Mol Biol Evol-1998-Williams-600-7

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					Gene and Domain Duplication in the Chordate Otx Gene Family: Insights
from Amphioxus Otx
Nic A. Williams and Peter W. H. Holland
School of Animal and Microbial Sciences, The University of Reading, U.K.

       We report the genomic organization and deduced protein sequence of a cephalochordate member of the Otx hom-
       eobox gene family (AmphiOtx) and show its probable single-copy state in the genome. We also present molecular
       phylogenetic analysis indicating that there was a single ancestral Otx gene in the first chordates which was duplicated
       in the vertebrate lineage after it had split from the lineage leading to the cephalochordates. Duplication of a C-
       terminal protein domain has occurred specifically in the vertebrate lineage, strengthening the case for a single Otx
       gene in an ancestral chordate whose gene structure has been retained in an extant cephalochordate. Comparative
       analysis of protein sequences and published gene expression patterns suggest that the ancestral chordate Otx gene
       had roles in patterning the anterior mesendoderm and central nervous system. These roles were elaborated following
       Otx gene duplication in vertebrates, accompanied by regulatory and structural divergence, particularly of Otx1
       descendant genes.

      Comparison of expression patterns of homologous                        partial overlap (redundancy) in function between Otx1
genes during embryonic and adult life can provide use-                       and the more widely expressed Otx2. Deletion of the
ful clues to the identification of homologous body                            mouse Otx2 gene causes embryonic lethality; embryos
regions or structures in different animal groups (Abou-                      show a perturbation in the gastrulation process and a
heif et al. 1997). This is a necessary foundation for un-                    complete lack of head structures normally found anterior
derstanding evolutionary changes in morphology or em-                        to rhombomere 3 (Acampora et al. 1995; Matsuo et al.
bryonic development. However, in many cases, identi-                         1995; Ang et al. 1996). This absence of a large region
fication of directly homologous (orthologous) genes, and                      of the anterior head is probably a result of the lack of
hence comparable gene expression patterns, is hampered                       an underlying prechordal plate which expresses Otx2
by gene duplication events in one or another lineage.                        (but not Otx1) during gastrulation. The Otx gene family
After duplication, daughter genes may have lost an an-                       seems to control similar developmental processes in
cestral function or acquired new gene-specific functions.                     Xenopus and zebrafish, although there are at least three
Thus, whenever gene expression patterns are compared                         Otx gene family members in each of these species (Mori
between taxa in studies of developmental evolution, it                       et al. 1994; Kablar et al. 1996). The expression patterns
is necessary to identify gene duplication events and to                      of the vertebrate genes are broadly comparable to that
relate these to putative roles of each gene within the                       of the Drosophila homolog orthodenticle (otd) (Finkel-
gene phylogeny. Only then can ancestral and derived                          stein et al. 1990), which is also anteriorly restricted dur-
roles of members of genes be distinguished. Here, we                         ing embryonic development. Phenotypes resulting from
used this approach to gain insight into the origin and                       mutations at the Drosophila otd locus are also broadly
development of the vertebrate head and brain by cloning                      similar to those arising from gene targeting of Otx
and analyzing a cephalochordate member of the Otx                            genes, ranging from deletion of the ocelli of the adult
homeobox gene family (AmphiOtx). Members of the                              fly (the ocelliless mutant) to elimination of specific an-
vertebrate Otx homeobox gene family play important                           terior head segments in the loss-of-function mutant otd
roles in the specification and patterning of anterior neu-                    (Finkelstein et al. 1990). The discovery that expression
ral and head structures. The mouse genome has two ev-                        patterns and developmental roles were comparable be-
olutionarily related Otx genes with spatially and tem-                       tween arthropods and vertebrates raised the intriguing
porally overlapping expression patterns in embryogen-                        scenario that their common ancestor already possessed
esis (Simeone et al. 1992, 1993). Mice lacking Otx1                          a patterned head region, the development of which was
through gene targeting show spontaneous epileptic be-                        dependent on an ancestral otd/Otx gene (Holland, Ing-
havior and have defects in the patterning of the telen-                      ham, and Krauss 1992; Slack, Holland, and Graham
cephalic and mesencephalic regions, cerebellum, eyes,                        1993). This would imply that the origin and specializa-
and ears (Acampora et al. 1996). This is a less drastic                      tion of a head region did not occur independently in the
phenotype than might be predicted from the broad dis-                        coelomate protostome and deuterostome lineages.
