DNA hypomethylation restricted to the murine
forebrain induces cortical degeneration and impairs
postnatal neuronal maturation
Leah K. Hutnick1,2, Peyman Golshani3,4, Masakasu Namihira1, Zhigang Xue1, Anna Matynia3,
X. William Yang5, Alcino J. Silva3,5,6, Felix E. Schweizer3and Guoping Fan1,2,?
1Department of Human Genetics and2Department of Neuroscience Interdepartmental Program, David Geffen School
of Medicine, University of California at Los Angeles, 695 Charles Young Drive South, Los Angeles, CA 90095, USA,
3Department of Neurobiology,4Department of Neurology,5Department of Psychiatry and Behavioral Sciences and
6Department of Psychology, UCLA, Los Angeles, CA 90095, USA
Received February 27, 2009; Revised April 18, 2009; Accepted May 6, 2009
DNA methylation is a major epigenetic factor regulating genome reprogramming, cell differentiation and
developmental gene expression. To understand the role of DNA methylation in central nervous system
(CNS) neurons, we generated conditional Dnmt1 mutant mice that possess ?90% hypomethylated cortical
and hippocampal cells in the dorsal forebrain from E13.5 on. The mutant mice were viable with a normal life-
span, but displayed severe neuronal cell death between E14.5 and three weeks postnatally. Accompanied
with the striking cortical and hippocampal degeneration, adult mutant mice exhibited neurobehavioral
defects in learning and memory in adulthood. Unexpectedly, a fraction of Dnmt12/2cortical neurons survived
throughout postnatal development, so that the residual cortex in mutant mice contained 20–30% of hypo-
methylated neurons across the lifespan. Hypomethylated excitatory neurons exhibited multiple defects in
postnatal maturation including abnormal dendritic arborization and impaired neuronal excitability. The
mutant phenotypes are coupled with deregulation of those genes involved in neuronal layer-specification,
cell death and the function of ion channels. Our results suggest that DNA methylation, through its role
in modulating neuronal gene expression, plays multiple roles in regulating cell survival and neuronal
maturation in the CNS.
Neural development is composed of a cascade of genetic pro-
grams that precisely control stage-specific gene activities
required for neural patterning, cell migration and neuronal
connectivity. Recent studies indicate that the temporally-
and spatially-controlled gene expression in the nervous
system is not only regulated by the transcriptional machinery,
but also subject to modulation by epigenetic mechanisms such
as DNA methylation and chromatin remodeling. DNA methyl-
ation is catalyzed by a family of DNA methyltransferases
(Dnmts) that include de novo (Dnmt3a and Dnmt3b) and
maintenance methyltransferases (Dnmt1) (1–3). All three
enzymes are expressed in the central nervous system (CNS)
and are dynamically regulated during development and
inhibits gene expression by either directly interfering with
transcription factor binding to DNA (6) or recruitment of
methyl CpG binding proteins (MBDs) which complex with
co-repressor(s) and histone modification enzymes such as his-
totone deacetylases and methyltransferases to transform chro-
matin to a repressive state (reviewed in 5 and 7).
The important role for DNA methylation in CNS develop-
ment and function is first implicated by the identification of
MeCP2 mutations in mental retardation disorder Rett syn-
drome (8). As a prototype of the MBD proteins, MeCP2 is
highly expressed in post-mitotic neurons and is involved in
regulating neuronal gene expression including neurotrophin
BDNF and transcription factor Dlx5/6 (9–11). MeCP2-
deficient mice exhibit many typical Rett phenotypes including
(4,5). Mechanistically, DNA methylation
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Human Molecular Genetics, 2009, Vol. 18, No. 15
Advance Access published on May 10, 2009
neuronal atrophy and stereotypic symptom such as irregular
breathing pattern (12,13). To directly examine the role of
DNA methylation in the CNS, we have previously created a
mouse model with DNA hypomethylation in the entire devel-
oping CNS (14). By conditional deletion of Dnmt1 in neural
precursor cells, we demonstrated that DNA hypomethylation
in the CNS disrupts neural control of breathing at birth,
leading to neonatal lethality of mutant mice (14). Furthermore,
DNA hypomethylation in neural precursor/stem cells triggers
precocious glial differentiation by activating the JAK-STAT
pathway and the expression of glial marker genes such as
glial fibrillary acidic protein (Gfap) (15–17). Thus, DNA
methylation serves as a key epigenetic mechanism in the tem-
poral control of neural stem cell differentiation.
In the current study, we attempted to address the role of
DNA methylation during cortical neuronal maturation at the
perinatal stages. We have crossed the Emx1-cre transgene
(18) with the Dnmt1 conditional allele (Dnmt12lox) (14) to
generate a strain of Dnmt1 mutant mice that are viable in
adulthood but exhibit DNA hypomethylation exclusively in
the dorsal forebrain. Although mutant mice have a normal life-
span, they develop cortical degeneration during early postnatal
development and show obvious behavioral defects such as
hyperactivity and impaired learning and memory. Surpris-
ingly, a significant portion of hypomethylated cortical
neurons was retained in the mutant brain, allowing us to
address the effect of DNA hypomethylation on neural gene
expression and the maturation of cortical neurons postnatally.
Mutant mice are viable with conditional Dnmt1 gene
deletion in the dorsal forebrain
We used Emx1-cre to trigger in vivo deletion of Dnmt1 exclu-
sively in telencephalic precursors, inducing DNA hypomethy-
lation in excitatory neurons and astroglia of the cortex and
hippocampus but not in inhibitory interneurons (18,19). We
confirmed the gene deletion pattern in Emx1-cre; Dnmt12lox
mice by crossing onto the R26R-lacZ reporter strain (20).
