Conservation and Diversification of Msx Protein in Metazoan Evolution
Hirokazu Takahashi,* Akiko Kamiya,* Akira Ishiguro,* Atsushi C. Suzuki, Naruya Saitou,àAtsushi Toyoda,§ and Jun Aruga**Laboratory for Comparative Neurogenesis, RIKEN Brain Science Institute, Wako, Saitama 351-0198, Japan; Department of
Biology, Keio University, Hiyoshi, Yokohama 223-8521, Japan; àDivision of Population Genetics, National Institute of Genetics,
Mishima 4511-8540, Japan; and §Sequence Technology Team, RIKEN Genomic Sciences Center, Yokohama 230-0045, Japan
Msx (/msh) family genes encode homeodomain (HD) proteins that control ontogeny in many animal species. We
compared the structures of Msx genes from a wide range of Metazoa (Porifera, Cnidaria, Nematoda, Arthropoda,
Tardigrada, Platyhelminthes, Mollusca, Brachiopoda, Annelida, Echiura, Echinodermata, Hemichordata, and Chordata)
to gain an understanding of the role of these genes in phylogeny. Exon–intron boundary analysis suggested that theposition of the intron located N-terminally to the HDs was widely conserved in all the genes examined, including thoseof cnidarians. Amino acid (aa) sequence comparison revealed 3 new evolutionarily conserved domains, as well as verystrong conservation of the HDs. Two of the three domains were associated with Groucho-like protein binding in botha vertebrate and a cnidarian Msx homolog, suggesting that the interaction between Groucho-like proteins and Msx
proteins was established in eumetazoan ancestors. Pairwise comparison among the collected HDs and their C-flanking aa
sequences revealed that the degree of sequence conservation varied depending on the animal taxa from which thesequences were derived. Highly conserved Msx genes were identified in the Vertebrata, Cephalochordata, Hemichordata,
Echinodermata, Mollusca, Brachiopoda, and Anthozoa. The wide distribution of the conserved sequences in the animal
phylogenetic tree suggested that metazoan ancestors had already acquired a set of conserved domains of the current Msx
family genes. Interestingly, although strongly conserved sequences were recovered from the Vertebrata, Cephalochordata, and Anthozoa, the sequences from the Urochordata and Hydrozoa showed weak conservation. Because theVertebrata–Cephalochordata–Urochordata and Anthozoa–Hydrozoa represent sister groups in the Chordata and Cnidaria,
respectively, Msx sequence diversification may have occurred differentially in the course of evolution. We speculatethat selective loss of the conserved domains in Msx family proteins contributed to the diversification of animal
body organization.

Introduction
Msx family proteins are critical regulators of metazoan
ontogeny, as has been revealed in many studies (reviewed
in Davidson 1995; Bendall and Abate-Shen 2000; Ramosand Robert 2005). In the Ecdysozoa, Drosophila muscle
segment homeobox (msh) has a role in the regional specification of neuroectoderm and muscle progenitors (Isshiki
et al. 1997; Nose et al. 1998), and Caenorhabditis Vab-15
has a role in touch receptor neuron development (Du andChalfie 2001). (Here, we use the term ‘‘Msx family’’ to denote a gene family orthologous to those encoding vertebrateMsx, including msh and vab-15.) In the Echinodermata,Strongylocentrotus and Heliocidaris Msx homologs may
control gastrulation and skeletal patterning (Tan et al.
1998; Wilson et al. 2005). In the Vertebrata, Msx1 orMsx2 plays roles in dorsoventral patterning of embryos, regionalization of the neural tube, establishment of neuralcrest tissue, and the genesis of various types of organs, including the palate, tooth, skull, middle ear, jaw, hair, mammary gland, and limbs, both in mammals (Satokata andMaas 1994; Bendall and Abate-Shen 2000; Satokata
et al. 2000; Lallemand et al. 2005; Ramos and Robert2005) and amphibians (Suzuki et al. 1997; Monsoro-Burq
et al. 2005; Khadka et al. 2006). Mutations in human MSX1
are responsible for selective tooth agenesis (STA) (Vastardis
et al. 1996), cleft palate, cleft lip (van den Boogaard et al.
2000), and nail dysplasia (Jumlongras et al. 2001), andthose in MSX2 are responsible for parietal foramina
Key words: Msx, homeodomain, protein conserved domain,
Groucho, protein–protein interaction, exon–intron boundary.
E-mail: jaruga@brain.riken.jp.
Mol. Biol. Evol. 25(1):69–82. 2008
doi:10.1093/molbev/msm228
Advance Access publication October 16, 2007

(PFM) (Wuyts et al. 2000) and craniosynostosis (CSO)(Wilkie et al. 2000). These facts enlighten us about not only
the importance of Msx family genes in each animal but also
the similarities and differences of their roles in different animal groups.
In phylogenetic terms, the structural features and/or
expression profiles of Msx homologs have been describedin insects (Walldorf et al. 1989), ascidians (Holland 1991),
sponges (Seimiya et al. 1994), and leeches (Master et al.
1996). Some researchers have noticed that the role of theMsx family in neuroectodermal patterning is similar in
the fruit fly and vertebrates (Isshiki et al. 1997; Arendt
and Nubler-Jung 1999); Msx family genes specify laterallongitudinal columns of neuroectoderm in both types of animals, raising the possibility that the bilaterian ancestors
had already used Msx family genes in establishing their nervous systems. The same expression pattern is essentiallyconserved in another insect, Tribolium castaneum (Wheeler
et al. 2005). However, this hypothesis is still uncertain andawaits verification by examination in other animal species.
In a previous study, we performed a molecular phylogenetic analysis of wide-ranging groups of animals to determine the role of Zic family zinc finger proteins inevolution. We compared both the amino acid (aa) sequenceand the exon–intron organization of many interspecies orthologs from major metazoan phyla (Aruga et al. 2006).
The study revealed novel evolutionarily conserved domains
and gave us a broad understanding of the processes of protein evolution and the traits involved in evolutionarychange. We therefore applied the same strategy to Msx family proteins. Some earlier works had pointed out a promising
direction for an analysis of the molecular phylogeny of Msx
family genes. A pioneering study by Holland (1991) compared vertebrate, ascidian (Ciona), and Drosophila Msx homologs. The relationship between the Msx genes ofÓ 2007 The Authors.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

