A new perspective on neural tube defects: folic acid and microRNA misexpression.
ABSTRACT Neural tube defects (NTDs) are the second most common birth defects in the United States. It is well known that folic acid supplementation decreases about 70% of all NTDs, although the mechanism by which this occurs is still relatively unknown. The current theory is that folic acid deficiency ultimately leads to depletion of the methyl pool, leaving critical genes unmethylated, and, in turn, their improper expression leads to failure of normal neural tube development. Recently, new studies in human cell lines have shown that folic acid deficiency and DNA hypomethylation can lead to misexpression of microRNAs (miRNAs). Misexpression of critical miRNAs during neural development may lead to a subtle effect on neural gene regulation, causing the sometimes mild to severely debilitating range of phenotypes exhibited in NTDs. This review seeks to cohesively integrate current information regarding folic acid deficiency, methylation cycles, neural development, and miRNAs to propose a potential model of NTD formation. In addition, we have examined the relevant gene pathways and miRNAs that are predicted to affect them, and based on our investigation, we have devised a basic template of experiments for exploring the idea that miRNA misregulation may be linked to folic acid deficiency and NTDs.
- SourceAvailable from: Ahmed Fazary[Show abstract] [Hide abstract]
ABSTRACT: The protonation equilibria of vitamin B9 (folic acid) was studied at 298.15 K in a different water–dioxane mixtures [100 wdioxane = 20 %, 40 %, 60 %, and 80 %] with an ionic strength of 0.15 mol·dm–3 NaNO3. The influence of dioxane content on the protonation processes was explained. Also, four protonation constants have been determined in 60 % dioxane with an ionic strengths of (0.15, 0.20, 0.25, and 0.30) mol·dm–3 NaNO3 using the pH-potentiometric technique. HYPERQUAD 2008, a program based on nonlinear least-squares curve fitting was used to determine these four stepwise protonation constants of folic acid from the analysis of pH-potentiometric data. From the determined protonation constants, the representative folate species distribution diagrams were provided by HYSS 2009 program and discussed.Journal of Chemical & Engineering Data 07/2013; 58(8):2219–2223. · 2.05 Impact Factor
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ABSTRACT: Pemetrexed, a new multitarget antifolate antineoplastic agent, has significantly improved the overall survival in nonsquamous non-small-cell lung cancer patients. Presently, pemetrexed is recommended for first line treatment in combination with platinum derivatives, for second line treatment as a single agent and, more recently, as maintenance treatment after first line chemotherapy. In this article we critically appraise the status of pemetrexed including pharmacodynamics, pharmacokinetics, toxicity, and the cost effectiveness of pemetrexed, as well as the predictive biomarkers for pemetrexed based chemotherapy.OncoTargets and Therapy 01/2014; 7:937-945. · 1.34 Impact Factor
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ABSTRACT: Maternal folate intake has reduced the incidence of human neural tube defects by 60-70 %. However, 30-40 % of cases remain nonresponsive to folate intake. The main purpose of this study was to understand the molecular mechanism of folate nonresponsiveness in a mouse model of neural tube defect. We used a folate-nonresponsive Fkbp8 knockout mouse model to elucidate the molecular mechanism(s) of folate nonresponsiveness. Neurospheres were grown from neural stem cells isolated from the lumbar neural tube of E9.5 Fkbp8 (-/-) and wild-type embryos. Immunostaining was used to determine the protein levels of oligodendrocyte transcription factor 2 (Olig2), Nkx6.1, class III beta-tubulin (TuJ1), O4, glial fibrillary acidic protein (GFAP), histone H3 Lys27 trimethylation (H3K27me3), ubiquitously transcribed tetratricopeptide repeat (UTX), and Msx2, and quantitative real-time (RT)-PCR was used to determine the message levels of Olig2, Nkx6.1, Msx2, and noggin in neural stem cells differentiated in the presence and absence of folic acid. Fkbp8 (-/-)-derived neural stem cells showed (i) increased noggin expression; (ii) decreased Msx2 expression; (iii) premature differentiation-neurogenesis, oligodendrogenesis (Olig2 expression), and gliogenesis (GFAP expression); and (iv) increased UTX expression and decreased H3K27me3 polycomb modification. Exogenous folic acid did not reverse these markers. Folate nonresponsiveness could be attributed in part to increased noggin expression in Fkbp8 (-/-) embryos, resulting in decreased Msx2 expression. Folate treatment further increases Olig2 and noggin expression, thereby exacerbating ventralization.Child s Nervous System 05/2014; · 1.24 Impact Factor
A New Perspective on Neural Tube Defects: Folic Acid
and MicroRNA Misexpression
J.M. Shookhoff and G. Ian Gallicano*
Department of Biochemistry and Molecular & Cellular Biology, Georgetown University Medical Center, Washington, DC
Received 2 December 2009; Revised 1 March 2010; Accepted 7 March 2010
Summary: Neural tube defects (NTDs) are the second
most common birth defects in the United States. It is
well known that folic acid supplementation decreases
about 70% of all NTDs, although the mechanism by
which this occurs is still relatively unknown. The current
theory is that folic acid deficiency ultimately leads to
depletion of the methyl pool, leaving critical genes
unmethylated, and, in turn, their improper expression
leads to failure of normal neural tube development.
Recently, new studies in human cell lines have shown
that folic acid deficiency and DNA hypomethylation can
lead to misexpression of microRNAs (miRNAs). Misex-
pression of critical miRNAs during neural development
may lead to a subtle effect on neural gene regulation,
causing the sometimes mild to severely debilitating
range of phenotypes exhibited in NTDs. This review
regarding folic acid deficiency, methylation cycles, neu-
ral development, and miRNAs to propose a potential
model of NTD formation. In addition, we have examined
the relevant gene pathways and miRNAs that are pre-
dicted to affect them, and based on our investigation,
we have devised a basic template of experiments for
exploring the idea that miRNA misregulation may be
linked to folic acid deficiency and NTDs. genesis 48:282–
C 2010 Wiley-Liss, Inc.
Key words: neural
development; neural tube defects; spina bifida
Neural tube defects (NTDs) are the second most com-
mon form of birth defects and affect ?300,000 new-
borns worldwide each year (CDC, 2005; Copp et al.,
2003; Wong et al., 2008). The two most common NTDs
are spina bifida (17.9 per 100,000 live births in the
United States) and anencephaly (11.11 per 100,000 live
births in the United States; CDC, 2007). Spina bifida,
which affects ?1,500–2,000 newborns every year in the
United States, results from a failure of the neural tube to
close properly, a process that is normally completed at
28 days of gestation in humans (AACC, 2007; Keller-Peck
and Mullen, 1997; Nakatsu et al., 2000).
