Genomics 87 (2006) 783 – 792
www.elsevier.com/locate/ygeno

A wide-range phylogenetic analysis of Zic proteins:
Implications for correlations between protein structure conservation
and body plan complexity☆
Jun Aruga a,⁎, Akiko Kamiya a , Hirokazu Takahashi a , Takahiko J. Fujimi a , Yuri Shimizu a ,Keiko Ohkawa a , Shigenobu Yazawa b,c , Yoshihiko Umesono b,c , Hideki Noguchi d ,Takashi Shimizu e , Naruya Saitou f , Katsuhiko Mikoshiba g , Yoshiyuki Sakaki d ,Kiyokazu Agata b,c , Atsushi Toyoda daLaboratory for Comparative Neurogenesis, RIKEN Brain Science Institute, Wako 351-0198, Japanb
RIKEN Center for Developmental Biology, Kobe 650-0047, Japanc
Laboratory for Molecular Developmental Biology, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japand
Sequence Technology Team, RIKEN Genomic Science Center, Yokohama 230-0045, Japane
Graduate School of Science, Hokkaido University, Sapporo 060-0810, Japanf
Division of Population Genetics, National Institute of Genetics, Mishima 4511-8540, Japang
Laboratory for Developmental Neurobiology, RIKEN Brain Science Institute, Wako 351-0198, Japan
Received 4 October 2005; accepted 16 February 2006
Available online 29 March 2006

Abstract
We compared Zic homologues from a wide range of animals. Striking conservation was found in the zinc finger domains, in which an exon–intron
boundary has been kept in all bilateralians but not cnidarians, suggesting that all of the bilateralian Zic genes are derived from a single gene in abilateralian ancestor. There were additional conserved amino acid sequences, ZOC and ZF-NC. Combined analysis of the zinc finger, ZOC, and ZFNC revealed the presence of two classes of Zic, based on the degree of protein structure conservation. The “conserved” class includes Zic proteinsfrom the Arthropoda, Mollusca, Annelida, Echinodermata, and Chordata (vertebrates and cephalochordates), whereas the “diverged” class contains
those from the Platyhelminthes, Cnidaria, Nematoda, and Chordata (urochordates). The result indicates that the ancestral bilateralian Zic protein had
already acquired an entire set of conserved domains, but that this was lost and diverged in the platyhelminthes, nematodes, and urochordates.
© 2006 Elsevier Inc. All rights reserved.
Keywords: Comparative genomics; Evolution; Body plan; Exon–intron boundary; Transcription factor; Protein structure; Conserved domain; Zic; Zinc finger

Introduction
Recent studies of Zic family genes have revealed their rolesin the development of various animals. In vertebrates, they are
involved in neural and neural crest development, skeletal
patterning, and left–right axis establishment (reviewed in
Abbreviations: ZF, zinc finger; ZOC, Zic-Opa conserved domain; ZF-NC,
zinc finger–N-flanking conserved region; AA, amino acid residue(s); ORF, open
reading frame.
☆
Sequence data from this article have been deposited with the EMBL/
GenBank/DDBJ Data Libraries under Accession Nos. AB231864–AB231884.
⁎ Corresponding author. Fax: +81 48 467 9792.
E-mail address: jaruga@brain.riken.jp (J. Aruga).
0888-7543/$ - see front matter © 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.ygeno.2006.02.011[1,2]). In urochordates, they participate in the determination ofcell fate toward neural, notochord, and muscle cells [3–5]. In
the Protostomia, a fly Zic homologue, Opa, regulates
segmentation and midgut morphogenesis [6,7], whereas thenematode Zic homologue, Ref-2, has a role in vulval
development [8]. Although the Zic proteins commonly haveroles in cell fate decision in the early embryonic stages, there
seems to be significant phylogenetic variability in these roles.
For example, their role in neural development has been
demonstrated in the Deuterostomia, but not in the Protostomia.
However, in the Cnidaria, hydra Zic (Hyzic) is expressed in a
subset of neural cell precursors, suggesting the involvement ofthese proteins in neural development, as is the case in

