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

HACD1基因突变与人类和狗的自然发生的遗传性肌肉疾病有关。这种犬类疾病很普遍，在过去的15年里，人们选择性地培育了拉布拉多犬，以产生受感染的犬群用于研究。hacd1缺陷先天性肌病最近在人类中被描述，转基因小鼠也被开发-所有这些都具有许多临床和病理特征。患者和患病动物逐渐表现出明显的虚弱，运动耐受性差，肌肉量减少和进食困难。目前还没有治疗方法，人们对这种疾病的机制也知之甚少。HACD1编码一种在发育和成熟肌肉中特异性表达的蛋白质，被认为在长链脂肪（VLCFA）的合成中起重要作用。研究已经记录了肌肉生长、发育和肌肉膜系统修复和维护的缺陷，这些缺陷值得进一步探索；然而，现有的狗和老鼠动物模型有缺点，尤其是从3r的角度来看。这项工作将旨在开发和验证胚胎斑马鱼hacd1缺陷的新模型。这个模型将在曼彻斯特大学产生，那里有基因组编辑和产生突变系的专业知识。这个模型、进一步工作所需的经验和技术将在利物浦大学的水族馆中建立起来，这条线将提供给巴黎的合作者，以部分取代他们的哺乳动物模型。发展中的斑马鱼被提倡用于应用3R原理的研究应用，并且是一个成熟的实验系统，用于研究肌肉发育和疾病，包括一些先天性肌病。斑马鱼在受精后3天（dpf）内肌肉发育迅速，肌肉纤维成熟，此时它们仍处于神经发育不成熟的阶段，确实因为这个原因，在5dpf之前，斑马鱼不受动物（科学程序）法的保护，因为它们不被认为能够经历痛苦。胚胎在母体外发育，很容易注射用于基因操作，因此很容易成像。它们作为一个封闭的系统存在，直到5dpf开始喂养，因此它们不受外部因素的影响，例如细胞等培养基的差异或母体通过胎盘（哺乳动物）输送营养物质。在本研究的准备工作中，已经鉴定并证实了斑马鱼中HACD1的等同物及其在该物种发育中的肌肉表达。通过将hacd1突变引入胚胎斑马鱼获得的初步证据证实了所需的技术，并证明它们显示出与受影响的狗和人类相同的肌肉异常。我现在建议建立携带hacd1突变的鱼种，并产生纯合突变胚胎，以研究hacd1突变对肌肉的影响。这对于产生具有相同突变和一致表型的胚胎用于研究非常重要，并且允许我们与其他实验室共享这些细胞系。在突变体胚胎发育过程中，将分析had1缺乏对肌肉结构和功能以及脂质组成（特别是VLCFAs）的影响。因此，hacd1突变斑马鱼将是这项研究的主要和直接的成果，它将减少和取代hacd1缺乏的哺乳动物模型的使用，同时使我们能够回答使用细胞和哺乳动物模型无法轻易探索的问题。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HACD1-CNM疾病机制的理解，这是未来治疗策略发展的关键一步，最终可能使狗和人类都受益。

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.