Department of Anatomy and Developmental Biology, University College London, Gower Street, London WC1E 6BT, UK
* Author for correspondence (e-mail: c.stern{at}ucl.ac.uk)
Accepted 2 September 2004
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SUMMARY |
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Key words: Xenopus, Chick, Neural induction, FGF, BMP, Wnt, Smad6, Default model
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Introduction |
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The search for neural inducing factors was unsuccessful for decades, and it
is only recently that some have been found. To date, all identified molecules
with this activity in Xenopus have in common that they inhibit BMP
signalling. This led to the widely accepted `default model', which proposes
that ectodermal cells are fated to become neural `by default' but are normally
inhibited from a neural fate by BMPs expressed throughout the ectoderm, from
which they must be released for neural induction to occur
(Hemmati-Brivanlou and Melton,
1997; Weinstein and
Hemmati-Brivanlou, 1999
;
Muñoz-Sanjuán and Brivanlou,
2002
). Consistent with this, the organizer expresses a number of
BMP antagonists, which include Chordin
(Sasai et al., 1994
);
Noggin (Lamb et al.,
1993
); Follistatin
(Hemmati-Brivanlou et al.,
1994
); DAN, Gremlin and Drm
(Hsu et al., 1998
);
Xnr3 (Hansen et al.,
1997
); Cerberus
(Bouwmeester et al., 1996
);
and Dickkopf (Glinka et al.,
1998
). In Xenopus, misexpression of any of these
antagonists leads to neural induction in animal caps, while overexpression of
BMPs has the opposite effect.
However, studies in other species (especially chick and mouse) have
questioned this simple interpretation
(Streit and Stern, 1999c). In
the chick, the expression patterns of BMPs and BMP antagonists do not fit this
model so neatly, and misexpression of neither BMPs nor BMP antagonists
produces the effects expected from the model
(Streit et al., 1998
). In the
mouse, mutants lacking the BMP antagonists Cerberus, Dickkopf-1, Noggin and/or
Chordin still develop a nervous system
(McMahon et al., 1998
;
Bachiller et al., 2000
;
Belo et al., 2000
;
Mukhopadhyay et al., 2001
).
These results suggest that BMP inhibition may not be absolutely required for
neural induction and that other molecules may be implicated in the process.
Recently, it has been shown that FGF signalling is required as an early step
for neural induction in the chick (Streit
et al., 2000
; Wilson et al.,
2000
), consistent with earlier findings in Xenopus that
BMP antagonists cannot induce neural markers when FGF signalling is blocked
(Launay et al., 1996
;
Sasai et al., 1996
). However
FGF is not a sufficient neural inducer
(Streit et al., 2000
;
Wilson et al., 2000
), and it
has been proposed that cooperation of FGF with inhibition of Wnt signalling
can repress BMP transcription (Bainter et
al., 2001
; Wilson and Edlund,
2001
; Wilson et al.,
2001
), and FGF and related factors have been shown to
phosphorylate the linker region of the BMP effector Smad1
(Pera et al., 2003
). With this
interpretation, BMP inhibition remains as playing a central role in neural
induction.
Here, we have re-evaluated the participation of BMP and BMP antagonism
during neural induction. We chose to manipulate the BMP pathway
intracellularly in a cell-autonomous way, taking advantage of Smad6, the
inhibitory Smad (Imamura et al.,
1997; Hata et al.,
1998
). BMP signalling starts with the binding of extracellular BMP
dimer to BMP receptor type II (BMPRII), which is then able to recognize BMP
receptor type I (BMPRI) forming a tertiary complex, where BMPRII activates
BMPRI by phosphorylation. In turn, active BMPRI recruits Smad1/Smad5/Smad8
proteins to the membrane and activates them by phosphorylation, which allows
them to bind to Smad4; the complex then translocates to the nucleus to
regulate transcription (von Bubnoff and
Cho, 2001
). A more divergent group of Smad proteins has been
described: the inhibitory Smads (Smad6/Smad7/Smad10). Although Smad7 inhibits
both activin/nodal-related and BMP/TGFß signalling, Smad6 is a potent and
specific antagonist of the BMP pathway. It acts by actively associating with
and blocking BMPRI, as well as by competing with Smad4 to bind phosphorylated
Smad1/5/8. In addition, Smad6 can inhibit an alternative BMP intracellular
signalling pathway involving TCF3/Lef1/ß-catenin
(von Bubnoff and Cho,
2001
).