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vertebrates [9]. It is possible that structural change in the Zic
genes contributed to the establishment of a body plan uniqueto each animal.
Although the phylogenetic roles of Zic genes areinteresting, our understanding may not be sufficient. This
seems to be partly due to the fact that previous studies havedealt with small numbers of representative model animals.
These circumstances led us to investigate the structure of Zic
genes in a broad range of animals, particularly in animal phyla
from which Zic homologues have not been reported. Here, wereport the genomic structures and predicted amino acidsequences of newly identified Zic genes in the Cnidaria,
Platyhelminthes, Annelida, Mollusca, Arthropoda, and Echinodermata, and we compare them with the sequence data
currently available in public databases. The results reveal an
overall picture of the Zic genes' evolutionary process,suggesting the involvement of Zic protein structure diversification in body plan simplification.
Results
Zic gene homologues are widely distributed in animal genomes
We first searched Zic gene homologues in each kingdomof living organisms by performing a homology searchagainst current databases. We found no obvious Zic

homologues in bacteria, algae, plants, fungi, and protists,
although there are a number of ZF domain-encoding genes
in these organisms. The highest level of similarity, with the
exception of the metazoans, was found in some fungal
proteins (data not shown). However, a homology searchusing these fungal sequences revealed that they were moreclosely related to GLIS, another subfamily of ZF proteins in
the metazoans, raising the possibility that the fungal ZF
protein was derived from a common ancestor with GLI/
GLIS/ZIC ZF superfamily proteins [1]. On the basis of theseresults, we assumed that the presence of Zic homologues
was limited to metazoans.
To identify Zic gene homologues in metazoans, weestablished PCR conditions under which we could amplifya region in the ZF domain of Zic-related genes by using
nested degenerate primers. PCR allowed us to clone the Zic
homologues from a broad range of animals, including
cnidarians, platyhelminthes, annelids, molluscs, nematodes,
arthropods, echinoderms, and chordates (Table 1). In addition
to our cloning and sequence determination, the rapidly
increasing whole genome sequence project benefited searching and comparing Zic homologues. However, our attempt toclone a sponge homologue of Zic was not successful (J.A.,
Naoyuki Iwabe, K.A., Takashi Miyata, unpublished observation). As a whole, these results strongly suggest that Zic
genes are retained very widely in the Eumetazoa.

Table 1
Animals used in this study
PhylumAnimal species

Abbr.

IntronGenomeGenome clone (accession no.)cDNA accession
no. [reference]

Cnidaria

Scolionema suvaense
Hydra vulgaris
Nematostella vectensis

Ssu
Hv
Nv

D
0
0

FosmidDatabase
BAC

Sco26, Sco36 (AB231882)

AB231883[9]Dugesia japonica
Schmidtea mediterranea
Schistosoma mansoni
Tubifex tubifex
Loligo bleekeri
Octopus ocellatus
Corbicula sp.
Spisula solidissima
Caenorhabditis elegans
Drosophila melanogaster
Anopheles gambiae
Pandinus imperator
Artemia franciscana
Strongylocentrotus
purpuratus
Asterina pectinifera
Ciona intestinalis
Ciona savignyi
Halocynthia roretzi
Branchiostoma floridae
Xenopus sp.
Mus musculus
Homo sapiens

Dj
Sme
Sma
Tt
Lb
Oo
Cj
Sso
Ce
Dm
Ag
Pi
Af
Sp

A
A

Database
BAC
FosmidPlatyhelminthes

Annelida
Mollusca

Nematoda
Arthropoda

Echinodermata

Chordata

Ap
Ci
Cs
Hr
Bf
Xl
Mm
Hs

CH314-49A19 (AB231867)
CH314-55K22 (AB231868)
AB231880, AB231881

A
A
ACBE
AB
A
AB
A
A
AC

A
A, AB
A, AB
A, AB

FosmidBAC
Database
Database
Database
PCR
Fosmid, PCR
Database
FosmidDatabase
Database
Database
BAC
Database
Database
Database

CH103-42N16 (AB231864)
Tub8, Tub12 (AB231869)

AB231870
AB231874
AB231875

Cor8, Cor10 (AB231873)
CH312-9E16 (AB231865)(AB231876)
Art1 (AB231878)

Ast1 (AB231871)

CH302-61N11 (AB231866)

[8]
[6,7]
XM_321856
AB231877
AB231879

AB231872
[31]
[5]
[3,4]
[19]
Ref. in [1]Ref. in [1]Ref. in [1]Database means that sequence information was obtained from public databases. Intron indicates the types of intron. Intron 0 indicates no introns in the zinc finger
domain.