tribution of Otx1 transcripts throughout much of the em-                           We have previously reported the expression pattern
bryonic forebrain and midbrain, suggesting that there is                     of an Otx gene from the cephalochordate amphioxus
                                                                             (Williams and Holland 1996); the probable closest in-
    Key words: Otx, homeobox, gene duplication, amphioxus, head
                                                                             vertebrate relative of the vertebrates. This gene, desig-
                                                                             nated AmphiOtx, is expressed in a manner temporally
     Address for correspondence and reprints: Peter W. H. Holland,
School of Animal and Microbial Sciences, The University of Reading,
                                                                             and spatially comparable to that of the vertebrate Otx
Whiteknights, Reading RG6 6AJ, United Kingdom. E-mail:                       genes, and we have used it as a molecular marker of                                                  forebrain homologs in the amphioxus neural tube. Here,
Mol. Biol. Evol. 15(5):600–607. 1998                                         we describe the sequence and genomic organization of
  1998 by the Society for Molecular Biology and Evolution. ISSN: 0737-4038   AmphiOtx and present data in support of a single-copy

                                                                                                  Amphioxus Otx   601

state. We also present molecular phylogenetic analysis      tions (X.l Otx1 and X.l Otx4—Kablar et al. 1996; H.r
and gene domain duplication data which strongly sup-        Otx—Wada et al. 1996). These sequences were aligned
port the existence of a single Otx gene in an ancestral     with AmphiOtx using the CLUSTAL W program (GCG)
chordate. We propose ancestral roles for this gene in       and adjusted by eye to maximize continuous stretches
patterning of the anterior mesendoderm and central ner-     of sequence similarity. Drosophila or Tribolium otd pro-
vous system (CNS) of chordates. These roles were fur-       teins could not be included in the alignment because of
ther refined following Otx gene duplication on the ver-      extreme divergence outside the homeodomain. A dis-
tebrate lineage accompanied by regulatory and structural    tance matrix was calculated using 219 confidently align-
divergence of the Otx genes and proteins.                   able sites, 119 of which were variable. The distance
                                                            measure was the mean character difference, calculated
Materials and Methods                                       using a step matrix which gives the minimum number
Library Screening                                           of nucleotide replacement substitutions needed to con-
                                                            vert one amino acid to another (based on the genetic
      A Branchiostoma floridae genomic library (Garcia-
                                                            code used in nuclear genes of most organisms). A neigh-
Fernandez and Holland 1994) was screened with a 139-
                                                            bor-joining tree was constructed using PAUP (version
bp partial homeobox probe isolated from a 3.8-kb cDNA
                                                            4.0.0d54), and confidence in each node was assessed by
clone of Drosophila otd (kindly provided by N. Perri-
                                                            100 bootstrap replicates. This step matrix was also used
mon). Three overlapping genomic phages were isolated,
                                                            for the maximum-parsimony tree, performed with a heu-
and two of these, Bfg395–1 and Bfg398–2, were further
                                                            ristic search with 100 replicates of the random-addition
restriction mapped and subcloned. A region showing se-
                                                            sequence in order to improve the chance of finding the
quence similarity to the second exon of the vertebrate
                                                            most-parsimonious tree(s). Six equally most-parsimoni-
Otx genes was fully sequenced from both phages and
                                                            ous trees of length 381 were found; a strict consensus
was found to be identical. A subclone from Bfg398–2
                                                            was constructed.
was found to contain sequence identity to the third Otx
exon of vertebrate genes, and this was also fully se-       PEST Sequence Analysis
quenced. A probe prepared from the same Drosophila
                                                                 The deduced AmphiOtx protein sequence and chor-
otd region was also used to screen a B. floridae 5–24-h
                                                            date Otx protein sequences were searched for putative
embryonic cDNA library kindly provided by J. Lange-
                                                            PEST regions using the PESTfind program available at
land. A single positive recombinant was subcloned and
fully sequenced in both directions; the DNA sequence
                                                            menu.cgi. The minimum number of amino acids form-
is available in the EMBL and GenBank databases (ac-
                                                            ing a PEST region between positive flanks was set at
cession number AF043740). The 5 region of this
AmphiOtx cDNA was used as a probe to isolate the
missing 5 exon from the Bfg398–2 phage. The genomic
and cDNA sequences were compared and were found to          Results
code for an identical homeodomain sequence within an        Genomic Organization of AmphiOtx
open reading frame varying by just three amino acid
                                                                 Since the Otx homeobox is well conserved between
sequence polymorphisms outside the homeodomain.
