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First published online August 10, 2007
doi: 10.1242/10.1242/dev.008177
1 Neural Development Unit, Institute of Child Health, University College London,
30 Guilford Street, London WC1N 1EH, UK.
2 Department of Cell Biology, Duke University Medical Center, Durham, NC 27710,
USA.
3 Molecular Genetics and Evolution Group, Research School of Biological
Sciences, Australian National University, Canberra ACT 0200, Australia.
* Authors for correspondence (e-mails: p.ybot-gonzalez{at}ich.ucl.ac.uk; a.copp{at}ich.ucl.ac.uk)
Accepted 27 June 2007
 |
SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Neurulation, Morphogenesis, Neural tube defects, Noggin, Sonic hedgehog, Mouse, Zic genes
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), and form
part of the phenotype of over 100 mutant mouse strains
(Copp et al., 2003).
Mouse primary neurulation is characterised by a stereotypical pattern of
neural plate bending along the spinal neuraxis
(Shum and Copp, 1996). Neural
tube closure begins at the level of the cervical/hindbrain boundary at
embryonic day (E)8.5 (6-somite stage), and finishes at E10.5 (30-somite stage)
when closure is completed at the upper sacral level. In the intervening
48-hour period, a wave of neural tube closure propagates in a cranio-caudal
direction down the spine. Just caudal to the closure propagation front, a
region of open, elevating neural folds comprises the `posterior neuropore'
(PNP). This represents the next axial region to undergo neural tube closure
(Van Straaten et al., 1992).
Previously, we divided the continuous process of spinal neurulation into three
modes, according to the morphology of neural plate bending within the PNP
(Shum and Copp, 1996). In mode
1 (E8.5-E9), the closing neural plate has a V-shaped cross section, with
bending solely at the median hinge point (MHP), overlying the notochord. As
neurulation progresses to lower spinal levels, mode 2 (E9-E9.75) becomes
recognisable, in which the closing neural folds adopt a different morphology
with paired dorsolateral hinge points (DLHPs) in addition to the MHP. At the
most-caudal level of the spinal axis, just prior to completion of spinal
neurulation, MHP bending disappears and the neural plate bends solely at the
DLHPs (mode 3; E9.75-E10.5).
Although the cell shape changes that comprise bending of the
neuroepithelium at MHP and DLHPs have been documented
(Schoenwolf, 1985;
Smith et al., 1994), the
identity of the dorsoventral molecular signals that regulate these cell shape
changes remains unknown. Previously, we and others showed that MHP bending
requires the influence of the adjacent notochord: suppression of notochordal
development results in the absence of midline bending
(Davidson et al., 1999;
Smith and Schoenwolf, 1989;
Ybot-Gonzalez et al., 2002).
In an analogous way, removal of the surface ectoderm, which normally covers
the outer aspect of the dorsal neural fold, results in the absence of DLHPs,
whereas just a small surface ectodermal remnant is capable of inducing a DLHP
(Jacobson and Moury, 1995;
Moury and Schoenwolf, 1995;
Ybot-Gonzalez et al., 2002).
Hence, signals from the notochord and surface ectoderm are required for MHP
and DLHP formation, respectively. By contrast, the paraxial mesoderm can be
removed without influencing MHP or DLHP formation
(Ybot-Gonzalez et al., 2002).
Further studies have demonstrated a negative influence of Shh, emanating from
the notochord, on the presence of DLHPs. Shh is both necessary and sufficient
to inhibit DLHP formation, as demonstrated by the occurrence of DLHPs at an
abnormally rostral level in Shh-/- mice, and the
inhibition of DLHP formation by beads releasing N-terminal Shh (Shh-N) peptide
implanted adjacent to the dorsolateral neural plate at low spinal levels
(Ybot-Gonzalez et al.,
2002).
