Human ESC-Derived Neural Crest Model Reveals a Key Role for SOX2 in Sensory Neurogenesis

Article (PDF Available)inCell stem cell 8(5):538-51 · May 2011with49 Reads
DOI: 10.1016/j.stem.2011.03.011 · Source: PubMed
The transcription factor SOX2 is widely known to play a critical role in the central nervous system; however, its role in peripheral neurogenesis remains poorly understood. We recently developed an hESC-based model in which migratory cells undergo epithelial to mesenchymal transition (EMT) to acquire properties of neural crest (NC) cells. In this model, we found that migratory NC progenitors downregulate SOX2, but then start re-expressing SOX2 as they differentiate to form neurogenic dorsal root ganglion (DRG)-like clusters. SOX2 downregulation was sufficient to induce EMT and resulted in massive apoptosis when neuronal differentiation was induced. In vivo, downregulation of SOX2 in chick and mouse NC cells significantly reduced the numbers of neurons within DRG. We found that SOX2 binds directly to NGN1 and MASH1 promoters and is required for their expression. Our data suggest that SOX2 plays a key role for NGN1-dependent acquisition of neuronal fates in sensory ganglia.
Cell Stem Cell
Human ESC-Derived Neural Crest Model Reveals
a Key Role for SOX2 in Sensory Neurogenesis
Flavio Cimadamore,
Katherine Fishwick,
Elena Giusto,
Ksenia Gnedeva,
Giulio Cattarossi,
Amber Miller,
Stefano Pluchino,
Laurence M. Brill,
Marianne Bronner-Fraser,
and Alexey V. Terskikh
Sanford-Burnham Medical Research Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA
California Institute of Technology, Pasadena, CA 91125, USA
Institute of Experimental Neurology, IRCCS, San Raffaele, Milan 20132, Italy
*Correspondence: (F.C.), (A.V.T.)
DOI 10.1016/j.stem.2011.03.011
The transcription factor SOX2 is widely known to play
a critical role in the central nervous system; however,
its role in peripheral neurogenesis remains poorly
understood. We recently developed an hESC-based
model in which migratory cells undergo epithelial to
mesenchymal transition (EMT) to acquire properties
of neural crest (NC) cells. In this model, we found
that migratory NC progenitors downregulate SOX2,
but then start re-expressing SOX2 as they differen-
tiate to form neurogenic dorsal root ganglion (DRG)-
like clusters. SOX2 downregulation was sufficient to
induce EMT and resulted in massive apoptosis
when neuronal differentiation was induced. In vivo,
downregulation of SOX2 in chick and mouse NC cells
significantly reduced the numbers of neuro ns within
DRG. We found that SOX2 binds directly to NGN1
and MASH1 promoters and is required for their
expression. Our data suggest that SOX2 plays a
key role for NGN1-dependent acquisition of neuronal
fates in sensory ganglia.
The SRY (sex-determining region)-box 2 (SOX2) gene encodes
a transcription factor required for maintenance of the pluripotent
state of human embryonic stem cells (hESCs) (Adachi et al.,
2010). Because a morphologically recognizable neural plate
expresses SOX2, it is considered one of the earliest functional
markers of neuroectodermal specification (Rex et al., 1997).
The role of SOX2 in central nervous system (CNS) development
and adult neurogenesis has been extensively investigated
(Cavallaro et al., 2008; Ferri et al., 2004; Graham et al., 2003;
Miyagi et al., 2008). However, its function in the peripheral
nervous system (PNS) is less studied. At E12.5, prospective
satellite glial cells and Schwann cell precursors express SOX2,
where it blocks myelination and terminal differentiation; SOX2
is downregulated in both of the mature glial cell lineages (Le
et al., 2005).
NC delamination from dorsal neuroepithelium is a classic
example of epithelial-mesenchymal transition (EMT) (Meule-
mans and Bronner-Fraser, 2004). SOX2 is downregulated in
delaminating dorsal neuroepithelial cells (migratory NC), and
its enforced expression in the avian prospective NC impedes
EMT and the acquisition of NC fates (Wakamatsu et al., 2004;
Wegner and Stolt, 2005). However, migratory NC cells transiently
re-express SOX2 when they reach the dorsal root ganglia (DRG)
(Wakamatsu et al., 2000). The DRG neurons are formed in three
successive overlapping waves. Ngn2 initiates the first wave and
Ngn1 initiates the second (Ma et al., 1999). The Ngn2-dependent
wave mainly gives rise to the small clusters of TrkB
and TrkC
neurons (mechanoreceptors and proprioceptors), whereas
Ngn1-dependent wave gives rise to the large clusters of TrkA
neurons (peptidergic and nonpeptidergic neurons) as well as
peripheral glia (Marmige
re and Ernfors, 2007). The third wave
of neurogenesis arises from the boundary cap cells and contrib-
utes to TrkA
neurons and glia (Marmige
re and Ernfors, 2007).
We have previously described a rapid and efficient protocol for
differentiation of hESC into NC lineages, including sensory and
autonomic neurons, Schwann cells, smooth muscle cells, mela-
nocytes, and cartilage (Curchoe et al., 2010). Here we used
hESCs as an in vitro model of human NC cells. In agreement
with previous studies, we observed that SOX2 is a strong inhib-
itor of EMT and human NC delamination. We showed that SOX2
downregulation is sufficient to induce EMT and acquisition of
migratory phenotypes in culture. Surprisingly, re-expression of
SOX2 in NC cells is essential for generation of peripheral
neurons, likely through its direct as well as indirect modulation
of proneural gene expression.
Human ESC-Derived Model of Neural Crest
The neuroepithelial cells were derived from hESCs as previously
described (Bajpai et al., 2009; Cimadamore et al., 2009) followed
by the NC cultures (Curchoe et al., 2010). Analysis of neuroepi-
thelial cells demonstrated the expression of NESTIN, MUSASHI1,
PAX6, and SOX2 (Figures 1A–1C) as well as markers of dorsal
neuroepithelium, such as PAX3 and SOX9 (Figures 1 E and 1F).
Adherent neuroepithelial clusters were also positive for the NC
marker p75
(Figure 1D), which is expressed in the human
neural tube (Thomas et al., 2008). Little or no expression of
ventral neural tube markers NKX2.2 and HNF3b (Figures 1G
and 1H) was seen. The addition of a Sonic Hedgehog (SHH)
agonist (Purmorphamine) ventralized the cultures, decreased
the expression of dorsal markers (GDF7, SOX9, and SLUG),
538 Cell Stem Cell 8, 538–551, May 6, 2011 ª2011 Elsevier Inc.
and increased the expression of ventral markers (HNF3b,
NKX2.2, and SHH) (Figure 1I). These data are consistent with
the dorsal identity of hESC-derived neuroepithelium (dNE), which
harbors premigratory neural crest cells.
