1 Departament de Genètica, Facultat de Biologia, Universitat de
Barcelona, Avenida Diagonal 645, E-08028 Barcelona, Spain
2 Evolution and Development Group, Depart. Prof. Hans Lehrach,
Max-Planck-Institut fur Molekulare Genetik, 14195 Berlin (Dahlem),
Germany
Author for correspondence (e-mail:
jgarcia{at}bio.ub.es)
Accepted 21 August 2003
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SUMMARY |
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Key words: Hairy, bHLH, amphioxus, DDC model, Subfunctionalisation, Duplication
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Introduction |
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In vertebrates, there are two Hairy genes in chicken [Hairy1 and
Hairy2 (Palmeirim et al.,
1997; Jouve et al.,
2000
)], in zebrafish [her6 and her9
(Pasini et al., 2001
;
Leve et al., 2001
)] and in the
frog [X-Hairy1 and X-Hairy2
(Davis et al., 2001
)]. However,
in mice, only a single Hairy gene has been so far identified [Hes1
(Sasai et al., 1992
)]. Other
HES and HER genes (for Hairy-enhancer of split related) belong to the Enhancer
of split [E(spl)] subfamily and not to the Hairy subfamily of the bHLH-O
family of transcription factors (Davis and
Turner, 2001
).
For mouse Hes1 and both chicken Hairy genes, a striking dynamic
expression pattern in the presomitic mesoderm (PSM), where they cycle with a
temporal periodicity that corresponds to the formation of one somite, has been
observed (Palmeirim et al.,
1997; Jouve et al.,
2000
). Besides this cycling pattern in PSM, their expression is
also detected in several endoderm-derived tissues, the notochord, and the
central nervous system (CNS) (Sasai et
al., 1992
). Accordingly, mice mutant for the Hes1 gene
exhibit severe defects in neural and endocrine development. Briefly, these
defects are thought to be due to the premature differentiation of postmitotic
neurons or endocrine cells, respectively
(Ishibashi et al., 1995
;
Jensen et al., 2000
). In
neither the zebrafish nor Xenopus is there a dynamic expression
pattern of Hairy genes in the PSM comparable with that in amniotes. During
somitogenesis, the zebrafish Hairy gene her6 is expressed in the
posterior part of each segmented somite and in stripes in the anterior PSM.
Within the CNS, her6 is expressed first in the prospective forebrain,
and later in hindbrain segmentation with a very dynamic, segmentally
restricted pattern. Low levels of her6 expression are also present in
the notochord (Pasini et al.,
2001
). The zebrafish Hairy gene her9 is also expressed
during CNS development but in contrast to its paralogue, neither in segmented
somites nor in the PSM. Within the CNS, her9 is predominantly
expressed in the fore- and midbrain, and transiently in the hindbrain, leaving
a non-expressing gap at the midbrain-hindbrain boundary (MHB) while it is also
expressed in the midline mesoderm. However, other bHLH genes closely related
to the Hairy subfamily, i.e. E(spl) subfamily members, exhibit a cyclic
expression comparable with that of amniote Hairy genes [e.g. her1
(Holley et al., 2000
)]. This
similar behaviour has led some authors to propose that these genes are
behaving as (or represent) functional Hairy genes, although they do not
represent ortologous genes (Gajewski and
Voolstra, 2002
). In the frog, the two Hairy homologues are
expressed in the CNS, somites and PSM. Both genes have identical expression
patterns as a band prefiguring a new somite formation in the anterior PSM and
are also transcribed weakly in segmented somites. Within the neuroectoderm,
they exhibit a non-overlapping expression pattern
(Davis et al., 2001
).
A characteristic of vertebrate Hairy genes is therefore to be very
pleiotropic, in contrast to the protostome Hairy subfamily members. To
understand when this multiplicity of functions was acquired during evolution,
we studied the Hairy subfamily in the cephalochordate amphioxus. Amphioxus is
the closest living invertebrate relative to vertebrates and has not undergone
the massive gene duplications events (up to polyploidization) that took place
early during vertebrate evolution (see
Spring, 2002). It is believed
that amphioxus represents a direct descendant of the
cephalochordate/vertebrate ancestor. Hence, amphioxus has been widely used as
a model system to study the ancestral function of a gene family at vertebrate
origins, represented by a single gene in the chordate ancestor that may be
very similar to that of modern amphioxus. Surprisingly, we have isolated eight
canonical Hairy family members from the `pre-duplicative' amphioxus genome
that we have called Amphi-hairyA to Amphi-hairyH. At least
six of them are linked in the genome, supporting their origin by tandem
duplication. We have analysed their expression pattern during amphioxus
development by whole-mount in situ hybridisation, and by RT-PCR for those
genes for which no expression was detected by the former technique.
