Different distribution of melanophores and xanthophores in early tailbud and larval stages inTriturus alpestris

Summary The subepidermal distribution of xanthophores and melanophores is investigated in embryos ofTriturus alpestris with a uniform (stage 28+) and a banded melanophore pattern (stage 35/36). In ultrathin head and trunk sections from stage 35/36 embryos which externally show longitudinal dorsal and lateral melanophore bands in the trunk and less compact continuations of the dorsal bands in the head, xanthophores were discovered in addition to melanophores. Melanophores contain melanosomes while xanthophores which are not externally visible, are recognized by their pterinosomes. Both chromatophore cell types are mutually exclusively distributed on the epidermal basement membrane (bm). Mesenchymal cells seemed not to be able to replace them, except on the bm of the corneal epithelium where there were only mesenchymal cells. In head and trunk sections from stage 28+ embryos which externally show a distribution of uniformly scattered melanophores on the dorsolateral halves, melanophores were found on the dorsolateral neural crest migration route. No epidermal bm was present and xanthophores were undetectable. In ventrolateral and ventral portions of embryos of both stages no chromatophores occurred. This investigation defines the histological localization of melanophores and xanthophores in embryos with a typical uniform and banded melanophore arrangement; a subsequent study analyzes when xanthophores appear and how they arrange with melanophores in alternating zones.     

An in vitro reconstitution system for the assessment of chromatin protein fluidity during Xenopus development

Chromatin fluidity, which is one of the indicators of higher-order structures in chromatin, is associated with cell differentiation. However, little is known about the relationships between chromatin fluidity and cell differentiation status in embryonic development. We established an in vitro reconstitution system that uses isolated nuclei and cytoplasmic extracts of Xenopus embryos and a fluorescence recovery after photobleaching assay to measure the fluidities of heterochromatin protein 1 (HP1) and histone H1 during development. The HP1 and H1 fluidities of nuclei isolated from the tailbuds of early tadpole stage (stage 32) embryos in the cytoplasmic extracts of eggs and of late blastula stage (stage 9) embryos were higher than those in the cytoplasmic extracts of mid-neurula stage (stage 15) embryos. The HP1 fluidities of nuclei isolated from animal cap cells of early gastrula stage (stage 10) embryos and from the neural plates of neural stage (stage 20) embryos were higher than those isolated from the tailbuds of stage 32 embryos in egg extracts, whereas the HP1 fluidities of these nuclei were the same in the cytoplasmic extracts of stage 15 embryos. These results suggest that chromatin fluidity is dependent upon both cytoplasmic and nuclear factors and decreases during development.     

Changes in the distribution of melanophores and xanthophores inTriturus alpestris embryos during their transition from the uniform to banded pattern

Summary The change in distribution of melanophores from stage 28+ (uniform melanophore pattern) to stage 34 (banded melanophore pattern) and the participation of xanthophores in these changes has been investigated inTriturus alpestris embryos by studying the social behaviour of single cells. While melanophores are clearly visible from outside the embryo at stage 28+, xanthophores cannot be recognized from the outside until after stage 34. In ultrathin sections of stage 34 embryos, xanthophores are observed alternating with melanophores in a zonal distribution (Epperlein 1982). Using detached pieces of dorsolateral trunk skin, which retain their chromatophores after detachment, the entire distribution of melanophores and xanthophores can be visualized in a scanning electron microscope (SEM). In ambiguous cases (early stages), cells were reprocessed for transmission electron microscopy (TEM) and the presence of the characteristic pigment organelles was assessed. In addition, xanthophores were specifically identified by pteridine fluorescence with dilute ammonia. Pteridines were also identified chromatographically in skin homogenates. The combination of these methods allowed precise identification and quantitative determination of melanophores and xanthophores. Both cell types were present as codistributed, biochemically differentiated cells at stage 28+. Changes in the pattern up to stage 34 were due to the rearrangement at the epidermal-mesodermal interface of a relatively fixed number of melanophores which became preferentially localised at the dorsal somite edge and at the lateral plate mesoderm, and to the distribution of an increasing number of xanthophores to subepidermal locations in the dorsal fin and between the melanophore bands. Concomitant was an increase in the thickness of the epidermal basement membrane and a change in shape of chromatophores from elongate via stellate to rosette shaped, which may be correlated with a shift from migratory to sessile phases.     