70 Takahashi et al.

zebrafish and other vertebrates were investigated in terms of
aa sequences and expression patterns (Ekker et al. 1997)
and chromosomal synteny (Postlethwait 2006). Perryet al. (2006) focused on phylogenetic comparisons among
primate Msx genes. However, these studies are not sufficient for our purpose because they only dealt with the limited evolutionary processes.
We report here the molecular phylogeny of Msx
family genes, as determined by a comparison of the Msx
sequences from 13 animal phyla. We compared the conserved domains of Msx proteins and the exon–intron organization of Msx genes, and we performed a functional
analysis of some of the conserved domains. The results indicated that the eumetazoan ancestor already possessed
Msx protein with a full set of conserved domains; these domains have diverged strongly in some animal groups.
Materials and Methods
Database Search
The similarity search against the current databases wasdone with the Blast algorithm (BlastP, TBlastN, Altschul
et al. 1990, 1997) against public databases (http://
www.ncbi.nlm.nih.gov/blast/,
http://www.ensembl.org/
index.html). Presence or absence was estimated by a database search with TBlastN algorithm against selected genome sequence databases that used key Mus musculus
Msx-1 or Nematostella Msx sequences. A decision on authentic homology was based on reverse Blast analysis inwhich the sequence that showed the highest score in a certain species was used as a key sequence for the homology
search by using the BlastX algorithm against National Center for Biotechnology Information (NCBI) eukaryotic databases. If the sequence that showed the highest Blast scoreamong the identified sequences contained the Msx proteins,
this labeled a member of Msx family (table 1).
The 18S ribosomal RNA sequences were collectedfrom the NCBI database (accession number, supplementary
text, see Supplementary Material online). These sequenceswere chosen because they were derived from animal species
that were identical, or closely related, to those used in theMsx phylogenetic analysis (table 1).
The search for Eh-like sequences was done as follows.
A local aa sequence database was constructed by DNASpace (Hitachi Software Engineering, Tokyo, Japan).
The database was subjected to a homology search by using
the Smith–Waterman algorithm (Smith and Waterman1981) and the consensus Eh sequences FSV[DE]  [IL][IL]as key sequences. Sequences that gave scores of more than
15 under set parameters (i.e., Matrix, BLOSUM62; initial
gap penalty, 5; extension gap penalty, 2) were listed as
candidate sequences for the alignment shown in table 2.
Animals
Genomic resources for Scolionema suvaense, Tubifex
tubifex, Loligo bleekeri, Octopus ocellatus, Corbicula fluminea, Pandinus imperator, Artemia franciscana, and Asterina pectinifera have been described (Aruga et al. 2006).
The maintenance and recovery of Milnesium tardigradum

species have been described (Suzuki 2003). Limulus polyphemus, Lingula anatina, Urechis caupo, and Anemonia erythraea were purchased from local vendors. Thysanozoon
sp. was collected from the seashore in Kanagawa Prefecture, Japan. Its picture can be seen at http://lcn.brain.
riken.jp/photos.html. Species identification was done bysequencing 18S ribosomal RNA genes (data not shown).

Polymerase Chain Reaction Cloning of Msx cDNA
RNAs were isolated by using TRIzol reagent (Invitrogen, CA) in accordance with the manufacturer’s recommendation. cDNAs were generated by using a 3#-Full RACECore Set (Takara Bio, Shiga, Japan). The homologs were
initially identified by nested polymerase chain reaction(PCR) on cDNA or genomic templates. The following primers were used for PCR amplification of the homeodomain(HD) region of Msx family genes. The first PCR was carriedout by using MshDF-1, 5#-YTIMGIAARCAYAARACIAAYMG-3,# and MshDR-1, 5#-TTIGCICKRTTYTGRAACCA-3#, and the second PCR was done by using
MshDF-2, 5#-AAYMGIAARCCIMSIACICCITT-3#, and
MshDR-2, 5#-CCAIATYTTIAYYTGIGTYTC-3#. Each
PCR consisted of 35 cycles of 94 °C for 1 min, 38 °C
for 1 min, and 72 °C for 2 min. The PCR was performed
with ExTaq DNA polymerase (Takara Bio) in the presenceof BD TaqStart anti-Taq antibody (BD Biosciences, San
Diego, CA). cDNAs corresponding to HD at their 3# ends
were cloned by using a 3#-Full RACE Core Set (Takara
Bio). The aa sequences were deduced from nucleotide sequencing of multiple PCR fragments.

Cloning of Genomic DNA
Isolation of high molecular weight DNA, fosmid library construction, and library screening were done as described (Aruga et al. 2006). Fosmid genomic libraries wereprepared with CopyControl pCC1FOS vector (Epicentre,
WI). In this study, we isolated 8 fosmid clones from A. pectinifera [Ast-1 (36,228 bp), Ast-3 (42,842 bp)], C. fluminea[Cor-2 (37,605 bp), Cor-3 (37,272 bp)], T. tubifex [Tub-1(38,378 bp), Tub-3 (35,661 bp)], and S. suvaense [Sco-1(42,994 bp), Sco-6 (38,643 bp)]. Cor-2 and Cor-3 wereoverlapping with a completely matching 34,763-bp sequence. Sco-1 and Sco-6 were overlapping with a completely matching 24,029-bp sequence. Ast-1 and Ast-3
and Tub-1 and Tub-3 were derived from independent
genes. Nematostella vectensis genomic DNA was obtainedby PCR amplification of N. vectensis genomic DNA using
following primers that are based on the draft genome sequences (http://www.ncbi.nlm.nih.gov/BLAST/tracemb.
shtml): 5#-CCACCATGGAGGCGGATCGCGATTTGCCT-3# and 5#-ATAAACTAATGCGGGTGCAGAAAACCTG-3#.

DNA Sequencing and Molecular Phylogenic Analysis
Sequencing and data assembly were done as described(Toyoda et al. 2002). Genomic sequences of Schmidtea

Metazoa-Wide Phylogenetic Analysis of Msx 71

Table 1
Animals Used in This Study
PhylumSubphylumClass
Chordata
Vertebrata
Mammalia
Aves
Amphibia
Osteichthyes
Cephalochordata
Leptocardia
Urochordata
Ascidiacea
Appendicularia
Hemichordata
Enteropneusta
Echinodermata
Echinoidea
Asteroidea
Arthropoda
Chelicerata
Crustacea
Insecta

Tardigrada
Nematoda
Mollusca
Cephalopoda
Bivalvia
Brachiopoda
Inarticulata
Annelida
Polychaeta
Oligochaeta
Hirudinida
EchiuraPlatyhelminthes
Tubellaria
Cnidaria
Hydrozoa

Anthozoa
Porifera
Demospongiae

SpeciesAbbreviation

GenomecDNA

Msx-Related Genes (Synonym, Accession Number)

Mus musculus
Gallus gallus
Xenopus laevis

Mm
Gg
Xl

DB
DB

DB
DB
DB

Danio rerio

Dr

DB

DB

Msx1 (AAH16426), Msx2 (Q03358), Msx3 (P70354)
Msx1 (Hox7, P50223), Msx2 (Hox8, P28362)
Msx1 (AAH81101)
Msh-A (Q03357), Msh-B (Q03356), Msh-C (Q01703),
Msh-D (Q01704), MsxE (AAB03273)

