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First published online November 9, 2007
doi: 10.1242/10.1242/dev.010173
1 Sars International Centre for Marine Molecular Biology, University of Bergen,
Thormøhlensgt. 55, 5008 Bergen, Norway.
2 Adolf-Butenandt-Institut, Ludwig-Maximilians-Universität, Schillerstrase
44, D-80336 München, Germany.
3 IBDM/LGPD Case 907, Campus de Luminy, 13288 Marseille, France.
4 Departamento de Bioquimica y Biologia Molecular, Facultad de Ciencias
Biológicas, Universidad de Concepción, Casilla 160-C,
Concepción, Chile.
5 Developmental Biology Center and Developmental and Cell Biology Department,
University of California at Irvine, Irvine, CA 92697, USA.
* Authors for correspondence (e-mails: ralph.rupp{at}med.uni-muenchen.de; ulrich.technau{at}sars.uib.no)
Accepted 4 September 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Brachyury, Hydra, Gene duplication, Head formation, Regeneration, Mesoderm, Evolution, Structure function analysis, Xenopus, Animal cap
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Kortschak et al., 2003;
Technau et al., 2005;
Miller et al., 2005;
Putnam et al., 2007). In
contrast to the apparent morphological simplicity of the Cnidarians it appears
that there is no significant change in the gene set between basal cnidarians
and vertebrates (Technau et al.,
2005; Putnam et al.,
2007). One factor that adds to the complexity of cnidarian genomes
is the relatively high number of independent gene duplications
(Technau et al., 2005;
Chourrout et al., 2006). Gene
duplications are thought to be one of the major driving forces of evolution.
Although many of the duplicated genes are predicted to end up as pseudogenes,
some contribute significantly to the evolution of developmental processes by
establishing novel interactions and functions. In principle, a fully
functional copy of a gene reduces the selection pressure significantly,
allowing for rapid divergence of one or both copies of the gene
(Ohno, 1970;
Cooke et al., 1997;
Krakauer and Nowak, 1999).
Comparisons of paralogous genes and dominance of mutations in yeast and humans
suggest that haploinsufficient genes, whose loss-of-function alleles are
dominant over wild-type alleles, are more likely to be fixed after gene
duplication than haplosufficient (i.e. dominant wild-type) genes
(Kondrashov and Koonin, 2004).
Interestingly, haploinsufficient genes include predominantly developmental
regulators, structural and regulatory proteins and transcription
regulators.
Predictions surrounding the fate of duplicate genes over long evolutionary
time scales have been made in theoretical studies
(Cooke et al., 1997;
Force et al., 1999). The
Duplication-Degeneration-Complementation model of Force and colleagues focuses
on the evolution of cis-regulatory regions after the duplication event,
although it does not explicitly exclude a divergence of the coding sequence
(Lynch and Force, 2000). The
detailed experimental analysis of paralogous genes and the degree of
conservation and diversification of functions might therefore provide insight
into metazoan evolution. Furthermore, in the case of important developmental
pathways, paralogous genes might contribute to the evolution of animal body
plans.
One of these important developmental genes encodes the T-box transcription
factor Brachyury (Herrmann et
al., 1990). Brachyury has a conserved role in mesoderm
differentiation, and elongation of the posterior body axis in all vertebrates
(for reviews, see Smith, 1997;
Smith, 1999;
Naiche et al., 2005).
Comparison of basal deuterostome and protostome larvae, as well as
diploblastic cnidarians, indicates that Brachyury has an ancestral
function in defining the blastopore (Arendt
et al., 2001; Scholz and
Technau, 2003) (for a review, see
Technau, 2001), although the
precise cellular function can vary in the different animal lineages (e.g.
Wilkinson et al., 1990;
Gross and McClay, 2001;
Lartillot et al., 2002).
Interference with Brachyury function in vertebrates inhibits
convergent extension, causes apoptosis, and reveals separate requirements in
the Fibroblast growth factor (FGF)- and Activin-signalling pathways
(Conlon and Smith, 1999).
Although Brachyury is considered an early panmesodermal marker in
vertebrates, this `mesodermal' gene also occurs in diploblasts, as first
reported in the freshwater cnidarian Hydra
(Technau and Bode, 1999). The
Hydra Brachyury homologue HyBra1 is expressed in the
hypostome, the most apical part of the polyp, between the tentacle ring and
the mouth opening (Technau and Bode,
1999). The hypostome is the location of the head organiser of the
animal (Browne, 1909;
Broun and Bode, 2002), which is
considered equivalent to the Spemann organiser in amphibians
(Spemann und Mangold, 1924).
