Transcript NCUR Poster Final

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