Branchiostoma floridae

BfDB

Msx (CAA10201)

Ciona intestinalis
Molgula oculata
Oikopleura dioica

Ci
Mo
Od

DB
DB
DB

CAD56691
AAA87223
AAW24005

Saccoglossus kowalevskii

SkDB

Msx (ABD97280)

Strongylocentrotus purpuratus
Heliocidaris erythrogramma
Heliocidaris tuberculata
Asterina pectinifera

Sp
Her
Ht
Ap

DB
DB
DB
PCR

SpMsx (AAB97688)
Msx (AAY86177)
Msx (AAY86178)
MsxA [Ast-1 (AB302953)]a, MsxB [Ast-3 (AB302954)]a

Limulus polyphemus
Pandinus imperator
Artemia franciscana
Tribolium castaneum
Drosophila melanogaster
Anopheles gambiae
Apis mellifera
Milnesium tardigradum
Caenorhabditis elegans
Caenorhabditis briggsae

Lp
Pi
Af
Tc
Dm
Ag
Ame
Mt
Ce
Cb

PCR
PCR
PCR

Loligo bleekeri
Octopus ocellatus
Corbicula fluminea

Lb
Oo
Cf

Lingula anatina

La

Platynereis dumerilii
Tubifex tubifex
Helobdella sp.

Pd
Tt
Hel

Urechis caupo

Uc

Schmidtea mediterranea
Thysanozoon sp.

Sm
Thy

Hydra viridis
Hydra vulgaris
Scolionema suvaense
Podocoryne carnea
Nematostella vectensis
Anemonia erythraea
Acropora millepora

Hvi
Hvu
Ss
Pc
Nv
Ae
Ami

Ephydatia fluviatilis

Ef

DB

DB
Fosmid

DB
DB
DB
DB

DB
DB

DB

PCR
DB
DB

MsxA (AB302959)a
MsxA (AB302960)a, MsxB (AB302961)a
Msx (AB302962)a
Muscle segment homeoprotein (AAW21975)
msh (Q03372)
EAA08817
Msx (AB362784)b
Msx (AB302966)a
vab-15 (Q09604)
CAE58718

PCR
PCR
PCR

Msx (AB302963)a
Msx (AB302964)a
Msx [Cor-2/Cor-3 (AB302955)]aPCR

Msx (AB302965)a

DB
PCR
DB

Msx (CAJ38810)
MsxA [Tt-1 (AB302956)]a, MsxB [Tt-3 (AB302957)]a
Msx (AAB37254)

PCR

Msx (AB302967)aPCR

Msx (AB362785)b
Msx (AB302968)a

FosmidFosmidDB

Fosmid

DB
DB
PCR
DB
PCR
DB

CAA45912
CAB88390
Msx [Sco-1/Sco-6 (AB302958)]a
AAX58756
Msx (AB362783)a
Msx (AB302969)a
msh3 (ABK41269)

DB

prox3 (AAA20151)

PCR

NOTE.—DB, sequence collected from NCBI databases; fosmid, isolated as fosmid genomic clones in this study; PCR, cloned by PCR amplification of the cDNA.
a
Sequence newly determined in this study.
b
Sequences edited from the publicized draft sequences (http://www.ncbi.nlm.nih.gov/BLAST/tracemb.shtml).

mediterranea, Hydra vulgaris, Caenorhabditis elegans,
Strongylocentrotus purpuratus, Ciona intestinalis, andN. vectensis were derived from public databases (http://
www.ncbi.nlm.nih.gov/BLAST/tracemb.shtml). Sequence
analysis was done with DNASISPro (Hitachi Software

Engineering, Tokyo, Japan), Sequencher (Gene Codes,
Ann Arbor, MI), and Genetyx (Genetyx, Tokyo, Japan)
software. Homology searching was performed against
a public database (http://www.ncbi.nlm.nih.gov/BLAST/)
by using Blast and discontinuous MEGA Blast.

72 Takahashi et al.

Table 2Eh1 Motifs Found in N-terminal Regions of Metazoan Msx
Proteins
Species
Gg_1
Mm_1
Xl_1
Dr_E
Bf
Ci
Od
Sk
Sp
Her
Ap_Ab
Dm
Ame
Ce
Cf
Pd
Tt_Ab
Ss
Pc
Nv
Ami
Consensus

Eh1N
55
61
67
44
46
76
6
73
61
51
59
134
43
14
79
132
119
53
30
29
14

FSVEALMAFSVEALMAFSVEALMAFSVEALMAFSVASLMAFSIEFLLSFSVDWIISFSVASLISFRVESLFSFRVESFFSFRVESLVGFSVASLLAFSVDSLLSFSVESLLTFGVDSIISFSVDSIISFSVAAIMAFSIDYILNFSIDYLLNFSVESLISFSVESLISFSV[D/E]S[L/I][L/I]S

Eh1C
106
118
120
68
91

FPSVGALGKFSVGGLL-K
YPVGAIM-Qa
FSVEVLQLPFSVEGILSK

112
100
104
108

FSVEGILSKHSVENILAKHSVENILAK
YTVEGILAS

151
180
170

FSMDEILGKFSVDGILSKFTVDGILGG

57
43

FSVESILEK
FSVERLLDKFSV[D/E]GILXKa
The sequence was highly diverged from other Eh1C sequences. In Xenopus
tropicalis (AAH62514), Eh1C sequence was FPVGGIMK.
b
Ap_B and Tt_B Eh1N were identical to those in Ap_A and Tt_A,
respectively.

The aa and nucleotide sequences were aligned by
ClustalW (Thompson et al. 1994). Some of the aligned sequences were corrected by visual inspection. Ancestral sequences were deduced from the present aa sequence data byusing ANCESCON, a distance-based program that gives
more accurate ancestral sequence reconstruction than do
PAML, PHYLIP, and PAUP* at large evolutionary distances (Cai et al. 2004). Phylogenetic tree analysis was donewith MEGA3.1 (Neighbor-Joining [NJ] tree and Maximal
Parsimony [MP] tree Kumar et al. 2004) and MrBayes 3.1.2(Bayesian Inference [BI] tree Huelsenbeck and Ronquist
2001; Ronquist and Huelsenbeck 2003). NJ tree was basedon the distance calculation with point accepted mutation(PAM) matrix (Dayhoff et al. 1978) after removing position
containing gaps (complete deletion option). In the NJ and
MP trees, the tree reliability was estimated by bootstrap test
(Felsenstein 1985) with 1,000 repetitions. In the BI analysis, we used an empirical model (WAG distances, Whelan
and Goldman 2001) with gamma, alpha shape parameter,
and aa frequencies estimated from the data. We ran
1,000,000 generations with 1 cold and 3 incrementallyheated Markov chains, random starting trees for each chain,and trees sampled every 100 generations. We constructed
a 50% major rule consensus tree from the last 15,000 treesthat were saved (burnin 5 2,500).
The evolutionary distances in figure 3C were calculated
by MEGA3.1. The distances of aa sequences (Msx) and nucleotide sequences (18S RNA) were determined by using thePAM matrix (Dayhoff et al. 1978) and the Tamura–Nei
model (Tamura and Nei 1993), respectively. Measurement
was done after removal of any alignment gap-containing
sites, assuming different evolutionary rates among sites(gamma distribution, a 5 0.37 for Msx aa sequences

