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From the Wilmer Ophthalmological Institute, The Johns Hopkins University School of Medicine, Baltimore, Maryland.
PURPOSE. The homeobox genes Pax6 and Chx10 are diffusely expressed in proliferating, undifferentiated retina neuroepithelial cells. Distinct, topographically specific expression patterns emerge, however, as postmitotic cells become organized into layers. The hypothesis that the product of each gene may be necessary for the differentiation of particular nonphotoreceptor neuron subsets and that their absence may be required for progenitor cells to differentiate as photoreceptors was tested in this study.
METHODS. Neural retinas from 5-day-old chick embryos were dissociated, cultured at low density, and cotransfected with a plasmid expressing the green fluorescent protein (GFP) reporter gene, and a plasmid expressing Pax6, Chx10, Optx2, or the control gene lacZ. After further culture, the cells were fixed and processed for the detection of cell-specific markers.
RESULTS. Nonphotoreceptor neurons increased threefold with Chx10 and almost sixfold with Pax6, compared with cells transfected with lacZ. The frequency of GFP(+) cells immunoreactive with the ganglion cell-specific antibody RA4 was unchanged by Chx10, but was increased twofold by Pax6. Conversely, Chx10 and Pax6 expression diminished the photoreceptor population to approximately 35% and 15% of control values, as determined by morphologic analysis, visinin immunocytochemistry, and peanut lectin binding. Optx2 had some inhibitory effects on photoreceptor differentiation, which were accompanied by marked increases in the frequency of morphologically undifferentiated cells.
CONCLUSIONS. These results are consistent with the hypothesis that Chx10 and Pax6 promote the differentiation of nonphotoreceptor neurons while inhibiting the differentiation of photoreceptor cells.
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