Eph receptor-ligand interactions are necessary for guidance of retinal ganglion cell axons in vitro

Previous results of an in vitro guidance test, the stripe assay, have demonstrated the presence of a repulsive axon guidance activity for temporal retinal axons in the posterior part of the vertebrate optic tectum. Ephrin-A5 and Ephrin-A2 are ligands for the EphA subfamily of Eph receptor tyrosine kinases, which are expressed in overlapping gradients in the posterior part of the tectum. When recombinantly expressed, both proteins have been shown to guide retinal ganglion cell axons in the stripe assay. While these results suggest that Ephrin-A5 and Ephrin-A2 form part of the posterior repulsive guidance activity, they do not elucidate whether they are necessary components. Here we report that soluble forms of the ligands at nanomolar concentrations completely abolish this repulsive activity. Similar results were obtained with the soluble extracellular domain of EphA3, which is a receptor for Ephrin-A2 and Ephrin-A5, but not with the corresponding domain of EphB3, a receptor for the transmembrane class of Eph ligands. These experiments show that the repulsive axon guidance activity seen in the stripe assay is mediated by Ephrin-A ligands.     

Group specific antibodies against the putative AMP-binding domain signature SGTTGXPKG in peptide synthetases and related enzymes

Ephrin-A5 (AL-1/RAGS), a ligand for Eph receptor tyrosine kinases, repels retinal axons in vitro and has a graded expression in the superior colliculus (SC), the major midbrain target of retinal ganglion cells. These properties implicate ephrin-A5 in the formation of topographic maps, a fundamental organizational feature of the nervous system. To test this hypothesis, we generated mice lacking ephrin-A5. The majority of ephrin-A5-/- mice develop to adulthood, are morphologically intact, and have normal anterior-posterior patterning of the midbrain. However, within the SC, retinal axons establish and maintain dense arborizations at topographically incorrect sites that correlate with locations of low expression of the related ligand ephrin-A2. In addition, retinal axons transiently overshoot the SC and extend aberrantly into the inferior colliculus (IC). This defect is consistent with the high level of ephrin-A5 expression in the IC and the finding that retinal axon growth on membranes from wild-type IC is inhibited relative to that on membranes from ephrin-A5-/- IC. These findings show that ephrin-A5 is required for the proper guidance and mapping of retinal axons in the mammalian midbrain.     

The cellular patterns of BDNF and trkB expression suggest multiple roles for BDNF during Xenopus visual system development

The temporal patterns of BDNF and trkB expression in the developing Xenopus laevis tadpole, and the responsiveness of retinal ganglion cells to BDNF, both in culture and in vivo, suggest significant roles for this neurotrophin during visual system development (Cohen-Cory and Fraser, Neuron 12, 747-761, 1994; Nature 378, 192-196, 1995). To examine the potential roles of this neurotrophin within the developing retina and in its target tissue, the optic tectum, we studied the cellular sites of BDNF expression by in situ hybridization. In the developing optic tectum, discrete groups of cells juxtaposed to the tectal neuropil where retinal axons arborize expressed BDNF, supporting the target-derived role commonly proposed for this neurotrophin. In the retina, retinal ganglion cells, ciliary margin cells, and a subset of cells in the inner nuclear layer expressed the BDNF gene. The expression of BDNF coincided with specific trkB expression by both retinal ganglion cells and amacrine cells, as well as with the localization of functional BDNF binding sites within the developing retina, as shown by in situ hybridization and BDNF cross-linking studies. To test for a possible role of endogenous retinal BDNF during development, we studied the effects of neutralizing antibodies to BDNF on the survival of retinal ganglion cells in culture. Exogenously administered BDNF increased survival, whereas neutralizing antibodies to BDNF significantly reduced baseline retinal ganglion cell survival and differentiation. This suggests the presence of an endogenous retinal source of neurotrophic support and that this is most likely BDNF itself. The retinal cellular patterns of BDNF and trkB expression as well as the effects of neutralizing antibodies to this neurotrophin suggest that, in addition to a target-derived role, BDNF plays both autocrine and/or paracrine roles during visual system development.     

