The enteric nervous system promotes intestinal health by constraining microbiota composition.

Sustaining a balanced intestinal microbial community is critical for maintaining intestinal health and preventing chronic inflammation. The gut is a highly dynamic environment, subject to periodic waves of peristaltic activity. We hypothesized that this dynamic environment is a prerequisite for a balanced microbial community and that the enteric nervous system (ENS), a chief regulator of physiological processes within the gut, profoundly influences gut microbiota composition. We found that zebrafish lacking an ENS due to a mutation in the Hirschsprung disease gene, sox10, develop microbiota-dependent inflammation that is transmissible between hosts. Profiling microbial communities across a spectrum of inflammatory phenotypes revealed that increased levels of inflammation were linked to an overabundance of pro-inflammatory bacterial lineages and a lack of anti-inflammatory bacterial lineages. Moreover, either administering a representative anti-inflammatory strain or restoring ENS function corrected the pathology. Thus, we demonstrate that the ENS modulates gut microbiota community membership to maintain intestinal health.

Strategies to Mitigate a Mycobacterium marinum Outbreak in a Zebrafish Research Facility.

In 2011, the zebrafish research facility at the University of Oregon experienced an outbreak of Mycobacterium marinum that affected both research fish and facility staff. A thorough review of risks to personnel, the zebrafish veterinary care program, and zebrafish husbandry procedures at the research facility followed. In the years since 2011, changes have been implemented throughout the research facility to protect the personnel, the fish colony, and ultimately the continued success of the zebrafish model research program. In this study, we present the history of the outbreak, the changes we implemented, and recommendations to mitigate pathogen outbreaks in zebrafish research facilities.

Innate immune responses to gut microbiota differ between oceanic and freshwater threespine stickleback populations.

Animal hosts must co-exist with beneficial microbes while simultaneously being able to mount rapid, non-specific, innate immune responses to pathogenic microbes. How this balance is achieved is not fully understood, and disruption of this relationship can lead to disease. Excessive inflammatory responses to resident microbes are characteristic of certain gastrointestinal pathologies such as inflammatory bowel disease (IBD). The immune dysregulation of IBD has complex genetic underpinnings that cannot be fully recapitulated with single-gene-knockout models. A deeper understanding of the genetic regulation of innate immune responses to resident microbes requires the ability to measure immune responses in the presence and absence of the microbiota using vertebrate models with complex genetic variation. Here, we describe a new gnotobiotic vertebrate model to explore the natural genetic variation that contributes to differences in innate immune responses to microbiota. Threespine stickleback, Gasterosteus aculeatus, has been used to study the developmental genetics of complex traits during the repeated evolution from ancestral oceanic to derived freshwater forms. We established methods to rear germ-free stickleback larvae and gnotobiotic animals monoassociated with single bacterial isolates. We characterized the innate immune response of these fish to resident gut microbes by quantifying the neutrophil cells in conventionally reared monoassociated or germ-free stickleback from both oceanic and freshwater populations grown in a common intermediate salinity environment. We found that oceanic and freshwater fish in the wild and in the laboratory share many intestinal microbial community members. However, oceanic fish mount a strong immune response to residential microbiota, whereas freshwater fish frequently do not. A strong innate immune response was uniformly observed across oceanic families, but this response varied among families of freshwater fish. The gnotobiotic stickleback model that we have developed therefore provides a platform for future studies mapping the natural genetic basis of the variation in immune response to microbes.

Evidence for a core gut microbiota in the zebrafish.

Experimental analysis of gut microbial communities and their interactions with vertebrate hosts is conducted predominantly in domesticated animals that have been maintained in laboratory facilities for many generations. These animal models are useful for studying coevolved relationships between host and microbiota only if the microbial communities that occur in animals in lab facilities are representative of those that occur in nature. We performed 16S rRNA gene sequence-based comparisons of gut bacterial communities in zebrafish collected recently from their natural habitat and those reared for generations in lab facilities in different geographic locations. Patterns of gut microbiota structure in domesticated zebrafish varied across different lab facilities in correlation with historical connections between those facilities. However, gut microbiota membership in domesticated and recently caught zebrafish was strikingly similar, with a shared core gut microbiota. The zebrafish intestinal habitat therefore selects for specific bacterial taxa despite radical differences in host provenance and domestication status.