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The copy numbers of Zic in each species varied from 1 to
5. Two Zic genes were identified in two planarians [Dj andSme (ZicA and ZicB)] and three urochordates [Ci, Cs, and Hr(ZicL/ZicN and macho1)]. Five Zic genes (ZicA, ZicB, ZicC,
ZicD, and ZicE) were present in a sea anemone (Nv).
Mammals (Hs, Mm) possessed five related genes. Five Zic
genes also were identified in other vertebrate species such as
Xl and Gallus gallus (T.J.F., Y.S., A.T., J.A., unpublishedobservation). Only a single Zic species was identified in eachof the remaining animals surveyed in this study.
The ZF domain amino acid sequence is highly conserved
We next deduced the amino acid (AA) sequence of each
Zic protein by sequencing cDNA and/or genomic clones andthen subjected the putative protein sequences to comparative
analysis (Fig. 1). The highest degree of conservation was
found in the ZF domain. The similarity of the AA
sequences of any two species was more than 75%, andthere were 60 absolutely conserved AA (yellow letters in
Fig. 1) within the region ranging from ZF2 to ZF5 (104AA). The conservation in this region was markedly higher
than that in the other regions. There were several clusters ofabsolutely conserved AA sequences in the ZF2–ZF5 region,and in many positions AA changes within equivalent AA
groups were observed.
Some new findings were revealed when we examinedthe variation within the highly conserved ZF1–ZF5 region.
First, vertebrate Zic1–3 had a unique structure in ZF5
whereby the number of AA between the first and thesecond cysteine residues was 2 instead of 4. Second, thenumber of AA between the two cysteines of the ZF1 C2H2
motif was highly variable, ranging from 6 (nematoda) to 38(mosquito). Third, ZF1 was not as strongly conserved as
ZF2–ZF5.
Comparison among entire amino acid sequences reveals thepresence of evolutionarily conserved domains other than the
ZF domain
Comparison of AA sequences in the entire ORFs
revealed that there were two additional evolutionarily
conserved sequences (ZF-NC and ZOC). ZF-NC could beseen in the N-terminal flanking region of the ZF domain(Fig. 1). The consensus sequence of ZF-NC can besummarized as GAF(F/L)RYMRQP-(0–7 AA)-IKQE.
Sequences perfectly matching the ZF-NC consensus could
be seen in molluscs, arthropods, annelids, and somechordates (vertebrates and cephalochordates), but not incnidarians, platyhelminthes, nematodes, or urochordates.
However, ZF-NC-lacking species contained minimal conservations that could have been residues of the ZF-NC
sequence. ZOC (Zic–Opa conserved domain, Fig. 2A) is
the N-terminally located domain, which was found as asequence conserved between mouse Zic1–3 and fly Oddpaired [10,11]. The consensus sequence can be summarizedas (S/T)RDFLxxxR. This sequence motif was conserved in

