FEBS Letters 580 (2006) 2803–2807
Excess methionine suppresses the methylation cycle and inhibits
neural tube closure in mouse embryos
Louisa P.E. Dunlevya, Katie A. Burrena, Lyn S. Chittyb,
Andrew J. Coppa, Nicholas D.E. Greenea,*
b
a
Neural Development Unit, Institute of Child Health, University College London, 30 Guilford Street, London WC1N 1EA, UK
Clinical and Molecular Genetics Unit, Institute of Child Health, University College London, 30 Guilford Street, London WC1N 1EA, UK
Received 17 March 2006; revised 4 April 2006; accepted 6 April 2006
Available online 21 April 2006
Edited by Jesus Avila
Abstract Suppression of one-carbon metabolism or insufficient
methionine intake are suggested to increase risk of neural tube
defects (NTD). Here, exogenous methionine unexpectedly
caused frequent NTD in cultured mouse embryos. NTD were
associated with reduced cranial mesenchyme cell density, which
may result from a preceding reduction in proliferation. The
abundance ratio of S-adenosylmethionine to S-adenosylhomocysteine was also decreased in treated embryos, suggesting methylation reactions may be suppressed. Such an effect is potentially
causative as NTD were also observed when DNA methylation
was specifically inhibited. Thus, reduced cranial mesenchyme
density and impairment of critical methylation reactions may
contribute to development of methionine-induced NTD.
2006 Published by Elsevier B.V. on behalf of the Federation of
European Biochemical Societies.
Keywords: Methionine; S-Adenosylmethionine;
S-Adenosylhomocysteine; Neural tube defect; Exencephaly
homocysteine levels, could also limit the production of methionine. Indeed, dietary studies indicate that low maternal methionine intake is associated with increased risk of an NTD
pregnancy [7,8], leading to the suggestion that a higher methionine intake may reduce risk [8]. In support of this idea, in vivo
administration of methionine to pregnant dams reduces the
incidence of spinal NTD in the Axial defects mutant mouse [9].
One essential function of methionine in neurulation may be
as precursor for S-adenosylmethionine (SAM), the methyl donor in transmethylation reactions. Inhibition of the conversion
of methionine to SAM in chick delays anterior neural tube closure [10], and completely prevents closure in mice (Dunlevy
et al., unpublished). Given that suppression of the methylation
cycle increases NTD risk, and the suggestion that supplemental methionine may promote neural tube closure, we decided to
check that exposure of mouse embryos to excess methionine
during neural tube closure would not cause any adverse effects.
2. Materials and methods
1. Introduction
One-carbon metabolism, comprising the methylation and folate cycles, is essential for many cellular processes including
methylation of DNA and proteins, homocysteine metabolism
and production of nucleotides [1,2]. Methionine and folate
metabolism are interlinked by the methionine synthase-mediated reaction in which homocysteine is remethylated to methionine. Evidence suggests that these metabolic cycles play a key
role in determining the risk of neural tube defects (NTD), severe congenital malformations in which the neural tube fails
to form correctly during embryogenesis [3]. Incomplete closure
of the cranial neural tube leads to an NTD termed exencephaly, a condition which is lethal due to subsequent in utero
degeneration of exposed brain tissue leading to anencephaly.
In humans the clinical importance of the folate/methylation
cycles in neurulation is suggested by studies showing that folic
acid supplementation prevents many cases of NTD [4], while
sub-optimal maternal levels of folate or vitamin B12 or elevated
levels of homocysteine are risk factors for NTD [2,5,6]. Factors
such as sub-optimal folate status that are associated with raised
*
Corresponding author. Fax: +44 20 7831 4366.
E-mail address: n.greene@ich.ucl.ac.uk (N.D.E. Greene).
Abbreviations: NTD, neural tube defects; SAH, S-adenosylhomocysteine; SAM, S-adenosylmethionine
2.1. Mouse embryo culture
Random-bred CD1 mice were purchased from Charles River, UK.
