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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 overexpression alters the topographic map formation.After infection of the optic tectum at E2 with RCAS-BP-B-EGFPF (control) (A) or with RCAS-BP-B-EphA3ΔC-EGFPN3 (B) and DiI labeling of the naso-dorsal retina at E16, the retina and the tectum were analyzed in whole mounts at E18 (A, B). Graphs represent retinal whole mounts labeled in naso-dorsal areas (ND) with DiI (black circles) (upper) and tectal whole mounts where the corresponding DiI labeled RGC axons (drawings) are shown in microphotographs. (A) Optic fibers (OFs) pass throughout the intermediate tectum (arrowheads) and form a termination zone (TZ) (arrow) over an area of EGFPF expression in the corresponding target area (caudal tectum) in a control tectum. (B) OFs pass through the target area (caudal tectum) without producing TZs (arrowheads) in a tectum where EphA3ΔC-EGFP was overexpressed. N: nasal, T: temporal, V: ventral, D: dorsal, R: rostral, C: caudal. Scale bars  = 50 µm. (C–F) Graphs show the proportions of temporal (C, E) and nasal (D, F) OFs which form TZs in the rostral and the caudal tectum respectively in EGFPF-expressing tecta versus EphA3ΔC-EGFP-overexpressed tecta. (C, D) represent the proportions of TZs observed after the remodeling period -E15 (HH41)-E19 (HH45)- whereas (E, F) represent the proportions of TZs observed during the remodeling period -E11 (HH37)-E14 (HH40)-. (D) Nasal RGC axons (N RGC) present a significantly lower proportion of TZs in the caudal area of EphA3ΔC-EGFP-overexpressed tecta between E15 and E19 whereas (C) temporal RGC axons (T RGC) do not present any significant difference in the rostral area of EphA3ΔC-EGFP-overexpressed tecta between E15 and E19 (Student t test, n: 6 EphA3ΔC-EGFP-overexpressed tecta versus 7 control tecta for nasal RGC; n: 4 EphA3ΔC-EGFP-overexpressed tecta versus 10 control tecta for temporal RGCs). (F) Nasal RGCs present a significantly lower proportion of TZs in the caudal area of EphA3ΔC-EGFP-overexpressed tecta between E11 and E14 whereas (E) temporal RGCs do not present any significant difference between the rostral areas of the EphA3ΔC-EGFP-overexpressed tecta and control tecta (Student t test; n: 6 EphA3ΔC-EGFP-overexpressed tecta versus 41 control tecta for nasal RGCs and 5 EphA3ΔC-EGFP-overexpressed tecta versus 53 control tecta for temporal RGCs). N: nasal, T: temporal, V: ventral, D: dorsal, R: rostral, C: caudal. Results are shown as mean +/− SE.
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pone-0038566-g007: EphA3 ectodomain overexpression alters the topographic map formation.After infection of the optic tectum at E2 with RCAS-BP-B-EGFPF (control) (A) or with RCAS-BP-B-EphA3ΔC-EGFPN3 (B) and DiI labeling of the naso-dorsal retina at E16, the retina and the tectum were analyzed in whole mounts at E18 (A, B). Graphs represent retinal whole mounts labeled in naso-dorsal areas (ND) with DiI (black circles) (upper) and tectal whole mounts where the corresponding DiI labeled RGC axons (drawings) are shown in microphotographs. (A) Optic fibers (OFs) pass throughout the intermediate tectum (arrowheads) and form a termination zone (TZ) (arrow) over an area of EGFPF expression in the corresponding target area (caudal tectum) in a control tectum. (B) OFs pass through the target area (caudal tectum) without producing TZs (arrowheads) in a tectum where EphA3ΔC-EGFP was overexpressed. N: nasal, T: temporal, V: ventral, D: dorsal, R: rostral, C: caudal. Scale bars  = 50 µm. (C–F) Graphs show the proportions of temporal (C, E) and nasal (D, F) OFs which form TZs in the rostral and the caudal tectum respectively in EGFPF-expressing tecta versus EphA3ΔC-EGFP-overexpressed tecta. (C, D) represent the proportions of TZs observed after the remodeling period -E15 (HH41)-E19 (HH45)- whereas (E, F) represent the proportions of TZs observed during the remodeling period -E11 (HH37)-E14 (HH40)-. (D) Nasal RGC axons (N RGC) present a significantly lower proportion of TZs in the caudal area of EphA3ΔC-EGFP-overexpressed tecta between E15 and E19 whereas (C) temporal RGC axons (T RGC) do not present any significant difference in the rostral area of EphA3ΔC-EGFP-overexpressed tecta between E15 and E19 (Student t test, n: 6 EphA3ΔC-EGFP-overexpressed tecta versus 7 control tecta for nasal RGC; n: 4 EphA3ΔC-EGFP-overexpressed tecta versus 10 control tecta for temporal RGCs). (F) Nasal RGCs present a significantly lower proportion of TZs in the caudal area of EphA3ΔC-EGFP-overexpressed tecta between E11 and E14 whereas (E) temporal RGCs do not present any significant difference between the rostral areas of the EphA3ΔC-EGFP-overexpressed tecta and control tecta (Student t test; n: 6 EphA3ΔC-EGFP-overexpressed tecta versus 41 control tecta for nasal RGCs and 5 EphA3ΔC-EGFP-overexpressed tecta versus 53 control tecta for temporal RGCs). N: nasal, T: temporal, V: ventral, D: dorsal, R: rostral, C: caudal. Results are shown as mean +/− SE.

