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60. Supported by the National Institute for Deafness and
other Communication Disorders; the National Sci-
ence Foundation; the McKnight, Alfred P. Sloan, and
Keck Foundations (G.L.); a Sloan and Swartz Founda-
tions fellowship (J.P.-O.); a Department of Defense
National Defense Science and Engineering graduate
fellowship (O.M.); the Elizabeth Ross fellowship
(G.C.T.); and a Helen Hay Whitney postdoctoral fel-
lowship (R.I.W.). We thank M. Westman for his intra-
cellular PN data; C. Pouzat for help with spike sorting;
S. Farivar for help with the immunocytochemistry;
the Laurent Lab; E. Schuman, A. Siapas, and C. Mead
for discussions; M. Roukes for help with silicon te-
trodes; I. Lubenov and A. Siapas for help with wire
tetrodes; M. Walsh for electronics; and the Caltech
Biological Imaging Center for their resources and
expertise. Multichannel silicon probes were provided
by the University of Michigan Center for Neural
Communication Technology sponsored by NIH NCRR
grant no. P41-RR09754.
Supporting Online Material
Materials and Methods
Figs. S1 to S5
4 February 2002; accepted 31 May 2002
Regulation of Cerebral Cortical
Size by Control of Cell Cycle
Exit in Neural Precursors
Anjen Chenn1,2* and Christopher A. Walsh2†
Transgenic mice expressing a stabilized ?-catenin in neural precursors develop
enlarged brains with increased cerebral cortical surface area and folds resem-
bling sulci and gyri of higher mammals. Brains from transgenic animals have
enlarged lateral ventricles lined with neuroepithelial precursor cells, reflecting
an expansion of the precursor population. Compared with wild-type precursors,
These results show that ?-catenin can function in the decision of precursors to
proliferate or differentiate during mammalian neuronal development and sug-
gest that ?-catenin can regulate cerebral cortical size by controlling the gen-
eration of neural precursor cells.
A massive increase in the size of the cerebral
cortex is thought to underlie the growth of
intellectual capacity during mammalian evo-
lution. The increased size of larger brains
results primarily from a disproportionate ex-
pansion of the surface area of the layered
sheet of neurons comprising the cerebral cor-
tex (1–7), with the appearance of convolu-
tions of the cortical surface (with crests
known as gyri and intervening grooves called
sulci) providing a means of increasing the
total cortical area in a given skull volume.
This horizontal expansion of the cerebral cor-
tex is not accompanied by a comparable in-
crease in cortical thickness; in fact, the 1000-
fold increase in cortical surface area between
human and mouse is only accompanied by an
?twofold increase in cortical thickness (8).
The cerebral cortex is organized into co-
lumnar functional units (9), and the expan-
sion of the cerebral cortex appears to result
from increases in the number of radial col-
umns rather than from increases in individual
column size (5, 10). These observations have
led to the proposal that increases in the num-
ber of columns result from a corresponding
increased number of progenitor cells (5). It
has been suggested that minor changes in the
relative production of progenitors and neu-
rons could produce dramatic increases in cor-
tical surface area (5, 11).
One protein that might regulate the pro-
duction of neural precursors is ?-catenin, an
integral component of adherens junctions
(12) that interacts with proteins of the T cell
(TCF/LEF) family to transduce Wnt signals
(13). Wnts (a family of secreted signaling
molecules that regulate cell growth and cell
fate) (14) and TCF/LEF family members (15,
16) are expressed in overlapping patterns in
the developing mammalian brain, and numer-
ous studies support the role of Wnt signaling
in cell fate regulation during development
(17). Inactivation of specific Wnts (18, 19),
TCF/LEF members (20), or ?-catenin (21)
results in specific developmental brain de-
fects, and persistent activation of ?-catenin
has been implicated in a variety of human
cancers (13), including some resembling neu-
ral precursors such as medulloblastoma (22).