Comparative evolutionary studies in basal cnidarians indicate that the oral
end of the Hydra polyp derives from the blastopore
(Scholz and Technau, 2003).
Under all developmental circumstances, i.e. bud formation, head regeneration,
reaggregation and embryogenesis, HyBra1 is expressed very early at
the site of future hypostome formation, suggesting an early role in head
organiser/head formation in Hydra
(Technau and Bode, 1999;
Technau et al., 2000;
Broun and Bode, 2002).
T-Box genes have never been reported from unicellular organisms. It is
therefore likely that this class of developmental control genes evolved with
multicellularity. Two T-box genes, tbx2 and Brachyury, have
been isolated from sponges (Adell et al.,
2003; Manuel et al.,
2004). T-box genes fall into a number of subfamilies with
Brachyury as the most distinct subfamily
(Papaioannou and Silver, 1998;
Papaioannou, 2001;
Wilson and Conlon, 2002). Most
animals appear to have only one Brachyury gene; however, a few
organisms, including vertebrates, have two or more paralogous
Brachyury genes. Where studied, these paralogues are highly similar
in sequence and have mostly overlapping expression domains
(Strong et al., 2000;
Knezevic et al., 1997;
Hayata et al., 1999),
suggesting rather recent gene duplication events.
In this paper, we report the isolation of a second Hydra Brachyury gene, which appears to be the result of an ancient gene duplication event. Our phylogenetic analysis suggests that the duplication event most likely occurred after the divergence of cnidarians. We show that HyBra1 and HyBra2 occupy distinct expression domains in the hypostome of the polyp, and show very different dynamics of expression during head regeneration. This indicates an evolution of cis-regulatory elements. To investigate the impact of coding-sequence evolution between the two paralogues, we tested the function of both Hydra genes in a heterologous assay system: the Xenopus animal caps. This assay revealed strikingly different inductive capacities for HyBra1 and HyBra2. Domain-swapping experiments indicate that their different activities are defined largely by their divergent C-terminal domains. Our data show that the Hydra Brachyury paralogues have undergone a mixture of subfunctionalisation and neofunctionalisation and the evolution of HyBra gene function occurred both at the cis-regulatory and the protein levels.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Xenopus embryos were in vitro fertilised,
de-jellied and cultured by standard methods
(Sive et al., 1989). At the
four-cell stage, each cell was microinjected with 2.5 nl volume into the
animal hemisphere. RNA doses ranged from 0.1 to 1.0 ng/embryo, and were
adjusted to result in roughly comparable protein amounts, as judged by western
blot analysis of embryos injected with Myc-tagged protein variants. Animal
caps were dissected using Dumont N°5 forceps between stages 8.5 and 9
(Nieuwkoop and Faber, 1967)
and cultivated at 23°C in 0.5x MBS containing 10 µg/ml Gentamycin
until stage 36 (hatching).
Cloning of HyBra1 and HyBra2
Fragments of the T-box were cloned by PCR using degenerate primers (forward
5'-TTYGGNGMNCAYTGGATG-3' and reverse
5'-RAANSCYTTNGCRAANGG-3') at low annealing temperatures.
Random-primed probes of these HyBra1 and HyBra2 T-box
fragments were used to screen a Lambda-Zap cDNA library (Stratagene) using
standard protocols (Sambrook et al.,
1989). A full-length clone of HyBra2 was obtained from
the screen of the cDNA library. To obtain the 3' end of HyBra1
3' RACE experiments were performed as described
(Frohman et al., 1988).
GenBank accession numbers of HyBra1 and HyBra2 full-length
clones are (AY366371, AY366372), respectively.
DNA constructs and in vitro transcription
For RNA microinjection experiments, the Xbra cDNA was subcloned
into the pRNA3 vector; Hybra1 and Hybra2 coding regions were
cloned by PCR into BamHI/XbaI sites of pCS2+ and
StuI/XbaI sites of pCS2+MT6 respectively. The
chimeric constructs XTH2A and H2TXA were generated by
subsequent subcloning of the respective PCR products. For both chimerae, the
conserved amino acid motif AKAFL-DAKER was used as a junction. Capped in vitro
transcripts were generated from SfiI-linearised plasmids and T3 RNA
polymerase (HyBra chimerae and Xbra), or by NotI
linearisation and Sp6 RNA polymerase (wt HyBra paralogues).
