Cortical Neural Precursors Inhibit
Their Own Differentiation via N-Cadherin
Maintenance of b-Catenin Signaling
Jianing Zhang,1,3Gregory J. Woodhead,1,3Sruthi K. Swaminathan,1Stephanie R. Noles,1Erin R. McQuinn,1
Anna J. Pisarek,1Adam M. Stocker,1Christopher A. Mutch,1Nobuo Funatsu,2and Anjen Chenn1,*
1Department of Pathology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA
2Department of Pathology, University of California, Irvine, CA 92697, USA
3These authors contributed equally to this work
Little is known about the architecture of cellular
microenvironments that support stem and precursor
cell niches are organized by specialized supporting
cells, in the developing cerebral cortex, neural stem/
precursor cells reside in a neurogenic niche lacking
distinct supporting cells. Here, we find that neural
precursors themselves comprise the niche and regu-
late their own development. Precursor-precursor
contact regulates b-catenin signaling and cell fate.
In vivo knockdown of N-cadherin reduces b-catenin
signaling, migration from the niche, and neuronal
differentiation in vivo. N-cadherin engagement acti-
anism through which cells in tissues can regulate
their development. These results suggest that neural
precursor cell interactions can generate a self-
supportive niche to regulate their own number.
Specialized microenvironments or niches coordinate the specifi-
cation, self-renewal, and differentiation of stem cells (Fuchs
et al., 2004). Adult stem cell niches are distinct anatomical struc-
tures organized by specialized cells that create a supportive
habitat for stem cells (Moore and Lemischka, 2006). In contrast,
whether similar microenvironment structures regulate stem and
precursor cells in developing tissues remains poorly understood
(Alvarez-Buylla and Lim, 2004).
In the developing cerebral cortex, neural precursors reside in
the ventricular zone (VZ), a neurogenic layer of epithelial cells
that lines the neural tube. Residence in the VZ appears to sustain
the proliferation of neural precursor cells, as when neural precur-
sors are removed from the VZ, nearly all exit the cell cycle and
differentiate (Temple, 2001). The observation that the VZ, unlike
adult stem cell niches, consists almost exclusively of dividing
precursors (Takahashi et al., 1993) suggested the possibility
that the cortical precursors themselves could provide their own
supportive environment in development.
Physical contact between stem cell and niche appears crucial
to stem cell self-renewal. Recent studies indicate that adhesion
mediated by adherens junctions anchor germline stem cells to
the Drosophila ovary stem cell niche (Song et al., 2002), while
similar adhesion complexes have been described between
hematopoietic stem cells and their niche in the bone marrow
(Zhang et al., 2003). In contrast, instead of adhering to special-
ized supporting cells, cerebral cortical neural precursors link to
adjacent precursors through adherens junctions (Chenn et al.,
1998). While adherens junctions appear to physically anchor
stem cells to their niches and cortical neural precursors to
each other, how adherens junctions might regulate signaling
pathways that control stem cell renewal remains poorly under-
stood. Here, we examine the role of the adherens junctions
proteins N-cadherin and b-catenin in the neurogenic niche of
the developing cerebral cortex.
b-Catenin Signaling Characterizes the Developing
Cortical Neural Precursor Niche
During cortical development, b-catenin signaling can regulate
neural precursor proliferation and differentiation (Chenn and
Walsh, 2002; Hirabayashi et al., 2004; Woodhead et al., 2006).
environment of developing neural precursors, we first character-
ized endogenous b-catenin signaling in the developing cortex. In
situ hybridization for b-gal in the BAT-Gal transgenic reporter
mouse line (Maretto et al., 2003) and GFP expression in the
Axin2-dGFP transgenic reporter mice (Jho et al., 2002) revealed
oping dorsal telencephalon in a medial high to lateral low
gradient, while activity was absent from ventral structures
(Figures 1A and 1B).
