Section of Integrative Biology, Section of Molecular, Cell and Developmental Biology, Institute for Cellular and Molecular Biology, University of Texas at Austin, 1 University Station C0930, Austin, TX 78712, USA
Author for correspondence (e-mail:
dparichy{at}mail.utexas.edu)
Accepted 28 October 2004
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SUMMARY |
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Key words: Zebrafish, Pigment pattern, Morphogenesis, Neural crest, fms, Csf1r, Xanthophore, Melanophore, Phylogeny, Evolution
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Introduction |
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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.
|
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 fms
activity 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 fms
activity 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.
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Materials and methods |
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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
).
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Results |
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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. rerio
and 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 fms
locus.
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. rerio
by 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).
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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.
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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. albolineatus
might 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).
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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. albolineatus
actually 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).
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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. albolineatus
during 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 xD. 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.
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Discussion |
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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. albolineatus
resembles 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. albolineatus
retain 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. rerio
and 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 fmsj4e1
mutant 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 fms
activity. 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
xD. albolineatus hybrids, and the reduction of xanthophores in
tester fms mutant D. rerio xD. albolineatus
hybrids 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 Kit
locus, 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.
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Supplementary material |
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ACKNOWLEDGMENTS |
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Footnotes |
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