Manipulation of second messengers by ectopic expression of receptors in transgenic Drosophila.
PhD thesis, University of Glasgow.
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The combination of physiology and genetics affords opportunities to perform experiments with greater precision, and with less risk of artefact, than heretofore possible. This is illustrated here by demonstrating the selective manipulation of the second messengers cyclic GMP (cGMP), cyclic AMP (cAMP) and calcium (Ca2+) in the renal (Malpighian) tubule of the genetic model organism, Drosophila melanogaster.
The importance of the second messengers cGMP, cAMP and Ca2+ in control of fluid transport makes the Malpighian tubule an ideal testbed for such technology. The actions of known hormones can be compared with the results obtained by manipulating levels of the second messenger through which these are thought to act.
Flies were constructed or obtained transgenic for the rat atrial natriuretic peptide receptor, GC-A, the Drosophila 5HT7Dro receptor, and the Drosophila 5HT1ADro receptor, under control of the UAS or heat-shock promoter. Tubules dissected from such flies were then demonstrated to show diuresis induced by application of rANP or 5HT, whereas controls showed no response. Second messenger measurements showed that the rat GC-A receptor acted to raise cGMP levels, the 5HT7Dro receptor to raise cAMP levels, and the 5HT1ADro receptor to raise intracellular calcium. In addition, modulation of both cell-specific cell nucleotide phosphodiesterase activity and cell-specific cyclic nucleotide-dependent kinase activity of tubules with elevated cyclic nucleotide levels was observed, implicating these enzymes as key regulators/effectors of signalling in tubules. Cross-talk between cGMP, cAMP and Ca2+ was also assessed and shown to be variable, depending on the particular cell type in which the signal was generated. This reveals further complexity in the control of tubules and suggests a distinct role for cyclic nucleotide-gated channels in principal cells.
This study has validated ectopic transgene expression as a generic technology with potential beyond Malpighian tubules: in principle, such transgenes can be expressed specifically in any population of cells that can be delineated by a GAL4 driver line.
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