Laboratorio de Embriología Molecular, Instituto de Biología Celular y Neurociencias, Facultad de Medicina, Universidad de Buenos Aires, Paraguay 2155, 3° piso (1121), Buenos Aires, Argentina
Author for correspondence (e-mail:
rqcarras{at}mail.retina.ar)
Accepted 13 February 2003
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SUMMARY |
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Key words: Notch, Sonic hedgehog, Chordin, Floor plate, Notochord, Presenilin, Xenopus laevis
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INTRODUCTION |
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Careful experiments revisited those studies performed on birds and found
that the reason why floor plate did not develop after the removal of the
notochord was that the floor plate precursors were removed also, since
Hensen's node (equivalent to the amphibian's Spemann's organiser) generates
both midline structures (Catala et al.,
1996; Teillet et al.,
1998
; Le Douarin and Halpern,
2000
). This agrees with several studies in vertebrates including
chicken, mouse, fish and amphibians, which show that precursors of the floor
plate, notochord and dorsal endoderm originate within the organiser
(Spemann and Mangold, 1924
;
Selleck and Stern, 1991
;
Catala et al., 1995
;
Catala et al., 1996
;
Wilson and Beddington, 1996
;
Shih and Fraser, 1995
;
Melby et al., 1996
;
Amacher et al., 2002
;
Latimer et al., 2002
).
Therefore, if the notochord and the floor plate share the same embryonic
origin, it will be necessary to reconcile this scenario with the role and the
hierarchy of the molecules described above. This is complicated by the
findings that in zebrafish embryos, only the development of the LFP seems to
be Shh dependent, while the medial cells appear to be dependent on Nodal
activity (Rebagliati et al.,
1998
; Sampath et al.,
1998
; Schauerte et al.,
1998
; Odenthal et al.,
2000
).
Notch signalling is best known from its central role in lateral inhibition
during neurogenesis. Within a proneural cluster, the future neuron is the
source of the membrane-bound ligand Delta, which interacts with the receptor
Notch on the surface of the neighbouring cells. The receptor is cleaved to
render the intracellular domain (NotchICD) that enters the nucleus
and, in association with CSL intracellular transducers such as Su(H),
activates the transcription of target genes that lead to the suppression of
the neuronal fate in the cells surrounding the neuronal precursor
(Schroeter et al., 1998;
Bray, 1998
; Wolfe and Haas,
2001; Kopan and Goate,
2002
).
Different models provided evidence that Presenilins facilitate the Notch
signalling pathway. Most authors favour the idea that their -secretase
activity, which mediates the proteolytic cleavage of the amyloid precursor
protein (APP), is also involved in the final proteolytic step that cleaves the
Notch receptor to produce NotchICD
(De Strooper et al., 1999
;
Struhl and Greenwald, 1999
,
Struhl and Greenwald, 2001
;
Chan and Jan., 1999
;
Taniguchi et al., 2002
). It
was also shown that a
-secretase-independent mechanism may play a
partial role in Notch signal transduction
(Berezovska et al., 2000
;
Berechid et al., 2002
) and
other research suggested that Presenilin is required before the generation of
NotchICD (Ye et al.,
1999
; Ray et al.,
1999
).
We have previously shown that both shh and presenilin
repress primary neurogenesis in Xenopus laevis embryos
(Franco et al., 1999;
Paganelli et al., 2001
). Since
presenilin stimulates shh expression in the floor plate, we
suggested that the effects of presenilin could be exerted through
shh. We also proposed that in the primary neurogenesis cascade,
shh acts very upstream of the lateral inhibition step mediated by
Notch, modulating the activity of prepattern genes. Bearing in mind the
relationship between Notch and Presenilin, and because lateral inhibition does
not account for the repression of primary neuron development after
presenilin overexpression, we wondered whether Notch signalling could
modulate shh expression.
To answer this question, we either activated or prevented Notch signalling in Xenopus embryos and found that Notch stimulates shh and plvs expression in the floor plate and represses the notochordal markers chordin (chd) and brachyury (bra). These changes are accompanied by an expansion of the floor plate and a reduction of the notochord size. We propose that Notch may execute a binary decision, favouring floor plate development at the expense of the notochord, and this preferentially occurs before mid gastrula. We also show that shh down-regulates chd and suggest that shh itself may be involved in reinforcing the binary decision executed by Notch. We present evidence that Presenilin can also modulate this switch in a Notch-dependent way.
