Histone deacetylases control neurogenesis in embryonic brain by inhibition of BMP2/4 signaling.

Abstract

Histone-modifying enzymes are essential for a wide variety of cellular processes dependent upon changes in gene expression. Histone deacetylases (HDACs) lead to the compaction of chromatin and subsequent silencing of gene transcription, and they have recently been implicated in a diversity of functions and dysfunctions in the postnatal and adult brain including ocular dominance plasticity, memory consolidation, drug addiction, and depression. Here we investigate the role of HDACs in the generation of neurons and astrocytes in the embryonic brain.
As a variety of HDACs are expressed in differentiating neural progenitor cells, we have taken a pharmacological approach to inhibit multiple family members. Inhibition of class I and II HDACs in developing mouse embryos with trichostatin A resulted in a dramatic reduction in neurogenesis in the ganglionic eminences and a modest increase in neurogenesis in the cortex. An identical effect was observed upon pharmacological inhibition of HDACs in in vitro-differentiating neural precursors derived from the same brain regions. A reduction in neurogenesis in ganglionic eminence-derived neural precursors was accompanied by an increase in the production of immature astrocytes. We show that HDACs control neurogenesis by inhibition of the bone morphogenetic protein BMP2/4 signaling pathway in radial glial cells. HDACs function at the transcriptional level by inhibiting and promoting, respectively, the expression of Bmp2 and Smad7, an intracellular inhibitor of BMP signaling. Inhibition of the BMP2/4 signaling pathway restored normal levels of neurogenesis and astrogliogenesis to both ganglionic eminence- and cortex-derived cultures in which HDACs were inhibited.
Our results demonstrate a transcriptionally-based regulation of BMP2/4 signaling by HDACs both in vivo and in vitro that is critical for neurogenesis in the ganglionic eminences and that modulates cortical neurogenesis. The results also suggest that HDACs may regulate the developmental switch from neurogenesis to astrogliogenesis that occurs in late gestation.

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Histone Deacetylases Control Neurogenesis in Embryonic
Brain by Inhibition of BMP2/4 Signaling
Maya Shake `d1, Kathrin Weissmu ¨ller1, Hanno Svoboda1¤, Peter Hortschansky2, Norikazu Nishino3, Stefan
Wo ¨lfl4, Kerry L. Tucker1*
1Interdisciplinary Center for Neurosciences, University of Heidelberg, Heidelberg, Germany, 2Leibniz Institute for Natural Product Research and Infection Biology, Hans
Kno ¨ll Institute, Jena, Germany, 3Graduate School of Life Science and Systems Engineering, Kyushu Institute of Technology, Kitakyushu, Japan, 4Institute of Pharmacy and
Molecular Biotechnology, University of Heidelberg, Heidelberg, Germany
Abstract
Background: Histone-modifying enzymes are essential for a wide variety of cellular processes dependent upon changes in
gene expression. Histone deacetylases (HDACs) lead to the compaction of chromatin and subsequent silencing of gene
transcription, and they have recently been implicated in a diversity of functions and dysfunctions in the postnatal and adult
brain including ocular dominance plasticity, memory consolidation, drug addiction, and depression. Here we investigate the
role of HDACs in the generation of neurons and astrocytes in the embryonic brain.
Principal Findings: As a variety of HDACs are expressed in differentiating neural progenitor cells, we have taken a
pharmacological approach to inhibit multiple family members. Inhibition of class I and II HDACs in developing mouse embryos
with trichostatin A resulted in a dramatic reduction in neurogenesis in the ganglionic eminences and a modest increase in
neurogenesis in the cortex. An identical effect was observed upon pharmacological inhibition of HDACs in in vitro-
differentiating neural precursors derived from the same brain regions. A reduction in neurogenesis in ganglionic eminence-
derived neural precursors was accompanied by an increase in the production of immature astrocytes. We show that HDACs
control neurogenesis by inhibition of the bone morphogenetic protein BMP2/4 signaling pathway in radial glial cells. HDACs
function at the transcriptional level by inhibiting and promoting, respectively, the expression of Bmp2 and Smad7, an
intracellular inhibitor of BMP signaling. Inhibition of the BMP2/4 signaling pathway restored normal levels of neurogenesis and
astrogliogenesis to both ganglionic eminence- and cortex-derived cultures in which HDACs were inhibited.
Conclusions: Our results demonstrate a transcriptionally-based regulation of BMP2/4 signaling by HDACs both in vivo and in
vitro that is critical for neurogenesis in the ganglionic eminences and that modulates cortical neurogenesis. The results also
suggest that HDACs may regulate the developmental switch from neurogenesis to astrogliogenesis that occurs in late
gestation.
Citation: Shake `d M, Weissmu ¨ller K, Svoboda H, Hortschansky P, Nishino N, et al. (2008) Histone Deacetylases Control Neurogenesis in Embryonic Brain by
Inhibition of BMP2/4 Signaling. PLoS ONE 3(7): e2668. doi:10.1371/journal.pone.0002668
Editor: Tailoi Chan-Ling, University of Sydney, Australia
Received February 27, 2008; Accepted June 11, 2008; Published July 16, 2008
Copyright: ? 2008 Shake `d et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was funded by the German Research Foundation (DFG, SFB 488, Teilprojekt B7), the University of Heidelberg, the BMBF (Grants 0311786 &
0312558 to P.H.), the Minerva Stiftung of the Max Planck Society (M.S.), and the Studienstiftung des deutschen Volkes (H.S.).
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: kerry.tucker@urz.uni-hd.de
¤ Current address: Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge, United Kingdom
Introduction
Neurons are the predominant terminally-differentiated cell type
produced in the brain during prenatal development in vertebrates
[1]. In the developing cortex, glutamatergic projection neurons are
generated that then migrate radially outward to assume their
proper position in one of the six layers of the postnatal neocortex.
Cortical neurons arise from proliferating radial glia cells [2,3],
either directly generated in an asymmetrical fashion at the
pseudostratified ventricular zone or deriving from a symmetrical-
ly-dividing basal progenitor, also generated by radial glia, in the
subventricular zone [4–6]. In the lateral and medial ganglionic
eminences (GE) of the embryonic striatum and pallidum,
respectively, GABAergic interneurons are generated to a large
extent in the subventricular zone, and many of these neurons
proceed to leave the GE, migrating tangentially and populating
the cortex (reviewed in [7,8]). Radial glia also contribute to
neurogenesis in the GE [2]. The progenitor populations in the
cortex and GE express a distinctly different palette of transcription
factors to direct regional-specific neurogenesis. For example, Pax6
and Ngn2 expression in the dorsal telencephalon leads to a cortical
differentiation program including the expression of the proneural
transcription factors Math2/3, NeuroD1/2, and Tbr1/2, whereas
Mash1 and Nkx2.1 expression in the ventral telencephalon leads
to a striatal / pallidal differentiation program marked by the
expression there of the homeobox genes Dlx1/2, Dlx5/6, and
Gsh1/2, respectively [7].
In mammals, astrocytes and oligodendrocytes are first born late
in gestation and continue to be produced well after birth [1]. The
developmental transition from a primarily neurogenic to an
astrogliogenic program is only partially understood. The emer-
gence of astrogliogenic precursors is actively suppressed by the
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basic helix-loop-helix transcription factors Ngn2 and Mash1
[9,10]. Two key signal transduction pathways are involved in
the promotion of astrogliogenesis. Leukemia inhibitory factor (LIF)
and ciliary neurotrophic factor (CNTF) activate the JAK-STAT
pathway through their common receptor gp130 [11,12], while the
bone morphogenetic proteins 2 and 4 (BMP2/4), binding to their
co-receptors BMPR1A/B and BMPR2, activate the Smad1/5/8
pathway (reviewed in [13]) Both of these pathways converge in vitro
at the promoter for glial fibrillary acidic protein (GFAP), a marker
of newborn, maturing astrocytes [12]. BMPs are a family of
secreted growth factors belonging to the TGFb superfamily.
BMP2 and BMP4 promote the generation of astrocytes both in
vitro and in vivo [14,15], partially by the induction of the
transcription factors Hes5, Id1, and Id3, which antagonize the
pro-neurogenic activity of Ngn1 [16]. However, the importance of
BMPs for neurogenesis in the brain is still unclear. Multiple BMPs
[17] and their receptors [18,19] are expressed in neurogenic
regions of the developing brain, starting as early as 10.5 d.p.c. in
the telencephalon [18] and extending throughout the major period
of neurogenesis in the developing cortex and the GE [19]. Initial
experiments indicated that ectopically applied BMP2 and -4
promote neurogenesis in the cortex [20,21], but BMP2 and -4
were shown to inhibit neurogenesis in the embryonic and adult
striatum [14,22] and interneuron development in the embryonic
cortex [23]. Analyses using mice deficient in the BMP receptor
Bmpr1a [24] suggested a minimal role in the regulation of cortical
neurogenesis. However, recent reports clearly documented the loss
of DI1 and DI2 interneurons in the spinal cord [25] and of
cerebellar granule neurons in the cerebellum [26] of mice lacking
both Bmpr1a and Bmpr1b. Ablation of Bmpr1a and Bmpr1b in the
telencephalon resulted in holoprosencephaly and embryonic death
by 11.5 d.p.c. [27], thereby precluding analysis of the importance
of BMP2 and 4 for the major periods of neurogenesis and
astrogliogenesis.
The modification of chromatin is a central aspect of the
regulation of gene transcription in eukaryotic cells. The covalent
modification of core histones by methylation, phosphorylation,
and acetylation forms a combinatorial ‘‘code’’ that governs the
transcriptional activity of DNA sequences wound around histone
octamers [28]. Acetylation of the e-amino groups of lysine residues
in the amino-termini of core histones by histone acetyltransferases
(HATs) neutralizes the positive charges that normally stabilize the
formation of the compacted 30-nm fiber and inter-fiber interac-
tions [29]. Thus, histone acetylation leads to a decondensed
nucleosomic structure that allows access of transcription factors to
the DNA. Conversely, transcriptional inactivity is associated with
the removal of acetyl groups by histone deacetylases (HDACs), a
large group of enzymes classified into three gene families [30].
