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L. J. Rizzolo, R. Sun, X. Chen, S. Peng, H. Zhang; Analysis of the Transcriptome Reveals Extensive Regulation of Cultured RPE by Secretions of the Neural Retina. Invest. Ophthalmol. Vis. Sci. 2008;49(13):2437.
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This study developed a genomic context in which neural retina-RPE interactions can be explored. The ability of the neural retina to regulate the barrier properties of the RPE was exploited to examine how the neural retina regulates the expression of the RPE transcriptome in general and genes related to barrier function in particular.
Chick RPE was isolated from embryonic day 7 (E7) and E14 and cultured on filters in a serum free medium, plus or minus retinal conditioned medium. The retinal conditioned medium (E14rCM) was prepared by organ culture of E14 neural retinas and applied to the apical surface of the culture RPE. In some experiments, 2 % fetal bovine serum was used to antagonize the effects of conditioned medium. After nine days in culture, the transepithelial electrical resistance (TER) was determined. Total RNA was isolated to probe the chick genome on Affymetrix gene microarrays. Expression of the transcriptome was compared to expression in vivo. Effects on tight junction proteins were confirmed by quantitative RT-PCR.
In E7 and E14, the TER increased 3-4X in the presence of E14rCM. Serum reduced this effect. Without E14rCM, the cultures expressed 89% (E7), or 86% (E14), of the transcriptome (20,494 probesets) that was expressed during normal development. Only 6% (E7), 5% (E14), of the probesets expressed in culture were not expressed in vivo. E14rCM regulated the expression of 7,201 (E7), 2460 (E14), probesets. Examination of 710 genes important for RPE functions including transcription regulation, signal transduction, the visual cycle, phagocytosis, blood-retinal barrier and extracellular matrix formation revealed 48% (E7), 51% (E14), of the mRNA’s were expressed within 2X of levels expressed in vivo. E14rCM brought the expression of many of the overexpressed or underexpressed genes closer to the levels of expression seen in native E14 RPE. In E7 cultures, these included claudin family members, which form tight junctional strands. In E14 cultures, the expression of structural proteins of tight junctions was minimally affected.
The E14 neural retina promotes the differentiation of cultured E7 RPE, but for E14 RPE, it functions to maintain the expression of a smaller set of proteins. The increase in TER of E7 RPE correlates with increased expression of tight junctional proteins, but to explain the TER increase for E14 RPE, we need to mine the data for regulators of tight junctions.
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