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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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Developmental patterns of expression of tectal EphA3 and retinal ephrin-As and Tyr-602 phosphorylated-EphA4.(A–D) Immunofluorescence of EphA3 was performed in sections which extend along the rostro-caudal axis of tecta obtained from chicken embryos and early postnatal chicks. Photographs of rostral (left), and caudal (right) tectum obtained from the same section are successively shown at 6 (A), 12 (B), 17 days of development (C) (E6, E12, E17) and 3 postnatal days (P3) (D). Immunofluorescence (left) and merge image of immunofluorescence and nuclear labeling with Hoechst (right) of each area are shown. The lamination process is more advanced in rostral (left) than in caudal (right) tectum, so different developmental stages can be appreciated in different areas analyzed in the same age [3]. As lamination progresses, EphA3 is expressed along all the radial tectum in both areas, but the expression of superficial layers is higher in rostral than in caudal tectum. This pattern of expression is appreciated at E6 (A) and presents the highest level around E12 (B), decreasing postnatally (D). Ventricular zone (VZ), subventricular zone (SVZ), premigratory zone (PMZ), the transitory cell compartment 1 (TCC1), TCC2, TCC3, TCC4, C “stratum griseum periventriculare” (C “SGP”), C “stratum album centrale” (C “SAC”), C “stratum griseum centrale” (C “SGC”), C “j”, C “i”; C “h”; layers a, b, c, d, e, f, g, h, i, and j of the stratum griseum et fibrosum superficiale (SGFS); stratum opticum (SO) (for nomenclature see Material and Methods, [3], [5]). Scale bars  = 50 µm. (E-H). Immunofluorescence of ephrin-A2 (E–H) was performed in sections which extend along the naso-temporal axis of retinas obtained from E6 (E), E12 (F), E17 (G) and P3 (H). Photographs of nasal (left) and temporal (right) retinal areas obtained from the same section are successively shown. Immunofluorescence (left), nuclear labeling with Hoechst (middle) and merge image (right) of each area are shown. Ephrin-A2 is expressed in neuroepithelial cells and RGCs at E6 (E). When development progresses plexiform layers, amacrine cells and lastly external segments of photoreceptors are labeled (F–H). RGCs show ephrin-A2 in the soma, dendrites and the axon (insets). The expression of ephrin-A2 extends along all the retina, presenting a decreasing naso-temporal gradient mainly in the RGCs. This gradient presents the highest level of expression between E12 (F) and E17 (G), but persists postnatally when the expression of the photoreceptors increases (H). (I, J) Immunofluorescence of Tyr-602 phosphorylated-EphA4 was performed in sections which extend along the naso-temporal axis of retinas obtained when the gradient of ephrin-A2 is highly expressed (E6 and E12). Photographs of nasal (left) and temporal (right) retinal areas obtained from the same section are successively shown. Immunofluorescence (left), nuclear labeling with Hoechst (middle) and merge image (right) of each area are shown. Tyr-602 phosphorylated-EphA4 mainly expresses in RGCs and extends throughout all the retina, in a decreasing naso-temporal gradient at E6 (I) and E12 (J). Neuroepithelial cells (NEC), retinal ganglion cells (RGCs), optic fibers (arrowhead), photoreceptors (asterisk), external nuclear layer (ENL), inner nuclear layer (INL), inner plexiform layer (IPL). Scale bars  = 50 µm, inset: 10 µm.
