1 Division of Developmental Biology, National Institute for Medical Research,
The Ridgeway, Mill Hill, London NW7 1AA, UK
2 Wellcome Trust/Cancer Research UK Institute of Cancer and Developmental
Biology and Department of Zoology, University of Cambridge, Tennis Court Road,
Cambridge CB2 1QR, UK
Author for correspondence (e-mail:
jim{at}welc.cam.ac.uk)
Accepted 12 June 2003
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SUMMARY |
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Key words: Xenopus, Cell adhesion, Gastrulation, Morphogenesis, Embryogenesis, Apoptosis
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Introduction |
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Although members of the Bix/Mix family are similar in their amino acid
sequences (particularly in their homeodomains), expression patterns and
biological functions, they do differ in some respects. Most recently, for
example, they have been found to differ in their abilities to interact with
Smad proteins; the Smad-interacting motif present in Bix2 (Milk) and Mixer is
also present in Bix3, but not in Bix1, Bix4, Mix.1 or Mix.2
(Germain et al., 2000;
Randall et al., 2002
). In the
first part of this paper we add to these differences by showing that Bix3
induces a different spectrum of genes to that induced by Bix1. More
significantly, however, we reveal a novel and unexpected property of members
of the Bix family: the ability to regulate apoptosis. Apoptosis is observed in
response to overexpression of Bix3, but not in response to Bix1; we show that
it does not, however, correlate with the possession of a Smad-interaction
motif. Interestingly, inhibition of Bix3 function by microinjection of an
antisense morpholino oligonucleotide also causes apoptosis, suggesting that
apoptosis might be regulated during normal development according to particular
concentrations of Bix3.
In investigating the molecular basis of this phenomenon we found that Bix1 has cryptic apoptotic activity, which is revealed following deletion of approximately 25 C-terminal amino acids. This observation suggests that the C-terminal region of Bix1 represses the apoptotic activity of the rest of the protein, a property that presumably is not shared with the C-terminal region of Bix3. Consistent with this idea, a chimeric version of Bix1 in which the C-terminal domain is derived from Bix3 is able to induce apoptosis. The domain of Bix1 that represses apoptosis contains an essential threonine residue; if this is mutated to alanine, Bix1 again acquires the ability to cause apoptosis. We discuss the significance of these observations for the control of apoptosis and for the regulation of Bix protein activity.
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Materials and methods |
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In situ hybridisation
In situ hybridisations were performed essentially as described
(Harland, 1991), except that BM
purple was used as a substrate. In an effort to improve probe penetration,
some embryos were cut in half before performing the procedure.
Expression constructs
Capped RNA was transcribed as described
(Smith, 1993). RNA was
synthesised from the following plasmid constructs; full details are available
on request: pBix1.64T, pBix1 (1-376).64T, pBix1 (1-315).64T, pBix1
(1-225).64T, pBix1 (1-225;315-401).64T, pBix1 (1-144;225-315).64T,
pBix1/3.64T, pBix1 (1-397).64T, pBix1 SS378.64T, pBix1 T384A.64T, pBix1
EE393/4AA.64T, pBix1 Q132E.64T, pBix1 (1-376) Q132E.64T, pBix3.64T, pBix3
(1-360).64T, pBix3 (1-302).64T, pBix3 (1-214).64T, pBix3 PP313/3AA.64T,
pBix1-HA.64T, pBix3-HA.64T, pBix1
UTR.64T, pBix3
UTR.64T, pHBcl2.
All Bix constructs were HA-tagged.
DNA expression constructs were as follows: Bix1. pcDNA3, Bix1 (1-376).
pcDNA3, Bix1 (1-315). pcDNA3, Bix1 (1-225). pcDNA3, Bix1 (1-397). pcDNA, Bix1
SS378. pcDNA3, Bix1 T384A. pcDNA3, Bix1 EE393/4AA. pcDNA3, Bix1 Q132E. pcDNA3,
Bix1 (1-376) Q132E. pcDNA3, Bix3. pcDNA3, Bix3 (1-360). pcDNA3, Bix3 (1-302).
pcDNA3, Bix3 (1-214). pcDNA3 and Bix3 PP313/3AA. pcDNA3. Details of these
plasmids, all of which carry an HA tag, are available on request. The P3
reporter construct comprises 6 copies of the P3 site (TAATTGAATTA)
(Wilson et al., 1993) cloned
into a luciferase reporter containing the E4 minimal promoter
(Latinkic and Smith,
1999
).
