Rapid Changes in Cortical and Subcortical Brain Regions after Early Bilateral Enucleation in the Mouse.

Functional sensory and motor areas in the developing mammalian neocortex are formed through a complex interaction of cortically intrinsic mechanisms, such as gene expression, and cortically extrinsic mechanisms such as those mediated by thalamic input from the senses. Both intrinsic and extrinsic mechanisms are believed to be involved in cortical patterning and the establishment of areal boundaries in early development; however, the nature of the interaction between intrinsic and extrinsic processes is not well understood. In a previous study, we used a perinatal bilateral enucleation mouse model to test some aspects of this interaction by reweighting sensory input to the developing cortex. Visual deprivation at birth resulted in a shift of intraneocortical connections (INCs) that aligned with ectopic ephrin A5 expression in the same location ten days later at postnatal day (P) 10. A prevailing question remained: Does visual deprivation first induce a change in gene expression, followed by a shift in INCs, or vice versa? In the present study, we address this question by investigating the neuroanatomy and patterns of gene expression in post-natal day (P) 1 and 4 mice following bilateral enucleation at birth. Our results demonstrate a rapid reduction in dorsal lateral geniculate nucleus (dLGN) size and ephrin A5 gene expression 24-hours post-enucleation, with more profound effects apparent at P4. The reduced nuclear size and diminished gene expression mirrors subtle changes in ephrin A5 expression evident in P1 and P4 enucleated neocortex, 11 and 8 days prior to natural eye opening, respectively. Somatosensory and visual INCs were indistinguishable between P1 and P4 mice bilaterally enucleated at birth, indicating that perinatal bilateral enucleation initiates a rapid change in gene expression (within one day) followed by an alteration of sensory INCs later on (second postnatal week). With these results, we gain a deeper understanding of how gene expression and sensory input together regulate cortical arealization and plasticity during early development.

In vivo expression of ephrinA5-Fc in mice results in cephalic neural crest agenesis and craniofacial abnormalities.

Eph receptors and their ligands ephrins have been implicated in guiding the directed migration of neural crest cells (NCCs). In this study, we found that Wnt1-Cre-mediated expression of ephrinA5-Fc along the dorsal midline of the dien- and mesencephalon resulted in severe craniofacial malformation of mouse embryo. Interestingly, expression of cephalic NCC markers decreased significantly in the frontonasal process and branchial arches 1 and 2, which are target areas for the migratory cephalic NCCs originating in the dien- and mesencephalon. In addition, these craniofacial tissues were much smaller in mutant embryos expressing ephrinA5-Fc. Importantly, EphA7-positive cephalic NCCs were absent along the dorsal dien- and mesencephalon of mutant embryos expressing ephrinA5-Fc, suggesting that the generation of cephalic NCCs is disrupted due to ephrinA5-Fc expression. NCC explant experiments suggested that ephrinA5-Fc perturbed survival of cephalic NCC precursors in the dorsal midline tissue rather than affecting their migratory capacity, which was consistent with our previous report that expression of ephrinA5-Fc in the dorsal midline is responsible for severe neuroepithelial cell apoptotic death. Taken together, our findings strongly suggest that expression of ephrinA5-Fc decreases a population of cephalic NCC precursors in the dorsal midline of the dien- and mesencephalon, thereby disrupting craniofacial development in the mouse embryos.

RhoA modulates signaling through the mechanistic target of rapamycin complex 1 (mTORC1) in mammalian cells.

The mechanistic target of rapamycin (mTOR) in complex 1 (mTORC1) pathway integrates signals generated by hormones and nutrients to control cell growth and metabolism. The activation state of mTORC1 is regulated by a variety of GTPases including Rheb and Rags. Recently, Rho1, the yeast ortholog of RhoA, was shown to interact directly with TORC1 and repress its activation state in yeast. Thus, the purpose of the present study was to test the hypothesis that the RhoA GTPase modulates signaling through mTORC1 in mammalian cells. In support of this hypothesis, exogenous overexpression of either wild type or constitutively active (ca)RhoA repressed mTORC1 signaling as assessed by phosphorylation of p70S6K1 (Thr389), 4E-BP1 (Ser65) and ULK1 (Ser757). Additionally, RhoA·GTP repressed phosphorylation of mTORC1-associated mTOR (Ser2481). The RhoA·GTP mediated repression of mTORC1 signaling occurred independent of insulin or leucine induced stimulation. In contrast to the action of Rho1 in yeast, no evidence was found to support a direct interaction of RhoA·GTP with mTORC1. Instead, expression of caRheb, but not caRags, was able to rescue the RhoA·GTP mediated repression of mTORC1 suggesting RhoA functions upstream of Rheb to repress mTORC1 activity. Consistent with this suggestion, RhoA·GTP repressed phosphorylation of TSC2 (Ser939), PRAS40 (Thr246), Akt (Ser473), and mTORC2-associated mTOR (Ser2481). Overall, the results support a model in which RhoA·GTP represses mTORC1 signaling upstream of Akt and mTORC2.

