1 Department of Zoology, University of Oxford, South Parks Road, Oxford OX1 3PS,
UK
2 Department of Hematology and Oncology, Graduate School of Medicine, University
of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan
* Author for correspondence (e-mail: peter.holland{at}zoo.ox.ac.uk)
Accepted 1 April 2004
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SUMMARY |
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Key words: Dmbx1, AmphiDmbx, CiDmbx, Ciona, Chordate, MHB, Isthmus
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Introduction |
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Comparison of gene expression patterns and developmental anatomy between
vertebrates, amphioxus and ascidians has yielded some surprises concerning the
evolutionary history of brain pattering. For example, both ascidians and
vertebrates show a basic tripartite ground plan along the anteroposterior axis
of the dorsal nerve cord, comprising an anterior region expressing Otx family
homeobox genes (marking the forebrain and midbrain of vertebrates), a central
region expressing Pax2/5/8 genes (marking the midbrain-hindbrain boundary of
vertebrates) and a Hox-expressing region (hindbrain and spinal cord of
vertebrates). These zones do not, of course, correspond to the classically
defined anatomical divisions of fore-, mid- and hindbrain of vertebrates, but
instead reveal the probable ancestral ground plan upon which chordate brain
development is based (Wada et al.,
1998). Very recently, a similar tripartite pattern of gene
expression has been reported for hemichordates
(Lowe et al., 2003
) and
arthropods (Hirth et al.,
2003
), suggesting that this ground plan may be even older even
than chordate origins.
There are several unresolved issues concerning the evolution of the
tripartite neural tube, particularly concerning the central zone marked by
Pax2/5/8 gene expression. In vertebrates, this zone is the site of the
midbrain-hindbrain boundary organiser (MHB organiser or isthmic organiser,
IsO). The MHB organiser was initially identified through transplantation
experiments in chick embryos (Gardner and
Barald, 1991; Martinez and
Alvarado-Mallart, 1990
;
Martinez et al., 1991
). These
experiments suggest that the vertebrate MHB acts an organising centre, with
inductive influences both anterior and posterior of its location. In
urochordates, a homologous region was first noted in Halocynthia as a
stripe of Pax2/5/8 expression, similar to Pax gene expression in the
vertebrate MHB (Wada et al.,
1998
). An FGF8/17/18 gene is expressed immediately caudal to this
stripe in Ciona, suggesting that ascidian embryos probably do have
organiser activity in this region within the visceral ganglion
(Imai et al., 2002
); however,
this organiser activity has not been tested functionally.
One important issue of uncertainty surrounding the MHB region concerns
amphioxus. Unlike vertebrates and ascidians, the cephalochordate amphioxus
does not show Pax2/5/8 expression posterior to Otx gene expression and
anterior to the Hox-expressing region
(Kozmik et al., 1999).
Furthermore, En and Wnt1 genes are also not expressed in
this region in amphioxus, despite their expression and function at the MHB in
vertebrates (Holland et al.,
1997
; Holland et al.,
2000
). Amphioxus, therefore, lacks the MHB. Taking into account
the comparative data from ascidians and hemichordates, it is most parsimonious
to conclude that tripartite regionalisation is an ancient character of the
vertebrate neural tube, and that secondary modification has occurred on the
cephalochordate lineage (Wada et al.,
1998
; Williams and Holland,
1998
). Such modifications might have occurred in concert with the
unusual rostral extension of the notochord in amphioxus, extending beyond the
tip of the cerebral vesicle.
A related issue of importance concerns the origin of the midbrain. In
vertebrates, the Otx-expressing domain encompasses both forebrain and
midbrain; consequently, comparative data on Otx gene expression cannot reveal
when the distinction between these two structures arose. Recently, we and
others reported cloning and expression of a novel PRD-class homeobox gene in
the mouse, Dmbx1 (diencephalon/mesencephalon-expressed brain homeobox
gene 1) (Broccoli et al., 2002;
Gogoi et al., 2002
;
Miyamoto et al., 2002
;
Ohtoshi et al., 2002
;
Takahashi et al., 2002
;
Zhang et al., 2002
).
