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 fmsmutants 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 fmsrequirements 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 fmsactivity 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. albolineatusresults 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.

An outstanding challenge for developmental biology is to elucidate the mechanisms underlying adult form, and how changes in these mechanisms produce variation within and between species. Over the last few years, substantial progress has been made in identifying genes associated with major morphological differences (Galant and Carroll, 2002; Tanaka et al.,2002; Shapiro et al.,2004). Nevertheless, determining how genetic differences are translated into morphological differences will require a thorough understanding of how cellular behaviors are altered in different genetic backgrounds. Of particular interest are the mechanisms underlying variation in traits that have relevance to human health and disease, clear adaptive significance in nature, or both.

One useful system for studying the genetic and cellular bases for variation in adult form is the pigment pattern expressed by Danio fishes(Parichy, 2003; Kelsh, 2004; Quigley et al., 2004). These patterns differ dramatically across species, and include horizontal stripes,vertical bars, spots, and uniform patterns resulting from the arrangements of several classes of pigment cells, including black melanophores, yellow-orange xanthophores and reflective iridophores. Pigment cells in teleosts and other vertebrates are derived from neural crest cells, which also contribute to neurons and glia of the peripheral nervous system, bone and cartilage of the craniofacial skeleton, adrenal chromaffin cells, endocardial cushion cells,and other tissues (Hörstadius,1950; Smith et al.,1994; Le Douarin,1999). Neural crest-derived lineages are associated with a variety of human disease syndromes (Matthay,1997; Amiel and Lyonnet,2001; Ahlgren et al.,2002; Widlund and Fisher,2003; Farlie et al.,2004) and have had major roles in the diversification of vertebrates (Gans and Northcutt,1983; Hall, 1999). Besides serving as a potential model for development and evolution of other neural crest-derived traits, pigment patterns are especially interesting because of their ecological and behavioral significance, with teleost pigment patterns having roles in shoaling, mate recognition, mate choice and predator avoidance (Endler, 1983; Houde, 1997; Couldridge and Alexander,2002; Allender et al.,2003; Engeszer et al.,2004).

One approach to identifying the genetic and cellular bases for pigment pattern diversity in danios has used hybrids between zebrafish, D. rerio, and other danio species(Parichy and Johnson, 2001; Quigley et al., 2004). Wild-type D. rerio exhibit four to five melanophore stripes(Fig. 1A,D). When crossed with other danios, hybrid offspring develop pigment patterns that typically resemble D. rerio more closely than the heterospecific danio. This finding suggested that complementation tests could be used to screen loci identified as recessive D. rerio pigment pattern mutants for contributions to pigment pattern differences between species: mutants for which hybrids have pigment patterns different from controls identify genes that may differ between species and thus identify candidates for further analyses.

Fig. 1.

Adult stripe development and loss in danios. (A) Adult D. rerioexhibit dark stripes of melanophores and iridophores (with few xanthophores),alternating with light stripes of xanthophores and iridophores (with few melanophores). (B) Adult D. albolineatus lack distinctive melanophore stripes and exhibit only a weak interstripe region posteriorly. (C) fms mutant D. rerio exhibit disrupted stripes in which metamorphic melanophores are reduced and xanthophores are absent. (D) Detail of stripes and interstripes in D. rerio. Dark cells are melanophores and yellow-orange cells are xanthophores (arrow). Reflective iridophores are found throughout, but are organized differently in interstripe regions(arrowheads); under this illumination, iridophores outside of the interstripe region are evident only by their blue iridescence over some melanophores. (E)Detail of pigment pattern in D. albolineatus adult. Melanophores are widely distributed over the flank. Adults exhibit reddish erythrophores(arrow) distributed widely over the flank and interstripe iridophores(arrowheads) form only a narrow, irregular band over part of the flank. Erythrophores are present in several other danios as well, although not in D. rerio. Adult fish are 25-30 mm in length. Scale bars in D: 200μm for D,E.

Fig. 1.

Adult stripe development and loss in danios. (A) Adult D. rerioexhibit dark stripes of melanophores and iridophores (with few xanthophores),alternating with light stripes of xanthophores and iridophores (with few melanophores). (B) Adult D. albolineatus lack distinctive melanophore stripes and exhibit only a weak interstripe region posteriorly. (C) fms mutant D. rerio exhibit disrupted stripes in which metamorphic melanophores are reduced and xanthophores are absent. (D) Detail of stripes and interstripes in D. rerio. Dark cells are melanophores and yellow-orange cells are xanthophores (arrow). Reflective iridophores are found throughout, but are organized differently in interstripe regions(arrowheads); under this illumination, iridophores outside of the interstripe region are evident only by their blue iridescence over some melanophores. (E)Detail of pigment pattern in D. albolineatus adult. Melanophores are widely distributed over the flank. Adults exhibit reddish erythrophores(arrow) distributed widely over the flank and interstripe iridophores(arrowheads) form only a narrow, irregular band over part of the flank. Erythrophores are present in several other danios as well, although not in D. rerio. Adult fish are 25-30 mm in length. Scale bars in D: 200μm for D,E.

A previous study used interspecific hybrids to investigate the genetic bases for the evolutionary loss of stripes in D. albolineatus, in which pigment cells are nearly uniformly dispersed(Fig. 1B,E)(Parichy and Johnson, 2001). Hybrids between wild-type D. rerio and D. albolineatusdevelop stripes similar to D. rerio but unlike D. albolineatus. By contrast, one of ten mutant loci tested yielded a non-complementation phenotype in which hybrids lacked stripes like D. albolineatus. This locus was identified as fms, which encodes a type III receptor tyrosine kinase (Parichy et al., 2000b) known previously for roles in hematopoiesis and osteoclast development (Yoshida et al.,1990; Stanley et al.,1997; Dai et al.,2002; Scheijen and Griffin,2002; Barreda et al.,2004). Analyses of D. rerio fms mutants(Fig. 1C) demonstrate that fms promotes the development of a late-appearing population of adult melanophores that differentiates from latent stem cells during the larval-to-adult transformation, or metamorphosis(Parichy et al., 2000b). fms also is essential for melanophore survival and migration into stripes, although melanophores themselves do not detectably express fms. Rather, fms is expressed by cells of the xanthophore lineage and is essential for recruiting xanthophores from latent precursors. In turn, interactions between melanophores and fms-dependent cells of the xanthophore lineage are required for melanophore stripe formation(Parichy and Turner,2003a).

In this study, we test whether changes in fms or fms-dependent cell lineages underlie pigment pattern differences between D. rerio and D. albolineatus, as well as other danios (Parichy and Johnson,2001). We first identify additional species for which pigment patterns of hybrids depend on fms, and show that stripe loss in D. albolineatus hybrids depends on fms and other modifier loci. We next ask whether pigment pattern development in D. albolineatus resembles that of fms mutant D. rerio, as would be predicted by the simplest model in which a loss of fmsactivity has contributed to the evolutionary loss of stripes in D. albolineatus. We find that melanophore deficits and behaviors in D. albolineatus are similar to fms mutant D. rerio, yet D. albolineatus exhibit a dramatic increase - rather than a decrease- in xanthophore numbers. These findings reject the simplest model in which stripe loss in D. albolineatus depends on a loss of fmsactivity and a corresponding loss of the xanthophore lineage. Finally, we use interspecific hybrids to test an alternative model in which evolutionary changes in pigment cell interactions are responsible for stripe loss. Together these results identify interspecific variation in the fms pathway or cellular requirements for fms activity, and support a model in which evolutionary changes in pigment patterns depend in part on alterations in melanophore interactions.

Fish stocks and rearing conditions

Fish were reared at 28.5°C (14 hour light: 10 hour dark). Danio rerio were the inbred mapping strain ABut or a mixed genetic background comprising ABut, wikut, ekkwill and other stocks. Danio aff. albolineatus, D. choprae, D. aff. dangila, D. `hikari' and D. aff. kyathit were from Transship Discounts (Jamaica, NY, USA). The precise taxonomic status of the stocks is uncertain. Danio albolineatus were derived from stocks provided by M. McClure (Cornell University).

Complementation tests between D. rerio and heterospecific danios were performed as described (Parichy and Johnson, 2001). When mutant loci were mapped, we examined hybrid phenotypes in crosses segregating the mutant allele to control for allelic variation at other unlinked loci, and we genotyped hybrid offspring by PCR. Danio rerio mutants used for interspecific complementation tests have been described: sox10 (colourless)(Dutton et al., 2001); endothelin receptor b1 (ednrb1, roseb140)(Parichy et al., 2000a); tfap2a (lockjawts213)(Knight et al., 2003; Knight et al., 2004); mitfa (nacrew2)(Lister et al., 1999); jaguarc7 (Fisher et al., 1997); and pumaj115e1(Parichy and Turner, 2003b; Parichy et al., 2003). Additional mutants were derived from on-going mutagenesis screens (D.M.P.,unpublished).

Nomenclature for pigment pattern elements

Previous studies defined elements of the adult pigment pattern in D. rerio (Parichy and Johnson,2001; Parichy and Turner,2003b), including the first developing or `primary' melanophore stripes (1D, 1V) that develop dorsal and ventral to the horizontal myoseptum,as well as later-developing `secondary' dorsal and ventral melanophore stripes(2D, 2V). We refer to the xanthophore-rich areas between melanophore stripes as `interstripe' regions.

Genotyping

fms genotyping was accomplished by primer extension assays using conditions described (Parichy and Turner,2003a). A 100 bp product was amplified from genomic DNA using forward and reverse primers flanking the fmsj4e1 mutant lesion (fms-j1f, ACT CTT GGT GCT GGT GCG TTT G; fms-j1r, CTT TGA GCA TTT TCA CAG CC) (Parichy et al.,2000b). Wild-type D. rerio or D. albolineatus fms alleles result in the addition of two nucleotides (ddCA), whereas the D. rerio fmsj4e1 allele results in addition of four nucleotides (ddCTTA) to the extension primer (fms-j1r). Genotyping methods for other loci used in interspecific complementation tests are available on request.

In situ hybridization and histology

Methods for in situ hybridization, as well as tyrosinase assays and controls followed those described previously(Quigley et al., 2004).

Imaging and quantitative analysis

We examined melanophore behaviors by imaging individual larvae once-daily or twice-daily beginning when melanophores first appear outside of early larval melanophore stripes (∼14 days post-fertilization, dpf)(Parichy et al., 2000b; Parichy and Turner, 2003b),through development of the adult pigment pattern (46 dpf; once-daily series)or middle stages of pigment pattern metamorphosis (35 dpf; twice-daily series). Images were acquired with a Zeiss Axiocam HRc digital camera mounted on an Olympus SZX12 stereozoom microscope then transferred to Adobe Photoshop for analysis with FoveaPro 3.0 (Reindeer Graphics).

To quantify melanophore numbers, three regions of the anterior flank in both D. rerio and D. albolineatus were defined to represent the location of the ventral primary melanophore stripe, the primary interstripe, and the region populated by dorsal and scale-associated melanophores in D. rerio. Regions were defined by measuring the height (h) of the flank at the anterior margin of the anal fin; the measurement areas were then placed 0.5 h anterior from this location, and extending 0.25 h further anteriorly. Regions were located dorsoventrally as functions of h, according to preliminary analyses of D. rerio and dorsoventral margins of each region were 0.1 h. All melanophores were counted within these regions for each day of imaging using semi-automated feature recognition. Melanophore densities were calculated according to the areas of each region. We examined three to six individuals of each species per image series.

