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

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

On the Importance of Countergradients for the Development of Retinotopy: Insights from a Generalised Gierer Model

During the development of the topographic map from vertebrate retina to superior colliculus (SC), EphA receptors are expressed in a gradient along the nasotemporal retinal axis. Their ligands, ephrin-As, are expressed in a gradient along the rostrocaudal axis of the SC. Countergradients of ephrin-As in the retina and EphAs in the SC are also expressed. Disruption of any of these gradients leads to mapping errors. Gierer''s (1981) model, which uses well-matched pairs of gradients and countergradients to establish the mapping, can account for the formation of wild type maps, but not the double maps found in EphA knock-in experiments. I show that these maps can be explained by models, such as Gierer''s (1983), which have gradients and no countergradients, together with a powerful compensatory mechanism that helps to distribute connections evenly over the target region. However, this type of model cannot explain mapping errors found when the countergradients are knocked out partially. I examine the relative importance of countergradients as against compensatory mechanisms by generalising Gierer''s (1983) model so that the strength of compensation is adjustable. Either matching gradients and countergradients alone or poorly matching gradients and countergradients together with a strong compensatory mechanism are sufficient to establish an ordered mapping. With a weaker compensatory mechanism, gradients without countergradients lead to a poorer map, but the addition of countergradients improves the mapping. This model produces the double maps in simulated EphA knock-in experiments and a map consistent with the Math5 knock-out phenotype. Simulations of a set of phenotypes from the literature substantiate the finding that countergradients and compensation can be traded off against each other to give similar maps. I conclude that a successful model of retinotopy should contain countergradients and some form of compensation mechanism, but not in the strong form put forward by Gierer.     

EphA4 catalytic activity causes inhibition of RhoA GTPase in Xenopus laevis embryos

The Eph family of receptor tyrosine kinases is involved in limiting cell and tissue interactions via a repulsive mechanism. The mechanism of repulsion involves reorganizing the actin cytoskeleton, but little is known of the molecular components that connect the receptor to the actin cytoskeleton. Recent studies in retinal ganglion cells have demonstrated that EphA4 activates the small GTPase Rho. We have investigated the involvement of Rho in signaling downstream from EphA4. As a model system, we have used a chimeric receptor called EPP that we express and activate in early Xenopus embryos. Previous studies demonstrated that EPP activation leads to loss of cell-cell adhesion and change in cell shape, plus loss of aspects of cell polarity in epithelial cells, such as apical microvilli and the apical/basolateral boundary. In this study, we show that injecting inhibitors of Rho GTPases into early Xenopus embryos produces a phenotype very similar to that resulting from EPP activation. More importantly, expression of a constitutively active form of Xenopus RhoA (XRhoA) concurrent with activated EPP rescued embryos from the loss of cell-cell adhesion and change in cell shape associated with EPP. These data argue that, in contrast to the case in retinal ganglion cells, EphA4 in early Xenopus embryos acts to inhibit RhoA, suggesting that this receptor may regulate Rho differently (and therefore affect the cytoskeleton differently) in neuronal and non-neuronal cells. Furthermore, overexpression of ephexin, a novel guanine nucleotide exchange factor for Rho family GTPases, also blocks EPP-induced dissociation. This suggests that EphA4, which has been demonstrated to activate ephexin in cultured neuronal cells, may also target Rho GTPase via an ephexin-independent pathway.     

The Caenorhabditis elegans Six/sine oculis class homeobox gene ceh-32 is required for head morphogenesis

Repulsion plays a fundamental role in the establishment of a topographic map of the chick retinotectal projections. This has been highlighted by studies demonstrating the role of opposing gradients of the EphA3 receptor tyrosine kinase on retinal axons and two of its ligands, ephrin-A2 and ephrin-A5, in the tectum. We have analyzed the distribution of these two ephrins in other retinorecipient structures in the chick diencephalon and mesencephalon during the period when visual connections are being established. We have found that both ephrin-A2 and ephrin-A5 and their receptors EphA4 and EphA7 are expressed in gradients whose orientation is consistent with the topography of the nasotemporal axis of the respective retinofugal projections. In addition, their distribution suggests that receptor-ligand interactions may be involved in the organization of connections between the different primary visual centers and, thus, in the topographic organization of secondary visual projections. Interestingly, where projections lack a clear topographic representation, a uniform expression of the Eph-ephrin molecules was observed. Finally, we also show that a similar patterning mechanism may be implicated in the transfer of visual information to the telencephalon. These results suggest a conserved function for EphA receptors and their ligands in the elaboration of topographic maps at multiple levels of the visual pathway.     