The lacZ-staining pattern is restricted to the dorsal cortex
and hippocampus in both E16.5 Emx1-cre; Dnmt12lox/þhetero-
zygous controls (designated as control group) and Emx1-cre;
Dnmt12lox/2loxmutants (mutant group hereafter), indicating
the pattern of Dnmt1 gene deletion in the CNS (Fig. 1A).
It has been reported that Cre-mediated recombination in
Figure 1. Hypomethylated cortical cells are detected in Emx1-cre; Dnmt12lox/2loxmutants throughout the normal life span (A) LacZ histology pattern shows brain
regions with Dnmt1 gene deletion via the R26R reporter (ref. 20). (B) The recombination efficiency was analyzed by Southern blotting analysis. The percentages
of Dnmt1 gene deletion are derived from the ratio of recombined null allele (1lox) over the sum of functional Dnmt12lox(2lox) and 1lox alleles. (C) The graph
view of the efficiency of Dnmt1 gene deletion in control and mutant mice from E11.5 to adulthood at 1.5 years old. (D) Southern blot analysis of DNA methyl-
ation in dorsal cortex. DNA was digested with methyl-sensitive enzyme HpaII (CCGG) and was hybridized to an IAP probe. CON, Emx1-cre; Dnmt1210x/þ
control; MUT, Emx1-cre; Dnmt1210x/210xmutant; Ctx, cortex; str, striatum.
2876 Human Molecular Genetics, 2009, Vol. 18, No. 15
Emx-cre mice was initiated between E8.5 and E10.5 as
characterized before (18,19). Southern blot analysis confirmed
that significant Dnmt1 gene deletion was observed in E11.5,
and the efficiency in mutant cortices peaks at 89% (E13.5)
and declines to 58% at P0, down to 39% at P7, and maintains
at ?30% during adulthood (Fig. 1B and C). The decrease in
Dnmt1 gene deletion efficiency in mutant brains when com-
pared with controls is likely caused by degeneration of hypo-
methylated cells (see below). In parallel to the time course of
Dnmt1 gene deletion, we found significant demethylation in
mutant cortical cells as assessed by Southern blot analysis
of the normally heavily methylated Intracisternal-A-Particle
(IAP) retroviral element (Fig. 1D). These results suggest
hypomethylated neurons are present in dorsal forebrain
throughout the mutant animal’s lifespan, but loss of hypo-
methylated neurons seems to occur at both pre- and post-natal
Emx1-cre; Dnmt1 mutant mice exhibit severe cortical
degeneration and neurobehavioral defects in learning
After conducting a careful histological examination of the
brain morphology of control and mutant littermates, we
found striking cortical degeneration in mutant brains at peri-
natal stages (Fig. 2A). Only a moderate decrease in cortical
thickness was observed in mutant brains at P0; however, a
severe loss of cortical and hippocampal volume was found
during early postnatal stages (Fig. 2B). Indeed, quantitative
measurements of cortical thickness indicate, as a percentage
of control, a reduction of 36.7+1.9% in P7 mutants and
further diminishment by 68.3+1.3% in adult mutants
(Fig. 2B). Immunostaining for the post-mitotic neuronal
marker NeuN confirms that the mutant cortex contains
fewer neurons thanheterozygouslittermatecontrols
Figure 2. Cortical degeneration in dorsal forebrain of Emx1-cre; Dnmt12lox/2loxmutants (A and B) Microscopic images of cresyl-violet staining in control and
mutant brain sections at newborn, early postnatal P7, and adult stages. (C) NeuN immunohistochemistry at P7 reveals a decrease in the density of neurons in the
cortex of the mutant. (D and E). Immunostaining for GFAP in control and mutant cortex. Ctx, cortex; vz, ventricular zone; str, striatum; hipp, hippocampus; lv,
lateral ventricle; fx, fornix; ic, internal capsule; I–IV, cortical layers; wm, white matter; mz, marginal zone; dcp, dense cortical plate. Con, Emx1-cre; Dnmt12lox/þ
control; mut, Emx1-cre; Dnmt12lox/2loxmutant.
Human Molecular Genetics, 2009, Vol. 18, No. 152877
(Fig. 2C). An increase of GFAP-positive reactive astroglia
was accompanied with neuronal loss in the cortical layers,
indicative of gliosis due to postnatal neuronal cell death
(Fig. 2D and E).
To visualize the distribution of hypomethylated neurons in
mutant brains, we employed immunohistochemical analysis
against the major core protein of IAP retroelements (21).
IAP expression is undetected in methylation proficient cells,
but is induced specifically in hypomethylated somatic cells
(Fig. 3A) (22,23). In the cortex of one-month-old mutant
mice, IAP immunoreactivity is restricted to the dorsal cortical
zone, precisely corresponding to the area of Dnmt1 gene del-
etion (Fig. 3B). Co-localization of IAP immunoreactivity and
NeuN marker showed that the majority of IAPþcells are
neurons. In one-month-old mutant brain, among the total
32.3% IAPþcells, 70.7% are NeuNþneurons (Fig. 3C and
D). The remaining IAPþcells (29.3%) are GFAPþastrocytes
that were also derived from Emx1-positive precursor cells.
Similar results were observed in the two-month-old mutant
mice (Fig. 3D). Thus, both demethylated neurons and
GFAPþastrocytes can survive in the adult mutant brain. In
contrast, methylation proficient parvalbumin- and calbindin-
positive GABAergic interneurons were negative for IAP
immunoreactivity (data not shown). Inhibitory GABAergic
interneurons originate from the medial ganglionic eminence
during embryonic development and they migrate into
dorsal cortex (24). Thus, interneurons are DNA methylation
proficient from the ontogeny and are spared from Emx1-cre-
mediated Dnmt1 gene deletion.