Strikingly, Amphi-Hairy genes seem to have undergone a
duplication-degeneration-complementation process (the DDC model). This model
is proposed as an explanation to account for the maintenance of duplicated
genes. Accordingly, duplicated copies of a single gene preserve their
maintenance in the genome by differential loss of cis-regulatory regions among
the duplicates. Hence, a complex expression pattern of a single gene is
subdivided in discrete components, and paralogue genes decouple a pleiotropic
role that was carried out by their ancestor. Thus, genes become only partially
redundant, and the unique roles of a given duplicate prevent its loss in the
genome by selection processes. Only the summation of the expression pattern of
Amphi-hairy A to Amphi-hairy D account for the expression
pattern of vertebrate Hairy genes (e.g. that of the single mouse Hairy-gene
Hes1).
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Materials and methods |
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Amphi-Hairy cDNA clones isolation
Degenerate oligonucleotides were designed over an alignment of the bHLH
domain of Hairy proteins (Palmeirim et
al., 1997). cDNA obtained from RNA of adult amphioxus was used as
a template for the PCR reaction (1 minute at 94°C, and 35 cycles of 10
seconds at 94°C, 20 seconds at 42°C, 30 seconds at 72°C) with the
degenerate primers h1 and h2 (see Table S1 at
http://dev.biologists.org/supplemental/).
As no bands of the expected size were obtained, a semi-nested PCR with the
degenerate primers h1 and h3 (see Table S1 at
http://dev.biologists.org/supplemental/)
under the same conditions was performed. A band of the expected size was
excised and subcloned. Sequencing showed its similarity to the Hairy subfamily
genes.
The cloned fragment was used to screen a cDNA library from 6-20 hours
postfertilization amphioxus embryos
(Langeland et al., 1998).
Approximately 4x105 pfu were screened at moderate stringency
[55°C, Church's buffer (Shifman and
Stein, 1995
)] with the PCR product. This led to the identification
of four Hairy homologues, which we named Amphi-hairyA, Amphi-hairyB,
Amphi-hairyC and Amphi-hairyD. The h1 and h2 primers were used
to amplify by PCR most of the bHLH domain of Amphi-hairyA to
Amphi-hairyD, and all four products were used in conjunction to
perform a further screening over the same cDNA library under less stringent
conditions (50°C). We thus isolated an additional Hairy homologue that we
denominated Amphi-hairyE. Amphi-hairyA to Amphi-hairyD plus
an additional Amphi-hairyF genes were also identified through an EST
project of gastrula and neurula stages
(Panopoulou et al., 2003
).
To ascertain whether we cloned all amphioxus Hairy genes, we amplified the
putative second intron of Amphi-Hairy genes by PCR with the degenerate primers
h1 (exon 2) and SPLIT R (exon 3), on multiple extractions of genomic DNA from
single individuals. As no bands were visible, after primer cleaning, we
performed a nested PCR reaction with the degenerate primers SPLIT F (exon 2)
and h2 (exon 3) (see Table S1 at
http://dev.biologists.org/supplemental/
for primers nomenclature and regions amplified). The PCR products were cloned
into plasmid using two different strategies. First, a nested PCR was resolved
on an agarose gel and the bands excised one by one. Second, a nested PCR
reaction was primer cleaned and directly used for ligation and subsequent
cloning. This yielded the isolation of all the former Amphi-Hairy genes plus
two new products that were used to screen a genomic library
(Ferrier et al., 2000
),
resulting in the isolation of Amphi-hairyG and
Amphi-hairyH.
In addition, we screened a PAC library gently provided by Chris Amemiya (Yale University, USA) with a mixture of Amphi-Hairy clones. The positive clone 51K5 was used as a template in PCR reactions (2 minutes at 94°C and then 35 cycles of 15 seconds at 94°C, 10 seconds at 60°C and 20 seconds at 72°C) with Amphi-Hairy specific oligonucleotides (hairyAF, hairyAR, hairyBF, hairyBR, RT-CF, RT-CR, hairyDF, hairyDR, RT-EF, RT-ER, RT-FF, RT-FR, RT-GF, RT-GR, RT-HF and RT-HR; see Table S1 at http://dev.biologists.org/supplemental/). Amphi-hairyA to Amphi-hairyF were contained in the PAC clone, whereas Amphi-hairyG and Amphi-hairyAmphi-hairyH were not.
Phylogenetic analysis
The putative protein sequences of the Amphi-Hairy genes were aligned with
their homologues from other organisms using ClustalX
(Thompson et al., 1994). The
complete proteins were used to construct a Neighbour-joining tree. Topology
robustness was assessed by 1000 bootstrap resampling of the data.
