Transcription factor MEF2C influences neural
stem/progenitor cell differentiation and
maturation in vivo
Hao Li*†, Jonathan C. Radford*†, Michael J. Ragusa‡, Katherine L. Shea‡, Scott R. McKercher*, Jeffrey D. Zaremba*,
Walid Soussou*, Zhiguo Nie*, Yeon-Joo Kang*, Nobuki Nakanishi*, Shu-ichi Okamoto*, Amanda J. Roberts§,
John J. Schwarz‡, and Stuart A. Lipton*§¶
*Center for Neuroscience, Aging, and Stem Cell Research, Burnham Institute for Medical Research, La Jolla, CA 92037;‡Center for Cardiovascular Sciences,
Albany Medical Center, Albany, NY 12208; and§Molecular and Integrative Neurosciences Department, The Scripps Research Institute, La Jolla, CA 92037
Edited by Eric N. Olson, University of Texas Southwestern Medical Center, Dallas, TX, and approved May 3, 2008 (received for review March 21, 2008)
Emerging evidence suggests that myocyte enhancer factor 2 (MEF2)
MEF2C the predominant isoform in developing cerebrocortex. Here,
we show that conditional knockout of Mef2c in nestin-expressing
in vivo, resulting in aberrant compaction and smaller somal size. NSC
proliferation and survival were not affected. Conditional null mice
surviving to adulthood manifested more immature electrophysiolog-
ical network properties and severe behavioral deficits reminiscent of
role for MEF2C in programming early neuronal differentiation and
proper distribution within the layers of the neocortex.
neurogenesis ? synaptogenesis ? autism ? Rett syndrome
activity (1). To facilitate analysis of MEF2C function in early
neuronal development, we engineered a conditional knockout in
NSCs by crossing floxed Mef2c mice with Nestin-Cre mice. In
alteration in the distribution of new neurons in the neocortex
and the opposite effect on synaptic activity, i.e., decreased neuro-
transmission persisting into adulthood.
MEF2C belongs to the myocyte enhancer factor 2 (MEF2)
subfamily of the MADS (MCM1-agamous-deficiens-serum re-
sponse factor) gene family (2, 3). We cloned MEF2C from devel-
oping mouse brain, and Eric Olson and colleagues then discovered
it in the heart (2, 4, 5). In cerebrocortex, MEF2 transcriptional
activity is restricted to differentiated cortical neurons in a specific
laminar pattern, and its distribution increases along the rostrocau-
dal axis (2, 4, 6). These features led to speculation on the potential
role of MEF2C in the architechtonics of the cerebral cortex (2).
Previous studies demonstrated an important role for MEF2C in
apoptosis (8) and synapse formation (1, 9) in vitro or in brain slices.
form of MEF2C induces embryonic stem cells to commit to a
neuronal fate in a virtually exclusive fashion (10). However, studies
on the effect of endogenous MEF2C on CNS neurons in vivo were
impeded by the embryonic lethality of conventional Mef2c-null
mice because of cardiovascular defects at embryonic day (E) 9.5,
before brain development (7). Here, we report that conditionally
aggregation and compaction of neurons migrating into the lower
layers of the neocortex during development. Knockout mice sur-
viving to adulthood manifest smaller, apparently less mature neu-
rons and smaller whole brain size, with resultant aberrant electro-
physiology and behavior.
MEF2C Conditional Knockout Mice. Knockout of the Mef2c gene is
embryonic lethal because of severe heart developmental defects
nockdown of the transcription factor MEF2C in mature cere-
brocortical neurons results in increased synaptic number and
(7). Therefore, to investigate the role of MEF2C in brain develop-
ment, we conditionally knocked out Mef2c in neural stem/
information (SI) Materials and Methods) (11). Heterozygous
n-Cre?/Mef2cloxp/?mice were indistinguishable from wild type
recombination included PCR, immunofluorescence, and immuno-
blotting. Normally, MEF2 activity (Fig. S1A) (5, 6) and protein of
in the brain. In adult n-Cre?/Mef2cloxp/?2-null mutant mice, we
observed a marked decrease in brain size, cortical thickness (Fig.