Pigment cell pattern formation in Taricha torosa: The role of the extracellular matrix in controlling pigment cell migration and differentiation

The neural crest is a population of highly migratory mesenchymal cells that ultimately localize in specific sites and differentiate into a variety of cell types. This report describes studies on the factors governing the migratory pathways, differentiation, and ultimate localization of the neural crest-derived pigment cells (black melanophores and yellow xanthophores) in the California newt, Taricha torosa. Melanophores first appear scattered in the dorsal portion of the lateral neural crest migratory pathway (between the somites and the ectoderm). These cells are eventually found in two stripes: a dorsal stripe that runs along the apex of the somites, and a midbody stripe near the somite-lateral plate mesoderm border. Melanophores are not seen in the dorsal fin of prehatching embryos. Xanthophores can be identified with the light microscope using NH₄OH-induced autofluorescence of pteridines and in the transmission electron microscope (TEM) by the presence of pterinosomes. Xanthophores first appear scattered among the melanophores over the surface of the somites; these cells eventually are found between the two melanophore stripes and in the dorsal fin. We were interested in determining the roles of the extracellular matrix (ECM) in controlling the formation of pigment cell patterns in T. torosa. Immunocytochemistry, Alcian blue staining of paraffin sections and ruthenium red staining of thin sections (accompanied by Streptomyces hyaluronidase and chondroitinase ABC digestion) were used to identify the composition and distribution of the ECM surrounding the pigment cells at various stages during development. The adhesive glycoprotein fibronectin is found in the dorsal portion of the lateral neural crest migratory pathway as well as in the dorsal fin matrix. Glycosaminoglycans (GAG) are found primarily in the dorsal fin and in the ECM surrounding the notochord. The dorsal fin ECM contains hyaluronate (HA), which was identified in the TEM as Streptomyces hyaluronidase-sensitive 3–5 nm microfibrils, as well as sulfated proteoglycan aggregates. We then confronted T. torosa neural crest cells in vitro with known ECM molecules. When neural folds are explanted onto tissue culture plastic in half-strength L-15 medium containing 10% fetal calf serum (FCS), cells migrate from the explant and differentiate into melanophores after 6 to 9 days. Xanthophores appear in the cultures 2 to 4 days after the appearance of melanophores. When cultured on three-dimensional collagen gels, xanthophores migrate significantly farther (P < 0.01) onto and into the collagen than melanophores (336 ± 183 vs 196 ± 160 μm from the edge of the explant). When 2.5 mg/ml chondroitin sulfate (CS) is present in the collagen gel, the distance that both pigment cell types migrate from the explant is reduced, with the result being that only xanthophores invade the GAG-rich matrix. When 1 mg/ml HA is present in the collagen gel, the differentiation of pigment cells is inhibited. Melanophores appear 48 hr later than in control gels without HA, and the number of melanophores in the explant after 10 days is significantly reduced (P < 0.01; 26.6 vs 1.1 melanophores/explant). When 1 mg/ml of HA is added to the FCS-enriched medium over neural crest cells spreading on tissue culture plastic, there is a similar delay and inhibition of pigment cell differentiation. With 2 mg/ml of CS there is no effect on pigment cell differentiation in vitro. Melanophores eventually appear in the dorsal fin of T. torosa several weeks after hatching. When fragments of dorsal fin that contain no apparent melanophores are transferred onto tissue culture plastic, melanophores appear in the explants after a few days in culture. These results suggest the following model of ECM-cell interactions during pigment cell pattern formation in T. torosa: Pigment cells differentiate in regions of the embryo that contain relatively little GAG. Xanthophores are able to invade the GAG-rich dorsal fin, but melanophores can not. The melanophores that eventually appear in the dorsal fin are derived from the neural crest cells that invaded the fin during early development, and were delayed in differentiating by the presence of HA.     

Acceleration of amphibian embryonic melanophore development by melanophore-stimulating hormone, N6,O2-dibutyryl adenosine 3',5'-monophosphate and theophylline.

