Dissecting a role for ZAK in skeletal muscle development using genetics and embryology in zebrafish and Xenopus

在骨骼肌发育和修复过程中，肌源性干细胞增殖，当被触发时，退出细胞周期并融合形成多核肌纤维。这个过程发生在胚胎发育和再生过程中，当肌肉受伤或处于疾病状态时，如肌肉萎缩症。细胞信号传导是调节肌原细胞从增殖到分化的重要触发器。该项目将研究一种蛋白质的作用，这种蛋白质是MAPK信号通路的一部分，称为ZAK。该蛋白的基因编码最近被认为与人类遗传性肌病有关，本项目将利用遗传学和分子胚胎学来确定ZAK在骨骼肌发育和修复中的任何作用。本项目的分析将使用非哺乳动物脊椎动物模型（非洲爪蟾和斑马鱼）在体内进行。使用CRISPR/Cas9进行基因编辑在青蛙和鱼类中非常有效，这种基因靶向方法将用于破坏青蛙和鱼类中的ZAK。利用显性负性和诱导型FGF受体调控FGF信号传导，抑制和过度刺激MAPK通路，将是剖析需要ZAK活性的通路的一种方法。IGF和EGF信号也将在ZAK调节肌源性发育，生长和修复的背景下进行研究。学生还将获得使用胚胎学操作蛙胚胎和产生斑马鱼遗传系的经验，以及使用细胞和分子测定来分析信号转导和基因表达。

During skeletal muscle development and repair, myogenic stem cells proliferateand, when triggered to do so, exit the cell cycle and fuse to formmultinucleated myofibres. This process occurs both during embryonicdevelopment and during regenerative processes that happen when muscle isinjured or in a disease-state, as occurs in muscular dystrophy. Cell signalling isan important trigger that regulates a myogenic cell's switch from proliferationto differentiation. This project will investigate the role of a protein that is partof the MAPK signalling pathway, called ZAK. The gene coding for this proteinhas recently been implicated in an inherited myopathy in humans and thisproject will use genetics and molecular embryology to determine any role forZAK in skeletal muscle development and repair.The analyses in this project will be done in vivo using non-mammalianvertebrate animal models (Xenopus and zebrafish). Gene editing usingCRISPR/Cas9 is very effective in frogs and fish and this gene targeting approachwill be used to disrupt ZAK in frogs and fish. Manipulating the FGF signallingwith dominant negative and inducible FGF receptors to inhibit andoverstimulate the MAPK pathway will be one approach to dissect the pathwaysthat require ZAK activity. IGF and EGF signalling will also be investigated in thecontext of ZAK regulation of myogenic development , growth and repair. Thestudent will also gain experience using embryological manipulations of frogsembryos and generating genetic lines in zebrafish, as well as using cell andmolecular assays to analyse signal transduction and gene expression.