pigment pattern formation in the medaka embryo

Pigment pattern formation in the medaka embryo

M. Lynn Lamoreux1,*, Robert N. Kelsh2,

Yuko Wakamatsu3 and Kenjiro Ozato3

12 Comparative Medicine Program, Texas A&M University, College

Station, TX, USA2Department of Biology and Biochemistry, Centre for Regenerative

Medicine, Developmental Biology Programme, University of Bath,

Bath, UK3Nagoya University, Chikusa-ku, Nagoya, Japan

*Address correspondence to M. Lynn Lamoreux,

e-mail: [email protected]

Summary

For the study of development of pigmentation,

compared with mammalian models, fish offer the

advantage of multiple chromatophore types and

ready access to the developing embryo for observa-

tion and experimental manipulation. Compared with

zebrafish embryos, medaka embryos have an addi-

tional unique chromatophore-type and superb prop-

erties for conditional mutation studies. The rich

resources of medaka mutants, combined with data

obtainable from other species, potentially offer

information not otherwise readily available regard-

ing chromatophore lineage. Here we summarize the

embryonic development of normal medaka pigment

pattern, based on observations using embryos of a

panel of wild type and mutant fish. A more detailed

description of development is available in the

appendix of the on-line version of this paper (see

Supplementary Material). We make some compari-

sons with zebrafish development to emphasize the

increased power of both systems when utilized

together. These two models will, in combination, be

a powerful system for studies of the embryogenesis

and evolution of pigmentation.

Key words: medaka/pigmentation/neural crest develop-

ment/chromatophores

Received 26 June 2004, revised and accepted for publi-

cation 7 November 2004

Introduction

The development of pigmentation, both ontogenic and

phylogenic, informs our understanding of embryogene-

sis in general and neural crest patterning in particular.

The origin of pigment cells from the neural crest

is amenable to research using a wide variety of well

established techniques; most mutations that impact pig-

mentation are visible and nonlethal; and many pigment

mutants are well characterized at the molecular level in

several species. Pigment cells also are the defective

element of several medical conditions involving their dif-

ferentiation, survival and replication. Examples include

melanoma, albinism, piebaldism (white spotting) and

vitiligo. And of course pigmentation is of cosmetic signi-

ficance.

Compared with mammals, study of pigmentation in

fish offers additional benefits. The most obvious is the

availability of all embryonic stages. Furthermore, fish

have multiple pigment cell types, whereas mammals

produce only melanocytes, and fish are easier and less

expensive to maintain. The domesticated fish models, of

course, avoid problems faced in working with mammals

or species that must be collected in the wild. The exist-

ence of two well characterized and easily managed fish

models, zebrafish and medaka, increases the potential

value of both model species for the evaluation, compar-

atively, of genetic control over phenotypic development.

The Japanese medaka offers particular advantages to

pigment cell research, both in itself and in comparison

with other species such as zebrafish (Furutani-Seiki and

Wittbrodt, 2004). At the same time, medaka uniquely

satisfies some specific needs for biological research.

This hardy little fish produces a dozen or more eggs at

a predictable time each day, and it retains them

attached to the female for several hours. Thus collection

is convenient and staging (Iwamatsu, 2004) simplified.

Medaka are easy and inexpensive to raise and to trans-

port. They are tolerant of wide ranges of temperature

and salinity. The embryonic chorion can be removed

manually or using hatching enzyme (Wakamatsu, 1997;

Medaka Web Site http://biol1.bio.nagoya-u.ac.jp:8000/

Welcome.html) for observation without interfering with

development. Viable technologies include cell culture

(Hong et al., 1996, 1998; Wakamatsu et al., 1993,

1994), cryopreservation (Strussman et al., 1999), nuclear

transfer (Ando and Wakamatsu, 1995; Niwa et al., 2000;

Wakamatsu et al., 2001a), cloning (Naruse et al., 1985)

and production of transgenics (Ozato et al., 1992). Many

natural mutants are available (Medaka Web Site http://

biol1.bio.nagoya-u.ac.jp:8000/Welcome.html, Kelsh et al.,

2004; Yamamoto, 1975).