These findings raise the possibility that
?-catenin influences cell number or cell fate
decisions in the developing nervous system.
?-catenin is widely expressed in many tis-
sues (23). To examine more closely the expres-
sion patterns of ?-catenin during mammalian
1Department of Pathology, Brigham and Women’s
Hospital, Boston, MA 02115, USA.2Division of Neu-
rogenetics, Department of Neurology, Beth Israel
Deaconess Medical Center, Boston, MA 02115, USA.
*Present address: Department of Pathology, North-
western University School of Medicine, 303 East Chi-
cago Avenue, Chicago, IL 60611–3008, USA.
†To whom correspondence should be addressed. E-
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www.sciencemag.orgSCIENCEVOL 29719 JULY 2002
neural development, in situ hybridization of
?-catenin was performed on embryonic mouse
brain sections. Strong hybridization was ob-
served for ?-catenin in neuroepithelial precur-
sors in the ventricular zone across the period
during which neurons were produced (Fig. 1A).
Immunostaining with a monoclonal antibody
indicates that, in neuroepithelial precursors,
?-catenin protein is enriched at adherens junc-
tions at the lumen of the ventricle, where it
colocalizes in rings with F-actin, highlighted by
rhodamine phalloidin (Fig. 1B).
To examine whether activating ?-catenin
signaling could regulate mammalian brain
development, we generated transgenic mice
overexpressing an NH2-terminally truncated
form of ?-catenin fused at the COOH-termi-
nal with green fluorescent protein (GFP)
(?N90?-catenin-GFP) in neuroepithelial pre-
cursors. NH2-terminally truncated ?-catenin
no longer requires Wnt signaling for sustain-
ing activity, because it lacks key phosphoryl-
ation sites for GSK3? that normally target it
for destruction in the absence of Wnts (24).
This form of ?-catenin is stabilized constitu-
tively in vivo and remains able to bind E-
cadherin and ?-catenin and to activate tran-
scription by binding with TCF/LEF cofactors
(24, 25) (Fig. 2B) [see supplementary online
material (SOM)]. The expression of ?N90?-
catenin-GFP was driven by the enhancer el-
ement contained in the second intron of the
nestin gene (Fig. 2C) (see SOM), which di-
rects expression in central nervous system
progenitor cells (26).
Transgenic embryos at embryonic day
15.5 (E15.5) have grossly enlarged brains,
with a considerable increase in the surface
area of the cerebral cortex, without a corre-
sponding increase in cortical thickness (n ?
10) (Fig. 3). Sections through the forebrain
revealed that, in transgenic brains, the hori-
zontal growth of the tissue is so extensive that
the normally smooth cerebral cortex of the
mouse forms undulating folds resembling the
gyri and sulci of higher mammals (Fig. 3B)
(27). Brains from E17.5 embryos showed
similar enlargement and folding (fig. S1). In
Fig. 1. Expression of
?-catenin transcript and
protein in neural pre-
cursors. (A) ?-catenin
in situ hybridization in
sections through de-
veloping mouse cere-
bral cortex. ?-catenin
is strongly expressed
in the ventricular zone
(VZ) precursor cells at
all ages during which
cortical neurons are gen-
present in the develop-
ing cortical plate. Bar,
200 ?m. (B) Immuno-
staining through E14.5
mouse ventricular zone
reveals ?-catenin immu-
noreactivity (green) con-
centrated in rings at the
of the same section with
rhodamine phalloidin re-
colocalized with adher-
distribution at the lumenal surface. The merged view indicates that ?-catenin colocalizes with phalloidin. Bar,
Fig. 2. Transcriptional activation by ?-catenin and expression and
transgenic construct design. (A) pTOPFLASH luciferase reporter assay
in NT-2 cells. NT-2 cells were transfected with pTOPFLASH, contain-
ing four consensus LEF-1/TCF-1 binding sites, a minimal Fos promot-
er, and a luciferase reporter (43). Transfections were performed with
and without cytomegalovirus (CMV)-?90?catenin-GFP. CMV-LacZ
was used to normalize for transfection efficiency. Twenty-four hours
later, cells were lysed and protein extracts were assayed for luciferase.