S1B), and body weight (Fig. S1C). Nonetheless, during brain
development, we found no change in cell proliferation (Fig. S1D)
or apoptosis (by TUNEL) in n-Cre?/Mef2cloxp/?2-null mice versus
control. Notably, only 60% of the conditional null mice survived to
adulthood (Fig. S1E).
Newly Formed Cortical Neurons Are Abnormally Compacted in MEF2C
Conditional Knockout Mice. In mouse neocortex, neuronal differen-
tiation commences at approximately E11.5, peaks at approximately
E15.5, and reaches completion approximately at birth (13). Exci-
tatory pyramidal neurons make up ?70–80% of the neuronal
population and migrate radially during corticogenesis. The other
20–30% of neocortical neurons are GABAergic interneurons gen-
tangential migration. Once within the neocortex, interneurons
undertake radial migration together with pyramidal neurons to
form the characteristic laminated cortical plate. In the n-Cre?/
Mef2cloxp/?2-null neocortex, we found that during this migration of
neurons, severe compaction occurred in the cortical plate with
variable phenotypic penetrance. In the severe manifestation, we
observed dramatically compacted clusters of neurons, resulting in
disrupted layer formation at a late embryonic stage (Figs. 1 and 2
and Fig. S2) (n ? 10 mice at E18.5).
We also investigated the number and distribution of newly
generated neurons. By stereological counting with the optical
dissector, 4 days after bromodeoxyuridine (BrdU) pulse labeling at
E14.5, the ratio of newborn neuronal nuclear antigen (NeuN)-
expressing neurons to total BrdU-labeled cells in the Mef2c con-
ditional null neocortex was similar to that of control (?3.9%
Author contributions: S.A.L. designed research; H.L., J.C.R., M.J.R., K.L.S., J.D.Z., W.S., Z.N.,
Y.-J.K., A.J.R., and J.J.S. performed research; M.J.R., K.L.S., S.R.M., J.D.Z., W.S., Z.N., Y.-J.K.,
N.N., S.-i.O., A.J.R., J.J.S., and S.A.L. analyzed data; and H.L., J.C.R., S.R.M., N.N., S.-i.O., and
S.A.L. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
†H.L. and J.C.R. contributed equally to this work.
¶To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2008 by The National Academy of Sciences of the USA
www.pnas.org?cgi?doi?10.1073?pnas.0802876105 PNAS ?
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difference), although the neurons were distributed in a more
compacted pattern (Fig. 2A and Fig. S2). Despite this compaction,
compared with control, the Mef2c conditional null neocortex
manifested a normal laminar pattern of cells born between E12.5
and E15.5 (Fig. S3).
null mice at E18.5, nestin- and PSA-NCAM-expressing neural
stem/progenitor cells appeared to be unaffected (Figs. 1 and 2C),
suggesting that despite compaction of a population of migrating
cells, the precursor cells were distributed normally throughout the
neocortex. In contrast, compacted cells had further committed to a
neuronal fate, expressing the early neuronal markers ?-III tubulin
(TuJ1) and doublecortin (DCX) (Figs. 1 and 2A); subsequently,
transcription factor (Er) 81, T-box brain gene (Tbr) 1, glutamate
decarboxylase (GAD) 65/67, and microtubule-associated protein 2
(MAP-2) (Fig. 2). Subplate formation appeared to be intact,
consistent with normal neural stem/progenitor cell (NSC) devel-
opment between E11 and E13. In the more severely affected cases,
radially migrating neurons in the E18.5 Mef2c-null neocortex
appeared to be hindered by compaction soon after crossing the
subplate (Fig. 2A Middle and Fig. S2). Both pyramidal neurons and
interneurons were affected, as indicated by the specific markers
Tbr1 and GAD 65/67, respectively (Fig. 2 B and C). In less severely
affected cases at E18.5, many cortical plate neurons remained
ectopically distributed between the subplate and cortical plate (Fig.
