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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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Ephrin-A2 and –A6 expression and EphA4 Tyr-602 phosphorylation decrease from nasal to temporal RGCs.(A–E) Expression gradients of ephrin-A2 and –A6 depend upon the level of expression of each RGC and the proportion of RGCs that express them. (A, B) Immunofluorescence against ephrin-A2 (left), phase-contrast (middle) and merge (right) of RGC axons grown during 24 hours from E7 nasal (A) and temporal (B) retinal explants are shown. Expression intensity and proportion of labeled axons are higher in nasal explants. Negative axons are depicted (arrowheads) in temporal explants. Scale bars  = 10 µm. (C) Quantification shows that a higher proportion of nasal axons express ephrin-A2 and –A6 (Student's t test, ephrin-A2: 4 independent experiments, 557 nasal and 733 temporal axons; ephrin-A6: 2 independent experiments, 326 nasal and 181 temporal axons). (D) Quantification of fluorescent intensity measured as integral optic density (IOD) in axons of nasal and temporal explants immunolabeled against ephrin-A2 shows that nasal axons present higher levels of expression (Mann Whitney test, 3 independent experiments, n: 52 nasal and 49 temporal axons). (E) Histogram of fluorescent intensity of one experiment shows that nasal and temporal axons form two partially overlapping populations where the major proportion of nasal axons presents higher levels of intensity than the major proportion of temporal axons. Similar results were obtained in the other experiments. (F, G) Immunofluorescence against Tyr-602 phosphorylated-EphA4 in nasal (F) and temporal (G) retinal explants are shown. Expression is higher in nasal axons. Scale bars  = 10 µm. (H) IOD is significantly higher in nasal axons (Mann Whitney test, 3 independent experiments, n: 75 nasal and 84 temporal axons). I. Histogram of fluorescent intensity shows that nasal and temporal axons form two partially overlapping populations where the major proportion of nasal axons presents higher levels of intensity than the major proportion of temporal axons. Similar results were obtained in the other experiments. (J) Western blot analysis against ephrin-A2, Tyr-602-phosphorylated-EphA4 and EphA4 of crude membrane fractions obtained from nasal and temporal retinas at E8. (K, L) Quantification of ephrin-A2/actin (K) and Tyr-602-phosphorylated-EphA4/actin ratios (L) from 3 similar experiments shows that nasal retina presents significantly higher levels of ephrin-A2 and Tyr-602-phosphorylated-EphA4 (Student's t test). (M, N) Confocal microphotographs of nasal (M) and temporal (N) axons grown from retinal explants immunolabeled against ephrin-A2 (red) and EphA4 (green) are shown. Right images are the merges. They show that there are more patches of overlapping (yellow) in the nasal axonal shaft and growth cone than in the temporal ones. Scale bars  = 5 µm. (Pearson's correlation coefficient: 0.62+/−0.011 versus 0.55+/−0.008, p: 0.045; Manders overlap coefficient: 0.82+/−0.002 versus 0.75+/−0.001, p: 9.56*10−6, 2 independent experiments; n: nasal RGCs: 30 axons, temporal RGCs: 26). Results are shown as mean +/− SE,. Nasal RGC axons (N), temporal RGC axons (T).
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pone-0038566-g002: Ephrin-A2 and –A6 expression and EphA4 Tyr-602 phosphorylation decrease from nasal to temporal RGCs.(A–E) Expression gradients of ephrin-A2 and –A6 depend upon the level of expression of each RGC and the proportion of RGCs that express them. (A, B) Immunofluorescence against ephrin-A2 (left), phase-contrast (middle) and merge (right) of RGC axons grown during 24 hours from E7 nasal (A) and temporal (B) retinal explants are shown. Expression intensity and proportion of labeled axons are higher in nasal explants. Negative axons are depicted (arrowheads) in temporal explants. Scale bars  = 10 µm. (C) Quantification shows that a higher proportion of nasal axons express ephrin-A2 and –A6 (Student's t test, ephrin-A2: 4 independent experiments, 557 nasal and 733 temporal axons; ephrin-A6: 2 independent experiments, 326 nasal and 181 temporal axons). (D) Quantification of fluorescent intensity measured as integral optic density (IOD) in axons of nasal and temporal explants immunolabeled against ephrin-A2 shows that nasal axons present higher levels of expression (Mann Whitney test, 3 independent experiments, n: 52 nasal and 49 temporal axons). (E) Histogram of fluorescent intensity of one experiment shows that nasal and temporal axons form two partially overlapping populations where the major proportion of nasal axons presents higher levels of intensity than the major proportion of temporal axons. Similar results were obtained in the other experiments. (F, G) Immunofluorescence against Tyr-602 phosphorylated-EphA4 in nasal (F) and temporal (G) retinal explants are shown. Expression is higher in nasal axons. Scale bars  = 10 µm. (H) IOD is significantly higher in nasal axons (Mann Whitney test, 3 independent experiments, n: 75 nasal and 84 temporal axons). I. Histogram of fluorescent intensity shows that nasal and temporal axons form two partially overlapping populations where the major proportion of nasal axons presents higher levels of intensity than the major proportion of temporal axons. Similar results were obtained in the other experiments. (J) Western blot analysis against ephrin-A2, Tyr-602-phosphorylated-EphA4 and EphA4 of crude membrane fractions obtained from nasal and temporal retinas at E8. (K, L) Quantification of ephrin-A2/actin (K) and Tyr-602-phosphorylated-EphA4/actin ratios (L) from 3 similar experiments shows that nasal retina presents significantly higher levels of ephrin-A2 and Tyr-602-phosphorylated-EphA4 (Student's t test). (M, N) Confocal microphotographs of nasal (M) and temporal (N) axons grown from retinal explants immunolabeled against ephrin-A2 (red) and EphA4 (green) are shown. Right images are the merges. They show that there are more patches of overlapping (yellow) in the nasal axonal shaft and growth cone than in the temporal ones. Scale bars  = 5 µm. (Pearson's correlation coefficient: 0.62+/−0.011 versus 0.55+/−0.008, p: 0.045; Manders overlap coefficient: 0.82+/−0.002 versus 0.75+/−0.001, p: 9.56*10−6, 2 independent experiments; n: nasal RGCs: 30 axons, temporal RGCs: 26). Results are shown as mean +/− SE,. Nasal RGC axons (N), temporal RGC axons (T).