Also available are mutagenesis (Fukamachi et al.,

2001), established molecular technologies (Matsumoto

et al., 1996) and a rapidly developing gene map

(Naruse et al., 2000, 2004a,b; Medaka EST database,

Copyright ª Blackwell Munksgaard 2005 Reviewdoi: 10.1111/j.1600-0749.2005.00216.x

64 Pigment Cell Res. 18; 64–73

http://medaka.lab.nig.ac.jp/; Medaka Genome Project

http://dolphin.lab.nig.ac.jp/medaka/; Medaka Genome Ini-

tiative http://medaka.dsp.jst.go.jp/MGI/; Medaka Expres-

sion Pattern Database http://www.embl-heidelberg.de/

mepd/; Medaka Developmental Mutants Group http://

medaka.dsp.jst.go.jp/DMG/). Finally, the completion of a

large-scale mutagenesis screen has further boosted the

genetic resources for this model, although sadly pig-

mentation was not a target of the screens (Furutani-

Seiki et al., 2004b). Thus medaka are suitable for

research projects at all levels from the classroom to the

sophisticated research laboratory (Ishikawa, 2000;

Iwamatsu, 1997; Kirchen and West3 , Carolina Biological;

Ozato and Wakamatsu, 1994; Packer, 2001). Here we

summarize the larval pigment pattern and its develop-

ment, as observed using wild-type and selected pig-

ment mutant stocks (listed in Table 1), and provide a

detailed description in the appendix of the online version

of this paper (see Supplementary Material). Materials

and methods are also presented in more detail in

Appendix A. This description supplements the excellent

general staging series of Iwamatsu (2004). We also

highlight some comparisons between pigment pattern

formation in medaka and zebrafish to enable more ready

appreciation of the value of these superb models.

Chromatophores

Presence or absence of chromatophores or pigment of

any of the four distinct types (and some subtypes) is

under tight genetic control. The mature embryonic wild

type pattern is shown in Figure 1. Critical evaluation of

the chromatophore types is possible because of the

availability of a panel of medaka mutants (Table 1) that

permit observation of each chromatophore type in

absence of the obscuring pigmentation of other chroma-

tophore types, or in various ‘made to order’ combina-

tions. Some explanation of the history of medaka

nomenclature may be helpful, and is provided in Appen-

dix A of the online version of this paper (see Supple-

mentary Material).

In the adult, the four types of medaka chromatoph-

ores are found in the scales and also beneath the

scales, as well as in the peritoneum, in the eyes and

around the brain and spinal column (Bagnara, 1998;

Bagnara and Hadley, 1973; Hama, 1975; Hama et al.,

1965; Inagaki et al., 1994; Menter et al., 1979; Obika

and Fukuzawa, 1993; Takeuchi, 1975, 1982). In general

the pigment in these chromatophores is contained

within membranous organelles, though the carotenoid

vesicles of xanthophores apparently lack membranous

envelopes (Obika, 1996). Three of the medaka chromat-

ophores – melanophores, xanthophores and iridophores

– appear to be similar to those of zebrafish. The fourth,

leucophores, have not been reported in zebrafish

embryos, although a few similar cells are seen in adult

tail (Johnson et al., 19954 ).

Melanophores are grey or black in colour and contain

melanin (Hishida et al., 1961; Ide and Hama, 1969;

Nakajima and Obika, 1986; Tomita and Hishida,

1961a,b). Because melanophores obscure developmen-

tal events in embryos and adults, the ‘albino’ (i-3/i-3)

and/or b/b fish that lack pigmented melanophores are

helpful for observing the other three types of chromat-

ophores during development (Figures 2 and 3).