We next wanted to rule out the involvement of known signaling
pathways that affect migration during neocortical development in
the Mef2c brain null phenotype. For example, DCX is involved in
cortical neuronal migration (14), but we found normal expression
the Reelin- and CDK5-signaling pathways are well known for
becomes inverted when either pathway is disrupted (15). So, we
examined Disabled 1 (Dab1), which mediates Reelin signaling, and
p35, which is involved in CDK5 signaling, but we found that both
null cortex (Fig. S5). These Dab1 and p35 findings are consistent
with the normal ordering of the layers after migration in our
conditional null neocortex (Fig. S3). Thus, the compacted cell
phenotype in the Mef2c-null mutant does not appear to be caused
by a cell intrinsic migrational defect of these molecules. Addition-
ally, because of the abnormal neuronal clustering in the more
two cell adhesion molecules known to be important in this process,
but they were expressed in the mutant mice as well (Fig. 2C for
PSA-NCAM and Fig. 3A for integrin ?5).
Classically, radial glia were thought to serve as a scaffold for
migrating neurons in radial migration. More recently, radial glia
the same time produce neural progenitor cells that stain positively
for brain lipid-binding protein (BLBP), glial acid fibrillary protein
(GFAP), vimentin, and nestin (16, 17). At E18.5, we found cells
staining for these markers that followed the severity of the pheno-
phenotype; Fig. S4 shows less clustering but clearly an altered
expression pattern of vimentin in mice with the less severe pheno-
type). Thus, the morphology of BLBP- and vimentin-labeled radial
glia reflected the abnormal compaction and neuronal distribution
in the Mef2c-null neocortex, suggesting that radial glia could
contribute to these defects.
Mef2c-Null Mice Manifest Disorganization of the Cortical Plate in
Postnatal/Adult Neocortex. In mice, neuronal migration is largely
day (P)7, although the brain is not considered to be fully mature
until 3 weeks of age (18). Here, we found that the developmental
abnormality of neuronal compaction that we observed in embry-
onic Mef2c-null mice (Fig. S6) led to disorganization of the cortical
plate by P7 and persisted into adulthood, as shown by neuron-
plate did not separate well from the subplate, and neuronal cell
distribution within the neocortex manifested a more compacted,
thinner cortical plate (Fig. 3). This compaction of neurons in the
laminar cortex persisted from postnatal through adult stages (num-
ber of mice examined, n ? 25 at P7 and n ? 43 for adult). In
particular, layer 5 (specifically labeled with Er81) was dramatically
thinned and more compacted in the Mef2c-null mutants (Fig. 3 B
and C). Furthermore, the neurons were smaller in size as assessed
quantitatively on Nissl-stained sections [18.52 ? 2.89 ?m in diam-
(n ? 92); mean ? SD, P ? 0.0001] (Fig. 3C), suggesting a less
mature phenotype in the knockout. By stereological counting, total
NeuN-labeled neurons in the entire adult cortex were decreased by
?30% in the Mef2c-null, compared to WT.
We next asked whether neurons in the adult cortex exhibit more
immature electrophysiological properties than their WT counter-
parts. MEF2-binding sites are present in the promoters of many
neuronally restricted genes. These include genes involved in neu-
ronal electrical activity, such as type II sodium channels, AMPA
receptor subunits, and NMDA receptor subunits (19, 20); thus, less
transcription could conceivably contribute to the observed lower
NR1 protein expression (Fig. S7). Therefore, lack of MEF2C early
in development might be expected to result in a more immature
array (MEA) recordings that adult hippocampal slices from Mef2c-
null brains compared with WT showed smaller field excitatory
postsynaptic potentials (fEPSPs) and smaller input/output (I/O)
TuJ1 (TuJ1?) in the cortical plate (cp) is shown. Graph quantifies immunofluo-
rescent markers in control versus Mef2c-nulls (*, P ? 0.001; see Materials and
www.pnas.org?cgi?doi?10.1073?pnas.0802876105Li et al.
