Morphogenetic Proteins in Embryogenesis and Tissue Regen

我们的主要目标是识别和定义蛋白质的作用，这些蛋白质可用于修复因创伤、辐射、肿瘤或其他病理损伤而受损的组织或结构。我们已经鉴定了十几种新的蛋白质。其中两种诱导软骨的形成，一种诱导骨和造血组织的形成，一种在定义发育胚胎的整体模式中起关键作用，另外三种功能未知。这些蛋白质之一，Frzb，是Wnt生长因子癌基因的信号传导的分泌抑制剂，并且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）家族的分泌性生长因子与这些蛋白质的分泌性拮抗剂之间的关键反馈相互作用。发现BMP 4和7、Wnt-8、BMP拮抗剂脊索蛋白和头蛋白以及Wnt拮抗剂Frzb的局部表达建立了引起脊椎动物肌肉最早前体的形态发生场。这些蛋白质的表达模式的关系似乎保持Wnt-8和BMPs 4和7之间的动态平衡，控制组织的命运。 为了研究这些信号的相互作用，使功能丧失实验成为可能的技术即使不是必不可少的，也是有用的。因此，我们正在从X中分离编码所研究蛋白质的基因。tropicalis，一个二倍体的相对X.laevis，可能允许建设的缺失使用位点特异性DNA修复和更容易应用的第三代反义技术。我们还准备了重组腺病毒编码Frzb和Aesthetics的高水平蛋白表达和评估骨骼修复的哺乳动物模型，后者的实验正在进行中。我们已经分离出两栖动物的同源基因的CMP 1/GDF-5和一种新的钙调素结合蛋白，并已确定其生物分布。功能研究正在进行中。我们还分离了一个cDNA编码的蛋白质的未知功能与序列相似的小鼠ky基因，与深刻的脊柱后凸，并已确定其表达域。我们也正在开发程序，以评估形态发生剂对信号转导途径的影响，使用新的蛋白质组学技术。这需要开发一种新的技术，用于使用II型限制性内切酶引入表位标签，该技术将具有普遍适用性。

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.