Here, we identify the molecular interactions regulating dorsolateral bending during mouse neurulation. Analysis of embryos lacking function of Bmp2 or noggin, and of wild-type embryos exposed to the local release of Bmp2 and noggin peptides, identifies Bmp2 as an inhibitor of DLHP formation. By contrast, noggin and probably another Bmp antagonist, neuralin (also called chordin-like 1), induce DLHP formation. DLHPs are restricted to low levels of the spinal neuraxis because, at upper levels, Shh concentrations are high and noggin expression is inhibited. DLHPs are absent from homozygous Zic2Ku embryos, which later develop severe spina bifida owing to the absence of Bmp antagonists in the dorsal neural plate. Hence, this study reveals a molecular mechanism of regulation of neural tube closure, based on inhibition of Bmp signalling, in the spinal region of the mouse embryo.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), noggin
(McMahon et al., 1998),
Shh (Chiang et al.,
1996) and Zic2 (Elms
et al., 2003) were genotyped as described previously. Whole-embryo
culture was performed as described (Copp et
al., 1999), with opening of the yolk sac and amnion to a minimum
extent compatible with access to the PNP region for bead insertion. AffiGel
blue beads (BioRad, Cat. No. 153-7302) were soaked for at least 2 hours at
4°C in 0.5 µg/µl Bmp2 (R&D Systems), 1 µg/µl noggin
(R&D Systems), 1 µg/µl Shh-N peptide (R&D Systems) or in PBS as
a vehicle control. Beads were held by suction on the end of a mouth-controlled
glass micropipette, inserted singly or in pairs through a slit in the surface
ectoderm overlying the neural fold and positioned as closely as possible to
the dorsolateral neural fold region. Following a variable period in culture,
depending on the experiment, embryos were removed from their extraembryonic
membranes, rinsed in PBS, and fixed in either Bouin's fluid for haematoxylin
and eosin (H&E) staining, 4% paraformaldehyde (PFA) in PBS for in situ
hybridisation, and either PFA or Sainte-Marie's fixative (95% ethanol, 1%
acetic acid) for immunohistochemistry. Presence or absence of a DLHP was
determined in transverse embryo sections using angle measurement criteria as
described previously (Ybot-Gonzalez et
al., 2002).
In situ hybridisation
Previously published probes were: Bmp2, Bmp4 and Bmp7
(Furuta et al., 1997),
cadherin 6 (Henderson et al.,
1997), chordin (Klingensmith
et al., 1999), Msx1
(Mackenzie et al., 1991),
Msx2 (Monaghan et al.,
1991), neuralin (Coffinier et
al., 2001) and Zic2
(Gaston-Massuet et al., 2005).
Additional Msx1 and Msx2 probes were as described
(Catron et al., 1996). To
prepare a cDNA probe for noggin, forward
5'-CCAGCACTATCTACACATCC-3' and reverse
5'-ACTTGGATGGCTTACACACC-3' primers were used to amplify a 518 bp
fragment corresponding to nucleotides 327-845 of the noggin cDNA sequence
(GenBank accession number u79163). Reverse transcriptase (RT)-PCR was
performed on total RNA from E10.5 CBA/Ca embryos using TRIzol reagent (Gibco
BRL). The amplified fragment was cloned into the pGEM-T vector (Promega, UK)
and sequenced to confirm its identity. Whole-mount in situ hybridisation with
preparation of 50 µm transverse vibratome sections was as described
(Copp et al., 1999). In situ
hybridisation on paraffin-embedded sections was performed using
digoxigenin-labelled cRNA probes
(Breitschopf et al., 1992).
Sense-strand cRNA probes were tested for all genes with no specific
hybridisation.
Immunohistochemistry, cell counting and statistical analysis
Fixed embryos were dehydrated, embedded in paraffin wax and sectioned at 7
µm. Sections were rehydrated and antigen retrieval was performed using
Declere (Cell Marque). Antibodies to the phosphorylated forms of Smad1, Smad5
and Smad8 combined (phospho-Smad1,5,8; dilution 1:100; Cell Signalling
Technology); caspase 3 (dilution 1:1000; Cell Signaling Technology);
phospho-histone H3 (dilution 1:250; Upstate Biotechnology); noggin (dilution
1:7; R&D systems); and Bmp2 (dilution 1:10; R&D Systems) were diluted
in 5% goat or rabbit serum, 0.15% glycine and 2 mg/ml BSA in Tris-buffered
saline. Primary antibodies were detected with a biotinylated goat anti-rabbit
or biotinylated rabbit anti-goat (both 1:250, DAKO), using a Vectastain ABC
kit (Vector) and diaminobenzidine (peroxidase substrate kit DAB, Vector).