Class I HDACs (HDAC1, -2, -3, and -8) are orthologs of the yeast
RPD3 protein and are almost exclusively localized in the nucleus.
They have been shown to co-localize in complexes such as Sin3,
NuRD, and Co-REST that potentiate HDAC activity and contain
transcriptional co-repressors [31,32]. HDAC1 also associates with
the methylCpG-binding protein MeCP2 [33], providing a
functional link between DNA methylation and histone modifica-
tion. Class II HDACs (HDAC4, -5, -6, -7, -9, and -10) are
homologous to the yeast HDA1 protein, are double the size of the
class I HDACs, and can shuttle between the nucleus and the
cytoplasm [34]. Class II HDACs interact with the transcriptional
co-repressors N-CoR and SMRT [35] and the MEF2 family of
transcription factors [36]. Class I and II HDACs share almost
identical catalytic domains, whereas the NAD+-dependent class III
HDACs form an evolutionarily distinct family homologous to yeast
Sir2 proteins [37].
The importance of histone acetylation for dynamic regulation of
gene expression during development has been intensively
investigated for skeletal and cardiac myogenesis. The class II
HDACs HDAC4, -5, and -7 associate with the myogenic
transcription factor MEF2 and thereby inhibit its activity in the
nucleus [36]. Myogenic signals lead to the phosphorylation of
these HDACs by CaM kinase [38] and, after binding of 14-3-3
proteins, export from the nucleus [39,40]. The HAT p300 can
then associate with MEF2, and also MyoD [41], to promote the
activation of muscle-specific genes. Evidence is now emerging that
HDACs are also important for the development of the nervous
system as well. HDACs have been shown to promote the genesis
[42] and maturation [43,44] of oligodendrocytes in the rat [43–45]
and in zebrafish [42]. Two reports have shown that the class I
hdac1 promotes the generation of neurons in the retina [46] and
the spinal cord [47] of the zebrafish. Evidence for the role of
HDACs in the control of neurogenesis and astrogliogenesis in the
mammalian brain, however, is lacking. Although ablation of Hdac4
can occasionally lead to exencephaly [48], other gene knockouts in
the mouse have resulted in either early embryonic lethality (Hdac1
[49]), or in no reported change in brain development (Hdac5 [50],
Hdac9 [51], Hdac7 [52], and Hdac2 [53]). This may be explained
by the large number of HDACs that are expressed in the
developing mammalian brain [44], which could allow for
functional redundancy.
We investigated the role of HDACs in the generation of neurons
and astrocytesindeveloping brain.AsalargenumberofHDACsare
expressed in differentiating neural stem cells and progenitors, we
employed a pharmacological approach to inhibit all members of the
class I and II families. Treatment of mouse embryos with the highly-
specific HDAC inhibitor trichostatin A caused a dramatic reduction
in neurogenesis in the GE and a modest increase in the cortex. An
identicaleffectwas observed ininvitro-differentiated neural precursor
cultures prepared from embryonic GE and cortex [54]. We
demonstrate that HDACs control the transcription of Bmp2 itself
and Smad7, an inhibitor of BMP signaling [55], whose expression is
inhibited and promoted by HDACs, respectively. HDACs promote
neurogenesis in the GE by downregulation of BMP2/4 signaling,
leading to the favored production of neurons over astrocytes,
whereas HDAC-mediated inhibition of BMP2/4 signaling in the
cortex leads to an opposite outcome.
Results
HDAC function is necessary for neurogenesis in the
ganglionic eminences in vitro
In order to perform a molecular biological and biochemical
analysis of the effects of HDAC inhibition upon neurogenesis in
the developing embryo, an in vitro-based approach was at first
selected. To generate a uniform population of neural precursors
directly from embryonic brain, we turned to the well-established
technique of neurosphere cultures [54]. We performed in vitro
differentiation upon precursor cells derived from embryonic lateral
and medial ganglionic eminences (GE). Neurosphere cultures were
prepared from the GE from mice of the inbred C57BL/6J
background at 15.5 d.p.c., cultured for seven days as floating cell
aggregates, and then differentiated for seven days in monolayer
culture on polyornithine-coated cover slips, according to standard
methods, in a medium that supports the differentiation of both
neurons and astrocytes [56]. To promote differentiation, the
mitogen basic FGF (bFGF) was withdrawn after 2.5 days in vitro
(DIV; Fig. 1A). We first utilized Western blots to examine protein
expression of class I (HDAC1, -2, -3) and class II HDACs
(HDAC4, -5, -6, -7) in these cultures. All examined HDACs were
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expressed throughout the period of differentiation, with a general
decrease in expression levels over the course of time, with the
exception of HDAC4 and 6, which maintained and increased,
respectively, their level of protein expression (Fig. S1A). We can
clearly attribute the expression of HDACs to neural precursors at
early stages of the cultures, as they comprise more than 90% of the
cell population after plating the neurospheres out for monolayer
culture, as judged by positive staining for nestin and cellular
morphology (Fig. S1B, C).
As neural precursors express a wide variety of class I and II
HDACs, we decided to adopt a pharmacological approach to
inhibit both families. We employed trichostatin A (TSA), a highly
specific Streptomyces-derived hydroxamic acid that interacts at
nanomolar concentration with the catalytic site of class I and II
HDACs, acting as a potent, reversible, competitive inhibitor [57].
10 nM TSA was added to the medium upon plating out of
dissociated neurospheres and again at 2.5 DIV, when bFGF was
withdrawn (Fig. 1A). After an additional 4.5 days (7 DIV) cells were
fixed and stained with the TuJ1 antibody to detect b-tubulin III
expression, a marker for newborn neurons, evaluating cell number
by counting DAPI-stained nuclei. The percentage of neurons
dramatically decreased from 35% in untreated cultures to 12%
upon 10 nM TSA treatment (Fig. 1B, C),. A statistically significant
decrease could be observed from the fourth day of differentiation
(Fig. 1C and Table 1). The percentage of neurons formed did not
increase if cultures were differentiated an additional 3 days in the
absence of TSA (Control: 31.261.5%; TSA: 7.960.6%, n=2).
We employed a wide range of HDAC inhibitors, including the
hydroxamic acids SAHA and SBHA and the cyclic tetrapeptide
apicidin. All of these inhibitors were able to inhibit neurogenesis, in
all cases completely eliminating neurogenesis at nanomolar or
micromolar concentrations (Fig. S2A). Interestingly, valproic acid
(VPA, 2-propylpentanoic acid), a short-chained fatty acid that
preferentially inhibits class I HDACs [58,59] and has long been used
as an anti-epileptic drug, did not show a reduction in neurogenesis
(Table 1), even when used at 1.0 mM, a concentration well above
the IC50range for inhibition of Class I HDACs [58].
To address the possibility that HDAC inhibition through TSA
may inhibit the expression of b-tubulin III in neurons, we also
prepared neurosphere cultures from transgenic mice (the tauGFP
line) with an insertion of EGFP at the tau locus, which is expressed
specifically in newborn neurons in a time frame similar to
induction of b-tubulin III [60]. Using antibodies to GFP to identify
neurons, a similar reduction in neurogenesis was observed upon
treatmentwith 10 nMTSA
10.864.1%, n=4). Finally, we prepared neurosphere cultures
from wild-type mice of the CBA/J background and examined
neurogenesis after treatment with TSA in order to test the
specificity of this effect for a given genetic background. Again,
10 nM TSA application reduced neurogenesis, from 33.8% in
control conditions to 10.8% upon application of TSA. The
inhibition of neuron production by TSA showed a concentration-
dependent effect, with increasing concentrations resulting in fewer
neurons, dropping down to only 4.3% at 50 nM (Table 2).
Neural precursors derived from neurospheres can differentiate
into neurons, oligodendrocytes and astrocytes, or they can remain
as proliferating neural precursors [61]. To determine if the HDAC
(Control:32.562.4%;TSA:
Figure 1. Inhibition of HDACs by TSA treatment blocks
neurogenesis in differentiating neural progenitor cultures
derived from embryonic GE. (A) Neurosphere cultures derived
from 15.5 d.p.c. mouse GE were dissociated, cultured on polyornithine,
and the mitogen bFGF was removed from the cultures at 2.5 days in
vitro (DIV). Neurogenesis was evaluated at 7 DIV. (B) 7 DIV cultures were
stained with the TuJ1 antibody (red) to detect b-tubulin III expression,
marking newborn neurons, and DAPI (green) to stain cell nuclei. (C)
Time course of suppression of neurogenesis by TSA. Cultures were
stained at each day of the 1-week differentiation period and the
percentage of neurons produced was calculated using a DAPI stain to
evaluate total cell number. Cultures were untreated (2) or treated with
10 nM TSA (+). (D) The cultures described in (B) were also stained for
astrocytes with an antibody recognizing GFAP (red) and DAPI (green) to
stain cell nuclei. (B, D) Scale bar=100 mm. (E) The percentage of cells
detected with the GFAP antibody is indicated. (C, E) Mean values +/2
SEM (n=5). *=p,0.05, **=p,0.01, Mann-Whitney U test.
doi:10.1371/journal.pone.0002668.g001
Table 1. Time-course of suppression of neurogenesis by TSA, showing percentage of cells that become neurons, as seen in Fig. 1C.
