1. Introduction

1.1. Background

An immense amount of progress has been made recently in our understanding of the basic mechanisms and function of the verte-brate circadian clock. The major catalysts for this progress have been the success of the genetic dissection of the clock mechanism in Drosophila [1] together with the fundamental similarities be-tween vertebrate and fruit fly clocks. Furthermore, by applying powerful mouse genetics tools, detailed insight into the precise function and organization of vertebrate circadian clock compo-nents has been gained [2]. It is therefore not intuitively obvious what we could gain at this point from studying the circadian clock molecular mechanism in an alternative vertebrate model such as the zebrafish. This review attempts to give an overview on the cur-rent knowledge that has been gathered in zebrafish and show how the biology of this model actually makes it eminently well suited to explore the circadian cloc.

1.2. Zebrafish as a model

The original interest in zebrafish as a vertebrate model system came not from the circadian clock – but from the field of embryol-ogy and developmental biology [3,4]. Several aspects of its basic biology make it inherently ideal for these areas of study. For one, it has small, completely transparent embryos that develop rapidly in an egg-shell or chorion. Development from the single fertilized cell to a moving, recognizable vertebrate embryo with a central nervous system and most organ systems normally takes just 24 h. Furthermore, the embryos develop externally and so the whole developmental process can be watched non-invasively in a petri dish under the microscope. Combined with the ability to establish transgenic lines expressing fluorescent reporter genes, the zebrafish is widely regarded as an excellent model for live imaging in vivo [5] (Fig. 1)

    Another major advantage of the zebrafish is its proven utility for large-scale forward genetic screens [3,4]. Again, certain features of its biology make it a more attractive model than mice for such experiments. Firstly, reproduction is relatively simple to establish in the lab. If a single pair of male and female zebrafish is left to-gether overnight in a small tank of water, soon after ‘‘lights on’’ the following morning, the fish typically lay hundreds of eggs. Sec-ondly, the adults are hardy, small (2–3 cm long), reach sexual maturity after 2–3 months and can be raised at high density at very low cost. Various protocols have now been established for muta-genesis including the use of chemical mutagens and retroviral insertion and also for the subsequent mapping of the mutated gene [5]

The last few years have seen the use of zebrafish expanding from its traditional user base and being applied to study perse biomedically relevant aspects of biology including behaviour and physiology [6,7]. This is primarily the result of its low cost, its proven utility for forward genetics, the many similarities in basic physiology between mammals and fish, and, for the early developmental stages, fewer ethical concerns. Furthermore, the impressive capacity of most zebrafish tissues to regenerate following injury has attracted considerable atten-tion from research aimed at understanding and treating certain human diseases such as heart and neurodegenerative diseases and cancer [7,8].

1.3. Chronobiology and the zebrafish

The attention of chronobiologists originally turned to zebra-fish many years ago [9,10]. This was during the ‘‘dark ages’’ of our knowledge of the molecular basis of the vertebrate clock. At that stage our understanding of the workings of the Drosoph-ila, Neurospora and cyanobacterial circadian clocks was far from complete. Furthermore, no vertebrate circadian clock genes had been cloned and so the nature of the molecular mechanism of the circadian clock in vertebrates was a complete mystery. Given the clear success of using forward genetics to identify the first clock mutants and genes in non-vertebrates, and the difficulties to perform large scale genetic screening on mice, the zebrafish seemed an ideal model to apply in the quest to identify and characterize the first clock mutants in a genetically tractable ver-tebrate species. Furthermore, given its extensive characterization in many developmental biology studies, it held great promise as a model to trace the origin of the circadian clock during embryo-genesis. However, with the availability of the first molecular tools to study the core circadian clock, it soon became apparent that zebrafish offered many more advantages. Notably, the peripheral clocks of this species are directly entrainable by light [11]. This contrasts with the situation in mammals but resem-bles the peripheral clocks of Drosophila [12]. This direct light sensing property was also encountered in cell lines derived from zebrafish embryos [11].