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From Alcohol to Zebrafish:
Ethanol as a source of DNA damage in fancl-/zebrafish
Vishesh Khanna
August 17th, 2012
Mentor: Catherine Wilson
PI: Dr. John Postlethwait
http://3inq.com/movies/avengers-and-zebra-fish, wikipedia.org
Outline
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Introduction to Fanconi anemia (FA)
Hypothesis and Methodology
Experimental Results
Conclusions and Future Directions
What is Fanconi anemia (FA)?
• Rare & inherited genomic instability
disorder causing:
– Bone marrow failure
– Developmental abnormalities
• Missing/underdeveloped thumb and radius
• Micropthalmia, microcephaly and hypogonadism
– Highly increased risk of acute myeloid
leukemia (AML) and head & neck cancers
http://blog.lib.umn.edu/mmf/news/assets_c/2010/11/Wagner-and-Molly_04661811.html;
http://images.radiopaedia.org/images/569184/5f582aadf79f75b627abd02b28e5ed.jpg
What causes FA?
• Biallelic mutations in 1 of 15
genes involved in DNA repair
• Comprise the Fanconi
anemia/Breast Cancer (FABRCA) pathway
Gene
FANCA
FANCB
FANCC
FANCD1 (BRCA2)
FANCD2
FANCE
FANCF
FANCG
FANCI
BRIP1 (FANCJ)
FANCL
FANCM
PALB2 (FANCN)
RAD51C (FANCO)
SLX4 (FANCP)
What does the FA pathway do?
Repairs DNA interstrand crosslinks (ICLs)
Deans and West,
Alcohol and Fanconi anemia
• Ethanol  Acetaldehyde (AA)  ICLs in vitro
• Aldh2  metabolizes acetaldehyde
• Patients have only have FA pathway mutations
• Sought to develop a zebrafish model
The zebrafish as a model of FA
• FA genes conserved from humans to zebrafish
• FA pathway mutants  more sensitive to
synthetic ICL-causing agents than WT siblings
brca2 =
fancd1
Acridine Orange : Cell death marker
DEB = synthetic ICL-causing agent
Rodriguez-Mari et al., 2011
Outline
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Introduction to Fanconi anemia (FA)
Hypothesis and Methodology
Experimental Results
Conclusions and Future Directions
The Research Question
Are fancl-/- zebrafish embryos more
sensitive to ethanol than their wild-type
siblings?
Hypothesis: fancl-/- zebrafish embryos are
more sensitive to ethanol than their wildtype siblings
Experimental Methodology
• Cross fancl+/- heterozygotes collect and expose embryos to
ethanol from 6-48 hours post-fertilization (hpf)
• Examine morphology at 5 days post-fertilization (dpf) and
genotype
• Prediction:
fancl-/- embryos will display more severe phenotypes than their
wild-type counterparts
http://www.news.cornell.edu/stories/Feb07/wholezfish.jpg
Outline
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Introduction to Fanconi anemia (FA)
Hypothesis and Methodology
Experimental Results
Summary and Conclusions
Future Directions
EtOH causes developmental
defects
5 dpf
0 mM
micropthalmia
100
mM
widespread edema
150
mM
[EtOH] (mM)
Percentage with
Widespread
Edema
0 mM
0% (0/32)
100 mM
16% (5/32)
150 mM
41% (13/32)
EtOH causes developmental defects
5 dpf
n=9
n=9
n=7
n=11
n=7
fancl-/- larvae are not specifically affected by EtOH
n=11
n=12
n=9
n=9
n=12
n=11
n=7
n=18
n=7
n=11
n=12
n=9
n=9
n=12
n=11
n=7
n=18
n=7
EtOH increases hemoglobin levels at 48 hpf
O-dianisidine: reacts
with hemoglobin to
produce a red stain
Control
fancl-/- embryos do not show reduced hemoglobin
0 mM EtOH
100 mM
50 mM
75 mM
48 hpf
fancl+/+
fancl+/Control
fancl-/-
EtOH does not exacerbate cell death in
fancl-/- embryos
Acridine Orange = marker
of cell death
N=2
N=3
N=3
N=7
N=2
Outline
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•
•
•
Introduction to Fanconi anemia (FA)
Hypothesis and Methodology
Experimental Results
Conclusions and Future Directions
Summary & Conclusions
• Embryonic ethanol exposure causes
morphological defects, but they are not
exacerbated in fancl-/- embryos
• Ethanol does not specifically affect hemoglobin
levels in fancl-/- embryos
• Ethanol exposure increases cell death, but not
specifically in fancl-/- embryos
• Overall, the data do not support my hypothesis
Future Directions
• Repeat exposure studies
– Add inhibitor of ethanol metabolism
• Direct embryonic acetaldehyde exposure
– Experiments in progress
• Later exposure windows (early larval stages)
– Helps rule out presence of maternal Fancl in early
embryonic stages
Acknowledgments
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Dr. John Postlethwait
Catherine Wilson
The Postlethwait Lab
Peter O’Day
Adam Unger
SPUR and R-25 Interns
The NIH-NICHD R25 Summer Research
Program
NIH-1R25HD070817