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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 increases the velocity of nasal RGCs axon growth and RGC axons choose growing on EphA3-expressing cells.(A) Quantification of axonal growth rate (during the first 100 minutes since axon appearance) of nasal explants exposed to soluble clustered EphA3-Fc or Fc (Student's t test, 3 experiments, n: 12 axons for explant, 3 explants for condition). Results are shown as mean +/− SE. (B) Quantification of distance covered by nasal axons exposed to soluble clustered EphA3-Fc and Fc during the first 100 minutes after their appearance. Nasal axons grow faster with EphA3-Fc (p: 3.62*10−8, Student's t test). (C–E) Representative microphotographs of a time-lapse experiment. They show the behavior of axonal growth cones of nasal and temporal explants making contact with EphA3ΔC-EGFP-transfected-HEK293 cells or EGFP-F-transfected-HEK293 cells (control) (see arrows). (C) Nasal growth cones indistinctly attach or retract from EGFP-F-transfected-293 cells (Total time: 147 minutes). Similar results were obtained with temporal growth cones. (D) Nasal axons tend to grow along EphA3ΔC-EGFP-transfected-293 cells (Total time: 183 minutes) whereas (E) temporal axons tend to adhere to EphA3ΔC-EGFP-transfected-293 cells (Total time: 471 minutes). Scale bars  = 20 µm (see Videos S1, S2, S3). (F) Proportions of positive events (elongation or adhesion) produced by growth cones making contact with EphA3ΔC-EGFP-transfected-HEK293 cells versus EGFP-F transfected-HEK293 cells. Positive events significantly increase with EphA3 expressing-cells (Fisher's exact test; n: 79 nasal growth cones, 39 temporal growth cones; 3 nasal and 3 temporal explants for condition). (G) Discrimination of positive events between elongation and adhesion produced by growth cones making contact with EphA3ΔC-EGFP-transfected-HEK293 cells. Nasal axons present a significantly higher proportion of elongation than temporal ones (Fisher's exact test; n: 58 growth cones; 3 nasal and 3 temporal explants). Nasal (N), temporal (T).
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pone-0038566-g004: EphA3 ectodomain increases the velocity of nasal RGCs axon growth and RGC axons choose growing on EphA3-expressing cells.(A) Quantification of axonal growth rate (during the first 100 minutes since axon appearance) of nasal explants exposed to soluble clustered EphA3-Fc or Fc (Student's t test, 3 experiments, n: 12 axons for explant, 3 explants for condition). Results are shown as mean +/− SE. (B) Quantification of distance covered by nasal axons exposed to soluble clustered EphA3-Fc and Fc during the first 100 minutes after their appearance. Nasal axons grow faster with EphA3-Fc (p: 3.62*10−8, Student's t test). (C–E) Representative microphotographs of a time-lapse experiment. They show the behavior of axonal growth cones of nasal and temporal explants making contact with EphA3ΔC-EGFP-transfected-HEK293 cells or EGFP-F-transfected-HEK293 cells (control) (see arrows). (C) Nasal growth cones indistinctly attach or retract from EGFP-F-transfected-293 cells (Total time: 147 minutes). Similar results were obtained with temporal growth cones. (D) Nasal axons tend to grow along EphA3ΔC-EGFP-transfected-293 cells (Total time: 183 minutes) whereas (E) temporal axons tend to adhere to EphA3ΔC-EGFP-transfected-293 cells (Total time: 471 minutes). Scale bars  = 20 µm (see Videos S1, S2, S3). (F) Proportions of positive events (elongation or adhesion) produced by growth cones making contact with EphA3ΔC-EGFP-transfected-HEK293 cells versus EGFP-F transfected-HEK293 cells. Positive events significantly increase with EphA3 expressing-cells (Fisher's exact test; n: 79 nasal growth cones, 39 temporal growth cones; 3 nasal and 3 temporal explants for condition). (G) Discrimination of positive events between elongation and adhesion produced by growth cones making contact with EphA3ΔC-EGFP-transfected-HEK293 cells. Nasal axons present a significantly higher proportion of elongation than temporal ones (Fisher's exact test; n: 58 growth cones; 3 nasal and 3 temporal explants). Nasal (N), temporal (T).

Mentions: To investigate the dynamics of axon growth in retinal explants, we performed time-lapse experiments. These assays revealed that nasal RGC axons started to grow after 9 hours of exposure to both the soluble clustered EphA3-Fc or Fc control, but axons grew faster when exposed to EphA3-Fc than to Fc (Fig. 4A, B). Hence, EphA3-Fc enhances nasal axon growth without modifying the time point of initial axon formation.


