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A. L. Akhtar, J. D. Brown, S. Dutta, I. B. Dawid, B. P. Brooks; Evaluation of Possible Candidate Genes Involved in Optic Fissure Closure. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3073.
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During the fifth week of normal human gestation, optic fissure closure occurs in a manner that is spatially and temporally regulated. We hypothesize that interruption of one or more of the genes involved in approximation and/or fusion of the edges of the optic fissure could lead to compromise of closure and subsequent uveal coloboma - a potentially blinding disease in humans.
Gene expression microarray analysis of optic fissure closure in the mouse and subsequent analysis of biological significance through Ingenuity Pathways yielded a subset of genes hypothesized to be involved in optic fissure closure. Gene expression was confirmed through real-time RT-PCR and in-situ hybridization. Two genes from this subset were selected for investigation: SFRP-2 (soluble Wnt antagonist involved in non-canonical Wnt signaling) and FAT-4 (non-classical giant cadherin, thought to affect planar call polarity in Drosophila). A reverse-genetics, morpholino (MO) knockdown strategy was employed in wild-type AB/TU zebrafish to assess any resultant physiologic phenotype whereby optic fissure closure was affected. Antisense MO oligos for SFRP-2 and FAT-4 were targeted to the translation start site. Dose dependent concentrations (2, 4 and 8 ng) of each MO were singularly injected in zebrafish embryos at the 1-4 cell stage and compared to controls.
Ocular phenotype was assessed at 24, 48 and 72 hpf using DIC optics. SFRP-2 MO-injected embryos exhibited no evidence of optic fissure closure compromise at any MO concentration as compared to controls. FAT-4 MO injection proved to be lethal at all concentrations. Two surviving FAT-4 MO-injected zebrafish larvae (at 4 ng) assessed at 24 hpf appeared to exhibit a developmentally abnormal ocular phenotype with possible microphthalmia. However, they did not live past 30+ hpf.
Although both SFRP-2 and FAT-4 appear to be significantly regulated in the mouse at the mRNA level in the correct spatial and temporal pattern to cause optic fissure closure defects, morpholino knockdown of these genes in zebrafish failed to produce a convincing ocular phenotype. Lack of any kind of compromised phenotype in SFRP-2 MO-injected zebrafish larvae may be due to biological redundancy of the soluble Wnt antagonist family. The general lethality of FAT-4 knockdown in zebrafish indicates that this gene is likely globally critical in terms of early development and patterning. Current work is focusing on evaluating other candidate genes using a similar strategy.
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