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Dickinson College (Pennsylvania)
Sara Patterson1, Paul Cohen1, Jennifer Strykowski2, Harry Burgess2, and Ralph Nelson,1
1Neural Circuitry Unit, National Institute of Neurological Disorders and Stroke; 2Behavioral Genetics, National Institute of Child Health and Development
Forward geneticsprovides an avenue to explore the expression and function of genes both previously recognized, and as yet unrecognized in nervous system circuitry, development and function. The Burgess laboratory created a library of zebrafish lines with Gal4 insertions throughout the genome. Gal4 activates UAS constructs separately bred into the strains. One of these insertionsEt(SCP1:Gal4ff)y245 (y245) selectively caused photoreceptors to fluoresce, when expressed in the line y245;UAS:kaede, in particular, zebrafish red and green cones in both larvae and adults, and additionally, rods in adults. The transgene insertion mapped to the promoter of Musashi1 (Msi1). The insertion also appeared to trigger a partial loss of UV cone electrical responses in adults (Nelson et al, 2013-ARVO abstract). This project examines the spectral changes induced by the transgene inthe isolated cone responses of 5dpf and 6dpf zebrafish larvae. It also revisits UV cone morphology of the adults using the triple transgenic line SWS1:GFP;y245;UAS:ntrMCherry.
Microelectrodes were used to study the ERG response in larval zebrafish eyesat 5 and 6 days post fertilization (dpf). Zebrafish eyes were perfused in MEM containing 20mM L-Aspartate which blocks glutamatergic receptors and isolates the photoreceptor PIII response. The experimental protocol consisted of ten wavelengths, each presented four times at seven different brightness levels for a total of 280 stimuli.
Confocal images of the zebrafish retinas were taken with a Zeiss confocal microscope 510 Meta. The SWS1:GFP;y245;UAS:ntrmCherry line labeled UV cones with GFP and red/green cones with ntrMCherry.
Wild types followed a normal developmental pattern with an increase in spectral sensitivity from 5dpf to 6dpf and y245 heterozygotes increased slightly. However, y245 homozygotes showed a marked decrease in sensitivity between 5 and 6dpf. In addition, amplitude of the mean PIII response was lower in y245 homozygotes and heterozygotes than in wild types.
Confocal images showed considerable degeneration of the entire UV cone mosaic as well as malformation in individual UV cones. The inconsistent line of UV puncta suggests connections with ON-bipolar cells may be affected. Accordingly, preliminary ERG recordings using CNQX to isolate the photoreceptor and ON-bipolar response show that zebrafish with a y245;UAS:kaede gene lack the characteristic ON-bipolar cell b-wave.
The PIII responses show the transgene insertion near Musashi1 causes deficiencies in photoreceptor responses. Confocal images show structural abnormalities not only in UV cones but also in the connections to ON –bipolar cells. Taking the lack of a ON-bipolar b-wave in zebrafish with a y245;UAS:kaede phenotype into consideration, these results suggest that the effects of Musashi1 in the retina are not limited to the photoreceptors.
Last Modified November 27, 2013