Stage 14 (Gallien and Durocher, 1957)Pleurodeles waltlii embryos were treated with α- or β-melanophore-stimulating hormone (MSH), ACTH, dbc-AMP, c-AMP plus theophylline, theophylline, 5′-AMP, or 2′,3′-AMP. The development of melanophores was accelerated (appearing two stages earlier than in control embryos) by α- or β-MSH, ACTH, dbc-AMP, c-AMP plus theophylline, or theophylline alone. By the time embryos developed to stage 28, the control and treated embryos were indistinguishable in the number, distribution, and general morphology of their melanophores, suggesting these agents do not induce melanophore formation or mitosis. Cyclic AMP alone, 5′-AMP or 2′,3′-AMP were ineffective. Accelerated cytodifferentiation apparently requires preinduction by the invaginating chordo-mesoderm during stages 13 and 14 as presumptive neural plate explants cultured before chordo-mesoderm induction (stage 8) failed to produce melanophores with or without α-MSH or dbc-AMP. Explants from stage 14 embryos treated with α-MSH or dbc-AMP developed the same number of melanophores at the approximate time that treated whole embryos developed melanophores. Whole embryo experiments involving the faster developing embryos of Xenopus laevis were similar to those described for P. waltlii.     

Xanthophores in chromatophore groups of the premigratory neural crest initiate the pigment pattern of the axolotl larva

Summary The barred pigment pattern (Lehman 1957) of the axolotl larva is best observed from stage 41 onwards, where it already consists of alternating transverse bands of melanophores and xanthophores along the dorsal side of the trunk. The present study investigateswhen the two populations of neural crest derived chromatophores, melanophores and xanthophores become determined andhow they interact to create the barred pigment pattern. The presence of phenol oxidase (tyrosinase) in melanophores (revealed by dopa incubation) and pteridines in xanthophores (visualized by fluorescence) were used as markers for cell differentiation in order to recognize melanophores and xanthophores before they became externally visible. It was found that melanophores and xanthophores were already determined in the premigratory neural crest, at stages 30/31 and 35–36, respectively. Between stages 35–36 and 38 they were arranged in a prepattern of several distinct, mixed chromatophore groups along the dorsal trunk, morphologically correlated in the scanning electron microscope with humps on the original crest cell string. While the occurrence of xanthophores was restricted to the chromatophore groups and around them, melanophores were already uniformly distributed in the dorsolateral flank area, having migrated from trunk neural crest portions including the groups. The bar component of the pigment pattern was subsequently initiated by xanthophores, which caused melanophores in and around the chromatophore groups to fade or become invisible. The barred pattern was established by the formation of alternating clusters of like cells, melanophores and xanthophores.     

Ultraviolet effects on presumptive primordial germ cells (pPGCs) in Xenopus laevis after the cleavage stage

The presumptive primordial germ cell (pPGC) number with development after the cleavage stage and the fate of pPGCs damaged by uv irradiation were studied in successive Epon sections (0.5 μm thick) with the light microscope in both uv-irradiated and unirradiated Xenopus embryos. taking survival rate and sterility into consideration. The pPGCs of the uv-irradiated embryos occupy nearly the same location in the embryos as those of the unirradiated embryos at stages 12, 17, 23, and 28 [see Ikenishi, K., and Kotani, M. (1975). Develop. Growth Different. 17, 101–110]. At stage $\text{33}\text{34}$ they are found in the central part of the endoderm cell mass in the uv-irradiated embryos, while they are situated in the lateral or dorsal part of the endoderm cell mass in the unirradiated. In the uv-irradiated embryos, a cavity which was never found in the unirradiated embryos was observed in the endoderm cell mass beneath the archenteron cavity and in the almost-median part of the posterior endoderm cell mass at stages 17 and 23, respectively, and some vacuoles in pPGCs as well as in somatic cells around those pPGCs were noticed at stages $\text{17–}\text{33}\text{34}$. The number of pPGCs of the unirradiated enbryos increases about three- or fourfold during stages 12–46, while the pPGCs of the uv-irradiated embryos slowly increase in number from stage 17 to stage 28, indicating that the division occurs in pPGCs, then decrease with development and finally disappear from the tadpole.     

