The establishment of neuronal properties is controlled by Sox4 and Sox11

Abstract

The progression of neurogenesis relies on proneural basic helix-loop-helix (bHLH) transcription factors. These factors operate in undifferentiated neural stem cells and induce cell cycle exit and the initiation of a neurogenic program. However, the transient expression of proneural bHLH proteins in neural progenitors indicates that expression of neuronal traits must rely on previously unexplored mechanisms operating downstream from proneural bHLH proteins. Here we show that the HMG-box transcription factors Sox4 and Sox11 are of critical importance, downstream from proneural bHLH proteins, for the establishment of pan-neuronal protein expression. Examination of a neuronal gene promoter reveals that Sox4 and Sox11 exert their functions as transcriptional activators. Interestingly, the capacity of Sox4 and Sox11 to induce the expression of neuronal traits is independent of mechanisms regulating the exit of neural progenitors from the cell cycle. The transcriptional repressor protein REST/NRSF has been demonstrated to block neuronal gene expression in undifferentiated neural cells. We now show that REST/NRSF restricts the expression of Sox4 and Sox11, explaining how REST/NRSF can prevent precocious expression of neuronal proteins. Together, these findings demonstrate a central regulatory role of Sox4 and Sox11 during neuronal maturation and mechanistically separate cell cycle withdrawal from the establishment of neuronal properties.

Full text

The establishment of neuronal properties
is controlled by Sox4 and Sox11
Maria Bergsland, Martin Werme,
1
Michal Malewicz,
1
Thomas Perlmann, and Jonas Muhr
2
Ludwig Institute for Cancer Research, Karolinska Institute, SE-171 77 Stockholm, Sweden
The progression of neurogenesis relies on proneural basic helix–loop–helix (bHLH) transcription factors. These
factors operate in undifferentiated neural stem cells and induce cell cycle exit and the initiation of a
neurogenic program. However, the transient expression of proneural bHLH proteins in neural progenitors
indicates that expression of neuronal traits must rely on previously unexplored mechanisms operating
downstream from proneural bHLH proteins. Here we show that the HMG-box transcription factors Sox4 and
Sox11 are of critical importance, downstream from proneural bHLH proteins, for the establishment of
pan-neuronal protein expression. Examination of a neuronal gene promoter reveals that Sox4 and Sox11 exert
their functions as transcriptional activators. Interestingly, the capacity of Sox4 and Sox11 to induce the
expression of neuronal traits is independent of mechanisms regulating the exit of neural progenitors from the
cell cycle. The transcriptional repressor protein REST/NRSF has been demonstrated to block neuronal gene
expression in undifferentiated neural cells. We now show that REST/NRSF restricts the expression of Sox4
and Sox11, explaining how REST/NRSF can prevent precocious expression of neuronal proteins. Together,
these findings demonstrate a central regulatory role of Sox4 and Sox11 during neuronal maturation and
mechanistically separate cell cycle withdrawal from the establishment of neuronal properties.
[Keywords: Sox proteins; proneural bHLH proteins; REST/NRSF; neurogenesis; development; CNS]
Supplemental material is available at http://www.genesdev.org.
Received July 27, 2006; revised version accepted October 26, 2006.
The progression of vertebrate neurogenesis relies on
mechanisms that in an orderly fashion direct precursor
cells to exit the cell cycle, down-regulate progenitor cell
identities, and to subsequently initiate the expression of
neuronal properties. Despite advances in defining
mechanisms that control the initiation of neurogenesis,
the genetic program that drives the acquisition of the
neuronal phenotype of post-mitotic neurons remains to
be characterized.
Insights into the mechanisms that regulate pan-neu-
ronal gene expression have been derived from studies of
the zinc finger repressor protein REST (RE1 silencing
transcription factor, also known as NRSF) (Chong et al.
1995; Schoenherr and Anderson 1995), which is ubiqui-
tously expressed in neural precursors and has the capac-
ity to bind and repress a large number of genes encoding
neuronal proteins. In contrast, the proneural basic helix–
loop–helix (bHLH) transcription factors, including Ngn1,
Ngn2, and Mash1, function in neural stem cells to ini-
tiate the progression of neurogenesis (Bertrand et al.
2002). While proneural bHLH proteins mediate this
function by committing stem cells to the neuronal lin-
age and by inducing cell cycle exit, their expression is
generally suppressed before progenitor cells exit the pro-
liferative zone and begin to express neuronal properties
(Gradwohl et al. 1996; Fode et al. 2000). Thus, the ability
of proneural proteins to promote the terminal steps of
neurogenesis must rely on downstream regulatory pro-
grams that subsequently establish the expression of neu-
ronal properties in post-mitotic neural cells.
The bHLH genes Math3 and NeuroD (Lee et al. 1995;
Perron et al. 1999) and the non-basic HLH gene Ebf1
(Garcia-Dominguez et al. 2003) are examples of tran-
scription factors that have been suggested to function
downstream from proneural bHLH proteins during the
maturation steps of neurogenesis. Despite the fact that
these proteins can induce ectopic formation of neurons
in Xenopus (Lee et al. 1995; Perron et al. 1999; Garcia-
Dominguez et al. 2003), mice deficient for NeuroD,
Math3,orEbf1 display only minor neurogenic defects
(Naya et al. 1997; Garel et al. 1999; Tomita et al. 2000),
and their role during neurogenesis remains unclear. Fur-
thermore, a substantial number of neurons are generated
prior to the induction of NeuroD expression (Lee et al.
1995; Roztocil et al. 1997). Thus, the molecular mecha-
nism that controls the terminal steps of neurogenesis
and the expression of neuronal properties has not yet
been identified.
The HMG-box transcription factors of the Sox gene
1
These authors contributed equally to this work.
2
Corresponding author.
E-MAIL jonas.muhr@licr.ki.se; FAX 46-8-332812.
Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.403406.
GENES & DEVELOPMENT 20:3475–3486 © 2006 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/06; www.genesdev.org 3475

