Date of Award
Spring 5-15-2010
Degree Name
Doctor of Philosophy (PhD)
Degree Type
Dissertation
Abstract
Daily rhythms of behavior are controlled by a circuit of circadian pacemaking neurons. In Drosophila, 150 pacemakers participate in this network, and recent observations suggest the network is composed of heterogeneous oscillators which normally interact and synchronize. Sixteen oscillator neurons (the small and large LNvs) express a neuropeptide called pigment dispersing factor (PDF) whose signaling is required for the synchrony between oscillators. Given the significance of PDF signaling for numerous aspects of behavioral and molecular rhythms, determining precisely where and how signaling via the PDF receptor (PDFR) occurs is now a central question in the field.
In this study, I pursued the expression patterns of PDFR via two independent approaches. I utilized GAL4 mediated rescue of PDFR, and also generated a large transgene of PDFR through recombineering methodology. I found that GAL4-mediated rescue of pdfr phenotypes using a UAS-PDFR transgene is insufficient to provide complete behavioral rescue. In contrast, a ~70 kB PDF receptor (pdfr) transgene does rescue the entire pdfr circadian behavioral phenotype. The transgene (pdfr-myc) is widely but heterogeneously expressed among pacemakers, and also among a limited number of non-pacemakers. My results support an important hypothesis: the small LNv cells directly target a subset of the other crucial pacemaker neurons cells. Furthermore, expression of pdfr-myc confirms an autocrine feedback signaling by PDF back to PDF-expressing cells. Finally, the results present an unexpected PDF receptor site: the large LNv cells appear to target a subset of glia that resides at the base of the eye.
Next, using this authentic pdfr reporter, the 70 kB pdfr-myc, I observed precise coincidence of PDFR expression and high CRYPTOCHROME (CRY) expression, the circadian photoreceptor, within the circadian clock neurons. PDFR and CRY are highly co-expressed in the 5th small LNv, three LNd, and six to seven DN1s. This observation of precise co-expression of these two genes prompted me to test potential genetic interactions of pdf signaling and cry. Flies doubly mutant for pdfr and cry display severely disrupted locomotor rhythms with an absence of both morning and evening anticipatory peaks. Moreover, these double mutant flies were also arrhythmic under constant dark or light conditions, further suggesting the lack of a functional clock. I also observed an unexpected genetic interaction of PDF and CRY signaling in over-expression conditions. An extra copy of pdfr gene in a cryb background causes long period phenotype, whereas an extra copy of pdfr in a wild-type background does not change any aspect of circadian behavior. This suggests that these two signaling pathways are not simply redundancy since over-expression of one signaling cannot replace the lack of the other: instead of restoring normal behavior that genetic combination generates abnormalities in behavior.
Finally, I tested how these behavioral phenotypes can be related to the changes in the cellular properties of the clock network. In pdfr;; cryb double mutants, which display arrhythmic behavior under LD and constant conditions, molecular rhythms were abnormal in a critical subset of clock neurons (the 5th small LNv and all LNd). In these pacemakers, I observed sustained levels of high nuclear PERIOD. I conclude that this arrested PER rhythms in the subset of clock neurons is the principle basis for the lack of circadian coordination of behavior in the double mutant flies. In the gain-of-function of pdfr flies, I observed cell fate changes from PDF-expressing oscillators to PDFR-expressing slow-clock oscillators. When this same phenomenon was studied in a cryb mutant background, I saw clear period extension in the flies' circadian behavior. This is a new finding in the field: that the balance of different clock cell types may be a point of physiological regulation and may affect the period of circadian rhythms.
Overall, this thesis work reveals an unexpected convergence of Cry-mediated and PDF signaling pathways. This convergence is required to support circadian behavioral rhythms, and initiates a possible link between the balance of cell types and behavioral phenotypes.
Language
English (en)
Chair and Committee
Fanxin Long
Committee Members
David C Beebe, Kyunghee Choi
Recommended Citation
Im, Seol Hee, "The convergence of PDF signaling and CRYPTOCHROME in the neuronal network underlying Drosophila circadian behavior" (2010). Arts & Sciences Electronic Theses and Dissertations. 504.
https://openscholarship.wustl.edu/art_sci_etds/504
Comments
Permanent URL: https://doi.org/10.7936/K7TH8JW5