Topographic guidance labels in a sensory projection to the forebrain

Visual connections to the mammalian forebrain are known to be patterned by neural activity, but it remains unknown whether the map topography of such higher sensory projections depends on axon guidance labels. Here, we show complementary expression and binding for the receptor EphA5 in mouse retina and its ligands ephrin-A2 and ephrin-A5 in multiple retinal targets, including the major forebrain target, the dorsal lateral geniculate nucleus (dLGN). These ligands can act in vitro as topographically specific repellents for mammalian retinal axons and are necessary for normal dLGN mapping in vivo. The results suggest a general and economic modular mechanism for brain mapping whereby a projecting field is mapped onto multiple targets by repeated use of the same labels. They also indicate the nature of a coordinate system for the mapping of sensory connections to the forebrain.     

Neurotrophins in the developing and regenerating visual system

The neurotrophins NGF, BDNF, NT-3 and NT-4 have a wide range of effects in the development and regeneration of neural circuits in the visual system of vertebrates. This review focuses on the localization and functions of neurotrophins in the retina, lateral geniculate nucleus, suprachiasmatic nucleus, superior colliculus/optic tectum, and isthmic nuclei. Research of the past 20 years has shown that neurotrophins and their receptors are localized in numerous visual centers from the retina to the visual cortex, and that neurotrophins influence proliferation, neurite outgrowth and survival of cells in the visual system in vitro and in vivo. A relationship between electrical activity and neurotrophic functions has been established in several visual centers in the CNS, and neurotrophins have been implicated in synaptic plasticity in the visual cortex. Besides functions of neurotrophins as retrograde, target-derived trophic factors, recent data indicate that neurotrophins may have anterograde, afferent as well as local, paracrine actions in the retina, optic nerve and the visual cortex. Some neurotrophins appear to regulate proliferation and survival of glial cells in the optic pathways. Neurotrophins increase the survival of retinal ganglion cells after axotomy or ischemia and they promote the regeneration of retinal ganglion cell axons in some vertebration. Neurotrophins also rescue photoreceptors from degeneration. These findings implicate the neurotrophins not only as important regulators during development, but also as potential therapeutic agents in degenerative retinal diseases and after optic nerve injury.     

Glutamate receptor binding in juvenile and adult Rana pipiens CNS

Autoradiographic methods were used to map NMDA- and quisqualate-sensitive glutamate binding sites in the brain of mature and juvenile Rana pipiens frogs. NMDA- and quisqualate-sensitive sites were consistently co-localized in the CNS. The highest glutamate binding occurred in the telencephalon, hypothalamus, and cerebellum. Glutamate binding sites were also specifically localized in visual pathways, including the superficial neuropil of the optic tectum, consistent with glutamate being the retinal ganglion cell neurotransmitter. The distribution of glutamate binding sites in the brain of juvenile postmetamorphic frogs was similar to that in adults. In general, Quis binding increased about twofold in adults compared to juveniles, whereas NMDA binding did not show a comparable developmental increase. To test whether glutamate binding sites are located on retinal axon terminals or on tectal cell dendrites in the optic tectum, juvenile postmetamorphic frogs were enucleated unilaterally, and receptor binding was performed following 1, 3, 7, and 14 days survival. The denervated tectal neuropil showed a delayed decrease in NMDA- and quisqualate-sensitive binding, consistent with the receptors being located on postsynaptic tectal cell dendrites.     

Differential withdrawal of retinal axons induced by a secreted factor

To understand the development of the topographic map in the chick retinotectal projection, we studied the long-term interactions between retinal axons and tectal cell processes using a novel coculture system, the ryomen chamber. Both nasal and temporal retinal axons initially grew equally well on a substrate consisting of posterior tectal cell processes; however, subsequently most temporal axons withdrew from this surface, whereas most nasal axons did not. Experiments using conditioned media indicate that posterior tectal cells induced withdrawal of the temporal axons by secreting a soluble factor. This withdrawal seems to be distinct from the immediate repulsive effect of ephrin-A2 (ELF-1) and ephrin-A5 (RAGS) seen in the stripe assay because (1) the withdrawal-inducing factor was diffusible, whereas ephrin-A2 and -A5 are membrane-bound, and (2) the withdrawal-inducing factor appeared later in development than ephrin-A2 and -A5. Furthermore, sensitivity to the withdrawal-inducing factor decreased continuously from the temporal to nasal retina. These results suggest that target cell-induced axonal withdrawal may be involved during a late stage of the development of the retinotectal map.     