To examine melanophore behaviors, we followed individual melanophores throughout pigment pattern metamorphosis(Parichy et al., 2000b; Parichy and Turner, 2003b). This approach allows quantification of new melanophores that arise by differentiation or proliferation ('births') and loss of melanophores by death or de-differentiation; we refer to losses as `deaths' based on additional histological evidence, although we cannot formally exclude the possibility that some melanophores disappear by de-differentiation.

We assessed melanophore movements in twice-daily image series by determining the relative dorsal-ventral position of each melanophore followed,with the dorsal edge of the flank receiving a value of 0, and the ventral edge of the flank receiving a value of 1. We then examined the distances moved by melanophores relative to flank height, and calculated net dorsal-ventral changes in melanophore position as the difference between final and initial positions. Negative dorsal-ventral changes reflect dorsal movements, whereas positive dorsal-ventral changes reflect ventral movements. Total movements were calculated as the absolute values of these displacements. In once-daily image series, we overlayed sequential images that had been rescaled to correct for growth and aligned to minimize overall melanophore displacements and we calculated changes in melanophore position in any direction as proportions of flank height. In both approaches, using relative as opposed to absolute distances controls to some degree for passive movements due to growth, but cannot control entirely for potential differences in growth pattern between species. Thus, we further verified the magnitude of melanophore movements between species by examining relative changes in melanophore position that cannot be accounted for simply by passive movements. These rearrangements are consistent with quantitative analyses and are most easily viewed in animations(see below).

Statistical analyses were performed with JMP 5.0.1a for Macintosh (SAS Institute, Cary NC, USA). Residuals were examined for normality and homoscedasticity (Sokal and Rohlf,1994). Total melanophore numbers, melanophore births and melanophore deaths were examined by nested analyses of variance, in which individuals were nested within species and day of development was treated as a categorical variable and main effect. Births and deaths were square-root transformed prior to analyses to normalize residuals. Melanophore movements were examined by nested analyses of variance or covariance in which species differences were tested after controlling for variation among individuals(nested within species). To assess species differences in absolute movements of melanophores, we calculated absolute values for net directional movements. To assess species differences in directional melanophore movements, we controlled additionally for variation among anteroposterior regions (anterior,middle, posterior; nested within individuals), and we treated melanophore starting position as a covariate; dorsal and ventral regions of the flank were analyzed separately owing to differences suggested by preliminary analyses. Absolute and directional movements were arcsine-transformed prior to analyses. Least squares means from these analyses are reported below. Alternative parameterizations of statistical models yielded qualitatively similar results.

Phylogenetic analysis

Phylogenetic relationships were reconstructed from mitochondrial 12S and 16S rDNA sequences (12S: H1478, 5′-TGA CTG CAG AGG GTG ACG GGC GGT GTG T-3′; L1091, 5′-AAA AAG CTT CAA ACT GGG ATT AGA TAC CCC ACT AT-3′; 16S: 16Sar-L, 5′-CGC CTG TTT ATC AAA AAC AT-3′;16Sbr-H, 5′-CCG GTC TGA ACT CAG ATC ACG T-3′)(Kocher et al., 1989; Palumbi et al., 1991). Analyses were performed as described(Quigley et al., 2004) using PAUP* 4.0b10 and MrBayes(Huelsenbeck and Ronquist,2001; Swofford,2002).

Comparative fms dependence of hybrid pigment patterns

Hybrids between wild-type D. rerio and D. albolineatusdevelop stripes, whereas hybrids between fms mutant D. rerioand D. albolineatus lack stripes(Parichy and Johnson, 2001). Given this association between fms and hybrid stripe loss in D. albolineatus, and the critical role for fms in melanophore stripe formation in D. rerio, we examined the fms dependence of hybrid pigment patterns for other danios. For each species, melanophore patterns of control hybrids with wild-type D. rerio resembled D. rerio more closely than the heterospecific danio(Fig. 2).

Fig. 2.

Comparative analyses reveal fms-dependence of hybrid pigment pattern formation across Danio species. (A) Phylogenetic relationships. The traditional grouping of danios comprises at least two major clades, `Danio' and `Devario'(Kullander, 2001; Fang, 2003). Heterospecific danios with names in color were used for interspecific complementation tests with fmsj4e1 mutant D. rerio. Colors of names indicate fms-dependence of hybrid (or heterozygous) pigment pattern: red, strong fms-dependence; green, mild fms-dependence; blue, no apparent fms-dependence(see text). (B,E,H,K,N,Q) Danio wild-type pigment patterns.(C,F,I,L,O,R) Control hybrids between wild-type D. rerio and heterospecific danios. (D,G,J,M,P,S) Hybrids between fmsj4e1 mutant D. rerio and heterospecific danios. (B-D) Danio albolineatus lack distinctive stripes whereas control hybrids develop stripes with irregular borders; fms mutant hybrids lack stripes. (E-G) Danio aff. albolineatusresembles D. albolineatus but has a reduced interstripe region posteriorly and the anal fin lacks a melanophore stripe; these fish may represent a divergent population of D. albolineatus or a closely related species (Fang and Kottelat,2000). Control hybrids develop stripes with irregular borders like D. albolineatus control hybrids; fms mutant hybrids lack distinctive stripes. (H-J) Danio `hikari' resembles D. kerri(Parichy and Johnson, 2001):melanophore stripes are broad and diffuse and include very few xanthophores;interstripe regions are narrow and irregular by comparison with D. rerio (Fig. 1A). Control hybrids develop distinctive melanophore stripes and interstripes with regular borders. Tester fms mutants have fewer xanthophores than controls.(K-M) Danio aff. kyathit resembles D. rerioinitially but develops fissures in melanophore stripes as the fish grows; D. aff. kyathit also exhibit red erythrophores, particularly in the fins. Control hybrids lack stripe fissures, and fms mutant hybrids are not discernibly different (minor differences between control and tester hybrids are within the range of variation exhibited by different families within genotypes). (N-P) Danio choprae transiently develop horizontal stripes, then lose these stripes as a uniform pattern of melanophores emerges; vertical bars of melanophores arise in adults. Control hybrids develop and maintain horizontal stripes resembling those of D. rerio whereas fms mutant hybrids have fewer melanophores and xanthophores and less organized patterns. (Q-S) Danio aff. dangila adults have melanophore stripes interrupted by lighter spots and interstripes, and are morphologically indistinguishable from D. dangila (Parichy and Johnson,2001). Control hybrids resemble D. rerio, and fms mutant hybrids exhibit no clear difference from controls. Data not shown: Danio nigrofasciatus exhibit well-defined stripes and control hybrids are intermediate between D. rerio and D. nigrofasciatus parental species(Parichy and Johnson, 2001; Quigley et al., 2004); tester hybrids do not differ from controls for either fmsj4blue(Parichy and Johnson, 2001) or fmsj4e1. Tester fmsj4e1 hybrids could not be obtained for D. kerri; tester fmsj4bluehybrids resemble control hybrids, similar to tester fmsj4blue hybrids with D. albolineatus, likely reflecting modifier loci in the fmsj4blue background (see text). Hybrids with species outside of the Danio clade (A) were not viable, consistent with previous observations(Parichy and Johnson, 2001). All fish are between 25-35 mm standard length, except D. dangila and its hybrids, which are 50-80 mm. GenBank accession numbers for 12S and 16S sequences used in phylogeny reconstruction were: AY707450, AY707456; U21372,U21381; AF322658, AF322663; AY707446, AY707452; AF322663; AY707449, AY707455;AY707447, AY707453; AF322656, AF322661; U21376, U21384; AF322659, AF322664;U21377, U21377; U21375, U21370; AY707448, AY707454; U21553, U21554; AF322660,AF322665; U21378, U21386; AY707445, AY707451; AY37481, AY37482; AY37483,AY37484.

Fig. 2.

Comparative analyses reveal fms-dependence of hybrid pigment pattern formation across Danio species. (A) Phylogenetic relationships. The traditional grouping of danios comprises at least two major clades, `Danio' and `Devario'(Kullander, 2001; Fang, 2003). Heterospecific danios with names in color were used for interspecific complementation tests with fmsj4e1 mutant D. rerio. Colors of names indicate fms-dependence of hybrid (or heterozygous) pigment pattern: red, strong fms-dependence; green, mild fms-dependence; blue, no apparent fms-dependence(see text). (B,E,H,K,N,Q) Danio wild-type pigment patterns.(C,F,I,L,O,R) Control hybrids between wild-type D. rerio and heterospecific danios. (D,G,J,M,P,S) Hybrids between fmsj4e1 mutant D. rerio and heterospecific danios. (B-D) Danio albolineatus lack distinctive stripes whereas control hybrids develop stripes with irregular borders; fms mutant hybrids lack stripes. (E-G) Danio aff. albolineatusresembles D. albolineatus but has a reduced interstripe region posteriorly and the anal fin lacks a melanophore stripe; these fish may represent a divergent population of D. albolineatus or a closely related species (Fang and Kottelat,2000). Control hybrids develop stripes with irregular borders like D. albolineatus control hybrids; fms mutant hybrids lack distinctive stripes. (H-J) Danio `hikari' resembles D. kerri(Parichy and Johnson, 2001):melanophore stripes are broad and diffuse and include very few xanthophores;interstripe regions are narrow and irregular by comparison with D. rerio (Fig. 1A). Control hybrids develop distinctive melanophore stripes and interstripes with regular borders. Tester fms mutants have fewer xanthophores than controls.(K-M) Danio aff. kyathit resembles D. rerioinitially but develops fissures in melanophore stripes as the fish grows; D. aff. kyathit also exhibit red erythrophores, particularly in the fins. Control hybrids lack stripe fissures, and fms mutant hybrids are not discernibly different (minor differences between control and tester hybrids are within the range of variation exhibited by different families within genotypes). (N-P) Danio choprae transiently develop horizontal stripes, then lose these stripes as a uniform pattern of melanophores emerges; vertical bars of melanophores arise in adults. Control hybrids develop and maintain horizontal stripes resembling those of D. rerio whereas fms mutant hybrids have fewer melanophores and xanthophores and less organized patterns. (Q-S) Danio aff. dangila adults have melanophore stripes interrupted by lighter spots and interstripes, and are morphologically indistinguishable from D. dangila (Parichy and Johnson,2001). Control hybrids resemble D. rerio, and fms mutant hybrids exhibit no clear difference from controls. Data not shown: Danio nigrofasciatus exhibit well-defined stripes and control hybrids are intermediate between D. rerio and D. nigrofasciatus parental species(Parichy and Johnson, 2001; Quigley et al., 2004); tester hybrids do not differ from controls for either fmsj4blue(Parichy and Johnson, 2001) or fmsj4e1. Tester fmsj4e1 hybrids could not be obtained for D. kerri; tester fmsj4bluehybrids resemble control hybrids, similar to tester fmsj4blue hybrids with D. albolineatus, likely reflecting modifier loci in the fmsj4blue background (see text). Hybrids with species outside of the Danio clade (A) were not viable, consistent with previous observations(Parichy and Johnson, 2001). All fish are between 25-35 mm standard length, except D. dangila and its hybrids, which are 50-80 mm. GenBank accession numbers for 12S and 16S sequences used in phylogeny reconstruction were: AY707450, AY707456; U21372,U21381; AF322658, AF322663; AY707446, AY707452; AF322663; AY707449, AY707455;AY707447, AY707453; AF322656, AF322661; U21376, U21384; AF322659, AF322664;U21377, U21377; U21375, U21370; AY707448, AY707454; U21553, U21554; AF322660,AF322665; U21378, U21386; AY707445, AY707451; AY37481, AY37482; AY37483,AY37484.

