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In the vertebrate eye, the retinal pigment epithelium (RPE) and the neural retina arise from a single layer of neuroectoderm. Factors influencing the differentiation of retinal neurons have been identified; however, little is known about molecules directing the differentiation of the RPE. Here we have found that the neurotrophin brain-derived neurotrophic factor (BDNF) plays an autocrine role in the differentiation and survival of Xenopus laevis RPE. Fluorescent in situ hybridization studies showed a precise co-expression of BDNF and its receptor trkB in the retinal neuroepithelium and actively differentiating RPE; in vitro studies demonstrated survival- and differentiation-promoting effects in serum-free explants and dissociated cultures. When a dominant negative mutant of the trkB receptor was expressed in developing embryos, severe arrest of RPE differentiation was seen with persistence of nestin- and Notch-positive neuroblasts.
Fig. 1.
Co-Localization of Notch, TrkB, and BDNF in the developing optic cup. Top panels, FISH of stage 26 Xenopus optic cups (sagittal sections, en face). In A–C, a single section was probed with cRNA antisense probes incorporating fluorescein-12-UTP (Boeringer Mannheim) for Xenopus Notch (A) or TMR-5-UTP (Molecular Probes) fluorescein isothiocianate for the kinase-containing domain of Xenopus trkB (B) andNotch plus trkB (C). D–F, FISH of stage 28 retinas (coronal sections) probed with FITC antisense riboprobes for trkB (D), RITC probes for BDNF (E), and FITC-trkB plus RITC-BDNF (F). G–J, en face views of stage 30 Xenopus retinas probed with FITC-trkB (G), RITC-BDNF (H), FITC-trkB plus RITC-BDNF (I), and FITCNotch (J). K, Bright-field exposure of a stage 32 Xenopus retina probed with FITC-trkB (L). M, Bright-field exposure of a stage 32 Xenopus retina probed with FITC-Notch (N). Scale bar, 0.1 mm.
Fig. 2.
BDNF-blocking antibody prevents RPE differentiation in vitro. A–C, Representative control stage 26 explant at days 1, 3, and 5 after plating. D–F, Representative explant cultured in the presence of 15 μg/ml BDNF antibody at days 1, 3, and 5.G–I, Represented explant cultured in the presence of 50 μg/ml BDNF antibody.
Fig. 3.
BDNF promotes RPE survival. The survival rates of dissociated RPE in the presence (black squares) or absence (white circles) of 100 ng/ml BDNF are shown.Points represent the average of eight wells. Error bars indicate SD.
Fig. 4.
Expression of the Myc-tagged TrkB mutant.A, Whole-mount immunohistochemistry of stage 32 embryos using an anti-myc antibody (9E10). At left is an embryo injected with sterile water, at right, three mutants expressing the myc-tagged trkB mutant. Scale bar, 0.1 mm.B, Immunofluorescent staining (using the 9E10 antibody) of the eyes of stage 26 embryos injected either with water (WT) or cRNA encoding the trkB mutant (TrkBMut). Scale bar, 10 μm. C, Western blot of stage 26 embryo lysates injected with either sterile water (WT) or cRNA encoding the myc-tagged trkB mutant (TrkBMut). A band at 145 kDa corresponds to the mutant trkB receptor.
ig. 5.
Retinal pigment abnormalities in dominant negative TrkB mutants. Representative photographs of the eyes of water-injected controls (A) and the trkB dominant negative mutants (B, C). D–F, Representative sections from the central retinas of cresyl violet–eosin-stained tissues. Embryos were generated simultaneously and killed at stage 45. A water-injected embryo is shown in D; mildly and severely affected embryos are seen in E and F, respectively. Scale bar, 50 μm. G, H, Immunofluorescent sections from a stage 45 water-injected control embryo (G) or mildly affected mutant (H) probed with a polyclonal antibody to CRALBP (Bunt-Milam and Saari, 1983). Arrows indicate the RPE layer. I,J, Immunofluorescent sections from stage 45 water-injected controls (I) or a severely affected mutant (J). Sections were probed with a polyclonal antibody raised against the intermediate filament nestin (Tohyama et al., 1992). K, L, Sections from a stage 45 water-injected control (K) or severely affected trkB mutant (L) probed with RITC antisense riboprobe for Xenopus Notch. Scale bar, 0.1 mm.
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