Spina bifida can occur from either failures in primary
neurulation (failure of the posterior neural pore to close)
or secondary neurulation (when the surface ectoderm
fails to separate from the neural tube; Copp and Brook,
1989; Copp et al., 2003). Primary neurulation, which
forms the brain and majority of the spinal cord, results in
the formation of the neural tube and midline fusion of
the neural plate. Secondary neurulation is continuation
of the neural tube to create the posterior portion of the
spinal cord without neural folding. Open NTDs are due
to improper closure during primary neurulation (menin-
gocele and myelomeningocele), whereas closed NTDs
are due to improper closure during secondary neurula-
tion (spina bifida occulta). This improper closure may be
the result of an abnormally reduced rate of cell prolifera-
tion in the notochord but not the neuroepithelium (as
found in curly tail mouse mutants).
*Correspondence to: G. Ian Gallicano, Department of Biochemistry and
Molecular & Cellular Biology, Georgetown University Medical Center, 3900
Reservoir Rd. NW, Washington, DC 20057.
Contract grant sponsor: Samueli Institute, Contract grant sponsor: US
Army Medical Research and Materiel Command, Contract grant numbers:
W81XWH-06-1-0279 and W81XWH-07-1-0240
Published online 12 March 2010 in
Wiley InterScience (www.interscience.wiley.com).
Additional Supporting Information may be found in the online version of
Abbreviations: BCL-2, B-cell CLL/lymphoma 2; CBS, cystathione-b-syn-
thase; CSL, CBF1, Su(H), Lag-1 (regulator of Notch responsive genes);
DBX1, developing brain homeobox 1; DHFR, dihyrdofolate reductase;
DLL1, delta-like 1; DNMT1, DNA methyltransferase 1; DNMT3a, DNA meth-
yltransferase 3a; DNMT3b, DNA methyltransferase 3b; E2F3, E2F transcrip-
tion factor 3; ELAV2, embryonic lethal, abnormal vision, Drosophila-like 2;
EMX2, empty spiracles homeobox 2; FGF8, fibroblast growth factor 8;
FGF15, fibroblast growth factor 15; FOXA2, forkhead box A2; G9a, euchro-
matic histone-lysine N-methyltransferase 2; GBX2, gastrulation brain
homeobox 2; GRG4, transducin-like enhancer of split 4 homolog of Dro-
sophila E; GSK3, glycogen synthase kinase 3; HES1, hairy and enhancer of
split 1; HES3, hairy and enhancer of split 3; HES5, hairy and enhancer of
split 5; HOXA1, homeobox A1; JAG1, Jagged-1; MTHFR, N5N10-methylenete-
trahydrofolate reductase; NGN1, neurogenin 1; NGN2, neurogenin 2; P53,
tumor protein p53; PAX3, paired box 3; RBL2, retinoblastoma-like 2; RIZ1,
PR domain containing 2, with ZNF domain.
' 2010 Wiley-Liss, Inc.genesis 48:282–294 (2010)
Based on the current mouse models, it is clear that no
one overarching mechanism results in NTDs. Examina-
tion of the cells present at the site of the lesions shows
that NTDs are due to a wide range of reasons: increased/
decreased apoptosis, altered cellular proliferation, and
premature differentiation into neural cells (Copp et al.,
2003 includes an online table that is comprehensive in
listing mouse models of NTDs and the believed or
known primary cause; Hatakeyama et al., 2004; Hirata
et al., 2001; Keller-Peck and Mullen, 1997; Wong et al.,
2008; Xu et al., 1999; Yang et al., 2006). It is unknown if
human NTDs share a similar range in diverse etiology,
which makes pinpointing of all the contributing factors
With respect to spina bifida research, mouse models
have been produced in which a gene or series of genes
are knocked out or conditionally knocked out (Copp
et al., 2003). However, these mouse models often die in
utero or shortly after birth and, additionally, few mutant
models of caudal closed NTDs exist (Bell et al., 2003;
Bulgakov et al., 2004; Finnell et al., 2002; Friedman and
Kaester, 2006; Hatakeyama et al., 2004; Hirata et al.,
2001; Nagai et al., 2000; Pierani et al., 2001; Sahara
et al., 2007; Tachibana et al., 2002; Xu et al., 1999). This
is in sharp contrast to what is seen in humans in which
spina bifida occulta (a caudal closed NTD) is the most
common. Thus, there is a strong indication that modified
gene expression and not complete loss of gene expres-
sion is a driving force of NTDs. Current research, while
comprehensive, still has not pinpointed the exact mech-
anism of NTD formation.
UNDERSTANDING THE CAUSES OF NTDs
Folic Acid, DNA Methylation, and NTDs
It has been known for sometime that folic acid defi-
ciency contributes to NTDs but the mechanism cur-
rently remains vague (Bohnsack and Hirschi, 2004; Boot
et al., 2003; Finnell et al., 2002; Johnson et al., 1999;
Rosenquist et al., 1996; Whitehead et al., 1995). Folate
deficiency occurs in about 10% of the US population
and low folic acid has been associated with chromo-
somal damage, including strand breaks and misincorpo-
ration of uracil into DNA (Blount et al., 1997; Duthie
and Hawdon, 1998; Pogribny et al., 2006b). Folic acid
deficiency was linked to the formation of NTDs by the
observation that pregnant women who took medica-
tion that interfered or antagonized folate metabolism
(aminopterin, carbamazpine, and valproic acid) had a
greater number of children with spina bifida (Johnson
et al., 1999).
Why the developing central nervous system (CNS) is
more vulnerable to folate deficiency than other tissues
remains a mystery, but folate, along with Dnmt1, has
been found in high concentrations in the CNS during
normal development (Blount et al., 1997; Finnell et al.,
2002). The precursor cells for both neural crest cells and
the neuroepithelial cells of the neural tube also have a
higher level of folate receptor expression (Boot et al.,
2003). Research has shown that folic acid levels directly
correlate to genomic DNA methylation and deficiency is
associated with global genomic hypomethylation, which
is reversible with repletion therapy (Bohnsack and Hir-
schi, 2004; Choi et al., 2005; Pufulete et al., 2005; Ram-
persaud et al., 2000).
