ORIGINAL ARTICLE

Pigment Cell Melanoma Res. 21; 677–686

Melanophores in the stripes of adult zebrafish do nothave the nature to gather, but disperse when they
have the space to move
Go Takahashi and Shigeru Kondo
Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-Ku, Nagoya,Japan

KEYWORDS
melanophore ⁄ xanthophore ⁄ pigment
pattern ⁄ migration ⁄ laser ablation

CORRESPONDENCE Shigeru Kondo, e-mail: skondo@bio.nagoya-u.ac.jp

PUBLICATION DATA Received 27 March 2008,
revised and accepted for publication 18 August
2008
doi: 10.1111/j.1755-148X.2008.00504.x

Summary
Animal skin pattern is one of the good model systems used to study the mechanism of pattern formation.
Molecular genetic studies with zebrafish have shown that pigment cells play a major role in the mechanismof stripe formation. Among the variety of cellular events that may be involved in the mechanism, aggregationof melanophores has been suggested as an important factor for pattern formation. However, only a few
experimental studies detected the migration ability of melanophores in vivo. Here, we tried to determine
whether melanophores really have the ability to aggregate in the skin of zebrafish. Melanophores in the adult
stripes are packed densely and they rarely move. However, when the neighboring pigment cells are killed,they move and regenerate the stripe pattern, suggesting that melanophores retain the migration ability. To
analyze the migration, we ablated a part of the melanophores by laser to give free space to the remaining
cells; we then traced the migration. Contrary to our expectation, we found that melanophores repulsed one
another and dispersed from the aggregated condition in the absence of xanthophores. Apparent aggregation
may be forced by the stronger repulsive effect against the xanthophores, which excludes melanophores fromthe yellow stripe region.

Introduction
Many vertebrates have a variety of color patterns on
their skin. How these beautiful patterns benefit the natural selection of animals is one of the major questionsof evolutionary biology. On the other hand, these patterns are also of interest from the perspective of spatial
pattern formation in animal development. Animal skinpatterns are generally not similar at all to the shape ofthe tissues under the skin. In the cases of some tropicalfish, pigment patterns in the skin regenerate when they
are perturbed by the growth of the fish or artificial
manipulation of the pigment cells (Kondo and Asai,
1995; Yamaguchi et al., 2007), suggesting that thesepatterns form autonomously in the skin without any prepattern. As most of the known patterning mechanisms
identified in early development are dependent on the‘prepattern’, the discovery of a mechanism of skin pattern formation may lead to finding a new principle that
controls the late-stage morphogenesis where the positional information derived from the egg becomes lessimportant.
Among the model organisms used in molecular
genetic studies, only zebrafish has a very clear pigment
pattern in its skin and fins. Recent studies of skin patterning are thus mainly focused on zebrafish. The
stripes of zebrafish are composed of three types of pigment cells (melanophores, xanthophores, and iridophores) localized in the hypodermis of the skin. The
black stripe mainly consists of melanophores and thin
iridophores. The yellow stripe consists of xanthophores
and thick iridophores (Hirata et al., 2003, 2005).
Screening of artificially induced mutants or the pet
shop lines has identified many kinds of mutant linesª 2008 The Authors, Journal Compilation ª 2008 Blackwell Munksgaard