To examine b-catenin signaling at a cellular level, we used a
b-catenin reporter construct that expresses a destabilized green
acterized b-catenin responsive promoter, TOPdGFP (Dorsky
et al., 2002), and confirmed that cells in the VZ throughout the
lateral, dorsal, and medial cortical walls showed expression of
destabilized GFP (Figure 1C). Electroporation of a modified
construct containing both mCherry and TOPdGFP (pDual,
Figure 1D) on a single plasmid revealed that signaling neural
472 Developmental Cell 18, 472–479, March 16, 2010 ª2010 Elsevier Inc.
precursors had prominent apical endfeet connecting the cell
body to the ventricular lumen (Figure 1E).
This observation supported the possibility that components
an expression construct driving an N-cadherin-GFP fusion re-
electroporation) N-cadherin in individual precursors enriched at
the apical endfeet, consistent with localization at adherens junc-
tions (Figure 1F, arrowhead). Together, these observations
reveal (1) the presence of b-catenin signaling in the developing
cortical VZ, and (2) coexpression of N-cadherin with b-catenin
signaling in the VZ.
N-Cadherin Regulates b-Catenin Signaling in Cortical
Previous studies have shown that b-catenin signaling in VZ
precursors regulates precursor number by influencing cell-cycle
exit (Chenn and Walsh, 2002; Woodhead et al., 2006). What
regulates b-catenin signaling in the ventricular zone niche?
The observation that neural precursor contact with other
precursors stimulates proliferation (Temple and Davis, 1994)
suggested to us the possibility that cell contact in the niche
might promote b-catenin signaling. To test whether neural
precursor cell contact regulates b-catenin signaling, we exam-
ined the activity of the b-catenin-responsive reporter construct
pSuper8TOPFLASH (DasGupta et al., 2005) in primary cortical
cultures at varying cell densities and observed that high cortical
cell density upregulates b-catenin signaling (Figure 2A). Western
blot analysis confirmed that increased b-catenin signaling was
accompanied by increased active (unphosphorylated) b-catenin
(van Noort et al., 2002) with higher cell densities (Figure 2B).
b-catenin signaling in low-density cells was not restored by
coculture, with a high density of cells separated by a Transwell
compartment (Shen et al., 2004) despite an equivalent overall
density of cells in the culture, suggesting that cell contact or
close proximity is necessary to stimulate signaling (Figure 2C).
Because many factors might also regulate density-dependent
changes in signaling in neural precursors, we decided to
examine the specific role of N-cadherin in b-catenin signaling
in high-density cultures of primary neural precursors. We found
that blocking N-cadherin function with 5 mM EGTA (Figure 2D),
shRNA (Figures 2E and 2G), or function-blocking N-cadherin
antibody (Figure 2F) reduced b-catenin signaling. Finally, we
also examined the regulation of Axin2, an endogenous target
of b-catenin signaling, using an Axin2 reporter construct driving
luciferase (Jho et al., 2002). Cotransfection of this reporter
construct along with three different shRNA’s against N-cadherin
(versus their respective controls) confirmed the findings that
N-cadherin knockdown reduced b-catenin-mediated signaling
To examine the requirement for N-cadherin in b-catenin
signaling in vivo, we performed loss-of-function studies using
in utero electroporation of short hairpin constructs targeting
construct with the N-cadherin shRNA knockdown construct re-
vealed that b-catenin signaling was reduced by N-cadherin
these studies (calcium dependence, function-blocking antibody,
shRNA knockdown, and Axin2 responsiveness) suggest that the
endogenous activation of b-catenin by neural precursor cell
contact utilizes N-cadherin.
Figure 1. Relationship of N-Cadherin Expression and b-Cat-
enin Signaling in Cortical Precursors
(A) In situ hybridization for b-gal transcript from transgenic BAT-Gal
reporter mouse (E14.5 cortex).
(B)Expression ofGFPincortexofAxin2-dGFP reporter mouse.Higher
power confocal images shows enrichment of b-gal and GFP signal in
VZ, with reduced expression in IZ and CP.