family have diverse regulatory functions during the for-
mation of the vertebrate CNS (Pevny and Placzek 2005).
Sox1, Sox2, and Sox3, which are expressed by most pre-
cursor cells, act to maintain the expression of progenitor
identities and thus preserve cells in an undifferentiated
state (Bylund et al. 2003; Graham et al. 2003), whereas
another HMG-box protein, Sox21, has the opposite ac-
tivity and allows cells to initiate a differentiation pro-
gram (Sandberg et al. 2005). Hence, B-group Sox proteins
appear to have key regulatory roles in the commitment
of progenitors to neurogenesis. In contrast to Sox1–3 and
Sox21, Sox4 and Sox11, which constitute the C-group of
the Sox gene family (Kamachi et al. 2000), are mainly
expressed in neural cells that have already been commit-
ted to neuronal differentiation (Uwanogho et al. 1995;
Cheung et al. 2000), raising the possibility that these
proteins control later aspects of neurogenesis. Mice, in
which either the function of Sox4 or Sox11 has been
inactivated, do not reveal any significant role of C-group
Sox proteins during neurogenesis (Cheung et al. 2000;
Sock et al. 2004), but structural similarities and the con-
served expression patterns among these proteins indicate
that functional redundancy may compensate for the loss
of an individual Sox4 or Sox11 gene.
In this study, we have examined the role of Sox4 and
Sox11 in the formation of neurons in the vertebrate
CNS. We report that Sox4 and Sox11 operate down-
stream from proneural bHLH proteins and are vital for
the establishment of pan-neuronal protein expression.
Interestingly, misexpression of Sox4 and Sox11 does not
cause progenitor cells to exit the division cycle or com-
mit to a neuronal differentiation program. Instead, Sox4
and Sox11 can induce precocious expression of neuronal
markers in self-renewing precursors. Examination of a
neuronal gene promoter indicates that Sox4 and Sox11
can mediate their functions as transcriptional activators.
Collectively, these findings establish an essential role of
Sox4 and Sox11 in neuronal maturation and separate
mechanistically cell cycle exit and the induction of pan-
neuronal protein expression.
Results
The expression of Sox4 and Sox11 is confined
to post-mitotic differentiating neural cells
To examine the role of group-C Sox proteins during neu-
rogenesis, we defined the expression of Sox11 in the spi-
nal cord of chick embryos from Hamburger-Hamilton
(HH) stages 10–22. At forelimb levels, Sox11 protein
could first be detected at stage 11, which coincides with
the appearance of differentiated neurons expressing the
neuronal marker Tuj1 (Fig. 1A; Moody and Stein 1988).
Between stages 11 and 22, the expression of Sox11 in-
creased significantly (Fig. 1B,C), and at stage 22, the ex-
pression of Sox11 could be detected both medial to and
within the domain of Tuj1
+
neurons (Fig. 1C).
To better characterize how the distribution of Sox11
protein varies during the course of neurogenesis, we next
compared the expression of Sox11 with molecular mark-
ers defining progenitor cells or differentiating neurons.
In the ventricular zone, expression of Sox11 protein was
restricted to post-mitotic cells expressing the neural pro-
genitor marker Sox3 (Fig. 1D,E; Pevny et al. 1998; Bylund
et al. 2003). Furthermore, most of these cells coexpressed
the proneural bHLH transcription factor Ngn2 (Fig. 1F;
Fode et al. 1998), whereas Sox11
+
cells in the intermedi-
ate zone expressed the bHLH protein NeuroM/Math3
(Fig. 1G), a marker for post-mitotic neural cells that are
in the process of down-regulating progenitor markers
and initiating the expression of neuronal genes (Roztocil
et al. 1997). Sox11 protein could also be detected in more
differentiated neurons that had up-regulated expression
of the pan-neuronal marker NF1 (Fig. 1H; Karlsson et al.
1987). Thus, the expression of Sox11 is initiated as post-
mitotic Ngn2
+
cells up-regulate NeuroM/Math3 and is
maintained in differentiating neurons as these start to
express neuronal properties (Fig. 1L). In addition, the
other group-C member, Sox4, was expressed in a similar
pattern as Sox11 (Fig. 1I,J), and in the stage 24 chick
neural tube, their expression could be detected at most
positions along the dorsoventral axis (Fig. 1K). However,
while the distribution of Sox4 and Sox11 mRNAs over-
lapped extensively, the expression of Sox4 became pro-
gressively weaker in differentiated neurons located in
the lateral aspect of the marginal zone (Fig. 1K; Cheung
et al. 2000).
Sox4 and Sox11 can direct the expression
of neuronal properties
As the expression of Sox4/11 is restricted to post-mitotic
differentiating neurons, it is possible that Sox4/11 have
regulatory roles during the terminal steps of neurogen-
esis. To begin to address this issue, we examined the
expression of pan-neuronal markers after misexpression
of Sox11 in the ventricular zone. Misexpression of Sox11
for 24 h resulted in strong ectopic expression of the neu-
ronal proteins Tuj1 and MAP2 (Fig. 2A–C,W; Riederer
and Matus 1985). However, no premature or ectopic ex-
pression could be detected of the neuronal markers NF1
and SCG10 (Fig. 2D,E,W; Curmi et al. 1997). Sox4 had
similar activity as Sox11 and caused transfected cells to
up-regulate the expression of Tuj1 and MAP2 but not
that of NF1 or SCG10 (Supplementary Fig. 1; data not
shown). Thus, Sox4 and Sox11 have redundant functions
and can promote precursor cells to up-regulate the ex-
pression of pan-neuronal markers.
The induced expression of neuronal proteins in Sox4/
11-electroporated cells could either reflect that these
cells have completed a neurogenic program prematurely
or that the transfected progenitor cells have bypassed the
initial steps of neurogenesis and up-regulated the expres-
sion of neuronal markers in an ectopic fashion. To ad-
dress these different possibilities, we next analyzed
Sox11-electroporated cells with markers discriminating
between self-renewing progenitors and cells that have
initiated a neurogenic program. Transfection of Sox11
for 24 h did not induce any significant alteration in the
expression of the bHLH proteins Ngn2 or NeuroM/
Bergsland et al.
3476 GENES & DEVELOPMENT

Math3 (Fig. 2F,G), markers characteristic of cells that
have initiated a neurogenic program. Furthermore, many
electroporated cells expressed the progenitor marker
Sox3 (Fig. 2H,K,X), and the number of transfected cells
that were in a self-renewing state was comparable to
progenitor cells electroporated with a GFP-expressing
control vector (Fig. 2I,J,L,X). Comparable results were
obtained with Sox4 (data not shown). Thus, misexpres-
sion of Sox4 and Sox11 does not promote progenitor cells
to exit the division cycle or initiate a neuronal differen-
tiation program. Instead, a high incidence of the ectopic
Tuj1
+
cells coexpressed the progenitor marker Sox3 (Fig.
2M,P,X), and several of these cells were also in a self-
renewing state (Fig. 2N,O,Q,X). Together, these results
indicate that Sox4/11 function during the terminal steps
of neurogenesis and can activate the expression of neu-
ronal traits independently of mechanisms directing the
exit of progenitors from the cell cycle.
Sox11-electroporated neural tubes, which were al-
lowed to develop for 48 h instead of 24 h, contained
many transfected cells that were post-mitotic, located in
the marginal zone, and expressed all pan-neuronal mark-
ers examined, including NeuN and NF1 (Fig. 2R–T). In
line with this, Sox11-transfected cells located in the
marginal zone also expressed the interneuron marker
Lim2 and the motor neuron marker Isl1/2 (Tsuchida et
al. 1994) in a normal dorsoventral pattern (Fig. 2U,V).
Thus, although misexpression of Sox4 and Sox11 causes
self-renewing progenitor cells to up-regulate premature
ectopic expression of neuronal properties, the trans-
fected cells are still capable of undergoing the normal
pathway of neurogenesis and establishing a complete
neuronal phenotype.
The establishment of pan-neuronal gene expression
requires Sox4 and Sox11 function
To further examine the role of group-C Sox proteins dur-
ing the formation of neurons, we blocked the expression
of Sox4 and Sox11 using RNA interference (siRNA)
(Grishok and Mello 2002). Electroporation of siRNAs
(Rao et al. 2004) directed against chick Sox4 or Sox11
(␣-Sox4 and ␣-Sox11) decreased the level of Sox4 and
Sox11 mRNA transcripts, whereas scrambled control
Figure 1. Expression of Sox4 and Sox11 in the
developing chick spinal cord. (A–C) Expression
of Sox11 and Tuj1 protein in spinal cords from
HH stages 11 to 22. (D–H) Expression of Sox11 in
comparison with Sox3
+
(D), BrdU
+
(E), Ngn2
+
(F),
NeuroM/Math3
+
(G), and NF1
+
(H) cells. The
white box in Dindicates the analyzed area in
E–H.(I–K) Expression of Sox4 (I) and Sox11 (J)
mRNA. (K) The image represents an overlay of
images Iand J.(L) The summary figure indicates
the distribution of Sox1–3, Ngn2, NeuroM/
Math3, Sox4, and Sox11 and neuronal proteins in
the developing neural tube. Bars: A,B,40µm;
C,D, 60 µm;, E–H), 15 µm; I–K, 100 µm.
Neuronal properties and Sox4/11
GENES & DEVELOPMENT 3477

