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 22 December 2004
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SUMMARY |
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Key words: Delta, Notch, Hairy2a, Floor plate, Notochord, Xenopus laevis
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Introduction |
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The origin of the FP has been subject of a recent controversy, which can be
synthesized in three models, based on those described by Strähle and
colleagues (Strähle et al.,
2004). (1) The `induction' model postulates that the FP derives
from the neural ectoderm and is induced by secreted Shh from the notochord
(Placzek et al., 2000
;
Tanabe and Jessell, 1996
;
Chiang et al., 1996
). (2) The
`allocation' model postulates a common origin for notochord and MFP in the
vertebrate's organiser, with MFP precursors invading the midline of the
overlying neural plate (Spemann and
Mangold, 1924
; Selleck and
Stern, 1991
; Gont et al.,
1993
; Wilson and Beddington,
1996
; Catala et al.,
1995
; Catala et al.,
1996
; Shih and Fraser,
1995
; Melby et al.,
1996
; Teillet et al.,
1998
; Amacher et al.,
2002
; Latimer et al.,
2002
) (see also Le Douarin and
Halpern, 2000
). In this model, Shh is regarded as a factor
necessary for survival of FP cells and for maintenance of their phenotype, and
as an inducer of anterior and lateral FP
(Le Douarin and Halpern, 2000
;
Thibert et al., 2003
;
Charrier et al., 2002
;
Patten et al., 2003
;
Rebagliati et al., 1998
;
Sampath et al., 1998
;
Schauerte et al., 1998
;
Odenthal et al., 2000
). (3)
The `induction and allocation' model tends to reconcile the experimental
evidence from the first two models by proposing that the inductive step for
the MFP [involving Shh and/or Nodal signalling, which varies among vertebrate
species (see Strähle et al.,
2004
)] takes place before the segregation of MFP and notochord
precursors emerging from the organiser. Then, specified MFP precursors
populate the midline of the neural plate.
In Xenopus embryos the term notoplate designates the midline
neural plate cells that later become the FP of the neural tube. It was
reported that the notoplate arises from the dorsal non-involuting marginal
zone (DNIMZ), a region of ectoderm located just above the dorsal involuting
marginal zone (DIMZ) or dorsal lip. The latter contains mesodermal cells that
enter through the blastopore during gastrulation and extend along the
anterior-posterior (AP) axis, giving rise to the prechordal plate and
notochord. The notoplate precursors remain in the ectodermal layer but,
together with the notochord precursors, undergo convergent-extension movements
during gastrulation, resulting in the midline extension of the future FP along
the AP axis (Jacobson, 1981;
Keller et al., 1985
;
Keller and Danilchik,
1988
).
We have recently described that before mid-gastrula Notch executes a binary
cell-fate switch that favours FP development at the expense of the notochord,
leading to the specification of the different cell populations that contribute
to the dorsal midline (DML) in Xenopus
(López et al., 2003).
As a corollary, we proposed that the early organiser indeed contains cells
that have the potential to develop either as notochord or FP, but the question
of whether they constitute a mixed population or occupy different compartments
within the organiser remained unanswered. In addition, we described that Notch
signalling activates shh expression, and secreted Shh would amplify
the effects of the binary decision by inhibiting notochord specification. By
this means, Shh would refine the segregation of both cell-populations
initially started by Notch. This mechanism could in part underlie the role of
shh as a FP inducer during the early event proposed by the third
model of FP formation.
The main goal of the present work is to understand the molecular and cellular mechanisms that govern the development of the DML structures, beginning from their precursors in the Spemann's organiser. In particular, we wanted to answer the following questions: (1) Which is the ligand(s) for the Notch receptor that triggers the cell-fate switch FP vs. notochord? (2) Which is the Notch-target gene(s) that executes this switch? (3) How do cells from the Spemann's organiser give rise to the FP?
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Materials and methods |
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Samples were injected as previously described
(López et al., 2003).