Asymmetric mRNA expression in the retinal neuroepithelium of the chick embryo assessed by differential display polymerase chain reaction

In the normal retinotectal topography established during the embryonic development of the chick visual system, retinal ganglion cell axons from the nasal retina connect to the posterior part and temporal retinal axons connect to the anterior part of the optic tectum. For the investigation of position-specific gene expression along the nasal-temporal axis of the retinal neuroepithelium (RN), differential display PCR was carried out from the nasal or temporal part of the RN at HH11 (E2). We found several genes that are differentially expressed either in the nasal or in the temporal part of the RN and the analysis of the asymmetrically expressed fragments showed at least one cDNA fragment to be exclusively expressed in the nasal RN. This fragment was 550 bp in size.     

Serotonin modulates induced synaptic activity in the optic tectum of the frog

The extent to which retinal signals are modulated at central sites is unknown. We sought to determine the effects of serotonin, a neurotransmitter present in the retinorecipient layers of the frog tectum, on retinotectal transmission. Acute electrical stimulation delivered to the retinorecipient layer of optic tectum brain slices was used to model the activation of tectal neurons by visual inputs. This stimulation evoked either a monosynaptic or a polysynaptic current response in patch-clamped tectal neurons. External application of serotonin blocked both of these induced currents as did 5-carbotryptamine (5-CT), a nonselective agonist of 5-HT1 receptors. Alpha-methylserotonin, a nonselective agonist of 5-HT2 receptors, also blocked polysynaptic responses but was less effective than either serotonin or 5-CT in blocking monosynaptic ones. Lateral synaptic interactions between tectal cells, modeled by acute electrical stimulation in the main cellular layer of the tectum, were also blocked by serotonin, 5-CT or alpha-methylserotonin. The presented data suggest that endogenous serotonin may strongly affect visual signal processing by modulating synaptic transmission between both the retina and the tectum as well as between tectal neurons. This modulation is likely to be due, at least in part, to a demonstrated outward current induced by serotonin in a subpopulation of tectal cells.     

The Eph family in the patterning of neural development

The Eph family represents the largest subfamily of receptor tyrosine kinases. Its members are predominantly expressed in the developing and adult nervous system. Besides playing an important role in the contact-mediated repulsion of axons, they have recently also been implicated in the control of cell migration. Characteristics of the Eph family are extended promiscuity in the interaction between receptors and ligands, the necessity of membrane attachment of the ligands to exert their function, the lack of induction of mitogenic responses, and the bi-directional signalling of receptors and ligands.     

Inhibition of protein tyrosine kinases impairs axon extension in the embryonic optic tract

The role of protein tyrosine kinase (PTK) activity in the development of the retinal projection was examined in vivo by applying inhibitors of cytoplasmic PTKs, herbimycin A and lavendustin A, to intact brain preparations of Xenopus embryos. The inhibitors were present during the period when retinal ganglion cell axons first navigate through the optic tract to reach their target, the optic tectum. A majority of inhibitor-treated retinal axons stalled at the beginning of the optic tract, leading to an 80% reduction in projection length at the highest doses. All inhibitor-treated axons that did extend into the optic tract exhibited normal pathfinding behavior. Tyrosine kinase assays of inhibitor-treated brains demonstrated that at doses at which retinal axon extension was severely impaired, PTK activity, including that of src family proteins, was reduced by 50-60%. Consistent with the in vivo findings, PTK inhibitors reduced neurite outgrowth from cultured retinal neurons by 70-80%. This contrasts with the strong enhancement of outgrowth induced by the same inhibitors in cultured chick ciliary ganglion neurons and suggests that the mediation of outgrowth by PTK activity may vary in different neuronal types. Inhibitor-treated growth cones cultured on laminin were larger than normal, suggesting that tyrosine phosphorylation can modulate growth cone-substrate adhesive interactions. Our results in vivo and in vitro provide complementary evidence that retinal axon outgrowth is inhibited by pharmacological blockers of PTK activity and indicate that inhibitor-sensitive PTKs normally play a role in promoting retinal neurite extension.     