We propose to consider hESC-derived dNE clusters as
a surrogate model for the EMT transition from dNE to migratory
NC (Figure 1J). Indeed, when hES-derived neurospheres were
plated on fibronectin (FN)—a permissive substrate for neural
crest (NC) migration (Henderson and Copp, 1997)—a migratory
cell population emerged ( Figure 1K), which was positive for
SOX10 (Figure 1L), a classic marker of migratory NC cells (Para-
tore et al., 2001). Compared with cells in neuroepithelial clus-
ters, we found that migratory NC cells strongly downregulated
the neural adhesion molecule N-CADHERIN (N-CAD) (Akitaya
and Bronner-Fraser, 1992)(Figure 1M) and lost nuclear expres-
sion of SOX2 (Figure 1N), consistent with observations in chick
embryos (Wakamatsu et al., 2004). Furthermore, while the
spheres were positive for the CNS marker PAX6, we observed
the loss of PAX6 in the migratory cells and an acquisition of
the NC marker Integrin-a4(Figure 1O). Integrin-a4 is expressed
in migrating cells and in DRG (Kil et al., 1998) and is a classic
cellular receptor for fibronectin (Mould et al., 1994). Thus the
expression patterns of SOX2, SOX10, N-CAD, PAX6, and
INT-a4 allowed us to delineate the dNE clusters from the migra-
tory NC cells, suggesting that hESC-based cultures faithfully
reproduce early events of NC delamination from dNE. Flow
cytometry revealed 5% of cells positive for INT-a4 in the early
cultures (Figure 1P) and up to 80% afterward ( Figure 1Q). These
results suggest that under the present culture conditions, the
original CNS identity of primary dNE cells was rapidly lost in
favor of the NC identity. In agreement with the strong upregula-
tion of INT-a4, the CNS marker PAX6 was almost complete ly
absent in NC cells from the first passage on (see Figure S1D
available online). The INT- a4 expression remained approxi-
mately at the same level for at least six passages (Figures
S1A and S1B) along with NESTIN expression (Figure S1C).
We conclude that under current culture conditions, the dNE
cells rapidly emigrate from clusters and acquire characteristics
of NC cells.
SOX2 Downregulation Induces EMT
Forced expression of SOX2 blocks EMT and inhibits NC
delamination and NC migration in chick and quail embryos
(Wakamatsu et al., 2004), thus overriding the activity of Bone
Morphogenetic Protein 4 (BMP4), a well-known NC inducer
(Sela-Donenfeld and Kalcheim, 1999; Shoval et al., 2007). We
investigated whether downregulation of SOX2 (loss of function)
is sufficient to induce EMT using the ESC-based model of
human NC.
Human ESCs were stably transduced with lentivirus carrying
the inducible SOX2 shRNA and the efficiency of SOX2 downre-
upon addition of dox was verified (75% reduction;
Figures S2A and S2B). The specificity of knockdown was verified
by assessing the transcript levels of other SOXB1 members (i.e.,
SOX1 and SOX3)(Figure S2C). Human ESCs stably transduced
with inducible SOX2 shRNA-expressing lentivirus were neural-
ized for 5 days and cells were allowed to emigrate from dNE neu-
rospheres for 4 days in the presence of dox. SOX2 downregula-
tion significantly enhanced cell migration when compared to
control cells carrying inducible scrambled shRNA (Figures 2A–
2C), suggesting that SOX2 downregulation in dNE cells in the
absence of exogenous BMP4 is sufficient to induce migratory
We observed that dNE cells within the clusters tend to
organize into N-CAD
neuroepithelial rosettes (Figure 2D). The
neuroepithelial rosette cytoarchitecture was almost completely
disrupted and N-CAD staining was largely eliminated following
SOX2 knockdown (Figures 2D–2F). We also investigated
whether SOX2 downregulation altered expression of SLUG and
ADAM10, two well-established inducers of EMT in neural crest
cells (Carl et al., 1999; Shoval et al., 2007). As shown in Figure 2G,
RT-PCR analysis showed that SLUG expression remained
unchanged and was abundant in cells treated with either SOX2
shRNA or scrambled control shRNA. SLUG expression in vivo
is required but not sufficient for EMT in dNE cells, since not all
SLUG-expressing dNE cells migrate (Linker et al., 2000). Thus,
our results suggest that hES-derived dNE cells are competent
for EMT and that SOX2 downregulation does not alter SLUG
expression. The ADAM10 metalloprotease can enhance migra-
tion in dNE cells via a mechanism involving N-CAD cleavage
and internalization that, in turn, results in loss of intercellular
adhesion and enhanced Wnt signaling (Shoval et al., 2007). In
contrast to SLUG, ADAM10 expression was upregulated upon
SOX2 knockdown (Figure 2G), consistent with the role of
ADAM10 in promoting NC delamination.
We also investigated whether the NC inducer BMP4 alters the
level of SOX2 expression in human dNE cells. To this end, we
seeded dNE cells in the presence of BMP4 and determined the
percentage of cells positive for nuclear SOX2 after 4 days of
treatment (Figure 2H). BMP4 treatment efficiently downregu-
lated SOX2 expression when compared to untreated cells, indi-
cating that SOX2 is downstream of BMP signaling during early
phases of NC specification (Figure 2I). Our results suggest that
SOX2 downregulation is sufficient to initiate delamination from
dNE clusters and that BMP4 may induce NC delamination, at
least in part, through the downregulation of SOX2.
NC Cells Re-Express SOX2 within DRG-Like
Secondary Clusters
Although SOX2 expression antagonizes EMT and NC delamina-
tion, it is re-expressed in nascent chicken DRG (Wakamatsu
et al., 2000) along with N-CAD (Akitaya and Bronner-Fraser,
1992). We found that SOX2 and N-CAD are reacquired in
nascent embryonic day 10 (E10) murine DRG (Figures 3A and
3B) along with SOX10 (Figure S3A). In mouse and rat DRG,
SOX10 marks multipotent cells able to give rise to both glia
and neurons (Kim et al., 2003; Montelius et al., 2007). These
observations suggest that gangliogenesis involves upregulation
of SOX2 and N-CAD by migratory NC cells coalescing into
ganglia (Figure 3C). We asked whether hESC-NC cells could
re-express SOX2 and N-CAD. We cultured single cell dNE clus-
ters in the presence of BMP4 for 4 days and obtained near homo-
geneous SOX2
population (Figure 3D, left panel). Exposure to
Leukemia Inhibitory Factor (LIF), shown to promote Mesen-
chymal to Epithelial Transition (MET) in the rat metanephros
(Barasch et al., 1999) resulted in SOX2
clusters (Figure 3D, right
panel). We have also used a modified collagen invasion assay
(Voroteliak et al., 2002). Single dNE cells seeded on top of 1%
Cell Stem Cell
Key Role of SOX2 in Sensory Neurogenesis
Cell Stem Cell 8, 538–551, May 6, 2011 ª2011 Elsevier Inc. 539
Neuroepithelial markers
Dorsal markers Ventral markers
NC marker
Fold change
P0 (Primary spheres)
4 INT- 4
INT- 4
Figure 1. Dorsal Identity of hESC-Derived Neuroepithelium
(A–H) Neural clusters at day 6 of neural induction express NESTIN, MUSASHI1 (A), PAX6 (B), SOX2 (C), p75 (D), and dorsal markers PAX3 (E) and SOX9 (F) and
lack the ventral marker NKX2.2 (G) and the floor plate marker HNF3b (H).
(I) Quantitative PCR of dorsal (GDF7, SOX9, SLUG) and ventral (HNF3b, NKX2.2, SHH) markers in hESCs neuralized for 6 days without (black bars, set to 1) or with
10 mM of Purmorphamine (open bars). All values were normalized to 18S expression; *p < 0.005.
(J and K) Schematic representation of NC delamination from the dorsal neural tube (J) and a model of human NC delamination from hESC-derived dNE clusters
(K). NT, neural tube; dNT, dorsal neural tube; dNE, dorsal neuroepithelium; EMT, epithelial to mesenchymal transition; NC, Neural Crest.