Omission of primary antibody served as a negative control. Embryos for
comparison of combined phospho-Smad1, -Smad5 and -Smad8 staining were
processed together in the same paraffin block and on the same slide, to enable
accurate comparison. Sections were counter-stained with methylene green. For
analysis of apoptotic cell frequency, the number of cells positive for caspase
3 was determined in the dorsal and ventral halves of the neural plate in 7-12
sections through the PNP of three to four embryos at each of modes 1 and 3.
The mean number of apoptotic cells per section was compared by two-way
analysis of variance, using neurulation mode and neural plate region as
variables. For analysis of cell proliferation, cells positive for
phospho-histone H3 were counted in the dorsal two thirds of the neural plate
in five sections of three embryos at each of modes 1 and 3. The mean number of
mitotic cells per section was compared by t-test.
Scanning electron microscopy
Embryos were rinsed in PBS and fixed overnight in 2% glutaraldehyde, 2% PFA
in PBS. Tissues were rinsed in phosphate buffer and post-fixed in 1%
OsO4 for 1 hour. Samples were dehydrated through an ascending
alcohol series, using three changes of acetone to displace the alcohol, then
CO2 critical point dried, mounted on specimen stubs, gold
sputter-coated and examined in a JEOL SEM 5410 LV scanning electron
microscope.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Diminished Bmp signalling in association with DLHP formation
We compared mode 1, in which DLHPs are absent, with mode 3, in which DLHPs
are present (Fig. 1A).
Bmp2 and Bmp7 mRNA transcripts occurred with similar
intensity in the surface ectoderm overlying the spinal neural folds of mode 1
(Fig. 1B,D) and mode 3
(Fig. 1C,E and data not shown)
embryos. Bmp2 was localised specifically to the dorsal-most ectoderm,
whereas Bmp7 was expressed throughout the surface ectoderm at this
axial level. Bmp4, Bmp5 and Bmp6 were expressed in
more-ventral or anterior embryonic regions, but not in the vicinity of the
neural folds (data not shown). In the absence of any apparent difference in
Bmp2 or Bmp7 expression between modes 1 and 3, we questioned
whether the presence or absence of DLHPs might be associated with differences
in the activity of downstream Bmp signalling. Cadherin 6, a gene regulated by
Bmp signalling (Sela-Donenfeld and
Kalcheim, 1999), was expressed intensely throughout the neural
plate in mode 1 (Fig. 1F), but
appeared downregulated at mode 3 (Fig.
1G and data not shown). By contrast, Msx1, which is also
regulated by Bmps in some systems, was not detectable in the neural folds of
mode 1 embryos (Fig. 1H), and
was only weakly expressed at the neural fold tips in mode 3
(Fig. 1I and data not shown).
Msx2 was not detected in the PNP region (data not shown).
Immunohistochemistry for phospho-Smad1,5,8, which are immediately downstream
of Bmp receptor activation (Massague and
Wotton, 2000), revealed markedly stronger expression in the dorsal
neural folds at mode 1 than at mode 3 (Fig.
1J,K), consistent with the pattern of cadherin 6 expression. It
seems, therefore, that, although Bmp gene expression per se does not differ
along the neuraxis, downstream Bmp signalling is strongest in locations where
DLHPs are absent. This suggests an inhibitory effect of Bmps on neural plate
bending.
