Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of chicken to investigate regulation of the early embryonic RPE (retinal pigment epithelium) transcriptome by the neural retina

ABSTRACT: Purpose: The retinal pigment epithelium (RPE) forms the outer blood-retinal barrier. Primary cultures of RPE can model the barrier, but are very sensitive to culture conditions. We examined how the neural retina regulates the RPE transcriptome in a culture model of embryonic development. Attention focused on the tight junctional genes essential for barrier function. Methods: Chick RPE from embryonic day 7 (E7) or embryonic day 14 (E14) was cultured on filters in a serum free medium. Media conditioned by organ culture of neural retinas was applied to the apical surface of the cultured RPE. Fetal bovine serum (2%) was included in some experiments. When the transepithelial electrical resistance (TER) reached a plateau, total RNA was isolated to probe the chick genome on Affymetrix microarrays. The expression tight junctional mRNAs was confirmed by real-time PCR and immunoblotting the protein. Results: The TER was slightly increased by serum, but increased 2-3X by retinal conditioned medium. Serum diminished the effect of retinal conditioned medium. In basal conditions, 86% of the transcriptome expressed during development in vivo was also expressed in culture. Approximately 5% of the transcriptome expressed in culture was absent in vivo. E14 retinal conditioned medium affected 15% of the transcriptome in E7 cultures (24% if serum was included), but only 1.9% in E14 cultures (12% with serum). Examination of 610 genes important for RPE function revealed that mRNAs for 17% were regulated by retinal conditioned medium alone in E7 cultures, compared to 6.2% for E14. For tight junctions, retinal conditioned medium had the most affect on members of the claudin family. These results were confirmed by quantitative real-time PCR. Besides regulating mRNA levels, immunoblotting and immunocytochemistry suggested additional mechanisms whereby retinal secretions regulated protein expression and localization. Conclusions: Gene expression in primary cultures of embryonic RPE resembled the native tissue, but expression, and differentiation, is improved when elements of the normal extracellular environment are replicated in culture. For a small group of proteins, retinal secretions were required to maintain in vivo-like expression in culture. Albeit insufficient, retinal secretions promoted differentiation of immature RPE and helped maintain the properties of more mature RPE. Experiment Overall Design: RPE were isolated from chicken embryos on embryonic day 7 or 14 and cultured, as described . E7 is when tight junctions of the RPE begin to form; E14 is when tight junctions achieve their mature morphological appearance in vivo. The cells were cultured in medium that contains or lacks E14 retinal conditioned medium. Because serum inhibits the effect of retinal conditioned medium, serum was added to some cultures, resulting in 4 culture conditions for each age. To isolate total RNA, the RNeasy Protect kit (Qiagen) was used according to the manufacturer's protocols. For each culture, 3 independent preparations were used for analysis on Affymetrix microarrays of the chicken genome (Santa Clara, CA). The quality of the total RNA was assessed by the Keck Center, Yale University using formamide gels and a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA)

