Date of Award
Doctor of Philosophy (PhD)
Musculoskeletal disorders in humans present with a broad range of phenotypes that vary in severity, onset, and genetic cause. The musculoskeletal system is comprised of complex and diverse tissues that make up the majority of total body mass. Thus, mutations in many different genes with differing structure and function can produce phenotypically similar developmental and morphological defects in the muscle and skeleton. In addition, the muscular and skeletal systems develop non-autonomously, wherein development and function of each will affect development of the other. Thus, mutations in genes responsible for development of this complex system have the potential to influence morphogenesis outside of their tissue of expression. Many different gene defects have the potential to cause musculoskeletal disorders and to properly treat afflicted patients it is necessary to understand the underlying causes of pathogenesis. One such example of pathogenic morphogenesis is Distal Arthrogryposis (DA), a group of ten congenital disorders characterized by contractures of the joints of the distal limbs that presents at birth. The most severe form of DA, Freeman-Sheldon syndrome (DA2A), is most commonly associated with autosomal dominant missense mutations in the motor domain of MYH3, a sarcomeric embryonic myosin heavy chain gene expressed in developing skeletal muscle. While this gene is expressed only transiently in skeletal muscle tissue, symptoms present in the structure of skeletal joints and persist beyond birth. To examine the disease mechanism responsible for developing skeletal muscle and joint contracture and to explore treatment options for afflicted patients, we created the most commonly observed human DA2A pathogenic variant (MYH3R672H) in the analogous gene in zebrafish (smyhc1R673H). The heterozygous zebrafish had contractures of the spine that modeled the phenotype of DA2A patient’s muscle contractures. We observed shortened muscle fibers and sarcomeres, disorganized skeletal muscle tissue, and disrupted motor activity. The phenotype was rescued with an actin-myosin inhibitor, para-aminoblebbistatin, suggesting that contractures are caused by constitutive muscle contraction. While we were able to characterize the phenotype of this pathogenic variant, many variants of unknown significance have been recorded in the motor domain of MYH3. Given the labor and time required to generate missense mutants using traditional methods, a higher throughput means of mutant generation is necessary to examine the function of dozens of reported variants. To develop a higher throughput method of analyzing single variants, we created zebrafish lines that would be expected to enable high-throughput screening. A GFP reporter tag at the end of smyhc1 is used as a marker for successful homology directed repair. A deletion is induced where reported variants cluster in the human analog that also serves to put GFP out of frame. In-frame repair and integration of a variant of interest via a repair template returns GFP into frame, allowing for efficient screening of F0 embryos. Finally, we analyzed a fibronectin domain containing 1 (fndc1) zebrafish knockout to better understand its association with human scoliosis. fndc1 zebrafish mutants displayed a higher bone mineral density and slightly larger vertebral centra, though scoliosis was not present. Using zebrafish to model musculoskeletal disease, we were able to elucidate disease mechanisms of distal arthrogryposis and scoliosis, as well as develop more efficient methods for variant analysis in zebrafish.
Chair and Committee
Whittle, Julia Nicole, "Generation, Characterization, and Treatment of Functional Zebrafish Models for Musculoskeletal Disorders" (2022). Arts & Sciences Electronic Theses and Dissertations. 2756.