Expression of Circadan Clock Genes in Multiple Cell Types in the Retina

Abstract

Abstract: : Purpose: In vitro studies have demonstrated that Xenopus laevis photoreceptor layers are capable of self–sustained circadian oscillations in vitro, indicating that photoreceptors are the site of an endogenous circadian oscillator. Recently, we demonstrated that two paralogs (xPer1 and xPer2) of the Drosophila central clock gene period exhibited different temporal patterns of expression in the Xenopus retina, and that one (Per2) was directly responsive to light and dopamine. The goal of this study was to determine the cellular site of light, cAMP and dopamine regulated xPer2 expression. Methods:Isolated Xenopus laevis eye cups were maintain in constant darkness in culture and exposed at specific times of day to light or quinpirole, a dopamine agonist, in the presence or absence of CPT–cAMP. Experiments were terminated by isolation of whole retinal RNA for Northern analysis of Per1 and Per2 expression, or by fixation for conventional in situ hybridization using Per1 and Per2 specific probes. Results:Retinal xPer2 expression increased in response to both light and quinpirole; xPer1 expression was not influenced light or quinpirole. CPT–cAMP had no effect on the light–evoked change in Per2 expression, but dramatically blocked the effect of quinpirole. In situ hybridization demonstrated that xPer2 was expressed in multiple cell types in the retina, but both the light and quinpirole regulated signals occurred in photoreceptors. In contrast, xPer1 was expressed throughout the retina without enrichment in photoreceptors. Conclusions:Our data demonstrate that xPer2 in photoreceptors is regulated by light and dopaminergic pathways that are differentially affected by cAMP, and are consistent with a model in which xPer2 plays a critical role in entrainment of the photoreceptor circadian oscillator by light and dopamine. More generally, our data, along with our previous demonstration of per gene expression in RPE, show that both xPer2 and xPer1 mRNAs are expressed in most if not all cell types within the retina and that xPer1 mRNA is abundant within cells of the inner nuclear and ganglion cell layers. Per gene expression, therefore, is consistent with a role for multiple clocks within the retina.

Keywords: circadian rhythms • gene/expression • photoreceptors