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Diane M. Cockburn, Nardos Tassew, Jason Charish, Philippe Monnier; Identification Of A Novel Protein Involved In Microphthalmia. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1820.
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During the development of the visual system, proper eye growth relies on cellular mechanisms such as protein synthesis. Because mRNA translation may occur in remote cell compartments, we decided to screen for genes involved in eye size with the goal of identifying translation factors that regulate this process. Therefore, we initiated an in vivo screen of factors that alter eye size.
To do so, we used RNA interference to knockdown endogenous proteins in the developing chick eye. The right eye of embryonic day 2 (E2) chicks was electroporated with either an miRNA fused to RFP, or a control plasmid containing GFPmiRNA. At E4 the diameter of both the left and right eyes was measured to acquire a right/left eye ratio, as the left eye serves as an internal control. Immunohistochemistry for TUNEL or BrdU markers was performed on sections to analyze the amount of retinal cell death or proliferation, respectively.
Knockdown with the first miRNA induced a reduction of eye size by half (p<0.5, n=9), whereas control miRNA did not significantly change eye size. In agreement with this data, a second miRNA also caused a small eye. To confirm that the effect observed with the miRNAs did not result from an off target effect, we also performed rescue experiments in which a construct that contained both miRNA and exogenous protein was used. Not only did the rescue constructs restore eye size, but they also lead to the formation of a slightly bigger eye (22% increase in size compared to controls, p<0.5, n=9). This may most likely be the result of higher levels of the tested proteins when compared to wild type. To gain further insight into the mechanisms underlying small eye phenotype, we decided to test whether the small eye is the result of increased cell death or reduced cell proliferation. TUNEL staining showed no increase in cell death in knockdown cells compared to controls. However, BrdU experiments showed that 49% of control cells were undergoing proliferation, and only 34% of retinal cells with a knockdown of our protein of interest were undergoing proliferation (p<0.05).
This is, the first ever indication that the factor we identified is involved in eye size. Because we were able to rescue the phenotype, we provided further strong evidence that this yet uncharacterized factor is indeed regulating eye size. Furthermore, the first mechanistic analysis indicates that size alteration results from altered levels of proliferation, rather than cell death. Our research shines a light on novel factors that may play a role in the development of microphthalmia.
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