Modeling Inflammatory Bowel Disease in Zebrafish

Abstract

Inflammatory bowel disease (IBD) is a chronic inflammatory condition in the gastrointestinal tract. The etiology of IBD remains elusive; however, IBD is thought to be caused by the dysregulated intestinal immune system, which mounts excessive responses to commensal flora. Over 40 susceptibility genes for IBD have been identified by genome wide association studies; many of them are central players in maintaining epithelial integrity, and innate and adaptive immunity. Over the past two decades, transgenic and chemical mouse models of IBD have offered insight into the molecular pathways that have been linked to IBD pathogenesis. The mouse has been regarded as the gold standard of model species for disease modeling due to the conservation of disease pathways and processes between the organism and humans. The research described here proposes the zebrafish as a critical vertebrate species for the modeling of IBD. Bioinformatic approaches have identified zebrafish orthologues of three genes that have been associated with human IBD: interleukin-10 (illO'), nucleotide oligomerisation domain 2 (nod2), and ATG16 autophagy related 16-like 1 (atgl6ll). Further bioinformatic analyses also established the orthologous relationship of these genes with human equivalents. Expression of illO, nod2, and atgl6ll was determined temporally and spatially. The bioinformatic work provided the background for undertaking gene-targeting studies. There are limited tools available for gene-targeting studies in zebrafish. In view of this, this work presents the development and investigation of microRNA based directed gene silencing methods in zebrafish. Unlike small interfering RNAs (siRNAs), the microRNA based method offers temporal and spatial regulation of the silencing effectors. Proof-of-concept experiments demonstrates the efficacy of the method in zebrafish embryos, however, the gene silencing on the engogenous gene atgl6ll was less effective. The vectors for transgenic expression of silencing microRNA (s) were constructed. The work described above provides the foundation for future microRNA-based gene-targeting disease models. In addition to attempts to generate genetic models of IBD in zebrafish, the work on the chemical induction of intestinal inflammation using dextran sulfate sodium (DSS), which has been widely used in mouse colitis models, showed limited success. This research also presented studies that investigated the commercial proprietary molecules for real-time in vivo imaging of inflammation. Commensal bacteria also play a critical role in IBD pathogenesis. The work on community analysis of zebrafish intestinal bacteria indicated that subtle differences in spatial distribution and diversity of intestinal bacteria at intra-and inter-individual levels. This research also demonstrated the manipulability of the intestinal flora by deriving germ-free larval zebrafish. Overall, the thesis indicates the promising future of the zebrafish as an alternative model species for human IBD.