Developmental patterns of expression of tectal EphA3 and retinal ephrin-As and Tyr-602 phosphorylated-EphA4.

<p>(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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038566#pone.0038566-Rapacioli1" target="_blank">[3]</a>. 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038566#s4" target="_blank">Material and Methods</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038566#pone.0038566-Rapacioli1" target="_blank">[3]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038566#pone.0038566-Scicolone3" target="_blank">[5]</a>). 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.