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Topographic mapping: Organising by repulsion and competition?
Abstract
The establishment of topographic maps of neuronal connections is believed to involve graded repulsion mediated by EphA receptors and ephrin-A ligands. Gene knockouts show that ephrin-A ligands do indeed have a crucial role in mapping, and that mechanisms in addition to graded repulsion must also be at work.
... The Eph receptors are a large subfamily of tyrosine kinase receptors that provide molecular guidance cues for axon targeting. These guidance factors, and their natural ligands the ephrins, are widely distributed in the developing central nervous system (CNS) and are involved in the development of the topography of innervation during neuronal development (for reviews see Flanagan and Vanderhaeghen, 1998; Zhou, 1998; Wilkinson, 2000 Wilkinson, , 2001 Knoll and Drescher, 2002). Most members of the Eph receptor family, including EphA2 to A5 and EphB1 to B6, are expressed in the adult central nervous system (Flanagan and Vanderhaeghen, 1998; Zhou, 1998; Gerlai, 2001). ...
... Thus, understanding the underlying mechanisms of such sprouting phenomena is of great importance in furthering our understanding of epilepsy, learning and memory, and repair processes. Given the relatively high expression of Eph receptors/ephrins in the hippocampal region ( Gao et al., 1998; Moreno-Flores and Wandosell, 1999; Gerlai et al., 1999; Buchert et al., 1999; Yue et al., 2002; Murai et al., 2003), their expression in gradients during development (Zhang et al., 1996; Gao et al., 1996; Yue et al., 2002), and their roles in axonal guidance during development (Flanagan and Vanderhaeghen, 1998; Zhou, 1998; Wilkinson, 2000 Wilkinson, , 2001 Knoll and Drescher, 2002), we postulated that these axon guidance molecules might be involved in directing activity-dependent sprouted mossy fiber collaterals to their destinations in adult CNS. In the present study, we examined the distribution of the receptor EphA5 and one of its ligands, ephrin-A3, in the rat. ...
... We show that complementary gradients of the ligand ephrin-A3 and receptor EphA5 are expressed in the mossy fiber system in the adult rat CNS. The membrane-bound Eph receptors and ephrin ligands are widely expressed in the developing nervous system and serve as location signals for the formation of topographic maps (Flanagan and Vanderhaeghen, 1998; Zhou, 1998; Wilkinson, 2000 Wilkinson, , 2001 Klein, 2001; Knoll and Drescher, 2002; Pittman and Chien, 2002). There are often complementary gradients of Eph receptors and ephrins in the source and target regions of the axons. ...
The Eph family of tyrosine kinase receptors and their ligands, ephrins, are distributed in gradients and serve as molecular guidance cues for axonal patterning during neuronal development. Most of these molecules are also expressed in mature brain. Thus, we examine here the potential roles of such molecules in plasticity and activity-dependent mossy fiber sprouting of adult CNS. We show that the ligand ephrin-A3 and the receptor EphA5 are expressed in complementary gradients in the adult rat mossy fiber system. Using the kindling model, we demonstrate that exogenous immunoadhesins that affect the interaction of endogenous EphA receptors and ephrin-A ligands modulate the development of kindling, one type of long-term plasticity, in mature rat brain. These immunoadhesins, combined with epileptogenic stimulations, alter both the extent and the pattern of collateral axonal sprouting in the mossy fiber pathway. Our results suggest that EphA receptors and ephrin-A ligands modify neuronal plasticity and may serve as spatial cues that modulate the development and pattern of activation-dependent axonal growth in adult CNS.
... Reverse signaling (efn-mediated) is generally regarded as an attractive and stabilizing stimulus for neuronal extensions and stabilizing synaptic connections such as ephrin-B3 [28,29], whereas activated forward signaling (Eph-mediated) is repulsive to outgrowing neurites (EphA3, EphA4, EphA5, and EphB2) [7,30] and is responsible for inducing axonal growth cone collapse (EphA4, EphA5, EphB1, EphB2, and EphB3) [31][32][33][34][35][36][37], restricting mid-line crossing of axons (EphA4, EphB1, EphB2, and EphB3) [38][39][40][41][42], and destabilizing synaptic connections (EphA1, EphA4, EphB1, EphB2, and EphB3) [42][43][44][45][46]. During development, Eph/ efn signaling is responsible for establishing guiding gradients that direct the retinotopic projections of retinal ganglion cell (RGC) axons onto the visual centers of the brain [11][12][13][14][47][48][49][50][51]. This guidance for the outgrowing RGC neurites established by Eph/efn signaling is achieved through graded repulsion rather than attraction [7,31,32,38]. ...
... Ephrin signaling is highly relevant to the visual system and retina, as counter-gradients of the various members in this ligand-receptor family mediate the establishment of the dorso-ventral and naso-temporal axes during retinotopic map formation in the retina and superior colliculus, as well as guide the decussation of RGC projections through the optic chiasm [11,13,14,47,[49][50][51][84][85][86]. Ephrin signaling through Eph receptors on RGC projections achieve their guidance by exerting a graded repulsive stimulus on the actin cytoskeletal dynamics across the neuronal cells [7,31,32,38]. ...
Background
Optic neuropathy is a major cause of irreversible blindness, yet the molecular determinants that contribute to neuronal demise have not been fully elucidated. Several studies have identified ‘ephrin signaling’ as one of the most dysregulated pathways in the early pathophysiology of optic neuropathy with varied etiologies. Developmentally, gradients in ephrin signaling coordinate retinotopic mapping via repulsive modulation of cytoskeletal dynamics in neuronal membranes. Little is known about the role ephrin signaling plays in the post-natal visual system and its correlation with the onset of optic neuropathy.
Methods
Postnatal mouse retinas were collected for mass spectrometry analysis for erythropoietin-producing human hepatocellular (Eph) receptors. Optic nerve crush (ONC) model was employed to induce optic neuropathy, and proteomic changes during the acute phase of neuropathic onset were analyzed. Confocal and super-resolution microscopy determined the cellular localization of activated Eph receptors after ONC injury. Eph receptor inhibitors assessed the neuroprotective effect of ephrin signaling modulation.
Results
Mass spectrometry revealed expression of seven Eph receptors (EphA2, A4, A5, B1, B2, B3, and B6) in postnatal mouse retinal tissue. Immunoblotting analysis indicated a significant increase in phosphorylation of these Eph receptors 48 h after ONC. Confocal microscopy demonstrated the presence of both subclasses of Eph receptors within the retina. Stochastic optical reconstruction microscopy (STORM) super-resolution imaging combined with optimal transport colocalization analysis revealed a significant co-localization of activated Eph receptors with injured neuronal cells, compared to uninjured neuronal and/or injured glial cells, 48 h post-ONC. Eph receptor inhibitors displayed notable neuroprotective effects for retinal ganglion cells (RGCs) after six days of ONC injury.
Conclusions
Our findings demonstrate the functional presence of diverse Eph receptors in the postnatal mammalian retina, capable of modulating multiple biological processes. Pan-Eph receptor activation contributes to the onset of neuropathy in optic neuropathies, with preferential activation of Eph receptors on neuronal processes in the inner retina following optic nerve injury. Notably, Eph receptor activation precedes neuronal loss. We observed a neuroprotective effect on RGCs upon inhibiting Eph receptors. Our study highlights the importance of investigating this repulsive pathway in early optic neuropathies and provides a comprehensive characterization of the receptors present in the developed retina of mice, relevant to both homeostasis and disease processes.
... Reverse signaling (efn-mediated) is generally regarded as an attractive and stabilizing stimulus for neuronal extensions and stabilizing synaptic connections [28,29], whereas activated forward signaling (Eph-mediated) is repulsive to outgrowing neurites [7,30] and is responsible for inducing axonal growth cone collapse [31][32][33][34][35][36][37], restricting mid-line crossing of axons [38][39][40][41][42], and destabilizing synaptic connections [42][43][44][45][46]. During development, Eph/efn signaling is responsible for establishing guiding gradients that direct the retinotopic projections of RGC axons onto the visual centers of the brain [11][12][13][14][47][48][49][50][51]. This guidance for the outgrowing RGC neurites established by Eph/efn signaling is achieved through graded repulsion rather than attraction [7,31,32,38]. ...
... Ephrin signaling is highly relevant to the visual system and retina, as counter-gradients of the various members in this ligand-receptor family mediate establish the dorso-ventral and nasotemporal axes during retinotopic map formation in the retina and superior colliculus, as well as guide the decussation of RGC projections through the optic chiasm [11,13,14,47,[49][50][51][85][86][87]. ...
Background: Optic neuropathy (ON) is a major cause of irreversible blindness, yet the molecular determinants that contribute to neuronal demise have not been fully elucidated. Several studies have identified ephrin signaling as one of the most dysregulated pathways in the early pathophysiology of ON with varied etiologies. Developmentally, gradients in ephrin signaling coordinate retinotopic mapping via repulsive modulation of cytoskeletal dynamics in neuronal membranes. Little is known about the role ephrin signaling played in the post-natal visual system and its correlation with the onset of optic neuropathy.
Methods: Postnatal mouse retinas were collected for mass spectrometry analysis for Eph receptors. Optic nerve crush (ONC) model was employed to induce optic neuropathy, and proteomic changes during the acute phase of neuropathic onset were analyzed. Confocal and super-resolution microscopy determined the cellular localization of activated Eph receptors after ONC injury. Eph receptor inhibitors assessed the neuroprotective effect of ephrin signaling modulation.
Results: Mass spectrometry revealed expression of seven Eph receptors (EphA2, A4, A5, B1, B2, B3, and B6) in postnatal mouse retinal tissue. Immunoblotting analysis indicated a significant increase in phosphorylation of these Eph receptors 48 hours after ONC. Confocal microscopy demonstrated the presence of both subclasses of Eph receptors in the inner retinal layers. STORM super-resolution imaging combined with optimal transport co-localization analysis revealed a significant co-localization of activated Eph receptors with injured neuronal processes, compared to uninjured neuronal and/or injured glial cells, 48 hours post-ONC. Eph receptor inhibitors displayed notable neuroprotective effects after 6 days of ONC injury.
Conclusions: Our findings demonstrate the functional presence of diverse Eph receptors in the postnatal mammalian retina, capable of modulating multiple biological processes. Pan-Eph receptor activation contributes to the onset of neuropathy in ONs, with preferential activation of Eph receptors on neuronal processes in the inner retina following optic nerve injury. Notably, Eph receptor activation precedes neuronal loss. We observed neuroprotective effects upon inhibiting Eph receptors. Our study highlights the importance of investigating this repulsive pathway in early optic neuropathies and provides a comprehensive characterization of the receptors present in the developed retina of mice, relevant to both homeostasis and disease processes.