The current prevailing theory is that folic acid defi-
ciency ultimately causes hypomethylation of genomic
DNA because it is the source of methyl groups used in
numerous methylating reactions (Duthie and Hawdon,
1998; Mathers, 2005). Folic acid donates methyl groups
by a complex pathway where it is first converted to tet-
rahydrofolate (THF) by dihydrofolate reductase (DHFR)
and, through a series of steps, from THF to 5-methyl-THF
by N5N10-methylenetetrahydrofolate reductase (MTHFR;
Chen et al., 2001). 5-methyl-THF serves to replenish the
methylated form of Vitamin B12, a cofactor for methio-
nine synthase. Methionine synthase is the enzyme that
converts homocysteine into methionine, which in turn
is converted to S-adenosyl-L-methionine (SAM), the pri-
mary methyl donor for DNA methyltransferases (Choi
et al., 2005).
It is important to comprehend the relationship
between folic acid and methylation due to their key roles
in development. Methylation is essential for synthesis of
membrane phospholipids, myelin basic protein, and
neurotransmitters as well as for establishing proper gene
expression in embryogenesis (Biniszkiewicz et al., 2002;
Chen et al., 2001). During embryogenesis, all three
major DNA methyltransferases (Dnmt1, Dnmt3a, and
Dnmt3b) are expressed, the most important being
Dnmt1 and Dnmt3b in establishing and maintaining
proper genomic methylation (Fan et al., 2001; Jackson
et al., 2004). In mammalian embryonic development,
prior to implantation, the genome becomes demethy-
lated by the 8- to 16-cell stage, with the inner cell mass
undergoing global remethylation at discrete CpG sites
before onset of gastrulation (Finnell et al., 2002; Kafri
et al., 1992; Santos et al., 2002). This is then followed by
the demethylation and transcription of tissue-specific
genes during organogenesis (Jackson-Grusby et al.,
2001). Studies have shown that improper methylation
patterns can lead to multiple developmental malforma-
tions and embryonic lethality.
Global DNA hypomethylation can also be caused by
an increase in S-adenosyl-homocysteine (SAH). After
donating its methyl group, SAM becomes SAH, which is
converted to homocysteine via SAH hydrolase by a re-
versible reaction (Bohnsack and Hirschi, 2004; Chen
et al., 2001). If homocysteine is not converted to methi-
onine then it may be converted back to SAH, which
inhibits DNA methyltransferases because they have an
increased affinity for SAH over SAM (Jackson et al.,
2004). Cystathione-b-synthase (CBS) is the first enzyme
of the transsulfuration pathway that irreversibly removes
cellular homocysteine; in the CBS knockout mouse
model, increased levels of SAH leads to genomic DNA
hypomethylation (Pufulete et al., 2005). Rosenquist
A NEW PERSPECTIVE ON NEURAL TUBE DEFECTS
et al. were able to produce NTDs and cardiac abnormal-
ities by the addition of homocysteine to developing
chick embryos. Folate-specific defects seem to corre-
spond to increased homocysteine levels and women
with normal folate levels but increased plasma homocys-
teine are more likely to have infants with NTDs (Blount
et al., 1997; Johnson et al., 1999; Whitehead et al.,
Another complication in trying to develop a compre-
hensive theory regarding the origins of NTDs is that
MTHFR-null mice, mice whose methionine synthesis
has been disrupted chemically, do not develop spinal
defects (Copp et al., 2003). Clearly, the relationship
between folate metabolism, methylation, and NTDs is
ambiguous and is further convoluted by the fact that
other deficiencies, such as Vitamin B12, can present
clinically in a similar manner to folic acid deficiency
Protein Methylation and NTDs
Histones offer another site of methylation that may be
affected by decreases in the methyl-donor pool and may
also contribute to NTDs if improperly methylated. There
are currently 24 known sites of methylation: 17 on lysine
residues and seven on arginine residues (Pogribny et al.,
2006b). Pogribny et al. fed rats a methyl deficient diet
(MDD) and found a significant decrease in trimethylation
(H4K20); H3K9 trimethylation is necessary for cells to
differentiate and its loss favors proliferation. Progribny
et al. also found a MDD was concomitant with a
decrease in global DNA methylation and a 40 and 44%
decrease in H3K9 and H4K20 trimethylation, respec-
tively (Pogribny et al., 2006a).
In another study, Tachibana et al. established G9a2/2
embryonic stem cells (ESCs; G9a is a mammalian histone
methylase that methylates H3K9) and differentiated
them with all-trans retinoic acid (RA), which is known
to promote differentiation along a neural lineage (Bohn-
sack and Hirschi, 2004; Diez del Corral et al., 2003;
Pierani et al., 2001; Wong et al., 2008). The G9a2/2
ESCs were found to have reduced proliferative capabil-
ities and, additionally, it was shown that loss of H3K9
methylation led to an increase of H3K9 acetylation,
while conversely, methylation of H3K9 via G9a caused
gene silencing and was essential for normal early
Methylated DNA also attracts methyl-binding proteins
that recruit histone deacetylase protein complexes (Pog-
ribny et al., 2006b; Santos et al., 2002) and loss of DNA
methylation has been shown to increase histone acetyla-
tion. Histone hypoacetylation prevents differentiation of
ESCs in vitro, and histone methylation can influence
DNA methylation indicating that the methylation status
of DNA and histones are intricately linked, and pertur-
bances in proper methylation of either may alter normal
repression or expression of genes during development
(Jackson et al., 2004).
miRNA Misregulation and NTDs
MicroRNAs (miRNAs) are small, noncoding tran-
scripts, and ?22 nucleotides long, which belong to a
regulatory class of RNAs that repress expression of target
mRNAs (Bak et al., 2008; Krichevsky et al., 2003, 2006;
Miska et al., 2004). miRNAs have been shown to be a
driving force in embryonic development in multiple spe-
cies (such as zebrafish, xenopus, and mouse) (Foshay
and Gallicano, 2009; Leucht et al., 2008) and have dis-
tinct expression patterns within the developing brain
and CNS (Bak et al., 2008). Because of the prediction
that miRNAs are highly regulated by genomic methyla-
tion, it is a logical question to ask if alteration in methyl-
group availability and methylation patterns affects
expression of miRNAs (Han et al., 2007; Weber et al.,
2007). Recent evidence supports this line of reasoning.
It is interesting to note that at least half of the promoter
regions for miRNAs are predicted to be in close proxim-
ity to CpG islands and their methylation frequency is
predicted to be at least an order of magnitude higher
than that of protein-coding genes.
