The respiratory system of Drosophila develops as a fluid-filled epithelium. Late in embryonic development, the fluid is rapidly replaced with gas, an event that occurs within minutes and is independent of contact with outside air. The mechanisms underlying this phase transition remain enigmatic, but carbonic anhydrases may play a crucial role in the process by producing CO2 gas while simultaneously promoting reabsorption of fluid by the surrounding epithelium.
A bioinformatics study of the carbonic anhydrase gene family in Drosophila showed that though humans and flies possess similar numbers of carbonic anhydrase genes (13 and 15 respectively), most of these have evolved after divergence. Based on our analyses, we elected to study two genes that are unique to insect lineages, cah2 and CG6074. Both genes are expressed in the trachea, and have probable signal peptides, suggesting that they are secreted. Also consistent with secretion, Cah2 protein, is glycosylated and has a likely GPI linkage site. These two genes are developmentally and tissue specifically regulated, showing high expression in the tracheal epithelium at the time of tracheal filling.
To functionally characterize these genes, fly lines containing mutagenic MiMIC insertions in both CG6074 and cah2 were obtained from the Bloomington Stock center. Both insertions are in introns and are oriented properly to create early terminations in each of the proteins. The cah2 mutation proved to be lethal in the late embryonic to early larval stages; presenting with a variety of defects in tracheal filling and maintenance of the open airway. Excision lines completely revert the phenotypes indicating that the MiMIC insertion is the cause of the observed defects. We are in the process of generating lines to rescue mutants by tissue specific expression, and generating an endogenously tagged version of the cah2 gene to allow us to directly observe the temporal and spatial expression of the protein. These experiments will enable us to directly test the hypothesis that carbonic anhydrases mediate transitions between fluid-filled and gas-filled states.