Obtaining embryos and in situ hybridisation
Ripe adults of the Florida lancelet, Branchiostoma floridae, were
collected from Old Tampa Bay (Florida, USA) during the summer breeding season.
The males and females were spawned electrically in the laboratory, and
selected developmental stages were raised as described in Holland and Holland
(Holland and Holland,
1993).
In situ hybridisation was performed according to previously published
methods (Holland et al.,
1996). When available, the 3' coding region of each
Amphi-Hairy gene (region that included neither the conserved bHLH domain nor
the orange domain), which was amplified using primers h5 and h3rep (see Table
S1 at
http://dev.biologists.org/supplemental/),
was used as a template for the DIG-labelled antisense probe.
After photographed as wholemounts, selected embryos were contrasted in 1% Poinceau S, 1% acetic acid, dehydrated through an ethanol series and embedded in Spurr's resin. Serial 3 µm sections were obtained with a glass knife, mounted in DePeX and photographed under Nomarski optics.
RT-PCR
We performed RT-PCR experiments with the Amphi-Hairy genes for which we did
not detect any expression in the whole-mount hybridisation, i.e.
Amphi-hairyE to Amphi-hairyH. Specific primers (RT-EF,
RT-ER, RT-FF, RT-FR, RT-GF, RT-GR, RT-HF and RT-HR; see Table S1 at
http://dev.biologists.org/supplemental/)
were designed and multiplex separate reactions were performed for each gene in
conjunction with the use of Amphi-hairyC as an internal positive
control. The primers used for Amphi-hairyC (RT-CF and RT-CR; see
Table S1 at
http://dev.biologists.org/supplemental/)
were designed in such a way that the region amplified was larger than the
region amplified for Amphi-hairyE to Amphi-hairyH.
cDNA from 12-, 15-, 18- and 21-hour embryos and adults was obtained by standard methods (J. R. Bayascas, PhD Thesis, University of Barcelona, 1997). Samples were used as a template for the PCR. PCR conditions were 1 minute at 94°C, and 30 cycles of 20 seconds at 94°C, 20 seconds at 55°C, 20 seconds at 72°C. The full-length cDNA clones of Amphi-hairyC, Amphi-hairyE and Amphi-hairyF, and genomic clones for Amphi-hairyG and Amphi-hairyH were used as templates for positive controls. After electrophoresis, gels were blotted and hybridised with Amphi-hairyC plus Amphi-hairyE-, Amphi-hairyF-, Amphi-hairyG- or Amphi-hairyH-specific probes.
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Results |
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Phylogenetic analysis
To gain more insight into the relationships between Hairy genes, we
conducted a molecular phylogenetic analysis by the neighbour-joining method on
the complete Hairy proteins, using mouse and human HES2 sequences [E(spl)
subfamily of bHLH-O] as outgroups (Fig.
1B). Vertebrate Hairy proteins clustered together (bootstrap value
100%) into two well-supported groups (values 100 and 78%), in agreement with
other phylogenetic studies (Gajewski and
Voolstra, 2002). All the Amphi-Hairy proteins form a monophyletic
group (73%) that branches immediately outside these groups (as their sister
group; 80%) suggesting that all they have originated by independent
duplication in the cephalochordate lineage, in harmony with the hypothesis
that vertebrate genes have originated by duplication after the
cephalochordate-vertebrate divergence.
In a recent study (Gajewski and
Voolstra, 2002), the existence of 5 Hairy-type genes in the
Fugu rubripes genome, named FrHer6.1, FrHer6.2, FrHer9,
FrHer10.1 and FrHer10.2 is highlighted. The authors conclude
that more Hairy genes should exist in the zebrafish genome and that at least
one of those types (the Her10 type) in other vertebrate genomes. To further
analyse the phylogenetic relationships among the HES/HER subfamilies of bHLH
transcription factors, we conducted similar phylogenetic trees with the whole
set of human (Ledent et al.,
2002
), mouse, Drosophila, zebrafish and Fugu
proteins. In our analyses, all vertebrate and invertebrate Hairy genes group
together (71% bootstrap value), whereas the Her10 Fugu class groups with the
enhancer of split representative HES2 (92%). Thus, only the fish Her6 and 9
classes represent truly Hairy-subfamily homologues, whereas the rest group
with enhancer-of-split related sequences (see Fig. S2 at
http://dev.biologists.org/supplemental/).
We thus argue that there are only two classes of Hairy genes in vertebrates.