Analysis of gene expression by real-time RT-PCR
RNA was extracted using the Roche TriPure reagent according to the
manufacturer's protocol. Briefly, animal caps were homogenised in 800 µl
TriPure reagent, extracted with 200 µl chloroform and incubated at room
temperature for 15 minutes. Debris was removed by centrifugation and nucleic
acids were precipitated by addition of isopropanol, following addition of 10
µg glycogen as carrier. DNA was removed by incubation for 30 minutes at
37°C in RNAase-free DNAase followed by Proteinase K treatment for a
further 30 minutes. Samples were phenol/chloroform extracted and RNA was
precipitated with 2 volumes of ethanol and 1/6 volume 5M LiCl at -20°C for
30 minutes.
Gene expression analyses were performed by real-time RT-PCR and a
LightCycler SystemTM (Roche). Primer pairs for cerberus, goosecoid,
XHex and Sox17 are described by Xanthos et al.
(Xanthos et al., 2001
).
plakoglobin primers are described by Kofron et al.
(Kofron et al., 1999
) and
ornithine decarboxylase primers by Heasman et al.
(Heasman et al., 2000
).
TUNEL assay
Whole-mount TUNEL staining was performed essentially as described
(Hensey and Gautier, 1998).
Briefly, embryos were fixed in MEMFA (100 mM MOPS pH 7.4, 2 mM EGTA, 1 mM
MgSO4, 4% formaldehyde) and stored in methanol at -20°C. They
were rehydrated in PBT (0.2% Tween 20 in PBS), washed in PBS, and incubated in
150 U/ml terminal deoxynucleotidyltransferase (Gibco BRL) and 0.5 µM
digoxigenin-dUTP (Boehringer Mannheim). The reaction was terminated in PBS/1
mM EDTA for 2 to 4 hours at 65°C, followed by extensive washes in PBS. The
embryos were then blocked in PBT/20% heat-inactivated lamb serum, followed by
incubation in a 1:2000 dilution of anti-digoxigenin antibody coupled to
alkaline phosphatase (Boehringer Mannheim). Embryos were extensively washed in
PBS and stained using nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl
phosphate as substrates. The reaction product was visible within approximately
30 minutes. In some cases, embryos were bleached overnight in 70% methanol and
10% hydrogen peroxide and viewed following dehydration in methanol and
mounting in 2:1 benzyl benzoate:benzyl alcohol.
tPARP cleavage assay
The poly(ADP) ribose polymerase (tPARP) cleavage assay was performed
essentially as described by Hensey and Gautier
(Hensey and Gautier, 1997).
Briefly, embryos were homogenised in EB buffer [80 mM ß-glycerophosphate
(pH 7.3), 15 mM MgCl2, 20 mM EGTA, 10 mM DTT plus protease
inhibitors] and the homogenate was centrifuged three times at 4°C, to
remove particulate matter. Five µl of each homogenate was mixed with 3
µl of 35S-labelled PARP and incubated at 24°C for 15
minutes. Samples were denatured and run on a 12% PAGE gel at 130 V for one
hour.
In vitro translation
pBix1.64T and pBix3.64T (both of which include the 5' untranslated
regions of the cDNAs) were transcribed and translated in the Promega
TNT-coupled Reticulocyte Lysate System. Briefly, 1 µg DNA was incubated
with lysate, buffer, SP6 RNA polymerase, amino acids,
[35S]-methionine and RNAase inhibitor in the presence or absence of
10 µM antisense Bix3 morpholino oligonucleotide (see
Fig. 5). Reactions were
incubated at 30°C for 90 minutes. A 1 µl aliquot from each reaction was
run on a 12% polyacrylamide gel and developed overnight.
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Western blots were performed as described
(Tada et al., 1997), using
monoclonal antibody 12CA5 to detect the HA epitope.
Transient transfections in cell culture
COS cells were transiently transfected by lipofection with variable amounts
of effector DNA, 100 ng of firefly luciferase reporter construct, 100 ng
reference plasmid pRL-TK (Promega), and appropriate amounts of pcDNA3
(InVitrogen) to a total of 800-1000 ng. Each experiment was performed in
duplicate. Cells were harvested approximately 48 hours after transfection and
Renilla and firefly luciferase activities were determined according to the
manufacturer's instructions (Dual luciferase kit, Promega). For western blots,
cells were transiently transfected with 1 µg effector DNA and harvested as
described (Sambrook et al.,
1989). Western blots to detect the HA epitope were performed as
described above.