Our analyses reveal fms-dependent hybrid pigment patterns for additional taxa (Fig. 2A). For both D. albolineatus and D. aff. albolineatus,fmsj4e1 hybrids lacked stripes(Fig. 2B-G). For D.`hikari', fmsj4e1 hybrids developed normal melanophore stripes but fewer xanthophores than controls(Fig. 2B-J, Fig. 3A,B). Within the clade that includes D. rerio, D. kyathit and D. nigrofasciatus,fmsj4e1 hybrids did not differ consistently from controls(Fig. 2K-M)(Parichy and Johnson, 2001)(data not shown). Finally, D. choprae fmsj4e1 hybrids exhibited disrupted adult stripes and a severe xanthophore deficit(Fig. 2N-P, Fig. 3C-H), but D. dangila fmsj4e1 hybrids were not discernibly different from controls(Fig. 2Q-S).

Fig. 3.

Mild and strong fms-dependence of hybrid pigment patterns for D. `hikari' and D. choprae. (A,B) Danio `hikari'hybrids. (A) Wild-type control hybrids develop distinctive melanophore stripes and interstripe regions (arrow, A), whereas fms mutant hybrids have about half as many xanthophores as controls (B). (C-F) Danio chopraehybrids at larval stages. (C,E) Wild-type control hybrids have numerous xanthophores both on the body (arrow, C) and in the fins (arrowheads, E).(D,F) fms mutant hybrids have fewer xanthophores than controls. (G,H)Adult D. choprae control hybrids (G) have more xanthophores (arrows)than tester fms mutant hybrids (H). Scale bars: in A, 500 μm for A,B,G,H); in C, 150 μm for C-F.

Fig. 3.

Mild and strong fms-dependence of hybrid pigment patterns for D. `hikari' and D. choprae. (A,B) Danio `hikari'hybrids. (A) Wild-type control hybrids develop distinctive melanophore stripes and interstripe regions (arrow, A), whereas fms mutant hybrids have about half as many xanthophores as controls (B). (C-F) Danio chopraehybrids at larval stages. (C,E) Wild-type control hybrids have numerous xanthophores both on the body (arrow, C) and in the fins (arrowheads, E).(D,F) fms mutant hybrids have fewer xanthophores than controls. (G,H)Adult D. choprae control hybrids (G) have more xanthophores (arrows)than tester fms mutant hybrids (H). Scale bars: in A, 500 μm for A,B,G,H); in C, 150 μm for C-F.

Thus, four out of seven danios exhibited noncomplementation phenotypes with fmsj4e1 mutant D. rerio(Fig. 2A); one of these was mild (D. `hikari') and three were severe (D. albolineatus,D. aff. albolineatus, D. choprae). Whether mild non-complementation for D. `hikari' represents a unique derived change for this species, or a basal change within the D.`hikari'-D. albolineatus clade is uncertain without deeper taxonomic sampling. Given the close phylogenetic relationship of D. albolineatus and D. aff. albolineatus, these results imply at least two evolutionary changes resulting in severe noncomplementation phenotypes, one separating D. albolineatus-D. aff. albolineatus from other danios, and another separating D. choprae from other danios.

fms-dependent hybrid stripe disruption in D. albolineatus

Given the broader variation in fms-dependence across danios, we sought to further test evolutionary roles for fms and fms-dependent pathways, focusing on D. albolineatus because of the simplicity of its pattern. Previous analyses tested D. albolineatus hybrids for non-complementation of fmsj4e1, fmsj4e3 and fmsj4blue, all of which are recessive in D. rerioand exhibit presumptive null phenotypes(Parichy et al., 2000b; Parichy and Johnson, 2001). Hybrids for fmsj4e1 and fmsj4e3 lacked stripes, whereas hybrids for fmsj4blue developed stripes. Since fmsj4e1 and fmsj4e3 were maintained in the inbred AB* (ABut) genetic background,whereas fmsj4blue was maintained in a different background, the formal possibility exists that other loci in the ABut background were responsible. Alternatively, modifier loci affecting the penetrance of a fms effect could differ across backgrounds. Thus, we asked whether pigment pattern variation in hybrids with D. albolineatus segregates with alleles at the fmslocus.

Our analyses support a model in which stripe disruption in hybrids depends on fms, with the magnitude of this effect determined by additional modifier loci. We generated heterozygous fms mutant D. rerioby crossing fmsj4e1, maintained in the inbred background ABut, with another inbred mapping strain, wikut. We then crossed these fmsj4e1(AB)/fms+(wik)D. rerio to D. albolineatus. Hybrid offspring segregated two phenotypes in ∼1:1 ratios: either well-organized, `strong' melanophore stripes, or a poorly organized, `weak' stripe pattern, with significantly fewer melanophores and xanthophores (Fig. 4A-F). We categorized fish into alternative `strong' and `weak'stripe classes, then asked whether individuals carried the fms+(wik) wild-type D. rerio allele or the fmsj4e1(AB) mutant D. rerio allele. Primer extension genotyping for the fmsj4e1 lesion demonstrates that, in every instance, hybrids with `strong' stripes carried the wild-type allele whereas hybrids with `weak' stripes carried the mutant allele (n=105; Fig. 4G,H).

Fig. 4.

Segregation analysis supports an essential role for fms in D. albolineatus ×D. rerio hybrid stripe development.(A,B,D,E) Tester fmsj4e1/+ hybrids exhibit either of two easily distinguishable phenotypes, one in which stripes are well organized(A,B), and another in which stripes are weakly organized with irregular borders and fewer xanthophores (D,E). Differences are evident during pigment pattern metamorphosis (A,D) but are most apparent in adults (B,E). Details showing well-organized (C) and weakly organized (F) stripes in hybrid adults. Pigment cell complements in hybrids with weak stripes were significantly reduced compared with hybrids with strong stripes, with 84% of the number of melanophores (F1,16=8.76, P<0.01) and only 14%of the number of xanthophores (F1,16=20.85, P<0.0005). The reduction in melanophore numbers is comparable to that observed in homozygous fmsj4e1 mutant D. rerio and fms174 mutant D. rerio at restrictive temperature; however, neither D. rerio mutant allele retains xanthophores (Parichy et al.,2000b; Parichy and Turner,2003a). (G,H). Primer extension genotyping for fmsalleles. (G) In D. rerio, an extension primer adjacent to the fmsj4e1 lesion yields additions of two nucleotides in homozygous wild-type individuals, additions of four nucleotides in homozygous fmsj4e1 individuals, and additions of both two and four nucleotides in heterozygous fms+/fmsj4e1 individuals. (H) Primer extension genotyping for tester fmsj4e1/+ hybrids that have been classified as having `strong' or `weak' stripes. All hybrids with strong stripes carry the D. rerio fms+(wik) wild-type allele and the D. albolineatus fms allele, resulting in two nucleotide additions to the extension primers. All hybrids with weak stripes carry the D. rerio fmsj4e1 mutant allele and a D. albolineatus fms allele, resulting in the addition of four and two nucleotides to the extension primer, respectively. Scale bars: in A, 200 μm for A,D; in B, 500 μm for B,E; in C, 200 μm for C,F.

Fig. 4.

Segregation analysis supports an essential role for fms in D. albolineatus ×D. rerio hybrid stripe development.(A,B,D,E) Tester fmsj4e1/+ hybrids exhibit either of two easily distinguishable phenotypes, one in which stripes are well organized(A,B), and another in which stripes are weakly organized with irregular borders and fewer xanthophores (D,E). Differences are evident during pigment pattern metamorphosis (A,D) but are most apparent in adults (B,E). Details showing well-organized (C) and weakly organized (F) stripes in hybrid adults. Pigment cell complements in hybrids with weak stripes were significantly reduced compared with hybrids with strong stripes, with 84% of the number of melanophores (F1,16=8.76, P<0.01) and only 14%of the number of xanthophores (F1,16=20.85, P<0.0005). The reduction in melanophore numbers is comparable to that observed in homozygous fmsj4e1 mutant D. rerio and fms174 mutant D. rerio at restrictive temperature; however, neither D. rerio mutant allele retains xanthophores (Parichy et al.,2000b; Parichy and Turner,2003a). (G,H). Primer extension genotyping for fmsalleles. (G) In D. rerio, an extension primer adjacent to the fmsj4e1 lesion yields additions of two nucleotides in homozygous wild-type individuals, additions of four nucleotides in homozygous fmsj4e1 individuals, and additions of both two and four nucleotides in heterozygous fms+/fmsj4e1 individuals. (H) Primer extension genotyping for tester fmsj4e1/+ hybrids that have been classified as having `strong' or `weak' stripes. All hybrids with strong stripes carry the D. rerio fms+(wik) wild-type allele and the D. albolineatus fms allele, resulting in two nucleotide additions to the extension primers. All hybrids with weak stripes carry the D. rerio fmsj4e1 mutant allele and a D. albolineatus fms allele, resulting in the addition of four and two nucleotides to the extension primer, respectively. Scale bars: in A, 200 μm for A,D; in B, 500 μm for B,E; in C, 200 μm for C,F.

These data confirm that a hybrid non-complementation phenotype segregates with fms. Yet, this phenotype is less severe than that of fmsj4e1 (Fig. 2D) and fmsj4e3 in the AButbackground, and more severe than that for fmsj4blue in a different background (Parichy and Johnson,2001). This suggests that modifier loci contribute to the phenotype, and that these modifiers differ across genetic backgrounds.

Segregation analyses thus place the non-complementing locus in the vicinity of fms, and suggest roles for modifier loci in determining hybrid pigment patterns.

Temperature-sensitive fmsallele confirms role in hybrid stripe loss

We used a temperature-sensitive fms allele to further confirm the requirement for fms in D. albolineatus hybrid pigment pattern development. Segregation analyses placed the non-complementing locus within ∼1 cM of fms, a region likely to include several other genes. We reasoned that a fms allele demonstrated previously to exhibit temperature sensitivity could be used to exclude roles for these neighboring loci: a fms-specific effect should be manifested as a complementation phenotype at the permissive temperature, and a noncomplementation phenotype at the restrictive temperature. Thus, we used the temperature-sensitive allele fmsut.r4e174A(fms174), which exhibits a wild-type phenotype at 24°C and a fms null phenotype at 33°C(Parichy and Turner, 2003a). We crossed homozygous fms174 mutant D. rerio to D. albolineatus and reared hybrid siblings at either 24°C or 33°C. Tester fms174 hybrids reared at 24°C were indistinguishable from control hybrids(Fig. 5A,B), as were wild-type hybrids reared at 33°C (data not shown). By contrast, fms174 hybrids reared at 33°C developed poorly organized melanophore stripes and fewer xanthophores(Fig. 5C,D). These results provide compelling additional evidence that hybrid non-complementation phenotypes depend on fms, rather than on other closely linked loci.

