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 11 October 2004
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SUMMARY |
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Key words: Zebrafish, Pigment pattern, Evolution, Morphogenesis, Neural crest, Stem cell, Phylogeny
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Introduction |
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Given the biomedical and evolutionary significance of neural crest cells
and their derivatives, it is of paramount importance to identify the
mechanisms by which these cells are patterned to generate the particular forms
expressed by juveniles and adults. Most studies have focused on the early
patterning of neural crest cells during embryogenesis. Yet, recent studies
have demonstrated post-embryonic neural crest-derived stem cells in peripheral
nerves, gut and skin (Morrison et al.,
1999; Bixby et al.,
2002
; Kruger et al.,
2002
; Nishimura et al.,
2002
; Iwashita et al.,
2003
; Sieber-Blum and Grim,
2004
; Sieber-Blum et al.,
2004
; Joseph et al.,
2004
). These findings suggest that the development and maintenance
of adult traits, as well as the evolution of these traits, may depend on
contributions from latent stem cells in addition to direct contributions from
neural crest cells at embryonic stages.
A useful system for studying the development and evolution of neural
crest-derived traits is the pigment pattern of teleost fishes
(Quigley and Parichy, 2002;
Parichy, 2003
;
Kelsh, 2004
). In the zebrafish
Danio rerio, an early larval pigment pattern develops during
embryogenesis as neural crest cells differentiate into early larval
melanophores and other pigment cell classes. This pattern is largely completed
by 3 days post-fertilization (dpf), and includes melanophore stripes along the
dorsal and ventral edges of the myotomes, and along the horizontal myoseptum
(Milos and Dingle, 1978a
;
Kelsh et al., 2000
). The early
larval pigment pattern remains essentially unchanged for about two weeks,
until the onset of pigment pattern metamorphosis. At this time, new
melanophores appear over the flank in regions not previously occupied by these
cells, and during the following two weeks, the pigment pattern is transformed
into that of the adult (Fig. 1)
(Kirschbaum, 1975
;
Johnson et al., 1995
;
Parichy et al., 2000b
).
Genetic and cellular analyses demonstrate that new melanophores arising at
metamorphosis differentiate from latent precursors or stem cells of
presumptive neural crest origin (Johnson
et al., 1995
; Parichy and
Turner, 2003b
); such melanophores also play a crucial role in
pigment pattern regeneration (Goodrich and
Nichols, 1931
; Rawls and
Johnson, 2000
; Rawls and
Johnson, 2001
). Although previous studies provide compelling
evidence that metamorphic and regenerative melanophores are derived from
post-embryonic latent precursors, specific markers for these cells have not
been demonstrated, and their locations, potencies and developmental
requirements remain largely unknown. Given these caveats, we refer to these
post-embryonic melanophores simply as `metamorphic' melanophores.
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In this study, we ask whether adult melanophore stripes develop similarly across species, and in particular, whether the relative roles of neural crest-derived early larval melanophores and metamorphic melanophores have been maintained during evolution. To address this question, we first examine D. nigrofasciatus (Fig. 1), a species having fewer melanophores and stripes than D. rerio, but in which stripes that do develop are similar to those of D. rerio. Whereas stripes in D. rerio arise almost entirely from metamorphic melanophores, we show that stripes in D. nigrofasciatus arise from fewer metamorphic melanophores and an increased number of neural crest-derived early larval melanophores that persist into the adult. This interspecific variation led us to test the relative roles of these melanophore lineages during pigment pattern development in several additional species. These analyses demonstrate that a primary role for metamorphic melanophores in adult pigment pattern formation is likely to be ancestral for Danio, and that D. nigrofasciatus exhibits a unique, derived reduction in these cells, with a corresponding increased contribution of early larval melanophores to the adult pigment pattern. We further demonstrate that evolutionary changes within D. nigrofasciatus are extrinsic (non-autonomous) to the melanophore lineages, and we identify a candidate genetic pathway for mediating this change. These analyses highlight the potential for studies of D. rerio and its relatives to reveal basic mechanisms of post-embryonic neural crest development.
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Materials and methods |
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Nomenclature for pigment pattern elements at larval and adult stages
Previous studies defined pigment pattern elements in D. rerio
(Parichy and Johnson, 2001;
Parichy and Turner, 2003b
),
including: early larval dorsal, lateral and ventral melanophore
stripes (ELD, ELL, ELV); adult first-developing (primary) dorsal and
ventral melanophore stripes (1D, 1V); and later-developing (secondary) dorsal
and ventral melanophore stripes (2D, 2V). Additionally, between adult
melanophore stripes are xanthophore-rich `interstripe' regions. For
simplicity, we use the term `stripes' to refer exclusively to the adult
primary melanophore stripes (1D, 1V), unless indicated otherwise.