However, and in agreement with Gajewski and Voolstra data, it is still
plausible that further Hairy homologues remain to be discovered in the
zebrafish genome, as it is accepted that extensive gene duplications up to
tetraploidisation occurred in the fish lineage
(Amores et al., 1998
;
Robinson-Rechavi et al.,
2001
). This fact is represented here by the Fugu specific
duplication of the Her6 gene (FrHer6.1 and FrHer6.2)
(Gajewski and Voolstra, 2002
).
Furthermore, the misplacement of the chicken Hairy genes at the base of the
rest of tetrapode genes was noted previously
(Gajewski and Voolstra, 2002
)
and may be due to a slight higher divergence rate of the chicken genes.
Amphi-hairyA to F are clustered in the lancelet
genome
In order to ascertain whether amphioxus Hairy specific gene duplications
occurred by tandem or transgene duplication, we isolated and analysed a
positive clone (51K5) from an amphioxus PAC library. We performed PCR on the
PAC DNA with specific Amphi-Hairy primers to determine their presence in the
genomic region isolated. We thus deduced that six of the eight Hairy genes
(Amphi-hairyA to Amphi-hairyF) are linked in the amphioxus
genome and contained in the same PAC (Fig.
2). However, we were unable to amplify Amphi-hairyG or
Amphi-hairyH with the same strategy, concluding that they are absent
in the genomic region contained in the PAC isolated.
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No signal is detected at the blastula or early gastrula stages for any of the Amphi-Hairy genes. During mid-gastrula stage, Amphi-hairyA is expressed in two domains: in the anterior endoderm (Fig. 3A) and just outside the dorsal lip of the blastopore in the presumptive neural plate (arrow in Fig. 3A).
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The expression pattern in the presumptive somitic mesoderm is very similar in Amphi-hairyB, Amphi-hairyC and Amphi-hairyD. For example, a three-stripe pattern is shown for Amphi-hairyC (Fig. 3F) and a four-stripe pattern is shown for Amphi-hairyD (Fig. 3G,H).
Amphi-hairyB, Amphi-hairyC and Amphi-hairyD are also expressed in the presumptive neural plate of the late gastrula. For Amphi-hairyB, expression is at about the level of the first and second somites (Fig. 3C, arrow) and is strongest laterally (Fig. 3D, arrows). For Amphi-hairyC, the relatively weak signal in the neural plate is in two regions: an anterior one between the first and second somites (Fig. 3F, arrow) and a posterior one from third somitic stripe towards the posterior-most part of the gastrula (Fig. 3F, twin arrows). And last, Amphi-hairyD is expressed only in a more posterior region of the neural plate between the third and the fourth somite (Fig. 3G, arrow).
In summary, Amphi-hairyA was the only gene expressed in the endoderm and not in the presumptive somitic mesoderm, region where the rest of Amphi-Hairy genes seem to be co-expressed. In the medial neural plate all four genes have a striking pattern, being all them expressed there but in complementary patterns. Amphi-hairyA is expressed in the posterior-most region, then is expressed Amphi-hairyC but leaving a gap between somitic stripes 2 and 3 that is filled by Amphi-hairyD expression, and anteriorly are Amphi-hairyB and again Amphi-hairyC expressed.
Amphi-hairyA, B, C and D genes expression in
neurula stages
During neurula stage, the expression of the four Amphi-Hairy genes becomes
more gene-specific. Amphi-hairyA gene behaves according to the
pattern observed for earliest stages: it is expressed in the endoderm and the
posterior-most part of the neural tube. In early neurula stages the signal is
conspicuous along most of the gut and also in the posterior-most third of the
dorsal nerve cord (Fig. 4A,
arrow). The nascent Hatschek's left diverticulum also expresses the gene
(arrowhead in Fig. 4A). In
later neurulae, the signal remains in the posterior third of the neural tube
(arrow in Fig. 4B), whereas the
signal in the gut is now restricted to specific regions. It is confined to its
ventral posterior-most part, a middle part, and in the anterior-most part, the
signal is restricted dorsally and in the Hatschek's left anterior diverticulum
(arrowhead in Fig. 4B) and the
anterior wall of the gut.
