Reduction of ephrin-A5 aggravates disease progression in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that affects motor neurons in the brainstem, spinal cord and motor cortex. ALS is characterized by genetic and clinical heterogeneity, suggesting the existence of genetic factors that modify the phenotypic expression of the disease. We previously identified the axonal guidance EphA4 receptor, member of the Eph-ephrin system, as an ALS disease-modifying factor. EphA4 genetic inhibition rescued the motor neuron phenotype in zebrafish and a rodent model of ALS. Preventing ligands from binding to the EphA4 receptor also successfully improved disease, suggesting a role for EphA4 ligands in ALS. One particular ligand, ephrin-A5, is upregulated in reactive astrocytes after acute neuronal injury and inhibits axonal regeneration. Moreover, it plays a role during development in the correct pathfinding of motor axons towards their target limb muscles. We hypothesized that a constitutive reduction of ephrin-A5 signalling would benefit disease progression in a rodent model for ALS. We discovered that in the spinal cord of control and symptomatic ALS mice ephrin-A5 was predominantly expressed in neurons. Surprisingly, reduction of ephrin-A5 levels in SOD1G93A mice accelerated disease progression and reduced survival without affecting disease onset, motor neuron numbers or innervated neuromuscular junctions in symptomatic mice. These findings suggest ephrin-A5 as a modifier of disease progression that might play a role in the later stages of the disease. Similarly, we identified a more aggressive disease progression in patients with lower ephrin-A5 protein levels in the cerebrospinal fluid without modifying disease onset. In summary, we identified reduced expression of ephrin-A5 to accelerate disease progression in a mouse model of ALS as well as in humans. Combined with our previous findings on the role of EphA4 in ALS our current data suggests different contribution for various members of the Eph-ephrin system in the pathophysiology of a motor neuron disease.

Epha2 and Efna5 participate in lens cell pattern-formation.

Ephrin type-A receptor 2 (EPHA2) and one of its ligands, ephrin-A5 (EFNA5), have been associated with loss of eye lens transparency, or cataract, - an important cause of visual impairment. Here we show that mice functionally lacking EPHA2 (Epha2-null), EFNA5 (Efna5-null), or both receptor and ligand (Epha2/Efna5-null) consistently develop mostly transparent lenses with an internal refractive disturbance and a grossly disturbed cellular architecture. In situ hybridization localized Epha2 and Efna5 transcripts to lens epithelial cells and nascent fiber cells at the lens equator. In vivo labeling of Epha2-null lenses with a thymidine analog detected a significant decrease in lens epithelial cell proliferation within the germinative zone resulting in impaired early lens growth. Ex vivo imaging of Epha2-null, Efna5-null, and Epha2/Efna5-null lenses labelled in vivo with a membrane-targeted red fluorescent protein revealed misalignment of elongating fiber cells at the lens equator and loss of Y-suture pattern formation near the anterior and posterior poles of the lens. Immuno-fluorescent labeling of lens major intrinsic protein or aquaporin-0 (MIP/AQP0) showed that the precise, radial column patterning of hexagonal fiber cells throughout the cortex region was disrupted in Epha2-null, Efna5-null and Epha2/Efna5-null lenses. Collectively, these data suggest that Epha2 and Efna5 participate in the complex, global patterning of lens fiber cells that is necessary for maximal optical quality.

Decreased maternal behavior and anxiety in ephrin-A5-/- mice.

During development of the nervous system, molecular signals mediating cell-cell interactions play critical roles in the guidance of axonal growth and establishment of synaptic functions. The Eph family of tyrosine kinase receptors and their ephrin ligands has been shown to mediate neuronal interactions in the development of topographic axon projection maps in several brain regions, and the loss of Eph activities result in defects in select axonal pathways. However, effects of deficiencies of the Eph signals on animal behavior have not been well documented. In this study, we showed that inactivation of a ligand of the Eph receptors, ephrin-A5, resulted in defects in maternal behavior and alterations in anxiety. Female ephrin-A5 -/- mice show significant defects in nest building and pup retrieval. In addition, lower levels of anxiety were observed in both male and female null mice. These changes were not due to deficiencies in estradiol, progesterone or corticosterone levels. Our observations suggest that ephrin-A5 plays a key role in the development and/or function of neural pathways mediating mouse maternal care and anxiety.

Proper closure of the optic fissure requires ephrin A5-EphB2-JNK signaling.