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pone-0038566-g001: Developmental patterns of expression of tectal EphA3 and retinal ephrin-As and Tyr-602 phosphorylated-EphA4.(A–D) Immunofluorescence of EphA3 was performed in sections which extend along the rostro-caudal axis of tecta obtained from chicken embryos and early postnatal chicks. Photographs of rostral (left), and caudal (right) tectum obtained from the same section are successively shown at 6 (A), 12 (B), 17 days of development (C) (E6, E12, E17) and 3 postnatal days (P3) (D). Immunofluorescence (left) and merge image of immunofluorescence and nuclear labeling with Hoechst (right) of each area are shown. The lamination process is more advanced in rostral (left) than in caudal (right) tectum, so different developmental stages can be appreciated in different areas analyzed in the same age [3]. As lamination progresses, EphA3 is expressed along all the radial tectum in both areas, but the expression of superficial layers is higher in rostral than in caudal tectum. This pattern of expression is appreciated at E6 (A) and presents the highest level around E12 (B), decreasing postnatally (D). Ventricular zone (VZ), subventricular zone (SVZ), premigratory zone (PMZ), the transitory cell compartment 1 (TCC1), TCC2, TCC3, TCC4, C “stratum griseum periventriculare” (C “SGP”), C “stratum album centrale” (C “SAC”), C “stratum griseum centrale” (C “SGC”), C “j”, C “i”; C “h”; layers a, b, c, d, e, f, g, h, i, and j of the stratum griseum et fibrosum superficiale (SGFS); stratum opticum (SO) (for nomenclature see Material and Methods, [3], [5]). Scale bars  = 50 µm. (E-H). Immunofluorescence of ephrin-A2 (E–H) was performed in sections which extend along the naso-temporal axis of retinas obtained from E6 (E), E12 (F), E17 (G) and P3 (H). Photographs of nasal (left) and temporal (right) retinal areas obtained from the same section are successively shown. Immunofluorescence (left), nuclear labeling with Hoechst (middle) and merge image (right) of each area are shown. Ephrin-A2 is expressed in neuroepithelial cells and RGCs at E6 (E). When development progresses plexiform layers, amacrine cells and lastly external segments of photoreceptors are labeled (F–H). RGCs show ephrin-A2 in the soma, dendrites and the axon (insets). The expression of ephrin-A2 extends along all the retina, presenting a decreasing naso-temporal gradient mainly in the RGCs. This gradient presents the highest level of expression between E12 (F) and E17 (G), but persists postnatally when the expression of the photoreceptors increases (H). (I, J) Immunofluorescence of Tyr-602 phosphorylated-EphA4 was performed in sections which extend along the naso-temporal axis of retinas obtained when the gradient of ephrin-A2 is highly expressed (E6 and E12). Photographs of nasal (left) and temporal (right) retinal areas obtained from the same section are successively shown. Immunofluorescence (left), nuclear labeling with Hoechst (middle) and merge image (right) of each area are shown. Tyr-602 phosphorylated-EphA4 mainly expresses in RGCs and extends throughout all the retina, in a decreasing naso-temporal gradient at E6 (I) and E12 (J). Neuroepithelial cells (NEC), retinal ganglion cells (RGCs), optic fibers (arrowhead), photoreceptors (asterisk), external nuclear layer (ENL), inner nuclear layer (INL), inner plexiform layer (IPL). Scale bars  = 50 µm, inset: 10 µm.

Mentions: To investigate the function of tectal EphA3 we first analyzed its developmental pattern of expression by performing immunohistochemistry during the embryonic development −4 to 18 days of incubation (E4 to E18)- and the early postnatal period –newly hatched, postnatal days 3 and 7 (P3 and P7)-. The study was achieved on sections coinciding with the rostro-caudal developmental gradient axis of the chicken optic tectum. This allowed us to distinguish between the most developed rostral pole (where the highest level of EphA3 is expressed), and the less developed caudal pole (where the lowest level of EphA3 is expressed) [3], [5], [18]. We showed that EphA3 is expressed along the main cellular layers during the embryonic development (Fig. 1A–D). The stratum opticum (SO) formed by the optic fibers is also labeled mainly in the rostral tectum where the EphA3-positive temporal fibers arrive. The levels of expression of EphA3 in the superficial layers – which include the SO and the retino-recipient layers- produce a decreasing rostro-caudal gradient which extends along the entire tectal axis. This gradient can be appreciated at E5–E6 –when the EphA3-positive temporal RGC axons have not yet invaded the tectum- and presents the highest level of expression around E12 when the retinotectal mapping is taking place. The EphA3 expression decreases postnatally when the retinotectal mapping has finished.


