A Rapid Screenable Assay for Compounds That Protect Against Intestinal Injury in Zebrafish Larva

The predominantly anaerobic microbiota of the distal ileum and colon contain an extraordinarily complex variety of metabolically active bacteria and fungi that intimately interact with the host's epithelial cells and mucosal immune system. Crohn's disease, ulcerative colitis, and pouchitis are the result of continuous microbial antigenic stimulation of pathogenic immune responses as a consequence of host genetic defects in mucosal barrier function, innate bacterial killing, or immunoregulation. Altered microbial composition and function in inflammatory bowel diseases result in increased immune stimulation, epithelial dysfunction, or enhanced mucosal permeability. Although traditional pathogens probably are not responsible for these disorders, increased virulence of commensal bacterial species, particularly Escherichia coli, enhance their mucosal attachment, invasion, and intracellular persistence, thereby stimulating pathogenic immune responses. Host genetic polymorphisms most likely interact with functional bacterial changes to stimulate aggressive immune responses that lead to chronic tissue injury. Identification of these host and microbial alterations in individual patients should lead to selective targeted interventions that correct underlying abnormalities and induce sustained and predictable therapeutic responses.

Inflammatory bowel disease (IBD) has been attributed to aberrant mucosal immunity to the intestinal microbiota. The transcription factor XBP1, a key component of the endoplasmic reticulum (ER) stress response, is required for development and maintenance of secretory cells and linked to JNK activation. We hypothesized that a stressful environmental milieu in a rapidly proliferating tissue might instigate a proinflammatory response. We report that Xbp1 deletion in intestinal epithelial cells (IECs) results in spontaneous enteritis and increased susceptibility to induced colitis secondary to both Paneth cell dysfunction and an epithelium that is overly reactive to inducers of IBD such as bacterial products (flagellin) and TNFalpha. An association of XBP1 variants with both forms of human IBD (Crohn's disease and ulcerative colitis) was identified and replicated (rs35873774; p value 1.6 x 10(-5)) with novel, private hypomorphic variants identified as susceptibility factors. Hence, intestinal inflammation can originate solely from XBP1 abnormalities in IECs, thus linking cell-specific ER stress to the induction of organ-specific inflammation.

Intestinal development in amniotes is driven by interactions between progenitor cells derived from the three primary germ layers. Genetic analyses and gene targeting experiments in zebrafish offer a novel approach to dissect such interactions at a molecular level. Here we show that intestinal anatomy and architecture in zebrafish closely resembles the anatomy and architecture of the mammalian small intestine. The zebrafish intestine is regionalized and the various segments can be identified by epithelial markers whose expression is already segregated at the onset of intestinal differentiation. Differentiation of cells derived from the three primary germ layers begins more or less contemporaneously, and is preceded by a stage in which there is rapid cell proliferation and maturation of epithelial cell polarization. Analysis of zebrafish mutants with altered epithelial survival reveals that seemingly related single gene defects have different effects on epithelial differentiation and smooth muscle and enteric nervous system development.