and a 5 0.42 for 18S ribosomal RNA nucleotide sequences). The a parameters were estimated by Tree-Puzzle program (Schmidt et al. 2002). For the evolutionary distanceanalysis, we omitted the paralogs and sequences from identical genera (those in Heliocidaris, Caenorhabditis, and Hydra species), except for one representative sequence. The
representative sequences were the most strongly conservedin each group. According to these criteria, Mm_2, Gg_2,
Dr_E, Ap_A, Pi_B, Tt_A, Her, Ce, and Hvu were usedin the distance analysis, whereas Mm_1, Mm_3, Dr_A,
Dr_B, Dr_C, Dr_D, Gg_1, Ap_B, Pi_A, Tt_B, Ht, Cb,
and Hvi were not used.
Comparison of evolutionary rates among the chordateand cnidarian subgroups (fig. 3D and E) were done bycounting different aa residues between the target aa sequences and a reference sequence (Anc_Msx) by using
MEGA3.1. Counting was performed for the sequencesfrom the Chordata and Cnidaria. Subsequently, the numbers of residues nonidentical and identical to the reference
sequence were placed in 2  2 tables to compare the differences between 2 sequences from distinct sister subgroups.
Fisher’s exact test was performed to evaluate the statisticalsignificance of the differences in the ratios of nonidentical
to identical residues.
Plasmid Construction and Immunoprecipitation
Xl_Msx1 cDNA (a gift from Dr Atsushi Suzuki) wascloned into pCS2 þ Myc tag vector (Turner and Weintraub
1994). Nv_Msx genomic DNA and mouse Grg1 cDNA
were cloned into pcDNA3.1/His (Invitrogen) that was modified to have an initiation methionine, and either 2 Flag epitope tags, 3 HA tags at their HindIII/KpnI sites. The Grg1
cDNA was obtained from Riken FANTOM clones (http://
www.gsc.riken.go.jp/e/FANTOM/) (Carninci et al. 2005).
Site-directed mutations were introduced into the proteincoding region according to the method of Ito et al.
(1991). The primer sequences will be provided upon request. A truncation (stop) mutant of Xl_Msx1 was generated by inserting a stop linker (5#-TGAATATCA-3#) intoa unique SmaI site in the open reading frame (ORF) of
Xl_Msx cDNA.
Immunoprecipitation was performed essentially as described (Ishiguro et al. 2007). Briefly, COS7 cells weretransfected with the epitope-tagged Msx and Grg1 expression vectors with Lipofectamine Plus reagent (Invitrogen).
The transfected cells were washed and harvested in PBS()containing 1 mM phenylmethylsulfonylfluoride (PMSF),
and total cell extracts were prepared with a lysis wash bufferconsisting of 20 mM HEPES–KOH, pH 7.8, 10% glycerol,
150 mM NaCl, 0.5 mM dithiothreitol, 0.1 mM ethylenediaminetetraacetic acid, 0.5% NP-40, and 1 mM PMSF. The
extracts were incubated with anti-FLAG or anti-HA affinity
beads (Sigma, St Louis, MO) at 4 °C for 6 h. The beads
were subsequently washed with the buffer. The bound proteins were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis, transferred onto polyvinylidene difluoride membranes (Nihon Millipore, Tokyo,
Japan), and detected by antibodies against the epitope tags
using ECL western blotting detection reagent (GE Healthcare, Tokyo, Japan).

Metazoa-Wide Phylogenetic Analysis of Msx 73

Results
Collection of Msx-Related Sequences from Metazoa
A homology search against current sequence databases revealed Msx orthologs could only be detected inmetazoans. We did not find definitive Msx orthologs infungi, algae, plants, and protists. This result is consistent
with a previous finding that homeobox genes belonging
to the ANTP class, in which Msx is included, are confinedto the Metazoa (Holland and Takahashi 2005).
We first collected Msx family gene sequences througha database search. The homology search, which covered
both complete and incomplete nucleotides and protein sequence databases, revealed the presence of Msx homologs
in the Porifera, Cnidaria, Nematoda, Arthropoda, Annelida,
Echinodermata, Hemichordata, and Chordata (table 1). Porifera prox3 (Seimiya et al. 1994) was an Msx ortholog, assuggested by the phylogenetic tree analysis in Galle et al.
(2005). Genomic information on exon–intron boundaries
was available for vertebrates, insects, and a urochordate.
To improve the comprehensiveness of our analysis, wenewly cloned partial cDNA fragments of Msx homologs
from a wide range of animals in the Platyhelminthes, Mollusca, Echiura, Tardigrada, Brachiopoda, Cnidaria, Annelida,
Arthropoda, and Echinodermata. In addition, fosmid genomic clones were isolated from starfish (A. pectinifera),sludgeworm (T. tubifex), bivalve (C. fluminea), and jellyfish(S. suvaense), and their entire nucleotide sequence was determined. The number of Msx genes collected in a species
varied from 1 to 5. We observed closely related, but significantly diverged, genes (paralogs) in T. tubifex, A. pectinifera, and P. imperator, besides those described earlier invertebrates (Ekker et al. 1997). In total, we obtained 17 additional Msx sequences from 14 animal species from 9 animal phyla (table 1).
The aa Residues Functionally Important in MammalianMsx HDs are Strongly Conserved in Metazoan Orthologs
After sequencing the collected cDNA and genomic
clones, the aa sequences were deduced. We first compared
the aa sequences of the HD and its C-terminally flanking(CF) region (fig. 1). The alignment revealed that the
HDs were strongly conserved. The Msx HD has DNAbinding activity. The residues that are responsible for themolecular interaction with DNA bases (R2, R5, K46,
I47, Q50, N51, and R58) or DNA phosphoribosyl backbones (K3, T6, F8, Y25, R31, W48, R53, K55, andK57) (Hovde et al. 2001) were absolutely conserved among
the collected sequences. Some residues (F8 and R58) wereconserved in Msx, but not in Dlx family proteins that contained HDs most similar to Msx (fig. 1) (Gauchat et al.
2000). The Msx HD is also known to physically interact
with proteins that are essential for the molecular functionof Msx. K3, R5, and F8 are required for interactions andtranscriptional repression by the general transcription factor
TFIIF (Zhang et al. 1996). Mutations in human MSX HDs,
including R31P (STA, Hu et al. 1998), L13P (corresponding to MSX2 L154P in PFM, Wuyts et al. 2000), RK1819del (corresponding to MSX2 RK159-160del in PFM,
Wuyts et al. 2000), P7H (corresponding to MSX2

P148H in CSO, Jabs et al. 1993) are missense mutations
that cause genetic disorders. These sites were conserved
among the metazoan Msx proteins, except that RK18-19
had different residues in urochordates, insects, and cnidarian species (fig. 1). These results indicate that functionally
important residues in mammalian Msx proteins are strongly
conserved in all metazoan Msx HDs.