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Takahashi & Kondo

having different pigment patterns, and the responsible
genes have been cloned. These mutant genes are classified into two categories: those with a defect in thedevelopment of pigment cells and those with normal
development of pigment cells at the embryonic stage
but a disrupted pattern in adulthood. Molecular genetic
studies of the mutants in the first category have shownthat nacre (mitf; Lister et al., 1999), sparse (kit; Parichy
et al., 1999), and rose (ednrb1; Parichy et al., 2000a) are
required for the development of melanophores, and that
panther (fms; Parichy et al., 2000b) is required for thedevelopment of xanthophores. Interestingly, wheneither type of pigment cell ‘mis-develops’, other pigment cells fail to localize to their normal positions. This
fact suggests that the interaction between pigment cells
plays a critical role in the generation of the skin pigmentation pattern (Maderspacher and Nüsslein-Volhard,2003). Mutants of leopard (Kirschbaum, 1975) and jaguar ⁄ obelix (Haffter et al., 1996) belong to the second
category. In these mutants, both types of pigment cells
develop normally, but the arrangement of the cells ischanged from stripes to spots (leopard) or wider stripes(jaguar ⁄ obelix), suggesting that these genes are required
specifically for pattern formation. Recently, positional
cloning of these mutants revealed that the responsible
genes code a gap junction (Connexin41.8; Watanabe
et al., 2006), and an inwardly rectifying potassium channel 7.1 (Kir7.1; Iwashita et al., 2006), respectively. However, little is known about their functions in theinteractions between the pigment cells.
Parichy and Turner (2003a) utilized the temperature
specific allele of the panther gene and killed both melanophores and xanthophores temporally. They haveobserved that the pigment cells re-developed to form
the stripe pattern in the caudal fin. Recently, Yamaguchi
et al. (2007) also found a similar regeneration phenomenon in the body trunk using laser ablation of the pigment cells. Interestingly, in both cases, the regenerated
patterns are not always in the original direction,
whereas the spacing between the lines is identical tothe original. These facts suggest that the stripe pattern
is not fixed, but that the pigment cells in the adult fishretain the ability to generate the stripe pattern. Therefore, by investigating the behavior of the pigment cells
in the adult fish, it should be possible to identify the cellular mechanisms involved in pattern formation.
From the observation of melanophore behavior in thewild-type and mutant lines, it has been suggested thatthe migration of melanophores may play an important rolein pattern formation (McClure, 1999; Kelsh, 2004). In larval pigment pattern formation, Svetic et al. (2007) haveshown Sdf1a, which is an attractant for melanophore, isnecessary for normal pattern formation. At the earlystage of adult pattern formation, melanophores are distributed almost randomly in the body trunk. Then dorsal
melanophores tend to move ventrally, and ventral melanophores tend to move dorsally, to form the initial two
678

black stripes. Once the melanophores are integrated in
the black stripes, migration of the cell becomes rare. Parichy and Turner reported that this movement is impairedin fms (which has dispersed melanophores all over thetrunk) and puma (which has mottled pattern), suggestingthat the defect of such directed migration caused theirabnormal patterns (Parichy et al., 2000b; Parichy andTurner, 2003b). Maderspacher and Nüsslein-Volhard(2003) also pointed the importance of the migration fromtheir chimera experiments and proposed a hypotheticalmechanism that includes the autonomous aggregation
activity of melanophores. However, these suggestions
are mainly from the simple observation of the resulting
pattern in normal and chimera fish. To determine themigration activity of the pigment cells, a more extensive
elucidation is required.
In this research, we analyzed the migration of melanophores to determine whether they have the ability toaggregate. As Yamaguchi et al. (2007) have shown intheir laser experiment, pigment cells in the adult stripe
retain the ability to generate the pigment pattern. If they
still retain the migration ability, it should be possible toassess the migration of the melanophore by eliminating
the surrounding cells to give space to move to. Using
laser ablation, we performed a series of experiments todetermine if the melanophore can move and, if theycan, to which direction. The result was contrary to our
expectation and previous suggestions. We found thatmelanophores in the adult skin repulsed one anotherand dispersed from the aggregated condition.

Results
Migration of melanophore during adult pigment
pattern formation
First, we observed melanophore migration during theearly formation of adult stripe by tracing the position ofindividual cells (Figure 1). In zebrafish, the transition
from larval pigment pattern (Milos and Dingle, 1978a,b;
Milos et al., 1983) to adult pigment pattern began about
2 week post-fertilization. At the onset [14–21 day postfertilization (dpf)], only melanophores were distributed
almost randomly over the flank. One week later (21–
28 dpf), xanthophores developed around the horizontal
myoseptum to form the first yellow stripe that contains
the isolated melanophores. At the dorsal and ventral
regions of the yellow stripe, xanthophores did notappear, but melanophores continuously developed,
resulting in two black stripes composed of only melanophores. One-fourth (14 of 54) of the melanophores
remained in the yellow stripe and died soon after. However, three-fourths (40 of 54) of them survived andmigrated dorsally or ventrally to join the black stripes(Figure 1). Some of the melanophores migrated along
the distance of a half stripe width (Figure 1 cell numbers 1, 2, 3, 7 and 11), implying that there is somelong-range attracting effect that gathers melanophores.