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)

Developmental patterns of expression of tectal EphA3 and retinal ephrin-As and Tyr-602 phosphorylated-EphA4.(A–D) Immunofluorescence of EphA3 was performed in sections which extend along the rostro-caudal axis of tecta obtained from chicken embryos and early postnatal chicks. Photographs of rostral (left), and caudal (right) tectum obtained from the same section are successively shown at 6 (A), 12 (B), 17 days of development (C) (E6, E12, E17) and 3 postnatal days (P3) (D). Immunofluorescence (left) and merge image of immunofluorescence and nuclear labeling with Hoechst (right) of each area are shown. The lamination process is more advanced in rostral (left) than in caudal (right) tectum, so different developmental stages can be appreciated in different areas analyzed in the same age [3]. As lamination progresses, EphA3 is expressed along all the radial tectum in both areas, but the expression of superficial layers is higher in rostral than in caudal tectum. This pattern of expression is appreciated at E6 (A) and presents the highest level around E12 (B), decreasing postnatally (D). Ventricular zone (VZ), subventricular zone (SVZ), premigratory zone (PMZ), the transitory cell compartment 1 (TCC1), TCC2, TCC3, TCC4, C “stratum griseum periventriculare” (C “SGP”), C “stratum album centrale” (C “SAC”), C “stratum griseum centrale” (C “SGC”), C “j”, C “i”; C “h”; layers a, b, c, d, e, f, g, h, i, and j of the stratum griseum et fibrosum superficiale (SGFS); stratum opticum (SO) (for nomenclature see Material and Methods, [3], [5]). Scale bars  = 50 µm. (E-H). Immunofluorescence of ephrin-A2 (E–H) was performed in sections which extend along the naso-temporal axis of retinas obtained from E6 (E), E12 (F), E17 (G) and P3 (H). Photographs of nasal (left) and temporal (right) retinal areas obtained from the same section are successively shown. Immunofluorescence (left), nuclear labeling with Hoechst (middle) and merge image (right) of each area are shown. Ephrin-A2 is expressed in neuroepithelial cells and RGCs at E6 (E). When development progresses plexiform layers, amacrine cells and lastly external segments of photoreceptors are labeled (F–H). RGCs show ephrin-A2 in the soma, dendrites and the axon (insets). The expression of ephrin-A2 extends along all the retina, presenting a decreasing naso-temporal gradient mainly in the RGCs. This gradient presents the highest level of expression between E12 (F) and E17 (G), but persists postnatally when the expression of the photoreceptors increases (H). (I, J) Immunofluorescence of Tyr-602 phosphorylated-EphA4 was performed in sections which extend along the naso-temporal axis of retinas obtained when the gradient of ephrin-A2 is highly expressed (E6 and E12). Photographs of nasal (left) and temporal (right) retinal areas obtained from the same section are successively shown. Immunofluorescence (left), nuclear labeling with Hoechst (middle) and merge image (right) of each area are shown. Tyr-602 phosphorylated-EphA4 mainly expresses in RGCs and extends throughout all the retina, in a decreasing naso-temporal gradient at E6 (I) and E12 (J). Neuroepithelial cells (NEC), retinal ganglion cells (RGCs), optic fibers (arrowhead), photoreceptors (asterisk), external nuclear layer (ENL), inner nuclear layer (INL), inner plexiform layer (IPL). Scale bars  = 50 µm, inset: 10 µm.
