Morphogenetic Proteins in Embryogenesis and Tissue Regen

胚胎发生和组织再生中的形态发生蛋白

• 批准号: 6839730
• 负责人: MALCOLM MOOS
• 金额: --
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6839730
• 关键词: Adenoviridae; Mammalia; Xenopus; biological signal transduction; bone morphogenetic proteins; developmental genetics; embryogenesis; gene expression; glycoproteins; growth factor; histogenesis; inhibitor /antagonist; oncogenes; protein localization; protein structure function; proteomics; regeneration; restriction endonucleases; site directed mutagenesis

Our primary goal is to identify and define the actions of proteins that can be used to repair tissues or structures that have been damaged by trauma, radiation, neoplasia, or other pathologic insult. We have identified over a dozen novel proteins. Two of these induce the formation of cartilage, one induces the formation of bone and hematopoietic tissue, one plays a key role in defining the overall pattern of the developing embryo, and three others have unknown functions. One of these proteins, Frzb, is a secreted inhibitor of signaling by the Wnt growth factor oncogenes, and is the prototype of a growing family of related proteins. Key feedback interactions between secreted growth factors belonging to the Wnt and Bone Morphogenetic Protein (BMP) families and secreted antagonists of these proteins were elucidated. Localized expression of BMPs 4 and 7, Wnt-8, the BMP antagonists chordin and noggin, and the Wnt antagonist Frzb were found to establish the morphogenetic field giving rise to the earliest precursors of vertebrate muscle. The relationship of expression pattern of these proteins appears to maintain a dynamic balance between Wnt-8 and BMPs 4 and 7 pathways that controls tissue fate. To examine the interactions of these signals, technologies to enable loss-of-function experiments will be useful if not essential. We are therefore isolating genes encoding the proteins under study from X. tropicalis, a diploid relative of X.laevis that may allow construction of deletions using site-specific DNA repair and more facile application of third-generation antisense technologies. We have also prepared recombinant adenoviruses encoding Frzb and ADMP for both high-level protein expression and evaluation in mammalian models of skeletal repair; the latter experiments are in progress. We have isolated amphibian orthologs of CDMP-1/GDF-5 and a novel calmodulin binding protein and have characterized their biological distributions. Functional studies are in progress. We have also isolated a cDNA encoding a protein of unknown function with sequence similar to that of the murine ky gene, associated with profound kyphosis, and have defined its domain of expression. We are also developing procedures to evaluate the effects of morphogens on signal transduction pathways using novel proteomic techniques. This has required development of a novel technique for introducing epitope tags using Type II restriction endonucleases that will be of general applicability.

我们的主要目标是识别和确定可用于修复因创伤、辐射、肿瘤或其他病理性侮辱而受损的组织或结构的蛋白质的作用。我们已经鉴定了十几种新的蛋白质。其中两个诱导软骨的形成，一个诱导骨和造血组织的形成，一个在定义发育中胚胎的整体模式中起关键作用，另外三个功能未知。其中一种蛋白质Frzb是由Wnt生长因子癌基因分泌的信号抑制因子，是一个不断增长的相关蛋白质家族的原型。阐明了属于Wnt和骨形态发生蛋白(BMP)家族的分泌型生长因子与这些蛋白的分泌型拮抗剂之间的关键反馈作用。BMP4和7、Wnt-8、BMP拮抗剂Chordin和noggin以及Wnt拮抗剂Frzb的定位表达为脊椎动物肌肉的早期前体建立了形态发生场。这些蛋白表达模式的关系似乎维持了WNT-8和BMPs4和7途径之间的动态平衡，这些途径控制着组织的命运。 为了研究这些信号的相互作用，能够进行功能丧失实验的技术即使不是必要的，也是有用的。因此，我们正在从热带X.tropicalis分离编码正在研究的蛋白质的基因，X.laevis是X.laevis的二倍体亲属，它可能允许使用定点DNA修复和更容易地应用第三代反义技术来构建缺失。我们还制备了编码Frzb和ADMP的重组腺病毒，用于在哺乳动物骨骼修复模型中进行高水平蛋白表达和评估；后一项实验正在进行中。我们分离了CDMP-1/GDF-5的两栖同源物和一种新的钙调蛋白结合蛋白，并表征了它们的生物学分布。功能研究正在进行中。我们还分离到了一个编码未知功能蛋白的cDNA，其序列与小鼠Ky基因相似，与深度后凸相关，并确定了其表达结构域。我们还在利用新的蛋白质组学技术开发程序来评估形态致病因子对信号转导途径的影响。这需要开发一种新的技术，利用具有普遍适用性的II型限制性内切酶引入表位标签。