(C) b-catenin signaling revealed by coelectroporation of TOPdGFP
reporter construct (green) and mCherry (red) of E14.5 brain. Low-
power image reveals that electroporated regions of lateral, dorsal,
and medial cortical VZ (highlighted by coelectroporation of mCherry)
are characterized by b-catenin signaling (green).
(D) Schematic of electroporation protocol used in (C): DNA is injected
into lateral ventricle and is introduced into the adjacent neuroepithelial
cells directed toward the positive electrode, aligned outside the brain
as shown. Cartoons of reporter constructs used in these experiments
(E)Dual TOPdGFP/mCherry reporter (Figures 1D;Figure S1)highlights
b-catenin signaling in precursor cell bodies and apical foot processes
(arrowheads). Bar = 20 mm.
(F) Antibody staining for N-cadherin in the E13.5 mouse cortical plate
reveals staining throughout the developing cortical plate. Acute anal-
ysis of coelectroporation with GFP-tagged N-cadherin (right panels)
and red fluorescent protein reveals N-cadherin labels VZ precursor
cell membranes and is enriched at apical junctions (arrowhead)
14 hr after electroporation. Bars = 20 mm.
N-Cadherin Maintains the Cortical Progenitor Niche
Developmental Cell 18, 472–479, March 16, 2010 ª2010 Elsevier Inc. 473
N-Cadherin Knockdown In Vivo Causes Premature
Migration and Neuronal Differentiation
To examine the effects of reducing N-cadherin on neural
precursor cell fate in vivo, we examined the migration and differ-
entiation of cells following in utero electroporation of shRNA
against N-cadherin. We observed that reduction of N-cadherin
resulted in increased cell migration from the VZ (Figures
3A–3D), premature neuronal differentiation (Figures 3A–3C and
3E), and increased cell cycle exit (Figure 3F, G). We found that
restoring b-catenin signaling by introducing a stabilized, active
form of the protein (D90b-catenin) along with N-cadherin shRNA
found no evidence that N-cadherin reduction led to non-cell-
autonomous effects on cell proliferation when nonelectropo-
rated cells were analyzed (Figure S3). Together, the findings
that N-cadherin reduction leads to reduction of b-catenin
signaling, increased neuronal differentiation, and increased
cell-cycle exit suggest that N-cadherin interactions between
Figure 2. N-Cadherin Maintains b-Catenin Signaling in Cortical Precursors
(A) b-catenin reporter assays in E14.5 rat primary cortical cultures transfected with pSUPER8TOPFLASH, with fold induction relative to signal from lowest cell
density condition. Cell densities of 1, 2.5, 5, 10, and 20 3 105/ml represent 2.5, 6.25, 12.5, 25, and 50 3 104/cm2, respectively. (A) p < 0.001 by ANOVA;
N = 11 experiments (1, 10, 20 3 105/ml), N = 2 experiments (2.5, 5 3 105/ml). ***p < 0.001 for 20 3 105/ml versus all other densities except p < 0.01 versus
5 3 105condition. Neuman Keuls Post-hoc test.
(B) High cell density increases the amount of dephosphorylated b-catenin.
(C) Signaling is not stimulated in cultures of 1 3 105/ml cells with 2 3 106/ml cocultured above in a Transwell chamber. p = 0.0016, N = 3 experiments; all pairwise
comparisons p < 0.05 except **p < 0.01 for 2 3 106/ml versus 1 3 105/ml or 1 3 105/ml + coculture conditions and p > 0.05 1 3 105/ml versus 1 3 105/ml +
(D) b-catenin signaling at high cell densities requires extracellular calcium. p = 0.0041; N = 3 experiments. **p < 0.01 2 3 106/ml versus 2 3 106/ml + 5 mM EGTA;
p > 0.05 for 1 3 105/ml versus 2 3 106/ml + 5 mM EGTA.
(E) shRNA to N-cadherin reduces b-catenin signaling. p = 0.0226; N = 3 experiments. * indicates p < 0.05 for NCAD shRNA versus EGFP or shRNA against EGFP.
(F) Function-blocking antibody to N-cadherin inhibits b-catenin signaling. p = 0.0036; N = 3 experiments. ** indicates p < 0.01 for function-blocking antibody
versus untreated or control IgG.