Phylogenetic analysis
ClustalW was used to align amino acid sequences of the T-box proteins.
Maximum likelihood program PUZZLE (Schmidt
et al., 2002) and MrBayes
(Huelsenbeck and Ronquist,
2001) were used to perform a phylogenetic tree analysis. In the
Puzzle analysis 1000 replica were computed using JTT as a substitution model,
assuming a strong rate heterogeneity in the gamma distribution. In MrBayes
3.1, 1,7 Mio generations were run using the WAG model of substitution and
eight gamma rate categories.
RNA in situ hybridisation
Hydra in situ hybridisation was carried out as previously
described (Grens et al., 1995)
with minor modifications. We omitted the elevated temperature step [80°C,
and for detection we used NBT/BCIP instead of BMpurple (Roche)].
Xenopus double in situ hybridisations were performed as described
(Wittenberg et al., 1999) with a primary Fast Red stain for the
fluorescein-labelled probe terminated by heat inactivation for 30 minutes at
65°C in 0.1 M EDTA-TBS buffer, followed by a BM-Purple (Roche) stain for
the digoxygenin-labelled probe.
Protein synthesis assay
To assay for protein synthesis, 50 µCi [35S]methionine was
injected into the gastric cavity of adult polyps, and incubated for 1 hour.
Thereafter, five polyps were dissolved in 100 µl lysis buffer (50 mM
Tris-HCl, pH 8.0, 2% SDS, 100 mM ß-mercaptoethanol). After lysis, an
equal volume of 1 mg/ml BSA was added and 100 µl lysate was spotted and
air-dried on a glass fibre filter (Whatman GF/A 2.4 cm; #1820024). Then the
filter was rinsed twice with 3 ml ice-cold 20% trichloroacetic acid (TCA) for
5 minutes, followed by two washes in 12.5% TCA to remove unincorporated
labeled amino acids. Filters were then washed twice with 95% ethanol followed
by one wash with acetone, air dried, placed in scintillation vials with 1 ml
scintillation fluid and the total number of counts determined. As a negative
control, five unlabeled animals were lysed and then 50 µCi
[35S]methionine was added to the lysate.
Inhibition of translation by cycloheximide
Polyps were incubated in 5 or 50 µg/ml cycloheximide (Sigma) in
Hydra medium (HM) for 15 minutes, 1 hour or 2 hours. After treatment,
polyps were washed five times in HM to remove traces of cycloheximide. To
inhibit translation during regeneration, animals were pretreated with
cycloheximide for 15 minutes and subsequently decapitated at t0
beneath the tentacles. Regenerates were further incubated in cycloheximide for
2 hours, washed five times and allowed to regenerate for the times
indicated.
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) with some
minor modifications. Polyps were treated for 24 hours in 5 µM
alsterpaullone (Sigma), then washed extensively and further incubated in HM
for 15 hours and 40 hours, respectively. Specimens were then fixed and
subjected to in situ hybridisation. DMSO at the corresponding concentration
was used as a control with no effect. |
RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Using 3' RACE we have now obtained the 1845-bp-long full-length clone
with an open reading frame of 364 amino acids. By degenerate PCR and cDNA
library screening, we isolated an additional full-length T-box gene. The clone
consists of 1423 bp, encoding a protein of 388 amino acids
(Fig. 1C). Phylogenetic
analysis revealed that this gene also clearly belongs to the
Brachyury subfamily of T-box genes and we therefore named it
HyBra2 (Fig. 2). The
T-domains of the two HyBra genes show about 70% identity
(Fig. 1A). When compared with
the T-domain of vertebrate Brachyury proteins, HyBra1 and HyBra2 show about
80% and 70% identities, respectively (Fig.
1A,C). By contrast, the remaining domains are far less conserved,
with an overall amino acid identity of <20% (data not shown).
Interestingly, two Brachyury protein domains, one positioned N-terminally of
the T-box and the other one named R1, which is embedded within the activation
domain, have been gained and lost during evolution
(Marcellini et al., 2003;
Kispert et al., 1995). Whereas
the N-terminal domain is absent in non-bilaterian Brachyury proteins, and was
therefore probably acquired at the base of the bilaterians
(Marcellini, 2006), the
ancient R1 fragment in the C-terminal domain is already present in cnidarians,
where it is found in HyBra1 and Nematostella Brachyury
(Scholz and Technau, 2003),
but not in HyBra2. Strikingly, both domains have been lost in Tunicates and
arthropods but are conserved in all other bilaterian paralogues
(Marcellini, 2006).
