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David Whitmore

Publications and source records attributed to David Whitmore.

7 recordsLinked to original sources
PubMed2006

Light reaches the very heart of the zebrafish clock.

Zebrafish are typically used as a model system to study various aspects of developmental biology, largely as a consequence of their ex vivo development, high degree of transparency, and, of course, ability to perform forward genetic mutant screens. More recently, zebrafish have been developed as a model system with which to study circadian clocks. Cell lines generated from early-stage zebrafish embryos contain clocks that are directly light-responsive. We describe recent experiments using single-cell luminescent imaging approaches to study clock function in this novel cell line system. Furthermore, studies examining the process of entrainment to light pulses within this cell population are described in this review, as are experiments examining light-responsiveness of early-stage zebrafish embryos.

Animals↗
PubMed2005

Imaging of single light-responsive clock cells reveals fluctuating free-running periods.

Zebrafish tissues and cell lines contain circadian clocks that respond directly to light. Using fluorescence-activated cell sorting, we have isolated clonal cell lines that contain the reporter construct, zfperiod4-luciferase. Bioluminescent assays show that oscillations within cell populations are dampened in constant darkness. However, single-cell imaging reveals that individual cells continue to oscillate, but with widely distributed phases and marked stochastic fluctuations in free-running period. Because these cells are directly light responsive, we can easily follow phase shifts to single light pulses. Here we show that light acts to reset desynchronous cellular oscillations to a common phase, as well as stabilize the subsequent free-running period.

Animals↗
PubMed2004

E-box function in a period gene repressed by light.

In most organisms, light plays a key role in the synchronization of the circadian timing system with the environmental day-night cycle. Light pulses that phase-shift the circadian clock also induce the expression of period (per) genes in vertebrates. Here, we report the cloning of a zebrafish per gene, zfper4, which is remarkable in being repressed by light. We have developed an in vivo luciferase reporter assay for this gene in cells that contain a light-entrainable clock. High-definition bioluminescence traces have enabled us to accurately measure phase-shifting of the clock by light. We have also exploited this model to study how four E-box elements in the zfper4 promoter regulate expression. Mutagenesis reveals that the integrity of these four E-boxes is crucial for maintaining low basal expression together with robust rhythmicity and repression by light. Importantly, in the context of a minimal heterologous promoter, the E-box elements also direct a robust circadian rhythm of expression that is significantly phase-advanced compared with the original zfper4 promoter and lacks the light-repression property. Thus, these results reveal flexibility in the phase and light responsiveness of E-box-directed rhythmic expression, depending on the promoter context.

Animals↗
PubMed2003

Light regulates the cell cycle in zebrafish.

The timing of cell proliferation is a key factor contributing to the regulation of normal growth. Daily rhythms of cell cycle progression have been documented in a wide range of organisms. However, little is known about how environmental, humoral, and cell-autonomous factors contribute to these rhythms. Here, we demonstrate that light plays a key role in cell cycle regulation in the zebrafish. Exposure of larvae to light-dark (LD) cycles causes a range of different cell types to enter S phase predominantly at the end of the day. When larvae are raised in constant darkness (DD), a low level of arrhythmic S phase is observed. In addition, light-entrained cell cycle rhythms persist for several days after transfer to DD, both observations pointing to the involvement of the circadian clock. We show that the number of LD cycles experienced is essential for establishing this rhythm during larval development. Furthermore, we reveal that the same phenomenon exists in a zebrafish cell line. This represents the first example of a vertebrate cell culture system where circadian rhythms of the cell cycle are observed. Thus, we implicate the cell-autonomous circadian clock in the regulation of the vertebrate cell cycle by light.

Animals↗
PubMed2003

Teleost multiple tissue (tmt) opsin: a candidate photopigment regulating the peripheral clocks of zebrafish?

Isolated organs and cell lines from zebrafish exhibit circadian oscillations in clock gene expression that can be entrained to a 24-h light/dark cycle. The mechanism underlying this cellular photosensitivity is unknown. We report the identification of a novel opsin family, tmt-opsin, that has a genomic structure characteristic of vertebrate photopigments, an amino acid identity equivalent to the known photopigment opsins, and the essential residues required for photopigment function. Significantly, tmt-opsin is expressed in a wide variety of neural and non-neural tissues, including a zebrafish embryonic cell line that exhibits a light entrainable clock. Collectively the data suggest that tmt-opsin is a strong candidate for the photic regulation of zebrafish peripheral clocks.

Amino Acid Sequence↗
PubMed2002

Zebrafish melanopsin: isolation, tissue localisation and phylogenetic position.

Photoreception is best understood in retinal rods and cones, but it is not confined to these cells. In non-mammals, intrinsically photosensitive cells have been identified within several structures including the pineal, hypothalamus and skin. More recently novel light sensitive cells have been identified in the inner/basal retina of both teleosts and rodents. Melanopsin has been proposed as the photopigment mediating many of these non-rod, non-cone responses to light. However, much about the melanopsin gene family remains to be clarified including their potential role as photopigments, and taxonomic distribution. We have isolated the first orthologue of melanopsin from a teleost fish and show expression of this gene in a sub-set of retinal horizontal cells (type B). Zebrafish melanopsin, and orthologues of this gene, differ markedly from the vertebrate photopigment opsins. The putative counterion is not a glutamate but a tyrosine, the putative G-protein binding domain in the third cytoplasmic loop is not conserved, and they show low levels of amino acid identity (approximately 27%) to both the known photopigment opsins and to other members of the melanopsin family. Mouse melanopsin is only 58% identical to Xenopus, and 68% identical to zebrafish. By contrast, the photosensory opsin families show approximately 75% conservation. On the basis of their structure, genomic organisation, discrete evolutionary lineage, and their co-expression with other opsins, the melanopins are not obvious photosensory opsins. They might represent a separate branch of photopigment evolution in the vertebrates or they may have a non-direct photosensory function, perhaps as a photoisomerase, in non-rod, non-cone light detection.

Animals↗