosis, yet little is known about ephrin and Eph expression patterns beyond stage 39 when retinal axons first arrive in the tectum. We used alkaline phosphatase fusion proteins of EphA3, ephrin-A5, EphB2, and ephrin-B1 as affinity probes to reveal the expression patterns of ephrin-As, EphAs, ephrin-Bs, and EphBs, respectively. Analysis of brains from stage 40 to adult frog revealed that ephrins and Eph receptors are expressed throughout development. As observed in other species, staining for ephrin-As displayed a high caudal to low rostral expression pattern across the tectum, roughly complementary to the expression of EphAs. In contrast with the prevailing model, EphBs were found to be expressed in the tectum in a high dorsal to low ventral gradient in young animals. In animals with induced binocular tectal innervation, ocular dominance bands of alternating input from the two eyes formed in the tectum; however, ephrin-A and EphA expression patterns were unmodulated and similar to those in normal frogs, confirming that the segregation of axons into eye-specific stripes is not the consequence of a respecification of molecular guidance cues in the tectum.