Gene-editing HNF1B in zebrafish to decipher disease mechanisms by MODY5

Zebrafish has become an increasing valuable as a translational research model, and used world wide in studies of human disease mechanisms, as well as model for studying pathology of human disease like diabetes. We have previously reported on mutations in monogenic diabetes genes (MODYs) causing autosomal dominantly inherited diaetes, as well as gene variants in the same MODY genes as risk factors for type 2 diabetes. Some forms of MODY are associated with disease in not only pancreas, but in kidney and other organs. We will establish a MODY zebrafish harbouring mutation in the HNF1B gene associated with MODY5 disease. The aim with this zebrafish line is to study the molecular mechanisms behind organ specific pathology (pancreas hyper plasia, kidneycysts) as a result of mutation in the HNF1B gene, encoding the hepatocyte nuclear factor 1B.

Heterozygous loss-of-function mutations in the hepatocyte nuclear factor genes HNF4A, HNF1A and HNF1B give rise to clinically distinct subtypes of monogenic diabetes: Maturity-onset diabetes of the young (MODY1, MODY3 and MODY5, respectively). Although rare compared to type 1 and type 2 diabetes, MODY is a serious disease which lasts through a person’s lifetime, and with an increased risk of heart disease, kidney failure and blindness. In addition to early-onset diabetes by reduced pancreatic insulin, patients may also suffer from disease in other non-pancreatic organ tissues including the kidney (renal dysplasia/cysts/nephrocalcinosis), liver (liver dysfunction/cancer), or reproductive organs (uterus-/ovary-/testis pathology). As studies on molecular cause and disease mechanisms in such tissues have been severely limited, it is still unclear how mutations in these HNF genes result in such a broad spectrum of tissue specific MODY diseases. The objective of this project is to characterize the underlying disease mechanism by which the molecular drivers (HNF1B mutation) cause tissue specific disease in the kidneys. As zebrafish have shown to represent a powerful model system to study the basic mechanisms of disease in multiple organs and in diabetes, we propose to use zebrafish as animal model system for MODY5. By gene-editing HNF1B, disease characterization will be performed by an interdisciplinary team of researchers with expertise in MODY, in kidney disease, in zebrafish modelling, in organ/tissue imaging (in vivo/in situ), in molecular (transcriptome) investigations and complex data analyses by machine learning. Long term project discoveries may guide future targeted treatment investigations of MODY5 and kidney disease, with relevance to other more common forms of diabetes with phenotypic overlaps.

The MODY5 model will be made by CRISPR/Cas9 technology in tissue specific GFP-transgenic lines (relevant lines available at ZIRC: ZL1733 (nkx2.2a:GFP) or ZL1i669(ins:GFP), for pancreas endocrine and beta-cell content), and the Tg(wt1b:EGFP) zebrafish line for kidney development, to facilitate in vivo monitoring (by fluorescence microscopy) of tissue-based organ development. In these zebrafish lines, naturally occurring patient specific mutation (larger deletion- or premature termination mutations) in HNF1B A/B orthologue genes will also be incorporated. CRISPR efficiency will be tested by embryo genomic DNA isolation (fin-clips), PCR and genomic sequencing. Phenotype screening will be performed in the injected F0 individuals. If no phenotype is visible in F0 generations, we will cross F0 founders to obtain F1 heterozygous carrier lines. Stable heterozygous F1 carriers will be crossed to obtain homozygous progeny. Measurements of signs of diabetes by fasting and postprandial glucose/insulin measurements will be performed after blood collection in adult fish. Blood collection protocols are available for adult fish