Days in vitro1 DIV 2 DIV3 DIV4 DIV 5 DIV 6 DIV 7 DIV
Control 0.660.82.361.15.961.310.262.1 16.762.226.862.434.063.1
TSA 10 nM0.460.61.461.03.061.1 4.061.4 *3.061.4*4.161.6**11.862.6**
VPA 0.3 mM 0.660.72.761.28.361. 715.162.013.762.225.162.134.062.6
Mean values +/2 SEM (n=3–6).*=p,0.05,**=p,0.01,***=p,0.001, Mann-Whitney U test.
doi:10.1371/journal.pone.0002668.t001
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inhibitors had an effect on the production of the latter three cell
types, we stained cells after a week of differentiation with antibodies
against glial fibrillary acidic protein (GFAP), which recognizes
astrocytes; nestin, a marker for neural precursors; and O4, a marker
for newborn oligodendrocytes. Astrocytic differentiation almost
doubled in TSA-treated cells, changing from 11.962.0% in
untreated cells to 22.362.4% in 10 nM TSA-treated cells (Fig. 1D,
E; n=5). In addition, three other HDAC inhibitors, the hydroxamic
acids SAHA and SBHA and the cyclic tetrapeptide apicidin, all
increased astrogliogenesis in a dose-dependent fashion (Fig. S2B).
During the first 3 days of differentiation over 80% of the cells
expressed nestin in both TSA-treated and untreated cells (Fig. S1B,
C, D). This expression was reduced over the course of a week to
19.162.1% nestin (+) in the control cells (Fig. S3A, C), reflecting the
production of differentiated neurons, astrocytes, and oligodendro-
cytes. In contrast, 10 nM TSA treatment resulted in 25.963.3% of
the cells remaining nestin positive (Fig. S3A, C; n=3). Finally, after
treatment with 10 nM TSA, the percentage of oligodendrocytes
decreased from 7.361.6% to 1.760.5% (Fig. S3B, D; n=3). The
oligodendrocytes produced upon TSA treatment were not only
reduced in number but also demonstrated a less elaborate
morphology, as has previously been shown upon in vitro differenti-
ation of oligodendrocyte precursors [43].
Inhibition of HDACs for 7 days might lead to a large number of
transcriptional changes in neural precursor cells. Since TSA can
affect histone acetylation levels [57] and gene transcription [62]
already several hours after application, we determined the critical
time window for inhibition of HDAC function in order to inhibit
neurogenesis in cultures derived from GE. We tested 4 different
treatment periods (Fig. 2A): 1) 10 nM TSA applied first 24 hours
after cells were plated out, to eliminate the possibility that TSA
inhibits cells from adhering. 2) 2.5 days treatment of 10 nM TSA,
from the time that cells are plated out until bFGF is withdrawn. 3)
10 nM TSA applied 24 hours before bFGF withdrawal and for a
further 3.5 days until cells were fixed. 4) 10 nM TSA added only
24 hours before bFGF withdrawal. All 4 of these treatments resulted
in a decrease of neurogenesis similar in magnitude to continuous
treatment over 7 days. Importantly, only a 24-hour treatment of
TSA before bFGF was withdrawn was necessary to reduce
neurogenesis from 36% to 9%. This reduction in neurogenesis was
accompanied with an increase of nestin (+) precursors (Control:
23.761.8%; 10 nM TSA : 41.2613.5%) and GFAP (+) astrocytes
(Control: 18.368.1%; 10 nM TSA: 32.062.0%, n=2), as seen
before (Fig. S3A, C and Fig. 1D, E, respectively). Interestingly,
10 nM TSA treatment after bFGF withdrawal did not significantly
affect neurogenesis (data not shown).
To assess the acetylation levels of core histones in response to
TSA, we employed antibodies specifically recognizing the
acetylated forms of histone H3 and H4, using Western blots upon
protein lysates. Application of 10 and 50 nM TSA to neurosphere-
derived cultures one day before bFGF-withdrawal resulted in a
Table 2. Dose-dependent suppression of neurogenesis by
TSA.
Treatment ControlTSA 2 nMTSA 10 nMTSA 50 nM
% neurons30.861.922.662.3 9.761.2 **4.361.5 ***
Treatment of neurosphere cultures with the indicated TSA concentrations for 1
week during in vitro differentiation revealed a dose-dependent effect upon the
production of neurons. Mean values +/2 SEM (n=3). p values are indicated as
in Table 1.
doi:10.1371/journal.pone.0002668.t002
Figure 2. A 24-hour inhibition of HDACs before mitogen
withdrawal is sufficient to suppress neurogenesis, indepen-
dently of changes in proliferation and apoptosis, in differen-
tiating neural progenitor cultures derived from embryonic GE.
(A) Dissociated neurospheres were plated onto coverslips, and at 2.5
DIV bFGF was withdrawn (-bFGF), followed by 4.5 days of differenti-
ation. Neurogenesis was evaluated at 7 DIV. Neurosphere cultures were
treated for the indicated time periods (lines under the time line) with
10 nM TSA, then stained with the TuJ1 antibody to detect newborn
neurons. DAPI staining of nuclei was used to count cells, and the
percentage of neurons formed is shown. Mean values +/2 SEM (n.3).
***=p,0.001, Mann-Whitney U test. (B) Western blot analysis of
dissociated neurosphere cultures derived from embryonic GE treated
with 10 nM TSA for 1 or 24 hours before bFGF withdrawal show a
marked increase in acetylation levels of histone H3 and H4, using an
antibody specifically recognizing acetylated lysine residues on the
amino termini of histone H3 and H4. Loading levels were confirmed by
reprobing each blot with an antibody recognizing b-actin (below each
respective anti- acetylated histone panel). (C) Dissociated neurospheres
were treated with BrdU (10 mM) alone or with 10 nM TSA and BrdU for
24 hours. Cells were then fixed and stained with an anti-BrdU antibody.
DAPI staining was used to count cells, and the percentage of BrdU-
positive cells is shown (n=3). (D) After treatment of dissociated
neurosphere cultures for 24 hours with 10 nM TSA, bFGF was
withdrawn at 2.5 days in vitro, and apoptosis rates were measured
one day later using the TUNEL assay. DAPI staining was used to count
cells, and the percentage of TUNEL-positive cells is shown (n=3).
doi:10.1371/journal.pone.0002668.g002
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detectable increase in acetylation levels of both histone H3 and H4
already within one hour, with an even higher increase after
24 hours (Fig. 2B).
TSA is known to inhibit cell cycle progression at G1 and G2
phases [63]. In order to assess the effects that TSA may have upon
the cell cycle, we used BrdU labeling to assess the number of cells
in the culture that had replicated their DNA. Dissociated
neurospheres were treated with BrdU (10 mM) alone or with
10 nM TSA and BrdU for 24 hours. Cells were then fixed and
stained with an anti-BrdU antibody. No significant differences
were observed between control and TSA-treated cultures (Fig. 2C).
TSA can induce apoptosis in cancer cell lines and tumors, and
synthetic analogues of TSA are employed in cancer therapy [64].
We addressed the possibility that neurons were being produced
upon TSA treatment but then dying. Visual inspection of 10 nM
TSA-treated cultures did not indicate an increase in cell death,
and a DAPI staining of the nuclei of TSA-treated cells did not
reveal an increase in pyknotic nuclei (Control: 6.961.4%, TSA:
5.960.8%, n=4). We next examined apoptosis levels with a
TUNEL assay. After treatment of cultures for 24 hours with
10 nM TSA, bFGF was withdrawn at 2.5 days in vitro, and
apoptosis rates were measured one day later using the TUNEL
assay. The percent of TUNEL (+) apoptotic cells was similar in
control condition and upon TSA treatment (Fig. 2D).
HDACs downregulate BMP2/4 signaling to promote GE
neurogenesis in vitro
To elucidate the molecular mechanism by which HDACs
regulate neurogenesis, we investigated various developmental
pathways known to control neurogenesis in vivo. We first examined
the BMP2/4 signal transduction pathway. BMP2, BMP4, and
their receptors are expressed in neurosphere cultures [14] and
have been shown to promote astrocytic differentiation in vitro [14]
and in vivo [15]. BMP2 is highly similar to BMP4 (93% amino acid
identity), activates the same signaling pathway as BMP4, is also
expressed in the subventricular zone of embryonic striatum [14],
and like BMP4 was found to be expressed in differentiating neural
stem cell cultures (Fig. 3C, D, 4B).
As we expected HDACs to act upon a transcriptional level to
affect neurogenesis and astrogliogenesis in GE, we performed
quantitative, real-time RT-PCR upon mRNA extracted from
TSA-treated neurosphere cultures, comparing gene expression
levels to untreated cultures. 12 hours after treatment of 1.5 DIV
cultures with TSA, the expression of the BMP2/4-specific
receptors Bmpr1a, Bmpr 1b, and Bmpr2 were unchanged in GE-
derived cultures (Fig. 3A). In contrast, the expression of Bmp2 was
upregulated dramatically in GE-derived cultures (Fig. 3C) after
12 hours of TSA treatment, whereas Bmp4 was downregulated
(Fig. 3C). We next examined the expression of the inhibitory factor
Smad7 [55] in our cultures. Smad7 expression was downregulated
by TSA application at both concentrations (Fig. 3E). In addition,
the expression of the astrogliogenesis-promoting transcription
factors Stat1 and Stat3 was upregulated by TSA-treatment
(Fig. 3G). Curiously, the expression of the neurogenesis-promoting
transcription factor Ngn1 was also upregulated by TSA-treatment,
while that of Ngn2 remained unaffected (Fig. 3I). All of these effects
showed a TSA dose-dependence (Fig. 3C, E, G, I).
In order to test the relevance of the transcriptional control of
Bmp2 and Smad7 gene expression by HDACs, we decided to
influence the BMP2/4 signaling pathway by boosting or inhibiting
the extracellular signal. Recombinant BMP2 was applied to
differentiating neurosphere-derived cultures one day before the
withdrawal of bFGF at concentrations ranging from 10–100 ng/
ml. This resulted in a dramatic effect, in that over 50% of the cells
differentiated into astrocytes, as previously reported (Fig. 4C, D;
[14]). The GFAP+ cells were clustered together in what resembled
astrocytic ‘‘islands’’ (Fig. 4C, panel 7). We also observed similar
clusters of GFAP+ cells in cultures in which 10 nM TSA was
withdrawn after 5 days of treatment and then cultured for an
additional 3 to 4 days, for a total of 10–11 days (Fig. 4C, panel 8).
