Regulation of expression and role of the GDNF family receptors in neuronal development

Abstract

The aim of this project was to determine the temporal and spatial pattern of expression of GDNF family receptors in the developing embryo, with particular emphasis on expression in the peripheral nervous system, and to investigate how expression of receptor mRNAs is regulated in developing neurons. It was hoped that the data obtained would prove useful in further characterizing the role that the GDNF family of neurotrophic factors play in embryonic development. Semi- quantitative PCR revealed that GFRα-1, GFRα-2, GFRα-4 and ret mRNAs are widely distributed with both complementary and overlapping, though distinct, patterns of expression in the chicken embryo during development. Different populations of PNS neurons display different levels of responsiveness to GDNF and NTN and their sensitivity to these factors change throughout development. Examination of receptor expression by quantitative RT-PCR revealed that neurons that are more sensitive to GDNF express higher levels of GFRα-1 mRNA than GFRα-2 mRNA, and neurons that are more sensitive to NTN express higher levels of GFRα-2 mRNA compared to GFRα-1 mRNA. However, developmental changes in responsiveness of a population of neurons to these factors are not consistently paralleled by changes in the relative levels of GFRα transcripts. Furthermore, all neuronal populations express relatively high levels of ret mRNA. These results indicate the responsiveness of PNS neurons to GDNF and NTN is in part governed by the relative levels of expression of their GPI-linked receptors. To determine how the expression of the GDNF family receptors is regulated, embryonic neurons were cultured under different experimental conditions. I found that GFRα-1, GFRα-2, GFRα-4 and ret mRNAs are not significantly regulated by GDNF and/or NTN. However, depolarizing levels of KC1 cause marked changes in the expression of GFRα mRNAs. The effects of KCl are inhibited by L-type Ca2+ channel antagonists, suggesting that they were mediated by elevation of intracellular free Ca2+. KCl treatment increases the response of neurons to GDNF and decreases their response to NTN. There is no marked effect of depolarization on ret mRNA expression.