B). In a parallel fashion, patch–clamp experiments of single neu-
rons in acute brain slices of Mef2c-null mice showed that layer 5 of
the adult cortex displayed a decrease in the frequency and ampli-
tude of miniature (m)EPSCs (Fig. 4C), smaller evoked excitatory
postsynaptic currents (EPSCs), and a decrease in the I/O ratio
compared with WT (Fig. S8). One possibility to explain the smaller
amplitude of EPSC(P)s, decreased frequency, and amplitude of
mEPSCs, and smaller I/O curves is that fewer synapses and
postsynaptic receptors/channels are found in the adult Mef2c-nulls,
for example, as encountered in the early stages of initial synapse
formation. To investigate further the potential presynaptic conse-
quences of early knockout of MEF2C, we measured paired pulse
facilitation (PPF) in cortical neurons. PPF represents short-term
enhancement of presynaptic function in response to the second of
two paired stimuli caused by residual Ca2?in the presynaptic
terminal after the first stimulation. For example, increased PPF is
were expressed by aberrantly clustered neurons in Mef2c-null neocortex. Arrowheads indicate the subplate. (B) Layer 5-specific marker Er81 and layer 6-specific
marker Tbr1 were expressed by aberrantly clustered neurons in Mef2c-null neocortex. (C) GAD 65/67-positive mature interneurons clustered within the cortical
plate; PSA-NCAM-positive proliferating neuronal progenitor cells showed a normal distribution in the Mef2c-null. (D) The morphology of radial glia labeled by
?5 staining of neurons, labeled with BrdU upon their generation at E14.5, reveals the disorganized cortical plate in the null mice. (B) Layer 5 labeled with Er81
showed aberrant neuronal migration and little colocalization with Tbr1. (C) Similar to P7, in adult mice Nissl and Er81 staining reveal that Mef2c-null neocortex
manifested a disorganized and compacted cortical plate, with layer 5 most affected. cp, cortical plate; sp, subplate. (Scale bars, 200 ?m.)
Li et al.
July 8, 2008 ?
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associated with a decrease in the probability of neurotransmitter
release. We observed no statistical difference in PPF in the
Mef2c-null and WT mice but a trend toward increased PPF in the
nulls (Fig. S9), suggesting that a decrease in neurotransmitter
release might contribute to the decrease in mEPSC frequency in a
minor way, whereas the major effect might be accounted for by a
decrease in synaptic number. Quantitative synaptophysin staining
showed that fewer synaptic sites were indeed present in knockout
mice (Fig. S10). At the same time, MAP-2 staining of the neuropil
held constant between Mef2c-null and WT mice, suggesting that
frank neurodegeneration was not responsible for the decrease in
synapse number. These data are consistent with the notion that
adult Mef2c-null neuronal networks manifest a more immature
phenotype than WT.
Mef2c-Null Mice Display Behavioral Phenotypes. To determine
whether the apparent electrical immaturity during electrophysio-
logical recordings was reflected in neurological function of Mef2c
brain nulls, we performed a series of behavioral tests on mice that
survived to adulthood. The behavioral tests demonstrated that
clasping stereotypy. For example, the Elevated Plus Maze Test
predicts how animals respond to an approach–avoidance situation
involving open and elevated spaces versus enclosed ‘‘safe’’ areas.
Mef2c conditional null mice spent significantly more time than
littermate controls on the open arms [F(1,36) ? 7.5;*, P ? 0.01 by
ANOVA] while showing no difference in total arm entries (Fig.
5A). These data suggest that the KO mice have altered anxiety-like
behavior with no overall impairment of mobility. This conclusion is
also supported by the results of the Locomotor Activity Test in
which there was no overall effect of genotype or genotype ? sex in
ambulatory scores across the test session (Fig. 5B). Interestingly,
Mef2c conditional null mice had less activity in the center of the
apparatus and less rearing behavior than littermate control mice
anxiety response in these mice (Fig. S11).
