Deconstructing evolution of adult phenotypes: genetic analyses of kit reveal homology and evolutionary novelty during adult pigment pattern development of Danio fishes

The cellular bases for evolutionary changes in adult form remain largely unknown. Pigment patterns of Danio fishes are a convenient system for studying these issues because of their diversity and accessibility and because one species, the zebrafish D. rerio, is a model organism for biomedical research. Previous studies have shown that in zebrafish, stripes form by migration and differentiation of distinct populations of melanophores: early metamorphic (EM) melanophores arise widely dispersed and then migrate into stripes, whereas late metamorphic (LM) melanophores arise already within stripes. EM melanophores require the kit receptor tyrosine kinase, as kit mutants lack these cells but retain LM melanophores, which form a residual stripe pattern. To see if similar cell populations and genetic requirements are present in other species, we examined D. albolineatus, which has relatively few, nearly uniform melanophores. We isolated a D. albolineatus kit mutant and asked whether residual, LM melanophores develop in this species, as in D. rerio. We found that kit mutant D. albolineatus lack EM melanophores, yet retain LM melanophores. Histological analyses further show that kit functions during a late step in metamorphic melanophore development in both species. Interestingly, kit mutant D. albolineatus develop a striped melanophore pattern similar to kit mutant D. rerio, revealing latent stripe-forming potential in this species, despite its normally uniform pattern. Comparisons of wild types and kit mutants of the two species further show that species differences in pigment pattern reflect: (1) changes in the behavior of kit-dependent EM melanophores that arise in a dispersed pattern and then migrate into stripes in D. rerio, but fail to migrate in D. albolineatus; and (2) a change in the number of kit-independent LM melanophores that arise already in stripes and are numerous in D. rerio, but few in D. albolineatus. Our results show how genetic analyses of a species closely related to a biomedical model organism can reveal both conservatism and innovation in developmental mechanisms underlying evolutionary changes in adult form.     

Evolutionary diversification of pigment pattern in Danio fishes: differential fms dependence and stripe loss in D. albolineatus

The developmental bases for species differences in adult phenotypes remain largely unknown. An emerging system for studying such variation is the adult pigment pattern expressed by Danio fishes. These patterns result from several classes of pigment cells including black melanophores and yellow xanthophores, which differentiate during metamorphosis from latent stem cells of presumptive neural crest origin. In the zebrafish D. rerio, alternating light and dark horizontal stripes develop, in part, owing to interactions between melanophores and cells of the xanthophore lineage that depend on the fms receptor tyrosine kinase; zebrafish fms mutants lack xanthophores and have disrupted melanophore stripes. By contrast, the closely related species D. albolineatus exhibits a uniform pattern of melanophores, and previous interspecific complementation tests identified fms as a potential contributor to this difference between species. Here, we survey additional species and demonstrate marked variation in the fms-dependence of hybrid pigment patterns, suggesting interspecific variation in the fms pathway or fms requirements during pigment pattern formation. We next examine the cellular bases for the evolutionary loss of stripes in D. albolineatus and test the simplest model to explain this transformation, a loss of fms activity in D. albolineatus relative to D. rerio. Within D. albolineatus, we demonstrate increased rates of melanophore death and decreased melanophore migration, different from wild-type D. rerio but similar to fms mutant D. rerio. Yet, we also find persistent fms expression in D. albolineatus and enhanced xanthophore development compared with wild-type D. rerio, and in stark contrast to fms mutant D. rerio. These findings exclude the simplest model in which stripe loss in D. albolineatus results from a loss of fms-dependent xanthophores and their interactions with melanophores. Rather, our results suggest an alternative model in which evolutionary changes in pigment cell interactions themselves have contributed to stripe loss, and we test this model by manipulating melanophore numbers in interspecific hybrids. Together, these data suggest evolutionary changes in the fms pathway or fms requirements, and identify changes in cellular interactions as a likely mechanism of evolutionary change in Danio pigment patterns.     

