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

HACD 1基因的突变与人类和狗的自然发生的遗传性肌肉疾病有关。这种犬的情况很普遍，在过去的15年里，拉布拉多犬被选择性地繁殖，以产生受影响的犬群用于研究。最近在人类中描述了HACD 1缺陷型先天性肌病，并且还开发了转基因小鼠品系-所有这些都具有许多临床和病理特征。患者和受影响的动物逐渐表现出明显的虚弱，运动耐量差，肌肉质量减少和进食困难。没有治疗方法，疾病机制知之甚少。HACD 1编码一种在发育和成熟肌肉中特异性表达的蛋白质，被认为在极长链脂肪（VLCFA）的合成中很重要。研究已经记录了肌肉生长、发育以及肌肉膜系统的修复和维护方面的缺陷，这些缺陷值得进一步探索;然而，狗和小鼠中现有的动物模型具有缺点，尤其是从3Rs的角度来看。这项工作的目的是开发和验证一种新的胚胎斑马鱼HACD 1缺陷模型。该模型将在曼彻斯特大学生成，该大学拥有基因组编辑和生成突变株系的专业知识。这个模型，经验和技术需要进一步的工作，然后将建立在水族馆在利物浦大学和线将提供给合作者在巴黎，以部分取代使用他们的哺乳动物模型。发展中的斑马鱼被提倡用于应用3R原则的研究应用，并且是用于研究肌肉发育和疾病（包括一些先天性肌病）的完善的实验系统。斑马鱼在受精后3天（dpf）内经历快速的肌肉发育，并存在成熟的肌纤维，当时它们仍然处于神经学上不成熟的阶段，事实上，由于这个原因，在5dpf之前，它们不受动物（科学程序）法案的保护，因为它们被认为不能经历痛苦。胚胎在母体外发育，易于注射进行遗传操作，并且是透明的，因此易于成像。它们作为一个封闭的系统存在，直到5dpf喂养开始时-因此它们不受外部因素的影响，例如细胞或母体通过胎盘输送营养物质的差异。在这项研究的准备中，已经鉴定并证实了斑马鱼相当于HACD 1及其在该物种发育中肌肉中的表达。通过将hacd 1突变引入胚胎斑马鱼获得的初步证据已经验证了所需的技术，并证明它们显示出与受影响的狗和人类相似的肌肉异常。我现在建议建立携带hacd 1突变的鱼类品系，并产生纯合突变胚胎，以研究hacd 1突变对肌肉的影响。这对于生产具有相同突变和一致表型的胚胎进行研究非常重要，并允许我们与其他实验室共享这些细胞系。Hacd 1缺陷对肌肉结构和功能以及脂质成分（特别是VLCFA）的影响将在突变胚胎发育过程中进行分析。因此，Hacd 1突变斑马鱼将成为本研究的主要和直接输出，这将减少和取代使用哺乳动物模型的Hacd 1缺陷，同时使我们能够回答无法使用细胞和哺乳动物模型轻松探索的问题。这项工作旨在回答一个基本的生物学问题，并深入了解VLCFA在肌肉中的作用。这将提高对HACD 1-CNM疾病机制的理解，这是未来开发治疗策略的关键一步，最终可能使狗和人类受益。

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.