To determine whether hypomethylated neurons in mutant
mice undergo apoptotic cell death during the pre- and
post-natal development, we performed TUNEL assays in
brain sections at ages E11.5, E14.5, P0 and P7 (Fig. 4). At
E11.5, IAP staining showed the presence of hypomethylated
neural precursor cells in mutant cortex; however, no difference
in the number of TUNEL-positive cells was found between
controls and mutants. In contrast, by E14.5 at the peak of neu-
rogenesis, TUNEL-positive cells were significantly increased
in the mutant cortical plate and ventricular zone, which
co-localized with many IAP-positive cells (arrowhead in the
insets of Fig. 4). Our results indicated that apoptotic cell
death of hypomethylated cortical neurons and precursor cells
commences at E14.5. Immunostaining with an antibody
against activated caspase-3 confirmed that hypomethylated
cells undergo a caspase-activated apoptosis at E14.5 (Fig. 4).
Apoptotic cell death was also obvious in the dorsal cortex of
mutant mice between P0 and P7, during this period when
we observed a most dramatic reduction of cortical volume
(Fig. 4). Taken together, our results indicated that severe cor-
tical degeneration in Emx1-cre; Dnmt1 mutant mice is at least
in part mediated by hypomethylation-induced apoptotic cell
death at perinatal stages.
Given the observation of severe cortical degeneration in
mutants, we next assessed the neurobehavioral defects using
a battery of motor skill tasks and learning and memory trials
on a cohort of age-matched, sex-matched mutant and control
mice (age 6–8 months). We found that while mutant mice
can perform just as well as controls in motor learning and
Figure 3. Localization of demethylated cells in mutant brains by IAP immunostaining. (A) Western blotting of IAP protein in the dorsal cortex of wild type (con)
and Emx1-cre; Dnmt12lox/2loxmutants mice (mut) and Nestin-cre; Dnmt12lox/2loxmutant mice (Nestin Dnmt1, see ref. 20). (B) Immunohistochemical detection of
IAP in the dorsal cortex at 1 month of age in coronal sections. (scale bar ¼ 100 microns) (C) Co-staining with NeuN (a pan-neuronal marker) reveals an IAP-
positive staining pattern in mutant cortex only. (scale bar ¼ 25 microns) (D) Cell counts show that approximately 28–32% of the total cells are IAP-positive in 1
or 2 months (mo) old mutant mice. Among those IAPþ cells, 70.67+1.8% are NeuNþ neurons.
2878 Human Molecular Genetics, 2009, Vol. 18, No. 15
coordination (Supplementary Material, Fig. S1), they exhib-
ited severe deficits in learning and memory behaviors in
both paradigms of fear conditioning and Morris hidden
platform water maze (Fig. 5). In fact, the neurobehavioral
defect in Emx1-cre; Dnmt1 mice resembles the deficit
observed in hippocampal lesioned animals, which also show
severely impaired learning and memory (Fig. 5B). Taken
together, our data demonstrated that Dnmt1 deficiency in the
developing dorsal forebrain leads to cortical and hippocampal
degeneration in adult mutants, which exhibit behavioral
defects in learning and memory.
Deregulation of neuronal gene expression
in hypomethylated forebrain
The generation of viable mutant mice containing hypomethy-
lated cortical neurons allows us to profile the potential altera-
tion of neuronal gene expression and further investigate
cellular defects in dorsal cortex of Emx1-cre; Dnmt1 mutant
mice. We choose to analyze potential alteration of neuronal
gene expression in both prenatal E14.5 and early postnatal
P5 when mutant neurons are still abundant in the dorsal
cortex. At E14.5, the peak stage of neurogenesis, the cortical
Figure 4. Hypomethylated cells in the dorsal cortex undergo apoptotic cell death perinatally TUNEL immunohistochemistry were performed with E11.5, E14.5,
P0 and P7 brain samples. No difference in levels of apoptotic index at E11.5 even though demethylated cells IAP-positive at this time point. By E14.5, there is a
dramatic increase in apoptosis in the ventricular zone and the emerging cortical plate of the mutant embryo. This region is positive for strong IAP staining and
activated caspase-3 immunoreactivity. The neurotubulin marker TUJ1 (green) is used to show the cortical plate (cp) boundary from the ventricular zone (vz).
Note high magnification images from boxed region shows co-labeling of TUNEL-positive cells with IAP reactivity (arrowhead). Apoptotic signals are also easily
detected in mutant cortex at P0 and P7. con, Emx1-cre; Dnmt12lox/þcontrol; mut, Emx1-cre; Dnmt12lox/2loxmutant; Lv, lateral ventricle; str, striatum; cc, corpus
callosum; ctx, cortex.
Human Molecular Genetics, 2009, Vol. 18, No. 152879
structure is by and large intact and demethylated neurons
account for a majority of cells (82%). Thus, any gene deregu-
lation at E14.5 mutant cortex reflects the effect of DNA hypo-
methylatoin on immature CNS neurons. On the other hand,
P5 is a critical stage of neuronal maturation when proper
regulation of genes involved in neural plasticity is established.
Furthermore, a comparison of the altered gene expression pro-
files in these two stages also allows us to determine whether
expression changes caused by DNA hypomethylation are
showed that in the P5 dorsal cortex of mutant mice, 1047
genes (6.1%) are up-regulated and 444 (2.6%) genes that
are downregulated with a statistically significant 2-fold
threshold (Supplementary Material, Tables S1 and S2).
