A study of the distribution and function of P2 receptors during chick embryogenesis

The aim of this thesis is to investigate the possibility that extracellular nucleotides, and their receptors (P2 receptors) are involved in regulating developmental processes during chick embryogenesis. In the first chapter, the classification, structure, and signal transduction mechanisms of P2 receptors are described. The evidence that suggests that this type of signalling has the potential to be important developmentally is also discussed. In the second chapter, in-situ hybridisation is used to describe the developmentally regulated pattern of expression of cP2Y1, a G protein-coupled P2 receptor, in multiple tissue systems during chick embryogenesis. This is the first description of P2 receptor subtype expression during embryonic development of the chick, and only the second time that expression of a specific P2 receptor has been described during the embryonic development of an organism. The following two chapters are devoted to investigating the function of cP2Y1, in one of the regions in which it is expressed, the developing limb. In chapter 3 monitoring of intracellular calcium is used to characterise the response of early limb cells to extracellular nucleotides. This technique is used to show that in addition to cP2Y1, at least one other P2 receptor subtype is expressed in early limb cells. Monitoring of intracellular calcium, and RNase protection is also used to show that within 24 hours of culturing, expression of cP2Y1 is lost from limb cells. Monitoring of intracellular calcium was then used as an assay to demonstrate that expression of functional cP2Y1 could be rescued in these cells by transfection with an expression vector carrying the cP2Y1 cDNA. In the fourth chapter, the loss of cP2Y1 in-vitro is exploited, using a gain-of- function approach, to investigate a function for cP2Y1 in limb development. Early limb bud cells were transfected as described and characterised above, and an in vitro assay of cartilage formation was used to provide evidence that cP2Y1 may have an important role to play in regulating the formation of the limb skeleton. In the final chapter immunohistochemistry is used to show that two members of the P2X, ligand-gated ion channel family of P2 receptors are expressed during chick skeletal muscle development, and in the embryonic chick nervous system