CNRS UMR 6061, IFR 97, Faculté de Médecine, Université Rennes 1, 2 avenue Léon Bernard, CS 34317, 35043 Rennes Cedex, France
Author for correspondence (e-mail:
luc.paillard{at}univ-rennes1.fr)
Accepted 12 October 2004
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SUMMARY |
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Key words: mRNA stability, Poly(A) tail, Metamerization, Notch, Suppressor of Hairless/RBP-J
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Introduction |
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A recent report demonstrated a role for the Wnt/ß-catenin signalling
pathway in somite segmentation (Aulehla et
al., 2003). However, the segmentation process largely relies on
the dynamic expression of genes of the Notch signalling pathway
(Maroto and Pourquie, 2001
;
Saga and Takeda, 2001
). Notch
protein is a cell surface receptor that, when bound by its ligand from the
Delta family, undergoes a proteolytic cleavage that releases the intracellular
domain (ICD) in the cell. Notch ICD then associates with a CSL
(CBF1/RBP-J
, Suppressor of Hairless,
Lag1) protein. The Notch ICD-CSL complex transactivates a number of
genes, including enhancer of split/hairy-related genes. CSL proteins
that are not associated with Notch ICD repress the transcription of these
target genes (for a review, see Mumm and
Kopan, 2000
).
Some segmentation genes belonging to the Notch pathway display particular
expression patterns in the PSM of chick or mouse. Their corresponding mRNAs
are cyclically expressed, progressing as a wave in the PSM toward the anterior
part of the embryo once during the formation of each somite. This wave of
expression does not rely on cell migration. Rather, cells in the PSM are able
to successively express and repress segmentation mRNAs, and this periodicity
is thought to be the cause for periodic somite formation
(Maroto and Pourquie, 2001;
Saga and Takeda, 2001
). The
combination of the expression of these genes with the local concentration of
FGF8 commits certain cells to form the boundary between two adjacent somites
(Dubrulle et al., 2001
).
FGF8 mRNA is transcribed only at the posterior extremity of the
embryo, leading to a protein gradient that decreases toward the anterior
extremity (Dubrulle and Pourquié,
2004
).
In Xenopus laevis, the only cycling mRNAs described to date are
ESR9 and the closely related mRNA ESR10
(Li et al., 2003). Other
segmentation mRNAs show a different expression pattern in the PSM
(Jen et al., 1999
;
Jen et al., 1997
;
Lamar et al., 2001
;
Sparrow et al., 1998
). Some
are expressed in the caudal-most region of the PSM (tailbud domain). They are
also expressed very transiently in the anterior or posterior part of one or a
few presumptive somites (somitomeres). These segmentation mRNAs may be
expressed in a given somitomere and completely absent in the following one,
demonstrating a very dynamic expression pattern.
This dynamic expression pattern of segmentation genes requires: (1) that
the promoters controlling the expression of these genes can rapidly be
switched on and off; (2) that the corresponding mRNAs are unstable, so that
their expression patterns follow the transient activity of the promoters; and
(3) that the encoded proteins are unstable for the same reason. The first
point was illustrated for the lunatic fringe gene. In mice, the
transcription of this gene is activated in a periodic fashion, and elements
within the promoter that are responsible for cyclic expression were identified
(Cole et al., 2002;
Morales et al., 2002
).
Abolishing the cyclic pattern of expression of lunatic fringe strongly affects
somitic segmentation (Dale et al.,
2003
; Serth et al.,
2003
). The second point was observed for the Xenopus
Hairy2a gene. In transgenic Xenopus embryos, only the
combination of the Hairy2a gene promoter with a mRNA destabilizing
element [originating from Hairy2a 3' untranslated region (3'UTR)
or from the 3'UTR of another unstable mRNA] was able to confer to a
reporter gene an expression pattern similar to that of endogenous Hairy2a
(Davis et al., 2001
). It was
also noticed in mice that lunatic fringe mRNA was almost as unstable as intron
sequences (Cole et al., 2002
;
Morales et al., 2002
).
Finally, both lunatic fringe and Hes7 proteins show a dynamic expression
pattern, and, in the case of Hes7, this relies on
ubiquitin-proteasome-mediated degradation of the protein
(Bessho et al., 2003
;
Dale et al., 2003
).
The molecular mechanisms leading to instability of the segmentation mRNA
have not been investigated. However, in vertebrates, mRNA degradation is
frequently triggered by mRNA deadenylation (shortening of the poly(A) tail)
(Beelman and Parker, 1995).