... Finally, we asked whether Rac1 plays a role in development of the topography of the retinotectal projection in vivo. Development of normal topography is thought to depend on the response of growing retinal axons to ephrins expressed by tectal cells (Flanagan and Vanderhaeghen, 1998;O'Leary et al., 1999;Wilkinson, 2000). We found previously that injection into the developing eye of an antisense oligonucleotide targeting EphA3, Figure 4. Inhibition of Rac1 signaling but not expression of constitutively active Rac1 blocks DRG growth cone collapse in response to ephrin-A2. ...
... Previous work established that ephrin signaling via Eph receptors is involved in development of the normal pattern of axonal projections in the primary visual system (Flanagan and Vanderhaeghen, 1998;O'Leary et al., 1999;Wilkinson, 2000;Yates et al., 2001). We reasoned that if Rac1 is a required effector of ephrin signaling in vivo, as indicated by the in vitro data, then reducing Rac1 expression should mimic the effect of reduced EphA3 expression on development of the retinal projection. ...
Negative guidance molecules are important for guiding the growth of axons and ultimately for determining the wiring pattern in the developing nervous system. In tissue culture, growth cones at the tips of growing axons collapse in response to negative guidance molecules, such as ephrin-A2 and semaphorin 3A. The small GTPase Rac1 is involved in growth cone collapse, but the nature of its role is not clear. Rac1 activity assays showed that Rac1 is transiently inactivated after treatment with ephrin-A2. Ephrin-induced growth cone collapse, however, correlated with resumption of Rac1 activity. We demonstrate that Rac1 is required for endocytosis of the growth cone plasma membrane and reorganization of F-actin but not for the depolymerization of F-actin during growth cone collapse in response to ephrin-A2 and semaphorin 3A. Rac1, however, does not regulate constitutive endocytosis in growth cones. These findings show that in response to negative guidance molecules, the function of Rac1 changes from promoting actin polymerization associated with axon growth to driving endocytosis of the plasma membrane, resulting in growth cone collapse. Furthermore, Rac1 antisense injected into the embryonic chick eye in vivo caused the retinotectal projection to develop without normal topography in a manner consistent with Rac1 having an obligatory role in mediating ephrin signaling.
... It is B- type Ephrins which are transmembrane molecules. Ephrins are known for their role in topographic mapping of retinal ganglion cell axons on the optic tectum (O'Leary and Wilkinson, 1999;Wilkinson, 2000). EphrinB2 is known as a repellant cue for axon guidance and also has been found in a retinorecipient layer of chick tectum where RGC axons make synapses ( Braisted et al., 1997). ...
... Friendrich Bonhoeffer and colleagues showed that cues in the posterior tectum had a repellant activity on temporal axons retinal in vitro ( . Subsequent molecular characterization revealed that positional information, which is critical for topographic mapping, is provided by gradients of ligands and receptors of the Eph family (O' Leary and Wilkinson, 1999;Wilkinson, 2000), which are present on endfeet of radial glial cells (the substrata for migration) and retinal ganglion cell axons. GPI-anchored ligands such as EphrinA5 ( Drescher et al., 1995) and EphrinA2 responsible for their termination zone in the tectum ( Goodhill and Richards, 1999;Brown et al., 2000). ...
... Ephrins have been subdivided into two subclasses, the ephrin-As which are GPI-linked to the cell membrane, and the ephrin-Bs which are transmembrane proteins; likewise, the Eph receptors are also divided into two subclasses based on binding affinities to the ephrins (Eph Nomenclature Committee, 1997). Studies have shown that EphA receptor interactions with ephrin-A ligands underlie retinotectal topographic mapping (Feldheim et al., 2000;Brown et al., 2000;Wilkinson, 2000), and EphB2 and EphB3 receptors are involved in formation of brain commissures as well as retinal axon pathfinding to the optic disc Orioli et al., 1996;Birgbauer et al., 2000). ...
... Intriguingly, in the visual system, RGC axons appear to express both Ephs and ephrins (Marcus et al., 1996;Holash et al., 1997;Braisted et al., 1997;Connor et al., 1998;Hornberger et al., 1999;Birgbauer et al., 2000) leading to the possibility that Ephs and ephrins may serve as both receptors and ligands within the same cell. In the retina, RGC axons navigate to the optic disc in response to EphB proteins acting as guidance cues, while in the visual target, the superior colliculus, RGC axons have been shown to respond to ephrins, likely using EphA proteins as guidance receptors, during the formation of the retinotopic map (Wilkinson, 2000). Although EphA and EphB proteins may differ in their direction of signaling, recent studies have suggested that the EphA/ephrin-A system may also show reverse signaling (Davy et al., 1999;Davy and Robbins, 2000;Huai and Drescher, 2001;Knoll et al., 2001), and therefore EphA as well as EphB proteins may act both as receptors and guidance cues. ...
Axon pathfinding relies on cellular signaling mediated by growth cone receptor proteins responding to ligands, or guidance cues, in the environment. Eph proteins are a family of receptor tyrosine kinases that govern axon pathway development, including retinal axon projections to CNS targets. Recent examination of EphB mutant mice, however, has shown that axon pathfinding within the retina to the optic disc is dependent on EphB receptors, but independent of their kinase activity. Here we show a function for EphB1, B2 and B3 receptor extracellular domains (ECDs) in inhibiting mouse retinal axons when presented either as substratum-bound proteins or as soluble proteins directly applied to growth cones via micropipettes. In substratum choice assays, retinal axons tended to avoid EphB-ECDs, while time-lapse microscopy showed that exposure to soluble EphB-ECD led to growth cone collapse or other inhibitory responses. These results demonstrate that, in addition to the conventional role of Eph proteins signaling as receptors, EphB receptor ECDs can also function in the opposite role as guidance cues to alter axon behavior. Furthermore, the data support a model in which dorsal retinal ganglion cell axons heading to the optic disc encounter a gradient of inhibitory EphB proteins which helps maintain tight axon fasciculation and prevents aberrant axon growth into ventral retina. In conclusion, development of neuronal connectivity may involve the combined activity of Eph proteins serving as guidance receptors and as axon guidance cues.
... The retinotectal projection serves as a model system for understanding the development of topographic projections, which are numerous in the nervous system. The Eph family plays a prominent role in the development of this projection (for review, see [26,27]) and has been investigated in several species, from Drosophila, Xenopus, marsupials, chick, and zebrafish to mouse and rat. To obtain an "orderly cytochemical mapping", R Sperry and others postulated the existence of two or more gradients with their axes roughly perpendicular [28,29]. ...
... EphA receptors are expressed more highly in temporal retina and anterior tectum, while ephrin-As are expressed more highly in nasal retina and posterior tectum (see main text for further details). The differential expression patterns of the EphB family are not indicated here [26]. On the basis of repulsive interactions, axons with a high Eph receptor concentration project to regions of the tectum with low ephrin concentration and axons with a low Eph concentration project to regions of the tectum with high ephrin concentration (for review, see [47,48]). ...
Eph receptor tyrosine kinases and ephrins have been identified in organisms ranging from sponges to flies and worms to chick, mice and humans, thus allowing their function to be approached also from an evolutionary perspective. The structural analysis of Eph/ephrin crystals is providing hints for the existence of Eph and ephrin folds in plants and suggests a mechanism for triggering bi-directional signalling.
... Finally, we asked whether Rac1 plays a role in development of the topography of the retinotectal projection in vivo. Development of normal topography is thought to depend on the response of growing retinal axons to ephrins expressed by tectal cells (Flanagan and Vanderhaeghen, 1998; O'Leary et al., 1999; Wilkinson, 2000). We found previously that injection into the developing eye of an antisense oligonucleotide targeting EphA3, a receptor for ephrin-A2, altered the topography of the retinotectal projection (W. ...
... Thus, Rac1-mediated endocytosis may be required for ephrin-or semaphorin-induced growth cone collapse, but endocytosis does not appear to be essential or sufficient to induce collapse. Previous work established that ephrin signaling via Eph receptors is involved in development of the normal pattern of axonal projections in the primary visual system (Flanagan and Vanderhaeghen, 1998; O'Leary et al., 1999; Wilkinson, 2000; Yates et al., 2001). We reasoned that if Rac1 is a required effector of ephrin signaling in vivo, as indicated by the in vitro data, then reducing Rac1 expression should mimic the effect of reduced EphA3 expression on development of the retinal projection. ...
Negative guidance molecules are important for guiding the growth of axons and ultimately for determining the wiring pattern in the developing nervous system. In tissue culture, growth cones at the tips of growing axons collapse in response to negative guidance molecules, such as ephrin-A2 and semaphorin 3A. The small GTPase Rac1 is involved in growth cone collapse, but the nature of its role is not clear. Rac1 activity assays showed that Rac1 is transiently inactivated after treatment with ephrin-A2. Ephrin-induced growth cone collapse, however, correlated with resumption of Rac1 activity. We demonstrate that Rac1 is required for endocytosis of the growth cone plasma membrane and reorganization of F-actin but not for the depolymerization of F-actin during growth cone collapse in response to ephrin-A2 and semaphorin 3A. Rac1, however, does not regulate constitutive endocytosis in growth cones. These findings show that in response to negative guidance molecules, the function of Rac1 changes from promoting actin polymerization associated with axon growth to driving endocytosis of the plasma membrane, resulting in growth cone collapse. Furthermore, Rac1 antisense injected into the embryonic chick eye in vivo caused the retinotectal projection to develop without normal topography in a manner consistent with Rac1 having an obligatory role in mediating ephrin signaling.
... Anterior–posterior (A–P) topographic targeting of retinal axons is controlled by a graded distribution of EphAs and ephrinAs in retina and midbrain, respectively (for review , see Flanagan and Vanderhaeghen 1998; O'Leary and Wilkinson 1999; Wilkinson 2000; Klein 2001; McLaughlin et al. 2003a ). Axons expressing EphA receptors at high density project to the anterior midbrain SC with low expression of ephrinA2/A5 repellents, whereas axons with low EphA density project to more posterior regions of the SC, where they encounter higher levels of ephrinA repellents. ...
... O'Leary and coworkers (Yates et al. 2001) recently proposed models in which the principle role of ephrinAs in chick retinotectal mapping is to inhibit branch formation along the overshooting segment of RGC axons posterior to their termination zone. In agreement with previous models, it was proposed that the ephrinA repellent gradient cooperates with other gradients, either a countergradient of another repellent or a parallel gradient of an attractant (for review, see Flanagan and Vanderhaeghen 1998; O'Leary and Wilkinson 1999; Wilkinson 2000; McLaughlin et al. 2003a). O'Leary and coworkers (Yates et al. 2001) modified the model and suggested the presence of either a countergradient of another branch repellent or a parallel gradient of a branch promoting factor. ...