Cell Stem Cell
Key Role of SOX2 in Sensory Neurogenesis
540 Cell Stem Cell 8, 538–551, May 6, 2011 ª2011 Elsevier Inc.
collagen gel were allowed to migrate through the gel and the
cells adhered to the bottom of the well were assessed for
SOX2/N-CAD expression (Figures 3E–3G). Many cells reac-
quired both SOX2 and N-CAD expression and formed tight clus-
ters surrounded by N-CAD
, SOX2
cells (Figures 3E and 3F).
hESC-NC cultured in the presence of EGF (see Experimental
Procedures) routinely yielded a similar clear segregation
between migratory cells and secondary clusters forming polar-
ized neuroepithelial rosettes (Figure 3H) or tight clusters (Fig-
ure 3I) surrounded by the SOX2
cells. These clusters were posi-
tive for Integrin-a4(Figure 1Q and data not shown) and SOX10
(Figure S3) and negative for the human CNS marker PAX6
(Figure 4F).
Taken together, these results suggest that secondary clusters
routinely observed in monolayer cultures of hESC-NC cells after
single cell dissociation of primary dNE clusters resembles the
in vivo ganglia (DRG) with respect to their expression of SOX2,
N-CAD, Integrin-a4, SOX10, and PAX6.
SOX2 Expression is Associated with Neuronal Markers
Upon spontaneous differentiation of NC cultures, smooth
muscle actin (SMA)
cells were always found among the mesen-
chymal SOX2
cells (Figure 4A), whereas Peripherin or the
pan-neuronal marker Microtubule Associated Protein-2 (MAP2)
were strictly associated with the SOX2
aggregates (Figures
4B and 4C). Reminiscent of proliferating neurogenic DRG
in vivo (Kahane and Kalcheim, 1998; Wakamatsu et al., 2000)
and in accordance with the well-known role of SOX2 in prolifer-
ation (Ferri et al., 2004), cells in secondary neurogenic clusters
actively proliferated (Figure 4D), whereas surrounding mesen-
chymal cells were mostly postmitotic (Figure 4D). The vast
majority of the cells were positive for BRN3A (Figure 4E),
a marker for both early migrating neural crest and sensory
neurons (Fedtsova and Turner, 1995). In sharp contrast to cells
found in primary spheres, SOX2
cells in secondary neurogenic
clusters did not coexpress the CNS marker PAX6 (Figure 4F),
therefore suggesting their peripheral identity. High-resolution
imaging showed that weakly MAP2
cells were strongly
, whereas some MAP2
cells with mature neuronal
morphology were often weakly positive for SOX2 (Figure 4G).
Similar results were obtained for SOX2 and Peripherin coexpres-
sion (Figure 4H). Approximately 80% of MAP2
cells were also
positive (strongly or weakly) for SOX2 under this condition
(Figure 4I).
We investigated if SOX2 was coexpressed with neuronal
markers in vivo. Confocal analysis of E11 DRG showed clearly
detectable levels of Sox2 coexpressed with an early neuronal
marker, TuJ1, in the same cells (Figure S4). We also found that
in vivo, SOX2 tends to be coexpressed with SOX10 in E10
DRG neural progenitors (Figure S3A). Similarly, SOX2
cells in
secondary hESC-derived neurogenic clusters also showed
SOX10 expression (Figure S3B).
Taken together, these data suggest that SOX2 expression is
associated with peripheral neurogenesis and provide evidence
that secondary hESC-derived neurogenic clusters resemble
embryonic DRG.
Rosette lumen
area (
Migration area
(normalized to
sphere area)
- +
Figure 2. SOX2 Counteracts Epithelial
Mesenchymal Transition
(A and B) Bright field images of neurospheres
carrying dox-inducible control scrambled (shCTRL,
[A]) or SOX2 shRNA (shSOX2, [B]). Spheres were
plated on laminin/polyornithine and allowed to
migrate for 4 days in the presence of dox (1 mg/ml).
(C) Quantification of migration in conditions shown
in (A) and (B). Migration area for each sphere was
normalized to the sphere size.
(D–F) N-CAD staining showing rosette-epithelial
organization in the center of neurospheres flat-
tened for 4 days on lamin/polyornithine. shCTRL
(D) and shSOX2 (E) neurospheres plated in the
presence of dox; quantified in (F).
(G) RT-PCR analysis of SLUG and ADAM10 in
dNE cells expressing scrambled control and
(H) The percentage of SOX2
cells after 4 days in
the presence or the absence of 100 ng/ml BMP4.
(I) A model proposing the role of SO X2 in delami-
nation of dorsal neuroepithelial cells. Scale bars,
100 mm; Hoechst nuclear dye (Blue) was used
to stain cell nuclei; *p < 0.05; **p < 0.005. Error
bars ± SE.
(L–O) Cells emigrating from human dNE clusters acquire SOX10 (L) and lose N-CAD (M) and SOX2 (N). Cytoplasmic staining in (N) is a nonspecific staining.
Expression of NC marker Integrin-a4 (INT-a4) and the absence of the CNS marker PAX6 separate human NC and dNE clusters (O).
(P and Q) Flow cytometry analysis of INT-a4 showing the switch from CNS to NC identity when primary spheres (P) were cultured for one passage on FN-coated
plates (Q). Normal mouse IgG was used as a negative control; scale bars, 100 mm; blue, Hoechst. Error bars ± SE.
Cell Stem Cell
Key Role of SOX2 in Sensory Neurogenesis
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SOX2 is Required for Sensory Neurogenesis in Human
ESC-Derived NC Cultures
Culturing of hESC-NC cells with LIF for 7 days enriches for
and Peripherin
cells and depletes SMA
and GFAP
cells from the cultures. Addition of BMP4 had the opposite effect
(Figures 5A and 5B). These results suggest that the enrichment
for neuronal outcomes correlates with the levels of SOX2 (Fig-
ure 3D), consistent with our hypothesis that SOX2 is required
for neurogenesis, but not for glial or smooth muscle cell
Next we engineered hESC lines harboring SOX2-specific
inducible shRNA and investigated the differentiation outcomes
under neurogenic, myogenic, or gliogenic conditions upon
downregulation of SOX2.
The differentiation of NC cells under neurogenic conditions
(see Experimental Procedures) results in a high proportion of
cells expressing neuronal markers (Figures 5C and 5D, upper
panels). The downregulation of SOX2 under these conditions
resulted in dramatic loss of Peripherin
and MAP2
cells when
compared to control scrambled shRNA (Figures 5C and 5D,
lower panels). Immunodetection of the active form of Caspase3
(AC3) revealed extensive cell death upon SOX2 knockdown (Fig-
ure 5E). To determine the time when SOX2 function is required
during neuronal differentiation, we knocked down SOX2 expres-
sion at various time points after the onset of neuronal differenti-
ation, but before any mature Peripherin
neurons can be
detected (Figure S5). SOX2 downregulation at day 3 following
neuronal induction dramatically impaired neuronal differentia-
tion, whereas SOX2 downregulation at day 7 had no significant
effect on the total number of Peripherin
cells detected at day
14 (Figure S5). These data suggest that SOX2 plays a critical
role during the early commitment to the peripheral neuronal
We identified gliogenic culture conditions yielding almost
exclusively P0
double-positive putative early Schwann
cells (see Experimental Procedures). SOX2 knockdown under
such conditions had no significant effect on the number of
differentiated P0
cells (Figure 5F and data not shown
for GFAP). In sharp contrast to neurogenic culture conditions,
we did not observe increased cell death upon downregulation
of SOX2 under gliogenic conditions (data not shown). Next we
investigated the effect of SOX2 knockdown under differentia-
tion conditions yielding mixed myogenic/neurogenic or glio-
genic/neurogenic cultures (see Experimental Procedures).
SOX2 downregulation under mixed conditions significantly
increased the proportion of SMA
cells (Figure 5G) and P0
cells (Figure 5H and data not shown for GFAP expres-
sion). In both cases, the observed increase in SMA
and P0
cells happened at the expense of neuronal differentiation
(data not shown).