Microscopy, imaging and quantitative analyses
To examine melanophore behavior, we repeatedly imaged individual larvae
during pigment pattern metamorphosis, allowing us to follow the appearance,
disappearance and migration of individual melanophores
(Parichy et al., 2000b;
Parichy and Turner, 2003b
).
Individually reared fish were anesthetized with MS222 (Sigma) and imaged every
24 hours using an Olympus SZX-12 stereozoom microscope. To ensure that we
could follow cells at the edges of the flank, all fish were imaged lying
parallel to the camera, and also on a specially constructed stand providing an
angle 30° from normal. Images were transferred to Adobe Photoshop CS for
analysis, in some cases in conjunction with the FoveaPro 3.0 image processing
and analysis package (Reindeer Graphics).
Individual melanophores were tracked as previously described
(Parichy et al., 2000b;
Parichy and Turner, 2003b
),
with newly differentiated melanophores clearly distinguishable from
pre-existing melanophores by their initially lighter melanization (and in some
instances different color, see below). We identified individual melanophores
present in the early larval pigment patterns, then examined the fates of these
cells by examining their positions in sequential images. In following early
larval melanophores through metamorphosis, we could not formally observe cell
division in static image series, so we tracked only one presumptive daughter
following likely mitoses. Thus, our counts and estimated proportions of early
larval melanophore contributions to later stages in D. nigrofasciatus
are conservative and may underestimate true values to some degree. For
comparisons of early larval and metamorphic melanophore numbers between
species, we defined an area of interest bounded anteriorly by the anteriormost
anal fin ray insertion and posteriorly at two myotomes anterior to the caudal
peduncle. We counted individual early larval melanophores unilaterally within
this region. We determined total adult melanophore numbers within this region,
either within the adult ventral primary melanophore stripe, if present, or at
an equivalent dorsoventral position as observed in D. rerio, with a
height defined arbitrarily as one-quarter the flank height, as measured at the
anterior boundary. In final images from each individual, all melanophores in
the region of interest were marked and counted, either by eye or by the Count
plug-in of FoveaPro 3.0. Numbers of metamorphic melanophores were thus
calculated as the difference between the total numbers of melanophores
identified in the final images, and the numbers of melanophores that had been
followed into the region of interest from early larval stages. Statistical
analyses were performed with JMP 5.0.1a Statistical Software (SAS Institute,
Cary, NC). Additional information on quantitative image analyses is available
on request.
Cell transplantation and genetic mosaic analysis
We transplanted cells between mid-blastula stage [3.3-3.8 hours
post-fertilization (hpf)] D. rerio and D. nigrofasciatus
embryos, using a Narishige IM-9B micrometer-driven microinjection apparatus
mounted on a Narishige micromanipulator. We placed embryos in agar-lined
dishes containing 10% Hanks solution plus 1% penicillin/streptomycin, and
dechorionated embryos with fine forceps. We transplanted 20-100 cells into
each recipient and reared chimeric individuals through adult stages. To
identify donor D. rerio cells in D. nigrofasciatus hosts, we
used donors that were transgenic for EGFP driven by a ubiquitously expressed
D. rerio ß-actin promoter, kindly provided by Ken Poss
(Parichy and Turner, 2003a;
Parichy et al., 2003
). To
identify donor D. nigrofasciatus melanophores in D. rerio
hosts, we used hosts mutant for albino or nacre
(mitfa), which fail to develop melanin and melanophores,
respectively. Both of these mutant loci normally act autonomously to the
melanophore lineage, as revealed previously
(Lin et al., 1992
;
Lister et al., 1999
;
Parichy and Turner, 2003a
) and
confirmed in control experiments performed for the present analyses (data not
shown). Previous studies reveal minimal local correlation between the
distributions of pigment cells and other tissues in genetic mosaics examined
at metamorphic and adult stages
(Maderspacher and Nusslein-Volhard,
2003
; Parichy and Turner,
2003a
; Parichy et al.,
2003
). We confirmed that donor melanophores typically develop
independently of other local donor tissues in a subset of chimeras in which
donor embryos were injected with rhodamine dextran prior to the four-cell
stage, then were examined for the distribution of melanophores and other
tissues at 4 dpf (data not shown). We sorted chimeras at 3 dpf for the
presence or absence of donor melanophores, and as larvae approached
metamorphosis, we repeatedly imaged individual larvae to follow the behavior
of early larval melanophores and to assess the distribution of metamorphic
melanophores. Survival rates for interspecific chimeras were typically 5-10%
of that observed for comparable experiments involving only D. rerio
(Parichy and Turner, 2003a
;
Parichy et al., 2003
),
suggesting some species incompatibilities;
1% of chimeras were
informative for analyses of pigment pattern formation (see Results).