The amounts of synthetic mRNAs and morpholino injected are indicated in the
figures. Some injections included 0.5 ng of nuc-lacZ mRNA as
tracer.
X-gal staining, in situ hybridisation, immunohistochemistry and histology
X-gal staining, preparation of digoxigenin-labelled antisense RNA probes
and whole-mount in situ hybridisation were performed as described previously
(Franco et al., 1999;
Pizard et al., 2004
), except
that the proteinase K step was omitted in in situ hybridization. The
hairy2a template (Turner and
Weintraub, 1994
) was digested with BamHI and transcribed
with T7 RNA polymerase. For double in situ hybridization, fluorescein-labelled
antisense RNA probes were prepared with fluorescein-12-UTP (Amersham). Embryos
were hybridised with digoxigenin and fluorescein-labelled probes
simultaneously, washed and blocked according to the standard protocol and
incubated first with one of the antibodies conjugated with alkaline
phosphatase (AP) (1/2000 of anti-digoxigenin-AP or 1/5000 of
anti-fluorescein-AP, Fab fragments; Amersham). The corresponding probe was
revealed with 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) or Magenta Phos
(Sigma). Inactivation of the AP was carried out (65°C 30 minutes in
methanol, two 5-minute washes in methanol at room temperature), followed by
two 5-minute washes in MAB buffer and 15 minutes incubation in blocking
reagent before adding the second AP-conjugated antibody, which was revealed
with NBT+BCIP or BCIP alone (Sigma).
The c-myc epitope harboured by the notchICD or su(H)DBM constructs used for mRNAs injections and the fluorescein tag of AMOh were detected by immunohistochemistry. For this purpose, after in situ hybridization we performed the AP-inactivation, washing and blocking steps as described above. Then, embryos were incubated for 4 hours at room temperature with mouse 9E10 anti-Myc monoclonal antibody (Santa Cruz) diluted 1/500 or with anti-fluorescein-AP (Fab fragments, Amersham) diluted 1/5000, both in blocking reagent (the same as for in situ hybridization). The unbound antibodies were washed three times with MAB, 10 minutes each, at room temperature, and overnight at 4°C. The following day, embryos injected with AMOh were revealed with BCIP or Magenta Phos, as in the in situ hybridization protocol, and embryos injected with notchICD or su(H)DBM were incubated for 4 hours at room temperature with anti-mouse IgG-AP (Santa Cruz) diluted 1/500 or with anti-mouse IgG-HRP (Dako) diluted 1/100 in blocking reagent. After washing the excess of antibody as before, the anti-mouse IgG-AP was revealed with BCIP or Magenta Phos, as in the in situ hybridization protocol. Embryos incubated with the HRP-conjugated antibody were washed twice with TBS (10 mM Tris-HCl pH 6.5, 150 mM NaCl), 5 minutes each, equilibrated 30 minutes at room temperature with DAB solution (0.5 mg/ml of 3,3'-diaminobenzidine (Sigma) in 10 mM Tris-HCl pH 6.5) and revealed with 0.009% of H2O2 in DAB solution.
For histology, 50 µm sections were taken in an Oxford Vibratome and
mounted onto gelatine coated slides, as described by Hollemann et al.
(Hollemann et al., 1996).
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Results |
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In the dorsal marginal zone, evident transcription of both genes appears in the organiser region around stage 10.5 (Fig. 1). delta1-positive (delta1+) cells are scattered in the dorsal lip (asterisks, Fig. 1B,B'), and strongest expression is found in the rest of the marginal zone (arrow, Fig. 1B,B'). Sagittal sections of these embryos show that, in the organiser, delta1 transcripts are only found in the dorsal mesoderm that has not yet involuted (arrow, Fig. 1B''), whereas involuted axial mesoderm does not express this gene (asterisk, Fig. 1B''). At the same time, hairy2a+ cells are distributed in an arc on the DNIMZ (arrowheads, Fig. 1D-F''). This arc gradually accumulates more hairy2a+ cells and ultimately converges and extends along the AP axis, forming the notoplate (prospective FP) (G-K,M). Interestingly, at early and mid-gastrula stages several hairy2a-expressing cells are scattered on the DIMZ (asterisks, Fig. 1F-F''), where scattered delta1 cells are also found (Fig. 1L). Later, at neural plate stage, hairy2a is strongly expressed in the prospective FP, flanked by bilateral stripes of delta1 corresponding to the proneural domains of ventral motor neurons. Among the involuted DML cells, hairy2a is only found in the head mesoderm, while the notochord is devoid of transcripts (Fig. 1G-M).