Xanthophores are yellow or orange and contain pteri-

dine pigments and carotenes (Hama and Hasegawa,

1967; Hirao et al., 1969; Ide and Hama, 1969; Kamai-

Takeuchi and Hama, 19715 ; Obika, 1993; Takeuchi,

1960, 1961, 1968). The pteridines include principally

Table 1. Mutant medaka stocks (Kimmel

et al., 1995). The genotypes of fish that

were used to evaluate the development of

the four types of chromatophore in

presence or absence of differentiated

chromatophores of the other types. A plus

symbol (+) indicates that the fish of this

stock are homozygous for the wild-type

normal allele at the gene locus shown

above the symbol. A minus symbol ())indicates the gene locus is homozygous

for the mutant allele. Many other medaka

mutations have been identified that

influence pigmentation

Stock symbol Name of Mutant Stock

Genotype

Lf I-3 Gu Il-1 R B

‘wild type’ + + + + + +

lf ‘Leucophoreless’ stock ) + + + + +

i-3 ‘albino-3’ stock + ) + + + )gu ‘guanine-free’ stock + + ) + + )il-1 ‘iridophore-less’ stock + + + ) + +

STI ‘See Through I’ + ) ) + + +

STII ‘See Through II’ ) ) ) + + +

STII.YB Males + ) ) + + +

Females ) ) ) + + +

STIII ‘See Through III’ ) ) ) ) + +

FLFII* ‘Female Leucophore-free’

Males leucophore positive + + + + + )Females leucophore free ) + + + ) )

F2 (FLFII · STIII) All loci segregating

HO5 Inbred ‘orange-red’ or ‘golden’ + + + + + )White ‘white’ stock + + + + ) )

VASA stock ) + ) + + )

*Medaka web site http://biol1.bio.nagoya-u.ac.jp:8000/Welcome.html.

Pigment pattern formation1

Pigment Cell Res. 18; 64–73 65

sepiapterin, as is also the case with zebrafish xanthoph-

ores (Kamei-Takeuchi et al., 1968; Ziegler, 2003). Homo-

zygosity for the recessive allele at the R locus results in

dramatic reduction in numbers of xanthophores or in

their pigmentation (Figures 2 and 3).

Iridophores (also referred to in the literature as

guanophores) produce iridescent effects as a result of

their reflecting platelets or precisely aligned crystals of

purines, guanine, hypoxanthine and in some cases aden-

ine (Kawaguti and Takeuchi, 1968; Menter et al., 1979;

Ziegler, 2003) (Figure 3).

Leucophores are cream or whitish in colour. With inci-

dent lighting they lack the iridescence of iridophores.

Similar to xanthophores, they contain pteridine deriva-

tives, primarily drosopterinosomes (Hama, 19706 ; Seiji

et al., 1963). Several mutant loci differentially impact

leucophore phenotype (Table 2) and their study would

surely be rewarding. Within this study, all the fish

stocks that lack differentiated leucophores are of the

genotype lf/lf (Tomita, 1992; Wada et al., 1998). Medaka

of the genotype lf/lf (‘leucophore free’) completely lack

differentiated leucophores (Figures 2 and 3) (Tomita,

1992; Wada et al., 1995). The lf gene has no effect on

the pigmentation of melanophores and xanthophores,

Figure 1. (A) Wild type medaka at hatching, around 10 dpf. (B)

Wild type medaka at 13 dpf. This figure illustrates the major

features of pigmentation at hatching and just after hatching. Note

the normal distribution of the leucophores (yellow to cream

coloured) that are characteristically surrounded by black

melanophores in the dorsal and ventral stripes and elsewhere in

the embryo are independent of melanophores. Embryonic

melanophores form a dorsal stripe posterior to the head and a

ventral stripe posterior to the anus. The golden sheen of the eyes

results from xanthophores that overlie iridophores. Beneath the

xanthophores and iridophores the eyes are melanized. Similarly, the

peritoneum will be covered with black melanophores that are

overlain by reflective iridophores, and these to some extent by

golden xanthophores. In this picture the xanthophores are evident

only in the golden reflections on the eyes. See Figures 2 and 3 for

xanthophores. Other cream-coloured cells are leucophores.