Orthologues of this gene have also been reported from zebrafish
(Kawahara et al., 2002
),
chicken (Gogoi et al., 2002
)
and human (Zhang et al.,
2002
), although not from any invertebrate. The human and mouse
genes are also referred to as Otx3, which is erroneous as the gene is
not part of the Otx gene family. In all studies, a predominant site of
Dmbx1 expression is the prospective midbrain at early embryonic
stages. Indeed, Dmbx1 gene inactivation using morpholino antisense
oligonucleotide results in substantial reduction in the size of tectum and
eyes in zebrafish (Kawahara et al.,
2002
). These results suggest that Dmbx1 is a useful
marker for the presumptive midbrain before the MHB organiser is
established.
To clarify how midbrain patterning emerged during chordate evolution, we have cloned orthologues of Dmbx1 gene from amphioxus and ascidians. We report their full sequences, phylogeny, exon-intron organisation and spatiotemporal expression patterns. There are both similarities and differences in expression pattern between ascidians and vertebrates, but the expression in amphioxus is strikingly different. These data further strengthen the case for homology between ascidian and vertebrate tripartite organisations, with secondary modification in amphioxus. Furthermore, the data suggest that midbrain development is a novel character that evolved specifically in the vertebrate lineage, superimposed onto the ancestral tripartite organisation.
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Materials and methods |
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Isolation of the amphioxus Dmbx gene
For cloning the amphioxus homologue of the Dmbx1 gene, nested
degenerate oligonucleotide primers were designed to amplify a fragment of 129
bp conserved between vertebrate Dmbx1 genes. Initial primer sequences were:
F-out, 5'-CAACGTCGGAG(CT)(AC)GNACNGC-3 with SO2T
5'-C(GT)NC(GT)-(AG)TT(CT)TT(AG)AACCA-3'; the nested reaction used
F-mid, 5'-ATGCA(AG)CTNGANGCN(CT)TNGA-3' with SO2T. A cDNA library
from B. floridae embryos (reverse-transcribed with both random and
oligo dT primers) (Langeland et al.,
1998) was used as a PCR template. After cloning of the amplified
band, five recombinant clones were sequenced, of which three showed high
sequence similarity to vertebrate Dmbx1 genes. Screening of the B.
floridae cDNA library with this PCR-derived clone yielded one cDNA clone
including a 2.66 kb EcoRI fragment. This fragment was fully sequenced
on both strands.
Isolation of the ascidian Dmbx gene
We identified a presumptive orthologue of Dmbx1 through tblastn
searches of the Ciona intestinalis genome assembly v. 1.0
(Dehal et al., 2002), accessed
at the Joint Genome Institute server
(http://genome.jgi-psf.org/ciona4/ciona4.home.html).
It was not possible, however, to predict and assemble the complete gene
sequence from the surrounding genomic sequence information alone, without cDNA
information. To obtain this, we first designed four specific PCR primers
(For1, For2, Rev3 and Rev4) to amplify a fragment of 319 bp from the 5'
part of the coding region of the predicted gene. Nested PCR was performed
using primers For1 and Rev4, followed by For2 and Rev3, using as a template
C. intestinalis cDNA reverse-transcribed from mRNA extracted from 5
hour embryos. After cloning of the amplified band, four recombinant clones
were sequenced, all of which showed high sequence similarity to vertebrate
Dmbx1 genes. To clone the 3' half of the gene, sequence-specific primers
For2 and For3 were used in conjunction with vector primers M13 Forward and T7
to amplify from a C. intestinalis cDNA library constructed in
pBluescript (kindly provided by Dr Patrick Lemaire, Marseille, France). After
PCR amplification and cloning of the amplified band, two recombinant clones
were sequenced, both with high sequence similarity to vertebrate Dmbx1 genes.
After combining the sequence information from these two experiments, a new
sequence specific primer NewRev1 was designed near the 3' end of the
gene, and used in conjunction with For1 to amplify a band covering most of the
coding sequence, from a mixture of 6 hour embryo cDNA and the cDNA library.
This was cloned and two recombinants completely sequenced to verify that the
5' and 3' clones are naturally contiguous in mRNA. The sequence
was compatible with all sequence information obtained from the preceding
experiments. Primer sequences were: For1,
5'-ATGAATTATTATGACGCAAT-3'; For2,
5'-TCGTGCAATGTCAGTGTTCA-3'; For3,
5'-CTCTGGCAGATTTAATACTC-3'; Rev3,
5'-CAACATTCTCTGTTGTTTTC-3'; Rev4,
5'-CTGTTGTTTTCGATATTTTG-3'; NewRev1,
5'-AATTGAAGATTCCAAGGTTG-3'.