versions of ␣-Sox4 and ␣-Sox11 had no effect on the ex-
pression of Sox4 or Sox11 (Supplementary Fig. 2). Co-
transfection of ␣-Sox4 together with ␣-Sox11 resulted in
a simultaneous reduction of both Sox4 and Sox11 mRNA
transcripts and decreased the number of cells expressing
Sox11 protein (Fig. 3A; Supplementary Fig. 2). Notably,
45 h after ␣-Sox4 and ␣-Sox11 coelectroporation, the
number of cells expressing the neuronal markers Tuj1,
NF1, SCG10, Lim2, and Isl1/2 was significantly reduced
(Fig. 3B–E,V), suggesting that Sox4/11 function is re-
quired for the formation of cells expressing neuronal
properties. In accordance with this, the presence of co-
Figure 2. Sox4 and Sox11 can induce the expression of neuronal proteins. (A–E,W) Misexpression of Sox11 for 24 h (A) increased the
number of Tuj1
+
(B,W) and MAP2
+
cells (C,W), whereas no change in the expression levels of NF1 (D,W)orSCG10 (E) could be detected
(nⱖ6 embryos; [*] p< 0.05; [**] p< 0.01; [***] p< 0.001). (F,G) Electroporation of Sox11 did not alter the expression of the proneural
bHLH protein Ngn2 (F) or the bHLH protein NeuroM/Math3 (G). (H–L,X) Many Sox11-transfected cells expressed the progenitor
marker Sox3 (H,X), and the number of Sox11-transfected cells that incorporated BrdU (I,X) or expressed the cell cycle marker PCNA
(J) was comparable with cells electroporated with a GFP control vector (K,L,X)(nⱖ6 embryos; [***] p< 0.001). (M–Q,X) Most of the
ectopic Tuj1
+
cells coexpressed Sox3 (M,X), and several of these also incorporated BrdU (N,X) and expressed PCNA (O). No or very few
Tuj1
+
/Sox3
+
-incorporating cells (P,X) or Tuj1
+
/BrdU-incorporating cells (Q,X) could be detected in embryos electroporated with a GFP
control vector. (R–T) Forty-eight hours after Sox11 transfection (R), many of the electroporated cells were post-mitotic and also
expressed NeuN (S) and NF1 (T). (U,V) Misexpression of Sox11 for 48 h (U) did not disrupt the expression pattern of the subtype-specific
neuronal markers Lim2 and Isl1 (V). Results are represented as mean ± SD. Bars: A–E,H–Q,R,U,V, 60 µm; F,G, 75 µm; S,T,40µm.
Bergsland et al.
3478 GENES & DEVELOPMENT

transfected vectors, expressing high levels of Sox11, res-
cued the generation of neurons in ␣-Sox4- and ␣-Sox11-
electroporated neural tubes (Fig. 3F–J,V).
Next we determined at which step during neurogen-
esis a reduction in Sox4 and Sox11 expression obstructs
the establishment of neuronal properties. Transfection
of ␣-Sox4 and ␣-Sox11 did not affect the general expres-
sion level of the progenitor marker Sox3 or the propor-
tion of cells incorporating BrdU (Fig. 3K,L,V). Further-
more, the expression of the proneural protein Ngn2 was
comparable with the nontransfected control side (Fig.
3M,V). However, although a fraction of the transfected
cells expressed the post-mitotic marker p27
Kip1
(Fig. 3N;
El Wakil et al. 2006) and NeuroM/Math3 (Fig. 3O), their
expression was decreased by 61% and 65%, respectively
(Fig. 3U,V), a reduction that could be rescued by the pres-
ence of cotransfected Sox11 (Fig. 3P,U,V).
NeuroM/Math3 is expressed during the transition pe-
riod when differentiating cells are in the process of sup-
pressing progenitor specific markers and initiating the
expression of neuronal proteins. In the nontransfected
control side of the neural tube, 27% of the NeuroM/
Figure 3. Decreased levels of Sox4 and Sox11 block the establishment of a neuronal phenotype. (A–E,V) Forty-five hours after siRNA
transfection, the expression of Sox11 (A), Tuj1 (B,V), NF1 (C), SCG10 (D), and Lim2 and Isl1 (E) was considerably reduced compared
with the nontransfected control side (nⱖ6 embryos; [**] p< 0.01; [***] p< 0.001). (F–J,V) The generation of Tuj1
+
(G,V), NF1
+
(H),
SCG10
+
(I), and Lim2
+
and Isl1
+
(J) neurons could be rescued in
␣
-Sox4- and
␣
-Sox11-electroporated neural tubes by the presence of
Sox11 expression vectors (F). (K–M,V) Transfection of
␣
-Sox4 and
␣
-Sox11 siRNAs did not alter the expression of Sox3 (K), the rate of
BrdU incorporation (L,V), or the expression of Ngn2 (M,V). (N–P,U,V)
␣
-Sox4 and
␣
-Sox11 siRNAs reduced the number of p27
Kip1+
and
NeuroM/Math3
+
cells (N,O,U,V), a reduction that could be rescued by the presence of cotransfected Sox11 expression vectors (P,U,V)
(nⱖ6 embryos; [**] p< 0.01; [***] p< 0.001). (Q–U)In
␣
-Sox4 and
␣
-Sox11 siRNA-transfected neural tubes, most NeuroM/Math3
+
cells coexpressed the progenitor marker Sox3 (Q,U) but had failed to up-regulate NF1 expression (R,U). In contrast, in the nonelec-
troporated control side, 27% of the NeuroM/Math3
+
cells coexpressed Sox3 (S,U) and >70% coexpressed NF1 (T,U). Results are
represented as mean ± SEM. Bars: A–J, 60 µm; K–P,90µm;Q–T, 120 µm.
Neuronal properties and Sox4/11
GENES & DEVELOPMENT 3479