Using PANTHER classification
ontology analysis indicated that up-regulated genes exhibit
an overrepresentation of genes involved in apoptosis and
developmental process, consistent with the observed pheno-
type (Tables 1 and 2 and Supplementary Material, Fig. S2).
up-regulation of the Magea3 transcript in both E14.5 and P5
mutant cortex (Fig. 6A). This result is consistent with the
demethylation state of mutant cortical cells because Magea3,
an X-linked gene family member, is known to be repressed
by DNA methylation in both somatic cells and embryonic
stem cells (25) (Table 1). We further confirmed that the
up-regulation of apoptosis-related genes such as Casp4,
Gadd45a and Ngfr in P5 mutant cortex but not in E14.5 by
real-time RT–PCR, consistent with a dramatic cortical
degeneration in early postnatal stages but a relatively minor
defect of neuronal cell death in embryonic development
(Fig. 6B). Interestingly, deregulated gene list contained
several transcriptional factors including Lhx2 (Fig. 6C) that
regulates cortical layer specification and neuronal migration,
suggesting a potential defect in cortical architecture in the
dorsal cortex of mutant mice (24).
We also found that a number of genes implicated in regulat-
ing neural activity and neurogenesis were down-regulated
(Fig. 6C). For example, the neurotrophin gene Nt3 as well
as many channel genes (Scnn1a, Kcnh5, Kcnj9) were
significantly down-regulated, particularly in P5 mutant
brain, which is a critical stage of neuronal maturation
(Fig. 6D). These results suggest that mutant mice likely have
defects in electrophysiological properties and neural cell
Figure 5. Locomotion, thigmotaxis, and Morris Water Maze behavioral tests of control and mutant mice. (A) Open field test assesses animal exploratory be-
havior. Mutants exhibit hyperactivity as a function of increased locomotor activity; however, mutants do not appear to have altered anxiety levels as measured by
thigmotatic behavior. (B) Hippocampal dependent contextual fear tasks show a severe reduction of long term memory in the mutants, consistent with the loss of
the hippocampal structure in mutants. Re-exposure to the fear conditioning chamber (7 day) elicited no fear response in mutant animals, akin to the lack of
freezing seen in hippocampal lesioned animals. (C) Morris water maze testing the ability of mice to recall spatial navigation is a hippocampal specific learning
and memory task. Over continuous learning trial days, mutants do not improve in finding the hidden platform, nor do they exhibit a learning curve for the task of
locating the platform. (D) Conditional mutants score below chance (25%) in probe trials after a 14-day learning protocol, indicative of poor spatial memory in the
mutants. For each category of mice, more than 11 adult mice/genotype around the age of 6 months were used in behavioral tests.
2880Human Molecular Genetics, 2009, Vol. 18, No. 15
Hypomethylation leads to defects in neuronal morphology
Given the above results, we subsequently examined the
defects in morphology and neuronal excitability in mutant
mice. Although hypomethylated neurons were retained in
mutant brains at postnatal ages, cresyl violet histology
denoted the failure of distinct cortical layer development in
the mutant (Fig. 2B). To further analyze the morphological
and physiological properties of a subset of cortical excitatory
neurons, we crossed Dnmt1 mutants with BAC transgenic
mice (M4-eGFP) expressing green fluorescent protein in a
subset of cortical excitatory neurons of layers 2/3 and 5, as
well as a subset of striatal neurons (Fig. 7A–D) (26). ?95%
of the cortical M4-eGFP neuronal population were IAP-
positive in the mutant cortex (Fig. 7E), indicating that
mutant neurons expressing M4-eGFP were indeed a subpopu-
lation of hypomethylated excitatory neurons. Confocal images
of M4þ-EGFP-positive neurons demonstrated that mutant
neurons do not maintain proper layer specificity and display
abnormal branching (Fig. 7F). Consistent with the observed
cell death in the cortex of one-week-old mutants, the
number of demethylated eGFP-positive neurons decreased
between P0 and P7, and the remaining neurons exhibited
dystrophic neurites with loss of proper radial projections
to the pial surface (Fig. 7F). Camera Lucida drawings of
biocytin filled M4-eGFPþ control and mutant neurons
revealed a significantly larger soma area in mutant neurons
Table 1. Up-regulation of genes involving in cortical layer-specification,cell death and other functions in dorsal cortex in P5 Emx-Cre; Dnmt1 mutant mice
Gene symbolGenebankGene name Fold change
Layer-specific or area-specific expression genes
Apoptosis- or survival-related genes
LIM homeobox protein 5
distal-less homeobox 5 (Dlx5), transcript variant 1
NK6 transcription factor related, locus 2
cartilage homeo protein 1
oligodendrocyte transcription factor 2
distal-less homeobox 2
nerve growth factor receptor (TNFR superfamily, member 16)
caspase 4, apoptosis-related cysteine peptidase
growth arrest and DNA-damage-inducible 45 alpha
Fas (TNF receptor superfamily member)
histocompatibility 2, M region locus 3
histocompatibility 2, class II antigen A, beta 1
reproductive homeobox 2
X-linked lymphocyte-regulated 3B
X-linked lymphocyte-regulated complex
X-linked lymphocyte-regulated 4B
maelstrom homolog (Drosophila)
melanoma antigen, family A, 3
melanoma antigen, family A, 8
Table 2. Down-regulation of genes involving in cortical layer-specification, ion channels, and cell survival in dorsal cortex in P5 Emx-Cre; Dnmt1 mutant mice
Gene symbolGenebankGene name Fold change
Layer-specific or area-specific expression genes
Ion channel or neuronal activity
basic helix-loop-helix domain containing, class B5
LIM homeobox protein 2
LIM domain binding 2
LIM domain only 4
B-cell CLL/lymphoma 11A (zinc finger protein)
potassium voltage-gated channel
potassium inwardly-rectifying channel
potassium voltage-gated channel
potassium voltage-gated channel
potassium inwardly-rectifying channel
transient receptor potential cation channel
B-cell CLL/lymphoma 11A (zinc finger protein)
Human Molecular Genetics, 2009, Vol. 18, No. 152881
(con ¼ 84.12+10.88 mm2, mut ¼ 140.4+15.48 mm2; P ,
0.01) and an increase in both basal and apical dendritic
branching in demethylated neurons (Fig. 7G). These data
suggest DNA methylation is important for the morphological
maturation of neurons.