Sequence comparisons and molecular phylogenetic analyses
Deduced amino acid sequences were aligned using ClustalX
(Thompson et al., 1997) and
edited using GeneDoc (Nicholas et al.,
1997
) to remove regions of ambiguous alignment. Phylogenetic
analyses of amino acid sequences were performed using the neighbour-joining
method implemented in ClustalX, with outputs displayed using TreeView
(Page, 1996
). For the analysis
of PRD-class homeobox genes, we restricted the analysis to the homeodomain to
maximise representation of PRD class genes. Confidence in the phylogeny was
assessed by bootstrap re-sampling of the data.
Genomic organisation of Dmbx genes
A genomic clone of the amphioxus Dmbx gene was obtained from amphioxus
cosmid library MPMGc117 (distributed by the Resource Centre and Primary
Database,
www.rzpd.de)
by screening using the cDNA clone under high stringency conditions. Cosmid
clone MPMGc117 G1048 was found to contain the amphioxus Dmbx gene, as
confirmed by direct sequencing. Exon-intron organisation was determined by
direct sequencing of the cosmid clone, using primers based on cDNA sequence.
Exon-intron organisation of the ascidian Dmbx gene was determined by
comparison of the cDNA sequence with the Ciona intestinalis genome
assembly v. 1.0 (Dehal et al., 2000).
Whole-mount in situ hybridisation and sectioning
In situ hybridisation of whole-mount specimens were carried out as
described by Holland (Holland,
1999) for amphioxus, and by Mazet et al.
(Mazet et al., 2003
) for
ascidians. Probes for amphioxus and ascidian Dmbx were synthesised by in vitro
transcription using a DIG RNA Labeling Mix (Roche), following the supplier's
instructions. The amphioxus Dmbx template was a 1.1 kb cDNA subclone in
pBluescript, containing the full open reading frame, linearised with
NotI. The ascidian Dmbx template was a 681 bp cDNA subclone in pGEM-T
Easy, covering most of the open reading frame, linearised with SalI.
To enable double staining with other genes, we identified cDNA clones of
Ci-Pax2/5/8-A, Ci-Fgf8/17/18 and CiHox3 from a
Ciona EST collection assembled at Kyoto University by Professor N.
Satoh and collaborators
(http://ghost.zool.kyoto-u.ac.jp/indexr1.html).
Identity of the clones (Gene Collection ID: R1CiGC01B13 for
Ci-Pax2/5/8-A, R1CiGC28f14 for Ci-Fgf8/17/18 and R1CiGC02c13
for CiHox3) was verified by sequencing and comparison to published
sequence (Satou et al., 2002
).
A Ci-Pax2/5/8-A riboprobe was synthesised with a fluorescein RNA
Labeling Mix (Roche), and double staining performed
(Mazet et al., 2003
) using
this probe in conjunction with DIG-labelled Ciona Dmbx.
Ci-Fgf8/17/18 and a CiHox3 riboprobes were labelled with DIG
and double staining performed using fluorescein-labelled Ciona Dmbx.
Briefly, specimens were simultaneously hybridised with the DIG and
fluorescein-labelled probes, before washing and sequential detection of the
two labels. First, alkaline phosphatase-conjugated anti-fluorescein antibody
was used, followed by staining with Fast-Red (Roche). Specimens with definite
signal were treated with 0.1 M glycine-HCl (pH2.2), 0.1% Tween 20 for 10
minutes at room temperature to inactivate the first alkaline phosphatase,
washed, incubated with alkaline phosphatase-conjugated anti-digoxigenin
antibody and stained blue with NBT-BCIP (Roche). After being photographed as
whole mounts, amphioxus embryos were counterstained pink in 1% Ponceau S in 1%
aqueous acetic acid, dehydrated in ethanol, embedded in LR White resin (TAAB)
and prepared as 7.0 µm sections.
