Working with zebrafish at postembryonic stages.

As the processes of embryogenesis become increasingly well understood, there is growing interest in the development that occurs at later, postembryonic stages. Postembryonic development holds tremendous potential for discoveries of both fundamental and translational importance. Zebrafish, which are small, rapidly and externally developing, and which boast a wealth of genetic resources, are an outstanding model of vertebrate postembryonic development. Nonetheless, there are specific challenges posed by working with zebrafish at these stages, and this chapter is meant to serve as a primer for those working with larval and juvenile zebrafish. Since accurate staging is critical for high-quality results and experimental reproducibility, we outline best practices for reporting postembryonic developmental progress. Emphasizing the importance of accurate staging, we present new data showing that rates of growth and size-stage relationships can differ even between wild-type strains. Finally, since rapid and uniform development is particularly critical when working at postembryonic stages, we briefly describe methods that we use to achieve high rates of growth and developmental uniformity through postembryonic stages in both wild-type and growth-compromised zebrafish.

Evolution of danio pigment pattern development.

Pigment patterns of danio fishes are emerging as a useful system for studying the evolution of developmental mechanisms underlying adult form. Different closely related species within the genera Danio and Devario exhibit a range of pigment patterns including horizontal stripes, vertical bars, and others. In this review, I summarize recent work identifying the genetic and cellular bases for adult pigment pattern formation in the zebrafish Danio rerio, as well as studies of how these mechanisms have evolved in other danios. Together, these analyses highlight the importance of latent precursors at post-embrynoic stages, as well as interactions within and among pigment cell classes, for both pigment pattern development and evolution.

A comprehensive expressed sequence tag linkage map for tiger salamander and Mexican axolotl: enabling gene mapping and comparative genomics in Ambystoma.

Expressed sequence tag (EST) markers were developed for Ambystoma tigrinum tigrinum (Eastern tiger salamander) and for A. mexicanum (Mexican axolotl) to generate the first comprehensive linkage map for these model amphibians. We identified 14 large linkage groups (125.5-836.7 cM) that presumably correspond to the 14 haploid chromosomes in the Ambystoma genome. The extent of genome coverage for these linkage groups is apparently high because the total map size (5251 cM) falls within the range of theoretical estimates and is consistent with independent empirical estimates. Unlike most vertebrate species, linkage map size in Ambystoma is not strongly correlated with chromosome arm number. Presumably, the large physical genome size ( approximately 30 Gbp) is a major determinant of map size in Ambystoma. To demonstrate the utility of this resource, we mapped the position of two historically significant A. mexicanum mutants, white and melanoid, and also met, a quantitative trait locus (QTL) that contributes to variation in metamorphic timing. This new collection of EST-based PCR markers will better enable the Ambystoma system by facilitating development of new molecular probes, and the linkage map will allow comparative studies of this important vertebrate group.

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.

Conserved vertebrate chromosome segments in the large salamander genome.

Urodele amphibians (salamanders) are important models for embryological, physiological, and natural history research and are also a biomedically important group because they are the only vertebrates capable of regenerating entire organ systems. To enhance the utility of salamanders for biomedical research and for understanding genome evolution, genetic linkage analysis was used to identify chromosome segments that are homologous between ambystomatid salamanders and distantly related vertebrate model organisms. A total of 347 loci (AFLPs, RAPDs, and protein-coding loci) were mapped using an interspecific meiotic mapping panel (Ambystoma mexicanum and A. tigrinum tigrinum; family Ambystomatidae). Genome size in Ambystoma was estimated to be 7291 cM, the largest linkage map estimate reported for any organism. However, the relatively large size of the salamander genome did not hinder efforts to map and identify conserved syntenies from a small sample of 24 protein-coding loci. Chromosomal segments that are conserved between fishes and mammals are also conserved in these salamanders. Thus, comparative gene mapping appears to be an efficient strategy for identifying orthologous loci between ambystomatid salamanders and genomically well-characterized vertebrate model organisms.

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.

Mutational analysis of endothelin receptor b1 (rose) during neural crest and pigment pattern development in the zebrafish Danio rerio.