Embryos were generated by timed matings, explanted at embryonic
day (E) 8.5 and cultured in rat serum as described previously [11,12].
Methionine, ethionine, cycloleucine, folic acid or 5-azacytidine (Sigma–Aldrich) were dissolved in PBS and added to cultures as 1% (v/v)
additions. The same volume of PBS alone was added to control groups.
2.2. Assay of SAM and S-adenosylhomocysteine (SAH)
SAM and SAH were quantified in samples consisting of 2–4 embryos, using liquid chromatography tandem mass spectrometry [13].
2.3. Immunohistochemistry and quantitative analysis of cell density and
labelling
After culture, embryos were fixed in 4% paraformaldehyde (PFA),
embedded in paraffin wax and sectioned at 7 lm thickness. Quantitative analysis of cell density and neuroepithelial thickness was performed using haematoxylin and eosin-stained sections. For analysis
of proliferation and apoptosis, sections were processed for antibody
staining by anti-phospho-histone H3 (Upstate Biotechnology) or
anti-activated caspase-3 (Cell Signalling Technology). Labelling indices (number of labelled cells/total cell number · 100) were obtained
from coronal sections through the cranial region.
2.4. Statistical analysis
Proportions of affected and unaffected embryos were compared by
v2 test with pair-wise comparison by Fisher exact test, using SigmaStat
Version 2.03 (SPSS Inc.). Quantitative measurements were compared
by One Way Analysis of Variance on Ranks and pair-wise comparison
was made by Mann–Whitney Rank Sum Test.
0014-5793/$32.00 2006 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.
doi:10.1016/j.febslet.2006.04.020
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L.P.E. Dunlevy et al. / FEBS Letters 580 (2006) 2803–2807
3. Results
Exencephaly (% embryos)
80
PBS only
Methionine
∗
60
8000
2
PBS
Methionine; closed
Methionine; NTD
6000
4000
2000
0
∗
40
Fig. 2. Open neural tube phenotypes in methionine-treated embryos.
Representative control PBS-treated embryos (a) and 5mM methioninetreated (d,g–i) embryos. The extent of the region of open neural folds is
delineated by arrowheads. Examples show defects encompassing the
hindbrain and part or all of the midbrain (d,g), a region of midbrain
only (h), or forebrain only (i). Histological sections reveal the open
hindbrain neural folds and reduced cranial mesenchyme density (* in e)
in methionine-treated embryos (e,f) compared to PBS-controls (b,c).
Scale bars represent 200 lm.
Cell density (cells/mm )
In order to evaluate potential effects of methionine on cranial neurulation, mouse embryos were cultured from E8.5
for 24 h in the presence of methionine. Cranial neural tube closure occurs during this period, and is completed by the 16 somite stage in the normal CD1 mouse strain [14]. Thus, as
expected, the great majority of PBS-treated control embryos
completed closure during the culture (Figs. 1, 2a; Supplemental Table 1). In contrast, methionine treatment caused a dosedependent occurrence of exencephaly, in which the cranial
neural folds remained persistently open (Figs. 1, 2d, g–i).
The extent of the open region varied between embryos
(Fig. 2d, g–i), sometimes including the entire forebrain, midbrain and hindbrain (27% of affected embryos) but in other
cases involving only one of these regions (for proportions in
each category, see Supplemental Table 2).