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Results |
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Conservation of Dmbx proteins
The homeodomain is highly conserved between amphioxus and human Dmbx genes
(91.7% identity; Fig. 1),
although rather divergent in Ciona (78.3% with human;
Fig. 1). Unusually, these
levels of similarity are not confined to the homeodomain, but are also seen in
the N-terminal part of the protein. AmphiDmbx and human DMBX1 proteins are 88%
identical from the beginning of the ORF to the end of homeodomain, while
CiDmbx and human DMBX1 show 47% identity over the same region. The C-terminal
half of the protein is less conserved and particularly divergent in
Ciona. The AmphiDmbx protein does have several scattered conserved
residues in this region, plus a well conserved OAR domain
(Furukawa et al., 1997) near
the C terminus. The OAR domain was first described in the mouse Rax protein,
and is seen in several aristaless-related proteins and some other PRD-class
homeodomain proteins. It functions as an intra-molecular switch to regulate
the activity of the transcription factor
(Brouwer et al., 2003
).
One conserved motif of interest near the N terminus (data not shown), is a
six amino acid stretch [LAD(IL)IL]. This motif is similar to the GEH/en-1
consensus region (FSIDNIL) seen in several homeodomain proteins, known to
function as a repressor domain (Mailhos et
al., 1998). It is relevant to note that vertebrate Dmbx1
genes are also reported to function as repressors
(Kawahara et al., 2002
;
Zhang et al., 2002
).
Interestingly, an alternatively spliced form of Dmbx1 in human,
mouse, chicken and zebrafish has a five amino acid insertion (GCTFQ) within
this motif. The two splice variants may interact with different partner
proteins, and have different repressor activities.
Evolutionary history of Dmbx genes
To examine the phylogenetic distribution of Dmbx gene family, we conducted
blast searches of GenBank. We detected no Drosophila, Anopheles or
nematode genes that can be classified in the Dmbx gene family. Indeed, we
detected only one gene that could be an invertebrate Dmbx gene (in addition to
the amphioxus and ascidian genes reported here). This is the Hydra
vulgaris homeobox gene manacle (GenBank Accession Number
AF126249), which has regions of high sequence similarity to CiDmbx
(71.7% identity over the homeodomain; Fig.
1). The manacle cDNA sequence was deposited on GenBank by
D. M. Bridge and R. E. Steele (unpublished); expression of this gene in
differentiating basal disc ectoderm is described by Bridge et al.
(Bridge et al., 2000). This
finding reveals that the Dmbx gene family originated before the divergence of
the cnidarian and bilaterian lineages, very early in animal evolution.
The homeobox gene superfamily can be divided on the basis of homeodomain
sequence into the ANTP class, the PRD class and several divergent lineages
such as LIM and TALE (Galliot et al.,
1999). The Dmbx homeobox gene family is clearly a member of the
PRD class. For example, the human DMBX1 homeodomain has 60-65% identity with
human OTX1, PTX1 and GSC (members of the PRD class), but only 38-42% identity
with human HOXA1, EN1 and MSX1 (members of the ANTP class).
We conducted phylogenetic analysis using a diversity of PRD-class homeodomains. The analysis clearly groups manacle, AmphiDmbx, CiDmbx and vertebrate Dmbx genes together, confirming that they form a distinct gene family (Fig. 2A). They are not part of the Otx gene family. The most closely related gene families are probably Ptx, Otx and Gsc, although low support values make this conclusion tentative. This analysis is based on only the homeodomain, so should not be used to infer precise relationships, particularly within the Dmbx gene family. To address this, we undertook a second phylogenetic analysis using the full amino acid sequences of only Dmbx genes (other PRD class genes cannot be included), assigning manacle as the outgroup. This gave a gene phylogeny of CiDmbx, AmphiDmbx and vertebrates Dmbx genes that is entirely consistent with known relationships between the seven species (Fig. 2B). The implication is that these genes are orthologues, and that we are not sampling paralogous genes from a larger gene family.