                                                            Drosophila and vertebrates, we predicted that a gene or
Comparison of the genomic and cDNA sequences was
                                                            genes containing this motif would also be conserved in
used to identify the intron : exon boundaries of
                                                            amphioxus. An amphioxus gene was identified by
                                                            screening B. floridae genomic and cDNA libraries with
Southern Hybridization                                      a Drosophila otd homeobox probe. We designated this
     DNA from five individual adult amphioxus (Bran-         gene AmphiOtx because of its high sequence similarity
chiostoma floridae) was isolated as described by Shi-        to the vertebrate Otx genes, particularly over the hom-
meld (1997) and digested with EcoRI or HindIII. A 233-      eodomain (93%–98% amino acid identity).
bp radiolabeled probe containing the entire amphioxus            The AmphiOtx cDNA is 2,436 nt in length; the lon-
Otx homeobox was prepared from the cDNA by random           gest open reading frame has the potential to encode a
priming (Boehringer Mannheim). Hybridization was            protein of 322 amino acids. A homeobox is close to the
carried out in 0.25 M Na2HPO4 (pH 7.2), 7% SDS, 1%          5 end, typical of genes of the Otx gene family, and a
BSA, 1 mM EDTA at 65 C for 20 h, and the filter was          long 3 untranslated region is present. There is no ca-
washed in 2 SSC, 0.1% SDS at 65 C before exposing           nonical polyA adenylation signal (AATAAA), although
to film.                                                     the similar sequence ATTAAA lies only 15 nucleotides
                                                            5 of 10 adenine residues at the extreme 3 end of the
Molecular Phylogenetic Analysis                             clone. Such slight divergence from the normally highly
     Mouse, human, zebrafish (Danio rerio), frog (Xen-       conserved AATAAA signal has also been observed in a
opus laevis), and ascidian (Halocynthia roretzi) Otx pro-   number of other amphioxus cDNAs (unpublished data;
tein sequences were obtained from the EMBL database         S. M. Shimeld, personal communication); any functional
(accession numbers: mouse Otx1—P80205; mouse                significance is unknown.
Otx2—P80206; human Otx1—P32242; human Otx2—                      AmphiOtx intron : exon boundaries were deter-
P32243; D.r. Otx1—D26172; D.r. Otx2—D26173; D.r.            mined by comparison of cDNA and genomic DNA se-
Otx3—D26174; X.l Otx2—U19813) or from publica-              quences, shown schematically in figure 1. In common
602   Williams and Holland

     FIG. 1.—Genomic and cDNA organization of AmphiOtx. The black boxes represent the homeobox and white boxes represent other coding
regions. Intron sites are indicated, and restriction sites are shown below the genomic clone: P (Pvu II), R (EcoR I), H (Hind III).

with vertebrate Otx genes, the short 5 coding region is             AmphiOtx. The two other bands observed in the EcoRI
separated from the homeodomain by an intron (2.5 kb),               digests were weaker in intensity than the 3.3 kb band
and a second intron (4 kb) is present between the codons            and varied in size between individuals. We interpret this
for glutamine and valine residues at positions 46 and 47            as showing polymorphism around the region of the sec-
of the homeodomain (fig. 2). The position of this second             ond exon. Nonpolymorphic sites should have resulted in
intron is identical to that in mouse or human Otx and               a single band of equal intensity to the 3.3 kb band,
Drosophila otd, strongly suggesting that this intron site           which was not seen, while if two Otx-related genes were
existed before the divergence of the arthropod and chor-            present in the genome then three or four strong bands
date lineages.                                                      would have been seen (depending on intron : exon or-
      The more 5 intron in mouse, humans, and Dro-                  ganization). If two polymorphic genes were present,
sophila is found just 3 to a tyrosine-proline (YP) se-              even more bands, varying in intensity and size, should
quence, possibly a diverged version of the hexapeptide              be observed in each lane.