The Degree of HD Sequence Conservation Varies among
Taxonomic Groups
To determine the structural features of the Msx proteins in each taxon, we first drew a phylogenetic tree, basedon the alignment shown in figure 1, by using the NJ (fig. 2),
BI (supplementary fig. 1, Supplementary Material online),and MP (supplementary fig. 2, Supplementary Material online) methods. Monophyly of the Hydrozoa (Hvu, Ss, andPc) was recovered by the NJ/BI/MP trees, and those of theDiptera (Dm and Ag) and Cephalopoda (Oo and Lb) wererecovered by the BI/MP trees. However, with the exceptionof paralogs, there were no other strong groupings supported
by multiple trees. Instead, there was a high level of similarity between several evolutionarily distant animal species[e.g., only 2 changes in the 69 aa (9–77) sequences between
Mm_2 (Chordata) and La (Brachiopoda) (fig. 1)]. We therefore speculated that the metazoan ancestral Msx sequence
was strongly retained in some animal groups but not inothers.
To test this idea, we determined the mean distances ofthe HD þ CF sequence between all pairs of 12 taxa
(fig. 3A). In this grouping, the Tardigrada were grouped with
the Arthropoda in light of their sister-group relationship, as
determined by morphological and molecular analyses(Brusca and Brusca 2003); the Echiura were grouped with
the Annelida (McHugh 1997; Bleidorn et al. 2003); and the
Hemichordata were grouped with the Echinodermata on thebasis of recent molecular phylogenetic analyses (Cameron
et al. 2000; Peterson 2004). Two classes in the Cnidaria—Hydrozoa and Anthozoa—were tested separately because
analysis of the phylogenetic trees suggested that there
was a strong difference in tree length between these two
taxa. For reference sequence comparison, we deduced
the metazoan ancestor Msx sequence (Anc_Msx in fig.
1) by using ANCESCON, a program for the reconstructionof ancestral protein sequences that takes into account theobserved variations in evolutionary rates between positions(Cai et al. 2004). We first determined the aa number where
substitutions occurred in the Anc_Msx sequence for eachtaxon (fig. 3B). The sequences from the Vertebrata–
Cephalochordata (1 [group no. in fig. 3]), Echinodermata–Hemichordata (3), Mollusca (6), Brachiopoda (7), and Anthozoa (11) had low values (no greater than 6 aa substitutions), whereas those from the Urochordata (2), Hydrozoa(10), and Porifera (12) had large values (22–27 aa substitutions). The mean distances between all pairs of 2 taxa
were then determined concerning the Msx and 18S ribosomal RNA sequences (fig. 3C, Supplementary table 1,
Supplementary Material online). There were strongly deviated pairs along the distances of the Msx sequences. The
lowest group (pairs under the oblique line in fig. 3C)

74 Takahashi et al.

FIG. 1.—The aa alignment of HD and a C-terminal flanking region (HD þ CF). ‘‘Dots’’ indicate identical aa residues to those in the top sequence(Mm_2), and ‘‘hyphens’’ indicate the spaces artificially inserted in the multiple alignment program. The abbreviations for the animal species names are
given in table 1. Anc_Msx indicates the metazoan ancestral Msx sequence deduced by ANCESCON. ‘‘Plus’’ indicates the aa residues that are
responsible for molecular interaction with DNA bases, and ‘‘asterisk’’ indicates DNA phosphoribosyl backbones. The locations of residues responsible
for human genetic disorders (CSO, PFM, and STA; see text) are indicated at the top of the alignment. S206A indicates an artificial missense mutation
introduced into Xl-Msx1 in figure 6. ‘‘Shading’’ indicates the locations of DNA-associated or disease-causative aa residues in the alignment. ‘‘Openboxes’’ and ‘‘open circle’’ indicate the presence of phase-0 and phase-2 introns, respectively. The genes subjected for the exon–intron boundary analysis
are shown in figure 4. Bottom horizontal line indicates the region used in the calculation of evolutionary distances.

Metazoa-Wide Phylogenetic Analysis of Msx 75

Evolutionary Rates Differ between Two Independent
Pairs of Sister Groups
The evolutionary rates of 2 representative phyla, theCnidaria and Chordata, were compared. After comparison
between the ancestor sequence and each sequence from the
Cnidaria, we compared the diversification rates in any pairof sequences between the Anthozoa and Hydrozoa (fig. 3D,
supplementary table 2, Supplementary Material online). In
the Chordata, we compared the diversification rates between the Vertebrata, Cephalochordata, and Urochordata(fig. 3E, supplementary table 2, Supplementary Material
online). Fisher’s exact test of the results rejected the null
hypothesis that evolutionary rates were equal between
the 2 tested sequences in both the Anthozoa–Hydrozoa
and the Vertebrata–Cephalochordata–Urochordata comparisons (all P values were less than 0.01). The analysis indicated clear differences in the evolutionary rates among
sister groups in the Cnidaria and Chordata.
Comparison of Exon–Intron Organization of Msx FamilyProteins

FIG. 2.—NJ tree used for the metazoan Msx HD þ CF aa sequences.
The tree was rooted with mouse Dlx1 (Mm Dlx1) as an outgroup. Internallabels indicate bootstrap values (1,000 replicates). PAM matrix was used
for distance calculation by using the alignment shown in figure 1.
Branches with less than 50% bootstrap values were condensed.
Abbreviations for animal species names are indicated in table 1.

included pairs among the Vertebrata–Cephalochordata–
Hemichordata, Echinodermata, Mollusca, Brachiopoda,
and Anthozoa groups, whereas the highest one (pairs above
the broken line in fig. 3C) contained pairs among mainly the
Urochordata, Hydrozoa, and Porifera. From these results,
we postulated that the Vertebrata, Cephalochordata, Hemichordata, Echinodermata, Mollusca, Brachiopoda, and Anthozoa Msx sequences shared highly conserved features
among all the metazoan Msx sequences. On the other hand,the sequences belonging to the Urochordata, Hydrozoa, andPorifera diverged strongly.