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|
Expression of the AmphiDmbx gene
Using whole-mount in situ hybridisation and sectioning, we examined the
spatiotemporal pattern of AmphiDmbx expression during amphioxus
development. No expression was detected in blastulae, early gastrulae or mid
gastrulae (data not shown). At the end of the gastrula stage,
AmphiDmbx expression was first detected in the anterior mesendoderm
(Fig. 4A). This is also clearly
evident at the mid neurula stage (13 hours post fertilisation), when
expression is seen to be dorsal within this tissue
(Fig. 4B). At the mid neurula
stage (24 hours) and late neurula stage (36 hours), the anterior mesendoderm
expression extends rostrally into Hatschek's diverticula
(Fig. 4D-G). Expression in the
most rostral tip of the notochord is also evident at 36 hours, although this
expression does not overlie the pharyngeal expression
(Fig. 4E,G). Expression in
anterior endoderm persists at least for a few days, and is clearly evident in
swimming larvae (60 hours; Fig.
4H). At this stage, the expressing cells include part of the
club-shaped gland, endostyle, pharyngeal endoderm and the preoral ciliated pit
that develops from Hatschek's left diverticulum. Expression in notochord has
faded at this stage. No expression is detected in the neural tube at any stage
of development examined.
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Discussion |
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The latter interpretation is the one we proposed when reporting the cloning
of mouse Dmbx1 (Takahashi et al.,
2002). Our evidence at the time was circumstantial and based
primarily on molecular phylogenetics of homeobox genes. The cloning of
definitive Dmbx genes from amphioxus and ascidians, reported here, has
confirmed this deduction. Furthermore, our analyses show that the
Hydra homeobox gene, manacle
(Bridge et al., 2000
), also
belongs to Dmbx gene family. These findings demonstrate that the Dmbx genes
form a distinct, ancient, homeobox gene family. Members of this gene family
have been secondarily lost in the evolutionary lineages leading to D.
melanogaster, C. elegans and Anopheles gambiae.
Comparison of ascidian, amphioxus and vertebrate Dmbx genes reveals some
unusual patterns of conservation. First, although exon-intron organisation of
the gene is well conserved between human, mouse, pufferfish and amphioxus
(with an extra intron in amphioxus), the ascidian CiDmbx gene has a
very different genomic organisation. CiDmbx has eight exons, compared
with four in human, most probably the result of four additional intron
insertions. This change in gene organisation is mirrored by high levels of
sequence divergence within the Ciona gene. We also detected a large
amount of sequence polymorphism within the Ciona CiDmbx gene,
including numerous substitutions that cause variation in the encoded protein
sequence between alleles, especially in the C-terminal half of the protein.
These findings provide another example of the high rate of sequence evolution
reported for the ascidian genome (Ferrier
and Holland, 2002; Holland and
Gibson-Brown, 2003
).
Evolutionary history of chordate brain patterning
The mouse Dmbx1 gene shows a particularly interesting expression
pattern in the context of the tripartite organisation of the chordate neural
tube. The earliest expression site is the presumptive midbrain (at day E7.5 to
E8.5), with expression extending rostrally into the diencephalon by E10.
Caudally, however, this expression has a very clear spatial limit at the MHB
(Broccoli et al., 2002;
Gogoi et al., 2002
;
Miyamoto et al., 2002
;
Ohtoshi et al., 2002
;
Takahashi et al., 2002
;
Zhang et al., 2002
).
Dmbx1 transcripts also appear in the hindbrain, where they resolve
into bilateral, longitudinal stripes by E11 (possibly in specific neuronal
populations), but the MHB region is strikingly devoid of Dmbx1
expression (Takahashi et al.,
2002
). We hypothesise that Dmbx1 expression is activated
at a distance from the MHB, but repressed by the highest levels of FGF
activity at the MHB organiser; indirect support for this comes from the
observation that Dmbx1 is also expressed immediately subjacent to the
apical ectodermal ridge in limb development
(Takahashi et al., 2002
).
We reasoned that comparison with Dmbx gene expression in amphioxus and
ascidian development would be informative in two key respects. First, the
apparent absence of an MHB in amphioxus (or at least absence of a zone of
Pax2/5/8, En and Wnt1 activity between the Otx and Hox
domains) (Kozmik et al., 1999;
Holland et al., 1997
;
Holland et al., 2000
), allows
a test of the relation between Dmbx expression and the MHB. In short, we
expect a rather different expression pattern in amphioxus. Second, early
expression of Dmbx1 is a useful midbrain marker, dividing the
Otx-positive zone into anterior (forebrain) and posterior (midbrain) regions.
We were curious to see whether ascidian embryos also showed this expression
pattern, which may help resolve the origins of midbrain development in
chordate evolution. These comparisons are facilitated by the fact that
Dmbx1 is not part of a gene family in mouse; we can be sure we are
comparing the expression of single, directly orthologous genes.
