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H. M. El-Hodiri, R. I. Martinez-De Luna, L. E. Kelly, H. E. Moose; Expression of the Retinal Homeobox (Rx) Gene in Xenopus Retinal Progenitor Cells Requires Cooperative Activity of Conserved Genomic Elements. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5398.
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© ARVO (1962-2015); The Authors (2016-present)
TThe retinal homeobox (Rx) gene is expressed in all phases of retinal development. It is expressed in the prospective eye fields in the anterior neural plate and continues to be expressed in retinal progenitor cells (RPCs) in the ciliary marginal zone (CMZ) of the maturing Xenopus tadpole retina. Rx expression is essential for eye development. The goal of this study is to identify transcriptional regulatory elements that direct expression of Rx in RPCs.
Genomic sequences were analyzed and compared using the UCSC Genome Browser. Genomic DNA fragments were amplified from X. tropicalis genomic DNA and used to construct GFP reporter transgenes. These constructs were introduced into X. laevis embryos by intra-cytosolic sperm injection. Transgene expression was analyzed in the maturing retina (st 41) by in situ hybridization performed on 8 Î¼M sections of paraffin-embedded embryos using non-radioactively labeled antisense riboprobes. Transcription factor binding site prediction was performed using the UCSC Genome Browser and the Transcription Element Search System (TESS). Transcription factor binding was determined by electrophoretic mobility shift assay using radiolabeled probes and in vitro translated proteins.
We identified three ultra-conserved elements (UCEs) flanking the Rx coding region. UCE-3 is located on the 5â€TM-flank of the Rx coding region. We found that 3.1 kb X. laevis Rx1A promoter lacks UCE-3 and is not active in the CMZ. However, a 2.8 kb X. laevis Rx2A promoter containing UCE-3 is active in all but the most distal portion of the CMZ. Similarly, a 3 kb X. tropicalis Rx promoter containing UCE-3 is active in most of the CMZ while a 1.6 kb fragment lacking UCE-3 is not. Addition of a second UCE, UCE-2, to a transgene containing UCE-3 (the 3 kb X. tropicalis Rx promoter) results in activity of the promoter throughout the entire CMZ. We have identified several putative cis-acting response elements in UCE-2 and -3. We have identified transcription factors that interact with these response elements in vitro. All of these factors are expressed in the CMZ.
Expression of Rx in retinal progenitor cells is dependent on cooperative activity of highly conserved genomic regions. These progenitor cell enhancers may regulate Rx expression by interaction with a set of transcription factors expressed in progenitor cells.
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