Next, we studied whether cell death or cell proliferation differ in the
neural plate of mode 1 and mode 3 embryos. Immunohistochemistry for activated
caspase 3 revealed considerable numbers of dying cells dorsally in the neural
folds of mode 1, whereas cell death was only rarely observed in the mode 3
neural plate (Fig. 1L,M). The
incidence of cell death was significantly increased in the dorsal region of
the mode 1 neural plate (2.6±0.5 cells positive for caspase 3 per
section) compared with the ventral region at mode 1 (0.3±0.1 cells per
section; P<0.05), the dorsal region at mode 3 (0.4±0.01
cells per section; P<0.05) and the ventral region at mode 3
(0.2±0.1 cells per section; P<0.05). By contrast,
phospho-histone H3 immunostaining showed no difference in the frequency of
proliferating cells in the dorsal two thirds of the neural plate at mode 1 and
3 (Fig. 1N,O; mean number of
H3-positive cells: 1.5±0.2 for mode 1; 1.2±0.1 for mode 3;
P>0.05). Hence, programmed cell death, which is associated with
active Bmp signalling in the early embryonic hindbrain
(Graham et al., 1994;
Yokouchi et al., 1996;
Jernvall et al., 1998), also
appears to correlate specifically with mode 1 neurulation in the mouse embryo,
consistent with the idea that, at mode 1, Bmp signalling is strongest in the
dorsal neural plate, in which DLHPs are absent.
|
), we found that neural fold morphology at the PNP was clearly
identifiable in both E8.5 and E9.5 embryos. At the 7-somite stage, immediately
after neural tube closure was initiated, Bmp2-/- embryos
exhibited mode 1 neurulation (n=5), as did wild-type littermates
(Fig. 2A,B). Just a few hours
later, at the 9-somite stage, whereas wild-type embryos continued in mode 1
(Fig. 2C), all
Bmp2-/- embryos studied (n=7) were found to
exhibit striking DLHPs, indicating premature entry into mode 2/3
(Fig. 2D). This is despite the
growth retardation that characterises null embryos at this stage
(Zhang and Bradley, 1996). At
15 somites, the latest stage that could be examined owing to their imminent
demise, Bmp2-/- embryos (n=4) exhibited an
entirely closed spinal neural tube, consistent with the premature formation of
DLHPs (Fig. 2F)
(Yip et al., 2002). By
contrast, wild-type littermates with 15 somites were in mode 2, with prominent
DLHPs (Fig. 2E). We conclude
that Bmp2 is necessary for the inhibition of DLHPs early in mouse spinal
neurulation and that, in its absence, null embryos show premature, exaggerated
formation of DLHPs. To determine whether Bmp2 is sufficient for the inhibition of DLHP formation, we implanted AffiGel blue beads, soaked in either Bmp2 or PBS, within the presomitic mesoderm adjacent to one spinal neural fold, in wild-type embryos at either E8.5 (mode 1) or E9.5 (mode 2/3). After 5 hours culture, embryos were harvested and sectioned transversely to determine whether DLHPs had been induced (at mode 1, when DLHPs are normally absent) or inhibited (at mode 2/3, when DLHPs are normally present). Local release of either Bmp2 or PBS had no discernible effect on the mode 1 neural fold (Fig. 2G,I,K,M), whereas, in the mode 2/3 neuropore, Bmp2 inhibited dorsolateral bending in 76% of cases (n=25; Fig. 3Q) compared with PBS beads, which had no effect (compare Fig. 2H,L with Fig. 2J,N). Immunohistochemistry on sections of embryos implanted with Bmp2 beads showed a striking upregulation of phospho-Smad1,5,8 in tissues on the same side as the bead, but not contralaterally (Fig. 2K,L). By contrast, embryos with implanted PBS beads demonstrated only background Smad staining (Fig. 2M,N). Hence, local release of Bmp2 stimulates downstream signalling and is sufficient to inhibit dorsolateral bending of the neural plate in mouse spinal neurulation.
|
),
are necessary and sufficient to inhibit DLHPs, we next considered the
hypothesis that Bmp antagonists including noggin, neuralin
(Coffinier et al., 2001) and
chordin might be in vivo inducers of dorsolateral bending during mouse
neurulation. In situ hybridisation showed that neuralin was expressed in the
neural plate at modes 1, 2 and 3 of spinal neurulation
(Fig. 3A,B and data not shown),
whereas chordin was expressed solely in the notochord
(Fig. 3C,D and data not shown).
