Mechanisms of Development 121 (2004) 873–882
www.elsevier.com/locate/modo
Research paper
Possible roles of zic1 and zic4, identified within the medaka
Double anal fin (Da) locus, in dorsoventral patterning of the trunk-tail
region (related to phenotypes of the Da mutant)
Masato Ohtsukaa,*, Natsuko Kikuchia, Hayato Yokoib, Masato Kinoshitac, Yuko Wakamatsud,
Kenjiro Ozatod, Hiroyuki Takedab, Hidetoshi Inokoa, Minoru Kimuraa
a
Division of Basic Molecular Science and Molecular Medicine, Tokai University School of Medicine, Bohseidai, Isehara, Kanagawa 259-1193, Japan
b
Division of Biological Science, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku 113-0033, Japan
c
Division of Applied Biosciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan
d
Laboratory of Freshwater Fish Stocks, Bioscience Center, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan
Received 30 December 2003; received in revised form 26 March 2004; accepted 6 April 2004
Abstract
Double anal fin (Da) is a spontaneous medaka mutant that exhibits an unique ventralizing phenotype, a mirror-image duplication across
the lateral midline in the dorsal trunk-tail region. In the mutant, early D-V specification appears normal but the altered phenotype becomes
evident during late embryogenesis. In this study, we genetically specified the mutation to a 174-kb region harboring two zinc-finger type
transcription factors, zic1 and zic4, and compared the genomic structures of this region between wild-type and Da mutant fish. No mutation
was found in the coding regions of either gene of the mutant, while two fragments, 324 bp and 3 – 4 kb long, were found inserted downstream
of zic1 and zic4, respectively. Probably as a result of this, the expression of both genes is lost in the derivatives of the dorsal (epaxial) somite
and the region dorsal to the terminal axis bending. All these tissues are morphologically affected or become ventralized in the mutants. In
contrast, the expression in the head region and dorsal spinal cord remained unchanged. Detailed characterization of Da phenotypes revealed a
novel defect in the axial skeleton (spina bifida occulta) that was also found in zic1-deficient mice. Finally, zic1-morpholino injection partially
phenocopied early Da phenotypes. These findings strongly suggest that zic1 and/or zic4 are required for dorsal identity in the trunk-tail region
and that loss of their expression in the epaxial somite derivatives and tail region causes the Da phenotypes.
q 2004 Elsevier Ireland Ltd. All rights reserved.
Keywords: Medaka; Mutant; Dorsoventral; Mirror-image; Trunk-tail region; zic; Somite; Positional cloning; Spina bifida occulta
1. Introduction
A medaka mutant, Double anal fin (Da), which
exhibits autosomal semidominant inheritance, was spontaneously isolated from wild-type populations collected at
Toyokawa, Aichi Prefecture, Japan in 1966. Homozygous
Da 2 /2 exhibits unique phenotypes related to dorsoventral (D-V) patterning in fry to adult fish (Fig. 1).
Adult Da mutants have anal fins resembling dorsal fins,
* Corresponding author. Address: Division of Basic Molecular Science
and Molecular Medicine, Tokai University School of Medicine, Bohseidai,
Isehara, Kanagawa 259-1193, Japan. Tel.: þ 81-463-93-1121x2682;
fax: þ 81-463-96-2892.
E-mail address: [email protected] (M. Ohtsuka).
0925-4773/$ - see front matter q 2004 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.mod.2004.04.006
and rhombic caudal fins (Fig. 1A,B). Ishikawa (1990)
described ventralization of musculoskeletal patterns in
the caudal fin (Fig. 1C,D). The pigmentation pattern in
the dorsal trunk is also ventralized: iridophores, which
are normally present only on the belly, are distributed in
the dorsal trunk region (Tomita, 1975). The mutant
phenotype can be recognized during late embryogenesis
by the pattern of melanocyte distribution and the
morphology of the dorsal fin fold, dorsal myotomes
and terminal axis bending of the notochord (Tamiya et al.,
1997, Fig. 1E – L): the melanophores are distributed in
two rows (ventral-type) instead of a single (dorsal-type)
in the dorsal line (Fig. 1E –H), and the epaxial (dorsal)
myotome exhibits a ventral pattern in shape (transformed
from round to oval shape). Based on these phenotypes, it
874
M. Ohtsuka et al. / Mechanisms of Development 121 (2004) 873–882
Fig. 1. Phenotypes of the Da mutant. The wild-type medaka (A,C,E,G,I,K,M,O,Q) are compared with the mutants (B,D,F,H,J,L,N,P,R). (A –J, M– P)
Anterior to the left. (A,B) Adult medaka fish. Phenotypes of the mutant are
presented for dorsal fin, caudal fin and iridophore localization. (C,D)
Skeletal elements of the adult caudal fin. In the mutant, epural 3 is
differentiated (arrow; epural 2 þ 3) as described by Ishikawa (1990).
(E–H) Chromatophore arrangements of the tail in stage 39 embryos. Dorsal
view (E,F) and ventral view (G,H). Dorsal melanophores of the mutant are
arranged in two rows (F), similar to the ventral side (H). Numerous
xanthophores and leucophores appear in a single row along the dorsal
midline of the wild-type (yellow arrow in E), while only a few of these
chromatophores are seen in the mutant (F). (I,J) Lateral views of the tail
region in stage 39 embryos. Mutated phenotypes can be seen in the anterior
end of the dorsal fin fold (white arrowhead) and termini of the notochord
(arrow). While the notochord turns dorsally in the wild-type, no bending is
observed in the mutant. (K,L) Transverse sections of the trunk region of
has been postulated that the dorsal half of the caudal
region becomes a mirror-image of the ventral half across
the lateral midline. Consequently, adult Da medaka
mutants exhibit a streamlined (or tear-drop) body
shape, which is more similar to that of fast-swimming
fish in the middle layer of the sea (such as tuna) than
that of slowly swimming fish near the surface. Despite
these drastic phenotypic changes, the homozygote
matures, behaves, and reproduces normally.
