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EphA3 expressed in the chicken tectum stimulates nasal retinal ganglion cell axon growth and is required for retinotectal topographic map formation.

Ortalli AL, Fiore L, Di Napoli J, Rapacioli M, Salierno M, Etchenique R, Flores V, Sanchez V, Carri NG, Scicolone G - PLoS ONE (2012)

Bottom Line: We demonstrated in vitro and in vivo that EphA3 ectodomain (which is expressed in a decreasing rostro-caudal gradient in the tectum) is necessary for topographic mapping by stimulating the nasal axon growth toward the caudal tectum and inhibiting their branching in the rostral tectum.Furthermore, the ability of EphA3 of stimulating axon growth allows understanding how optic fibers invade the tectum growing throughout this molecular gradient.Therefore, opposing tectal gradients of repellent ephrin-As and of axon growth stimulating EphA3 complement each other to map optic fibers along the rostro-caudal tectal axis.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Developmental Neurobiology, Institute of Cell Biology and Neurosciences Prof. E. De Robertis (UBA-CONICET), School of Medicine, University of Buenos Aires, Buenos Aires, Argentina.

ABSTRACT

Background: Retinotopic projection onto the tectum/colliculus constitutes the most studied model of topographic mapping and Eph receptors and their ligands, the ephrins, are the best characterized molecular system involved in this process. Ephrin-As, expressed in an increasing rostro-caudal gradient in the tectum/colliculus, repel temporal retinal ganglion cell (RGC) axons from the caudal tectum and inhibit their branching posterior to their termination zones. However, there are conflicting data regarding the nature of the second force that guides nasal axons to invade and branch only in the caudal tectum/colliculus. The predominant model postulates that this second force is produced by a decreasing rostro-caudal gradient of EphA7 which repels nasal optic fibers and prevents their branching in the rostral tectum/colliculus. However, as optic fibers invade the tectum/colliculus growing throughout this gradient, this model cannot explain how the axons grow throughout this repellent molecule.

Methodology/principal findings: By using chicken retinal cultures we showed that EphA3 ectodomain stimulates nasal RGC axon growth in a concentration dependent way. Moreover, we showed that nasal axons choose growing on EphA3-expressing cells and that EphA3 diminishes the density of interstitial filopodia in nasal RGC axons. Accordingly, in vivo EphA3 ectodomain misexpression directs nasal optic fibers toward the caudal tectum preventing their branching in the rostral tectum.

Conclusions: We demonstrated in vitro and in vivo that EphA3 ectodomain (which is expressed in a decreasing rostro-caudal gradient in the tectum) is necessary for topographic mapping by stimulating the nasal axon growth toward the caudal tectum and inhibiting their branching in the rostral tectum. Furthermore, the ability of EphA3 of stimulating axon growth allows understanding how optic fibers invade the tectum growing throughout this molecular gradient. Therefore, opposing tectal gradients of repellent ephrin-As and of axon growth stimulating EphA3 complement each other to map optic fibers along the rostro-caudal tectal axis.

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EphA3 ectodomain decreases the density of interstitial filopodia in nasal RGC axons.A–D. Representative microphotographs of axons grown from nasal (A, C) and temporal (B, D) retinal explants exposed to soluble clustered Fc (A, B) or EphA3-Fc (C, D). Axons are labeled with Alexa 488-phalloidin. Arrows depict representative interstitial filopodia. Insets show filopodia at higher magnification. Scale bars  = 20 µm. (E) Quantification of filopodia number/100 µm of axon shafts. Nasal axons present higher density of interstitial filopodia and EphA3-Fc significantly decreases the density of interstitial filopodia in nasal RGC (ANOVA and Tukey postest, 3 experiments, n: 8 axons for explant, 4 explants for condition). Results are shown as mean +/− SE.
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pone-0038566-g005: EphA3 ectodomain decreases the density of interstitial filopodia in nasal RGC axons.A–D. Representative microphotographs of axons grown from nasal (A, C) and temporal (B, D) retinal explants exposed to soluble clustered Fc (A, B) or EphA3-Fc (C, D). Axons are labeled with Alexa 488-phalloidin. Arrows depict representative interstitial filopodia. Insets show filopodia at higher magnification. Scale bars  = 20 µm. (E) Quantification of filopodia number/100 µm of axon shafts. Nasal axons present higher density of interstitial filopodia and EphA3-Fc significantly decreases the density of interstitial filopodia in nasal RGC (ANOVA and Tukey postest, 3 experiments, n: 8 axons for explant, 4 explants for condition). Results are shown as mean +/− SE.

Mentions: As the topographic-specific formation of interstitial branches is considered a critical event in chicken and mice retinotopic mapping [2], [6], we investigated whether the EphA3 ectodomain regulates the density of axonal interstitial filopodia -the precursors of axonal branches [48], [49]- by performing an in vitro assay [37], [39], [49]. We exposed axons of nasal and temporal retinal explants to soluble clustered Fc or EphA3-Fc (2 nM) for 2 hours and quantified the axonal filopodia observed in phase-contrast or stained with Alexa 488-conjugated phalloidin after fixation. Axons with a similar low degree of interaxonal contacts were selected. We showed that nasal axons presented 17.49 %+/−6.58 % higher density of interstitial filopodia than the temporal ones and that EphA3-Fc significantly decreased the density of interstitial filopodia by 43.2%+/−5.6% in nasal RGC axons (Fig. 5A–E). This demonstrates that EphA3 ectodomain also reduces the density of axonal filopodia of the nasal RGCs in vitro.