Experimental hypoxia does not influence gene expression and protein synthesis of Eph receptors and ephrin ligands in human melanoma cells in vitro.

Eph receptor tyrosine kinases and their ephrin ligands are considered to play important roles in melanoma progression and metastasis. Moreover, hypoxia is known to contribute to melanoma metastasis. In this study, the influence of experimental hypoxia on the expression and synthesis of EphA2 and EphB4, and their corresponding ligands ephrinA1, ephrinA5, and ephrinB2 was studied systematically in four human melanoma cell lines in vitro. Melanoma cell monolayer and spheroid cultures were used as both extrinsic and intrinsic hypoxia models. Hypoxic conditions were confirmed by analyzing hypoxia-inducible factors 1α or 2α expression, vascular endothelial growth factor expression, and cellular uptake of [F]fluoromisonidazole. In normoxia, EphA2, EphB4, ephrinA1, ephrinA5, and ephrinB2 expression was detectable in all cell lines to varying extents. Considerable protein synthesis of EphA2 was detected in all cell lines. However, no effect of experimental hypoxia on both Eph/ephrin expression and protein synthesis was observed. This contributes critically to the debate on the hypothesis that hypoxia regulates the Eph/ephrin system in melanoma.

Regulation of ephrin-A expression in compressed retinocollicular maps.

Retinotopic maps can undergo compression and expansion in response to changes in target size, but the mechanism underlying this compensatory process has remained a mystery. The discovery of ephrins as molecular mediators of Sperry's chemoaffinity process allows a mechanistic approach to this important issue. In Syrian hamsters, neonatal, partial (PT) ablation of posterior superior colliculus (SC) leads to compression of the retinotopic map, independent of neural activity. Graded, repulsive EphA receptor/ephrin-A ligand interactions direct the formation of the retinocollicular map, but whether ephrins might also be involved in map compression is unknown. To examine whether map compression might be directed by changes in the ephrin expression pattern, we compared ephrin-A2 and ephrin-A5 mRNA expression between normal SC and PT SC using in situ hybridization and quantitative real-time PCR. We found that ephrin-A ligand expression in the compressed maps was low anteriorly and high posteriorly, as in normal animals. Consistent with our hypothesis, the steepness of the ephrin gradient increased in the lesioned colliculi. Interestingly, overall levels of ephrin-A2 and -A5 expression declined immediately after neonatal target damage, perhaps promoting axon outgrowth. These data establish a correlation between changes in ephrin-A gradients and map compression, and suggest that ephrin-A expression gradients may be regulated by target size. This in turn could lead to compression of the retinocollicular map onto the reduced target. These findings have important implications for mechanisms of recovery from traumatic brain injury.

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.

Expression of the tyrosine kinase receptor EphA5 and its ligand ephrin-A5 during mouse spinal cord development.

OBJECTIVES: To study the expression patterns of two Eph family molecules, the receptor EphA5, and the ligand ephrin-A5, during spinal cord development. METHODS: The receptor expression was analyzed using beta-galactosidase knockin mice, and affinity ligand probe binding. The ligand expression was assessed using two different affinity probes, and knockout mouse tissues as controls. RESULTS: EphA5 was expressed in the ventral spinal cord, while ephrin-A5 was located in the dorsolateral regions of the spinal cord throughout development. CONCLUSIONS: These results show that EphA5 and ephrin-A5 are expressed over broad developmental stages and may play important roles in establishing the dorsoventral organization of the spinal cord.

Ephrin-As and patterned retinal activity act together in the development of topographic maps in the primary visual system.

The development of topographic maps in the primary visual system is thought to rely on a combination of EphA/ephrin-A interactions and patterned neural activity. Here, we characterize the retinogeniculate and retinocollicular maps of mice mutant for ephrins-A2, -A3, and -A5 (the three ephrin-As expressed in the mouse visual system), mice mutant for the beta2 subunit of the nicotinic acetylcholine receptor (that lack early patterned retinal activity), and mice mutant for both ephrin-As and beta2. We also provide the first comprehensive anatomical description of the topographic connections between the retina and the dorsal lateral geniculate nucleus. We find that, although ephrin-A2/A3/A5 triple knock-out mice have severe mapping defects in both projections, they do not completely lack topography. Mice lacking beta2-dependent retinal activity have nearly normal topography but fail to refine axonal arbors. Mice mutant for both ephrin-As and beta2 have synergistic mapping defects that result in a near absence of map in the retinocollicular projection; however, the retinogeniculate projection is not as severely disrupted as the retinocollicular projection is in these mutants. These results show that ephrin-As and patterned retinal activity act together to establish topographic maps, and demonstrate that midbrain and forebrain connections have a differential requirement for ephrin-As and patterned retinal activity in topographic map development.