Figure 2. Two littermate fish of the ST-II.YB stock at 10 dpf. Both

are ‘albino’ in phenotype (genotype i-3/i-3), thus lack all

differentiated melanophores in skin and eyes, making it easier to

evaluate the other chromatophore types. The above fish also lacks

leucophores at all locations because of lf/lf genotype and is

deficient in xanthophore pigmentation due to its r/r genotype.

Figure 3. Two fish of hatching age. These fish are from a large

backcross mating that segregated at i-3, lf, gu, il-1 and r. Both

littermate fish in the figure lack melanin because of mutation at i-3.

The lower fish is r/r Gu/+lf/lf i-3/i-3. Iridescence of eyes and lateral

patches are evident; the eyes and lateral patches appear silvery

rather than gold because of the reduction in xanthophore

pigmentation overlying the iridophores. The above fish is deficient

in iridophore pigmentation because of its gu/gu genotye, but it

does have normal representation of xanthophores and leukophores.

Its genotype is R/-gu/gu Lf/-i-3/i-3.

Lamoreux et al.


nor on the physiological or morphological colour chan-

ges of chromatophores (Tomita, page 265 in Hama,

1975; Obika, 1988).

Embryonic development of medakapigmentation

The life cycle of the medaka begins with a clutch of one

or two dozen eggs that are produced each morning

within about an hour of first light. For several hours, the

eggs remain attached by a sticky substance to the

female, after which they normally drop from the female

and develop in the bottom of the tank or pond. The

embryos then proceed with their development within

the chorion for the first 9 d, when they hatch out as lar-

vae. Development of the embryonic pigment pattern

progresses smoothly to its completion at about 9 d

postfertilization (dpf) at 26�C (Figure 1). At this stage

some aspects of the larval pattern are beginning to

form. By 9 dpf in wild type fish, the eyes are black with

melanin under the iridophores and xanthophores, and all

four chromatophore types are present in an intricate

and consistent pattern on the body. We evaluated this

early embryonic pattern of pigmentation, and its devel-

opment, using wild type fish and mutations that ablate

one or more of the pigment cell types (Table 1). An

overview is presented below; the detailed description is

available Appendix B of the online version of this paper

(see Supplementary Material ).

The most obvious difference between the medaka

embryonic pigmentation, and that of the better-known

zebrafish, with which we made some comparisons, is

the essential absence of leucophores from the latter

and their prominent participation in medaka pigmenta-

tion. This difference notwithstanding, the pigment pat-

tern of medaka embryonic development is very similar

Table 2. A list of phenotypes resulting from medaka mutations that affect the leucophores*

Categories of defect Mutant stock/gene

Early differentiation of leucophores at brain ci, dp-2, rs-2, vc, Va

Early appearance on the body ci, dp-2, rs-2, vc

Brain-associated leucophore numbers high ci, dp2, ml3, rs2, vc, Va

Brain-associated leucophore fewer than normal lf, lf-2

lf-2 is very interesting comparator to lf. lf completely lacks differentiated leucophores.

lf-2 does have leucophores associated with the brain at the normal time; they fail to replicate, and eventually they disappear. Occasionally a

few make it to the outer surface of the body, supporting our assumption that the leucophores associated with the brain contribute to the

population of surface leucophores, but none survive to hatching in lf-2.

Numbers of leucophores on the body of the embryo

Normal dp-2

Ci did not have a normal body

Few or none lf, lf-2

Variable vc

High r-s2, ml-3

ml-3 has additional leucophores in the dorsal stripe pattern so that there are three parallel dorsal lines of leucophores rather than the normal

one.