Developmental morphology of the cyprinid fish Tanichthys albonubes

Embryonic, larval, and juvenile development of a small cyprinid species, Tanichthys albonubes, is described from laboratory-reared specimens. The eggs, measuring 1.0–1.2 mm in diameter, were demersal, almost spherical in shape, transparent and unpigmented, with a pale yolk without oil globules. Hatching occurred 45–53 h after fertilization at 25.5°–26.9°C. The newly hatched larvae, measuring 2.2–2.6 mm in body length (BL), had melanophores on the head and body. In particular, a dark vertical streak occurring posterior to the otic capsule and melanophores above the eyes were distinctive. The yolk was completely absorbed at 3.4 mm BL. Notochord flexion was initiated at 5.0 mm BL and finished at 6.0 mm BL. Aggregate numbers of all fin rays were completed at 11 mm BL. Squamation was initiated at 8.4 mm BL and completed at 13 mm BL. Although the eggs of T. albonubes resembled those of other small danionin species, including Aphyocypris chinensis, Chela dadiburjori, Danio rerio, Devario malabaricus, Gobiocypris rarus, Hemigrammocypris rasborella, and Horadandia atukorali, they differed from those of A. chinensis, C. dadiburjori, G. rarus, and Horadandia atukorali in having a wider perivitelline space. The larvae and juveniles of T. albonubes were similar to those of the aforementioned seven species plus Danio albolineatus, Danio kerri, and Devario sp. (cf. D. aequipinnatus) in general morphology. However, the early life stage morphology of T. albonubes differed from them in having a dark vertical streak posterior to the otic capsule and melanophores above the eyes in the yolk sac larval stage, and a dark lateral streak with an unpigmented area just above the former on the body, a dark blotch on the caudal fin, and reddish dorsal, anal, and caudal fins during the postflexion larval and juvenile stages.     

Pigment pattern evolution by differential deployment of neural crest and post-embryonic melanophore lineages in Danio fishes

Latent precursors or stem cells of neural crest origin are present in a variety of post-embryonic tissues. Although these cells are of biomedical interest for roles in human health and disease, their potential evolutionary significance has been underappreciated. As a first step towards elucidating the contributions of such cells to the evolution of vertebrate form, we investigated the relative roles of neural crest cells and post-embryonic latent precursors during the evolutionary diversification of adult pigment patterns in Danio fishes. These pigment patterns result from the numbers and arrangements of embryonic melanophores that are derived from embryonic neural crest cells, as well as from post-embryonic metamorphic melanophores that are derived from latent precursors of presumptive neural crest origin. In the zebrafish D. rerio, a pattern of melanophore stripes arises during the larval-to-adult transformation by the recruitment of metamorphic melanophores from latent precursors. Using a comparative approach in the context of new phylogenetic data, we show that adult pigment patterns in five additional species also arise from metamorphic melanophores, identifying this as an ancestral mode of adult pigment pattern development. By contrast, superficially similar adult stripes of D. nigrofasciatus (a sister species to D. rerio) arise by the reorganization of melanophores that differentiated at embryonic stages, with a diminished contribution from metamorphic melanophores. Genetic mosaic and molecular marker analyses reveal evolutionary changes that are extrinsic to D. nigrofasciatus melanophore lineages, including a dramatic reduction of metamorphic melanophore precursors. Finally, interspecific complementation tests identify a candidate genetic pathway for contributing to the evolutionary reduction in metamorphic melanophores and the increased contribution of early larval melanophores to D. nigrofasciatus adult pigment pattern development. These results demonstrate an important role for latent precursors in the diversification of pigment patterns across danios. More generally, differences in the deployment of post-embryonic neural crest-derived stem cells or their specified progeny may contribute substantially to the evolutionary diversification of adult form in vertebrates, particularly in species that undergo a metamorphosis.     

The Role of Early Learning in Determining Shoaling Preferences Based on Visual Cues in the Zebrafish, Danio rerio

Social preferences for specific colour patterns learned in early development, termed imprinting, may influence social and mating decisions. Imprinting has been demonstrated in both birds and fish and has been proposed to play a role in speciation. We raised wild‐type zebrafish, Danio rerio, either with individuals of the same colour pattern, an alternative colour pattern (leopard), or a closely related, unpatterned species, D. albolineatus. We also cross‐reared individual ‘leopard’D. rerio and D. albolineatus with wild‐type D. rerio. We tested the prediction that Danio would prefer to shoal with fish of the colour pattern with which they had been raised, irrespective of their own appearance. Rearing condition affected shoaling preferences between D. rerio and D. albolineatus, with individuals of both colour patterns preferring to associate with shoals of the colour pattern with which they had been raised. The more subtle distinction between the wild‐type and ‘leopard’D. rerio colour patterns did not elicit a shoaling preference. Thus, zebrafish exhibit shoaling preferences based on visual cues, the effect being stronger when patterns are more distinct. There is a strong learned component to these preferences, although the extent to which they may influence mating decisions is unclear.     