Accordingly, a deadenylation mechanism may be required for somitic
segmentation. We have described a sequence-specific mRNA deadenylation
mechanism that is active in early Xenopus embryos. A family of
Xenopus maternal mRNAs (the Eg/c-mos family) is deadenylated after
fertilization of Xenopus eggs. This is mediated by a short sequence,
EDEN (Embryo Deadenylation ElemeNt) that is present within the 3'UTR of
Eg/c-mos mRNAs. The associated RNA-binding protein, EDEN-BP (EDEN-binding
protein), was purified and shown by immunodepletion and rescue experiments to
be required for EDEN-dependent deadenylation
(Paillard et al., 2003
;
Paillard et al., 1998
). In
this article, we adressed the requirement of the EDEN-BP-dependent mechanism
for somitic segmentation in Xenopus embryos.
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Materials and methods |
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Plasmid construction and RNA synthesis
ESR5, X-Delta2 and XSu(H) 3'UTRs were PCR-amplified from
Xenopus embryos cDNA using the primers 289 and 291 (ESR5), 279 and
280 (X-Delta-2), and 283 and 284 [XSu(H)]. The PCR products were directly
cloned in pGEM-T (Promega) to be used as matrices for cross-linking analysis.
The XSu(H) plasmid was digested with BglII and XbaI, and
cloned into the same sites of pGbEg2-497
(Bouvet et al., 1994) (between
the 5'UTR of Xenopus ß-globin and a 65-nucleotide poly(A)
track) for deadenylation analysis. The resulting plasmid pGbXSu(H) used for
deadenylation analysis was linearized by restriction digest using
EcoRV for the synthesis of poly(A)+ RNAs.
32P-labelled and capped in vitro transcripts were obtained from
these matrices using the Riboprobe transcription kit (Promega) in the presence
of cap analog (Biolabs). pCS2-GFPmut plasmid and pCS2-XSu(H)1 plasmid were
linearized with NotI, and the resulting matrices were used to obtain
capped mRNA with the Message Machine kit (Ambion). EDEN-BP mRNA for rescue
experiments was transcribed from pT7T-EDEN-BP
(Paillard et al., 1998
).
Morpholinos
Morpholinos sequences were:
The CAT bases shown in bold are complementary to the initiating ATG codons. Morpholino oligonucleotides were dissolved in water.
Embryo manipulations, embryo staining and histological methods
Injections were made into the animal hemisphere of Xenopus laevis
embryos, into one or two blastomeres at the 2-cell stage, or one blastomere at
the 4-cell stage. Depending on the experiment, the injected solution contained
one or several of the following: 0.5-1.0 fmol of reporter transcript, 0.5-2.0
fmol of EDEN-BP, GFP or Xsu(H) mRNA, 100 or 200 ng of antibodies, and 1 to 50
ng of morpholinos, in a final volume of 9.2 to 36 nl. Embryos were allowed to
develop at 22°C. Following injection with a GFP mRNA-containing solution,
embryos were sorted, using a fluorescence microscope, at about stage 22
according to injection site (left/right, dorsal/ventral).
Whole-mount in situ hybridization was performed as described
(Harland, 1991). The EDEN-BP
probe corresponds to the whole open-reading frame cloned into the pT7TS vector
(Paillard et al., 1998
). The
sense probe was obtained by in vitro transcription from the T7 promoter after
linearization with EcoRI, and the antisense probe from the SP6
promoter after linearization with BglII. The ESR5, ESR9 and X-Delta2
probes were made from plasmids that were either constructed by RT-PCR or
provided by the IMAGE consortium. The MHC4 antisense probe was obtained from
the pGEM-MHC4 plasmid (a kind gift of Anna Philpott, Cambridge, UK).
Whole-mount immunohistochemistry was performed as described
(Jen et al., 1999
), using the
12/101 (Kintner and Brockes,
1985
) hybridoma (obtained from the Developmental Studies Hybridoma
Bank, University of Iowa) supernatant at a dilution of 1/10. The secondary
antibodies used were HRP-conjugated and staining was performed with
Diaminobenzidine.
Stage 39 embryos were fixed and embedded in paraffin wax and horizontal sections were stained by Hematoxylin and Eosin. Microscopy analysis was performed through the serial sections made on several different whole embryos.
Analytical methods
Cross-linking experiments were as described
(Paillard et al., 1998). For
deadenylation analysis, RNA from five injected embryos was extracted using
Tri-reagent (Euromedex). Samples were analyzed by electrophoresis on
polyacrylamide/urea gels, followed by autoradiography and/or STORM
analysis.