The development of complex organs such as the eye requires a delicate and coordinated balance of cell division and cell death. Although apoptosis is prevalent in the proximoventral optic cup, the precise role it plays in eye development needs to be investigated further. In this study, we show that reduced apoptosis in the proximoventral optic cup prevents closure of the optic fissure. We also show that expression of ephrin A5 (Efna5) partially overlaps with Eph receptor B2 (Ephb2) expression in the proximoventral optic cup and that binding of EphB2 to ephrin A5 induces a sustained activation of JNK. This prolonged JNK signal promotes apoptosis and prevents cell proliferation. Thus, we propose that the unique cross-subclass interaction of EphB2 with ephrin A5 has evolved to function upstream of JNK signaling for the purpose of maintaining an adequate pool of progenitor cells to ensure proper closure of the optic fissure.

Thalamic afferents influence cortical progenitors via ephrin A5-EphA4 interactions.

The phenotype of excitatory cerebral cortex neurons is specified at the progenitor level, orchestrated by various intrinsic and extrinsic factors. Here, we provide evidence for a subcortical contribution to cortical progenitor regulation by thalamic axons via ephrin A5-EphA4 interactions. Ephrin A5 is expressed by thalamic axons and represents a high-affinity ligand for EphA4 receptors detected in cortical precursors. Recombinant ephrin A5-Fc protein, as well as ephrin A ligand-expressing, thalamic axons affect the output of cortical progenitor division in vitro. Ephrin A5-deficient mice show an altered division mode of radial glial cells (RGCs) accompanied by increased numbers of intermediate progenitor cells (IPCs) and an elevated neuronal production for the deep cortical layers at E13.5. In turn, at E16.5 the pool of IPCs is diminished, accompanied by reduced rates of generated neurons destined for the upper cortical layers. This correlates with extended infragranular layers at the expense of superficial cortical layers in adult ephrin A5-deficient and EphA4-deficient mice. We suggest that ephrin A5 ligands imported by invading thalamic axons interact with EphA4-expressing RGCs, thereby contributing to the fine-tuning of IPC generation and thus the proper neuronal output for cortical layers.

The role of ephrin-A2 and ephrin-A5 in sensorimotor control and gating.

Many factors influence neurodevelopment. However, their contribution to adult neural function is often unclear. This is often due to complex expression profiles, cell signalling, neuroanatomy, and a lack of effective tests to assess the function of neural circuits in vivo. Ephrin-A2 and ephrin-A5 are cell surface proteins implicated in multiple aspects of neurodevelopment. While the role of ephrin-As in visual, auditory and learning behaviours has been explored, little is known about their role in dopaminergic and neuromotor pathways, despite expression in associated brain regions. Here we probe the function of ephrin-A2 and ephrin-A5 in the development of the dopaminergic and neuromotor pathways using counts of tyrosine hydroxylase (TH) positive cells in the substantia nigra pars compacta (SNpc) and the ventral tegmental area (VTA), the acoustic startle reflex (ASR), and a measure of sensorimotor gating, prepulse inhibition (PPI). Analysis of the ASR and PPI in ephrin-A2 and/or ephrin-A5 knock-out mice revealed that both genes play distinct roles in mediating ASR circuits, but are unlikely to play a role in PPI. Knock-out of either gene resulted in robust changes in startle response magnitude and measures of startle onset and peak latencies. However, ephrin-A2 and ephrin-A5 regulate aspects of the ASR differently: ephrin-A2 KO mice have increased startle amplitude, increased sensitivity and reduced latency to startle, whilst ephrin-A5 KO mice show opposite effects. Neither of the gene knock outs affected PPI, despite ephrin-A5 KO mice showing changes in dopamine cell numbers in nuclei thought to regulate PPI. We propose that majority of the changes observed ephrin-A2 and ephrin-A5 KO mice appear to be mediated by the effects on motor neurons and their muscle targets, rather than changes in auditory sensitivity.

Low-intensity repetitive transcranial magnetic stimulation improves abnormal visual cortical circuit topography and upregulates BDNF in mice.