We next compared the exon–intron organization of
Msx genes (fig. 4). All the Msx genes examined contained
at least 1 intron in the protein-coding region. All of the exon–
intron boundaries followed the GT/AG rule. Eighteen Msx
genes possessed only 1 intron, whereas the other 5 (AmeMsx, Ci-Msh, Ce-VAB15, Tt-MsxA, and Tt-MsxB) possessed
additional introns. The one in the N-terminal region flanking
the HD was located in a region with little sequence similarity(fig. 5). However, marked similarities were observed in the
distance from the most N-terminal end of the HD (11–28 aa),and the phases of the intron insertion site in the ORF were‘‘1’’ without exception. Furthermore, Seimiya et al. (1994)
putatively assigned a ‘‘phase-1’’ splicing acceptor site in
the 17 aa from the N-terminal end of the Ef-Msx (prox3)
HD, although the preceding exon was not identified. When
we aligned the N-terminal region aa sequence by adjusting
the intron position as the cardinal point, there was a weakly
conserved sequence near the intron position (fig. 5). The
conserved sequence was summarized as FPWMQ, where
tryptophan was strongly conserved (hereafter, we call theconserved sequence ‘‘PWM’’).
On the basis of these observations, we speculated thatthe intron was acquired in a common ancestor of a eumetazoa, or possibly a metazoan, Msx, and has been retained in
the all Msx family genes; we call this intron the AC (absolutely conserved) intron. The other conserved intron position was located near the C-terminal end of the HD at V45(phase 0) in 4 genes [Tt-MsxA, Tt-MsxB (Annelida), AmeMsx (Arthropoda), and Ci-Msh (Urochordata)] (fig. 1).
Comparison of Non–HD Region aa Sequences
The N-terminal region aa sequences have been evolutionarily conserved in a wide range of metazoan animals(table 2). The most conserved ones were located at the
N-terminal end, and the consensus sequence can be summarized as FSV[D/E]S[L/I][L/I]S, an Engrailed Homology
1 motif (Eh1), termed Eh1N in this paper. The other one

76 Takahashi et al.

FIG. 3.—Evolutionary distances computed from metazoan Msx aa sequences. (A) Taxonomic groups used in the analysis. Numbers correspond to
those in (B and C). (B) aa substations in comparison with ancestral Msx (Anc_Msx in fig. 1). The numbers of substituted aa residues were counted foreach Msx HD þ CF sequence. Solid bars indicate mean numbers of substituted aa in each taxon shown in (A). Error bars indicate SDs. Error bars areshown if there is more than one sequence; the number of sequences in each group (n) are indicated in the right column. The within-group distances wereas follows: Vertebrata–Cephalochordata–Hemichordata, 0.044; Urochordata, 1.106; Echinodermata, 0.032; Arthropoda–Tardigrada, 0.464; Mollusca,
0.021; Annelida–Echiura, 0.152; Platyhelminthes, 0.657; Hydrozoa, 0.889; and Anthozoa, 0.066. The values for the Vertebrata–Cephalochordata–
Hemichordata, Echinodermata, Mollusca, and Anthozoa were clustered below 0.05. (C) Between-group means of evolutionary distances of all pairs of
the 12 taxa in (A). Scatter graph indicates distances based on the Msx aa sequences [y axis, normal scale (left graph); log scale (right graph)] and those
based on the 18S RNA gene nucleotide sequences (x axis). The ‘oblique lines’’ are arbitrarily placed for the indication of the pairs with the low (below
the continuous line) and high (above the broken line) values along the distance of Msx sequences. The sequences subjected to the analysis in (B) and(C) were selected according to the criteria in Materials and Methods. (D and E) Probabilities of equal molecular evolutionary rate, as determined by
Fisher’s exact test. The tests were done between pairs of sequence from Cnidaria sister groups (D) or Chordata sister groups (E). The abbreviation of thesequence name can be seen in table 1. In both analyses, the presumptive ancestral Msx sequence (Anc_Msx) was used as a reference sequence.
Deuterostomia ancestral sequence was identical to Anc_Msx in the ANCESCON analysis.

Metazoa-Wide Phylogenetic Analysis of Msx 77(supplementary fig. 3B, Supplementary Material online).
The conservation of the 2 regions was limited to the sequences from Vertebrata, Cephalochordata, Echinodermata,Hemichordata, Mollusca, and Annelida. However, theirfunctional significance was not clear at this point.
Eh1N and Eh1C can Act as Binding Sites for GrouchoRelated Proteins

FIG. 4.—Schematic drawing of exon–intron structures of Msx genes
from various animals. ‘‘Open boxes’’ and ‘‘closed boxes’’ indicate non–HD and HD areas, respectively, of the Msx protein-coding region.
‘‘Horizontal bars’’ indicate introns, and ‘‘asterisks’’ above the horizontal
bars indicate AC introns.

was located between the Eh1N and PWM sequences. The
generalized sequence motif of this domain was FSV[D/E]GILXK; it was thus similar to Eh1N and was termed
Eh1C. Whereas Eh1N was conserved in all species,
Eh1C showed scattered distribution among the invertebrate
Msx proteins examined. When we mapped the presence of
the Eh1C sequence and PWM sequence in the animalgroups, it became clear that the Eh1C sequence was retained in the Vertebrata, Cephalochordata, Hemichordata,
Echinodermata, Mollusca, and Anthozoa (Cnidaria) but
not in the Urochordata and Hydrozoa (Cnidaria) Msx homologs isolated so far. In the Vertebrata, 2–5 paralogues
existed in the examined species, and Eh1C-related sequence could be observed at least in 1 paralogue, but notalways in the others. An examination of the current NCBI
database revealed that, in the Vertebrata, all the examined
Msx1 homologs in Xenopus, Ambyostoma, Notophthalamus, Gallus, Mus, Rattus, Bos, and all primate species kept
the same Eh1N sequence, LPFSVEALMAD. All the Msx2
homologs in Gallus, Mus, Rattus, Canis, Bos, and all primates had LPFSVEALMSD (data not shown).
In addition to the Eh1-related motifs, short conserved
sequence stretches were found in the N-terminal region(supplementary fig. 3A, Supplementary Material online)
and in C-terminal flanking of the region shown in fig. 1