Han et al. created a double-knockout Dnmt1 and
Dnmt3b cell line (DKO cells) from HCT-116 cells, result-
ing in a 95% reduction of global genomic methylation.
The consequence of this hypomethylation was differen-
tial expression of 13 miRNAs: seven were overexpressed
and six were underexpressed. miR-10a and miR-200a
had the greatest increase (2.85 and 2.35 times, respec-
tively); miR-125b, miR-221, and miR-222 were also over-
expressed (1.72, 1.34, and 1.34 times, respectively).
These five miRNAs are also found in the developing
mouse CNS and have a high homology to their corre-
sponding human miRNA sequences (Supporting Infor-
mation Tables 1–3; Betel et al., 2008; John et al., 2005;
Griffiths-Jones et al., 2004, 2006, 2008; Keller-Peck and
Mullen, 1997; MicroRNAdb, 2004). Additionally, miR-17,
miR-20a, and miR-106a were found to be underex-
pressed (0.71-, 0.77-, and 0.57-fold, respectively).
Marsit et al. addressed specifically if folate-depleted
media would change miRNA expression levels in a
human cell line. Using TK-6 cells (a human lymphoblas-
toid cell line) they demonstrated a profound global
increase in miRNA expression in response to folic acid
deficient (FAD) media. Three of the miRNAs overex-
pressed in this study were the same as those overex-
pressed in the DKO cells: miR-125b, miR-221, and miR-
222 (increased by 2.89, 2.50, and 2.09 times, respec-
tively). miR-22 and miR-34a, which also have roles in the
developing mouse CNS, were also overexpressed (1.93
and 2.08 times, respectively; see Supporting Information
Tables 2–4; Bak et al., 2008; Han et al., 2007; Krichevsky
et al., 2006; Miska et al., 2004). Marsit et al. confirmed
this effect in vivo by comparing the expression of miR-
222 between human patients with normal folic acid lev-
els to those who were FAD, and found that the patients
with folic acid deficiency had statistically significant
increased expression of miR-222. While the cell lines
used were not of embryonic lineage, they provide
SHOOKHOFF AND GALLICANO
powerful evidence that loss of genomic methylation
(DKO cells) and folic acid deficiency (TK-6 cells) can sig-
nificantly alter miRNA expression in human cells.
Two studies have also highlighted the role that etha-
nol, known to cause hypomethylation in the fetal mouse
genome, plays in the misexpression of miRNAs in the
developing mouse cerebral cortex. One study by Sathyan
et al. (2007) isolated fetal mouse cerebral cortical neural
precursors and exposed them to social and chronic levels
of ethanol, which altered expression of miR-9, miR-21,
miR-153, and miR-335, significantly impacting predicted
downstream targets Jagged-1 (a Notch receptor ligand)
and ELAV2. Another study by Wang et al. (2009) actually
examined the effects of ethanol on in vivo mouse brain
development and found that at least 14 different miRNAs
were misexpressed. These included, in addition to miR-9
(as found by Sathyan et al., 2007), miR-10a and miR-200a
(as in DKO cells) as well as miR-145 (as in FAD cells)
(Han et al., 2007; Marsit et al., 2006). These embryos also
displayed, amongst other abnormalities, defects in neural
tube closure starting at concentrations of 2.0 mg/ml
of ethanol. Wang et al. (2009) were able to show that on
folic acid administration, one overexpressed miRNA,
miR-10a, was downregulated and that its downstream
target, Hoxa1 protein, was subsequently upregulated in
a manner that appeared to be dose dependent. These
studies show a striking relationship between ethanol,
NTDs, misexpression of miRNAs, and folic acid.
DOWNSTREAM TARGETS of miRNA
MISEXPRESSION AND NTDs
Because of the large number of genes and miRNAs
potentially involved in neural tube and spinal develop-
ment (some miRNAs are predicted to have hundreds of
targets), sorting through which ones may or may not be
relevant to NTDs is a Herculean task. We decided to
compare genes known to be involved in neural tube,
CNS, and spinal development to miRNAs shown to be
affected by changes in folic acid levels or DNA methyla-
tion, to narrow down the overwhelming number of pos-
sibilities to a few concise, logical starting points, on
which future inquiry may be based.
We then divided the miRNAs into two groups: those
overexpressed or those underexpressed in response to
FAD media or by double knockout of Dnmt1 and
Dnmt3b. We divided genes into groups based on how
many hits for a particular miRNA they had. Our findings
uncovered a number of genes of high interest. For exam-
ple, two genes in particular, Dnmt1 and Foxa2, were pre-
dicted to be targets of miR-221 by two databases, micro-
rna.org and miRBase, for both humans and mice (Betel
et al., 2008; John et al., 2005; Griffiths-Jones et al., 2004,
2006, 2008; MicroRNAdb, 2004). Other genes were pre-
dicted as targets by both databases for humans and by
one database for mice [delta-like 1 (Dll1), Rbl2, and
Grg4], whereas others were predicted as targets by both
databases for mice and by one database for humans
(Dnmt1, MTHFR, Ngn1, and Rbl2).
Finally, there were genes that were predicted to be tar-
gets by both databases for humans or mice with no over-
lap between species (Riz1 and Grg4 in humans; Dbx1,
Emx2, Fgf15, and Gbx2 in mice; Tables 1 and 2; and see
Supporting Information Section for a more detailed
description of the genes and miRNAs considered, and
how they were selected). These genes were then ana-
lyzed in context of their relevant pathways to see how
alterations in miRNA expression might impact neural
tube development. Then, if possible, they were com-
pared with mutant mouse models where expression of
the gene of interest was either altered or repressed
Based on our in-depth analyses, the leading candidate
genes to consider are Dll1, Dnmt1, MTHFR, Rbl2, Dbx1,
Emx2, Fgf15, Foxa2, Gbx2, Grg4, Ngn1, and Riz1. Of
these, Dll1, Dnmt1, MTHFR, and Rbl2 are the most com-
pelling for further research. Dbx1, Emx2, Fgf15, Foxa2,
Gbx2, Grg4, Ngn1, and Riz1 are promising candidates
but at this time, either too little is known regarding their
roles in spinal cord development or there is insufficient
evidence in mouse models to justify further inquiry. To
strengthen our method of analysis, we further confirmed
the predictions of miRBase and microrna.org by running
the mRNA sequences of our strongest candidates
through two different algorithms based on pattern rec-
ognition, RNA22, and FindTar (Miranda et al., 2006; Ye
et al., 2008).