Noggin was expressed in the notochord, but also in the neural fold tips,
immediately underlying the Bmp2- and Bmp7-positive surface
ectoderm. Strikingly, in mode 1, noggin was expressed most-intensely in the
notochord, but only weakly at the neural fold tips
(Fig. 3E), whereas, in mode 3,
the reverse was true: noggin transcripts were intense at the neural fold tips
but weak in the notochord (Fig.
3F and data not shown). Mode 2 gave an intermediate result, in
which noggin expression was strong in both neural folds tips and notochord
(Fig. 3E,F, inset). Neuralin
was also expressed more-intensely at the tips of the neural folds in mode 3
than it was in mode 1, and also had an intermediate appearance in mode 2
(Fig. 3A,B). Hence, the dorsal
neural plate expression of noggin and neuralin correlates with the formation
of DLHPs at mode 3.
To test whether Bmp antagonism is necessary for DLHP formation, we examined
the PNP region of embryos homozygous for a loss-of-function allele of noggin,
in the presence of which spina bifida is observed at high frequency
(McMahon et al., 1998;
Stottmann et al., 2006). On
this genetic background, normal littermates displayed prominent DLHPs at mode
2 (17- to 18-somite stage), whereas stage-matched noggin-/-
(Nog-/-) embryos exhibited markedly reduced dorsolateral
bending, with neural plate morphology closely resembling mode 1
(Fig. 3G,H). This finding is
the opposite of that observed with Bmp2-/- embryos and is
consistent with the idea that noggin is required for dorsolateral bending
during PNP development.
To determine whether noggin is sufficient to induce DLHP formation, we inserted noggin-soaked beads adjacent to the neural fold in wild-type embryos. After 4-5 hours culture, in 43% of cases (n=58; Fig. 3Q), an ectopic DLHP was observed in the mode 1 neural plate (Fig. 3I,K,M), whereas noggin beads had no discernible effect on mode 2/3 neural folds (Fig. 3J,L,O). Immunohistochemistry for phospho-Smad1,5,8 confirmed that the local release of noggin markedly diminished downstream Bmp activation (Fig. 3M-P), although with variation between embryos. It is possible that this variable inhibition of phospho-Smad signalling by exogenous noggin can explain why noggin is not able to induct DLHPs in all bead-implanted embryos. We conclude that noggin is both necessary and sufficient to induce dorsolateral bending in mode 1 neural plate during mouse spinal neurulation.
Interestingly, exogenous noggin induced a contralateral DLHP in some embryos (13/58; Fig. 3Q), although it more frequently produced an ipsilateral DLHP (16/58; Fig. 3I,K,M). By contrast, the local release of Bmp2 inhibited DLHP formation and activated downstream Smad signalling solely on the same side as the implanted bead. Because the release of peptides from implanted beads is difficult to quantitate, we cannot rule out differential loading or diffusion of noggin and Bmp2 as an explanation for this observation. Alternatively, the strong expression of the Bmp antagonist chordin in the notochord (Fig. 3C,D) might serve to neutralise Bmp2 diffusing to the midline, whereas noggin is able to cross the midline unopposed.
Regulation of noggin expression by Bmp2 and Shh during spinal neurulation
The analysis of Bmp2- and noggin-null embryos, and of wild-type
embryos implanted with Bmp2 and noggin beads, suggests a mechanism in which
Bmp signalling negatively regulates DLHP formation, with alleviation of this
inhibition by noggin. At mode 1, noggin is expressed at low intensity in the
dorsal neural plate so that Bmp signalling is dominant, preventing DLHP
formation. By contrast, at mode 2/3, noggin expression is upregulated,
preventing Bmp-mediated DLHP inhibition and enabling dorsolateral bending to
occur. Two further questions arise: first, how is noggin expression regulated
spatially, so that transcripts are found specifically at the tips of the
neural folds? Second, how is noggin expression regulated temporally, so that
noggin transcripts are more plentiful in the dorsal neural plate at mode 2/3
than at mode 1?
|
).