Zebrafish puma mutant decouples pigment pattern and somatic metamorphosis

The genetic and developmental bases for trait expression and variation in adults are largely unknown. One system in which genes and cell behaviors underlying adult traits can be elucidated is the larval-to-adult transformation of zebrafish, Danio rerio. Metamorphosis in this and many other teleost fishes resembles amphibian metamorphosis, as a variety of larval traits (e.g., fins, skin, digestive tract, sensory systems) are remodeled in a coordinated manner to generate the adult form. Among these traits is the pigment pattern, which comprises several neural crest-derived pigment cell classes, including black melanophores, yellow xanthophores, and iridescent iridophores. D. rerio embryos and early larvae exhibit a relatively simple pattern of melanophore stripes, but this pattern is transformed during metamorphosis into the more complex pattern of the adult, consisting of alternating dark (melanophore, iridophore) and light (xanthophore, iridophore) horizontal stripes. While it is clear that some pigment cells differentiate de novo during pigment pattern metamorphosis, the extent to which larval and adult pigment patterns are developmentally independent has not been known. In this study, we show that a subset of embryonic/early larval melanophores persists into adult stages in wild-type fish; thus, larval and adult pigment patterns are not completely independent in this species. We also analyze puma mutant zebrafish, derived from a forward genetic screen to isolate mutations affecting postembryonic development. In puma mutants, a wild-type embryonic/early larval pigment pattern forms, but supernumerary early larval melanophores persist in ectopic locations through juvenile and adult stages. We then show that, although puma mutants undergo a somatic metamorphosis at the same time as wild-type fish, metamorphic melanophores that normally appear during these stages are absent. The puma mutation thus decouples metamorphosis of the pigment pattern from the metamorphosis of many other traits. Nevertheless, puma mutants ultimately recover large numbers of melanophores and exhibit extensive pattern regulation during juvenile development, when the wild-type pigment pattern already would be completed. Finally, we demonstrate that the puma mutant is both temperature-sensitive and growth-sensitive: extremely severe pigment pattern defects result at a high temperature, a high growth rate, or both; whereas a wild-type pigment pattern can be rescued at a low temperature and a low growth rate. Taken together, these results provide new insights into zebrafish pigment pattern metamorphosis and the capacity for pattern regulation when normal patterning mechanisms go awry.     

Zebrafish hybrids suggest genetic mechanisms for pigment pattern diversification in Danio

Pigment patterns of Danio fishes are a tractable system for assessing the developmental genetic bases for the evolution of adult form in vertebrates. These pigment patterns include multiple horizontal melanophore stripes in the zebrafish D. rerio, a complete absence of stripes in D. albolineatus, a few broad stripes in D. kerri, and a combination of stripes and spots in D. nigrofasciatus. Here we assess the genetics of pigment pattern development and evolution using interspecific hybrids. We first reconstruct the phylogenetic relationships of these species by analyzing mitochondrial 12S and 16S rDNA sequences. We find a clade comprising several small species of danio, and within this clade a sister taxon relationship between D. rerio and D. nigrofasciatus. We also find that the large bodied D. dangila is more closely related to the clade of small danios than other large bodied species. As a first step in evaluating the genetics of pigment pattern diversification in the group, we then examine the phenotypes of interspecific hybrids. Adult pigment patterns of hybrids between D. rerio and other danios are in many respects more similar to D. rerio than the heterospecific danio, demonstrating that alleles of pigment pattern genes in other species typically are recessive to D. rerio alleles. Furthermore, hybrids between two additional striped species (D. kerri, D. nigrofasciatus) and D. albolineatus suggest that striped patterns are dominant or semi-dominant over an absence of stripes. Together, these analyses support a model in which pigment pattern differences between D. rerio and other species result from gain-of-function alleles in D. rerio, or loss-of-function alleles in other danios. Finally, because several D. rerio pigment pattern mutants resemble heterospecific danios, we use interspecific complementation tests to assess potential roles for these loci in pigment pattern diversification. Crosses between other danios and most D. rerio pigment pattern mutants develop stripes, similar to control hybrids with wild-type D. rerio. These complementation phenotypes allow us to exclude most of these loci as having major effect roles in generating pigment pattern differences between species. In contrast, hybrids between fms mutant D. rerio and D. albolineatus fail to develop stripes, similar to D. albolineatus. This non-complementation phenotype identifies changes in fms, or the pathway in which it acts, as candidates for contributing to the evolutionary loss of stripes in D. albolineatus.     