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During neurula stage, Amphi-hairyC (Fig. 4J) and Amphi-hairyD (Fig. 4R) have slightly different expression patterns. Although both where expressed in the neural plate (arrowheads in Fig. 4K,S, respectively), they were complementarily expressed in the dorsal portion of the anterior gut. Amphi-hairyC was conspicuously expressed as two patches at the immediate lateral to the midline anterior endoderm (arrows in Fig. 4K), whereas Amphi-hairyD mRNA was present in the dorsal-most part (arrow in Fig. 4S) and in two lateral domains of the anterior endoderm (double arrow in Fig. 4S). In a medial section, both genes were also similarly transcribed in the neural plate (arrowhead in Fig. 4L). Amphi-hairyC was also highly expressed in the segmented somites (arrows in Fig. 4L). In a posterior transverse section, another difference came to light. Although both genes where highly expressed in the forming somites, Amphi-hairyC was the only one expressed in the neural plate (arrowhead in Fig. 4M and asterisk in Fig. 4T). Moreover, whereas Amphi-hairyC is weakly expressed in the forming notochord (asterisk in Fig. 4M), Amphi-hairyD is expressed at high levels in this territory (arrow in Fig. 4T). During the late neurula stage, both genes are expressed in dorsal structures (Fig. 4N for Amphi-hairyC and Fig. 4V for Amphi-hairyD) with several differences. Amphi-hairyC signal is mainly seen in the neural tube (arrowhead in Fig. 4P), the gut, the segmented somites (arrows in Fig. 4P) and only at low levels within the dorsal notochord (asterisk in Fig. 4P). Amphi-hairyD signal is also detected in the neural tube (arrowhead in Fig. 4W) and in the gut (Fig. 4W, arrow). However, Amphi-hairyD signal is not detected within the segmented somites, but is conspicuous in the ventral notochord (asterisk in Fig. 4W). In a posterior section through level ii in Fig. 4N and 4V, the signal is detected in the forming somites for both genes (Fig. 4Q,X, respectively). However, Amphi-hairyD signal is stronger detected in the forming notochord (asterisk in Fig. 4X) than Amphi-hairyC signal (Fig. 4Q).
Interestingly, all four genes are similarly expressed within the neural tube of late neurulae as they where expressed in the neural plate of gastrula stages. That is, Amphi-hairyA being the Hairy gene expressed in a posterior-most domain of the neural tube (Fig. 4B), Amphi-hairyB being highly expressed in the anterior-most domain (Fig. 4H), and Amphi-hairyC and Amphi-hairyD similarly expressed in between (Fig. 4N,V).
Amphi-hairyA, Amphi-hairyB, Amphi-hairyC and
Amphi-hairyD genes expression in larvae
During larval stages, Amphi-hairyA expression is similar to that
observed in late neurulae. Its mRNA continues to be restricted to the
posterior-most part of the dorsal nerve cord. Within the gut,
Amphi-hairyA is strongly expressed in three regions
(Fig. 5A): in the most
posterior gut it has a two-domain pattern; in a middle region there are
scattered positive cells (double arrow in
Fig. 5A); and in the anterior
gut it is expressed dorsally in the left anterior gut diverticulum that will
become the Hatschek's pit (arrow in Fig.
5A), in the anterior wall of the gut, and in the pharynx endoderm
more ventrally. Cross-sections through level i in
Fig. 5A show
Amphi-hairyA expression in the left anterior gut diverticulum (arrow
in Fig. 5B). A cross section
through a more posterior level (ii in Fig.
5A) shows the signal within the posterior neural tube (asterisk in
Fig. 5C) and the gut (arrow in
Fig. 5C).
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In contrast to earlier stages, Amphi-hairyC is no longer detected in neural tissues. Its expression is mainly observed in the posterior paraxial mesoderm associated with the tail bud, and in the anterior endoderm, where it is conspicuously expressed in a region ventral to the mouth and the branchial anlage (arrow in Fig. 5F), and in the ventral part of the left anterior gut diverticulum or preoral pit (asterisk in Fig. 5F). A cross-section through level i in Fig. 5F shows the expression in the ventral pharyngeal endoderm (arrow in Fig. 5G), and in the posterior mesoderm through a more posterior level (Fig. 5H). In a dorsal view, the signal in the posterior tail bud is better observed (Fig. 5I).
Last, Amphi-hairyD mRNA is not longer present in the neural tube during larval stages. It is expressed only in the anterior endoderm and in the posterior tail bud, similar to Amphi-hairyC. In the anterior region of the larva, it is expressed in a three-stripe pattern. The first one marks the region where the ventral duct of the club-shaped gland is developing (asterisk in Fig. 5J), and the two posterior ones that are in the branchial anlage, may prefigure the first two gill slits (arrows in Fig. 5J). This pattern is clearly seen from a ventral view (Fig. 5K). A section through level i in Fig. 5J makes further visible Amphi-hairyD expression within the club-shaped gland (arrow in Fig. 5L), and in the posterior mesoderm through a more posterior level (Fig. 5M). It is also noticeable from a dorsal view, the signal within the entire tail bud, although a bit higher in its anterior part, the chordoneural hinge of the tail bud (Fig. 5N).
