Luciferase clock reporters in zebrafish cell lines
We have developed an in vivo bioluminescent assay for the circadian clock in zebrafish embryo-derived cell lines. We have cloned the promoters from various zebrafish clock genes into luciferase reporter vectors and used these plasmids to stably transfect various cell lines. Following addition of luciferin we then monitor bioluminescence using a scintillation counter as a real time assay for clock gene expression. Illumination during the intervals between counting also allows us to explore how light influences the expression of individual clock genes as well as the function of the clock as a whole. Using this cell model system, we have performed a systematic promoter analysis of several clock genes in order to identify promoter elements involved in their regulation by light. Furthermore, we have explored the signaling pathways that mediate the effects of light by a detailed pharmacological analysis. Finally, we are using these reporter cells to test which wavelengths of light regulate the clock as a first step towards identifying the widely expressed photopigment.
Function of the clock during early zebrafish development and growth
We are investigating the effects of the clock upon cell proliferation and growth of zebrafish larvae. We have described a striking daily rhythm in the number of cells entering the cell cycle in 6 day old larvae that are maintained under a LD cycle. The majority of proliferating cells in a range of tissues preferentially enter S phase during the late afternoon. In embryos raised under DD, a significantly lower level of S phase is observed with no daily rhythm, predicting a much lower level of cell proliferation. Exposure of the zebrafish cell lines to LD cycles also leads to a day night rhythm of cell proliferation. Our data point to light influencing cell cycle progression via the circadian clock. Our future goals are to exploit the advantages of the zebrafish for genetic analysis and the availability of a cell culture model system well suited for more biochemical analysis to dissect the molecular basis of this phenomenon and its biological significance.
Temperature and the clock
Zebrafish, being poikilotherms, are an ideal model to study the entrainment of the vertebrate circadian clock by temperature changes. We are studying how clock gene expression responds to shifts in temperature in both larvae and the zebrafish cell lines. Also, we are testing whether temperature might influence the clock’s response to light. This could provide insight into how information from different zeitgebers is integrated by the clock. Finally, using the zebrafish cell lines and the various promoter luciferase reporter constructs we hope to identify which promoter elements mediating the gene expression response to temperature changes.
The effect of light on gene expression
Our data points to the widespread expression of a photopigment in zebrafish cells and tissues. This also raises the intriguing possibility that other aspects of physiology might be directly responsive to light in this vertebrate. Already, evidence points to repair of UV damaged DNA being under direct effect of light exposure. We have explored the effects of light on cell physiology in more detail by comparing global gene expression in light pulsed versus constant dark adapted zebrafish larvae, organ cultures and cell lines, using DNA chip technology. To complement this approach, we are also identifying light responsive enhancer modules in the promoter sequences of light inducible genes. By this approach, we aim to build a comprehensive map of the mechanisms whereby light signals are relayed to changes in gene expression and associated cellular functions.
Circadian clock, injury and cell proliferation
In zebrafish, most tissues and organs including the heart and central nervous system possess the remarkable ability to regenerate following severe injury. Both spatial and temporal control of cell proliferation and differentiation is essential for the successful repair and re-growth of damaged tissues. Using the regenerating adult zebrafish caudal fin as a model, we have demonstrated an involvement of the circadian clock in timing epidermal cell proliferation following injury. Peak numbers of S-phase cells occur at the end of the light period while lowest levels are observed at the end of the dark period. Remarkably, immediately following amputation the basal level of epidermal cell proliferation increases significantly with kinetics that depends upon the time of day when the amputation is performed. In sharp contrast, we fail to detect circadian rhythms of S-phase in the highly proliferative mesenchymal cells of the blastema. Now we are interested to understand why and how cell proliferation is strongly clock regulated in certain cell types but not in others.
Circadian Clock in Cavefish
We have performed a comparative, functional analysis of the circadian clock involving the zebrafish that is normally exposed to the day-night cycle and a cavefish species Phreatichthys andruzzii that has evolved in perpetual darkness. We have shown that this cavefish retains a food-entrainable clock that oscillates with an infradian period. However, this clock is not regulated by light. This comparative study pinpoints the two extra-retinal photoreceptors Melanopsin (Opn4m2) and TMT-opsin as essential upstream elements of the peripheral clock light input pathway. Using this comparative approach, we are interested to explore why and how several different photoreceptors contribute to the photic entrainment properties of fish peripheral tissues.
Updated: November 23, 2012