Developmental morphology of the cyprinid fish Inlecypris auropurpureus

Embryonic, larval, and juvenile development of a Myanmarese cyprinid fish, Inlecypris auropurpureus, is described from laboratory-reared specimens. The eggs, measuring 0.9–1.0 mm in diameter, were demersal, almost spherical in shape, transparent and unpigmented, with a pale yellow yolk without oil globules. Hatching occurred 49–56 h after fertilization at 26.2°–27.3°C. The newly hatched larvae, measuring 2.9–3.1 mm in body length (BL) with 17 + 19–20 = 36–37 myomeres, had melanophores on the head and body. A cement organ on the forehead for adhering to objects during the yolk sac and early preflexion larval stages was distinctive. The yolk was completely absorbed at 3.6–4.0 mm BL. Notochord flexion was initiated at 5.1–5.6 mm BL and finished at 7.1 mm BL. Aggregate numbers of all fin rays were completed at 14 mm BL. Squamation was initiated midlaterally on the anterior trunk at 14 mm BL and completed at 27 mm BL. Although the eggs of I. auropurpureus resembled those of the closely related species Chela dadiburjori, Danio rerio, and Devario malabaricus, they differed from those of Danio rerio and Devario malabaricus in having a narrower perivitelline space. The larvae and juveniles of I. auropurpureus were also similar to those of C. dadiburjori, Danio rerio, and Devario malabaricus in general morphology, but they differed from the latter three species in having a series of dark blotches laterally on the body in the juvenile stage. Moreover, I. auropurpureus differed from C. dadiburjori in having more myomeres and a near-single row of melanophores on the body along the dorsal midline from the yolk-sac to early postflexion larval stages, from Danio rerio in having a cement organ on the forehead during the yolk-sac and early preflexion larvae, and a single melanophore on the lower eye margin in the early yolk-sac larvae, and from Devario malabaricus in having a single melanophore on the lower eye margin in the early yolk-sac larvae. The presence of a cement organ on the forehead indicates a close relationship among the genera Inlecypris, Chela, and Devario.     

Temporal and cellular requirements for Fms signaling during zebrafish adult pigment pattern development

Ectothermic vertebrates exhibit a diverse array of adult pigment patterns. A common element of these patterns is alternating dark and light stripes each comprising different classes of neural crest-derived pigment cells. In the zebrafish, Danio rerio, alternating horizontal stripes of black melanophores and yellow xanthophores are a prominent feature of the adult pigment pattern. In fms mutant zebrafish, however, xanthophores fail to develop and melanophore stripes are severely disrupted. fms encodes a type III receptor tyrosine kinase expressed by xanthophores and their precursors and is the closest known homologue of kit, which has long been studied for roles in pigment pattern development in amniotes. In this study we assess the cellular and temporal requirements for Fms activity in promoting adult pigment pattern development. By transplanting cells between fms mutants and either wild-type or nacre mutant zebrafish, we show that fms acts autonomously to the xanthophore lineage in promoting the striped arrangement of adult melanophores. To identify critical periods for fms activity, we isolated temperature sensitive alleles of fms and performed reciprocal temperature shift experiments at a range of stages from embryo to adult. These analyses demonstrate that Fms is essential for maintaining cells of the xanthophore lineage as well as maintaining the organization of melanophore stripes throughout development. Finally, we show that restoring Fms activity even at late larval stages allows essentially complete recovery of xanthophores and the development of a normal melanophore stripe pattern. Our findings suggest that fms is not required for establishing a population of precursor cells during embryogenesis but is required for recruiting pigment cell precursors to xanthophore fates, with concomitant effects on melanophore organization.     