To directly examine whether genomic demethylation in
excitatory neurons alters electrophysiological parameters, we
performed whole-cell current clamp recordings in somatosen-
sory cortical slices from mutant (age P3–P53) and control
mice (age P1–P75). Excitatory mutant neurons possessed
more depolarized resting membrane potentials after P9 and
exhibited significantly broadened action potential (AP) half-
widths when compared with controls at most ages examined
(Fig. 8A–C). To rule out the possibility that the differences
in electrophysiological parameters are caused by recording
from different types of neurons, whole-cell recordings were
also performed in the same subset of M4-EGFP-positive
neurons in control and mutant mice. At P11–P12, the
M4-eGFPþ mutant neurons possessed significantly increased
AP half-widths [Value ¼ mean+SEM: con ¼ 1.8+0.1 ms
(n ¼ 6), mut ¼ 2.9+0.9 ms (n ¼ 15); P , 0.05], demonstrat-
ing that prolonged APs are characteristic of hypomethylated
neurons (Fig. 8B and C). The AP prolongation in mutant
neurons occurred via decreased rate of maximal rise and,
more prominently, a reduction of the maximal rate of fall,
suggesting perturbations to both sodium and potassium
channel function (Fig. 8D). Voltage-gated potassium currents
recorded in somatic outside-out patches
neurons indicated similar voltage-dependence of activation
and inactivation when compared with control (P9–P28).
However, potassium currents in somatic patches from
mutant neurons showed an absence of fast activation and inac-
tivation, signifying the absence of the rapidly inactivating pot-
assium current (IA) (Fig. 8D).
Figure 6. Hypomethylation alters neuronal gene expression in mutant cortex in both pre- and postnatal stages. (A and B) The expression of X-linked gene
(Magea3, A) and apoptosis-related genes (Gadd45, Casp4, Ngfr, B) in the dorsal cortex of wild type (WT, open bar) or Emx1-cre; Dnmt1 mutant (mut,
black bar) mice. (C and D) The expression of layer specific gene (Lhx2, C) and neuronal channels genes (Kcnh5, Kcnj9 and Scnn1a, D) in the dorsal cortex
of wild type and mutant mice. Quantitative real-time PCR was performed in cDNAs derived from E14.5 and P5 dorsal cortex of each mouse by using the specific
primers. Statistical significance was evaluated by the t-test. Mean+SEM (n ¼ 3).?P , 0.05.
2882 Human Molecular Genetics, 2009, Vol. 18, No. 15
Alterations of potassium channel Kv4.2 protein expression
and mRNA expression of Kchips in hypomethylated
A variety of studies suggest that the A-type fast inactivating
potassium current is mediated by the Kv4.x channel family
(27), and Kv4.2 is present both in the dendrites and in the
neuronal soma of excitatory neurons (28). However, real-time
RT–PCR assays did not find any significant differences in
mRNA expression of Kv4.2 in mutant cortex (Supplementary
Material, Fig. S3A). To determine whether there is a potential
discordance between mRNA and protein expression of Kv4.2
potassium channel in hypomethylated neurons, we performed
immunoblots for Kv4.2 using whole-cell cortical lysate from
both control and mutant dorsal cortex region. We saw a
decrease of Kv4.2 protein levels at both 2 weeks and
3 weeks of age, with an ?30% reduction at P12 and 60%
reduction at P21 (Supplementary Material, Fig. S3B). To
verify the decrease seen in the immunoblots, we also exam-
ined the overall levels of Kv4.2 immunostaining in control
and mutant cortex. We also see a decrease in Kv4.2 immunor-
eactivity in the mutant cortex with a loss of laminar-specific
patterning seen in the control cortex (Supplementary Material,
Fig. S3C). Because the mRNA levels of Kv4.2 are similar
between control and mutant cortices, the reduced expression
of Kv4.2 protein in Dnmt12/2 neurons suggests that an
indirect mechanism, such as translational inhibition or
protein instability may mediate the effect of DNA hypomethy-
lation on the reduction of Kv4.2 protein expression. It is worth
noting that the expression levels of many regulatory proteins
for potassium channel such as Kchip1, Kchip2 and Kchip 4a
were also altered in mutant cortex (Supplementary Material,
Fig. S4). We suggest that the combined defects in the
expression of Kv4.2 and Kchips as well as other types of
potassium and sodium ion channels (Fig. 6) may underlie
the defectin neuronal excitability
As one of the major epigenetic modifications in the mamma-
lian genome, DNA methylation is known to play multiple
roles in embryogenesis including developmental gene regu-
lation, genomic imprinting and X-inactivation. During the
development of CNS, we have demonstrated that DNA
Figure 7. Hypomethylated cortical neurons exhibit defects in dendritic arborization (A–D). Morphology of M4-egfp-positive neurons in control and mutant
cortices at P0 (A and B) and P7 (C and D). (E) A high percentage of M4-egfpþ cells colocalize with IAP reactivity in mutant cortices (94.98+0.85%).
(F) Camera lucida drawings of individual M4-egfp-positive neurons filled with biocytin from either control or mutant cortex. (G) Sholl analysis to analyze den-
dritic branching reveals an increase in basal and apical branching patterns (P , 0.01, student t-tests).