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Migration activity of adult zebrafish melanophore

20dpf

24dpf

26dpf

30dpf

Figure 1. Movement of the melanophores during the normal stripe formation. Time course of the stripe development in the body trunk ofwild type zebrafish was recorded under the normal lighting (upper panels) and UV dark field lighting (lower panels). Melanophores existed in
the 20 dpf fish were labeled by the number (111) and traced in the afterwards images. Xanthophores were visible by auto-fluorescence. The
daily images and the movie to show the migration trait are in the supplementary data (Figure S1 and Movie S1). To compensate the growthof the fish, each image was rescaled before the stacking. See the Method section and Figure S5 for the procedure of rescaling. (20 dpf)
melanophores were differentiated almost the all over the flank. Xanthophores are developing in the prospective yellow stripe region.
(24–30 dpf) Melanophores (numbered) in the yellow stripe region migrated to join the black stripe (40 ⁄ 54) or disappeared (14 ⁄ 54).

Isolated melanophore does not migrate toward thecluster of melanophores
To test whether the isolated melanophore really has thetendency to migrate to the cluster of the same cell
type, we ablated both melanophores and xanthophores
to set up a situation where the isolated cell was in thevicinity (about half the distance of a stripe width) to thecluster of melanophores (Figure 2). If the cluster of melanophores has some attracting effect, the isolated melanophore should move toward the cluster. Contrary towhat has been observed in normal stripe development(Figure 1), the isolated cell moved little and did not
approach the cluster (Figure 2). Instead, some of themelanophores in the cluster migrated toward the vacant
space where the pigment cells were ablated. This result
suggested that the melanophores do not have the ability
to gather, but that they actively disperse when they
have a space to move to.
Melanophores tend to disperse from a crowded
condition
To confirm that the movement of the melanophores
observed in Figure 2 is not by the attracting effect fromthe isolated cells, we performed a similar experiment
without the isolated cell (Figure 3). We made a vacant
space at the horizontal and vertical regions of the black
stripe to see whether this movement is related to theoverall directionality of the fish body or not. As shownin Figure 3, the melanophores always dispersed into thevacant space regardless of the directionality in the fish
body. This result showed that the melanophores simply
have the tendency to increase the distance from theneighboring melanophores.

Melanophores migrate more actively when they arein crowded condition
In the results shown in Figures 2 and 3, the isolated
melanophore moved little, whereas the melanophores in
the cluster moved actively. From this observation, we
expected that the migration activity of melanophores isenhanced when they are in a crowded condition. To
confirm this hypothesis, we compared the movementof melanophores set in high and in low cell densities(Figure 4A–D, O). Five melanophores were selected in
the middle of a black stripe, and the pigment cells surrounding the selected cells were ablated by laser tomake a space where they can move. Then we traced
the migration of all cells for 7 day and measured thetotal length of the migration path (Figure 4O). In Figures 4A, C, the selected cells are pointed in the whitecircles. The path of each cell is highlighted in Figures
4B, D. As shown in Figures 4B, O, the high-density cells
moved actively to be apart from the other cells. On theother hand, the movement of the low-density cells wasless active and did not have any directionality at all(Figure 4D, O).
Using the same method, we also analyzed the melanophore migration in two mutant lines, leopard (leo; Figure 4M) and jaguar ⁄ obelix (jag ⁄ obe; Figure 4N). The
leopard fish have spotted patterns. The jaguar ⁄ obelix
fish have wider stripes in which the melanophore density is less than that of the wild type. Close to the anal
fin of the jaguar ⁄ obelix fish, there is a region in which
the melanophores and xanthophores are distributed in amixed manner. The experimental results with the leopard fish were identical to those with the wild type, suggesting that melanophore migration is not affected byª 2008 The Authors, Journal Compilation ª 2008 Blackwell Munksgaard

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Takahashi & Kondo
A-P axis

D-V axis

B

Day 3

Day 1

A

Day 10Figure 2. In vivo migration of the isolated
melanophore. We ablated all pigment cells
that were left of the blue line (A) or above
the blue line (B), except the circled celland made a situation where the isolated
cell (red circle) was in the vicinity of thecluster of melanophores (day 3). The
yellow and green lines show the migration
path of the isolated cell and the cells in
the cluster, respectively. Daily images are
in Figure S2. Each image was rescaled tocompensate the fish growth by themethod described in Method section and
Figure S5.