Homology of murine and human miRNAs were also
compared to gain a better understanding of the rele-
vancy of extrapolating conclusions regarding pathways
between the two species. Homology for the miRNAs of
interest (miR-10a, miR-17, miR-20a, miR-22, miR-34a,
miR-106a, miR-125b, miR-200a, miR-221, and miR-222)
was either 100% or close to 100% for all except miR-
106a (Supporting Information Table 5; Griffiths-Jones
et al., 2006; MicroRNAdb, 2004).
Dll1 is involved in the Notch signaling pathway and
mediates its effects by binding to the Notch receptor,
causing its proteolytic cleavage and release of the Notch
intracellular domain (ICD) that continues the Notch sig-
naling cascade (Hatakeyama et al., 2006; Machka et al.,
2005; Yang et al., 2006). Notch has been shown to play
a role in the developing CNS by suppressing neuronal
differentiation and maintaining neural precursor cells
(NPCs) and is highly expressed in the ventral spinal cord
(Ivey et al., 2008). Ivey et al. showed that depletion of
Dll1 by miR-1 influenced ESCs to adopt a cardiac fate,
while suppressing endoderm and neuroectoderm differ-
entiation. Loss of Dll1 also resulted in maintenance of
neural progenitor gene expression. In humans, Dll1 was
predicted by miRBase and microrna.org to be a target of
miR-34a and by microrna.org for mice.
A NEW PERSPECTIVE ON NEURAL TUBE DEFECTS
An increase in the expression of miR-34a would lead
to a decrease of Dll1, which in turn would decrease
cleavage of the Notch receptor and subsequent release
of the Notch ICD; Figures 1 and 6. Three downstream
targets of Notch that are important in CNS development
include Hes1, Hes3, and Hes5, which serves to activate
their transcription by forming a complex with CSL, a
transcriptional inhibitor, and removing it from their Hes
promoters (Hirata et al., 2001; Jukkola et al., 2006; Yang
et al., 2006). Hes1, Hes3, and Hes5 suppress neuronal
differentiation by repressing expression of, amongst
other targets, Ngn1 and Ngn2 via activation of Math1
(Atoh1) (Kriks et al., 2005). As downregulation of Hes1,
Hes3, and Hes5 leads to premature neuronal differentia-
tion (Hatakeyama et al., 2004; Wong et al., 2008) and
their decrease would lead to upregulation of Ngn1 and
Ngn2, which in turn would lead to an increase in neuro-
genesis (Leucht et al., 2008).
Reduced Notch signaling has been shown to result in
premature differentiation of NPCs and increases the ra-
tio of V0-2 interneurons in the ventral spinal cord (Yang
et al., 2006). Conditional knockout mice for Notch1
(N1cKO) had premature differentiation of NPCs in the
ventral-to-dorsal direction, leading to the disappearance
of the ventral half of the central canal. It is interesting
to note that N1cKO mutants displayed an increase in
the neuronal population relative to wild type mice in the
ventral most region of the spinal cord, just above the
floorplate, similar to what is observed in two other mu-
tant mouse models that display features resembling the
human presentation of spina bifida: the splotch-delayed
(Pax3 mutant) embryos and curly tail mutants (Keller-
Peck and Mullen, 1997). N1cKO mutants and Dll1-defi-
cient mutants show a reduction in Hes1 and some Hes1-
null mice display NTDs (Hatakeyama et al., 2004; Hirata
et al., 2001; Machka et al., 2005). Fkbp8-null mice,
which present with spina bifida in a nearly identical
manner to humans, have also been shown to have a
decrease in neural differentiation antagonists, like Hes3,
although Hes3-null mutants are phenotypically normal
(Wong et al., 2008).
Hes12/2Hes32/2and Hes12/2Hes52/2double mutants,
as well as Hes12/2Hes32/2Hes52/2triple mutants, all
display NTDs with premature differentiation of NPCs
present in the neural tube. This pathway represents an
intriguing scenario, one in which a moderate decrease
of Dll1 may lead to a more moderate phenotype than
that displayed by N1cKO, which completely lacked the
ventral half of the central canal.
Dnmt1 is important for remethylation of the genome
in early embryogenesis, with Dnmt12/2mutants usually
dying by E8.0 (Biniszkiewicz et al., 2002; Jackson et al.,
2004; Jackson-Grusby et al., 2001). Dnmt1 is a predicted
target of miR-221 in both humans and mice by both data-
bases, as well as a predicted target for miR-222 by both
the databases for mice and by miRBase for humans. An
increase of both miR-221 and miR-222 may downregu-
late Dnmt1, which would lead to a decrease in mainte-
nance methylation, and this, in turn, would lead to a
decrease in differentiation (Fig. 2; Mathers, 2005).
(between E8.0 and 10.5; Fan et al., 2001), so the use of a
conditional Dnmt1 knockout is necessary to study the
impact its loss has on later stages of development. In
one study, primary mouse embryonic fibroblasts were
extracted and analyzed for global methylation and gene
expression. The conditional Dnmt1 knockouts under-
went p53-dependent apoptosis, global hypomethylation,
and altered gene expression. Three hundred seventy-
three of the genes analyzed were altered, including
an increase in SAH hydrolase, placental lactogen1, and
Genes of Interest With More Than One Predicated Hit for the miRNAs of Interest in Humans
List of microRNAs: Human
miRNAs overexpressedmiRNAs underexpressed
2 human/2 mouse
2 human/1 mouse
1 human/2 mouse
2 human/2 mouse
2 human/1 mouse
1 human/2 mouse
The miRNA is listed beneath the gene it is predicated to target.
Genes of Interest With More Than One Predicted Hit for the miRNAs of Interest in Mouse
List of micro RNAs: Mouse
miRNAs overexpressed miRNAs underexpressed
2 human/2 mouse 2 human/1 mouse 1 human/2 mouse 2 mouse
Dnmt1 miR-221 Dll1 miR-34a
Foxa2 miR-221Rbl2 miR-200a
2 human/2 mouse 2 human/1 mouse 1 human/2 mouse
Dnmt1 miR Grg4 miR-20a
Gbx2 miR-221 miR-200a
The miRNA is listed beneath the gene it is predicated to target.