Figure 3. HDACs control expression of genes in the BMP2/4
signaling pathway and regulatory genes involved in neuro-
genesis and astrogliogenesis. (A–J) Quantitative real time RT-PCR
was performed upon mRNA extracted from TSA-treated neurosphere
cultures, comparing gene expression levels to untreated cultures.
Dissociated neurosphere cultures derived from embryonic GE (A, C, E,
G, I) or cortex (B, D, F, H, J) were plated out onto 10-cm dishes and
treated 1.5 days later with vehicle (0.008% ethanol) or TSA (10 or
50 nM). RNA was extracted 12 hours later, and reverse-transcribed
cDNA was analyzed using TaqMan probes recognizing the following
genes: (A, B) Bmpr1a, Bmpr1b, Bmpr2; (C, D) Bmp2, Bmp4; (E, F) Smad7;
(G, H) Stat1, Stat3; (I, J) Ngn1, Ngn2. cDNA was normalized using probes
for GAPDH and HPRT. Mean values +/2 SEM (n=4–8). *=p,0.05,
**=p,0.01, ***=p,0.001, Student’s T-test. (D) p=0.06 for Bmp2
expression increase in cortex treated with 50 nM TSA.
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In order to test the involvement of endogenous BMP signaling in
normal and TSA-treated cultures, cells were treated with two
different BMP2/4 inhibitors. Alk3-ECD is a recombinant protein
consisting of the extracellular domain of the BMPR1A receptor
that can bind to BMP2 and -4 with high affinity, preventing them
from binding to the endogenous BMPR1A receptor [65]. Noggin
is a secreted polypeptide that binds to BMPs and prevent their
binding to and activation of their receptors [66]. Each inhibitor
was added for 24 hours before bFGF withdrawal, with or without
10 nM TSA, and then removed when bFGF was withdrawn. The
cultures were analyzed 4.5 days later, at 7 DIV. The use of either
Alk3-ECD or noggin (each at a concentration of 250 ng/ml) alone
did not affect neurogenesis (Fig. 4A, D) or astrocyte production
(Fig. 4C, D). However, addition of either BMP2/4 inhibitor in
combination with 10 nM TSA completely restored normal levels
of neurogenesis (Fig. 4A, D). In addition, the production of
astrocytes was reduced to levels seen in untreated cells (Fig. 4C, D).
Longer treatment of cultures with these reagents, starting 24 hours
before bFGF withdrawal but continuing for an additional 4.5 days,
produced similar results (Neurogenesis: Control: 31.160.7%;
TSA: 8.261.1%;Alk3-ECD:
27.961.4%; Noggin: 30.063.9%; Noggin & TSA: 24.862.3%,
n=2, reagent concentrations as above).
In order to investigate the effect of TSA upon neural stem cell
cultures, we next examined the activation of the BMP2/4 signaling
pathway. Binding of BMP2/4 to their cognate receptors results in
thephosphorylationoftwoadjacentserineresiduesintheSmad1/5/
8 proteins, followed by their translocation to the nucleus where they
act as transcriptional regulators of BMP2/4-responsive genes [67].
We examined the cultures with an antibody that specifically
recognizes Smad1/5/8 proteins phosphorylated at these serine
residues, staining cultures 1, 4, 12, and 24 hours after reagents were
added (Fig. 4E). Addition of 10 ng/ml BMP2 to cultures 24 hours
before bFGF withdrawal caused an accumulation of phosphorylated
Smad1/5/8 in the nucleus after just 1 hour of treatment (Fig. 4E).
This localization was sustained at 4 hours, whereas by 12 and
24 hours it was no longer detectable. A nuclear localization of
phosphorylated Smad1/5/8 became visible after 12 hours of
treatment with 50 nM TSA and after 24 hours of treatment with
10 nM TSA(Fig. 4E). This delay inSmad1/5/8 nuclear localization
compared to BMP2 application may reflect the need to induce a
change in transcription patterns of genes involved in BMP signaling,
such as Bmp2 (Fig. 3C) itself or Smad7 (Fig. 3E).
Additionally, we investigated two other pathways that are
known to regulate neurogenesis in mammals and that had been
implicated in the inhibition of retinal neurogenesis in a mutation
in the zebrafish hdac1 gene [46]. Examination by Western blot of
the protein levels of the intracellular domain of Notch1,
representing the cleaved, activated domain [68], revealed no
change in its expression upon a 7-day treatment with 10 nM TSA,
comparing cultures at 1, 2.5, 3.5, and 7 DIV (Fig. S4A). To
examine a possible involvement of the Wnt signaling pathway, we
treated neurosphere-derived cultures with inhibitors of Wnt
signaling, Dkk1 (0.5 mg/ml) and sFRP2 (0.2 mg/ml). These
inhibitors were added 24 hours before bFGF withdrawal, and
neurogenesis examined after a further 4.5 days of culture. In the
presence of the two inhibitors, no changes were seen in
neurogenesis, in comparison with no TSA or with 10 nM TSA
treatment, respectively (Fig. S4B). Although neither of these
experiments definitively rule out the involvement of Notch or Wnt
signaling in HDAC-mediated neurogenesis, the full complemen-
tation of neurogenesis seen with BMP2/4 inhibitors in combina-
tion with TSA treatment suggests that this is the most important
neurogenic pathway controlled by HDACs.
30.461.1%;Alk-3&TSA:
HDACs inhibit cortical neurogenesis in vitro through a
BMP2/4-dependent pathway
Previous reports have described differing effects of BMP2/4
signaling upon neurogenesis in different parts of the developing
brain, in that these growth factors seem to promote neurogenesis in
embryonic cortex [20,21] but inhibit neurogenesis in GE [14]. In
order to compare the effect of HDAC inhibition upon cultures
prepared from GE, we performed in vitro differentiation upon
precursorcellsderivedfromembryoniccortex.Neurospherecultures
Figure 4. TSA-induced inhibition of neurogenesis from GE-
derived precursors is restored by inhibition of the BMP2/4
signaling pathway. (A) Dissociated neurosphere cultures were plated
out onto coverslips and treated 1.5 days later with either BMP2 (10 ng/
ml), the BMP2/4 inhibitors Alk3-ECD (250 ng/ml) or Noggin (250 ng/ml),
alone or in combination with TSA (10 nM). All growth factors, inhibitors,
and bFGF were withdrawn 24 hours later. Cells were then cultured for
an additional 4.5 days and analyzed by immunofluorescence, staining
with the TuJ1 antibody (red) to detect neurons, and DAPI (green) to
stain cell nuclei. Scale bar=100 mm. (B) RT-PCR analysis of Bmp2 and
Bmp4 expression in dissociated neurosphere cultures. Control PCR
reactions lacking template cDNA are to the right. A 100-bp marker (M)
with an indicated 500-bp band is to the left. (C) The identical cultures
shown in (A) were stained with an anti-GFAP antibody (red) to detect
astrocytes, and DAPI (green) to stain cell nuclei. Panel number 8 shows
a culture from a separate experiment in which TSA was withdrawn at 7
DIV after 5 days of treatment and then cultured for an additional 4 days.
(A, C) The numbers in the lower right corners of the figures correspond
to the numbers beneath the graphs in (D). (D) Mean values +/2 SEM
(n.5). **=p,0.01, Mann-Whitney U test. (E) Dissociated neurospheres
were plated onto coverslips and were treated with BMP2 (10 ng/ml), or
TSA (10 nM or 50 nM) after 1.5 days of in-vitro differentiation, and fixed
after 1, 12, and 24 hours. Cells were stained with an antibody
recognizing phosphorylated-Smad1/5/8. Displayed here are control
conditions (left panels) and treatments (right panels, with indicated
compound and concentrations). Scale bar=50 mm.
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were prepared from C57BL/6J embryonic cortex at 15.5 d.p.c.,
cultured for seven days as floating cell aggregates, dissociated
mechanically, and then differentiated upon coverslips using
conditions identical to those used for GE-derived cultures. 10 nM
TSA was added to the medium upon plating out of dissociated
neurospheres and again after 2.5 days when basic FGF was
withdrawn (Fig. 1A). After an additional 4.5 days cells were fixed
and stained with the TuJ1 antibody to detect b-tubulin III
expression. In contrast to GE-derived cultures, the percentage of
neurons increased from 6.060.9% in untreated cultures to
10.861.9% upon TSA treatment (Fig. 5A), evaluating cell number
by counting DAPI-stained nuclei. In contrast, astrocytic differenti-
ation was reduced in TSA-treated cells, changing from 18.061.3%
in untreated cells to 6.961.4% in TSA-treated cells (Fig. 5B).
In cortical cultures treated with TSA, similar transcriptional
effects upon members of the BMP2/4 signaling pathway were
observed as in cultures derived from GE. In both culture types, 10
and 50 nM TSA treatment resulted in an increase in Bmp2
(Fig. 3C, D), Stat1, Stat3 (Fig. 3G, H), and Ngn1 (Fig. 3I, J), and a
decrease in Smad7 mRNA levels (Fig. 3E, F). In contrast to GE-
derived cultures, all three BMP2/4 receptors showed a reduction
in expression in cortical cultures at 50 nM TSA (Fig. 3B). In order
to determine if BMP2/4 signaling was also involved in the TSA-
mediated changes in neurogenesis and astrogliogenesis in the
cortex, recombinant BMP2 (10 ng/ml) was applied to differenti-
ating cortex-derived cultures one day before the withdrawal of
bFGF. In contrast to GE-derived cultures, BMP2 was able to
promote neurogenesis to the same extent that 50 nM TSA
application had (Fig. 5A). Interestingly, BMP2 treatment did not
lead to a statistically significant increase in astrogliogenesis, in
contrast to the GE-derived cultures (Fig. 5B). In order to test the
involvement of endogenous BMP2/4 signaling, cells were treated
with the extracellular BMP2/4 inhibitors Alk3-ECD and noggin.