Fold inductions of luciferase activity represent the average of three
experiments, with error bars representing one SEM. (B) ?90?-catenin
activates transcription in primary cortical cells. Primary cells from E17
cortex were transfected with the pTOPFLASH luciferase reporter
construct and the expression vectors as indicated. Luciferase activity
was assayed 48 hours after transfection. Fold inductions represent the
average of six experiments, with error bars indicating one SEM. (C)
Expression and transgenic constructs. Constructs removing the NH2-
terminal 90 amino acids of mouse ?-catenin are fused either to EGFP
or the kt3 epitope tag. For expression in transient transcription
assays, ?-catenin constructs are placed behind the CMV promoter.
The nestin second intron coupled with the thymidine kinase minimal
promoter are used to generate transgenic mice. The first intron from
the rat insulin II gene is incorporated to enhance expression levels.
The same ?-catenin alleles were used in both in vitro and transgenic
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19 JULY 2002VOL 297SCIENCE www.sciencemag.org
cresyl violet–stained sections, a densely
stained layer of cells adjacent to the enlarged
ventricular lumen morphologically resembled
the proliferative zone of wild-type brains but
was greatly expanded in surface area in the
transgenic animals (E15.5, n ? 10; E17.5,
n ? 6; E19.5, n ? 2). Because we observed
marked expansion of the cortical neuroepi-
thelium, we focused our further studies on
this population of cells at E15.5, an age mid-
way through mouse cortical neurogenesis.
To determine the identity of the cells that
may account for the expansion of the trans-
genic brains, we examined the expression of
markers specific for neuroepithelial precur-
sors and differentiating neurons. The basic
helix-loop-helix transcription factors Hes5
and Hes1 are downstream effectors of the
Notch signaling pathway and regulate neuro-
nal differentiation (28). Hes5 is expressed
specifically by neuroepithelial precursors,
whereas Hes1 is highly expressed in precur-
sors, with lower expression in more differen-
tiated cortical plate neurons (29). In situ hy-
bridization for Hes5 of comparable coronal
sections through wild-type and transgenic
brains suggests that the neural precursor pop-
ulation in transgenic animals is expanded
(Fig. 4A). The expression of both Hes1 (fig.
S1) and Ki67 (Fig. 5), a protein expressed in
all dividing cells (30, 31), highlighted the
ventricular zone and confirmed the findings
seen with Hes5, providing further support
that the precursor zone is expanded in trans-
genic animals. Finally, we used the thymidine
analog BrdU to label dividing neural precur-
sor cells by exposing embryos to BrdU for 30
min before killing them. Sections through
wild-type and transgenic brains show that the
same cells lining the ventricle also incorpo-
rate BrdU, confirming that the population of
cells labeled with the precursor markers is
composed of dividing cells (Fig. 3, E and F).
To investigate the spatial patterns of neuro-
nal differentiation in transgenic animals, we
examined the expression of three different
markers of cortical neuron populations—Reelin
(Reln), T-box brain gene 1 (Tbr-1), and TuJ1.