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MATERIALS AND METHODS |
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Synthetic capped mRNAs for microinjection were obtained as described
previously (Franco et al.,
1999). To direct the material to the future dorsal midline
populations, injections were delivered into the animal hemisphere of one
blastomere at the 2-cell stage at approximately 30-40° from the equator
and close to the cleavage plane, following the observations of Vodicka and
Gerhart (Vodicka and Gerhart,
1995
). The Xotch antisense oligodeoxynucleotide
(Xotch Mo) used was a 25-mer morpholino oligo (Gene Tools, LLC) with
the base composition 5'-GCACAGCCAGCCCTATCCGATCCAT-3'. The
X-ps-
antisense morpholino oligonucleotide
(X-ps-
Mo), the standard control morpholino oligo
(Control Mo) and the X-ps-
construction used for
in-vitro transcription were the same as those employed by Paganelli et al.
(Paganelli et al., 2001
). The
templates for mRNA synthesis has been described previously: X-shh
(Franco et al., 1999
);
notchICD, hGR/ICD22 and
X-su(H)DBM (Wettstein
et al., 1997
). Nuclear translocation of hGR/ICD22 was
induced with dexamethasone (Sigma, D4902).
For RNA interference of shh function, a deletion was made in the
construct used for making X-shh antisense probes described by Franco
et al. (Franco et al., 1999)
by digestion with Eco0109 I and BstEII, followed by fill-in
and ligation. The resulting construct contains a 935 bp insert encoding the
N-terminal region of X-Shh, from bp 51 to bp 986 of the cDNA sequence
(Ekker et al., 1995
). Sense
and antisense RNA were obtained after linearisation with EcoRI or
KpnI and transcription with T7 or T3 mRNA polymerase, respectively. 1
µg of each template was used for in vitro transcription, which was carried
out with the Megascript kit (Ambion). Synthetic RNAs were purified with the
Qiagen RNeasy mini kit. Equimolar amounts of sense and antisense RNAs were
annealed in 1 mM MgSO4, 30 mM NaCl at 70°C for 15 minutes and
then at 37°C for 45 minutes. The quality of double strand RNA (ds-RNA) was
tested by native agarose gel electrophoresis in TBE in the presence of
ethidium bromide. Gel mobility was shifted according to ds-RNA of the expected
length.
The amounts of synthetic mRNAs, ds-RNA and morpholinos injected are indicated in the table and figures. All injections included 0.5 ng of nuc-lacZ mRNA as tracer. For comparison, equal amounts of nuc-lacZ mRNA were delivered in each set of experiments.
Semi-quantitative RT-PCR, X-gal staining, in situ hybridisation,
C-myc immunohistochemistry and histology
Semi-quantitative RT-PCR analysis was performed essentially as described by
Paganelli et al. (Paganelli et al.,
2001). The number of cycles and the template input for PCR were
determined empirically in each case, within the linear range of amplification.
The forward (F) and reverse (R) primer sequences, the product sizes, and the
number of cycles were as follows: ef1
: F
5'-CAGATTGGTCCTGGATATGC-3', R
5'-ACTGCCTTGATGACTCCTAG-3', 268 bp., 26 cycles for stage 12, 25
cycles for stage 15; X-shh: F 5'-ATGCTGGTTGCGACTC-3'; R
5'-CCCGCCAGACTTGG-3', 581 bp., 36 cycles for stage 12, 32 cycles
for stage 15.
X-gal staining, preparation of digoxigenin-labeled antisense RNA probes and
whole-mount in situ hybridisation were performed as described previously
(Franco et al., 1999), except
that the proteinase K step was omitted in in situ hybridisation when embryos
would be further processed for immunohistochemistry.
50 µm sections were cut using an Oxford Vibratome and mounted onto
gelatine coated slides as described by Hollemann et al.
(Hollemann et al., 1996),
except that 4% formaldehyde was used instead of glutaraldehyde during the
embedding, in order to reduce background fluorescence. For immunolocalization
of the C-myc epitope, slides were washed three times in PBS, for 10 minutes
each, incubated for 1 hour at room temperature in blocking buffer containing
5% nonfat milk (Molico, Nestlé) in PBS, then overnight at 4°C with
mouse 9E10 anti-Myc monoclonal antibody (Santa Cruz) diluted 1/500 in blocking
buffer, in a wet chamber. The following day, slides were washed three times at
room temperature with PBS containing 0.1% Tween 20 (10 minutes each), twice
with PBS (10 minutes each), and incubated for 2 hours at room temperature in a
wet chamber in the dark with anti-mouse immunoglobulins-FITC (Dako F0232)
diluted 1/200 in blocking buffer. After washing with TBS (pH 7.5) containing
0.1% Tween 20, slides were mounted in PBS:glycerol (1:2).
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RESULTS |
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To corroborate whether endogenous Notch activity was indeed involved in this modulation, we prevented Notch signalling by injecting X-su(H)DBM mRNA, which encodes a dominant-negative variant of the Notch transducer X-Su(H). Whole embryos at the neural plate stage showed that shh transcripts were down-regulated (82%, n=90; Fig. 1D).