Each inhibitor was added for 24 hours before bFGF withdrawal,
with or without 10 nM TSA application, and was removed when
bFGF was withdrawn. The cultures were analyzed 4.5 days later,
at 7 DIV. As was seen in GE-derived cultures, neither Alk3-ECD
nor noggin alone influenced neurogenesis (Fig. 5A) or astro-
gliogenesis (Fig. 5B). However, addition of either of these BMP
inhibitors in combination with TSA restored neurogenesis to levels
seen in the control samples (Fig. 5A). Again, as seen in GE,
astrogliogenesis was also returned to control levels upon treatment
with both TSA and either inhibitor (Fig. 5B).
Bidirectional control of neurogenesis by HDACs in vivo
In order to determine whether the HDAC-mediated modula-
tion of neurogenesis and astrogliogenesis observed in vitro is of
physiological relevance, TSA was employed to inhibit HDACs
during embryogenesis in vivo. Timed-pregnant C57BL/6J female
mice were injected every 12 hours with vehicle (8% ethanol in 16
PBS) or with 12.5 mg TSA in vehicle, an amount of TSA that had
been previously shown to be nontoxic to murine embryos [69].
Injections began at 13.5 days post coitum (d.p.c.), a time point when
neurogenesis has already begun in GE and cortex, and embryos
were examined two days later (Fig. 6A). In order to quantify
neurogenesis, we performed FACS analysis upon dissociated
cortical and GE-derived cells from TSA-injected mice of the
tauGFP line, in which the cDNA encoding EGFP has been inserted
at the tau locus [60]. In this line, EGFP is expressed at high levels
in all neurons shortly after their birth in a time frame similar to
induction of b-tubulin III, and both markers can therefore be used
for the quantification of neurogenesis [70]. As judged by the
fraction of cells that were GFP-positive, neurogenesis in GE was
reduced by 45% (Fig. 6B, D), whereas neurogenesis increased in
the cortex modestly by 10% (Fig. 6C, E). The validity of FACS-
sorting of the cortical and GE-derived cells was subsequently
confirmed. Cells were collected with the same gating conditions
that were used for the determination of the neuronal fraction of
the populations, and the GFP-positive populations were examined
by staining for TuJ1, a standard marker for newborn neurons that
recognizes b tubulin III [71]. In the cortex and GE, over 90% and
80%, respectively, of the cells judged by FACS to be GFP-positive
were found to be TuJ1 positive (Fig. 6F). TuJ1 staining of acutely-
dissociated cortex and GE from embryos exposed to TSA
confirmed the FACS results (data not shown).
We next examined the influence upon telencephalic develop-
ment of HDAC inhibition, using immunohistofluorescence upon
embryos after one or two days of injections starting at 13.5 d.p.c.
Inspection of the entire brain revealed no apparent anatomical
abnormalities. As expected, acetylation of histone H3 increased
strongly in cells throughout the cortex and GE upon TSA
treatment (Fig. 7A), using an antibody that specifically recognizes
acetylated-histone H3. Using TuJ1 as a marker for newborn
neurons, we could clearly observe a decrease in signal in GE,
reflecting the decrease in neurogenesis seen with FACS analysis
(Fig. 7B). Many of the newborn neurons in the medial GE start
migrating tangentially into the cortex, using a route through the
cortical intermediate zone [8]. Using an antibody against GABA
Figure 5. HDAC inhibition by TSA treatment promotes
neurogenesis and inhibits astrogliogenesis in a BMP2/4-
dependent manner in differentiating neural progenitor cul-
tures derived from embryonic cortex. Dissociated neurosphere
cultures derived from 15.5 d.p.c. cortex were plated onto coverslips and
treated 1.5 days later with either BMP2 (10 ng/ml), the BMP2/4
inhibitors Alk3-ECD (250 ng/ml) or Noggin (250 ng/ml), alone or in
combination with TSA (10 nM). All inhibitors and bFGF were withdrawn
24 hours later. Cells were then cultured for an additional 4.5 days and
analyzed by immunofluorescence, using either the TuJ1 antibody to
detect neurons (A) or an anti-GFAP antibody to detect mature
astrocytes (B). DAPI staining of nuclei was used to count cells, and
the percentage of neurons (A) and astrocytes (B) formed is shown.
Mean values +/2 SEM (n=3). *=p,0.05, **=p,0.01, ***=p,0.001,
Mann-Whitney U test.
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to label these migrating inhibitory interneurons, we were clearly
able to see a reduction in staining of both the GE and also the
cortical intermediate and marginal zones in TSA-treated embryos,
reflective of a decrease in neurogenesis in the GE (Fig. 7C). To
quantitate this decrease, we counted the number of GABA-positive
neurons in the ventrolateral cortex (at the boundary with the mantle
zone ofthe lateralGE),whereindividualGABA-positiveneuronsare
easy to identify. We observed a 50% reduction in the numbers of
GABA-positive neurons in TSA-treated cultures, corresponding to
the overall decrease in neurogenesis seen with FACS analysis and
TuJ1 staining (Fig. 7D). This decrease in GE-derived neurons was
not caused by an increase in apoptosis of precursors or newborn
neurons, as we observed very few apoptotic figures in either vehicle-
or TSA-injected GE or the cortex at 15.75 d.p.c, as judged by
immunolabeling of activated caspase 3 (Fig. 8D). As would be
expectedfor the modest increaseincorticalneurogenesisrevealed by
FACS analysis (Fig. 6C, E), no obvious change in TuJ1 staining
could be observed in the cortex (Fig. 7B).
Interestingly, the number of mitotic cells increased both in the
ventricular (VZ) and subventricular zones (SVZ) of GE, but not in
the VZ / SVZ of the cortex (Fig. 8A), as judged by the use of an
antibody recognizing phosphorylated-histone H3, which becomes
phosphorylated specifically during mitosis [72]. In order to
quantify phosphorylated-histone H3-positive cells, the VZ was
delimited with a 30 mm line normal to the ventricle, and all
positive cells within this area were counted and displayed as the
number of cells within this area along the length of the ventricular
surface (per 100 mm, Fig. 8A). SVZ cells were counted as those
cells lying between 30 and 200 mm basal to the ventricular surface,
and displayed as the number of cells within this area (per 104mm2,
Fig. 8A).To identify the cells whose proliferation was increasing as
a result of TSA treatment, markers for radial glial cells were
examined, as radial glia are known to be precursors for both
neurons as well as astrocytes in the developing cortex and GE
[2,3]. An RC2 antibody, which detects neural precursors and
radial glia during embryogenesis [73], revealed a distinct
upregulation upon TSA treatment in both GE and the cortex
(Fig. 8B). Similarly, we observed an increase in the staining
intensity of the transcription factor Pax6 in the cortex of TSA-
treated animals, most prominently in the ventromedial cortex
(Fig. 8C), and an increase in the staining intensity of BLBP,
another marker for radial glia, in both GE and cortex after TSA
treatment (Fig. 8E). In order to test if these changes reflected an
increase in the numbers of radial glia in the TSA-treated embryos,
or just an increase in the expression level in cells already
expressing these markers, we plated out acutely-dissociated cells
from control and TSA-treated embryos and fixed them after
2 hours, followed by a subsequent staining with the corresponding
markers. Here, we clearly saw an increase in the number of cells
staining positive for both RC2 and Pax6 in the cortex (Fig. 8C),
with no change in GE (data not shown).
As had been seen in vitro, a reduction in neurogenesis in GE may
be explained by a precocious astrogliogenesis, which had been
previously seen in a genetic ablation of DNA methyltransferase 1
(Dnmt1) [74]. To examine this, an antibody recognizing S100b, a
marker for newborn astrocytes, was used to examine TSA-treated
Figure 6. HDAC inhibition by TSA in utero restricts GE-derived neurogenesis and promotes cortical neurogenesis. (A) Timed-pregnant
female tauGFP mice were injected every 12 hours with vehicle (B, C, upper diagrams) or with 12.5 mg TSA (B, C, lower diagrams), starting from 13.5
d.p.c., and embryos were examined 54 hours later at 15.75 d.p.c. FACS analysis was performed upon dissociated GE-derived (B) and cortical (C) cells,
using GFP as a marker for newborn neurons. For each treatment and tissue (B, C), a scatter plot showing forward (FSC-H) versus side scatter (SSC-H) is
shown on the left, and a histogram displaying GFP intensity versus the number of events is shown on the right. The population of cells analysed for
green fluorescence is indicated by the gate in the scatter plots (left panel). All cells to the right of the vertical line in the histogram (right panel) were
judged to be GFP-positive and therefore neurons. (D, E) Quantitation of the GFP-positive neuronal population in the developing GE (D) and cortex
(E), treated with vehicle (Control) or with TSA (TSA). Mean values +/2 SEM (n.6). **=p,0.01, ***=p,0.001, Mann-Whitney U test. (F) GFP-positive
cells were collected from embryonic cortex and GE by FACS sorting, plated out, and cultured for 3 hours, and then stained for expression of b-tubulin
III to verify their neuronal identity (n=2).
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embryos. An increase in staining could be observed specifically in
GE (Fig. 9A), which was accompanied by an increase in the
number of S100b-positive cells detected in acutely dissociated
cultures (Fig. 9B). The high background in S100b (7–9%) staining
came from endothelial cells of blood vessels, seen as tubular
structures in Fig. 9A, whereas the increase in staining in GE of
TSA-treated embryos is derived from small punctuate cells with
one or two processes (Fig. 9A, arrows). Whether this change
reflects the birth of newborn astrocytes was difficult to ascertain, as
we could not confirm their identity in vivo with a stain for GFAP, a
marker for more mature astrocytes (data not shown). In order to
see if the in situ environment is prohibitive for the production of
astrocytes at this time point, acutely-dissociated cultures were
made from both GE and cortex of vehicle- and TSA-injected
embryos. The freshly dissociated cultures were treated for 2 days
with either BMP2 (10 ng/ml), LIF (25 ng/ml), or both factors,
both of which have been previously shown to promote
astrogliogenesis in vitro from embryonic brain precursors [12,14].