785

arthropods, echinoderms, molluscs, annelids, and somechordates (vertebrates and cephalochordates), but not incnidarians, platyhelminthes, nematodes, or urochordates(Fig. 2B).
Molecular phylogenetic tree analysis reveals two Zic protein
clusters
To understand their relationships in light of these similarities,
we constructed a molecular phylogenetic tree on the basis of theamino acid sequences of those ZF domains with 20 ZF-NCcontaining amino acids at the N-terminal and 10 amino acids in
the C-terminal flanking sequences (Fig. 3). We found that Zic
proteins were clustered into two classes: “conserved” and“diverged.” The conserved class included the Vertebrata,
Cephalochordata, Mollusca, Arthropoda, Echinodermata, andAnnelida. The diverged class, characterized by longer branches
in the tree, included the Cnidaria, Nematoda, Platyhelminthes,and Urochordata. Interestingly, the ZOC domain was confinedto the conserved class, not the diverged class (Figs. 2B and 3).
Exon–intron organization of Zic genes reveals the existence ofa single ancestral bilateralian Zic geneIn our examination of genomic organization we focused on
the exon–intron boundaries in the ZF domain (Figs. 1 and 4).
As indicated in Fig. 1, exon–intron borders were recognized infive locations (A–E). Among them, an exon–intron borderlocated between ZF3 and ZF4, which we refer to hereafter as Aintron (border), was extraordinarily conserved and was
distributed in all of the Zic homologues except seven cnidarian
Zics, suggesting that the A-intron is a bilateralian-specificstructure of Zic genes.
In contrast to the A-intron, the remaining exon–intron
borders, designated here as B-, C-, D-, or E-intron, were eachfound in a small number of animals. B-intron was found in thevertebrate Zic1–3 and certain groups (Hexapoda and Crustacea)of the Arthropoda. C-intron was seen in the two types ofascidian Zic and nematode Ref-2. Nematode Ref-2 uniquely
contained another intron (E-intron) in addition to the A-, B-, andC-introns in its ZF domain. In cnidarians such as the jellyfish(Ssu), introns (D-introns) occurred in positions of the Zic genes
different from those mentioned above. In the hydra (Hv) and thesea anemone (Nv), however, there were no insertions of introns
within the ZF domain [0 (zero)-intron] at all.
A tandem array of multiple Zic genes was revealed in Nv
and Hs. In the case of the cnidarian Nv, the three genes
ZicC, ZicA, and ZicB were tandemly arrayed over anapproximately 80-kb region (Fig. 4). A tandem array of Zic
genes was also seen in vertebrate Zic1/Zic4 pairs as well as
Zic2/Zic5 pairs; these genes were arrayed in a head-to-headorientation (Fig. 4).
Discussion
Recent molecular phylogenetic analysis indicates that there
are three main taxa in bilateralian animals: Deuterostomia,

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their significance in animal evolution in the light of theabove-mentioned background.
Evolutionary processes of Zic genes

Fig. 2. (A) Amino acid alignment of the ZOC domain and (B) its distribution
in a phylogenetic tree. Phylogenetic trees were adapted and modified from
Tessmar-Rable and Arendt [37]. Types of exon–intron borders are also
indicated.

Ecdysozoa, and Lophotrochozoa (reviewed in [12,13]). The
Deuterostomia include echinoderms and the chordates, thelatter of which have three groups: vertebrates, cephalochordates (lancelets), and urochordates (ascidians). Ecdysozoa and
Lophotrochozoa are two sister groups in the Protostomia.
Ecdysozoa include arthropods and nematodes. The Lophotrochozoa contain annelids, molluscs, and platyhelminthes.
Phylogenetic trees based on molecular phylogeny are notalways consistent with classical phylogenetic classifications
based on morphological criteria, such as the presence orabsence of a coelom. In this regard, pseudocoelomic animals(nematode) and acoelomic animals (platyhelminthes) havebeen placed near the roots of bilateralian phylogenetic trees.
Accumulating evidence raises the possibility that nematodes
and a majority of the platyhelminthes are derived from apresumptive complex bilateralian ancestor that already had acoelom with a mesodermal lining. This bilateralian ancestor
possessed the various genes required for a complex bodyplan, such as numerous transcription factors including
homeobox genes and genes required for intercellular signaling(TGF-β, Hedgehog, Notch, and EGF) (reviewed in [13]). We
should discuss the evolutionary processes of Zic genes andFrom our current results, we can envisage the evolutionary
process of Zic genes as follows (Fig. 5A). The earliest Zic
genes that appeared in ancestral Eumetazoa or metazoans,probably in the pre-Cambrian era, may have shared common
structural features, namely the ZF and ZF-NC domains.
Exon–intron boundaries are not conserved between Eumetazoa and cnidarians. Therefore, we postulate that the earliest
Zic genes may not have had any introns in their ZF regions.
The identity of the direct ancestor of the earliest Zic gene is
not certain, but the presence of Gli/Nkl/Zic superfamilyrelated genes in fungi suggests that the Zic genes havediverged from a common ancestor of the Gli/Nkl/Zic
superfamily genes.
In a bilateralian ancestor, a single Zic gene would haveacquired an A-intron in the ZF3 region. All of the bilateralian
Zic genes are descendents of this bilateralian common ancestral
Zic (urbilateralian Zic), as supported by the presence of an Aintron in all the bilateralian Zic genes that we examined. The
urbilateralian Zic may have already possessed the ZOC domain,
as is supported by the widespread occurrence of ZOC in
bilateralian progenies. Therefore, the full set of conserved
domains (ZOC, ZF-NC, and ZF) had probably already appearedin the urbilateralian Zic.
Additional variation may have been given to urbilateralian Zic later in the evolutionary process. Intron sequenceswere inserted into Zic genes in vertebrates, urochordates,
arthropods, and nematodes; the positions of the introns
diverged but seem to have been conserved in each phylum.
The urochordate (ascidian) has a unique C-intron in bothtypes of Ci-Zic genes, macho-1 and ZicL, suggesting that agene duplication event occurred after insertion of the Cintron in the urochordate clade. In nematodes, five
additional introns, three of which were located within the
ZF domain, seem to have been inserted.
In the case of vertebrate Zic1–3 and insect Zic
homologues, there is an apparently common exon–intron
boundary (referred to as B-intron). The intron sequence islocated in the carboxy-terminally located histidine residue inthe five tandemly repeated C2H2 motifs. Two hypotheses
could explain the occurrence of B-intron in the evolutionary
process. One is the insertion of B-intron before thedivergence of the Deuterostomia and Protostomia. The
other assumes the independent occurrence of intron insertionin vertebrates and arthropods. We think that the latter ismore likely because there are no B-intron-containing Zic

