The Regulation of Neuropeptide Corazonin and Its Functional Analyses in Drosophila Melanogaster

Neuropeptides regulate diverse physiological processes, including homeostatic metabolism, behavior, reproduction, and development. The neuropeptide Corazonin (Crz), was first isolated from American cockroach, P. americana, as a potent cardioactive substance, and has been shown to exert diverse functions in different insects. In Drosophila, Crz expression is limited to three groups of neurons; totaling only 26 neurons out of ~10,000 neurons in a third instar larval central nervous system (CNS). In adults, Crz is expressed in 6-8 pairs of protocerebral neurons and 2 pairs of male specific abdominal ganglion. To gain insight into such tight regulatory mechanisms of Crz gene transcription, Crz promoter activity was dissected in vivo. The promoter bashing experiments yielding various 5‘-upstream sequences show that there are separate cis-acting elements that are highly conserved phylogenetically, which speaks to its functional significance in the activation of Crz transcription. In larval stage, a 504-bp upstream region is sufficient to activate Crz in all endogenous neurons.

Further dissection revealed two important regions; one between -419-bp and -504-bp region for the expression in dorsal medial neuron (DM). The other located between - 241-bp and -380-bp is responsible for dorsal lateral (DL) and ventral nerve cord (VNC) expression. The latter region can be subdivided into three DL-specific and two VNCspecific cis-acting elements. For DL-specific expression, two out of any three combination were needed; however, VNC needed two elements altogether.

Interestingly, basal transcription factor binding site TATA box showed minor role for Crz expression. In contrast to the larval expression, 321-bp upstream region is sufficient to activate Crz in all adult neurons. For the male-specific abdominal ganglion (ms-aCrz) expression, the cis-acting element was found to be in a region between -250-bp and - 290-bp. Overall, the data show that transcriptional regulatory mechanisms for Crz expression are not uniformed among Crz-containing neurons, which further indicates that their neuronal functions might be different.

To identify the roles of Crz in Drosophila, several fly behaviors were tested; ethanol-related responses, olfactory sensing responses and circadian rhythmic behaviors. Crz cell deficient (Crz-CD) flies and Crz receptor knock down (CrzR-KD) flies showed significantly delayed recovery from ethanol-induced sedation compared to control flies. Such hangover phenotype was ethanol specific. This result suggests that Drosophila Crz involves in ethanol-related responses. Further analyses suggest that Crz-CD, CrzR-KD and CrzR mutation did not affect aldehyde dehydrogenase (ALDH) at transcription level, but reduced ALDH enzyme activity. Crz is also associated with olfactory signaling, as Crz-CD and CrzR-KD flies are unable to find odor source, such as live yeast paste. Previously, Crz neurons located in the vicinity of nerve terminals originated from circadian pacemaker Pdf-expressing neurons, which indicate Crz neurons as part of circadian circuit. However, circadian locomotor rhythmic behavior of Crz-CD, Crz over-expression and CrzR-KD flies show normal circadian rhythmic behavior.