Perhaps Bmp2 and/or Bmp7 in the surface ectoderm are responsible for inducing
noggin expression, thereby explaining this dependence on surface ectoderm
attachment. To investigate this possibility, we examined the expression of
noggin mRNA in Bmp2-/- embryos compared with non-mutant
littermates. At all stages prior to the completion of PNP closure, at around
the 15-somite stage, we observed marked downregulation of noggin expression in
Bmp2-/- embryos, with a total absence of transcripts from
the dorsal neural plate (Fig.
4A-C). By contrast, notochordal expression of noggin continued to
be detected in Bmp2-/- embryos, although its expression
was less-intense caudally. We also implanted Bmp2 beads into wild-type
embryos, and observed a massive upregulation of noggin transcripts ipsilateral
to the bead (Fig. 4D,E). Hence,
dorsal neuroepithelial expression of noggin is stimulated by Bmp2 from the
overlying surface ectoderm, whereas notochordal noggin seems likely to be
regulated by factors other than Bmp2.
The expression of neither Bmp2 nor Bmp7 varied along the
spinal axis (Fig. 1B-E)
suggesting that other factor(s) must be responsible for the temporal
regulation of noggin expression from mode 1 to 3. We found previously that the
strength of Shh signalling from the notochord diminishes as the wave of spinal
neurulation passes down the body axis
(Ybot-Gonzalez et al., 2002),
raising the possibility that Shh might negatively regulate noggin expression
in the dorsal neural plate. If confirmed, this would be an example of
Shh-mediated dorsoventral regulation of neural tube gene expression at a
particularly early developmental stage. Most of the known Shh-regulated genes
are expressed after spinal neural tube closure
(Jessell, 2000). We examined
Shh-/- embryos and found the spread of the dorsal domain
of noggin expression to a more ventral level, both in the PNP and closed
neural tube (Fig. 4H-K).
Moreover, the implantation of beads soaked in Shh-N peptide led to marked
inhibition of noggin expression at the tip of the ipsilateral neural fold in
all embryos examined (n=6; Fig.
4F,G). Hence, it seems likely that noggin expression is confined
to low spinal levels (modes 2 and 3) as a result of negative regulation by Shh
during upper spinal neurulation. This finding provides a molecular explanation
for the inhibitory effect of Shh on DLHP formation, demonstrated in our
previous study (Ybot-Gonzalez et al.,
2002).
|
) fail to close their neural tube in both cranial and caudal
regions (Fig. 5A,B), with a 69%
frequency of exencephaly and 100% penetrant spina bifida later in gestation
(Elms et al., 2003). Scanning
electron microscopy of the PNP at E9.5 showed that dorsolateral bending was
present in wild-type littermates (Fig.
5C), whereas Zic2Ku/Ku embryos had completely
straight neural folds throughout the enlarged PNP
(Fig. 5D,E). Sections at the
rostral end of the neuropore confirmed that DLHPs are present in E9.5
wild-type embryos, early in mode 2, but absent from stage-matched
Zic2Ku/Ku mutants (Fig.
5F,G). To examine further the correlation between the absence of
DLHPs and failure of spinal neural tube closure, we determined the
rostral-most somite level at which the neural tube is persistently open in
Zic2Ku/Ku embryos. Remarkably little inter-embryo
variation was observed, with a mean somite level of 13.3±0.1 (range:
12-14; n=30). Wild-type embryos on the CBA/Ca and closely related
C3H/He (Zic2Ku) genetic backgrounds first exhibit DLHPs at
the 14-somite stage (Shum and Copp,
1996). Hence, our findings support the idea that Zic2 function is
required for the onset of mode 2 spinal neurulation, as marked by the first
appearance of DLHPs, but is not necessary for mode 1 neurulation. The
transition from mode 1 to mode 2 neurulation appears blocked in
Zic2Ku/Ku embryos, and neural tube closure fails from this
point onwards, leading to severe spina bifida.
