The first 24-hours of a circadian clock: development of the zebrafish circadian pacemaker and a role for microRNAs in clock function.

Most tissues and cells in an animal, including humans, contain a circadian or daily biological clock. These clocks regulate the behaviour and physiology of the animal, including sleep-wake times, body temperature etc. These clocks also regulate events within each cell, such as, for example, what time of day certain genes get turned on. In comparison, very few studies have looked at clocks in developing embryos, and even fewer at how the clock might actually start 'ticking'. This proposal aims to explore this issue, taking advantage of the zebrafish model system, which is well established as a tool for the study of early developmental events. We know that the mother deposits the products of key clock genes into the developing embryo. These RNA products breakdown with perfect timing, so as to generate a circadian oscillation on the first day of life. How is this achieved? We believe that molecules, called microRNAs, play a key role in this event, by timing the degradation of these clock molecules. Using newly discovered mutants, we aim to see if this is really the case, and how these microRNAs function to make the clock itself work and tell time. We also aim to make new cell lines in culture, from these mutant embryos, allowing us to explore in great detail how these microRNAs control clock genes to produce these daily changes.

When and how does the circadian clock start to oscillate in a developing embryo? Such a question is remarkably difficult to answer in a mammalian model system, but quite straightforward to address in a model like zebrafish, where embryos develop outside of the female in large numbers. Our previous studies have shown when the clock starts in zebrafish, on the very first day of development, and we have precisely mapped out the expression patterns of most key clock genes during this developmental process. What is very apparent from our work is that the RNA for most core clock genes is maternally deposited in the zebrafish egg. Upon fertilization these maternal transcripts are degraded with perfect timing, such as to establish a precisely timed circadian oscillation on the first day of development. How is this maternal RNA degradation controlled, and is it a critical event for starting the embryonic clock? Most maternal transcripts are regulated by microRNAs (miRNAs), and we intend to explore if this is also the case for clock gene transcripts. miRNAs are processed to a mature, functional state in part by dicer proteins in the cell. We will use dicer mutant zebrafish to assess the impact of a lack of miRNA function on how the clock starts to oscillate. Furthermore, we will employ a strategy with which we have had considerable success, which is to generate cell lines from these mutants. Using luminescent reporter constructs, we can then follow the clock oscillation in detail in cells that lack miRNA processing. By adding back, or over expressing, specific miRNAs in this mutant background, we can determine which specific molecules play a key role in clock function. Using a complimentary 'knock-down' strategy, using 'antagomirs' we will eliminate specific miRNAs in our oscillating zebrafish cell lines, and determine the exact consequences on clock function. Furthermore, we will determine which miRNAs are both rhythmic and light-induced in our cell line system.

Circadian rhythms, or the biological clock is a topic that has caught the interest of the general public in a significant way. The reasons for this are readily apparent, as almost all aspects of human behaviour, physiology and cell biology are controlled by the clock, and this idea is easily accessible to everyone. We all sleep for approximately a third of our lives, and most people can relate to getting drunk more easily at lunchtime than in the evening. This topic is, therefore, frequently discussed in newspapers, online sites and TV documentaries. A critical part of our research is appropriate dissemination of data and public engagement. In the case of projects like this we have found this a relatively small problem. The issues are quite simple, and easily accessible to the general public. This is true not only for adults, but also children and politicians, and we have found our work very accessible as an educational tool to explore basic aspects of evolution, and molecular biology. The issue simply remains how to best publicise the effort. Obviously, we intend to publish results in the usual peer reviewed scientific journals. Gene sequences etc. will be deposited in the appropriate databases. For a more general audience, the project will be discussed on our own lab web page, but also through UCL's media resources and communications/Press Office, as well as the media offices of the BBSRC. Several times each year I give general outreach talks to high school level students on this topic, from within the UK and also Europe. Interest levels are always high. Are there any translational or clinical consequences of this research? At its heart, of course, this project represents a basic science study of how a biological clock starts, and what makes it run smoothly. It is not immediately aimed at curing a specific, single disease. However, it is becoming very apparent that disorders in clock function can lead to increased health risks. These include increased risks of cancer, diabetes and heart disease. The consequences of clock disruption during embryo development have never been studied, but it is highly likely that there will be similarly severe health consequences to the developing foetus. This fact is of growing importance as people have children whilst living evermore temporally disrupted lifestyles, with shift work, international travel and other factors disrupting the daily timing systems of the embryo. We believe therefore that establishing a basic understanding of the clock in the developing embryo, and what it regulates, is a critical first step to identifying and preventing these health risks.