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 谱系与肌肉祖细胞的发育分化 细胞 虽然理解骨骼的主要动力 肌原性调节是由激活剂的发现带来的， 肌生成调节因子（MRF），MyoD，myf-5，肌生成素，MRF-4， 现在越来越清楚的是，其他机制，其中肌源性 监管可以归因于各种类别之间的协同作用， 转录的激活剂和抑制剂，参与了关键的 肌原性编程的步骤。 利用鸡胚， 一个非常有用的功能分析实验系统， 发育重要的基因，我们已经分离和克隆了一个 Nished蛋白在鸡骨骼肌中的表达 在早期的胚胎中似乎是准确编程的先决条件 骨骼肌的发生这一概念得到了进一步的实验 我们实验室的明确结果支持， 基因标记，结蛋白，肌球蛋白和肌细胞生成素，在成纤维细胞中被激活 10 T+稳定转染Nished沿着获得 肌肉细胞的独特形态特征。 该设施 这个实验范例决定了不久的将来 进一步研究的方向，本文提出，这是进行 严格控制的评估是否激活骨骼 肌肉基因与Nished介导的任何 已知肌原性调节网络。 该提案将测试 假设Nished对表达有直接影响， 反应基因的内源性网络， Nished在胚胎组织中的生理水平会产生影响 对整个动物内肌肉组织表型的发育 上下文 Nished的精确功能预测将通过 包括控制异位表达和基因转移在内的多种方法， 瞄准实验 我们的具体目标是：（一）全面开展 光谱研究，以测试有待探索的结构-功能相关性 在功能获得型实验中，（ii）建立功能 通过鉴定Nished在体内表达的意义， Nished响应基因网络的内源网络，以及（iii）确定 Nished在转基因小鼠中生理作用以及通过靶向 在小鼠中切除选定的基因座。 这项研究的结果是 预计将有助于深入了解负责的机制 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.