Melanophore lineage and clonal organization of the epidermis in Xenopus embryos as revealed by expression of a biogenic marker, GFP

Melanophore lineage during embryogenesis of Xenopus laevis was traced using the overexpression of a biogenic marker, green fluorescent protein (GFP). Two different approaches were applied after injection of GFP mRNA (hence a marker construct) into each blastomere at the 16-cell stage. In in vivo experiments, the embryos injected with a marker construct were grown until stage 45, in which melanophores were distributed over the whole body and were good enough for checking GFP expression at their migratory destination. In in vitro experiments, neural tubes of the embryos injected with a marker construct were isolated and cultured at stage 21 to examine by virtue of GFP expression how neural crest cells differentiate into melanophores. The results obtained from both in vivo and in vitro experiments indicated the following: 1) selected animal blastomeres vastly contribute to the development of melanophores, whereas other animal blastomeres do so slightly at a limited pace; and 2) vegetal blastomeres never contribute to melanophores in normal development, whereas certain vegetal blastomeres have a potential to give rise to melanophores in vitro. The analyses using GFP also disclosed that the dorsal and ventral epidermis derive from the restricted animal blastomeres in the normal development. Since the dorso-ventrality of the epidermis has been inseparably coupled with integumental pigmentation, the clonal organization of the epidermis observed in the present study is discussed in the light of pigment pattern formation attributed by melanophores.     

Secondary coverage of the yolk by the body wall in the direct developing frog, Eleutherodactylus coqui: an unusual process for amphibian embryos

 Eleutherodactylus coqui develops directly from a large 3.5-mm egg to a froglet, without an intervening tadpole stage. We have examined the development of the body wall, a structure whose behavior has been altered in this derived development. In an event that is unusual for amphibian embryos, the yolk mass is secondarily surrounded by the body wall, which originates near the embryo’s trunk. The epidermis of the body wall is marked by melanophores, and the rectus abdominis, which will form the ventral musculature, is near its leading edge. As the body wall expands, the epidermis, melanophores, and rectus abdominis all move from the dorsal side to close over the yolk at the ventral midline. The original ectoderm over the yolk undergoes apoptosis, as it is replaced by body wall epidermis. Intact muscles are not required for ventral closure of the body wall, despite their normal presence near the advancing edge. Comparative examination of embryos of Xenopus laevis and Rana pipiens suggests that ventral closure does not occur in species with tadpoles. The expansion of dorsal tissues over the yolk, as illustrated by E. coqui, may have been important in the origin of amniote embryos. Received: 23 April 1998 / Accepted: 28 June 1998     

Neural crest cell behavior in white and dark embryos of Ambystoma mexicanum: Epidermal inhibition of pigment cell migration in the white axolotl

Melanophores in larvae of the white (dd) strain of the Mexican axolotl (Ambystoma mexicanum) are confined to the dorsal midline of the trunk and dorsal posterior part of the head, whereas those in dark larvae (D_-) are distributed over the flank as well. Our results show that this phenotype of white larvae is the result of the failure of the melanophores or their neural crest precursor cells to migrate laterally due to an inhibition of or a failure in the support of their migration in the subepidermal space by the overlying epidermis. Correlated light and scanning electron microscopy of dissected larvae showed melanophores occupying the subepidermal space on the flank of dark larvae, whereas these cells were restricted to the dorsal midline of white larvae. Grafting experiments in which patches of epidermis, the underlying mesoderm, or both, were exchanged between dark and white embryos suggested that white epidermis alone can prevent the integration of pigment cells on the flank of dark larvae and, conversely, that grafts of dark epidermis alone can support their migration on the flank of white larvae. Mesoderm, when grafted alone, could not be shown to have similar effects.     

Identification of pigment cells during early amphibian development (Triturus alpestris,Ambystoma mexicanum)

Summary The purpose of the present investigation was to provide and apply a methodological manual with which the distribution, patterning and relationship of melanophores and xanthophores can be analyzed during early amphibian development. For demonstration of the methods, which include ultrastructural, histochemical and biochemical approaches, Triturus alpestris and Ambystoma mexicanum (axolotl) embryos are used. These two species differ conspicuously in their larval pigment patterns, showing alternating melanophore bands in horizontal (T. alpestris) and vertical (axolotl) arrangements. With transmission- and scanning electron microscopy melanophores and xanthophores were distinguished by their different pigment organelles and surface structures. The presence of phenol oxidase (tyrosinase) was used to reveal externally invisible or faintly visible melanophores by applying an excess of 3,4 dihydroxy-phenylalanine (dopa). Xanthophores were made visible in fixed and living embryos by demonstrating their pterin fluorescence. In addition, pterins were analyzed by HPLC in embryos before and after pigmentation was visible.Abbreviations DOPA dihydroxy-phenylalanine - FCS fetal calf serum - FIF formaldehyde-induced fluorescence - FITC fluorescein isothiocyanate - HPLC high performance liquid chromatography Dedicated to the memory of Dr. Michael Claviez     

