|dc.description.abstracteng||System biology approaches, coupled with emerging omics tools, have been widely used and greatly accelerated biological and medical research and discovery. In this thesis, I will focus on applying those tools and technologies to the post-transcriptional regulation of the circadian clock by miRNAs in mice and the development of a single-cell clock model in zebrafish, respectively.
The circadian clock is an endogenous oscillator that regulates a wide range of cellular, metabolic, physiological, and behavioral rhythms in animals, plants and micro-organisms. Gene regulatory circuits driving circadian rhythms in higher vertebrates and insects are mainly generated through transcriptional/translational feedback loops of core clock genes (Bmal1, Clock, Per gene family, Cry gene family, and Rev-erb α/β (Nr1d1/2)). Recent advance in clock regulation suggests that post-transcriptional mechanisms play essential roles in the regulation of circadian gene expression. By integrative analysis of various types of published data, we identified 57 circadian miRNA primary transcripts and in mouse liver 56 of them are regulated by one or more core circadian transcription regulator. We found that the mature miRNAs arising from these circadian primary transcripts were either not oscillating at all or oscillating but with low amplitudes. The over-expression of one of them, miR-378, in mouse liver by adenovirus injection downregulated a significant proportion of the circadian oscillating target genes. In particular, we observed that miR-378 participates in the circadian control of both, cell cycle and metabolism. It does so by forming either coherent or incoherent feed-forward loops with different circadian regulators.
Although the molecular mechanism of circadian clock is well studied, how and when these cellular rhythms arise during development is largely unknown. We sought to study the development of circadian clock using zebrafish (Danio rerio), a well-established model organism for the study of animal development and circadian rhythms. Due to the transparency of the fish larval stages, zebrafish is also a suitable system for in vivo live imaging. We constructed a transgenic zebrafish line, Venus-NLS-PEST (VNP), driven by the promoter of nr1d1, which encodes the core circadian regulator Rev-Erbα. This system allows us to monitor single-cell circadian rhythm in vivo for the first time. We carried out single-cell in vivo imaging of nr1d1:VNP fish over embryonic development, and found that circadian expression of pineal nr1d1:VNP positive cells was superimposed on their cell-type-specific developmental expression trends. Furthermore, the fish line helped us to address a long-standing issue in circadian clock ontogeny: is the clock development by synchronizing existing oscillators or by initializing coherent single-cell clocks? We found that early exposure to the light-dark (LD) cycle during development initializes the oscillators of individual cells rather than synchronizing existing oscillators of individual cells as researchers in the field have previously believed. This shows the necessity of the single-cell circadian reporter.||de