Pigment patterns of fishes are a tractable system for studying the genetic and cellular bases for postembryonic phenotypes. In the zebrafish Danio rerio, neural crest-derived pigment cells generate different pigment patterns during different phases of the life cycle. Whereas early larvae exhibit simple stripes of melanocytes and silver iridophores in a background of yellow xanthophores, this pigment pattern is transformed at metamorphosis into that of the adult, comprising a series of dark melanocyte and iridophore stripes, alternating with light stripes of iridophores and xanthophores. Although several genes have been identified in D. rerio that contribute to the development of both early larval and adult pigment patterns, comparatively little is known about genes that are essential for pattern formation during just one or the other life cycle phase. In this study, we identify the gene responsible for the rose mutant phenotype in D. rerio. rose mutants have wild-type early larval pigment patterns, but fail to develop normal numbers of melanocytes and iridophores during pigment pattern metamorphosis and exhibit a disrupted pattern of these cells. We show that rose corresponds to endothelin receptor b1 (ednrb1), an orthologue of amniote Ednrb genes that have long been studied for their roles in neural crest and pigment cell development. Furthermore, we demonstrate that D. rerio ednrb1 is expressed both during pigment pattern metamorphosis and during embryogenesis, and cells of melanocyte, iridophore, and xanthophore lineages all express this gene. These analyses suggest a phylogenetic conservation of roles for Ednrb signaling in the development of amniote and teleost pigment cell precursors. As murine Ednrb is essential for the development of all neural crest derived melanocytes, and D. rerio ednrb1 is required only by a subset of adult melanocytes and iridophores, these analyses also reveal variation among vertebrates in the cellular requirements for Ednrb signaling, and suggest alternative models for the cellular and genetic bases of pigment pattern metamorphosis in D. rerio.

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.

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 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.

Zebrafish sparse corresponds to an orthologue of c-kit and is required for the morphogenesis of a subpopulation of melanocytes, but is not essential for hematopoiesis or primordial germ cell development.

The relative roles of the Kit receptor in promoting the migration and survival of amniote melanocytes are unresolved. We show that, in the zebrafish, Danio rerio, the pigment pattern mutation sparse corresponds to an orthologue of c-kit. This finding allows us to further elucidate morphogenetic roles for this c-kit-related gene in melanocyte morphogenesis. Our analyses of zebrafish melanocyte development demonstrate that the c-kit orthologue identified in this study is required both for normal migration and for survival of embryonic melanocytes. We also find that, in contrast to mouse, the zebrafish c-kit gene that we have identified is not essential for hematopoiesis or primordial germ cell development. These unexpected differences may reflect evolutionary divergence in c-kit functions following gene duplication events in teleosts.

Genetic analysis of steel and the PG-M/versican-encoding gene AxPG as candidates for the white (d) pigmentation mutant in the salamander Ambystoma mexicanum.

Vertebrate non-retinal pigment cells are derived from neural crest (NC) cells, and several mutations have been identified in the Mexican axolotl Ambystoma mexicanum (Ambystomatidae) that affect the development of these cell lineages. In "white" (d) mutant axolotls, premigratory NC cells differentiate as pigment cells, yet fail to disperse, survive, or both, and this leads to a nearly complete absence of pigment cells in the skin. Previous studies revealed that d affects pigment cell development non-autonomously, and have reported differences between white and wild-type axolotls in the structure and composition of the extracellular matrix through which NC and pigment cells migrate. Here we test the correspondence of d and two candidate genes: steel and AxPG. In amniotes, Steel encodes the cytokine Steel factor (mast cell growth factor; stem cell factor; kit ligand), which is expressed along the migratory pathways of melanocyte precursors and is required by these cells for their migration and survival; mammalian Steel mutants resemble white mutant axolotls in having a deficit or complete absence of pigment cells. In contrast, AxPG encodes a PG-M/versican-like proteoglycan that may promote the migration of A. mexicanum pigment cells, and AxPG expression is reduced in white mutant axolotls. We cloned a salamander orthologue of steel and used a partial genetic linkage map of Ambystoma to determine the genomic locations of steel, AxPG, and d. We show that the three genes map to different linkage groups, excluding steel and AxPG as candidates for d.

Experimental analysis of character coupling across a complex life cycle: pigment pattern metamorphosis in the tiger salamander, Ambystoma tigrinum tigrinum.

Developmental relationships among characters are expected to bias patterns of morphological variation at the population level. Studies of character development thus can provide insights into processes of adaptation and the evolutionary diversification of morphologies. Here I use experimental manipulations to test whether larval and adult pigment patterns are coupled across metamorphosis in the tiger salamander, Ambystoma tigrinum tigrinum (Ambystomatidae). Previous investigations showed that the early larval pigment pattern depends on interactions between pigment cells and the lateral line sensory system. In contrast, the results of this study demonstrate that the major features of the adult pigment pattern develop largely independently of both the early larval pattern and the lateral lines. These results suggest that ontogenetic changes that occur across metamorphosis decouple larval and adult pigment patterns and could thereby facilitate independent evolutionary modifications to the patterns during different stages of the life cycle.