Fig. 5.

A temperature-sensitive allele uniquely implicates fms in melanophore stripe disorganization and xanthophore reduction in hybrids. Tester fms174 mutant D. rerio ×D. albolineatus hybrids were reared at 28.5°C through middle metamorphosis, then were transferred either to 24°C or 33°C until adult pigment patterns had formed. (A,B) fms174 hybrids reared at 24°C develop well-organized stripes and numerous xanthophores on the body (A) and fins (B, arrows). (C,D) fms174 hybrids reared at 33°C develop poorly organized melanophore stripes and have fewer xanthophores on the body (C) and fins (D, arrows). Pigment patterns of control wild-type hybrids do not differ between 24°C and 33°C (data not shown). Scale bars: in A, 1 mm for A,C; in B, 150 μm for B,D.

Fig. 5.

A temperature-sensitive allele uniquely implicates fms in melanophore stripe disorganization and xanthophore reduction in hybrids. Tester fms174 mutant D. rerio ×D. albolineatus hybrids were reared at 28.5°C through middle metamorphosis, then were transferred either to 24°C or 33°C until adult pigment patterns had formed. (A,B) fms174 hybrids reared at 24°C develop well-organized stripes and numerous xanthophores on the body (A) and fins (B, arrows). (C,D) fms174 hybrids reared at 33°C develop poorly organized melanophore stripes and have fewer xanthophores on the body (C) and fins (D, arrows). Pigment patterns of control wild-type hybrids do not differ between 24°C and 33°C (data not shown). Scale bars: in A, 1 mm for A,C; in B, 150 μm for B,D.

Finally, D. albolineatus hybrids did not reveal noncomplementation phenotypes for any of 16 other recessive D. rerio mutants affecting pigment cell numbers and arrangements (ednrb1b140, kitb5, mitfaw2, sox10ut.r13e1, tfap2ats213, cezanneut.r17e1, degasut.r18e1, jaguarc7, leopardt1, oberonj198e1, pissarrout.r8e1, picassout.r2e1, primrosej199, pumaj115e1, or seuratut.r15e1),including bonaparteut.r16e1, which has melanophore and xanthophore defects similar to fms mutants (D.M.P. and E.L.M.,unpublished). Thus, D. albolineatus hybrid pigment patterns are uniquely fms-dependent within this broader collection of loci required for pigment pattern development.

Altered melanophore lineage development during D. albolineatus adult pigment pattern formation

Genetic analyses above reveal a strong fms dependence of hybrid pigment pattern development for D. albolineatus (as well as D. choprae) but do not indicate how this dependence reflects natural variation between species. Given the noncomplementation phenotype of fms hybrids, we reasoned initially that D. albolineatusmight exhibit a loss of fms activity relative to D. rerio. This simple model predicts that pigment pattern metamorphosis in D. albolineatus should resemble that of fms mutant D. rerio. By comparison to wild-type D. rerio, fms mutants have fewer metamorphic melanophores, increased melanophore death, decreased melanophore movement into stripes, and an absence of xanthophores(Parichy et al., 2000b; Parichy and Turner, 2003a). We thus asked whether D. albolineatus pigment pattern metamorphosis entails some or all of these differences relative to wild-type D. rerio. Our analyses reveal dramatic differences between wild-type D. rerio and D. albolineatus(Fig. 6; see Movies 1-4 in supplementary material). Although some similarities are seen between D. albolineatus and fms mutant D. rerio, there are major differences as well (next section).

Fig. 6.

Development of adult pigment patterns in D. rerio (A-F) and D. albolineatus (G-L). Panels shown are of selected days from a complete image series for individual, representative larvae. (A) In D. rerio,pigment pattern metamorphosis is marked by the differentiation of metamorphic melanophores (arrow, showing one of many) over the myotomes. The white box in each image delineates the horizontal myoseptum; the single arrowhead in A indicates one of several early larval melanophores that have persisted along the horizontal myoseptum through the beginning of metamorphosis. Double arrowhead indicates deep melanophores along the dorsal aspect of the neural tube; triple arrowhead, deep melanophores lining the dorsal surface of the peritoneum. sb, swim bladder. (B) As metamorphosis proceeds, additional metamorphic melanophores (small arrows) develop over the myotomes, and iridophores and xanthophores differentiate ventral to the horizontal myoseptum in the prospective primary interstripe region (large arrow). (C) By middle stages of pigment pattern metamorphosis, melanophores have started to organize into stripes in the region of the prospective dorsal primary melanophore stripe (p1D) and prospective ventral primary melanophore stripe (p1V). (D) As the dorsal and ventral melanophore stripes become increasingly distinctive,additional late metamorphic melanophores differentiate already within these stripes (e.g. arrow). (E) Near the completion of pigment pattern metamorphosis, distinctive dorsal and ventral primary melanophore stripes (1D,1V) border a well-defined interstripe region. (F) Pigment pattern metamorphosis is completed with the development of scales and scale-associated melanophores (s). In D. albolineatus, melanophores typically do not persist along the horizontal myoseptum from earlier stages, and instead melanophores, initially deeper between the myotomes, migrate to the surface(arrowhead, data not shown). (H) Metamorphic melanophores (e.g. small arrow)differentiate widely scattered over the myotomes, as in D. rerio, but fewer iridophores (large arrow) develop ventral to the horizontal myoseptum.(I,J) As metamorphosis proceeds, additional metamorphic melanophores appear over the myotomes, yet these cells typically do not migrate far from their site of differentiation. Relatively few late metamorphic melanophores appear within the regions where stripes form in D. rerio (compare with D).(K) Near the completion of metamorphosis, melanophores remain relatively dispersed compared with D. rerio, and only a weak pattern of melanophore stripes (arrowheads) borders an irregular and narrow interstripe region on the posterior trunk. (L) At the end of metamorphosis, D. albolineatus have far fewer sub-dermal melanophores than D. rerio, and these cells are widely distributed where the interstripe region develops in D. rerio. The interstripe region extends only to the middle of the flank (arrow) and reddish erythrophores have started to differentiate within this region. Images shown have been rescaled across stages to maintain the same approximate field of view. Larval standard lengths in mm, (A-F) 7.1, 7.7, 8.3, 9.5, 12.0, 13.3, (G-L) 8.1, 9.3, 10.1, 11.0, 12.8,13.4. Scale bars: in A, 500 μm for A,G; in F, 1 mm for F,L.

Fig. 6.

Development of adult pigment patterns in D. rerio (A-F) and D. albolineatus (G-L). Panels shown are of selected days from a complete image series for individual, representative larvae. (A) In D. rerio,pigment pattern metamorphosis is marked by the differentiation of metamorphic melanophores (arrow, showing one of many) over the myotomes. The white box in each image delineates the horizontal myoseptum; the single arrowhead in A indicates one of several early larval melanophores that have persisted along the horizontal myoseptum through the beginning of metamorphosis. Double arrowhead indicates deep melanophores along the dorsal aspect of the neural tube; triple arrowhead, deep melanophores lining the dorsal surface of the peritoneum. sb, swim bladder. (B) As metamorphosis proceeds, additional metamorphic melanophores (small arrows) develop over the myotomes, and iridophores and xanthophores differentiate ventral to the horizontal myoseptum in the prospective primary interstripe region (large arrow). (C) By middle stages of pigment pattern metamorphosis, melanophores have started to organize into stripes in the region of the prospective dorsal primary melanophore stripe (p1D) and prospective ventral primary melanophore stripe (p1V). (D) As the dorsal and ventral melanophore stripes become increasingly distinctive,additional late metamorphic melanophores differentiate already within these stripes (e.g. arrow). (E) Near the completion of pigment pattern metamorphosis, distinctive dorsal and ventral primary melanophore stripes (1D,1V) border a well-defined interstripe region. (F) Pigment pattern metamorphosis is completed with the development of scales and scale-associated melanophores (s). In D. albolineatus, melanophores typically do not persist along the horizontal myoseptum from earlier stages, and instead melanophores, initially deeper between the myotomes, migrate to the surface(arrowhead, data not shown). (H) Metamorphic melanophores (e.g. small arrow)differentiate widely scattered over the myotomes, as in D. rerio, but fewer iridophores (large arrow) develop ventral to the horizontal myoseptum.(I,J) As metamorphosis proceeds, additional metamorphic melanophores appear over the myotomes, yet these cells typically do not migrate far from their site of differentiation. Relatively few late metamorphic melanophores appear within the regions where stripes form in D. rerio (compare with D).(K) Near the completion of metamorphosis, melanophores remain relatively dispersed compared with D. rerio, and only a weak pattern of melanophore stripes (arrowheads) borders an irregular and narrow interstripe region on the posterior trunk. (L) At the end of metamorphosis, D. albolineatus have far fewer sub-dermal melanophores than D. rerio, and these cells are widely distributed where the interstripe region develops in D. rerio. The interstripe region extends only to the middle of the flank (arrow) and reddish erythrophores have started to differentiate within this region. Images shown have been rescaled across stages to maintain the same approximate field of view. Larval standard lengths in mm, (A-F) 7.1, 7.7, 8.3, 9.5, 12.0, 13.3, (G-L) 8.1, 9.3, 10.1, 11.0, 12.8,13.4. Scale bars: in A, 500 μm for A,G; in F, 1 mm for F,L.

We first examined the temporal accumulation of melanophores. Daily image series show that D. albolineatus exhibited only ∼67% as many melanophores as D. rerio (F1,142=245.56, P<0.0001) across the entire image series. The deficit was most evident where melanophore stripes form in D. rerio, and became increasingly severe at later stages (Fig. 6, Fig. 7A). Only where the primary interstripe forms in D. rerio were melanophore numbers greater in D. albolineatus, reflecting the more uniform pattern of melanophores (Fig. 7B).

Fig. 7.