There have been no reports on the mutations that
cause phenotypes similar to Da. Large-scale screening of
zebrafish mutants identified many mutations with defects
in early D-V axis specification such as dino and swirl
(Hammerschmidt et al., 1996; Mullins et al., 1996).
Subsequent analyses have shown that they are mostly
related to the pathway of organizer-derived factors and
BMPs (for review, see Schier (2001)). Indeed, they
exhibit ventralized or dorsalized phenotypes that are
evident during gastrulation. In contrast, the Da mutant is
phenotypically normal during the gastrulation to segmentation stages, and the first distinct sign of the phenotype
is a ventral-type pattern of melanophore distribution on
the dorsal trunk (Tamiya et al., 1997). This phenotype
becomes evident by hatching stages. More importantly,
only the trunk-tail structures (skeleton, fin shape and
pigmentation pattern) exhibit a mirror-image duplicated
pattern, while the head region appears normal. Thus, the
Da mutant provides a unique model system for study of
the mechanisms controlling the overall D-V patterning of
vertebrate posterior trunk and tail.
We have for many years been attempting to isolate the
gene responsible for the Da mutant. In a previous study, we
genetically mapped the Da locus to linkage group (LG) VIII
(corresponding to LG 20 on the current map (Naruse et al.,
2000)) of the medaka chromosome, and obtained polymorphic markers located in close proximity to the Da
mutation (Ohtsuka et al., 1999). More recently, the genomic
region containing the mutation was completely covered by
Bacterial Artificial Chromosome (BAC) and cosmid clones
(Ohtsuka et al., 2004). Subsequent genetic analysis in this
study specified the critical region to a 174 kb region in
which only two zinc finger of cerebellum (zic) genes, zic1
and zic4, are located. Although no mutation was found in the
zic1 and zic4 coding regions of the Da mutant, the
expression of both genes is specifically lost in the tissues
stage 39 embryos. Dorsal myotomes do not cover the spinal cord in the
mutant (white arrows). (M–P) Phenotypes in the trunk axial skeleton.
Lateral (M,N) and dorsolateral views (O,P). Neural arches of the mutant are
irregularly shaped (N,P), and neural spines are deformed and larger (N)
than those of the wild-type (M). Epipleurals (arrows in O,P) of the mutant
are missing or truncated. (Q,R; lateral view) Acetylated tubulin staining of
stage 39 embryos. Neuromasts are positioned laterally and ventrally in the
wild-type (Q), while some neuromasts are dorsally mislocated in the mutant
(arrows in R).
M. Ohtsuka et al. / Mechanisms of Development 121 (2004) 873–882
that are affected in the mutant. Further knockdown and
phenotypic analyses indicated the involvement of zic gene
in the unique Da phenotypes.
2. Results
2.1. zic1 and zic4, candidates for the Da gene
As shown in Fig. 2, our positional cloning approach
enabled covering of the genomic region containing the Da
mutation by BAC clones (Ohtsuka et al., 1999, 2004). We
have identified three genes, zinc finger of cerebellum 1
(zic1), zic4 and phospholipid scramblase 1 ( plscr1) within
the BAC clone, B180F18, by exon prediction programs
875
and comparison with the Fugu, mouse and human genomes
(Ohtsuka et al., 2004). Detailed genetic mapping conducted
in this study excluded the plscr1 gene from the smallest Da
region (173,992 bp), defined by stsMA019 and C134M15R
(shown in red in Fig. 2B), leaving two genes (zic1, zic4) as
candidates. zic1 and zic4 were amplified from cDNA
libraries of 2 –3-day and stage 18 –23 embryos by PCR
(data not shown). In mice and zebrafish, zic1 is expressed in
the dorsal region of the spinal cord and somites as well as
the anterior head region (Nagai et al., 1997; Rohr et al.,
1999). Therefore, two zic genes were considered strong
candidates for the causative gene, although the head region
appears unaffected in the Da mutant.
The zic1 and zic4 genes (Fig. 2D) exhibit a bidirectional
(head-to-head) gene configuration (Ohtsuka et al., 2004).
Fig. 2. Physical mapping of the Da region. (A) Chromosome walking and specification of the minimum Da region. Chromosome walking was initiated from
STS markers shown in blue letters. The genomic region around the Da region was covered by a BAC contig. STS markers shown in black letters were derived
from BAC end sequences. Using these markers and recombinant progeny, the region critical for the Da mutation was specified to a region of approximately
200 kb (Da region; shown in red). Regarding the chromosomes of recombinant progeny, regions derived from the HNI strain and the Da mutant are shown in
blue and red, respectively. Chromosomal regions shown in gray are the recombined regions. (B) BAC clone B180F18 containing the 228,508 bp insert.