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 increases the velocity of nasal RGCs axon growth and RGC axons choose growing on EphA3-expressing cells.(A) Quantification of axonal growth rate (during the first 100 minutes since axon appearance) of nasal explants exposed to soluble clustered EphA3-Fc or Fc (Student's t test, 3 experiments, n: 12 axons for explant, 3 explants for condition). Results are shown as mean +/− SE. (B) Quantification of distance covered by nasal axons exposed to soluble clustered EphA3-Fc and Fc during the first 100 minutes after their appearance. Nasal axons grow faster with EphA3-Fc (p: 3.62*10−8, Student's t test). (C–E) Representative microphotographs of a time-lapse experiment. They show the behavior of axonal growth cones of nasal and temporal explants making contact with EphA3ΔC-EGFP-transfected-HEK293 cells or EGFP-F-transfected-HEK293 cells (control) (see arrows). (C) Nasal growth cones indistinctly attach or retract from EGFP-F-transfected-293 cells (Total time: 147 minutes). Similar results were obtained with temporal growth cones. (D) Nasal axons tend to grow along EphA3ΔC-EGFP-transfected-293 cells (Total time: 183 minutes) whereas (E) temporal axons tend to adhere to EphA3ΔC-EGFP-transfected-293 cells (Total time: 471 minutes). Scale bars  = 20 µm (see Videos S1, S2, S3). (F) Proportions of positive events (elongation or adhesion) produced by growth cones making contact with EphA3ΔC-EGFP-transfected-HEK293 cells versus EGFP-F transfected-HEK293 cells. Positive events significantly increase with EphA3 expressing-cells (Fisher's exact test; n: 79 nasal growth cones, 39 temporal growth cones; 3 nasal and 3 temporal explants for condition). (G) Discrimination of positive events between elongation and adhesion produced by growth cones making contact with EphA3ΔC-EGFP-transfected-HEK293 cells. Nasal axons present a significantly higher proportion of elongation than temporal ones (Fisher's exact test; n: 58 growth cones; 3 nasal and 3 temporal explants). Nasal (N), temporal (T).
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pone-0038566-g004: EphA3 ectodomain increases the velocity of nasal RGCs axon growth and RGC axons choose growing on EphA3-expressing cells.(A) Quantification of axonal growth rate (during the first 100 minutes since axon appearance) of nasal explants exposed to soluble clustered EphA3-Fc or Fc (Student's t test, 3 experiments, n: 12 axons for explant, 3 explants for condition). Results are shown as mean +/− SE. (B) Quantification of distance covered by nasal axons exposed to soluble clustered EphA3-Fc and Fc during the first 100 minutes after their appearance. Nasal axons grow faster with EphA3-Fc (p: 3.62*10−8, Student's t test). (C–E) Representative microphotographs of a time-lapse experiment. They show the behavior of axonal growth cones of nasal and temporal explants making contact with EphA3ΔC-EGFP-transfected-HEK293 cells or EGFP-F-transfected-HEK293 cells (control) (see arrows). (C) Nasal growth cones indistinctly attach or retract from EGFP-F-transfected-293 cells (Total time: 147 minutes). Similar results were obtained with temporal growth cones. (D) Nasal axons tend to grow along EphA3ΔC-EGFP-transfected-293 cells (Total time: 183 minutes) whereas (E) temporal axons tend to adhere to EphA3ΔC-EGFP-transfected-293 cells (Total time: 471 minutes). Scale bars  = 20 µm (see Videos S1, S2, S3). (F) Proportions of positive events (elongation or adhesion) produced by growth cones making contact with EphA3ΔC-EGFP-transfected-HEK293 cells versus EGFP-F transfected-HEK293 cells. Positive events significantly increase with EphA3 expressing-cells (Fisher's exact test; n: 79 nasal growth cones, 39 temporal growth cones; 3 nasal and 3 temporal explants for condition). (G) Discrimination of positive events between elongation and adhesion produced by growth cones making contact with EphA3ΔC-EGFP-transfected-HEK293 cells. Nasal axons present a significantly higher proportion of elongation than temporal ones (Fisher's exact test; n: 58 growth cones; 3 nasal and 3 temporal explants). Nasal (N), temporal (T).
Mentions: To investigate the dynamics of axon growth in retinal explants, we performed time-lapse experiments. These assays revealed that nasal RGC axons started to grow after 9 hours of exposure to both the soluble clustered EphA3-Fc or Fc control, but axons grew faster when exposed to EphA3-Fc than to Fc (Fig. 4A, B). Hence, EphA3-Fc enhances nasal axon growth without modifying the time point of initial axon formation.

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