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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 stimulates nasal RGC axon growth in vitro.(A, B) Microphotographs of nasal retinal explants grown on clustered Fc (A) or clustered EphA3-Fc at 2 nM (B). RGC axons grow longer on EphA3-Fc. Scale bars  = 20 µm. (C–D) Quantification of axon length of nasal (C) and temporal explants (D) grown on substrates formed by laminin and clustered Fc or EphA3-Fc at different concentrations. Axon length is indicated in µm and concentrations are indicated in nM of Fc or EphA3-Fc. Temporal explants grow longer axons than nasal ones in control conditions (p: 0.0002, compare first bar of nasal RGCs in C with first bar of temporal RGCs in D). EphA3-Fc increased nasal RGCs axon growth from 1 to 4 nM showing a peak at 2 nM. Temporal RGCs did not present any significant change in axon growth on EphA3-Fc between 0.5 and 4 nM and presented a significant decrease at 8 nM (ANOVA and Tukey postest, 3 independent experiments, n: 20 longer axons for explant, 3 explants for condition). (E) Quantification of axon length of nasal and temporal explants exposed to soluble clustered Fc or EphA3-Fc at 2 nM. Nasal RGC axons grow significantly longer with EphA3-Fc (ANOVA and Tukey postest, 3 independent experiments, n: 50 longer axons for explant, 3 explants for condition). (F–G) Dissociated nasal retinal neurons immunolabeled against neuron specific βIII tubulin. They present longer axons on clustered EphA3-Fc at 2 nM (G) than on clustered Fc (F). Scale bars  = 10 µm. (H) Quantification of axon length of nasal and temporal dissociated retinal neurons grown on clustered Fc or EphA3-Fc at 2 nM. Nasal retinal neurons grow significantly longer axons on EphA3-Fc. (ANOVA and Tukey postest, 3 independent experiments, n: nasal retinal neurons on EphA3-Fc: 288, nasal retinal neurons on Fc: 328, temporal retinal neurons on EphA3-Fc: 636, temporal retinal neurons on Fc: 646). (I) The plot depicts the distribution of axon length of nasal and temporal dissociated retinal neurons (NRN and TRN) grown on EphA3-Fc versus Fc. Values given on the y-axis indicate the proportion of retinal neurons which axons reach the length shown on the x-axis. Nasal retinal neurons present a higher proportion of axons between 20 and 40 µm (n: nasal retinal neurons on EphA3-Fc: 288, nasal retinal neurons on Fc: 328, temporal retinal neurons on EphA3-Fc: 636, temporal retinal neurons on Fc: 646). Results are shown as mean +/– SE.
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pone-0038566-g003: EphA3 ectodomain stimulates nasal RGC axon growth in vitro.(A, B) Microphotographs of nasal retinal explants grown on clustered Fc (A) or clustered EphA3-Fc at 2 nM (B). RGC axons grow longer on EphA3-Fc. Scale bars  = 20 µm. (C–D) Quantification of axon length of nasal (C) and temporal explants (D) grown on substrates formed by laminin and clustered Fc or EphA3-Fc at different concentrations. Axon length is indicated in µm and concentrations are indicated in nM of Fc or EphA3-Fc. Temporal explants grow longer axons than nasal ones in control conditions (p: 0.0002, compare first bar of nasal RGCs in C with first bar of temporal RGCs in D). EphA3-Fc increased nasal RGCs axon growth from 1 to 4 nM showing a peak at 2 nM. Temporal RGCs did not present any significant change in axon growth on EphA3-Fc between 0.5 and 4 nM and presented a significant decrease at 8 nM (ANOVA and Tukey postest, 3 independent experiments, n: 20 longer axons for explant, 3 explants for condition). (E) Quantification of axon length of nasal and temporal explants exposed to soluble clustered Fc or EphA3-Fc at 2 nM. Nasal RGC axons grow significantly longer with EphA3-Fc (ANOVA and Tukey postest, 3 independent experiments, n: 50 longer axons for explant, 3 explants for condition). (F–G) Dissociated nasal retinal neurons immunolabeled against neuron specific βIII tubulin. They present longer axons on clustered EphA3-Fc at 2 nM (G) than on clustered Fc (F). Scale bars  = 10 µm. (H) Quantification of axon length of nasal and temporal dissociated retinal neurons grown on clustered Fc or EphA3-Fc at 2 nM. Nasal retinal neurons grow significantly longer axons on EphA3-Fc. (ANOVA and Tukey postest, 3 independent experiments, n: nasal retinal neurons on EphA3-Fc: 288, nasal retinal neurons on Fc: 328, temporal retinal neurons on EphA3-Fc: 636, temporal retinal neurons on Fc: 646). (I) The plot depicts the distribution of axon length of nasal and temporal dissociated retinal neurons (NRN and TRN) grown on EphA3-Fc versus Fc. Values given on the y-axis indicate the proportion of retinal neurons which axons reach the length shown on the x-axis. Nasal retinal neurons present a higher proportion of axons between 20 and 40 µm (n: nasal retinal neurons on EphA3-Fc: 288, nasal retinal neurons on Fc: 328, temporal retinal neurons on EphA3-Fc: 636, temporal retinal neurons on Fc: 646). Results are shown as mean +/– SE.