Detailed genetic mapping narrowed down the Da region (indicated in red) to 174 kb defined by stsMA019 and C134M15R. One backcross progeny recombined
between stsMA019 and C269M19F, and another recombined between B172F24M4 and C134M15R. STS markers C134M15R and C269M19F were derived
from cosmid ends (Ohtsuka et al., 2002), and design of stsMA019 was based on the BAC sequence. (C) Genes located within the BAC clone. After
determination of the complete BAC sequence, we identified only three genes within the BAC clone. Genetic mapping excluded the plscr1 gene from the Da
region, and therefore only the zic1 and zic4 genes remained within this region. Insertions specific to the Da mutant (shown in red arrows) are candidates for the
Da mutation. The scale is the same as in (B). (D) Genomic organization of the zic1 and zic4 genes. Exons of each gene are shown as E1, E2, and E3. The coding
regions are shown in the blue boxes (dark blue regions are zinc finger motifs). Positions of SNPs detected in the Da mutant are indicated in purple arrows. The
single-nucleotide insertion and deletion in the Da mutant are indicated by blue and green arrows, respectively.
876
M. Ohtsuka et al. / Mechanisms of Development 121 (2004) 873–882
Both genes are transcribed, as confirmed by RT-PCR (3-day
embryos; data not shown) and whole-mount in situ
hybridization analyses (described below). Genomic DNA
sequences of zic1, zic4 and their intergenic region in the
mutant were compared with those of the wild-type BR strain
evolutionarily closest to the Da mutant background. No
obvious mutation was found in the coding region of the two
genes. Although several single nucleotide polymorphisms
(SNPs) were detected within the non-coding regions
(Fig. 2D), none of these are located in the evolutionally
conserved region among species (medaka, Fugu, humans
and mice) (data not shown). In parallel, we searched several
regions that were specifically rearranged in some Da
mutants, by LA-PCR and genomic southern hybridization
analyses, since most of the spontaneous medaka mutations
isolated so far were caused by insertion of putative
transposable elements (Fukamachi et al., 2001; Koga et al.,
1995; Loosli et al., 2001). We finally found two insertions in
the mutant Da region (arrows in Fig. 2C); one is a 324-bp
insertion (AB164417) located 15.8 kb downstream of the
zic1 gene, while the other is a 3– 4 kb insertion located 7 kb
downstream of the zic4 gene. The 324-bp insertion shows
sequence similarity with some medaka genomic fragments
by BLASTN analysis. Thus, this must be a medaka repeat.
The nucleotide sequence of the 3 – 4 kb insertion has
remained to be determined, since it could not be amplified
by LA-PCR possibly due to the presence of secondary
structural element. These regions are candidates for the
mutation that could affect transcription of the genes.
2.2. Patterns of expression of zic1 and zic4 in wild-type
embryos
Since positional cloning of the Da locus has identified
zic1 and zic4 as candidate genes, we first examined the
pattern of expression of the two genes in medaka embryos
by whole-mount in situ hybridization. Early pattern of
expression in the head region of the medaka was almost the
same as that in the zebrafish (data not shown; Rohr et al.,
1999).
In the trunk-tail region, zic1 expression is detected in the
dorsal spinal cord and the somite derivatives. Expression in
the somite becomes detectable between stages 19 (2-somite
stage; data not shown) and 21 (6-somite stage; Fig. 3A).
Expression is confined to the dorsal half (we refer to this as
the epaxial somite in accordance with Sporle (2001)). This
expression continues (Fig. 3E,I,O) and gradually becomes
restricted to the dorsal-most region and the region
surrounding the spinal cord (Fig. 3S). By analogy with the
pattern of expression of murine zic1, the positive cells
around the spinal cord could derive from the dorsal
sclerotome.
zic1 exhibits high levels of expression in the region
dorsal to the terminal axis bending (Fig. 1I) in the tail at
stage 35 (Fig. 3K). At stage 39 (hatching stage), cells
expressing zic1 are observed between the myotomes,
dorsally to the spinal cord, underneath the dorsal epidermis
(Fig. 3W). The origin of these mesenchymal cells is not
clear at present, because lineage analyses of the dermis and
epaxial myotome are not available for fish. However, based
on the description in amniote somites (Sporle, 2001), we
reasoned that they are derived from the dorsal bud of the
epaxial myotome during its dorsal elongation. At the level
of the dorsal fin fold, zic1-expressing cells are present in the
fin mesenchyme (Fig. 3Y).
There have been no reports on the precise spatiotemporal expression of the zic4 gene during embryogenesis
(Aruga et al., 1996). Whole-mount staining of zic4 mRNA
in this study revealed that the zic4 expression in the epaxial
somite derivatives was almost the same as that of zic1,
although staining was weaker even when a longer probe was
used (Fig. 3C,G,M,Q,U).
2.3. Patterns of expression of zic1 and zic4 in Da mutants
We then examined zic1 and zic4 expression in Da
mutants. RT-PCR analyses demonstrated that the two genes
are expressed equally in both wild-type and mutant embryos
(data not shown). Indeed, the expression in the head and
dorsal spinal cord is almost unaffected (Fig. 3F,H,P,T,V),
except for zic4 expression in head region (Fig. 3H; zic4
expression in the Da is weaker than that in wild-type
especially in rhombomere). However, the expression in the
somites, sclerotome, and the mesenchyme above the spinal
chord and in the tail region is greatly reduced or lost in the
Da mutant (Fig. 3B,D,F,H,J,L,N,P,R,T,V,X). When staining time was prolonged, weak and patchy expression of zic1
was detectable in the mutants especially in anterior somites
(Fig. 3F,J).
2.4. zic-morpholino injection
Expression analysis suggested that loss of zic1 and zic4
expression in the above-mentioned tissues may cause Da
phenotypes. To test this hypothesis, a morpholino-based
knockdown experiment was performed. The effect was
assessed at stage 39 (hatching stage) by examination of the
earliest phenotypes of Da, abnormal distribution of
melanophores (Fig. 1F; two rows of dorsal melanophore
arrangement instead of a single in wild-type embryos),
anterior shift in the dorsal fin fold (Fig. 1J), and a failure in
dorsal myotome elongation (Fig. 1L) (Tamiya et al., 1997).