In many cases exogenous agents that induce NTD also cause
non-specific growth retardation or toxicity [15]. However,
mean crown-rump length, number of somites and yolk sac circulation score after culture were comparable for treated and
PBS-control embryos (Supplemental Table 1), indicating that
exposure to these doses of methionine did not adversely affect
embryonic growth, developmental progression or viability,
respectively. With the exception of persistently open cranial
neural folds, methionine-treated embryos did not exhibit gross
morphological abnormalities (Fig. 2). Moreover, on histological sections there were no obvious alterations in the neuroepithelial thickness (Fig. 2, Supplemental Fig. 1), unlike previous
findings using a methonine analogue, ethionine (Dunlevy et al.,
unpublished). However, abnormalities were observed in the
cranial mesenchyme of embryos exposed to methionine at
doses of 5 mM or higher. In exencephalic embryos the cranial
mesenchyme subjacent to the open neural folds appeared less
dense than in comparable sections of control embryos (compare Fig. 2b–c to e–f). Subsequent quantification of cell density, in transverse sections at matched axial levels, revealed a
significant reduction in mesenchymal cell density in methionine-treated embryos, compared to PBS-controls (Fig. 3). This
reduction in density was present throughout the cranial mesen-
Central
Lateral
Concentration (mM)
Fig. 3. Methionine-induced NTD are associated with reduced density
of cranial mesenchyme. At matched axial levels, cell density was
calculated in defined areas of the cranial mesenchyme; in the midline
subjacent to the neural tube (central) and in lateral areas of the same
section close to the surface ectoderm (lateral). Values are means ±
S.E.M. and * indicates significant difference to the value for PBScontrols in the equivalent region (P < 0.05). Number of sections
analysed was 12 for PBS-controls, 12 for methionine; closed and 8 for
methionine; NTD.
Fig. 1. Inhibition of the methylation cycle causes cranial NTD
(exencephaly) in mouse embryos. Embryos were cultured from E8.5
to 9.5 in the presence of methionine and scored for the presence of
exencephaly, defined as persistently open cranial neural folds in an
embryo with 16 or more somites. Asterisks indicate significantly
increased incidence compared to PBS controls (*P < 0.05).
chyme of affected embryos, but not in apparently unaffected
embryos, in which the cranial neural tube had closed normally.
If the reduction in density of cranial mesenchyme following
methionine treatment is a potential contributory factor in
20
0
0.0
0.5
1.5
2.0
5.0
10.0
L.P.E. Dunlevy et al. / FEBS Letters 580 (2006) 2803–2807
development of exencephaly, changes should be detectable
prior to failure of neural fold closure. To address this possibility, additional embryos were cultured in the presence of 5 mM
methionine or PBS, and analysed after 12 h in culture, by
which time they had developed to the stage immediately preceding closure. We hypothesised that depletion of mesenchyme
could be a consequence of reduced cellular proliferation and/or
increased rate of apoptosis and these were assessed using
immunohistochemistry for phosphorylated histone-3 and
cleaved caspase-3, respectively. Labelling indices were determined in alternate transverse sections at matched axial levels
corresponding to the rostral limit of the optic vesicle
(Fig. 4). Analysis revealed a significantly lower percentage of
phosphorylated histone-3 positive cells in treated embryos
than in controls at the 12–13 somite stage, indicative of a reduced rate of proliferation. The rate of apoptosis, as indicated
by labelling for cleaved caspase-3, appears to increase with
12
Phosphorylated Histone H3
Cleaved caspasea 3
PBS
Methionine
PBS
Methionine
Labelling index (%)
10
8
6
4
2
0
12-13
14-15
12-13
Somite stage
14-15
Fig. 4. Methionine causes a reduced rate of cranial mesenchyme
proliferation prior to failure of neural tube closure. Staining for
phosphorylated histone H3 and cleaved caspase-3 are presented as
mean labelling index ± S.E.M. * indicates significant difference from
controls at the equivalent stage (P < 0.05). Number of sections (4–5
per embryo) were 29, 36, 54, 31, 34, 25, 34 and 24 for bars 1–8,
respectively.
2805
development in control and treatment groups (Fig. 4). Interestingly, at both the 12–13 and 14–15 somite stages the labelling
index for cleaved caspase-3 appeared higher in methioninetreated embryos, although this was not statistically significant.
