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Abstract

The genetic basis of many muscle diseases is known but an understanding of the mechanism underlying muscle weakness is often missing hence a gap remains for the development of effective treatments of these pathologies. Since the process of muscle development as well as function is highly conserved throughout evolution, the skeletal muscles of zebrafish (Danio rerio) show remarkable structural and molecular similarities to those of humans. At the same time, they also make up a considerable portion of its body. Therefore, investigating the developmentally relevant motility genes in zebrafish could help to decipher essential but poorly understood aspects of myogenesis. In this project, I adopted two distinct experimental approaches. The first part of the thesis deals with creating a genetic knockout model to understand the molecular function of the genes apobec2a and 2b which were shown to be relevant for muscle development by our lab. In the course of creating knockouts, we applied a novel, efficient and cost-effective method to predict guide RNA efficiency in zebrafish. The quantitative assessment of gRNAs was provided by the web tool, PCR-F-SEQ which was developed and optimized by us especially for the zebrafish model system. This tool represents a simple but powerful method to screen injected batches of embryos before sending them for raising. Although apobec2a/2b morphants show a dystrophic phenotype, the genetic knockouts do not exhibit any muscle phenotype indicating a possible genetic compensation. In addition, an unbiased approach of investigating the motility mutants isolated from Tübingen screens was used. The rate of retrieval of mutant couples from sperm samples of the Ist and IIIrd Tübingen screens were around 66% and 40% respectively. Following the revival and phenotypic characterization of these lines, we developed a pipeline using next generation sequencing to accurately identify the disease-causing alleles. Mapping of mutations and validation of candidate genes were successfully done for all the six revived lines. Amongst which, we reported a missense mutation in choline-O-acetyltransferase a (chata) gene, encoding an enzyme essential for the synthesis of a major neurotransmitter, acetylcholine (ACh). The in-silico analysis showed that the substitution of serine to arginine might affect the protein stability disrupting the catalysis of acetyl CoA and choline to form ACh. In conclusion, this thesis showcases the challenges and strengths of both reverse and forward genetic approaches to study vertebrate development and also highlights the importance of strategies and tools now available for making genetic models.