Amphi-hairyE to H expression
Although we tried extensively in situ whole-mount hybridisations with
Amphi-hairyE and Amphi-hairyF probes, we were unable to
detect any expression on amphioxus embryos. Similarly, we failed to detect
these genes by RT-PCR using specific primers for each of the two genes, in
conjunction with specific primers for Amphi-hairyC
(Fig. 6A for
Amphi-hairyE and Amphi-hairyC, and
Fig. 6B for
Amphi-hairy-F and Amphi-hairyC). As only single clones for
Amphi-hairyE and Amphi-hairyF were isolated from gastrula
and neurula embryonic libraries (see Table S2 at
http://dev.biologists.org/supplemental/),
we concluded that either they are expressed during embryogenesis at levels
which are very low to be detected with whole-mount in situ hybridisation or
RT-PCR or they are present due to transcription leakage. We also performed
RT-PCR experiments on adult mRNA and were unable to detect their expression
(data not shown).
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Discussion |
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All eight amphioxus Hairy genes are closely related to both vertebrate and
protostome Hairy genes (Fig.
1B; see Fig. S1 at
http://dev.biologists.org/supplemental/).
The existence of multiple copies of Hairy genes in the amphioxus genome is not
in conflict with the `pre-duplicative' state of its genome. All the copies put
together would constitute the pro-orthologue of vertebrate Hairy genes
(Fig. 1B) and each Amphi-Hairy
gene is a trans-orthologue of each vertebrate gene, which means that they have
arose by independent duplication in the B. floridae genome. This is
enhanced by the finding that at least six of the Hairy genes are closely
linked in the genome, suggesting their origin by tandem duplication. The fact
that the cephalochordate lineage predated the massive gene/genome duplications
at the origins of vertebrates (Spring,
2002) does not imply that its genome is not evolving, meaning that
it can undergo specific gene duplications, gene losses, etc. Amphioxus may
resemble the ancestor of the vertebrates, but it is not the ancestor, only its
closest living relative, a privileged position that did not include the
freezing of its genome.
Differential fate after duplication of the amphioxus Hairy genes: the
DDC model
Several mathematical models have been developed to explain the future of
paralogue genes after duplication from a single ancestral gene. These models
predict that duplicate genes initially have fully overlapping, redundant
functions, such that one copy may shield the second from natural selection, if
gene dose is not crucial. Because deleterious mutations occur more frequently
than beneficial ones (Lynch and Walsh,
1998), the classical model predicts that one of the duplicate loci
should most commonly deteriorate into a pseudogene
(Watterson, 1983
). The
classical model also considers a rarer alternative: maintenance of duplicate
copies, owing to the fixation of a rare beneficial mutation in one copy that
endows it with a novel function, while the other maintains the original role
(Ohno, 1970
). The DDC model
(for duplication-degeneration-complementation) was proposed by Force et al.
(Force et al., 1999
) as a
possible explanation for the higher maintenance of duplicate genes in
duplicated genomes observed than that expected under the classical
mathematical models. According to this model, duplicate copies of an ancient
single gene preserve their maintenance in the genome by subfunctionalisation,
i.e. by differential degeneration of regulatory regions among the duplicate
genes. Hence, paralogue genes decouple a pleiotropic role that was carried out
by their single ancestor gene and become thus all copies necessary for
carrying out all the functions that the ancestral pre-duplicate gene carried
out.
Amphi-hairyA, Amphi-hairyB, Amphi-hairyC and Amphi-hairyD genes have expression patterns that generally do not overlap, although some of the differences are subtle. Briefly, Amphi-hairyA is mainly expressed within the endoderm and to a lesser extent at the posterior neural tube, Amphi-hairyB in the neural tube, posterior paraxial mesoderm and somites, and Amphi-hairyC and Amphi-hairyD are more widely expressed in all three embryonic layers. There are several cases of combinatorial patterns among the Amphi-Hairy genes all along amphioxus development. More noticeable are those within the neural plate first and the neural tube latter, and those in some anterior endoderm-derived structures in amphioxus larvae. The single mouse gene Hes1 is expressed all along the neural tube, but strikingly, only the summation of expression for all four Hairy genes of amphioxus covers the entire amphioxus neural plate and neural tube. Amphi-hairyB is expressed at the very front of the animal, Amphi-hairyA at the posterior, and Amphi-hairyC and Amphi-hairyD genes in between. It is also interesting that a combination of two Hairy genes (Amphi-hairyA plus Amphi-hairyB) is required to include all the cells of Hatschek's left diverticulum, a suspected homologue of the vertebrate adenohypophysis. By contrast, zebrafish her9 is expressed in the whole pituitary. Hence, if we assume that this subfunctionalisation has originated by differential losses of cis regulatory sequences, one must be struck by the high degree of complexity in the regulatory regions of Hairy genes. Only for these two examples, we have to think in at least three distinct regulatory elements driving the expression of an ancestral pre-duplicative Hairy gene within the neural tube, and at least two driving the expression in the left anterior diverticulum, that have been differentially lost by the duplicate copies during or after the process of duplication. Fig. 7A summarises Amphi-hairyA to Amphi-hairyD expression data. This points out for a complex organisation of Hairy regulatory sequences. Based on the partitioned expression of Amphi-Hairy genes, distinct regulatory elements must be invoked to account for particular places of expression, which have been individualised in amphioxus duplicates. A plausible schematic representation of territorial enhancers deduced from Amphi-hairyA-Amphi-hairyD expression data is shown in Fig. 7B.