the leopard mutation (Figure 4E–H, O), whereas the
migration activity of melanophores in the jaguar ⁄ obelix
fish was apparently diminished in both high-density andlow-density conditions (Figure 4I–L, O), suggesting thatthe migration ability of melanophores are restricted by
the mutation.
Xanthophores may exclude melanophores from theyellow stripe region
As shown above, melanophores do not have ability togather but have the tendency to go away from nearby
melanophores when they have a space to move into.
This conclusion apparently contradicts the observation
with respect to natural stripe formation (Figure 1) inwhich the isolated melanophores come to join the cluster of melanophores in the black stripes. This difference
may be due to the existence of xanthophores. If therepulsive effect between xanthophore–melanophore isstronger than that between melanophore–melanophore,
the exclusion of melanophores may cause to move theisolated melanophore into the black stripe.
To test this hypothesis, we used the day 30 fish inwhich the initial melanophores remained in the xanthophore stripe and ablated the surrounding xanthophore to
see whether the xanthophores affect melanophore
680

migration. As we expected, the melanophores in theprospective yellow stripe region remained there when
the surrounding xanthophores were lost, whereas themelanophores in the control region went into the black
stripe (Figure 5). This result shows that xanthophores
affect the migration of melanophores and may exclude
the melanophores from the yellow stripe region.

Discussion
Melanophores in the adult stripe are distributed thickly
and rarely move during normal growth. However, ourstudy has shown that they cannot stay in a crowded
condition when there is a room to migrate to and they
move to widen the distance from the neighboring melanophores. Although this conclusion is contrary to thatpredicted in previous studies (Maderspacher and Nüsslein-Volhard, 2003), a similar nature of melanophore(melanocytes) has been reported in other experiments.
Milos and Dingle (1978a,b) have shown that melanophores in zebrafish larvae have the natural tendency to
avoid the close proximity of the neighboring cells. Melanoblasts or young melanophores isolated from theamphibian skin repel one another in culture condition(Twitty and Niu, 1948, 1954). This repulsive property ofª 2008 The Authors, Journal Compilation ª 2008 Blackwell Munksgaard

Migration activity of adult zebrafish melanophore
D-V axis

A-P axis

B

Figure 3. Migration of the melanophore
located at the edge of the cluster. To givethe space to move, we ablated a group of
melanophores (A) or xanthophores (B) andrecorded the migration of themelanophores located near the vacant
space. Green lines show migration path.
Daily images are in Figure S3. Rescalingof the images was performed as
described in Method section and
Figure S5.

Day 10

Day 3

Day 1

A

the pigment cells may be functionally reasonablebecause this effect can give rise to a uniform distribution in the two-dimensional spaces. Holbrook et al.
(1989) have shown that the melanocytes in the epidermal sheets of fetuses appear to be regularly distributed
without making contact with one another. The uniform
distribution of melanocytes in the skin should be a benefit in terms of protection from UV light.
In spite of their nature to disperse, melanophores in
the adult stripes stay unmoved in a crowded condition.
This suggests that there is some external force thatpacks melanophores densely in the black stripe region.
From observations that melanophores moved to the yellow stripe region when xanthophores were absent (Figure 3B), and that the isolated melanophore joined theblack stripe when xanthophores were present (Figure 5),
we surmise that the yellow stripe acts as the wall topack the melanophores into the black stripes. There areat least two kinds of simple mechanisms that explain
the wall effect of xanthophores.
The first is that melanophores were excluded by thestronger repulsion between melanophores and xanthophores. In the completed adult pattern, the gaps
between black stripe and yellow stripe look larger thanthose between homogeneous cells (Figure 6A). We

measured the distances between the center of cells andfound that the distance between melanophore–xanthophore (82 lm) is much larger than that of melanophore–melanophore (50 lm) or xanthophore–xanthophore(36 lm; Figure 6B, C). Therefore, there should some
gaps between the melanophores and xanthophores. In adetailed study using electron microscopy, Hirata et al.
(2003) found that some remaining xanthophores exist in
the adult black stripe. However, a layer of iridophores
always exists between the melanophores and xanthophores to preclude direct interaction between them,suggesting that the direct contact of these two cell types
is not preferable in the skin. A similar repulsive effectbetween melanophores and xanthophores has also beenreported in the culture cell from larval axolotl. Lehman(1957) showed that both melanophores and xanthophores in cultures tended to avoid close contact with adifferent cell type. These observations also suggest thestrong repulsion between melanophores and xanthophores.
The second possibility is that melanophores were
sorted out via strong aggregation of the xanthophores.
In Oryzias latipes, N-CAM and N-cadherin are specifically expressed in xanthophores but not in melanophores or iridophores (Fukuzawa and Obika, 1995).