Sequence comparison further revealed that HyBra2 carries a spliced
leader (SL-B) at the 5' end (Stover
and Steele, 2001), whereas no spliced leader is present in the
isolated HyBra1 cDNA (data not shown). Genome walking analysis of the
publicly available shotgun sequences of the Hydra genome project
(Craig J. Venter Institute, MD, USA) showed that the two HyBra
paralogues are not closely linked in the genome. Interestingly, when comparing
HyBra1 and Xbra, five out of six intron positions in the
coding sequence are conserved, four of which reside in the T-box and are
conserved throughout the animal kingdom. These intron sites are also conserved
in HyBra2 except the last one linking the T-box to the C-terminal
domain (Fig. 1D). In summary,
the structural similarities in the T-box, the conserved R1 motif and the
arrangement of introns indicate that the ancestral Brachyury gene
structure of HyBra1 has been conserved throughout evolution and the
HyBra2 paralogue is derivative in various aspects.
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)
(Fig. 2). In this more
extensive analysis, the cnidarian Brachyury proteins are divided in two
separate clusters. HyBra1 clusters with Brachyury homologues from
Nematostella (Scholz and Technau,
2003) and from a marine relative, the hydrozoan Hydractinia
echinata, whereas HyBra2 forms a cluster with a Brachyury from the
hydrozoan Podocoryne carnea
(Spring et al., 2002).
However, none of them were grouped with any of the paralogues from the
chordates, nor did the bilaterian Brachyury proteins fall into two distinct
(bra1 and bra2) subclasses, although lower Deuterostomes tended to cluster
with HyBra2, whereas vertebrate sequences are all closer to HyBra1. However,
both HyBra1 and HyBra2 are more similar to deuterostome than protostome
Brachyury proteins, reflecting the divergence in the different lineages. Since
the basal cnidarian Nematostella vectensis has only a
Bra1-type gene (Scholz and
Technau, 2003), this analysis is consistent with the view that
HyBra1 and HyBra2 represent two ancient paralogues that
evolved by a duplication event. Most likely, this event occurred early in
cnidarian evolution, after the divergence of the hydrozoan lineage
approximately 550 million years ago. It is also clear, that according to this
scenario, HyBra2 has undergone a more rapid rate of sequence
evolution.
Expression pattern of HyBra1 and HyBra2 in adult polyps and during bud formation
We previously showed that HyBra1 is expressed in the hypostome,
the apical tip of Hydra (Technau
and Bode, 1999). During various developmental conditions, such as
head regeneration, bud formation (the asexual form of Hydra
reproduction), the formation of hydra from aggregates of cells, and
embryogenesis, it is expressed very early during head formation
(Technau and Bode, 1999;
Technau et al., 2000). In situ
hybridisation showed that HyBra2 was expressed in the hypostome of
Hydra polyps in a very similar pattern to that of HyBra1
(Fig. 3A,C). However, vibratome
sections of adult polyps revealed that HyBra2 was expressed primarily
in the ectodermal layer whereas HyBra1 was expressed predominantly in
the endoderm (Fig. 3B,D). A
comparison of various bud stages showed that HyBra2 was expressed
just as early as HyBra1 as a spot in the ectoderm at stage 1
(Fig. 3E,H). In subsequent
stages of bud formation, HyBra2, like HyBra1, remained
restricted to the apical tip of the bud, which is the future hypostome
(Fig. 3F,I). Early stages, as
well as vibratome sections of later bud stages, confirmed that HyBra1
was primarily expressed in the endoderm
(Fig. 3G), whereas
HyBra2 was predominantly expressed in the ectoderm
(Fig. 3J). This suggests that
HyBra2, like HyBra1, plays an early role in head formation
during bud formation, but acts mainly in the other germ layer.
HyBra1 and HyBra2 expression is differently regulated during regeneration
We next analysed HyBra1 and HyBra2 expression during head
regeneration in animals cut at apical and mid-gastric positions of the body
column. As shown previously (Technau and
Bode, 1999), HyBra1 appears in the regenerating head of
animals bisected at the apical end of the body column within 1-3 hours of head
removal (Fig. 4A;
Fig. 5A-C). In sharp contrast,
HyBra2 was first expressed at apical levels after roughly 8-10 hours
(Fig. 4A;
Fig. 5G-I). There was a
substantial variability from animal to animal in the onset of HyBra2
expression.