Adult-type pigment cells,which color the ocular sides of flounders at metamorphosis,localize as precursor cells at the proximal parts of the dorsal and anal fins in early larvae

Flounders form left-right asymmetry in body coloration during metamorphosis through differentiation of adult-type melanophores and xanthophores on the ocular side. As the first step in investigating the formation of flounder body coloration asymmetry, in this study, we aimed to determine where the precursors of adult-type chromatophores distribute in larvae before metamorphosis. In Paralichthys olivaceus and Verasper variegatus, GTP cyclohydrolase 2 (gch2), a common marker of melanoblasts and xanthoblasts, was found to be transiently expressed in cells located along the bilateral skeletal muscles at the basal parts of the dorsal and anal fins of premetamorphic larvae. When V. variegatus larvae were fed with a strain of Artemia collected in Brazil, this gch2 expression was abolished and the differentiation of adult-type melanophores was completely inhibited, while the density of larval melanophores was not affected. In a cell trace test in which the cells at the basal part of the dorsal fin were labeled with DiI at the premetamorphic stage, adult-type melanophores labeled with DiI were found in the skin on the ocular side after metamorphosis. These data suggest that, in flounder larvae, adult-type melanophores are distributed at the basal parts of the dorsal and anal fins as unpigmented precursor cells.     

Role of svp in Drosophila Pericardial Cell Growth

The Drosophila dorsal vessel is a segmentally repeated linear organ, in which seven-up (svp) is expressed in two pairs of cardioblasts and two pairs of pericardial cells in each segment. Under the control of hedgehog (hh) signaling from the dorsal ectoderm, svp participates in diversifying cardioblast identities within each segment. In this experiment, the homozygous embryos of svp mutants exhibited an increase in cell size of Eve positive pericardial cells (EPCs) and a disarranged expression pattern, while the cardioblasts pattern of svp-lacZ expression was normal. In the meantime, the DA1 muscle founders were absent in some segments in svp mutant embryos, and the dorsal somatic muscle patterning was also severely damaged in the late stage mutant embryos, suggesting that svp is required for the differentiation of Eve-positive pericardial cells and DA1 muscle founders and may have a role in EPC cell growth.     

Grafting analysis of the periodic albino mutant of Xenopus laevis

The differentiation of normal and mutant (a^P/a^P) Xenopus laevis melanophores in chimerae was analyzed to determine the tissues affected by this mutation. Normal melanophores in mutant host tissue differentiate in mutant host tissue prior to those of the mutant host. These normal melanophores were initially normal in appearance, but, after the differentiation of the mutant host's melanophores, they became indistinguishable from their host's melanophores. These normal melanophores persist in more than normally punctate form after the disappearance of the mutant host's melanophores in late larval life. Parabiosis and head transplants between mutant and normal embryos did not affect the character of either type of melanophore developing in tissue of its own genotype, indicating that the hormonal control of melanophore differentiation is not affected by the mutation. Therefore, the periodic albino mutant affects the capacity of the mutant melanophore to differentiate and the ability of the mutant skin to support normal melanophore differentiation.     

Production of rhesus monkey cloned embryos expressing monomeric red fluorescent protein by interspecies somatic cell nuclear transfer

Interspecies somatic cell nuclear transfer (iSCNT) is a promising method to clone endangered animals from which oocytes are difficult to obtain. Monomeric red fluorescent protein 1 (mRFP1) is an excellent selection marker for transgenically modified cloned embryos during somatic cell nuclear transfer (SCNT). In this study, mRFP-expressing rhesus monkey cells or porcine cells were transferred into enucleated porcine oocytes to generate iSCNT and SCNT embryos, respectively. The development of these embryos was studied in vitro. The percentage of embryos that underwent cleavage did not significantly differ between iSCNT and SCNT embryos (P > 0.05; 71.53% vs. 80.30%). However, significantly fewer iSCNT embryos than SCNT embryos reached the blastocyst stage (2.04% vs. 10.19%, P < 0.05). Valproic acid was used in an attempt to increase the percentage of iSCNT embryos that developed to the blastocyst stage. However, the percentages of embryos that underwent cleavage and reached the blastocyst stage were similar between untreated iSCNT embryos and iSCNT embryos treated with 2 mM valproic acid for 24 h (72.12% vs. 70.83% and 2.67% vs. 2.35%, respectively). These data suggest that porcine-rhesus monkey interspecies embryos can be generated that efficiently express mRFP1. However, a significantly lower proportion of iSCNT embryos than SCNT embryos reach the blastocyst stage. Valproic acid does not increase the percentage of porcine-rhesus monkey iSCNT embryos that reach the blastocyst stage. The mechanisms underling nuclear reprogramming and epigenetic modifications in iSCNT need to be investigated further.     

