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V. C. Fleisch, H. B. Schoenthaler, J. von Lintig, S. C. F. Neuhauss; Subfunctionalization of a Retinoid Binding Protein Provides Evidence for Two Parallel Visual Cycles in the Cone-Dominant Zebrafish Retina. Invest. Ophthalmol. Vis. Sci. 2008;49(13):4532.
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© ARVO (1962-2015); The Authors (2016-present)
The vertebrate visual cycle serves the purpose of recycling 11-cis retinal in the retina in order to restore vision after bleaching. In recent years evidence has accumulated that cones might employ an alternative pathway for recycling these retinoids as compared to rods. Retinoid binding proteins (CRBP, CRALBP, IRBP) are known to be essential for the canonical (rod) visual cycle. In order to assess the function of Cralbp in cone visual pigment regeneration, we made use of zebrafish behavioral genetics.
Two orthologues of the CRALBP gene (CRALBP-a, CRALBP-b) were cloned in the zebrafish. Spatial and temporal expression patterns were investigated. The antisense morpholino technique was employed to create zebrafish larvae lacking Cralbp-a or -b protein. Visual system performance of injected larvae was evaluated in OKR and ERG measurements. Furthermore, a HPLC analysis of retinoids was performed in order to assess 11-cis retinal regeneration capabilities of larvae devoid of Cralbp-a or -b.
The zebrafish genome harbors two orthologues of Cralbp, which we denoted as cralbp-a and -b. These paralogues are differentially expressed in the zebrafish retina. While Cralbp-a is found in the RPE, Cralbp-b expression is restricted to Müller glia cells. Targeted downregulation of Cralbp-a does not have any effect on visual behavior, as assessed by OKR recordings. However, in ERG measurements, a slight decrease in b-wave amplitude was observed. In contrast, injection of Cralbp-b morpholino resulted in a highly significant reduction of the ERG b-wave amplitude as well as in severely impaired contrast sensitivity in the OKR assay. Analysis of 11-cis retinal levels employing a regeneration paradigm revealed reduced levels of visual chromophore in both Cralbp morphants.
Differential expression of Cralbp paralogues in the zebrafish retina as well as results obtained from behavioral and physiological studies of Cralbp-a/-b deficient larvae provide strong evidence for the existence of two pathways of visual pigment recycling in the zebrafish. We show that Cralbp-a is implicated in the canonical visual cycle located to the RPE, whereas Cralbp-b is part of a cone-specific visual cycle located to Müller glia cells. Moreover, our data suggest that cones use both pathways for recycling 11-cis retinal, however with a predominant usage of the visual cycle located to Müller glia cells.
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