Interestingly, HyBra1 initially appears several hours later in
animals bisected in the middle of the body column
(Fig. 4B) compared with animals
bisected at the apical end (Fig.
4A) (Technau and Bode,
1999). This delay is consistent with the delay in head formation
at this level and a lower level of the head activation gradient
(Bode and Bode, 1984). By
contrast, there was only a slight delay in the onset of HyBra2
expression in regenerating heads following mid-column bisection compared with
those bisected near the head (Fig.
4B). Furthermore, the apical half of these bisected animals
transiently, but strongly expressed HyBra1 at the site of foot
regeneration, whereas HyBra2 is expressed very weakly, and only in a
few individuals (Fig. 4C; H.B.
and U.T., unpublished). Hence, HyBra1 is among the earliest genes
expressed during head regeneration, and is transiently upregulated during foot
regeneration, whereas HyBra2 appears somewhat later and is expressed
only where a head is being formed.
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HyBra1 is an immediate-early response gene during head regeneration
HyBra1 is one of the earliest genes expressed during head
regeneration and bud formation (Technau
and Bode, 1999). This raised the question whether initiation of
HyBra1 expression depends on the synthesis of another factor to
activate its expression. To address this question we analysed the expression
of HyBra1 and HyBra2 during regeneration in the presence of
the translational inhibitor cycloheximide (CHX)
(Cascio and Gurdon, 1987). We
first established that a treatment with 5-50 µg/ml CHX for up to 2 hours
leads to slight but reversible phenotypic effects. Under these conditions, the
reappearance of tentacles after head removal is delayed by 12-18 hours (data
not shown). Treatment for up to 6 hours was also tolerated by the animals, but
had more severe effects. We then measured the inhibition of translation by
incorporation of [35S]methionine. We found that after treatment
with either 5 or 50 µg/ml CHX for 15 minutes, translation was inhibited by
80-90%. Thereafter, the rate of protein synthesis slowly recovered over the
next 2 days (data not shown). Treatment for 2 hours resulted in the 80-90%
level of inhibition being maintained for 6 hours, followed by a slow recovery
(data not shown).
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One possible candidate protein to regulate HyBra1 expression is
ß-catenin (Hobmayer et al.,
2000). It is expressed ubiquitously, transcriptionally upregulated
during regeneration and stabilised in the nucleus at the apical end of
Hydra (Hobmayer et al.,
2000; Broun et al.,
2005). Indeed, we obtained support for such a role of
ß-catenin by treatment with the Glycogen synthase kinase 3 ß
(GSK3ß) inhibitor alsterpaullone
(Broun et al., 2005).
Inhibition of GSK3ß results in stabilisation and nuclear translocation of
ß-catenin, where it can act, in concert with T-cell factor (TCF), as a
transcriptional regulator (Leost et al.,
2000; Bain et al., 2003). Treatment with alsterpaullone for 24
hours followed by incubation in Hydra medium for 15-40 hours leads to
an ectopic upregulation of both HyBra1 and HyWnt genes in
the body column (Fig. 6),
confirming earlier results (Broun et al.,
2005). Like HyBra1, HyBra2 is also upregulated broadly in
response to alsterpaullone treatment (Fig.
6E-H). HyWnt and both HyBra genes are
downregulated at the same time in the hypostome
(Fig. 6; data not shown).
Interestingly, 15 hours after treatment ectopic upregulation of
HyBra1 and HyBra2, but not of HyWnt, in the body
column is detectable in single cells, which appear to be interstitial cells
(Fig. 6B,C,F,G). After 40 hours
of alsterpaullone treatment, both HyBra1 and HyBra2 are
strongly upregulated throughout the endoderm of the gastric cavity, with peaks
of expression of regular spacing consisting of 10-15 epithelial cells also
visible in the ectoderm (Fig.
6D,H). Ectopic HyWnt expression is found in similar spots
but does not expand to ubiquitous upregulation in the endoderm
(Fig. 6L)
(Broun et al., 2005). This
suggests that HyBra1 and HyBra2 are direct or indirect
targets of the Wnt-ß-catenin pathway.