We engineered dNE cells carrying the dox-inducible SOX2
shRNA (targeting the mRNA 3
UTR) with lentivirus express-
ing a SOX2-GFP fusion protein (which lacks the 3
UTR and
therefore is not affected by SOX2 shRNA). Enforced expression
DRG-like clusters
Figure 3. hESC-Derived NC Cells Form DRG-Like Clusters
(A–C) Migratory NC cells reacquire both SOX2 (A) and N-CAD expression (B) after coalescing into embryonic day 10 (E10) dorsal root ganglia (DRG); (d) and (v)
denote the dorsal-ventral orientation of the neural tube schematically illustrated (C).
(D) dNE cells reacquire SOX2 expression following LIF treatment. Left panel shows SOX2 staining in cells treated for 4 days with 100 ng/ml BMP4. 6.69 ± 1.25% of
total cells were SOX2
. Four days of treatment with 100 ng/ml LIF (right panel) resulting in formation of numerous SOX2
clusters with 34.15 ± 3.1% of total cells
expressing SOX2.
(E and F) hES-derived migratory neural crest cells reform clusters after migration through the collagen gel; low (E) and high (F) magnifications.
(G) Schematic of human dNE migrating through collagen to form colonies at the bottom of the plate.
(H and I) Clusters of cultured dNE cells. SOX2
cells are found in the polarized epithelial rosettes identified by the apical accumulation of clusters positive for the
tight junction protein ZO-1 (H) and N-CAD (I). Scale bar, 100 mm; blue, Hoechst dye. Error bars ± SE.
Cell Stem Cell
Key Role of SOX2 in Sensory Neurogenesis
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of SOX2-GFP, but not GFP alone, rescued neuronal differentia-
tion as assessed by staining for the early neuronal marker Tuj1
(Figure 5I). Expression of a more mature marker MAP2 was not
observed, suggesting that SOX2 may promote early, but not
late, phases of neuronal commitment. This is consistent with
the previous reports in chick embryos, where the overexpression
of SOX2 blocked differentiation of neural precursors into mature
neurons (Graham et al., 2003).
Taken together, these results suggest that in hESC-NC cells,
SOX2 function is critical at the early stages of neuronal differen-
tiation, but dispensable for the differentiation into SMA
and P0
SOX2 is Required for the Generation of a Subset of DRG
Neurons In Vivo
To test the role of Sox2 in vivo, we performed a targeted knock-
down in chick embryos with shRNA under the control of an
enhancer that mediates expression of the neural crest marker
FoxD3 (Fishwick et al., unpublished data). The chicken Sox2-
specific shRNA or control shRNA were electroporated into
the trunk neural tube of HH10 (10 somite stage; Hamburger,
1988) chick embryos, efficiently targeting a large percentage
of the premigratory NC. Embryos were fixed 2 days later, by
which time neural crest cells had completed migration and
condensed to form DRG. Consistent with the fact that neuronal
Figure 4. SOX2 Expression is Associated with Peripheral Neuronal Differentiation
(A–C) Spontaneous differentiation in SMA
smooth muscle cells was always seen outside of SOX2
clusters (B and C). SOX2
aggregates are associated wit h
Peripherin and MAP2.
(D and E) Secondary neurogenic clusters immunostained for MAP2 and Ki67 (D) and sensory neuronal marker BRN3A (E).
(F) SOX2 and PAX6 coexpression in dNE cells (primary spheres [P0], left panel). NC cells in secondary neurogenic clusters are SOX2
(passage 1 [P1], right
(G and H) SOX2 is expressed in cells lacking neuronal morphology and weakly positive for Peripherin or MAP2 (solid arrows); neurons with mature morphology
and strong MAP2, Peripherin staining are weak positive / negative for SOX2 (open and solid arrowheads, respectively).
(I) Quantification of MAP2/SOX2 colabeling from the experiment illustrated in (H). In all images blue, Hoechst dye. Scale bars: (A–F) 100 mm, and (G–H) 10 mm;
**p < 0.005. Error bars ± SE.
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Key Role of SOX2 in Sensory Neurogenesis
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Normalized Area
MAP2 Peri
Peripherin MAP2
Peri+ area
MAP2+ area
AC3+ area
P0+ area
SMA+ area
Tuj1+ (%)
Normalized Area
P0+ area
Figure 5. SOX2 is Required for Peripheral Neurona l Differentiation
(A and B) hESC-NC cultured with LIF (upper row) are enriched for Peripherin and MAP2; the addition of BMP4 (lower row) enriches for the smooth muscle (SMA)
and glial (GFAP) markers.
(C and D) Under the neurogenic conditions, differentiation into Peripherin- (C) and MAP2- (D) positive neurons is abolished when SOX2 expression is down-
regulated using dox-inducible SOX2 shRNA, but not control scrambled shCTRL.
(E) Immunostaining for active Caspase3 (AC3) under neurogenic culture conditions in shCTRL (upper panel) and shSOX2 cells (lower panel) cultured in the
presence of dox.
(F–H) Expression of SOX2 shRNA versus scrambled shCTRL under gliogenic conditions did not affect P0
cells (F) and resulted in the increase in SMA
cells (G)
and P0
cells (H) under the mixed conditions.
(I) SOX2 overexpression rescues neuronal differentiation. The dNE cells carrying dox-inducible SOX2 shRNA were transduced with lentivirus expressing GFP or
a SOX2-GFP fusion protein (lacking the endogenous SOX2 3
UTR targeted by SOX2-specific shRNA) and cultured under neurogenic conditions in the presence of
dox. After 14 days, neuronal differentiation was assayed by Tuj1 immunostaining. Arrows point to TuJ1
neurons coexpressing exogenous SOX2-GFP. Blue,
Hoechst. Area values for a given marker were normalized to total Hoechst area (see Experimental Procedures for details). Scale bars, 100 mm; *p < 0.05,
**p < 0.005. Error bars ± SE.
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Key Role of SOX2 in Sensory Neurogenesis
544 Cell Stem Cell 8, 538–551, May 6, 2011 ª2011 Elsevier Inc.
differentiation is ongoing, we observed that, on average, 53% of
neural crest-derived cells transfected with control constructs ex-
pressed the definitive neuronal marker HUC/D within the DRG of
control electroporated embryos (Figure 6A). In contrast, only an
average of 26% of neural crest-derived cells transfected with
Sox2 shRNA stained positive for HUC/D. These results suggest
that Sox2 downregulation in the migratory NC in chick embryos
inhibits neuronal differentiation with DRG.
Next, we used conditional knockout of Sox2 in the mouse NC
lineage using classic Cre recombinase under the control of Wnt1,
which is expressed in NC cells at the time of their initial emigra-
tion (Hollyday et al., 1995). Mice carrying homozygous Sox2
alleles (Favaro et al., 2009), Wnt1:Cre (Danielian et al., 1998), and
Z/EG reporter (Novak et al., 2000) (which activates GFP upon Cre
recombination) revealed a homogeneous GFP expression in E11
DRG cells (Figure 6B). Indeed, the Wnt1-controlled Cre activa-
tion resulted in efficient elimination of Sox2 in E11 ganglionic
cells of Wnt1:Cre/Sox2
mice (Figure 6C). Note that
Sox2 is expressed in E10 DRG predominantly in the dorsal part
of the ganglion. Neuronal differentiation was assessed by immu-
nostaining for the neuronal markers Tuj1 and HuC/D in the DRG
of wild-type (WT) and Sox2 conditional knockout (cKO) mice at
E11 and E14.5. Quantification of serial sections starting from
the trunk region revealed a 25% and 29% reduction respectively
in Tuj1
and HuC/D
neurons at E11 (Figures 6D and 6E) in DRG
of Sox2 cKO mice. At E14.5, serial sections aligned at the level of
the forelimbs were stained for Tuj1 and quantified. Compared to
WT, Sox2 cKO mice displayed a 45% reduction in Tuj1
onic neurons (Figure 6F). Together, changes in Sox2 expression
in NC decreased the numbers of DRG neurons by half both in
chick and in mouse embryos and supported the critical role of
SOX2 in sensory neurogenesis.