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Takahashi & Kondo

Day 1

Day 2

Day 1

Day 7
B

C

D

E

F

G

H

I

J

K

L

Day 7

obetd15

leotq270

WT

A

Leopard

O 140
120

***

High
Low

***
**
**

100

***
**

µm

80

M

NS

60

Obelix
40
20
0

N

682

WT

leo

obe

ª 2008 The Authors, Journal Compilation ª 2008 Blackwell Munksgaard

Migration activity of adult zebrafish melanophore
Figure 4. Repulsive effect among the melanophores in the cluster. (A–L) Migration of five melanophores those distributed in a crowded (A,E, I) or dispersed (C, G, K) condition were recorded after the ablation of the surrounding pigment cells. B, D, F, H, J and L show the original
distribution and the migration path of each melanophore. For the details of the path recording, see the Method section and Figure S5. (M, N)
Pigment Pattern of leo mutant and jag ⁄ obe mutant, respectively. (O) The total length of the migration path (green lines) were measured andrepresented in the graph. Values are the means of the 19 (for wild type) or 20 (for leopard and jag ⁄ obe) cells. Statistical analyses were
performed by t-test (one-tailed) in WT and leo, and by Welch’s t test (one-tailed) in obe (**P < 0.01 ***P < 0.001 NS: not significant). To
assess the difference in migration activity among WT, leo, and obe, we performed multiple comparisons by Tukey’s test (**P < 0.01 ***:
P < 0.001). Daily images of this experiment are in Figure S4. For the rescaling of the images to compensate the fish growth, see the Method
section and Figure S5. Scale bar: 100 lm. Error bars represent ±SEM.

ment (Steinberg, 2007) and a highly possible
mechanism may be involved in the segregation of different cells.
As migration of pigment cells is expected as one of
the important factors in the development of pigment
patterns, the resulting pattern may change when the
migrating ability is affected by mutation. In this study,
we found that the motility of melanophores is reducedin the jaguar ⁄ obelix mutant (Figure 4I–L, O), which has
wider stripes. The gene responsible for the mutation
was identified as kir7.1 (Iwashita et al., 2006), and it
was shown through mosaic analysis that gene activity isrequired in the melanophores (Maderspacher and Nüsslein-Volhard, 2003). Loss of the ion channel may change

Therefore, xanthophores can gather by these binding
molecules and, as result, exclude the neutral melanophores. However, there are some circumstantial evidences that deny this possibility. Our detailed electron
microscopy analysis (Hirata et al., 2003, 2005) did notdetected overlaps or tight junctions between two adjacent xanthophores, suggesting that aggregation of xanthophores is unlikely. The wider gap between the black
stripes and yellow stripes cannot be explained by thisxanthophore aggregation effect either.
Temporarily, we cannot exclude one of these possibilities as we have not succeeded to test the cell–cellaffinity in vitro. However, sorting derived from the difference in cell–cell affinity is common in animal develop-

A

B

C

D

7

24

31

12

Figure 5. Migration of melanophores in the presence or absence of xanthophores. (A, B) Xanthophores in the red square were ablated, andthe migration of melanophores with or without the xanthophore was recorded by bright field lighting (A) and UV lighting (B). (C) The migration
paths of the melanophores recorded from the (A) and (B). In the region where xanthophores were removed many melanophores remained in
the yellow stripe region. On the other hand, with the xanthophores, most of the melanophores migrated into the black stripes. (D) The
proportion of cells migrated into the black stripe region and those remained in the yellow stripe region. Values are sum of the melanophores
from five individuals. The statistical significance of differences between each group was assessed by chi-squared test (P < 0.0001). We
rescaled the images: day 5 (95%), day 10 (84%). Scale bar: 100 lm.

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683

Takahashi & Kondo

A

B

Figure 6. Distance between
melanophore–melanophore, xanthophore–
xanthophore and melanophore–
xanthophore. (A) An image from bodytrunk of adult (3 months) zebrafish. There
are gaps between the black and yellow
stripes. (B) The procedure to measure thedistances between the pigment cells.
(C) The average distances between thecenter of neighboring cells (M:melanophore X: xanthophore). The
distance between M–X is much larger
than that of M–M or X–X, suggesting theexistence of gap between themelanophores and xanthophores. To
assess the difference of distances
between homogeneous cells (M–M or
X–X) and heterogeneous cells (M–X), we
performed Welch’s t-test (one-tailed;**P < 0.001).