Since shh mRNA is present both in the prospective floor plate and notochord at this stage, we wanted to know which structures were affected. For this purpose, we analysed transverse sections of more advanced neurulae, when the notochord and floor plate are more clearly distinguished from each other. Control embryos show shh transcripts in the floor plate and a dorsal-ventral gradient in the notochord, with highest levels close to the floor plate (Fig. 1B'). Embryos injected with notchICD revealed an expansion of the shh domain corresponding to the floor plate and a concomitant reduction of the notochord size on the injected side (Fig. 1C',C''). Conversely, X-su(H)DBM-injected embryos showed a decrease in shh transcripts in the floor plate domain, while the notochord size was simultaneously augmented on the injected side (Fig. 1D',D'').
The Notch-induced up-regulation of shh in the floor plate domain
may happen by a direct or indirect regulation and/or by favouring the
development of the floor plate. Therefore, we studied the consequences of
activating the Notch pathway on the expression of another gene that was
described to participate in floor plate development. Plvs mRNA
precedes and overlaps shh expression and is first detected in the
dorsal marginal zone of late blastulae. During gastrulation it is expressed at
the dorsal midline by cells that undergo convergent-extension movements. At
the early neurula stage, transcripts are distributed throughout the dorsal
midline in the three germ layers, i.e. the prospective floor plate, the
notochord and the dorsal endodermal cells lining the archenteron
(Ruiz i Altaba and Jessell,
1992) (Fig.
1E,E').
Whole embryos at the neural plate stage show that plvs expression also increases on the injected side when the Notch pathway is stimulated (75%, n=16; Fig. 1F) and transverse sections confirm that this occurs in the floor plate domain, which appears expanded (Fig. 1F',F''). Conversely, X-su(H)DBM-injected embryos showed a decrease in plvs expression (95%, n=19; Fig. 1H). Thus, Notch signalling positively regulates the expression of two genes that were shown to be able of promoting floor plate development.
Notch decreases chd and bra expression and restricts
notochord development
To further study the effect of Notch signalling on notochord development,
we analysed whether activating or blocking the pathway could change the
expression of the mRNA encoding the secreted polypeptide Chd. In early
neurulae, chd transcripts are normally present in the notochord and
the prechordal mesoderm (Sasai et al.,
1994) (Fig. 2A,D).
At this stage we found that notchICD strongly reduced
chd expression on the injected side (66%, n=35;
Fig. 2B). Transverse sections
of slightly more advanced embryos again showed that the notochord size, now
visualised by the expression of chd, was reduced on the injected
side, while the overlying layer containing the prospective floor plate was
concomitantly thickened (Fig.
2E). In contrast, when Notch signalling was interfered with by
injecting X-su(H)DBM or an antisense morpholino
oligonucleotide against Xenopus notch-1 (Xotch Mo), chd expression
was increased, appearing as a more dense and superficial staining in whole
embryos (63%, n=49 for X-su(H)DBM; 75%,
n=16 for Xotch Mo; Fig.
2C,I). Transverse sections revealed that this effect was the
result of an increase of chd-positive cells, which adopted more
dorsal positions, as if they were occupying the normal place of floor plate
cells (arrow, Fig. 2F).
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In Xenopus, transcription of bra begins at mid-blastula
transition, but strongest levels are achieved when gastrulation starts. At
this stage, transcripts are distributed throughout the entire marginal zone
(presumptive mesodermal cells). During gastrulation, mesodermal cells that
migrate anteriorly turn-off bra expression with the exception of the
notochord. During neurula stages, transcripts persist only in the notochord
and in a circumblastoporal ring (Smith et
al., 1991) (Fig.
2J,L).
Neurula stage embryos showed that notchICD strongly reduced the notochordal expression of bra (86%, n=42; Fig. 2K,M). We conclude that activation of the Notch pathway down-regulates the expression of two notochordal markers, the transcription factor Bra and the secreted polypeptide Chd, and concomitantly decreases the notochord size; at the same time, Notch up-regulates two molecules expressed by the floor plate, the transcription factor Plvs and the secreted factor Shh, and increases the floor plate size. All this evidence suggests that a binary choice determines between floor plate and notochordal fates and when Notch signalling is active, favours floor plate development at the expense of the notochord.
The floor plate versus notochord switch executed by Notch mainly
occurs before mid gastrula
To determine the time of highest competence for the proposed binary
decision in response to Notch signalling, we performed time-course experiments
by injecting hGR/ICD22 mRNA, a construction encoding the
ligand-binding domain of the human glucocorticoid receptor fused to the amino
termini of NotchICD. Thus, nuclear translocation of
NotchICD can be induced upon dexamethasone administration
(Wettstein et al., 1997).