Epithelial cell proliferation in the developing zebrafish intestine is regulated by the Wnt pathway and microbial signaling via Myd88.

Rates of cell proliferation in the vertebrate intestinal epithelium are modulated by intrinsic signaling pathways and extrinsic cues. Here, we report that epithelial cell proliferation in the developing zebrafish intestine is stimulated both by the presence of the resident microbiota and by activation of Wnt signaling. We find that the response to microbial proliferation-promoting signals requires Myd88 but not TNF receptor, implicating host innate immune pathways but not inflammation in the establishment of homeostasis in the developing intestinal epithelium. We show that loss of axin1, a component of the ß-catenin destruction complex, results in greater than WT levels of intestinal epithelial cell proliferation. Compared with conventionally reared axin1 mutants, germ-free axin1 mutants exhibit decreased intestinal epithelial cell proliferation, whereas monoassociation with the resident intestinal bacterium Aeromonas veronii results in elevated epithelial cell proliferation. Disruption of ß-catenin signaling by deletion of the ß-catenin coactivator tcf4 partially decreases the proliferation-promoting capacity of A. veronii. We show that numbers of intestinal epithelial cells with cytoplasmic ß-catenin are reduced in the absence of the microbiota in both WT and axin1 mutants and elevated in animals' monoassociated A. veronii. Collectively, these data demonstrate that resident intestinal bacteria enhance the stability of ß-catenin in intestinal epithelial cells and promote cell proliferation in the developing vertebrate intestine.

Intestinal alkaline phosphatase detoxifies lipopolysaccharide and prevents inflammation in zebrafish in response to the gut microbiota.

Vertebrates harbor abundant lipopolysaccharide (LPS) in their gut microbiota. Alkaline phosphatases can dephosphorylate and detoxify the endotoxin component of LPS. Here, we show that expression of the zebrafish intestinal alkaline phosphatase (Iap), localized to the intestinal lumen brush border, is induced during establishment of the gut microbiota. Iap-deficient zebrafish are hypersensitive to LPS toxicity and exhibit the excessive intestinal neutrophil influx characteristic of wild-type zebrafish exposed to LPS. Both of these Iap mutant phenotypes are dependent on Myd88 and Tumor Necrosis Factor Receptor (Tnfr), proteins also involved in LPS sensitivity in mammals. When reared germ-free, the intestines of Iap-deficient zebrafish are devoid of neutrophils. Together, these findings demonstrate that the endogenous microbiota establish the normal homeostatic level of neutrophils in the zebrafish intestine through a process involving Iap, Myd88, and Tnfr. Thus, by preventing inflammatory responses, Iap plays a crucial role in promoting mucosal tolerance to resident gut bacteria.

Distinct signals from the microbiota promote different aspects of zebrafish gut differentiation.

All animals exist in intimate associations with microorganisms that play important roles in the hosts' normal development and tissue physiology. In vertebrates, the most populous and complex community of microbes resides in the digestive tract. Here, we describe the establishment of the gut microbiota and its role in digestive tract differentiation in the zebrafish model vertebrate, Danio rerio. We find that in the absence of the microbiota, the gut epithelium is arrested in aspects of its differentiation, as revealed by the lack of brush border intestinal alkaline phosphatase activity, the maintenance of immature patterns of glycan expression and a paucity of goblet and enteroendocrine cells. In addition, germ-free intestines fail to take up protein macromolecules in the distal intestine and exhibit faster motility. Reintroduction of a complex microbiota at later stages of development or mono-association of germ-free larvae with individual constituents of the microbiota reverses all of these germ-free phenotypes. Exposure of germ-free zebrafish to heat-killed preparations of the microbiota or bacterial lipopolysaccharide is sufficient to restore alkaline phosphatase activity but not mature patterns of Gal alpha1,3Gal containing glycans, indicating that the host perceives and responds to its associated microbiota by at least two distinct pathways.