As shown in Fig. 4, injection of zic1 morpholinos (MO), but
not zic4 MO, was able to phenocopy the Da early
phenotypes, although the rate of occurrence was not high
at 16.2% [11/68; 7/56 for Mo-zic1 and 4/12 for Mozic1SP1], probably due to limited effectiveness of MO at
later stages. In those affected embryos, the pattern of
melanophore distribution in the dorsal trunk became
ventral-type (two rows, Fig. 4A,B) and this was confirmed
by histological sections (arrows in Fig. 4C,D). Histological
analysis further revealed that the MO injection inhibited
M. Ohtsuka et al. / Mechanisms of Development 121 (2004) 873–882
877
Fig. 3. Patterns of expression of zic1 and zic4. Lateral views (A–N; anterior to the left) and transverse sections (O–Y; dorsal to the top) of zic1 and zic4
expression in wild-type and Da mutant embryos. (A–D) Somite expression (arrows) is initiated in wild-type embryos until stage 21, but not in Da embryos.
(E–H) Obvious differences in the pattern of expression in somites between wild-type and Da embryos at stage 25. In contrast, expression in the head regions is
unaffected in the Da mutants. (I) The epaxial region is clearly distinguishable from the hypaxial region by staining of stage 30 wild-type embryos with the zic1
probe. (J) In the stage 30 Da embryo, spinal cord expression is intact, and subtle patchy expression can be seen in the epaxial region. (K–N) Intense zic1 and
zic4 expression is observed in termini of the tail in wild-type embryos at stage 35, but not in Da embryos. (O–R) Sections of stage 25 embryos reveal that
epaxial expression of somites is affected in the mutants. (S– V) In stage 33 –34 embryos, zic1 and zic4 expression is restricted to the dorsal-most region of the
somite derivatives and region surrounding the spinal cord. Expression in the spinal cord is unaffected in Da. (W–Y) In stage 39 embryos, zic1 expression is
detected in the region dorsal to myotome elongation (W), and is not detected in Da (X). Spinal cords are enhanced by dashed lines. Cells expressing zic1
contribute to the mesenchyme of the dorsal fin fold (Y).
878
M. Ohtsuka et al. / Mechanisms of Development 121 (2004) 873–882
Fig. 4. Phenotypes of morpholino (MO)-injected embryos. (A,B) Dorsal melanophores of the tail are arranged in two rows (red arrows in B) in some of the zic1
MO-injected wild-type embryos, similar to those of Da mutants. This phenotype is not seen in the control-MO-injected embryos (A). (C,D) Dorsal myotome
phenotype. The melanocytes are indicated by arrows, and the myotomes are outlined by dashed lines. In zic1 MO-injected embryo with abnormal melanocyte
distribution, the dorsal myotome in the trunk region fails to cover the dorsal neural tube (D), while control embryo does (C). The induced phenotype is nearly
identical to that in Da homozygous embryos (Fig. 1L). (E) MO-injecion partially phenocopies the Da phenotype, anterior expansion of the dorsal fin fold.
(Lateral view). In this picture, the anterior end of the dorsal fin fold in the injected embryo is located at the fourth myotome level posterior to the anus
(arrowhead), the position between normal (the seventh myotome) and Da mutant (the same level of the anal myotome) (see also Fig. 1I,J). Arrow indicates the
position of the anus.
dorsal elongation of myotome (Fig. 4C,D). Furthermore,
the injected embryos tended to show anterior shift in the
position of dorsal fin fold: the anterior end of dorsal fin fold
normally lies at the seventh myotome located posterior to
the anal myotome (Fig. 1I), while it lies in the fourth to the
sixth after MO injection (Fig. 4E). Since the dorsal fin fold
in Da homozygous mutants extends anteriorly and usually
reaches the level of the anal myotome (Fig. 1J; Tamiya et al.,
1997), the MO injection in the present study partially
phenocopied the Da mutant. All these phenotypes are found
in Da homozygous embryos. These early Da phenotypes
often appeared simultaneously in MO-injected embryos.
We also examined MO-injected embryos for a defect in
the terminal axis bending of the notochord, one of the early
Da phenotypes (Fig. 1I,J). However, we were unable to
obtain reproducible results because control embryos
sometimes failed to exhibit the terminal axis bending due
to the high level of MO injection. As expected, most of
the injected embryos also exhibited malformation of the
head structures that may reflect the expression of zic1 in the
head region. In contrast, none of the embryos injected with
control MO exhibited either the Da phenotypes or an
abnormal head (0%, [0/47]; Fig. 4A). The results indicate
that loss of zic1 expression causes most of the early Da
phenotypes. It is worth noting that co-injection of zic1 and
zic4 MO, and two different zic1 MO did not increase the
penetrance of the phenotypes.