Homology and evolutionary novelty in the deployment of extracellular matrix molecules during pigment pattern formation in the salamanders Taricha torosa and T. rivularis (Salamandridae)

Salamander larvae exhibit a diverse array of pigment patterns shortly after hatching. Previous studies have identified roles for the extracellular matrix and lateral line sensory system in promoting the development of a phylogenetically common pattern of horizontal melanophore stripes. In contrast, salamanders in the genus Taricha exhibit evolutionarily derived pigment patterns and pattern-forming mechanisms. Taricha torosa larvae exhibit compact melanophore stripes that develop via redundant, lateral line-independent mechanisms, whereas T. rivularis larvae lack stripes and instead have melanophores uniformly distributed over the flank. In this study, I test roles for candidate patterning molecules of the extracellular matrix in promoting the development of species-specific pigment patterns in Taricha. I show that tenascin deposition is negatively correlated with melanophore distributions both intraspecifically and interspecifically: this matrix molecule is present where melanophores do not localize in T. torosa and is absent from these same regions where melanophores are abundant in T. rivularis. Embryological manipulations further indicate that transient expression of tenascin in a prospective interstripe region of T. torosa reflects a phylogenetically conserved effect of lateral line development. Finally, anti-laminin immunoreactivity is negatively correlated with melanophore distributions in T. torosa, and this species exhibits a general retardation of extracellular matrix development that may allow persistent, evolutionarily novel melanophore motility in this species. Together these findings identify tenascin and laminin, or molecules co-regulated with these matrix components, as candidates for promoting early larval pigment pattern development in Taricha.     

Expression patterns of the creatine metabolism‐related molecules AGAT,GAMT and CT1 in adult zebrafish Danio rerio

AGAT, GAMT and CT1, three creatine synthesis and transport‐related molecules, have been widely studied in mammals. To explore their homologous genes in adult zebrafish Danio rerio, the gene expression patterns of these three genes in D. rerio were investigated. The results reveal that AGAT, GAMT and CT1 are expressed widely in diverse tissues of D. rerio where the homologous genes in mammals are also expressed.     

Cytophysiological basis of disruptive pigmentary patterns in the leopard frog Rana pipiens. I. Chromatophore densities and cytophysiology

Statistical analyses of chromatophore densities and morphological states in wild type, burnsi, and kandiyohi: adult Rana pipiens integument yielded the conclusion that disruptive pigmentary pattern is permanently defined by regional chromatophore densities. Spatial chromatophore patterning is enhanced and rendered more visible by the morphological and physiological differences among chromatophores within the various pattern regions. Specifically, (1) chromatophores were differentialy distributed among pattern regions, (2) greater complexity of gross disruptive patterning had greater underlying melanophores densities, (3) there were significant density differences among non-sib individual animals, among fertilization, and among shipment batches, but not among full-sibs, and (4)kandiyohi individuals had more while burnsi individuals had fewer total melanophores than wild type. A stellate morphology was significantly correlated with high melanophore density, although the relationship of melanophore density to melanophore morphology differed among pattern regions and among genotypes. The functional interrelationships of density and morphology, and their role in disruptive patterning and coloration, are discussed.     

Pigment pattern formation in larval ambystomatid salamanders: Ambystoma tigrinum tigrinum

We have begun a comparative study of pigment patterns and their mechanisms of formation in ambystomatid salamanders in an attempt to elucidate the evolution of these traits in this family. In Ambystoma t. tigrinum, the migration of the prospective pigment cells was followed by using scanning electron microscopy and light microscopy combined with markers (dopa incubation for detecting melanophores, ammonia-induced pterin fluorescence for detecting xanthophores). The pigment pattern resulting from the cell migration shares features both with the alternating vertical xanthophore and melanophore bars of A. mexicanum and the horizontal stripes of certain salamandrids and ambystomatids. The pigment pattern of A. t. tigrinum is interpreted here as an intermediate evolutionary step between a primitive horizontal stripe pattern and a derived vertical bar pattern. The initiation of pigment pattern formation resembles the situation in A. mexicanum, probably reflecting the close phylogenetic relationship between the two taxa.     