Reggie-1 and reggie-2, two cell surface proteins expressed by retinal ganglion cells during axon regeneration

Fish--in contrast to mammals--regenerate retinal ganglion cell axons when the optic nerve is severed. Optic nerve injury leads to reexpression of proteins, which typically are first expressed in newly differentiated retinal ganglion cells and axons. Here we identified two new proteins of fish retinal ganglion cells, reggie-1 and reggie-2, with monoclonal antibody M802 and molecular cloning techniques. In normal fish, M802 stained the few retinal axons derived from newborn ganglion cells which in fish are added lifelong to the retinal margin. After optic nerve injury, however, M802 labeled all retinal ganglion cells and retinal axons throughout their path into tectum. Consistent with M802 staining, reggie-1 and reggie-2 mRNAs were present in lesioned retinal ganglion cells, as demonstrated by in situ hybridization, but were not detectable in their normal mature counterparts. In western blots with membrane proteins of the adult goldfish brain, M802 recognizes a 48x10(3) Mr protein band. At the amino acid level, 48x10(3) Mr reggie-1 and reggie-2 are 44% identical, lack transmembrane and membrane anchor domains, but appear membrane associated by ionic interactions. Reggie-1 and reggie-2 are homologous to 35x10(3) Mr ESA (human epidermal surface antigen) but are here identified as neuronal surface proteins, present on newly differentiated ganglion cells at the retinal margin and which are reexpressed in mature ganglion cells upon injury and during axonal regeneration.     

Thermoregulatory responses of the newborn pig during experimentally induced hypothermia and rewarming

Eph family receptor tyrosine kinases (including EphA3, EphB4) direct pathfinding of neurons within migratory fields of cells expressing gradients of their membrane-bound ligands. Others (EphB1 and EphA2) direct vascular network assembly, affecting endothelial migration, capillary morphogenesis, and angiogenesis. To explore how ephrins could provide positional labels for cell targeting, we tested whether endogenous endothelial and P19 cell EphB1 (ELK) and EphB2 (Nuk) receptors discriminate between different oligomeric forms of an ephrin-B1/Fc fusion ligand. Receptor tyrosine phosphorylation was stimulated by both dimeric and clustered multimeric ephrin-B1, yet only ephrin-B1 multimers (tetramers) promoted endothelial capillary-like assembly, cell attachment, and the recruitment of low-molecular-weight phosphotyrosine phosphatase (LMW-PTP) to receptor complexes. Cell-cell contact among cells expressing both EphB1 and ephrin-B1 was required for EphB1 activation and recruitment of LMW-PTP to EphB1 complexes. The EphB1-binding site for LMW-PTP was mapped and shown to be required for tetrameric ephrin-B1 to recruit LMW-PTP and to promote attachment. Thus, distinct EphB1-signaling complexes are assembled and different cellular attachment responses are determined by a receptor switch mechanism responsive to distinct ephrin-B1 oligomers.     

Stability of vancomycin-resistant enterococcal genotypes isolated from long-term-colonized patients

The Eph-related receptor tyrosine kinases constitute a large family of receptors with most members displaying specific expression patterns in the developing embryo. Ligands for Eph receptor tyrosine kinases, recently renamed ephrins, comprise a family of at least 8 membrane-bound members that display promiscuous binding to Eph receptors. Here we report the characterization of a human cDNA clone with high homology to the gene encoding the murine ephrin-A2 ligand. The human gene encodes a single 2.4-kb mRNA with a restricted and developmentally-regulated tissue distribution pattern. In the fetus, ephrin-A2 mRNA is expressed in brain and intestine, whereas in the adult, high levels of ephrin-A2 mRNA are detectable in lung and intestine. Using PCR-based screening of genomic DNA from human x rodent hybrid cell lines, the gene encoding ephrin-A2 (EFNA2) was assigned to chromosome 19. Fluorescence in situ hybridization to metaphase chromosome preparations refined this localization to band p13.3.     