Structure-function relationship in animal cap assays for mesoderm induction
We were interested whether the high degree of sequence divergence between
the two HyBra paralogues reflects two different functions. To test
this, we used a well-defined in vivo system, the Xenopus animal cap.
Untreated animal caps differentiate into epidermal tissue, but can be forced
to form mesoderm if injected at the two- to four-cell stage with Xenopus
bra mRNA (Smith et al.,
1991). Injection of mRNAs encoding other T-box genes such as
VegT or eomesodermin, induces endoderm in addition to
mesoderm, indicative of the endogenous functions of these proteins
(Marcellini et al., 2003;
Conlon et al., 2001;
Zhang and King, 1996;
Zhang et al., 1998). Other
factors, such as otx2 and ath-3 have been shown to induce
differentiation of neural tissue in animal caps
(Gammill and Sive, 1997;
Takebayashi et al., 1997).
Hence, animal caps can display a variety of very distinct and specific
differentiation fates in response to an exogenous transcription factor
provided by mRNA microinjection. Notably, Brachyury mRNAs from a wide
range of bilaterian species induce mesoderm under these assay conditions
(Marcellini et al., 2003).
Thus, this assay can be used to rapidly identify functionally crucial domains
or motifs generated by sequence divergence during animal evolution.
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Intriguingly, it turned out that HyBra2 is a highly potent cement
gland inducer, even when very low RNA concentrations were injected
(Fig. 7H,K;
Fig. 8G,M). The
Xenopus cement gland, which is temporarily formed during the tadpole
stage, consists of palisade-like epithelial cells. These cells have a distinct
morphological structure and are therefore easy to identify. Of all
Hybra2-injected caps, 86% contained one or several cement gland-like
areas characterised by their regular arrangement of strongly pigmented cells
(Fig. 7H,K). Additional
evidence that these structures are cement glands comes from two observations.
First, like their endogenous counterparts at the anterior end of sibling
embryos (Fig. 7C), their
surface was highly adhesive, indicating mucus secretion (data not shown).
Second, 84% of Hybra2-injected caps expressed Xcg-1
(Sive et al., 1989), a
cement-gland-specific marker (Fig.
8G,M). Since there is evidence that cement glands, although
epithelial in nature, develop as a consequence of neural induction
(Sasai and De Robertis, 1997),
we analysed the animal caps for expression of the pan-neural marker
N-CAM (see Fig. S1 in the supplementary material) and
ß-tubulin, a marker for differentiated neurons
(Fig. 8). Although these neural
mRNAs were hardly detectable in Hybra1- and Xbra-injected
caps, both markers were strongly expressed in a significant fraction of the
Hybra2-injected explant (Fig.
8F,H,M and see Fig. S1 in the supplementary material). The absence
of muscle and endoderm suggests that this induction results from the direct
conversion of ectoderm into neural tissue in response to HyBra2.
Together, the Xenopus animal cap experiments reveal that the
activities of HyBra1 and vertebrate Xbra are very similar,
but that HyBra2 has significantly diverged and acquired cement gland
and neural-inducing properties.
This unexpectedly distinct, qualitative difference in activity motivated us to look into the domains of the proteins in more detail. We therefore constructed two chimeras, swapping the T-Box and C-terminal domains of Xbra and HyBra2, as shown in Fig. 7A. Surprisingly, animal caps injected with the H2TXA chimera contained not a single cement gland, whereas 40% of them were elongated (Fig. 7I,K). Although the latter suggests the induction of mesoderm, muscle actin was only rarely induced (Fig. 8I,M). Moreover, no Xcg-1 or ß-tubulin expression was detected (Fig. 8I,J,M). The converse fusion, XTH2A, induced cement glands and neural ß-tubulin approximately to the same extent as HyBra2, but neither induced elongation or muscle actin expression (Fig. 7J,K; Fig. 8K,L,M). Thus, the XTH2A variant phenocopies the activity of HyBra2 and is unable to induce mesoderm.
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|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). We
now report the identification of a second Brachyury gene from this
basal metazoan, termed HyBra2. The phylogenetic analysis strongly
suggests that the duplication event occurred at the base of the Hydrozoan
lineage, at least 550 million years ago
(Chen et al., 2002). Hydrozoa
have retained two Brachyury genes, Bra1 and Bra2.
