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Research
Changing clothes easily: connexin41.8
regulates skin pattern variation
Masakatsu Watanabe and Shigeru Kondo

DOI: 10.1111/j.1755-148X.2012.00984.x

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Pigment Cell Melanoma Res.

SHORT COMMUNICATION

Changing clothes easily: connexin41.8 regulates skinpattern variation
Masakatsu Watanabe and Shigeru Kondo
Graduate School of Frontier Biosciences, Osaka University, Osaka, JapanKEYWORDS connexin ⁄ gap junction ⁄ reaction–diffusion ⁄ skin pattern ⁄ zebrafish

CORRESPONDENCE M. Watanabe, e-mail: watanabe-m@fbs.osaka-u.ac.jp
PUBLICATION DATA Received 5 January 2012,
revised and accepted for publication 2 February
2012, published online 7 February 2012
doi: 10.1111/j.1755-148X.2012.00984.x

Summary
The skin patterns of animals are very important for their survival, yet the mechanisms involved in skin patternformation remain unresolved. Turing’s reaction–diffusion model presents a well-known mathematical explanation of how animal skin patterns are formed, and this model can predict various animal patterns that are
observed in nature. In this study, we used transgenic zebrafish to generate various artificial skin patterns
including a narrow stripe with a wide interstripe, a narrow stripe with a narrow interstripe, a labyrinth, and a‘leopard’ pattern (or donut-like ring pattern). In this process, connexin41.8 (or its mutant form) was ectopically
expressed using the mitfa promoter. Specifically, the leopard pattern was generated as predicted by Turing’s
model. Our results demonstrate that the pigment cells in animal skin have the potential and plasticity toestablish various patterns and that the reaction–diffusion principle can predict skin patterns of animals.

Skin pattern is an important feature when measuring an
animal’s likelihood for survival and mating, and it is also
an important marker for the evolutionary adaptation by aspecies to its environment. The fact that even closely
related animals show extremely divergent patterns suggests that animal skin has a plasticity that can lead togreat variability. Such variability can be described mathematically by Turing’s reaction–diffusion (RD) model,
which has been frequently cited to explain the diversity
and robustness of naturally occurring patterns (Kondo
and Asai, 1995; Kondo and Miura, 2010; Meinhardt,
1982; Turing, 1952). This diversity is achieved by changing the parameters in a series of partial differential equations, as demonstrated by the re-creation of naturally
arising endophenotypic patterns (Miyazawa et al., 2010).

To understand the roles that development and genetics play in skin pattern formation, zebrafish is the preferred model organism. Previous studies have revealed
the genetic factors related to the development and differentiation of pigment cells (Kelsh et al., 2009; Parichy
et al., 1999; Rawls and Johnson, 2001), and interactions
among these pigment cells have been proposed to be
sufficient for skin pattern formation (Nakamasu et al.,2009; Takahashi and Kondo, 2008). Indeed, laser ablation experiments successfully reconstructed the RD
phenomenon on zebrafish skin (Yamaguchi et al., 2007).
However, the molecular factors that regulate theseinteractions remain largely unknown. One candidate isConnexin41.8 encoded by cx41.8. This gene is responsible for the skin pattern of leopard zebrafish, which has

SignificanceIn nature, we see various animal skin patterns such as stripes, spots, waves, donut rings, and tiles.
Researchers are studying how the spatial patterning is determined. Theoretical explanations for how such
patterns arise have been proposed, but the molecular mechanisms remain unsolved. In this report, we
generated various skin patterns artificially on zebrafish skin using the connexin41.8 gene and showedthat connexin41.8 functions to impart variation to animal skin patterns.

ª 2012 John Wiley & Sons A/S

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Watanabe & Kondo

a spot pattern distinct from the stripe pattern typicallyseen in wild-type (WT) zebrafish (Figure S1) (Maderspacher and Nusslein-Volhard, 2003; Watanabe et al.,2006). In fact, different cx41.8 mutants show a variety
of stripe–spot patterns (Figure S1). Furthermore theimportance of Cx41.8 for pattern formation is supported
by its function as one of the components of gap junctions, which connect neighboring cells and mediate cell–
cell interactions (Kumar and Gilula, 1996). The interactions between two hypothetical factors are consistent
with both the RD model and experimental observations(Kondo and Miura, 2010). In this study, we appliedtransgenic techniques to test whether the RD model
could predict how cx41.8 is related to skin patterning.
The 4.5-kb fragment upstream of exon 1 in cx41.8
was cloned into a pTol2 plasmid (Figure 1A) (Kawakami,2005), resulting in a pTol2-cx41.8 promoter vector.
Transgenic experiments using the pTol2-cx41.8pro:
cx41.8 plasmid revealed that the 4.5-kb fragment can