(G) shRNA to N-cadherin reduces N-cadherin protein levels; shRNA constructs (NCAD, from Maeda et al., 2005) versus shRNA against EGFP (middle) or clone
13176 from Open Biosystems versus corresponding nonsilencing shRNA control in primary neural precursors. Western blots performed on E13.5 mouse primary
cortical precursors nucleofected with either control or N-cadherin knockdown shRNA constructs and were cultured for 24 hr before lysing cells. Western blots
repeated three independent replicates with similar results.
(H–J) Axin2 Luciferase reporter is regulated identically as SUPER8TOPFLASH. E14.5 primary cortical cultures at 2 3 106/ml transfected with Axin2 Luciferase
reporter show activation of reporter by stabilized b-catenin (D90b-catenin versus GFP) (H), showing responsiveness of this reporter to b-catenin signaling. Three
repeated-measures ANOVA; ** indicates p < 0.01 versus nonsilencing shRNA.
(K) Control E14.5 mouseforebrain coelectroporated with TOPdGFP (green),mCherry (red),and nonsilencing shRNA construct. Green (yellow upon merge) repre-
by Student’s t test; n = 4 brains.
images; Bar = 200 mm (A and B), 20 mm. Error bars represent 1 SEM.
N-Cadherin Maintains the Cortical Progenitor Niche
474 Developmental Cell 18, 472–479, March 16, 2010 ª2010 Elsevier Inc.
ing b-catenin signaling.
N-Cadherin Regulates b-Catenin Signaling through Akt
We observed that N-cadherin shRNA, function-blocking anti-
bodies (Figure 2), and inhibitors of canonical Wnt signaling
(Figure S4A) could all reduce endogenous b-catenin signaling.
However, blocking N-cadherin did not inhibit Wnt-stimulated
activation of b-catenin (Figure S4B), suggesting that N-cadherin
function in b-catenin signaling might utilize an alternative non-
Wnt-mediated mechanism. Increasing evidence suggests that
specific posttranslational modifications (such as specific phos-
creases Neuronal Differentiation and Cell
Cycle Exit In Vivo
(A–E) E13.5 mouse forebrain coelectroporated in
uterowithpCAG-GFP and 4-fold (by mass)excess
of nonsilencing shRNA construct (control) or
glial marker Pax6 (A), intermediate progenitor
marker Tbr2 (B), and early postmitotic neuronal
marker Tbr1 (C). Electroporated cells are green,
and the respective antigens, red. Bar = 50 mm.
Cell histograms represent mean fraction of total
electroporated cells found in each brain region,
showing increased exit from the VZ (D) or the frac-
poration, showing premature neuronal differentia-
tion (E). For distribution, shRNA versus control
c2= 58.4, p < 0.0001 (control n = 4 brains,
1060 cells; shRNA n = 3 brains, 527 cells). For
cell identity, shRNA versus control c2= 172,
p < 0.001 (control n = 2 brains, 1887 cells; shRNA
n = 4 brains, 2226 cells). Error bars = 1 SEM.
(F and G) N-cadherin knockdown reduces cell
cycle re-entry and is rescued by stabilized
b-catenin. E13.5 embryos electroporated with
control nonsilencing shRNA, N-cadherin shRNA,
or N-cadherin shRNA and stabilized b-catenin
labeled green, and Ki67 staining, red. Bottom
images in (F) show the visual representation of
the individual cells where all cells targeted by elec-
troporation that also express Ki67 are labeled red,
whileelectroporated cellsthatdo notexpressKi67
are white. (G) Histogram displaying the proportion
of electroporated cells expressing Ki67 ± SEM
(n = 3 brains each, 2490 cells total). p = 0.0028
by repeated-measures ANOVA; **p < 0.01 by
Newman Keuls post-hoc test.