Fig. 1. (A) Domain structure of typical Zic protein. (B) Amino acid alignment of the ZF domain and ZF-NC domain. Letters with yellow background indicatecompletely conserved AA residues, and those with green indicate AA residues conserved more than 49% among the listed sequences. Bars with ZF-NC or numbers(1–5) indicate the extent of the ZF-NC domain and C2H2 ZF motifs 1–5, respectively. Asterisks indicate the cysteine and histidine residues that form C2H2 ZF motifs.
A–E with arrowheads indicate the positions of the evolutionarily conserved exon–intron boundaries in the ZF. The phases of the exon–intron boundaries in thetranslation frames are A, 1; B, 0; C, 0; D, 2; E, 0. Abbreviations of the animal species are given in Table 1.

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Fig. 3. Molecular phylogenetic tree using ZF and ZF-NC domains. Neighbor-joining tree was drawn by MEGA3.1 [32] after a multiple alignment by CLUSTALW[38]. Amino acid sequences containing ZF domains with 20 AA at the N-terminal and 10 AA at the C-terminal flanking sequences were used for the analysis. Animals
in the same phyla or subphyla are encircled by the gray lines. The numbers near the internal branches indicate the statistical significance calculated by bootstrap test.
Values of more than 94% are indicated in the tree.

genes in the other Protostomia and Deuterostomia species.
In addition, it seems plausible that B-intron-containing
vertebrate Zic1, Zic2, and Zic3 homologues have been
generated after unique gene duplication events in this animal
taxon (see below).
To explain the complex organization of vertebrate Zic
genes, we propose the model outlined in Fig. 5B. The
ancestral Zic in the ancestral chordates may have been similar
to the urbilateralian Zic, which is represented by acephalochordate (Bf) Zic gene. First, gene duplication
occurred, resulting in head-to-head array of Zic genes with
A-intron. Then, B-intron was inserted in one of the two Zic
genes; this was followed by duplication twice (quadruplication) of the duplicated Zic, concomitant with the entire
genome duplication events postulated in accordance with the
DNA dose per haploid genome and the molecular phylogenetic analysis of many genes [14,15]. Of the resulting eight
Zic genes, three (one AB-intron gene and two A-intron genes)
may have disappeared in the course of evolution. The
vertebrate Zic1, Zic2, and Zic3 have conserved sequences
within this subfamily of Zic genes. In particular, theconservation of their carboxy-terminal sequence region,
NFNEWYV, is clear. Partly similar sequences were foundin the midst of the region between the ZF domain and the Cterminal ends of vertebrate Zic4 and Zic5 and the
cephalochordate Zic gene, suggesting that truncation of theopen reading frame occurred in the prototype Zic genes after
the initial duplication and before the quadruplication. It is
possible that this domain was endowed with a new property
that was functionally or structurally important for Zic1, Zic2,
and Zic3 proteins.
The presence of multiple types of exon–intron boundaries
in the highly conserved ZF domains is the most prominent
feature of Zic genes. Both intron loss and intron gain occur
in the course of evolution [16,17]. Although the exon–intron
structure is highly dynamic [18,19], the introns are useful as
phylogenetic characters as well as nucleotide and protein
sequences [20–22]. In the case of Zic genes, the distributionof A-intron attracts our attention. A-intron was identified in
all of the identified 27 bilateralian Zic genes compared inthis study, but was not in the seven cnidarian Zic genes. In
addition, the presence of A-intron can be predicted in thedraft genome sequences of four insect species (Aedes
aegypti, Tribolium castaneum, Bombyx mori, Apis mellifera)
and in six vertebrate species (G gallus, Xenopus tropicalis,
Takifugu rubripes, Tetraodon nigroviridis, Danio rerio,