SOX2 Regulates Proneural bHLH Genes
Reminiscent of the effects of SOX2 downregulation in hESC-NC
cultures, the absence of proneural factors can trigger apoptosis
during early phases of neurogenesis in vivo (Guillemot et al.,
1993; Ma et al., 1999). We therefore asked whether SOX2 loss
altered expression of bHLH factors such as NGN1 and
MASH1, critical for the development of sensory and autonomic
(sympathetic, parasympathetic, enteric) neurons (Guillemot
et al., 1993; Ma et al., 1999). First, we determined the kinetics
of cell death upon downregulation of SOX2 under neurogenic
conditions using the activation of Caspase3 as readout. No
substantial cell death was observed for the first 3 days, but
massive apoptosis was observed thereafter (Figures 7A and
7B). We then isolated total RNA from day 3 cultures, prior to
the onset of apoptosis, and compared the expression of NGN1
Wnt1:CRE; Z/EG
TUJ1+ neurons/
ganglionic section
HuC/D+ neurons/
ganglionic section
TUJ1+ neurons/
ganglionic section
Wnt1:CRE; Sox2
% GFP+ HuC/D+
in ganglia
Wnt1:CRE; Sox2
Wnt1:CRE; Sox2
Wnt1:CRE; Sox2
Wnt1:CRE; Sox2
Wnt1:CRE; Sox2
Wnt1:CRE; Sox2
Wnt1:CRE; Sox2
Figure 6. SOX2 Downregulation In Vivo Reduces the Number of DRG
(A) Immunohistochemistry for the neuronal marker HuC/D and GFP (labeling
shRNA expressing cells) in electroporated chick embryos; arrows point to
cells coexpressing HuC/D and GFP within DRG. Quantification of HuC/
D-GFP coexpression (graph) revealed a reduction of HuCD
cells in the
embryos electroporated with Sox2-specific shRNA compared to control
shRNA (shCTRL). *p < 0.01 Student’s t test. Scale bar, 30 mm. Error
bars ± SD.
(B) Cre activation in DRG was probed in Wnt1:Cre x Z/EG mice by GFP
staining. At E11, ganglia were homogeneously positive for GFP, demonstrating
Cre activation.
(C) Sox2 expression is efficiently eliminated in murine E11 ganglia of
Wnt1:CRE x Sox2
mice (right panel, compare with Sox2 wild-type
mice, left panel).
(D) Tuj1 expression in E11 ganglia of the wild-type (left panel) and Sox2
conditional knockout mice (right panel); quantification of Tuj1
neurons in
ganglionic sections. n = 23.
(E) HuC/D expression in E11 ganglia of WT (left panel) and Sox2 cKO mice
(right panel); quantification of HuC/D
neurons in ganglionic sections. n = 32.
(F) Tuj1 expression in E14.5 ganglia of the wild-type (left panel) and Sox2
conditional knockout mice (right panel); note the smaller size of the ganglia in
Sox2 mutants at this time point. Quantification of Tuj1
neurons in ganglionic
sections. n = 50. (WT, wild-type; cKO, conditional knock-out). Scale bars:
(B–F), 100 mm; *p < 0.05, **p < 0.005. Error bars ± SE.
Cell Stem Cell
Key Role of SOX2 in Sensory Neurogenesis
Cell Stem Cell 8, 538–551, May 6, 2011 ª2011 Elsevier Inc. 545
Figure 7. SOX2 Regulates Proneural bHLH Genes at the Onset of Neurogenesis
(A and B) Representative images and (B) quantification of active Caspase3 (AC3) staining in NC cells carrying the DOX-inducible SOX2 shRNA at various time
points during neuronal differentiation. SOX2 was knocked down by dox administration on day 0. Day 3 was the last time point tested before a detectable increase
in cell death. All SOX2 knockdown experiments (C and D and H and I) were performed with the cells harvested at day 3 after dox administration.
(C) RT-PCR gene expression analysis for the bHLH proneuronal genes NGN1 and MASH1 in scrambled control (shCTRL) and SOX2 (shSOX2) shRNA-transduced
NC cells (day 3).
(D) qPCR confirmation of the results in (C); normalized to 18S transcripts.
(E) Position of the putative SOX binding sites (black boxes) on the human NGN1 and MASH1 promoters. Transcriptio n initiation sites are shown as +1.
(F and G) Chromatin immunoprecipitation using SOX2-specific antibody demonstrated significant enrichment over the isotype controls, suggest ing a direct
binding of SOX2 at two sites of the NGN1 (F) and MASH1 (G) promoters; *p < 0.05, **p < 0.005.
(H) qPCR analysis of the NOTCH pathway in shSOX2 and shCTRL cells 3 days after dox administration (the onset of neuronal differentiation). Values are
normalized to 18S. Error bars ± SE.
(I) Representative examples from the microarray analysis of gene expression upon downregulat ion of SOX2 by shRNA (day 3).
Cell Stem Cell
Key Role of SOX2 in Sensory Neurogenesis
546 Cell Stem Cell 8, 538–551, May 6, 2011 ª2011 Elsevier Inc.
and MASH1 in cells treated with SOX2 shRNA or control
scrambled shRNA. Both RT-PCR and quantitative PCR (qPCR)
analyses revealed that SOX2 downregulation dramatically
reduced the expression of both of the bHLH transcription factors
analyzed (Figures 7C and 7D). These findings are consistent with
the massive neuronal death upon downregulation of SOX2. Next,
we determined whether SOX2 protein is actually bound to the
promoters of proneural bHLH transcription factors in human
ESC-derived NC cells. We identified three putative SOX2 binding
sites on both the NGN1 and MASH1 promoters (Figure 7E).
Using chromatin immunoprecipitation (ChIP) coupled to qPCR,
we found statistically significant evidence that SOX2 is bound
to NGN1 and MASH1 promoters at least at two out of three puta-
tive SOX binding sites (Figures 7F and 7G). These results suggest
that at the onset of neuronal differentiation, SOX2 functions to
promote cell survival by facilitating the expression of proneural
bHLH factors, likely via direct interaction with their promoter
On the other hand, the NOTCH pathway has long been impli-
cated in the repression of proneural genes (Cau et al., 2000;
Chen et al., 1997; Ishibashi et al., 1994). Upon binding of
NOTCH transmembrane receptors to JAGGED and DELTA
ligands, the NOTCH intracellular domain (NOTCH-ICD) cleaves,
translocates to the nucleus, and forms a complex with RBPJ
transcriptional modulator. NOTCH-ICD/RBPJ complex triggers
the expression of bHLH proteins HES1 and HES5, known to
repress the expression of proneural genes such as MASH1
and NGN1 (Cau et al., 2000; Chen et al., 1997) and block
neuronal differentiation (Ishibashi et al., 1994). We therefore
investigated the effect of SOX2 knockdown on several compo-
nents of the NOTCH pathway. The qPCR analysis revealed that
SOX2 downregulation in dNE cells cultured for 3 days under
neurogenic conditions resulted in 2 fold upregulation of
NOTCH2, JAGGED1, and RBPJ (Figure 7H). The expression of
HES1 and HES5 was upregulated 3- and 2-fold, respectively
(Figure 7H). The NOTCH1 and DELTA1 (DLL1) transcripts were
slightly (30%) downregulated, compared to control shRNA.