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pone-0038566-g001: Developmental patterns of expression of tectal EphA3 and retinal ephrin-As and Tyr-602 phosphorylated-EphA4.(A–D) Immunofluorescence of EphA3 was performed in sections which extend along the rostro-caudal axis of tecta obtained from chicken embryos and early postnatal chicks. Photographs of rostral (left), and caudal (right) tectum obtained from the same section are successively shown at 6 (A), 12 (B), 17 days of development (C) (E6, E12, E17) and 3 postnatal days (P3) (D). Immunofluorescence (left) and merge image of immunofluorescence and nuclear labeling with Hoechst (right) of each area are shown. The lamination process is more advanced in rostral (left) than in caudal (right) tectum, so different developmental stages can be appreciated in different areas analyzed in the same age [3]. As lamination progresses, EphA3 is expressed along all the radial tectum in both areas, but the expression of superficial layers is higher in rostral than in caudal tectum. This pattern of expression is appreciated at E6 (A) and presents the highest level around E12 (B), decreasing postnatally (D). Ventricular zone (VZ), subventricular zone (SVZ), premigratory zone (PMZ), the transitory cell compartment 1 (TCC1), TCC2, TCC3, TCC4, C “stratum griseum periventriculare” (C “SGP”), C “stratum album centrale” (C “SAC”), C “stratum griseum centrale” (C “SGC”), C “j”, C “i”; C “h”; layers a, b, c, d, e, f, g, h, i, and j of the stratum griseum et fibrosum superficiale (SGFS); stratum opticum (SO) (for nomenclature see Material and Methods, [3], [5]). Scale bars  = 50 µm. (E-H). Immunofluorescence of ephrin-A2 (E–H) was performed in sections which extend along the naso-temporal axis of retinas obtained from E6 (E), E12 (F), E17 (G) and P3 (H). Photographs of nasal (left) and temporal (right) retinal areas obtained from the same section are successively shown. Immunofluorescence (left), nuclear labeling with Hoechst (middle) and merge image (right) of each area are shown. Ephrin-A2 is expressed in neuroepithelial cells and RGCs at E6 (E). When development progresses plexiform layers, amacrine cells and lastly external segments of photoreceptors are labeled (F–H). RGCs show ephrin-A2 in the soma, dendrites and the axon (insets). The expression of ephrin-A2 extends along all the retina, presenting a decreasing naso-temporal gradient mainly in the RGCs. This gradient presents the highest level of expression between E12 (F) and E17 (G), but persists postnatally when the expression of the photoreceptors increases (H). (I, J) Immunofluorescence of Tyr-602 phosphorylated-EphA4 was performed in sections which extend along the naso-temporal axis of retinas obtained when the gradient of ephrin-A2 is highly expressed (E6 and E12). Photographs of nasal (left) and temporal (right) retinal areas obtained from the same section are successively shown. Immunofluorescence (left), nuclear labeling with Hoechst (middle) and merge image (right) of each area are shown. Tyr-602 phosphorylated-EphA4 mainly expresses in RGCs and extends throughout all the retina, in a decreasing naso-temporal gradient at E6 (I) and E12 (J). Neuroepithelial cells (NEC), retinal ganglion cells (RGCs), optic fibers (arrowhead), photoreceptors (asterisk), external nuclear layer (ENL), inner nuclear layer (INL), inner plexiform layer (IPL). Scale bars  = 50 µm, inset: 10 µm.
Mentions: To investigate the function of tectal EphA3 we first analyzed its developmental pattern of expression by performing immunohistochemistry during the embryonic development −4 to 18 days of incubation (E4 to E18)- and the early postnatal period –newly hatched, postnatal days 3 and 7 (P3 and P7)-. The study was achieved on sections coinciding with the rostro-caudal developmental gradient axis of the chicken optic tectum. This allowed us to distinguish between the most developed rostral pole (where the highest level of EphA3 is expressed), and the less developed caudal pole (where the lowest level of EphA3 is expressed) [3], [5], [18]. We showed that EphA3 is expressed along the main cellular layers during the embryonic development (Fig. 1A–D). The stratum opticum (SO) formed by the optic fibers is also labeled mainly in the rostral tectum where the EphA3-positive temporal fibers arrive. The levels of expression of EphA3 in the superficial layers – which include the SO and the retino-recipient layers- produce a decreasing rostro-caudal gradient which extends along the entire tectal axis. This gradient can be appreciated at E5–E6 –when the EphA3-positive temporal RGC axons have not yet invaded the tectum- and presents the highest level of expression around E12 when the retinotectal mapping is taking place. The EphA3 expression decreases postnatally when the retinotectal mapping has finished.

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