Gene-editing of the monogenic diabetes associated gene hnf1ba to model multisystemic MODY5 disease mechanisms in zebrafish (DR)

Maturity-Onset Diabetes of the Young (MODY) is a rare form of monogenic diabetes, characterized by autosomal dominant inheritance, non-obesity, and early-onset diabetes due to reduced pancreatic beta-cell secretion. Precision medicine increasingly relies on an accurate interpretation of the consequence of genetic mutations in diseases like MODY. Clinical investigations of patients with MODY5 associated mutations in the gene encoding hepatocyte nuclear factor 1-beta (HNF1B), have revealed disease in not only the pancreas, but one or more additional organ systems, including the kidney, liver, and the reproductive system. In this thesis, using CRISPR-Cas9 mediated knockout technology, we targeted the paralog of the human HNF1B, hnf1ba in zebrafish. To understand the expression patterns of hnf1ba in wild-types, we performed a spatial localization using in situ hybridization conducted on 24 and 96 hpf larvae and confirmed that hnf1ba is expressed in the pancreas and pronephros (early kidneys. Triple immuno- histochemistry was used to explore the two predominating cell types in the endocrine pancreas, the insulin-producing beta-cells and the glucagon-producing alpha-cells, as well as the exocrine pancreas targeted by EGFP in the Tg(wt1b:EGFP) zebrafish line. The results of the pancreas immunostaining showed a significant reduction in both beta- and alpha-cell numbers in CRISPR-Cas9 injected zebrafish larvae. Further studies are needed to truly understand the vast scope of the transcription factor HNF1B, and its molecular mechanisms connected to MODY5 disease. This thesis provides several avenues for further research regarding the establishment of zebrafish as a stable MODY5 model.Masteroppgave i molekylærbiologiMOL399MAMN-MO