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.
