Transcript Inflammation in Fanconi Anemia Zebrafish Mutants

Characterizing Intestinal
Inflammation in Fanconi Anemia
Mutants
Sam Carpentier
University of Oregon SPUR 2009
http://blogs.nature.com/nm/spoonful/zebrafish.jpg
Zebrafish Models of Intestinal
Inflammation
•Intestinal Alkaline Phosphatase Mutant
•Unable to detoxify LPS from Gram-negative
bacteria.
•Sox 10 Mutant
•Defective gut peristalsis that leads to
increased intestinal inflammation.
Why Fanconi Anemia?
 Fanconi Anemia is a DNA-repair disease that
leads to problems such as cancer and bloodrelated diseases.
Inflammatory Bowel Disease Symptoms have been
associated with some cases of Fanconi Anemia.
http://wpcontent.answers.com/wikipedia/commons/thumb/7/
78/Fanconi%27s_anemia_101.jpg/190pxFanconi%27s_ane
mia_101.jpg
Aim
 Test the hypothesis that Fanconi mutants
have increased intestinal inflammation.
Assay
 We will count Myeloperoxidase (MPO) positive
neutrophils in the zebrafish intestine.
Wild-Type
Mutant
MPO Positive Cells Per Fish
Fanconi D1 Mutants Do Not
Have Elevated Inflammation
20
15
p=.64
10
Data from 2
Independent
trials
5
0
WT
FancD1 Mutant
MPO Positive Cells Per Fish
Fanconi G Mutants Do Not Have
Increased Inflammation
25
20
15
10
p= .40
5
0
WT
Fanc G Mutant
MPO Positive Cells Per Fish
Fanconi C Mutants May Have
Increased Inflammation
12
11
10
9
8
7
6
5
4
3
2
1
0
p=.007
p=.70
Modifications of MPO Counting
Protocol
• MPO-positive cells were counted that may
have been outside of the gut.
•Further studies quantified the MPO-positive
cells in sectioned tissue.
MPO Positive Cells Per Fish
Fanconi C Mutants May Have
Increased Inflammation
20
15
10
P=.18
5
0
WT
FanC C Mutant
MPO Positive Cells Per Fish
Fanc G Appear to Have
Increased Inflammation
20
15
10
P=.05
5
0
WT
FancG Mut
Why is There Increased
Inflammation in the Fanconi C
Mutants?
•Immune imbalance, independent of
microbiota.
•Increased inflammatory response to the
microbiota.
Raise Fanc C Mutants
Germ- Free
(Without Microbiota)
derive
germ-free
inflamed mutant
?
MPO Positive Cells Per Fish
Fanc C Mutant Inflammation is
a Response to the Microbiota
20
15
10
5
0
WT p=.0003
Mutant p=.0001
Do Fanc C Mutants Exhibit
Defective Peristalsis and
Bacterial Imbalance?
Fanc C fish were fed fluorescent green food for three
days, followed by a fluorescent red food.
5dpf
6dpf
7dpf
8dpf
Fanc C WT Movie
QuickTime™ and a
H.264 decompressor
are needed to see this picture.
Fanc C Mutant Movie
QuickTime™ and a
H.264 decompressor
are needed to see this picture.
Conclusions
 There does not appear to be increased
intestinal inflammation in the Fanc D1.
 Intestinal Inflammation may be increased in
both the Fanc C and G mutant fish.
 Intestinal inflammation in the Fanc C mutants is
a response to the microbiota.
 Gut motility appears to be unimpaired in the
Fanc C mutant fish.
Future Directions
Gut Microbiota Reciprocal Transplantation
 Transfer CV Fanc C Mutant microbiota into
GF Wild-Type Fish
inflamed mutant
donor
Acknowledgements
Erika Mittge
Karen Guillemin
Kate Woronowicz
Kristin Alligood
Judith Eisen
The Guillemin Lab
Peter O’Day
SPUR Program
Tom Titus
Rose
Chelsie Fish
Catharine Wilson
Poh Kheng Loi
UO Zebrafish Facility