Modulation of reproductive behaviours by neuropeptides in drosophila

All animals must adapt their behaviour according to their environment and intrinsic state. At the level of the nervous system, this demands flexibility in the output of neural circuits. Neuropeptides, the largest class of signalling molecule in the nervous system, are crucial for engendering this flexibility, signalling through G-protein coupled receptors to modulate neuronal and synaptic properties. This study investigated the role of neuropeptides in the modulation of reproductive behaviours in Drosophila melanogaster. These behaviours are ‘hard-wired’ into the nervous system by the action of developmental genes yet display considerable flexibility in response to environmental and physiological factors. Though several neuropeptides are thought to modulate reproductive behaviour, the underlying mechanisms remain poorly understood. Knowledge of where neuropeptides act represents a particularly significant gap in our understanding. Thus, a key objective of this study was to elucidate the distribution of neuropeptide receptors (NPRs). Intersectional genetic approaches were used to target and label cells expressing NPRs, revealing broad expression across distinct compartments of the nervous system. Crucially, expression of several NPRs was evidenced in the neural circuits governing reproductive behaviour, facilitating direct modulation of these circuits. Analysis of single-cell RNA sequencing data corroborated these findings and provided additional insights, for example: revealing low expression-levels of NPRs, expression in transcriptionally distinct cell types, and co-expression in single cells.
Subsequent investigations focused on SIFaR, the receptor for SIFamide: a neuropeptide thought to modulate reproduction, feeding and sleep. Newly generated SIFaR mutant alleles produced abnormal reproductive behaviours, supporting a role for SIFaR signalling in their regulation. RNA interference was used to disrupt SIFaR exclusively in the neural circuits underlying these behaviours. This, too, produced robust phenotypes, suggesting that SIFamide modulates reproductive behaviour via direct signalling through these neurons. To study downstream effects of SIFaR signalling, FRET and calcium imaging experiments were designed and piloted, and an optimal paradigm for further investigation was established.
A range of approaches were employed to investigate how SIFamide performs its integrative functions. Immunohistochemical investigation, and analysis of hemibrain connectome data, supported the existence of just 4 SIFamide-producing neurons in the brain, with the latter providing evidence of heterogeneity in their morphologies, uncovering a potential mechanism for the modulation of discrete behaviours by SIFamide. Finally, approaches to assess the function of SIFamide in specific physiological contexts were tested. This included generation of a ‘FlpSTOP’ allele, facilitating temporally and spatially restricted disruption of SIFaR, and the design and validation of experiments to assay reproductive behaviours in the context of starvation.
Collectively, this data elucidates the expression profile of key neuropeptide receptors in Drosophila and, by establishing novel tools and approaches, paves the way for future investigation into neuropeptide signalling mechanisms.

Advisor: Goodwin, S