In amphioxus, AmphiDmbx gene expression is strongest in the anterior endoderm, notochord, endostyle and part of the club-shaped gland. We did not detect any AmphiDmbx transcripts in the dorsal nerve cord of amphioxus at any stage of development. This finding is consistent with the suggestion, made from other gene expression data, that the anterior nerve cord of amphioxus has been secondarily modified from the ancestral tripartite organisation. Amphioxus has Otx and Hox-expressing neural domains, in regions homologues to those of vertebrates, but the intervening zone (equivalent to the MHB in vertebrates) is missing. When considered alongside the Ciona data (discussed below), we conclude that Dmbx gene expression in the nerve cord has been secondarily lost in the cephalochordate lineage, alongside loss of the MHB region (Fig. 6).
|
Data from ascidians, Drosophila, hemichordates and vertebrates now
concur that tripartite regionalisation of the neural tube is very ancient, and
that the vertebrate fore/midbrain, MHB and hindbrain/spinal cord evolved from
this tripartite organisation. In ascidians, the rostral (Otx-expressing) and
caudal (Hox-expressing) zones have an intervening zone expressing a Pax2/5/8
gene (Wada et al., 1998) and
an En family homeobox gene (Imai et al.,
2002
). In hemichordates, the central zone is marked at least by
En (Lowe et al.,
2003
). In Drosophila, Poxn and Pax2 (Pax-2/5/8
family) are each expressed at the interface of otd (Otx family) and
unpg (Gbx family) genes, anterior to the Hox-expressing region
(Hirth et al., 2003
). However,
possession of the tripartite organisation is not sufficient to indicate the
existence of organiser activity; it could simply mark three distinct spatial
regions. In the Drosophila embryo, no obvious brain phenotypes are
described after mutational inactivation of fly homologues of the key genes of
the vertebrate MHB, such as pax2 and Poxn (Pax-2/5/8
homologues) or branchless (Fgf homologue)
(Hirth et al., 2003
). This
suggests that although Drosophila has the tripartite organisation, it
lacks MHB organiser activity; the former preceded the latter in evolution.
Turning to chordates, it is likely that MHB organiser activity had emerged
by the time that the ascidian and vertebrate lineages diverged. The Ciona
Ci-Fgf8/17/18 gene is expressed immediately posterior to Pax2/5/8
(marking the middle of the three regions), in a manner reminiscent of
Fgf8 expression and function in the vertebrate MHB organiser. There
are subtle differences, notably that in Ciona Ci-Fgf8/17/18, CiDmbx
and CiHox3 transcripts are co-localised posterior to Pax2/5/8, at the
visceral ganglion (Imai et al.,
2002). In vertebrates, Fgf8 gene expression is caudal to
the MHB isthmus, but slightly rostral to Dmbx1 and Hox gene
expression which each start in rhomobomere 2
(Irving and Mason, 2000
;
Takahashi et al., 2002
). This
difference might reflect a recently evolved inhibitory effect of FGF8 activity
on Hox gene expression in vertebrates (see
Irving and Mason, 2000
). Until
now, there is no direct evidence for the Ciona FGF8/17/18 protein
conferring organiser activity. However, if ascidians do have MHB organiser
activity, as these data suggest, then it is very interesting that
Ciona lacks Dmbx expression anterior to the Pax2/5/8-positive cells.
The implication is that the evolution of the MHB organiser preceded the
evolution of a distinct midbrain.
In summary, comparative data indicate that a tripartite ground plan for brain development existed in the common ancestor of the Bilateria. At some point in the evolution of vertebrates, probably before the divergence of ascidians and vertebrates, the central region of this tripartite plan acquired MHB organiser activity, which acts to refine the developmental patterning of the adjacent regions. At the same time, these regions must have obtained competency to respond to signals from the organiser. The lineage leading to amphioxus, which diverged a little later, most probably lost the MHB region, with concomitant modification of adjacent tissues. In the evolutionary lineage leading to vertebrates, we conclude that the zone immediately rostral to the MHB organiser became further subdivided, to include a specific midbrain region marked by Dmbx1 gene expression.
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ACKNOWLEDGMENTS |
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