3. N-CadherinKnockdown In-
phorylation) of b-catenin can contribute
tinal stem cells, b-catenin phosphoryla-
tion at Ser552 (via AKT) leads to its stabi-
lization and nuclear localization (He et al.,
2004). As N-cadherin adhesion can lead
to activation of the phosphatidylinositol
3-kinase/AKT pathway (Tran et al., 2002),
we reasoned that this pathway might contribute to the N-cad-
herin-dependent activation of b-catenin we observed in neural
Using an antibody that recognizes b-catenin phosphorylated
at Ser552, we observed that b-catenin Ser552P was found in
dividing neural precursors in the VZ (Figure 4A). Costaining
with the mitotic marker phosphorylated histone H3 (pHH3) re-
vealed that the b-catenin Ser552P-expressing cells were mitotic
cells (Figure 4A). These findings suggested that N-cadherin
could activate b-catenin via AKT-mediated phosphorylation at
We investigated whether Akt might link N-cadherin to b-cate-
nin activation in cortical precursors. We found that function-
blocking antibodies to N-cadherin (Figure 4B) or shRNA to
N-Cadherin Maintains the Cortical Progenitor Niche
Developmental Cell 18, 472–479, March 16, 2010 ª2010 Elsevier Inc. 475
N-cadherin (Figures S4C and S4D) led to a significant reduction
in phosphorylated (active) Akt in primary cortical precursors. To
test the link between Akt activation and phosphorylation of
b-catenin at Ser552, we inhibited Akt in neural precursors using
triciribine (API-2), a small molecule Akt pathway inhibitor (Yang
et al., 2004). Triciribine treatment of primary cortical precursors
reduced the fraction of cells expressing b-catenin Ser552 in
a dose-dependent fashion (Figure 4C). Finally, expression of
a dominant-negative (kinase-dead) Akt also reduced both base-
(Figure 4D). To confirm whether Akt functions downstream of
N-cadherin to mediate b-catenin signaling, we coexpressed
myristoylated (active) Akt along with shRNA to N-cadherin and
measured b-catenin signaling by TOP-flash reporters. We found
that that myrAkt rescued b-catenin signaling following N-cad-
herin knockdown (Figure 4E). We also found that myrAkt alone
could increase b-catenin signaling, a finding consistent with
Figure 4. b-Catenin Ser552P in the Developing
Ventricular Zone and Interactions of N-Cad-
herin, Akt, and b-Catenin Signaling
(A) Staining (purple) for b-catenin Ser552P shows
nuclear staining in mitotic cells along the ventricle
(higher magnification of inset shown). Immunofluores-
cence reveals b-catenin Ser552P-expressing cells
(green) costain for the mitotic marker phosphorylated
Histone H3 (pHH3, red; costained cells, yellow).
Higher magnification confocal optical sections of inset
area (bottom) confirm colocalization. Bar = 200 mm
and 20 mm.
(B) Inhibition of N-cadherin engagement with function-
blocking antibody on primary neural precursors leads
to reduction of Ser473P (active) Akt by western blot;
quantitation, right. N = 3; p = 0.0467 by paired t test.
(C) Inhibition of Akt activity with triciribine reduces
number of b-catenin Ser552P in primary cortical
precursor culture at 24 hr; N = 3; p = 0.0094 by
repeated-measures ANOVA; *p < 0.05; **p < 0.01
versus untreated, Newman Keuls post-hoc.
(D) Dominant-negative (kinase dead, HA-Akt-K179M)
Akt inhibits endogenous (N = 4; p = 0.0402) and Wnt-
stimulated b-catenin signaling (N = 4; p = 0.0284) in
TOPFlash reporter assay (Paired t test).
(E) Inhibition of b-catenin signaling by N-cadherin
shRNA is rescued by myristoylated (active) Akt. N = 3;
p < 0.0001 by repeated-measures ANOVA; **p < 0.01;
***p < 0.001 by Newman-Keuls post-hoc. In (D)
and (E), luciferase signal from pSUPER8TOPFLASH
is normalized tocotransfected
TCF-1 binding sites, and is unresponsive to b-catenin
signaling (as in Figure 2; see also Figure S1).