Induction of NTD by methionine was rather unexpected,
and we therefore quantitated SAM and SAH in cultured embryos, in order to determine the effect of treatment on the
methylation cycle. In methionine-treated embryos there was
a significant reduction in the ratio of abundance of SAM to
SAH (Table 1). For comparison, we also analysed embryos
that had been cultured in the presence of an established methylation cycle inhibitor, cycloleucine [10], at a dose that is
known to cause NTD (Dunlevy et al., unpublished). As expected from a previous study (Dunlevy et al., unpublished),
cycloleucine-treated embryos displayed a significant reduction
in the SAM/SAH ratio, but this was not of greater magnitude
than that caused by methionine (Table 1). The reduction of the
SAM/SAH ratio that we detected in cultured embryos is expected to result in suppression of methyltransferase activity,
such as methylation of genomic DNA which uses SAM as
methyl donor. We therefore tested the specific requirement
for adequate DNA methylation during neurulation by culturing mouse embryos in the presence of 5-azacytidine, a cytidine
analogue that is incorporated into DNA but not methylated.
As for methionine and methylation cycle inhibitors, a significant proportion of embryos treated with 50 ng/ml 5-azacytidine exhibited cranial NTD (Table 2), suggesting that
inhibition of DNA methylation is sufficient to prevent neural
tube closure. In the case of 5-azacytidine, the SAM/SAH ratio
was not disturbed in treated embryos (Table 1), as this inhibitor acts downstream of the methylation cycle.
Potential mechanisms by which folic acid supplementation
has been proposed to prevent NTD include provision of nucleotides for cellular proliferation or stimulation of the methylation cycle [1,16]. As our data suggested that reduced
proliferation and/or suppression of methylation may both be
involved in the mechanism underlying methionine-induced
NTD, we tested the ability of folic acid to prevent these defects. Embryos were cultured for 24 h with methionine or a
methylation cycle inhibitor, ethionine, at doses that specifically
caused exencephaly (Table 3). Additional embryos were con-
Table 1
Quantification of SAM and SAH in cultured mouse embryos
Treatment
No. samples
SAM (nmoles/mg protein)
SAH (nmoles/mg protein)
Ratio SAM/SAH
PBS
Methionine (5 mM)
Cycloleucine (15 mM)
PBS
5-Azacytidine (50 ng/ml)
5
5
5
7
5
3.79 ± 0.54
4.40 ± 0.46
3.68 ± 0.45
4.29 ± 0.74
5.40 ± 0.78
0.024 ± 0.004
0.043 ± 0.006
0.031 ± 0.004
0.026 ± 0.005
0.035 ± 0.005
167.0 ± 12.4
104.0 ± 4.7*
120.0 ± 7.5*
165.8 ± 8.5
154.6 ± 8.3
Embryos were cultured from E8.5 for 17 h, prior to quantification of SAM and SAH. A separate control group was cultured concurrently with
5-azacytidine treated embryos. Values are expressed as means ± S.E.M. * Indicates significant difference from values for PBS-controls, P < 0.05.
Table 2
Development of embryos cultured in the presence of 5-azacytidine
5-Azacytidine conc. (ng/ml)
No. embryos
Yolk sac circulation
Somites
Crown-rump length (mm)
No. cranial NTD (%)
0
50
10
19
2.9 ± 0.1
2.8 ± 0.1
19.9 ± 0.5
19.5 ± 0.7
2.42 ± 0.11
2.27 ± 0.07
0 (0)
13 (68.4)*
NTD were assessed as in Fig. 1. Values are given as means ± S.E.M. Yolk sac circulation was scored from 0 (no circulation) to 3 (full circulation
throughout the yolk sac). * Indicates significant difference from control group (P < 0.05).
