Does intercellular communication through gap junctions influence the zebrafish circadian clock?

Most animals and plants contain a biological or circadian clock that times events in the body to match the day-night changes in the environment. This clock was once thought to be present only in the brain, but more recent experiments have shown that most tissues and cells in the body also contain a clock. To examine this process, we have developed cells in culture, made from zebrafish embryos, where each cell contains a daily clock, and can directly see when the lights come on at dawn. By using luminescent reporter genes, we can now monitor when genes involved in the clock mechanism switch on by the fact that the cells start to glow. Now cells don't work alone in the body. They influence each other through hormonal signals, and through direct cell contact. Cells form special channels, called gap junctions, which allow small molecules to pass from one cell to another. The aim of this project is to examine whether the amount of cell communication changes across the day, and to then change the number of gap junctions, both increasing and decreasing their levels, in order to see how this may alter how the clock measures time. Does making cells talk to each other more strongly make the circadian clock run more precisely in the light, as well as in constant darkness? Or, in contrast, by stopping cells directly talking to each other, does the cellular clock run less accurately?

Peripheral tissues, as well as single cells, contain circadian oscillators. We have been able to examine this phenomenon very clearly in zebrafish cell lines by virtue of the fact that not only does each cell contain a clock, but they are all directly light responsive. This makes our zebrafish cell lines a unique model system with which to study clock function. Moreover, we have been able to automate this analysis by using luminescent reporter gene constructs, which allow for monitoring of clock function at both the population and single cell level. Our single cell imaging experiments on newly plated cells have revealed quite rapid clock dampening and dramatic stochastic changes in cellular clock period. Of course, this situation does not perfectly reflect the natural in vivo situation for several reasons, including the fact that these cells have had insufficient time to form functional gap junctions. The aim of this collaborative proposal is to explore the role of such intercellular communication in our luminescent clock-containing cell lines. We will examine the types of gap junctions present in these cells and whether they undergo daily or light driven changes. Functional tests will reveal whether or not there are daily changes in the extent of cellular communication through this mechanism. In addition, we will directly explore their role on clock function by both overexpressing particular gap junction components, as well as using dominant negative constructs to reduce cellular communication. This represents the first extensive study of the role of gap junctions in clock function in a peripheral, cell line circadian system.