Distribution of leucophores associated with the brain

Normal ci, ml-3, Va, lf

dp-2 could not tell there were too many or too poorly defined?

Rs and vc had many little differentiated leucophores fleck-looking down the neural tube, giving the impression that these may represent

migration of leucophore precursors.

Distribution of leucophores on the body

Ventral stripe abnormality ci

Variable vc

Gaps, empty locations, on the tail. dp-2

Described above lf, lf-2 and ml-3

Phenotype of leucophores associated with the brain

Normal ci, lf

Pale dp-2, Va

Small lf-2, ml-3, rs-2

Variable vc

Phenotype of leucophores on body

Normal ci, dp-2 a little off fuzzy, ml-3, lf

Absent lf-2

Small rs-2, vc, Va

Pale rs-2

*Medaka is a similarly rich genetic resource for the study of melanophores, xanthophores and iridophores. Medaka web site

http://biol1.bio.nagoya-u.ac.jp:8000/Welcome.html.32



to that of zebrafish, up to about 5 d of age (Figure 5),

with dorsal stripe (DS), lateral stripe (LS) and ventral

stripe (VS) (Kimmel et al., 1995; Milos and Dingle,

1978a,b). The fourth melanophore stripe in zebrafish,

the Yolk Sac Stripe is substituted by a melanophore

cluster that we term the Yolk Sac Cluster (YSC, see

also Kelsh et al., 2004). The embryonic origin of these

stripes in medaka and zebrafish appears to be homolog-

ous, but the mechanistic basis of their patterning

remains to be determined. The most striking (and dispu-

ted) difference between the two species seems pheno-

typically to be that developing iridophores occupy the

locations in zebrafish where leucophores are found in

medaka. (M.L. Lamoreux, personal observation). We

use terminology that emphasizes observed similarities.

Pigment cells differentiate in the following sequence:

melanophores (from Stage 22; 9-somite stage; Iwama-

tsu, 2004), leucophores (from Stage 25, 20-somite

stage, very soon after appearance of otoliths), iridoph-

ores and xanthophores (from Stage 34, 5.25 dpf). The

pattern of development is under genetic control, is pre-

cise and repeatable and proceeds generally in a rostral-

caudal gradient.

Melanophores

Using a dissecting microscope the first melanophores

are seen on the yolk sac at stage 22 (8–9 somite stage).

Within 1–3 h, melanophores appear on the embryonic

body, and begin formation of the DS over the mid and

hindbrain. By stage 23 (12-somite stage) the yolk sac

melanophores are evenly dispersed as individual cells

around the whole yolk sac. On the third day the num-

bers of melanophores on the yolk sac have reached

their maximum. As the yolk sac blood vessels (Cuvierian

ducts and median yolk vein) form, melanophores rapidly

become associated with them, beginning at Stage 26

(22-somite stage) and noticeably concentrated by Stage

29 (34-somite stage). By Stage 36 (6 dpf) most of the

yolk sac melanophores are associated with blood ves-

sels. By the end of development, the yolk sac mel-

anophores have clumped together ventrally, below the

yolk, forming a YSC.

The DS melanophores, in the meantime, by Stage 23

(12-somite stage) are taking up patterned positions over

the anterior trunk regions, as well as over the mid and

hindbrain. By Stage 29 (34-somite stage), a few mel-

anophores are found in the skin dorsolateral to the fore-

brain, many are above the mid- and hindbrain (although

on the rostral hindbrain most melanophores are in fact

on the lateral faces) and a few may be over the trunk.

The net result is two lateral tracts of melanophores on

the surface of the head. The two tracts converge at the

posterior margin of the head. Posterior to the conver-

gence of the two tracts, the rather well defined double

line of melanophores seems to contain one melano-

phore unit, consisting of one to three melanophores,

per segment.

