Characterization of Hypocretin Expression in Zebrafish (2023)
Undergraduate: Yingning Sang
Faculty Advisor: Christina Graves
Department:
a. Objectives: Hypocretin modulates the circadian sleep/wake cycle, feeding behavior, and how organisms respond to stress. While hypocretin-containing neurons are found in the lateral hypothalamic (LH) area of the central nervous system, debate remains as to whether bona fide hypocretin producing neurons can be found in the enteric nervous system. Therefore, we are interested in determining whether hypocretin is only made by hypothalamus or also by other neurons. Instead of employing antibodies, which might be leaky or recognizing something else sending false signals, we obtained a transgenic hypocretin gfp expressing fish and looked its expression in the gut.
b. Experimental Methods: We used fluorescent microscopy to image transgenic hcrt:egfp+ zebrafish at different time points and also performed wholemount immunostaining (anti-gfp retrieval of endogenous gfp expression and co-labelled with a pan-neuronal antibody) and high resolution confocal imaging of ex vivo adult gut.
c. Results: Image analysis revealed abundant gfp+ hcrt expressing cells in the gut of adult zebrafish in the myenteric plexus layer, consistent with enteric nervous system expression of hcrt. The gfp+ cells also exhibit maximal-neighbor patterning and more densely populate the proximal gut.
d. Conclusions: To date, research on the existence of hypocretin-producing neurons in the enteric nervous system has been limited. Decades-old findings suggest a gut hcrt+ population may be present, but these studies have largely relied on antibody retrieval at a time when rigorous antibody validation was less commonplace than today. Our data, by relying on endogenous expression of gfp under the hcrt promoter, provides evidence for a bona fide hcrt+ population in the gut.