EphA3 expressed in the chicken tectum stimulates nasal retinal ganglion cell axon growth and is required for retinotectal topographic map formation.

Ortalli AL, Fiore L, Di Napoli J, Rapacioli M, Salierno M, Etchenique R, Flores V, Sanchez V, Carri NG, Scicolone G - PLoS ONE (2012)

EphA3 ectodomain decreases the density of interstitial filopodia in nasal RGC axons.A–D. Representative microphotographs of axons grown from nasal (A, C) and temporal (B, D) retinal explants exposed to soluble clustered Fc (A, B) or EphA3-Fc (C, D). Axons are labeled with Alexa 488-phalloidin. Arrows depict representative interstitial filopodia. Insets show filopodia at higher magnification. Scale bars  = 20 µm. (E) Quantification of filopodia number/100 µm of axon shafts. Nasal axons present higher density of interstitial filopodia and EphA3-Fc significantly decreases the density of interstitial filopodia in nasal RGC (ANOVA and Tukey postest, 3 experiments, n: 8 axons for explant, 4 explants for condition). Results are shown as mean +/− SE.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3369860&req=5

pone-0038566-g005: EphA3 ectodomain decreases the density of interstitial filopodia in nasal RGC axons.A–D. Representative microphotographs of axons grown from nasal (A, C) and temporal (B, D) retinal explants exposed to soluble clustered Fc (A, B) or EphA3-Fc (C, D). Axons are labeled with Alexa 488-phalloidin. Arrows depict representative interstitial filopodia. Insets show filopodia at higher magnification. Scale bars  = 20 µm. (E) Quantification of filopodia number/100 µm of axon shafts. Nasal axons present higher density of interstitial filopodia and EphA3-Fc significantly decreases the density of interstitial filopodia in nasal RGC (ANOVA and Tukey postest, 3 experiments, n: 8 axons for explant, 4 explants for condition). Results are shown as mean +/− SE.
Mentions: As the topographic-specific formation of interstitial branches is considered a critical event in chicken and mice retinotopic mapping [2], [6], we investigated whether the EphA3 ectodomain regulates the density of axonal interstitial filopodia -the precursors of axonal branches [48], [49]- by performing an in vitro assay [37], [39], [49]. We exposed axons of nasal and temporal retinal explants to soluble clustered Fc or EphA3-Fc (2 nM) for 2 hours and quantified the axonal filopodia observed in phase-contrast or stained with Alexa 488-conjugated phalloidin after fixation. Axons with a similar low degree of interaxonal contacts were selected. We showed that nasal axons presented 17.49 %+/−6.58 % higher density of interstitial filopodia than the temporal ones and that EphA3-Fc significantly decreased the density of interstitial filopodia by 43.2%+/−5.6% in nasal RGC axons (Fig. 5A–E). This demonstrates that EphA3 ectodomain also reduces the density of axonal filopodia of the nasal RGCs in vitro.

Bottom Line: We demonstrated in vitro and in vivo that EphA3 ectodomain (which is expressed in a decreasing rostro-caudal gradient in the tectum) is necessary for topographic mapping by stimulating the nasal axon growth toward the caudal tectum and inhibiting their branching in the rostral tectum.Furthermore, the ability of EphA3 of stimulating axon growth allows understanding how optic fibers invade the tectum growing throughout this molecular gradient.Therefore, opposing tectal gradients of repellent ephrin-As and of axon growth stimulating EphA3 complement each other to map optic fibers along the rostro-caudal tectal axis.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Developmental Neurobiology, Institute of Cell Biology and Neurosciences Prof. E. De Robertis (UBA-CONICET), School of Medicine, University of Buenos Aires, Buenos Aires, Argentina.

ABSTRACT

Background: Retinotopic projection onto the tectum/colliculus constitutes the most studied model of topographic mapping and Eph receptors and their ligands, the ephrins, are the best characterized molecular system involved in this process. Ephrin-As, expressed in an increasing rostro-caudal gradient in the tectum/colliculus, repel temporal retinal ganglion cell (RGC) axons from the caudal tectum and inhibit their branching posterior to their termination zones. However, there are conflicting data regarding the nature of the second force that guides nasal axons to invade and branch only in the caudal tectum/colliculus. The predominant model postulates that this second force is produced by a decreasing rostro-caudal gradient of EphA7 which repels nasal optic fibers and prevents their branching in the rostral tectum/colliculus. However, as optic fibers invade the tectum/colliculus growing throughout this gradient, this model cannot explain how the axons grow throughout this repellent molecule.

Methodology/principal findings: By using chicken retinal cultures we showed that EphA3 ectodomain stimulates nasal RGC axon growth in a concentration dependent way. Moreover, we showed that nasal axons choose growing on EphA3-expressing cells and that EphA3 diminishes the density of interstitial filopodia in nasal RGC axons. Accordingly, in vivo EphA3 ectodomain misexpression directs nasal optic fibers toward the caudal tectum preventing their branching in the rostral tectum.

Conclusions: We demonstrated in vitro and in vivo that EphA3 ectodomain (which is expressed in a decreasing rostro-caudal gradient in the tectum) is necessary for topographic mapping by stimulating the nasal axon growth toward the caudal tectum and inhibiting their branching in the rostral tectum. Furthermore, the ability of EphA3 of stimulating axon growth allows understanding how optic fibers invade the tectum growing throughout this molecular gradient. Therefore, opposing tectal gradients of repellent ephrin-As and of axon growth stimulating EphA3 complement each other to map optic fibers along the rostro-caudal tectal axis.

Show MeSH
Related in: MedlinePlus