Characterizing Intestinal Inflammation in Fanconi Anemia Mutants Sam Carpentier University of Oregon SPUR 2009 Sam Carpentier University of Oregon SPUR

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 blood- related diseases. 78/Fanconi%27s_anemia_101.jpg/190pxFanconi%27s_ane mia_101.jpg  Inflammatory Bowel Disease Symptoms have been associated with some cases of Fanconi Anemia.

Aim  Test the hypothesis that Fanconi mutants have increased intestinal inflammation.  We will count Myeloperoxidase (MPO) positive neutrophils in the zebrafish intestine. Assay

Wild-TypeMutant

Fanconi D1 Mutants Do Not Have Elevated Inflammation Data from 2 Independent trials p=.64

Fanconi G Mutants Do Not Have Increased Inflammation p=.40

Fanconi C Mutants May Have Increased Inflammation WT 1WT 2Mut 2 Mut 1 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.

Fanconi C Mutants May Have Increased Inflammation P=.18

Fanc G Appear to Have Increased Inflammation P=.05

Why is There Increased Inflammation in the Fanconi C Mutants? Immune imbalance, independent of microbiota. Increased inflammatory response to the microbiota.

inflamed mutant derive germ-free ? Raise Fanc C Mutants Germ- Free (Without Microbiota)

Fanc C Mutant Inflammation is a Response to the Microbiota CV WT Fanc C CV Mut GF WT Fanc C GF Mut WT p=.0003 Mutant p=.0001

Do Fanc C Mutants Exhibit Defective Peristalsis and Bacterial Imbalance? 5dpf6dpf7dpf8dpf Fanc C fish were fed fluorescent green food for three days, followed by a fluorescent red food.

Fanc C WT Movie

Fanc C Mutant Movie

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.  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 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 Chelsie Fish SPUR Program Catharine Wilson Tom Titus Poh Kheng Loi Rose UO Zebrafish Facility