By Stage 35 (5.25 dpf) the DS melanophores begin to

form something of the final pattern and the DS extends

to almost the full length of the tail. By Stage 37 (7 dpf),

most embryos have rostral hindbrain melanophores in

the mature (medial) pattern, although a few still have

them in a mediolateral position; by Stage 38 (8 dpf), the

pattern is complete as shown in Figure 1.

In the meantime, leucophores have been developing,

having appeared at stage 25 (20-somite stage) as dis-

cussed below.

The VS is significantly advanced by Stage 23 (12-

somite stage), with melanophores scattered under the

hindbrain between and posterior to the otic vesicles. By

Stage 24 (16-somite stage) VS melanophores are at a

somewhat higher concentration just caudal to the otic

vesicle, where five to seven melanophores remain

throughout larval development but do not overlap or

intermingle with leucophores that are present more

anteriorly. Melanophores are absent from the posterior-

most hindbrain region, but a connection to the trunk

melanophores remains in the form of at least one cell

process on each side that provides contact between the

otic and trunk groups of VS melanophores and remains

through embryonic development.

By Stage 29 (34-somite stage), melanophores form a

prominent medial plexus between the otic vesicles and

extend from around the base of the pectoral fin through

the length of the trunk as a bilaterally symmetric pair of

lateral bands, but are as yet absent from the tail. At

Stage 32 (4.25 dpf), melanophores form a similar pat-

tern, but more densely populate the anterior regions.

They form dense plexi between/behind the otic vesicles

and behind the pectoral fin. If the melanophores are con-

tracted, the otic plexus can be seen to consist of seven

or eight melanophores and the trunk plexus of two paral-

lel lines of melanophores on each side of the fish in the

region that overlies the developing peritoneum, also

referred to as the developing Lateral Patches (LP). The

VS continues to develop caudad from around the base of

pectoral fins through the length of the trunk. From the

anus posteriorly the width of the band is reduced to a

bilaterally symmetric pair of ‘tramtracks’ through the

remaining length of the trunk and into the rostral tail.

At Stage 35 (5.25 dpf), melanophores in the region of

the forming LPs are concentrated laterally and are

sparse medially and they now form a broad medial band

the length of the trunk. In the tail, the bilaterally sym-

metric bands extend to the same position as the LS

melanophores, around segment 30–34. By Stage 36

(6 dpf) the pattern in the VS is essentially complete,

with bilaterally symmetric bands of melanophores

extending to near the tail tip, and with the ventral gap

visible in some embryos.

Leucophores

An interesting characteristic of leucophore development

in the embryo/larval stages is their very tight association

Lamoreux et al.


with melanophores in some locations, notably the DS,

but their isolation from melanophores at first appear-

ance, where they are associated with the hindbrain,

always anterior to the otic placodes. Using embryos that

lack melanophores, so that their development may be

visualized, leucophores are first seen as six bilaterally

symmetrical flecks, then dull white spots at Stage 25

(20-somite stage). By Stage 29 (34-somite stage) these

cells are highly stellate, cream-coloured and form a

mesh-like plexus from inside the eyes to the anterior

edge of the otic vesicles. Figure 4 shows this stage; we

selected an example in which the lecophores were con-

densed, so that they appear individually rather than as

the meshlike plexus that would be the more usual

observation. The leucophores are clearly more dorsad

than the otic plexus of melanophores and anterior to

them. As the embryo develops, this plexus becomes

yet more pronounced. The pattern of leucophore distri-

bution seems to be maintained; leucophores remain in

the original locations, while additional leucophores take

up positions as far anterior as the medial face of the

eye by Stage 33 (late 4 dpf). By Stage 33 they appear

to be migrating to the dorsal surface of the body, ini-

tially in the regions of the nose, eyes and ears, exitting

around these structures, and thence both dorsad to join

the DS over the brain, and ventrad to the LP and VS. As

the leucophores take up positions on the head, each

becomes surrounded by two or three melanophores to

form the melanophore units that are characteristic of

the DS as described above.