Repetitive transcranial magnetic stimulation (rTMS) is increasingly used as a treatment for neurological and psychiatric disorders. Although the induced field is focused on a target region during rTMS, adjacent areas also receive stimulation at a lower intensity and the contribution of this perifocal stimulation to network-wide effects is poorly defined. Here, we examined low-intensity rTMS (LI-rTMS)-induced changes on a model neural network using the visual systems of normal (C57Bl/6J wild-type, n = 22) and ephrin-A2A5(-/-) (n = 22) mice, the latter possessing visuotopic anomalies. Mice were treated with LI-rTMS or sham (handling control) daily for 14 d, then fluorojade and fluororuby were injected into visual cortex. The distribution of dorsal LGN (dLGN) neurons and corticotectal terminal zones (TZs) was mapped and disorder defined by comparing their actual location with that predicted by injection sites. In the afferent geniculocortical projection, LI-rTMS decreased the abnormally high dispersion of retrogradely labeled neurons in the dLGN of ephrin-A2A5(-/-) mice, indicating geniculocortical map refinement. In the corticotectal efferents, LI-rTMS improved topography of the most abnormal TZs in ephrin-A2A5(-/-) mice without altering topographically normal TZs. To investigate a possible molecular mechanism for LI-rTMS-induced structural plasticity, we measured brain derived neurotrophic factor (BDNF) in the visual cortex and superior colliculus after single and multiple stimulations. BDNF was upregulated after a single stimulation for all groups, but only sustained in the superior colliculus of ephrin-A2A5(-/-) mice. Our results show that LI-rTMS upregulates BDNF, promoting a plastic environment conducive to beneficial reorganization of abnormal cortical circuits, information that has important implications for clinical rTMS.

Ephrin-A5/EphA4 signalling controls specific afferent targeting to cochlear hair cells.

Hearing requires an optimal afferent innervation of sensory hair cells by spiral ganglion neurons in the cochlea. Here we report that complementary expression of ephrin-A5 in hair cells and EphA4 receptor among spiral ganglion neuron populations controls the targeting of type I and type II afferent fibres to inner and outer hair cells, respectively. In the absence of ephrin-A5 or EphA4 forward signalling, a subset of type I projections aberrantly overshoot the inner hair cell layer and invade the outer hair cell area. Lack of type I afferent synapses impairs neurotransmission from inner hair cells to the auditory nerve. By contrast, radial shift of type I projections coincides with a gain of presynaptic ribbons that could enhance the afferent signalling from outer hair cells. Ephexin-1, cofilin and myosin light chain kinase act downstream of EphA4 to induce type I spiral ganglion neuron growth cone collapse. Our findings constitute the first identification of an Eph/ephrin-mediated mutual repulsion mechanism responsible for specific sorting of auditory projections in the cochlea.

Further analysis of the lens of ephrin-A5-/- mice: development of postnatal defects.

PURPOSE: The cells of the mammalian lens must be carefully organized and regulated to maintain clarity. Recent studies have identified the Eph receptor ligand ephrin-A5 as a major contributor to lens development, as mice lacking ephrin-A5 develop abnormal lenses, resulting in cataracts. As a follow-up to our previous study on the cataracts observed in ephrin-A5(-/-) animals, we have further examined the morphological and molecular changes in the ephrin-A5(-/-) lens. METHODS: Wild-type and ephrin-A5(-/-) eyes at various ages were fixed, sectioned, and examined using histological techniques. Protein expression and localization were determined using immunohistochemistry and western blot analysis. RESULTS: Lens abnormalities in the ephrin-A5(-/-) animals are observed at postnatal stages, with lens opacity occurring by postnatal day 21. Structural defects in the lens are first observed in the outer lens fiber cell region where cells in the ephrin-A5(-/-) lens are severely disorganized. Ephrin-A5 and the Eph receptor EphA2 are expressed during early ocular development and continue to be expressed into postnatal stages. The cataracts in the ephrin-A5(-/-) mutants occur regardless of the presence of the CP49 mutation. CONCLUSIONS: In this follow-up study, we have uncovered additional details explicating the mechanisms underlying ephrin-A5 function in the lens. Furthermore, elucidation of the expression of ephrin-A5 and the Eph receptor EphA2 in the lens supports a fundamental role for this receptor-ligand complex in lens development. These observations, in concert with our previous study, strongly suggest that ephrin-A5 has a critical role in postnatal lens fiber organization to maintain lens transparency.

Ephrin-A5 deficiency alters sensorimotor and monoaminergic development.