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Results |
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Defects in cell adhesion caused by Bix3 can also be observed in isolated animal pole regions. When Bix3-expressing animal caps are dissected from Xenopus embryos at the late blastula stage, they fail to adhere and disaggregate into single cells, whereas control caps, or those expressing Bix1, form compact spheres (Fig. 2D,H,L).
Bix1 and Bix3 activate different genes to different
extents
The results described above suggest that Bix1 and Bix3
exert different biological effects. In support of this observation we find
that although Bix3, similar to Bix1, activates the
expression of endoderm-specific genes, the spectra of genes activated by
Bix1 and Bix3 differ
(Fig. 3). In assessing these
experiments we note that the levels of our reference gene,
plakoglobin, are reduced by Bix3
(Fig. 3). This is probably
because of loss of cells before the samples are harvested (see
Fig. 2); a similar effect is
seen using another reference gene, ornithine decarboxylase (data not
shown). When corrections are made for this loss (see inset,
Fig. 3), Bix3 proves
to be a particularly powerful inducer of Sox17 and
goosecoid, but it cannot induce expression of Xhex or
cerberus. By contrast, Bix1 induces XHex strongly
and cerberus to some extent. We reach the same conclusion following
experiments in which animal caps are harvested before the onset of apoptosis
and in which levels of the reference gene are similar in Bix1- and
Bix3-injected animal caps (data not shown). Bix1 and
Bix3 therefore differ in their inducing activities as well as in
their effects on cell adhesion.
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We note that TUNEL-positive cells only appear after the decrease in embryonic cell adhesion illustrated in Fig. 2, and even the more sensitive tPARP assay only detects caspase activity at mid-gastrula stage 11 (Fig. 4J). One interpretation of these observations is that the primary effect of Bix3 is to reduce cell adhesion during gastrulation and that this leads to apoptosis through anoikis. Interestingly, however, the anti-apoptotic protein human Bcl2 can delay the onset of caspase activity (Fig. 4K), and in five out of seven experiments it also delayed the onset of cell disaggregation (Fig. 4L-M), suggesting that loss of adhesion is a consequence of apoptosis and that the later appearance of TUNEL-positive cells and caspase activity are because of lower sensitivities of these techniques.
Loss of Bix3 function by antisense morpholino oligonucleotides also
causes apoptosis
Our results show that overexpression of Bix3 causes apoptosis:
what is the effect of loss of function of this gene? To investigate this
question we designed an antisense morpholino oligonucleotide directed against
the Bix3 5' untranslated region. The Bix family is
highly conserved (Tada et al.,
1998), and it proved difficult to design an oligonucleotide
specific for Bix3. In particular, it seemed probable that our Bix3
oligonucleotide might also inhibit translation of Bix1
(Fig. 5A), and indeed the
antisense Bix3 oligonucleotide blocked translation of Bix1 as well as Bix3
both in an in vitro coupled transcription/translation system
(Fig. 5B) and after injection
of RNA encoding HA-tagged forms of Bix1 and Bix3 into Xenopus embryos
(Fig. 5C). No effect was
observed on translation of Bix2 or Bix4
(Fig. 5B). Although these
observations complicate the interpretation of our experiments, we believe the
apoptotic phenotype described below is because of depletion of Bix3 and not
Bix1, because cell death can be 'rescued' by injection of the former and not
the latter (see Fig. 7).
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Although the Bix3 antisense morpholino oligonucleotide caused apoptosis in the Xenopus embryo, it had little effect on regional specification. Patterns of expression of Sox3, Otx2 and Krox20 were normal in morpholino-injected embryos, although expression levels were considerably lower (data not shown).
To confirm that the observed phenotype is a specific response to the depletion of Bix3 protein, embryos were injected with Bix3 antisense morpholino oligonucleotide together with RNA encoding Bix1 or Bix3 (Fig. 7). The Bix constructs lacked the 5' untranslated region against which the morpholino oligonucleotide was directed. Coinjection of Bix3 RNA 'rescued' apoptosis caused by the Bix3 morpholino oligonucleotide in a dose-dependent manner. Thus 100 pg Bix3 RNA had little effect (Fig. 7D), whereas 200 pg RNA elicited good rescue (Fig. 7E). Embryos coinjected with 400 pg Bix3 RNA did undergo apoptosis (Fig. 7F), perhaps because Bix3 levels were now too high. By contrast, no concentration of RNA encoding Bix1 was able to rescue the apoptotic effects of the Bix3 morpholino (Fig. 7G-I). These results suggest that the phenotype described in Fig. 6 is specifically because of the absence of Bix3 protein.