We show that although forced BMP expression in the neural plate inhibits
the expression of the definitive neural marker Sox2, it does not
affect expression of the earlier marker Sox3. Moreover, BMP
inhibition is not sufficient for neural induction, either in competent chick
epiblast or in the prospective ventral epidermis of Xenopus. These
results suggest that BMP inhibition is a relatively late step in a molecular
cascade leading to the acquisition of neural identity. We tested the proposal
of Wilson and Edlund (Wilson and Edlund,
2001) for the chick embryo by investigating whether combinations
of FGF, BMP inhibition and Wnt inhibition might suffice; we find that no
combination of these can induce neural tissue in vivo.
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Materials and methods |
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Anti-phospho Smad1 antibody was a kind gift of P. ten Dijke
(Chang et al., 2002). In situ
hybridization and whole-mount immunocytochemistry was performed as described
(Stern, 1993
;
Streit and Stern, 2001
).
Xenopus experiments
Xenopus oocytes were fertilized in vitro and the embryos staged
according to Nieuwkoop and Faber
(Nieuwkoop and Faber, 1967).
The cSmad6 (a kind gift from P. Szendro and G. Eichele)
(Yamada et al., 1999
) coding
sequence in pCS2+, FGF8 pCS2+ (a kind gift from R. Mayor)
(Christen and Slack, 1997
) and
eFGF pCS2+ (Xenopus FGF4, a kind gift from J Slack)
(Lombardo and Slack, 1997
)
were used to produce mRNA. Capped mRNA was made with mMessage mMachine
(Ambion). Microinjection was performed as described
(Marchant et al., 1998
). The
capped mRNA was injected into the animal zone of two-cell stage embryos, into
the ventral marginal zone of four-cell stage embryos or into blastomere A4 at
the 32-cell stage together with 5-10 ng lysine-fixable fluorescein dextran
(FDX, 40,000 Mr; Molecular Probes) as a lineage tracer in
most experiments. Animal caps were dissected with eyebrow knives at stage 8-10
with the embryos in 0.75x NAM, and were allowed to grow until their
siblings reached stage 21. The accuracy of injection into the A4 blastomere
was assessed by the fate of its progeny. Embryos in which labelled cells were
found in regions other than the ventral epidermis were discarded without
further processing. The proportion of embryos discarded was almost identical
in control (18/51=35% embryos with incorrect labelling) and
Smad6-injected embryos (29/79=37%). In situ hybridization was carried
out as described (Linker et al.,
2000
).
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Results |
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Despite published reports that Smad6 should inhibit all BMP signalling
(reviewed by von Bubnoff and Cho,
2001), it is conceivable that some escapes inhibition in our
experimental setup. To overcome this, we misexpressed a dominant-negative form
of the BMP receptor (Suzuki et al.,
1994
), Chordin or Noggin, or a combination of all of the above,
with Smad6. In all cases (dnBMPR 0/6; Noggin 0/6; Chordin 0/2;
Smad6+dnBMPR+Noggin+Chordin 0/9), no expression of Sox2 was seen in
the electroporated region (Fig.
2).
|
BMP inhibition is required as a late event in formation of the neural plate
The above experiments suggest that BMP inhibition is not sufficient for
neural induction but is it necessary? To address this, we
electroporated Xenopus BMP4
(pCAß-XBMP4-IRESGFP) into the prospective neural plate
of stage 3+ embryos, and analysed the effects in time course. After 12 and 15
hours of incubation, the early marker Sox3 is not affected (0/14 at
12 hours, 0/8 at 15 hours; Fig.
3A,B,G,H), while the later marker Sox2 is strongly
downregulated in the neural plate (9/9 at 12 hours, 10/10 at 15 hours;
Fig. 3C,D,I,J). At 20 hours,
both Sox3 (10/10, Fig.
