A novel embryonic zebrafish model to replace mammals in the study of 3-hydroxyacyl CoA dehydratase 1-associated muscle disorders

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Mutations in the HACD1 gene are associated with naturally-occurring inherited muscle diseases in humans and dogs. The canine condition is widespread and Labradors have been selectively bred over the last 15 years to produce colonies of affected dogs for research. HACD1-deficient congenital myopathies are more recently described in humans and lines of transgenic mice have also been developed - all share many clinical and pathological features. Patients and affected animals progressively display marked weakness, poor exercise tolerance, reduced muscle mass and difficulties eating. There is no treatment and the disease mechanisms are poorly understood. HACD1 encodes a protein that is specifically expressed in developing and mature muscles and is thought to be important in the synthesis of fats with very long chain lengths (VLCFA). Research has documented defects in muscle growth, development and repair and maintenance of muscle membrane systems which warrant further exploration; however, existing animal models in dogs and mice have disadvantages not least from a 3Rs perspective. This work will aim to develop and validate a novel model of HACD1-deficiency in embryonic zebrafish. This model will be generated at the University of Manchester where there is expertise in genome editing and generating mutant lines. This model, the experience and techniques required for further work will then be established in the aquarium at the University of Liverpool and the line will be made available to collaborators in Paris to partially replace use of their mammalian models. The developing zebrafish is advocated for research applications where 3R principles are applied and is a well-established experimental system for the study of muscle development and diseases, including some congenital myopathies. Zebrafish undergo rapid muscle development with the presence of mature muscle fibres within 3 days post fertilisation (dpf) when they are still otherwise at a neurologically immature stage, indeed for this reason prior to 5dpf they are not covered by the Animal (Scientific Procedures) Act as they are not thought to be able to experience suffering. Embryos develop outside the mother, are simple to inject for genetic manipulation and are transparent therefore easy to image. They exist as a closed system until 5dpf when feeding starts - they are therefore unaffected by external factors such as differences in culture media like cells or maternal delivery of nutrients via the placenta as in mammals. In preparation for this study the zebrafish equivalent of HACD1 and its expression in developing muscle in this species has been identified and confirmed. Preliminary evidence obtained by introducing hacd1 mutations into embryonic zebrafish has validated the techniques needed and demonstrated that they display muscle abnormalities that replicate those seen in affected dogs and humans. I now propose to establish lines of fish carrying mutations in hacd1 and produce homozygous mutant embryos to investigate the effects of HACD1 mutations in muscle. This is important to produce embryos with the same mutation and consistent phenotype for research, and to allow us to share the lines with other laboratories. The effect of Hacd1-deficiency on muscle structure and function and on lipid composition (particularly VLCFAs) will be analysed during development of mutant embryos.The hacd1-mutant zebrafish will hence be a major, and immediate, output of this study that will reduce and replace use of mammalian models of HACD1-deficiency whilst allowing us to answer questions that cannot easily be explored using cellular and mammalian animal models. This work aims to answer a fundamental biological question and provide insight into the roles of VLCFA in muscle. This will improve understanding of the disease mechanisms in HACD1-CNM, a critical step for future development of treatment strategies that may ultimately benefit both dogs and humans.