Embryos used for western analysis were homogenized in 100 mM Tris-HCl (pH
7.5), 4 mM EDTA, 16 mM KCl, 0.01% Triton X100, 10 µg/ml Pepstatin, 10
µg/ml Leupeptin, 10 µg/ml Chymostatin. Unsoluble particles were removed
by centrifugation (10 minutes at 12,000 g) and proteins were
extracted by one volume of 1,1,2-Trichloro-trifluoroethane (Fluka). Proteins
were resolved by 10 or 12% SDS-PAGE, blotted onto nitrocellulose, probed by a
primary antibody and revealed either by alkaline phosphatase-coupled secondary
antibodies using ECF for analysis on a STORM 860, or peroxydase-coupled
secondary antibodies using ECL. Primary antibodies used were 83 antiserum
against EDEN-BP (Paillard et al.,
1998) or the cdc2 A17 monoclonal antibody (Abcam).
Real-time PCR was performed with an ABI Prism 7000 apparatus (Applied),
using SybrGreen mastermix and the primers 482 and 483 [XSu(H)], or 19 and 20
(EF1). For each mRNA sample from individual embryos, quantifications
were done in triplicate. Relative mRNA quantities were given by the difference
of the Ct with a standard Ct (Ct0) according to the formula [relative
quantity=2 exp(Ct0-Ct)]. The results are expressed as the ratio of relative
quantity of XSu(H) to relative quantity of EF1
.
Immunoprecipitations were made using purified antibodies against EDEN-BP,
bound and chemically cross-linked (with dimethyl pimelimidate) to magnetic
protein A-Dynabeads according to the manufacturer's instructions (Dynal).
After the incubation of embryo extract (2 hours, 4°C), beads were
abundantly rinsed, and bound proteins were eluted by boiling in SDS buffer.
RNA was extracted with Tri-reagent (Euromedex), reverse-transcribed and
amplified with primers 449 and 474 [XSu(H)], or 141 and 142 (EF1).
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Results |
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The expression level of EDEN-BP in tailbud (stage 25) embryos after injection of E-Mo into both blastomeres of two-cell embryos was analyzed by western blotting (Fig. 2A, upper panel). Similar loading in all lanes was attested by reprobing the same blot with anti-cdc2 antibodies (lower panel). As compared with control sibling embryos (lane 1), E-Mo strongly decreased the expression level of EDEN-BP (lane 2). A higher expression level was restored by the co-injection of 0.5 or 2 fmol of EDEN-BP mRNA with E-Mo (lanes 3, 4). Accordingly, morpholinos inhibit the expression of EDEN-BP in tailbuds and the microinjection of EDEN-BP mRNA can rescue this inhibition to an almost normal level of expression.
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|
In addition to the rescue experiments, several experiments were performed to test specificity. First, as the effects of the morpholinos are observed in dorsal regions of the embryo, it is expected that injecting morpholinos into presumptive ventral regions should yield reduced effects. Accordingly, injections of morpholinos were repeated in one blastomere at the four-cell stage, together with a GFP mRNA. A smaller amount of morpholinos (12.5 ng of each) was injected, so as to account for the reduced volume of the injected cells. Embryos were sorted at early tailbud according to ventral or dorsal expression of GFP. Injection of E-Mo in a presumptive dorsal blastomere at the four-cell stage yielded a similar percentage of affected embryos as did injection at the 2-cell stage. By contrast, after injection into a ventral blastomere, the percentage of affected embryos was much lower (Table 1). Second, the percentage of affected embryos was dependent on the quantity of injected morpholinos; injecting 12.5 ng (instead of 25 ng) of each morpholino reduced the percentage of affected embryos from 90% to 42% (Table 1). Finally, anti EDEN-BP antibodies specifically inhibited the rapid deadenylation of EDEN-containing RNAs, when injected into Xenopus embryos (see Data S1 in supplementary material). We therefore analyzed the developmental consequences of injecting these antibodies into one blastomere of two-cell embryos. Anti EDEN-BP, but not control non-immune antibodies, yielded segmentation defects similar to those provoked by the injection of morpholinos (Fig. 3A,C; Table 1). As for morpholinos, only the injected side of the embryo was affected (Fig. 3B,D). Also, both sides of the antibody-injected embryos that showed defects in somitic segmentation were similarly stained with the 12/101 monoclonal antibody (Fig. 3C,D). If the segmentation defects caused by the morpholinos were just side effects, it would be highly improbable that the antibodies would cause the same effect, as molecularly different tools are expected to cause different side effects.