In situ hybridization and histology
We used in situ hybridization to detect transcripts for melanophore lineage
markers, as described previously (Parichy
et al., 2000a; Parichy et al.,
2000b
; Parichy et al.,
2003
). Larvae were fixed briefly in 4% paraformaldehyde, 1% DMSO
in PBS, decapitated, and then fixed overnight at 4°C. Larvae were
transferred to methanol, rehydrated to PBST (PBS with 0.2% Tween-20), then
treated for 20 minutes at room temperature with 20 µg/ml proteinase-K in
PBST containing 1% DMSO. Larvae were postfixed for 20 minutes at room
temperature in 4% paraformaldehyde, 0.005% glutaraldehyde, washed in PBST,
then washed three times in hybridization solution lacking tRNA and heparin.
Prehybridizations were performed overnight at 60°C in hybridization
solution (50% formamide, 5xSSC, 500 µg/ml yeast tRNA, 50 µg/ml
heparin, 0.2% Tween-20, 9.2 mM citric acid). Hybridizations were performed at
60°C over two nights, in fresh hybridization solution containing
digoxigenin-labeled riboprobes fractionated to
300 nucleotides. Larvae
were then washed twice, for 15 minutes each, in 2xSSCT, and three times,
for 2 hours each, in 0.2xSSCT at 60°C. After graded changes to PBST,
larvae were blocked overnight at 4°C in 2 mg/ml BSA, 5% heat-inactivated
calf serum in PBST, then incubated at 4°C over two nights in fresh
blocking reagent containing 1:5000 anti-digoxigenin alkaline
phosphatase-conjugated Fab fragments (Roche). Larvae were washed over two
nights in PBST, transferred to alkaline phosphatase buffer [100 mM Tris (pH
9.5), 50 mM MgCl2, 100 mM NaCl, 0.1% Tween-20], and the color
developed with NBT/BCIP.
To assay for tyrosinase activity, larvae were fixed for 2 hours in 4%
paraformaldehyde in PBS, rinsed three times in PBS, incubated in 0.1%
L-dopa (Sigma) for 1 hour to overnight, rinsed in PBS, then stored
in glycerol (Camp and Lardelli,
2001; McCauley et al.,
2004
). We verified the specificity of the assay for melanoblasts
by the reduced staining on the flanks of metamorphosing nacre mutant
D. rerio, which have defects in the melanophore lineage
(Lister et al., 1999
;
Parichy et al., 2000b
), and we
verified that newly melanized (tyrosinase+) cells are not
macrophages by Neutral Red staining
(Herbomel et al., 1999
) (data
not shown).
Phylogenetic analysis
We reconstructed phylogenetic relationships based on mitochondrial 12S and
16S rDNA sequences, obtained using standard methods and universal primers
(Kocher et al., 1989;
Palumbi et al., 1991
).
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'.
Sequences were aligned using CLUSTAL-W, inspected by eye and edited as
necessary. We then analyzed combined 12S and 16S sequences (784 nucleotides)
using maximum likelihood estimation in PAUP* 4.0b10 for Macintosh
(Swofford, 2002). Maximum
likelihood analyses used a general time-reversible plus gamma model.
Substitution rate matrix, nucleotide frequencies, and among site rate
variation were estimated from the data by preliminary parsimony analyses using
a heuristic search strategy. Maximum likelihood, parsimony and distance
methods produced trees with the same topology. To estimate confidence values
for reconstructed nodes, we performed two independent analyses. First, we
performed 100 nonparametric bootstrap replicates using PAUP*.
Second, we performed a Bayesian analysis of the data using MrBayes
(Larget and Simon, 1999
;
Huelsenbeck and Ronquist,
2001
; Wilcox et al.,
2002
), with 3000 replicate trees from 300,000 generations
following the approach to asymptotic likelihood values. Both approaches gave
nearly identical confidence values, which we report as percentages of
recovered trees in the phylogram (see Results).
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Results |
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Closer inspection reveals about twice as many melanophores in D.
rerio than in D. nigrofasciatus
(Fig. 2A,C; see below).
Melanophore colors differ as well. In D. rerio, the dorsal and
ventral stripes consist almost entirely of grey-black melanophores. Yet,
occasional brownish melanophores occur at the ventral edge of the dorsal
stripe (Fig. 2A,B), where a few
melanophores derive not from latent precursors at metamorphosis, but from the
rearrangement of embryo-derived melanophores originally present in the early
larval lateral stripe along the horizontal myoseptum
(Parichy and Turner, 2003b).
