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S. Yoshikawa, X. C. Zhao; Ventral-Directed Outflow of the Ocular Fluid in the Zebrafish Eyes. Invest. Ophthalmol. Vis. Sci. 2009;50(13):3637.
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Our long-term goal is to generate genetically manipulated zebrafish to study human eye diseases, especially the glaucoma. This study investigates if the annular ligament (AL), a unique connective tissue at the iridocorneal angle of the fish, has a physiological role to maintain the intraocular pressure (IOP) and modifications of the AL cells by molecular and genetic approaches can alter the IOP in transgenic zebrafisih.
To visualize the outflow of the ocular fluid, rhodamine-labeled microbeads were injected to the zebrafish eye. The bead movement was recorded by time-lapse, fluorescence video microscopy. To study the role of AL, a targeted cell ablation experiment was designed. Stable transgenic zebrafish of the fusion protein of nitroreductase (NTR) and red fluorescent protein mCherry under the control of the AL-specific promoter of the zebrafish gelsolin-like 1 gene was established. NTR is a bacterial enzyme that converts non-toxic prodrug Metronidazole (Mtz) to toxic. NTR is not harmful before the Mtz treatment and the targeted cell death can be controlled by the timing of the Mtz administration and the cell type-specific promoter of the transgene, in this case, AL.
All 34 bead-injected eyes showed ventral-directed outflow. Most of the beads were trapped at the ventral position of the iridocorneal angle while some passed out from the anterior chamber. The same observation was obtained from 2 other fish species, goldfish and minnow. In the stable transgenic zebrafish, the red fluorescent signal of the NTR-mCherry fusion protein was detected specifically detected in AL at 3 days post-fertilization (dpf) and there after. Nearly 99% mCherry-positive cells in AL disappeared by 5 dpf by the 10 mM Mtz treatment starting on 1 dpf.
The fish eye has a ventrally-unidirected outflow of the ocular fluid. The existence of the outflow suggests that the fish eye has a similar IOP regulatory system to the human eye. It also suggests that the zebrafish AL has a special anatomical structure at the ventral position that allows liquid pass from the anterior chamber to the extra-ocular spaces, which would be functionally identical to the human Schlemm’s canal. Stable transgenic zebrafish carrying the conditional suicide gene NTR are generated. They can be used for the further loss-of-function studies of the zebrafish AL in the IOP regulation.
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