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

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.

HACD enzymes are essential for very long chain fatty acid (VLCFA) biosynthesis and HACD1 is specifically expressed in striated muscles. Mutations in HACD1 cause progressive congenital myopathies in humans and dogs. The roles of HACD1 and VLCFA in muscle development and disease are poorly understood; however, our recent work in Labrador Retrievers with HACD1-associated centronuclear myopathy and cellular and mouse models has documented defects in muscle growth, development, repair and maintenance of muscle membrane systems. Existing models have their limitations: cellular models of myopathies are too simplistic and we have shown them to be highly influenced by their lipid environment. There are ethical and moral implications for using murine and canine models in research, in addition placental mammals develop in utero where maternal factors can influence muscle development.
An embryonic zebrafish model overcomes many of these issues and presents an ideal system for studying the roles of HACD1 and VLCFA in developing and mature muscle. They develop external to the mother and mature muscle structure is present within 3 days of fertilisation. I have established a novel F0 embryonic zebrafish model with mutations in hacd1 that exhibits a myopathic phenotype. In this project I will develop this and generate a zebrafish line carrying clinically relevant mutations in hacd1 and provide detailed characterisation of the effects on homozygous hacd1-mutant embryos at 72hpf including muscle membrane structure, ultrastructure and functional assessments of excitation-contraction coupling. In addition, I will evaluate a developmental time-course of lipidomic changes in wildtype zebrafish and quantify the impact of Hacd1-deficiency on the lipidome. The major output of this work from an NC3Rs perspective is a validated model of HACD1 deficiency to be shared with collaborators that will have an immediate and long-term impact on the field and a partial replacement of mammalian models.

Cellular models are often inadequate to study later stages of muscle development and diseases affecting mature muscles - crucially for this research cellular models lack an organised tubuloreticular membrane system which is central to the pathogenesis of CNMs. Hence, currently the only animal models available for researchers to study HACD1-associated congenital myopathies are dogs and mice.
Labradors have been specifically bred for this research since 2003 and affected dogs in colonies have a poor quality of life due to this environment and their disease (a disabling phenotype with marked weakness, poor exercise capacity, fatigue, collapse and eating difficulties). Lines of transgenic mice have been more recently developed which are used frequently for HACD1 research, their use could be partially replaced with the generation of an embryonic zebrafish model.
The major output from this fellowship will be to generate and validate two novel zebrafish hacd1-mutant lines that will deliver on the NC3R's remit by partially replacing these mammalian models with embryonic zebrafish prior to the onset of neurological sensitivity. The lines will be initially developed and I will receive training at Manchester University which has a Home Office license application in progress and established facilities and experience in genome editing and generating mutant lines. During this project, the mutant lines and associated techniques will be transferred to the University of Liverpool BSU to make these techniques widely available at this institution. Lines will be available for use by our collaborators at École nationale vétérinaire d'Alfort, also established researchers in the field and a number of avenues for enquiry are already planned together. Firstly, during this fellowship we will produce novel data regarding the impact of Hacd-1 deficiency on muscle development and the lipidome (replacing the use of 50+ mice). In the short term we plan to perform a therapeutic screen of molecules identified through these lipidomic strategies, this would otherwise involve the use of 100 mice per molecule. Once established the lines will be made publicly available thereby allowing other researchers to utilise this model which has significant advantages over mammalian models not least from a 3Rs perspective. I anticipate a significant, long-term 3Rs impact in this field through the availability of this new model and improved animal welfare through partial replacement of protected species with zebrafish embryos.
Data from this fellowship will presented at two different international conferences, therefore educating other researchers into the benefits of using embryonic zebrafish as well as making them aware of a line they could utilise for. Data will then be publicised in open access journals, with preprint prior to publication to ensure the data is available to both the scientific community and the general public.
Continuing on from previous outreach activities I will continue to aid in educating members of the public, charities and policy makers about general muscle biology, the cardiovascular system and responsible animal use in research. The Institute of Ageing and Chronic Disease has an active outreach program with the general public and local children's charities that provide an opportunity for disseminating this information.
I myself will also be impacted by this fellowship. During this fellowship I will gain expertise in the generation of mutant zebrafish lines and enhance my knowledge on the genetics behind which this is possible. I will learn single fibre dissociation and associated techniques: which are highly utilised within muscle biology laboratories and therefore highly desirable and transferable. Through my collaboration with Laurent Tiret I will further my knowledge in lipidomics and analysis of such datasets. Directing this fellowship will allow my further development as an independent researcher and prepare me for leading my own group.