2.5. Detailed gene expression analyses in Da mutants
To further characterize early Da phenotypes, we carried
out thorough gene expression analyses in wild-type and
mutant embryos at stage 25, when expression of zic1 and
zic4 in the epaxial somite is undetectable in the mutant. The
genes we chose were wnt1, wnt4, wnt5b, wnt11r, myf5,
myoD, pax2, pax6, pax7, gbx2, gsh1, c-ret, bmp4, zic2
M. Ohtsuka et al. / Mechanisms of Development 121 (2004) 873–882
and zic5, which are expressed in various tissues in the trunk
region at this stage. Unexpectedly, none of the genes
examined exhibited a clear difference in pattern of
expression between wild-type and Da embryos (this figure
is supplied in our web site: http://bunsei2.med.u-tokai.ac.
jp:8080/, ohtsuka/supplemental_figure.html). For example, wnt11r and wnt5b, which are mainly expressed in the
dorsal and ventral regions of the posterior trunk, respectively, exhibit normal expression in the mutants (P, P0 , Q, Q0
in a supplemental figure). As indicated by the normal
expression of wnt1, wnt4, wnt11r, pax2, pax6, pax7, gbx2,
gsh1, c-ret, bmp4, zic2 and zic5, dorsoventral polarity is
normally established in the spinal cord of the mutants.
Furthermore, the normal expression of wnt5b, wnt11r, myf5,
myoD, pax7, bmp4, zic2 and zic5 in the somite and its
derivatives implies that regional specification and differentiation of the somite proceed normally in Da mutants at
stage 25. We also examined the expression of wnt11r, myf5,
bmp4, zic2 and zic5 at stages 30 –31, but failed to detect any
difference between wild-type and mutant embryos (data not
shown).
2.6. Defects in axial skeleton and other tissues
zic1-knockout mice exhibit multiple abnormalities in the
axial skeleton (Aruga et al., 1999). Despite of a detailed
report on the skeletal structures of mutant caudal fin
(Ishikawa, 1990), the axial skeleton of the mutant has
received little attention. Thus, we re-examined the skeletal
defects in Da mutants, focusing on the axial skeleton. The
mutants possess trunk neural arches with irregular shapes
(Fig. 1M,N). In some cases, midline fusion of several neural
arches is disturbed, resulting in spina bifida occulta. This
phenotype could be comparable to that in zic1-knockout
mouse (Aruga et al., 1999). Neural spines are also enlarged
and deformed in the mutants (Fig. 1N). While the
epipleurals are attached to the proximal region of the ribs
in the wild-type (arrow in Fig. 1O), they are truncated or
missing in the mutant (arrow in Fig. 1P). In contrast to other
phenotypes, these skeletal defects do not seem to be a
ventralization. The centrums, ribs and hemal spines appear
to be unaffected in Da mutants (data not shown).
In addition to the axial skeleton, we detected undescribed
phenotypic changes in the distribution of xanthophores and
leucophores (Fig. 1E,F), and in positioning of neuromasts in
the tail region of Da mutants (Fig. 1Q,R) of the hatching
embryo.
3. Discussion
Genetic and physical mapping and comparative analyses
have revealed that the Da region contains only 2 genes, zic1
and zic4 (Ohtsuka et al., 2004). These genes are expressed in
wild-type medaka embryos, as confirmed by RT-PCR and in
situ hybridization. However, in the mutant genome, there is
879
no mutation such as a deletion and/or non-synonymous
substitution found in the coding regions, and instead, 324 bp
and 3 – 4 kb fragments are inserted 15.8 and 7 kb downstream of zic1 and zic4, respectively. Despite of this, the
present findings strongly suggest that zic1 and/or zic4 are
causative genes for the Da mutation.
3.1. zic1 and zic4 expression related to the Da phenotype
The patterns of expression of medaka zic1 and zic4 are
nearly identical, though zic4 expression tends to be much
weaker. Expression is mainly detected in the anterior head,
dorsal spinal chord, epaxial somite and its derivatives, and is
very similar to that of mammalian zic1(Nagai et al., 1997).
The phenotype of Da has been characterized as a mirrorimage duplication of the trunk-tail region, including the
muscular pattern, fin morphology and pigmentation pattern
(Ishikawa, 1990; Tamiya et al., 1997). All of this
pleiotrophic Da phenotype could be explained by the loss
of zic1 and zic4 expression in these affected tissues. The
sites of expression in the trunk-tail region include the
derivatives of the epaxial somites (dorsal myotome and
screlotomal cells surrounding the spinal cord), the mesenchymal cells above the dorsal spinal chord, and mesenchymal cells in the dorsal fin hold and the tail termini. All of
these tissues lose the expression of zic1 and zic4, and their
development is affected in Da mutants, with abnormal
morphology of the trunk neural arches (Fig. 1N), a failure in
dorsal myotome elongation (Fig. 1L; Tamiya et al., 1997)
and terminal axis bending (Fig. 1J), and a ventral-type
transformation of the dorsal fin morphology (Fig. 1B,J).
Thus, zic1 and zic4 expression is observed in the right place
and with the correct timing to account for the Da phenotype.
The loss of zic1 and zic4 expression in these affected
tissues could be caused by one of the SNPs or insertions
found near zic1 and zic4. Considering that all SNPs detected
are located in non-conserved regions among species, the
two insertions could cause the Da mutation. Since the
expression in neural tissues remains largely unaffected,
these insertions may disrupt the activity of a somite-specific
enhancer or function as an insulator between the enhancer
and the genes. Tandem bidirectional organization and
identical expression in the somite derivatives suggest that
the two genes could be regulated by the same enhancer.
Regarding the murine zic1gene, the enhancer of the dorsal
spinal cord lies between 2.0 and 0.9 kb of the 50 -flanking
region of zic1 (Aruga et al., 2000). However, the somite
enhancer of zic1 remains unidentified.