The Eph receptors and their ligands, the ephrins, play an important role during neural development. In particular, ephrin-A5 is highly expressed in the developing nervous system in several brain regions including the olfactory bulb, frontal cortex, striatum and hypothalamus. Although a number of studies have characterized the expression of ephrin-A5 in these regions, very little is known about the functional consequences that might follow alterations in the expression of this ligand. Previously, we demonstrated that ephrin-A5 acts as a guidance molecule regulating the trajectory of the ascending midbrain dopaminergic pathways. In light of this finding and the critical role of dopamine in modulating a number of behaviors, we sought to determine whether loss of ephrin-A5 altered neurobehavioral development. Our results indicate that ephrin-A5-null mice exhibit delays in reaching developmental milestones and in the maturation of motor skills. In addition, they exhibit increased locomotor activity and reduced levels of brain monoamines. Therefore, we conclude that ephrin-A5 expression appears to be critical for proper development of central monoaminergic pathways and that its loss results in a number of neurodevelopmental abnormalities. Because alterations in monoamine function are associated with a variety of neurodevelopmental disorders, these data suggest that further study on the potential role of ephrin-A5 in such disorders is warranted.

Diverse roles of Eph/ephrin signaling in the mouse lens.

Recent genetic studies show that the Eph/ephrin bidirectional signaling pathway is associated with both congenital and age-related cataracts in mice and humans. We have investigated the molecular mechanisms of cataractogenesis and the roles of ephrin-A5 and EphA2 in the lens. Ephrin-A5 knockout ⁻/⁻ mice often display anterior polar cataracts while EphA2⁻/⁻ lenses show very mild cortical or nuclear cataracts at weaning age. The anterior polar cataract of ephrin-A5⁻/⁻ lenses is correlated with multilayers of aberrant cells that express alpha smooth muscle actin, a marker for mesenchymal cells. Only select fiber cells are altered in ephrin-A5⁻/⁻ lenses. Moreover, the disruption of membrane-associated ß-catenin and E-cadherin junctions is observed in ephrin-A5⁻/⁻ lens central epithelial cells. In contrast, EphA2⁻/⁻ lenses display normal monolayer epithelium while disorganization is apparent in all lens fiber cells. Immunostaining of ephrin-A5 proteins, highly expressed in lens epithelial cells, were not colocalized with EphA2 proteins, mainly expressed in lens fiber cells. Besides the previously reported function of ephrin-A5 in lens fiber cells, this work suggests that ephrin-A5 regulates ß-catenin signaling and E-cadherin to prevent lens anterior epithelial cells from undergoing the epithelial-to-mesenchymal transition while EphA2 is essential for controlling the organization of lens fiber cells through an unknown mechanism. Ephrin-A5 and EphA2 likely interacting with other members of Eph/ephrin family to play diverse functions in lens epithelial cells and/or fiber cells.

Expression of ephrinA5 during development and potential involvement in the guidance of the mesostriatal pathway.

Identifying guidance cues that direct axon growth to their final connections during development is of crucial interest if we aim to repair circuits damaged in adulthood following neurodegenerative disorders or common traumatic injuries. In this work, we set out to determine the ephrinA5 guidance molecule involvement in the establishment of the mouse mesostriatal pathway during development. We showed, in vitro and in vivo, that a proportion of mesencephalic dopaminergic cells express the ephrinA5 receptor, EphA5. Moreover, we observed, using stripe assays, that ephrinA5 purified protein has a repulsive effect on most of the mesencephalic dopaminergic projections. In vivo, we detected rostro-caudal and ventro-dorsal ephrinA5 protein expression gradients in the vicinity of the dopaminergic axons in the ventral telencephalon and in the striatum, during the embryonic and early postnatal development. In addition, other EphA5 ligands were also detected in the mesostriatal pathway. Together, these expression patterns suggest that, ephrinAs and more specifically ephrinA5, may be actors in the guidance of dopaminergic projections. Further studies will focus on identifying the molecular specificity of these guidance cues, taking into account the mesencephalic dopaminergic heterogeneous neuronal population. This may help increase the integration of neuronal transplants in the mature lesioned brain or provide tools to re-establish mesostriatal circuits in vivo.

Loss of ephrin-A5 function disrupts lens fiber cell packing and leads to cataract.

Cell-cell interactions organize lens fiber cells into highly ordered structures to maintain transparency. However, signals regulating such interactions have not been well characterized. We report here that ephrin-A5, a ligand of the Eph receptor tyrosine kinases, plays a key role in lens fiber cell shape and cell-cell interactions. Lens fiber cells in mice lacking ephrin-A5 function appear rounded and irregular in cross-section, in contrast to their normal hexagonal appearance in WT lenses. Cataracts eventually develop in 87% of ephrin-A5 KO mice. We further demonstrate that ephrin-A5 interacts with the EphA2 receptor to regulate the adherens junction complex by enhancing recruitment of beta-catenin to N-cadherin. These results indicate that the Eph receptors and their ligands are critical regulators of lens development and maintenance.