The cultures were then examined five days later at 7 DIV, using
an anti-GFAP antibody to detect newborn astrocytes. In both
acutely-dissociated GE-derived and cortical cells, both BMP2 and
LIF could promote the production of GFAP-positive astrocytes,
and the combination of the two factors showed an even stronger
effect, as reported previously [12] (Fig. 9C). However, in cultures
derived from GE of TSA-treated embryos, astrogliogenesis was
substantially promoted not only in the presence of LIF and BMP2
but also in their absence (Fig. 9C). In distinct contrast, BMP2 and
LIF-promoted astrogliogenesis was significantly inhibited in
cortical cultures derived from TSA-treated embryos (Fig. 9C).
Therefore, HDAC inhibition showed the same effect upon
astrogliogenesis from acute cultures derived from TSA-injected
embryos as it did upon in vitro-differentiated neurosphere cultures
prepared from these two brain regions.
In order to see if the effects of HDAC inhibition upon
neurogenesis and astrogliogenesis were occurring through a
BMP2/4-dependent pathway, as demonstrated in vitro, the nuclear
localization of phosphorylated Smad1/5/8 was examined in
vehicle- and TSA-treated embryos. In both the cortex (Fig. 10A,
arrows) and GE (Fig. 10B, arrows) of vehicle-treated embryos,
faint staining could be seen in cells of the ventricular zone of the
cortex and GE and the subventricular zone of GE. In contrast,
nuclear localization of phosphorylated Smad1/5/8, as revealed by
DAPI colocalization (data not shown), was increased in these
regions in TSA-treated embryos (Fig. 10A, B, left panels).
Quantitation of the cells with nuclear localization of phosphory-
lated Smad1/5/8 revealed a large increase in these three regions
(Fig. 10C, D, E), using the same counting method as for
phosphorylated-histone H3 (Fig. 8A). Most of the cells responding
to TSA-treatment were also seen to be co-labelled for the neural
precursor marker RC2 (Fig. 10A, B, middle and right panels),
Figure 7. HDAC inhibition by TSA in utero results in a decrease of neurogenesis in the GE and a corresponding reduction in GE-
derived GABA-positive cells undergoing tangential migration in the cortex. Timed-pregnant tauGFP mice were injected every 12 hours
with vehicle (VEH) or with TSA (TSA) starting from 13.5 d.p.c., and embryos were examined at 15.75 d.p.c., using immunohistofluorescence with the
indicated antibodies (upper right). In all panels, dorsal is at the top. Lateral is to the left (A, B, C) or the right (D) of each panel, respectively, and ‘‘V’’
indicates the ventricle. (A) Both cortex (upper portion of figure and inset) and GE (lower portion of figure to the right of the inset) show an increase in
the acetylation of histone H3 upon TSA treatment, as revealed with an anti-acetylated-histone H3 antibody. Inset: red=anti-acetylated-histone H3
antibody, green=DAPI. (B) Using the TuJ1 antibody to detect newborn neurons, the GE (Str) shows a decrease in signal in TSA-treated embryos,
reflecting a decrease in neurogenesis, while the cortex (Crx) looks essentially the same upon TSA treatment. (C) An antibody recognizing GABA labels
GE-derived interneurons migrating tangentially in the intermediate zone of the cortex (Crx, arrow). This migrating population is substantially
decreased in TSA-treated embryos. GABA-positive cells are also substantially decreased in GE (Str). (D) The number of GABA-positive cells (photos left,
inset: red=anti-GABA, green=DAPI) were quantified in the ventrolateral cortex (Crx), reflecting the number of cells per slide in one hemisphere
(graph: Mean values +/2 SEM (n=3). ***=p,0.001, Student’s T-test. Scale bars: (A, C, D) 200 mm, (B) 100 mm, (A: inset) 25 mm, (D: inset) 20 mm.
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Figure 8. HDAC inhibition by TSA in utero results in an increase in proliferation in the ventricular (VZ) and subventricular zones
(SVZ) in embryonic GE, and an increase in the number of radial glia in the cortex. Timed-pregnant tauGFP mice were injected every
12 hours with vehicle (VEH) or with TSA (TSA) starting from 13.5 d.p.c., and embryos were examined at 15.75 d.p.c., using immunohistofluorescence
with the indicated antibodies (upper right). (A) An antibody detecting phosphorylated-histone H3 (green) was used to detect mitotic cells, and TSA
can be seen to increase mitotic figures in the ventricular and subventricular zones of GE (Str, left) but not in the cortex (Crx, right). In order to
quantify phosphorylated-histone H3-positive cells, the VZ was delimited with a 30 mm line normal to the ventricle, and all positive cells within this
area were counted and displayed as the number of cells within this area along the length of the ventricular surface (per 100 mm). SVZ cells were
counted as those cells lying between 30 and 200 mm basal to the ventricular surface, and displayed as the number of cells within this area (per
104mm2). This quantitation is shown (below photos). (B) The RC2 antibody was used to label neural precursors / radial glia, and an increase in signal
can be seen in TSA-treated embryos in both the cortex (Crx) and GE (Str) (middle panels, red). Many of the RC2-positive cells were identified as mitotic
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indicating that neural precursors / radial glia are the cell
population responding to HDAC inhibition by nuclear localization
of the downstream effector of BMP2/4 signaling, phosphorylated
Smad1/5/8.
To see which cells were producing BMP2 and BMP4 in the brain,
in situ hybridization was coupled with immunohistofluorescence for
the radial glial marker BLBP (Fig. 11). As before, 13.5 d.p.c. timed-
pregnant mice were injected every 12 hours with vehicle or with
12.5 mg TSA invehicle, and embryoswere examined 24 hours later.
In vehicle-treated 14.5 d.p.c. embryos, low levels of Bmp2 expression
couldbeseenthroughoutthe cortexandGE(Fig.11A,toppanel).At
the ventricular zone of the cortex and the GE, some BLBP-positive
radial glia were seen to express Bmp2 (Fig. 11A, thin arrows, middle
andbottompanels),butmanyBmp2-expressingcellswerenotlabeled
with the BLBP-antibody, especially in the subventricular zones of
GE and cortex (Fig. 11A, thick arrows, middle and bottom panels).
Upon 24-hour treatment with TSA, a strong up-regulation of Bmp2
could be observed in both GE and cortex (Fig. 11A9, top panel).
Although some of the cells strongly-expressing Bmp2 co-labelled for
BLBP (Fig. 11A9, thin arrows, middle and bottom panels), most of
them did not (Fig. 11A9, thick arrows, middle and bottom panels). In
contrast, the expression pattern of Bmp4 did not appear to change
significantly upon TSA treatment (Cf. Fig. 11B with Fig. 11B9,
labeled as above). Together, these data are in correlation with that
seen in in vitro cultures, in which HDAC inhibition upregulated the
mRNA expression of Bmp2 but not of Bmp4. It also supports the idea
that not only are radial glia expressing BMP2/4, but they are also
responding to these factors, as seen with the nuclear localization of
phosphorylated Smad1/5/8 after HDAC inhibition (Fig. 10).
Discussion
We report here a requirement of HDAC activity for the
generation of neurons from the GE of the embryonic mouse
telencephalon and a modulatory role of HDACs in cortical
neurogenesis. TSA, a potent and specific inhibitor of class I and II
HDACs, was seen in the GE to greatly reduce neurogenesis in vivo
and almost completely block neurogenesis in vitro. In contrast, in
the cortex neurogenesis was actually enhanced upon HDAC
inhibition. We have identified the BMP2/4 signaling pathway as
the target of HDAC activity, both in the GE and the cortex.
During brain development HDACs inhibit BMP2/4 signaling, as
this signaling pathway is upregulated, beginning at the transcrip-
tional level, upon inhibition of HDACs by TSA. We observed that
TSA inhibition of HDAC activity resulted in an increase of histone
H3 and H4 acetylation levels already within one hour of drug
application, suggesting that corresponding changes in gene
expression could take place very quickly, as has been reported
before [62]. In principle, the suppression of BMP signaling could
be accomplished at the transcriptional level in a number of ways,
including the ligands BMP2/4, their receptors, and genes in the
signaling pathway downstream of the BMP ligands, such as the
Smad family members. Indeed, within 12 hours of TSA
application to cortical or GE-derived precursor cultures, we saw
an upregulation in the expression of Bmp2, and a down-regulation
in the inhibitory cytoplasmic factor Smad7. Both of these changes
could contribute to an upregulation in the BMP2/4 signaling
pathway. To prove this point, extracellular inhibition of BMP2/4
activity by noggin or Alk3-ECD restored both neurogenesis and
astrogliogenesis to normal levels in TSA-treated cultures derived
from both cortex and GE. Although noggin can inhibit other BMP
family members, the binding and inhibition of BMP2 and -4 by
Alk3-ECD, the high affinity receptor for these growth factors,
identifies this BMP subfamily as responsible for the reported effects.
Together, the two complementary transcriptional effects in Bmp2
and Smad7 may act in a synergistic fashion to tip the balance from
neurogenesis to astrogliogenesis in the GE, and to further reinforce
neurogenesis in the cortex. We propose that an important role of
HDACs in GE-derived neural progenitor cells is to suppress the
responsiveness of the progenitors to astrocyte fate-promoting signals
from BMP2 and BMP4. Two lines of evidence suggestthat these two
factors are produced by the progenitors themselves. In vitro, the
culturesarehomogenousatthelevelofnestinstainingintheminimal
time period in which TSA exerts its effects, i.e. in the 24 hour period
before bFGF withdrawal (Fig. 4C, D, E). The observed levels of
Bmp2 and Bmp4 gene expression are therefore probably reflective of
production by the nestin-positive progenitors. In vivo, direct
identification of Bmp2- and Bmp4-expressing cells by in situ
hybridization, followed by the labeling of radial glia cells with an
antibody recognizing BLBP, clearly identifies radial glia as a
significant minority of the cells in the ventricular zone of both GE
and cortex that respond to HDAC inhibition by upregulation of
Bmp2 (Fig. 11A, A9).