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Delta1 down-regulates chd and activates hairy2a, and the latter behaves as a Notch target in the DML precursors
In order to determine whether hairy2a is a target of Notch in the
DML precursors, we activated or prevented Notch signalling with
notchICD or su(H)DBM mRNA,
respectively, and analysed the expression of hairy2a. To identify
those cells that inherited and translated the injected mRNAs, we revealed by
immunohistochemistry the c-myc epitope fused as a tag to the
notchICD and su(H)DBM fragments.
Injection of notchICD mRNA, which encodes a constitutively
active form of the receptor independent of ligand binding, produced an
enlargement of the hairy2a domain on the injected side, both in
gastrulae and neurulae (85%, n=122,
Fig. 3A,A',C-C'',
green asterisks). To corroborate whether endogenous Notch activity was indeed
involved in this modulation, we injected su(H)DBM mRNA,
which encodes a dominant negative variant of the Notch transducer Su(H). We
observed a decrease of hairy2a+ cells both in gastrulae and neurulae
(70%, n=114, Fig.
3B,B',D-D'', red asterisks). Transverse sections of
neural-plate stage embryos confirmed that these changes occurred in the FP
precursors (Fig. 3C'', green asterisk; D'', red asterisk). Therefore, hairy2a, whose
midline expression at the trunk level exclusively marks FP cells, corroborates
that Notch signalling increases the FP size, as previously shown with other
markers that are co-expressed by FP and notochord
(López et al.,
2003).
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From these results, we conclude that hairy2a behaves as a Notch target in the DML precursors and that Delta1 is the ligand capable of switching on the binary decision executed by Notch in the DML precursors.
hairy-2a represses the notochordal fate
To assess whether hairy2a is able to execute the cell-fate switch
triggered by Notch in the DML, we firstly analysed the effects of
overexpressing or blocking hairy2a on notochord development by
looking at the expression of two notochordal markers: chd and
brachyury (bra). When we injected 1 ng of hairy2a
mRNA, chd was drastically repressed in the Spemann's organiser after
dorsal injections (94%, n=33, Fig.
4A, red asterisk). Paradoxically, ventral or lateral injections
resulted in ectopic chd transcription in the rest of the marginal
zone (100%, n=27; arrowhead, Fig.
4B). When sibling control embryos reached the neural plate stage,
all dorsally injected embryos were arrested at gastrulation. Chd+
cells were reduced in number, they could not migrate anteriorly and remained
mostly in the outer layer encircling the blastopore, which was unable to
complete its closing (100%, n=23;
Fig. 4C, red asterisk).
Interestingly, at the neurula stage, ventrally injected embryos developed a
normal dorsal axis (white arrow, Fig.
4D), but the ectopic chd+ cells remained close to the
ventral blastopore lip and were unable to migrate and extend anteriorly (100%,
n=20, arrowhead, Fig.
4D). Lower doses of hairy2a mRNA also repressed dorsal
chd expression and resulted in ectopic chd+ cells on ventral
or lateral mesodermal locations in a similar proportion of injected embryos
(e.g. for chd repression in dorsally injected embryos: 86% with 0.5
ng of hairy2a, n=14; 95% with 0.25 ng of hairy2a, n=20) but
qualitatively, the effects were gradually weaker (not shown). However, with
0.25 ng injections, gastrulation could better proceed, allowing us to examine
other markers at neural plate stages (see below). To block hairy2a
function we injected an antisense morpholino oligonucleotide complementary to
a sequence of hairy2a comprising the initiation codon (AMOh). 10 ng
of AMOh increased the number of chd+ cells on the injected side, both
in gastrulae (Fig.