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of the conserved class, comprising the Vertebrata, Cephalochordata, Echinodermata, Annelida, Mollusca, and Arthropoda; the other is the diverged class that includes the
Cnidaria, Platyhelminthes, Nematoda, and Urochordata.
Widespread occurrence of the conserved class Zic genes
in the Ecdysozoa, Lophotrochozoa, and Deuterostomia
suggests that the bilateralian ancestor gene belongs to theFig. 4. Schematic drawing of exon–intron structures of Zic genes from various
animals. Gray and black boxes indicate non-ZF and ZF parts of the Zic protein
coding region, respectively. Horizontal bars indicate introns; capital letters (A–E) above the introns indicate the types of exon–intron borders. Generaltranscription direction is left to right; for Nv_ZicB/ZicC/ZicD/ZicE and Hs
ZIC1–5, the direction of the transcription is indicated by arrows. Each arrowalso indicates the extent of one Zic protein-coding exon.

Oryzias latipes) (J.A., unpublished observation). The extent
of exon–intron structure conservation seems to be higher
than that of the genes for animal elongation factor-1α[18,19] and animal translation initiation factor 2 [23], bothof which have been well characterized as to the evolutionary
conservation of the exon–intron structure. The location ofA-intron in Zic genes may be noted to be highly conservedin bilateralian animals, and its distribution in the animal
kingdom should be further investigated. These results alsosuggest that the extent of phylogenetic conservation of theirlocations considerably varies among introns. However, thisspeculation awaits further verification since the number ofgenes or animal species that have been subjected to thewide-range phylogenetic analysis of exon–intron structure is
still limited.
Implications for body plan evolution
The distribution of the ZOC domain and the molecularphylogenetic tree of the Zic family suggest the presence of
two classes of Zic genes (Figs. 2 and 3). One is composed

Fig. 5. Evolutionary processes of Zic genes. (A) Hypothetical model that
explains the diversity of Zic genes in the Eumetazoa. (B) Establishment ofvertebrate Zic1–5 genes. Boxes indicate protein-coding regions. Thickhorizontal bars indicate intron and flanking region. Green indicates protein
coding regions without clearly conserved domain structures; red indicates zincfinger domains; dark blue indicates ZOC; light blue indicates ZF-NC. Thin
arrows in B represent the direction of transcription.