Math3
+
cells coexpressed the progenitor marker Sox3,
and 70% of the NeuroM/Math3
+
cells coexpressed the
neuronal marker NF1 (Fig. 3S–U). In the siRNA-treated
side of the neural tube, most of the remaining NeuroM/
Math3
+
cells coexpressed Sox3 (Fig. 3Q,U), and very few
NeuroM/Math3
+
cells that expressed NF1 could be de-
tected (Fig. 3R,U). Thus, decreased levels of Sox4/11 ex-
pression appear to block the differentiation step at which
post-mitotic NeuroM/Math3
+
cells up-regulate the ex-
pression of neuronal properties. Notably, in the siRNA-
treated neural tubes, but not in embryos coelectropor-
ated with siRNAs together with a Sox11 expression vec-
tor, we could detect a small increase in the number of
apoptotic cells (Supplementary Fig. 3). Thus, one possi-
bility is that the failure of NeuroM/Math3
+
cells to up-
regulate the expression of neuronal proteins results in
cell degeneration. Furthermore, although the expression
of Sox4/11 and NeuroM/Math3 appears to be initiated
simultaneously (Fig. 1G), we cannot rule out the possi-
bility that the reduction of NeuroM/Math3
+
cells is
reflecting an additional role of Sox4/11 upstream of
NeuroM/Math3 expression.
Repression of Sox11 downstream targets blocks
the expression of neuronal properties
In vitro studies have revealed that group-C Sox proteins
can function as transcriptional activators (van de Weter-
ing et al. 1993; Kuhlbrodt et al. 1998; Schmidt et al. 2003;
Wiebe et al. 2003), an activity that has been mapped to
their C-terminal regions (Hargrave et al. 1997; Kuhlbrodt
et al. 1998; Wegner 1999). To further determine the
mechanism by which group-C Sox proteins direct the
expression of neuronal characters, we generated obligate
repressor or activator variants of Sox11. Sox11 cDNA,
lacking the C-terminal putative activation domain, was
either fused to the transactivation domain of the cDNA
encoding the viral protein VP16 (Sox11
⌬C-term
–VP16)
(Berk et al. 1998) or to the transcriptional repressor do-
main of the Drosophila Engrailed gene (Sox11
⌬C-term
–EnR)
(Fig. 4A; Smith and Jaynes 1996). Misexpression of
Sox11
⌬C-term
–VP16 mimicked the activity of full-length
Sox11 and induced ectopic expression of Tuj1 and MAP2
but not the expression of NF1 or SCG10 (Fig. 4B–F; data
not shown). In contrast, the repressor form of Sox11,
Sox11
⌬C-term
–EnR, had the opposite activity and sup-
pressed the endogenous expression of both Tuj1 and
MAP2 (Fig. 4B,G–I). In addition, Sox11
⌬C-term
–EnR also
blocked the expression of NF1 and SCG10 (Fig. 4B,J; data
not shown). Thus, active repression of Sox11 target genes
perturbs the induction of neuronal properties. Together,
these results indicate that the ability of Sox11 to direct
the expression of neuronal traits relies on its function as
a transcriptional activator.
Sox4 and Sox11 can function as transcriptional
activators of a neuronal gene promoter
To further examine the mechanism by which group-C
Sox proteins control neuronal gene expression, we fo-
cused on the upstream 622-base-pair (bp) non-coding re-
gion of the mouse Tubb3 gene (Tubb3
622
; also known as
classIII ␤-tubulin or Tuj1) (Fig. 5A), as this DNA segment
is sufficient to drive the expression of a reporter gene in
EC cells during neuronal differentiation (Dennis et al.
2002). To determine whether group-C Sox proteins func-
tionally interact with this genomic element, COS1 cells
were transfected with expression constructs encoding
Sox4 and Sox11 proteins, together with a LacZ reporter
containing the isolated Tubb3 genomic fragment and a
minimal ␤-globin promoter (Tubb3
622
-LacZ) (Fig. 5A).
Sox4 and Sox11 displayed similar activities in this sys-
tem and activated the Tubb3
622
-LacZ reporter nearly
ninefold (Fig. 5B). Notably, examination of the Tubb3
622
sequence revealed that three potential binding sites for
Sox4 and Sox11 (van Beest et al. 2000) were located
within a domain −91 to −207 bp upstream of the Tubb3
transcriptional start site (Tubb3
166
) (Fig. 5A). In a DNA-
binding gel shift assay, both recombinant Sox4 and
Sox11 proteins interacted with these sites (Fig. 5C) in a
sequence-specific manner (Fig. 5C; Supplementary Fig.
4). In line with these findings, the Tubb3
166
-LacZ re-
Figure 4. Active repression of Sox11 target genes prevents the
expression of pan-neuronal properties. (A) The constructs used
were as follows: The C-terminal part of Sox11 was replaced with
the transactivation domain of VP16 (Sox11
⌬C-term
–VP16), or
with the repressor domain of the Drosophila Engrailed gene
(Sox11
⌬C-term
–EnR). (B–F) Sox11
⌬C-term
–VP16 (C) behaved like
full-length Sox11 and induced the expression of Tuj1 (B,D) and
MAP2 (B,E) but not that of NF1 (B,F). (B,G–J) Forced expression
of Sox11
⌬C-term
–EnR (G) prevented neuronal protein expression,
including Tuj1 (B,H), MAP2 (B,I), and NF1 (B,J)(nⱖ6 embryos;
[**] p< 0.01; [***] p< 0.001). Results are represented as
mean ± SD. Bars: C–F,60µm;G–J,80µm.
Bergsland et al.
3480 GENES & DEVELOPMENT

porter (Fig. 5A) could be activated by Sox4 and Sox11 in
a dose-dependent manner (Fig. 5B), and mutations of
each individual Sox4/11-binding site reduced the trans-
activation of the Tubb3
166
-LacZ reporter more than six-
fold (Fig. 5D). Moreover, Ngn2 or Sox3, which have been
demonstrated to induce and repress Tuj1 expression, re-
spectively (Novitch et al. 2001; Bylund et al. 2003; Gra-
ham et al. 2003), could not regulate the Tubb3
166
-LacZ
reporter through the defined Sox4/11-binding sites
(Supplementary Fig. 5). Hence, using Tubb3 as an ex-
ample, this set of experiments indicates that Sox4 and
Sox11 can function as transcriptional activators of a neu-
ronal gene promoter.
REST and proneural proteins regulate the expression
of Sox4 and Sox11
Proneural bHLH proteins can promote the generation of
neurons from precursor cells (Sun et al. 2001; Lo et al.
2002). However, as the expression of proneural proteins
is suppressed before neural progenitors exit the prolifera-
tive zone (Gradwohl et al. 1996; Fode et al. 2000), their
ability to induce the expression of pan-neuronal markers
must rely on downstream transcriptional programs.
Since the expression of Sox11 is initiated in differentiat-
ing Ngn2
+
cells (Fig. 1F), the expression of group-C Sox
genes may be controlled by proneural protein activity.
To examine this possibility, we misexpressed either
Ngn2 or Id2 in the developing chick neural tube. Indeed,
electroporation of Ngn2 for 10 h induced high levels of
Sox11 expression (Fig. 6A) before transfected cells had
up-regulated high levels of Tuj1 (Fig. 6B,C). Comparable
results were obtained with another proneural gene,
Ascl1 (also known as Mash1) (Guillemot and Joyner
1993; data not shown). Id2, which functions as a passive
repressor of proneural protein activity (Yokota 2001), had
the opposite function compared with Ngn2 and de-
creased the level of Sox11 gene expression 48 h after
electroporation (Fig. 6D). In addition, similar to Sox11,
the expression of Sox4 was induced by Ngn2 and re-
pressed by Id2 activity (data not shown). Thus, the in-
duction of Sox4/11 expression in differentiating neural
cells appears to depend on proneural protein activity.
Since the expression of group-C Sox genes is regulated
by proneural proteins, we next examined whether the
ability of Ngn2 to direct the establishment of a neuronal
phenotype is dependent on the activity of Sox4/11. To
address this issue, Ngn2 was misexpressed either alone
or together with coelectroporated ␣-Sox4 and ␣-Sox11
siRNAs. Twenty-four hours after Ngn2 electroporation,
a majority of the transfected cells had down-regulated
the expression of Sox3 and up-regulated the expression of
Sox11 (Fig. 6E,F,U). At this stage, many of the cells had
exited the division cycle and up-regulated the expression
of p27
Kip1
(Fig. 6G,H,U) and the definitive neuronal
markers Tuj1 and NF1 (Fig. 6I,J,U). The presence of
␣-Sox4 and ␣-Sox11 siRNAs reduced the ability of Ngn2
to suppress the expression of Sox3 (Fig. 6K,U) and
blocked the establishment of Sox11 protein expression
(Fig. 6L). However, a high proportion of the electropor-
ated cells were post-mitotic, and many cells had also
up-regulated the expression of p27
Kip1
(Fig. 6M,N,U). In
contrast, only few cells coelectroporated with Ngn2 and
␣-Sox4 and ␣-Sox11 siRNAs had induced the expression
of Tuj1 and NF1 (Fig. 6O,P,U). Thus, in the absence of
Sox4/11 expression, Ngn2 can force cells to exit the di-
vision cycle but is unable to direct cells to up-regulate
the expression of neuronal markers.
Neuronal gene expression is subjected to negative
regulation by REST (Chong et al. 1995; Schoenherr and
Figure 5. Sox4 and Sox11 interact with
promoter elements of the neuronal gene
Tubb3. (A)Tubb3 promoter constructs
used in transactivation studies. S1–S3 in-
dicate potential Sox4- and Sox11-binding
sites, S1 (TTCTATTGTCCCC), S2, and
S3 (CCGCATTGTGCGG). X marked in
red indicates mutated sites. (B) Transacti-
vation of the Tubb3
166
-LacZ and Tubb3
622
-
LacZ reporters by Sox4 or Sox11. (C) Re-
combinant Sox4 and Sox11 proteins were
able to bind to S1, S2, and S3 (S23) in a
DNA-binding gel shift assay. No binding
could be detected when these sites were
mutated: S1mut (TTCTCCCGTCCCC)
and S23mut (CCGCGGGGTGCGG). (D)
The ability of Sox4 and Sox11 to transac-
tivate the Tubb3
166
-LacZ reporter con-
struct was reduced more than sixfold
when S1 and S23 were mutated (S1M and
S23M). The error bars in Band Dindicate
the standard deviation (SD) of three inde-
pendent transfections. Each experiment
was repeated six times. (**) p< 0.01; (***)
p< 0.001.
Neuronal properties and Sox4/11
GENES & DEVELOPMENT 3481