These data suggest that, at the population level, SOX2 knock-
down results in activation of the NOTCH pathway, including
HES1 and HES5 genes, providing an alternative mechanism
for the observed downregulation of proneural genes and the
lack of neuronal differentiation.
Identification of SOX2-Interacting Proteins in hESC-NC
To identify potential cofactors of SOX2 during peripheral neuro-
genesis, we performed immunoprecipitation (IP) of the endoge-
nous SOX2-containing complexes from hESC-NC cultures. As
demonstrated in these cultures, SOX2 is only expressed in the
DRG-like clusters (Figures 3 and 4; Figures S1 and S3). Tryptic
peptides from the IP material were subjected to liquid chroma-
tography-tandem mass spectrometry (LC-MS/MS) using an
LTQ Orbitrap Velos mass spectrometer equipped with electron
transfer dissociation (Thermo Fisher Scientific). SOX2 protein
was identified in both biological duplicate IP samples that used
SOX2 specific antibodies and was absent from both biological
replicate IP samples with isotype-matched IgG controls. Meta-
analysis using the NextBio search engine (
identified biogroups and pathways potentially regulated by the
SOX2 interacting proteins (Tables S1–S3). We detected SIX4
and CREB nuclear factors, known to be important in early
neuronal survival during development. Six4 is expressed in
cranial sensory placodes (Kobayashi et al., 2000) and olfactory
placode (Chen et al., 2009); however, Six4 is not expressed in
the neural tube at any time point in development, and Pax3,
a marker of dorsal neuroepithelium, is expressed normally in
the dorsal neural tube of Six4 null embryos, suggesting that pre-
migratory NC field is not perturbed (Grifone et al., 2005). The loss
of Six4 results in massive loss of the sensory neurons in devel-
oping trigeminal ganglia (Konishi et al., 2006) and contributes
to the block of neurogenesis in olfactory placode (
Chen et al.,
Curiously, this is accompanied by the loss of proneuro-
genic transcription factors Ngn1 and Mash1 (Chen et al.,
2009). Therefore, it appears that Sox2-Six4 complex is unique
to peripheral ganglia and, taken together with previously pub-
lished data, our results suggest that Sox2-Six4 complex plays
a critical role in sensory/olfactory neurogenesis. In mouse
CNS, Creb1 deletion leads to neurodegeneration only in a
background (Mantamadiotis et al., 2002). However,
the peripheral nervous system is more sensitive to Creb1 loss
since extensive apoptosis and peripheral neuron loss was seen
during early gangliogenesis in Creb1-deficient mice even in the
presence of the wild-type Crem (Lonze et al., 2002). These
data hint at the existence of critical cofactors of SOX2, with
exclusive or contextual role in DRG compared to CNS tissue.
Meta-Analysis of Gene Expression upon SOX2
To reveal transcriptional changes associated with SOX2 knock-
down in hESC-NC cells, we performed global gene expression
analysis (Illumina microarrays, 24K human referenced genes).
Volcano algorithm (GeneSpring software, Agilent Technologies),
identified 200 transcripts altered over 2-fold upon SOX2
knockdown. NextBio meta-analysis ( identi-
fied biogroups and pathways altered by SOX2 downregulation
(Figure S6). Downregulated biogroups included cell cycle
progression genes, neuronal differentiation and maturation
factors, and cell-cell adhesion genes. Upregulated biogroups
included cell-extracellular matrix adhesion, muscle differentia-
tion pathways, and genes regulated by Serum Response Factor
(SRF). Representative genes of each biogroup are shown in
Figure 7I. Well-known G1/S regulators (CyclinD1, CCND1;
Cyclin-dependent kinase 2, CDK2; and DNA replication regula-
tors, MCM2 and MCM6) were among downregulated genes in
the cell cycle-related biogroups. Several integrins—such as
ITGA5, ITGA10, and ITGA11, which are important for cell adhe-
sion to the fibronectin and collagen matrices—were upregu-
lated, whereas transcripts encoding cell-cell adhesion mole-
cules—such as CDH5 and NINJURIN2 (NINJ2), constitutively
expressed in peripheral sensory and enteric neurons (Araki
and Milbrandt, 2000)—were downregulated upon SOX2
knockdown. Because NC migration is dependent upon the
increased affinity for the extracellular matrix and decreased
cell-cell adhesion (Perris and Perissinotto, 2000), these results
are consistent with the increased delamination and migration
of human dNE cells following SOX2 knockdown (Figure 2).
SOX2 knockdown triggered the downregulation of neurofilament
light and medium polypeptides NEFL and NEFM. Mutations in
the NEFL gene are associated with the peripheral neuropathies
Cell Stem Cell
Key Role of SOX2 in Sensory Neurogenesis
Cell Stem Cell 8, 538–551, May 6, 2011 ª2011 Elsevier Inc. 547
Charcot-Marie-Tooth types 2E and 1F (Jordanova et al., 2003;
Mersiyanova et al., 2000 ). In summary, SOX2 knockdown under
neurogenic conditions triggers the downregulation of molecular
signatures indicative of cell proliferation, cell-cell adhesion and
neuronal differentiation, whereas the genes promoting muscle
differentiation and cell-matrix adhesion were upregulated.
Early stages of human gestation are virtually inaccessible for
experimental research, making hESC cultures a unique model
from which to study the development of human NC lineages.
Here we used hESC-NC to model NC delamination and differen-
tiation into neurons, glia, and smooth muscle. We demonstrated
that downregulation of SOX2 in dNE clusters is sufficient to
induce EMT/migratory NC. These loss-of-function data are
consistent with the previously published gain-of-function data
in which overexpression of SOX2 in chick dNE (premigratory
NC cells) blocks NC delamination (Wakamatsu et al., 2004). Our
results suggest that SOX2 may function downstream of BMP4,
a classical inducer of EMT (Sela-Donenfeld and Kalcheim,
1999), but upstream of ADAM10, a protease known to cleave
the extracellular portion of N-CAD (Shoval et al., 2007).
We found that SOX2 function is required during the onset of
neuronal differentiation, such that downregulation of SOX2 in
hESC-NC cells interfered with their ability to acquire neuronal,
but not glial or mesenchymal, fates. Chromatin IP suggested
that under the culture conditions optimized for neuronal differen-
tiation, SOX2 is bound to the promoter of proneural genes NGN1
and MASH1 in the progenitors of peripheral neurons. The knock-
down of SOX2 under these conditions resulted in dramatic
downregulation of proneural bHLH transcription factors and
massive apoptotic cell death. This is reminiscent of the situation
documented during CNS development where proneural genes
are critical for both the acquisition of neuronal (but not glial) fates
and the survival of young neuroblasts (Cai et al., 2000; Olson
et al., 2001). Moreover, in the periphery, the lack of Ngn1 resulted
in massive neuronal death in the cranial sensory ganglia (Ma
et al., 1999).
In chick, specific downregulation of Sox2 in the migratory NC
cells reduced the numbers of neurons in DRG by 50%.
Because Sox2 was downregulated using shRNA, it is possible
that partial loss of neurons is due to the incomplete elimination
of Sox2 protein. Alternatively, Sox2 might be required for the
generation of a subset of peripheral neurons in DRG. The mouse
conditional knockout data is consistent with the latter scenario.