Human Molecular Genetics, 2009, Vol. 18, No. 15 2883
methylation is not only essential for the vital postnatal CNS
functions, but also controls the timing of astrogliogenesis
(14,16). However, specific roles for DNA methylation in post-
natal neuronal maturation are largely unknown. In this line of
Emx1-cre; Dnmt1 mice, we demonstrated that a wave of cell
death occurs in the developing CNS between E14.5 to three
weeks postnatally, consistent with the previous findings that
substantial DNA demethylation in somatic cells, but not in
embryonicstem cells, leads
(14,29,31). Furthermore, our data showed that DNA hypo-
growth. Our results may offer an explanation for the pheno-
types in Nestin-cre; Dnmt1 mutant mice, where DNA hypo-
methylation in the CNS disrupts neural control of respiration
and causes neonatal lethality (14). Perhaps defects in either
neuronal excitability or connectivity in the hypomethylated
CNS could perturb the generation and/or relay of respiratory
drive, thus causing respiratory failure in neonatal mutant mice.
excitability and dendritic
Multiple physiological defects in hypomethylated forebrain
In this study, we showed that Dnmt1 deletion in mutant cor-
tices peaks at 89% on E13.5, declines thereafter, and finally
stays at ?30% in adulthood. Because we observed severe
neuronal cell loss in mutant brain (Figs 3, 4 and 7), the
decrease of Dnmt1 gene deletion certainly reflects on the
degeneration of hypomethylated neurons. It is worth noting
that methylation proficient GABAergic interneurons are
present in postnatal mutant brains. Therefore, we cannot rule
out the possibility that the preferential survival of GABAergic
interneurons over hypomethylated excitatory neurons and
astrocytes in postnatal CNS may also contributes to the
observed decrease in the efficiency of Dnmt1 gene deletion.
One of the surprising observations in this study is that 20–
30% of hypomethylated excitatory cortical neurons survive in
Emx1-cre; Dnmt1 mutant mice throughout their lifespan,
suggesting that a subset of hypomethylated neurons escape
Figure 8. Hypomethylated neurons exhibit defects in resting membrane and action potentials. (A) Significant changes in resting membrane potential (RMP) were
graphed (?P , 0.05, student t-test). (B) Action potential wave forms in control and mutant cells including M4-eGFP-positive neurons. (C) Action potential half-
width and maximum rate of repolarization in P1–4, 5–8, 9–12, 13–16, 17–20 and P21–adult regular spiking cortical neurons in control and mutant slices. Note
the increased half-width resulting from decrease in the maximal rate of repolarization at all ages. (D) Voltage schematic and leak subtracted currents elicited by
stepping from 2110 mV to þ40 mV, with or without a 50 ms pre-step to 240 mV in outside out patches pulled from control and mutant RS cortical neurons.
Figures are averages of currents obtained from 23 control patches and 8 mutant patches. Subtraction of pulses with the pre-step from pulses without the pre-step
result in isolation of the rapidly inactivating potassium conductance (IA) in control neurons but not mutant neurons. Note that potassium currents in mutant
somatic outside out patches inactivate much more slowly than those obtained from control neurons.
2884 Human Molecular Genetics, 2009, Vol. 18, No. 15
selection pressure during postnatal maturation. In previous
models of Dnmt1/DNMT1 deletion in mouse fibroblasts and
human colon cancer cells, apoptosis was partly triggered by
a p53-dependent pathway (29,31). It remains to be determined
whether loss of hypomethylated neurons in Emx1-cre; Dnmt1
mice is also mediated by p53-dependent pathway. Further-
more, it will be of interests to determine whether the survival
of a subset of excitory neurons in Emx1-cre; Dnmt1 mutant
mice is triggered by the cell autonomous inhibition of p53
or via a novel cell survival pathway.
Although we found massive neural degeneration and behav-
ioral defects in learning and memory in mutant mice, these
mice have a normal lifespan in a laboratory environment.
Another group has also reported that mutant mice with the
loss of dorsal forebrain can survive into adulthood, even
though they display behavioral abnormality (32). These obser-
vations suggest that degeneration of the forebrain is not
directly incompatible with viability of mutant mice in a lab-
Deregulation of multiple neuronal genes
in hypomethylated neurons
Previous studies have demonstrated that activity-dependent
regulation of the neurotrophin gene Bdnf in cortical neurons
involves a DNA methylation-related chromatin remodeling
mechanism (9,10). However, misregulation of Bdnf alone
cannot not explain the pleiotropic phenotypes observed in
this Emx1-cre; Dnmt1 model. It is likely that multiple genes
are deregulated in the absence of Dnmt1 in the developing
CNS. The availability of hypomethylated cortical neurons in
Emx1-cre; Dnmt1 mutant mice at all developmental stages
provides a unique opportunity to identify critical genes that
are subjected to regulation by DNA methylation in a stage-
and cell type-specific manner. Our microarray study showed
that ?1500 genes (8.5% of all annotated genes) that are
either up-regulated or down-regulated in P5 mutant cortex
postnatally. We further showed that the altered expression of
a subset of neuronal genes commences in E14.5 mutant
cortex, which including genes involved in neuronal survival,
cell patterning as well as neuronal excitability. It is worth
noting that not all deregulated genes are directly caused by
DNA demethylation. First, while demethylation is compatible
with gene up-regulation, the observed down-regulation of
neuronal genes is most likely via a secondary effect. Second,
when loss of excitatory neurons is severe in the dorsal
cortex postnatally, we cannot rule out the possibility that the
up-regulation of a subset of genes, such as those genes
highly expressed in the inhibitory interneurons, is attributed
to the shift of cell population towards the enrichment of inter-
In demethylated embryonic stem cells, we recently showed
up-deregulated genes are related to lineage-specific differen-
tiation (25). Thus, the number of deregulated genes in hypo-
methylated and differentiated cells are significantly higher
than that in undifferentiated embryonic stem cells. It is
likely that the profiles of gene deregulation in different
types of somatic cells are different. In demethylated
mouse fibroblasts, ?10% of genes are deregulated (29).
of genesarederegulated andmost of
In hypomethylated CNS neurons, up-regulated genes are
over-represented by homeobox genes, other DNA-binding
proteins, receptors and signal molecules for developmental
processes. In the category of down-regulated genes, which
are presumably through indirect mechanisms in hypomethy-
lated cells, ion channel genes and cytoskeleton genes are
over-represented. Our data support the notion that DNA
methylation contributes to the tissue- and cell type-specific
gene expression in the CNS. Recently, it has been reported
that neuronal activity regulates gene expression through the
induction of DNA demethylation (33,34), and these activity-
dependent demethylation modulates excitatory neurotrans-
mission and adult neurogenesis. Thus, our genetic model of
Dnmt1 gene deletion in cortical excitatory neurons will be
very useful to analyze the effect of DNA demethylation on
activity-dependent neuronal gene regulation.
