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D. J. Cameron, J. E. Dowling; Temporal Elimination of Retinoic Acid Signaling Alters Eye Development in Zebrafish. Invest. Ophthalmol. Vis. Sci. 2009;50(13):4002.
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Retinoic acid (RA) plays a vital role in embryonic development. Studies examining RA effects in mice require double and triple gene knockouts, whereas zebrafish embryos are easily modulated by biochemical perturbations that affect RA levels or synthesis. The relative size of the eye and its well documented embryogenesis make zebrafish an ideal candidate for studying the role of RA during eye development. We are using simple biochemical ways to alter the amount of RA during early stages of zebrafish eye development to determine the role of RA during early eye development.
Zebrafish are bred and maintained under standard conditions at 28.5°C. Morphological features are used to stage the embryos in hours and days post-fertilization. Embryos at the prebud, 2, 6, and 9 somite stages are treated for two hours with 250uM citral, a potent inhibitor of retinal aldehyde dehydrogenases, diluted into DMSO or an equivalent amount of DMSO as a control. Embryos are fixed, sectioned and stained using a Lees’ methylene blue-basic fuchsin dye procedure. The retina lamination and organization of citral treated eyes was examined and compared to control eyes.
The timing of the chemical perturbations is critical. Inhibiting RA with citral at the prebud stage, the earliest time of treatment, leads to the development of a much smaller eye compared to other time points. Interestingly, treating at a later time, 6-9 somites, also results in a smaller eye, but with a different cytoarchitecture. A smaller percentage of these embryos also lack a ventral retina - the predominant phenotype for treatment at the 2 somite stage. Of the citral treated fish that survive to the larval stage, nearly all are able to swim, respond to touch and live to at least 7.5 days post-fertilization.
Retinoic acid is a key molecule involved in zebrafish early eye development. Temporal control of RA signaling is vital for normal eye development. Subtle changes in the timing of RA availability dramatically alter the developmental outcome of the eye. Understanding the various outcomes of RA elimination during early eye development may prove helpful in determining the factors involved in RA signaling in the developing eye.
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