Control embryos were injected with the construction but left untreated. Two
windows of induction with 10 µM dexamethasone were assayed: one that
included the first half of gastrulation (from stage 9+ to stage 11) and the
other included the second half (from stage 11 to stage 13). In situ
hybridisation revealed that in response to Notch signalling, nearly three-fold
more embryos were able to up-regulate shh before stage 11 than after
this stage. Meanwhile, chd down-regulation occurred in nearly
two-fold more embryos before stage 11 than after this stage
(Table 1). These results
suggest that the Notch-mediated binary decision that chooses floor plate fate
in preference to the notochord for the trunk region mainly takes place around
early gastrulation. To further test this hypothesis, we analysed whether the
effect of activating or blocking Notch signalling on chd expression
could also be detected at early gastrula. We chose chd instead of
shh because of its stronger and broader expression in the early
organiser (see below), so that unambiguous differences in transcripts levels
between the injected and non-injected side could be distinguished. We found
that notchICD decreased chd transcripts in the
organiser on the injected side (90%, n=21;
Fig. 3B; see also
Fig. 5B,C), while
X-su(H)DBM produced the opposite effect (63%,
n=49; Fig. 3C).
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Xenopus notch-1 transcripts are present in the early
organiser and later in floor plate precursors
It was reported that Xenopus notch-1 (Xotch) transcripts are
present in domains of primary neurogenesis from late gastrula stages
(Chitnis et al., 1995). RNAse
protection assays revealed maternal and zygotic Xotch mRNA and its
presence in the three germ layers, especially in the dorsal mesoderm, when
gastrulation starts (Coffman et al.,
1990
). A more precise study of the early distribution would help
to understand whether Xotch transcripts are present at the right time
and place to elicit the effects suggested above. Therefore, we performed in
situ hybridisation of early and late gastrula/early neurula stages and
attempted to correlate Xotch mRNA distribution with the expression
patterns of chd and shh.
At early gastrula, Xotch transcripts are present both in the
involuting and non-involuting dorsal marginal zone
(Fig. 4A) and extend towards
the animal pole but are absent from the ventral marginal zone and the vegetal
yolk mass (not shown). Sagittal sections reveal Xotch transcripts in
the epithelial and subepithelial layers of the dorsal blastopore lip
(Fig. 4D), where chd
is strongly expressed (Fig.
4B,E). Shh transcripts are first detected at this time
(Fig. 4C), but are delayed in
relation to chd, which began to be expressed in the dorsal marginal
zone shortly before gastrulation started
(Sasai et al., 1994). It is
noticeable that shh expression is more confined than chd and
Xotch: transcripts are mainly found in several cells in the
subepithelial layer of the organiser, with some faint distribution in the
epithelial layer (Fig. 4F).
Neither Xotch nor shh mRNA are detected in the deep zone of
the organiser, which mostly contains the involuted precursors of the
prechordal mesoderm and expresses chd. Therefore, when gastrulation
starts, a population of cells containing notochord precursors expresses the
three markers analysed.
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Thus, at early gastrula, Xotch transcripts are present in a population of cells in the organiser which is known to contain notochord precursors and co-express chd and shh, giving support to the idea that Notch signalling may be involved in the regulation of these genes and in cell-fate decisions during the specification of the dorsal midline structures. Later on, among the dorsal midline layers, Xotch transcripts are only present in the developing floor plate, suggesting that notochord cells in the trunk can no longer respond to Notch ligands to divert towards floor plate fate.
Notch activity down-regulates chd expression both in
cell-autonomous and non cell-autonomous ways
Early gastrulae injected with notchICD showed that the
patches of X-gal staining coincided with the territories of chd
repression but in some embryos, repression of chd was also seen
towards more lateral regions at a distance from the X-gal patch
(Fig. 5B,C). Because X-gal
staining could be underestimating the cells harbouring the injected
notchICD mRNA, we made use of the c-Myc epitope fused to
the NotchICD fragment encoded by the synthetic mRNA that we
injected in our experiments. Thus, we were able to identify more precisely
those cells that inherited and translated the notchICD
mRNA by immunofluorescence for the Myc-tag and compared their localisation
with the expression of chd.