Anderson 1995), a transcriptional repressor protein ex-
pressed in the ventricular zone. REST has been demon-
strated to exert its function through a direct repressive
interaction with neuronal gene enhancers. However, it
has not been addressed whether REST also represses the
expression of genes encoding activators of neuronal gene
expression. To examine this possibility, chick embryos
were transfected with full-length REST and analyzed for
the expression of Sox4 and Sox11. Indeed, 48 h after
REST overexpression the number of cells expressing
Sox4 and Sox11 was significantly reduced (Fig. 6Q; data
not shown). Moreover, studies in both chick and mouse
embryos indicate that loss of REST function leads to
derepression of neuronal markers within the ventricular
zone (Chen et al. 1998). To examine whether the loss of
REST activity also is followed by a derepression of Sox4/
11, we next misexpressed a dominant-negative form of
REST (dnREST) (Chen et al. 1998). Twenty hours after
dnREST transfection, many electroporated cells had ini-
tiated the expression of Sox4/11 (Fig. 6R; data not
shown), and this was followed by an up-regulation of
neuronal markers (Fig. 6S; data not shown). Notably, the
Figure 6. The expression of Sox4 and
Sox11 is controlled by REST and proneural
proteins. (A–C) Electroporation of Ngn2 in-
duced high levels of ectopic Sox11 expres-
sion already 10 h after transfection (A),
whereas the expression of Tuj1 first could
be readily detected 24 h after electropora-
tion (B,C). (D) Electroporation of Id2 for 48
h reduced the expression of Sox11.(E–J,U)
Overexpression of Ngn2 for 24 h promoted
cells to suppress progenitor characters
(E,G,U) and up-regulate the expression of
Sox11 (F), p27
Kip1
(H,U), and neuronal pro-
teins (I,J,U)(nⱖ6 embryos; [*] p< 0.05;
[***] p< 0.001). (K–P,U) Cells cotrans-
fected with Ngn2 and ␣-Sox4 and ␣-Sox11
siRNAs for 24 h expressed reduced levels of
Sox3 (K,U) and had exited the cell cycle
(M,U) and up-regulated p27
Kip1
(N,U). Co-
transfected cells did not up-regulate the ex-
pression of Sox11 (L) or neuronal markers
(O,P,U). (Q–T) Misexpression of REST for
48 h decreased the expression of Sox11 (Q)
(48% ± 11% reduction; [***] p< 0.001),
whereas a dominant-negative version of
REST, dnREST, 20 h after electroporation
had induced high levels of ectopic Sox11 (R)
and Tuj1 expression (S). (T)dnREST-trans-
fected cells could not up-regulate the ex-
pression of Tuj1 when the expression of
Sox4 and Sox11 was prevented by siRNAs.
Results are represented as mean ± SD. Bars:
A–C,R–T,60µm;E–P,80µm;D,Q,50µm.
(V) Proposed molecular network regulating
the establishment of pan-neuronal proper-
ties. Proneural bHLH proteins drive the ini-
tial steps of neurogenesis and direct the
exit of neural cells from the division cycle.
Proneural proteins also induce the expres-
sion of Sox4 and Sox11, which in turn ac-
tivate neuronal gene expression. According
to this model, proneural proteins induce
the expression of an additional genetic pro-
gram (designated X) that together with
Sox4 and Sox11 activates a complete neu-
ronal phenotype in differentiating neurons.
REST/NRSF prevents precocious expres-
sion of neuronal proteins in undifferenti-
ated neural cells both by a direct repression
of neuronal genes and by restricting the ex-
pression of Sox4 and Sox11 to neural cells
that have exited the cell cycle.
Bergsland et al.
3482 GENES & DEVELOPMENT

expression level of the proneural genes Ngn2 and Cash1
was not altered by misexpressed full-length REST or
dnREST (Supplementary Fig. 6), and transfected progeni-
tor cells responded similarly to dnREST as to Sox4/11
and up-regulated only a partial array of neuronal mark-
ers. Hence, dnREST-transfected cells up-regulated the
expression of Tuj1 and MAP2 but not that of NF1 or
NeuN (data not shown). In addition, the ability of elec-
troporated dnREST to derepress neuronal marker expres-
sion was blocked when the accompanying up-regulation
of Sox4/11 expression was prevented by coelectroporated
␣-Sox4 and ␣-Sox11 (Fig. 6T). Thus, the ability of REST
to suppress neuronal gene expression appears, at least in
part, to be dependent on its capacity to restrict the ex-
pression of Sox4/11 (Fig. 6V).
Discussion
Proneural bHLH transcription factors are essential for
the progression of neurogenesis and can induce cell cycle
exit and commit progenitors to a neurogenic program
(Farah et al. 2000; Bertrand et al. 2002; Kintner 2002; Lo
et al. 2002), but how these proteins promote differenti-
ated progeny to obtain a neuronal phenotype has re-
mained elusive. We have shown here that Sox4 and
Sox11 function downstream from proneural bHLH pro-
tein as critical activators of both generic and subtype-
specific neuronal properties. Elimination of Sox4 and
Sox11 activity did not disrupt the ability of proneural
bHLH proteins to promote cell cycle exit, but blocked
their capacity to establish the expression of neuronal
properties. Together, these data reveal a central regula-
tory role of group-C Sox proteins during neuronal matu-
ration and suggest that the induction of Sox4 and Sox11
expression reflects a critical step in the acquisition of a
neuronal phenotype.
The bHLH proteins Math3 and NeuroD represent ex-
amples of transcription factors that are expressed in post-
mitotic neurons, downstream from proneural proteins.
Mice deficient for Math3 or NeuroD display only minor
neurogenic defects (Lee et al. 1995; Naya et al. 1997;
Tomita et al. 2000). However, misexpression of these
proteins in Xenopus embryos can convert epiblast and
neural precursor cells into differentiated neurons (Lee et
al. 1995; Perron et al. 1999). Hence, even if Math3 and
NeuroD are expressed exclusively in post-mitotic neural
cells, these factors appear to have the capacity to induce
progenitor cells to complete a neurogenic program. In
contrast, Sox4/11 could induce self-renewing precursor
cells to up-regulate the expression of neuronal markers,
but misexpression of these proteins did not promote
cells to exit the cell cycle or suppress progenitor specific
gene expression. Thus, the activity of Math3 and
NeuroD differs from that of Sox4/11 and more resembles
the function of proneural bHLH proteins. These obser-
vations also establish that the ability of Sox4/11 to in-
duce the expression of neuronal proteins can be func-
tionally separated from mechanisms whereby proneural
bHLH proteins promote the initial steps of neurogenesis.
Apart from Math3 and NeuroD, additional basic and
non-basic HLH proteins are expressed in differentiating
neurons, downstream from proneural proteins (Bertrand
et al. 2002). While the activity of Sox4/11 is distinct from
that of Math3 and NeuroD, our results do not argue
against the possibility that other members of the HLH
transcription factor family are required, together with
Sox4/11, for the induction of neuronal traits in post-mi-
totic neurons (Fig. 6V).
Although Sox4/11 appear to be essential for neuronal
protein expression, they could only induce a partial array
of neuronal markers in self-renewing progenitors. The
inductive capacity of Sox4/11 was not, however, depen-
dent on the proliferative status of the transfected pro-
genitor cells. For instance, Sox11 induced the same set of
neuronal markers in cells that were in a self-renewing
state, as in cells that had been forced to exit the division
cycle by the CDK inhibitor p27
Kip1
(Supplementary Fig.
7). Another possibility, which may explain why Sox4/
11 could induce only a subset of neuronal markers at
ectopic locations, is that neuronal genes may be sub-
jected to active repression in progenitor cells. Thus, a
relief of such repressive program may be a prerequisite
for the establishment of a full neuronal phenotype. Fi-
nally, Sox proteins have been reported to act in concert
with heterodimerizing partner factors (Kamachi et al.
2000). Hence, it is possible, and even likely, that Sox4/11
normally act in synergy with other factors during neu-
ronal maturation and that the absence of such partner
factor(s) in undifferentiated progenitor cells limits the
inductive capacity of Sox4/11 (Fig. 6V).
We found that the expression of Sox4/11 is directed to
post-mitotic neural cells by a combinatorial function of
proneural proteins and REST/NRSF. The expression of
Sox4/11 was induced by proneural proteins and sup-
pressed by Id2, an inhibitor of proneural protein function
(Yokota 2001). Moreover, full-length REST/NRSF re-
pressed the expression of Sox4/11, whereas its dominant-
negative form (dnREST) induced high levels of ectopic
Sox4/11 expression, followed by an up-regulation of neu-
ronal markers (Chen et al. 1998). Interestingly, in these
experiments, the capacity of dnREST to activate the ex-
pression of neuronal proteins was dependent on Sox4/11
expression and was limited to neuronal markers that
could be ectopically induced by Sox4/11 misexpression
(e.g., Tuj1 and MAP2 but not NF1 or NeuN). Hence, in
addition to its ability to directly repress neuronal gene
enhancers (Lunyak et al. 2002), REST/NRSF also appears
to prevent precocious expression of neuronal proteins in
undifferentiated neural cells through its capacity to sup-
press the expression of Sox4/11 (Fig. 6V).
The expression of group-C Sox genes is not restricted
to the developing CNS but can be detected in cells from
several distinct origins (Uwanogho et al. 1995; de Mar-
tino et al. 2000; Maschhoff et al. 2003; Sock et al. 2004).
For instance, Sox11 has been demonstrated to activate
the muscle differentiation gene myogenin in C2C12
myoblasts (Schmidt et al. 2003). Furthermore, in ze-
brafish, one of the two Sox4 orthologs (Sox4b) is neces-
sary for pancreatic endocrine cell differentiation and the
expression of the hormone glucagon (Mavropoulos et al.
Neuronal properties and Sox4/11
GENES & DEVELOPMENT 3483

