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Stella Glasauer, Matthias Gesemann, Stephan C F Neuhauss; A Genetic Approach to Elucidate Visual Processing in the Zebrafish Retina. Invest. Ophthalmol. Vis. Sci. 2014;55(13):4165.
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Each photoreceptor is contacted by several bipolar cell types, marked by distinct morphologies and responses to visual inputs. Transgenic zebrafish lines provide powerful tools to study connectivity and function of cells. The goal of this study is to transgenically label subtypes of retinal bipolar cells and study their functions. As we aim to use regulatory regions of genes specifically expressed in bipolar cells to drive transgene expression, it is necessary to identify new markers of bipolar cells. One such gene encodes a Ca2+ channel subunit mutated in patients suffering from progressive cone dystrophy. We have characterized the orthologous genes (cacna2d4a and b) in zebrafish.
Candidate genes have been identified by RNA in-situ hybridization on developing and adult zebrafish retinae. In order to isolate the regulatory elements of these candidate genes, we cloned the more compact regulatory regions of the puffer fish. Those regulatory regions were used to drive expression of membrane-tagged reporters in zebrafish, which visualize cell shapes and target cells for electrophysiology.To address functions of cacna2d4a and b, we used Morpholino-mediated knockdown followed by histological examination and electroretinographiy.
We found that genes encoding Calcium binding proteins (cabp2a, 2b, 5a and 5b) show strong and restricted, but distinct expression patterns in bipolar cells. Similar to mice, genes encoding accessory Calcium channel subunits α2δ (cacna2d4a and b) are expressed in bipolar cells and additionally in photoreceptors. Knockdown of either cacna2d4a or b does not change morphology of 5 day old zebrafish retinae and also resulted in no alterations in electroretinography.
We were able to find promising candidate genes to use in our transgenic approach. The restricted expression of cabps to few cell types reflects their specialized roles, probably in visual processing. The fact that knockdown of the zebrafish cacna2d4 genes does not show a phenotype with the methods employed may be due to the progressive nature of the related cone dystrophy.
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