ORGANISM(S): Gallus gallus

SUBMITTER: Lawrence Rizzolo

PROVIDER: E-GEOD-10538 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Purpose: The retinal pigment epithelium (RPE) forms the outer blood-retinal barrier. Primary cultures of RPE can model the barrier, but are very sensitive to culture conditions. We examined how the neural retina regulates the RPE transcriptome in a culture model of embryonic development. Attention focused on the tight junctional genes essential for barrier function. Methods: Chick RPE from embryonic day 7 (E7) or embryonic day 14 (E14) was cultured on filters in a serum free medium. Media conditioned by organ culture of neural retinas was applied to the apical surface of the cultured RPE. Fetal bovine serum (2%) was included in some experiments. When the transepithelial electrical resistance (TER) reached a plateau, total RNA was isolated to probe the chick genome on Affymetrix microarrays. The expression tight junctional mRNAs was confirmed by real-time PCR and immunoblotting the protein. Results: The TER was slightly increased by serum, but increased 2-3X by retinal conditioned medium. Serum diminished the effect of retinal conditioned medium. In basal conditions, 86% of the transcriptome expressed during development in vivo was also expressed in culture. Approximately 5% of the transcriptome expressed in culture was absent in vivo. E14 retinal conditioned medium affected 15% of the transcriptome in E7 cultures (24% if serum was included), but only 1.9% in E14 cultures (12% with serum). Examination of 610 genes important for RPE function revealed that mRNAs for 17% were regulated by retinal conditioned medium alone in E7 cultures, compared to 6.2% for E14. For tight junctions, retinal conditioned medium had the most affect on members of the claudin family. These results were confirmed by quantitative real-time PCR. Besides regulating mRNA levels, immunoblotting and immunocytochemistry suggested additional mechanisms whereby retinal secretions regulated protein expression and localization. Conclusions: Gene expression in primary cultures of embryonic RPE resembled the native tissue, but expression, and differentiation, is improved when elements of the normal extracellular environment are replicated in culture. For a small group of proteins, retinal secretions were required to maintain in vivo-like expression in culture. Albeit insufficient, retinal secretions promoted differentiation of immature RPE and helped maintain the properties of more mature RPE. Keywords: Tissue interactions, retina, retinal pigment epithelium, blood-retinal barrier, tight junctions

Project description:Purpose: The morphology of the RPE shows minimal change as the neural retina and choriocapillaris differentiate. Nonetheless, initial studies of barrier-related proteins suggest extensive remodeling of the RPE in response to this changing environment. A genomic approach was used to investigate the extent of this remodeling. Methods: RPE was isolated from E7, E10, E14 and E18 chick embryos and total RNA extracted for probing the entire genome on Affymetrix microarray chips. Statistical parameters using ANOVA were adjusted to yield a theoretical false discovery rate of 5%. STEM software was used to cluster genes into statistically related patterns of expression. Gene ontology clustering, using Affymetrix software was used for functional clustering. The proteinlounge.com database was used as a source of known biological pathways. Results: Of the 37,694 probe sets on the microarray, 17,199 were not absent. Of the 20,495 expressed probes, the expression of 8,889 was developmentally regulated. 4814 of these could be clustered into 12 patterns of expression that were statistically significant. The developmental patterns of expression for 22 tight and adherens junction proteins have been reported. Only two showed small variations from the patterns revealed by the microarray. Further, as would be predicted, expression of genes that promote progression through the cell cycle decreased and genes that inhibit progression increased with age. The data indicate extensive remodeling of the extracellular matrix, cell surface receptors, cell-cell junctions, transcellular ion transport and signal transduction pathways throughout development. Notably, the appearance of claudin 20, ZO-3 and cadherins 13 and 20 very late in development suggest barrier properties continue to change after functional junctions are formed. Conclusions: The data reveal a far more dynamic view of the RPE and its interactions with its environment than would be expected from morphological examination. The remodeling of junctional complexes, extracellular matrix interactions and transcellular transport capabilities indicates a continuous remodeling of the blood-retinal barrier. These data provide a standard whereby culture models of RPE function and regulation may be judged. SUBMITTER_CITATION: Rizzolo LJ, Chen X, Weitzman M, Sun R and Zhang H (2007) Analysis of the RPE transcriptome reveals dynamic changes during the development of the outer blood-retinal barrier. Mol. Vis. 13: in press. Experiment Overall Design: Sheets of RPE were isolated from chicken embryos on Embryonic day 7 (E7), E10, E14 and E18 and stored in RNAlatter (Qiagen, Valencia, CA), as described <Rahner C, Fukuhara M, Peng S, Kojima S, Rizzolo LJ. The apical and basal environments of the retinal pigment epithelium regulate the maturation of tight junctions during development. J Cell Sci. 2004;117:3307-18>. To isolate total RNA, the RNeasy Protect kit (Qiagen) was used according to the manufacturer's protocols. For each age, 3-4 independent preparations were used for analysis on Affymetrix microarrays of the chicken genome (Santa Clara, CA). For each preparation, sheets of RPE were pooled from 20-30 eyes. The quality of the total RNA was assessed by the Keck Center, Yale University using formamide gels and a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA)

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Transcription profiling of chicken to investigate regulation of the early embryonic RPE (retinal pigment epithelium) transcriptome by the neural retina

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