RUI: Interrogating circadian gene regulation of metabolism and gene expression during dorsal mesoderm specification in Xenopus laevis

University Of Wisconsin-Whitewater, Whitewater WI

Investigators

Abstract

In adults, the circadian clock functions to coordinate or synchronize physiology and behavior (e.g. sleep/wake) with the time of day. Disruption of this coordination (e.g. shift work, jet lag) can disrupt an organism’s overall health and well-being. Circadian clock components are not known to keep time in the early embryo, yet the PI has recently identified two components (Bmal1 and Nr1d1) that alter changes in gene expression and metabolism during early frog development and influence skeletal muscle formation. The PI hypothesizes that these components are influencing a key molecule in metabolic reactions that helps cells convert nutrients into energy and can also affect other cellular functions, such as gene expression and protein function. Proposed experiments will investigate the effects of loss of these two circadian clock components on various early aspects of muscle development. The PI will also investigate whether downstream targets of the adult circadian clock impact muscle development, thus elucidating early impacts of circadian clock components with early developmental decisions. Broader Impact activities include creation of undergraduate laboratory experiences in genome editing and metabolism; training undergraduates in cutting edge research techniques at the University of Wisconsin Whitewater and the Marine Biology Laboratory (Woods Hole, Massachusetts); mentoring of a post-doctoral researcher in both research and teaching; and outreach to K-12 students and the public. Most mechanistic studies linking circadian gene expression to early cell fate decisions assess differentiation of ESCs in culture. These experiments fail to identify changes in gene expression and metabolism during the decision to adopt a specific cell fate. The PI hypothesizes that, like in adults, two circadian clock components Bmal1 and Nr1d1 impact the production of NAD+ in the developing Xenopus leavis embryo. Aim1 proposes to clearly define changes in gene expression and metabolism during dorsal mesoderm specification to identify key targets of Bmal1 and Nr1d1 that impact the metabolic shift. This will be accomplished through RNA-seq comparing the response of wild-type and bmal1 knockout animal cap ectoderm during dorsal mesoderm induction and specification by activin. Similarly, RNA-seq will be used to compare the response of wild type animal caps treated with DMSO or SR8278 (a Nr1d1/2 antagonist) during dorsal mesoderm specification. In both experiments metabolic outputs such as oxygen consumption rate (OCR), and NAD+, NAD+/NADH, and NADP+/NADPH levels will be monitored in parallel with RNA-Seq sample collection. Aim 2 will test whether NAD+ and Sirtuins regulate the response of cells to TGFbeta signaling by exposing activin induced animal caps to a sirtuin 1/2/5 inhibitor (suramin) or a sirtuin 1/5 activator (melatonin). During dorsal mesoderm induction and specification, it will be tested at what point the tissue is sensitive to suramin and melatonin treatment, and whether each drug affects NAD(P)+/NAD(P)H, OCR, and gene expression (oscillator, clock-controlled genes, and mesodermal genes). Lastly, the research will investigate whether Sirtuins negatively feedback on Bmal1 protein via deacetylation during dorsal mesoderm specification. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.