Alarm substance from adult zebrafish alters early embryonic development in offspring

Alarm substances elicit behavioural responses in a wide range of animals but effects on early embryonic development are virtually unknown. Here we investigated whether skin injury-induced alarm substances caused physiological responses in embryos produced by two Danio species (Danio rerio and Danio albolineatus). Both species showed more rapid physiological development in the presence of alarm substance, although there were subtle differences between them: D. rerio had advanced muscle contraction and heart function, whereas D. albolineatus had advanced heart function only. Hence, alarm cues from injured or dying fish may be of benefit to their offspring, inducing physiological responses and potentially increasing their inclusive fitness.     

oca2 regulation of chromatophore differentiation and number is cell type specific in zebrafish

We characterized a zebrafish mutant that displays defects in melanin synthesis and in the differentiation of melanophores and iridophores of the skin and retinal pigment epithelium. Positional cloning and candidate gene sequencing link this mutation to a 410‐kb region on chromosome 6, containing the oculocutaneous albinism 2 (oca2) gene. Quantification of oca2 mutant melanophores shows a reduction in the number of differentiated melanophores compared with wildtype siblings. Consistent with the analysis of mouse Oca2‐deficient melanocytes, zebrafish mutant melanophores have immature melanosomes which are partially rescued following treatment with vacuolar‐type ATPase inhibitor/cytoplasmic pH modifier, bafilomycin A1. Melanophore‐specific gene expression is detected at the correct time and in anticipated locations. While oca2 zebrafish display unpigmented gaps on the head region of mutants 3 days post‐fertilization, melanoblast quantification indicates that oca2 mutants have the correct number of melanoblasts, suggesting a differentiation defect explains the reduced melanophore number. Unlike melanophores, which are reduced in number in oca2 mutants, differentiated iridophores are present at significantly higher numbers. These data suggest distinct mechanisms for oca2 in establishing differentiated chromatophore number in developing zebrafish.     

Pigment pattern formation in larval ambystomatid salamanders: Ambystoma talpoideum,Ambystoma barbouri,and Ambystoma annulatum

The pigment pattern formation in embryos and larvae of three ambystomatid salamanders was investigated in an evolutionary context. Early neural crest development was studied with scanning electron microscopy. Pigment cell migration and pattern formation were investigated at the light microscopy level with markers that labelled the two pigment cell types specifically before they were fully differentiated. In all three species, the pigment pattern formation started when xanthophores that had first formed aggregates in the crest migrated ventrally. As previously observed in other species, vertical bars always form by a mechanism involving earlier onset of migration in melanophores than in xanthophores and aggregate formation in the crest. In Ambystoma talpoideum and A. annulatum, a pattern of vertical chromatophore bars formed, which was superimposed on a pattern of horizontal stripes. In Ambystoma barbouri, the tendency to form this pattern was obscured by the high density of melanophores. It is suggested that variation among species may be due to differences in the chromatophore density and in the melanophore/xanthophore ratio. Mapping of the evolution of vertical bars onto existing phylogenies for the group was confounded by controversies about how to interpret the phylogenetic data. On the phylogeny that takes all the available evidence into account, there are two equally parsimonious mappings. Vertical bars have either evolved only once and been lost twice, or evolved twice and been lost once. This rather conservative pattern can be explained both as an effect of stabilizing selection and as a result of developmental constraints. © 1994 Wiley-Liss, Inc.     

Pigment pattern formation in the larval salamander Ambystoma maculatum

As part of an ongoing comparative study of pigment patterns and their formation in embryos and larvae of ambystomatid salamanders, Ambystoma maculatum from two differnt populations, one in the northern (New York) and one in the central (Tennessee) United States, were investigated. Scanning electron microscopy was used to study early neural crest development. Light microscopy in combination with markers for the two pigment cell types (xanthophores and melanophores) made it possible to follow pigment cell migration before the pigment cells were fully differentiated. A bilateral pigment pattern consisting of two horizontal melanophore stripes surrounding an interstripe area populated by xanthophores formed in the larvae. In both populations, some variation was present in the form of a continuum ranging from clear horizontal stripes to extreme cases with a random pattern. Unlike the other ambystomatids that have been investigated, the neural crest cells in A. maculatum do not form aggregates and no vertical bars are formed. Instead, both the pattern and its formation are very similar to what has been reported for salamandrids. If pattern formation mechanisms can act as developmental constraints we would expect the A. maculatum pattern to be the primitive condition in the Ambystomatidae, using the Salamandridae as the outgroup. There is no strong support for this when aggregate formation is used as a character and mapped onto phylogenies for the group. The aggregate formation mechanism, and the pigment pattern that it leads to, have most likely been secondarily lost in A. maculatum. © 1993 Wiley-Liss, Inc.     

