The enteric nervous system (ENS) innervates the gut and plays a role in numerous gut functions including digestion, motility, and immune responses. Patients having an aberrant ENS (i.e. Hirschsrpung disease) have increased susceptibility to gut inflammation and altered microbiota. It is unknown why some patients with aberrant ENS have inflammation. The most common mutation causing Hirschsprung disease is Ret, which has an ortholog in zebrafish (ret) and when mutated decreases the number of enteric neurons. The zebrafish model is ideal because it is genetically tractable, transparent at larval stages, and microbial composition can be controlled (gnotobiotology). Hypothesis: The enteric nervous system regulates gut inflammation Specific Aims: 1.Identify the differences in inflammation (neutrophil abundance) in ret mutant zebrafish. 2.Determine the role of the microbiota in neutrophil abundance. Introduction Role of the Enteric Nervous System in Gut Inflammation Joshua Gil, Kristi Hamilton, Judith Eisen Institute of Neuroscience, University of Oregon Experimental Design We compared differences in neutrophil number in three different gnotobiotic conditions: 1. Conventional (CV, normal microbiota) 2. Vibrio cholera (pro- inflammatory bacteria) 3. Germfree (GF, sterile) Figure 1. Experimental flow chart. Cross fish and collect eggs 0 days post fertilization (dpf). 3 dpf pre-sort for transgenic fish with green neurons and red neutrophils. 6 dpf dissect gut quantify neutrophils and bacteria. Figure 2. (Top) 6 dpf zebrafish. (Bottom) wildtype (wt) and ret mutant guts. Neurons identified by green florescence. wt ret ret Mutants have Decreased Inflammation A. ret mutants have fewer neutrophils when reared with conventional microbiota B. ret mutants have fewer neutrophils even with when reared with pro- inflammatory Vibrio C. Wildtype zebrafish reared with Vibrio have increased inflammation D. ret mutants reared with Vibrio do not have increased inflammation The Microbiota is the Source of Inflammation A. Wildtype and ret mutants reared germfree have the same number of neutrophils B. Conventionally reared wildtypes have microbial induced inflammation * * * Microbial Colonization of the Gut does not Explain Inflammation A. Vibrio colonize both wt and ret mutant zebrafish B. Neutrophil number is not correlated with number of Vibrio ret mutant zebrafish show less inflammation than wildtypes Vibrio cholera causes inflammation in wt but not ret mutants wildtype and ret mutants zebrafish have equivalent amounts of bacteria colonization in their guts Summary The ENS is required to regulate microbial inflammation. Conclusions Identify microbial composition in conventionally reared ret mutants Determine mechanism of ENS regulation of inflammation by using multiple ENS mutants, such as ret and sox10 Future Directions Values are means ± SEM Two-tailed t-test, letters denote significance p<0.05. n=7-37 /group. ENS Regulated Inflammation Microbiota ENS Unregulated Inflammation Microbiota Acknowledgements Eisen Lab: Ellie Melancon, Levi Simonson, Charlotte Taylor, Guillemin Lab: Sophie Sichel, Annah Rolig, Keaton Stagaman SPUR Program: Peter O’Day, Marylin Drennan, NSF: NSF DBI/BIO CV GF Values are means ± SEM Two-tailed t-test, letters denote significance p<0.05. n=10 /group. Values are means ± SEM Two-tailed t-test, (*) denote significance p<0.05. n=13-47 /group.