(F) Illustration of the interaction of the N-cadherin and
Wnt regulation of b-catenin signaling. N-cadherin
engagement activates Akt via phosphorylation at
Ser473. Active Akt can lead to increased b-catenin
stability indirectly by Ser9 phosphorylation and inhibi-
tion of GSK3b or directly by phosphorylation of b-cat-
enin at Ser552. Wnt signaling leads to inactivation of
phosphorylated b-catenin. Wnts may also regulate
N-cadherin levels (Toyofuku et al., 2000; Tufan and
Tuan, 2001). N-cadherin engagement may also facili-
association of neural precursor cells.
(G) Model of the VZ niche in development. VZ precur-
sors exhibit b-catenin signaling (green) and are joined
to each other via N-cadherin adherens junctions (red).
Following N-cadherin reduction, b-catenin signaling is
also reduced, causing increased neuronal differentia-
tion and migration into the intermediate zone (IZ)
toward the cortical plate.
Error bars represent 1 SEM.
N-Cadherin Maintains the Cortical Progenitor Niche
476 Developmental Cell 18, 472–479, March 16, 2010 ª2010 Elsevier Inc.
the idea that this pathway may exist in parallel with the canonical
Wnt signaling pathway. Together, these observations suggest
that N-cadherin engagement leads to phosphorylation of Akt
and subsequent Akt-mediated phosphoryation and activation
Here, we demonstrate (1) a type of niche regulation where neural
precursor cells generate their own self-supportive niche and (2)
signaling through Akt in neural precursors. Our observations
suggest that neural precursors comprise a niche in which the
precursor cells themselves function as supporting cells to
provide evidence that neural stem/precursor cell self-renewal is
promoted by signals produced by the cells themselves and
suggest that N-cadherin is a crucial mediator of precursor-
precursor signaling. Inhibition of N-cadherin leads to reduction
of b-catenin signaling, premature neuronal differentiation, cell
cycle exit, and increased migration toward the developing
Although it has been suggested that cell contact and cadherin
stabilization leads to reduction of b-catenin signaling by titration
of cytoplasmic b-catenin (Nelson and Nusse, 2004), we found
that high cortical cell density surprisingly upregulates b-catenin
signaling in a fashion requiring N-cadherin. Our studies suggest
a model in which N-cadherin engagement leads to Akt activa-
tion. Akt-dependent phosphorylation of b-catenin at Ser552
results in stabilization of b-catenin and increased transcriptional
activation (Figure 4F).
Our observations that N-cadherin regulates maintenance of
precursors in the niche support studies of other stem cell types
(Zhang et al., 2003), suggesting that N-cadherin mediates
attachment of stem cells to their niches. However, the devel-
oping cortical VZ is unusual among most well-characterized
ing cells, but of other precursor cells. Instead of facilitating inter-
actions between stem cells and supporting cells, N-cadherin in
the VZ mediates interactions between proliferating precursors
with each other. While our studies support findings that cell-
cell contact in tissues regulates key signaling pathways during
differentiation by mediating a ‘‘community effect’’ (Gurdon,
1988), adult stem cell niches are characterized by distinct sup-
porting cells and microenvironmental structures (Fuchs et al.,
2004; Moore and Lemischka, 2006). Although whether adult
stem cell self-interactions also have self-supportive function or
share molecular mechanisms is not known, high local cell
density of human embryonic stem cells promotes self-renewal
and inhibits differentiation (Peerani et al., 2007).
Other factors that regulate neural precursors may function
similarly; endothelial factors that promote neural precursor
self-renewal lead to increased precursor cell contact and
increased b-catenin levels (Shen et al., 2004), and Numb and
Numblike proteins can regulate N-cadherin function (Rasin
et al., 2007). Although disruption of epithelial integrity and cell
polarity can cause hyperproliferation (Bilder et al., 2000), leading
to suggestions that cell contact negatively regulates proliferation
(Lien et al., 2006), our findings that N-cadherin can regulate cell
fate and signaling even in areas without tissue disorganization
suggest a more complex regulation of proliferation and cell
fate bycell adhesion molecules. Our studies support recent find-
ings suggesting that disruptions of adherens junctions and cell
polarity in VZ progenitors lead to premature differentiation (Cap-
pello et al., 2006; Yokota et al., 2009). Alterations in N-cadherin
signaling may mediate the alterations in cell fate observed
when adherens junctions are disrupted and cell polarity is lost.