Temporal regulation of ephrin/Eph signalling is required for the spatial patterning of the mammalian striatum.

Brain structures, whether mature or developing, display a wide diversity of pattern and shape, such as layers, nuclei or segments. The striatum in the mammalian forebrain displays a unique mosaic organization (subdivided into two morphologically and functionally defined neuronal compartments: the matrix and the striosomes) that underlies important functional features of the basal ganglia. Matrix and striosome neurons are generated sequentially during embryonic development, and segregate from each other to form a mosaic of distinct compartments. However, the molecular mechanisms that underlie this time-dependent process of neuronal segregation remain largely unknown. Using a novel organotypic assay, we identified ephrin/Eph family members as guidance cues that regulate matrix/striosome compartmentalization. We found that EphA4 and its ephrin ligands displayed specific temporal patterns of expression and function that play a significant role in the spatial segregation of matrix and striosome neurons. Analysis of the striatal patterning in ephrin A5/EphA4 mutant mice further revealed the requirement of EphA4 signalling for the proper sorting of matrix and striosome neuronal populations in vivo. These data constitute the first identification of genes involved in striatal compartmentalization, and reveal a novel mechanism by which the temporal control of guidance cues enables neuronal segregation, and thereby the generation of complex cellular patterns in the brain.

Functional topography and integration of the contralateral and ipsilateral retinocollicular projections of ephrin-A-/- mice.

Topographically ordered projections are established by molecular guidance cues and refined by neuronal activity. Retinal input to a primary visual center, the superior colliculus (SC), is bilateral with a dense contralateral projection and a sparse ipsilateral one. Both projections are topographically organized, but in opposing anterior-posterior orientations. This arrangement provides functionally coherent input to each colliculus from the binocular visual field, supporting visual function. When guidance cues involved in contralateral topography (ephrin-As) are absent, crossed retinal ganglion cell (RGC) axons form inappropriate terminations within the SC. However, the organization of the ipsilateral projection relative to the abnormal contralateral input remains unknown, as does the functional capacity of both projections. We show here that in ephrin-A(-/-) mice, the SC contains an expanded, diffuse ipsilateral projection. Electrophysiological recording demonstrated that topography of visually evoked responses recorded from the contralateral superior colliculus of ephrin-A(-/-) mice displayed similar functional disorder in all genotypes, contrasting with their different degrees of anatomical disorder. In contrast, ipsilateral responses were retinotopic in ephrin-A2(-/-) but disorganized in ephrin-A2/A5(-/-) mice. The lack of integration of binocular input resulted in specific visual deficits, which could be reversed by occlusion of one eye. The discrepancy between anatomical and functional topography in both the ipsilateral and contralateral projections implies suppression of inappropriately located terminals. Moreover, the misalignment of ipsilateral and contralateral visual information in ephrin-A2/A5(-/-) mice suggests a role for ephrin-As in integrating convergent visual inputs.

Ephrin-A5 modulates the topographic mapping and connectivity of commissural axons in murine hippocampus.

Entorhinal and commissural/associational projections show a non-overlapping distribution in the hippocampus proper and the dentate gyrus. The expression of Ephrins and their Eph receptors in the developing hippocampus indicates that this family of axonal guidance molecules may modulate the formation of these connections. Here we focused on the role of the ephrin-A5 ligand in the development of the main hippocampal afferents. In situ hybridization showed that ephrin-A5 mRNA was detected mainly in the principal cells of the hippocampus proper and in the dentate gyrus throughout postnatal development. Immunocytochemical analyses revealed prominent expression of the EphA3 receptor, a putative receptor for ephrin-A5, in the main cells and the neuropil of the developing hippocampus. Tracing experiments in ephrin-A5(-/-) mice showed that commissural projections were transiently altered in the hippocampus proper at P5, but they were mistargeted throughout the postnatal development in the dentate gyrus. Immunocytochemistry with anti-calbindin antibodies revealed that the dentate mossy fiber projection was not altered in ephrin-A5(-/-) mice. Electron microscopy studies showed alterations in the density of synapses and spines in commissural/associational layers, but not in entorhinal layers, and in the mossy fibers in these animals. Taken together, these findings indicate that ephrin-A5 signaling is involved in the formation and maturation of synapses in the hippocampus.

Ephrin-as guide the formation of functional maps in the visual cortex.