Deregulation of ion channel expression underlies defects in
electrophysiological parameters in hypomethylated
The survival of hypomethylated neurons in postnatal CNS
allows us to probe electriphysiological defects in these cells.
We have previously recorded thalmo-cortical projection path-
ways in this line of Emx1-cre; Dnmt1 mutant mice (35). We
showed that hypomethylated neurons are unable to form soma-
tosensory barrel maps in the cortex despite the ability for
hypomethylated neurons to receive and elicit excitatory post-
synaptic current upon thalamocortical projection stimulation.
neurons revealed severely reduced long-term potentiation
upon thalamocortical pathway stimulation. Our current study
indicated that post-synaptic cortical neurons exhibit defects
in neuronal excitability, which may underlie the observed
defect in thalmo-cortical long-term potentiation.
Changes in ion channel expression, specifically Kv4.2, may
result from direct cell-autonomous effects of demethylation or
indirectly result from failure of maturation of intrinsic ionic
currents because of degeneration of cortical neurons in the
demethylated cortex. We favor a direct effect because electro-
physiological studies in animals with severely disturbed corti-
cal lamination do not show changes in AP repolarization
(36,37). One explanation for the decrease in Kv4.2 protein
without the concomitant decrease in mRNA levels is post-
However, increased degradation, or perturbation of channel
protein integration into cell membrane could also leads to a
decrease in protein without alternation of mRNA level.
Further molecular characterization of the expression of ion
channels will help elucidate molecular mechanisms underlying
the aberrant electrophysiological profiles of Dnmt12/2
It has been known that the maturation of interneuronal gene
expression depends on neuronal activity and trophic support
(38,39). Moreover, the effect of excitatory neurons on inter-
neurons is not only through neurotransmitters but only via
neurotrophic factors, such as BDNF (40). Thus, although inter-
neurons are present in the mutant brain, their physiological
functions may be impaired as a consequence of the loss of
demethylated excitatory neurons.
by microRNA pathways.
Human Molecular Genetics, 2009, Vol. 18, No. 152885
DNA methylation in neurodegeneration and aging
Recently, improper DNA methylation has been indirectly
linked to neurodegenerative diseases. A pathological hallmark
of these diseases is targeted neuronal degeneration leading to
cell death. Apoptotic cell death pathways appear to be acti-
vated in neurodegenerative disorders (41). It is conceivable
that genes activated during apoptosis are preferentially
silenced in healthy cells. However, upon injury or insult
de-repression of pro-apoptotic genes is necessary to trigger
programmed cell death. Consistent with this hypothesis, indu-
cing hypomethylation via depletion of the methionine pool in
cultured primary neurons results in apoptosis (42). Interest-
ingly, it has been postulated that folate deficiency, and by
extension DNA hypomethylation, may enhance neural vulner-
ability to excitotoxic insults and oxidative stress in neurodegen-
erative diseases such as Alzheimer’s disease and Parkinson’s
disease (42,43). Thus, biochemical evidence suggests a putative
link between hypomethylation-induced gene de-regulation and
apoptosis in neurodegenerative disorders.
The generation of Emx1-cre; Dnmt1 mutant mice creates a
model system of severe cortical and hippocampal degeneration
during early postnatal stages in mutant mice. Thus, our animal
model will be valuable to study both behavioral and physiologi-
cal parameters in association with cortical and hippcampal
functions.Moreover, theseveredisruptionof corticaland hippo-
campal structures will provide a system to test the ability of in
vitrogenerated neural precursorcellstoreplenishthesedegener-
ated structures via transplant therapy. While Emx1-cre; Dnmt1
mutant cortex is degenerated, the thalamus and striatum struc-
tures are still present (Fig. 2). We propose that Emx1-cre;
Dnmt1 mutant mice is also a useful model system to examine
the effects of severe cortical degeneration on target structures
in thalmo-cortical and cortico-striatal pathways.
MATERIALS AND METHODS
Generation of Emx1-cre; Dnmt1 conditional mutants
and southern analysis
The use of transgenic mice is approved by UCLA Institutional
Animal Research Committee. Using the cre-loxP binary gene
deletion strategy, we crossed female mice homozygous for the
Dnmt1 conditional allele Dnmt12lox(14) with male mice carry-
ing the Emx1-cre transgene (kindly provided by Dr S. Itohara
at RIKEN Brain Research Institute, Japan). Control heterozy-
gous and mutant offspring were obtained inexpected Mendelian
Southern hybridization using genomic DNA samples procured
from dorsal cortex at multiple ages. We also used the IAP
repeat probe to detect global methylation levels in the same
DNA samples. Detailed explanation of probes used and
methods were previously described (14). This conditional line
was crossed to two reporter strains, the R26R ßgeo line (20)
and the M4-egfp bac transgenic (26) for further analysis.
