Functional analysis of Pannexin 3 during mammalian and avian osteogenesis, and the physiology of Pannexin 1 ohnologs in teleost fish

• Main Author: Bond, Stephen Raymond
• Language: English
• Published: University of British Columbia 2013
• Online Access: http://hdl.handle.net/2429/43759

The pannexins (Panx) are a family of chordate channel proteins with ancient homology to the invertebrate gap junction proteins called innexins. Only three distinct pannexin paralogs have currently been described in the Vertebrata, although all three are represented by orthologs in each of the major vertebrate clades. As public sequence databases have expanded however, a fourth pannexin sequence has revealed itself in teleost fish, apparently representing a duplication of Panx1. The first set of objectives for this thesis was to assess the likely genetic source of this duplication event, determine if and where the two Panx1 paralogs (denoted panx1a and panx1b) are expressed in a model teleost (zebrafish), and characterize some of the physiological properties that differ between the two gene products. Data will be presented indicating that the whole genome duplication event punctuating the teleost radiation ~350 million years ago is the likely source of this duplication, panx1a is widely distributed while panx1b is expressed primarily in the brain and eye, and neo- and/or subfunctionalization is discernible through differences in subcellular trafficking and distinct electrophysiological characteristics. The second set of objectives was the generation and dissemination of a public web based graphical user interface for the automatic design of restriction-free cloning primers, which is a method heavily used for the generation of custom DNA plasmids in this thesis. The third set of objectives focused on characterizing Panx3 expression in osteogenic tissues. In particular, determining how Panx3 expression is regulated in osteoblasts and hypertrophic chondrocytes, and describing the physiological consequences of this expression. Using in vitro techniques, it was determined that the osteogenic transcription factor Runx2 is necessary, but not necessarily sufficient, to promote expression of Panx3. To identify the functional importance of the protein in vivo, a replication competent avian retrovirus was used to force over-expression and to deliver shRNA into embryonic chicken forelimbs. Increased expression had no overt impact on bone development, while knockdown resulted in a bone dysplasia, manifested as a reduction in total mineralized bone volume, and correlated with reduced expression of the hypertrophic chondrocyte marker COL10A1.