Date of Award

Summer 8-18-2023

Level of Access Assigned by Author

Open-Access Thesis

Degree Name

Master of Science (MS)

Department

Biological Sciences

Advisor

Jared Talbot

Second Committee Member

Clarissa Henry

Third Committee Member

Joshua Kelley

Abstract

Distal Arthrogryposis Type 1 (DA1) involves mild muscle weakness and limb skeletal abnormalities thought to be caused by paralysis in utero. Why the limbs are particularly affected in DA1 and the degree of paralysis that leads to these skeletal deformities in utero remains unclear. Several muscle genes are known to cause DA1, including MYLPF (myosin light chain phosphorylatable), which encodes a myosin light chain protein that binds close to the force-generating head of myosin heavy chains. The zebrafish mylpfa-/- mutant displays a phenotype consistent with DA1, including impaired myosin activity, reduced muscle force overall, and complete fin paralysis. I began my work by analyzing myofibril structure in the mylpfa-/- mutant, as well as the mylpfa-/-; mylpfb-/- double mutant which had not been previously characterized carefully. We developed new quantitative techniques to analyze sarcomere formation in high-resolution images, tested the role of Mylpf in sarcomere assembly with statistical rigor, and investigated how these defects impact pectoral fin development. The quantitative suite we developed extends prior image analysis methods by determining the degree to which proteins localize to sarcomere-length repeats within images selected regions of interest from an image are sarcomeric in high-resolution images. To independently verify the technique, we examined wild-type sarcomere formation and growth from 24-72 hours post fertilization (hpf) and identify unique phases of muscle maturation in this time frame. Then, we applied the method to the mylpfa/mylpfb datasets, showing quantitatively found that sarcomeres are entirely unaffected by mylpfb mutation, are partially organized in the mylpfa-/- mutant, and are consistently absent when both genes are mutated (mylpfa; mylpfb double mutants). Analysis of the pectoral fin region showed that sarcomere defects in the mylpfa mutant are nearly as severe as in the mylpfa-/-; mylpfb-/- double. Consistent with the model that DA arises by limb paralysis and the observations that fins are completely paralyzed and lack sarcomeres in the mylpfa-/- mutant, I find that the pectoral fin cartilage size is reduced by 25% in the mylpfa-/- mutant, a defect that I confirmed using two alleles; this degree of the defect was also consistent across multiple stages of development. Consistent with the observation that both the mylpfa-/- mutant and the mylpfa-/-; mylpfb-/- double mutants have complete fin paralysis, I similarly find a 25% reduction in the double mutant. Intriguingly, preliminary analysis of a mutant completely lacking muscle in the fin (∆six1a;4a-/-;∆six1b;4b-/-) has an even more severe fin cartilage defect. These findings suggest that Mylpf gene function is required for sarcomeric protein organization to promote normal fast-twitch muscle and cartilage development.

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