Sagittal sections of early gastrulae showed down-regulation of chd expression in patches of Myc-tag-positive cells (compare Fig. 5E,F with 5D). In addition, chd down-regulation was also observed in Myc-tag-negative cells that were surrounded by Myc-tag-positive cells (compare Fig. 5H,I with 5G). Transverse sections of early neurulae also revealed Myc-tag-positive cells displaying a strong down-regulation of chd intermingled with Myc-tag-negative cells that did express chd, although in much lower levels than cells on the non-injected side (Fig. 5J-M). Therefore, since chd expression was repressed in the same cells that inherited the notchICD mRNA and also in neighbouring cells, our results suggest that active Notch has the ability to down-regulate chd expression in both cell-autonomous and non-cell-autonomous ways. Transverse sections of more advanced neurulae showed that Myc-tag-positive cells were chd negative and rarely contributed to the notochord; instead, they were located in the overlying layer containing the floor plate (Fig. 5N,O; see also Fig. 2E), which appeared thickened, while the notochord was concomitantly reduced in size and chd-positive cells were decreased on the injected side as shown before, indicating that cells that inherited notchICD mRNA diverted their notochord fate and became incorporated into the floor plate. This developmental series suggests that cells within the organiser are able to repress chd expression in response to active Notch and later segregate to the prospective floor plate.
Shh down-regulates chd
Bearing in mind the preceding results and the proposed role of Shh as
inducer of floor plate development, we wanted to analyse whether an
enhancement of Shh signalling could contribute to reduce the number of
chd-positive cells. Gastrulae unilaterally injected with
X-shh mRNA showed a decrease in chd expression in the early
organiser (83%, n=36; Fig.
3D), in a manner reminiscent to that obtained after Notch
activation (Fig. 3B). Next, we
wanted to know what happened if we interfered with shh function.
Double-stranded RNA has been successfully used as a potent and specific
reagent for silencing different genes in Xenopus embryos
(Oelgeschläger et al.,
2000; Nakano et al.,
2000
; Lau et al.,
2001
; Zhou et al.,
2002
). When we specifically degraded the endogenous transcripts
with X-shh double-stranded RNA (X-shh-ds) (compare
Fig. 3G,G' with
F,F'), we observed a significant increase of
chd-positive cells on the injected side (44%, n=43;
Fig. 3E), resembling the effect
of blocking Notch signalling with X-su(H)DBM
(Fig. 3C). Because the X-gal
staining was not reduced in X-shh-ds-injected embryos when compared
with those injected with nuc-lacZ mRNA alone (compare
Fig. 3E with 3A), we conclude
that the phenotype that we observe is not due to non-specific mRNA degradation
or to non-specific interference of protein translation. To verify the
specificity of the effects produced by X-shh-ds, we analysed the
expression of N-tubulin and gli3, which were shown to be
down-regulated by X-shh overexpression
(Franco et al., 1999
). We also
examined the distance between the optic vesicles in tadpoles, since targeted
disruption of the mouse shh gene leads to cyclopia
(Chiang et al., 1996
). As
expected, X-shh-ds increased the number of primary neurons (55%,
n=38; Fig. 3H),
expanded the gli3 domain (55%, n=38;
Fig. 3I) and produced several
grades of cyclopia (80%, n=35;
Fig. 3J). Moreover, another
hint that the action of X-shh-ds RNA is specific is that it does not
deplete other unrelated endogenous transcripts, for example N-tubulin
or even chd (notice in Fig.
3H that the X-gal staining is extensively distributed on the
injected side of the embryo, indicating that X-shh-ds RNA, although
present in the domains of N-tubulin expression, does not promote the
degradation of N-tubulin mRNA; for chd see
Fig. 3E). Thus, Shh signalling
specifically down-regulates chd and this effect is already evident
when the proposed switch controlling notochordal and floor plate fates
modulated by Notch mainly takes place. This correlation suggests that Shh
signalling may be in part mediating the effect of Notch in the specification
of the different cell populations that configure the dorsal midline.
Presenilin up-regulates shh and down-regulates chd
in a Notch-dependent way
Because Presenilins have been implicated in Notch signalling, we wanted to
know whether Notch mediates the up-regulation of shh promoted by
presenilin. Therefore, we unilaterally co-injected Xenopus
embryos with presenilin- (X-ps-
) and
X-su(H)DBM mRNAs and analysed the expression of
shh at neurula stages. When injected alone, X-ps-
expands shh expression on the injected side (84%, n=19;
Fig. 6B), as we have previously
described (Paganelli et al.,
2001
). Transverse sections show a lateral expansion of the floor
plate domain and a concomitant reduction of the notochord domain on the
injected side (Fig. 6E),
resembling the consequences of Notch activation. When Notch signalling was
prevented by co-injection of X-su(H)DBM (i.e. down-stream
of the processing step of the receptor where Presenilin is thought to
intervene), X-ps-
was unable to up-regulate shh.
Moreover, the effect of blocking the Notch pathway prevailed, and we observed
a reduction of shh in the floor plate domain (82%, n=34;
Fig. 6C,F).
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Together, these results suggest that Notch signalling requires Presenilin activity to modulate the binary switch that decides between notochord and floor plate fates.
