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AR Caffe, S Azadi, Y Zhang, I Holmqvist, T van Veen; RAR-alpha, RXR-gamma and ROR-beta2 Are Differentially Affected in Mouse Retinal Explants . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2686.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: In retinal explants (RE) cultured from postnatal day (PN) 2 green cones are missing. Recently it was demonstrated that thyroid beta2 receptors (TRs-beta2) are essential for green cone differentiation. However, the nuclear TRs function as heterodimers with retinoid receptors (RRs). Thus, to gain insight into the role of TR/RR heterodimers in the missing green cone phenomenon, the fate of RRs in RE needs to be analyzed. Methods: C3H +/+ mice were used to generate RE and in vivo retinal samples; PN2+div1 (1 day in vitro), PN3, PN2+div12 with and without RPE, PN14 light- or dark-adapted, PN-1-year light- or dark-adapted. Semi-quantitative RT-PCR was performed. Six retinas or RE per condition were pooled and total RNA extracted. RNA amounts were determined using spectrophotometry and gel electrophoresis (GE). Densitometry verified equal amounts of sample RNA. Using random primers first strand cDNA was synthesized from same amounts of sample RNA. The calibration curve for each specific primer pair was determined from pooled cDNA. Out of the linear region the same volume cDNA of all samples was PCR amplified with specific primers for RAR-alpha, RXR-gamma, and ROR-beta2. These RRs are linked to cone differentiation. The PCR products were separated by GE, visualized by ethidium bromide, quantified by densitometry, and normalized against the internal control (18S rRNA). Data was analyzed statistically. Results: In PN2+div1 RE level of RXR-gamma transcript was greatly depressed compared to PN3. ROR-beta2 and RAR-alpha levels were less affected. In PN2+div12 RE RRs transcript levels were further decreased, however, the presence of the RPE attenuated this effect. In PN14 retina RXR-gamma and ROR-beta2 transcripts displayed circadian rhythms. The former peaked in the dark-adapted whereas the latter in the light-adapted tissue. In the aged retina the RXR-gamma circadian rhythm was maintained whereas this was reversed for ROR-beta2. Conclusion: RRs are differentially affected in mouse RE; the RPE partially protects against loss of RRs gene expression. Rapid down regulation of RXR-gamma is likely involved in absence of green cone differentiation in mouse RE. Retinal RRs show differential circadian rhythms, some of which depend on age.
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