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INITIAL CHARACTERIZATION OF WHEN A FUNCTIONAL CIRCADIAN SYSTEM IS PRESENT IN THE EYE OF XENOPUS LAEVIS Researcher: Nicole Sarver Mentor: Dr. Kristen Curran Department of Biological Sciences – University of Wisconsin at Whitewater Materials and Methods Circadian Rhythm 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. For circadian rhythm to occur there must be both a central oscillator gene, which serves as an internal clock when there are no external stimuli and several output genes Results Fertilize frog eggs Dissect out 10 eyes from similarly staged embryos at 4 different circadian times; dawn (ZT0), midday (ZT6), dusk (ZT 12), and midnight (ZT 18). Figure 2. Quantitative analysis of xBmal1 expression in Stage 31 eyes. Background By using Real time RT PCR, genes may be quantitatively measured at different times during the day, which can allow us to determine when the circadian oscillator becomes fully functional in the eye. * Extract RNA Circadian Genes of Interest Reverse Transcription to obtain cDNA Figure 3. Quantitative analysis of xBmal1 expression in Stage 40 eyes. Real-Time RT-PCR: Figure 1. Model of oscillator function and link to outputs xBmal1: • Central oscillator gene • Transcription factor • Expressed during the night This technique is used to amplify and quantify a selected DNA molecule. A fluorescent dye (Sybergreen) is then used to intercalate between double stranded DNA, allowing for quantitative detection and relative quantitation. Conclusions Stage 31 Arrhythmic Expression (ANOVA; p=0.176) Stage 40 Rhythmic Expression Sybergreen Question (* ANOVA; p=.0004, when ZT18 was compared to ZT0) What stage is the circadian oscillator fully functional in the eye? Future Directions Development of Xenopus laevis tadpoles Acknowledgements: Chancellor Telfer Dean Pinkerton Dean Stone UWW Undergraduate Research Dean Barnett Dr. Kris Curran Jessica Solis Aaron Trow Beta Beta Beta Biological Honors Society Zygote Cleavage Blastula Gastrula St 1 St 2-5 St 6-9 St 10-13 Neurula Tailbud Tadpole St 14-20 St 21-39 St 40+ Find a more exact stage of when the circadian oscillator is fully functional Find the stage at which the output gene (NAT) is first rhythmic Find out if we can use less eyes, yet still get enough amplification to see a pattern of expression