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DISCUSSION |
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Our data show that Notch signalling promotes the expression of shh and plvs, two markers of floor plate specification, and together expands the floor plate. At the same time it represses the expression of both notochordal markers examined, chd and bra, while concomitantly reduces the notochord size.
Grafting and ablation experiments in birds have shown that the notochord
and the floor plate derive from the Hensen's node during primary neurulation,
and from the cordoneural hinge (the remains of Hensen's node) during secondary
neurulation [(Catala et al.,
1995; Catala et al.,
1996
; Teillet et al.,
1998
) for revision see Le Douarin and Halpern
(Le Douarin and Halpern,
2000
)]. Lineage tracing revealed that the Xenopus late
organiser, which is the equivalent of the avian cordoneural hinge, also
originates both structures during tail formation
(Gont et al., 1993
) and the
pioneer work of Spemann and Mangold in amphibians clearly demonstrated that
the implanted dorsal lip differentiates into notochord and floor plate in the
trunk [see figure 25 in Spemann and Mangold
(Spemann and Mangold, 1924
)].
When the cellular origin of the Xenopus organiser was traced in the
32-cell embryo, the progeny of the B1 blastomere gave rise to 70% of the
organiser, and descendants were found both in the notochord and the floor
plate [see figure 6 in Vodicka and Gerhart
(Vodicka and Gerhart, 1995
)].
Fate maps of the embryonic shield, the teleost equivalent of the Spemann's
organiser, also established that this region contributes to both structures
(Shih et al., 1995; Melby et al.,
1996
; Amacher et al.,
2002
; Latimer et al.,
2002
). Therefore, if the floor plate and the notochord derive from
a common cell population, our results indicate that Notch may be executing a
binary cell-fate decision: when active, it promotes floor plate specification
at the expense of the notochord. This is consistent with other findings in
zebrafish embryos: missense mutants of the Notch ligand delta A (dlA)
develop excess notochord and reduced numbers of floor plate and hypochord
cells, while overexpression of dlA leads to the opposite effect
(Appel et al., 1999
).
For the trunk region, the proposed binary decision appears to take place
mainly around the beginning of gastrulation, since the effect of activating or
blocking Notch signalling on chd expression is already evident at
early gastrula and the competence of chd and shh to respond
to active Notch is highest from stage 9+ to stage 11. Therefore, at least two
parallel mechanisms seem to contribute to stop this Notch- mediated
cell-fate switch in the dorsal midline precursors while gastrulation proceeds:
first, Xotch transcripts ultimately disappear from the developing
notochord, suggesting that these cells become refractory to divert to floor
plate in response to Notch ligands; second, the competence of the binary
switch for responding to active Notch decreases throughout gastrulation.
Interestingly, X-delta-1 transcripts are present in the dorsal
blastopore lip at stage 10.5 (Ma et al.,
1996) and then disappear from the involuted cells
(Wittenberger et al., 1999
).
Functional experiments should elucidate whether X-Delta-1 is the ligand that
operates the proposed binary switch, but its down-regulation in the involuted
cells may underlie another key component in the mechanisms that contribute to
stop Notch signalling in the dorsal midline.
Besides the previously reported up-regulation of shh, we show here
that presenilin also down-regulates chd. Because both
activities were prevented when the transduction of the Notch pathway was
impeded, we conclude that Presenilin is required by Notch signalling during
the binary switch that specifies dorsal midline fates. X-ps-
expression appears to be ubiquitous, but transcripts are present at the right
time to modulate Notch signalling during dorsal midline development
(Tsujimura et al., 1997
).
Notch signalling up-regulates the expression of molecules involved in
floor plate specification
Evidence from several sources suggest that Notch activation may trigger a
cascade linking shh and plvs, which ultimately results in
favouring floor plate development. First, notchICD
injection increases both transcripts in floor plate precursors (this paper).
Second, their expression domains overlap, but plvs precedes
shh, beginning at late blastula
(Ruiz i Altaba and Jessell,
1992) (this work). After neural tube closure plvs mRNA is
replaced by transcripts from the closely related gene hnf3ß, and
it was proposed that the combined expression of both transcription factors in
Xenopus is equivalent to that of hnf3ß in rats and mice
(Ruiz i Altaba et al., 1993a
).