SHOOKHOFF AND GALLICANO
b-catenin (3.0, 4.0, and 2.3 times, respectively). An
increase in SAH hydrolase should lead to a decrease in
SAH (as a decrease in SAH hydrolase leads to an increase
in SAH) but that is not what is observed when NTDs are
present (Blount et al., 1997; Han et al., 2007; Johnson
et al., 1999; Whitehead et al., 1995). An increase in pla-
cental lactogen1 is interesting because it is a marker of
extraembryonic tissues and it is also overexpressed in
Dnmt [3a2/2and 3b2/2] double knockouts (Jackson
et al., 2004; Santos et al., 2002), which also have global
DNA hypomethylation, and spontaneously differentiate
into extraembryonic tissue in the presence of Leukemia
Inhibitory Factor (LIF).
b-Catenin is an effector in the Wnt pathway and plays
a role in the neural development and methylation path-
ways (Foshay and Gallicano, 2008; Hirata et al., 2001;
Litorchick et al., 2004; Shimogori et al., 2004; Ye et al.,
2001). During early murine NPC proliferation, a b-cate-
nin-TCF complex binds directly to the Ngn1 promoter,
upregulating its expression, initiating neurogenesis. Mis-
expression of b-catenin has been shown to increase cel-
lular proliferation and delay differentiation in the brain,
increasing the number of NPCs, as well as the number of
neurons (Hatakeyama et al., 2004). Despite a potential
increase in SAH hydrolase (and thus a decrease in SAH),
downregulation of Dnmt1 by misexpression of miR-221
and miR-222 seems a good candidate for investigation as
Dnmt1 is highly expressed in the developing CNS (Fan
et al., 2001), its overexpression has been shown to
decrease differentiation, and may increase proliferation
via an increase in b-catenin (Jackson et al., 2004; Jack-
son-Grusby et al., 2001).
Dnmt1 is also a predicted target of miR-17 in both
humans and mice by both databases. A decrease in miR-17
may lead to an increase in Dnmt1 leading to premature
differentiation (Mathers, 2005; Fig. 2). In a study that
created Dnmt1 overexpressing chimeric mice, none of
the embryos survived past E14.5, with three malformed
of the Notch receptor (ICD) where it migrates to the nucleus and relieves repression of the Hes1 and Hes3 promoters by binding to the
repressor CSL (CSL 5 CBF1 and Rbp-Jj). Expression of Hes1 and Hes3 leads to repression of Ngn1, Ngn2, and Ngn3 via Math1. Ngn1 is
known to promote neurogenesis in the mid-hind brain and the formation of dI2 interneurons. Dll1 is a predicted target of miR-34a by both
databases for human and by microrna.org for mice; miR-34a was shown to be overexpressed in FAD TK-6 cells. Misexpression of miR-34a
may lead to the downregulation of Dll1, which would decrease expression of Hes1 and Hes3 ultimately leading to upregulation of Ngn1 and
Ngn2, and an increase in neurogenesis. dI, dorsal interneurons; SNS, sympathetic nervous system.
Dll1 pathway and consequence if miR-34a is misexpressed. Normal Dll1 expression results in cleavage of the intracellular domain
A NEW PERSPECTIVE ON NEURAL TUBE DEFECTS
between E10.5 and E12.5 (unfortunately, the authors did
not describe the nature of these malformations, so it is
unknown if any NTDs were present; Biniszkiewicz et al.,
2002). Although both underexpression and overexpres-
sion of Dnmt1 has been shown to result in embryonic
lethality, it seems unlikely that a scenario of Dnmt1 over-
expression could confidently be explored as a cause of
NTDs because the mechanism is not currently specific
enough to show a likely link.
Rbl2, which is normally expressed at low levels in
mouse ESCs and during neuronal differentiation, acts to
suppress expression of Dnmt3a and Dnmt3b, and when
dephosphorylated inhibits cellular growth by sequester-
ing the transcription factor E2F4 (Benetti et al., 2008;
Bohnsack and Hirschi, 2004; Sinkkonen et al., 2008).
Rbl2 is decreased in proliferating cells and is elevated in
quiescent cells via phosphorylation by GSK3 on a
unique loop region, which stabilizes it (Litorchick et al.,
2004). Suppression of Rbl2 by the miR-290 cluster in
mouse ESCs has been shown to lead to a marked
increase in Dnmt3a and Dnmt3b expression. Rbl2 was a
predicted target of miR-200a by both databases for
human and by microrna.org for mice.
When Rbl2 is suppressed, expression of Dnmt3a and
Dnmt3b increases, so downregulation of miR-200a would
result in an increase of de novo methylation. This seems
unlikely to contribute to the etiology of NTDs in folate
deplete conditions, as there would be a dearth of methyl
donors. However, Benetti et al. predicted that Rbl2 is a
target of miR-106a, which was confirmed by microrna.org
for both humans and mice, and by miRBase for mice (Sup-
porting Information Tables 2 and 3; Fig. 3). A decrease in
miR-106a expression during neurogenesis, when the miR-
290 cluster is already downregulated, may lead to an
increase in Rbl2, decreasing expression of Dnmt3a and
Dnmt3b, which could contribute to the development of
NTDs by further decreasing DNA methylation, and
increasing misexpression of miRNAs (Han et al., 2007). In
dicer-null cells, the decrease in Dnmt3a and Dnmt3b
expression was shown to be directly related to an
increase in Rbl2 protein expression (Sinkkonen et al.,
2008) and maternal-zygotic dicer mutant zebrafish exhibit
abnormal brain morphogenesis and neural differentiation
(Bak et al., 2008). This scenario is a strong candidate for
exploration since Rbl2 is expressed at low levels during
neuronal differentiation and, therefore, its overexpression
at that time could alter normal neural development.
Research is conflicting in regards to polymorphisms of
the MTHFR gene and their contribution to NTDs (Choi
et al., 2005; Mathers, 2005; Pogribny et al., 2006b; Sa-
hara et al., 2007, Shang et al., 2008; Stegmann et al.,
role in the maintenance of methylation patterns established by Dnmt3a and Dnmt3b. Improper maintenance of methylation can lead to alter-
ations in proliferation and differentiation. Dnmt1 is a predicted target of miR-221 by both databases for humans and mice and miR-222 by
both databases for mice and miRBASE for humans. Overexpression of miR-221 and miR-222 may lead to downregulation of Dnmt1 and a
subsequent decrease in maintenance methylation. A decrease in Dnmt1 has been shown to prevent differentiation of ESCs as well as
increase expression of SAH hydrolase, placental lactogen1, and b-catenin. Placental lactogen1 is a marker of extraembryonic tissue and is
also overexpressed in Dnmt [3a2/2and 3b2/2] double knockouts. Overexpression of b-catenin has been shown to increase cellular prolifer-
ation, delay differentiation in the brain, and increase the number of NPCs and subsequent neurons. SAH hydrolase has also been shown to
be upregulated when maintenance methylation is decreased, but this does not fit in entirely with what is known of NTDs, as its overexpres-
sion would lead to a decrease in SAH, and in NTDs, SAH levels are usually elevated. Dnmt1 is also a predicted target of miR-17 by both
databases for humans and mice. A decrease in miR-17 may lead to a reciprocal increase in Dnmt1 expression, leading to an increase in
maintenance methylation, which has been shown in chimeric mice to lead to premature differentiation. Both underexpression and overex-
pression of Dnmt1 can result in murine embryonic lethality.