Whether the duplicated Brachyury genes in Hydractinia
(Bra2-type) and Podocoryne (Bra1-type) have been preserved as in
Hydra or have been lost is not yet clear. Our sequence analysis
further suggests that bilaterian, in particular all vertebrate Brachyury
proteins, share more features with HyBra1 than with HyBra2. This suggests that
a HyBra1-like molecule was present in the common ancestor of cnidarians and
bilaterians and that it has retained more of its ancestral structural features
than HyBra2. Interestingly, HyBra1 shares a small conserved motif in
the activation domain with the vertebrate Brachyurys, corresponding to the
core of the repression module 1 (R1) as described previously
(Kispert et al., 1995). The
conservation of this motif suggests that it might play a role as a conserved
interface for protein-protein interaction.
The role of Brachyury during head formation in Hydra
HyBra1 and HyBra2 are expressed in endoderm and ectoderm,
respectively, of the hypostome, which is considered equivalent to the Spemann
organiser (Browne, 1909;
Broun and Bode, 2002).
Interestingly, although HyBra1 and HyBra2 mRNA expression is
activated at the same stage during budding, the kinetics of expression of
HyBra1 and HyBra2 differs significantly during regeneration:
HyBra1 is an immediate-early gene, in the sense that its onset of
expression does not require protein synthesis, whereas HyBra2
expression does require protein synthesis and occurs significantly later. This
points to some crucial differences in the molecular regulatory network between
regeneration (for reviews, see Holstein et
al., 2003; Galliot et al.,
2006; Bosch, 2007)
and budding, despite the fact that overall the same genes are used. Head
formation is thought to be suppressed in the body column of a normal polyp by
an inhibitor that forms a concentration gradient from the head to the budding
region (MacWilliams, 1983).
While the nature of this postulated inhibitor is still unclear, we propose
that decapitation rapidly removes the head inhibitor from the tissue, which
leads to a local post-translational modification of a transcriptional
regulator present in the tissue that activates HyBra1 (and other
immediate-early response genes). Members of the MAPK and CREB pathway are
implicated in early head regeneration and possibly involved in the regulation
of HyBra1 (Cardenas et al.,
2000; Cardenas and Salgado,
2003; Manuel et al.,
2006; Kaloulis et al.,
2004). Another candidate molecule to activate HyBra1
expression upon post-translational modification is ß-catenin.
ß-catenin is expressed throughout the animal, but upregulated at
the apical tip during regeneration
(Hobmayer et al., 2000) and
stabilised in the apical region in nuclei
(Broun et al., 2005). In
accordance with a role in regulating HyBra expression,
HyBra1 and HyBra2 expression is ectopically upregulated in
animals where ß-catenin has been ectopically stabilised by alsterpaullone
treatment.
Hence, inhibition of early head-specific genes in the body column might be lifted in response to regeneration signals, preparing the tissue for rapid regeneration, irrespective of its future fate. By contrast, HyBra2 does not belong to the immediate-early-response genes during head regeneration and is not detectable during foot regeneration. After the immediate-early response, additional factors, which stabilise the polarity of the tissue and tell the regenerating end whether to differentiate a head or a foot, must be involved.
The evolutionary divergence of the Hydra Brachyury paralogues occurred at diverse levels
It has been repeatedly proposed that divergence and evolution of
cis-regulatory regions may drive such increasing complexity as observed in the
lineage of the vertebrates (Levine and
Tijan, 2003; Davidson and
Erwin, 2006). The duplication-degeneration-complementation (DDC)
model provides a theoretical framework for the fate of paralogues upon
duplication (Force et al.,
1999; Lynch and Force,
2000). Essentially, paralogues that are retained over long
evolutionary time scales underwent either subfunctionalisation or
neofunctionalisation. Subfunctionalisation mainly emphasises the divergence
and subdivision of the cis-regulatory elements of the parental gene. Indeed,
there are cases where the gene expression domains of both duplicates together
make up for the expression domain of a single orthologue in a species, which
has branched off before the duplication event
(De Martino et al., 2000). In
the case of HyBra1 and HyBra2 the acquired cis-regulatory
elements resulted in a paralogue-specific spatial and temporal regulation of
the two Hydra Brachyury genes. If the combined expression of both
paralogues reflects the expression of the ancestral gene, this would mean that
Brachyury is marking a domain in Hydra, rather than a
particular germ layer.