Third, functional correlation from overexpression experiments in frog embryos
associates plvs/hnf3ß and shh with the specification of
floor plate fate: hnf3ß promotes the ectopic expression of floor
plate markers including shh and hnf3ß itself, in the
neural tube; plvs promotes the ectopic expression of
F-spondin, a marker of differentiated floor plate, in the dorsal neural
tube, and shh induces the ectopic expression of F-spondin,
hnf3ß and shh itself
(Ruiz i Altaba et al., 1993b
;
Ruiz i Altaba et al., 1995
;
Roelink et al., 1994
). It
could be argued that in these studies, the ectopic expression of floor plate
markers within the neural ectoderm was obtained at tadpole stages and could
not be detected earlier, when floor plate is normally specified. Furthermore,
in Xenopus embryos the regulatory relationship between plvs
and shh was not directly tested and the ability of
hnf3ß/plvs and shh to promote floor plate
development was not analysed in the context of their normal functional domain.
Therefore, functional experiments in this context should be carried out to
test the hierarchy of the relationship between shh and plvs
upon Notch activation.
Evidence from other vertebrate models also indicate that shh and
hnf3ß are functionally linked. Mice lacking shh
activity did not develop a distinct floor plate despite the presence of a
differentiated notochord during early stages, and hnf3ß
expression in the ventral neural tube was never initiated
(Chiang et al., 1996).
However, mice homozygous for targeted mutations of the hnf3ß
gene failed to develop the notochord and the floor plate and lacked
shh expression, but the severe impairment in the development of the
node and its derivatives did not allow us to determine whether
hnf3ß is directly required for shh expression
(Ang and Rossant, 1994
;
Weinstein et al., 1994
).
However, HNF3-binding sites have been found in the promoter and other
regulatory regions of the mouse and zebrafish shh gene, including
intronic enhancers that direct the expression in floor plate and notochord,
and it was suggested that shh expression in both structures is
regulated by HNF3-dependent and independent mechanisms
(Chang et al., 1997
;
Müller et al., 1999
;
Epstein et al., 1999
).
However, the enhancer that directs hnf3ß expression in floor
plate cells in mice contains a Gli binding site, which was proposed to respond
to Shh signalling (Sasaki et al.,
1997
). Thus, evidence collected from mice suggest that Shh protein
from the notochord induces HNF3ß during specification of the floor plate
and HNF3ß in turn activates shh expression in floor plate cells.
Analysis in zebrafish support the idea that shh is a target of
HNF3ß, nevertheless, it was proposed that, unlike floor plate development
in the mouse, zebrafish embryos employ two distinct mechanisms for floor plate
specification, one dependent on Nodal activity, which induces MFP, and the
other dependent on Shh, which induces LFP
(Schauerte et al., 1998
;
Odenthal et al., 2000
).
Notch down-regulates the expression of molecules involved in dorsal
axial mesoderm development
In Xenopus, chd is able to promote notochord development in
mesodermalised animal caps and in u.v.-ventralised embryos, both in
cell-autonomous and non cell-autonomous ways
(Sasai et al., 1994). Chd is a
potent antagonist of ventralising BMPs
(Sasai et al., 1995
;
Piccolo et al., 1996
), and it
was suggested that notochord formation requires co-repression of both BMP and
Wnt signalling (Yasuo and Lemaire,
2001
). Inactivation of Chd protein by the metalloprotease Xolloid
leads to strong ventralised phenotypes up to neurula stages, and later the
notochord is frequently absent (Piccolo et
al., 1997
). Lack of Chd activity in zebrafish embryos disrupts
posterior notochord development, and Ntl (Bra) protein is absent from the
posterior notochord (Hammerschmidt et al.,
1996
; Schulte-Merker et al.,
1997
). Thus, Chd has been mainly regarded as an inhibitor of
ventralising signals during mesodermal patterning and its requirement for
notochord development may reflect this fact.
Experiments conducted in Xenopus embryos showed that bra
is one of the direct targets of mesoderm-inducing factors and promotes
development of posterior mesoderm in ectodermal explants
(Smith et al., 1991). The
dorsal-ventral character of this mesoderm depends on bra
concentration, the highest dose being able to promote somitic muscle but never
notochord formation (Cunliffe and Smith,
1992
; Cunliffe and Smith,
1994
), and hence, it is unable to induce chd in this kind
of explants (Taira et al.,
1997
). However, when co-expressed with either plvs or the
BMP antagonist noggin (which is also present in dorsal mesoderm),
bra can promote notochord development
(Cunliffe and Smith, 1994
;
O'Reilly et al., 1995
).
Besides, lack of bra function in mouse and zebrafish or changing its
behaviour from transcriptional activator to repressor in Xenopus
results in the absence of posterior mesoderm and failure of notochord
differentiation (Chesley,
1935
; Halpern et al.,
1993
; Conlon et al.,
1996
). It appears therefore that the transcription factor Bra is
necessary but not sufficient in the pathway that leads to notochord
differentiation, and other molecules such as Plvs and BMP antagonists may
cooperate in this process. In this context, it will be interesting to test
whether the combined action of bra and the BMP antagonist
chd is sufficient to promote notochord formation. In conclusion, our
results show that Notch signalling down-regulates the expression of two
molecules required for notochord development.