During mammalian brain development, neural progenitors at
first generate primarily neurons and toward the end of gestation
switch to the production of astrocytes and oligodendrocytes [1].
How do BMP2/4 affect this switch? In our study, a 24-hour
treatment with TSA just before bFGF withdrawal is sufficient to
inhibit neurogenesis in GE-derived precursor cultures (Fig. 6A),
and up to 4 cell divisions could occur between the cell type that is
affected by TSA in this 24-hour period and the generation of
neurons or astrocytes 3–4 days later, based upon a calculated cell-
cycle length of 21 hours (Maya Shake `d, unpublished data; [75]).
The affected cell type could in principle be a bipotential progenitor,
but it is more likely to represent a progenitor such as a radial glial
cell [76], that gives rise to separate neuroblasts and glioblasts, with
BMP2/4 favoring the formation of glioblasts. Indeed, the cell type
shown to respond to an upregulation of Bmp2 transcription in the
developing cortex and GE proved to be almost exclusively RC2-
positive neural precursor cells, as identified by nuclear staining for
phosphorylated Smad1/5/8 (Fig. 10). Interestingly, we saw an
increase in vivo in the cortex in the RC2-positive population, with a
corresponding slight increase in neurogenesis. This would be
consistent with a neurogenic role of these precursors at this
developmental time point. Indeed, it has been shown that ectopic
application of BMP2 or BMP4 to acute telencephalic slices [21] or
dissociated cultures [20] from this time period (14.5 d.p.c. in the
mouse, E16 in the rat) promotes neurogenesis.
Interestingly, after HDAC inhibition in the GE we saw an
opposite effect upon neurogenesis and no such increase in the
radial glial population. In contrast to the cortex, radial glia in the
GE are poised to start generating astrocytic precursors at 14.5–
by phospho-histone H3 staining (left panels, green, arrows), as indicated by an overlap of the two images (right panels, arrows) (C, E) A similar
increase was seen using an antibody recognizing the radial glial markers Pax6 (C, red) or BLBP (E, red). (C, graphs) The percentage of cells expressing
RC2 (left) or Pax6 (right) in the cortex was quantitated by staining acutely dissociated cultures with the respective antibodies, using a nuclear DAPI
stain to evaluate total cell number. (D) An antibody detecting cleaved caspase 3 recognizes very few positive nuclei (red, arrows) in GE of either
vehicle- or TSA-treated embryos. In all panels, dorsal is at the top, lateral is to the left, and ‘‘V’’ marks the ventricle. Scale bars: (A, D, E) 100 mm, (B)
50 mm, (C) 200 mm. (C) Mean values +/2 SEM (n.3). *=p,0.05, **=p,0.01, Mann-Whitney U test.
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15.5 d.p.c., the timeframe of our investigation [2]. The advanced
developmental state of the radial glia in the GE could explain the
relative ease with which HDAC inhibition, leading to an
upregulation of BMP2/4 signaling, results in a switch to an
astrogliogenic program. In contrast, neocortical radial glia are in
the middle of their neurogenic program in the time frame we
investigated. In this context, the strong expression of the
neurogenic factor Ngn1 has been shown to actively inhibit
astrogliogenesis in the neocortex at this time [77], and indeed,
Ngn1 was seen to be upregulated upon HDAC inhibition in
precursor cultures derived from cortical progenitors (Fig. 3J).
However, Ngn1 was also seen to be upregulated by HDAC
inhibition in GE-derived precursor cultures (Fig. 3I), but it clearly
did not show a neurogenic effect upon the cultures, perhaps
explained by the fact that its basal level of transcription is much
lower in the GE than in neocortex. Finally, HDACs have catalytic
activity upon proteins other than histones, including alpha-tubulin
[78], p53, and CBP/p300 [36], so there is also the possibility that
HDACs regulate the acetylation of non-histone proteins important
for BMP signaling. Clearly, other factors must be at work to
explain the difference in response to HDAC inhibition in the
cortex and the GE, and these differences will best be investigated
by performing an unbiased gene expression analysis using
microarray gene chips.
It is unclear why GE precursors do not precociously generate
GFAP+ astrocytes in vivo after HDAC inhibition, as they certainly
do so in precursor cultures derived from the GE (Fig. 5C, D).
Premature astrogliogenesis in vivo has been seen in several mouse
mutants in neurogenic genes [9,79] and for the DNA-methylating
enzyme Dnmt1 [74]. In the Dnmt1 mutant, the astrogliogenic
transcription factors Stat1 and Stat3 are upregulated at the
transcriptional level, a change correlating with a demethylation
of the Stat1 gene promoter. Interestingly, we also observed an
upregulation of both Stat1 and Stat3 in both cortical and GE
cultures upon TSA treatment (Fig. 3G, H), without seeing any
increase in astrogliogenesis in cortical cultures, which could be
explained by the anti-astrogliogenic effect of Ngn1 in cortical
precursors [77]. Similarly, it could be that pharmacological
modulation of histone acetylation levels is not sufficiently strong
to overcome inhibitory signals to astrogliogenesis present in the
embryonic GE, and that these inhibitory signals are either absent
or reduced in in vitro cultures. Indeed, astrogliogenesis was clearly
promoted in dissociated cultures prepared from the GE of
embryos that had been treated with TSA in utero and then treated
with either BMP2, LIF, or both factors together (Fig. 9C).
However, the nature of these signals remains unclear.
Which HDAC is responsible for the control of neurogenesis in
the cortex and GE? Of the five different HDAC inhibitors used in
this study, only VPA, which specifically inhibits class I HDACs at
the concentrations we have used [58], did not show any affect
upon neurogenesis, although it did inhibit the production and
maturation of oligodendrocytes, as reported previously [43]. This
suggests that class II HDACs may be responsible for controlling
neurogenesis in the telencephalon. Of the class II HDACs that we
examined, we detected protein expression of HDAC4, -5, -6, and -
7 (Fig. 4). The expression of HDAC9 has also been reported at the
mRNA level in differentiating neurosphere cultures [80]. As it is
possible that these gene products may perform similar functions in
Figure 9. HDAC inhibition by TSA in utero potentiates
premature astrogliogenesis in embryonic GE. Timed-pregnant
tauGFP mice were injected every 12 hours with vehicle (VEH) or with
TSA (TSA) starting from 13.5 d.p.c., and embryos were examined at
15.75 d.p.c. (A) An anti S100-b antibody (red) demonstrates a
precocious production of immature astrocytes in GE (Str, arrows) but
not the cortex (Crx) of TSA-treated embryos. In all panels, dorsal is at the
top, lateral is to the left, and ‘‘V’’ marks the ventricle. Scale bar: 200 mm.
(B) Quantification performed upon stainings of acutely-dissociated
cultures prepared from GE and cortex of 15.75 d.p.c. embryos indicated
an increase in the number of S100-b-positive cells from GE but not the
cortex. (C) Potentiation of astrogliogenesis in GE was revealed by the
culture of acutely dissociated cells from GE and cortex, respectively, of
15.75 d.p.c. vehicle- and TSA-treated embryos. Dissociated cultures
were treated for 2 days with the astrocyte-promoting factors BMP2
(10 ng/ml) and / or LIF (25 ng/ml). Cultures were fixed after an
additional 5 days of culture and stained for astrogliogenesis with an
antibody recognizing GFAP, using a DAPI stain to evaluate total cell
number. (B, C) Mean values +/2 SEM (n=4). *=p,0.05, ***=p,0.01,
Mann-Whitney U test, p values were all determined by comparison of
control and TSA-treated for any given condition.
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the neural progenitors, identification of the individual HDACs
involved in neurogenesis may be difficult. In this respect,
comparison with myogenesis may prove useful. In the mouse,
individual knock-outs of Hdac4 [48], Hdac5 [50] and Hdac7 [52]
have not been reported to show a disruption in the skeletal
myogenesis program, although all three proteins are clearly
Figure 10. HDAC inhibition by TSA in utero promotes translocation of phosphorylated Smad1/5/8 to the nucleus of neural
precursor / radial glial cells. Timed-pregnant tauGFP mice were injected every 12 hours with vehicle (VEH) or with TSA (TSA) starting from 13.5
d.p.c., and embryos were examined at 15.75 d.p.c., using immunohistofluorescence with the indicated antibodies (upper right). (A, B) An antibody
recognizing phosphorylated-Smad1/5/8 (green, left panels) shows an increase in the nuclear localization of these transcription factors in the
ventricular zone of the cortex (A) and the ventricular and subventricular zone of GE (B) upon treatment with TSA. Amost all of the cells showing
nuclear phosphorylated-Smad1/5/8 were identified as radial glia by positive staining using the neural precursor / radial glial marker RC2 (red, middle
panels, arrows), as indicated by an overlap of the two images (right panels, arrows). In all panels, dorsal is at the top, lateral is to the right, and ‘‘V’’
marks the ventricle. Scale bar: 50 mm. (C, D, E) Quantitation of cells positive for nuclear phosphorylated-Smad1/5/8 in the ventricular zone of the
cortex (C), and the ventricular (D) and subventricular zone (E) of GE, after 2 days of TSA treatment, using a DAPI stain to quantitate cell number. For
the purpose of counting phosphorylated-Smad1/5/8-positive cells in GE and cortex, the VZ and SVZ of the respective regions were defined as above
for the phosphorylated-histone H3 stains (Figure 8A). Mean values +/2 SEM (n=5). ***=p,0.001, Mann-Whitney U test.