4E-E''', green asterisks) and neurulae (not shown) (82%,
n=45). When co-injected with 0.5 ng of hairy2a mRNA, 10 ng
of AMOh reversed the down-regulation of chd that produces
hairy2a mRNA alone, demonstrating that this antisense morpholino
specifically interferes with the translation of hairy2a transcripts
[chd+ cells decreased in only 10% of the dorsally injected embryos,
the remaining ones evidenced an increase of chd+ cells (67%) or were
unaffected (23%), n=30; Fig.
4F,F', green asterisk].
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Together, these results show that hairy2a, like Notch signalling, represses bra and chd in the Spemann's organiser. Therefore, hairy2a may be the mediator of Notch in the repression of the notochordal fate that takes place during the DML cell-fate switch. In addition, an excess of hairy2a activity interferes with the normal movement of chd+ (notochordal) cells, blocking their involution.
hairy 2a favours the FP fate
We next addressed the question of whether hairy2a was able to
promote the FP fate as does Notch signalling. Therefore, we overexpressed or
blocked hairy2a and looked at the expression of two FP markers:
foxa4a and shh. However, these genes are also expressed in
the notochord progenitors. Although at stage 11 it is possible to discern the
contribution of foxa4a to the arc of the notoplate, the low
expression of shh at this time does not allow the determination of
the FP and notochord components that contribute to the shh domain.
Thus, we were interested in analysing the effects of hairy2a at
neural plate stages, when both components can be clearly distinguished.
Although 1 ng of hairy2a mRNA had the strongest effects on mesodermal
markers at gastrula stages and also promoted ectopic expression of
foxa4a on ventral locations (arrowhead,
Fig. 5A''), this dose
severely interfered with gastrulation movements, and we were unable to
distinguish between the FP and the notochordal components of shh
expression in these extremely affected embryos. Thus, we decided to lower the
dose until we were able to analyse shh in neurulae, but preserving
the effects, although milder, on notochordal markers. This compromise could be
reached with 0.25 ng of hairy2a mRNA, as described above. In these
conditions, overexpression of hairy2a increased foxa4a+
cells in the notoplate precursors (80%, n=15,
Fig. 5A,A', green
asterisk) and increased shh+ cells in the prospective FP (67%,
n=27, Fig.
5D-D'', green asterisks). In contrast, blocking
hairy2a with 10 ng of AMOh decreased foxa4a+ cells in the
notoplate in gastrulae and in the prospective FP in neurulae (54%,
n=44, Fig.
5B-C'', red asterisks) and shh+ cells in the
prospective FP in neural plate stage embryos (58%, n=19,
Fig. 5E-E'', red
asterisks). In conclusion, hairy2a, like Notch signalling, increases
foxa4a+ and shh+ cells within the prospective FP domain.
These results suggest that hairy2a is a mediator of Notch in the
promotion of FP specification that takes place during the DML cell-fate
switch. Overall, hairy2a is able to promote the FP fate at the
expense of the notochord, and this was confirmed by double in situ
hybridization of chd and foxa4a in injected embryos: 0.25 ng
of hairy2a mRNA increase the domain of chd-foxa4a+ cells (FP
precursors, green asterisk) and concomitantly decrease the domain of chd+
foxa4a+ cells (notochord precursors, red asterisk) (79% and 92%,
respectively; n=14) (Fig.
5F).