Concerning the defects in the arrangement of melanophores, it is not clear whether melanophores express zic
genes or whether the underlying dorsal mesenchyme affects
their arrangement. Since development of pigment cells
(e.g. migration and differentiation) is thought to be affected
by their environment (Kunisada et al., 2001), pigmentation
defects in Da may be secondarily caused by a defect in
880
M. Ohtsuka et al. / Mechanisms of Development 121 (2004) 873–882
the dorsal mesenchyme or myotome. The same is probably
true of mis-location of the neuromasts (Fig. 1R).
3.2. MO-knockdown phenocopied early phenotype
of the Da mutant
The results of our morpholino-based knockdown experiment suggest that zic1 is responsible for the Da phenotypes.
Furthermore, the finding that injection of zic4 MO did not
induce this phenotype and co-injection of zic1 and zic4 MOs
did not increase the penetrance of the phenotypes suggests
that Zic4 has little function in the determination of the Da
phenotype. Since the mutant phenotype can be detected at
relatively later stages (more than 5 days after fertilization),
we had to inject a maximum concentration of MO. But, even
under these experimental conditions, the significant number
(16.2%) of the injected embryos exhibited the clear Da
phenotypes, abnormal pigmentation pattern, anterior shift in
the position of the dorsal fin fold, and a failure in dorsal
myotome elongation. These phenotypes are very specific to
the Da mutantion and are never obtained under normal
conditions or in control injections. Of course, zic1-MO
injection did not faithfully mimic the Da mutation in which
somite-related expression is specifically lost, since, as in
zic1-knockout mice, zic1-MO inhibits the activity of Zic1 in
nearly all tissues from the beginning of its expression.
Taken together, it is reasonable to conclude that zic1 is a
causative gene for the Da mutation.
The ENU induced allele of the Da mutant (da35a) was
previously reported (Loosli et al., 2000). Thus, we have
sequenced 10 kb-genomic region containing zic genes in
da35a (Fig. 2D). In this region, the genomic sequence of
da35a is identical to that of the spontaneous allele examined
in the present study, indicating that this region in both
alleles comes from the same genetic background.
Expression patterns of zic1, zic4 and other genes examined
so far were also identical between both alleles (data not
shown). These results indicate that the ENU mutation, like
the spontaneous Da mutation discussed above, should be
located outside of the 10 kb region and affects the activity of
the somite enhancer.
3.3. The Da mutant shares phenotypic features with
the zic1-knockout mouse
The skeletal phenotype of the Da mutant resembles that
of the zic1-knockout mouse (Aruga et al., 1999). Neural
arch defects in the Da mutant are presumably homologous
to vertebral arch phenotypes of the knockouts leading to
spina bifida occulta. Unlike the knockout mice, the neural
spines, probably corresponding to the spinous processes of
mice, are present in the Da mutants but are irregular in
shape. This discrepancy could be explained as follows. In
the knockout mice, zic1 expression in both the somite and
spinal chord is lost from early stages of development, while
in the Da mutants only later somite-related expression is
affected. As discussed by Aruga et al. (1999), zic1
expression in the dorsal spinal chord appears essential for
the formation of spinous processes. Thus, the phenotype of
the Da mutants suggests that late patterning of spinous
processes requires zic expression in the somite derivatives.
Furthermore, the abnormal hair color over the spina bifida
occulta found in the knockout mice could be homologous to
the abnormal patterning of iridophores in Da mutants. No
description of the tail phenotype in the knockout mice is yet
available. This may be due to a novel function of zic genes
in fish or to a subtle undetectable change in the mutant
mouse tail.
3.4. Implications of the Da mutant
The Da mutation causes a mirror-image duplication
rather than hypogenesis or dysgenesis of the dorsal trunk
structures. Thus, the expression of zic1 and/or zic4 in somite
derivatives is required for their correct patterning on the
dorsal side, but not for differentiation of certain cell types
such as those of the skeletons and muscles. The Da
phenotype further suggests that the trunk-trail region is born
as ‘ventral’ and that it is later given a dorsal identity by
dorsal-specific genes such as zic1. We carried out detailed
expression analyses using various markers, but failed to
observe any change at the molecular level in somite and
neural tissues except for the expression of zic genes.
Analysis of gene cascades that function downstream of zic
genes in each affected tissue will clearly be required.
The Da phenotype is consistent with the hypothesis of
Sporle (2001): a mechanism exists that establishes approximate mirror-image duplication, primarily in somite derivatives. According to this hypothesis, the epaxial somatic bud
arises as a mirror-image duplication of the hypaxial
(ventral) somatic bud by establishing polarized borders,
resulting in tripartite regionalization of somite derivatives
into the epaxial – intercalated – hypaxial regions. Intriguingly, the Da phenotype suggests that the external
appearance, namely the fin morphology and pigmentation
pattern, reflects the regionalization of the myotome. If this is
the case, there must be tissue interaction between the somite
(or its derivatives) and the surface ectoderm. Since no such
interaction has been reported so far, it would be of interest to
examine how the somite affects the pattering of the surface
ectoderm using the Da mutants.
3.5. Conclusion
Genetic and expression analyses identified zic1 and zic4
as candidates for the Da gene. In the present study, two
insertions were found in the Da region near the two genes.
However, they do not disrupt the proteins of Zic1 and Zic4,
but may disrupt the tissue-specific enhancer of the two
genes, leading to the loss of somite-specific expression of
zic1 and zic4 in the mutant. Subsequent analyses suggested
that the loss of zic1 and/or zic4 in somite-derived tissues is
M. Ohtsuka et al. / Mechanisms of Development 121 (2004) 873–882
responsible for the Da phenotype. Transgenic rescue
experiments, identification of a somite-specific enhancer
and transplantation experiments will aid further understanding of the mechanisms underlying D-V patterning of
the trunk-tail region through the activity of Zic proteins.