Over-expression of Eph and ephrin genes in advanced ovarian cancer: ephrin gene expression correlates with shortened survival.

BACKGROUND: Increased expression of Eph receptor tyrosine kinases and their ephrin ligands has been implicated in tumor progression in a number of malignancies. This report describes aberrant expression of these genes in ovarian cancer, the commonest cause of death amongst gynaecological malignancies. METHODS: Eph and ephrin expression was determined using quantitative real time RT-PCR. Correlation of gene expression was measured using Spearman's rho statistic. Survival was analysed using log-rank analysis and (was visualised by) Kaplan-Meier survival curves. RESULTS: Greater than 10 fold over-expression of EphA1 and a more modest over-expression of EphA2 were observed in partially overlapping subsets of tumors. Over-expression of EphA1 strongly correlated (r = 0.801; p < 0.01) with the high affinity ligand ephrin A1. A similar trend was observed between EphA2 and ephrin A1 (r = 0.387; p = 0.06). A striking correlation of both ephrin A1 and ephrin A5 expression with poor survival (r = -0.470; p = 0.02 and r = -0.562; p < 0.01) was observed. Intriguingly, there was no correlation between survival and other clinical parameters or Eph expression. CONCLUSION: These data imply that increased levels of ephrins A1 and A5 in the presence of high expression of Ephs A1 and A2 lead to a more aggressive tumor phenotype. The known functions of Eph/ephrin signalling in cell de-adhesion and movement may explain the observed correlation of ephrin expression with poor prognosis.

EphA7-ephrin-A5 signaling in mouse somatosensory cortex: developmental restriction of molecular domains and postnatal maintenance of functional compartments.

Members of the Eph family of receptor tyrosine kinases and their ligands, the ephrins, are expressed in distinct patterns in the forming cortex. EphA7 is expressed early in cortical development, becoming concentrated in anterior and posterior domains, whereas ephrin-A5 is expressed later in corticogenesis, highest in the middle region that has low levels of EphA7. The EphA7 gene produces full-length and truncated isoforms, which are repulsive and adhesive, respectively. Analysis of cortical RNA expression demonstrates that proportions of these isoforms change with time, from a more repulsive mix during embryogenesis to a more permissive mix postnatally. To examine how EphA7 and ephrin-A5 influence the formation of cortical regions, EphA7-/- mice were analyzed. Within the cortex of EphA7-/- mice, the distribution of ephrin-A5 was more extensive, encompassing its usual medial domain but also extending more posteriorly toward the occipital pole. Moreover, relative levels of ephrin-A5 along the cortex's anatomical axes changed in EphA7-/- animals, creating less striking shifts in ligand abundance. Furthermore, in vivo functional studies revealed that EphA7 exerts a repulsive influence on ephrin-A5-expressing cells during corticogenesis. In contrast, EphA7 appears to mediate permissive interactions in the postnatal cortex: the area of somatosensory cortex was significantly reduced in EphA7-/- mice. A similar reduction was present in ephrin-A5-/- animals and a more pronounced decrease was observed in EphA7/ephrin-A5-/- cortex. Taken together, this study supports a role for EphA7 and ephrin-A5 in the establishment and maintenance of certain cortical domains and suggests that the nature of their interactions changes with cortical maturity.

Ephrin-A5 inhibits growth of embryonic sensory neurons.

EphA-ephrin signaling has recently been implicated in the establishment of motor innervation patterns, in particular in determining whether motor axons project into dorsal versus ventral nerve trunks in the limb. We investigated whether sensory axons, which grow out together with and can be guided by motor axons, are also influenced by Eph-ephrin signaling. We show that multiple EphA receptors are expressed in DRGs when limb innervation is being established, and EphA receptors are present on growth cones of both NGF-dependent (predominantly cutaneous) and NT3-dependent (predominantly proprioceptive) afferents. Both soluble and membrane-attached ephrin-A5 inhibited growth of approximately half of each population of sensory axons in vitro. On average, growth cones that collapsed in response to soluble ephrin-A5 extended more slowly than those that did not, and ephrin-A5 significantly slowed the extension of NGF-dependent growth cones that did not collapse. Finally, we show that ectopic expression of ephrin-A5 in ovo reduced arborization of cutaneous axons in skin on the limb. Together these results suggest that sensory neurons respond directly to A-class ephrins in the limb. Thus, ephrins appear to pattern sensory axon growth in two ways-both directly, and indirectly via their inhibitory effects on neighboring motor axons.

Ephrin-A5 overexpression degrades topographic specificity in the mouse gluteus maximus muscle.