Ephrin-As and their receptors, EphAs, are expressed in the developing cortex where they may act to organize thalamic inputs. Here, we map the visual cortex (V1) in mice deficient for ephrin-A2, -A3, and -A5 functionally, using intrinsic signal optical imaging and microelectrode recording, and structurally, by anatomical tracing of thalamocortical projections. V1 is shifted medially, rotated, and compressed and its internal organization is degraded. Expressing ephrin-A5 ectopically by in utero electroporation in the lateral cortex shifts the map of V1 medially, and expression within V1 disrupts its internal organization. These findings indicate that interactions between gradients of EphA/ephrin-A in the cortex guide map formation, but that factors other than redundant ephrin-As are responsible for the remnant map. Together with earlier work on the retinogeniculate map, the current findings show that the same molecular interactions may operate at successive stages of the visual pathway to organize maps.

Ephrin-As and neural activity are required for eye-specific patterning during retinogeniculate mapping.

In mammals, retinal ganglion cell (RGC) projections initially intermingle and then segregate into a stereotyped pattern of eye-specific layers in the dorsal lateral geniculate nucleus (dLGN). Here we found that in mice deficient for ephrin-A2, ephrin-A3 and ephrin-A5, eye-specific inputs segregated but the shape and location of eye-specific layers were profoundly disrupted. In contrast, mice that lacked correlated retinal activity did not segregate eye-specific inputs. Inhibition of correlated neural activity in ephrin mutants led to overlapping retinal projections that were located in inappropriate regions of the dLGN. Thus, ephrin-As and neural activity act together to control patterning of eye-specific retinogeniculate layers.

Ephrin-A2 and -A5 influence patterning of normal and novel retinal projections to the thalamus: conserved mapping mechanisms in visual and auditory thalamic targets.

Sensory axons are targeted to modality-specific nuclei in the thalamus. Retinal ganglion cell axons project retinotopically to their principal thalamic target, the dorsal lateral geniculate nucleus (LGd), in a pattern likely dictated by the expression of molecular gradients in the LGd. Deafferenting the auditory thalamus induces retinal axons to innervate the medial geniculate nucleus (MGN). These retino-MGN projections also show retinotopic organization. Here we show that ephrin-A2 and -A5, which are expressed in similar gradients in the MGN and LGd, can be used to pattern novel retinal projections in the MGN. As in the LGd, retinal axons from each eye terminate in discrete eye-specific zones in the MGN of rewired wild-type and ephrin-A2/A5 knockout mice. However, ipsilateral eye axons, which arise from retinal regions of high EphA5 receptor expression and represent central visual field, terminate in markedly different ways in the two mice. In rewired wild-type mice, ipsilateral axons specifically avoid areas of high ephrin expression in the MGN. In rewired ephrin knockout mice, ipsilateral projections shift in location and spread more broadly, leading to an expanded representation of the ipsilateral eye in the MGN. Similarly, ipsilateral projections to the LGd in ephrin knockout mice are shifted and are more widespread than in the LGd of wild-type mice. In the MGN, as in the LGd, terminations from the two eyes show little overlap even in the knockout mice, suggesting that local interocular segregation occurs regardless of other patterning determinants. Our data demonstrate that graded topographic labels, such as the ephrins, can serve to shape multiple related aspects of afferent patterning, including topographic mapping and the extent and spread of eye-specific projections. Furthermore, when mapping labels and other cues are expressed in multiple target zones, novel projections are patterned according to rules that operate in their canonical targets.

Miswiring of limbic thalamocortical projections in the absence of ephrin-A5.

Axon guidance cues of the ephrin ligand family have been hypothesized to regulate the formation of thalamocortical connections, but in vivo evidence for such a role has not been examined directly. To test whether ephrin-mediated repulsive cues participate in sorting the projections originating from distinct thalamic nuclei, we analyzed the organization of somatosensory and anterior cingulate afferents postnatally in mice lacking ephrin-A5 gene expression. Projections from ventrobasal and laterodorsal nuclei to their respective sensory and limbic cortical areas developed normally. However, a portion of limbic thalamic neurons from the laterodorsal nucleus also formed additional projections to somatosensory cortical territories, thus maintaining inappropriate dual projections to multiple cortical regions. These results suggest that ephrin-A5 is not required for the formation of normal cortical projections from the appropriate thalamic nuclei, but rather acts as a guidance cue that restricts limbic thalamic axons from inappropriate neocortical regions.