motif found in many classes of homeodomain proteins.                      Two bands are present in each HindIII digest lane
The AmphiOtx sequence lacks such a motif, but its most              which are equal in intensity. We mapped a HindIII site
5 intron is found just 3 of a tyrosine-threonine (YT)               within the second intron of the genomic Bfg398–2 clone
sequence (fig. 2). In Drosophila, alternative splicing at            and infer that the two HindIII bands observed center on
this intron site results in the deletion of six codons, in-         this site. Hence, the genomic Southern blot results pro-
cluding those encoding the YP motif, in the majority of             vide strong support for the existence of a single amphi-
RNA transcripts (Vandendries, Johnson, and Reinke                   oxus Otx-related gene.
1996). Thus, although this intron site is found in a di-
verged sequence region in amphioxus, its position may               AmphiOtx Sequence Analysis
be homologous to that of Drosophila otd and vertebrate                    To investigate whether AmphiOtx is an ortholog of
Otx genes. A further two introns are present in Dro-                one of the multiple vertebrate Otx genes or whether it
sophila otd near the N-terminus which are not present               is directly descendent from a precursor of these genes,
in either AmphiOtx or human or mouse Otx genes.                     we analyzed the deduced protein sequences of the Otx
                                                                    gene family. The Otx homeodomain has been highly
Genomic Southern Blot                                               conserved through evolution; only two differences are
     A Southern blot of genomic DNA isolated from                   seen between the AmphiOtx and Drosophila otd hom-
five individual amphioxus was probed with a 233-bp                   eodomains (not shown), with up to four differences
AmphiOtx cDNA probe that spanned the second intron                  when compared with other chordate Otx genes (fig. 2).
site in the homeobox. Between two and three bands                   The complete AmphiOtx, ascidian Otx (Hroth) and ver-
could be seen in each lane (fig. 3), with the most con-              tebrate Otx deduced protein sequences are aligned in
sistent being approximately 3.3 kb in size in the EcoRI             figure 2 to show regions of sequence similarity. Some
digest (four out of five individuals). This band corre-              regions, including the homeodomain but also substantial
sponds to a 3.3-kb EcoRI fragment cloned from both the              regions outside this, are clearly alignable; these sequenc-
Bfg398–2 and the Bfg395–1 phage clones (see fig. 1 for               es are marked in bold in the figure and can be used
genomic map) which contains part of the homeobox in                 confidently in phylogenetic analysis. Intervening se-
the third exon of AmphiOtx. The probe used in the                   quences between these regions are more variable and
screening should also hybridize to the second exon of               cannot be aligned with any degree of confidence; these
                                                                                                                      Amphioxus Otx     603

     FIG. 2.—Alignment of the deduced amino acid sequences of AmphiOtx, Hroth, and the vertebrate Otx genes. Dashes represent gaps
introduced into the alignment to maximize sequence similarity. Residues marked in bold were used in the molecular phylogenetic analysis. The
homeodomain is boxed, and the repeated protein domain at the C-terminus is bracketed and labeled A or B. Intron sites in AmphiOtx are
indicated with a triangle.
604   Williams and Holland

                                                                               FIG. 4.—Phylogenetic tree based on analysis of the amino acid
                                                                         sequences of the chordate Otx gene family. The values at the nodes
     FIG. 3.—Southern blot of B. floridae genomic DNA digested with       refer to a score from 100 bootstrap resamplings of the data; only scores
either EcoR I (R) or Hind III (H) and hybridized with a probe prepared   above 75 are shown. The upper figure refers to neighbor-joining anal-
from the AmphiOtx homeobox sequence. DNA from five individuals,           ysis, and the lower figure refers to maximum-parsimony analysis. Both
1–5, is shown. Size markers are indicated by arrows.                     analyses gave an identical tree topology except that within the Otx1-
                                                                         like clade, zebrafish Otx1 and Otx3 proteins were placed as sister genes
                                                                         in the maximum-parsimony consensus.