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Discussion |
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We present evidence that Delta1 is the ligand that triggers this cell-fate
switch, and that hairy2a is the Notch target that mediates the
repression of the notochordal fate and the promotion of FP development. We
found that hairy2a is able to repress genes that are involved in
dorsal axial mesoderm development, such as chd and bra
(Chesley, 1935;
Smith et al., 1991
;
Cunliffe and Smith, 1992
;
Cunliffe and Smith, 1994
;
Halpern et al., 1993
;
Sasai et al., 1994
;
Sasai et al., 1995
;
O'Reilly et al., 1995
;
Piccolo et al., 1996
;
Piccolo et al., 1997
;
Hammerschmidt et al., 1996
;
Conlon et al., 1996
;
Schulte-Merker et al., 1997
),
and to promote the expression of genes related to FP specification, such as
shh and foxa4a (Ruiz i
Altaba et al., 1993
; Ruiz i
Altaba et al., 1995
; Roelink
et al., 1994
; Chiang et al.,
1996
; Chang et al.,
1997
; Sasaki et al.,
1997
; Epstein et al.,
1999
; Müller et al.,
1999
). Since hairy2a is a transcriptional repressor, at
least two alternative explanations for its role in DML development arise. (1)
A permissive role for FP development, which implies that hairy2a
represses genes that specify the notochordal fate and allows the development
of FP identity through some default mechanism. In this context, it is
intriguing that markers of FP specification are also expressed by the
notochord (e.g. shh, foxa4a), the only exception being
hairy2a itself, whereas notochordal markers seem to be exclusively
present in the notochord (e.g. bra, chd). hairy2a may thus
deplete the DML precursors of molecules required for the specification of
notochord, allowing FP to develop. (2) An instructive role, which implies
that, apart from the repression of genes required for notochord development,
hairy2a may indirectly promote FP specification by repressing a
negative regulator of genes that specify FP fate (e.g. shh,
foxa4a).
Bra behaves as a transcriptional activator
(Conlon et al., 1996), and the
zebrafish homologue is no tail (ntl). Notably, while the
notochord does not differentiate in ntl mutant embryos, the FP is
widened (Halpern et al.,
1997
). This suggests that Bra activity antagonises FP development
while promoting notochord formation. Therefore, bra may constitute a
key target in the Notch-dependent binary switch, and it may be directly
repressed by hairy2a. Further experimentation will be needed to
elucidate whether a hierarchical relationship links hairy2a and
bra in this switch in Xenopus. Interestingly, although
hairy2a is able to activate chd ectopically in ventral or
lateral mesoderm, it represses bra in all locations. Since chd+
bra- cells are unable to involute and migrate properly and bra
is involved in convergent-extension movements
(Conlon and Smith, 1999
;
Kwan and Kirschner, 2003
), it
is conceivable that hairy2a interferes with them by repressing
bra. We suggest that the specification of FP or notochord fates is
intimately linked to cell movements during gastrulation and that
hairy2a constitutes a master gene operating on both events.
It was recently described that the zinc finger transcriptional activator
Churchill (ChCh) stops the ingression of cells through the primitive
streak in chicken embryos (Sheng et al.,
2003). The authors postulated that during normal embryogenesis a
decision between paraxial mesodermal and neural fates is made by establishing
the boundary that restricts cell ingression during gastrulation. We propose
that an analogous mechanism may take place during DML development, with
hairy2a stopping the ingression of notoplate cells that, otherwise,
would have been incorporated into the notochord. This specialised neural vs.
mesodermal switch for DML precursors is envisaged through the observation that
the Xenopus FP retains the potential to differentiate into neurons,
since the ectopic expression of the proneural gene X-ngnr1 in the FP
precursors turns on N-tubulin expression, and the bHLH-O
transcriptional repressor XHRT1, which is expressed in the
prospective FP, represses neurogenesis
(Taelman et al., 2004
).
Interestingly, XHRT1 is able to heterodimerize with Hairy proteins,
suggesting that they may be biologically relevant partners. Although
XHRT1 responds to Notch and can inhibit chd and
bra, it is unlikely to be involved in the notochord vs. FP switch
because it appears after mid-gastrula, later than hairy2a (this work)
and hairy2b (Taelman et al.,
2004
). However, the three transcripts ultimately co-localise in
the FP precursors. This fact, together with the presence of notch1
transcripts in FP at neural plate stages
(López et al., 2003
)
and of delta1 transcripts flanking the hairy2a FP domain
(Fig. 1M of this work) support
the idea of XHRT1 having a role in preventing FP cells from adopting
a neuronal fate (Taelman et al.,
2004
), perhaps in collaboration with hairy2a/b.