... For instance, ephrinA5 and ephrinA2 are expressed in an increasing anterior to posterior gradient in the developing tectum (Drescher et al, 1995;Monschau et al, 1997). Such gradients are known to play a critical role in the precise positioning of cells, such as occurs during the establishment of retinotectal connections (reviewed in Wilkinson, 2000b; see also Section 1.1.7.1). ...
Members of the Erythropoietin-producing hepatoma (Eph) family of receptor tyrosine kinases are dynamically expressed during embryogenesis, with roles in topographic mapping, hindbrain segmentation and neural crest cell migration thought to be mediated by repulsive/anti-adhesive cues induced by interaction with their membrane-bound ligands, known as ephrins. Formerly known as epithelial cell kinase (eck), EphA2 is expressed primarily in cells of epithelial origin in the adult, including the mammary gland. Results presented in this thesis demonstrate that elevated steady-state levels of catalytically active EphA2 protein were characteristic of mammary tumours arising in WAPras transgenic mice. These mice express v-Ha-Ras under the control of the mammary-specific whey acidic protein (WAP) promoter. Furthermore, a mouse mammary epithelial cell line expressing v-Ha- Ras also displayed elevated levels of EphA2 mRNA and protein. This implies a potential role for EphA2 in tumourigenesis, and provided a highly relevant in vitro cell culture model in which further investigation revealed downregulation of EphA2 in response to mitogen-activated protein kinase (MAPK) pathway inhibition. Analysis of mice bearing an insertional mutation at the EphA2 locus crossed with WAPras transgenic mice revealed continued upregulation of EphA2 protein in mammary tumours, in contrast to the significant deficiency in EphA2 protein in other adult tissues of these double mutant mice. This suggests a potentially complex mechanism for the transcriptional regulation of EphA2. Overexpression of recombinant EphA2 in a non-transformed mammary epithelial cell line resulted in a 'rounded' cellular morphology, implicating EphA2 in the regulation of cell-extracellular matrix interactions. Furthermore, recombinant ephrinA1, cloned and expressed as a source of cognate ligand for EphA2, also influenced the cytoskeleton. Finally, receptor activation by ephrinA1 induced transient inhibition of MAPK activity, suggesting the presence of an EphA2-MAPK regulatory feedback loop, which may be overcome by oncogenic Ras to upregulate EphA2 and thus confer anti-adhesive properties on tumourigenic cells.
... Colognato and Krushel (2000); ll. Brose and Tessier-Lavigne (2000); 12. Niclou et al. (2000); 13. Wilkinson (2000); 14. ; 15.Kn6ll and Drescher (2002); 16. Plump et al. (2002); 17. ...
... Foxg1 activity leads to high levels of ephrin-A5 in the nasal retina which ultimately results in anterior retinal ganglion cell axons crossing the optic chiasm, whereas high levels of ephrin-A3 induced by Foxd1 activity leads to temporal axons remaining as ipsilateral projections. The molecular mechanism of opposing gradients of Eph receptors and their Ephrin ligands that guides retinal ganglion cell axons can be found in several excellent reviews (Wilkinson, 2000;Torborg and Feller, 2005;Scicolone et al., 2009). ...
... It is generally believed that mechanisms and molecules, which act in axon guidance during development, are reactivated and re-used in the adult after lesioning axon tracts (Aubert et al., 1995). Based on this assumption, we have chosen the well-characterized retinotectal projection (Flanagan and Vanderhaeghen, 1998;O'Leary et al., 1999;Wilkinson, 2000) as a`model' system to study the reestablishment of proper axonal connections. ...
The idea has been put forward that molecules and mechanisms acting during development are re-used during regeneration in the adult, for example in response to traumatic injury in order to re-establish the functional integrity of neuronal circuits. Members of the Eph family of receptor tyrosine kinases and their ‘ligands’, the ephrins, play a prominent role during development of the retinocollicular projection in rodents, where EphA receptors and ephrin-As are expressed in gradients in both the retina and the superior colliculi (SC). We were interested in investigating whether EphA family members are also expressed or re-expressed in the adult after optic nerve lesion, since the presence of axon guidance information is an important prerequisite for a topographically appropriate re-connection by retinal ganglion cell (RGC) axons. This analysis was encouraged by results showing that RGC axons do not exert guidance preferences in response to membranes from adult unlesioned SC, but in response to membranes from the adult deafferented SC. We found a graded expression pattern of ephrin-As in the SC both before and after deafferentation, which was remarkably similar to those found during development. EphA receptor levels were reduced in the SC after deafferentation and the expression patterns of the EphB family were not changed. In particular, the presence of a graded ephrin-A expression in the deafferented SC suggests that – if robust regeneration of RGC axons can be achieved – topographic guidance information as a likely requirement for a functionally successful re-establishment of the retinocollicular projection is available.
... For example, increased sprouting of intact primary somatosensory axonal arbors occurs after denervation of neighboring inputs in immature and mature systems (Chiaia et al., 1993; Renehan et al., 1994); these changes do not appear to be activity dependent (Henderson et al., 1992; Jacquin et al., 1993). In contrast, work in the visual system showed that the age-related refinement of retinal ganglion cell arbors is dependent both on activity-dependent (Sretavan and Shatz, 1986; Karmarkar and Dan, 2006) and activity-independent mechanisms (Wilkinson, 2000; Gosse et al., 2008). Although not tested directly here, the density results are consistent with outcomes that would be attributable to competition. ...
Neural competition among multiple inputs can affect the refinement and maintenance of terminal fields in sensory systems. In the rat gustatory system, the chorda tympani, greater superficial petrosal, and glossopharyngeal nerves have distinct but overlapping terminal fields in the first central relay, the nucleus of the solitary tract. This overlap is largest at early postnatal ages followed by a significant refinement and pruning of the fields over a 3 week period, suggesting that competitive mechanisms underlie the pruning. Here, we manipulated the putative competitive interactions among the three nerves by sectioning the greater superficial petrosal and glossopharyngeal nerves at postnatal day 15 (P15), P25, or at adulthood, while leaving the chorda tympani nerve intact. The terminal field of the chorda tympani nerve was assessed 35 d following nerve sections, a period before the sectioned nerves functionally regenerated. Regardless of the age when the nerves were cut, the chorda tympani nerve terminal field expanded to a volume four times larger than sham controls. Terminal field density measurements revealed that the expanded terminal field was similar to P15 control rats. Thus, it appears that the chorda tympani nerve terminal field defaults to its early postnatal field size and shape when the nerves with overlapping fields are cut, and this anatomical plasticity is retained into adulthood. These findings not only demonstrate the dramatic and lifelong plasticity in the central gustatory system, but also suggest that corresponding changes in functional and taste-related behaviors will accompany injury-induced changes in brainstem circuits.
... In general, Eph-ephrin interactions trigger cell-cell repulsion (Flanagan and Vanderhaeghen, 1998;Wilkinson, 2000Wilkinson, , 2001Pasquale, 2005). This effect is typically observed in the retina T-N axis /SC A-P axis topographic mapping system. ...
Endocytosis of Eph-ephrin complexes may be an important mechanism for converting cell-cell adhesion to a repulsive interaction. Here, we show that an endocytosis-defective EphA8 mutant forms a complex with EphAs and blocks their endocytosis in cultured cells. Further, we used bacterial artificial chromosome transgenic (Tg) mice to recapitulate the anterior>posterior gradient of EphA in the superior colliculus (SC). In mice expressing the endocytosis-defective EphA8 mutant, the nasal axons were aberrantly shifted to the anterior SC. In contrast, in Tg mice expressing wild-type EphA8, the nasal axons were shifted to the posterior SC, as predicted for the enhanced repellent effect of ephrinA reverse signalling. Importantly, Rac signalling was shown to be essential for EphA-ephrinA internalization and the subsequent nasal axonal repulsion in the SC. These results indicate that endocytosis of the Eph-ephrin complex is a key mechanism by which axonal repulsion is generated for proper guidance and topographic mapping.
... 18,19 Eph/ephrin interactions are often repulsive, preventing growing axons or migrating cells from entering inappropriate areas (reviewed in refs. [20][21][22]. However, there is also evidence for attractive effects of the Eph/ephrin system on neuronal cells. ...
Cortical interneurons are born in the proliferative zones of the ganglionic eminences in the subpallium and migrate to the developing cortex along well-defined tangential routes. The mechanisms regulating interneuron migration are not completely understood. Here we examine the role of class-A members of the Eph/ephrin system in directing the migration of interneurons. In situ hybridizations demonstrated that ephrin-A3 is expressed in the developing striatum, an area that is strictly avoided by migrating cortical interneurons in vivo, which express the EphA4 receptor. We then examined interneuron migration in grafting experiments, where explants of the medial ganglionic eminence (MGE) from enhanced green fluorescent protein-expressing transgenic mice were homotopically grafted into host slices from wildtype littermate embryos. After blocking ephrin-A ligands, many interneurons invaded the striatal anlage. Moreover, stripe assay experiments revealed that ephrin-A3 acts as a repellent cue for neurons from the medial ganglionic eminence. Downregulation of the EphA4 receptor via siRNA transfection reduced the repulsive effect of ephrin-A3, indicating that EphA4 mediates at least in part the repulsive effect of ephrin-A3 on these cells. Together, these results suggest that ephrin-A3 acts as a repulsive cue that restricts cortical interneurons from entering inappropriate regions and thus contributes to define the migratory route of cortical interneurons.
... As a result, models of this generation generally have not replicated the systemsbased results of the previous generation. Indeed certain features of the newer molecular-based experiments remain unexplained by these recent models as well, so that incorporating features from earlier models may be of benefit (e.g., see Goodhill, 2000; Wilkinson, 2000). ...
Proper wiring up of the nervous system is critical to the development of organisms capable of complex and adaptable behaviors. Besides the many experimental advances in determining the cellular and molecular machinery that carries out this remarkable task precisely and robustly, theoretical approaches have also proven to be useful tools in analyzing this machinery. A quantitative understanding of these processes can allow us to make predictions, test hypotheses, and appraise established concepts in a new light. Three areas that have been fruitful in this regard are axon guidance, retinotectal mapping, and activity-dependent development. This chapter reviews some of the contributions made by mathematical modeling in these areas, illustrated by important examples of models in each section. For axon guidance, we discuss models of how growth cones respond to their environment, and how this environment can place constraints on growth cone behavior. Retinotectal mapping looks at computational models for how topography can be generated in populations of neurons based on molecular gradients and other mechanisms such as competition. In activity-dependent development, we discuss theoretical approaches largely based on Hebbian synaptic plasticity rules, and how they can generate maps in the visual cortex very similar to those seen in vivo. We show how theoretical approaches have substantially contributed to the advancement of developmental neuroscience, and discuss future directions for mathematical modeling in the field.