positions were excluded from phylogenetic analysis. In
total, 219 amino acid positions were analyzed from the                   Otx gene in the ancestral vertebrate-cephalochordate lin-
464 sites of the alignment. Molecular phylogenetic trees                 eage and that this condition has been retained in the
were constructed by the neighbor-joining method, and                     cephalochordates.
confidence was assessed by bootstrap resampling of the                          In addition to molecular phylogeny reconstruction,
data (fig. 4). We root the tree using Hroth as the out-                   we also analyzed the AmphiOtx translated protein for
group, because the longest branch length leads to this                   PEST sequences, putative indicators of rapidly degraded
group and also because urochordates are generally ac-                    proteins (Rogers, Wells, and Rechsteiner 1986). Such a
cepted as being the sister group to the vertebrate–ceph-                 sequence is present in AmphiOtx (PESTfind score 10.3)
alochordate clade. We analyzed the same alignment us-                    between residues at positions 160 and 288 in figure 2.
ing maximum parsimony. Six most-parsimonious trees                       This region appears to be quite divergent between the
were found; bootstrap values are superimposed onto the                   chordate Otx proteins, and was excluded from the mo-
neighbor-joining tree shown in figure 4. The consensus                    lecular phylogeny analysis, as the sequences could not
maximum-parsimony tree indicated a position for                          be confidently aligned. However, the equivalent region
AmphiOtx identical to that of neighbor-joining analysis.                 (by position) also encodes putative PEST sequences in
      These analyses confirm that the vertebrate genes                    all three zebrafish Otx proteins (Mori et al. 1994). We
group into two very well supported classes: the Otx2                     also find a putative PEST sequence in this region in
genes and the Otx1-like genes. Interestingly, the Otx2                   mouse Otx1and Otx2 proteins (PESTfind scores 4.8 and
genes have shorter branch lengths than those of the                      8.3, respectively) and two putative PEST sequences in
Otx1-like genes. This implies a higher rate of sequence                  this region in Hroth protein (PESTfind scores 18.9 and
evolution within the Otx1 family compared with the                       6, respectively). The presence of one or more putative
Otx2 genes. AmphiOtx is placed on a separate branch                      PEST sequences in all chordate Otx proteins at the same
from those leading to the vertebrate Otx genes, diverging                relative position indicates the conservation of an ancient
before the separation of the Otx1 and Otx2 genes. This                   biochemical function, even though sequence divergence
evolutionary position is supported by a bootstrap values                 has occurred. A short intracellular half-life could be im-
of 96% using neighbor-joining and 91% using maxi-                        portant for the Otx protein family in order to fine tune
mum-parsimony. This is strong evidence for the exis-                     their developmental expression and resultant transcrip-
tence of a single ancestral chordate Otx gene which was                  tional regulation of other genes. This would be partic-
duplicated along the vertebrate lineage after it split from              ularly advantageous during the complex genetic inter-
the lineage leading to the cephalochordates. Even if a                   actions during early embryonic development.
second Otx gene does exist in amphioxus (and our data
argue that it does not), such a gene would have arisen                   Domain Duplication in Vertebrate Otx Genes
from an independent duplication in the amphioxus lin-                          Molecular phylogenetic reconstruction traditionally
eage. We conclude that there was a single copy of an                     treats DNA or protein sequences as one-dimensional
                                                                                                     Amphioxus Otx   605

strings of characters, homologous between taxa. While        sequence; it also has a genomic organization similar to
powerful, this approach tends to overlook larger-scale       that of the vertebrate Otx genes. Molecular phylogenetic
features or motifs that hold historical information. In      analysis strongly argues that a single ancestral chordate
particular, regions excluded from phylogenetic analysis      Otx gene was duplicated in the vertebrate lineage after
due to alignment difficulties can still be a source of his-   it split from the lineage leading to the cephalochordates.
torical information.                                         The cephalochordate amphioxus retains the ancestral
      We noticed that in all vertebrate Otx proteins, a      condition. This conclusion is further strengthened by the
motif of approximately 18–23 amino acids at the C-           identification of an internal protein motif duplication in
terminus of the gene is present as an imperfect tandem       the vertebrate lineage; this motif is present as two copies
repeat (boxed and named in fig. 2). To our knowledge,         in vertebrate Otx proteins but as one in AmphiOtx,
this repeat has not previously been noted. We suggest        Hroth, and SpOtx. These analyses imply that the am-
that it is likely to have arisen by the tandem duplication   phioxus and ascidian Otx genes are direct ‘‘descen-
of a single copy of this motif. Searches against the         dants’’ of the ancestral chordate Otx gene, uncompli-
EMBL and GenBank databases reveal no regions in oth-         cated by gene duplication events.
