Materials and Methods

1
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
Results
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
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 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 p0.176) Stage 40 Rhythmic
Expression ( ANOVA p.0004, when ZT18 was
compared to ZT0)
Figure 1. Model of oscillator function and link
to outputs

• xBmal1
• Central oscillator gene
• Transcription factor
• Expressed during the night

Sybergreen
Question What stage is the circadian oscillator
fully functional in the eye?

• Future Directions
• 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

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
Neurula Tailbud Tadpole
St 1 St 2-5 St 6-9 St 10-13
St 14-20 St 21-39 St 40