Smad-binding and transcriptional activation are not required for
Bix3-induced apoptosis
The results described above indicate that Bix3 plays a role in
cell survival during early Xenopus development and in this respect it
resembles Bix2 (data not shown) but differs from Bix1 and
Bix4 (Fig. 2 and data
not shown). In an effort to map the domain of Bix3 responsible for
the induction of apoptosis, RNA encoding C-terminal truncations of Bix3 was
injected into the animal pole regions of Xenopus embryos at the
one-cell stage. All truncated Bix3 constructs proved to cause
apoptosis (Fig. 8A), indicating
that apoptotic activity is not localised to amino acids 214-389. We note that
Bix3(1-302) and Bix3(1-214) lack the Smad interaction motif (SIM)
(Germain et al., 2000;
Randall et al., 2002
),
suggesting that interaction with Smads is not necessary for
Bix3-induced apoptosis. To reinforce this conclusion, a mutant
version of Bix3 was constructed in which two conserved prolines of the SIM
(amino acids 313 and 314) are replaced by alanines, thus creating Bix3
PP313/4AA. RNA encoding this protein also caused apoptosis, confirming that
interaction with Smad proteins is not necessary for Bix3-induced cell death
(Fig. 8A).
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Fig. 8B shows that Bix3 is an activator of transcription and that this activity is distributed throughout the C-terminal region. Mutation of the SIM (Bix3 PP313/4AA) does not affect transcriptional activation by Bix3, indicating that interaction with Smad proteins is not required for this aspect of Bix3 function. We note, however, that although transcriptional activation is greatly reduced in (for example) the construct Bix3(1-214), this is not associated with a decrease in the ability of the protein to induce apoptosis. There is, therefore, no strong correlation between transcriptional activation and the ability to cause apoptosis.
There was no evidence of apoptosis in cultured cells transfected with any of the Bix3 constructs described above (data not shown), suggesting that Bix3 apoptotic activity is context-dependent.
Bix1 has cryptic apoptotic activity that is revealed after deleting
C-terminal amino acids
In the course of experiments designed to ask why Bix1 differs from Bix3 in
its ability to cause apoptosis, we prepared a series of constructs encoding
C-terminally truncated forms of Bix1. Consistent with previous work
(Tada et al., 1998), injection
of full-length Bix1 RNA did not induce apoptosis
(Fig. 9A). To our surprise,
however, all C-terminally truncated versions of Bix1, including Bix1 (1-376)
which lacks just 25 amino acids, did cause apoptosis
(Fig. 9A). These observations
suggest that Bix1 has cryptic apoptotic activity which is usually masked by
its 25 most C-terminal amino acids.
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The apoptotic activity of truncated Bix1 requires DNA binding but not
the ability to activate transcription
We also assessed the ability of Bix1 to activate transcription, using the
methods employed for Bix3 (see Fig.
8). Bix1 proved to be a powerful activator
(Fig. 9A), and, as observed
with Bix3, activity was distributed throughout the C-terminal region (although
deletion of amino acids 376-401 and of amino acids 225-315 caused a
particularly marked decrease in activation). These results were confirmed in
experiments in which Bix1 constructs were fused to the GAL4 DNA-binding domain
and co-transfected into cultured cells together with a reporter containing
GAL4-binding sites (data not shown). This analysis revealed strong
transcriptional activity between amino acids 225 and 255 and between amino
acids 376 and 401 of Bix1.
Deletion of amino acids 377-401 caused a slight decrease in transcriptional activation activity and conferred on Bix1 the ability to induce apoptosis (Fig. 9A). There is no correlation, however, between the loss of transcriptional activity and the loss of ability to repress apoptosis because Bix1 (1-376) does retain significant transcriptional activity (Fig. 9A) and, as discussed below, Bix1 T384A has full transcription activation function but also causes cell disaggregation (Fig. 10C).
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The ability of Bix1 to prevent apoptosis is associated with threonine
384
Our results suggest that there is a C-terminal domain of Bix1 that is
capable of suppressing Bix-induced apoptosis and that this domain is
absent in Bix3. To confirm this conclusion, we made a chimeric version of Bix1
in which the 45 most C-terminal amino acids are derived from Bix3. As
predicted, this chimeric molecule is capable of causing apoptosis
(Fig. 10A).