er proteins with a similar sequence, and it remains to be           In the last few years, many examples have been
seen whether this motif confers any particular function.     reported of genes undergoing duplication during the ear-
The tandem repeat is only present in vertebrate Otx pro-     ly evolution of vertebrates. Examples include the Hox
teins, but we note that both AmphiOtx and the ascidian       gene cluster, Wnt genes, and the Pax 1/9 genes (for re-
Hroth proteins contain a single copy of this motif (see      views see Holland et al. 1994; Holland and Garcia-Fer-
fig. 2). The sea urchin Otx protein (named SpOtx or             `
                                                             nandez 1996; Sharman and Holland 1996; Sidow 1996).
HpOtx, depending on species; Gan et al. 1995; Saka-          Hence, the Otx genes can now be added to the growing
moto et al. 1997) also has a similar sequence near its       list. It has been proposed by several authors that dupli-
C-terminus (not shown). We could not identify similar-       cation of genes and gene networks during vertebrate
ity in arthropod otd proteins. The most parsimonious         evolution may have contributed to, or permitted, the in-
explanation is that this motif arose in the deuterostome     creased complexity of the vertebrate body plan. For ex-
lineage after it split from the coelomate protostome lin-    ample, new genes arising from duplication may have
eage; the ancestral single-copy state for the motif is re-   permitted the evolution of a more elaborate brain, the
tained in echinoderms, ascidians, and amphioxus. Tan-        skeleton, extensive mesodermal organogenesis, and the
dem duplication of this region occurred in the vertebrate    neural crest-derived tissues. With regard to the Otx
lineage after splitting the vertebrate and cephalochordate   genes, however, there is no simple relation between du-
lineages. Furthermore, we note that the first motif (Do-      plication of Otx genes and the origin of new brain
main A) of Otx1 proteins shows a higher degree of se-        regions. Traditional theories supported a ‘‘simple’’ or
quence conservation with Domain A of Otx2 proteins           rudimentary brain in ancestral chordates, but such a no-
than it does with the second motif (Domain B) of the         tion is rapidly being eroded with molecular and mor-
same gene, and vice versa. This suggests that the inter-     phological evidence of distinct forebrain, midbrain, and
nal tandem duplication event predated duplication of the     hindbrain regions in amphioxus (Holland et al. 1992,
whole Otx gene. This is further strong evidence that         1996; Lacalli, Holland, and West 1994; Lacalli 1996;
there was just a single Otx gene in the ancestral chordate   Williams and Holland 1996).
which underwent gene duplication in the vertebrates af-             If amphioxus and vertebrates possess the same ma-
ter the splitting of the vertebrate and cephalochordate      jor brain regions, what might be the evolutionary sig-
lineages.                                                    nificance of Otx gene duplication? Comparison of gene
                                                             expression patterns in ascidia, amphioxus, and verte-
Discussion                                                   brates is informative. The ascidian Otx gene, Hroth
                                                             (Wada et al. 1996), has an early expression phase in
     Members of the Otx gene family are intimately in-       involuting anterior mesendoderm and later expression in
volved in the specification and patterning of anterior        the sensory vesicle, containing sensory and pigment
neural and head structures in vertebrates and arthropods.    cells. This pattern is similar to the expression pattern of
They are also expressed in other regions, however, such      AmphiOtx apart from the fine detail of the later expres-
as the ventral midline of arthropods and the anterior gut    sion in anterior neural structures (Williams and Holland
of vertebrates and amphioxus. This raises questions          1996). Taken together with our phylogenetic analyses,
about the role of the ancestral Otx gene and whether         these expression patterns point to an ancestral role for
functional or regulatory changes were associated with        chordate Otx in patterning the anterior mesendoderm
duplication of an ancestral Otx gene. To address these       during early development and in the involvement of this
questions, expression and function must be viewed in         tissue in the induction of specific neural regions in the
the context of a robust phylogeny of the gene family,        overlying ectoderm. This is followed by a later specific
including all gene duplication events. To facilitate this,   patterning role in the anterior CNS. What happened to
we cloned a homolog of the Otx gene family from am-          these roles after Otx gene duplication? The mouse Otx2
phioxus, the probable sister group of the vertebrates.       gene retains the widespread early and late developmen-
     We isolated a single Otx-related gene from amphi-       tal expression domains; interestingly, its paralog, Otx1,
oxus, designated AmphiOtx. This gene is clearly assign-      has much more localized expression, both spatially and
able to the Otx gene family by its conserved homeobox        temporally. This suggests that following gene duplica-
606    Williams and Holland

tion, the regulation of Otx2 has altered little from the      at the C-terminus. This motif is included within Domain
ancestral state, while Otx1 gene expression has diverged.     A and Domain B of vertebrate Otx genes, noted in this
In this context, it is also worth noting that the rate of     study, and has been termed the OTX tail.
