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J. D. Brown, S. Dutta, I. B. Dawid, A. L. Akhtar, R. F. Bonner, R. P. Alur, J. M. Gross, W.-Y. Chan, B. P. Brooks; Two Putative Zinc-Finger Containing Proteins Are Necessary for Proper Optic Fissure Closure in a Zebrafish Model. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3074.
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© ARVO (1962-2015); The Authors (2016-present)
We seek to identify novel participants in optic fissure closure and relate them to human uveal coloboma. We have previously identified 297 genes differentially regulated in the closing optic fissure. Our current study characterizes the role of two putative zinc-finger containing protein paralogs and establishes a potential mechanism for their action in fissure closure.
Wild type AB/TL zebrafish were injected at the 1-4 cell stage with 4-8ng antisense morpholino oligos, targeted to the translation start site or splice site, and/or 100pg of mRNA. Live fish were observed using DIC optics at 24, 48, and 72hpf. RT-PCR was performed using intron-spanning primers. Whole-mount in situ hybridization was performed at 18 and 24hpf using digoxigenin or fluorescein-labeled transcripts.
Morpholino knockdown of the two putative zinc finger proteins yields an optic fissure closure defect in 98.6% (224/227) of morphant zebrafish. A synergistic effect is observed when both transcripts are reduced simultaneously. To confirm specificity, two morpholinos were designed for each transcript, yielding identical phenotypes. A reduction in spliced mRNA by RT-PCR also confirms the knockdown of the specific targets. mRNA rescue, by co-injection of wild-type mRNA with morpholino increases the number of normal eyes and decreases the overall phenotype severity: 73.9% (34/46) high grade defect in morpholino only and 31.4% (16/51) in morpholino + mRNA. Whole mount in situ hybridization reveals that morphant fish have a marked change in the expression level and patterning of pax2.1, a key determinant of the ventral optic fissure and stalk identity. Other ventral markers of eye development, vax1 and vax2, are largely unaffected.
The two putative zinc-finger containing genes identified play an important role in normal optic fissure closure. Reduction of viable transcripts in zebrafish leads to a coloboma phenotype which recapitulates the malformation seen in human uveal coloboma. The experimental phenotype is also associated with misregulation of pax2.1, a transcription factor normally expressed in the closing optic fissure and itself a cause of coloboma where deficient. This interaction is being investigated further in vitro. The relevance of these findings to human patients with uveal coloboma is being explored.
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