Dnmt1 pathway and consequences if miR-221 and miR-222 are overexpressed or if miR-17 is underexpressed. Dnmt1 plays a vital
SHOOKHOFF AND GALLICANO
1999). While studies show a correlation between the
MTHFR C677T allele and NTDs in Polish, German, Ital-
ian, and Brazilian populations, no correlation was seen
in French, Spanish, or Shanxi Chinese populations.
MTHFR is a predicted target of miR-34a by both data-
bases for mice and by microrna.org for humans (Sup-
porting Information Table 3). An increase in miR-34a
may lead to a decrease in MTHFR expression, which in
turn would lead to a decrease in the level of 5-methyl-
THF (Bohnsack and Hirschi, 2004; Pufulete et al., 2005;
Fig. 4). A decrease in methionine and, thus, a decrease
in SAM would follow, decreasing DNA methylation,
while simultaneously increasing homocysteine levels
(Mathers, 2005; Whitehead et al., 1995). Concomitant
with rising homocysteine and SAH levels would be inhi-
bition of DNA methyltransferases, further decreasing
miR-34a by both databases for mice and by microrna.org for humans. Overexpression of miR-34a in response to folic acid deficiency may
lead to a reduction of MTHFR, which would drastically reduce the available 5-methyl-THF required to methylate the cofactor of methionine
synthase, vitamin B12. DHFR was also found to be a target of miR-24, which converts FA to THF; after FA is converted to THF, the next step
is the conversion of THF to 5-methyl-THF by MTHFR (N5N10-methylenetetrahydrofolate reductase). Methionine synthase converts homo-
cysteine into methionine, which is then converted to SAM (S-adenosyl-L-methionine), the primary methyl donor for DNA methyltransferases
(Dnmts). If homocysteine is not converted to methionine it can be converted to SAH (S-adenosyl-homocysteine) in a reversible reaction by
SAH hydrolase. This would further contribute to hypomethylation because SAH is an inhibitor of Dnmts as well as Riz1, which methylates
H3K9 (histone 3, lysine 9), the trimethylation of which is necessary for differentiation.
There are consequences to the MTHFR and DHFR pathway if miR-34a is overexpressed. MTHFR was predicted as a target of
ing neuronal differentiation. Its function is to repress expression of Dnmt3a and Dnmt3b, which normally remethylate the genome of the ICM
(inner cell mass). Recent experiments have shown that Rbl2 is targeted and suppressed by the miR-290 cluster in murine ESCs, leading to
significant upregulation of Dnmt3a and Dnmt3b. Rbl2 was predicted by one experiment to be a target of miR-106a by microrna.org for both
humans and mice and by miRBASE for mice. Underexpression of miR-106a may lead to the overexpression of Rbl2 resulting in suppression
of Dnmt3a and Dnmt3b, which would further exacerbate hypomethylation and contribute to a loss of differentiation and further misexpres-
sion of miRNAs.
Rbl2 pathway and consequences if miR-106a is underexpressed. Rbl2 is normally expressed at low levels in murine ESCs and dur-
A NEW PERSPECTIVE ON NEURAL TUBE DEFECTS
DNA methylation (Chen et al., 2001; Kim et al., 2003).
In addition, research has shown that MDDs increase SAH
levels and decrease H3K9 methylation, possibly altering
gene expression (Pogribny et al., 2006a). SAH is also an
inhibitor of Riz1, which methylates H3K9, and was pre-
dicted by both the databases to be a target of miR-221 for
humans (but not mice). However, in MTHFR-null mice,
although they displayed a wide range of abnormalities,
none of them displayed NTDs (although some presented
with kyphosis, a hunched back, and kinked tails similar to
curly tail mutants who do display spina bifida). This situa-
tion is interesting because miR-34a is also predicted to tar-
get Dll1. In concert, simultaneous downregulation of Dll1
and MTHFR by overexpression of miR-34a could lead to
NTDs by disruption of two different pathways.
One more important relationship to discuss is that of
DHFR and miR-24. Although DHFR did not turn up any
significant hits in the database search and miR-24 was
not considered an miRNA of interest in this review (nei-
ther it is neural specific nor was it predicted to target
Dll1, Dnmt1, Mthfr, or Rbl2), Mishra et al., 2004.
showed that DHFR is targeted by miR-24, which was
found to be upregulated 1.36 times greater than control
in TK-6 cells grown in FAD media (Marsit et al., 2006).
This suggests that folate deficiency could initiate a nega-
tive feedback loop where overexpression of miR-24 sup-
presses DHFR, which would further decrease methyl-do-
nor pools, exacerbating methyl deficiency (Bohnsack
and Hirschi, 2004; Pufulete et al., 2005; Fig. 4).
DHFR can also be indirectly affected by miR-34a over-
expression: miR-34a targets and downregulates the tran-
scription factor E2F3, as shown by Welch et al., (2007)
in several human neuroblastoma cell lines. E2F3 is a
member of the E2F family that regulates differentiation
and proliferation by activating many downstream tar-
gets, including DHFR (Humbert et al., 2000; Fig. 5).
Overexpression of miR-34a in three neuroblastoma lines,
Kelly, NGP, and SK-N-AS, led to significant downregula-
tion of E2F3 expression, which was confirmed by both
Western and luciferase constructs (Fig. 5). This illus-
trates yet another way in which perturbing folic acid me-
tabolism may initiate a feedback loop of increasing folate
deficiency via misexpression of miRNAs. Along with
Dll1, Dnmt1, MTHFR, and Rbl2, DHFR should also be
included in a list of strong candidates to explore the
association between folic acid deficiency, methylation,
misexpression of miRNAs, and development of NTDs.