sequence divergence over alignable regions is higher in
Otx1-like genes than in Otx2 genes (fig. 4), suggesting        Acknowledgments
that structural divergence of the protein has accompa-
nied regulatory divergence. We suggest, therefore, that            We thank Jim Langeland for the cDNA library,
it is the unique roles of Otx1 (those not shared by Otx2)     Max Telford for advice on constructing molecular phy-
that provide insight into the evolutionary consequence        logenies, and Norbert Perrimon for the Drosophila otd
of Otx gene duplication. Targeted mutagenesis of mouse        cDNA clone. This work was supported by BBSRC grant
Otx1 results in a reduction in the size of the cortex and     number G04203.
an increase in the size of the cerebellum (Acampora et
al. 1996). This is not a simple deletion of structures, but   LITERATURE CITED
alteration of their organization. We suggest, therefore,
that after duplication of the Otx gene in the vertebrate      ABOUHEIF, E., M. AKAM, W. J. DICKINSON, P. W. H. HOLLAND,
                                                                 A. MEYER, N. H. PATEL, R. A. RAFF, V. L. ROTH, and G.
lineage, Otx2 largely retained the ancestral roles, while        A. WRAY. 1997. Homology and developmental genes.
Otx1 was co-opted for more complex interactions with             Trends Genet. 13:432–433.
other genes, both temporally and spatially. This allowed      ACAMPORA, D., S. MAZAN, Y. LALLEMAND, V. AVANTAGGIATO,
an increase in the potential to specify discrete/novel neu-      M. MAURY, A. SIMEONE, and P. BRULET. 1995. Forebrain
ronal cell groups, particularly in the cerebellum and ce-        and midbrain regions are deleted in Otx2 / mutants due to
rebral cortex.                                                   defective neurectoderm specification during gastrulation.
       To make inferences about the roles of Otx genes           Development 121:3279–3290.
earlier in evolution, before the emergence of chordates,      ACAMPORA, D., S. MAZAN, V. AVANTAGGIATO, P. BARONE, F.
                                                                 TUORTO, Y. LALLEMAND, P. BRULET, and A. SIMONE. 1996.
we must include other phyla in the comparison. Two
otd-like genes have been cloned recently from the beetle         Epilepsy and brain abnormalities in mice lacking the Otx1
                                                                 gene. Nat. Genet. 14:218–222.
Tribolium castaneum (Li et al. 1996), denoted Tc otd-1        ANG, S.-L., O. JIN, M. RHINN, N. DAIGLE, L. STEVENSON, and
and Tc otd-2. Tc otd-1 has a spatiotemporal expression           J. ROSSANT. 1996. A targeted mouse Otx2 mutation leads
pattern which is similar to that of Drosophila otd. A            to severe defects in gastrulation and formation of axial me-
‘‘cap’’ of Tc otd-1 expression, which includes the an-           soderm and to deletion of rostral brain. Development 122:
terior third of the embryo, is found in the early blasto-        243–252.
derm, but expression quickly fades from the anterior          FINKELSTEIN, R., D. SMOUSE, T. M. CAPACI, A. C. SPRADLING,
pole during cellularization. Expression remains in a             and N. PERRIMON. 1990. The orthodenticle gene encodes a
large preantennal region of the developing head lobes            novel homeo domain protein involved in the development
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