3M,N) and Sox2 (14/14;
Fig. 3O,P) are downregulated.
At this time, histological analysis showed that neural plate morphology is
lost in the electroporated region (not shown). By contrast, control embryos
electroporated with GFP show no downregulation of either marker at
any time point (0/11; Fig.
3E,F,K,L,Q,R). These results show that BMP inhibition is necessary
for expression of definitive neural plate markers and for neural plate
formation, but does not appear to affect the early steps of this process.
|
|
|
BMP inhibition by Smad6 is not sufficient for neural induction in Xenopus
The above results in the chick are in direct conflict with the dominant
`default model' (Hemmati-Brivanlou and
Melton, 1997; Weinstein and
Hemmati-Brivanlou, 1999
;
Muñoz-Sanjuán and Brivanlou,
2002
) that was based on experiments in Xenopus embryos.
In these experiments, mRNA encoding BMP antagonists is usually injected at the
animal pole of early embryos (one- to four-cell stage), where the RNA may be
inherited not only by prospective epidermal cells but also by presumptive
neural plate or crest cells. To overcome this problem and to generate an assay
more directly comparable with those in chick embryos, we injected a lineage
tracer together with cSmad6 mRNA (400 pg-1 ng) into the A4 (most
ventral animal) blastomere at the 32-cell stage
(Fig. 6A): this is the only
blastomere that does not consistently contribute progeny to neural plate or
neural crest in intact embryos (Dale and
Slack, 1987
; Moody,
1987a
). Embryos were then grown to the neurula stage and probed
with Sox3 (a definitive neural marker in Xenopus). Both in
Smad6-injected embryos and in GFP-injected controls, normal
expression of Sox3 was seen in the neural plate, but no ectopic
expression was ever seen in the injected cells, which contributed to the most
ventral epidermis (Smad6, 0/26,
Fig. 6B-E; control, 0/21,
Fig. 6F-I). An identical result
was obtained using NCAM as a neural marker (0/5; not shown).
|
We then tested whether FGFs alone or with Smad6 might induce Sox3. Neither FGF8 (10 pg) nor FGF4 (0.16 pg) alone induced brachyury (FGF8: 0/13; FGF4: 0/12; not shown) or Sox3 (FGF8, 0/13, Fig. 7D-F; FGF4, 0/13, Fig. 7G-I). In combination, FGF8+Smad6 (10 pg+1 ng) also failed to induce either marker (bra, 0/51; Sox3, 0/42), while FGF4+Smad6 (0.16 pg+ 1ng) induced Sox3 (45/49; Fig. 7J-L) but not brachyury (0/38; Fig. 7A-C) or other mesoderm markers tested (Chordin, 0/8; MyoD, 0/8; Goosecoid, 0/21; Vent1, 0/9; not shown).
|
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Discussion |
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Although several objections have been raised to the model and to the
interpretation of experiments that led to it (see
Streit and Stern, 1999c), this
model is generally so dominant that it is described in all current
developmental biology textbooks as the accepted mechanism for neural
induction.
Conflicting data from chick and other species
The first major objections to the default model as a sufficient explanation
for neural induction were raised as a result of observations in the chick
(Streit et al., 1998): BMP4,
BMP7 and their antagonists Chordin, Noggin and Follistatin are not expressed
with the correct spatial and temporal patterns to fit neatly with the
proposals of the model, misexpression of Chordin or Noggin in competent
epiblast using grafts of secreting cells do not induce neural tissue, and
misexpression of BMP4 or BMP7 by the same method in the prospective neural
plate does not block neural induction. Furthermore cell dissociation of chick
epiblast does not induce neural differentiation but rather muscle
(George-Weinstein et al.,
1996
; George-Weinstein et al.,
1997
). Consistent with these results in the chick, mouse mutants
that lack Chordin, Noggin or both BMP antagonists still have a nervous system,
although they lack the most anterior structures
(McMahon et al., 1998
;
Bachiller et al., 2000
;
Belo et al., 2000
;
Mukhopadhyay et al.,
2001
).
