Regulation of pigmentation in zebrafish melanophores Darren W. Logan, Sally F. Burn and Ian J. Jackson* MRC Human Genetics Unit, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, UK *Address correspondence to Ian J. Jackson, e-mail: ian.jackson@hgu.mrc.ac.uk Present address: Darren W. Logan, Department of Cell Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, ICND 222, La Jolla, CA 92037, USA Summary In comparison with the molecular genetics of melan- ogenesis in mammals, the regulation of pigmenta- tion in poikilothermic vertebrates is poorly understood. Mammals undergo morphological col- our change under hormonal control, but strikingly, many lower vertebrates display a rapid physiological colour change in response to the same hormones. The recent provision of extensive genome sequen- cing data from teleost zebrafish, Danio rerio, pro- vides the opportunity to define the genes and proteins mediating this physiological pigment response and characterise their function biologically. Here, we illustrate the background adaptation pro- cess in adults and larvae and describe a novel assay to visualize and directly quantify the rate of zebrafish melanophore pigment translocation in unpreceden- ted detail. We demonstrate the resolution of this assay system; quantifying the zebrafish melano- phore response to melanin-concentrating and mel- anocyte-stimulating hormones. Furthermore, we investigate the intracellular signalling downstream of hormone stimulation and the biomechanical pro- cesses involved in zebrafish pigment translocation, confirming the importance of cyclic adenosine monophosphate (cAMP) as a mediator of pigment translocation and finding intact microtubules are essential for both melanin dispersion and aggrega- tion in zebrafish, but that microfilament disruption affects aggregation only. In conclusion, we propose these data establish the zebrafish as an experimental model for studying both physiological colour change and the molecular basis of pigment translocation. Key words: zebrafish/background adaptation/hormonal regulation/melanophore Received 29 September 2005, revised and accepted for publication 14 February 2006 Introduction The physiological colour change observed in poikilother- mic vertebrates, defined as rapid changes in colour caused by intracellular movement of pigment, has intri- gued scientists for over 80 yr. This pigment transloca- tion can have wide ranging visual effects, from the dramatic social colour changes in chameleons, to the more subtle background adaptation observed in most fish and amphibians. Since the discovery that the frog pituitary contains a factor that acts on pigment cells (Allen, 1916; Smith, 1916), it has become clear that physiological colour change is largely under hormonal control. However, almost 50 yr passed before it was first demonstrated that melanocyte-stimulating hormones (MSHs) dis- persed pigment containing melanosomes in amphibians (Bagnara, 1964). Originally championed by Hogben in the 1930s (Waring, 1963), a bihumoral theory of colour control was leant credence by the extraction of melanin- concentrating hormone (MCH) from the catfish (Enami, 1955). However, it was not until much later that the melanosome aggregating activity of MCH was des- cribed (Kawauchi et al., 1983). Although the melanocyte-stimulating and MCHs were first identified in cold-blooded vertebrate species, the focus of the resulting body of research has been firmly on their function in a mammalian context. A range of physiological processes mediated by MSHs (a, b and c) via their high-affinity receptors, was uncovered initially in mouse models (Chen et al., 1997, 2000; Huszar et al., 1997; Robbins et al., 1993). Subsequent analysis identified orthologous genes with comparable functions in humans. One such receptor, the melanocortin-1- receptor (MC1R), has been shown to mediate the pig- mentary effects of MSHs in a number of mammalian and avian species (reviewed in Garcia-Borro ´n et al., 2005; Jackson, 1997). Melanin-concentrating hormone receptors were also first identified in mammals (An et al., 2001; Chambers et al., 1999). The discovery that MCH, in contrast to MSH, has an antagonistic effect on appetite in animal models suggests a conserved link between the two sig- nalling systems (Ludwig et al., 1998; Shimada et al., 1998). However, there is no evidence for MCH having a role in mammalian pigmentation. Instead, the agouti sig- nalling protein (ASP) appears to be a functional antagon- ist of MSH (Lu et al., 1994). Copyright ª 2006 The Authors doi: 10.1111/j.1600-0749.2006.00307.x Journal compilation ª 2006 Blackwell Munksgaard 206 Pigment Cell Res. 19; 206–213