act as a promoter for cx41.8 and results in a stripe pattern in leopard zebrafish (Figure 1A–D), confirming that
this system can be applied to pigment cells in adultzebrafish. Next, we replaced the cx41.8 fragment in the
pTol2-cx41.8pro:cx41.8 plasmid with an EGFP cassetteand used EGFP expression as a reporter for cx41.8,
finding EGFP signals in melanophores and xanthophores(Figure 1E–L; see legend). We next compared the EGFP
expression driven by the cx41.8 promoter (Figure 1M)
with that of DsRed driven by the mitfa promoter(Figure 1N) (Lister et al., 1999) in young zebrafish(11 mmSL; Parichy et al., 2009). mitfa is a master control gene for the development and pigmentation of melanophores and is expressed in melanoblasts andmelanophores. We named the cx41.8 promoter as
cx41.8pro and mitfa promoter as mitfapro here. We
found that EGFP signals were expressed in the same
cells as DsRed signals, suggesting that Cx41.8 may
have a role in melanophore development and ⁄ or differ-

A

B

F

G

H

I

J

K

C

D

E

L

M

N

Figure 1. Expression of cx41.8. Design of plasmids for the transgenic experiments. (A) Upper line indicates the genome structure around thezebrafish cx41.8 locus. Lower lines indicate the pTol2 regions of the plasmid constructs: (top to bottom) pTol2-cx41.8pro:cx41.8, pTol2cx41.8pro:EGFP, and pTol2-mitfapro:DsRed. Purple boxes indicate the cx41.8 exons; blue boxes, open reading frame; yellow boxes, theupstream region of cx41.8, which includes the cx41.8 5’ untranslated region; black boxes, transposon sequences (TP); and green and redboxes, the EGFP and DsRed cassettes, respectively. ‘cx41.8pro’ indicates the cx41.8 promoter, and ‘mitfapro’ indicates the mitfa promoter.
Panels B–E show skin patterns of WT zebrafish (B), cx41.8t1 allele (C), leopard zebrafish rescued with pTol2-cx41.8pro:cx41.8 (D; cx41.8t1 ⁄ t1
background, F1 generation), and transgenic fish with pTol2-cx41.8pro:EGFP (E; WT background, F1 generation). EGFP signals were detected
in melanophores and xanthophores of untreated caudal fin (F–H; magnified image of the yellow boxed region in E) and trypsin-treated caudal
fin (I–K). F, I: color images of the melanophores and xanthophores; G, J: EGFP signals derived from the cx41.8 promoter; H, K: overlaid
images of F and G and of I and J, respectively. White arrows and yellow arrows in H and K indicate EGFP signals in melanophores andxanthophores, respectively. EGFP signals were also detected in the trunk region of adult zebrafish (L), a region that is indicated by the whitebox in E. (M and N). EGFP expression (M) and DsRed expression (N) driven by the cx41.8pro promoter and mitfapro promoter, respectively,
were compared at the trunk region in young zebrafish (11 mmSL).

2ª 2012 John Wiley & Sons A/SSkin pattern variations on zebrafish

entiation and that the mitfa promoter can be used toectopically express cx41.8 in melanophores of adultzebrafish. Recent studies have revealed that mitfa
expression also can be detected in precursor cells for
iridophores and xanthophores in zebrafish embryos (Curran et al., 2011), indicating the possibility that cx41.8
activated by the mitfa promoter was also expressed inthese precursor cells (see below). On the other hand,the zebrafish skin pattern is changeable even after the
adult phenotype is fixed (Yamaguchi et al., 2007). Furthermore, DsRed expression driven by the mitfa promoter was detected only in melanophores (not in
xanthophores) in adult fin tissue (Figure S2). Theseobservations prompted us to consider the mitfa promoter as a melanophore-specific promoter for our studyof adult phenotypes, although further analysis inembryos might be required to determine the Cx41.8
function in the precursor cells.
Next, cx41.8 was expressed in melanophores using
the mitfa promoter in several genetic backgrounds. The
transgene causes presumably mixed expression of theendogenous protein and transgenic protein in melanophores, whereas the xanthophores express only theendogenous protein. The observed differences in thepatterns obtained imply that this transgenic expression