In addition to cell-cell adhesion, cell-to-extracellular-matrix
adhesion also appears critical in maintaining VZ architecture
(Loulier et al., 2009). Further understanding of how precursor
cells integrate signals from cell density and their microenviron-
ment will lend insight into the mechanisms that underlie the
growth of cells during development as well as the regulation of
Statistical analysis was performed with Graphpad Prism, with error bars on all
graphs representing 1 SEM.
In Utero Electroporation
In utero electroporation and cell analysis performed as in Woodhead et al.,
2006 (plasmid and antibody information listed in Supplemental Information).
For in vivo TOP-d-GFP signaling studies, coelectroporation with DN-TCF4
and ICAT confirmed specificity (Woodhead et al., 2006).
Cell Culture and Luciferase Assays
Primary cortical cultures and luciferase assays performed as described in
Noles and Chenn, 2007. For coculture assays, 1 3 106cells were placed
onto 6.5 mm transwell membrane inserts with 0.4 mm pore size (Costar)
above freshly plated and transfected cells at 1 3 105/ml (2.5 3 104/cm2) as
in Shen et al., 2004. For function-blocking antibodies, cultures at high density
blocking antibody (De Wever et al., 2004; Makrigiannakis et al., 1999; Waller-
and et al., 2008) (ACAM, GC-4, Sigma) or control IgG1k isotype control (BD
PharMingen catalog number 554721) 5 hr after transfection of reporter
constructs. For protein analysis, 5 million primary cortical precursors were
transfected by AMAXA Nucleofection (Amaxa Biosystems) following manufac-
turer protocols. For triciribine treatment, primary cortical cells were plated at
a density of 2 million/ml and immediately treated with 0, 1, 5, or 10 mM triciri-
bine (EMD) for 24 hr.
Cell-Cycle Re-Entry Studies
E13.5 embryonic cortices were electroporated with Ncad-shRNA or control
shRNA, together with pCAG-EGFP as control. For the rescue experiment,
Ncad-shRNA, pCAG-D90b-catenin-GFP or pCAG-EGFP was electroporated.
After 48 hr, the E15.5 embryonic brains were dissected, fixed, cryosectioned,
and stained for GFP and ki67 as in Stocker and Chenn, 2009.
Supplemental Information includes four figures and Supplemental Experi-
mental Procedures and can be found with this article online at doi:10.1016/
The first two authors contributed equally to this study. Supported by the
NINDS RO1 NS047191, Searle Scholars, Sontag Foundation Distinguished
Scientist Award, and March of Dimes Research Grant 6-FY07-401 (A.C.),
N-Cadherin Maintains the Cortical Progenitor Niche
Developmental Cell 18, 472–479, March 16, 2010 ª2010 Elsevier Inc. 477
F30NS053303 (G.W.), F30NS051864 (C.M), NIH T32 GM08061 (A.S.), and the
Katten Muchin Rosenman Travel Scholarship (G.W. and S.N.). We thank R.
Dorsky (Utah) for pTOP-dGFP, X. He (Harvard) for DN-LRP6, A. Kenney
(Memorial Sloan Kettering) for DN-AKT, L. Li (Stowers) for b-catenin Ser552
antibody, R.T.Moon (UW, Seattle)for Super8xTOPflash and Super8xFOPflash
of California, San Diego) for mCherry, M. Wheelock (Nebraska) for pSuper N-
cadherin shRNA and control shRNA constructs, Ed Monuki (Irvine) for in situ
hybridization expertise, and C. J. Gottardi (Northwestern) for advice.
Received: December 5, 2008
Revised: October 5, 2009
Accepted: December 22, 2009
Published: March 15, 2010
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