In D. nigrofasciatus, however, both dorsal and ventral stripes
contain numerous brown melanophores (Fig.
2C,D), and melanophores are not present along the ventral myotome
edge (where the early larval ventral stripe had been). Melanophore color
variation is apparent transiently after metamorphosis, and is not equally
pronounced in all families; whether this variation reflects the age of the
melanin contained within the cells or some other biochemical difference is not
clear. Nevertheless, the differences in melanophore colors and their relative
frequencies in the adult pigment patterns of D. rerio and D.
nigrofasciatus led us to hypothesize that cryptic patterning variation
might underlie the superficially similar stripes between these species.
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In D. rerio, the onset of pigment pattern metamorphosis is marked
by the differentiation of single `pioneer' metamorphic melanophores over the
middle of most ventral myotomes (Fig.
3A). Subsequently, metamorphic melanophores differentiate widely
over the myotomes, between the early larval stripes
(Fig. 3B,C). The adult primary
stripes become increasingly apparent (Fig.
3D), as initially dispersed metamorphic melanophores migrate short
distances to the sites of stripe formation, and as additional metamorphic
melanophores differentiate within the stripes themselves
(Fig. 3C,D). A few early larval
melanophores migrate from the horizontal myoseptum to join the dorsal adult
primary melanophore stripe (Fig.
3D,E), but most remain in place and eventually are lost
(Parichy and Turner, 2003b).
As fish approach the end of metamorphosis, a juvenile pattern emerges, with
adult dorsal and ventral primary melanophore stripes consisting almost
entirely of melanophores that have differentiated from latent precursors
during metamorphosis (Fig.
3E,E').
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Ancestral role for metamorphic melanophores in adult pigment pattern development and derived patterning mechanisms in D. nigrofasciatus
The relative contributions to adult pigment patterns of early larval
melanophores and metamorphic melanophores could vary continuously across
species. Alternatively, either the D. rerio or the D.
nigrofasciatus mode could be typical. To distinguish between these
possibilities, and to determine which, if either, mode is ancestral and which
is derived, we sought to examine pigment pattern metamorphosis in additional
species.
Because danio relationships remain poorly understood, we first sequenced
12S and 16S rDNA from additional taxa to infer phylogenetic relationships
(Fig. 5). These analyses
confirm the close relationship between D. rerio and D.
nigrofasciatus, as well as D. kyathit
(Fig. 1). The phylogeny also
supports a split between Danio and Devario [formerly within
Danio (Fang, 2003)].
Moreover, these data reveal additional pigment pattern diversity within
Danio (as defined in Fig.
5): these fish have been known to have horizontal stripes, spots,
uniform patterns, and more complex pigment patterns; Danio choprae
adds vertical barring to the repertoire
(Fig. 1).
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D. rerio mutants identify a candidate pathway for metamorphic melanophore reduction and early larval melanophore morphogenesis in D. nigrofasciatus
D. rerio mutants can identify genes and pathways that contribute
to interspecific pigment pattern differences
(Parichy and Johnson, 2001).
Given the reduced number of metamorphic melanophores in D.
nigrofasciatus compared to D. rerio, we investigated whether
genes isolated as D. rerio mutants with defects in melanophore
development also contribute to the difference between species. We used
interspecific hybrids to test for complementation of D. rerio mutant
alleles by crossing mutant D. rerio to D. nigrofasciatus and
comparing these tester (mutant) hybrids to control (wild-type) hybrids. Tester
hybrids exhibiting fewer melanophores than controls identify genes that may
contribute to the interspecific difference, whereas tester hybrids that have
similar melanophore numbers to controls identify genes less likely to have
major effect roles.
Control hybrids between wild-type D. rerio and D.
nigrofasciatus have phenotypes intermediate between species. Whereas
melanophore numbers in primary adult stripes are increased over D.
nigrofasciatus and are closer to D. rerio, melanophore numbers
in secondary adult stripes, and the total numbers of stripes, are closer to
D. nigrofasciatus than D. rerio
(Fig. 8A)
(Parichy and Johnson, 2001).
Comparing adult hybrid phenotypes does not reveal gross non-complementation of
the recessive melanophore mutants sox10ut.r13e1,
tfap2ats213, bonaparteut.r16e1,
cezanneut.r17e1, degasut.r18e1,
oberonj198e1, pissarrout.r8e1,
picassout.r2e1, primrosej199, pumaj115e1
or seuratut.r15e1 (e.g.