results in heteromeric gap junctions having modified
properties. Our transgenic approach yielded several skinpatterns (Figure 2), which we compared with those generated in simulations (Figures 2, 3, and S3). For example, the skin pattern of WT zebrafish resembled thestripe pattern seen in Chapman’s zebra (Figures 2A andS3A), whereas the spot pattern in leopard zebrafish
resembled that seen in cheetah (Figs 2B and S3B). The
transgene mitfapro:cx41.8 expressed in a cx41.8t1 ⁄ t1
background generated a bongo pattern consisting ofbroad stripes interlaced with narrow ones (Figures 2C
and S3C), whereas a labyrinth pattern was generated
when the mutant Cx41.8M7, in which six amino acidresidues were deleted at the N terminus of Cx41.8,
was used in the WT background (Figures 2D, S3D,
and S4, Cx41.8M7). Furthermore, an insertion of threeresidues at the N terminus of Cx41.8 (Figure S4,MKLCx41.8) changed the skin pattern in WT zebrafish,
as the black stripes of the melanophores were graduallycleaved and replaced with xanthophores during growth,
resulting in a narrow-stripe pattern (Figure 2E). Thisphenomenon is easily constructed in the two-stage Turing model described by Liu et al. (2006). Initially, parameters were set to produce the WT stripe pattern(Figure 3A, left column). Du and Dv were then changed

A

B

C

D

E

F

G

Figure 2. Natural skin patterns observed in zebrafish. Animals showing classic pigment patterns: (A) Chapman’s zebra (Equus quagga
chapmani), (B) cheetah (Acinonyx jubatus), (C) bongo (Tragelaphus eurycerus eurycerus), (D) puffer fish (Tetraodon mbu), (E) Grevy’s zebra(Equus grevyi), and (F) leopard (Panthera pardus). Zebrafish: (A) WT (cx41.8+), (B) leopard zebrafish (cx41.8t1 ⁄ t1), (C) Tg(mitfapro:
cx41.8)cx41.8t1 ⁄ t1, (D) Tg(mitfapro:M7cx41.8)cx41.8+, (E) Tg(mitfapro:MKLcx41.8)cx41.8+, and (F) Tg(mitfapro:MKLcx41.8)cx41.8tq270 ⁄ +.
Simulation conditions are described in Data S1 and Fig. S3.

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Watanabe & Kondo

A

B

C

Figure 3. Skin patterns predicted by the RD model. (A) Cleavedstripe and (B) ring patterns generated by the RD model, where u
and v are concentrations of hypothetical factors, au + bv + c andeu + fv + g are the respective reaction kinetics, and Du and Dv are
the respective hypothetical diffusion coefficients for u and v (C).
Parameters were as follows: a = 0.08, b = )0.08, d = )0.03,
e = 0.1, f = )0.06, g = )0.12, Du = 0.02, Dv = 0.5, 0 £ au + bv + x£ synUmax = 0.2, and 0 £ eu + fv + g £ synVmax = 0.5. Finally,c = 0.16 for (A) and 0.02 for (B). The starting pattern is one thinline (A) or random dots (B). After the initial patterns were fixed (leftcolumns in A and B), Du and Dv were changed to 0.002 and 0.05,
respectively, in both simulations.

such that cleaved stripes appeared (Figure 3A). Similarly, a donut ring pattern could be theoretically generated if the initial conditions led to the spot pattern(Figure 3B). To confirm this donut phenomenon experimentally, we inserted the MKLcx41.8 transgene into
the cx41.8tq270 ⁄ + background because this mutant fish
had the largest spot pattern among the fish with leopard
alleles (Figure S1). The resulting ring pattern resembled
those seen on leopard (Figure 2F). We therefore named
this pattern the ‘leopard pattern’.
As observed in transgenic zebrafish shown in
Figures 1 and 2, during pattern formation gap junctions
were predicted to activate melanophore production andrepress xanthophore production because without
cx41.8, the number of melanophores decreased andthe number of xanthophores increased. In addition, thenumber of melanophores increased when the transgene
mitfapro:cx41.8 was expressed only in melanophores
whereas the number of xanthophores did not change(Figure 2C). In this study, some of the simulations werecarried out considering the known function of cx41.8 inpigment cells. Namely, for the spot and bongo patterns,
parameter c (concentration of a hypothetical component) was changed, which may affect the number or
ratio of pigment cells (Figure S3). Furthermore, the simulated cleaved-stripe pattern (Figure 2E, F) was
obtained by changing the diffusion constants Du and Dv
after the initial patterns were produced, which was consistent with the cleaved-stripe pattern observed during