In the Fear Conditioning Test, Mef2c conditional null mice
(Fig. 5C). This effect was not caused by a generalized freezing
afferent pathway and recorded in CA1 stratum radiatum. Responses of WT (ctrl) pyramidal neurons, as measured by initial slope, were significantly larger than
bar, 100 ms and 50 mV.) (Lower) Representative evoked EPSCs from layer 5 neocortex in whole-cell recordings at a holding potential of ?70 mV (Scale bar, 50
ms and 100 pA.) (B) I/O curves of hippocampal fEPSP initial slope in response to increasing stimulation intensity. (C) Representative mEPSCs recorded from
Mef2c-null and WT layer 5 pyramidal cells voltage clamped at ?70 mV. Bar graphs show quantification of results. mEPSC frequency was decreased in Mef2c-null
compared with WT cortical neurons (n ? 33;*, P ? 0.005), consistent with a decrease in synaptic release sites or probability of release.
Reduced excitability of Mef2c-null adult brain slices compared with control. (A) fEPSPs from hippocampal slices stimulated at the Schaeffer collateral
www.pnas.org?cgi?doi?10.1073?pnas.0802876105 Li et al.
of freezing during the habituation trial. These data may be ex-
plained by the shock (the unconditioned stimulus) resulting in a
generalized fear behavior. This confounded the analysis of cued
conditioning because levels of freezing before cue exposure were
elevated. The results of the context portion of the Fear Condition-
ing Test were consistent with a deficit in spatial memory (Fig. 5C).
Although there was an overall increase in freezing in the context
previously associated with shock (P ? 0.05), if the genotypes were
investigated separately, this effect was significant in WT controls
(P ? 0.01) but not in the Mef2c-null mice (P ? 0.2).
We performed the Y Maze Test for Spontaneous Alternations,
manner in a Y maze. This is a paradigm for studying working
memory. Mef2c conditional null mice showed a decrease in spon-
taneous alternations [F(1,36) ? 11.2;*, P ? 0.01], with no signif-
icant difference in total arm entries (Fig. 5D). These results suggest
that Mef2c conditional null mice have impaired spatial working
memory. We also performed the Novel Object Exploration Test to
measure the ability of mice to build up spatial representations of
their environment and react to the introduction of novel stimuli.
Littermate control mice showed the classic pattern of habituation
to the object over the four initial trials and then renewed interest
conditional null mice displayed low levels of object exploration,
again consistent with altered anxiety-like behavior. The nulls
showed no evidence of renewed interest in the object when it was
moved, suggesting decreased spatial memory functioning in these
to occur spontaneously in the Mef2c-null mice when lifted by the
tail. Therefore, we examined this behavior more quantitatively.
than littermate control mice (?2? 7.6;*, P ? 0.05 by ?2test) (Fig.
in humans, as seen in Rett syndrome.
The processes by which neurons are born, migrate, differentiate,
and integrate into neural circuits are central problems in the study
of brain development. An understanding of these processes would
advance knowledge of general neurodevelopment and provide
in total arm entries, calculated over a 5-min period. (B) Locomotor activity was not different among null and control groups measured for 30 min as horizontal
(locomotion) behavior. (C) Cued and contextual fear conditioning tests revealed that Mef2c-null mice manifest an altered anxiety phenotype, displaying freezing
behavior in the altered context before tone onset [t(31) ? 2.2, P ? 0.05 by ANOVA; n ? 10 mice for each paradigm in A–F). Values are mean ? SEM. (D) Total number
10 s, and rated for clasping.
Mef2c-null mice display altered anxiety-like behavior and decreased cognitive function. (A) The Elevated Plus Maze showed an apparent lowered state of
Li et al.
July 8, 2008 ?
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no. 27 ?
valuable knowledge concerning how such processes might produce
pathology when alterations occur. We report here that the tran-
scription factor MEF2C is crucial for normal neuronal develop-
ment, distribution, and electrical activity in the neocortex. When
MEF2C was absent early in brain development, we observed an
abnormal distribution of neurons in the cortical plate with conse-
quent developmental abnormalities. Hippocampal and cortical
electrophysiological responses coupled with quantitative immuno-
histofluorescence suggested the presence of a more immature
exact opposite of that obtained by knocking down MEF2 activity in
more mature neurons, which resulted in an increase in synapse
activity and number (1).