CONCLUSIONS: FUTURE DIRECTIONS
The results of this review show the intertwined and
complex pathways of folic acid metabolism, methyla-
tion, miRNA expression, and gene expression in neural
development. The above miRNAs and genes examined
present a possible explanation of the etiology of NTDs
and may explain folate and inositol independent NTD
Excessive RA is also known to cause NTDs, spina
bifida in particular, and can induce lesions similar to
those found in humans in both mice and rats (Bohnsack
and Hirschi, 2004; Wong et al., 2008; Zhao et al., 2008).
RA is required for normal neuronal differentiation, and
lation of the previous four figures and serves to integrate the various proposed pathways tying in folic acid metabolism, methylation, neuro-
genesis, and misexpression of miRNAs.
A possible cellular model of NTDs incorporating miRNA misexpression and their known or predicted targets. This figure is a compi-
SHOOKHOFF AND GALLICANO
the most active site of RA signaling during development
is the spinal cord (Diez del Corral et al., 2003). RA
pushes cells toward a neural fate, inhibits proliferation,
and attenuates Fgf8 expression in the neuroepithelium.
RA was also found to increase miRNA expression in Hl-
60 myeloid leukemia cells (Weber et al., 2007), increase
miR-34a expression in SK-N-AS neuroblastoma cells (Kim
et al., 2003), and a recent study in a spina bifida rat
model showed that RA not only led to increased cell
death but was responsible for the downregulation of
miR-9/9*, miR-124a, and miR-125b as well as a decrease
in the expression of Bcl-2 and p53 in the affected spinal
cords. This link between RA, spina bifida, and miRNA
downregulation further strengthens the likelihood that
misexpression of miRNAs plays a part in the develop-
ment of NTDs (Fig. 6).
The ultimate purpose of this review is to explore how
miRNA misexpression may alter normal neural tube devel-
opment, contrasting with current genetic investigations
that explore one or two genes at a time then try to extrap-
olate how loss of these genes may affect downstream tar-
gets that result in a mutant phenotype. Misexpression of
miRNAs leads to a very different model: one in which mul-
tiple miRNAs target different genes belonging to several
different pathways that may not be directly connected,
complicating identification of the genes whose altered
expression is involved in the development of NTDs.
Each of the genes discussed when knocked out do not
lead to spina bifida as it is observed in humans, which is
why a miRNA model of NTDs is an attractive alternative
to gene knockouts. By collating the previously discussed
emerges that may explain how even mild depletion of fo-
lic acid can lead to genomic hypomethylation and possi-
bly NTDs (Fig. 5).
A more thorough understanding of how these factors
connect is hampered by a lack of information on folic
acid deficiency and miRNA misexpression in mouse and
human neural stem cells. Because of their abnormal
pathophysiology, cancer cell lines are not reliable mod-
els of the correlation between folic acid deficiency,
methylation, and misexpression of miRNAs, and,
clearly, lymphoblastoid cells do not contribute to nor-
mal neural tube formation. Other cellular models need
to be established to carry out further exploration and
to determine if the association between folic acid defi-
ciency and alteration in miRNA expression holds true
in neural cell lines. Verification of putative miRNA tar-
gets need to be conducted and the methylation status
of any misexpressed miRNA should be assayed for com-
parison with a control. If it can be established that folic
acid deficiency alters miRNA expression in murine
cells, then mouse models incorporating this informa-
tion should be made by injecting mimics to the miRNAs
liferation is tempered by the proper timing of cellular differentiation. The result is a bending at the dorsolateral hinge points that brings the
apical tips of the neural tube in close proximity so that they can fuse and form a closed, hollow structure. A general model of microRNA mis-
regulation involves altered miRNA expression due to low folic acid levels that perturb genomic methylation, specifically microRNAs genes.
As microRNAs can act to promote differentiation, one possible consequence is premature differentiation (b, left) resulting in shortened sides
unable to fold inward and fuse. A specific example involving miR-34a overexpression during neural tube development illustrates how the op-
posite result can also lead to a neural tube defect (b, right). miR-34a overexpression may induce significant downregulation of Dll1, the end
result being a prolonged state of ventral progenitor expression, instead of appropriately timed differentiation. Stuck in a progenitor state the
affected cells would keep proliferating, resulting in an overgrowth that mechanically inhibits bending at the dorsolateral hinge points or even
their formation. The inability to close would expose the developing neural tube to amniotic fluid, further damaging the afflicted area.
A hypothetical consequence of miR-34a overexpression targeting Dll1. In normal neural tube closure (a), the amount of cellular pro-
A NEW PERSPECTIVE ON NEURAL TUBE DEFECTS
of interest that were found to be overexpressed in con-
junction with inhibitors for miRNAs that were shown
to be underexpressed into embryos before neural tube
closure (around E9.0).
In closing, the idea of folic acid deficiency contribut-
ing to NTDs via misexpression of miRNAs is an intrigu-
ing idea and one that should be explored. Because so
many current mouse models display extreme pheno-
types or embryonic lethality, and heterozygotes are usu-
ally unaffected, mutations in genes, especially null muta-
tions, do not seem sufficient to explain NTDs (Bell et al.,
2003; Bulgakov et al., 2004; Finnell et al., 2002; Fried-
man and Kaestrer, 2006; Hatakeyama et al., 2004; Hirata
et al., 2001; Mathers, 2005; Nagai et al., 2000; Pierani
et al., 2001; Sahara et al., 2007; Tachibana et al., 2002;
Xu et al., 1999). miRNAs may offer a more subtle deregu-
lation, and there is enough evidence that miRNA genes
are under regulation by methylation to add plausibility
to the idea (Bak et al., 2008; Han et al., 2007; Lujambio
et al., 2007; Weber et al., 2007). A better understanding
of how folic acid deficiency may contribute to misex-
pression of miRNAs and whether this can lead to NTDs
will broaden our knowledge of CNS development as
well as possibly lead us to new treatments to further
decrease their incidence.
The authors acknowledge Dr. Partha Banerjee, Dr.
Andrew Gajda, and Dr. Tammy Gallicano for their techni-
cal expertise, advice, and critical reading of the manu-
script. The views, opinions and/or findings contained in
this report are those of the author and should not be
construed as the opinion or policy of the Samueli Insti-
tute. The views, opinions and/or findings contained in
this report are those of the author and should not be
construed as an official Department of the Army posi-
tion, policy or decision unless so designated by other
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