Eh1N and Eh1C were very close to the sequences forbinding of the transcriptional corepressor Groucho (Paroush
et al. 1994; Fisher et al. 1996; Tolkunova et al. 1998). To
evaluate the functional significance of the conserved domains in the N-terminal regions, we generated mutant
Msx proteins that had alanine substitutions in the conserved
Eh1N, Eh1C, and PWM regions and analyzed their
Groucho-related protein-binding abilities. For this purpose,expression constructs for FLAG or HA epitope-taggedmouse Groucho-related-gene1 (Grg1), Myc-tagged XlMsx1, and FLAG-tagged Nv-Msx were generated and used
for a coimmunoprecipitation assay. When these expression
vectors were transfected into COS7 cells, we detected each
epitope-tagged protein with the expected molecular weightin an immunoblot assay (fig. 6 and data not shown). FLAGor HA-Grg1 expression vector was then cotransfected with
Myc-Xl-Msx1 or HA-Nv-Msx, respectively, and the Grg1
proteins were immunoprecipitated with anti-epitope tag antibodies. Both Myc-Xl-Msx1 and FLAG-Nv-Msx were coprecipitated with Grg1 (fig. 6B and C).
We also tested the physical interaction between Grg1
and mutant Msx1 proteins that contained substitution mutations in their conserved domains (fig. 6A). We prepared 5
expression vectors with substituted mutant Xl-Msx1 (Stop,
Eh1N, PWM, Eh1N&PWM, HD-S206A) (fig. 6A). The
mutants lacking Eh1N (Eh1N, Eh1N&PWM) lost binding
to Grg1 protein, whereas the PWM and HD substitution
mutants retained Grg1-binding abilities comparable tothose of wild-type Xl-Msx1 (fig. 6B).
Because Nv-Msx contained 2 Eh1-like sequences(Eh1N and Eh1C), we generated expression vectors for
4 Nv-Msx mutant proteins, including either single or combined mutants of the Eh1 domains (fig. 6A). Immunoprecipitation experiments showed that a mutation in Eh1N or
Eh1C gave a strong or weak decrement, respectively, in
Grg1-binding ability (fig. 6C), whereas the PWM mutants
were unaffected. The Eh1N and Eh1C combined mutant(NvMsxEh1N&C) completely lacked the ability to bindto Grg1, suggesting that both domains bind to Grg1. Collectively, these results indicated that the Eh1-like domains
can be bound by Grg1 protein in both vertebrate and anthozoan animals.
Discussion
Functional Significance of Msx Conserved Domains
Msx1 HD binds specific DNA target sequences(Hovde et al. 2001). In addition, many proteins bind mammalian Msx1 or Msx2 proteins [Lhx1 to HD (Bendall et al.
1998); Dlx to HD (Zhang et al. 1997); Grg to the N-terminal
region (Rave-Harel et al. 2005); PIAS to the C-terminal region (Lee et al. 2006); Pax3 (Bendall et al. 1999), Pax9

78 Takahashi et al.

FIG. 5.—The aa alignment of the HD N-terminal flanking region. PWM indicates the newly identified conserved domain (shaded, PWM). All the
AC introns were located in phase 1 of the ‘‘boxed’’ residues. ‘‘Long arrow’’ indicates the HD region. ‘‘Hyphens’’ and ‘‘equals marks’’ indicate the spaces
and deletions, respectively, that were introduced for the alignment. The equals marks in Dm and Ame represent deletions of PPGMFPGAGFGG and
HGHLYTPHGGPTSPN, respectively. The ‘‘asterisk’’ in the Ef line indicates the presence of a putative splicing acceptor site in phase-1 of the following
HD-containing exon.

(Ogawa et al. 2006), and Histone H1b to the N-terminal
region (Lee et al. 2004); and Dlxin-1 (Masuda et al.
2001) and TFIIF (RAP74 and RAP30) to the N-terminal
region of the HD (Newberry et al. 1997)]. The physical interaction between these molecules may have acted as a formof evolutionary constraint. The overall conservation of the
Msx HD suggests that interaction with these molecules iscritical for the molecular functions of Msx. At the aa residue
level, all the DNA-interacting and TFIIF-interacting residues in the Msx HD were conserved without exception,suggesting that interaction with these molecules, in particular, is essential for many metazoans to survive against natural selection.
Other structure–function information can be obtained
through an analysis of the point mutations in human MSX1
and MSX2. Some missense mutations in the HD are associated with STA, PFM, and CSO (fig. 1). Besides these HD
mutations, it is noteworthy that a mutation in the methionine of Eh1N (L61K in human MSX1) is associated witholigodontia, a congenital form of tooth agenesis (Lidral
and Reising 2002). Our results revealed that these residues
are strongly conserved not only in primates, as revealed by
Perry et al. (2006), but also in the Msx of many metazoans.
The characteristic signs of these congenital anomalies suggest that the disease-associated residues are essential, atleast for cranial and tooth development in humans. However, the strong conservation of the residues in invertebrate
animals suggests that the same sequences can be used invarious developmental and/or survival contexts of invertebrates. This view supports an idea that, in many metazoans,the gene encoding Msx protein is among the most fundamental and versatile.

Our results revealed that the Eh1N and Eh1C domains
are required for the interaction of Msx with Grg1 in mammalian cells. The Eh1 domain was originally identified in
the domain mediating the transcriptional repression of Drosophila engrailed protein through interaction with the transcriptional corepressor, Groucho. Although the Eh1-likedomains have been identified in various transcription factors (Copley 2005), a recent study showed that Grg1 canphysically interact with Msx1 and can regulate an Msx target gene (Rave-Harel et al. 2005). Our study revealed thatGrg1 binding was mediated by an Eh1-like domain in XlMsx1 protein and that the 2 Eh1-like sequences in anthozoan Nv-Msx were able to bind to Grg1 in mammaliancells. These results suggest that Groucho-like–proteinbinding activity is retained in metazoan Msx proteins. Interestingly, the Nematostella genome contains sequences
highly similar to Grg1 (H.T. and J.A., unpublished data).
Furthermore, vertebrate, Drosophila, and nematoda
Groucho-related genes play critical roles in animal development (Pflugrad et al. 1997; Gasperowicz and Otto
2005). It would be interesting to see how putative Msxbinding domains in Groucho family proteins are conservedin metazoans. Collectively, these results suggest that thephysical interaction between Msx and Groucho-like protein
was established in the eumetazoan ancestor and has beenutilized in various ways in the course of evolution.
Variation in the Degree of Protein Structure
Conservation in the Msx Family
We focused here on the conserved domains in Msx
proteins based on the phylogeny of species. Phylogenetic

Metazoa-Wide Phylogenetic Analysis of Msx 79

FIG. 6.—Functional characterization of the conserved domains. (A)Schematic drawing of conserved domains in the Msx proteins and the aasequences in the mutant and wild-type Msx proteins. Stop(TGA),
a truncation mutant generated by inserting a stop codon just after proline
62 in Xl_Msx1 aa sequence (AAH81101); S206A, a substitution mutationin which serine 206 in the Xl_Msx1 was changed into alanine (position of
the targeted serine residue is indicated in fig. 1). Substitution mutations in
Eh1N, Eh1C, and PWM sequences of Xl_Msx1 or Nv_Msx are indicatedin below the diagram. The highly diverged Eh1C sequence Xl_Msx1(table 2) was not changed. (B) Immunoprecipitation assay between
FLAG-Mm-Grg1– and Myc-Xl-Msx1–derived proteins. (C) Immunoprecipitation assay between HA-Mm-Grg1– and FLAG-Nv-Msx–derived
proteins. In (B) and (C), the immunoprecipitation experiments werecarried out by using cell extracts from COS7 cells cotransfected with
Grg1 and Msx expression vectors. The results of immunoblot using theantibodies indicated on the left side of the panels are shown. The
combinations of transfected vectors in each cell lysate are indicated at thetops of the pictures. Immunoblot analyses were performed both on the 6%of cell lysates subjected to the immunoprecipitation experiments (IB, 6%
input) and the immunoprecipitated (IP) proteins.