The ability of Bix1 to repress cell disaggregation is contained within the 25 most C-terminal amino acids (Fig. 9A). The amino acid sequences of Bix1 and Bix3 are very similar within this region, but there are three positions of interest (Fig. 10B). First, Bix1 lacks a pair of serines between amino acids 377 and 378; second, Bix1 contains a threonine at position 384 whereas Bix3 contains an alanine; and finally (and perhaps of less significance), amino acids 396-399 of Bix1 comprise the residues IIRT whereas Bix3 consists of LLKS. To determine which of these groups of amino acids might be involved in repressing cell disaggregation, we designed a series of mutated versions of Bix1 (Fig. 10C). RNAs encoding Bix1 (1-397), which lacks the four C-terminal amino acids of Bix1, or Bix1 SS378, which contains two additional serines at position 378, behave in a similar manner to wild-type Bix1 when injected into Xenopus embryos at the one-cell stage. However, mutation of threonine 384 into an alanine, creating Bix1 T384A, caused apoptosis, as did mutation to alanine of two conserved glutamate residues at positions 393 and 394 of Bix1, creating Bix1 EE393/4AA (Fig. 10C).
These results first suggest that the ability of the C terminus of Bix1 to
repress apoptosis resides in part in threonine 384. In this respect, it is
interesting that Bix4 (which, similar to Bix1, does not cause apoptosis) also
contains a threonine at this position, whereas Bix2 (which does cause
apoptosis) contains an alanine. Mix.1 and Mix.2 also contain a threonine
residue at this position, and to our knowledge these proteins have not been
reported to cause cell disaggregation or apoptosis. Mixer contains a glycine
residue at the equivalent position; it has not been reported to cause
apoptosis, although Henry and Melton
(Henry and Melton, 1998)
comment on the dumbbell shape of animal caps derived from embryos injected
with RNA encoding Mixer, and the fact that one end is yolky in appearance. The
significance of threonine 384 is discussed below.
The pair of glutamate residues at positions 393 and 394 is part of the DWEEN motif, which is conserved in all Bix family members as well as in Mix.1 and Mix.2 (but not in Mixer). It is probable that this motif represents an essential structural element of the Bix family, and that mutation impairs its function.
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Discussion |
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The function of Bix3 during normal development was studied by microinjection of an antisense morpholino oligonucleotide. The results indicate that loss of Bix3 function in the Xenopus embryo also causes apoptosis, and 'rescue' experiments with different concentrations of Bix3 RNA suggest that cell survival requires Bix3 levels to be maintained within a certain range. These results, and others, are discussed below.
Different effects of Bix family members: Bix3 causes apoptosis
Members of the Bix family of homeodomain-containing proteins have similar
expression patterns (Fig. 1)
but different biological actions. One example described in this paper concerns
the inductive activities of the two genes, which differ markedly
(Fig. 3), but more significant
are their effects on apoptosis (Figs
2,
4). Overexpression of
Bix3 causes dramatic cell death in the Xenopus embryo,
whereas Bix1 has no apparent effect on cell survival whatsoever. That
the cell death caused by Bix3 is indeed because of apoptosis is confirmed by
TUNEL staining (Fig. 4A-I), by
a tPARP cleavage assay (Fig.
4J), and by the fact that death can frequently be rescued, at
least in part, by co-expression of human Bcl2
(Fig. 4K-N). We do not know
whether the apoptotic effects of Bix3 are related to its inducing activity,
which differs from that of Bix1; one way to address this question might be to
compare the inducing activities of Bix1 and Bix1(T384A) or those of chimeric
proteins in which the C-terminal regions of Bix1 and Bix3 are swapped.
Depletion of Bix3 causes apoptosis
The overexpression experiments described in this paper are complemented by
experiments using antisense morpholino oligonucleotides, which indicate that
depletion of Bix3 protein also causes apoptosis (Figs
6,
7). TUNEL staining revealed
that apoptosis occurred in dorsal tissue and in the endodermal mass, both of
which are derived from Bix3-expressing cells, and also in the neural
plate, at least some of which may derive from Bix3-expressing cells
(see Keller, 1975). Bix3
depletion had no effect on patterning of the early embryo; the spatial
expression patterns of three neural markers were normal, although their levels
were low. These experiments, together with those described above, indicate
that one function of Bix3 during normal development is to protect cells from
apoptosis, but that too much Bix3 is not a good thing, because this also
causes cells to die.
This dose-dependent effect of Bix3 is reminiscent of the
dose-dependent effects of Fgf8 in regulating apoptosis in the
developing forebrain (Storm et al.,
2003). Complete absence of Fgf8, or higher-than-normal
levels of transcription, both increase apoptosis, whereas a reduction from
normal levels has the opposite effect. This phenomenon is not completely
understood, but the authors suggest a mechanism in which cell survival
requires FGF8 (which is why apoptosis is high in the absence of signalling)
but that FGF8 also induces, in a concentration-dependent manner, inhibitors of
cell survival (which is why high concentrations of FGF8 also increase cell
death). The regulation of apoptosis by Bix3 may involve a similar mechanism,
and we are searching for targets of this transcription factor that may
function in such a scheme.
