Gap Junctions/Limb Outgrowth, Patterning, Skeletogenesis

描述（由申请人提供）：本申请的目的是研究间隙连接通信在脊椎动物肢体生长和模式以及肢体骨骼元件形成过程中的作用。我)。肢体的生长和模式是由顶端外胚层脊（AER）和极化活动区（ZPA）之间的相互作用控制的。顶端外胚层脊是一种特殊的外胚层结构，位于肢体的远端尖端，指导肢体中胚层的增殖和近远端外胚层的生长，极化活动区是一组位于肢体后周的中胚层细胞，被认为指定了肢体前后轴的位置值。我们之前的研究表明，广泛的间隙连接通信发生在AER本身的细胞之间，以及响应AER和ZPA信号的后中胚层和远中胚层细胞之间。我们的研究还表明，间隙连接基因Cxn43的丰富表达与这些区域有关。在这些拟议的研究中，我们将验证由Cxn43组成的间隙连接在AER活动，肢体生长和模式中发挥重要作用的假设。我们将研究通过错表达Cxn43、反义Cxn43，或通过鸡蛋中鸡肢体中的逆转录病毒载体，或通过转基因小鼠中的AER特异性增强元件，突变或修饰的Cxn43，来调节AER和肢体中胚层间隙连接通讯的影响。(二)。肢体骨元形成的初始阶段以软骨前间质凝聚的形成为特征，并分化成为肢体的软骨模型。软骨模型的软骨细胞在软骨内成骨过程中逐渐成熟，导致软骨被骨取代。我们前期的研究表明，肢体软骨前间质凝聚过程中间隙连接通讯显著增加，并且Cxn43在软骨凝聚过程中短暂表达。其他研究也提示含有cxn43的间隙连接参与软骨细胞成熟和成骨分化。在这些拟议的研究中，我们将验证由Cxn43组成的间隙连接参与肢体骨骼元件的软骨形成和/或成骨的假设。我们将研究在骨骼形成过程中缝隙连接通讯的调节作用，上述调节是通过鸡蛋中鸡肢体中的逆转录病毒载体，或通过转基因小鼠肢体中的软骨特异性增强元件实现的。

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.