Zebrafish Chronobiology

Using the zebrafish as a model system to study the vertebrate circadian clock

This research group was originally established an exchange program between the CNRS (France) and the Max-Planck-Gesellschaft. Dr. Nick Foulkes is a CNRS scientist and heads this research group. The aim of the group is to study the molecular biology and genetics of the circadian clock in zebrafish. Research overview: Circadian Rhythms Clock genes Peripheral clocks Why use zebrafish to study circadian rhythms? Rhythmic clock gene expression and light - entrainable pacemakers in vertebrate organs and cells Current lines of research Selected Publications Open positions

Circadian Rhythms

Almost every aspect of plant and animal biology shows day - night rhythms. Many persist even under constant conditions however with period lengths that are not precisely 24 hours and for this reason they are termed "circadian" (Circa - diem). Central to the generation of circadian rhythms is an endogenous circadian clock which is constantly reset ("entrained") by environmental factors such as light to ensure that it remains synchronised with the natural 24 hour cycle.

Where are clocks located? Are clocks generated by cell-cell interactions or are they cell autonomous? How are they entrained by light - dark cycles and how do they ultimately control physiology?

Classically, the circadian clock in vertebrates was shown to reside in so-called central "oscillator" or "pacemaker" structures. In mammals the suprachiasmatic nucleus (SCN) of the hypothalamus and the retina are both the sites of pacemakers while in lower vertebrates, the pineal gland also appears to contain an additional pacemaker. Within these structures, individual cells have been shown to contain clocks which are synchronised in the context of the tissue. In mammals, the SCN clock is entrained by light via photoreceptors in the retina which appear to be distinct from the rod and cone ocular photoreceptor cells. However, more recently this centralized model for the vertebrate clock has been challenged by the discovery of clock functions in diverse tissues and cell types.

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Clock genes

Genetic analysis of mutations affecting the clock in Neurospora, Drosophila, Cyanobacteria and Arabidopsis and most recently the mouse have lead to the identification of clock component genes. Details of the molecular organization of the circadian clock have now started to emerge and it is clear that the basic features are highly conserved between animals, plants and fungi. Interestingly, many clock gene products function as transcription factors which ultimately activate or repress their own expression and thus constitute what are termed autoregulatory transcriptional feedback loops. Changes in the subcellular localization, posttranslational modifications, and delays between transcription and translation of these proteins seem to be ultimately crucial for clock function.

In the mouse, the clock components identified to date include CLOCK and BMAL1, two bHLH-PAS transcription factors which are able to form heterodimers and activate transcription upon binding to E-box promoter elements. Amongst the regulatory targets of these factors are the three period genes (mper1, 2 and 3) which encode closely related PAS domain factors. The PERs function as negative regulators, blocking activation by CLOCK:BMAL1. Other negative regulatory factors which appear to function as heterodimers with the PER proteins are the cryptochromes (CRY 1 and 2), which are close relatives of the 6-4 photolyase DNA repair enzymes but lack the DNA repair enzymatic function. Finally, the kinase, Casein Kinase I epsilon also seems to represent a central clock component. It is thought to phosphorylate the PER proteins and so regulate their stability and subcellular localization. The current favourite model for how these factors are organized into an autoregulatory feedback loop, is summarized in the following figure.



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Peripheral clocks

One unexpected discovery which came from the first characterisation of the expression of clock genes in mouse is that their expression was detected in many organs and was not restricted to the central pacemaker structures such as the SCN. Furthermore, the expression of the mper genes oscillates with a circadian rhythm in many different tissues. It has also been shown that circadian oscillations in gene expression can be detected even in immortalized mammalian tissue culture cell lines following serum shock - starvation treatments. The situation is comparable to that in Drosophila where rhythmic expression of the period gene is detected in many different tissues, even when these are detatched from the fly and placed in primary culture. These observations suggest that the "system level" organization of the clock as well as its molecular components are well conserved between insects and vertebrates. Furthermore, in Drosophila, the peripheral tissue clocks have been shown to be directly entrained by light-dark cycles in culture, suggesting that the photopigments, capable of entraining the clock, may be widely expressed in peripheral tissues . That this might also be a property of some vertebrate clocks is hinted at from our own work with zebrafish.

Why use zebrafish to study circadian rhythms ?

Attractions of using zebrafish for studying the vertebrate clock:

1. Large-scale forward genetic analysis to identify new clock components.
2. Stable or transient transgenic lines for mechanistic studies.
3. Examine the established and function of the clock during embryogenesis and early development.
4. Peripheral clocks in zebrafish as well as zebrafish cell lines are directly light entrainable.

Rhythmic clock gene expression and light-entrainable pacemakers in vertebrate organs and cells

We originally demonstrated that the Clock gene's mRNA levels oscillate strongly in the pineal gland and retina as well as in all adult zebrafish tissues tested (for example, heart, kidney, gill, skin and even blood), with the exception of the testis. This is remarkable since the expression of the murine homologue does not oscillate.

The observation of clock oscillation in the majority of peripheral tissues raised the question as to whether these oscillations were being driven from a central pacemaker(s) or if they reflected the existence of multiple, independent clocks in the different tissues. In order to address this question, we established primary organ cultures of the zebrafish heart and kidney under constant conditions. These cultures were maintained for 2 to 3 days and we were still able to visualize a circadian rhythm of Clock expression through this period. This observation constitutes strong evidence that different fish organs do indeed contain there own clocks. Subsequently in vitro cultures of transgenic rat tissues were shown to maintain rhythms of mper1 expression demonstrating that peripheral clocks may be widespread in vertebrates.

One central question is how are these different clocks synchronised. Is there a coordinating role for "central" pacemakers or can these peripheral tissues detect external timing signals such as light directly, and entrain their clocks? Our subsequent findings would seem to support the later possibility. We showed that the Clock rhythm measured in the cultured hearts and kidneys can be directly entrained by a light : dark cycle. The possibility that vertebrate tissues generally might contain light responsive circadian clocks has many profound implications.

We have extended our experiments by examining the effects of light - dark cycles on Clock expression in primary zebrafish cell lines. A number of cell lines have been derived from early zebrafish embryos. In PAC-2 cells, we demonstrated that we could induce a circadian rhythm of Clock mRNA expression by placing cells in a light-dark cycle. Furthermore, this rhythm persisted for several cycles even when the cells were subsequently returned to constant darkness. A light entrainable circadian clock in the PAC-2 cell line provides a powerful tool to further explore the function of the zebrafish circadian clock and the mechanisms which enable it to be entrained directly by light.

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Current lines of research

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 are comparing global gene expression in light pulsed versus constant dark adapted zebrafish larvae, organ cultures and cell lines, using DNA chip technology. In collaboration with Georg Otto (Genetics) we hope to use this approach to provide clues as to the wider physiological effects of light.

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Selected Publications

Open Positions

Applications from potential diploma students and postdocs are always welcome.