Osteoporosis is an orthopedic disease in which old bone begins to reabsorb but is not replaced by new bone. WNT16 is a critical gene influencing genetic risk for osteoporosis. The WNT16 locus harbors genetic variants associated with bone mineral density (BMD). Mice with global or conditional null mutations in Wnt16 exhibit reduced cortical bone mass and strength. Recently, our lab has shown that zebrafish with null mutations in wnt16 exhibit reduced vertebral bone length with otherwise normal bone morphology. My objective was to generate somatic mutant zebrafish with protein-truncating mutations in wnt16 and assess the effects of these mutations on adult zebrafish spine morphology and mineralization. My hypothesis was that protein-truncating mutations in wnt16 induce severe alterations in adult vertebral bone mass and morphology that are not observed in wnt16 null mutants. CRISPR-based gene editing was used to generate somatic mutants, wnt16^trunc, by targeting the terminal exon of wnt16, resulting in a prematurely truncated gene product. FishCuT was used to assess measures of bone morphology and mineralization in the spine; this included the volume, thickness, and tissue mineral density of the centrum, and neural and haemal arches. FishCuT analysis revealed wnt16^trunc mutants had significantly reduced centrum volume and length. Our results indicate that zebrafish somatic mutants with wnt16 protein-truncating mutants exhibit severe phenotypic changes and morphological abnormalities not apparent in wnt16 null mutants. This could reveal potential targets with regards to the development of therapeutic agents for manipulating WNT16 signaling in humans. These results also allow for interpretation of the functional consequences of human genetic variants in WNT16 that are predicted to result in truncated protein products. Future studies are focused on generating germline mutants with wnt16 protein-truncating mutations and determining the molecular mechanism by which such mutations give rise to severe phenotypes not present in wnt16 knockout animals.

Osteoporosis is a condition in which bones become weak and brittle which leads to increased risk of bone fractures. Approximately 60-80% of bone mineral density is determined by genetics. One gene in humans that is known to contribute to bone resorption is TNFSF11. In normal conditions, TNFSF11 encodes for receptor activator of nuclear factor kappa-B ligand (RANKL). When this ligand binds its receptor RANK, this stimulates osteoclast activity and bone resorption occurs. Zebrafish are an emerging model organism for bone biomedical research. My question is: are zebrafish an appropriate model organism to study human bone biology regarding TNFSF11 and other genes that it interacts with? If so, we can use zebrafish to study this gene and eventually apply our findings to humans. My hypothesis was that zebrafish with loss-of-function mutations in the TNFSF11 gene would exhibit high bone mass, due to a decreased level of RANKL released resulting in decreased osteoclast activity. To test this, CRISPR technology was used to mutate the TNFSF11 gene rendering it functionless. 12 wildtype and 12 mutant fish that were scanned via microcomputed tomography. I generated maximum intensity projections using a program called FIJI and then analyzed the fish projections using a computer program called FishCuT (developed by Dr. Kwon and his colleagues) which analyzes bone mineral density and measurements related to bone microarchitecture. When analyzing the data using R, I was able to find statistically significant difference between bone measurements in the wildtype and mutant fish. The mutant fish had bones that were more mineralized in comparison to the wildtype fish. These data support that TNFSF11 in humans and fish serves the same function because hyper-mineralized bones are expected from decreased bone resorption. Future studies will be directed at investigating the effects of these mutations in germline mutant fish.