The Eek receptor, a member of the Eph family of tyrosine protein kinases, can be activated by three different Eph family ligands

The Eph family of receptors, the largest subgroup within the tyrosine protein kinase receptor family, are comprised of at least thirteen members, many of which are predominantly expressed in the developing and adult nervous system. In this study, we have isolated a full-length cDNA, encoding the mouse homologue of a previous partially characterized Eek protein, a member of Eph receptor tyrosine kinase family. In a comparison of the amino acid sequences of various Eph family members, Eek is most similar to Ehk-3/MDK1, Sek/Cek8, Ehk-2, Hek/Mek4/Cek4, and Bsk/Ehk1/Rek7/Cek7, which are predominantly expressed in the nervous system. Additionally, we have used a low-stringency PCR cloning technique to identify ligands, related to B61, that may interact with Eek. Three different GPI-linked ligands, namely Elf-1/Cek7-L, Ehk1-L/Efl-2/Lerk3 and AL-1/RAGS, were isolated from mouse brain. To study the functional interactions between these ligands and the Eek receptors, we have constructed chimeric ligands consisting of the Fc portion of human IgG fused to their carboxyl-terminus. These chimeric ligands bound to, and activated both the Eek receptors and the Eek-TrkB chimeric receptors expressed in NIH3T3 cells. These findings suggest that Eek receptor can be activated by at least three different GPI-linked ligands.     

Neurotrophins and their receptors in the tench retina during optic nerve regeneration

To understand the role of neurotrophins in the visual system, we investigated the distribution of both neurotrophins and their receptors within the retina of a fish that has the capacity to spontaneously regenerate its optic nerve axons after lesion. Intact retinas and retinas from tench, whose optic nerve had been crushed, were analyzed by immunohistochemistry and in situ hybridization. Trk receptors were mainly immunolocalized in cells of the inner nuclear and ganglion cell layers, a distribution coincident with that of their mRNAs. Nerve growth factor (NGF) immunoreactivity was detected exclusively in Müller cell processes, and brain-derived neurotrophic factor (BDNF) was found in both neuronal bodies and Müller cell processes. Neurotrophin-3 (NT-3) was detected in most of the cell nuclei, and neurotrophin-4/5 (NT-4/5) was localized in fibers and in a few cells in the inner retina. An increase in both TrkA protein and mRNA was detected during axonal regeneration within the retinal ganglion cell layer, reaching a maximum 30 days postcrush and returning to normal levels by day 90, when optic nerve regeneration is almost completed in this fish. None of the other neurotrophins and receptors showed appreciable changes. The heterogeneous distribution patterns of neurotrophins and their receptors in fish retina, their differences from the distribution observed in other species, and the TrkA changes after optic nerve crush suggest an important role for these molecules in the normal physiology of the fish retina and during the regeneration process.     

Fibroblast growth factor receptor function is required for the orderly projection of ganglion cell axons in the developing mammalian retina

During the early stages of development various cell adhesion molecules (CAMs) and fibroblast growth factor receptors (FGFR) are expressed throughout the retinal neuroepithelium. The ability of retinal ganglion cells to project their axons to the optic fissure depends, in part, on cell-cell interactions mediated by cell adhesion molecules. In the present study we show that the ability of the firstborn rat retinal ganglion cells to extend axons in vitro can be stimulated by NCAM and L1, but not N-cadherin. Both CAM responses can be fully inhibited by antibodies that block neuronal fibroblast growth factor receptor function and by agents that block defined steps in the FGFR signal transduction cascade. When added to living E13.5 rat retinal whole-mount preparations the same agents induced errors in the orderly establishment of young axon patterns in the retinal periphery and caused axons in the retinal center to defasciculate. These results suggest that the activation of the fibroblast growth factor receptor signal cascade not only promotes survival and proliferation of various cell types but can also mediate intraretinal axon guidance.     

Two distinct populations of tectal neurons have unique connections within the retinotectorotundal pathway of the pigeon (Columba livia)