Formation of the adult pigment pattern in zebrafish requires leopard and obelix dependent cell interactions

Colour patterns are a prominent feature of many animals and are of high evolutionary relevance. In zebrafish, the adult pigment pattern comprises alternating stripes of two pigment cell types, melanophores and xanthophores. How the stripes are defined and a straight boundary is formed remains elusive. We find that mutants lacking one pigment cell type lack a striped pattern. Instead, cells of one type form characteristic patterns by homotypic interactions. Using mosaic analysis, we show that juxtaposition of melanophores and xanthophores suffices to restore stripe formation locally. Based on this, we have analysed the pigment pattern of two adult specific mutants: leopard and obelix. We demonstrate that obelix is required in melanophores to promote their aggregation and controls boundary integrity. By contrast, leopard regulates homotypic interaction within both melanophores and xanthophores, and interaction between the two, thus controlling boundary shape. These findings support a view in which cell-cell interactions among pigment cells are the major driving force for adult pigment pattern formation.     

Histology of melanic flank and opercular color pattern elements in the firemouth cichlid,Thorichthys meeki

Dark melanic color pattern elements, such as bars, stripes, and spots, are common in the skin of fishes, and result from the differential distribution and activity of melanin‐containing chromatophores (melanophores). We determined the histological basis of two melanic color pattern elements in the integument of the Firemouth Cichlid, Thorichthys meeki. Vertical bars on the flanks were formed by three layers of dermal melanophores, whereas opercular spots were formed by four layers (two lateral and two medial) in the integument surrounding the opercular bones. Pretreatment of opercular tissue with potassium and sodium salts effectively concentrated or dispersed intracellular melanosomes. Regional differences in epidermal structure, scale distribution, and connective tissues were also identified. J. Morphol. 2013. © 2013 Wiley Periodicals, Inc.     

Developmental morphology of the cyprinid fish Horadandia atukorali

Embryonic, larval, and juvenile development of a small Indian cyprinid, Horadandia atukorali, is described from laboratory-reared specimens. The eggs, measuring 0.7–0.8mm in diameter, were demersal, almost spherical in shape, transparent and unpigmented, with a pale yellow yolk without oil globules. Hatching occurred 47–54h after fertilization at 26.3–27.5°C. The newly hatched larvae, measuring 2.3–2.6mm in body length (BL) with 16+13=29 myomeres, had no melanophores, except on the eye, a single melanophore occurring on the lower margin, and xanthophores surrounding the pupil. The yolk was completely absorbed at 3.0mm BL. Notochord flexion was initiated at 4.0mm BL and finished at 4.4mm BL. Aggregate numbers of all fin rays were completed at 8.0mm BL. Squamation was initiated at 6.4mm BL and completed at 9.5mm BL. Although the eggs of Horadandia atukorali resembled those of other small danionin species, including Aphyocypris chinensis, Chela dadiburjori, Danio rerio, Devario malabaricus, and Hemigrammocypris rasborella, they differed from those of Danio rerio and Devario malabaricus in having a narrower perivitelline space. The larvae and juveniles of Horadandia atukorali were also similar to those of the latter five species in general morphology, especially in the presence of a melanophore on the lower margin of the eye at hatching, as in C. dadiburjori. However, the early life stage morphology of Horadandia atukorali differed from the other danionin species in having a conical yolk sac at hatching, no cement organ on the forehead in the yolk-sac larval stage, a divided gas bladder in the flexion larval stage, two dark lateral streaks on the head and chevron-like melanophores on the ventral body surface from the preflexion to postflexion larval stages, and xanthophores on the eyes at hatching.