Opposing gradients of ephrin-As and EphA7 in the superior colliculus are essential for topographic mapping in the mammalian visual system

During development of the retinocollicular projection in mouse, retinal axons initially overshoot their future termination zones (TZs) in the superior colliculus (SC). The formation of TZs is initiated by interstitial branching at topographically appropriate positions. Ephrin-As are expressed in a decreasing posterior-to-anterior gradient in the SC, and they suppress branching posterior to future TZs. Here we investigate the role of an EphA7 gradient in the SC, which has the reverse orientation to the ephrin-A gradient. We find that in EphA7 mutant mice the retinocollicular map is disrupted, with nasal and temporal axons forming additional or extended TZs, respectively. In vitro, retinal axons are repelled from growing on EphA7-containing stripes. Our data support the idea that EphA7 is involved in suppressing branching anterior to future TZs. These findings suggest that opposing ephrin-A and EphA gradients are required for the proper development of the retinocollicular projection.     

Analysis of mouse EphA knockins and knockouts suggests that retinal axons programme target cells to form ordered retinotopic maps

I present a novel analysis of abnormal retinocollicular maps in mice in which the distribution of EphA receptors over the retina has been modified by knockin and/or knockout of these receptor types. My analysis shows that in all these cases, whereas the maps themselves are discontinuous, the graded distribution of EphA over the nasotemporal axis of the retina is recreated within the pattern of axonal terminations across rostrocaudal colliculus. This suggests that the guiding principle behind the formation of ordered maps of nerve connections between vertebrate retina and superior colliculus, or optic tectum, is that axons carrying similar amounts of Eph receptor terminate near to one another on the target structure. I show how the previously proposed marker induction model embodies this principle and predicts these results. I then describe a new version of the model in which the properties of the markers, or labels, are based on those of the Eph receptors and their associated ligands, the ephrins. I present new simulation results, showing the development of maps between two-dimensional structures, exploring the role of counter-gradients of labels across the target and confirming that the model reproduces the retinocollicular maps found in EphA knockin/knockout mice. I predict that abnormal distributions of label within the retina lead to abnormal distributions of label over the target, so that in each of the types of knockin/knockout mice analysed, there will be a different distribution of labels over the target structure. This mechanism could be responsible for the flexibility with which neurons reorganise their connections during development and the degree of precision in the final map. Activity-based mechanisms would play a role only at a later stage of development to remove the overlap between individual retinal projection fields, such as in the development of patterns of ocular dominance stripes.     

Small molecules can selectively inhibit ephrin binding to the EphA4 and EphA2 receptors

The erythropoietin-producing hepatocellular (Eph) family of receptor tyrosine kinases regulates a multitude of physiological and pathological processes. Despite the numerous possible research and therapeutic applications of agents capable of modulating Eph receptor function, no small molecule inhibitors targeting the extracellular domain of these receptors have been identified. We have performed a high throughput screen to search for small molecules that inhibit ligand binding to the extracellular domain of the EphA4 receptor. This yielded a 2,5-dimethylpyrrolyl benzoic acid derivative able to inhibit the interaction of EphA4 with a peptide ligand as well as the natural ephrin ligands. Evaluation of a series of analogs identified an isomer with similar inhibitory properties and other less potent compounds. The two isomeric compounds act as competitive inhibitors, suggesting that they target the high affinity ligand-binding pocket of EphA4 and inhibit ephrin-A5 binding to EphA4 with K(i) values of 7 and 9 mum in enzyme-linked immunosorbent assays. Interestingly, despite the ability of each ephrin ligand to promiscuously bind many Eph receptors, the two compounds selectively target EphA4 and the closely related EphA2 receptor. The compounds also inhibit ephrin-induced phosphorylation of EphA4 and EphA2 in cells, without affecting cell viability or the phosphorylation of other receptor tyrosine kinases. Furthermore, the compounds inhibit EphA4-mediated growth cone collapse in retinal explants and EphA2-dependent retraction of the cell periphery in prostate cancer cells. These data demonstrate that the Eph receptor-ephrin interface can be targeted by inhibitory small molecules and suggest that the two compounds identified will be useful to discriminate the activities of EphA4 and EphA2 from those of other co-expressed Eph receptors that are activated by the same ephrin ligands. Furthermore, the newly identified inhibitors represent possible leads for the development of therapies to treat pathologies in which EphA4 and EphA2 are involved, including nerve injuries and cancer.     