The tectofugal pathway is a massive ascending polysynaptic pathway from the tectum to the thalamus and then to the telencephalon. In birds, the initial component of this pathway is known as the tectorotundal pathway; in mammals, it is known as the tectopulvinar pathway. The avian tectorotundal pathway is highly developed; thus, it provides a particularly appropriate model for exploring the fundamental properties of this system in all amniotes. To further define the connectivity of the tectorotundal projections of the tectofugal pathway, we injected cholera toxin B fragment into various rotundal divisions, the tectobulbar projection, and the ventral supraoptic decussation of the pigeon. We found intense bilateral retrograde labeling of neurons that stratified within layer 13 and, in certain cases, granular staining in layer 5b of the optic tectum. Based on these results, we propose that there are two distinct types of layer 13 neurons that project to the rotundus: 1) type I neurons, which are found in the outer sublamina of layer 13 (closer to layer 12) and which project to the anterior and centralis rotundal divisions, and 2) type II neurons, which are found in the inner sublamina of layer 13 (closer to layer 14) and which project to the posterior and triangularis rotundal divisions. Only the labeling of type I neurons produced the granular dendritic staining in layer 5b. An additional type of tectal neuron was also found that projected to the tectobulbar system. We then injected Phaseolus vulgaris-leucoagglutinin in the optic tract and found that the retinal axons terminating within tectal layer 5b formed narrow radial arbors (7-10 microm in diameter) that were confined to layer 5b. Based on these results, we propose that these axons are derived from a population of small retinal ganglion cells (4.5-6.0 microm in diameter) that terminate on the distal dendrites of type I neurons. This study strongly indicated the presence of a major bilateral oligosynaptic retinotectorotundal pathway arising from small retinal ganglion cells projecting to the rotundus with only a single intervening tectal neuron, the proposed type I neuron. We suggest that a similar organization of retinotectopulvinar connections exist in reptiles and in many mammals.     

The expression and regulation of chick EphA7 suggests roles in limb patterning and innervation

Eph receptors and their ligands, the ephrins, have been implicated in early patterning and axon guidance in vertebrate embryos. Members of these families play pivotal roles in the formation of topographic maps in the central nervous system, the formation of brain commissures, and in the guidance of neural crest cells and motor axons through the anterior half of the somites. Here, we report a highly dynamic expression pattern of the chick EphA7 gene in the developing limb. Expression is detected in discrete domains of the dorsal mesenchyme from 3 days of incubation. The expressing cells are adjacent to the routes where axons grow to innervate the limb at several key points: the region of plexus formation, the bifurcation between dorsal and ventral fascicles, and the pathway followed by axons innervating the dorsal muscle mass. These results suggested a role for EphA7 in cell-cell contact-mediated signalling in dorsal limb patterning and/or axon guidance. We carried out experimental manipulations in the chick embryo wing bud to alter the dorsoventral patterning of the limb. The analyses of EphA7 expression and innervation in the operated wings indicate that a signal emanating from the dorsal ectoderm regulates EphA7 in such a way that, in its absence, the wing bud lacks EphA7 expression and shows innervation defects at the regions where the gene was downregulated. EphA7 downregulation in the dorsal mesenchyme after dorsal ectoderm removal is more rapid than that of Lmx-1, the gene known to mediate dorsalisation in response to the ectodermal signal. These results add a new gene to the dorsalisation signalling pathway in the limb. Moreover, they implicate the Eph receptor family in the patterning and innervation of the developing limb, extending its role in axon pathfinding to the distal periphery.     

Expression of multiple cadherins and catenins in the chick optic tectum

Cadherins form a large family of homophilic cell adhesion molecules that are involved in numerous aspects of neural development. The best-studied neural cadherin, N-cadherin, is concentrated at synapses made by retinal axons in the chick optic tectum and is required for the arborization of retinal axons in their target (retinorecipient) laminae. By analogy, other cadherins might mediate arborization or synaptogenesis in other tectal laminae. Here we consider which cadherins are expressed in tectum, which cells express them, and how their expression is regulated. First, using N-cadherin as a model, we show that synaptic input regulates both cadherin gene expression and the subcellular distribution of cadherin protein. Second, we demonstrate that N-, R-, and T-cadherin are each expressed in distinct laminar patterns during retinotectal synaptogenesis and that N- and R- are enriched in nonoverlapping synaptic subsets. Third, we show that over 20 cadherin superfamily genes are expressed in the tectum during the time that synapses are forming and that many of them are expressed in restricted groups of cells. Finally, we report that both beta-catenin and gamma-catenin (plakoglobin), cytoplasmic proteins required for cadherin signaling, are enriched at synapses and associated with N-cadherin. However, beta- and gamma-catenins are differentially distributed and regulated, and form mutually exclusive complexes. This result suggests that cadherin-based specificity involves multiple cadherin-dependent signaling pathways as well as multiple cadherins.