4. Experimental procedures
4.1. Medaka strains
4.1.1. Wild-type
The commercially available orange-red strain was used
for whole-mount in situ hybridization and skeletal specimens. The Cab inbred strain was used for whole-mount in
situ hybridization analyses and antibody staining. For
morpholino injections, the HNI inbred strain was used.
For genetic mapping, recombinant backcross progeny
obtained from a cross between the HNI strain and the Da
mutant were used as previously described (Ohtsuka et al.,
1999). We used DNA isolated from HNI, orange-red, i, BR,
Hd-rR, and HO5 as PCR templates to select the strain most
evolutionarily close to the Da mutant. Genomic DNA of the
BR strain, which was closest, was used for subsequent
analyses.
4.1.2. Da mutant
The original allele of the Da mutant, isolated spontaneously and kept as a closed colony (Ohtsuka et al., 1999),
was used.
4.2. Chromosome walking
Chromosome walking was initiated from STS markers
(stsM02-5 and stsB07-3; (Ohtsuka et al., 1999) as described
(Ohtsuka et al., 2004). Isolated BAC clones were ordered
based on Hind III digested patterns. This was confirmed by
PCR analyses using STS markers derived from BAC ends.
Simultaneously, we selected STS markers located at both
ends of the contigs. We determined the nucleotide
sequences of these markers from both the HNI strain and
the Da mutant to find polymorphisms. These polymorphisms were used to type the recombinant backcross
progeny and determined the direction of walking. Next,
we conducted chromosome walking by screening the BAC
library with STS markers that located on the Da side. To
determine the smallest region critical for the Da phenotype,
typings of recombinant backcross progeny were conducted
using polymorphic STS markers.
4.3. Sequencing
BAC clone B180F18 was shotgun-sequenced as
described (Ohtsuka et al., 2004). After determination of
the complete sequence of the BAC clone, genes contained
within this BAC clone were searched as described
881
(Ohtsuka et al., 2004), and using NIX software (http://
www.hgmp.mrc.ac.uk/NIX/).
For nucleotide sequences of the Da mutant and other
strains, we designed primer sets to amplify regions of
interest. Direct sequencing of PCR products was performed
using a ABI Prism Big Dye Terminator Cycle Sequencing
Kit (Applied Biosystems) on an ABI PRISM 310 DNA
sequencer.
4.4. Whole-mount in situ hybridization and antibody
staining
Whole-mount in situ hybridization was performed using
digoxigenin-labeled RNA riboprobes as described (Loosli
et al., 1998). Stained specimens were then subjected to
cryostat sectioning.
Whole-mount staining with acetylated tubulin antibody
was carried out as described (Koster et al., 1997).
4.5. Morpholino injection
zic1, zic4 and control morpholino oligos were injected
into the cytoplasm of one- or two-cell stage embryos
obtained from an HNI inbred strain. Injected embryos were
reared until the phenotype (arrangement of the dorsal
melanophore) could be clarity observed (stage 39; hatching
stage). In addition to single morpholino injection, co-injections of zic1 and zic4 morpholinos, two zic1 morpholinos
and two zic4 morpholinos were conducted. Concentrations
of the injected morpholino solution were 600 mM for single
morpholino injection, and 300 mM each for co-injection.
Sequences of morpholino oligos were Mo-zic1
(5 0 -CTGCGTCCAGGAGCATCTTCAGTCT-3 0 )
and
Mo-zic1SP1 (50 -GTCTGCGCTGCTTACCGGTGTGAGT-30 )
for zic1, Mo-zic4 (50 -CTTCCCAAAGCATCCACGCTCATTA-30 ) and Mo-zic4SP1 (50 -GATCTCCACTGACCTGTGTGCGTTC-30 ) for zic4 and Mo-cont (50 -TCTGACTTCTACGAGGACCTGCGTC-30 ) for control.
4.6. Preparation of skeletal specimens
Adult medaka were fixed in 10% formaldehyde and
rinsed in water. Cartilage was stained in a 0.1% Alcian blue
solution (in 80% ethanol/20% glacial acetic acid) for two
days. The specimens were transferred consecutively to 100,
75, and 40% ethanol, and then to deionized sterilized water.
They were next treated several times with a trypsin solution
until transparent. Scales were then removed. To stain bony
tissues, specimens were treated for 24 h with a 0.5%
potassium hydroxide solution containing enough alizarin
red to tint the solution deep purple. Next, the medaka were
subjected to treatment with a 0.5% potassium hydroxide:
glycerol series, at ratios of 1:3 (containing several drops of
H2O2), 1:1, and 3:1. Samples were then observed under a
stereoscopic microscope.
882
M. Ohtsuka et al. / Mechanisms of Development 121 (2004) 873–882
Acknowledgements
We thank Drs Hiroshi Hori, Daisuke Kobayashi and
Masao Hyodo for providing the BAC library, bmp4
probe and hatching enzyme, respectively. We also thank
Dr Atsuko Shimada for her help and advice in injection
experiments. We are grateful to Dr Jochen Wittbrodt and
members of his laboratory for technical advice in in situ
hybridization and antibody staining. We thank Drs Kenji
Imai, Lionel Larue, Rolf Kemler and Yasushi Taniguchi for
valuable comments. We thank Dr Masafumi Tanaka for
critical reading of the manuscript. This work was supported
by Grant-in-Aid for Scientific Research (C) by the Ministry
of Education, Science, Sports and Culture of Japan, and
Research Fellowships of the Japan Society for the
promotion of Science for Young Scientists to M.O.