However, chick epiblast cells previously exposed to a grafted organizer for
at least 5 hours (13 hours are required for full induction)
(Gallera and Ivanov, 1964;
Gallera, 1971
) can respond to
Chordin by stabilizing the expression of the early marker Sox3 (but
still do not express the definitive neural marker Sox2)
(Streit et al., 1998
;
Streit et al., 2000
). This
finding led to the hypothesis that signals other than BMP antagonists are
required to confer sensitivity to epiblast cells to BMP signalling, and
therefore that BMP inhibition may be a relatively downstream step in the
induction process, if it is required at all.
But these experiments are open to three major criticisms. First, they cannot address whether it is necessary to inhibit BMP signalling at all for neural induction to occur. Second, it is possible that misexpression of BMP or its antagonists using a graft of secreting cells does not deliver enough active protein to overcome the endogenous signals. Third, it is possible that any one of the BMP antagonists is not sufficient to inhibit all BMP signalling.
Here we have addressed the first question using electroporation of BMP4 in
an expression construct directly into the epiblast; we find that BMP
inhibition is indeed necessary for neural plate development. However, although
misexpression of BMP4 affects the definitive neural marker Sox2, it
does not alter the expression of the early marker Sox3 [which at
early stages is not restricted to prospective neural cells but is also
expressed in future epidermis and mesoderm cells (see
Sheng et al., 2003)],
consistent with the idea that BMP inhibition is a relatively late step in a
cascade leading to neural induction in the chick.
To address the latter two criticisms, we electroporated the cell-autonomous BMP antagonist Smad6, either alone or together with other BMP antagonists (Chordin, Noggin and/or dnBMPR). In none of these cases do we see induction of either Sox3 or Sox2 in competent epiblast of the area opaca, now strongly suggesting that BMP inhibition is not sufficient for neural induction in the chick.
FGF signalling and neural induction
The finding (see above) that signals from the organizer other than BMP
antagonists are required as an upstream step before chick epiblast cells can
respond to the antagonists led to a screen for genes that are activated during
the initial signalling period. To date, two genes have been described from
this screen: ERNI (which is induced in just 1 hour) and
Churchill (induced after 4-5 hours)
(Streit et al., 2000;
Sheng et al., 2003
). In turn,
the use of ERNI as a marker led to the identification of FGF
signalling as both necessary and sufficient to induce all the known early
markers (ERNI, Sox3 and Churchill) as well as to sensitize
cells to Chordin; however, it is not sufficient to induce Sox2 or a
neural plate (Streit et al.,
2000
; Sheng et al.,
2003
). It was also shown that this early FGF step takes place
during very early stages of development, even before gastrulation begins
(Streit et al., 2000
;
Wilson et al., 2000
).
A requirement for FGF signalling has now also been demonstrated in the
ascidian Ciona (Kim and Nishida,
2001; Minokawa et al.,
2001
; Bertrand et al.,
2003
; Hudson et al.,
2003
), where it appears to be the main neural inducing signal. In
Xenopus, however, the evidence for FGF in neural induction has been
controversial (Kengaku and Okamoto,
1995
; Lamb and Harland,
1995
; Launay et al.,
1996
; Sasai et al.,
1996
; Xu et al.,
1997
; Holowacz and Sokol,
1999
; Hongo et al.,
1999
; Curran and Grainger,
2000
; Ribisi et al.,
2000
; Umbhauer et al.,
2000
). One of the reasons for this may be that, in
Xenopus, FGF signalling has mainly been inhibited using a
dominant-negative version of the FGF receptor 1 (FGFR1), which may not inhibit
all FGF signalling as FGF signals in neural induction appear to be transmitted
at least in part by FGFR4 (Hardcastle et
al., 2000
; Umbhauer et al.,
2000
).
Recently, it has been shown that signalling by FGF and by other secreted
proteins that work through MAP kinase act in part by phosphorylating the
linker region of Smad1, rather than the C terminus as does BMP signalling
(Pera et al., 2003). A
double-phosphorylation mechanism was therefore proposed as a molecular basis
for the cooperation between FGF and BMP in neural induction and other
embryonic signalling events (Pera et al.,
2003
). However, a more recent study in mouse by Soriano and
colleagues has elegantly demonstrated that mice carrying mutations in these
two distinct domains of Smad1 show different and additive phenotypes, and that
the mutations cannot complement each other, suggesting that linker and
C-terminal phosphorylation of Smad1 (and thus MAPK and BMP signalling) have
different functions during early development
(Aubin et al., 2004
).