... Among these, the Eph family of receptor tyrosine kinases and their membrane bound ligands, the ephrins, has recently been shown to fulfill the functions of the cytochemical tags proposed by Sperry. Eph family receptors and their ligands are widely expressed during embryonic development and play important roles in cell-cell recognition events (recent reviews: Flanagan and Vanderhaeghen, 1998; Frisen et al., 1999; Wilkinson, 2000). During development of the visual system, ephrin A2 and ephrin A5 are expressed in nested caudal to rostral gradients in the optic tectum (Cheng et al., 1995; Drescher et al., 1995; Monschau et al., 1997). ...
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.
... A characteristic of the ephrin–Eph signaling system is the ability to elicit bidirectional signaling, that is classical forward signaling by the Eph receptor via its intrinsic tyrosine kinase activity and reverse signaling by the transmembrane ephrinB ligand via its cytoplasmic domain (reviewed in567 ; see also Update). Ephrin functions in axon guidance , topographic map formation and angiogenesis have been covered by recent reviews [1,3,891011. Here, I will review work on ephrins published over the course of the past year in the established fields of axon guidance and topographic map formation, and in new areas such as synaptogenesis and neural stem cell biology. ...
Ephrins are cell surface associated ligands for Eph receptor tyrosine kinases and are implicated in repulsive axon guidance, cell migration, topographic mapping and angiogenesis. During the past year, Eph receptors have been shown to associate with glutamate receptors in excitatory neurons, suggesting a role in synapse formation or function. Moreover, ephrin/Eph signaling appears to regulate neural stem cell proliferation and migration in adult mouse brains. The mode of action of ephrin/Ephs has been expanded from repulsion to adhesion and from cell surface attachment to regulated cleavage.
... The members of the engrailed gene family are thought to be involved in the formation of tectal polarity (Itasaki et al., 1991; Itasaki and Nakamura, 1992). In chick embryos, ectopic expression of en-2 induces anterior tectum to express several characteristics of the posterior tectum; arborization of axon termini from the nasal retinal ganglion cells (RGCs; Itasaki and Nakamura, 1996), enhanced mitotic activity as compared to the cells in the normal anterior tectum (Lee et al., 1997), and strong expression of ephrin-A2 and ephrin-A5 (Logan et al., 1996; Shigetani et al., 1997), which are both expressed in the posterior tectum with a gradient that decreases rostrally (Cheng and Flanagan, 1994; Drescher et al., 1995), and play crucial roles as repulsive guidance molecules for the temporal RGC axons in the retinotectal topographic projection (reviewed by Holder and Klein, 1999; O'Leary and Wilkinson, 1999; Mellitzer et al., 2000; Wilkinson, 2000). Zebra®sh Islet-3 is a LIM (Lin-11, Islet-1, and Mec-3) homeodomain-containing transcription factor which is expressed in the central nervous system, especially in the eyes and the tectum (Tokumoto et al., 1995; Kikuchi et al., 1997). ...
Development of the tectum and the cerebellum is induced by a reciprocal inductive signaling between their respective primordia, the midbrain and the midbrain/hindbrain boundary (MHB). We set out to identify molecules that function in and downstream of this reciprocal signaling. Overexpression of LIM domain of the transcription factor Islet-3 (LIM(Isl-3)) leads to inhibition of this reciprocal signaling and to resultant defects in tectal and cerebellar development. We therefore searched for genes that may be either up- or down-regulated by overexpression of LIM(Isl-3) by comparing the gene expression profiles in the midbrain and the MHB of normal embryos and embryos in which Islet-3 function was repressed, using a combination of ordered differential display and whole-mount in situ hybridization. Among genes identified in this search, two cDNA fragments encoded Wnt1 and FGF8, which are already known to be essential for the reciprocal signaling between the midbrain and the MHB, confirming the effectiveness of our strategy. We identified four other partial cDNA clones that were specifically expressed around the MHB, ten cDNAs specifically expressed in the tectum, and three cDNAs expressed in neural crest cells including those derived from the midbrain level. The ephrin-A3 gene was specifically expressed in posterior tectum in a gradient that decreased anteriorly. Although ephrin-A2 and ephrin-A5 have been reported to be expressed in the corresponding region in mouse embryos, the superior/inferior colliculi, mouse ephrin-A3 is not expressed prominently in this region, suggesting that the role of ephrin-A3 in brain development may have been altered in the process of brain evolution.
... For example, a number of cadherins and ephrins and their receptors are differentially expressed in the developing cortex (Suzuki et al., 1997;Nakagawa et al., 1999;Gao et al., 1998;Donoghue and Rakic, 1999;Mackarehtschian et al., 1999;Sestan et al., 2001). Ephrin signaling is involved in the establishment of other connectivity maps, such as the retinotectal map (reviewed in Flanagan and Vanderhaeghen, 1998;Wilkinson, 2000) and it is therefore possible that it is involved in the establishment of the thalamocortical map. If so, this raises the question of how any early molecular regionalization may or may not be related to later functional arealization. ...
Understanding the development of the vertebrate brain and in particular that of the neocortex, where high brain functions reside, remains one of the most difficult and exciting tasks in biology. In this review, we discuss recent experimental evidence as well as different possibilities for the intrinsic regionalization of the embryonic dorsal telencephalon, which may be related to the formation of distinct functional areas in the adult neocortex.
... The role of the Eph family in the establishment of neuronal connections has been studied in a number of different model systems, in particular the formation of the retinotectal projection. It is now believed that this topographically organised projection is set up to a large extent on the basis of complementary gradients of Eph receptors and ephrins in both the retina and the tectum, involving repulsive but also attractive interactions between Eph receptors and ephrins (reviewed by Knöll and Drescher, 2002;Wilkinson, 2000). In ephrin-A mutant mice, mapping across the anteroposterior axis is affected (Feldheim et al., 1998;Feldheim et al., 2000;Frisen et al., 1998), while recent data from in vivo analyses showed an important role of EphB family members in mapping across the mediolateral axis Mann et al., 2002). ...
The Eph family of receptor tyrosine kinases and their ligands, the ephrins,play important roles during development of the nervous system. Frequently they exert their functions through a repellent mechanism, so that, for example, an axon expressing an Eph receptor does not invade a territory in which an ephrin is expressed. Eph receptor activation requires membrane-associated ligands. This feature discriminates ephrins from other molecules sculpturing the nervous system such as netrins, slits and class 3 semaphorins, which are secreted molecules. While the ability of secreted molecules to guide axons,i.e. to change their growth direction, is well established in vitro, little is known about this for the membrane-bound ephrins. Here we set out to investigate – using Xenopus laevis retinal axons – the properties of substratum-bound and (artificially) soluble forms of ephrin-A5(ephrin-A5-Fc) to guide axons.
We find – as expected on the basis of chick experiments – that,when immobilised in the stripe assay, ephrin-A5 has a repellent effect such that retinal axons avoid ephrin-A5-Fc-containing lanes. Also, retinal axons react with repulsive turning or growth cone collapse when confronted with ephrin-A5-Fc bound to beads. However, when added in soluble form to the medium, ephrin-A5 induces growth cone collapse, comparable to data from chick.
The analysis of growth cone behaviour in a gradient of soluble ephrin-A5 in the `turning assay' revealed a substratum-dependent reaction ofXenopus retinal axons. On fibronectin, we observed a repulsive response, with the turning of growth cones away from higher concentrations of ephrin-A5. On laminin, retinal axons turned towards higher concentrations,indicating an attractive effect. In both cases the turning response occurred at a high background level of growth cone collapse. In sum, our data indicate that ephrin-As are able to guide axons in immobilised bound form as well as in the form of soluble molecules. To what degree this type of guidance is relevant for the in vivo situation remains to be shown.
... Furthermore, ephrins may downregulate functional receptors in the nasal retina (Hornberger et al., 1999). On the other hand, ephrins are considered to reduce the sensitivity of Eph receptors (desensitization model; Wilkinson, 2000). Therefore, it was assumed that the functional graded receptor density is determined by an overlap of the graded ligand density and the receptor density. ...
Topographic mapping of retinal ganglion axons to the midbrain is computed by the servomechanism model, which is based on the experimental result of cell attachment. Cells expressing a certain level of Eph proteins (receptors for ephrin ligands) optimally attach to a surface that expresses a specific level of ephrin ligand density. The retina has an increasing nasal-to-temporal gradient of Eph receptor density, and the optic tectum/superior colliculus has an increasing 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. Quantitatively, a retinal axon has a receptor density (R) that is determined by its retinal position, and the axon terminal is induced to adhere to the tectal site of ligand density (L = S/R), where S is a constant. Consequently, the servomechanism model defines positions of axon terminals on the midbrain. Abnormal topographic maps are reported in a knock-in experiment with elevated density of Eph receptors and a knock-out experiment lacking ephrin ligands using gene-targeting technology. By adding competition between axon terminals for target sites to the servomechanism model, the abnormal maps became easy to understand. Furthermore, the servomechanism-competition model allowed conjecture of the gradient shapes of receptor and ligand densities and estimation of the capacity of the midbrain surface to accept retinal axon terminals.
... The roles of the Eph family of receptor tyrosine kinases and their membrane-bound ephrin ligands in guiding axons to their distant targets have been studied intensively, for example in topographically organized sensory systems such as the retinotectal projection Flanagan and Vanderhaeghen, 1998;Wilkinson, 2000;Knöll and Drescher, 2002;Kullander and Klein, 2002;McLaughlin et al., 2003). Activation of EphA receptors triggers a repulsive response, ultimately leading to a turning or a collapse of the growth cone (Weinl et al., 2003). ...
EphA receptor tyrosine kinases and their ephrin ligands play important roles in wiring of the developing nervous system. We have investigated here the function of Src family kinases (SFKs) in the retinotectal projection to dissect the signaling pathways by which EphA receptors control actin/microtubule rearrangements that underlie growth cone guidance and collapse. Both EphAs and SFKs are expressed broadly in retinal growth cones, and SFKs are recruited to EphA receptors after ephrinA stimulation. In the stripe and growth cone collapse assays we observe an abolition of EphA-mediated repulsion after inhibiting SFKs, either pharmacologically or enzymatically via electroporation-mediated overexpression of the SFK inhibitor Csk. In addition, we identify cortactin and the RhoGEF ephexin, which interact with EphA receptors in retinal axons, as targets of SFK-dependent tyrosine phosphorylation. In sum, our data suggest an important role of SFKs as downstream signaling molecules in EphA receptor-mediated repulsive axon guidance.
