Circadian Regulation of Iodopsin Expression in the Developing Retina ---In Vitro and In Ovo Studies

Abstract

Abstract: : Purpose:To compare the developmental onset and circadian rhythms of iodopsin expression in embryonic chicken retinal explant cultures to those in retinas from chicken embryos. Methods:All eggs were incubated under a 12Light :12Dark (12L:12D) cycle until utilized in the experiments. Retinal explants were prepared from embryonic day 9 (E9) chicken embryos and cultured in vitro. For the circadian experiments, the regulation of iodopsin expression was examined in ovo using retinas removed from E10 to 15 chicken embryos and in vitro utilizing retinas maintained in culture for 1 to 6 days (DIC). The eggs and retinal explant cultures were exposed to one of three lighting regimes: (1) 12L:12D cycle; (2) 12L:12D cycle followed by constant darkness; (3) 12L:12D cycle followed by reversal of the light cycle (12D:12L). Iodopsin transcript levels in both embryo retina and explant cultures were measured every 12 hr by northern or slot blot techniques. The onset of iodopsin expression in retina in ovo and in explant culture was determined using northern blot methods and immunohistochemistry. Results:Expression of iodopsin was detected in cultured retinal explants as early as 3DIC using both northern blot and immunohistochemical methods. In contrast, iodopsin expression was not observed in ovo until E15. Transcription of the iodopsin gene in 3DIC retinal explant cultures exhibited a robust rhythm under 12L:12D cycle for up to 9 days. In embryos maintained under 12L:12D conditions, rhythmic iodopsin transcription was detectable in retina on E17, 2 days after the onset of iodopsin expression, and the rhythm became much more robust from E18 to E20. Iodopsin transcript rhythms observed in both retinal explants and in the retinas of embryos maintained under 12L:12D conditions were maintained for at least 48 hours when the cultured retinas or embryos were placed in constant darkness. Reversal of the light/dark cycle produced a rapid reversal of the phase of the iodopsin rhythm (within 24 hr) in DIC6 retinal explants and in retinas of E18 embryos. Conclusion:The onset of iodopsin expression in explant cultures occurs earlier than observed in ovo and our data suggest that circadian oscillators play a significant role in controlling the transcription of this gene during development. The iodopsin mRNA rhythms observed in explant cultures accurately reflect the iodopsin rhythms observed in ovo and support the use of explant cultures in studies of retinal circadian clock function.

Keywords: 349 circadian rhythms • 560 retinal culture • 417 gene/expression