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L.P.E. Dunlevy et al. / FEBS Letters 580 (2006) 2803–2807
Table 3
Folic acid (500 lm) does not prevent NTD induced by methionine (5 mM) or ethionine (5 mM)
Reagent
No. embryos cultured
Yolk sac circulation
Somites
Crown-rump length (mm)
No. cranial NTD (%)
PBS
Methionine
Methionine + FA
13
27
25
2.92 ± 0.08
2.89 ± 0.08
2.60 ± 0.08
20.0 ± 0.52
20.3 ± 0.32
20.3 ± 0.45
2.41 ± 0.08
2.43 ± 0.05
2.40 ± 0.06
0
10 (37)*
12 (48)*
PBS
Ethionine
Ethionine + FA
6
23
26
3.00 ± 0.00
2.96 ± 0.04
2.89 ± 0.08
20.2 ± 0.60
19.6 ± 0.48
20.8 ± 0.35
2.40 ± 0.05
2.47 ± 0.06
2.48 ± 0.06
0
12 (57.1)*
17 (65.4)*
Embryos were cultured with or without folic acid and assessed following culture. Values are given as means ± S.E.M. * Indicates significant variation
from control embryos (P < 0.05).
currently treated with methionine or ethionine and folic acid,
at a concentration that significantly reduces NTD incidence
in the splotch (Sp2H) mouse [17]. However, folic acid had no
effect on the incidence of exencephaly induced by either methionine or ethionine (Table 3), nor was there any detectable
effect on growth or developmental progression.
4. Discussion
The methylation cycle appears essential for cranial neural
tube closure since inhibition prevents or delays closure in
mouse and chick embryos [10]. The finding, in the present
study, that methionine can also induce a significant incidence
of isolated NTD was therefore unexpected, as methionine
was predicted to increase flux through the methylation cycle.
However, quantification of SAM and SAH revealed that
although SAM abundance was slightly elevated by methionine
treatment, there was a proportionately greater increase in the
abundance of SAH. This effect may result from feedback suppression of the conversion of homocysteine to methionine. The
consequent increase in homocysteine availability would in turn
lead to the increase in SAH levels that we observe, since the
equilibrium of the reaction that interconverts SAH and homocysteine favours formation of SAH [18]. The overall effect, a
reduction in the SAM/SAH ratio is predicted to inhibit transmethylation reactions [18].
Suppression of methylation reactions could be envisaged to
cause NTD through several possible mechanisms. DNA methylation is an important mechanism for epigenetic regulation of
gene expression [19] and we observed NTD in mouse embryos
exposed to 5-azacytidine, as reported previously [20]. Since
numerous genes with a variety of functions are essential for
cranial neurulation [3], mis-regulation of one or more of these
has the potential to cause exencephaly. Mouse genetic models
and inhibitor studies also indicate the importance of cytoskeletal protein function during cranial neurulation [3,21,22]. Several cytoskeletal proteins are known to be methylated at this
stage of development [23] and it is therefore possible that altered regulation of cytoskeletal proteins through aberrant
methylation could influence closure.
In addition to likely methylation changes in methionine-exposed embryos, the reduction in cranial mesenchyme cell density potentially contributes to development of exencephaly, as
the mesenchyme is thought to play an important role in facilitating cranial neurulation by supporting the elevating neural
folds [3,24]. For example, depletion of the mesenchyme has
been associated with failure of closure in Cart1 null mice
[25]. In our study, reduced mesenchymal density was only pres-
ent in exencephalic embryos but not those that completed neural tube closure, such that this abnormality could be causative
or, alternatively, might be secondary to failure of closure.
Arguing for a contributory role, failure of closure is preceded
by reduced proliferation (and possibly increased apoptosis) in
cranial mesenchyme, which could explain the subsequent reduced density. We therefore propose that both suppression
of crucial methylation reactions and reduced proliferation of
cranial mesenchyme may contribute to failure of neurulation.
Overall, there is now considerable evidence, both from
experimental models and human studies, that suppression of
the methylation cycle could contribute to increased risk of
NTD. Supplementation with agents that promote adequate
flux through the methylation cycle may therefore be beneficial.
However, the present study indicates that excess levels of
methionine can suppress the methylation cycle, suggesting that
methionine may be contra-indicated as a protective agent.
Acknowledgements: The authors are grateful to John Scott for helpful
discussions and for financial support from BDF Newlife, Medical
Research Council and Wellcome Trust.
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.febslet.2006.04.
020.
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