The molecular basis of induction of apoptosis
The observation that Bix1 differs from Bix3 in its ability to cause
apoptosis offers an opportunity to study the molecular basis of the
phenomenon, and our results suggest that induction of apoptosis requires DNA
binding but not transcriptional activation
(Fig. 8, Fig. 9B). Use of a series of
deletion constructs, chimeras and point mutations (Figs
9,
10) suggests that Bix1
contains a cryptic apoptotic domain whose activity is suppressed by a short
sequence at the C terminus of the protein. The corresponding region of Bix3
lacks this activity, thus rendering the protein apoptotic, and we have
identified a crucial threonine residue in Bix1 that is required for suppressor
activity; mutation of this amino acid to an alanine turns Bix1 into an inducer
of apoptosis. We are now asking whether this residue is phosphorylated and
whether it interacts with components of the cell death (or anti-cell death)
apparatus.
Regulation of apoptosis during early Xenopus
development
Little is known about the regulation of apoptosis during Xenopus
embryogenesis, although three potential pathways have recently been
identified. The first involves the methylation status of the embryo, where
Stancheva and colleagues have shown that loss of a maintenance methylase
causes the onset of apoptosis (Stancheva
et al., 2001). They suggest that hypomethylation alters the state
of embryonic blastomeres such that apoptosis is triggered by cell
differentiation. A second pathway involves signalling by Xfz8, a member of the
Frizzled family of Wnt receptors, which triggers apoptosis through a pathway
involving activation of c-Jun N-terminal kinases (JNKs)
(Lisovsky et al., 2002
).
The third pathway, which may be the most relevant to the present data,
involves signalling by members of the BMP family, although, as with the other
pathways, the link to apoptosis is rather poorly understood. It is possible
that this pathway is linked to the Frizzled pathway described above. The first
piece of evidence concerns TAK1 (TGF-ß activated kinase), a MAP kinase
kinase kinase (MAPKKK) whose activity is stimulated by TGF-ß1 or BMP-4
(Yamaguchi et al., 1995).
Overexpression of xTAK1 in the Xenopus embryo causes
dramatic apoptosis which can be rescued by human BCL2 RNA, thereby
revealing the ventralising activity of xTAK1
(Shibuya et al., 1998
). It
seems probable that TAK1 participates in a kinase cascade culminating in the
activation of JNK family members that, as described above, are involved in the
regulation of apoptosis (Xia et al.,
1995
; Moriguchi et al.,
1996
; Davis,
2000
).
Recent evidence suggests that TAK1 might be activated not only by
TGF-ß family members, but also by non-canonical Wnt signalling
(Ishitani et al., 2003).
However, further evidence implicating BMP signalling in the regulation of cell
death comes from the observation that depletion of maternal Smad8 in the
Xenopus embryo also causes apoptosis
(Miyanaga et al., 2002
).
Smad8, similar to Smads 1 and 5, is thought to mediate BMP signalling, yet
other means of interfering with BMP function have not been reported to cause
apoptosis in Xenopus (see
Miyanaga et al., 2002
).
Interestingly, the effect of depleting the embryo of maternal Smad8 can be
rescued with Smad8 but not by Smad1, suggesting that different Smads of the
1/5/8 family have different functions
(Miyanaga et al., 2002
).
Why should depletion of Smad8 give the same phenotype as overexpression of TAK1? It is possible, of course, that the two pathways are completely independent, but another idea is that cell survival in the embryo requires a balance of signalling through the two pathways, and that disrupting, or enhancing, one of them changes the extent of signal through the other. We are investigating this possibility.
Bix3 may be involved in this pathway as a gene that is regulated
by BMP signalling. Bix1 is known to be activated by BMP-4
(Tada et al., 1998), and the
same may be true of Bix3. If so, Bix3 may not be the only
apoptotic transcription factor, for overexpression of Xenopus Fli, a
member of the ETS family, also causes apoptosis and its apoptotic effects can
be reduced by a dominant-negative BMP receptor
(Goltzené et al., 2000
).
The interactions between different BMP signalling pathways and genes such as
Bix3 and XFli require further study.
