Initial characterization of when a functional circadian system is present in the eye of Xenopus laevis

Abstract

Almost all organisms present in nature exhibit a circadian rhythm, a 24 hour cycle in which the organism undergoes both physiological and behavioral changes based on the time of day. The circadian control of physiological processes in organisms is important because it allows them to anticipate changes (physiological/molecular/behavioral) that need to occur at different times of the day. In these organisms there is both a central oscillator that serves as an internal clock when there are no external stimuli and several output genes which display rhythmic patterns based on the circadian cycle. By quantitatively analyzing two genes, an output gene (NAT) and a central oscillator gene (xBmal1), we aim to determine at what stage of development the circadian oscillator becomes fully functional in the eyes. In order to analyze these eyes we must first dissect out tadpole eyes, at different times of the day (dawn, midday, dusk, and midnight). We then extract RNA and convert it to cDNA (reverse transcription). Real-time RT- PCR is then used on the cDNA to quantitatively measure the amount of NAT and xBmal1. In previous experiments, we have found that both NAT and xBmal1 display rhythmic expression at stage 46. We are now in the process of analyzing stage 26 eyes. We anticipate that at stage 26 we will have arrhythmic expression of both the central oscillator gene and the output gene, which can then serve as a negative control. If we were to see any rhythmic expression at this stage, we would plan for future experiments testing for expression at an earlier stage.