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Y. Pan, A. Rorick, H. M. El-Hodiri; Identification of Rx-Dependent Genes by Microarray Approach. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3088.
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© ARVO (1962-2015); The Authors (2016-present)
The retinal homeobox (Rx) gene product is essential for eye development. However little is known regarding the Rx genetic pathway. Rx and other early eye genes can be induced in naive Xenopus ectoderm by insulin-like growth factor (IGF). In this study, we used a microarray approach to identify IGF-induced genes with expression dependant on Rx.
Rx or control antisense morpholino (ASMO or COMO respectively) was injected into one-celled Xenopus embryos. IGF RNA (4ng) was injected into all blastomeres of the same embryos at four-cell stage. Animal cap explants were cut at stage 8-9 and collected at stage 13. Total RNA was extracted from 30-40 pooled explants and used to generate cDNAs that were used to probe Affymetrix Xenopus laevis microarrays. Genes exhibiting a two-fold or greater change in expression level in response to Rx depletion were identified. Expression patterns of these genes were analyzed by in situ hybridization using whole or sectioned embryos. The change in expression level of each gene was confirmed by quantitative RT-PCR of RNA collected from animal caps prepared similarly to those used for microarray analysis.
Our results confirmed that IGF was able to induce early eye genes in Xenopus animal caps, and that a Rx ASMO, but not COMO could knock down both Rx1A and Rx2A. By microarry analysis, we identified 46 genes that were down-regulated when Rx was knocked down. We obtained ESTs for top 21 of these genes. Nine of these genes were expressed in the ciliary marginal zone (CMV) of the maturing retina, where Rx is expressed. These genes were further validated by quantitative RT-PCR to verify decreased expression upon Rx knock down. We also analyzed 12 genes that were up-regulated by knocking down Rx. Among them, five were expressed in the CMV or photoreceptor layer, where Rx is expressed. Included were retinal, novel and unidentified genes.
We have identified 14 genes that are likely to depend on Rx for expression. This set of genes includes nine genes that are positively regulated by Rx and five genes that are negatively regulated by Rx.
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