Zic2Ku/Ku mutants lack dorsal expression of Bmp antagonists
The absence of DLHPs from Zic2Ku/Ku embryos indicates a
requirement for Zic2 specifically in modes 2 and 3 of neurulation.
Consistent with this, we detected upregulation of Zic2 expression in
mode 3 compared with mode 1 (Fig.
6A,B). Because DLHP formation requires antagonism of the
inhibitory influence of Bmp2, we considered two possible explanations for the
absence of DLHPs in Zic2Ku/Ku embryos: overexpression of
Bmp2 or a lack of Bmp antagonism. Elevated expression of phospho-Smad1,5,8
suggested supra-normal levels of Bmp signalling in
Zic2Ku/Ku embryos compared with wild-type littermates
(Fig. 6C,D). On the other hand,
Bmp2 expression in the surface ectoderm overlying the neural folds
was similar in Zic2Ku/Ku and wild-type embryos
(Fig. 6E,H). By contrast, the
expression of noggin (Fig.
6F,I) and neuralin (Fig.
6G,J) was markedly downregulated in the dorsal neural tube of
Zic2Ku/Ku embryos compared with wild-type littermates,
strongly suggesting that Bmp antagonism is defective in the absence of
functional Zic2. Taken together, these observations suggest that the lack of
DLHPs in Zic2Ku/Ku embryos results from abnormally high
levels of Bmp signalling owing to the failure of expression of the Bmp
antagonists noggin and neuralin.
|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Shum and Copp, 1996). Whereas
neural tube closure in the upper spine of mouse embryos is achieved via
bending solely at the MHP (mode 1), closure in the low spine also involves
dorsolateral bending (modes 2 and 3). Our analysis of the
Zic2Ku mutant shows that the complete absence of DLHPs is
incompatible with the progression of spinal neural tube closure beyond the
level of the 14th somite. Hence, the formation of DLHPs is an
obligatory part of low spinal neurulation and, in its absence, severe spina
bifida results.
|
).
Noggin expression is de-inhibited and antagonises the negative influence of
Bmp2 on DLHPs, allowing bending to occur. Shh-null embryos exhibit
mode 3-type neurulation along the entire body axis
(Ybot-Gonzalez et al., 2002),
demonstrating that DLHPs are the `default' neural plate behaviour, in the
absence of Shh influence. Hence, the transition from mode 1 to mode 2 to mode
3 in mouse spinal neurulation is regulated by Shh, with Bmp and its
antagonists playing down-stream regulatory roles.
Evidence from other systems supports the existence of a negative-feedback
loop in which Bmp antagonists are induced by Bmps, but then serve to limit the
intensity of Bmp signalling. For example, Bmp4 induces noggin expression in
chick muscle (Amthor et al.,
1999) and somites
(Sela-Donenfeld and Kalcheim,
2002), whereas, in the mouse embryo, Bmp2 from the ventral
ectodermal ridge induces noggin expression in the adjacent ventral mesoderm
(Goldman et al., 2000).
Overexpression of noggin in the chick neural tube downregulates Bmp4 activity
and delays neural crest induction
(Sela-Donenfeld and Kalcheim,
2000; Sela-Donenfeld and
Kalcheim, 1999; Liem et al.,
1995; Liem, Jr et al.,
1997), demonstrating the quantitative nature of the effect of
noggin on Bmp signalling activity. Moreover, noggin behaves as a typical
example of a dorsal neural tube gene that is repressed, in a quantitative
manner, by the ventralising activity of Shh
(Jessell, 2000).