Mentions: EphA3-Fc induced a concentration-dependent response on axon growth which was specific for nasal and temporal RGCs (Fig. 3A–D). Overall, nasal RGCs presented shorter axons than temporal RGCs in control conditions. The former were 18.95 %+/−4.8 % shorter than temporal axons, (compare first bar of Fig. 3C with first bar of Fig. 3D). EphA3-Fc significantly increased nasal RGC axon growth between 1 nM and 4 nM (Fig. 3C), presenting the maximal effect at 2 nM (this concentration increased axon length in 128.14 %+/−12.17 % with respect to control). These concentrations of EphA3-Fc did not produce any significant effect on temporal RGC axon growth (Fig. 3D). However, higher concentrations of EphA3-Fc (8 nM) did not present any significant effect on nasal RGC axon growth (Fig. 3C) whereas decreased temporal RGC axon growth (Fig. 3D).


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 stimulates nasal RGC axon growth in vitro.(A, B) Microphotographs of nasal retinal explants grown on clustered Fc (A) or clustered EphA3-Fc at 2 nM (B). RGC axons grow longer on EphA3-Fc. Scale bars  = 20 µm. (C–D) Quantification of axon length of nasal (C) and temporal explants (D) grown on substrates formed by laminin and clustered Fc or EphA3-Fc at different concentrations. Axon length is indicated in µm and concentrations are indicated in nM of Fc or EphA3-Fc. Temporal explants grow longer axons than nasal ones in control conditions (p: 0.0002, compare first bar of nasal RGCs in C with first bar of temporal RGCs in D). EphA3-Fc increased nasal RGCs axon growth from 1 to 4 nM showing a peak at 2 nM. Temporal RGCs did not present any significant change in axon growth on EphA3-Fc between 0.5 and 4 nM and presented a significant decrease at 8 nM (ANOVA and Tukey postest, 3 independent experiments, n: 20 longer axons for explant, 3 explants for condition). (E) Quantification of axon length of nasal and temporal explants exposed to soluble clustered Fc or EphA3-Fc at 2 nM. Nasal RGC axons grow significantly longer with EphA3-Fc (ANOVA and Tukey postest, 3 independent experiments, n: 50 longer axons for explant, 3 explants for condition). (F–G) Dissociated nasal retinal neurons immunolabeled against neuron specific βIII tubulin. They present longer axons on clustered EphA3-Fc at 2 nM (G) than on clustered Fc (F). Scale bars  = 10 µm. (H) Quantification of axon length of nasal and temporal dissociated retinal neurons grown on clustered Fc or EphA3-Fc at 2 nM. Nasal retinal neurons grow significantly longer axons on EphA3-Fc. (ANOVA and Tukey postest, 3 independent experiments, n: nasal retinal neurons on EphA3-Fc: 288, nasal retinal neurons on Fc: 328, temporal retinal neurons on EphA3-Fc: 636, temporal retinal neurons on Fc: 646). (I) The plot depicts the distribution of axon length of nasal and temporal dissociated retinal neurons (NRN and TRN) grown on EphA3-Fc versus Fc. Values given on the y-axis indicate the proportion of retinal neurons which axons reach the length shown on the x-axis. Nasal retinal neurons present a higher proportion of axons between 20 and 40 µm (n: nasal retinal neurons on EphA3-Fc: 288, nasal retinal neurons on Fc: 328, temporal retinal neurons on EphA3-Fc: 636, temporal retinal neurons on Fc: 646). 