Fig. 8), adding to the
previously excluded loci ednrb1, fms, kit, mitfa, leopard, fritz and
jaguar (Parichy and Johnson,
2001
). Thus, genes contributing to the differences in the final
numbers of adult melanophores between species either are not likely to be
represented in this collection of 18 D. rerio pigment pattern
mutants, or differences in allelic strengths are not sufficient to reveal
non-complementation.
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We first investigated whether any of several D. rerio mutants
exhibiting stripes dorsally and spots ventrally, as in D.
nigrofasciatus, might have similar modes of pigment pattern metamorphosis
to D. nigrofasciatus. Examination of one of these mutants,
ednrb1 (Parichy et al.,
2000a), revealed little contribution of early larval melanophores
to the adult pigment pattern, unlike in D. nigrofasciatus
(Fig. 9A-D, and data not
shown). Thus, a similarity of pigment pattern elements does not predict the
underlying mode of pigment pattern metamorphosis.
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These results indicate that differences in total numbers of adult melanophores between D. rerio and D. nigrofasciatus are not likely to be explained by differences at loci already isolated as D. rerio melanophore mutants. Moreover, similarity of adult pigment pattern alone is not a good predictor for the underlying mode of pigment pattern metamorphosis. By contrast, interspecific complementation tests for melanophore morphogenesis suggest a role for puma or its pathway in determining the relative contributions of metamorphic melanophores and neural crest-derived early larval melanophores to the adult pigment patterns of D. rerio and D. nigrofasciatus.
Reduction of metamorphic melanophore lineage in D. nigrofasciatus
The reduction in metamorphic melanophores in D. nigrofasciatus
could reflect a failure to recruit committed melanophore precursors
(melanoblasts) from uncommitted latent precursors or stem cells during
metamorphosis. For example, puma mutant D. rerio exhibit
severe reductions in metamorphic melanoblasts compared with wild-type D.
rerio (Parichy et al.,
2003). If the same pathway affected in puma mutant D.
rerio has evolved between D. rerio and D.
nigrofasciatus, then fewer melanoblasts should be observed in D.
nigrofasciatus compared with wild-type D. rerio. Alternatively,
fewer metamorphic melanophores in D. nigrofasciatus could reflect a
later block in this lineage, with similar numbers of melanoblasts being
recruited from latent precursors then failing to terminally differentiate as
melanophores. To distinguish between these possibilities, we used molecular
markers and histological assays to compare D. rerio and D.
nigrofasciatus during metamorphosis.
Our examination of the melanophore lineage during metamorphosis reveals a
severe reduction in the number of melanoblasts in D. nigrofasciatus,
suggesting an early block in metamorphic melanophore development. We examined
the distribution of cells expressing transcripts for two molecular markers,
dopachrome tautomerase (dct) and tyrosinase
(tyr), which encode enzymes required for melanin synthesis and thus
identify committed melanophore precursors (as distinct from latent stem cells)
(Kelsh et al., 2000;
Camp and Lardelli, 2001
). We
observed fewer dct+ and tyr+ cells
throughout metamorphosis in D. nigrofasciatus compared to D.
rerio (Fig. 10).
Importantly, however, we observed strong staining for each marker in fully
differentiated melanophores, and in the more rare, unmelanized cells, in
D. nigrofasciatus, demonstrating the efficacy of these probes in this
cross-species comparison.
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|
Differences between D. rerio and D. nigrofasciatus are non-autonomous to melanophore lineages
The different modes of pigment pattern metamorphosis in D. rerio
and D. nigrofasciatus could reflect evolutionary changes that are
intrinsic (autonomous) or extrinsic (non-autonomous) to melanophore lineages.
Although species differences have been attributed to intrinsic factors
(Twitty and Bodenstein, 1939;
Rawles, 1948
;
Schneider and Helms, 2003
),
the extensive migrations and cellular interactions during neural crest and
melanophore development imply many opportunities for extrinsic factors to
generate differences in form as well
(Erickson and Perris, 1993
;
Parichy, 1996
;
Halloran and Berndt, 2003
). To
distinguish between these possibilities, we examined melanophore behaviors and
patterns in genetic mosaics. These analyses demonstrate a primary role for
extrinsic factors in determining early larval melanophore contributions to
adult stripes, as well as the positions of adult stripes on the flank.