References
Aruga, J., Yozu, A., Hayashizaki, Y., Okazaki, Y., Chapman, V.M.,
Mikoshiba, K., 1996. Identification and characterization of Zic4, a new
member of the mouse Zic gene family. Gene 172, 291–294.
Aruga, J., Mizugishi, K., Koseki, H., Imai, K., Balling, R., Noda, T.,
Mikoshiba, K., 1999. Zic1 regulates the patterning of vertebral arches in
cooperation with Gli3. Mech Dev 89, 141–150.
Aruga, J., Shimoda, K., Mikoshiba, K., 2000. A 50 segment of the mouse
Zic1 gene contains a region specific enhancer for dorsal hindbrain and
spinal cord. Brain Res Mol Brain Res 78, 15 –25.
Fukamachi, S., Shimada, A., Shima, A., 2001. Mutations in the gene
encoding B, a novel transporter protein, reduce melanin content in
medaka. Nat Genet 28, 381–385.
Hammerschmidt, M., Pelegri, F., Mullins, M.C., Kane, D.A., van Eeden,
F.J., Granato, M., et al., 1996. dino and mercedes, two genes regulating
dorsal development in the zebrafish embryo. Development 123, 95–102.
Ishikawa, Y., 1990. Development of caudal structures of a morphogenetic
mutant (Da) in the teleost fish, medaka (Oryzias latipes). J Morphol
205, 219–232.
Koga, A., Inagaki, H., Bessho, Y., Hori, H., 1995. Insertion of a novel
transposable element in the tyrosinase gene is responsible for an albino
mutation in the medaka fish, Oryzias latipes. Mol Gen Genet 249,
400– 405.
Koster, R., Stick, R., Loosli, F., Wittbrodt, J., 1997. Medaka spalt acts as a
target gene of hedgehog signaling. Development 124, 3147–3156.
Kunisada, T., Yamazaki, H., Hayashi, S.I., 2001. Review: ligands for
receptor tyrosine kinases expressed in the skin as environmental
factors for melanocyte development. J Investig Dermatol Symp Proc
6, 6–9.
Loosli, F., Koster, R.W., Carl, M., Krone, A., Wittbrodt, J., 1998. Six3, a
medaka homologue of the Drosophila homeobox gene sine oculis is
expressed in the anterior embryonic shield and the developing eye.
Mech Dev 74, 159–164.
Loosli, F., Koster, R.W., Carl, M., Kuhnlein, R., Henrich, T., Mucke, M.,
Krone, A., Wittbrodt, J., 2000. A genetic screen for mutations affecting
embryonic development in medaka fish (Oryzias latipes). Mech Dev 97,
133 –139.
Loosli, F., Winkler, S., Burgtorf, C., Wurmbach, E., Ansorge, W., Henrich,
T., et al., 2001. Medaka eyeless is the key factor linking retinal
determination and eye growth. Development 128, 4035–4044.
Mullins, M.C., Hammerschmidt, M., Kane, D.A., Odenthal, J., Brand, M.,
van Eeden, F.J., et al., 1996. Genes establishing dorsoventral pattern
formation in the zebrafish embryo: the ventral specifying genes.
Development 123, 81–93.
Nagai, T., Aruga, J., Takada, S., Gunther, T., Sporle, R., Schughart, K.,
Mikoshiba, K., 1997. The expression of the mouse Zic1, Zic2, and Zic3
gene suggests an essential role for Zic genes in body pattern formation.
Dev Biol 182, 299 –313.
Naruse, K., Fukamachi, S., Mitani, H., Kondo, M., Matsuoka, T., Kondo,
S., et al., 2000. A detailed linkage map of medaka, Oryzias latipes:
comparative genomics and genome evolution. Genetics 154,
1773–1784.
Ohtsuka, M., Makino, S., Yoda, K., Wada, H., Naruse, K., Mitani, H., et al.,
1999. Construction of a linkage map of the medaka (Oryzias latipes)
and mapping of the Da mutant locus defective in dorsoventral
patterning. Genome Res 9, 1277–1287.
Ohtsuka, M., Kikuchi, N., Nogami, M., Inoko, H., Ozato, K., Kimura, M.,
2002. Rapid screening of a novel arrayed medaka (Oryzias latipes)
cosmid library. Mar Biotechnol 4, 173–178.
Ohtsuka, M., Kikuchi, N., Ozato, K., Inoko, H., Kimura, M., 2004.
Comparative analysis of a 229 kb medaka genomic region, containing
the zic1 and zic4 genes, with fugu, human and mouse. Genomics 83,
1063–1071.
Rohr, K.B., Schulte-Merker, S., Tautz, D., 1999. Zebrafish zic1 expression
in brain and somites is affected by BMP and hedgehog signalling. Mech
Dev 85, 147–159.
Schier, A.F., 2001. Axis formation and patterning in zebrafish. Curr Opin
Genet Dev 11, 393–404.
Sporle, R., 2001. Epaxial – adaxial – hypaxial regionalisation of the
vertebrate somite: evidence for a somitic organiser and a mirrorimage duplication. Dev Genes Evol 211, 198–217.
Tamiya, G., Wakamatsu, Y., Ozato, K., 1997. An embryological study of
ventralization of dorsal structures in the tail of medaka (Oryzias latipes)
Da mutants. Dev Growth Differ 39, 531 –538.
Tomita, H., 1975. In: Yamamoto, T., (Ed.), Mutant genes in the medaka,
Medaka (killifish), biology and strains, Keigaku, Tokyo, Japan, pp.
251 –272.