Growth of the postnatal rat retina in vitro: quantitative RT-PCR analyses of mRNA expression for photoreceptor proteins.
OBJECTIVE
To investigate whether previously reported changes in protein expression of middle and long (M/L) and short (S) wavelength cone opsin pigments in cultured retina are correlated with changes in their gene expression. Additionally, to elucidate the importance of a functional retinal pigment epithelium for the development of photoreceptor outer segments.


METHODS
Neonatal rat retinas were maintained in culture for 11 days and either fixed in 4% paraformaldehyde for immunohistochemistry or prepared for RNA extraction, reverse transcription polymerase chain reaction (RT-PCR), and quantitative RT-PCR. S-cone and M/L-cone photoreceptors as well as rod photoreceptors were immunohistochemically identified using specific antibodies. Peanut agglutinin (PNA)-lectin histochemistry was used to identify interphotoreceptor matrix associated with cone photoreceptors. Immunolabeling for ED-1 and RPE65 was performed in combination with PNA-lectin staining to examine interactions between photoreceptor cells and the retinal pigment epithelium. Relative estimates of mRNA expression levels for M/L-opsin, S-opsin, recoverin, and rhodopsin in normal and cultured retina were determined by using quantitative RT-PCR.


RESULTS
Strong immunolabeling for recoverin and rhodopsin accumulated in outer segments as well as photoreceptor somata in vitro. Cultured and normal retinas showed similar relative expression levels of recoverin and rhodopsin mRNA. In cultured rat retina, the density of S-cones was high and M/L-cones could not be immunohistochemically detected. However, M/L-cone photoreceptor mRNA was detectable, but at a fourfold lower level in cultured than in vivo retinas. The S-cone photoreceptor mRNA level was almost twofold lower than in vivo. Retinal pigment epithelium cells in cultured specimens showed no RPE65 immunolabeling, but expressed immunolabeling for ED-1 indicating phagocytic activity of these cells in vitro.


CONCLUSIONS
We assume that the high density of S-cones and virtually no M/L-cones seen in in vitro retinas might represent an immature stage with numerous S-cones and suppressed transdifferentiation into M/L-cone phenotype. A non-functional relationship between photoreceptor cells and a dysfunctional retinal pigment epithelium may have severe consequences for the development of outer segments.
Liljekvist-Larsson
Ingela
I
Wallenberg Retina Center, Department of Ophthalmology, Lund University, Lund, Sweden.
Törngren
Marie
M
Abrahamson
Magnus
M
Johansson
Kjell
K
D000818:Animals; D000831:Animals, Newborn; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism; D002352:Carrier Proteins; D002454:Cell Differentiation; D053331:Ectodysplasins; D005136:Eye Proteins / Q000235:genetics* / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D018507:Gene Expression Regulation, Developmental; D051597:Hippocalcin; D008074:Lipoproteins; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D009419:Nerve Tissue Proteins; D009924:Organ Culture Techniques; D020419:Photoreceptor Cells, Vertebrate / Q000166:cytology / Q000378:metabolism*; D010857:Pigment Epithelium of Eye / Q000378:metabolism; D011506:Proteins / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D051381:Rats; D051603:Recoverin; D012160:Retina / Q000254:growth & development* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012243:Rhodopsin / Q000235:genetics / Q000378:metabolism; D019745:cis-trans-Isomerases; D000099178:Retinoid Isomerohydrolase
D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; D053331:Ectodysplasins; D005136:Eye Proteins; D051597:Hippocalcin; D008074:Lipoproteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D011506:Proteins; D012333:RNA, Messenger; D051603:Recoverin; D012243:Rhodopsin; D019745:cis-trans-Isomerases; D000099178:Retinoid Isomerohydrolase; C496733:Rcvrn protein, rat