MOLECULAR BASIS OF MYOGENIC RESPONSE IN DEVELOPMENT

拟议的实验是我们研究的继续，旨在 了解肌源性细胞建立的机制 谱系与肌祖细胞的发育分化 细胞。虽然了解骨骼的主要推动力 激活剂的发现带来了生肌调节。 生肌调节因子(MRF)，MyoD，Myf-5，Mygenin，MRF-4，是 现在越来越清楚的是，其他的机制，其中的肌原 监管可以归因于不同类别的 转录的激活物和抑制物，都参与了至关重要的 肌源性编程的步骤。使用鸡胚，它提供了 一种实用的人体机能分析实验系统 重要的发育基因，我们已经分离和克隆了 鸡骨骼肌调节蛋白(NADD)的表达 在早期胚胎中似乎是精确编程的先决条件 骨骼肌发生的基因。这一想法得到了进一步的实验 我们实验室的确凿结果支持肌源性 结蛋白、肌球蛋白和肌生成素等基因标记物在成纤维细胞中被激活 随着NED基因的获得，10T+基因稳定表达 肌细胞具有明显的形态特征。该设施 这一实验范式决定了不久的将来 在此提出了进一步研究的方向，即开展 严格控制评估骨骼是否被激活 肌肉基因与Ned介导的任何一种 已知的生肌调节网络。该提案将考验 假设NED对 反应基因的内源网络和细胞内的变化 NED在胚胎组织中的生理水平会产生影响 关于全动物肌肉组织表型发育的研究 背景。对NED的精确功能的预测将通过以下方式完成 多种方法，包括受控的异位表达和基因 靶向实验。我们的具体目标是：(一)进行全面的 光谱研究检验结构与功能的相关性有待探索 在函数增益型实验中，(Ii)建立函数 核仁组成区基因在体内表达的意义 Ned-Response基因网络的内源网络和(III)确定 Ned在转基因小鼠中的生理作用及靶向作用 消融小鼠选定的基因位点。这项研究的结果是 预计将对责任机制提供重大见解(S) 用于建立和维持骨骼肌表型。

The proposed experiments are a continuation of our studies designed to understand the mechanisms responsible for establishment of myogenic cell lineage and the development and differentiation of muscle progenitor cells. While the major impetus in understanding of the skeletal myogenic regulation was brought by the discovery of the activator myogenic regulatory factors (MRFs), MyoD, myf-5, myogenin, MRF-4, it is now becoming clear that other mechanisms, wherein the myogenic regulation can be attributed to the synergy between various classes of activators and inhibitors of transcription, are involved in crucial steps of myogenic programming. Using the chicken embryos, which offer a highly useful experimental system for functional analysis of developmentally important genes, we have isolated and cloned a regulatory protein (Nished) in chicken skeletal muscle whose expression in early embryos appears to be a prerequisite for accurate programming of skeletal myogenesis. This notion received further experimental support by the definitive results in our laboratory that the myogenic gene markers, desmin, myosin and myogenin, are activated in fibroblasts 10T + stably transfected with Nished along with the acquisition of distinct morphological characteristics of the muscle cell. The facility with this experimental paradigm determines the immediate future direction of further studies, proposed herein, which is to carry out a rigorously controlled assessment whether the activation of the skeletal muscle genes is linked with the Nished mediated derepression of any known myogenic regulatory network. The proposal will test the hypothesis that Nished has a direct consequence on the expression of the endogenous network of responsive genes and that alterations in the physiological level of Nished in embryonic tissues will have an impact on development of the muscle tissue phenotype within the whole animal context. Prediction of the precise function of Nished will be done by a variety of approaches including controlled ectopic expression and gene targeting experiments. Our specific aims are to: (i) undertake a full spectrum study to test the structure-function correlates to be explored in gain-in-function type experiments, (ii) establish the functional significance of Nished expression in vivo by identification of the endogenous network of Nished-responsive gene network and (iii) determine the physiological role of Nished in transgenic mice and via targeted ablation of selected loci in mice. The outcome of this study is expected to lend significant insights into the mechanism(s) responsible for establishment and maintenance of skeletal muscle phenotype.