Our present experiments demonstrate that in the chick, neither FGF2, FGF3,
FGF4 nor FGF8 is a sufficient neural inducer in the absence of
brachyury expression, even when any of these is combined with Smad6
as a BMP antagonist, consistent with the previous findings that FGF8+Chordin
do not induce Sox2 expression when administered as proteins
(Streit et al., 2000). These
results suggest that FGFs may only be able to induce definitive neural tissue
in cooperation with other signals in addition to BMP antagonists.
A role for Wnt signalling in neural induction?
More recent experiments in the chick, using NFz8 as the Wnt antagonist,
explant assays from the area pellucida and an antibody against Sox2, suggested
that Wnt inhibition together with FGF can act as a sufficient neural inducer,
and FGF3 was suggested as the endogenous factor
(Wilson et al., 2001). These
experiments were interpreted as indicating that BMP signalling can be
inhibited by these treatments through an alternative pathway and that the key
event may be downregulation of BMP at a transcriptional level
(Bainter et al., 2001
;
Wilson and Edlund, 2001
). In
Xenopus, however, Wnt signalling seems to promote neural induction
(Baker et al., 1999
), although
it is thought that the conflict is resolved by differential timing of these
events: Wnt signalling is required in early (pre-gastrula) stages of
development, while inhibition of Wnt may be important for acquisition of
neural fate at later stages (Bainter et
al., 2001
; Wilson and Edlund,
2001
).
To test whether inhibition of Wnt signalling can cooperate with BMP
antagonism and/or FGF signalling in vivo, we misexpressed combinations of
these agents in competent epiblast. Even a combination of FGF (FGF2, FGF3,
FGF4 or FGF8), Smad6 and four different Wnt antagonists (alone or in
combination) is unable to induce Sox2 expression in this assay. The
difference between our result and those obtained by Wilson et al.
(Wilson et al., 2001) are
difficult to explain, but it is possible either that isolated explants
cultured for 48 hours are somehow sensitized to neural inducing signals, or
that the Sox2 antibody might crossreact with Sox3 (which is induced by FGF8).
We have attempted to use this same antibody for our experiments with a variety
of protocols, but were unable to obtain reliable background-free staining
(data not shown). Based on the results of the present experiments, we can only
conclude that even a combination of FGF+Smad6+anti-Wnt is insufficient to
mimic the effects of a grafted organizer and induce Sox2 expression,
or an ectopic neural plate, in competent epiblast of the area opaca. We
suggest that neural induction is a multi-step process that begins very early
in development and involves other neural inducing factors, which remain to be
identified.
Do different vertebrates use different mechanisms to specify neural fate?
The results of our present experiments in the chick, along with those
previously published by ourselves and other groups, still raise the issue of
whether different vertebrates might use different molecular pathways to induce
the nervous system. We therefore tested whether inhibition of BMP signalling
with Smad6 is sufficient for neural induction in Xenopus.