trees of the 4 conserved domains that we characterized(Eh1N, Eh1C, PWM, and HD) showed a correlation between the degree of HD sequence divergence and the absence of Eh1C and PWM sequences (fig. 7). In the
Hydrozoa and Urochordata, which showed HD sequence

divergence in Msx, conservation of the Eh1C or PWM sequences was not evident. In contrast, the HD sequence of
Msx was conserved in the Anthozoa, Echinodermata,
Cephalochordata, Vertebrata, and Mollusca. HD in the Annelida, Arthropoda, and Nematoda showed an intermediate
degree of sequence diversification; these taxa lacked Eh1C
and had incomplete PWM sequences.
These results allow us to speculate on the overall Msx
evolutionary process (fig. 7). Ancestral Msx appeared in themetazoan ancestor, and this gene probably possessed the
AC intron. Because of the absence of the N-terminal regionof Ef-Msx, we do not know whether the metazoan ancestor
was already equipped with all the conserved domains.
However, the eumetazoan ancestor, at the latest, may havealready contained the Eh1N, Eh1C, PWM, and HD sequences. The 4 conserved sequences diverged differentially inthe course of evolution. The degree of divergence was relatively small in the Anthozoa, Echinodermata, Cephalochordata, Vertebrata, and Mollusca but large in the
Porifera, Hydrozoa, and Urochordata.
There are several potential pitfalls in this hypothesis.
First, we are assuming that the diverged-type Msx genes
were generated from conserved-type Msx genes. As an opposing idea, convergence to the one prototype sequence cannot be ruled out at this point. Second, roles of possible
phylum-specific or class-specific conserved domains remains unclear due to insufficient numbers of sequences
for the within-phyla comparison. In this regard, increasingcomplexity-style evolution is still possible in Msx family
evolution. Third, we might have missed highly diverged,
but functionally equivalent, sequences because conservationof the Eh1N, Eh1C, and PWM sequences relies on shortsequence stretches. Fourth, we cannot yet conclude thatthe variable divergence rates reflect general acceleration(or deceleration) of the molecular evolutionary rate or are
limited to particular molecular species. Although we utilized
the available 18S ribosomal RNA sequences as references,
a comprehensive evaluation should be done with additionalreferences. These points may be readily addressed by anextended analysis of additional molecular species.
Our hypothesis could also be evaluated by examining
whether or not other genes fundamental to the organizationof the animal body [so called ‘‘tool-kit’’ genes (Carroll et al.
2001)] show similar evolutionary tendencies. In a previous
study, we found that selective loss of the conserved domain
in the Zic family is found in certain animal taxa proteins(Aruga et al. 2006). In the case of Zic family proteins, conserved Zic can be seen in the Arthropoda, Mollusca, Annelida, Echinodermata, and Chordata (vertebrates andcephalochordates), whereas diverged Zic can be seen in
the Platyhelminthes, Cnidaria, Nematoda, and Chordata(urochordates). On the basis of the phylogenetic distribution of the conserved Zic proteins, we proposed that the bilaterian ancestors had already acquired the full set ofconserved domains that is found in currently living animals.
Thus, in the cases of both the Msx and the Zic family proteins, the ancestral genes may already have possessed a setof conserved domains that were selectively lost in thecourse of evolution.
This differential divergence rate among the taxa could
be involved in diversification of the organization of the

80 Takahashi et al.

FIG. 7.—Evolutionary process of Msx genes. Hypothetical model that explains the diversity of Msx genes in the Eumetazoa. ‘‘Thick horizontal
bars’’ indicate AC (absolutely conserved) introns. Dark, light, and intermediate gray boxes indicate conserved type, diverged type, and intermediatetype HD þ CF regions, respectively; black thick vertical lines indicate Eh1N; gray thick vertical lines indicate Eh1C; striped boxes indicate PWM; andhatched boxes indicate HD.

animal body. However, it is premature to conclude this because our understanding of the role of the Msx genes in eachanimal taxa is very limited. If we consider the Cnidaria sistergroups, Anthozoa and Hydrozoa, hydrozoan Msx is expressed in regenerating muscle tissues (Yanze et al. 1999;
Galle et al. 2005) and anthozoan Msx in the planula larval
ectoderm (de Jong et al. 2006). However, because these reports dealt with expression profiles in only limited stages of
the animals’ life cycles, we are deterred from considering further the similarities and differences between Msx usage in thetwo classes of animals. If we consider the chordate Msx homologs, ascidian Msx genes appear to be commonly expressed in muscle, notochord, neural plate precursor cells,
and the folding neural plate (Ma et al. 1996; Aniello et al.
1999). Some of these expression sites overlap with thoseof Msx gene expression in vertebrates (Davidson 1995), suggesting that some of the roles of Msx are shared between ascidians and vertebrates. However, vertebrate Msx genes are
expressed in the limb bud, mandibular process, tooth, uterus,
and other many organs that are not apparently found in ascidians. It is possible that the negative pressures for structural
diversification differ between ascidians and vertebrates.
Finally, our results revealed the differential diversification of the Msx protein functional domain that became
established in the eumetazoan ancestor in the course ofevolution. The similarity of this evolution to that of Zic
family proteins raises the possibility that a group of‘‘tool-kit’’ genes shared the same feature, that is, differential
diversification of the conserved domain. We await further
extension of the metazoan-wide phylogenetic analysis ofdevelopmentally critical genes.

Supplementary Material
Supplementary text, figures 1–3, and tables 1 and 2 areavailable at Molecular Biology and Evolution online (http://
www.mbe.oxfordjournals.org/). Database deposition: The
sequences reported in the paper have been deposited in
the GenBank/EMBL/DDBJ database accession numbers.
AB302953–AB302969, AB362783–AB362785.
Acknowledgments
We thank Yayoi Nozaki and all the technical staff of
the Sequence Technology Team of RIKEN GSC at the Research Resource Center, RIKEN BSI, for their technical assistance. We also thank Atsushi Suzuki (Hiroshima
University) for the plasmid. This study was done as a collaborative research project of the Strategic Research Program at
RIKEN. It was supported by grants-in-aid from the Japanese
Ministry of Education, Science, Sports, and Technology.
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William Martin, Associate Editor
Accepted October 11, 2007