Notably, it was demonstrated that the floor plate, revealed by shh
expression, is widened in zebrafish ntl mutant embryos
(Halpern et al., 1997),
suggesting that Bra activity antagonises floor plate development while
promoting notochord formation. Moreover, in line with our findings, it was
recently described that Notch represses ntl cell-autonomously
(Latimer et al., 2002
).
Therefore, bra may constitute a key target in the binary switch that
decides between notochord and floor plate fates under the control of Notch
signalling. Further experimentation will be needed to elucidate whether a
hierarchical relationship links chd and bra in this
switch.
Notch signalling may be required for medial floor plate
specification
The enhancement of shh and plvs transcripts that we
observe could be due to a Notch-dependent up-regulation of both genes or it
may be a consequence of favouring floor plate development. Analogously,
chd and bra down-regulation may be the result of their
repression by active Notch or a consequence of disfavouring notochord
development. However, as discussed above, shh and plvs have
been implicated in the specification of floor plate fate, and bra and
chd are necessary for proper notochord formation. Therefore, more
than being regarded as mere markers, changes in the expression of these genes
appear to be inherent in the fate decisions that are taking place. Thus, it
seems more likely that Notch signalling triggers floor plate specification at
the expense of the notochord through the opposite regulation of
plvs/shh and chd/bra. Although further experimentation
should elucidate if shh is required for Notch effects, and whether
the activation of shh is direct or indirect, we have shown that
shh down-regulates chd, and this can already be seen at
early gastrula, suggesting that an enhancement of Shh signalling may be
necessary for limiting the number of notochordal precursors. This mechanism
may underlie some aspects of the role of shh in promoting floor plate
development.
Classically, Notch activation has been considered as a mechanism by which a
cell remains in a progenitor state to be available for subsequent waves of
differentiation or as a way to repress the specification of certain cell types
in favour of others. However, recent evidence suggests that Notch may have an
instructive role in specifying glial fate
(Gaiano et al., 2000;
Morrison et al., 2000
;
Furukawa et al., 2000
;
Scheer et al., 2001
) (for a
review, see Gaiano and Fishell,
2002
; Lundkvist and Lendahl,
2001
). In this scenario, there could be at least two explanations
for the role of Notch signalling in the development of the dorsal midline. (1)
A permissive role for floor plate development, which implies that within the
population of dorsal midline precursors, Notch activation may repress the
notochordal fate and allow the development of floor plate identity through
some default mechanism. In this context, it is intriguing that some markers of
floor plate specification are also expressed by the notochord (e.g. shh,
plvs) whereas notochordal markers seem to be exclusively present in the
notochord (e.g. bra, chd), and depleting embryos of Bra activity, as
in the zebrafish mutant ntl
(Halpern et al., 1997
),
favours floor plate development at the expense of the notochord. Active Notch
may thus deplete the dorsal midline precursors of molecules required for the
specification of notochord, allowing floor plate to develop. (2) An
instructive role, implying that, apart from the repression of genes required
for notochord development, Notch signalling may actively promote floor plate
specification by increasing the expression of genes that specify floor plate
fate (e.g. shh, plvs).
In conclusion, we propose that the early organiser contains a population of cells with the potential to develop either as floor plate or notochord. Activation of Notch (which is present in the dorsal blastopore lip) in response to a ligand, which may be X-Delta-1 but this remains to be elucidated, may switch-off the genetic program for notochord specification in a subset of cells (evident by repression of bra and the cell-autonomous down-regulation of chd) and switch-on the program for floor plate development (instructive hypothesis), including the enhancement of plvs and shh expression, or allow this program to proceed (permissive hypothesis). In turn, secreted Shh could refine the segregation of both populations by limiting the number of notochordal (chd-positive) cells by a non cell-autonomous mechanism. This is consistent with the developmental profile of chd and shh expression. Their spatial patterns partially overlap in the early gastrula organiser but chd precedes shh and displays a broader domain. Later, when gastrulation finishes, this spatial relationship is reversed: while shh is expressed throughout the dorsal midline cell populations, chd expression is excluded from the prospective floor plate and the dorsal lining of the archenteron, and thus covers a subset of the shh expression domain. This dynamic profile and the results of our functional experiments may thus underlie a negative feed back of shh over chd (Fig. 7). Whether the cell-autonomous down-regulation of chd elicited by Notch is due to the activation of a transcriptional repressor or is mediated by secreted Shh acting on the same cell, where it is activated by Notch (i.e. in an autocrine way), remains to be elucidated.
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ACKNOWLEDGMENTS |
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Footnotes |
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