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important for the in vitro differentiation of myoblasts to myotubes
[36]. In addition, HDAC5 and HDAC9 have been reported to
play functionally redundant roles in heart development in vivo [50].
Possible redundancy of HDAC function in neurogenesis from
neural stem cells is currently under investigation through
sequential and combinatorial knockdown experiments using
RNA interference. It will also be important, however, to undertake
a closer examination of the various HDAC knockout strains for
defects in nervous system development.
Our results stand in agreement to a previous report [81]
showing that several HDAC inhibitors, including TSA, VPA, and
sodium butyrate, dramatically increase neurogenesis from neuro-
spheres derived from adult rat hippocampus, which develops as an
invagination of the dorsal telencephalon at the same time as the
cortex. We did not see such a large increase in neurogenesis in
embryonic cortex in vivo, but we did observe a doubling of
neurogenesis in vitro. In addition to the difference in species and
developmental stage examined in these two studies, these results
may reflect the different roles that BMPs play in neurogenesis in
various regions of the brain. In the developing cortex, BMP2/4
promote the formation of neurons [21], but in contrast they have
been shown to inhibit neurogenesis in neural stem cells derived
from the subventricular zone of the embryonic GE [14]. Our data
are consistent with an inhibitory role of BMP2/4 upon
Figure 11. HDAC inhibition by TSA in utero promotes an upregulation in the expression of Bmp2 but not Bmp4 in both radial glial
and non-radial glial cells of the embryonic cortex and GE. Timed-pregnant tauGFP mice were injected every 12 hours with vehicle (A, B: VEH)
or with TSA (A9, B9: TSA) starting from 13.5 d.p.c., and embryos were examined at 14.5 d.p.c. using in situ hybridization for mRNA expression of Bmp2
(A, A9: top and left panels) and Bmp4 (B, B9: top and left panels), combined with immunohistofluorescence for the radial glial marker BLBP (A, A9, B,
B9: green, right panels). The top photo in each panel (A, A9, B, B9) shows Bmp2 or Bmp4 expression in the cortex and GE, while the middle and
bottom panels are magnifications of the cortex (Crx) and GE (Str), respectively. Each in situ hybridization (middle and bottom rows, left panel) is
followed by a staining of this section for the radial glial marker BLBP (middle and bottom rows, right panel). Thin arrows indicate Bmp2- (A, A9) or
Bmp4-expressing (B, B9) cells staining positive for BLBP, while thick arrows indicate Bmp2- (A, A9) or Bmp4-expressing (B, B9) cells negative for BLBP.
In all panels, dorsal is at the top and lateral is to the left. Scale bars: (A, A9, B, B9: top row) 200 mm; (A, A9, B, B9: middle and bottom rows) 50 mm.
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neurogenesis in the ventral telencephalon. It is of interest to note
that ablation of hdac1 in the embryonic zebrafish also leads to a
reduction in neurogenesis, both in the retina [46] and in motor
neurons of the spinal cord [47]. In the former case, Hdac1 was
found to suppress both Wnt and Notch signaling. We investigated
both of these pathways and found that neither of them seem to
play a role in the HDAC-mediated promotion of neurogenesis, at
least in GE-derived neural progenitor cells. Whether this reflects a
vertebrate class difference between the two model organisms or
differences in HDAC function between various regions of the
nervous system is unclear.
Conclusions
In this study, HDACs are shown to play an important role in
neurogenesis in the developing GE and a modulatory role in
neocortical neurogenesis. HDAC control of Bmp2 and Smad7
transcription leads to an inhibition of BMP2/4 signaling and
thereby a neurogenic program in GE, and in contrast an inhibition
of neurogenesis in the cortex. Inhibition of class I and II HDACs
with TSA or other HDAC inhibitors causes an increase and a
decrease, respectively, in the expression of Bmp2 and Smad7,
leading to an elevation in BMP2/4 signaling and a switch to an
astrocytic program in the GE and a promotion of neurogenesis in
the cortex. Our study suggests that histone acetylation levels play a
crucial and similar role in the regulation of the transcription of
genes in the BMP signaling pathway in both dorsal and ventral
telencephalon, but that this similar modulation of transcription
results in opposite biological outcomes. The reason for this
difference remains unclear, and therefore future studies will
examine this hypothesis in detail, examining global changes as well
as specific modifications of core histones at the promoters of genes
relevant for BMP2/4 signaling.
Materials and Methods
All reagents were purchased from Sigma-Aldrich (Taufkirchen,
Germany) unless otherwise indicated.
Mouse lines
All animal experiments were conducted in compliance with the
regulations of the state of Baden-Wu ¨rttemberg, Germany. We
employed C57BL/6J and CBA/J mice (Charles River, Sulzfeld,
Germany) and the strain Mapttm1(GFP)Klt[60], which had been
backcrossed to wild-type C57BL/6J mice for more than ten
generations to generate a congenic line that would avoid possible
inconsistencies arising from mixed genetic backgrounds.
Neurosphere culture
We employed mice of the inbred backgrounds C57BL/6J or
CBA/J and the tauGFP mouse line. Neurospheres (NS) were
prepared from the cortex and the lateral and medial GEs of 15.5–
16.0 d.p.c. (Theiler Stage 23/24) embryos, essentially as described
[61], with the addition of bFGF at 10 ng/ml (full protocol
described in [56]). Embryos were dissected on ice in HBSS
(Invitrogen) with 1% HEPES and decapitated. The brain was
removed, the hemispheres separated, and the GE removed with
fine forceps. To prepare neurosphere cultures from the cortex, the
hippocampus and olfactory bulbs were removed from the cerebral
hemispheres, and the remaining cortex was dissected out using
forceps. GE or cortical cells were mechanically dissociated with a
fire-polished (fp) Pasteur pipette and plated out in cell culture flasks
with 100,000 cells per milliliter (ml) in NS Medium (F12/DMEM
(1:1) with B27 supplement (Invitrogen), penicillin/streptomycin
(100 U/ml, Invitrogen), human EGF (20 ng/ml; Sigma) and
human bFGF (10 ng/ml; R&D Systems, Wiesbaden, Germany)).
The NS were incubated in suspension at 37uC, 5% CO2for 1
week and fed on the 5thday with an equal volume of NS medium.
For differentiation, 7 day-old NS were collected into 50-ml tubes
and centrifuged for 3 minutes at 2006 g. The NS were
mechanically dissociated using a fp Pasteur pipette and plated
out with 200,000 cells per well in 12-well plates containing 18-mm
coverslips (pre-plated with 200 mg/ml polyornithine) in NS
medium without EGF and with 1% fetal calf serum (FCS,
Invitrogen), a medium that supports the differentiation of both
neurons and astrocytes. After 2.5 days of incubation the medium
was changed to NS medium without bFGF and EGF but with 1%
FCS. The following pharmacological reagents were added in
different experiments: trichostatin A (Sigma, Calbiochem), val-
proic acid (Sigma, Calbiochem), SAHA (N. Nishino), apicidin
(Sigma), and SBHA (Calbiochem). Cells were fixed in 4% PFA for
30 minutes at 4uC at different time points and then processed for
cytofluorescence.
Immunocytofluorescence
Fixed cells were blocked for 1 hour at room temperature
(Blocking buffer: 0.5% triton X-100, 1% BSA and 5% NGS in 16
PBS). Blocking buffer did not contain triton when O4 antibody
was used. Abs were diluted in blocking buffer, applied overnight,
and washed 4 times in PBS, followed by incubation with
appropriate secondary antibodies, washing, and mounting for
microscopy, as described below for immunohistofluorescence.
Primary Abs were diluted as follows: anti-nestin (Rat-401 clone;
BD Pharmingen) 1:1000, anti-b tubulin III (TuJ1 clone; Covance
and R&D Systems) 1:1500, anti-GFAP (GA5 clone; Sigma and
rabbit polyclonal Z0334; DAKO Cytomation, Hamburg, Ger-
many) 1:500, O4 (kind gift of Prof. J. Trotter, Mannheim,
Germany) 1:100, anti-GFP (kind gift of U. Mu ¨ller) 1:1000, and
anti-phosphorylated Smad1/5/8 (rabbit 9511; Cell Signaling)
1:1000. Proliferation was measured using BrdU labeling (10 mM)
for 24 hours followed by immunohistofluorescence detection using
an anti-BrdU Ab (clone BU33; Sigma) 1:1000. Apoptosis was
measured using ApopTag Red in situ apoptosis detection kit
(Chemicon). Secondary antibodies were used as described below
for immunohistofluorescence.
SAHA synthesis
SAHA was synthesized as described [82].
Recombinant proteins
The recombinant proteins human BMP2 (rhBMP2), human
Noggin (rhNoggin), and the extracellular domain of the human
BMP receptor Alk3 (BMPR1A, residues 24–152) (rhAlk3-ECD)
were expressed as maltose-binding protein (MBP) fusion proteins
in E. coli. The coding sequences were fused in frame to MBP into a
malE fusion vector derived from pMALc2X (New England
Biolabs, Frankfurt, Germany) in which the factor Xa site is
replaced with a 6x-His tag and a thrombin-cleavage site
(LVPRGS) [65]. rhBMP2 was expressed after IPTG induction
of transformed E. coli cultured in a glucose/mineral salt medium.
MBP-BMP2 fusion proteins were purified by amylose affinity
chromatography, followed by in vitro dimerization and final
purification of dimeric rhBMP2 after thrombin cleavage as
described [65,83]. Activity of rhBMP2 was tested by its ability to
induce alkaline phosphatase in the myoblast line C2C12, which is
associated with cellular differentiation [84]. rhNoggin and rhAlk3-
ECD were prepared similarly, with the exception that rhAlk3-
ECD did not require an in vitro dimerization step. The
functionality of rhAlk3-ECD was demonstrated by surface
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