We transplanted cells from D. nigrofasciatus to D. rerio,
and then reared chimeras through metamorphosis
(Parichy and Turner, 2003a;
Parichy et al., 2003
). To
identify donor D. nigrofasciatus melanophores, we used D.
rerio hosts mutant for the albino locus, which acts autonomously
to the melanophore lineage to promote melanization, but does not otherwise
affect melanophore development or pigment pattern formation
(Lin et al., 1992
); D.
nigrofasciatus melanophores thus appear as the only melanized cells in a
field of unmelanized but otherwise normal melanophores
(Lin et al., 1992
;
Parichy et al., 1999
;
Kelsh et al., 2000
). To assess
the mode of pigment pattern metamorphosis, we identified chimeras that
developed D. nigrofasciatus early larval melanophores, then we
repeatedly imaged these individuals through metamorphosis.
We predicted that if species differences are autonomous to the melanophore
lineages, then donor D. nigrofasciatus early larval melanophores
should contribute to the adult ventral melanophore stripe (as in D.
nigrofasciatus); if differences between species are non-autonomous to the
melanophore lineages, then donor D. nigrofasciatus early larval
melanophores should fail to contribute to this stripe (as in D.
rerio). Fig. 12A-D shows
a representative D. nigrofasciatusD. rerio chimera.
Donor D. nigrofasciatus early larval melanophores are present within
the early larval stripe along the ventral myotomes but do not contribute to
the adult ventral stripe. Thus, early larval melanophore morphogenesis
resembles that of D. rerio rather than that of D.
nigrofasciatus (compare with Fig.
3). Moreover, D. nigrofasciatus melanophores that
differentiated during metamorphosis did so at the normal location of D.
rerio stripes, rather than further ventrally as in D.
nigrofasciatus (compare with Fig.
3). These findings indicate that factors non-autonomous to the
melanophore lineages determine species differences in early larval melanophore
contributions to adult stripes, as well as the positions of adult stripes.
These results also obviate the identification of other donor D.
nigrofasciatus cells in D. rerio hosts, as the final
distributions of donor melanophores cannot easily be explained by a simple
coincidence of D. nigrofasciatus melanophores and other D.
nigrofasciatus donor tissues (which might have explained the alternative
result, had donor melanophores behaved like their own, donor species, rather
than the host species).
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Finally, we investigated whether evolutionary changes in interactions
between melanophores themselves might contribute to the different metamorphic
modes between species. We reasoned that a reduction in the numbers of
metamorphic melanophores, and thus reduced contact inhibition of movement
(Tucker and Erickson, 1986),
might allow early larval melanophores to leave their initial positions during
metamorphosis in D. nigrofasciatus. To test this possibility, we
transplanted D. nigrofasciatus cells to nacre mutant D.
rerio hosts. nacre mutants lack melanophores owing to an
inactivating mutation in mitfa, which normally acts autonomously to
the melanophore lineage (Lister et al.,
1999
). We predicted that if changes in melanophore-melanophore
interactions alone are responsible for species differences, then D.
nigrofasciatus early larval melanophores in nacre mutant hosts
should contribute to the adult ventral stripe (as in D.
nigrofasciatus). If other factors contribute to the species differences,
the D. nigrofasciatus early larval melanophores should fail to
contribute to this stripe (as in D. rerio).
Fig. 12F-I shows a D.
nigrofasciatus
nacre mutant D. rerio chimera.
Repeated imaging demonstrates that donor D. nigrofasciatus early
larval melanophores do not contribute to the adult ventral stripe, which forms
at a position similar to that seen in D. rerio. These data
demonstrate that factors extrinsic to melanophore lineages contribute to
differences in pigment pattern metamorphosis between D. rerio and
D. nigrofasciatus.
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Discussion |
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|
Our results suggest a major role for latent precursors, presumptively of
neural crest origin, during the development of adult pigment patterns in
danios and their relatives (Fig.
13). Lineage analyses revealed that in each species (except D.
nigrofasciatus), adult pigment patterns were formed principally by
metamorphic melanophores derived from latent precursors, rather than by early
larval melanophores derived from neural crest cells during embryogenesis. The
prevalence of this mode of pigment pattern metamorphosis strongly suggests
that this is a shared, ancestral trait for Danio. To our knowledge,
this represents the first systematic survey across species to define the
cellular origins for an adult neural crest-derived trait. Previous studies
have demonstrated roles for melanocytes derived from stem cells in the
development of mammalian pigmentation
(Nishimura et al., 2002), and
for melanophores derived from latent precursors in pigment pattern formation
of some teleosts, including D. rerio
(Johnson et al., 1995
;
Sugimoto, 2002
;
Parichy and Turner, 2003b
).
Latent precursors probably also generate adult pigment patterns of many
amphibians (Parichy, 1998
).