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conserved class of Zic. These two classes may reflect thedifference in diversification of the ancestor Zic genes. In
general terms, the complexity of body structure seems to bewell correlated with the extent of the Zic protein structure
conservation. Cnidarians are the bilayered animals; Platyhelminthes are acoelomatic animals, and nematodes arepseudocoelomatic. The animals belonging to these threephyla may have a relatively simple body structure incomparison to the other animals examined in this study. The
urochordates seem to be unique among the four members ofthe diverged class. However, their body structure is simpler
than that of other chordates in that they do not have either
a large CNS or segmented mesodermal structures, both ofwhich are retained in vertebrates and cephalochordates.
Therefore, the “complexity” of the body structures seems to
be a major determinant of the structural conservation of Zic
proteins.
There have been some indications that changes in protein
structure between different animal species result in qualitative
functional changes that can affect body plan. In botharthropod Hox proteins [24] and proneural proteins [25],structural changes determine their binding protein repertoires.
The ZOC domain has been shown to be necessary for thetranscriptional activation capacity of the mouse Zic2 protein[10]. I-mfa, an inhibitor of myogenic transcription factor
protein, binds the region including ZOC and inhibits Zic2mediated transcriptional activation by translocating Zic2
proteins from the nucleus to the cytoplasm [10]. It has been
proposed that the extent of ZOC domain conservation isrelated to functional diversity among mouse Zic1–5 proteins[26]. In addition, our recent study showed that an evolutionarily conserved transcription factor could specifically bind the
ZOC domain (T. Tohmonda, K. Mizugishi, K.M., J.A.,
unpublished observation), suggesting that ZOC has multifunctional properties. In contrast to ZOC, there are few
known facts that can explain the significance of the ZF-NC
domain. Its relevance to ZOC is intriguing, since both
domains are often colocalized in the animal phylogenetic tree.
It is possible that these two domains are closely located in amature protein or bind common partner molecules. Structuraland functional evaluation of the conserved domains from aphylogenetic point of view is needed to obtain a better
understanding.
Materials and methods
Animals
Dugesia japonica was derived from a clonal planarian strain that was
originally established by K. Watanabe (Himeji Technical Institute, Hyogo,
Japan) [27]. The asexually reproductive jellyfish Scolionema suvaense[28],which was originally purchased from a local pet dealer, has been kept in
artificial seawater for more than 1 year and fed brine shrimp. The
freshwater oligochaete Tubifex tubifex was obtained as described [29].
Loligo bleekeri was obtained from Aburatsubo Marine Park Aquarium(Kanagawa, Japan). Octopus ocellatus was obtained from Ushimado Marine
Laboratory, Okayama University. Corbicula sp. was purchased from a local
fish dealer. A scorpion, Pandinus imperator,and a brine shrimp, Artemia
franciscana, were purchased from local pet dealers. Asterina pectinifera wascollected from the seashore in Kanagawa Prefecture, Japan. To avoid
contamination with genetic materials from other animal species, embryos inpure water or saline or artificial seawater were collected in the case of T.
tubifex, P. imperator, L. bleekeri, O. ocellatus, and As. pectinifera. 18S
ribosomal DNA sequences were determined for S. suvaense (Accession No.
AB231884), Ar. franciscana, P. imperator, and Corbicula sp. for species
confirmation. The S. suvaense sequence was most similar to those derived
from animals in the phylum Cnidaria, class Hydrozoa, orders Limnomedusae and Trachymedusae, both of which orders are different from that of
Hydra vulgaris (class Hydrozoa, order Athecata).

PCR cloning of Zic cDNA
The following primers were used for PCR amplification of the ZF domain of
the Zic-related genes. The first PCR was carried out using ZF2F_1, 5′TTYAARGCIAARTAYAARYT-3′, and ZF3R, 5′-T(T/G)RTGIATYTTIARRTTYTC-3′, and the second PCR was done using ZF2F_2, 5′-GCIAARTAYAARYTIRTIAAYCA-3′, and ZF3R. Each PCR consisted of 35 cycles of 94°C for
1 min, 35°C for 1 min, and 72°C for 2 min. The PCR was carried out with ExTaq
DNA polymerase (Takara Bio, Shiga, Japan) in the presence of BD TaqStart antiTaqantibody (BD Biosciences, CA, USA). This PCR was highly specific to clone
Zic-related genes. RNAs were isolated by using TRIzol reagent (Invitrogen, CA,
USA) in accordance with the manufacturer's recommendation. The homologues
were initially identified by nested PCR on cDNA or genomic templates using
degenerate primers corresponding to the most ZF2 to ZF3 region. cDNAs
corresponding to ZF at their 3′ and 5′ ends were cloned by using a 3′-Full RACECore Set and 5′-Full RACE Core Set (Takara Bio). The entire open reading frame
region of the cDNAs was again cloned by the primers located outside the target
regions. The amino acid sequences were deduced from nucleotide sequencing ofmultiple PCR fragments.
To determine whether a gene belonged to the Zic gene family in structural
terms, we used the following criteria: (1) The ZF domain was composed of fiverepeats of C2H2 motifs. (2) The last four ZF motifs had an amino acid sequence that
had more than 75% similarity to other Zic family proteins. (3) The ZF domain
contained the ENLKIH, FANSSDR, and YTHPSSLRKH sequence motifs, which
are conserved in all of the Zic family proteins reported so far. (4) In the first ZF
region, the number of amino acid residues between the conserved cysteine residues
was greater than that in the other four ZF motifs in each of which four amino acidresidues were present.
The newly identified Zic genes were designated as “Zic” preceded by theabbreviation for each animal species. When multiple genes existed in one
species, ZicA, ZicB, ZicC, ZicD, and ZicE were used to discriminate each gene.
This style did not mandate structural similarity among A, B, C, D, and E fromdifferent phyla, in contrast to the Zic1–5 designation in vertebrates. However, inthe case of planarians, ZicA and ZicB are structurally related.