... ephrins and EphA receptors are required for anterior– posterior (A–P) topographic mapping [26,27], whereas B-type ephrins and EphB receptors are required for dorsal–ventral (D–V) mapping along the lateral–medial (L–M) axis of the midbrain target area [28] . Mechanistically , A–P mapping appears to involve repulsive interactions , including reverse signaling by ephrinAs [29]. ...
Ephrins are cell-surface-tethered ligands for Eph receptors, the largest family of receptor tyrosine kinases. During development, the Eph/ephrin cell communication system appears to influence cell behavior such as attraction/repulsion, adhesion/de-adhesion and migration, thereby influencing cell fate, morphogenesis and organogenesis. During adulthood, the Eph/ephrin system continues to play roles in tissue plasticity, for example in shaping dendritic spines during neuronal plasticity. Mechanistically, Eph-ephrin repulsive behavior appears to require ligand-receptor internalization and signaling to Rho GTPases.
... In rat ON explants, Sema5A, expressed by ON glial cells (oligodendrocytes in particular), induces collapse of RGC growth cones, inhibiting (repelling) axon re-growth: if this effect is blocked, axon growth is significantly increased. 44 On a wider and more sophisticated scale, gradients of inhibitory or repellent molecules, for instance, ephrins, 45 guide RGC axons to generate a retinotopic map in the colliculus or the lateral geniculate body. ...
In the 2004 Bowman Lecture, I give a panegyric for Sir William Bowman, an estimate of the importance and the epidemiology of anterior visual pathway developmental disorders, followed by a history of the anterior visual system. I review the normal development of the optic nerve and chiasm and the main developmental disorders: Optic Nerve Aplasia, Optic Nerve Hypoplasia and Achiasmia.
Two issues were examined regarding the trigeminal system in larval lampreys: (1) for normal animals, double labeling was used to confirm that the trigeminal system has a topological organization; (2) following trigeminal nerve root transections, double labeling was used to test whether the topological organization of the trigeminal system is restored. First, for normal animals, Alexa 488 dextran amine applied to the medial oral hood (anterior head) labeled trigeminal motoneurons (MNs) in the ventromedial part of the trigeminal motor nuclei (nVm) and axons of trigeminal sensory neurons (SNs) in the ventromedial part of the trigeminal descending tracts (dV). Also, Texas red dextran amine (TRDA) applied to the lateral oral hood labeled trigeminal MNs in the dorsolateral nVm and sensory axons in the dorsolateral dV. These results confirm the topological organization of the trigeminal system of normal lampreys. Second, following trigeminal nerve root transections, the physical integrity of the nerves was restored during growth of trigeminal sensory and motor axons. In addition, double labeling indicated a restoration and refinement of the topological organization of the trigeminal system with increasing recovery times, but mainly for nVm. Despite the paucity of growth of trigeminal sensory axons in dV even at long recovery times (12-16 wks), a substantial percentage of experimental animals recovered trigeminal-evoked swimming responses and trigeminal-evoked synaptic responses in reticulospinal (RS) neurons. Following trigeminal nerve root injury, several mechanisms, including axonal guidance cues, probably contribute to the substantial restoration of the topological organization of the lamprey trigeminal system.
The correct functioning of the nervous system, even from early embrio stages, requires that neurons establish
specific connections with other neurons or tissues. Since a neuronal network consists of an enormous number
neurons, finding the correct pathway for each one of them clearly constitutes a problem of morphogenesis. In
this article we study experimentally neurite growth using various culture media, and construct a theoretical model to explain
the experimental results. Numerical simulations reproduce the observations and allow to predict the existence and
functioning of chemicals which are important in neurite growth and guidance to attain connections.
The correct functioning of the nervous system, even from early embrio stages, requires that neurons establish specific connections with other neurons or tissues. Since a neuronal network consists of an enormous number neurons, finding the correct pathway for each one of them clearly constitutes a problem of morphogenesis. In this article we study experimentally neurite growth using various culture media, and construct a theoretical model to explain the experimental results. Numerical simulations reproduce the observations and allow to predict the existence and functioning of chemicals which are important in neurite growth and guidance to attain connections.
Eph receptors play pivotal roles in the axon guidance of retinal ganglion cells (RGCs) at the optic chiasm and the establishment of the topographic retinocollicular map. We previously demonstrated that protein tyrosine phosphatase receptor type O (PTPRO) is specifically involved in the control of retinotectal projections in chicks through the dephosphorylation of EphA and EphB receptors. We subsequently revealed that all the mouse R3 subfamily members (PTPRB, PTPRH, PTPRJ, and PTPRO) of the receptor protein tyrosine phosphatase (RPTP) family inhibited Eph receptors as their substrates in cultured mammalian cells. We herein investigated the functional roles of R3 RPTPs in the projection of mouse retinal axon of both sexes. Ptpro and Ptprj were expressed in mouse RGCs; however, Ptprj expression levels were markedly higher than those of Ptpro. Consistent with their expression levels, Eph receptor activity was significantly enhanced in Ptprj-knock-out (Ptprj-KO) retinas. In Ptprj-KO and Ptprj/Ptpro-double-KO (DKO) mice, the number of retinal axons that projected ipsilaterally or to the contralateral eye was significantly increased. Furthermore, retinal axons in Ptprj-KO and DKO mice formed anteriorly shifted ectopic terminal zones in the superior colliculus (SC). We found that c-Abl (Abelson tyrosine kinase) was downstream of ephrin–Eph signaling for the repulsion of retinal axons at the optic chiasm and in the SC. c-Abl was identified as a novel substrate for PTPRJ and PTPRO, and the phosphorylation of c-Abl was upregulated in Ptprj-KO and DKO retinas. Thus, PTPRJ regulates retinocollicular projections in mice by controlling the activity of Eph and c-Abl kinases.
This chapter discusses the vasculogenesis and angiogenesis. Formation of the vascular system is recognized as one of the most important events in development and has been a subject of intensive investigations. This chapter discusses many of the key principles of vascular development with a primary emphasis on the studies accomplished by the use of contemporary mouse genetics and embryological tools. Modem analyses of vascular development date back to the beginning of the 20th century; the primary focus was to provide morphological description of the process. Two sequential key morphogenic processes underlie vascular development: vasculogenesis and angiogenesis. Clusters of mesoderm-derived angioblastic cells differentiate to blood and endothelial cells, to form a structure referred to as a blood island within which clusters of blood cells are surrounded by a single layer of endothelial cells, which subsequently coalesce to form a number of initial vascular channels called the primary capillary plexus. This process, the formation of the primary capillary plexus, is referred to as vasculogenesis. The term angiogenesis is used to cover the entire vessel or the capillary plexus formation process following vasculogenesis including pruning, remodeling, and maturation. Recently, significant advancement in understanding of molecular mechanisms for these processes, as well as the emergence of new concepts, has been revolutionizing the field. Application of many mouse genetic tools plays a pivotal role in this recent revolution of the field.
Objective— The transmembrane ligand ephrin-B2 and its receptor tyrosine kinase EphB4 are specifically expressed on arterial and venous endothelial cells, respectively, and bidirectional signals mediated by both proteins play an important role in vascular development. However, how such bidirectional signals are required for cell-cell adhesion or repulsion remains unclear.
Methods and Results— Using a cell line and sorted primary endothelial cells, we show that ephrin-B2 forward signaling through the EphB4 receptor inhibits cell adhesion, whereas EphB4 reverse signaling by the transmembrane ephrin-B2 ligand does not. Cell migration is also inhibited on immobilized ephrin-B2-Fc but not on EphB4-Fc protein.
Conclusions— Ephrin-B2 forward signaling and EphB4 reverse signaling differentially affect cell adhesion and migration between arterial and venous endothelial cells.
Intense research over the past 15 years has demonstrated Eph receptors and their cell surface ephrin ligands to be one of the most prevalent and complex cell-cell communication systems; this system guides cell positioning and orchestrates tissue patterning in multicellular organisms by coordinating synchronised cell-cell adhesion or segregation of interacting cells. The expression of many Eph and ephrin family members together with their embryonic patterning functions often re-emerge during oncogenesis and have generated considerable interest as targets for anticancer therapies. The first generation of monoclonal antibodies, kinase inhibitors, and vaccines suggests considerable promise in preclinical and early clinical development, but there is little doubt that successful clinical use will rely on a comprehensive understanding of the complex and sometimes puzzling activities of Eph receptors during tumor progression.
Recent studies indicate that endocytosis of Eph-ephrin complexes may be one of the mechanisms by which a high affinity cell-cell adhesion is converted to a repulsive interaction. In this study, we show that EphA8 undergoes clathrin-mediated endocytosis upon treatment with ephrin-A5, and that EphA8 is associated tightly with Tiam-1, a Rac-specific guanine nucleotide exchange factor. Analysis of EphA8 deletion mutants revealed that a juxtamembrane region in EphA8 is critically involved in endocytosis of EphA8-ephrinA5 complexes. An EphA8 mutant lacking this juxtamembrane portion was defective for endocytosis with ephrinA5, and also displayed a weak association with Tiam-1. Expression of an endocytosis-defective version of EphA8 resulted in a low level of Rac activity in response to ephrin-A5 stimulation. More importantly, down-regulation of Tiam-1 resulted in inefficient endocytosis of EphA8-ephrinA5 complexes. These results suggest that Tiam-1 plays a role in clathrin-dependent endocytosis of EphA8-ephrinA5 complexes.
Thesis (Ph. D.)--University of Miami, 2006. Includes bibliographical references (leaves 127-146).
Die Proteine der EphrinB Familie spielen eine wichtige Rolle bei der Angiogenese und der Führung von Axonen und der Vasikulation. In der vorliegenden Arbeit konnte EphrinB mit Hilfe des Hefe-Zwei-Hybrid-Systems und durch Kopräzipitationsexperimente als Interaktionspartner der PTP-BL identifiziert werden. Die Interaktion erfolgt dabei über die PDZ4 Domäne der PTP-BL und den C-Terminus von EphrinB1. Eine Koexpression und Koregulation der beiden Proteine konnte im peripheren Nervensystem des embryonalen Huhnes mittels Western-Blot-Analyse nachgewiesen werden. Auf subzellulärer Ebene gelang es durch immuncytochemische Untersuchungen an kultivierten Hinterwurzelganglien-Neuronen eine Kolokalisation der beiden Proteine in den Wachstumskegeln der Neuriten zu zeigen. In Kotransfektionsexperimenten konnte gezeigt werden, dass durch Expression einer katalytisch aktiven, nicht aber einer katalytisch inaktiven Form der PTP-BL die ligandeninduzierte Phosphorylierung von EphrinB1 stark inhibiert wurde.