2005). Thus, as in the developing CNS, group-C Sox pro-
teins can drive the expression of differentiation proteins
both in mesodermal and endodermal cells. These find-
ings suggest that the selection of downstream genes con-
trolled by Sox4/11 ultimately depends on the linage from
which the progenitor cells are originating.
Sox proteins have several regulatory functions during
neurogenesis. The group-B Sox proteins (Sox1–3) main-
tain neural cells in an undifferentiated state by promot-
ing progenitor-specific gene expression (Bylund et al.
2003; Graham et al. 2003). Another group-B member,
Sox21, has the opposite activity and represses Sox1–3-
activated gene expression (Sandberg et al. 2005). Inter-
estingly, Sox21 operates downstream from proneural
bHLH proteins and is required for their capacity to com-
mit precursor cells to a neuronal differentiation program.
The findings in this study show that also group-C Sox
proteins are regulated by proneural bHLH proteins.
Thus, by regulating the expression of different groups of
Sox genes, proneural proteins can orchestrate both initial
and later steps of neurogenesis. Notably, Sox and bHLH
proteins have been demonstrated to play key roles in the
regulation of progenitor cell differentiation also outside
of the developing CNS (Kim et al. 2003; Schmidt et al.
2003). Thus, the functional interaction described in this
study may establish a general paradigm for the mecha-
nisms whereby progenitor cells are converted into more
mature cell types.
Materials and methods
Expression constructs and in ovo electroporation
cDNAs encoding full-length chick Sox11, mouse Sox4, mouse
REST, and the Zn-finger domain of REST (amino acids 203–440;
dnREST), mouse p27
kip1
were subcloned into the CMV-based
expression vector pCAGGS (Niwa et al. 1991). Sox4 and
Sox11 were tagged at their C termini with the myc epitope
(EQKLISEEDL). Other Sox11 constructs included cDNA encod-
ing amino acids 1–332 of chick Sox11 either fused to the VP16
activation domain (amino acids 400–488) or to a myc-tagged En
repressor (amino acids 2–298 of the Drosophila Engrailed pro-
tein). Other expression constructs, Sox3-myc and Ngn2-myc,
have been described previously (Bylund et al. 2003). RNA inter-
ference was performed by electroporation of 21-nucleotide (nt)
RNA duplexes (siRNA; http://www.invitrogen.com) at a con-
centration of 2 µg/µL together with pCAGGS nls-GFP (kindly
provided by Johan Ericson, Karolinska Institute, Stockholm,
Sweden). The targeted sequences were Sox4 mRNA, 5⬘-GGC
CCAGGAAGAAGGUGAA-3⬘; and Sox11 mRNA, 5⬘-GCUUU
CAUGGUGUGGUCUA-3⬘. Electroporation was performed in
chick embryos (Bylund et al. 2003) of HH stage 9–11 using ex-
pression vector concentration at 0.8–1.2 µg/µL. Electroporated
embryos were incubated for 10–48 h before fixation in 4% para-
formaldehyde and processing for in situ hybridization and im-
munohistochemistry. Statistical analysis was performed using
two-tailed Student’st-test based on six or more embryos from at
least three individual experiments.
Immunohistochemistry and in situ hybridization
Antibody staining was performed as described previously (Tsu-
chida et al. 1994). Labeling of apoptotic cells, using an Apoptosis
detection kit (Chemicon), was performed according to the
manufacturer’s recommendation. Guinea pig anti-sera were
generated against a chick Sox11 peptide (C terminus:
GGRLYYSFKNITKQ). The mouse MAP2 antibodies were Ab-
cam (ID no. Ab11267). Additional antibodies and BrdU labeling
methods are described elsewhere (Briscoe et al. 2000; Bylund et
al. 2003; Sandberg et al. 2005). In situ hybridization was per-
formed as described previously (Tsuchida et al. 1994) using
chick probes for Sox11,Sox4,SCG10 Cash1, and dnREST.
cDNAs encoding SCG10 and chick Sox4 were obtained from
MRC Geneservice (clone ID: chEST117f15 and chEST386n7).
Cell transfections and ␤-galactosidase assay
Mouse genomic fragments upstream of the Tubb3 transcrip-
tional start base pairs +54 to −566 (Tubb3
622
) and −50 to −215
(Tubb3
166
) were amplified by PCR and subcloned into the
pBGZA vector containing the minimal ␤-globin promoter and
the reporter gene LacZ. Three mutated versions of Tubb3
166
(S1M, S23M, and S123M) were generated by PCR. The distinct
reporter constructs were transfected (200 ng/200,000 cells),
with or without Sox3, Sox4, or Sox11 expression vectors (70–
280 ng/200,000 cells) into COS1 cells. Twenty-four hours after
cell transfection, the relative Luciferase and ␤-galactosidase ac-
tivity was measured. Cell transfections and activity assay meth-
ods have been described elsewhere (Wang et al. 2003).
Gel shift assays
The oligos used for the gel shift assay were ordered from Invit-
rogen: S1: sense, 5⬘-GCCTGGGTTCTATTGTCCCCACCAGA
GCGCTAG-3⬘; S1M: sense, 5⬘-GCCTGGGTTCTCCCGTCCC
CAGAGCGCTAG-3⬘; S23: sense, 5⬘-AGCCTGCCGCATTGT
GCGGCGCTCCACTAG-3⬘; S23M: sense, 5⬘-AGCCTGCCGC
GGGGTGCGGCGCTCCACTAG-3⬘. Sox3, Sox4, and Sox11
proteins were produced in bacteria Escherichia coli strain
BL21(DE3) Codon Plus (Stratagene) and purified according to
Wingate et al. (2005). The gel shift assay procedure has been
described previously (Aarnisalo et al. 2002).
Acknowledgments
We thank H. Kondoh for providing Sox11 cDNA, H. Cleavers for
providing Sox4 cDNA, D. Anderson for providing REST cDNA,
and B. Joseph for providing p27
kip1
cDNA. We are grateful to J.
Ericson and members of the Muhr laboratory for discussion and
comments on the manuscript. J.M. is supported by The Swedish
Natural Research Council, The Swedish Foundation for Strate-
gic Research, and the Ake Wibergs, Magnus Bergvalls, and
Jeanssons research foundations. J.M. and T.P. are supported by
the Ludwig Institute for Cancer Research. M.W. is supported by
the Swedish Brain Foundation.
References
Aarnisalo, P., Kim, C.H., Lee, J.W., and Perlmann, T. 2002. De-
fining requirements for heterodimerization between the ret-
inoid X receptor and the orphan nuclear receptor Nurr1. J.
Biol. Chem. 277: 35118–35123.
Berk, A.J., Boyer, T.G., Kapanidis, A.N., Ebright, R.H., Kobaya-
shi, N.N., Horn, P.J., Sullivan, S.M., Koop, R., Surby, M.A.,
and Triezenberg, S.J. 1998. Mechanisms of viral activators.
Cold Spring Harb. Symp. Quant. Biol. 63: 243–252.
Bertrand, N., Castro, D.S., and Guillemot, F. 2002. Proneural
genes and the specification of neural cell types. Nat. Rev.
Bergsland et al.
3484 GENES & DEVELOPMENT