Quantitative analyses of melanophore morphogenesis in D. rerio and D. albolineatus. (A) In regions where melanophore stripes develop in D. rerio, melanophore numbers are dramatically reduced in D. albolineatus and this deficit becomes more pronounced through pigment pattern metamorphosis. (B) In the middle of the flank, where the first interstripe region forms in D. rerio, melanophore numbers are increased in D. albolineatus, reflecting the absence of a distinctive and persistent melanophore-free region in the anterior of the flank. Despite the increased melanophore numbers, the overall complement of melanophores is dramatically reduced overall compared with D. rerio. (C) Analyses of twice daily image series reveal differential appearance ('births') and disappearance ('deaths') of melanophores between species. Shown are cumulative mean births and deaths recorded in each of three developing larvae through middle stages of pigment pattern metamorphosis, with `births' defined as the appearance of new melanophores (either by differentiation or proliferation)and `deaths' defined by the unambiguous loss of melanophores (see Materials and methods). Melanophore births were not significantly different between species (F1,107=1.87, P=0.2) after controlling for variation among individuals (F4,107=2.72, P<0.05) and across days (F21,107=5.43, P<0.001). By contrast, melanophore deaths were significantly greater in D. albolineatus than D. rerio(F1,81=3.04, P<0.005) after controlling for variation among individuals (F4,81=2.10, P=0.09)and across days (F29,81=2.47, P<0.001). Error bars are omitted for clarity. (D) Total melanophore movements are reduced in D. albolineatus compared with D. rerio, both during early and middle metamorphosis (left), and through later metamorphosis (right). Shown are mean (±s.e.m.) distances moved by individual melanophores,with distances expressed as percentages of the flank height. Left, species differences were significant (F1,696=14.23, P<0.0005; n=368, 344 melanophores in D. rerioand D. albolineatus) after controlling for variation associated with individuals (nested within species: F4,696=2.60, P<0.05), and anteroposterior region nested within individuals(F10,696=5.19, P<0.0001). Right, species differences were significant (F1,708=8.45, P<0.005; n=291, 419 melanophores in D. rerio and D. albolineatus) whereas inter-individual differences were not significant (P=0.2). (E,F) Directional movements of melanophores were significantly reduced in D. albolineatus compared with D. rerio. Each point represents a single melanophore followed from its first appearance to its final position at the end of the series or until it was lost(n=368, 344 melanophores in D. rerio and D. albolineatus, respectively). Plots show the initial dorsoventral positions at which melanophores first appeared, and the subsequent changes in dorsoventral positions by the end of the images series. The dorsal-most position on the flank is assigned a relative value of 0, and the ventral-most position on the flank is assigned a value of 1. Regression slopes are estimated separately for dorsal and ventral regions of the flank because of differences in shape and growth. (E) In D. rerio, melanophores that initially appear in more dorsal regions of the flank tend to move ventrally whereas melanophores that initially appear in more ventral regions of the flank tend to move dorsally (partial regression coefficients±s.e.m. for relationship between starting dorsoventral position and arcsine-transformed movements for dorsal and ventral regions of the flank, respectively:-0.17±0.02, -0.14±0.04). (F) In D. albolineatus,directional movements were significantly reduced compared with D. rerio in dorsal regions of the flank (F1,399=6.43, P<0.05), although species differences were not detectable in ventral regions (F1,399=1.40, P=0.2), as assessed by the magnitude of starting position ×species interactions [partial regression coefficients for dorsal and ventral, respectively:-0.10±0.02, -0.10±0.02; after controlling in both dorsal and ventral analyses for variation among individuals (nested within species, both P<0.005), variation among the three examined anteroposterior regions of the flank nested within individuals (both P<0.0005),main effects of species (P=0.7, P<0.005, respectively),and starting position independent of species (both P<0.0001)].

Fig. 7.

Quantitative analyses of melanophore morphogenesis in D. rerio and D. albolineatus. (A) In regions where melanophore stripes develop in D. rerio, melanophore numbers are dramatically reduced in D. albolineatus and this deficit becomes more pronounced through pigment pattern metamorphosis. (B) In the middle of the flank, where the first interstripe region forms in D. rerio, melanophore numbers are increased in D. albolineatus, reflecting the absence of a distinctive and persistent melanophore-free region in the anterior of the flank. Despite the increased melanophore numbers, the overall complement of melanophores is dramatically reduced overall compared with D. rerio. (C) Analyses of twice daily image series reveal differential appearance ('births') and disappearance ('deaths') of melanophores between species. Shown are cumulative mean births and deaths recorded in each of three developing larvae through middle stages of pigment pattern metamorphosis, with `births' defined as the appearance of new melanophores (either by differentiation or proliferation)and `deaths' defined by the unambiguous loss of melanophores (see Materials and methods). Melanophore births were not significantly different between species (F1,107=1.87, P=0.2) after controlling for variation among individuals (F4,107=2.72, P<0.05) and across days (F21,107=5.43, P<0.001). By contrast, melanophore deaths were significantly greater in D. albolineatus than D. rerio(F1,81=3.04, P<0.005) after controlling for variation among individuals (F4,81=2.10, P=0.09)and across days (F29,81=2.47, P<0.001). Error bars are omitted for clarity. (D) Total melanophore movements are reduced in D. albolineatus compared with D. rerio, both during early and middle metamorphosis (left), and through later metamorphosis (right). Shown are mean (±s.e.m.) distances moved by individual melanophores,with distances expressed as percentages of the flank height. Left, species differences were significant (F1,696=14.23, P<0.0005; n=368, 344 melanophores in D. rerioand D. albolineatus) after controlling for variation associated with individuals (nested within species: F4,696=2.60, P<0.05), and anteroposterior region nested within individuals(F10,696=5.19, P<0.0001). Right, species differences were significant (F1,708=8.45, P<0.005; n=291, 419 melanophores in D. rerio and D. albolineatus) whereas inter-individual differences were not significant (P=0.2). (E,F) Directional movements of melanophores were significantly reduced in D. albolineatus compared with D. rerio. Each point represents a single melanophore followed from its first appearance to its final position at the end of the series or until it was lost(n=368, 344 melanophores in D. rerio and D. albolineatus, respectively). Plots show the initial dorsoventral positions at which melanophores first appeared, and the subsequent changes in dorsoventral positions by the end of the images series. The dorsal-most position on the flank is assigned a relative value of 0, and the ventral-most position on the flank is assigned a value of 1. Regression slopes are estimated separately for dorsal and ventral regions of the flank because of differences in shape and growth. (E) In D. rerio, melanophores that initially appear in more dorsal regions of the flank tend to move ventrally whereas melanophores that initially appear in more ventral regions of the flank tend to move dorsally (partial regression coefficients±s.e.m. for relationship between starting dorsoventral position and arcsine-transformed movements for dorsal and ventral regions of the flank, respectively:-0.17±0.02, -0.14±0.04). (F) In D. albolineatus,directional movements were significantly reduced compared with D. rerio in dorsal regions of the flank (F1,399=6.43, P<0.05), although species differences were not detectable in ventral regions (F1,399=1.40, P=0.2), as assessed by the magnitude of starting position ×species interactions [partial regression coefficients for dorsal and ventral, respectively:-0.10±0.02, -0.10±0.02; after controlling in both dorsal and ventral analyses for variation among individuals (nested within species, both P<0.005), variation among the three examined anteroposterior regions of the flank nested within individuals (both P<0.0005),main effects of species (P=0.7, P<0.005, respectively),and starting position independent of species (both P<0.0001)].

Fewer metamorphic melanophores in D. albolineatus could reflect a change in melanophore specification, increased rates of melanophore or melanoblast death, or both. To distinguish among these possibilities, we first quantified the appearance and disappearance of melanophores in a temporally higher resolution series of images taken at 12-hour intervals. Averaged over entire series, the D. albolineatus larvae exhibited ∼91% as many melanophore `births' as the D. rerio larvae, but 376% as many`deaths' (Fig. 7C); suggesting that fewer melanophores in D. albolineatus does not reflect a failure to recruit stem cells into the melanophore lineage, but to some extent a reduction in the subsequent survival of these cells. Consistent with this inference, numbers of melanophore precursors were not obviously reduced in D. albolineatus, as revealed by molecular markers(Fig. 8A-H) and tyrosinase activity (Fig. 8I-L). Moreover, D. albolineatus melanophores frequently appeared, then disappeared,over short time intervals (Fig. 8M-O) and these cells, as well as l-dopastained,tyrosinase+ melanophore precursors, were common within the epidermis and in `extrusion bodies' at the epidermal surface, characteristic of teleost melanophore death (Parichy et al., 1999; Sugimoto,2002; Parichy and Turner,2003a) (Fig. 8P,R-T); only ∼10% as many epidermal, melanized cells occurred in D. rerio larvae (Fig. 8Q). These results indicate that fewer melanophores in D. albolineatus result, at least in part, from the death of these cells and their immediate precursors.

Fig. 8.

Altered melanophore lineage development in D. albolineatus. (A-H)Melanophore precursor abundance does not differ dramatically between species as revealed by distributions of cells expressing tyrosinase, dopachrome tautomerase (dct) and mitfa(Lister et al., 1999; Kelsh et al., 2000; Camp and Lardelli, 2001). tyrosinase and dct encode enzymes required for melanin synthesis, whereas mitfa encodes a transcription factor essential for melanophore specification. (A-D) In situ hybridization for tyrosinasemRNA in D. rerio (A,B) and D. albolineatus (C,D) during middle stages of pigment pattern metamorphosis (equivalent to larvae shown in Fig. 6C,I). (A) In D. rerio, numerous tyrosinase+ melanophores and unmelanized melanophore precursors (arrowhead) are observed in the region of the prospective dorsal primary melanophore stripe. (B) Higher magnification image of a different larvae showing tyrosinase+melanophores (arrow) and melanophore precursors (arrowhead). (C) In D. albolineatus, fewer melanophores are present but tyrosinase+ melanophore precursors (arrowhead) are not obviously reduced in number. (D) Higher magnification of a different larva showing tyrosinase+ melanophores (arrowhead) and unmelanized melanophore precursor (arrowhead). (E-H) Expression of other melanophore lineage markers also is similar between D. rerio and D. albolineatus. (E,F) Danio rerio exhibit unmelanized cells(arrowheads), and some melanized cells (arrow) expressing dct (E) and mitfa (F). (G,H) In D. albolineatus, the numbers of unmelanized cells expressing dct (G) and mitfa are similar to D. rerio. Results for kit and sox10-expressing cells were similar (data not shown)(Parichy et al., 1999; Dutton et al., 2001). (I-L)Tyrosinase-expressing melanophore precursors revealed by treating fixed larvae with the essential precursor for melanin synthesis, l-dopa(Camp and Lardelli, 2001; McCauley et al., 2004). Images in I and K are prior to l-dopa incubation, and images in J and L are the same fields of view after treatment with l-dopa for 5 hours. (I,J) In D. rerio, only a few tyrosinase+melanophore precursors are revealed by l-dopa incubation(arrowheads). (K,L) In D. albolineatus, however, numerous tyrosinase+ melanophore precursors are revealed by l-dopa treatment (arrowheads show only a few of these cells). These cells exhibit morphologies typical of melanoblasts and recently differentiated melanophores (inset). The increased number of l-dopa stained,tyrosinase+ cells as compared with molecular markers in D. albolineatus may reflect perduring protein in the absence of transcriptional activity. (M-T). Melanophores and melanophore precursors frequently are lost in D. albolineatus. (M-O) The same region of a D. albolineatus larvae imaged at 12-hour intervals reveals the transient appearance of several melanophores (arrowheads). Yellow-orange cells are xanthophores; these and other melanophores do not change positions between images. (P) High magnification image of D. albolineatus reveals melanin-containing debris (arrow) typical of melanophore death. Arrowhead,melanophore precursor that acquired melanin following l-dopa incubation of this larva. (Q-S) Cross-sections reveal the locations of melanophores and tyrosinase+ melanophore precursors in D. rerio (Q) and D. albolineatus (R,S). (Q) In D. rerio,few melanophores or melanophore precursors are located within the plane of the epidermis; one such melanophore is indicated by the arrowhead. e, epidermis;m, myotome. Arrow indicates iridophores of the developing interstripe region.(R) In D. albolineatus, numerous melanophores and tyrosinase+ melanophore precursors occur within the plane of the epidermis (arrowheads), although some melanophores are found subdermally, as in D. rerio (arrow). (S) Higher magnification of bracketed region in R, showing a melanin-containing extrusion body typical of teleost melanophore death. Arrowhead indicates bounding membrane. n, neuromast. Melanin-containing debris in P also is superficially located, as revealed by hexagonal outlines of adjacent epidermal cells. (T) High magnification image of whole-mount larva, showing extrusion body containing melanin granule (arrow) and staining for dct mRNA (arrowhead). Scale bars: in A, 80 μm for A,C; in B,40 μm for B,D; in E, 60 μm for E-H; in I, 80 μm for I-L; in P, 20μm for P; in Q, 60 μm for Q,R; in S, 20 μm for S,T.