In addition to subfunctionalisation, divergence of coding sequences in
paralogues can lead to neofunctionalisation. In the case of ancient
duplications, the paralogues may have diverged significantly and possibly
taken up novel functions. Therefore, ancient lineage-specific paralogues in
particular provide the opportunity to study the levels and target sites of the
diverging mutations. They also provide us with evolutionary variants of
proteins, which can then be studied for structure-function relationships and
help to rapidly identify crucial motifs for specific subfunctions. In the
absence of functional assays in Hydra with specific read-outs for
distinct cellular fates, we used a heterologous system - the Xenopus
animal caps - as an assay system to test whether both Hydra
paralogues have functionally diverged. This assay system is ideal, because
this naive tissue, which otherwise develops normally into undifferentiated
epidermis, but can be pushed to differentiate endoderm, mesoderm or neural
ectoderm, depending on the input. It is well established that
Brachyury induces mesoderm, both on the molecular level and in terms
of tissue properties such as convergent extension
(Smith et al., 1991). Although
Hydra does not contain mesoderm, HyBra1 is able to induce
mesoderm just like the Xenopus Brachyury gene
(Marcellini et al., 2003)
(this study). In contrast, HyBra2 behaves differently to other
bilaterian Brachyury genes in this assay by strongly inducing
formation of cement glands and neural tissue. Domain-swapping experiments
demonstrate that the HyBra2 C-terminal domain is necessary and sufficient
(when fused to a Brachyury T-box) to induce neural tissue formation.
Interestingly, Hybra2 is not the only Brachyury protein with neuralising
activity. The Xbra paralogue Xbra3 activates both mesodermal
and neural differentiation (Strong et al.,
2000). In animal caps, however, it induced only posterior neural
tissue and cement glands were not observed (Hartmann et al., 2002). It is
therefore not clear, whether this is a secondary effect of the neuralising
effect of embryonic FGF (eFGF), which forms a feedback loop with Brachyury
(Schulte-Merker and Smith,
1995). In addition, specific truncations of the Xbra
activation domain can convert the mesoderm inducer into a neuralising factor
(Rao, 1994). However,
C-terminal truncations of HyBra2 either did not alter the neuralising
phenotype or had no effect at all (data not shown). Further, the HyBra2
C-terminal domain is even longer than that of HyBra1, which is a mesoderm
inducer. We conclude that the similar phenotypes obtained with the HyBra2 and
with truncated version of Xbra in the study by Rao
(Rao, 1994) must be of
different origin, yet might reveal a common mechanism.
Brachyury has previously been shown to be a transcriptional activator, so
one possibility to explain the radical difference in the inductive behaviour
of HyBra2 in animal caps is that the C-terminal activation domain evolved into
a repression domain. However, this does not seem to be the case, as an
H2T-EnR variant (HyBra2 T-box fused to the
Engrailed repressor domain) did not mimic the neuralising activity of
HyBra2 (data not shown). Furthermore, repression of Brachyury
function by injection of a comparable XbraT-EnR construct caused
mesodermal defects in convergent extension and notochord formation and
induction of anterior endoderm (Conlon et
al., 1996; Conlon and Smith,
1999); however, the induction of cement glands had not been
reported in these studies. Therefore, it is unlikely that Hybra2 simply
represents an antimorphic variant of mesoderm-inducing Brachyury proteins.
Future studies aimed to identify proteins interacting with variants of
Brachyury might be key for our understanding of how transcriptional activity
and specificity is modulated.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/23/4187/DC1
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ACKNOWLEDGMENTS |
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3 independent repeats). (A,B)
GFP-injected control explants showed no staining of tested marker
genes. Injections of Xbra (C) and HyBra1 (E) induced
muscle actin, but not xcg-1 expression, whereas
HyBra2 (G) triggers the converse gene expression pattern.
Furthermore, mRNA of the differentiated neuronal marker
ß-tubulin was often present in HyBra2- (H), but never
in HyBra1- (F), and only rarely in Xbra-injected explants
(D). (I-L) Double in situ staining of animal caps injected with the
Hybra2/Xbra chimerae. (I,J) H2TXA-injected animal caps show no
xcg-1, ß-tubulin induction and very limited muscle
actin expression. (K,L) By contrast, XTH2A-injected caps clearly
show strong induction of xcg-1 and ß-tubulin, but no
muscle actin mRNA. (M) Statistical overview of induced markers
(xcg-1, muscle actin, ß-tubulin) by injection of
particular mRNAs (2-6 independent single and/or double in situ hybridisation
experiments).