Salamander pigment patterns: how can they be used to study developmental mechanisms and their evolutionary transformation?

Neural crest cells of ectothermic vertebrates give rise to three types of pigment cells: melanophores, xanthophores, and iridophores. In early larval salamanders, these cells can combine to generate a variety of pigment patterns across taxa, including vertical bars and horizontal stripes. Such patterns offer an opportunity to study neural crest morphogenesis and differentiation, as well as the evolution of these processes and the morphologies that arise from them. This review examines the phylogenetic distribution of specific pigment patterns, our current understanding of the mechanisms underlying these patterns, and how evolutionary transformations of these mechanisms may have resulted in alternative pigment patterns across taxa.

Pigment patterns of larval salamanders (Ambystomatidae, Salamandridae): the role of the lateral line sensory system and the evolution of pattern-forming mechanisms.

In many species of salamanders, pigment cells derived from the neural crest give rise to a horizontal stripe pattern in hatchling larvae. A defining element of these horizontal stripe patterns is a region over the middle of the myotomes that is relatively free of melanophores. This study shows that formation of a "melanophore-free region" and horizontal stripe pattern in Ambystoma tigrinum tigrinum (family Ambystomatidae) correlates with the development of the trunk lateral line sensory system. Moreover, prevention of lateral line development results in greater densities of melanophores in the middle of the flank, essentially eliminating the melanophore-free region in this taxon. A phylogenetic survey also revealed that ablation of the lateral lines has qualitatively similar effects on melanophores in seven of eight additional taxa (Ambystomatidae: A. barbouri, A. maculatum, A. talpoideum; Salamandridae: Notophthalmus viridescens, Pleurodeles waltl, Taricha granulosa, T. rivularis). In Taricha torosa, however, a superficially similar melanophore-free region forms prior to lateral line development, and ablation of the lateral lines does not perturb the horizontal stripe pattern. Finally, heterospecific grafting experiments demonstrated that T. torosa lateral lines are competent to generate a melanophore-free region, and T. torosa melanophores are competent to respond to cues associated with the lateral lines. These results indicate that lateral line-dependent pattern-forming mechanisms are common and probably ancestral within the families Ambystomatidae and Salamandridae and suggest that these ancestral mechanisms have been retained in T. torosa as redundant, lateral line-dependent mechanisms for stripe formation have evolved.

When neural crest and placodes collide: interactions between melanophores and the lateral lines that generate stripes in the salamander Ambystoma tigrinum tigrinum (Ambystomatidae).

A prominent element of the early larval pigment pattern in the salamander Ambystoma tigrinum tigrinum (family Ambystomatidae) is a horizontal stripe over the lateral surface of the myotomes where otherwise abundant, neural crest-derived melanophores are not found. This study examines the formation of this "melanophore-free region". When the trunk lateral lines were ablated (by removing cranial lateral line placodes), the melanophore-free region did not form; instead, melanophores populated the middle of the flank and the distribution of yellow, neural chest-derived zanthophores was perturbed. Time-lapse videomicrography demonstrated that during normal development, the melanophore-free region is established because melanophores retreat from the midbody lateral line primordium as it migrates caudally along the inner side of the epidermis. Melanophores do not repopulate the middle of the flank after primordium migration and heterochronic grafting experiments suggest that extracellular factors contribute to maintaining the melanophore-free region during these later stages. Finally, photographic series, microsurgical manipulations, electron microscopy, and staining for molecules of the extracellular matrix (peanut agglutinin-binding components, tenascin, chondroitin sulfate proteoglycans, fibronectin, laminin) suggest that several factors contribute to establishing and maintaining the melanophore-free region, including steric effects of the lateral lines, interactions between melanophores and xanthophores, lateral line-dependent alterations of the subepidermal basement membrane, and a general elaboration of the extracellular matrix. Lateral line effects on melanophores are inferred to be a shared, ancestral feature of pigment pattern development for the families Ambystomatidae and Salamandridae (D.M. Parichy, Dev. Biol. 174, 265-282. 1996). The results of this study thus provide insights into a phylogenetically primitive mechanism for stripe formation, and a context for interpreting evolutionary innovations in pattern-forming mechanisms.