Roles for extra-hypothalamic oscillators in the avian clock

Abstract

Avian circadian clocks are composed of a distributed network of neural and peripheral oscillators. Three neural pacemakers, located in the pineal, the eyes, and the hypothalamus, control circadian rhythms of many biological processes through complex interactions with slave oscillators located throughout the body. This system, an astonishing reflection of the life history of this diverse class of vertebrates, allows birds to coordinate biochemical and physiological processes and harmonize them with a dynamic environment. Much work has been done to understand what roles these pacemakers have in avian biology, how they function, and how they interact to generate overt circadian rhythms. The experimental work presented in this dissertation uses the domestic chicken, Gallus domesticus, as a model to address these questions and carry forward current understanding about circadian biology in this species. To do so, we utilized a custom DNA microarray to investigate rhythmic transcription in cultured chick pineal cells. We then sought to identify genes which might be a component of the pineal clock by screening for rhythmic transcripts that are sensitive to a phase-shifting light stimulus. Finally, we surgically removed the eyes or pineal from chickens to examine the roles of these extra-SCN pacemakers in regulating central and peripheral rhythms in metabolism and clock gene expression. Using these methods, we show that the oscillating transcriptome is diminished in the chick pineal ex vivo, while the functional clustering of clock controlled genes is similar. This distribution reveals multiple conserved circadian regulated pathways, and supports an endogenous role for the pineal as an immune organ. Moreover, the robustness of rhythmic melatonin biosysnthesis is maintained in vitro, demonstrating that a functional circadian clock is preserved in the reduced subset of the rhythmic pineal transcriptome. In addition, our genomic screen has yielded a list of 28 genes that are candidates for functional screening. These should be evaluated to determine any potential role they may have as a component of the pineal circadian clock. Finally, we report that the eyes and pineal similarly function to reinforce rhythms in brain and peripheral tissue, but that metabolism and clock gene expression are differentially regulated in chick.