INVESTIGATING THE ROLE OF THE NEUROPEPTIDE DIURETIC HORMONE 31 (Dh31) IN DROSOPHILA OOGENESIS
Markwei, Metabel Tori Naa Merle
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Background: Adult stem cells are essential for tissue homeostasis. The ability of stem cells to self-renew and generate differentiating progeny allows for the replacement of dying or damaged cells. In the Drosophila ovary, germline stem cell (GSC) number and proliferation, as well as later stages in oogenesis, are tightly regulated in response to diet and physiological changes. For example, insulin signaling, which controls cellular nutrient sensing, aging, and many other biological processes, regulates GSC proliferation and maintenance in response to diet. Drosophila insulin-like peptides (dILPs) are neuropeptides secreted from the brain, and our lab is currently investigating whether additional neuropeptides may influence GSC proliferation and/or maintenance, or later stages of oogenesis. Neuropeptides are important signaling molecules involved in many processes including metabolism and reproduction. Our lab previously performed a screen of 36 out of 44 Drosophila neuropeptides to identify neuropeptides that regulate oogenesis. The Drummond-Barbosa lab measured the rates of egg production within 15 days of brain-specific neuropeptide RNAi knockdown. Interestingly, pan-neuronal knockdown of the neuropeptide diuretic hormone 31 (Dh31) significantly decreased Drosophila egg production. These findings sparked our interest in investigating the role Dh31 plays in regulating egg production. Dh31 binds to a G-protein-coupled receptor, Dh31-R, a homolog to the human calcitonin receptor, to positively regulate adenylate cyclase activity. Furthermore, Dh31 is responsible for the regulation of water and NaCl homeostasis in the Malpighian tubules of the fly, in response to changes in diet and environment. Materials and Methods: RNAi-mediated knockdown experiments were conducted to disrupt Dh31 neuropeptide production, as well as its receptor function, Dh31-R, in a tissue-specific manner. Using immunohistochemistry together with confocal microscopy, we measured changes in the number of GSCs and their progeny (cysts), as well as the niche size (cap cell number) after Dh31 and Dh31-R RNAi knockdowns. Results: Dh31-R knockdowns in all somatic cells and Dh31 neuropeptide knockdown in the brain both result in a decrease in the number of eggs laid, no changes in the number of GSCs and CCs, and significant reductions in the number of 4-cell, 8-cell and 16-cell cysts. Conclusion: Dh31 likely neither affects GSC proliferation and number, nor niche size. Loss of Dh31 neuropeptide from the brain, and Dh31-R somatically, however, leads to a significant decrease in the number of GSC progeny (4-cell cysts, 8-cell cysts and 16 cell cysts). This loss is likely because of cyst death as there is no significant change in GSC proliferation as well. So far, our data suggests that Dh31 signaling is not required in the germline; however, further experiments need to be performed to rule out the possibility.