Dynamic Alterations of Retinal EphA5 Expression in Retinocollicular Map Plasticity

The topographically ordered retinocollicular projection is an excellent system for studying the mechanism of axon guidance. Gradients of EphA receptors in the retina and ephrin‐As in the superior colliculus (SC) pattern the anteroposterior axis of the retinocollicular map, but whether they are involved in map plasticity after injury is unknown. Partial damage to the caudal SC at birth creates a compressed, complete retinotopic map in the remaining SC without affecting visual response properties. Previously, we found that the gradient of ephrin‐A expression in compressed maps is steeper than normal, suggesting an instructive role in compression. Here we measured EphA5 mRNA and protein levels after caudal SC damage in order to test the hypothesis that changes in retinal EphA5 expression occur that are complementary to the changes in collicular ephrin‐A expression. We find that the nasotemporal gradient of EphA5 receptor expression steepens in the retina and overall expression levels change dynamically, especially in temporal retina, supporting the hypothesis. This change in receptor expression occurs after the change in ephrin‐A ligand expression. We propose that changes in the retinal EphA5 gradient guide recovery of the retinocollicular projection from early injury. This could occur directly through the change in EphA5 expression instructing retino‐SC map compression, or through ephrin‐A ligand signaling instructing a change in EphA5 receptor expression that in turn signals the retinocollicular map to compress. Understanding what molecular signals direct compensation for injury is essential to developing rehabilitative strategies and maximizing the potential for recovery.     

Graded expression patterns of ephrin-As in the superior colliculus after lesion of the adult mouse optic nerve

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.     

Regulation of axial patterning of the retina and its topographic mapping in the brain

Topographic maps are a fundamental organizational feature of axonal connections in the brain. A prominent model for studying axial polarity and topographic map development is the vertebrate retina and its projection to the optic tectum (or superior colliculus). Linked processes are controlled by molecules that are graded along the axes of the retina and its target fields. Recent studies indicate that ephrin-As control the temporal-nasal mapping of the retina in the optic tectum/superior colliculus by regulating the topographically-specific interstitial branching of retinal axons along the anterior-posterior tectal axis. This branching is mediated by relative levels of EphA receptor repellent signaling. A major recent advance is the demonstration that EphB receptor forward signaling and ephrin-B reverse signaling mediate axon attraction to control dorsal-ventral retinal mapping along the lateral-medial tectal axis. In addition, several classes of regulatory proteins have been implicated in the control of the axial patterning of the retina, and its ultimate readout of topographic mapping.     

Parcellation of the thalamus into distinct nuclei reflects EphA expression and function