The spatial cognitive deficits observed in Mef2c-null mice across
three behavioral tests are consistent with hippocampal deficits, and
the spontaneous paw-clasping behavior suggests motor control
deficits. Interestingly the behavioral findings are similar to those
observed in two mouse models of a mutant Mecp2 gene, encoding
methyl-CpG-binding protein 2 (MeCP2) (21–25). This pattern of
Mecp2 gene. MeCP2 is involved in gene silencing and known to
26). In turn, BDNF has recently been shown to activate MEF2
transcription factors (1, 9), raising the possibility of MEF2C in-
Rett phenotype. Additionally, the Mecp2 promoter contains multiple
provide feedback to activate Mecp2 transcription. Note, however, that
because Mef2c is autosomal, its deletion does not result in an X-linked
pattern of inheritance as seen in Rett syndrome.
The putative role of MEF2C in disease notwithstanding, our
produces a ‘‘compaction defect’’ during early neuronal differenti-
ation and results in a ‘‘decreased synaptic phenotype’’’ with behav-
ioral deficits in adulthood suggestive of an autism-like syndrome.
We speculate therefore that such early knockout of Mef2c contrib-
utes to an adult phenotype suggestive of immaturity or develop-
mental arrest in the neocortex, stunting somal size, number, dis-
tribution, and synapse formation. Intriguingly, Eric Olson and
colleagues (27) observed that when Mef2c is knocked out in vivo at
a later time point in development under the GFAP promoter-
driven Cre transgene, an ‘‘increased synaptic phenotype’’ results,
similar to reported MEF2 knockdown experiments performed on
of findings can be understood in the context of representing critical
pleiotropic effects of MEF2C on disparate temporal points in the
Materials and Methods
Generation of Mef2c Conditional Null Mice and MEF2 Reporter Mice. All
experiments were performed in accordance with institutional guidelines con-
cerning care and treatment of vertebrate animals. Refer to SI Materials and
Methods for additional details of the mouse strain crosses and other methods
used in this work.
Quantitative Immunohistochemistry for NSC and Neuronal Markers. Quantifi-
developing neocortex by deconvolution microscopy and dividing them into a
series of bands extending from the ventricular zone to the cortical surface.
Stereological Neuronal Cell Counts. Cells were pulse-labeled with BrdU at E14.5
cells that were also NeuN-positive. In the adult, total NeuN-positive cells were
counted with an optical dissector technique.
Quantitative Immunohistochemical Analysis for Volume of Dendritic Neuropil
and Synapse Number. For assessment of neuronal somal/dendritic and synaptic
changes, sections were immunolabeled with antibodies against MAP-2 and syn-
Electrophysiological Techniques. Brains of neonatal and adult male mice were
analyzed by using MEA or patch–clamp recordings.
Neurobehavioral Tests. We measured the effects on the mice of the Mef2c gene
knockout by assessing their paw-clasping response while being held by the tail
and by using well accepted behavioral tests.
Statistical Analysis. Data are presented as means ? SEM, unless otherwise
indicated. Comparisons were made by using a one-tailed Student’s t test or an
ANOVA for multiple comparisons. Values for n and significance limits are indi-
cated in figure legends or the text.
together with our work, this molecular link is consistent with the notion that
MEF2C may play a role in the pathogenesis of Rett syndrome and possibly other
forms of autism.
mice. S.A.L. was a Senior Scholar in Aging Research of the Ellison Medical Foun-
dation. This work was supported in part by National Institutes of Health (NIH)
Grants P01 HD29587 and R01 NS044326 (to S.A.L.) and Fellowship Award
0625088Y from the American Heart Association (to J.C.R.). The support of facil-
ities by the La Jolla Interdisciplinary Neuroscience Center Cores, NIH Blueprint
Grant P30 NS057096, is kindly acknowledged.
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