|
EDEN-BP morpholinos alter the segmented expression pattern of ESR5 and X-Delta2, but not ESR9, in the presomitic mesoderm
The PSM is not morphologically segmented, but harbors a segmented
expression pattern of a number of genes. Several zebrafish mutants display
somite segmentation defects that are associated with a complete loss of the
segmented expression of genes belonging to the Notch pathway in the PSM. By
contrast, the Tbx24 mutant fused somites also shows strong somite
segmentation defects, but Notch pathway genes retain some segmented expression
pattern in the PSM (Nikaido et al.,
2002; van Eeden et al.,
1998
). We therefore tested whether the segmentation defects caused
by the functional inhibition of EDEN-BP are associated with an altered
expression pattern of segmentation genes in the PSM.
The expression patterns of three segmentation markers (ESR5, ESR9 and
X-Delta2) in the PSM of E-Mo-injected embryos were analyzed by in situ
hybridization (Fig. 4). ESR5
and X-Delta2 are a Notch target and a Notch ligand, respectively.
ESR9 is the only Xenopus mRNA known to date to have a
cycling expression pattern comparable to what is described in higher
vertebrates (see Introduction) (Li et al.,
2003). As such, it may be the Xenopus oscillator, or a
direct output of the Xenopus oscillator, and is probably upstream of
most other gene products in the segmentation process.
|
The expression pattern of X-Delta2 on the non-injected side was also
similar to previous observations (Jen et
al., 1997). X-Delta2 was weakly expressed in the anterior parts of
S4, and more strongly in the TBD and the anterior parts of S1 to S3
(Fig. 4C). On the injected
side, X-Delta2 expression was weaker in the TBD. In the somitomeric region,
the expression level of this mRNA was not reduced, but a segmented pattern of
expression was no longer detected (Fig.
4D). Defects in the expression of ESR5 and X-Delta2 similar to
those shown in Fig. 4 were
detected in 48% (34/71) and 54% (36/67), respectively, of the embryos
examined.
As a cycling mRNA, ESR9 shows a dynamic mode of expression that
can be subdivided into three phases, named I to III
(Li et al., 2003;
Pourquie and Tam, 2001
). These
three phases were found in the PSM of embryos injected with E-Mo
(Fig. 4E-G'). The
distribution between the phases was not different in control and E-Mo-injected
embryos [Phase I, 31% (13/41) in control, 41% (44/107) in E-Mo; Phase II, 34%
(14/41) in control, 33% (35/107) in E-Mo; Phase III, 34% (14/41) in control,
26% (28/107) in E-Mo]. Furthermore, the embryos injected with E-Mo were fairly
symmetric regarding the expression of ESR9
(Fig. 4E-G'), indicating
that the injected and the uninjected sides of the embryos harbored the same
expression pattern for ESR9. We conclude from these data that EDEN-BP
morpholinos have no effect on the expression of ESR9. Therefore, they act
downstream of ESR9, but on or upstream of ESR5 and X-Delta2, in the
segmentation process.
XSu(H) mRNA is regulated by EDEN-BP
The above results show that a downregulation of EDEN-BP disrupts the
expression pattern of ESR5 and X-Delta2 mRNA. UV
cross-linking experiments were performed to test the ability of the
3'UTRs of these mRNAs to bind to EDEN-BP. GbORF-mosEDEN
(Paillard et al., 1998) was
used as a positive control for EDEN-BP binding
(Fig. 5A, lanes 1-4). This mRNA
produced a 53-55 kDa signal in the absence of competitor (lane 1), that was
efficiently competed by a 5-fold (lane 2) or 50-fold (lane 3) molar excess of
the same unlabelled RNA, but not by a 50-fold molar excess of the same
unlabelled RNA with the EDEN deleted (lane 4). That this signal corresponds to
EDEN-BP has been previously documented by immunoprecipitation and by
experiments cross-linking with recombinant protein
(Paillard et al., 1998
). No
signal was detected using either ESR5 3'UTR (lanes 5-8) or X-Delta2
3'UTR (lanes 9-12) as cross-linking probes, indicating that these
3'UTRs do not bind EDEN-BP and that they are not direct targets of the
EDEN-BP-dependent mechanism.
|
As EDEN-BP binds the XSu(H) 3'UTR in an in vitro (UV cross-linking)
assay, we next tested the in vivo interaction of EDEN-BP with XSu(H) mRNA.