Mentions: These studies showed that RGC axons (shafts and growth cones) express ephrin-A2 and –A6 in both nasal and temporal retinal thirds but presenting a decreasing naso-temporal gradient (Fig. 2A, B). To investigate whether this gradient is due to the number of axons which express ephrin-As or to the level of expression of each axon, we quantified the proportion of axons which express ephrin-A2 and –A6 and the intensity of labeling of each axon (Fig. 2A–E). We showed that not only temporal RGC axons express lower levels of ephrin-As with respect to nasal axons, but also a lower proportion of temporal RGC axons express ephrin-As if compared to nasal axons. The distribution profile appreciated in the histogram of fluorescent intensity (Fig. 2E) showed that nasal and temporal axons form two partially overlapping populations, the former presenting higher levels of expression.


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)

Ephrin-A2 and –A6 expression and EphA4 Tyr-602 phosphorylation decrease from nasal to temporal RGCs.(A–E) Expression gradients of ephrin-A2 and –A6 depend upon the level of expression of each RGC and the proportion of RGCs that express them. (A, B) Immunofluorescence against ephrin-A2 (left), phase-contrast (middle) and merge (right) of RGC axons grown during 24 hours from E7 nasal (A) and temporal (B) retinal explants are shown. Expression intensity and proportion of labeled axons are higher in nasal explants. Negative axons are depicted (arrowheads) in temporal explants. Scale bars  = 10 µm. (C) Quantification shows that a higher proportion of nasal axons express ephrin-A2 and –A6 (Student's t test, ephrin-A2: 4 independent experiments, 557 nasal and 733 temporal axons; ephrin-A6: 2 independent experiments, 326 nasal and 181 temporal axons). (D) Quantification of fluorescent intensity measured as integral optic density (IOD) in axons of nasal and temporal explants immunolabeled against ephrin-A2 shows that nasal axons present higher levels of expression (Mann Whitney test, 3 independent experiments, n: 52 nasal and 49 temporal axons). (E) Histogram of fluorescent intensity of one experiment shows that nasal and temporal axons form two partially overlapping populations where the major proportion of nasal axons presents higher levels of intensity than the major proportion of temporal axons. Similar results were obtained in the other experiments. (F, G) Immunofluorescence against Tyr-602 phosphorylated-EphA4 in nasal (F) and temporal (G) retinal explants are shown. Expression is higher in nasal axons. Scale bars  = 10 µm. (H) IOD is significantly higher in nasal axons (Mann Whitney test, 3 independent experiments, n: 75 nasal and 84 temporal axons). I. Histogram of fluorescent intensity shows that nasal and temporal axons form two partially overlapping populations where the major proportion of nasal axons presents higher levels of intensity than the major proportion of temporal axons. Similar results were obtained in the other experiments. (J) Western blot analysis against ephrin-A2, Tyr-602-phosphorylated-EphA4 and EphA4 of crude membrane fractions obtained from nasal and temporal retinas at E8. (K, L) Quantification of ephrin-A2/actin (K) and Tyr-602-phosphorylated-EphA4/actin ratios (L) from 3 similar experiments shows that nasal retina presents significantly higher levels of ephrin-A2 and Tyr-602-phosphorylated-EphA4 (Student's t test). (M, N) Confocal microphotographs of nasal (M) and temporal (N) axons grown from retinal explants immunolabeled against ephrin-A2 (red) and EphA4 (green) are shown. Right images are the merges. They show that there are more patches of overlapping (yellow) in the nasal axonal shaft and growth cone than in the temporal ones. Scale bars  = 5 µm. (Pearson's correlation coefficient: 0.62+/−0.011 versus 0.55+/−0.008, p: 0.045; Manders overlap coefficient: 0.82+/−0.002 versus 0.75+/−0.001, p: 9.56*10−6, 2 independent experiments; n: nasal RGCs: 30 axons, temporal RGCs: 26). Results are shown as mean +/− SE,. Nasal RGC axons (N), temporal RGC axons (T).
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getmorefigures.php?uid=PMC3369860&req=5