Conditional ablation of Sox2 using NC specific expression of
Cre (Wnt1-Cre) resulted in reduction of neurons in E14.5 DRG
by 45%, and the ganglia were also smaller in Sox2 mutant
animals at this time point. The requirement of SOX2 for NGN1
expression (as found in human ESC-derived NC cultures,
Figures 7C and 7D) could explain the loss of the Ngn1-depen-
dent subset of DRG neurons, mimicking the Ngn1 knockout
phenotype in the ganglia (Ma et al., 1999). Indeed, at earlier
time points (E11), when primarily the Ngn2-dependent neurons
are formed, only a 25% reduction of TuJ1
cells was seen in
the Sox2 ablated embryos. It will be informative to find out if
the trkA
DRG neurons are predominantly lost in Sox2 ablated
embryos compared to trkB
cells. The lack of NGN1 expres-
sion in human ESC-derived NC would explain a stronger in vitro
phenotype upon SOX2 knockdown, compared to conditional
ablation of Sox2 in mice. It remains possible that in the human
embryo, similarly to that documented in mice, NGN1-expressing
NC cells are able to generate peripheral neurons in the absence
of SOX2.
At E10, Sox2 staining was mainly detected within the dorsal
portion of the DRG, consistent with its role in Ngn1 induction
and initiation of the second wave of neurogenesis in the ganglia
(Montelius et al., 2007). Because the satellite cells, the glial cell
type associated with neuronal cell bodies in the DRG, are
thought to develop during a later period from E10.5–E13.5
(Farinas et al., 2002), it is unlikely that numerous Sox2
observed at E10 are committed glial cells. At E11, 11% of all
cells in ganglia expressed various levels of the early
neuronal marker TuJ1. Comprehensive lineage tracing experi-
ments will be required to determine if a transient upregulation
of Sox2 initiates the Ngn1 expression in the nascent sensory
In hESC-NC, SOX2 knockdown results in upregulation of
NOTCH2, JAG1, HES1, and HES5—known inhibitors of proneu-
ral genes. NOTCH signaling in this case seems to be mediated by
NOTCH2 and JAG1, which have been implicated in the cardiac
myogenic program of neural crest cells (Varadkar et al., 2008),
and in the CNS, Sox2 was shown to directly bind and repress
GFAP expression and glial fate ( Cavallaro et al., 2008). This
mechanism could also contribute to the glial differentiation in
human NC cultures upon knockdown of SOX2.
In developing CNS, both SOX2 overexpression (Graham
et al., 2003) and downregulation (Cavallaro et al., 2008; Miyagi
et al., 2008 ) negatively affect CNS neurogenesis. In chick
embryos, the overexpression of SOX2 DNA binding domain
fused to the Engrailed activator domain (SOX2ER) inhibits the
onset of neurogenesis in the developing CNS and prevents
delamination from the dorsal neural tube (Graham et al.,
2003). These discrepancies may reflect differences in model
organism and experimental approaches employed. For
instance, the overexpression of SOX2ER likely blocks the func-
tion of all SOXB1 members (Episkopou, 2005), while the induc-
tion of SOX2 shRNA did not significantly inhibit the level of
SOX1, and the expression of SOX3 was upregulated (Figure S2).
In addition, SOX2 is likely to function in a dose-dependent and
context-dependent fashion (Pevny and Nicolis, 2010). High
levels of SOX2 expression may reinforce the neural progenitor
characteristics, whereas low levels of SOX2 under neurogenic
conditions may promote the neuronal fates, while suppressing
nonneuronal fates. In our hands, the overexpression of SOX2-
GFP resulted in rescuing the early steps (TuJ1
) but not later
steps (Peripherin
, MAP2
) of peripheral neuronal differentiation
(Figure 5I).
A proneuronal role for SOX2 has been reported in the mamma-
lian CNS. Weak Sox2 expression was observed in Map2
cells in
cultures and neurospheres generated from Sox2 mouse hypo-
morphs in which Sox2 expression was reduced by 30%, and
these cells showed normal gliogenic potential in vitro but
severely reduced neuronal differentiation (Cavallaro et al.,
2008). These authors also found that Sox2 directly binds the
GFAP promoter and suppresses the expression of the GFAP
gene (Cavallaro et al., 2008). In vivo, such mutant mice show
Cell Stem Cell
Key Role of SOX2 in Sensory Neurogenesis
548 Cell Stem Cell 8, 538–551, May 6, 2011 ª2011 Elsevier Inc.
a 40%–60% decrease in GABAergic neurons. Furthermore,
reduced neuronal but not glial differentiation is also seen using
neurospheres derived from mice with conditional ablation of
Sox2 in the CNS (Miyagi et al., 2008).
Our findings imply that disrupted SOX2 function might
be linked to NC-related pathologies, or neurocristopathies.
Although most SOX2 mutations are likely to lead to early embry-
onic lethality (Avilion et al., 2003), some neurocristopathies might
be linked to nonlethal SOX2 loss-of-function mutations, or muta-
tions in genes encoding SOX2-interacting factors or down-
stream effectors. Indeed, nonlethal mutations in human SOX2
(a major cause of anophthalmia/microphthalmia; Fantes et al.,
2003) and the NC-related CHARGE syndrome (Sanlaville and
Verloes, 2007) are often marked by common defects, such as
sensorineural deafness (Hagstrom et al., 2005), which might be
linked to defective peripheral neurogenesis. In such cases,
modulating SOX2 expression and function might help develop
therapeutic applications.
Derivation, Maintenance, and Differentiation of Human dNE Cells
dNE was generated from hEC as described (Cimadamore et al., 2009).
propagation, cells were seeded onto Matrigel-coated plates (BD Biosciences,
final concentration = 1:30, 2h coating at room temperature) using base
medium (1:1 ratio of DMEM/F12 Glutamax-neurobasal medium [GIBCO], 2%
B27 supplement without vitamin A [GIBCO], 10% BIT 9500 [StemCell Technol-
ogies], and 1 mM glutamine [GIBCO]) supplemented with 20 ng/ml EGF
(Chemicon), 20 ng/ml bFGF, 5 mg/ml insulin (Sigma) and 5 mM nicotinamide
For segregation between epithelial and mesenchymal cells, accu-
tase-dissociated dNE cells were seeded onto FN-coated plates (1 mg/ml, over-
night coating) at a density of 45,000 cells/cm
in the presence of EGF 100 ng/
ml for 12 days. For differentiation into smooth muscle cells, dNE cells were
seeded onto FN-coated plates (1 mg/ml, overnight coating) at a density of
45,000 cells/cm
in base medium supplemented with 40 ng/ml EGF. Cells
were allowed to differentiate for 7 days.
Gliogenic conditions were obtained
by seeding dNE cells at 20,000 cells/cm
in FN-coated plates in base medium
supplemented with 1% horse serum. Cells were allowed to differentiate for
12 days. For neuronal differentiation (
neurogenic conditions), dNE cells were
seeded onto FN-coated plates at 45,000 cells/cm
in base medium supple-
mented with 40 ng/ml bFGF and 40 ng/ml BDNF and allowed to differentiate
for 14 days. Other conditions were as described in the text. For neuro-
sphere-based migration assays, hES-derived neurospheres were plated on
different substrates as described and cells allowed to migrate for a minimum
of 1 day to a maximum of 4 days on laminin/polyornithine-coated plates in
base media supplemented with 20 ng/ml EGF, 20 ng/ml bFGF, 5 mg/ml insulin,
and 5 mM nicotinamide.
ChIP and qPCR
ChIP was performed using the Ez-ChIP kit (Millipore) according to the manu-
facturer’s recommendations with the following modifications: 2 3 10
were used for each immunoprecipitation, cells were sonicated in order to yield
chromatin fragment of 200–500 bp, and 5 mg of immunoprecipitating anti-
bodies were employed in each ChIP. Antibodies were rabbit anti-SOX2
(Millipore AB5603) and normal rabbit IgG (Millipore PP64) as a nonspecific
control. qPCR amplification was performed with site-specific primers de-
signed to flank the putative SOX binding sites (Supplemental Experimental
Procedures). qPCR values were analyzed with the D(DCT) method and
normalized to the values obtained with the nonspecific antibody.
