Our research effort is primarily focused on the study of biological clocks, using zebrafish as a model system. Our early work was amongst the first to show that the majority of tissues and cells of the body contain independent clocks, which regulate the timing of fundamental aspects of cell biology. An unusual feature of the fish circadian system is that most cells are also directly light responsive. Some projects in the laboratory are aimed at identifying the molecules involved in this unusual light response, while others focus on the processes that the clock regulates. We employ a variety of molecular, cellular and biochemical techniques for these cell-based studies, including single cell luminescent imaging in the newly established Luminescent Imaging Facility here at UCL. Additional research projects include studies in the blind cavefish, Astyanax Mexicanus, where we are exploring both molecular and behavioural rhythms in the laboratory here in London, as well as in the caves of Northeastern Mexico.
ACCIDENTS AND GOOD LUCK. In 1996, Nick Foulkes and myself at the IGBMC in Strasbourg, France decided to develop zebrafish as a model system for a molecular examination of the circadian clock. Our starting position was quite clear. Following on from the earlier work of Greg Cahill in Houston (who sadly passed away in 2008), we believed the circadian clock was localized to both the eyes and pineal gland (Cahill, 1996). The first aim was to isolate a circadian clock gene, which could then be used as a marker of clock function. This we eventually achieved by cloning a zebrafish homolog of the then recently isolated mouse CLOCK gene (King et al., 1997). Initial expression studies showed that the zebrafish CLOCK transcript (now called CLOCK1) oscillated in both retina and pineal. We then examined CLOCK1 expression in a number of other tissues, mainly as a negative control for our eye and pineal data. These experiments were prior to the description of clock gene expression in mammalian tissues, and our expectation was for minimal or no expression of clock genes in the periphery. To our surprise, the CLOCK1 transcript was not only expressed, but also oscillated in zebrafish organs with the same timing as in the eye and pineal. The development of a simple organ culture system then allowed us to demonstrate that this oscillation continued in vitro, and so conclude that organs within the fish contained an autonomous circadian oscillator (Whitmore et al., 1998). The situation in fish is, therefore, similar to that described in Drosophila (Plautz et al., 1997).
The next obvious question was, how do these peripheral clocks entrain to the environmental light-dark cycle? Though it seemed somewhat insane, we went ahead and placed cultured organs (initially hearts and kidneys) on a light-dark cycle in an incubator, illuminated by a fibre optic. Matching organs from sibling fish on the same light-dark cycle were dissected and placed into a neighbouring incubator, but on a reverse light-dark cycle. The oscillation in CLOCK1 gene expression was then determined in both groups by RNase protection assays and showed that this organ clock could be re-entrained in vitro simply by changing the lighting regime. Therefore, these tissues not only contain a clock, but also the photopigments required to detect light, as well as the signal transduction machinery to set that clock (Whitmore et al., 2000).
Thanks to the kind help of Uwe Straehle (then in Strasbourg), we gained access to a number of embryonic zebrafish cell lines. The first of these we examined (PAC2 cells made initially by Nancy Hopkins' lab at MIT) showed a high level of CLOCK1 gene expression. When placed on a light-dark cycle, these cells oscillated with a circadian period and timing identical to that found in zebrafish organs. We now had access to a cell line that contained a clock and the phototransduction machinery necessary to set that clock (Whitmore et al., 2000).
Since these early studies, we have generated a number of luminescent zebrafish cell lines, which we use routinely to monitor clock function (Vallone et al., 2004). The entrainment study described above can now be performed with relative ease by maintaining these cells under different lighting conditions and monitoring bioluminescence in an automated fashion, as shown below, in our Per1-luciferase cells. These luminescent cell lines provide an invaluable tool for our clock studies, and allow students and postdocs in the laboratory to get plenty of sleep. Using very sensitive photon counting cameras to measure luminescence, we have gone on to image gene expression rhythms in single cells. These experiments have revealed an unexpected (at least, to us) level of stochastic noise within these cellular clocks (Carr and Whitmore, 2005). Additional studies have elucidated the key role played by one of the cryptochromes, Cry1a, in setting this clock to light (Tamai et al., 2007). Our studies in zebrafish embryos have shown the incredibly early stage at which the clock starts (Dekens and Whitmore, 2008), and even more fascinating, how extremely light responsive a blastula stage fish embryo is, only hours after fertilisation (Tamai et al., 2004). Our current studies build on these early observations and are leading us into exciting new directions, such as clock control of the cell cycle and, even more unexpectedly, studies on clock function in the caves of Northeastern Mexico.
Cahill G (1996)
Circadian regulation of melatonin production in cultured zebrafish pineal and retina.
Brain Res 708:177-181.
King DP, Zhao Y, Sangoram AM, Wilsbacher LD, Tanaka M, Antoch MP, Steeves TD,
Vitaterna MH, Kornhauser JM, Lowrey PL, Turek FW and Takahashi JS (1997)
Positional cloning of the mouse circadian clock gene.
Cell 89: 655-667.
Whitmore D, Foulkes NS, Straehle U and Sassone-Corsi P (1998)
Zebrafish Clock rhythmic expression reveals independent peripheral circadian oscillators.
Nat Neurosci 1: 701-707 (PDF).
Plautz JD, Kaneko M, Hall JC and Kay SA (1997)
Independent photoreceptive circadian clocks throughout Drosophila.
Science 278: 1632-1635.
Whitmore D, Foulkes NS and Sassone-Corsi P (2000)
Light acts directly on organs and cells in culture to set the vertebrate circadian clock.
Nature 404: 87-91 (PDF).
Vallone D, Gondi SB, Whitmore D and Foulkes NS (2004)
E-box function in a period gene repressed by light.
Proc Natl Acad Sci USA 101: 4106-4111 (PDF).
Carr A-JF and Whitmore D (2005)
Imaging of single light-responsive clock cells reveals fluctuating free-running periods.
Nat Cell Biol 7: 319-321 (PDF).
Tamai TK, Young LC and Whitmore D (2007)
Light signaling to the zebrafish circadian clock by Cryptochrome 1a.
Proc Natl Acad Sci USA 104: 2757-2765 (PDF).
Dekens MP and Whitmore D (2008)
Autonomous onset of the circadian clock in the zebrafish embryo.
EMBO J 27: 2757-2765 (PDF).
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