An orthologue of the kit-related gene fms is required for development of neural crest-derived xanthophores and a subpopulation of adult melanocytes in the zebrafish, Danio rerio

David M. Parichy ) 1 2 David G. Ransom 0 Barry Paw 0 Leonard I. Zon 0 Stephen L. Johnson 2 0 Department of Medicine, Childrens' Hospital of Boston, Howard Hughes Medical Institute , 300 Longwood Avenue, Enders 7, Boston MA 02115 , USA 1 Section of Integrative Biology and Institute for Cellular and Molecular Biology, University of Texas at Austin , Austin TX 78712 , USA 2 Department of Genetics, Washington University Medical School , St Louis, MO 63110 , USA An orthologue of the kit-related gene fms is required for development of neural crest-derived xanthophores and a subpopulation of adult melanocytes SUMMARY Developmental mechanisms underlying traits expressed in larval and adult vertebrates remain largely unknown. Pigment patterns of fishes provide an opportunity to identify genes and cell behaviors required for postembryonic morphogenesis and differentiation. In the zebrafish, Danio rerio, pigment patterns reflect the spatial arrangements of three classes of neural crest-derived pigment cells: black melanocytes, yellow xanthophores and silver iridophores. We show that the D. rerio pigment pattern mutant panther ablates xanthophores in embryos and adults and has defects in the development of the adult pattern of melanocyte stripes. We find that panther corresponds to an orthologue of the c-fms gene, which encodes a type III receptor tyrosine kinase and is the closest known homologue of the previously identified pigment pattern gene, kit. In mouse, fms is essential for the Recent years have seen dramatic advances in our understanding of the developmental genetic bases for the patterning of embryonic axes, tissues and organ rudiments. In contrast, we know relatively little about mechanisms underlying the expression of traits during later stages of development and in adults (Tata, 1993). Nevertheless, identifying the genes and cell behaviors underlying trait expression is an essential step in understanding the origins of naturally occurring trait variation and the evolution of form (Atchley and Hall, 1991; Phillips, 1999). One ecologically important trait that is particularly amenable to analysis is the externally visible pigment pattern of fishes in the genus Danio, which includes the zebrafish D. rerio. Pigment cells in Danio and other vertebrates are derived from neural crest cells that arise along the dorsal neural tube then disperse along stereotypical pathways throughout the embryo (Hrstadius, 1950; Erickson and Perris, 1993; Hall, development of macrophage and osteoclast lineages and has not been implicated in neural crest or pigment cell development. In contrast, our analyses demonstrate that fms is expressed and required by D. rerio xanthophore precursors and that fms promotes the normal patterning of melanocyte death and migration during adult stripe formation. Finally, we show that fms is required for the appearance of a late developing, kit-independent subpopulation of adult melanocytes. These findings reveal an unexpected role for fms in pigment pattern development and demonstrate that parallel neural crest-derived pigment cell populations depend on the activities of two essentially paralogous genes, kit and fms. 1999; Groves and Bronner-Fraser, 1999). In addition to pigment patterns, neural crest cells contribute to a host of other tissues and organ systems in vertebrates, including bones of the craniofacial skeleton, teeth, neurons and glia of the peripheral nervous system, endocardial cushion cells and endocrine glands. Indeed, Gans and Northcutt (1983) have argued that much of vertebrate morphology and its evolution can be understood in terms of the patterning of neural crest cells and their derivatives, and how these patterning mechanisms have changed phylogenetically. Elucidating the mechanisms by which the final form of larval and adult pigment patterns arise may thus shed light on more general mechanisms of trait variation and evolution in vertebrates. In danios and many other ectothermic vertebrates, pigment patterns result from the spatial arrangements and coloration of three major classes of neural crest-derived pigment cells: black melanocytes (or melanophores), yellow xanthophores and silver iridophores (Bagnara, 1998; Reedy et al., 1998). In D. rerio, these different classes of pigment cell combine to generate different pigment patterns during different phases of the life cycle (Kirschbaum, 1975; Johnson et al., 1995b). In larvae, a relatively simple pattern is evident by hatching (approx. 2.5 days; Fig. 1A). This early larval pigment pattern consists of several stripes of melanocytes and iridophores, as well as xanthophores that are widely distributed over the flank, giving an overall yellow cast to the body. This pattern persists until approx. 14 days, at which time a metamorphosis begins that ultimately results in the formation of the striped pigment pattern of the adult (Fig. 1C). Between 14 and 21 days, melanocytes increase in number and become visible dispersed throughout the skin in regions not previously occupied by these cells. Subsequently, between 21 and 28 days, melanocyte numbers increase more sharply and an adult stripe pattern begins to emerge. This pattern comprises two melanocyte stripes by 28 days, but additional stripes form as the fish grow. Dark stripes consist of melanocytes and iridophores, whereas light interstripe regions consist of xanthophores and iridophores (Fig. 2A). Finally, in addition to stripes that are found deep within the dermis adjacent to the myotomes (Hawkes, 1974), more superficial melanocytes and xanthophores cover the dorsal scales, and together give a dark cast to the dorsum of the fish. The mechanisms of adult stripe development remain largely unknown in D. rerio. Nevertheless, several mutants have started to provide insights into the genes and cell populations that are involved in pigment pattern metamorphosis in this species. A previous analysis (Johnson et al., 1995b) identified roles for three genes that are required for the development of adult stripes: sparse, rose and leopard. sparse mutant larvae have fewer melanocytes than wild type at 3 days. These cells then die, and the fish completely lack melanocytes until approx. 21 days, when a new population differentiates and contributes to an adult pigment pattern with one-half the wildtype complement of stripe melanocytes. In contrast, rose and leopard mutants each exhibit normal pigment patterns through 21 days, but melanocyte numbers increase less rapidly than wild type between 21 and 28 days, again resulting in one-half the wild-type complement of stripe melanocytes. These findings suggested that sparse on one hand, and rose and leopard on the other, identify genes required for the development of distinct classes of melanocytes in the adult pigment pattern: an early developing adult population dependent on sparse; and a later developing adult population dependent on rose and leopard. In support of this model, virtually all body stripe (...truncated)