Osteoporosis, a polygenic disease characterized by low bone mineral density (BMD) and increased fracture risk, is the most prevalent bone disease impacting over 200 million people worldwide. Because of the associated financial burdens and reductions in quality of life, there is an urgent need to determine the genetic causes of osteoporosis. The WNT family of proteins has been implicated in numerous developmental and disease pathways, with WNT16 specifically being linked to osteoporosis risk. WNT proteins contain 24 conserved cysteines, and mutations involving half of these cysteines are associated with human diseases or disrupted development in animal models. While modifications in cysteines 1-6, 8-9, 11-12, and 24 in different WNT proteins have been defined in vivo, the impact of cysteine 10 (c10) alteration remains unknown. Wnt crystal structure suggests that c10 creates a disulfide linkage with cysteine 11 and resides in a region that directly interacts with Frizzled receptors to initiate WNT signaling pathways. My hypothesis is that loss of c10 in WNT16 will result in altered BMD indicative of elevated osteoporosis risk. To test this, I outcrossed CRISPR-generated somatic zebrafish mutants harboring mutations at the wnt16 locus that target the c10 position, and isolated wnt16^w1012 mutants. Sanger sequencing and sequence alignment revealed a three amino acid deletion at Cys214, corresponding to c10 (p.Cys214_Gly216del). Analysis of micro-computed tomography scans showed significant decreases in wnt16^w1012 mutant centrum length, which matches wnt16 knockouts. My data indicates that wnt16^w1012 mutants phenocopy wnt16 knockouts, suggesting that c10 plays an essential role in WNT16 secretion and/or activity. My data further suggests that mutations that alter c10 have potential to contribute to osteoporosis pathogenesis. My ongoing studies are focused on further characterizing musculoskeletal phenotypes in wnt16^w1012 mutants and understanding the consequences of the mutation on protein structure through computational modeling.

Osteoporosis is a polygenic disease defined by low bone mineral density and is associated with increased rates for fractures and mortality. This condition commonly occurs in concert with sarcopenia, which is characterized by loss of muscle mass and function. When these occur in conjunction, a condition termed osteosarcopenia, there is an increased risk of falls which heightens the risk of fracture of already fragile osteoporotic bone. Genome wide association studies have identified genetic variants which influence osteosarcopenia-related traits. One such study identified pleiotropic effects on bone mineral density and lean mass at the CPED1/WNT16 lLocus. CPED1 has been hypothesized to be a causal gene, however there are very few studies characterizing CPED1, and it has no confirmed functions in humans or zebrafish. The goal of this study was twofold: to investigate the necessity of CPED1 for bone and lean mass in zebrafish. We analyzed a single-cell atlas of embryonic development and found that CPED1 is most strongly expressed in muscle. We generated two mutant alleles, CPED1w1003 and CPED1sa20221 via CRISPR gene editing. For analysis, 3 or 14 month old zebrafish were scanned using microCT, and ImageJ and FishCuT software were utilized to measure vertebral morphology and mineralization, lean mass, and standard length. Results showed no significant differences between mutant and control groups for both mutant alleles. The results of this study do not support CPED1 as being a causative gene underlying bone and muscle pleiotropy at the CPED1/WNT16 locus. This study also raises questions regarding the function of CPED1 in muscle and whether its loss may be compensated for by other genes.

WNT signaling plays an essential role in many developmental processes with WNT molecules functioning as directional and differentiation cues. The recapitulation of WNT signaling pathways during embryonic morphogenesis is a promising approach for the development of novel regenerative therapeutics to treat various conditions. Recent work from our lab has demonstrated wnt16, a wnt family member, to regulate zebrafish embryonic myogenesis. We showed that loss of function in wnt16 induced changes in zebrafish muscle morphology. Moreover, myogenic precursors coexpressed wnt16 and pax7, a gene that promotes muscle differentiation and is a marker of satellite cells that support skeletal muscle regeneration. These findings indicate that wnt16 is necessary for muscle morphogenesis, however, its specific function in muscle regeneration remains unclear. I hypothesize that wnt16 influences skeletal muscle regeneration by regulating pax7 in satellite cells. To test this, I am determining the time course of wnt16 expression in pax7+ satellite cells in injured zebrafish muscle. Additionally, I will determine whether wnt16 is necessary for activation of pax7 following muscle injury. Through this study, I expect to establish 1) that wnt16 is upregulated after a muscle injury as an injury response gene, and 2) that wnt16 is necessary for pax7 activation in satellite cells. If the outcomes are as expected, this will show that wnt16 influences skeletal muscle regeneration, mirroring its role in zebrafish myogenesis and that WNT16 may be a target for developing therapeutics to treat muscle injuries.

Osteoporosis is characterized by decreased bone mineral density (BMD) and increased bone fragility, putting patients at higher risk for fractures. Osteoporosis has a strong genetic component, as indicated by data suggesting that BMD is 50-85% heritable. The aim of genome-wide association studies (GWAS) is to identify genetic variants common in the population that influence disease-related traits. A prior GWAS identified 56 loci harboring variants associated with BMD, including the XKR9 locus. The XKR9 locus comprises several genes, and the causal gene at the locus is currently unknown. This project focuses on TRAM1, a gene in proximity to XKR9, and TRAM2, the homolog of TRAM1. TRAM1 plays an important role in stabilizing proteins during translocation into the endoplasmic reticulum. Though the function of TRAM2 is not well characterized, it is suggested to play a role in collagen translocation. Currently, no in vivo studies have been published investigating the TRAM genes. The goal of this project is to determine how TRAM genes impact bone morphology in zebrafish. We have isolated zebrafish with germline mutations for tram1, tram2, and a combination of the two. Micro-CT scans were generated at 90 days post fertilization, and ImageJ and FishCuT were used to quantify the impacts on vertebral bone morphology. tram1 mutants exhibited significant changes in tissue mineral density (TMD) while tram2 mutants showed no significant changes in bone morphology or mineralization. Additionally, fish with homozygous mutations in tram1 and heterozygous for tram2 showed no significant changes, however double mutants for both tram1 and tram2 were embryonically lethal. These findings identify overlapping and distinct roles for tram genes in vivo. They also provide evidence that variants at XKR9 could act through TRAM1 to influence BMD thereby introducing translocation as an important factor underlying genetic influence on osteoporosis risk.