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ACKNOWLEDGMENTS |
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Footnotes |
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Present address: Department of Anatomy and Developmental Biology,
University College London, Gower Street, London WC1E 6BT, UK
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Casey, E. S., Tada, M., Fairclough, L., Wylie, C. C., Heasman,
J. and Smith, J. C. (1999). Bix4 is activated
directly by VegT and mediates endoderm formation in Xenopus
development. Development
126,4193
-4200.
Cohen, J. M. (1997). Caspases: the executioners of apoptosis. Biochem. J. 326, 1-16.[Medline]
Davis, R. J. (2000). Signal transduction by the JNK group of MAP kinases. Cell 103,239 -252.[Medline]
Ecochard, V., Cayrol, C., Rey, S., Foulquier, F., Caillol, D.,
Lemaire, P. and Duprat, A. M. (1998). A novel
Xenopus Mix-like gene milk involved in the control of the
endomesodermal fates. Development
125,2577
-2585.
Frisch, S. M. and Francis, H. (1994). Disruption of epithelial cell-matrix interactions induces apoptosis. J. Cell Biol. 124,619 -626.[Abstract]
Germain, S., Howell, M., Esslemont, G. M. and Hill, C. S.
(2000). Homeodomain and winged-helix transcription factors
recruit activated Smads to distinct promoter elements via a common Smad
interaction motif. Genes Dev.
14,435
-451.
Goltzené, F., Skalski, M., Wolff, C.-M., Meyer, D., Mager-Heckel, A.-M., Darribère, T. and Remy, P. (2000). Heterotopic expression of the Xl-Fli transcription factor during Xenopus embryogenesis: modification of cell adhesion and engagement in the apoptotic pathway. Exp. Cell Res. 260,233 -247.[CrossRef][Medline]
Harland, R. M. (1991). In situ hybridization: an improved whole-mount method for Xenopus embryos. Methods Cell Biol. 36,685 -695.[Medline]
Heasman, J., Kofron, M. and Wylie, C. (2000). Beta-catenin signaling activity dissected in the early Xenopus embryo: a novel antisense approach. Dev. Biol. 222,124 -134.[CrossRef][Medline]
Henry, G. L. and Melton, D. A. (1998). Mixer, a
homeobox gene required for endoderm development.
Science 281,91
-96.
Hensey, C. and Gautier, J. (1997). A developmental timer that regulates apoptosis at the onset of gastrulation. Mech. Dev. 69,183 -195.[CrossRef][Medline]
Hensey, C. and Gautier, J. (1998). Programmed cell death during Xenopus development: a spatio-temporal analysis. Dev. Biol. 203,36 -48.[CrossRef][Medline]
Ishitani, T., Kishida, S., Hyodo-Miura, J., Ueno, N., Yasuda,
J., Waterman, M., Shibuya, H., Moon, R. T., Ninomiya-Tsuji, J. and Matsumoto,
K. (2003). The TAK1-NLK mitogen-activated protein kinase
cascade functions in the Wnt-5a/Ca(2+) pathway to antagonize Wnt/beta-catenin
signaling. Mol. Cell. Biol.
23,131
-139.
Keller, R. E. (1975). Vital dye mapping of the gastrula and neurula of Xenopus laevis. I. Prospective areas and morphogenetic movements of the superficial layer. Dev. Biol. 42,222 -241.[Medline]
Kofron, M., Demel, T., Xanthos, J., Lohr, J., Sun, B., Sive, H.,
Osada, S., Wright, C., Wylie, C. and Heasman, J. (1999).
Mesoderm induction in Xenopus is a zygotic event regulated by
maternal VegT via TGFbeta growth factors. Development
126,5759
-5770.
Lamb, T. M., Knecht, A. K., Smith, W. C., Stachel, S. E., Economides, A. N., Stahl, N., Yancopolous, G. D. and Harland, R. M. (1993). Neural induction by the secreted polypeptide noggin. Science 262,713 -718.[Medline]
Latinkic, B. V. and Smith, J. C. (1999).
Goosecoid and Mix.1 repress Brachyury expression and are required for
head formation in Xenopus. Development
126,1769
-1779.
Lisovsky, M., Itoh, K. and Sokol, S. Y. (2002). Frizzled receptors activate a novel JNK-dependent pathway that may lead to apoptosis. Curr. Biol. 12, 53-58.[CrossRef][Medline]
Mead, P. E., Brivanlou, I. H., Kelley, C. M. and Zon, L. I. (1996). BMP-4-responsive regulation of dorsal-ventral patterning by the homeobox protein Mix.1. Nature 382,357 -360.[CrossRef][Medline]
Mead, P. E., Zhou, Y., Lustig, K. D., Huber, T. L., Kirschner,
M. W. and Zon, L. I. (1998). Cloning of Mix-related
homeodomain proteins using fast retrieval of gel shift activities, (FROGS), a
technique for the isolation of DNA-binding proteins. Proc. Natl.