|
Insights into amphioxus early somitogenesis from Amphi-Hairy genes
expression
All three amphioxus Hairy genes that are expressed in the presumptive
somitic mesoderm (Amphi-hairyB, Amphi-hairyC and
Amphi-hairyD) shed light onto the long-standing debate regarding the
formation of the first four pair of amphioxus muscular somites. These somites
have the peculiarity that they bud off virtually simultaneously from the
dorsolateral walls of the archenteron. Hatschek
(Hatschek, 1893) claimed to
have observed embryos with a single pair of somites, whereas Conklin
(Conklin, 1932
) never detected
them and claimed that always more than one somite pair was present in all the
embryos he analyzed. Regardless of their simultaneous or sequential
anteroposterior appearance, they are molecularly prefigured one by one in the
dorsolateral wall of the archenteron, as we have detected gastrulae with a
two- to threestripe, three-stripe and four-stripe pattern
(Fig. 3). Moreover, the
maturation of those four first muscular somites also appears as a sequential
anteroposterior process, as the intensity of Amphi-hairyB,
Amphi-hairyC and Amphi-hairyD expression is stronger in
posterior (and thus younger) pre-somites than in the anterior (older) ones
(Fig. 3).
Similarities between amphioxus and vertebrate Hairy genes
Within the CNS, the mouse gene Hes1 is expressed in
undifferentiated neuronal precursor cells in the ventricular zone, and its
transcription decreases as neurogenesis proceeds until expression is no longer
detected in mature neurons or glial cells
(Sasai et al., 1992).
Accordingly, mutant mice for the Hes1 gene exhibit severe neural
defects. Moreover, in their brain there is an upregulation of some neural bHLH
factors and postmitotic neurons appear prematurely. It thus appears that
Hes1, like the Drosophila hairy gene, acts as a negative
regulator of neurogenesis, and that its downregulation is required for
precursor cells to enter the differentiation processes
(Ishibashi et al., 1995
). It
seems reasonable to think that amphioxus Hairy genes are carrying out a
similar function within the amphioxus nerve cord. In the mouse, Hes1
is expressed all along the neural tube, which corresponds to the summation of
the expression patterns of all four Amphi-Hairy genes. In larval stages, only
the anterior- and the posterior-most parts of the neural tube are positive for
a Hairy gene (Amphi-hairyB, Fig.
5D, and Amphi-hairyA,
Fig. 5A, respectively). It is
tempting to speculate that these differences may account for differential
maturation rates along the neural tube. In the zebrafish, the Hairy orthologue
her9 is expressed in the mid- and hindbrain but not in the
midbrain-hindbrain boundary (MHB) (Leve et
al., 2001
). It is striking therefore to note a gap also in the
anterior amphioxus neural tube (Amphi-hairyB expression in Figs
4,
5). The existence of a
tripartite brain in cephalochordates is still debatable
(Ferrier et al., 2001
) and the
gap of expression in this region suggests the presence of a MHB.
In endodermal derivatives, Hes1 is expressed in, and required for,
the proper development of the endocrine islet cells of the mouse pancreas as
well as other dispersed endocrine cells along the entire gut. It was suggested
that Hes1 functions as a general negative regulator of endodermal
endocrine differentiation, similar to its action within neural precursors
(Jensen et al., 2000).
Similarly, the amphioxus Hairy genes are expressed in the developing gut and
certain derivatives in the larva (Amphi-hairyA, Amphi-hairyC and
Amphi-hairyD). Amphioxus does not have a discrete pancreas but has
several types of endocrine cells incorporated into the gut epithelium, some of
which are possibly homologous of the pancreas-islet cells of mammals
(Holland et al., 1997b
).
Interestingly, patches of Amphi-hairyA expression in the gut may
represent regions with presumptive endocrine cell types. All Hairy genes but
Amphi-hairyB are also conspicuously expressed in combinatorial
patterns in some endoderm-derived glands. Briefly, in the left gut
diverticulum, Amphi-hairyA is predominantly expressed in the
dorsoposterior region and Amphi-hairyC in its ventral region, whereas
Amphi-hairyD is conspicuously expressed in the club-shaped gland.
