DEVELOPMENT 1011 RESEARCH ARTICLE INTRODUCTION The neural crest (NC) is a transient tissue that generates many important structures, including much of the peripheral nervous system, cranial skeleton and body pigmentation. Correct patterning of NC derivatives is achieved through regulation of cell migration and involves both environmental and cell-autonomous factors (Perris and Perissinotto, 2000; Krull, 2001). The somites have particular importance in patterning NC migration, as is perhaps best characterised in developing chick dorsal root ganglia (Lallier and Bronner-Fraser, 1988). Here, NC migrates on a ventral pathway (between somites and neural tube; called the medial pathway in zebrafish) only through the rostral portion of each sclerotome (Rickmann et al., 1985). This patterned migration results from differential expression of migration-promoting molecules such as thrombospondin, and migration-inhibiting molecules such as F- spondin and ephrin ligands, in rostral and caudal sclerotome, respectively (Davies et al., 1990). Mechanisms regulating migration on the dorsolateral (lateral pathway in zebrafish) pathway, between the skin and dermomyotome, are incompletely understood. Timelapse studies in zebrafish reveal that premigratory NC cells extend numerous protrusions that actively explore the lateral migration pathway. Before lateral pathway migration begins, these protrusions undergo rapid collapse, apparently because of somite-associated inhibitory activity (Jesuthasan, 1996). Later, the frequency of protrusion collapse decreases, allowing migration over the somite. Similarly, lateral pathway ‘maturation’ is indicated by heterologous extracellular matrix (ECM) transplantation studies in axolotl, with precocious NC migration initiated by ECM from older embryos (Löfberg et al., 1985; Löfberg et al., 1989). Two signals controlling the time when migration begins have been identified. Kit ligand attracts melanoblasts onto the dorsolateral pathway in mouse (Wehrle-Haller and Weston, 1995) and, in chick, ephrinB acting via EphB receptors regulates which neural crest cell types migrate on the dorsolateral pathway (Santiago and Erickson, 2002). In mouse and chick, only melanocytes utilise the dorsolateral pathway, and these become distributed ubiquitously, whereas in fish, several distinct types of chromatophores use this pathway and pigment pattern formation results from controlling their migration and final positioning (Kelsh, 2004). In zebrafish embryos, pigment patterns form from three chromatophore types. Black melanophores (equivalent to mammalian melanocytes) are principally arranged in four longitudinal stripes, including dorsal and ventral stripes extending from the head to the tip of the tail and the lateral stripe along the horizontal myoseptum of somites 6-26 (Kelsh et al., 1996). Iridescent iridophores are found within some melanophore stripes, whereas yellow xanthophores lie scattered throughout the embryo flank. The prominence and reproducibility of their pigment patterns make zebrafish ideally suited for mutant screens to identify NC patterning cues. Numerous loci necessary for correct development of embryonic (Kelsh et al., 1996; Odenthal et al., 1996) and adult (Haffter et al., 1996; Parichy et al., 2000a) zebrafish pigment cells have been identified. As with the pigment mutant collections in mice (Lyon and Searle, 1989), zebrafish pigmentation mutants exhibit a changed Sdf1a patterns zebrafish melanophores and links the somite and melanophore pattern defects in choker mutants Valentina Svetic 1, *, Georgina E. Hollway 2, *, Stone Elworthy 3 , Thomas R. Chipperfield 1 , Claire Davison 3 , Richard J. Adams 1,4 , Judith S. Eisen 5 , Philip W. Ingham 3 , Peter D. Currie 2 and Robert N. Kelsh 1,† Pigment pattern formation in zebrafish presents a tractable model system for studying the morphogenesis of neural crest derivatives. Embryos mutant for choker manifest a unique pigment pattern phenotype that combines a loss of lateral stripe melanophores with an ectopic melanophore ‘collar’ at the head-trunk border. We find that defects in neural crest migration are largely restricted to the lateral migration pathway, affecting both xanthophores (lost) and melanophores (gained) in choker mutants. Double mutant and timelapse analyses demonstrate that these defects are likely to be driven independently, the collar being formed by invasion of melanophores from the dorsal and ventral stripes. Using tissue transplantation, we show that melanophore patterning depends upon the underlying somitic cells, the myotomal derivatives of which – both slow- and fast-twitch muscle fibres – are themselves significantly disorganised in the region of the ectopic collar. In addition, we uncover an aberrant pattern of expression of the gene encoding the chemokine Sdf1a in choker mutant homozygotes that correlates with each aspect of the melanophore pattern defect. Using morpholino knock-down and ectopic expression experiments, we provide evidence to suggest that Sdf1a drives melanophore invasion in the choker mutant collar and normally plays an essential role in patterning the lateral stripe. We thus identify Sdf1 as a key molecule in pigment pattern formation, adding to the growing inventory of its roles in embryonic development. KEY WORDS: Neural crest, Migration, Patterning, Pigment pattern formation, Melanophore, Melanocyte, Xanthophore, Chromatophore, Slow muscle, Fast muscle, Horizontal myoseptum, cho, you-type, Sdf1a (Cxcl12a), Zebrafish Development 134, 1011-1022 (2007) doi:10.1242/dev.02789 1 Centre for Regenerative Medicine and Developmental Biology Programme, Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY, UK. 2 Victor Chang, Cardiac Research Institute, 384 Victoria Street, Darlinghurst, Sydney 2010, Australia. 3 MRC Centre for Developmental and Biomedical Genetics, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, UK. 4 Department of Anatomy, University of Cambridge, Downing Street, Cambridge CB2 3DY, UK. 5 Institute of Neuroscience, University of Oregon, Eugene, OR 97403, USA. *These authors contributed equally to this work † Author for correspondence (e-mail: bssrnk@bath.ac.uk) Accepted 18 December 2006