EDEN-BP was immunoprecipitated from embryo extracts, and the associated mRNAs
identified by RT-PCR. These analyses showed that XSu(H) mRNA was only
precipitated by the anti-EDEN-BP antibodies
(Fig. 5B, upper panel, lane 8),
and not with protein A alone or with non-immune antibodies (lanes 4, 6). An
unrelated mRNA, EF1 (Krieg et al.,
1989
), was not detected in the EDEN-BP immunoprecipitated mRNAs
(lower panel). Accordingly, EDEN-BP interacts with XSu(H) mRNA in vivo.
We next tested whether the XSu(H) 3'UTR confers a rapid,
EDEN-BP-dependent deadenylation to a reporter mRNA after injection into
Xenopus embryos. Reporter RNAs containing the XSu(H) 3'UTR
[GbXSu(H)] were injected with either control non-immune or anti-EDEN-BP
antibodies. The deadenylation behaviour of this reporter transcript was
analyzed by denaturing electrophoresis and autoradiography of RNAs extracted
at different times after injection (Fig.
5C). The deadenylation activity was quantified as the relative
amount of deadenylated transcript at each time point
(Fig. 5D). As for the
Gb-Eg2-410 transcript (see Fig. S1B in supplementary material), GbXSu(H) RNA
was rapidly deadenylated in the presence of control, non-immune antibodies
(lanes 6-10). Importantly, deadenylated RNAs are stable in early
Xenopus embryos until the blastula stage
(Audic et al., 1997;
Voeltz and Steitz, 1998
),
which allows the deadenylated transcript to be visualized and quantified. By
contrast, the injection of anti-EDEN-BP antibodies strongly reduced the
deadenylation of the GbXSu(H) (lanes 1-5). These data show that the XSu(H)
3'UTR contains the sequence information required to direct a reporter
RNA to the EDEN deadenylation pathway in Xenopus embryos.
As mRNA deadenylation leads to degradation after the blastula stage,
inhibiting the deadenylation of endogenous XSu(H) mRNA may increase its
steady-state level in tailbuds. Steady-state levels of XSu(H), as compared
with steady-state levels of EF1, were assessed in individual embryos by
real-time RT-PCR in stage-25 control (non-injected), E-Mo injected, and
rescued embryos (Fig. 5E). Some
variability occurs between individual embryos, but the average steady-state
level of XSu(H) mRNA is clearly higher in E-Mo-injected embryos than in
control or rescued embryos. These data show that the functional inhibition of
EDEN-BP in tailbuds causes the overexpression of XSu(H), probably via an
inhibition of its deadenylation.
Evidences for other targets of EDEN-BP in the segmentation process
The functional inhibition of EDEN-BP causes somitic segmentation defects
and overexpression of XSu(H). To test whether a causal relationship exists
between these events, the effect of overexpressing XSu(H) on segmentation was
analyzed. Embryos were injected into one blastomere at the two-cell stage with
an in vitro transcribed mRNA containing the XSu(H)1 ORF [but devoid of the
XSu(H)1 3'UTR], and were allowed to develop until stage 28. They were
then stained with the myotome-specific 12/101 antibody. Sixty-nine percent
(130/189) of the embryos injected with XSu(H)1 mRNA displayed
segmentation defects (Fig.
6A,B). These defects were similar to those provoked by
anti-EDEN-BP morpholinos or antibodies (compare with Figs
2,
3). Embryos injected with
XSu(H)1 RNA were also analyzed by in situ hybridization for the
expression of ESR5 and X-Delta2. Twenty-seven percent (21/79) and 44% (42/96)
of these embryos displayed an expression pattern of ESR5 and X-Delta2 similar
to that observed in E-Mo-injected embryos
(Fig. 6C-F, compare with
Fig. 4A-D).
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![]() |
Discussion |
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The role of post-transcriptional regulation in somitic segmentation has
been described very recently for a gene that is expressed following a precise
gradient. A decreasing gradient of FGF8 starting from the posterior extremity
of the embryo plays a key role in somitic segmentation and the acquisition of
segmental identity (Dubrulle et al., 2001b), and the slope of this gradient is
given by the stability of the corresponding mRNA
(Dubrulle and Pourquie, 2004).
The role of post-transcriptional regulation in segmentation has more often
been approached for genes that are cyclically or at least transiently
expressed. For instance, Hairy2a mRNA is transiently expressed in two
somitomeres of Xenopus embryos, and an mRNA-destabilizing element
localized within the 3'UTR is required to achieve this expression
pattern (Davis et al., 2001
).