Mentions: To analyze the retinotectal mapping along the rostro-caudal axis, we determined the proportions of temporal and nasal RGC axons which passed through or formed termination zones in the corresponding target area along the tectal rostro-caudal axis when a mature retinotectal projection was established (between E15 and E19). This analysis showed that EphA3 ectodomain overexpression significantly decreased the proportion of termination zones formed by nasal RGC axons in the caudal tectum (21.18%+/−9.47) when compared to the control-EGFPF-expressing tecta, (75.82%+/−4.84) (Fig. 7A, B, D). Thus, EphA3 ectodomain overexpression increased the proportion of nasal optic fibers which passed through the corresponding target areas and projected to the caudal end of the tectum. However, EphA3 ectodomain overexpression did not produce any significant change in the proportion of termination zones formed by temporal RGC axons in the rostral tectum (74.38%+/−9.89% in EGFPF-expressing tecta versus 49.29%+/−3.52% in EphA3ΔC-EGFP overexpressing tecta) (Fig. 7C). The penetrance of this phenotype was 100%. These results demonstrate that EphA3 ectodomain overexpression alters topographic map formation by stimulating nasal axon growth to the caudal tectum and decreasing branch 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 overexpression alters the topographic map formation.After infection of the optic tectum at E2 with RCAS-BP-B-EGFPF (control) (A) or with RCAS-BP-B-EphA3ΔC-EGFPN3 (B) and DiI labeling of the naso-dorsal retina at E16, the retina and the tectum were analyzed in whole mounts at E18 (A, B). Graphs represent retinal whole mounts labeled in naso-dorsal areas (ND) with DiI (black circles) (upper) and tectal whole mounts where the corresponding DiI labeled RGC axons (drawings) are shown in microphotographs. (A) Optic fibers (OFs) pass throughout the intermediate tectum (arrowheads) and form a termination zone (TZ) (arrow) over an area of EGFPF expression in the corresponding target area (caudal tectum) in a control tectum. (B) OFs pass through the target area (caudal tectum) without producing TZs (arrowheads) in a tectum where EphA3ΔC-EGFP was overexpressed. N: nasal, T: temporal, V: ventral, D: dorsal, R: rostral, C: caudal. Scale bars  = 50 µm. (C–F) Graphs show the proportions of temporal (C, E) and nasal (D, F) OFs which form TZs in the rostral and the caudal tectum respectively in EGFPF-expressing tecta versus EphA3ΔC-EGFP-overexpressed tecta. (C, D) represent the proportions of TZs observed after the remodeling period -E15 (HH41)-E19 (HH45)- whereas (E, F) represent the proportions of TZs observed during the remodeling period -E11 (HH37)-E14 (HH40)-. (D) Nasal RGC axons (N RGC) present a significantly lower proportion of TZs in the caudal area of EphA3ΔC-EGFP-overexpressed tecta between E15 and E19 whereas (C) temporal RGC axons (T RGC) do not present any significant difference in the rostral area of EphA3ΔC-EGFP-overexpressed tecta between E15 and E19 (Student t test, n: 6 EphA3ΔC-EGFP-overexpressed tecta versus 7 control tecta for nasal RGC; n: 4 EphA3ΔC-EGFP-overexpressed tecta versus 10 control tecta for temporal RGCs). (F) Nasal RGCs present a significantly lower proportion of TZs in the caudal area of EphA3ΔC-EGFP-overexpressed tecta between E11 and E14 whereas (E) temporal RGCs do not present any significant difference between the rostral areas of the EphA3ΔC-EGFP-overexpressed tecta and control tecta (Student t test; n: 6 EphA3ΔC-EGFP-overexpressed tecta versus 41 control tecta for nasal RGCs and 5 EphA3ΔC-EGFP-overexpressed tecta versus 53 control tecta for temporal RGCs). N: nasal, T: temporal, V: ventral, D: dorsal, R: rostral, C: caudal. Results are shown as mean +/− SE.