Fig. 8.

Altered melanophore lineage development in D. albolineatus. (A-H)Melanophore precursor abundance does not differ dramatically between species as revealed by distributions of cells expressing tyrosinase, dopachrome tautomerase (dct) and mitfa(Lister et al., 1999; Kelsh et al., 2000; Camp and Lardelli, 2001). tyrosinase and dct encode enzymes required for melanin synthesis, whereas mitfa encodes a transcription factor essential for melanophore specification. (A-D) In situ hybridization for tyrosinasemRNA in D. rerio (A,B) and D. albolineatus (C,D) during middle stages of pigment pattern metamorphosis (equivalent to larvae shown in Fig. 6C,I). (A) In D. rerio, numerous tyrosinase+ melanophores and unmelanized melanophore precursors (arrowhead) are observed in the region of the prospective dorsal primary melanophore stripe. (B) Higher magnification image of a different larvae showing tyrosinase+melanophores (arrow) and melanophore precursors (arrowhead). (C) In D. albolineatus, fewer melanophores are present but tyrosinase+ melanophore precursors (arrowhead) are not obviously reduced in number. (D) Higher magnification of a different larva showing tyrosinase+ melanophores (arrowhead) and unmelanized melanophore precursor (arrowhead). (E-H) Expression of other melanophore lineage markers also is similar between D. rerio and D. albolineatus. (E,F) Danio rerio exhibit unmelanized cells(arrowheads), and some melanized cells (arrow) expressing dct (E) and mitfa (F). (G,H) In D. albolineatus, the numbers of unmelanized cells expressing dct (G) and mitfa are similar to D. rerio. Results for kit and sox10-expressing cells were similar (data not shown)(Parichy et al., 1999; Dutton et al., 2001). (I-L)Tyrosinase-expressing melanophore precursors revealed by treating fixed larvae with the essential precursor for melanin synthesis, l-dopa(Camp and Lardelli, 2001; McCauley et al., 2004). Images in I and K are prior to l-dopa incubation, and images in J and L are the same fields of view after treatment with l-dopa for 5 hours. (I,J) In D. rerio, only a few tyrosinase+melanophore precursors are revealed by l-dopa incubation(arrowheads). (K,L) In D. albolineatus, however, numerous tyrosinase+ melanophore precursors are revealed by l-dopa treatment (arrowheads show only a few of these cells). These cells exhibit morphologies typical of melanoblasts and recently differentiated melanophores (inset). The increased number of l-dopa stained,tyrosinase+ cells as compared with molecular markers in D. albolineatus may reflect perduring protein in the absence of transcriptional activity. (M-T). Melanophores and melanophore precursors frequently are lost in D. albolineatus. (M-O) The same region of a D. albolineatus larvae imaged at 12-hour intervals reveals the transient appearance of several melanophores (arrowheads). Yellow-orange cells are xanthophores; these and other melanophores do not change positions between images. (P) High magnification image of D. albolineatus reveals melanin-containing debris (arrow) typical of melanophore death. Arrowhead,melanophore precursor that acquired melanin following l-dopa incubation of this larva. (Q-S) Cross-sections reveal the locations of melanophores and tyrosinase+ melanophore precursors in D. rerio (Q) and D. albolineatus (R,S). (Q) In D. rerio,few melanophores or melanophore precursors are located within the plane of the epidermis; one such melanophore is indicated by the arrowhead. e, epidermis;m, myotome. Arrow indicates iridophores of the developing interstripe region.(R) In D. albolineatus, numerous melanophores and tyrosinase+ melanophore precursors occur within the plane of the epidermis (arrowheads), although some melanophores are found subdermally, as in D. rerio (arrow). (S) Higher magnification of bracketed region in R, showing a melanin-containing extrusion body typical of teleost melanophore death. Arrowhead indicates bounding membrane. n, neuromast. Melanin-containing debris in P also is superficially located, as revealed by hexagonal outlines of adjacent epidermal cells. (T) High magnification image of whole-mount larva, showing extrusion body containing melanin granule (arrow) and staining for dct mRNA (arrowhead). Scale bars: in A, 80 μm for A,C; in B,40 μm for B,D; in E, 60 μm for E-H; in I, 80 μm for I-L; in P, 20μm for P; in Q, 60 μm for Q,R; in S, 20 μm for S,T.

Finally, we examined melanophore migration. Total distances moved by melanophores were reduced in D. albolineatus larvae compared with D. rerio larvae (Fig. 7D). Moreover, stripes in D. rerio develop in part through the directional migration of initially more dispersed melanophores to sites of stripe formation, and such movements were significantly reduced in D. albolineatus (Fig. 7E,F). Animations of developing larvae further support the interpretation that D. albolineatus melanophores move less than D. rerio melanophores (see Movies 5, 6 in supplementary material).

Melanophore morphogenesis in D. albolineatus thus resembles melanophore morphogenesis in fms mutant D. rerio, with fewer melanophores, increased death of cells in the melanophore lineage, and reduced melanophore migration as compared with wild-type D. rerio.

Enhanced xanthophore development in D. albolineatus

Melanophore morphogenesis in D. albolineatus is consistent with a model in which this species has evolved a loss of fms activity relative to wild-type D. rerio. This model also predicts that D. albolineatus should have fewer xanthophores, consistent with the reported absence of xanthophores in adult D. albolineatus(McClure, 1999). Yet, our analyses reject this notion: instead, we find that D. albolineatusactually have many more xanthophores than wild-type D. rerio.

During pigment pattern metamorphosis, D. albolineatus had greater numbers of xanthophores and these cells were distributed more widely than in D. rerio, in which xanthophores initially occur only near the horizontal myoseptum, and the dorsal and ventral margins of the flank(Fig. 9A-D,F,G). Xanthophores persist in older larvae and adult D. albolineatus, and are interspersed with melanophores (see Fig. S1 in supplementary material). Moreover, control hybrids between D. albolineatus and wild-type D. rerio had an intermediate number of xanthophores relative to parental species (Fig. 9E,H),in contrast to the severe xanthophore deficiency of fmsj4e1 mutant hybrids(Fig. 4D). Thus, xanthophore development is enhanced in D. albolineatus and this trait is dominant in hybrids but highly sensitive to reduced fms activity. Interestingly, D. choprae similarly exhibit enhanced xanthophore development and a strong fms noncomplementation phenotype(Fig. 2P; see Fig. S2 in supplementary material).

Fig. 9.

Excess xanthophores in D. albolineatus compared with D. rerio. (A-H) Xanthophores in D. rerio (A,B,F), D. albolineatus (C,D,G), and wild-type D. rerio ×D. albolineatus hybrids (E,H). (A) In D. rerio, few xanthophores(arrowhead) are visible during early stages of metamorphosis (stage equivalent to Fig. 6A) and most initially are found ventral to the horizontal myoseptum (box). Arrow indicates iridophores in the prospective primary interstripe region. (B) During middle stages of pigment pattern metamorphosis in D. rerio (e.g. Fig. 6C), xanthophores occur in the developing interstripe region (arrow) and a few faint xanthophores can be seen along the dorsal myotomes. (C) In D. albolineatus, xanthophores(arrowheads) are widely dispersed over the flank during early pigment pattern metamorphosis (e.g. Fig. 6G).(D) During later pigment pattern metamorphosis in D. albolineatus(e.g. Fig. 6I), xanthophores(arrowheads) persist widely scattered over the flank. Reddish erythrophores have started to develop in the interstripe region (arrow) and persist into the adult. Lineage relationships of erythrophores to xanthophores are unclear. (E)In hybrids between wild-type D. rerio and D. albolineatus,excess xanthophores (arrowheads) develop over the flank compared with D. rerio, and reddish erythrophores develop in the interstripe region(arrow). (F-H) Higher magnification images of larval D. rerio, D. albolineatus, and D. rerio ×D. albolineatushybrids shown in B-E. (I-L) In situ hybridization for early markers of the xanthophore lineage (Parichy et al.,2000b; Ziegler et al.,2000). Shown is GTP cyclohydrolase I (gch),which encodes an enzyme required for synthesizing pteridine pigments of xanthophores. Results for xanthine dehydrogenase (xdh),encoding a second pteridine synthesis enzyme were similar (data not shown).(I,J) D. rerio larvae; (K,L) D. albolineatus larvae. (I,K)In both species, gch+ cells occur over the myotomes during early metamorphosis. (J,L) At an optical plane medially within the same larvae shown in I and K, relatively few gch+ cells are observed in D. rerio (J), whereas many gch+ cells are present in D. albolineatus (L). (M,N) In situ hybridization for fms mRNA does not reveal clear differences in the numbers or distributions of fms+ cells between D. rerio (M)and D. albolineatus (N). Scale bars: in A, 100 μm for A,C; in B,160 μm in B,D,E; in F, 60 μm for F-H; in I, 60 μm for I-L; in M, 80μm for M,N.

Fig. 9.

Excess xanthophores in D. albolineatus compared with D. rerio. (A-H) Xanthophores in D. rerio (A,B,F), D. albolineatus (C,D,G), and wild-type D. rerio ×D. albolineatus hybrids (E,H). (A) In D. rerio, few xanthophores(arrowhead) are visible during early stages of metamorphosis (stage equivalent to Fig. 6A) and most initially are found ventral to the horizontal myoseptum (box). Arrow indicates iridophores in the prospective primary interstripe region. (B) During middle stages of pigment pattern metamorphosis in D. rerio (e.g. Fig. 6C), xanthophores occur in the developing interstripe region (arrow) and a few faint xanthophores can be seen along the dorsal myotomes. (C) In D. albolineatus, xanthophores(arrowheads) are widely dispersed over the flank during early pigment pattern metamorphosis (e.g. Fig. 6G).(D) During later pigment pattern metamorphosis in D. albolineatus(e.g. Fig. 6I), xanthophores(arrowheads) persist widely scattered over the flank. Reddish erythrophores have started to develop in the interstripe region (arrow) and persist into the adult. Lineage relationships of erythrophores to xanthophores are unclear. (E)In hybrids between wild-type D. rerio and D. albolineatus,excess xanthophores (arrowheads) develop over the flank compared with D. rerio, and reddish erythrophores develop in the interstripe region(arrow). (F-H) Higher magnification images of larval D. rerio, D. albolineatus, and D. rerio ×D. albolineatushybrids shown in B-E. (I-L) In situ hybridization for early markers of the xanthophore lineage (Parichy et al.,2000b; Ziegler et al.,2000). Shown is GTP cyclohydrolase I (gch),which encodes an enzyme required for synthesizing pteridine pigments of xanthophores. Results for xanthine dehydrogenase (xdh),encoding a second pteridine synthesis enzyme were similar (data not shown).(I,J) D. rerio larvae; (K,L) D. albolineatus larvae. (I,K)In both species, gch+ cells occur over the myotomes during early metamorphosis. (J,L) At an optical plane medially within the same larvae shown in I and K, relatively few gch+ cells are observed in D. rerio (J), whereas many gch+ cells are present in D. albolineatus (L). (M,N) In situ hybridization for fms mRNA does not reveal clear differences in the numbers or distributions of fms+ cells between D. rerio (M)and D. albolineatus (N). Scale bars: in A, 100 μm for A,C; in B,160 μm in B,D,E; in F, 60 μm for F-H; in I, 60 μm for I-L; in M, 80μm for M,N.