Neurosci. 3: 517–530.
Briscoe, J., Pierani, A., Jessell, T.M., and Ericson, J. 2000. A
homeodomain code specifies progenitor cell identity and
neuronal fate in the ventral neural tube. Cell 101: 435–445.
Bylund, M., Andersson, E., Novitch, B.G., and Muhr, J. 2003.
Vertebrate neurogenesis is counteracted by Sox1-3 activity.
Nat. Neurosci. 6: 1162–1168.
Chen, Z.F., Paquette, A.J., and Anderson, D.J. 1998. NRSF/
REST is required in vivo for repression of multiple neuronal
target genes during embryogenesis. Nat. Genet. 20: 136–142.
Cheung, M., Abu-Elmagd, M., Clevers, H., and Scotting, P.J.
2000. Roles of Sox4 in central nervous system development.
Brain Res. Mol. Brain Res. 79: 180–191.
Chong, J.A., Tapia-Ramirez, J., Kim, S., Toledo-Aral, J.J., Zheng,
Y., Boutros, M.C., Altshuller, Y.M., Frohman, M.A., Kraner,
S.D., and Mandel, G. 1995. REST: A mammalian silencer
protein that restricts sodium channel gene expression to
neurons. Cell 80: 949–957.
Curmi, P.A., Andersen, S.S., Lachkar, S., Gavet, O., Karsenti, E.,
Knossow, M., and Sobel, A. 1997. The stathmin/tubulin in-
teraction in vitro. J. Biol. Chem. 272: 25029–25036.
de Martino, S., Yan, Y.L., Jowett, T., Postlethwait, J.H., Varga,
Z.M., Ashworth, A., and Austin, C.A. 2000. Expression of
sox11 gene duplicates in zebrafish suggests the reciprocal
loss of ancestral gene expression patterns in development.
Dev. Dyn. 217: 279–292.
Dennis, K., Uittenbogaard, M., Chiaramello, A., and Moody,
S.A. 2002. Cloning and characterization of the 5⬘-flanking
region of the rat neuron-specific Class III ␤-tubulin gene.
Gene 294: 269–277.
El Wakil, A., Francius, C., Wolff, A., Pleau-Varet, J., and
Nardelli, J. 2006. The GATA2 transcription factor negatively
regulates the proliferation of neuronal progenitors. Develop-
ment 133: 2155–2165.
Farah, M.H., Olson, J.M., Sucic, H.B., Hume, R.I., Tapscott, S.J.,
and Turner, D.L. 2000. Generation of neurons by transient
expression of neural bHLH proteins in mammalian cells.
Development 127: 693–702.
Fode, C., Gradwohl, G., Morin, X., Dierich, A., LeMeur, M.,
Goridis, C., and Guillemot, F. 1998. The bHLH protein
NEUROGENIN 2 is a determination factor for epibranchial
placode-derived sensory neurons. Neuron 20: 483–494.
Fode, C., Ma, Q., Casarosa, S., Ang, S.L., Anderson, D.J., and
Guillemot, F. 2000. A role for neural determination genes in
specifying the dorsoventral identity of telencephalic neu-
rons. Genes &Dev. 14: 67–80.
Garcia-Dominguez, M., Poquet, C., Garel, S., and Charnay, P.
2003. Ebf gene function is required for coupling neuronal
differentiation and cell cycle exit. Development 130: 6013–
6025.
Garel, S., Marin, F., Grosschedl, R., and Charnay, P. 1999. Ebf1
controls early cell differentiation in the embryonic striatum.
Development 126: 5285–5294.
Gradwohl, G., Fode, C., and Guillemot, F. 1996. Restricted ex-
pression of a novel murine atonal-related bHLH protein in
undifferentiated neural precursors. Dev. Biol. 180: 227–241.
Graham, V., Khudyakov, J., Ellis, P., and Pevny, L. 2003. SOX2
functions to maintain neural progenitor identity. Neuron 39:
749–765.
Grishok, A. and Mello, C.C. 2002. RNAi (Nematodes: Cae-
norhabditis elegans). Adv. Genet. 46: 339–360.
Guillemot, F. and Joyner, A.L. 1993. Dynamic expression of the
murine Achaete-Scute homologue Mash-1 in the developing
nervous system. Mech. Dev. 42: 171–185.
Hargrave, M., Wright, E., Kun, J., Emery, J., Cooper, L., and
Koopman, P. 1997. Expression of the Sox11 gene in mouse
embryos suggests roles in neuronal maturation and epithe-
lio-mesenchymal induction. Dev. Dyn. 210: 79–86.
Kamachi, Y., Uchikawa, M., and Kondoh, H. 2000. Pairing SOX
off: With partners in the regulation of embryonic develop-
ment. Trends Genet. 16: 182–187.
Karlsson, J.E., Rosengren, L.E., and Haglid, K.G. 1987. A rapid
HPLC method to separate the triplet proteins of neurofila-
ment. J. Neurochem. 49: 1375–1378.
Kim, J., Lo, L., Dormand, E., and Anderson, D.J. 2003. SOX10
maintains multipotency and inhibits neuronal differentia-
tion of neural crest stem cells. Neuron 38: 17–31.
Kintner, C. 2002. Neurogenesis in embryos and in adult neural
stem cells. J. Neurosci. 22: 639–643.
Kuhlbrodt, K., Herbarth, B., Sock, E., Enderich, J., Hermans-
Borgmeyer, I., and Wegner, M. 1998. Cooperative function of
POU proteins and SOX proteins in glial cells. J. Biol. Chem.
273: 16050–16057.
Lee, J.E., Hollenberg, S.M., Snider, L., Turner, D.L., Lipnick, N.,
and Weintraub, H. 1995. Conversion of Xenopus ectoderm
into neurons by NeuroD, a basic helix–loop–helix protein.
Science 268: 836–844.
Lo, L., Dormand, E., Greenwood, A., and Anderson, D.J. 2002.
Comparison of the generic neuronal differentiation and neu-
ron subtype specification functions of mammalian achaete-
scute and atonal homologs in cultured neural progenitor
cells. Development 129: 1553–1567.
Lunyak, V.V., Burgess, R., Prefontaine, G.G., Nelson, C., Sze,
S.H., Chenoweth, J., Schwartz, P., Pevzner, P.A., Glass, C.,
Mandel, G., et al. 2002. Corepressor-dependent silencing of
chromosomal regions encoding neuronal genes. Science 298:
1747–1752.
Maschhoff, K.L., Anziano, P.Q., Ward, P., and Baldwin, H.S.
2003. Conservation of Sox4 gene structure and expression
during chicken embryogenesis. Gene 320: 23–30.
Mavropoulos, A., Devos, N., Biemar, F., Zecchin, E., Argenton,
F., Edlund, H., Motte, P., Martial, J.A., and Peers, B. 2005.
sox4b is a key player of pancreatic ␣cell differentiation in
zebrafish. Dev. Biol. 285: 211–223.
Moody, S.A. and Stein, D.B. 1988. The development of acetyl-
cholinesterase activity in the embryonic nervous system of
the frog, Xenopus laevis.Brain Res. 467: 225–232.
Naya, F.J., Huang, H.P., Qiu, Y., Mutoh, H., DeMayo, F.J., Le-
iter, A.B., and Tsai, M.J. 1997. Diabetes, defective pancreatic
morphogenesis, and abnormal enteroendocrine differentia-
tion in BETA2/neuroD-deficient mice. Genes &Dev. 11:
2323–2334.
Niwa, H., Yamamura, K., and Miyazaki, J. 1991. Efficient selec-
tion for high-expression transfectants with a novel eukary-
otic vector. Gene 108: 193–199.
Novitch, B.G., Chen, A.I., and Jessell, T.M. 2001. Coordinate
regulation of motor neuron subtype identity and pan-neuro-
nal properties by the bHLH repressor Olig2. Neuron 31: 773–
789.
Perron, M., Opdecamp, K., Butler, K., Harris, W.A., and Belle-
froid, E.J. 1999. X-ngnr-1 and Xath3 promote ectopic expres-
sion of sensory neuron markers in the neurula ectoderm and
have distinct inducing properties in the retina. Proc. Natl.
Acad. Sci. 96: 14996–15001.
Pevny, L. and Placzek, M. 2005. SOX genes and neural progeni-
tor identity. Curr. Opin. Neurobiol. 15: 7–13.
Pevny, L.H., Sockanathan, S., Placzek, M., and Lovell-Badge, R.
1998. A role for SOX1 in neural determination. Develop-
ment 125: 1967–1978.
Rao, M., Baraban, J.H., Rajaii, F., and Sockanathan, S. 2004. In
vivo comparative study of RNAi methodologies by in ovo
electroporation in the chick embryo. Dev. Dyn. 231: 592–
Neuronal properties and Sox4/11
GENES & DEVELOPMENT 3485