Histology, immunohistochemistry, TUNEL analysis
Histological examination was performed on mouse brain sec-
tions collected from samples fixed with 2 or 4% PFA via
cardiac perfusion (postnatal samples) or overnight immersion
fixation (embryonic samples), cryoprotected in two changes
of 30% sucrose, and cut in the coronal orientation using a
cryostat (Leica CM1900). Sections were stored at 2808C
until use. After thawing and hydration, sections were stained
using 0.1% cresyl violet, dehydrated through alcohols and
xylenes, and mounted with Permount (Fisher). LacZ detection
was performed as previously described (5). Immunohisto-
chemistry was performed from sections collected in the
manner described earlier.
For TUNEL analysis, sections fixed with 4% PFA/PBS and
stained using the Apoptag Fluoroscein In Situ Apoptosis
Detection Kit (Chemicon) following the supplied manufac-
turer’s protocol. For double labeling experiments, sections
were first incubated with tDT enzyme as per kit instructions,
then washed and incubated in 10% NGS in PBS to block non-
specific interactions for 1 h. Primary antibodies were added for
overnight incubation at room temperature, then secondary
antibodies were added in the dilution buffer supplied in the
Immunoblotting was performed as previously described
(16). Antibodies and dilutions used were rabbit anti-Dnmt1
(PATH52, a gift from Dr T. Bestor, Columbia University),
and mouse anti-GAPDH (Abcam, 1:5000).
Spatial learning was assessed with the hidden platform version
of the Morris water maze (44), as described previously (45).
Briefly, animals were trained with two trials per day for 10
days at which time a probe trial with the platform removed
was administered. As learning in all groups was low (data
not shown), an additional 4 days of four trials per day was
given at which time the final probe trial was administered.
Acquisition data are presented as the group averages, in
which escape latencies across four trials were averaged for
each animal. On the day following the final training/probe
trial, animals received three additional trials, in which the plat-
form was clearly marked (visible platform test). Escape
latencies for all trials during the visible-platform test are aver-
aged for each animal. Statistical comparisons were made
between groups (% time in training quadrant) with Student’s
t-tests. In addition, the percentage of time spent in the training
quadrant was compared with that of a chance performance
(25%) with a single group comparison. A selective search
strategy was indicated if animals performed significantly
above chance. The fear conditioning behavioral test follows
up the procedure as described in Costa et al. (30).
Detailed methods on patch-clamp recordings from neurons in
brain slices have been reported in the co-authors previous pub-
lications (46,47). P1–P73 control and mutant animals were
anesthetized with halothane. Cortical slices were cut at the
coronal angle or thalamocortical angle and incubated in
ACSF at 358C for 30 min and then at room temperature. Elec-
trodes for somatic recordings were pulled from borosilicate
glass capillaries to a final resistance of 3–6 mega Ohms
(Warner Instruments). Biocytin (3 mg/ml) was also added to
2886Human Molecular Genetics, 2009, Vol. 18, No. 15
a potassium gluconate-based internal solution during most
recordings. Somatic whole-cell recordings were performed
with an Optopatch patch-clamp amplifier (Cairn Research,
Kent, UK) in the current clamp configuration, from layer IV
neurons in control slices. As cortical lamination was disrupted
in mutant cortex, an attempt was made to record from neurons
at approximately similar proportional distances from the pial
surface, as in control cortical neurons. M4þ EGFP neurons
were located in layer V in control cortex and in the inferior
half of the cortical mantle in mutant cortex. Voltage-gated
potassium currents were recorded in the outside out patch
configuration in the presence of tetrodotoxin (500 nM).
Current and voltage clamp traces were analyzed using custom
made programs with Igor analysis software (Wavemetrics,
Lake Oswego, OR, USA). Biocytin and tetrodotoxin were pur-
chased from Sigma (St Louis, MO, USA). APs were elicited
after hyperpolarizing all cells to 270 mV to reduce the effect
of differing resting membrane potential on AP morphology.
Biocytin immunocytochemistry was performed as outlined
(48). Cells were reconstructed at ?100 using camera lucida
techniques. Sholl analysis was performed by superimposing
concentric circles of onto reconstructed neurons and counting
the number of dendritic intersections. Soma area was calcu-
lated using NIH Image software.
Gene expression microarrays were done with Agilent Whole-
Genome microarrays (G4122A) using the suggested protocol.
Briefly, we ran the isolated RNA through a Qiagen RNeasy
minElute column (Qiagen) and tested the quality of the RNA
on a NanoChip (Agilent). We converted the RNA into cDNA
and then the cDNA into cRNA using the Agilent Low RNA
Input Linear Amplification Kit (Agilent). We using a Nanodrop
(Nanodrop) to quantify the labeled cRNA and used 0.75 mg of
of labeled probes, 10? blocking reagent (Agilent) and 25?
Fragmentation buffer (Agilent). Reaction was stopped with the
addition of 2? Hybridization buffer (Agilent). We used
Agilent Whole-Genome microarrays for expression studies.
Slides were hybridized at 658 for 17 h at 4 revolutions per
minute and then washed once in Agilent Gene Expression wash
buffer 1 and once in Agilent Gene Expression wash buffer 2
before a quick washinacetonitrile. Slides were scanned immedi-
ately after washing to prevent ozone degradation. Arrays were
performed in triplicate. Microarray data described in this paper
can be accessed with GEO number GSE14216.
Quantitative reverse transcription polymerase
cDNA conversion was done using the iScript kit (Bio-Rad).
Quantitative PCR was done on an MyIQ Thermocycler
(Bio-Rad) using the Sybr Green Supermix (Bio-Rad).
Supplementary Material is available at HMG online.
We thank David Savin and Justin Sharim for processing and
analyzing the camera lucida drawings.
Conflict of Interest statement. None declared.
This work was supported by National Institutes of Health
(RO1 NS051411 to G.F.). L.K.H. was supported by NRSA
5F31NS051005 from NINDS and the ARCS Foundation.
G.F. is a Carol Moss Spivak Scholar in Neuroscience.
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