Intercellular signaling via the Eph receptor tyrosine kinases and their ligands, the ephrins, acts to shape many regions of the developing brain. One intriguing consequence of Eph signaling is the control of mixing between discrete cell populations in the developing hindbrain, contributing to the formation of segregated rhombomeres. Since the thalamus is also a parcellated structure comprised of discrete nuclei, might Eph signaling play a parallel role in cell segregation in this brain structure? Analyses of expression reveal that several Eph family members are expressed in the forming thalamus and that cells expressing particular receptors form cellular groupings as development proceeds. Specifically, expression of receptors EphA4 or EphA7 and ligand ephrin-A5 is localized to distinct thalamic domains. EphA4 and EphA7 are often coexpressed in regions of the forming thalamus, with each receptor marking discrete thalamic domains. In contrast, ephrin-A5 is expressed by a limited group of thalamic cells. Within the ventral thalamus, EphA4 is present broadly, occasionally overlapping with ephrin-A5 expression. EphA7 is more restricted in its expression and is largely nonoverlapping with ephrin-A5. In mutant mice lacking one or both receptors or ephrin-A5, the appearance of the venteroposterolateral (VPL) and venteroposteromedial (VPM) nuclear complex is altered compared to wild type mice. These in vivo results support a role for Eph family members in the definition of the thalamic nuclei. In parallel, in vitro analysis reveals a hierarchy of mixing among cells expressing ephrin-A5 with cells expressing EphA4 alone, EphA4 and EphA7 together, or EphA7 alone. Together, these data support a model in which EphA molecules promote the parcellation of discrete thalamic nuclei by limiting the extent of cell mixing.     

EphA4 and ephrin-B2 expression patterns during inferior colliculus projection shaping prior to experience

Central processing of complex auditory tasks requires elaborate circuitry. The auditory midbrain, or inferior colliculus (IC), epitomizes such precise organization, where converging inputs form discrete, tonotopically-arranged axonal layers. Previously in rat, we established that shaping of multiple afferent patterns in the IC central nucleus (CNIC) occurs prior to experience. This study implicates an Eph receptor tyrosine kinase and a corresponding ephrin ligand in signaling this early topographic registry. We report that EphA4 and ephrin-B2 expression patterns in the neonatal rat and mouse IC correlate temporally and spatially with that of developing axonal layers. DiI-labeling confirms projections arising from the lateral superior olive (LSO) form frequency-specific layers within the ipsilateral and contralateral mouse CNIC, as has been described in other species. Immunohistochemistry (EphA4 and ephrin-B2) and ephrin-B2 lacZ histochemistry reveal clear gradients in expression across the tonotopic axis, with most concentrated labeling observed in high-frequency, ventromedial aspects of the CNIC. Discrete patches of labeling were also discernible in the external cortex of the IC (ECIC; EphA4 patches in rat, ephrin-B2 patches in mouse). Observed gradients in the CNIC and compartmentalized ECIC expression persisted through the first postnatal week, before becoming less intense and more homogeneously distributed by the functional onset of hearing. EphA4 and ephrin-B2-positive neurons were evident in several auditory brainstem nuclei known to send patterened inputs to the IC. These findings suggest the involvement of cell-cell EphA4 and ephrin-B2 signaling in establishing order in the developing IC.     

E-cadherin regulates the function of the EphA2 receptor tyrosine kinase.

EphA2 is a member of the Eph family of receptor tyrosine kinases, which are increasingly understood to play critical roles in disease and development. We report here the regulation of EphA2 by E-cadherin. In nonneoplastic epithelia, EphA2 was tyrosine-phosphorylated and localized to sites of cell-cell contact. These properties required the proper expression and functioning of E-cadherin. In breast cancer cells that lack E-cadherin, the phosphotyrosine content of EphA2 was decreased, and EphA2 was redistributed into membrane ruffles. Expression of E-cadherin in metastatic cells restored a more normal pattern of EphA2 phosphorylation and localization. Activation of EphA2, either by E-cadherin expression or antibody-mediated aggregation, decreased cell-extracellular matrix adhesion and cell growth. Altogether, this demonstrates that EphA2 function is dependent on E-cadherin and suggests that loss of E-cadherin function may alter neoplastic cell growth and adhesion via effects on EphA2.     

Parcellation of the thalamus into distinct nuclei reflects EphA expression and function

