CONTROL OF MUSCLE PROTEIN SYNTHESIS DURING MYOGENESIS

骨骼肌为研究控制干细胞规格的分子遗传学机制提供了一个独特的机会 脊椎动物胚胎的细胞复杂性中的血统。拟议的研究调查了发育机制， 调节生肌调节基因MyfS和MyoD，它们控制着来自体节的骨骼肌谱系的规范 祖先。实验特别关注Sonic Hedgehog(Shh)和Wnt信号分子的分子机制， 由周围组织产生，诱导体细胞前体细胞转录激活MyfS和MyoD，从而启动 肌源性细胞定向化的过程。已经开发了互补的方法来研究这些机制 老鼠和鸟类的胚胎。首先，MyoD和MyfS转录增强子将通过转基因和转基因进行表征 报告基因分析，结合定点突变和核因子足迹，绘制调控序列和 确定相互作用的转录因子，假设介导Shh和Wnt诱导MyoD和MyfS。这些研究进行 为了区分介导Shh和Wnt信号转导的Gli和(3-catenin/LEF)转录因子是否相互作用 直接与MyoD和MyfS增强子一起控制它们的激活，或者替代地，这些信号是否在上游起作用 调节体节特异性转录因子。第二，一组体节激活的调节基因的发育功能， 将通过反义抑制和错误表达分析在禽类中进行鉴定 并通过对小鼠胚胎的基因打靶和遗传分析来确定它们在MyoD和MyfS中的特定功能 体节形成过程中的调控。这些研究将集中在两个新的硫脂酶家族上，这些酶在我们的屏幕上被确定为 体节激活基因，是激活鸡胚胎体节中MyoD所必需的。这些硫脂酶被假设为 细胞外基质中的脱硫酸盐蛋白多糖，从而控制发育信号分子的局部活性 对MyoD激活是必不可少的。在这些筛选中发现的其他新的体节激活调节基因将被研究 确定它们在控制体节形成、Shh信号转导以及MyoD和MyfS激活中的功能。调查结果： 这些研究有望导致新的发育调控基因的发现，这些基因控制着 脊椎动物胚胎中的肌源性细胞和其他谱系。这些知识将定义控制茎的基本机制 细胞的形成，这些信息可能会导致新技术的发展，以促进干细胞的更新 修复因疾病和创伤而受损的人体组织和器官。

Skeletal muscle provides a unique opportunity to investigate molecular genetic mechanisms that control specification of stem cell lineages within the cellular complexity of the vertebrate embryo. The proposed studies investigate developmental mechanisms that regulate the myogenic regulatory genes, MyfS and MyoD, which control the specification of skeletal muscle lineages from somite progenitors. Experiments focus specifically on the molecular mechanisms by which Sonic hedgehog (Shh) and Wnt signal molecules, produced by surrounding tissues, induce the transcriptional activation MyfS and MyoD in somitic progenitor cells, and thus initiate the process of myogenic cell specification. Complementary approaches have been developed to investigate these mechanisms in mouse and avian embryos. First, MyoD and MyfS transcriptional enhancers will be characterized using transgenic and transfection reporter gene assays, in combination with site-directed mutagenesis and nuclear factor footprinting, to map regulatory sequences and identify interacting transcription factors hypothesized to mediate Shh and Wnt induction of MyoD and MyfS.These studies undertake to distinguish whether the Gli and (3-catenin/LEFtranscription factors, which mediate Shh and Wnt signal transduction, interact directly with MyoD and MyfS enhancers to control their activation, or alternatively,whether these signals function upstream to regulate somite-specific transcription factors. Second, the developmental functions of a set of somite-activated regulatory genes, identified in differential cDNA cloning screens, will be investigated by antisense inhibition and misexpression analyses in avian embryos and by gene targeting and genetic analyses in the mouse embryo to determine their specific functions in MyoD and MyfS regulation during somite formation. These studies will focus on a family of two novel Sulfatases that were identified in our screen for somite-activated genes and are required for activation of MyoD in somites of avian embryos. These Sulfatases are hypothesized to desulfate proteoglycans in the extracellular matrix, thus controlling the localized activities of developmental signaling molecules essential for MyoD activation. Other novel somite-activated regulatory genes, identified in these screens, will be investigated to define their functions in the control of somite formation, Shh signal transduction, and MyoD and MyfS activation. The findings of these investigations are expected to lead to the discovery of novel developmental regulatory genes that control the specification of myogenic cells and other lineages in vertebrate embryos. Such knowledge will define fundamentalmechanisms that control of stem cell formation, and this information will likely lead to the development of new technologies to promote stem cell renewal in the repair of human tissues and organs damaged by disease and trauma.