Motor neurons project onto specific muscles with a distinct positional bias. We have previously shown using electrophysiological techniques that overexpression of ephrin-A5 degrades this topographic map. Here, we show that positional differences in axon terminal areas, an entirely different parameter of neuromuscular topography, are also eliminated with ephrin-A5 overexpression. Therefore, we now have both morphological and electrophysiological approaches to explore the mechanisms of neuromuscular topography.

Differential expression of two cell adhesion molecules, Ephrin-A5 and Integrin alpha6, during cranial neurulation in the chick embryo.

The formation of the neural tube (neurulation) depends on the physical properties of the cells and tissues both inside and outside the neural plate. One such important physical property is cell adhesion. Theoretical and biological evidence support a role for cell adhesion in neurulation, but few specific cell adhesion molecules have been identified during this process. Ephrin-A5 and Integrin alpha6 are two of the known genes encoding cell adhesion molecules that are likely to be directly involved in neurulation because neural tube defects result when they are knocked out in mice. Yet it remains unclear how they can act on the cell and tissue behaviors of neurulation, because their domains of expression in neurulating tissues have not been reported. We report here the detailed pattern of expression of these two cell adhesion molecules in the chick embryo throughout the stages of neurulation at the mRNA and protein level. We show that Ephrin-A5 and Integrin alpha6 are differentially expressed in the ectoderm, outside and inside the neural plate, respectively, and that they are both restricted to neurulation at cranial (brain) levels. We discuss the potential contribution of this differential expression to the cell adhesion mechanisms involved in cranial neurulation and anencephaly.

Area specificity and topography of thalamocortical projections are controlled by ephrin/Eph genes.

The mechanisms generating precise connections between specific thalamic nuclei and cortical areas remain poorly understood. Using axon tracing analysis of ephrin/Eph mutant mice, we provide in vivo evidence that Eph receptors in the thalamus and ephrins in the cortex control intra-areal topographic mapping of thalamocortical (TC) axons. In addition, we show that the same ephrin/Eph genes unexpectedly control the inter-areal specificity of TC projections through the early topographic sorting of TC axons in an intermediate target, the ventral telencephalon. Our results constitute the first identification of guidance cues involved in inter-areal specificity of TC projections and demonstrate that the same set of mapping labels is used differentially for the generation of topographic specificity of TC projections between and within individual cortical areas.

Alkaline phosphatase fusion proteins as affinity probes for protein localization studies.

The receptor affinity probe or receptor alkaline phosphatase (RAP) staining method uses soluble protein ectodomains fused to secreted placental alkaline phosphatase to locate ectodomain binding sites within cells or tissues. We have used this approach to identify expressing cells in tissue culture, in tissue sections, or in whole-mount embryos. The RAP method is especially useful in situations where a reliable monoclonal antibody is not available or if an orphan receptor is the focus of the study. The technique permits localization of both receptors and ligands and is readily quantifiable for cell-surface binding assays. Soluble ectodomain placental alkaline phosphatase fusion proteins are therefore highly sensitive reagents that permit the direct localization of available binding sites through simple chromogenic assays without purification, radioactive labeling, or secondary reagents.

EphA receptors and ephrin-A ligands exhibit highly regulated spatial and temporal expression patterns in the developing olfactory system.

The spatiotemporal expression patterns of the chemorepulsive EphA receptors, EphA4 and EphA7, and three ephrins-A2, A4 and A5, were examined in the developing rat primary olfactory system. Unlike the visual system that has simple and stable gradients of Ephs and ephrins, the olfactory system demonstrates complex spatiotemporal expression patterns of these molecules. Using immunohistochemistry, we demonstrate that expression of these molecules is dynamic and tightly regulated both within and between different cell types. We reveal restricted targeting of these proteins within subcellular compartments of some neurons. EphA4, ephrin-A2 and ephrin-A5 were expressed by primary olfactory axons during the embryonic formation of the olfactory nerve. There were no gradients in expression along the rostrocaudal or ventrodorsal axes in the nasal cavity and olfactory bulb. However, during the early neonatal period, axons expressing different levels of ephrin-A5 sorted out and terminated in a subpopulation of glomeruli that were mosaically dispersed throughout the bulb. The expression of EphA4 and ephrin-A2 was dramatically down-regulated on all axons during the early neonatal period of glomerular formation. The uniform co-expression of receptors and ligands before glomerular formation suggests they play a generic role in axon-axon interactions in the olfactory nerve and nerve fibre layer. In contrast, loss of EphA4 from axons during glomerular formation may facilitate the interaction of ephrin-A5 with Eph receptors on target cells in the bulb. While EphA4, EphA5 and EphA7 are not mosaically expressed by bulbar neurons, other Eph receptors may have expression patterns complementary to the ephrin-A5-positive subpopulation of glomeruli.