Acad. Sci. USA 95,11251
-11256.
Miyanaga, Y., Torregroza, I. and Evans, T.
(2002). A maternal Smad protein regulates early embryonic
apoptosis in Xenopus laevis. Mol. Cell. Biol.
22,1317
-1328.
Moriguchi, T., Kuroyanagi, N., Yamaguchi, K., Gotoh, Y., Irie,
K., Kano, T., Shirakabe, K., Muro, Y., Shibuya, H. and Matsumoto, K. et
al. (1996). A novel kinase cascade mediated by
mitogen-activated protein kinase kinase 6 and MKK3. J. Biol.
Chem. 271,13675
-13679.
Nieuwkoop, P. D. and Faber, J. (1975).Normal Table of Xenopus laevis (Daudin) . Amsterdam: North Holland.
Randall, R. A., Germain, S., Inman, G. J., Bates, P. A. and
Hill, C. S. (2002). Different Smad2 partners bind a common
hydrophobic pocket in Smad2 via a defined proline-rich motif. EMBO
J. 21,145
-156.
Rosa, F. M. (1989). Mix.1, a homeobox mRNA inducible by mesoderm inducers, is expressed mostly in the presumptive endodermal cells of Xenopus embryos. Cell 57,965 -974.[Medline]
Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual. New York: Cold Spring Harbor Press.
Shibuya, H., Iwata, H., Masuyama, N., Gotoh, Y., Yamaguchi, K.,
Irie, K., Matsumoto, K., Nishida, E. and Ueno, N. (1998).
Role of TAK1 and TAB1 in BMP signaling in early Xenopus development.
EMBO J. 17,1019
-1028.
Slack, J. M. W. (1984). Regional biosynthetic markers in the early amphibian embryo. J. Embryol. Exp. Morphol. 80,289 -319.[Medline]
Smith, J. C. (1993). Purifying and assaying mesoderm-inducing factors from vertebrate embryos. In Cellular Interactions in Development - A Practical Approach (ed. D. Hartley), pp. 181-204. Oxford: Oxford University Press.
Smith, J. C. and Slack, J. M. W. (1983). Dorsalization and neural induction: properties of the organizer in Xenopus laevis. J. Embryol. Exp. Morphol. 78,299 -317.[Medline]
Stancheva, I., Hensey, C. and Meehan, R. R.
(2001). Loss of the maintenance methyltransferase, xDnmt1,
induces apoptosis in Xenopus embryos. EMBO J.
20,1963
-1973.
Storm, E. E., Rubenstein, J. L. and Martin, G. R.
(2003). Dosage of Fgf8 determines whether cell survival is
positively or negatively regulated in the developing forebrain.
Proc. Natl. Acad. Sci. USA
100,1757
-1762.
Tada, M., Casey, E. S., Fairclough, L. and Smith, J. C.
(1998). Bix1, a direct target of Xenopus T-box
genes, causes formation of ventral mesoderm and endoderm.
Development 125,3997
-4006.
Tada, M., O'Reilly, M.-A. J. and Smith, J. C.
(1997). Analysis of competence and of Brachyury
autoinduction by use of hormone-inducible Xbra.
Development 124,2225
-2234.
Vize, P. D. (1996). DNA sequences mediating the transcriptional response of the Mix.2 homeobox gene to mesoderm induction. Dev. Biol. 177,226 -231.[CrossRef][Medline]
Wilson, D., Sheng, G., Lecuit, T., Dostatni, N. and Desplan, C. (1993). Cooperative dimerization of paired class homeo domains on DNA. Genes Dev. 7,2120 -2134.[Abstract]
Xanthos, J. B., Kofron, M., Wylie, C. and Heasman, J.
(2001). Maternal VegT is the initiator of a molecular network
specifying endoderm in Xenopus laevis.
Development 128,167
-180.
Xia, Z., Dickens, M., Raingeaud, J., Davis, R. J. and Greenberg, M. E. (1995). Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis. Science 270,1326 -1331.[Abstract]
Yamaguchi, K., Shirakabe, K., Shibuya, H., Irie, K., Oishi, I., Ueno, N., Taniguchi, T., Nishida, E. and Matsumoto, K. (1995). Identification of a member of the MAPKKK family as a potential mediator of TGF-ß signal transduction. Science 270,2008 -2011.[Abstract]