Thus, the FP fate would be specified and/or maintained in the course of at
least two binary decisions involving Notch signalling: (1) The early
notochord/FP decision, which appears to take place before mid-gastrula
(López et al., 2003).
Two parallel mechanisms seem to contribute to stop this switch: (a)
notch1 and delta1 transcripts disappear once the notochordal
cells involute (Wittenberger et al.,
1999
; López et al.,
2003
) (this work), suggesting that they become refractory to
divert to FP in response to Notch ligands, which, these cells do not produce
anymore; (b) the competence of the binary switch for responding to active
Notch decreases throughout gastrulation
(López et al., 2003
).
(2) The neuron/FP decision. In this event, Delta1 flanking the midline at
neural plate stages would activate the Notch receptor in the neighbouring FP
cells, which then would turn on hairy2/XHRT1 bHLH-O genes, thus
repressing the neuronal fate to maintain the FP phenotype.
Model interpreting the Notch-mediated cell-fate switch in the DML precursors
Fig. 7 (left column) shows
the dynamics of hairy2a expression in
notchICD-injected embryos in a developmental series from
early to late gastrula stages. Supernumerary hairy2a+ cells
(asterisks) first appear on the DIMZ on the injected-side and gradually
incorporate into the hairy2a-expressing arc on the DNIMZ that
demarcates the prospective notoplate. We propose that, in normal
embryogenesis, the early Xenopus organiser contains cells that have
the potential to develop either as notochord or FP
(Fig. 7 right column).
Delta1 expression starts at early gastrula in scattered cells on the
organiser and interacts with the Notch receptor in the surrounding cells,
leading to the activation of hairy2a and the repression of
chd and bra. hairy2a, in turn, impedes the movement of
involution, and hairy2a+ cells gradually incorporate into the growing
arc on the DNIMZ. This arc ultimately converges and extends along the AP axis
(white arrows), forming the notoplate (prospective FP). By this mechanism
involving notch and hairy2a, Delta signalling executes a
binary cell-fate switch that favours FP development at the expense of the
notochord, leading to the specification of the different cell populations that
contribute to the DML. This model reconciles the findings that the notoplate
arises from the DNIMZ with the hypothesis that FP cells arise from the
organiser (DIMZ). However, we cannot rule-out additional contributions to FP
development from the neural ectoderm (more specifically, to the anterior and
lateral FP) involving Shh as inducer, as it has been described for chicken and
zebrafish embryos (Le Douarin and Halpern,
2000; Thibert et al.,
2003
; Charrier et al.,
2002
; Patten et al.,
2003
; Rebagliati et al.,
1998
; Sampath et al.,
1998
; Schauerte et al.,
1998
; Odenthal et al.,
2000
). Interestingly, the Axolotl homologue of foxa4a,
unlike its Xenopus counterpart, has only a superficial expression in
the early organiser and later, it is only detected in the FP but never in the
notochord, thus resembling the expression of hairy2a in
Xenopus (Whiteley et al.,
1997
). Whiteley et al. provide two possible explanations: (a) the
superficial foxa4a+ cells in the axolotl organiser are future neural
FP cells, programmed very early at gastrulation; (b) the expressing cells are
a mixture of notochord and FP precursors, but later, foxa4a only
persists in FP cells. We presume that both explanations may not be mutually
exclusive and, as with Xenopus hairy2a, some cells may be
representing anterior FP cells, programmed at very early stages of
gastrulation, as those described in chicken
(Patten et al., 2003
), and
other cells may be posterior FP precursors being specified from a bipotential
population, which is also able to give rise to notochordal cells.
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ACKNOWLEDGMENTS |
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Footnotes |
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