Intercellular signaling via the Eph receptor tyrosine kinases and their ligands, the ephrins, acts to shape many regions of the developing brain. One intriguing consequence of Eph signaling is the control of mixing between discrete cell populations in the developing hindbrain, contributing to the formation of segregated rhombomeres. Since the thalamus is also a parcellated structure comprised of discrete nuclei, might Eph signaling play a parallel role in cell segregation in this brain structure? Analyses of expression reveal that several Eph family members are expressed in the forming thalamus and that cells expressing particular receptors form cellular groupings as development proceeds. Specifically, expression of receptors EphA4 or EphA7 and ligand ephrin-A5 is localized to distinct thalamic domains. EphA4 and EphA7 are often coexpressed in regions of the forming thalamus, with each receptor marking discrete thalamic domains. In contrast, ephrin-A5 is expressed by a limited group of thalamic cells. Within the ventral thalamus, EphA4 is present broadly, occasionally overlapping with ephrin-A5 expression. EphA7 is more restricted in its expression and is largely nonoverlapping with ephrin-A5. In mutant mice lacking one or both receptors or ephrin-A5, the appearance of the venteroposterolateral (VPL) and venteroposteromedial (VPM) nuclear complex is altered compared to wild type mice. These in vivo results support a role for Eph family members in the definition of the thalamic nuclei. In parallel, in vitro analysis reveals a hierarchy of mixing among cells expressing ephrin-A5 with cells expressing EphA4 alone, EphA4 and EphA7 together, or EphA7 alone. Together, these data support a model in which EphA molecules promote the parcellation of discrete thalamic nuclei by limiting the extent of cell mixing.     

Ephrin-A6, a new ligand for EphA receptors in the developing visual system

In the embryonic visual system, EphA receptors are expressed on both temporal and nasal retinal ganglion cell axons. Only the temporal axons, however, are sensitive to the low concentrations of ephrin-A ligands found in the anterior optic tectum. The poor responsiveness of nasal axons to ephrin-A ligands, which allows them to traverse the anterior tectum and reach their targets in the posterior tectum, has been attributed to constitutive activation of the EphA4 receptor expressed in these axons. EphA4 is highly expressed throughout the retina, but is preferentially phosphorylated on tyrosine (activated) in nasal retina. In a screen for EphA4 ligands expressed in chicken embryonic retina, we have identified a novel ephrin, ephrin-A6. Like ephrin-A5, ephrin-A6 has high affinity for EphA4 and activates this receptor in cultured retinal cells. In the embryonic day 8 (E8) chicken visual system, ephrin-A6 is predominantly expressed in the nasal retina and ephrin-A5 in the posterior tectum. Thus, ephrin-A6 has the properties of a ligand that activates the EphA4 receptor in nasal retinal cells. Ephrin-A6 binds with high affinity to several other EphA receptors as well and causes growth cone collapse in retinal explants, demonstrating that it can elicit biological responses in retinal neurons. Ephrin-A6 expression is high at E6 and E8, when retinal axons grow to their tectal targets, and gradually declines at later developmental stages. The asymmetric distribution of ephrin-A6 in retinal cells, and the time course of its expression, suggest that this new ephrin plays a role in the establishment of visual system topography.     

A critical role for the EphA3 receptor tyrosine kinase in heart development

Eph proteins are receptor tyrosine kinases that control changes in cell shape and migration during development. We now describe a critical role for EphA3 receptor signaling in heart development as revealed by the phenotype of EphA3 null mice. During heart development mesenchymal outgrowths, the atrioventricular endocardial cushions, form in the atrioventricular canal. This morphogenetic event requires endocardial cushion cells to undergo an epithelial to mesenchymal transformation (EMT), and results in the formation of the atrioventricular valves and membranous portions of the atrial and ventricular septa. We show that EphA3 knockouts have significant defects in the development of their atrial septa and atrioventricular endocardial cushions, and that these cardiac abnormalities lead to the death of approximately 75% of homozygous EphA3(-/-) mutants. We demonstrate that EphA3 and its ligand, ephrin-A1, are expressed in adjacent cells in the developing endocardial cushions. We further demonstrate that EphA3(-/-) atrioventricular endocardial cushions are hypoplastic compared to wildtype and that EphA3(-/-) endocardial cushion explants give rise to fewer migrating mesenchymal cells than wildtype explants. Thus our results indicate that EphA3 plays a crucial role in the development and morphogenesis of the cells that give rise to the atrioventricular valves and septa.     