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-g003: EphA3 ectodomain stimulates nasal RGC axon growth in vitro.(A, B) Microphotographs of nasal retinal explants grown on clustered Fc (A) or clustered EphA3-Fc at 2 nM (B). RGC axons grow longer on EphA3-Fc. Scale bars  = 20 µm. (C–D) Quantification of axon length of nasal (C) and temporal explants (D) grown on substrates formed by laminin and clustered Fc or EphA3-Fc at different concentrations. Axon length is indicated in µm and concentrations are indicated in nM of Fc or EphA3-Fc. Temporal explants grow longer axons than nasal ones in control conditions (p: 0.0002, compare first bar of nasal RGCs in C with first bar of temporal RGCs in D). EphA3-Fc increased nasal RGCs axon growth from 1 to 4 nM showing a peak at 2 nM. Temporal RGCs did not present any significant change in axon growth on EphA3-Fc between 0.5 and 4 nM and presented a significant decrease at 8 nM (ANOVA and Tukey postest, 3 independent experiments, n: 20 longer axons for explant, 3 explants for condition). (E) Quantification of axon length of nasal and temporal explants exposed to soluble clustered Fc or EphA3-Fc at 2 nM. Nasal RGC axons grow significantly longer with EphA3-Fc (ANOVA and Tukey postest, 3 independent experiments, n: 50 longer axons for explant, 3 explants for condition). (F–G) Dissociated nasal retinal neurons immunolabeled against neuron specific βIII tubulin. They present longer axons on clustered EphA3-Fc at 2 nM (G) than on clustered Fc (F). Scale bars  = 10 µm. (H) Quantification of axon length of nasal and temporal dissociated retinal neurons grown on clustered Fc or EphA3-Fc at 2 nM. Nasal retinal neurons grow significantly longer axons on EphA3-Fc. (ANOVA and Tukey postest, 3 independent experiments, n: nasal retinal neurons on EphA3-Fc: 288, nasal retinal neurons on Fc: 328, temporal retinal neurons on EphA3-Fc: 636, temporal retinal neurons on Fc: 646). (I) The plot depicts the distribution of axon length of nasal and temporal dissociated retinal neurons (NRN and TRN) grown on EphA3-Fc versus Fc. Values given on the y-axis indicate the proportion of retinal neurons which axons reach the length shown on the x-axis. Nasal retinal neurons present a higher proportion of axons between 20 and 40 µm (n: nasal retinal neurons on EphA3-Fc: 288, nasal retinal neurons on Fc: 328, temporal retinal neurons on EphA3-Fc: 636, temporal retinal neurons on Fc: 646). Results are shown as mean +/– SE.
Mentions: EphA3-Fc induced a concentration-dependent response on axon growth which was specific for nasal and temporal RGCs (Fig. 3A–D). Overall, nasal RGCs presented shorter axons than temporal RGCs in control conditions. The former were 18.95 %+/−4.8 % shorter than temporal axons, (compare first bar of Fig. 3C with first bar of Fig. 3D). EphA3-Fc significantly increased nasal RGC axon growth between 1 nM and 4 nM (Fig. 3C), presenting the maximal effect at 2 nM (this concentration increased axon length in 128.14 %+/−12.17 % with respect to control). These concentrations of EphA3-Fc did not produce any significant effect on temporal RGC axon growth (Fig. 3D). However, higher concentrations of EphA3-Fc (8 nM) did not present any significant effect on nasal RGC axon growth (Fig. 3C) whereas decreased temporal RGC axon growth (Fig. 3D).

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