Background: Duchenne muscular dystrophy (DMD) is a rare neuromuscular disease that occurs in 1 of every 3,500 live male births. DMD patients develop progressive muscle weakness, due to loss-of-function variants in the DYSTROPHIN (DMD) gene. The dedicator of cytokinesis (DOCK) family consists of 11 proteins that function as guanine nucleotide exchange factors (GEFs). We identified DOCK7 as increased in the skeletal muscles biopsies in DMD patients and mdx mice. We evaluated DOCK7’s functional impact on developing zebrafish using a novel CRISPR-generated knockout to evaluate DOCK7’s role in normal muscle and embryonic development.
Hypothesis: We hypothesized that dock7 mutant zebrafish have impaired muscle function, reduced locomotion, and impaired muscle structure as its expression is required muscle development.
Methods: We used CRISPR editing to generate dock7 mutant zebrafish lines and confirmed them using PCR genotyping to ultimately select 2 loss-of-function dock7 fish lines. We performed a skeletal muscle birefringence analysis; to observe the morphology of their dorsal muscles in 5 days-post-fertilization (dpf). We performed a touch-evoke stimulation assay to give insight into their muscle movement and function following a direct stimulus. Finally, we used the DanioVision locomotor tracking device to evaluate dock7 mutant locomotion for 15 minute intervals. Total locomotor activity and velocity were quantified.
Results: We observed reduced muscle birefringence muscle in our dock7 mutant fish at 5 dpf that was similar to those of the sapje. We observed impaired touch-evoke responses in our dock7 mutant fish suggesting a mild paralysis in comparison with our DMD (sapje; dystrophin-deficient). Finally, our DanioVision locomotion analysis revealed that the dock7 mutant lines had consistently reduced distance traveled and average velocity compared to sibling controls.
Conclusions: The dock7 mutant zebrafish larvae have reduced locomotion, impaired muscle birefringence, and reduced touch-evoke responses. The dock7 mutant zebrafish have disrupted skeletal muscle myofiber structure compared to the wild type and sibling controls. Future studies will involve mating our dock7 mutant zebrafish line to the sapje mutant zebrafish to determine if reducing Dock7 levels improves DMD outcomes.