Sox2 Manipulation in Chick Embry os
Fertilized chicken embryos (Gallus gallus domesticus) were obtained from
McIntyre Farms, Lakeside, CA, and incubated in a 38
C humidified incubator
until HH10 according to the staging of Hamburger (Hamburger, 1988). miR30
plasmid (4 mg/ml concentration) was injected by air pressure into the neural
tube of the embryo in ovo using a pulled glass needle. Platinum electrodes
were placed across the neural tube and a current of 3 3 21 V of 50 ms in
100 ms intervals was used to electroporate the cells on one half of the neural
tube. Embryos were resealed and reincubated a further 24 hr.
Transgenic Mice
Mice carrying Sox2
alleles (Favaro et al., 2009) were crossed with the mice
expressing Cre recombinase under the control Wnt1 promoter (wnt1:Cre). A
total of 23 sections were used for Tuj1 quantification at E11, 32 sections for
Hu/CD quantification E11, and 50 sections for Tuj1 quantification at E14.5.
mice were crossed to Z/EG mice (Jackson Laborato-
ries), which activates GFP upon Cre recombination to monitor Wnt1:Cre
activity in DRG. Additional details are available in Supplemental Experimental
Supplemental Information includes six figures, one table, and Supplemental
Experimental Procedures and can be found with this article online at doi:
We thank C.-T. Huang and K. Liu for their help with cloning, lentiviral produc-
tion, and microarray analysis. We thank Dr. S. Albini, Dr. S. Forcales, and
Professor L. Puri for sharing their expertise in chromatin immunoprecipitation
techniques. Sox2LoxP mice were kindly provided by Dr. Nicolis, and Wnt1-
CRE mice were kindly provided by Dr. Y. Yamaguchi. We thank Dr. J. Hou
for helping with IP-MS data analysis. This work has been supported by
CIRM postdoctoral fellowship to F.C., CIRM grant RS1004661 to A.T., and
transient research support to A.V. Terskikh from the Sanford-Burnham
Medical Research Institute and an NIH Blueprint core grant (PI, S.A. Lipton).
Received: July 16, 2010
Revised: January 26, 2011
Accepted: March 4, 2011
Published: May 5, 2011
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    • "SRY-related HMG-box 2 gene (Sox2), a member of the SoxB transcription factor family [45][46][47] , is a fundamental factor in self-renewal and multipotency of embryonic and adult neural stem cells (NSCs). It plays key roles during CNS development, such as in survival, proliferation and maintenance of NSCs [48][49][50], as well as in the acquisition of neural/glial identity [51][52][53][54][55][56][57][58][59][60][61]. As expected from the key role of Sox2 in neural progenitor cells (NPCs), previous studies have shown that early in neural tube development, Sox2 is expressed along the entire hindbrain [62, 63]. "
    [Show abstract] [Hide abstract] ABSTRACT: Background: Compartment boundaries are an essential developmental mechanism throughout evolution, designated to act as organizing centers and to regulate and localize differently fated cells. The hindbrain serves as a fascinating example for this phenomenon as its early development is devoted to the formation of repetitive rhombomeres and their well-defined boundaries in all vertebrates. Yet, the actual role of hindbrain boundaries remains unresolved, especially in amniotes. Results: Here, we report that hindbrain boundaries in the chick embryo consist of a subset of cells expressing the key neural stem cell (NSC) gene Sox2. These cells co-express other neural progenitor markers such as Transitin (the avian Nestin), GFAP, Pax6 and chondroitin sulfate proteoglycan. The majority of the Sox2(+) cells that reside within the boundary core are slow-dividing, whereas nearer to and within rhombomeres Sox2(+) cells are largely proliferating. In vivo analyses and cell tracing experiments revealed the contribution of boundary Sox2(+) cells to neurons in a ventricular-to-mantle manner within the boundaries, as well as their lateral contribution to proliferating Sox2(+) cells in rhombomeres. The generation of boundary-derived neurospheres from hindbrain cultures confirmed the typical NSC behavior of boundary cells as a multipotent and self-renewing Sox2(+) cell population. Inhibition of Sox2 in boundaries led to enhanced and aberrant neural differentiation together with inhibition in cell-proliferation, whereas Sox2 mis-expression attenuated neurogenesis, confirming its significant function in hindbrain neuronal organization. Conclusions: Data obtained in this study deciphers a novel role of hindbrain boundaries as repetitive pools of neural stem/progenitor cells, which provide proliferating progenitors and differentiating neurons in a Sox2-dependent regulation.
    Full-text · Article · Dec 2016
    Yuval PeretzYuval PeretzNoa ErenNoa ErenAyelet KohlAyelet Kohl+1more author...[...]
    • "From another point of view, in the processes of EMT and migration, the expression of Sox2 is downregulated, since SOX2 is a strong inhibitor of EMT and delamination [55]. However, migratory neural crest cells transiently re-express Sox2 when they reach the DRG, and re-downregulate it to differentiate for peripheral neurons [55]. In this manner, neural crest cells undergo a reversible EMT process, namely mesenchymal–epithelial transition (MET). "
    [Show abstract] [Hide abstract] ABSTRACT: Epithelial-mesenchymal transition (EMT) and cell migration are important processes in embryonic development of many tissues as well as oncogenesis. The pituitary gland is a master endocrine tissue and recent studies indicate that Sox2-expressing stem/progenitor cells actively migrate and develop this tissue during embryogenesis. Notably, although migration activity of stem/progenitor cells in the postnatal period seems to be reduced compared to that in the embryonic period, it is hypothesized that stem/progenitor cells in the adult pituitary re-migrate from their microenvironment niche to contribute to the regeneration system. Therefore, elucidation of EMT in the pituitary stem/progenitor cells will promote understanding of pituitary development and regeneration, as well as diseases such as pituitary adenoma. In this review, so as to gain more insights into the mechanisms of pituitary development and regeneration, we summarize the EMT in the pituitary by focusing on the migration of pituitary stem/progenitor cells during both embryonic and postnatal organogenesis.
    Article · Apr 2016
    • "SOX10 and FOXD3 are strongly expressed by premigratory neural crest (Figure 1A ) and associated with maintenance of multipotency (Kim et al., 2003Kim et al., , 2014 Nitzan et al., 2013; Teng et al., 2008). In contrast, neural stem cell marker SOX2 is downregulated in dorsal relative to ventral neural tube regions (Figure 1A), albeit required at low levels for neural crest EMT (Cimadamore et al., 2011). Transcript levels were compared with those in whole embryo lysates using qPCR (see the Supplemental Results;Figure S1A). "
    [Show abstract] [Hide abstract] ABSTRACT: Premigratory neural crest cells comprise a transient, embryonic population that arises within the CNS, but subsequently migrates away and differentiates into many derivatives. Previously, premigratory neural crest could not be maintained in a multipotent, adhesive state without spontaneous differentiation. Here, we report conditions that enable maintenance of neuroepithelial "crestospheres" that self-renew and retain multipotency for weeks. Moreover, under differentiation conditions, these cells can form multiple derivatives in vitro and in vivo after transplantation into chick embryos. Similarly, human embryonic stem cells directed to a neural crest fate can be maintained as crestospheres and subsequently differentiated into several derivatives. By devising conditions that maintain the premigratory state in vitro, these results demonstrate that neuroepithelial neural crest precursors are capable of long-term self-renewal. This approach will help uncover mechanisms underlying their developmental potential, differentiation and, together with the induced pluripotent stem cell techniques, the pathology of human neurocristopathies.
    Full-text · Article · Oct 2015
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