pone-0038566-g002: Ephrin-A2 and –A6 expression and EphA4 Tyr-602 phosphorylation decrease from nasal to temporal RGCs.(A–E) Expression gradients of ephrin-A2 and –A6 depend upon the level of expression of each RGC and the proportion of RGCs that express them. (A, B) Immunofluorescence against ephrin-A2 (left), phase-contrast (middle) and merge (right) of RGC axons grown during 24 hours from E7 nasal (A) and temporal (B) retinal explants are shown. Expression intensity and proportion of labeled axons are higher in nasal explants. Negative axons are depicted (arrowheads) in temporal explants. Scale bars  = 10 µm. (C) Quantification shows that a higher proportion of nasal axons express ephrin-A2 and –A6 (Student's t test, ephrin-A2: 4 independent experiments, 557 nasal and 733 temporal axons; ephrin-A6: 2 independent experiments, 326 nasal and 181 temporal axons). (D) Quantification of fluorescent intensity measured as integral optic density (IOD) in axons of nasal and temporal explants immunolabeled against ephrin-A2 shows that nasal axons present higher levels of expression (Mann Whitney test, 3 independent experiments, n: 52 nasal and 49 temporal axons). (E) Histogram of fluorescent intensity of one experiment shows that nasal and temporal axons form two partially overlapping populations where the major proportion of nasal axons presents higher levels of intensity than the major proportion of temporal axons. Similar results were obtained in the other experiments. (F, G) Immunofluorescence against Tyr-602 phosphorylated-EphA4 in nasal (F) and temporal (G) retinal explants are shown. Expression is higher in nasal axons. Scale bars  = 10 µm. (H) IOD is significantly higher in nasal axons (Mann Whitney test, 3 independent experiments, n: 75 nasal and 84 temporal axons). I. Histogram of fluorescent intensity shows that nasal and temporal axons form two partially overlapping populations where the major proportion of nasal axons presents higher levels of intensity than the major proportion of temporal axons. Similar results were obtained in the other experiments. (J) Western blot analysis against ephrin-A2, Tyr-602-phosphorylated-EphA4 and EphA4 of crude membrane fractions obtained from nasal and temporal retinas at E8. (K, L) Quantification of ephrin-A2/actin (K) and Tyr-602-phosphorylated-EphA4/actin ratios (L) from 3 similar experiments shows that nasal retina presents significantly higher levels of ephrin-A2 and Tyr-602-phosphorylated-EphA4 (Student's t test). (M, N) Confocal microphotographs of nasal (M) and temporal (N) axons grown from retinal explants immunolabeled against ephrin-A2 (red) and EphA4 (green) are shown. Right images are the merges. They show that there are more patches of overlapping (yellow) in the nasal axonal shaft and growth cone than in the temporal ones. Scale bars  = 5 µm. (Pearson's correlation coefficient: 0.62+/−0.011 versus 0.55+/−0.008, p: 0.045; Manders overlap coefficient: 0.82+/−0.002 versus 0.75+/−0.001, p: 9.56*10−6, 2 independent experiments; n: nasal RGCs: 30 axons, temporal RGCs: 26). Results are shown as mean +/− SE,. Nasal RGC axons (N), temporal RGC axons (T).
Mentions: These studies showed that RGC axons (shafts and growth cones) express ephrin-A2 and –A6 in both nasal and temporal retinal thirds but presenting a decreasing naso-temporal gradient (Fig. 2A, B). To investigate whether this gradient is due to the number of axons which express ephrin-As or to the level of expression of each axon, we quantified the proportion of axons which express ephrin-A2 and –A6 and the intensity of labeling of each axon (Fig. 2A–E). We showed that not only temporal RGC axons express lower levels of ephrin-As with respect to nasal axons, but also a lower proportion of temporal RGC axons express ephrin-As if compared to nasal axons. The distribution profile appreciated in the histogram of fluorescent intensity (Fig. 2E) showed that nasal and temporal axons form two partially overlapping populations, the former presenting higher levels of expression.

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