Most experiments on neural induction in Xenopus have been
conducted on animal caps cut from embryos injected at the animal pole at the
one- to four-cell stage. It is important to bear in mind that such animal caps
almost certainly include some prospective neural plate or neural crest cells
(Jacobson and Hirose, 1981;
Dale and Slack, 1987
;
Moody, 1987a
;
Moody, 1987b
;
Wetts and Fraser, 1989
;
Eagleson and Harris, 1990
;
Saint-Jeannet and Dawid, 1994
;
Delarue et al., 1997
). It is
conceivable that prior to their isolation from the embryo, these cells have
received some signals that, although not sufficient for neural induction, may
represent some early steps in the process. For this reason, we chose to target
Smad6 to the most ventral animal blastomere at the 32-cell stage, as this is
the only blastomere that does not consistently contribute to neural plate or
neural crest, but mainly to the most ventral (belly) epidermis. Using a
concentration of Smad6 mRNA that is sufficient to induce axial
duplications when targeted to the marginal zone (400 pg to 1 ng), and also
sufficient to induce neural tissue in animal caps (1 ng), we see no ectopic
expression of the neural markers Sox3 (which in Xenopus is a
definitive neural plate marker) or NCAM in the descendants of the
injected A4 cell. These findings suggest that, in the Xenopus embryo
as well as in the chick (Streit et al.,
1998
; Streit and Stern,
1999a
; Streit and Stern,
1999b
), BMP inhibition may only be sufficient to deviate the
border of the neural plate when the antagonists are targeted to its vicinity,
but is insufficient to cause prospective epidermis (cells fated for neither
the neural plate nor its border) to acquire neural traits. Therefore animal
cap assays are not a good test for whether a candidate molecule has neural
inducing activity.
Our chick and Xenopus results differ in one respect. Misexpression
of FGF4 (at concentrations that do not induce expression of
brachyury) with Smad6 induces Sox3 in the absence
of mesoderm markers in Xenopus, but does not elicit a comparable
response (Sox2) in chick. There are several possible interpretations
for this difference. (1) There may be real differences in the mechanism of
neural induction in the two species. We feel that this is unlikely as
cross-species grafts of the organizer work very well across all vertebrate
classes (Waddington, 1934;
Waddington, 1936
;
Waddington, 1937
;
Kintner and Dodd, 1991
;
Blum et al., 1992
;
Hatta and Takahashi, 1996
).
(2) The expression patterns of FGF4/eFGF are different in the two species
in chick FGF4 is expressed in mid-posterior streak but not in
the organizer (Streit and Stern,
1999b
), while in Xenopus, eFGF is expressed in a domain
including the dorsal lip (Isaacs et al.,
1992
; Isaacs et al.,
1995
); different FGFs may therefore fulfill this function in the
two species. (3) It is possible that either endoderm or lateral mesoderm
(which do not express any of the five markers tested) is induced in
Xenopus. (4) It is possible that the level of inductive signal
provided by Smad6+FGF4 is not sufficient to induce mesoderm, but enough to
cause injected cells to produce some products normally secreted by early
prospective mesendoderm cells. (5) It is possible that the
brachyury-expressing cells in the chick are prospective
caudal neural plate, which has been shown to express this marker in
chick (Storey et al., 1998
)
but not in Xenopus. Although this explanation seems the most likely,
we have observed that the induced Sox2 expression is restricted to
the Smad6-electroporated cells (marked by GFP), while brachyury is
also expressed in neighbouring cells; the possibility therefore remains that
the Sox2 induction in this experiment is indirect. Furthermore, this
does not explain the finding that in the presence of Wnt antagonists both
brachyury and Sox2 expression disappear. Finally, (6) in
Xenopus, the marker selected for assessing neural plate is
Sox3, while in chick the definitive marker is Sox2. These
genes (and the closely related Sox1) appear partly to have swapped
functions in evolution, even between birds and mammals
(Uwanogho et al., 1995
;
Collignon et al., 1996
); it is
therefore very likely that the enhancer elements regulating the expression of
these two markers differ in the two species. Furthermore, Sox3 is not
an exclusive neural marker in any of the vertebrate classes, and the induced
expression seen in Xenopus could correspond to a different cell
type.
Conclusion: neural induction as a multi-step process
Our experiments provide evidence that BMP inhibition is required for neural
induction in the chick, but only as a relatively late step in a molecular
cascade. They also strongly suggest that BMP inhibition is not sufficient to
cause competent ectodermal cells to acquire neural fates either in chick or in
Xenopus. In Xenopus, FGF synergizes with BMP inhibition to
induce neural markers (we cannot yet conclude definitively whether this
combination is sufficient). In chick, inhibition of BMP signalling, even
together with Wnt antagonists and/or FGF, is not sufficient for neural
induction. We propose that neural induction does not occur `by default' but
rather that it involves a succession of signalling events, where some players
remain to be identified.
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ACKNOWLEDGMENTS |
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