To further assess xanthophore development in D. albolineatus, we examined the distributions of cells expressing molecular markers of the xanthophore lineage. Between the epidermis and myotomes, where the adult pigment pattern develops, precursor distributions were similar between species(Fig. 9I,K). These data suggest that species differences in xanthophore development reflect differences in terminal differentiation of widely distributed precursors, rather than differences in the abundance or patterning of precursors themselves. In medial locations, however, precursors were more abundant in D. albolineatusthan D. rerio (Fig. 9J,L).

The many xanthophores and xanthophore precursors in D. albolineatus suggest that fms continues to be functional in this species. Consistent with this possibility, we could not detect differences in fms expression between D. rerio and D. albolineatusduring or after pigment pattern metamorphosis(Fig. 9M,N), and gross lesions are not apparent in the fms coding sequence(Parichy and Johnson,2001).

These results show that D. albolineatus develop xanthophores in greater numbers and over a broader area than D. rerio, tending to exclude a model in which the evolutionary loss of stripes in D. albolineatus results simply from a loss of fms activity.

Evolutionary changes in cell-cell interactions during pigment pattern formation

The persistence of xanthophores in D. albolineatus led us to seek other explanations for the similarity of melanophore behaviors between this species and fms mutant D. rerio. In wild-type D. rerio, melanophore survival and organization into stripes depends on interactions between melanophores and fms-dependent cells of the xanthophore lineage (Parichy and Turner,2003a), as well as interactions among melanophores. For example,the D. rerio leopard gene mediates both heterotypic interactions between melanophores and xanthophores, and homotypic interactions between melanophores (Maderspacher and Nusslein-Volhard, 2003), which we refer to collectively as`melanophore interactions'. The nature of these interactions is not yet known,but could include direct contacts between melanophores, xanthophores, or their precursors; alternatively, interactions could be indirect, involving secreted signaling molecules, trophic factors, or even intermediary cell types. Whatever their mechanism(s), the nearly uniform pigment pattern of D. albolineaneatus with interspersed melanophores and xanthophores(Fig. 1D; see Fig. S1C in supplementary material) and the irregular stripes of wild-type D. rerio ×D. albolineatus hybrids (compared with other danios, Fig. 2) resemble different D. rerio mutant alleles of leopard(Asai et al., 1999), as well as jaguar (obelix), which contribute to homotypic interactions among melanophores (Maderspacher and Nusslein-Volhard, 2003). Thus, we hypothesized that instead of a loss of xanthophores, stripe absence in D. albolineatus might reflect changes in melanophore interactions. In principle, a species difference in melanophore interactions could be revealed with genetic mosaics(Parichy and Turner, 2003a; Quigley et al., 2004), but incompatibilities during early embryogenesis have so far precluded cell transplantations between D. albolineatus and D. rerio(D.M.P., unpublished). Thus, we used an alternative approach.

We reasoned that variation in melanophore interactions would be revealed if melanophore numbers were reduced (by analogy with reduced xanthophores in fms mutant D. rerio and hybrids with D. albolineatus): with fewer melanophores, strong interactions should allow the emergence of an organized pattern of stripes or spots, whereas weak interactions should result in a failure to organize such pattern elements. To achieve this, we used the D. rerio mutant, duchamput.r19e1. A single mutant allele for duchamp reduces melanophores in heterozygous D. rerio to∼45% that of wild-type, yet the remaining melanophores form well-organized spots (Fig. 10A,B); D. rerio homozygous for duchamp exhibit more dispersed melanophores(see Fig. S3 in supplementary material). We predicted that for species with melanophore interactions equivalent to D. rerio, duchamp hybrids should develop spots similar to heterozygous duchamp mutant D. rerio. For species with weaker melanophore interactions than D. rerio, tester duchamp hybrids should fail to generate organized pattern elements and could exhibit more severe melanophore deficiencies.

Fig. 10.

Differential sensitivities across danios to melanophore reduction during hybrid pigment pattern development. An abbreviated phylogeny is shown on the left and wild-type danio pigment patterns are shown in A,C,E,G,I,K,M. Representative duchamp mutant hybrids are shown in B,D,F,H,J,L,N;schematics illustrate melanophore distributions (dorsal scale-associated melanophores are omitted for clarity). (A,B) Heterozygous duchampmutant D. rerio (B) exhibit spots and fewer melanophores than wild type (A). Xanthophore and iridophore deficits are not apparent. (C,D) duchamp hybrids for D. kyathit develop spots of melanophores, although these are somewhat less regular than in D. rerio, as is the wild-type D. kyathit stripe pattern. (E,F) duchamp hybrids for D. nigrofasciatus develop well-organized spots or even complete stripes. (G,H) duchamp hybrids for D. albolineatus exhibit a more severe melanophore reduction than observed in heterozygous duchamp mutant D. rerio or other hybrids, and these melanophores remain widely dispersed over the caudal flank. As in duchamp mutant D. rerio, gross deficits in xanthophore numbers were not apparent during pigment pattern metamorphosis (data not shown). (I,J) duchamp hybrids for D. `hikari' develop intermediate patterns, in which more melanophores are present than in D. albolineatus hybrids, but melanophore patterns range from weak clustering, to reticulation, to more uniform dispersion. (K-M) duchamp hybrids for the more distantly related D. chopraeand D. dangila develop spots similar to the D. rerio species group. Scale bars: in A, 1 mm for A-L; in M, 0.5 mm for M,N.

Fig. 10.

Differential sensitivities across danios to melanophore reduction during hybrid pigment pattern development. An abbreviated phylogeny is shown on the left and wild-type danio pigment patterns are shown in A,C,E,G,I,K,M. Representative duchamp mutant hybrids are shown in B,D,F,H,J,L,N;schematics illustrate melanophore distributions (dorsal scale-associated melanophores are omitted for clarity). (A,B) Heterozygous duchampmutant D. rerio (B) exhibit spots and fewer melanophores than wild type (A). Xanthophore and iridophore deficits are not apparent. (C,D) duchamp hybrids for D. kyathit develop spots of melanophores, although these are somewhat less regular than in D. rerio, as is the wild-type D. kyathit stripe pattern. (E,F) duchamp hybrids for D. nigrofasciatus develop well-organized spots or even complete stripes. (G,H) duchamp hybrids for D. albolineatus exhibit a more severe melanophore reduction than observed in heterozygous duchamp mutant D. rerio or other hybrids, and these melanophores remain widely dispersed over the caudal flank. As in duchamp mutant D. rerio, gross deficits in xanthophore numbers were not apparent during pigment pattern metamorphosis (data not shown). (I,J) duchamp hybrids for D. `hikari' develop intermediate patterns, in which more melanophores are present than in D. albolineatus hybrids, but melanophore patterns range from weak clustering, to reticulation, to more uniform dispersion. (K-M) duchamp hybrids for the more distantly related D. chopraeand D. dangila develop spots similar to the D. rerio species group. Scale bars: in A, 1 mm for A-L; in M, 0.5 mm for M,N.

Phenotypes of tester duchamp hybrids support a model in which variation in melanophore interactions contributes to pigment pattern differences among danios. Within the clade that includes D. rerio,duchamp hybrids for both D. kyathit and D. nigrofasciatus developed spots or stripes of melanophores(Fig. 10C-F). By contrast, duchamp hybrids for D. albolineatus failed to develop organized spots or stripes and also had a reduction in melanophore numbers that became increasingly severe as fish grew(Fig. 10G,H, Fig. 11C,D). duchamphybrids with D. `hikari' developed an intermediate phenotype between D. albolineatus and other danio hybrids, in which melanophores failed to organize into spots and remained either dispersed or in a reticulated pattern (Fig. 10I,J). Finally, duchamp hybrids for D. choprae and D. dangiladeveloped clusters of melanophores similar to D. rerio(Fig. 10K-N, Fig. 11A,B).

Fig. 11.

Melanophore and iridophore patterns in tester duchamp mutant hybrids. (A,B) Different illumination of the same fields of view reveals melanophore organization (A,C) and iridophore organization (B,D). (A,B) Tester duchamp hybrids for D. dangila exhibit clusters of melanophores (arrows) with iridophores (arrowheads) organized around these clusters, as for hybrids of the D. rerio species group. (C,D) Tester duchamp hybrids for D. albolineatus typically do not form clusters of melanophores (arrows), and iridophores (arrowheads) are poorly organized. Scale bars: in A, 200 μm for A,B; in C, 200 μm for C,D.

Fig. 11.

Melanophore and iridophore patterns in tester duchamp mutant hybrids. (A,B) Different illumination of the same fields of view reveals melanophore organization (A,C) and iridophore organization (B,D). (A,B) Tester duchamp hybrids for D. dangila exhibit clusters of melanophores (arrows) with iridophores (arrowheads) organized around these clusters, as for hybrids of the D. rerio species group. (C,D) Tester duchamp hybrids for D. albolineatus typically do not form clusters of melanophores (arrows), and iridophores (arrowheads) are poorly organized. Scale bars: in A, 200 μm for A,B; in C, 200 μm for C,D.

These analyses demonstrate that hybrid pigment patterns exhibit differential sensitivity across species to the duchamp mutant defect,with the greatest sensitivity in D. albolineatus. These findings are consistent with a model in which variation in melanophore interactions contribute to pigment pattern variation among danios.

Danio pigment patterns are emerging as a useful system for understanding both the development and evolution of adult form in vertebrates. Our study suggests a model for how evolutionary changes in pigment cell development have generated naturally occurring variation in this ecologically and behaviorally significant trait.