Our findings provide a striking parallel to the well-established
dorsoventral regulation of specific neuronal and glial cell types in the
spinal cord, in which Shh ventralises and Bmps dorsalise the neural tube,
promoting or inhibiting specific classes of downstream genes
(Jessell, 2000;
Rowitch, 2004). The
neuroepithelium at the stage of neural tube closure is pseudostratified, with
all cells remaining in the proliferative pool and no differentiated cells
types being present. The MHP and DLHPs, although morphologically distinct, do
not contain specific differentiated cell types. Therefore, the present study
shows that diffusible factors, including Bmps and Shh, can act over a distance
of several cell diameters to regulate the cell shape changes
(Schoenwolf, 1985;
Smith et al., 1994) that
mediate neural plate bending, prior to the onset of differentiation of
specific cell types.
Effect of loss of noggin function on spinal neurulation
Embryos homozygous for a null mutation of noggin show over-activation of
Bmp signalling (McMahon et al.,
1998), marked diminution of DLHPs (this study) and defects of
neural tube closure, with failure of cranial neurulation and late-appearing
spina bifida (McMahon et al.,
1998; Stottmann et al.,
2006). We observed residual DLHP activity in
Nog-/- embryos, probably explaining the ability of the
spinal neural tube to close in most homozygous embryos
(Stottmann et al., 2006).
Nevertheless, spinal neural tube closure in Nog-/- embryos
is only temporary, with re-opening in all fetuses by E14
(Stottmann et al., 2006). Our
findings suggest that Nog-/- embryos undergo `pseudo-mode
1' closure, with minimal formation of DLHPs, even at low levels of the spinal
neuraxis. This type of closure is probably unstable, in the highly curved
lower body, and subject to a high risk of re-opening to yield spina bifida at
later stages. Nog-/- embryos also exhibit defects of
sclerotomal differentiation (Stottmann et
al., 2006), which exacerbate the spinal malformations.
|
Role of Zic2 in spinal neurulation
Among the many mouse genetic models of spina bifida
(Copp et al., 2003),
Zic2Ku is the first in which the absence of DLHPs has been
described. Our analysis shows that, whereas Bmp2 expression is
similar in wild-type and mutant embryos, the expression of both noggin and
neuralin is undetectable in the Zic2Ku/Ku dorsal neural
plate. Significantly, we found that homozygotes for the curly tail
mutation, which also develop spina bifida, exhibit normal expression of noggin
and neuralin in the PNP region (data not shown), consistent with the presence
of normal DLHP formation in these embryos
(Shum and Copp, 1996). This
finding argues that the absence of noggin and neuralin expression is unlikely
to be a non-specific consequence of failed neural tube closure. Additional
developmental defects, particularly enhanced ventral curvature of the caudal
region, have been implicated in the failure of neural tube closure in the
curly tail mutant (Brook et al.,
1991).
Activation of the downstream Bmp signalling pathway appears enhanced in
Zic2Ku/Ku embryos, providing an explanation for the
complete absence of DLHPs, the formation of which requires Bmp antagonism.
Zic2 is expressed throughout the neural plate during neurulation,
whereas, following neural tube closure, its expression becomes restricted to
the dorsal neural tube and, eventually, to the roof plate
(Elms et al., 2003;
Gaston-Massuet et al., 2005).
In the chick, the expression of Zic proteins is enhanced by the overexpression
of Bmps, and is diminished by the overexpression of Shh
(Aruga et al., 2002). The
latter finding fits with the observations of the present study, in which
Zic2 expression was more-intense in mode 3, when Shh signalling is at
its weakest. It remains to be determined precisely how Zic2 enables DLHP
formation in the neural plate. The absence of noggin and neuralin expression
from Zic2Ku homozygotes suggests that Zic2 might
positively regulate the expression of these Bmp antagonists in response to
Bmp2. Alternatively, Zic2 might mediate a more downstream event in
dorsolateral bending, with a secondary feedback effect on noggin and neuralin
expression.
|
ACKNOWLEDGMENTS |
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|
Footnotes |
|---|
Present address: Department of Anatomy and Developmental Biology,
University College London, Gower Street, London, WC1E 6BT, UK 
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