Partial rescue of dorsal, a maternal effect mutation affecting the dorso-ventral pattern of the Drosophila embryo, by the injection of wild-type cytoplasm

Mutant alleles at the maternal effect locus dorsal cause a dorsalization of the Drosophila embryo. In extreme mutants, the embryos develop exclusively structures which derive from the dorsal-most region in normal eggs, in less strong phenotypes in addition to dorsal structures, structures normally derived from a dorso-lateral to lateral egg region are formed. Injection of cytoplasm from wild-type embryos into mutant embryos partially restores the dorso-ventral pattern in that injected embryos develop additional structures never formed in uninjected control embryos or embryos injected with mutant cytoplasm. The phenotype of injected embryos resembles that of weaker alleles at the dorsal locus indicating that the wild-type cytoplasm partially rescues the mutant phenotype. The response of the mutant embryos is restricted to the site of injection and occurs only when cytoplasm is injected into the ventral and not into the dorsal side of mutant embryos. The rescuing activity appears to be equally distributed in cleavage stage wild-type embryos, whereas, in syncytial blastoderm embryos, cytoplasm from the ventral side is about twice as effective as that taken from the dorsal side.     

α- and β-adrenoceptors of zebrafish in melanosome movement: a comparative study between embryo and adult melanophores

Zebrafish, like other teleosts, display rapid skin color change in response to the background through sympathetic nerves. Here, the α- and β-adrenoceptors of melanophores were studied pharmacologically both in zebrafish embryo and adult scale. In vitro experiments on adult scale melanophores demonstrated that both α₁- and α₂-adrenoceptors are functional in melanosome aggregation, the α₂ subtype being predominant. Most melanophores in zebrafish embryos were able to concentrate melanosomes to α₂-adrenergic agonist α-methylnorepinephrine when they first appeared. This ability increased at least in the following 48 h, showing melanophores at these stages have developed functional adrenoceptors and these receptors increase independently before sympathetic innervation. However, even high concentration (10⁻³ M) of α₁-adrenoceptor agonist phenylephrine was not able to evoke any paling of the embryos. In adult scales, propranolol enhanced the melanosome-aggregating response of epinephrine and isoproterenol, but not norepinephrine, indicating β-adrenoceptor mediates melanosome-dispersing response in adult zebrafish. Similar response was not observed in embryos until 60 h post-fertilization (hpf). The melanophore adrenoceptor blocking effects of phentolamine and propranolol in embryos were much lower than that in adult zebrafish, suggesting these adrenoceptors in developing melanophores are less sensitive to the classical antagonists.     

Sox5 Functions as a Fate Switch in Medaka Pigment Cell Development

Mechanisms generating diverse cell types from multipotent progenitors are crucial for normal development. Neural crest cells (NCCs) are multipotent stem cells that give rise to numerous cell-types, including pigment cells. Medaka has four types of NCC-derived pigment cells (xanthophores, leucophores, melanophores and iridophores), making medaka pigment cell development an excellent model for studying the mechanisms controlling specification of distinct cell types from a multipotent progenitor. Medaka many leucophores-3 (ml-3) mutant embryos exhibit a unique phenotype characterized by excessive formation of leucophores and absence of xanthophores. We show that ml-3 encodes sox5, which is expressed in premigratory NCCs and differentiating xanthophores. Cell transplantation studies reveal a cell-autonomous role of sox5 in the xanthophore lineage. pax7a is expressed in NCCs and required for both xanthophore and leucophore lineages; we demonstrate that Sox5 functions downstream of Pax7a. We propose a model in which multipotent NCCs first give rise to pax7a-positive partially fate-restricted intermediate progenitors for xanthophores and leucophores; some of these progenitors then express sox5, and as a result of Sox5 action develop into xanthophores. Our results provide the first demonstration that Sox5 can function as a molecular switch driving specification of a specific cell-fate (xanthophore) from a partially-restricted, but still multipotent, progenitor (the shared xanthophore-leucophore progenitor).