Evolution of melanophore patterning in Danio

Melanophore patterns vary markedly among danios, and our analyses demonstrate dramatic differences in melanophore morphogenesis underlying the uniform pattern of D. albolineatus and the striped pattern of D. rerio. Fewer melanophores accumulate during pigment pattern metamorphosis in D. albolineatus, largely due to increased death of melanophores and their immediate precursors. This late block in melanophore development resembles that seen in Astyanax cavefish(McCauley et al., 2004), but contrasts with an early block affecting melanophore specification in D. nigrofasciatus, which accounts for an equivalent total melanophore deficit as compared with D. rerio(Quigley et al., 2004). Interestingly, the mode of melanophore loss in D. albolineatusresembles that of kit mutant D. rerio(Parichy et al., 1999),raising the possibility of a difference in kit signaling between species. Finally, we also demonstrate that D. albolineatus melanophores move little and thus do not coalesce into distinctive stripes as in D. rerio. In these respects, pigment pattern metamorphosis of D. albolineatus resembles that of fms mutant D. rerio. However, this is where the similarities end, as D. albolineatusretain large numbers of xanthophores, in stark contrast to fms mutant D. rerio.

We propose a model in which changes in melanophore interactions underlie the evolutionary loss of stripes in D. albolineatus. In D. rerio, stripe formation depends on interactions between melanophores and fms-dependent cells of the xanthophore lineage, as well as on interactions among melanophores; in the absence of such interactions,initially dispersed melanophores fail to migrate into stripes and melanophore death is increased (Maderspacher and Nusslein-Volhard, 2003; Parichy and Turner, 2003a). Genetic analyses of D. albolineatus initially suggested that changes in melanophore behaviors might result from a fms-dependent loss of xanthophores. Yet, the persistence of xanthophores excludes this model. Rather, we favor an alternative scenario involving changes in interactions between melanophores and xanthophores, or between melanophores themselves. This model does not exclude the possibility that differences outside of pigment cell lineages also influence species differences in melanophore morphogenesis, either directly, or by modulating the competence of pigment cells to interact with one another.

Several lines of evidence support a model in which the loss of stripes in D. albolineatus results at least partly from changes in melanophore interactions. First, we find an interspersed arrangement of D. albolineatus melanophores and xanthophores, which resembles leopard mutant D. rerio that are defective for melanophore-melanophore and melanophorexanthophore interactions, as well as jaguar (obelix) mutant D. rerio that are defective for melanophore-melanophore interactions(Maderspacher and Nusslein-Volhard,2003). Moreover, hybrids between D. albolineatus and semi-dominant jaguarc5 mutant D. rerio develop uniform pigment patterns like D. albolineatus that are qualitatively more severe than the heterozygous jaguarc5 pigment pattern(Parichy and Johnson, 2001),suggesting a difference between species in the jaguar pathway or in jaguar-dependent cellular interactions. However, hybrids between D. rerio carrying a recessive jaguarc7 deficiency and D. albolineatus are indistinguishable from control hybrids,suggesting the jaguar gene of D. albolineatus is not grossly hypomorphic compared to wild-type D. rerio (this study, data not shown).

Second, phenotypes of hybrids between fms mutant D. rerioand D. albolineatus are consistent with evolutionary changes in melanophore interactions. In these hybrids, melanophore patterns more closely resemble the uniform pattern of D. albolineatus. fms acts autonomously to the xanthophore lineage in promoting melanophore stripe organization in D. rerio (Parichy and Turner, 2003a). Thus, the dramatically fewer xanthophores in hybrids between D. albolineatus and fms mutant D. rerio may phenocopy the actual difference between species; i.e. melanophore interactions likely to have been lost evolutionarily are restored in the control hybrid, but abrogated by the reduced xanthophore number of the fms mutant hybrid (14% of control; Fig. 4).

Third, phenotypes of hybrids between D. albolineatus and duchamp mutant D. rerio are consistent with interspecific changes in melanophore interactions. When we use the duchamp mutant allele of D. rerio to reduce melanophore numbers in hybrids, clusters of melanophores form on the flank of D. rerio and hybrids of four other species, but not D.albolineatus. The failure of D. albolineatus hybrids to organize melanophore spots does not reflect phylogenetic distance from D. rerio, as spots were formed in hybrids of the more distantly related D. dangila and D. choprae. Nor does the absence of spots result merely from a low starting number of melanophores in D. albolineatus, as the adult melanophore number is indistinguishable between this species and D. nigrofasciatus (Quigley et al.,2004). Finally, the lack of spots does not result from a higher growth rate that might carry melanophores passively away from one another, as D. dangila hybrids develop spots, despite growing more rapidly than D. albolineatus hybrids (D.M.P., unpublished). Our finding that duchamp hybrids for D. `hikari' have a phenotype intermediate to D. albolineatus and the other danios suggests that changes in melanophore interactions may have contributed to overall differences between the albolineatus-'hikari' clade and other danios,with a more extreme difference in D. albolineatus conceivably responsible for the absence of stripes in this species. Identification of the duchamp gene product will allow a fuller analysis of roles for this gene and pathway in melanophore interactions and their evolution.

Our analyses and previous studies of D. rerio highlight the potentially important role that intercellular interactions are likely to play in the development and evolution of neural crest derivatives. Such interactions have started to be characterized within melanocyte, neurogenic and rhombomeric lineages as well(Aubin-Houzelstein et al.,1998; Hagedorn et al.,1999; Trainor and Krumlauf,2000; Paratore et al.,2002; Hou et al.,2004). In principle, melanophore interactions could involve factors secreted or presented at the cell surface(Wehrle-Haller, 2003; Hou et al., 2004), or adhesive or junctional contacts among pigment cells or their precursors(Twitty, 1945; Tucker and Erickson, 1986; Parichy, 1996), although increased stratification of skin and pigment cell locations may preclude some direct contacts in adults (Hirata et al.,2003). By extension, evolutionary changes could reflect modifications to the interactions, if the competence to provide or receive signals is altered. Or, the same outcome could be effected by changes to the cellular context in which these interactions occur. For instance, simply the increased number of xanthophores in D. albolineatus may interfere with melanophore-melanophore interactions. In support of this notion,melanophores that become isolated within xanthophore-rich interstripe regions typically are lost in D. rerio(Goodrich et al., 1954; Parichy and Turner, 2003b),whereas in the anal fin of D. albolineatus, melanophores and xanthophores appear in temporally and spatially distinct waves, and a narrow melanophore stripe develops (Goodrich and Greene, 1959). Whatever their mechanisms, interactions within and among pigment cell classes suggest a rich source of variation for the evolutionary diversification of pigment patterns, without the necessity of correlated changes in other cell and tissue types. Such an independence of pigment pattern variation from other traits may in turn explain rapid and extensive pigment pattern evolution across species that are otherwise relatively similar in form.

Evolution of fms activity and function during xanthophore development

Interspecific complementation tests initially suggested that D. albolineatus could have reduced fms activity compared with D. rerio, as hybrids between wild-type D. rerio develop well-organized stripes, whereas hybrids with fmsj4e1mutant D. rerio either lack stripes or have poorly formed stripes,depending on genetic background (this study)(Parichy and Johnson, 2001). Despite this non-complementation phenotype, our analyses do not support a model in which D. albolineatus have evolved a loss of fmsactivity. Rather, these data suggest that species differ in their dosage sensitivity for fms during xanthophore development, or that evolutionary changes have occurred that affect molecular interactions within the fms pathway.

Despite a reported absence of xanthophores in adult D. albolineatus (McClure,1999), we find that larvae develop more xanthophores, these cells and their precursors arise in a broader range of locations than in D. rerio, and also persist into the adult. These observations contrast with the simplest loss-of-function model, in which xanthophores should be absent or reduced.

The excess of xanthophores in wild-type control D. rerio× D. albolineatus hybrids, and the reduction of xanthophores in tester fms mutant D. rerio ×D. albolineatushybrids reveals a genetic interaction not predicted by the recessive fms mutant phenotype in D. rerio. We envisage at least two complementary explanations for this interaction. First, xanthophore development may differ between species in its sensitivity to changes in Fms signaling. Genetic background effects are well known for mouse melanocyte mutants, including the structurally and functionally similar Kitlocus, and variable degrees of haploinsufficiency have been associated with modifier loci for mutants affecting neural crest derivatives more generally(Lamoreux, 1999; Ingram et al., 2000; Rhim et al., 2000; Nadeau, 2001; Cantrell et al., 2004). Conceivably, the more rapid and extensive development of xanthophores in D. albolineatus (and D. choprae) could entail a greater,continuous requirement for fms, such that any deficit results in pigment pattern defects.

A second explanation for fms hybrid non-complementation phenotypes lies in interspecific structural differences in fms, its ligand, csf1, or both. As fms and csf1 each act as dimers(Li and Stanley, 1991; Carlberg and Rohrschneider,1994; Ingram et al.,2000), signaling could be reduced if interspecific receptors or ligands dimerize less efficiently, or if structures of receptors and ligands co-evolve so that mismatched receptor-ligand pairings function less efficiently. To illustrate this point, we can imagine an extreme model in which any species mismatch in a receptor-ligand pairing ablates signaling. In a wild-type hybrid, functional receptor-ligand pairings would drop to one-eighth that of parental species (i.e. each species' pairing would comprise one-sixteenth of all combinations in the hybrid individual). In the tester fms mutant hybrid, functional receptor-ligand interactions would drop to one-sixteenth that of parental species. Given a fixed threshold of dosage sensitivity (here, between one-eighth and one-sixteenth of maximal), this model can easily account for the noncomplementation phenotype of tester fms mutant hybrids.

By extension, our analyses suggest evolution of cellular requirements for fms or rapid evolution of genes within the fms pathway. In fms hybrids, we observed strong noncomplementation phenotypes for D. albolineatus, D. aff. albolineatus and D. choprae, a weak non-complementation phenotype for D. `hikari',but complementation indistinguishable from wild-type D. rerio for D. kyathit, D.nigrofasciatus and D. dangila. These findings suggest most parsimoniously that: (1) changes have occurred that differentiate the D. albolineatus-D. `hikari' clade from the D. rerio-D. kyathit-D. nigrofasciatus clade; and (2)additional changes have occurred in the lineages leading either to D. choprae or D. dangila. This interspecific variation is striking,as similar tests across danios have failed to reveal noncomplementation for more than a dozen other D. rerio pigment pattern mutants, including the structurally and functionally similar kit locus(Parichy and Johnson, 2001)(D.M.P., unpublished). Thus, the fms pathway may be particularly useful for investigating the evolution of genetic dominance and developmental robustness, as well as the co-evolution of gene products within molecular pathways (Meir et al., 2002; Nijhout, 2002; Kondrashov and Koonin,2004).

Finally, this study reveals enhanced xanthophore development in both D. albolineatus and D. choprae compared with wild-type D. rerio. These findings raise the possibilities of differences in the distribution or abundance of csf1, or quantitative (as distinct from constitutive) gains of fms function in D. albolineatus and D. choprae compared with other danios. The latter possibility is opposite to initial predictions of allelic strengths(Parichy and Johnson, 2001),but is not inconsistent with the models proposed above. Immunohistochemical,transgenic, and other approaches should allow distinguishing between these models.

Thanks to C. Lee for help rearing fish, as well as M. Reedy, J. Wallingford and two anonymous reviewers for insightful discussions or comments on the manuscript. T. Wilcox provided valuable advice on phylogenetic methods. Mutant stocks were generously provided by M. Halpern, R. Knight, J. Lister, D. Raible, T. Schilling, S. Johnson, K. Poss and the Zebrafish International Resources Center at the University of Oregon. Supported by NIH R01 GM62182 to D.M.P.

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