Cellular and molecular mechanisms involved in the development of topographic ordered connections in the central nervous system (CNS) constitute a key issue in neurobiology because neural connectivities are the base of the CNS normal function. We discuss the roles of the Eph/ephrin system in the establishment of retinotopic projections onto the tectum/colliculus, the most detailed studied model of topographic mapping. The expression patterns of Ephs and ephrins in opposing gradients both in the retina and the tectum/colliculus, label the local addresses on the target and give specific sensitivities to growth cones according to their topographic origin in the retina. We postulate that the highest levels of these gradients could signal both the entry as well as the limiting boundaries of the target. Since Ephs and ephrins are membrane-bound molecules, they may function as both receptors and ligands producing repulsive or attractant responses according to their microenvironment and play central roles in a variety of developmental events such as axon guidance, synapse formation and remodeling. Due to different experimental approaches and the inherent species-specific differences, some results appear contradictory and should be reanalyzed. Nevertheless, these studies about the roles of the Eph/ephrin system in retinotectal/collicular mapping support general principles in order to understand CNS development and could be useful to design regeneration therapies.
To investigate the relationship among mossy fiber axon sprouting(MFS), synaptic reorganization, and the alteration of expression of Eph A5 and ephrin A3 in the dentate gyrus in rats with pilocarpine-induced chronic temporal lobe epilepsy.
Mossy fiber sprouting and synaptic formation in rats were observed by Neo-Timm staining, after the acute status epilepticus and chronic spontaneous temporal lobe epilepsy induced by lithium-chloride and pilocarpine. In situ hybridization was used to detect ephrin A3 mRNA and an immunohistochemical staining was applied to determine Eph A5 protein.
In entorhinal cortex, only Eph A5 mRNA and protein expressed, which significantly decreased on Day 7 after pilocarpine induced status epilepticus(P<0.01),and resumed to normal levels on Day 30 (P>0.05). In the dentate granule cells, ephrin A3 mRNA reduced obviously on Day 7 after pilocarpine-induced status epilepticus (P<0.01), and returned to normal levels on Day 30 (P>0.05).
The down-regulation of Eph A5 mRNA and protein in entorhinal cortex and dentate gyrus, and ephrin A3 mRNA in dentate gyrus after status epilepticus may be part of the endogenous molecular mechanism of mossy fiber sprouting to the inner molecular layer of dentate gyrus.
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.
Signals regulating diversification of olfactory sensory neurons to express odorant receptors and other genes necessary for correct assembly of the olfactory sensory map persist in the olfactory epithelium of adult mouse. We have screened for genes with an expression pattern correlating with the topography odorant receptor-expression zones. The Msx1 homeobox gene and a semaphorin receptor (Neuropilin-2) showed graded expression patterns in the olfactory epithelium. The gradients of Msx1 and Neuropilin-2 expression in basal cells and neurons, respectively, correlated with expression of a retinoic acid-synthesizing enzyme (RALDH2) in lamina propria. A BMP-type I receptor (Alk6) showed a reverse gradient of expression in the supporting cells of the epithelium. Considering known functions of identified genes in cell specification and axon guidance this suggests that zonal division of the olfactory sensory map is maintained, during continuous neurogenesis, as a consequence of topographic counter gradients of positional information.
During development, Eph receptors mediate the repulsive axon guidance function of ephrins, a family of membrane attached ligands with their own receptor-like signaling potential. In cultured glutamatergic neurons, EphB2 receptors were recently shown to associate with NMDA receptors at synaptic sites and were suggested to play a role in synaptogenesis. Here we show that Eph receptor stimulation in cultured neurons modulates signaling pathways implicated in synaptic plasticity, suggesting cross-talk with NMDA receptor-activated pathways. Mice lacking EphB2 have normal hippocampal synapse morphology, but display defects in synaptic plasticity. In EphB2(-/-) hippocampal slices, protein synthesis-dependent long-term potentiation (LTP) was impaired, and two forms of synaptic depression were completely extinguished. Interestingly, targeted expression of a carboxy-terminally truncated form of EphB2 rescued the EphB2 null phenotype, indicating that EphB2 kinase signaling is not required for these EphB2-mediated functions.
The Eph family of receptor tyrosine kinases and their 'ligands', the ephrins, have been implicated in a large number of developmental processes, such as boundary formation, cell migration, axon guidance and vasculogenesis. A characteristic of the EphB subclass is that both EphBs and transmembrane-anchored ephrin-Bs function as receptors and as ligands, a phenomenon commonly described as 'bi-directional signalling'. Here we review recent data indicating that EphA receptors and glycosylphosphatidylinositol (GPI)-anchored ephrin-As can also mediate bi-directional signalling. Moreover, characterization of the expression of ephrin-As on axons of the retinotectal and vomeronasal projections suggests that the EphA subfamily is involved in both repulsive and attractive guidance mechanisms during establishment of neuronal connections.
Thymus development and function are dependent on the definition of different and graded microenvironments that provide the maturing T cell with the different signals that drive its maturation to a functional T lymphocyte. In these processes, cell-cell interactions, cell migration, and positioning are clues for the correct functioning of the organ. The Eph family of receptor tyrosine kinases and their ligands, the ephrins, has been implicated in all these processes by regulating cytoskeleton and adhesion functioning, but a systemic analysis of their presence and possible functional role in thymus has not yet been conducted. In this regard, the current study combines different experimental approaches for analyzing the expression of four members of the Eph A family and their ligands, ephrins A, in the embryonic and adult rat thymus. The patterns of Eph and ephrin expression in the distinct thymic regions were different but overlapping. In general, the studied Eph A were expressed on thymic epithelial cells, whereas ephrins A seem to be more restricted to thymocytes, although Eph A1 and ephrin A1 are expressed on both cell types. Furthermore, the supply of either Eph A-Fc or ephrin A-Fc fusion proteins to fetal thymus organ cultures interferes with T cell development, suggesting an important role for this family of proteins in the cell mechanisms that drive intrathymic T cell development.
Retinal axons project to their central targets along two orthogonal topographic axes, anterior-posterior (A-P) and dorsal-ventral (D-V). While ephrin-A/EphA signaling determines A-P topography, little has been known about the molecular mechanisms guiding axons along the D-V axis. Two papers by Mann et al. and Hindges et al. in this issue of Neuron provide evidence for both forward and reverse ephrin-B/EphB signaling in regulating D-V topography.
In the small intestine, the progeny of stem cells migrate in precise patterns. Absorptive, enteroendocrine, and goblet cells migrate toward the villus while Paneth cells occupy the bottom of the crypts. We show here that beta-catenin and TCF inversely control the expression of the EphB2/EphB3 receptors and their ligand ephrin-B1 in colorectal cancer and along the crypt-villus axis. Disruption of EphB2 and EphB3 genes reveals that their gene products restrict cell intermingling and allocate cell populations within the intestinal epithelium. In EphB2/EphB3 null mice, the proliferative and differentiated populations intermingle. In adult EphB3(-/-) mice, Paneth cells do not follow their downward migratory path, but scatter along crypt and villus. We conclude that in the intestinal epithelium beta-catenin and TCF couple proliferation and differentiation to the sorting of cell populations through the EphB/ephrin-B system.
The transmembrane ligand ephrin-B2 and its receptor tyrosine kinase EphB4 are specifically expressed on arterial and venous endothelial cells, respectively, and bidirectional signals mediated by both proteins play an important role in vascular development. However, how such bidirectional signals are required for cell-cell adhesion or repulsion remains unclear.
Using a cell line and sorted primary endothelial cells, we show that ephrin-B2 forward signaling through the EphB4 receptor inhibits cell adhesion, whereas EphB4 reverse signaling by the transmembrane ephrin-B2 ligand does not. Cell migration is also inhibited on immobilized ephrin-B2-Fc but not on EphB4-Fc protein.
Ephrin-B2 forward signaling and EphB4 reverse signaling differentially affect cell adhesion and migration between arterial and venous endothelial cells.
In the past several years, a great deal has been learnt about the molecular basis through which specific neural pathways in the visual system are established during embryonic development. This review provides a framework for understanding the principles of retinal ganglion cell axon guidance, and introduces some of the families of axon guidance molecules involved. In addition, the potential relevance of retinal axon guidance to human visual developmental disorders, and to retinal axon regeneration, is discussed.
The Eph family tyrosine kinase receptors and their ligands have been linked to axon guidance and topographic mapping of the developing central nervous system. More specifically, the EphA5 receptor has been shown to play a role in development of hippocamposeptal, retinotectal and thalamocortical projections. Recently, a line of transgenic mice was developed which expresses a truncated EphA5 receptor lacking a functional tyrosine kinase domain. In a previous study, axonal tracing revealed that medial hippocampal axons in this strain projected laterally and ventrally away from their normal target area. In the current study, both transgenic and wild-type controls were evaluated in unconditioned (rotorod and locomotor activity) and conditioned (water maze and active avoidance) behavior tasks which tested hippocampal and striatal functioning. Compared to controls, the transgenic strain did not show differences in rotorod motor activity but did show a transient deficit in spatial navigation ability and a consistent impairment in active avoidance. The dominant-negative mutant receptor also resulted in a decrease in striatal dopamine and serotonin concentrations with no change in hippocampal monoamines. Collectively, these data suggest that animals expressing a truncated EphA5 receptor show deficits related to striatal functioning.
Receptors of the Eph family and their ligands (ephrins) mediate developmental vascular assembly and direct axonal guidance. Migrating cell processes identify appropriate targets within migratory fields based on topographically displayed ephrin gradients. Here, EphB1 regulated cell attachment by discriminating the density at which ephrin-B1 was displayed on a reconstituted surface. EphB1–ephrin-B1 engagement did not promote cell attachment through mechanical tethering, but did activate integrin-mediated attachment. In endothelial cells, attachment to RGD peptides or fibrinogen was mediated through v3 integrin. EphB1 transfection conferred ephrin-B1-responsive activation of 51 integrin-mediated cell attachment in human embryonic kidney cells. Activation-competent but signaling-defective EphB1 point mutants failed to stimulate ephrin-B1 dependent attachment. These findings lead us to propose that EphB1 functions as a 'ligand density sensor' to signal integrin-mediated cell–matrix attachment.