C

the cellular response to external signals that induce themigration of melanophores. In the body trunk of the jaguar ⁄ obelix fish, there are some regions where both melanophores and xanthophores exist in a mixed condition.
The reduced motility of melanophores could be one of
the causes of this incomplete segregation.
In this research, we have identified the migration ability of melanophores, which is related to the mechanismof stripe formation. However, this should only be a partof the mechanism and many other cellular behaviors
remain to be elucidated. The most important is the
behavior of the xanthophores. In contrast to melanophores, xanthophores mainly emerge at the prospective
yellow stripe region and rarely move, even when there
are vacant spaces (this study and Goodrich et al., 1954).
This fact implies that xanthophore migration is not
involved in the pattern-forming mechanism. If this is so,
how can the xanthophores find the position to make theyellow stripe? This is a question that needs to beaddressed. In nacre mutants that lack the melanophores, xanthophores cannot form the stripes, suggesting that melanophores are required for the correct
positioning of the xanthophores. As xanthophores do
not migrate, the influence from melanophores should be
related to the differentiation and cell death of mature
xanthophores. As well as cell migration, locally con684

trolled cell differentiation and death are the major factors that affect stripe formation. Identification of suchan interaction will uncover the mechanism that specifies
the pigment pattern, and we expect that our experimental methods used in this study can be utilized for such
purpose.

Methods
Zebrafish stock
In this research, the wild-type strain used was Tübingen. They
were maintained under standard laboratory conditions (Westerfield,2000). The age of the fish used in the experiments was about
40 day after hatching.

Laser ablation of pigment cells
Before ablation, the fish were anesthetized by using 0.01% MMS(ethyl-m-aminobenzoate methanesulfonate; Sigma, Munich, Germany) and were laid on a slide glass. During ablation, the fish werealways maintained under moist condition. Ablation was performedby using a 337-nm pulse laser (wavelength was changed to
365 nm by dye BPBD) from the VSL-337ND-S Laser Systems(Spectra-Physics, Mountain View, CA, USA), which was focused toa ·20 objective on a microscope.
We could confirm which cells were ablated to death or not bysome different methods. Before doing the ablation, fish were subjected to epinephrine to make the pigment cells compact. After
ablation, we put the fish in the dark environment (60min) to makeª 2008 The Authors, Journal Compilation ª 2008 Blackwell Munksgaard

Migration activity of adult zebrafish melanophore
the living melanophore disperse the melanosome. The living
melanophore became dendritic while the dead melanophores
remained in the round shape. More conveniently, we could detectsome signs of cell damage when doing the ablation. At the instantof cell ablation, the ablated cell slightly shrunk. Moreover, the damaged point in the cell was visible for approximately 10 s after theablation. Ablated cell showing these two signs died without exception. As the multiple image recording frequently kills the fish, we
did the melanosome dispersion test only when we were not surewhether the targeted cells were dead or not.

Time-lapse recording of cell migration
Migrations of melanophores were analyzed by image series thatwere recorded every day by CCD camera (LeicaDFC300FX; Leica,
Wetzlar, Germany) mounted on a stereoscopic microscope (Leica
MZ16FA; Leica) and software Leica Application Suite Version2.4.0
R1; Leica. To better visualize distinct melanophores, we treatedfish with two to three drops of 40 lM epinephrine, causing movement of melanosomes to the cell centers prior to viewing. The
images were transferred to Adobe Photoshop for analysis.
As the fish grows during the experiment, rescaling of each
image is required to determine the migration path of each pigmentcell. We took the horizontal myoseptum and non-ablated melanophores those located out of the ablated region as the reference
points, which were used for rescaling and positioning of each
image. [The explicit method is explained in the Figure S5. Thismethod was also described in other papers, (Parichy et al., 2000b;
Maderspacher and Nüsslein-Volhard, 2003; Parichy and Turner,
2003b; Quigley et al., 2005)]. To make sure the accuracy of thismethod, we applied the method to the normal growing zebrafish,
and found all the pigment cells looked unmoved compared to the
movement observed in the ablation experiments. Therefore, thismethod should be reliable enough to determine the migration path
recorded in this report.

Acknowledgements
This research is supported by Grant-in-Aid for Scientific Research
Gakujutu-Sousei and Genome Tokutei, and Riken Center for Developmental Biology.

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Fig. S1 Daily images of the experiment shown in
Figure 1.
Fig. S2 Daily images of the experiment shown in
Figure 2.
Fig. S3 Daily images of the experiment shown in
Figure 3.
Fig. S4 Daily images of the experiment shown in
Figure 4.
Fig. S5 The method for scaling the images.
Movie. S1 Normal pigment pattern formation (20-30
dpf).
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Supporting information
Additional Supporting Information may be found in the
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686ª 2008 The Authors, Journal Compilation ª 2008 Blackwell Munksgaard