By late 4 dpf or early 5 dpf one to three leucophores

appear in situ in the DS, that may or may not arise sep-

arately from those described above. They are approxi-

mately evenly spaced along the antero-posterior axis of

the body, and quickly develop the more mature plump

shape and cream-yellow colour. Six or eight hours later

a second wave of leucophores appears in the DS,

evenly spaced between those first few, and each leuco-

phore becomes positioned central to three or four DS

melanophores to form melanophores units (Figure 1

shows this pattern in wild type embryos). By Stage

38 (8 dpf), embryos with the melanophores in their

contracted state, or embryos that lack melanin pigmen-

tation (Figures 2 and 3), show abundant DS leucoph-

ores, approximately one per two melanophores in the

head and a near-continuous line in the trunk and tail. As

leucophores first appear they are initially duller and whi-

ter in colour, but share the spot-like morphology; thus,

unlike the leucophores associated with the brain and

those migrating across the surface of the body, they are

rounded and lack dendrites. The association of mel-

anophores with the leucophores is precise, and is under

genetic control as illustrated by the several mutant

stocks (Table 2) in which the pattern is modified.

Iridophores

Iridophores are most readily observed in fish that lack

melanophores (Figures 2 and 3), where one or a few

can be observed at Stage 34 (5 dpf) over the eyes

and LPs. In wild type fish they are first seen scattered

over the LPs from Stage 35 (5.5 dpf). They increase in

number, seen as a fine dusting of reflecting organ-

elles, using darkfield illumination, by Stage 37 (7 dpf)

and a dense dusting by Stage 39 (9 dpf). Overlying

xanthophores impart a golden yellow tint to the LPs

by Stage 38 (8 dpf) in wild type fish, but are first

clearly visible at Stage 34 (5 dpf) using fish that lack

melanophores.

Xanthophores

Xanthophores are more easily seen after several min-

utes of exposure to transmitted light. On wild-type fish

they are first detectable under the dissecting micro-

scope at around Stage 36 (6 dpf), but using mutant

stocks can be seen as early as 4 dpf as a yellow hue

dorsal to or lateral to the fore- and midbrains and on the

LP. The yellow hue becomes gradually more prominent,

but well-defined cells can be visualized only occasionally

before 9 dpf, first being obvious adjacent to the dorsal

third of the dorsal myotomes of trunk and tail during

Stage 39 (9 dpf). Visualization of xanthophores (and

iridophore cells, as opposed to their pigmented organ-

elles) is very difficult in wild-type fish. It is helpful to

evaluate xanthophores and iridophores using fish from

the stocks that lack both melanophores and leucophores

and are segregating at R and/or gu (Figures 2 and 3).

Eye

Melanization of the pigmented retinal epithelium begins

from around Stage 28 (30-somite stage) and becomes

progressively more intense (Iwamatsu, 2004). Choroidal

melanophores are seen from Stage 26 (22-somite stage)

and increase in number and melanization up to Stage 35

(5 dpf) when the eye becomes opaque. Choroidal irid-

ophores are first seen at Stage 32 (4.25 dpf) and then

rapidly increase in number, becoming a dense spotting

by late Stage 33 (late 4 dpf) and almost a solid block by

Stage 35 (5.25 dpf), with a particular concentration

around the iris visible from this stage onwards. The

Figure 4. A medaka embryo, within the chorion, stage 29,

showing early development of leucophores associated with the

brain. This embryo is albino, from the i-3 stock, thus the

leucophores can be visualized in absence of melanophores.



iridophore reflection is coloured yellow by associated

xanthophores by 5 dpf.

Discussion

Our characterization of the medaka raised a number of

questions regarding development of the four types of

chromatophores of medaka, as well as similarities and

differences of chromatophore embryogenesis compared

with zebrafish. Here we highlight four comparisons

between the phenotypes of medaka and of zebrafish

and raise a few of the questions that seem to offer rich

potential for further study (6 Table 3).

