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Series GSE10538 Query DataSets for GSE10538
Status Public on Sep 30, 2008
Title Regulation of the early embryonic RPE (retinal pigment epithelium) transcriptome by the neural retina
Organism Gallus gallus
Experiment type Expression profiling by array
Summary 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
 
Overall design RPE were isolated from chicken embryos on embryonic day 7 or 14 and cultured, 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>. 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)
 
Contributor(s) Sun R, Weitzman M, Chen X, Zhang H, Rizzolo LJ
Citation(s) 19057659
Submission date Feb 15, 2008
Last update date Mar 19, 2012
Contact name Lawrence J Rizzolo
E-mail(s) lawrence.rizzolo@yale.edu
Organization name Yale University
Department Dept of Surgery
Street address 333 Cedar St
City New Haven
State/province CT
ZIP/Postal code 06511
Country USA
 
Platforms (1)
GPL3213 [Chicken] Affymetrix Chicken Genome Array
Samples (24)
GSM264617 Regulation of the RPE ( retinal pigment epithelium) transcriptome by the neural retina (E7SF3-1)
GSM264754 Regulation of the RPE (retinal pigment epithelium) transcriptome by the neural retina (E7SF3-2)
GSM264759 Regulation of the RPE (retinal pigment epithelium) transcriptome by the neural retina (E7SF3-3)
Relations
BioProject PRJNA107893

Download family Format
SOFT formatted family file(s) SOFTHelp
MINiML formatted family file(s) MINiMLHelp
Series Matrix File(s) TXTHelp

Supplementary file Size Download File type/resource
GSE10538_RAW.tar 90.2 Mb (http)(custom) TAR (of CEL, CHP)
Raw data provided as supplementary file
Processed data included within Sample table

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