Gap Junctions/Limb Outgrowth, Patterning, Skeletogenesis

描述(申请人提供)：本申请的目的是研究缝隙连接通讯在脊椎动物肢体生长和构图以及肢体骨骼元素形成过程中的作用。i)。肢体突起和图案化是由顶端外胚层脊(AER)和极化活动区(ZPA)之间的相互作用控制的，顶端外胚层脊(AER)是一种特殊的外胚层结构，它指导着肢体中胚层的增殖和近端外胚层的生长，ZPA是位于肢体后缘的一组中胚层细胞，被认为是指定了肢体前后轴线上的位置值。我们以前的研究表明，在AER本身的细胞之间以及在AER和ZPA信号的响应下正在经历生长和图案化的后中胚层和远端中胚层的细胞之间，存在广泛的缝隙连接通讯。我们的研究还表明，缝隙连接基因Cxn43的大量表达与这些区域相关。在这些拟议的研究中，我们将检验由Cxn43组成的缝隙连接在AER活动、肢体伸展和图案形成中发挥重要作用的假设。我们将通过逆转录病毒载体将Cxn43、反义Cxn43或突变或修饰的Cxn43通过逆转录病毒载体或通过AER特异的增强子元件在转基因小鼠体内表达，来检测AER和肢体中胚层缝隙连接通讯的调控效果。Ii)。肢体骨骼元素形成的初始阶段以软骨前间充质凝聚物的形成为特征，这些软骨间充质凝聚体分化成为肢体的软骨模型。软骨模型的软骨细胞经历了渐进性成熟，导致软骨在软骨内成骨过程中被骨取代。我们以前的研究表明，在肢体前软骨间充质凝聚过程中，缝隙连接通讯显著增加，Cxn43通过凝聚软骨瞬时表达。其他研究也发现含有Cxn43的缝隙连接与软骨细胞成熟和成骨分化有关。在这些拟议的研究中，我们将检验由Cxn43组成的缝隙连接参与肢体骨骼元素的软骨形成和/或成骨的假设。我们将研究如上所述，通过逆转录病毒载体在卵子中的鸡肢或通过转基因小鼠肢体中的软骨特异性增强子元件，在骨骼形成过程中对缝隙连接通讯进行调制的效果。

DESCRIPTION (provided by applicant): The goal of this application is to study the role of gap junctional communication in vertebrate limb outgrowth and patterning, and during limb skeletal element formation. I). Limb outgrowth and patterning is controlled by interactions between the apical ectodermal ridge (AER), a specialized ectodermal structure located at the distal tip of the limb which directs the proliferation and proximodistal outgrowth of the limb mesoderm, and the zone of polarizing activity (ZPA), a group of mesoderm cells located at the posterior periphery of the limb which is thought to specify positional values across the anterior-posterior limb axis. Our previous studies indicate that extensive gap junctional communication occurs among the cells of the AER itself, and among the cells of the posterior and distal mesoderm which is undergoing outgrowth and patterning in response to signals from the AER and ZPA. Our studies also show that abundant expression of the gap junction gene Cxn43 correlates with these regions. In these proposed studies, we will test the hypothesis that gap junctions composed of Cxn43 play an important role in AER activity, limb outgrowth and patterning. We will examine the effects of modulation of gap junctional communication in the AER and limb mesoderm achieved by missexpression of Cxn43, antisense Cxn43, or of mutated or modified Cxn43 via retroviral vectors in the chick limb in ovo, or via an AER-specific enhancer element in transgenic mice. II). The initial phase of limb skeletal element formation is characterized by the formation of pre-cartilage mesenchymal condensations, which differentiate to become the cartilage models of the limb. The chondrocytes of the cartilage models undergo progressive maturation leading to the replacement of cartilage by bone during endochondral ossification. Our previous studies show that gap junctional communication increases dramatically during condensation of limb pre-cartilage mesenchyme, and that Cxn43 is transiently expressed by condensing cartilage. Other studies have also implicated Cxn43-containing gap junctions in chondrocyte maturation and osteogenic differentiation. In these proposed studies, we will test the hypothesis that gap junctions composed of Cxn43 are involved in chondrogenesis and/or osteogenesis of the limb skeletal elements. We will examine the effects of modulation of gap junctional communication during skeletogenesis achieved as above via retroviral vectors in the chick limb in ovo, or via a cartilage-specific enhancer element in the limbs of transgenic mice.