1 When Zebrafish embryos were raised in parallel

with the medaka, leucophores were not seen and no

equivalent chromatophores were seen associated with

the brain in early development of zebrafish in the

locations where leucophores occur in medaka. What

mechanisms account for the generation of a fourth chro-

matophore type in medaka?

2 Medaka leucophores show a distinctive association

with melanophores on the surface of the body, most

highly choreographed in the dorsal stripe, and a separ-

ation from melanophores elsewhere. What mechanisms

regulate their precise patterning within the body?

3 The general patterns of pigmentation development

in the two species strongly suggest homology. Larval

medaka have leucophores, whereas larval zebrafish do

not. Nevertheless, in the DS, the leucophore pattern in

developing medaka up to about 5 dpf shows strong par-

allels to the iridophore pattern of zebrafish (Figure 5),

and the timing of their ontogenies is similar. What are

the patterning mechanisms mediating chromatophore

maturation, specification and interactions in these two

species? In particular, what is the ontogenic and genetic

relationship between iridophores and leucophores?

Table 3. Developmental time line*

comparative time line of development of

zebra fish and medaka. Zebra fish data

obtained from Kimmel et al., 1995.

Medaka from our observations

Time Line 26o melanophoresMedaka - Egg fertilized leukophores

iridophoresZebra Fish xanthophores

- Mphores - 1 d Stage 17, 1 d 1 h Early neurula

-

- 2 d Stage 25, 2 d 2 h 18 Somite

-

Xanth & - 3 d Stage 29, 3 d 2 h 34 Somite- Iridphore

-

- 4 d Stage 31, 3 d 23 h Gill blood vessels

-

-Embryo - 5 d Stage 34, 5 d 1 h Pectoral fin blood Pattern Complete

-

6 d Stage 36 Heart development

-

7 d Stage 37 Pericardial cavity

-

8 d Stage 38 Spleen development

-

9 d Stage 39 Hatching

Medaka begin to shed embryonic melanophores and develop adult type

28.5o

Lamoreux et al.


Conversion of chromatophores from one type to another

has been documented in Bagnara et al. (1979); Ide

(1979) and Ide and Hama (1976)7 . However, a survey of

the medaka pigment mutants does not suggest a basis

for assuming their lateral conversion during normal

development, nor does it provide clues to vertical origin

of any one of the chromatophore types from another

type. The question is intriguing.

4 In subsequent embryonic development, the leu-

cophores of medaka essentially continue their cluster

relationship with melanophores, while the iridophores of

the zebrafish proceed to create the central stripe, bor-

dered on each side by melanophore stripes. What

mechanisms mediate these different behaviours? Inter-

estingly, in the medaka ci mutants leucophores of the

central stripe tend to elongate in such a way that they

appear to form a rather fragmented central leucophore

stripe bordered by melanophores. Understanding ci may

help to clarify this issue.

All the above observations warrant further study, and

we hope our accounting of them will make the means

more readily available. Clearly, availability of the medaka

model enhances and complements the zebrafish and

the mouse models.

Supplementary Material

The following material is available from: http://

www.blackwellpublishing.com/products/journals/suppmat/

PCR/PCR216/PCR216sm.htm

Appendix A1 Terminology, materials and methods.

Appendix A2 Embryonic development in detail.

Figure S1 Photography of medaka embryos.8

Acknowledgements

This work was funded by Visiting Research Fellowships (Nagoya

University) to LL and RNK. Zebrafish eggs were generously made

available by Dr. Hiroyuki Takeda of the National Institute of Genet-

ics, Mishima, Japan.

We thank Ms Chikako Inoue for preparation and arrangement of

experimental materials (fish). We wish to express our sincere

appreciation to Professor Jiro Matsumoto, who is and has been a

catalyst for changing lives as well as changing science.

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