Topographic maps with a defined spatial ordering of neuronal connections are a key feature of brain organization. Such maps are believed to develop in response to complementary position-specific labels in presynaptic and postsynaptic fields. However, the complementary labeling molecules are not known. In the well-studied visual map of retinal axons projecting to the tectum, the labels are hypothesized to be in gradients, without needing large numbers of cell-specific molecules. We recently cloned ELF-1 as a ligand for Eph family receptors. Here, RNA hybridization shows matching expression gradients for ELF-1 in the tectum and its receptor Mek4 in the retina. Binding activity detected with alkaline phosphatase fusions of ELF-1 and Mek4 also reveals gradients and provides direct evidence for molecular complementarity of gradients in reciprocal fields. ELF-1 and Mek4 may therefore play roles in retinotectal development and have properties predicted of topographic mapping labels.
The results of previous in vitro experiments indicate that a glycosylphosphatidylinositol (GPI)-anchored protein may play an important role in the guidance of temporal retinal axons during the formation of the topographically ordered retinotectal projection. We have purified and cloned a GPI-anchored, 25 kDa glycoprotein that is a good candidate for a molecule involved in this process. During the time of innervation by retinal ganglion cells, this protein is gradedly expressed in the posterior part of the developing tectum. In two different in vitro assay systems, the recombinant protein induces growth cone collapse and repulsion of retinal ganglion cell axons. These phenomena are observed for axons of temporal as well as nasal origin, indicating that an additional activity may be necessary to confer the nasotemporal specificity observed in previous assays. We named the protein RAGS (for repulsive axon guidance signal). The sequence of RAGS shows significant homology to recently identified ligands for receptor tyrosine kinases of the Eph subfamily.
Topographic maps, which maintain the spatial order of neurons in the order of their axonal connections, are found throughout the nervous system. In the visual retinotectal projection, ELF-1, a ligand in the tectum, and its receptors in the retina show complementary gradients in expression and binding, indicating they may be positional labels for map development. Here we show that ELF-1 acts as a repellent axon guidance factor in vitro. In vivo, when the tectal ELF-1 pattern is modified by retroviral overexpression, retinal axons avoid ectopic ELF-1 patches and map to abnormally anterior positions. All these effects were seen on axons from temporal but not nasal retina, indicating that ELF-1 could determine nasal versus temporal retinotectal specificity, and providing a direct demonstration of a cell recognition molecule with topographically specific effects on neural map development.
The Eph family of receptor tyrosine kinases and their ligands can be divided into two specificity subclasses: the Eck-related receptors and their GPI-anchored ligands, and the Elk-related receptors and their transmembrane ligands. Previous reports demonstrated that Eck- and Elk-related receptors in the retina distribute in high temporal-low nasal and high ventral-low dorsal gradients, respectively. While others have focused on complementary ligand gradients in the retinal axon target, the tectum, we report that ligands from each subclass also distribute in gradients opposing those of their corresponding receptors within the retina itself. Moreover, ligand gradients in the retina precede ganglion cell genesis. These results support an intraretinal role for Eph family members in addition to their previously proposed role in the development of retinotectal topography. The distinct distributions of Eph family members suggest that each subclass specifies positional information along independent retinal axes.
The isolation and characterisation of two zebrafish Eph receptor ligand cDNAs which we have called zfEphL3 and zfEphL4 is described. These genes are expressed in the presumptive midbrain of developing embryos from 6 somites. By 24 hours L3 is expressed throughout the midbrain including the region of the presumptive tectum whereas L4 is strongly expressed in the midbrain caudal to the presumptive tectum. At later stages of development L3 is expressed in a graded fashion throughout the tectum and L4 is maintained at its posterior margin. Growth cone collapse and pathway selection assays demonstrate that both these proteins have a collapse activity for retinal ganglion cells. When faced with a choice of substrate on which to grow, temporal axons from chick retinal ganglion cells selectively avoided membranes from Cos cells transfected with L3, whereas nasal axons did not. Both temporal and nasal axons avoided membranes from Cos cells transfected with L4. The expression patterns together with the functional data suggest that although both ligands may be able to guide retinal ganglion cells axons in vitro, they have different roles in the guidance of retinotectal projections in vivo. The expression of L3 is consistent with a role in the guidance of retinal ganglion cells to their targets on the tectum whereas that of L4 suggests a role in delineating the posterior boundary of the optic tectum.
Two ligands for Eph-related receptor tyrosine kinases, RAGS and ELF-1, have been implicated in the control of development of the retinotectal projection. Both molecules are expressed in overlapping gradients in the tectum, the target area of retinal ganglion cell axons. In two in vitro assays ELF-1 is shown to have a repellent axon guidance function for temporal, but apparently not for nasal axons. RAGS on the other hand is repellent for both types of axons, though to different degrees. Thus, RAGS and ELF-1 share some and differ in other properties. The biological activities of these molecules correlate with the strength of interaction with their receptors expressed on RGC axons. The meaning of these findings for guidance of retinal axons in the tectum is discussed.
The structural basis of cortical columns are radially oriented axon collaterals that form precise connections between distinct cortical layers. During development, these connections are highly specified from the initial outgrowth of collateral branches. Our previous work provided evidence for positional cues confined to individual layers that induce and/or prevent the formation of axon collaterals in specific populations of cortical neurons. Here we demonstrated with in situ hybridization techniques that mRNA of the Eph receptor tyrosine kinase EphA5 and one of its ligands, ephrin-A5, are present in distinct cortical layers, at a time when intrinsic connections are being formed in the cortex. Axonal guidance assays indicate that ephrin-A5 is a repellent signal for a populations of axons that in vivo avoid the cortical layer expressing ephrin-A5. In contrast to its established role as a repulsive axonal guidance signal, ephrin-A5 specifically mediates sprouting of those cortical axons that target the ephrin-A5-expressing layer in vivo. These results identify a novel function of ephrin-A5 on axonal arbor formation. The laminar distribution and the dual action on specific populations of axons suggest that ephrin-A5 plays a role in the assembly of local cortical circuits.
Eph receptor tyrosine kinases and their corresponding surface-bound ligands, the ephrins, provide cues to the migration of cells and growth cones during embryonic development. Here we show that ephrin-A5, which is attached to the outer leaflet of the plasma membrane by a glycosyl-phosphatidylinositol-anchor, induces compartmentalized signaling within a caveolae-like membrane microdomain when bound to the extracellular domain of its cognate Eph receptor. The physiological response induced by this signaling event is concomitant with a change in the cellular architecture and adhesion of the ephrin-A5-expressing cells and requires the activity of the Fyn protein tyrosine kinase. This study stresses the relevance of bidirectional signaling involving the ephrins and Eph receptors during brain development.
The results of previous in vitro experiments indicate that a glycosylphosphatidylinositol (GPI)-anchored protein may play an important role in the guidance of temporal retinal axons during the formation of the topographically ordered retinotectal projection. We have purified and cloned a GPI-anchored, 25 kDa glycoprotein that is a good candidate for a molecule involved in this process. During the time of innervation by retinal ganglion cells, this protein is gradedly expressed in the posterior part of the developing tectum. In two different in vitro assay systems, the recombinant protein induces growth cone collapse and repulsion of retinal ganglion cell axons. These phenomena are observed for axons of temporal as well as nasal origin, indicating that an additional activity may be necessary to confer the nasotemporal specificity observed in previous assays. We named the protein RAGS (for repulsive axon guidance signal). The sequence of RAGS shows significant homology to recently identified ligands for receptor tyrosine kinases of the Eph subfamily.
Molecular gradients have been postulated to control the topographic mapping of retinal axons in their central targets. Based initially on their expression patterns, and more recently on functional studies, members of the EphA subfamily of receptor tyrosine kinases and their ephrin-A ligands have been implicated in the guidance of retinal axons along the anterior–posterior axis of the chick optic tectum. The report that a receptor of the EphB subfamily, EphB2/Cek5/Nuk/Sek3, is expressed in a high ventral to low dorsal gradient in the developing chick retina and is present on ganglion cell axons suggests that it may be involved in the mapping of retinal axons along the corresponding dorsal–ventral axis of the tectum. To address this issue, we have determined the expression and distribution of ephrin-B1/LERK-2/Cek5-L and ephrin-B2/LERK-5/Htk-L/ELF-2, ligands for EphB2, in the developing chick retinotectal system using riboprobes, immunocytochemistry, and receptor affinity probes. Both ephrin-B1 and ephrin-B2 transcripts are expressed in a high dorsal to low ventral gradient in the developing retina, complementary to the distribution of EphB2. Ephrin-B1 and ephrin-B2 proteins are predominantly found in the developing plexiform layers, suggesting a role in the development of intraretinal connections. Neither protein is detected on ganglion cell axons. In tectum, ephrin-B1 transcripts are expressed in a high dorsal to low ventral gradient in the neuroepithelium and the protein is present along the processes of radial glia and is concentrated at their endfeet in the stratum opticum, at the time retinal axons are growing through it. This distribution of ephrin-B1 suggests that it influences retinal axon mapping along the dorsal–ventral tectal axis and may also be involved in intratectal development. In contrast, ephrin-B2 transcripts and protein are localized to the deeper retinorecipient laminae in the tectum at the time retinal axons begin to arborize in them, suggesting that this ligand may influence the laminar patterning of retinal axon terminations.
The Eph receptors are the largest known family of receptor tyrosine kinases. Initially all of them were identified as orphan receptors without known ligands, and their specific functions were not well understood. During the past few years, a corresponding family of ligands has been identified, called the ephrins, and specific functions have now been identified in neural development. The ephrins and Eph receptors are implicated as positional labels that may guide the development of neural topographic maps. They have also been implicated in pathway selection by axons, the guidance of cell migration, and the establishment of regional pattern in the nervous system. The ligands are anchored to cell surfaces, and most of the functions so far identified can be interpreted as precise guidance of cell or axon movement. This large family of ligands and receptors may make a major contribution to the accurate spatial patterning of connections and cell position in the nervous system.
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 stripes. 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 tectai innervation by retinal axons. These anterior-posterior differences disappear by embryonic day 14.
Axonal growth cones originating from explants of embryonic chick retina were simultaneously exposed to two different cell monolayers and their preference for particular monolayers as a substrate for growth was determined. These experiments show that: (1) nasal retinal axons can distinguish between retinal and tectal cells; (2) temporal retinal axons can distinguish between tectal cells that originated from different positions within the tectum along the antero-posterior axis; (3) axons originating from nasal parts of the retina have different recognizing capabilities from temporal axons; (4) the property of the tectal cells, which is attractive for temporal axons, has a graded distribution along the antero-posterior axis of the tectum; and (5) this gradient also exists in non-innervated tecta.