600.
Riederer, B. and Matus, A. 1985. Differential expression of dis-
tinct microtubule-associated proteins during brain develop-
ment. Proc. Natl. Acad. Sci. 82: 6006–6009.
Roztocil, T., Matter-Sadzinski, L., Alliod, C., Ballivet, M., and
Matter, J.M. 1997. NeuroM, a neural helix–loop–helix tran-
scription factor, defines a new transition stage in neurogen-
esis. Development 124: 3263–3272.
Sandberg, M., Kallstrom, M., and Muhr, J. 2005. Sox21 promotes
the progression of vertebrate neurogenesis. Nat. Neurosci. 8:
995–1001.
Schmidt, K., Glaser, G., Wernig, A., Wegner, M., and Rosorius,
O. 2003. Sox8 is a specific marker for muscle satellite cells
and inhibits myogenesis. J. Biol. Chem. 278: 29769–29775.
Schoenherr, C.J. and Anderson, D.J. 1995. The neuron-restric-
tive silencer factor (NRSF): A coordinate repressor of mul-
tiple neuron-specific genes. Science 267: 1360–1363.
Smith, S.T. and Jaynes, J.B. 1996. A conserved region of en-
grailed, shared among all en-, gsc-, Nk1-, Nk2- and msh-class
homeoproteins, mediates active transcriptional repression
in vivo. Development 122: 3141–3150.
Sock, E., Rettig, S.D., Enderich, J., Bosl, M.R., Tamm, E.R., and
Wegner, M. 2004. Gene targeting reveals a widespread role
for the high-mobility-group transcription factor Sox11 in tis-
sue remodeling. Mol. Cell. Biol. 24: 6635–6644.
Sun, Y., Nadal-Vicens, M., Misono, S., Lin, M.Z., Zubiaga, A.,
Hua, X., Fan, G., and Greenberg, M.E. 2001. Neurogenin pro-
motes neurogenesis and inhibits glial differentiation by in-
dependent mechanisms. Cell 104: 365–376.
Tomita, K., Moriyoshi, K., Nakanishi, S., Guillemot, F., and
Kageyama, R. 2000. Mammalian achaete-scute and atonal
homologs regulate neuronal versus glial fate determination
in the central nervous system. EMBO J. 19: 5460–5472.
Tsuchida, T., Ensini, M., Morton, S.B., Baldassare, M., Edlund,
T., Jessell, T.M., and Pfaff, S.L. 1994. Topographic organiza-
tion of embryonic motor neurons defined by expression of
LIM homeobox genes. Cell 79: 957–970.
Uwanogho, D., Rex, M., Cartwright, E.J., Pearl, G., Healy, C.,
Scotting, P.J., and Sharpe, P.T. 1995. Embryonic expression
of the chicken Sox2, Sox3 and Sox11 genes suggests an in-
teractive role in neuronal development. Mech. Dev. 49: 23–
36.
van Beest, M., Dooijes, D., van De Wetering, M., Kjaerulff, S.,
Bonvin, A., Nielsen, O., and Clevers, H. 2000. Sequence-
specific high mobility group box factors recognize 10–12-
base pair minor groove motifs. J. Biol. Chem. 275: 27266–
27273.
van de Wetering, M., Oosterwegel, M., van Norren, K., and
Clevers, H. 1993. Sox-4, an Sry-like HMG box protein, is a
transcriptional activator in lymphocytes. EMBO J. 12: 3847–
3854.
Wang, Z., Benoit, G., Liu, J., Prasad, S., Aarnisalo, P., Liu, X., Xu,
H., Walker, N.P., and Perlmann, T. 2003. Structure and func-
tion of Nurr1 identifies a class of ligand-independent nuclear
receptors. Nature 423: 555–560.
Wegner, M. 1999. From head to toes: The multiple facets of Sox
proteins. Nucleic Acids Res. 27: 1409–1420.
Wiebe, M.S., Nowling, T.K., and Rizzino, A. 2003. Identifica-
tion of novel domains within Sox-2 and Sox-11 involved in
autoinhibition of DNA binding and partnership specificity. J.
Biol. Chem. 278: 17901–17911.
Wingate, A.D., Campbell, D.G., Peggie, M., and Arthur, J.S.
2005. Nur77 is phosphorylated in cells by RSK in response to
mitogenic stimulation. Biochem J. 393: 715–724.
Yokota, Y. 2001. Id and development. Oncogene 20: 8290–8298.
Bergsland et al.
3486 GENES & DEVELOPMENT