4

the growth period of transgenic fish. We previously
reported that xanthophores enhance melanophore survival and that melanophores indeed cannot survive in
the absence of xanthophores (Nakamasu et al., 2009).
These observations led us to hypothesize that melanophores tend to maintain the stripe width because thesecells cannot survive at a distance from xanthophores.
Furthermore, a modified N-terminal domain in
MKLCx41.8 might affect the diffusion rate of small molecules between pigment cells, which may be related tomelanophore survival and cause stripe cleavage. On theother hand, it was unclear how and why the type of
pattern changed when the N-terminal domain encodedby cx41.8 was deleted (Figures 2D and S3D). To
address this issue, we next should investigate theimpact of the initial conditions in vivo, such as precursor
cell arrangement.
Our study reveals that unique skin patterns can begenerated and that animal skin patterning is indeed aplastic phenomenon. Interestingly, the skin patterns
obtained were generated by manipulating the expression (and therefore function) of a single gene, namely
cx41.8. Gap junctions constitute bidirectional channels
that directly connect neighboring cells. Recent studies
have revealed that many kinds of small molecules ofless than 1000 Da can be transferred through gap
junctions. However, it is still difficult to identify amolecule(s) that is specific to a particular cellular phenomenon because of the unselective properties of the
gap-junction channel. Our study suggests that the precise regulation of the N-terminal domain of connexin,
which works as a plug for the channel (Maeda et al.,2009; Oshima et al., 2007), is required for gap junction function. Interestingly, different types of mutant
connexins yield different zebrafish phenotypes(Figures 2D, E), which suggests that gap junctions
tune cell–cell interactions for pattern making and thatslight functional changes in the junctions are related
to pattern diversity. We recently found that not only
cx41.8 but also other connexins such cx44.1, cx45.6,
and cx48.5 also rescued the leopard phenotype
whereas cx43 did not. In addition, rat cx40, an
ortholog of zebrafish cx41.8, also rescued the leopard
phenotype (M. Watanabe, D. Watanabe and S.
Kondo, unpublished data); this information may prove
useful in future studies of connexin gene function inskin pattern formation.

Acknowledgements
We thank Mr. T. Watanabe from the Higashiyama Zoo, Nagoya,
Mr. H. Doi from the KAIKYOKAN Shimonoseki Marine Science Stadium, Ms. M. Ayumi from Zoo21st, and Mr. Koro for supplying theanimal pictures (T.W., bongo; H.D., puffer fish; M.A., Chapman’s
zebra, leopard, and Grevy’s zebra; and K., cheetah). This work was
supported by a Grant-in-Aid for Scientific Research on Innovative
Areas from the Ministry of Education, Culture, Sports, Science andTechnology of Japan.

ª 2012 John Wiley & Sons A/SSkin pattern variations on zebrafish

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Supporting information
Additional Supporting Information may be found in the
online version of this article:
Figure S1. Leopard alleles. (A) WT fish, (B) leopard
fish, (C) cx41.8tw28 heterozygous allele, (D) cx41.8tq270
heterozygous allele, (E) cx41.8tw28 homozygous allele,and (F) cx41.8tq270 homozygous allele. cx41.8t1 has anonsense mutation in the first extracellular loop domain;cx41.8tw28, an I31F mutation in transmembrane-1(TM-1), and cx41.8tq270, an I203F mutation in TM-4 of
Cx41.8. The phenotype of cx41.8t1 ⁄ + is the same asthat of WT fish.
Figure S2. DsRed signals driven by the mitfa promoter were detected only in melanophores of adult fish
fins. DsRed expression driven by the mitfa promoter
was examined in Tg(mitfapro:DsRed) in the F1 generation. (A) Intrinsic fluorescence of xanthophores (green);(B) melanophores expressed DsRed signals driven by
the mitfa promoter; (C) bright field image; and (D)merged image of A–C.
Figure S3. RD simulations. (A) Stripe, (B) spot, (C)stripe with wide interstripe, and (D) labyrinth. Simulation
conditions are given in the supplementary text.
Figure S4. Alignment of N-terminal region of Cx41.8.
Cx41.8M7 has its initial codon at the seventh positionof WT Cx41.8, which was generated by deleting thesecond through seventh amino acid residues (ADWSLL).
MKLCx41.8 has an extra three residues at the N-terminal end.
Data S1. Materials and Methods.
Please note: Wiley-Blackwell are not responsible forthe content or functionality of any supporting materials
supplied by the authors. Any queries (other than missing
material) should be directed to the corresponding author
for the article.

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