The axons of retinal ganglion neurons from a precise topographic map in the optic tectum in the midbrain, and the guidance of retinal axons by directional cues in the tectum is crucial in this process. Several in vitro systems have been developed in order to identify the molecular basis of these directional cues. Temporal, but not nasal, retinal axons avoid posterior tectal membranes and grow on anterior membranes as a result of repellent guidance activities that are linked by glycosylphosphatidylinositol (GPI) anchors to the posterior membranes. A putative GPI-anchored repulsive guidance molecule with a molecular weight of 33 kDa has previously been characterized. Indirect results from experiments in vitro support the hypothesis that this 33 kDa molecule guides temporal retinal axons.
To assess whether the 33 kDa protein is involved in axon guidance in vitro, we raised monoclonal antibodies against molecules that had been removed from tectal membranes by treatment with phosphatidylinositol-specific phospholipase C, which cleaves GPI anchors. A monoclonal immunoglobulin M, F3D4, recognized the 33 kDa molecule. In combination with chromophore-assisted laser inactivation, F3D4 caused a loss of the repellent activity from posterior tectal membranes in vitro. As a result, temporal retinal fibers were no longer repelled by posterior tectal membranes. This demonstrates that the F3D4 antigen, which we name RGM (repulsive guidance molecule) is involved in the guidance of retinal axons in an assay in vitro. In vivo, the expression of RGM increases from the anterior to the posterior pole of the optic tectum.
These findings not only support the hypothesis that retinal axons are guided by gradients of repulsive guidance molecules but, in combination with earlier studies of receptor kinases and their ligands that act during guidance, argue for the presence of several repellent guidance molecules with similar functions in vitro and expression patterns in vivo.
The continued functional characterization of Eph-related receptors and ligands has provided further information toward an understanding of the mechanisms controlling the retinotectal projection. Recent in vivo analyses have strengthened the idea that Engrailed defines the positional identity of the tectum along the anteroposterior axis, possibly by regulating the expression of Eph family members.
Recent evidence suggests that Eph receptor tyrosine kinases and their ligands provide positional information in the developing visual system. We previously found that the Eph receptor Cek5 is more highly expressed in the ventral than dorsal chicken embryonic retina. We now report the identification of a chicken ligand for Cek5 (cCek5-L) that is 75% identical to the ligand LERK2. In situ hybridization experiments do not reveal a dorsoventral gradient of cCek5-L transcripts in the optic tectum at Embryonic Day 8, suggesting that this ligand is not involved in guiding Cek5-expressing axons in the tectum. Surprisingly, it is in the retina that high levels of cCek5-L mRNA are present. In the early retina, cCek5-L is more highly expressed in the dorsal than the ventral aspect. Similarly, a Cek5 Ig chimera labels dorsal but not ventral retina, indicating that even if several Cek5 ligands are present, their overall distribution is complementary to that of Cek5. Hence, Cek5 and cCek5-L may both contribute to define anatomical compartments within the early retina. In contrast, in the 11-day embryonic retina the distributions of Cek5 and its ligand(s) show considerable overlap, suggesting changing functions as development progresses. In dissociated cultures of dorsal or ventral retinal cells seeded on plates coated with either receptor or ligand Ig chimeras, the interaction between Cek5 and its ligand(s) or cCek5-L and its receptor(s) is sufficient to mediate cell adhesion and allows neurite outgrowth.
Eph-like receptor tyrosine kinases have recently been identified as critical components in the development of the retinotectal system. Complementary gradients of receptors and ligands in the retina and tectum, and within the retina itself, have previously been described. Here, we present a novel centroperipheral gradient of expression for one member of this family of receptors, Cek9, suggesting that retinal patterning and axon guidance during the establishment of retinotectal projections may involve coordinate mapping along three axes. Furthermore, we show matching gradients of two cytoplasmic kinases, compatible with their putative involvement in the intracellular signaling pathways used by these receptors in the retina. We also demonstrate a dorsal to ventral expression gradient for Cek11, an Eck-like receptor, the Eph subclass previously suggested to specify positional information along the temporonasal axis.
The EphA3 receptor tyrosine kinase has been implicated in guiding the axons of retinal ganglion cells as they extend in the optic tectum. A repulsive mechanism involving opposing gradients of the EphA3 receptor on retinal axons and its ligands, ephrin-A2 and ephrin-A5, in the tectum influences topographic mapping of the retinotectal projection. To investigate the overall role of the Eph family in patterning of the visual system, we have used in situ hybridization to localize nine Eph receptors in the chicken retina and optic tectum at Embryonic Day 8. Three of the receptors examined correspond to the novel chicken homologs of EphA2, EphA6, and EphA7. Unexpectedly, we found that many Eph receptors are expressed not only in retinal ganglion cells, but also in tectal cells, In particular, EphA3 mRNA is prominently expressed in the anterior tectum, with a pattern reciprocal to that of ephrin-A2 and ephrin-A5. Similarly, ephrin-A5 is expressed not only in tectal cells but also in the nasal retina, with a pattern reciprocal to that of its receptor EphA3 and partially overlapping with that of its other receptor EphA4. Consistent with the even distribution of EphA4 and the polarized distribution of EphA4 ligands in the retina, probing EphA4 immunoprecipitates from different sectors of the retina with anti-phosphotyrosine antibodies revealed spatial differences in receptor phosphorylation. These complex patterns of expression and tyrosine phosphorylation suggest that Eph receptors and ephrins contribute to establishing topography of retinal axons through multiple mechanisms, in addition to playing a role in intraretinal and intratectal organization.
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.
Based on recent experimental studies of complementary gradients of receptor density (R) on the retinal surface and ligand density (L) on the tectal surface, and mapping of the high point on the receptor gradient to the low point on the ligand and vice versa, the servomechanism model was constructed involving a mechanism for the retinal axon to reach its target automatically sensing a difference between the signal strength (R.L) and the standard value (S). Computer simulations based on the model demonstrated desired two-dimensional topographic mapping of the retinal axons on the tectum, and explained three strange behaviors of the retinal axons that had been observed in stripe assays for retinal axons using stripes composed of tectal membrane fragments: repulsive behaviors of the retinal axons by the ligand substances, uncertainty of the nasal axons whether or not they show regional selectivity between substances of anterior and posterior tecta, and abrupt transition of growth of the axons originating at continuously varied retinal positions on the stripes having graded ligand density. Finally we suggested what is to be improved in stripe assays with the artificial gradient of the tectal membrane fragments.
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.
The Eph family is thought to exert its function through the complementary expression of receptors and ligands. Here, we show that EphA receptors colocalize on retinal ganglion cell (RGC) axons with EphA ligands, which are expressed in a high-nasal-to-low-temporal pattern. In the stripe assay, only temporal axons are normally sensitive for repellent axon guidance cues of the caudal tectum. However, overexpression of ephrinA ligands on temporal axons abolishes this sensitivity, whereas treatment with PI-PLC both removes ephrinA ligands from retinal axons and induces a striped outgrowth of formerly insensitive nasal axons. In vivo, retinal overexpression of ephrinA2 leads to topographic targeting errors of temporal axons. These data suggest that differential ligand expression on retinal axons is a major determinant of topographic targeting in the retinotectal projection.
The mechanisms underlying the formation of topographic maps in the retinotectal system have long been debated. Recently, members of the Eph and ephrin receptor-ligand family have been found to provide a molecular substrate for one type of mechanism, that of chemospecific gradient matching, as proposed by Sperry. However, experiments over several decades have demonstrated that there is more to map formation than gradient matching. This article briefly reviews the old and new findings, argues that these two types of data must be properly integrated in order to understand map formation fully, and suggests some experimental and theoretical ways to begin this process.
In the retinotectal projection, the Eph receptor tyrosine kinase ligands ephrinA2 and ephrinA5 are differentially expressed not only in the tectum, but also in a high-nasal-to-low-temporal pattern in the retina. Recently, we have shown that retrovirally driven overexpression of ephrinA2 on retinal axons leads to topographic targeting errors of temporal axons in that they overshoot their normal termination zones in the rostral tectum and project onto the mid- and caudal tectum. The behavior of nasal axons, however, was only marginally affected. Here, we show that overexpression of ephrinA5 affects the topographic targeting behavior of both temporal and nasal axons. These data reinforce the idea that differential ligand expression on retinal axons contributes to topographic targeting in the retinotectal projection. Additionally, we found that ectopic expression of ephrinA2 and ephrinA5 frequently leads to pathfinding errors at the chiasm, resulting in an increased stable ipsilateral projection.
In Xenopus tadpoles, all retinal ganglion cells (RGCs) send axons contralaterally across the optic chiasm. At metamorphosis, a subpopulation of EphB-expressing RGCs in the ventrotemporal retina begin to project ipsilaterally. However, when these metamorphic RGCs are grafted into embryos, they project contralaterally, suggesting that the embryonic chiasm lacks signals that guide axons ipsilaterally. Ephrin-B is expressed discretely at the chiasm of metamorphic but not premetamorphic Xenopus. When expressed prematurely in the embryonic chiasm, ephrin-B causes precocious ipsilateral projections from the EphB-expressing RGCs. Ephrin-B is also found in the chiasm of mammals, which have ipsilateral projections, but not in the chiasm of fish and birds, which do not. These results suggest that ephrin-B/EphB interactions play a key role in the sorting of axons at the vertebrate chiasm.
Ephrin-A2 and -A5 are thought to be anteroposterior mapping labels for the retinotectal/retinocollicular projection. Here, gene disruptions of both these ephrins are characterized. Focal retinal labeling reveals moderate map abnormalities when either gene is disrupted. Double heterozygotes also have a phenotype, showing an influence of absolute levels. In vitro assays indicate these ephrins are required for repellent activity in the target and also normal responsiveness in the retina. In double homozygotes, anteroposterior order is almost though not completely lost. Temporal or nasal retinal labelings reveal quantitatively similar but opposite shifts, with multiple terminations scattered widely over the target. These results indicate an axon competition mechanism for mapping, with a critical role for ephrins as anteroposterior topographic labels. Dorsoventral topography is also impaired, showing these ephrins are required in mapping both axes.
Chemoaffinity in the orderly growth of nerve fiber patterns and connections
- Sperry
Sperry RW: Chemoaffinity in the orderly growth of nerve fiber
patterns and connections. Proc Natl Acad Sci USA 1963,
50:703-710.