Fosmid and BAC cloning of genomic DNA
To isolate high-molecular-weight DNA, library screening was done as
described [30]. Fosmid genomic libraries were prepared using CopyControl
pCC1FOS vector (Epicentre, WI, USA). The genomic DNA was firstsheared by performing an appropriate number of pipettings. After being
blunted, the DNA fragments were size-fractionated by field-inversion gel
electrophoresis through a Genofield electrophoretic apparatus (ATTO,
Tokyo, Japan). The resultant 35- to 45-kb fraction was inserted into thefosmid vector. The BAC filter and clones were purchased from BACPAC
Resources, Children's Hospital Oakland Research Institute (OH, USA).
Some of the exon–intron boundary genomic sequences in P. imperator, L.
bleekeri,and As. pectinifera were determined by using a DNA Walking
SpeedUp Kit (Seegene, Seoul, Korea).

DNA sequencing and sequence analysis
Sequencing and data assembly were done as described [31]. Genomic
sequences of Schmidtea mediterranea, H. vulgaris, Caenorhabditis elegans,
Strongylocentrotus purpuratus,and Ciona intestinalis were derived from public
databases (http://www.ncbi.nlm.nih.gov/BLAST/tracemb.shtml). SequenceJ. Aruga et al. / Genomics 87 (2006) 783–792
analysis was done using DNASISPro (Hitachi Software Engineering, Tokyo,
Japan), Sequencher (Gene Codes, Ann Arbor, MI, USA), and Genetyx(Genetyx, Tokyo, Japan) software. Homology search was carried out against apublic database (http://www.ncbi.nlm.nih.gov/BLAST/) using BLAST anddiscontinuous Mega BLAST. The molecular phylogenetic tree was drawn
using MEGA version 3.1 [32] as follows: The amino acid sequences were firstaligned by CLUSTALW [33] under the following parameter settings: Gonnet
250 as protein weight matrix; gap opening and gap extension penalties for pairwise alignments, 35 and 0.75, respectively; gap opening and gap extension
penalties for multiple alignments, 15 and 0.3, respectively. The aligned
sequences were subjected to neighbor-joining tree construction using Poissoncorrection distance with bootstrap test (1000 replicates). For the multiplealignment shown in Fig. 1B, CLUSTALW parameters were changed as follows
so that cysteine and histidine residues in the C2H2 motifs were aligned correctly:
Gonnet 250 as protein weight matrix; gap opening and gap extension penalties
for pair-wise alignments, 10 and 0.1, respectively; gap opening and gap
extension penalties for multiple alignments, 10 and 0.2, respectively.
Exon–intron boundaries of Hs-ZIC2, Mm-Zic1-5, Dm-Opa, and Ce-Ref2
were reported [8,11,34,35,36]. The boundaries of Hs-ZIC1, ZIC3, ZIC4, andZIC5 were identified in the human genome databases (http://www.ncbi.nlm.nih.
gov/genome/seq/HsBlast.html). The other exon–intron boundaries were identified by a homology search against the genome sequences using originallydetermined (Af-Zic, Ap-Zic, Cj-Zic, Pi-Zic, Ssu-Zic, Tt-Zic) or reported (BfAmphiZic, Ci-macho-1, Ci-ZicL) cDNA sequences. In the case of Nv-ZicA-E,
Sma-Zic, Sp-Zic, and Sso-Zic, the homology search was done using cDNA
sequences of Ssu-Zic, Dj-ZicA, Ap-Zic, and Cj-Zic, respectively. All of theexon–intron boundaries followed the GT/AG rule.

Acknowledgments
We thank Ulrich Technau for correspondence concerning
Hyzic, Shin-ichi Nemoto for helpful comments on starfish
sampling, all the technical staff of Sequencing Technology
Team RIKEN GSC, Yayoi Nozaki, and Research Resource Center
RIKEN BSI for their technical assistance. This study was done asa collaborative research project of the Strategic Research Programin RIKEN. This work was supported by grants-in-aid from theJapanese Ministry of Education, Science, Sports, and Technology.
Appendix A. Supplementary data
Supplementary data associated with this article can be foundin the online version at doi:10.1016/j.ygeno. 2006.02.011.
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