Activation of the EGFR gene target EphA2 inhibits epidermal growth factor-induced cancer cell motility

EphA2 overexpression has been reported in many cancers and is believed to play an important role in tumor metastasis and angiogenesis. We show that the activated epidermal growth factor receptor (EGFR) and the cancer-specific constitutively active EGFR type III deletion mutant (EGFRvIII) induce the expression of EphA2 in mammalian cell lines, including the human cancer cell lines A431 and HN5. The regulation is partially dependent on downstream activation of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase and is a direct effect on the EphA2 promoter. Furthermore, EGFR and EphA2 both localize to the plasma membrane and EphA2 coimmunoprecipitates with activated EGFR and EGFRvIII. Ligand activation of EphA2 and EphA2 knockdown by small interfering RNA inhibit EGF-induced cell motility of EGFR-overexpressing human cancer cells, indicating a functional role of EphA2 in EGFR-expressing cancer cells.     

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. © 2012 Wiley Periodicals, Inc. Develop Neurobiol, 2013     

Target-derived and locally derived neurotrophins support retinal ganglion cell survival in the neonatal rat retina

Brain-derived neurotrophic factor (BDNF) and neurotrophin-4/5 (NT-4/5) protein and mRNA are found in the neonatal rat retina and also in target sites such as the superficial layers of the superior colliculus. Both neurotrophins support neonatal retinal ganglion cell survival in vitro. In vivo, injections of recombinant BDNF and NT-4/5 reduce naturally occurring cell death as well as death induced by removal of the contralateral superior colliculus. In the latter case, the peak of retinal ganglion cell death occurs about 24 h postlesion. We wished to determine: whether a similar time-course of degeneration occurs after selective removal of target cells or depletion of target-derived trophic factors, and whether ganglion cell viability also depends on intraretinally derived neurotrophins. Retinal ganglion cell death was measured 24 and 48 h following injections of kainic acid or a mixture of BDNF and NT-4/5 blocking antibodies into the superior colliculus and 24 h after intraocular injection of the same antibodies. Retinotectally projecting ganglion cells were identified by retrograde labeling with the nucleophilic dye diamidino yellow. We show that collicular injections of either kainic acid or BDNF and NT-4/5 blocking antibodies significantly increased retinal ganglion cell death in the neonatal rat 24 h postinjection, death rates returning to normal by 48 h. This increase in death was greatest following collicular injections; however, death was also significantly increased 24 h following intravitreal antibody injection. Thus retinal ganglion cell survival during postnatal development is not only dependent upon trophic factors produced by central targets but may also be influenced by local intraretinal neurotrophin release.     

Progranulin/EphA2 axis: A novel oncogenic mechanism in bladder cancer

The growth factor progranulin plays a critical role in bladder cancer by modulating tumor cell motility and invasion. Progranulin regulates remodeling of the actin cytoskeleton by interacting with drebrin, an actin binding protein that regulates tumor growth. We previously discovered that progranulin depletion inhibits epithelial-to-mesenchymal transition and markedly reduces in vivo tumor growth. Moreover, progranulin depletion sensitizes urothelial cancer cells to cisplatin treatment, further substantiating a pro-survival function of progranulin. Until recently, the progranulin signaling receptor remained unidentified, precluding a full understanding of progranulin action in tumor cell biology. We recently identified EphA2, a member of a large family of receptor tyrosine-kinases, as the functional receptor for progranulin. However, it is not established whether EphA2 plays an oncogenic role in bladder cancer. Here we demonstrate that progranulin, and not ephrin-A1, the canonical ligand for EphA2, is the predominant EphA2 ligand in bladder cancer. Progranulin evoked Akt- and Erk1/2-mediated EphA2 phosphorylation at Ser897, which could drive bladder tumorigenesis. We discovered that EphA2 depletion severely blunted progranulin-dependent motility and anchorage-independent growth, and sensitized bladder cancer cells to cisplatin treatment. We further defined the mechanisms of progranulin/EphA2-dependent motility by identifying liprin-α1 as a novel progranulin-dependent EphA2 interacting protein and establishing its critical role in cell motility. The discovery of EphA2 as the functional signaling receptor for progranulin and the identification of novel downstream effectors offer a new avenue for understanding the underlying mechanism of progranulin action and may constitute novel clinical and therapeutic targets in bladder cancer.