Computational modelling showed that mRNA instability is required to achieve
sustained oscillations of cyclic genes
(Lewis, 2003
). The
3'UTRs of several segmentation genes, from Xenopus or other
species, were able to replace that of Hairy2a, to confer a transient
expression pattern to a reporter gene, showing that they also contain
destabilization elements (Davis et al.,
2001
). This suggests that, in most species, the transient
expression pattern of most segmentation mRNAs requires destabilizing elements
that are localized within their 3'UTRs. The 3'UTRs of the genes
transiently expressed in Xenopus embryos that were tested here (ESR4,
ESR9, Hairy1, Hairy2a and NRARP) are not regulated by the EDEN-BP pathway.
This implies that the corresponding mRNAs are rapidly degraded by a mechanism
different from the EDEN-dependent deadenylation pathway. In Xenopus,
at least three deadenylation mechanisms have been described in addition to the
EDEN mechanism (Audic et al.,
2001
; Fox and Wickens,
1990
; Varnum and Wormington,
1990
; Voeltz and Steitz,
1998
) (reviewed by Paillard
and Osborne, 2003
), and different mRNA destabilization mechanisms
may also exist in other species.
In contrast to the above examples, XSu(H) mRNA is ubiquitously
expressed in Xenopus embryos
(Wettstein et al., 1997). How
then can XSu(H) overexpression account for the observed segmentation defects?
As for CSL proteins in other species (Mumm
and Kopan, 2000
), XSu(H) protein probably represses the
transcription of target genes when not associated with Notch ICD. Stimulating
the Notch pathway by overexpressing the Notch ligand X-Delta1 in
Xenopus embryos inhibits neuralization (a process termed lateral
inhibition), but this effect is reversed by also overexpressing XSu(H)
(Wettstein et al., 1997
),
confirming that, in that instance, overexpressing XSu(H) inhibits Notch
signalling. As somitic segmentation in Xenopus requires the periodic
stimulation/inhibition of Notch signalling
(Jen et al., 1999
), constant
inhibition of Notch signalling may be the main cause of the defects provoked
by overexpressing wild-type XSu(H). However, inhibiting the Notch pathway by
expressing a dominant-negative mutant of XSu(H) abolishes ESR5 expression and
stimulates X-Delta2 expression in the somitomeric region
(Jen et al., 1999
;
Jen et al., 1997
) (also our
unpublished data), which is different from what we observe after
overexpressing wild-type XSu(H). Consequently, inhibiting the Notch pathway by
overexpressing wild-type XSu(H) or expressing a dominant-negative mutant of
this protein leads to dissimilar consequences on the expression pattern of
these two segmentation markers. Mutant Xsu(H) has lost its capacity to
interact with DNA, and supposedly acts by sequestering Notch ICD. By contrast,
overexpressed wild-type Xsu(H) still interacts with DNA, and inhibits the
transcription of target genes (Wettstein
et al., 1997
). This difference may be used as a working hypothesis
in future experiments designed to explain the different expression patterns of
ESR5 and X-Delta2 after expression of wild-type or mutant XSu(H).
We show here, for the first time, the involvement of a molecular mechanism
of post-transcriptional regulation in vertebrate somitic segmentation. This
work was conducted in Xenopus, but its conclusions might be
generalized to other species. The zebrafish equivalent of EDEN-BP mRNA was
reported to be expressed uniformly in embryos up to 24 hours post-fecondation
(Suzuki et al., 2000). These
results are consistent with the zebrafish EDEN-BP protein being expressed in
PSM at the time of segmentation. Similarly, EST database searches reveal that
the murine equivalent of EDEN-BP is expressed during embryogenesis, and
preliminary experiments show that murine EDEN-BP is strongly expressed in the
PSM during segmentation (C.G.-C., unpublished). In addition, in in vitro
deadenylation assays, the human and murine equivalents of EDEN-BP can behave
as deadenylation factors (Paillard et al.,
2003
) (data not shown). From these results, it can be postulated
that the fish and mammalian equivalents of EDEN-BP could be responsible for
the destabilization of segmentation mRNAs in these species. If this were the
case, it would reveal a strong conservation of post-transcriptional control in
vertebrate somitic segmentation.
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ACKNOWLEDGMENTS |
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![]() |
Footnotes |
---|
Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/131/24/6107/DC1
* Present address: 16 Barker Hall, Department of Molecular and Cell Biology,
University of California Berkeley, Berkeley, CA 94720-3202, USA
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