Likewise, the adult epibranchial cartilage of the salamander Eurycea
bislineata arises from a discrete population of cells in the
perichondrium of the larval neural crest-derived epibranchial cartilage
(Alberch and Gale, 1986
). Given
the presence of post-embryonic neural crest stem cells and specified latent
precursors in a variety of tissues (see Introduction), it will be interesting
to determine the extent to which other adult traits depend on these cells (as
distinct from embryonic neural crest cells) for their initial patterning,
maintenance, and repair after injury.
The comparative approach we have taken also implicates post-embryonic,
latent precursors of presumptive neural crest origin in the generation of
organismal diversity. We examined species exhibiting a variety of adult
pigment patterns, including horizontal stripes that are compact (D. rerio,
D. kyathit) or diffuse (D. kerri, T. albonubes), as well as
vertical bars (D. choprae) and uniform patterns (D.
albolineatus). Despite this variation in adults, the larvae of these
species exhibit melanophore patterns that are indistinguishable from one
another, except for small differences in melanophore numbers (see Fig. S1 in
supplementary material; Fig.
7A). Our results demonstrate that much of the pigment pattern
diversity of adults reflects interspecific variation in the differentiation
and morphogenesis of metamorphic melanophores that are derived from latent
precursors or stem cells, rather than the reorganization of embryonic neural
crest-derived melanophores. That embryonic/early larval pigment patterns and
adult pigment patterns depend on different melanophore lineages suggests a
mechanism by which these pigment patterns may be relatively uncoupled across
life-cycle stages. Thus, evolutionary responses to selection on the adult
pigment pattern may be relatively unconstrained by features of the earlier
developing embryonic/early larval pigment pattern, if genetic controls differ
to some extent between neural crest-derived and metamorphic melanophore
lineages (Haldane, 1932;
Ebenman, 1992
;
Parichy, 1998
). Indeed,
several D. rerio pigment pattern mutants have defects limited to
particular embryonic or metamorphic melanophore lineages
(Johnson et al., 1995
;
Parichy et al., 1999
;
Parichy et al., 2000a
;
Parichy et al., 2000b
; Parichy
and Turner, 2003). Nevertheless, the extent of genetic independence across
life-cycle stages and its evolutionary consequences remains an
empirical question that deserves further analysis.
Evolution of pigment pattern metamorphosis in D. nigrofasciatus
A central problem in evolutionary developmental biology is the extent to
which similar phenotypes depend on the same or different underlying
mechanisms. Several recent analyses have demonstrated the repeated,
independent evolution of traits via common underlying genetic changes
(Sucena et al., 2003;
Mundy et al., 2004
;
Shapiro et al., 2004
). Such
cases of evolutionary parallelism suggest that pathways of evolutionary change
in development may be more limited than classical evolutionary theory might
suggest (Barton and Turelli,
1989
). By contrast, other analyses reveal divergent mechanisms
underlying repeated trait evolution
(Hoekstra and Nachman, 2003
;
Wittkopp et al., 2003
).
Despite this recent focus on traits that have evolved independently, we still
know little about developmental variation underlying traits having a common
evolutionary origin.
Our analyses reveal substantial differences in stripe development between
D. rerio and D. nigrofasciatus, despite the superficial
similarity of the final stripes that form, and the close phylogenetic
relationship of these species. Cryptic patterning variation has been observed
for other traits (Hall, 1984;
Minsuk and Keller, 1996
;
Jungblut and Sommer, 2000
),
and argues for the importance of a comparative approach in validating
conclusions gleaned from studies of model organisms
(Parichy, 2005
;
Bolker, 1995
;
Metscher and Ahlberg, 1999
).
Such variation may reflect selection to maintain a particular adult phenotype,
in the absence of selection for precisely how this phenotype is achieved. The
roles of teleost pigment patterns in predation avoidance, mate recognition,
mate choice and shoaling behavior suggests strong selection on adult
phenotypes (Endler, 1983
;
Houde, 1997
;
Couldridge and Alexander,
2002
; Engeszer et al.,
2004
; Allender et al.,
2003
); the behavioral roles and selective consequences of early
larval and metamorphic pigment patterns remain wholly unexplored.
Adult pigment pattern formation in D. nigrofasciatus differs from
that of D. rerio in having a lesser contribution from metamorphic
melanophores, and a correspondingly greater contribution from persisting early
larval melanophores (Fig. 13).
Thus, D. nigrofasciatus may be viewed as exhibiting a heterochronic
change in pigment pattern development, with a relatively paedomorphic (or
juvenilized) mode when compared with the inferred ancestral condition. This
uncoupling of pigment pattern and somatic metamorphosis is somewhat similar to
several species and subspecies of salamanders, in which adult spots and
stripes appear during the larval stage, prior to somatic metamorphosis
(Anderson, 1961;
Anderson and Worthington, 1971
;
Parichy, 1998
). Dissociability
of pigment pattern and somatic metamorphosis may be a generalized feature of
post-embryonic development in these ectothermic vertebrates.