Hatschek's left gut diverticulum contributes to the Hatschek's pit in the
adult, a structure thought to be homologous to the vertebrate adenohypophysis
(Whittaker, 1997
), an organ
where the zebrafish Hairy gene her9 is expressed
(Leve et al., 2001
).
In summary, the addition of the expression patterns of Amphi-hairyA to Amphi-hairyD genes closely resembles the expression of the single mouse Hes1 gene or the multiple Hairy genes in other vertebrate species. They are expressed in the CNS (all of them in complementary patterns), in the posterior paraxial mesoderm (Amphi-hairyB, Amphi-hairyC and Amphi-hairyD), in the posterior compartment of the segmented somites (Amphi-hairyB and Amphi-hairyC), in the gut (Amphi-hairyA always and Amphi-hairyC and Amphi-hairyD in neurula and larval stages), and in the notochord (Amphi-hairyD). Hence, the common ancestor of cephalochordates and vertebrates already possessed a single Hairy gene of a very pleitropic nature, in contrast to protostome Hairy genes. This pre-duplicative gene at the origins of vertebrates already functioned in similar territories as it does in extant vertebrates. In the amphioxus lineage, the highly pleiotropic gene suffered duplication and distinct regulatory regions were most probably maintained in particular copies.
Do the Amphi-Hairy genes cycle within the amphioxus posterior
paraxial mesoderm?
There is not a single technique available to demonstrate whether one (or
more) amphioxus Hairy genes are cycling within the PSM as they do in amniotes.
Amphioxus somitogenesis is divided in two different phases. During an earlier
phase, the somites originate by the budding off of the dorsolateral walls of
the archenteron (which could be considered a sort of PSM) forming their
coeloms by enterochoely. The first eight pairs of muscular somites are formed
during this early phase from paraxial mesoderm formed during gastrulation
(Holland et al., 1997a). By
contrast, during the second phase somites arise directly from the
proliferative tail bud by a schyzocoelic process
(Schubert et al., 2001
)
without the intervention of any visible PSM between the tail bud and the
nascent somites. Hence, if any Hairy gene has a cycling behaviour, it would
only be visible during the early phase of amphioxus somitogenesis or in the
tail bud itself. Only Amphi-hairyB, Amphi-hairyC and
Amphi-hairyD could in principle cycle during the first phase of
somitogenesis, as they are expressed at the right place at the right time.
Nevertheless, the first eight muscular somites arise very quickly (within a
few hours) and thus there is not much time for any cycling gene transcription.
Furthermore, the half-life of mRNA may mask short pulses of gene
transcription, and these would be missed in in situ hybridisation analyses of
hardly synchronous embryos.
In the chicken, the onset of the dynamic expression of Hairy genes
correlates with ingression of the paraxial mesoderm territory from the
epiblast into the primitive streak. Hence, the number of oscillations
experienced by somitic cells is correlated with their position along the
anteroposterior axis (Jouve et al.,
2002). If any Hairy gene is being regulated accordingly to this
cyclic behaviour, we should have been able to detect their `on and off'
expression within or around the blastopore, the equivalent structure to the
primitive streak. This was not the case, although again masking of cycling due
to fast development may had happened. Besides, we have detected Amphi-Hairy
genes expression within the paraxial mesoderm (Amphi-hairyB,
Amphi-hairyC and Amphi-hairyD) as stripes that prefigure the
first muscular somites. Moreover, we saw gastrulae with two to three, three or
four stripes (Fig. 3), which
may indicate that they are formed one by one, in the anterior PSM, similar to
Hairy genes in lower vertebrates such as the zebrafish her6 gene or
both Xenopus Hairy genes. The expression of these genes is seen as
one to three stripes at the anterior PSM that prefigure the regions where new
somites will be added (Pasini et al.,
2001
; Davis et al.,
2001
). Hence, we suggest that the cycling behaviour of the Hairy
subfamily may be an amniote novelty. However, as discussed above, a cycling
behavior of amphioxus Hairy genes cannot be totally discarded. Alternatively,
other genes belonging to the E(spl) subfamily may be cycling in amphioxus, as
her1 and her7 in zebrafish
(Holley et al., 2000
;
Leve et al., 2001
). In
addition, other non-bHLH genes related to the Notch signaling pathway, such as
the Notch ligand DeltaC, cycle in the zebrafish PSM
(Jiang et al., 2000
). Thus, it
is possible that other genes related to the Notch signalling pathway or other
genes from the E(spl) family cycle in the amphioxus PSM.
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ACKNOWLEDGMENTS |
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Footnotes |
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* Present address: Division of Developmental Biology, National Institute for
Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK
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