Diffusible retinal secretions regulate the expression of tight junctions and other diverse functions of the retinal pigment epithelium.
OBJECTIVE
The retinal pigment epithelium (RPE) forms the outer blood-retinal barrier. It is unclear how culture conditions might alter barrier properties of isolated RPE. We examined whether retinal secretions that increase the barrier functions of tight junctions in vitro also make gene expression in general more in vivo-like.


METHODS
Chick RPE from embryonic day 7 (E7) and E14 were cultured on filters. Media conditioned by organ culture of E14 neural retinas was added to the apical medium chamber. RNA was isolated to probe the chick genome on Affymetrix microarrays, and expression was compared to native E14 RPE. Expression was further analyzed by quantitative real-time PCR immunoblotting and immunocytochemistry.


RESULTS
More than 86% of the genes expressed in vivo were expressed in basal culture conditions, including RPE-specific markers such as RPE65 and bestrophin. 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 effect on members of the claudin family. 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 differentiation and the levels of gene expression became more in vivo-like when elements of the retinal environment were introduced into the medium bathing the apical side of the cultures. Albeit insufficient, retinal secretions promoted differentiation of immature RPE and helped maintain the properties of more mature RPE.
Sun
Ru
R
Department of Surgery, Yale University, New Haven, CT 06520-8062, USA.
Peng
Shaomin
S
Chen
Xiang
X
Zhang
Heping
H
Rizzolo
Lawrence J
LJ
D000199:Actins / Q000378:metabolism; D022005:Adherens Junctions / Q000378:metabolism; D000818:Animals; D002462:Cell Membrane / Q000235:genetics; D002478:Cells, Cultured; D002642:Chick Embryo; D016000:Cluster Analysis; D017077:Culture Media, Conditioned; D004058:Diffusion; D017097:Electric Impedance; D005109:Extracellular Matrix / Q000235:genetics; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental; D016678:Genome; D007473:Ion Channels / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008543:Melanins / Q000096:biosynthesis; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008870:Microtubules / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis; D010587:Phagocytosis / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D055213:Retinal Pigment Epithelium / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism; D019108:Tight Junctions / Q000378:metabolism*; D014795:Visual Pathways
D000199:Actins; D017077:Culture Media, Conditioned; D007473:Ion Channels; D008543:Melanins; D008565:Membrane Proteins; D012333:RNA, Messenger