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pone-0038566-g007: EphA3 ectodomain overexpression alters the topographic map formation.After infection of the optic tectum at E2 with RCAS-BP-B-EGFPF (control) (A) or with RCAS-BP-B-EphA3ΔC-EGFPN3 (B) and DiI labeling of the naso-dorsal retina at E16, the retina and the tectum were analyzed in whole mounts at E18 (A, B). Graphs represent retinal whole mounts labeled in naso-dorsal areas (ND) with DiI (black circles) (upper) and tectal whole mounts where the corresponding DiI labeled RGC axons (drawings) are shown in microphotographs. (A) Optic fibers (OFs) pass throughout the intermediate tectum (arrowheads) and form a termination zone (TZ) (arrow) over an area of EGFPF expression in the corresponding target area (caudal tectum) in a control tectum. (B) OFs pass through the target area (caudal tectum) without producing TZs (arrowheads) in a tectum where EphA3ΔC-EGFP was overexpressed. N: nasal, T: temporal, V: ventral, D: dorsal, R: rostral, C: caudal. Scale bars  = 50 µm. (C–F) Graphs show the proportions of temporal (C, E) and nasal (D, F) OFs which form TZs in the rostral and the caudal tectum respectively in EGFPF-expressing tecta versus EphA3ΔC-EGFP-overexpressed tecta. (C, D) represent the proportions of TZs observed after the remodeling period -E15 (HH41)-E19 (HH45)- whereas (E, F) represent the proportions of TZs observed during the remodeling period -E11 (HH37)-E14 (HH40)-. (D) Nasal RGC axons (N RGC) present a significantly lower proportion of TZs in the caudal area of EphA3ΔC-EGFP-overexpressed tecta between E15 and E19 whereas (C) temporal RGC axons (T RGC) do not present any significant difference in the rostral area of EphA3ΔC-EGFP-overexpressed tecta between E15 and E19 (Student t test, n: 6 EphA3ΔC-EGFP-overexpressed tecta versus 7 control tecta for nasal RGC; n: 4 EphA3ΔC-EGFP-overexpressed tecta versus 10 control tecta for temporal RGCs). (F) Nasal RGCs present a significantly lower proportion of TZs in the caudal area of EphA3ΔC-EGFP-overexpressed tecta between E11 and E14 whereas (E) temporal RGCs do not present any significant difference between the rostral areas of the EphA3ΔC-EGFP-overexpressed tecta and control tecta (Student t test; n: 6 EphA3ΔC-EGFP-overexpressed tecta versus 41 control tecta for nasal RGCs and 5 EphA3ΔC-EGFP-overexpressed tecta versus 53 control tecta for temporal RGCs). N: nasal, T: temporal, V: ventral, D: dorsal, R: rostral, C: caudal. Results are shown as mean +/− SE.
Mentions: To analyze the retinotectal mapping along the rostro-caudal axis, we determined the proportions of temporal and nasal RGC axons which passed through or formed termination zones in the corresponding target area along the tectal rostro-caudal axis when a mature retinotectal projection was established (between E15 and E19). This analysis showed that EphA3 ectodomain overexpression significantly decreased the proportion of termination zones formed by nasal RGC axons in the caudal tectum (21.18%+/−9.47) when compared to the control-EGFPF-expressing tecta, (75.82%+/−4.84) (Fig. 7A, B, D). Thus, EphA3 ectodomain overexpression increased the proportion of nasal optic fibers which passed through the corresponding target areas and projected to the caudal end of the tectum. However, EphA3 ectodomain overexpression did not produce any significant change in the proportion of termination zones formed by temporal RGC axons in the rostral tectum (74.38%+/−9.89% in EGFPF-expressing tecta versus 49.29%+/−3.52% in EphA3ΔC-EGFP overexpressing tecta) (Fig. 7C). The penetrance of this phenotype was 100%. These results demonstrate that EphA3 ectodomain overexpression alters topographic map formation by stimulating nasal axon growth to the caudal tectum and decreasing branch 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.

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Related in: MedlinePlus