The reduction of metamorphic melanophores and the persistence of early
larval melanophores in D. nigrofasciatus also is reminiscent of
several D. rerio mutants. This concordance highlights the utility of
D. rerio mutants both for understanding developmental mechanisms
within zebrafish, and for framing hypotheses that can be tested across species
to dissect mechanisms of evolutionary change. In this study, we examined roles
for several of these mutant loci in pigment pattern diversification using
interspecific complementation tests
(Parichy and Johnson, 2001).
Only hybrids between D. nigrofasciatus and puma mutant
D. rerio exhibited non-complementation phenotypes, with fewer
metamorphic melanophores and increased early larval melanophores persisting
into the adult pattern, when compared with control hybrids. This observation
raises the possibility that puma activity differs between species,
and might therefore explain the derived mode of pigment pattern metamorphosis
in D. nigrofasciatus. Nevertheless, puma acts autonomously
to the metamorphic melanophore lineage
(Parichy et al., 2003
),
whereas interspecific genetic mosaics constructed in this study reveal
differences that are non-autonomous to melanophore lineages (see below). Thus,
it seems unlikely that variation at the puma locus itself contributes
to these species differences. Rather, the non-complementation phenotype may
reflect interspecific variation in a sensitized puma-dependent
pathway. Consistent with this idea, our analyses demonstrate that both
puma mutant D. rerio
(Parichy et al., 2003
) and
D. nigrofasciatus exhibit far fewer metamorphic melanophore
precursors than wild-type D. rerio. These findings imply a change in
the early development or specification of the metamorphic melanophore lineage
in D. nigrofasciatus. This early block differs from the situation
found in several species of Astyanax cave fish
(McCauley et al., 2004
) and
D. albolineatus (Quigley et al.,
2005
), in which melanophore numbers are reduced owing to a later
block in this lineage, such that melanoblasts develop but then fail to
differentiate or survive. Examination of additional species should allow a
more complete reconstruction of the evolutionary history of melanophore
patterns and melanophore lineage modifications across taxa.
Besides the reduction in metamorphic melanophores, D.
nigrofasciatus exhibit a dramatic increase in the contribution of early
larval melanophores to the adult pigment pattern. By examining melanophore
behaviors and patterns in interspecific genetic mosaics, we demonstrate that
factors non-autonomous to melanophore lineages determine the different
behaviors of these cells between species. This contrasts with several studies
that have identified changes autonomous to neural crest or melanophore
lineages in determining species differences
(Twitty and Bodenstein, 1939;
Twitty, 1945
;
Rawles, 1948
;
Epperlein and Löfberg,
1990
; Schneider and Helms,
2003
), although some of these results are open to alternative
interpretations (Parichy,
1996
; Parichy,
2001
). Our findings suggest that the relative roles of intrinsic
and extrinsic factors differ both across species and across traits, and it is
too simplistic to ascribe evolutionary changes to intrinsic factors alone.
At least two models can be suggested for the non-autonomous factors
contributing to the differences in early larval melanophore morphogenesis
between D. rerio and D. nigrofasciatus. First, early larval
melanophores could reorganize in D. nigrofasciatus owing to the
reduced numbers of metamorphic melanophores, which might otherwise prevent the
cells from leaving their original positions within the early larval stripes by
contact inhibition of movement (Tucker and
Erickson, 1986). This explanation would suggest a relatively
simple pattern regulatory process, such that early larval melanophores fill
gaps resulting from reduced numbers of metamorphic melanophores
(Milos and Dingle, 1978b
). Our
examination of genetic mosaics between D. nigrofasciatus and
nacre mutant D. rerio, which lack melanophores, excludes the
loss of interactions between metamorphic melanophores and early larval
melanophores as being the sole factor underlying the difference between
species. Second, early larval melanophores could reorganize owing to changes
in other factors in the extracellular environment. Consistent with this idea,
the adult ventral melanophore stripe develops closer to the myotome edge in
D. nigrofasciatus than in D. rerio. Conceivably, D.
nigrofasciatus early larval melanophores may be close enough to respond
to stripe-forming cues that D. rerio early larval melanophores do not
encounter because these cues are situated further dorsally on the flank. The
difference between species also may reflect multiple partially redundant
changes in factors extrinsic to the melanophore lineage. These possibilities
are now being addressed by additional cell transplantation studies and by
seeking the nature of the stripe-forming cues themselves.
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ACKNOWLEDGMENTS |
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Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/131/24/6053/DC1
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