In wild-type mice at E15.5, Reln labels Cajal-
the developing cortical plate (Fig. 4). Similarly,
in the brains of transgenic animals, in situ hy-
bridization for Reln expression showed strong
labeling in its normal position at the margin of
the cortical plate. In wild-type mice at E15.5,
Tbr-1 is normally expressed in neurons of the
cortical preplate and subplate (Fig. 4). Similar-
ly, in situ hybridization for Tbr-1 in transgenic
animals indicates that cortical cells outside the
ventricular zone expressed Tbr-1 (Fig. 4). The
general pattern of Tbr-1 staining resembled that
of wild-type animals, with Tbr-1–expressing
cells situated in the region outside the progen-
itor zone in the developing cortical plate. How-
ever, much like those that express Reln, the
cells that express Tbr-1 were somewhat more
tical plate, as compared with cells with wild-
type expression. In E15.5 wild-type animals,
TuJ1 labels newly differentiated neurons out-
side the ventricular zone (Fig. 4). In transgenic
mice, TuJ1 immunoreactivity also labeled the
layer of cells outside the ventricular zone, sup-
localized outside the ventricular zone in trans-
genic animals. Despite the massive expansion
of cortical surface area, transgenic precursors
appear to differentiate into young neurons in an
approximately normal spatial pattern. Taken to-
gether, these expression studies suggest that
over-activating ?-catenin does not disrupt the
normal developmental sequence of neuronal
differentiation, and the horizontal expansion of
the cortical plate is a result of an increased
number of proliferative precursor cells.
Enlargement of the precursor pool in
transgenic brains can result from increased
mitotic rates, decreased cell death, changes in
cell fate choice (whether to differentiate or to
proliferate), or any combination of these fac-
tors. To examine whether the horizontal ex-
pansion of the progenitor pool in transgenic
animals results from increased mitotic rates,
we counted the proportion of precursor cells
that could be labeled by a 30-min pulse of
BrdU. To quantify the fraction of cells in S
phase, we obtained a labeling index (LI) by
counting the percentage of cortical progenitor
cells that were labeled by a single pulse of
Fig. 3. Enlarged brains and heads of ?-catenin transgenic animals with horizontal expansion of
precursor population. Mid-coronal section through the forebrain stained with cresyl violet of an
embryonic day 15.5 wild-type littermate control (A) and comparable section of a transgenic animal
(B) expressing a ?90?-catenin-GFP fusion protein in neural precursors. The forebrain of transgenic
animals is enlarged overall, with increased surface area and folding of the epithelial surface. Bar, 1
mm. Insets: Images of wild-type (a) and of transgenic (b) heads reveal gross enlargement of the
skull and forebrain vesicles protruding anteriorly (as indicated by the white arrowhead) over the
face of the embryo. Bar, 2 mm. (C and D) In situ hybridization for Hes5 in comparable coronal
sections through wild-type littermate control (C) and transgenic brain (D). Hes5 is expressed in
progenitor cells in the ventricular zone of wild-type and transgenic brains. Additional areas of
Hes5-expressing cells are located in ectopic regions away from the ventricular lumen in transgenic
animals (as indicated byt the black arrowheads). Bar, 1 mm. (E and F) BrdU-labeled cells in
transgenic animals after a 30-min exposure to BrdU. BrdU labels the same cells as the progenitor
markers Hes5 and Hes1. (F) Higher magnification image reveals that the overall organization of the
ventricular zone of transgenic animals is preserved, with S-phase progenitors occupying the outer
half of the ventricular zone, similar to wild-type progenitors. Bar, 1 mm (E), 200 ?m (F).
R E S E A R C H A R T I C L E S
www.sciencemag.org SCIENCEVOL 29719 JULY 2002
BrdU. Progenitor cells were identified by
Ki67 immunoreactivity (30, 31). Because in
mammalian cells the length of S phase re-
mains relatively constant while the length of
G1regulates proliferation (32), this LI pro-
vides an estimation of cell cycle length. If the
cell cycle is shortened, the relative fraction of
cells labeled by a brief BrdU pulse will in-
crease. Examination of random fields chosen
from six brains (three wild-type and three
transgenic brains) suggests that the transgenic
neural precursors did not divide significantly
faster than did normal wild-type precursors
[F(6,18)? 0.970, P ? 0.471] (Fig. 5A).
Programmed cell death (apoptosis) occurs
during normal development of the central ner-
vous system (33), and decreased programmed
cell death may be one mechanism underlying
the increased brain size of transgenic animals.
Apoptotic cell death was examined using
TUNEL staining in wild-type and transgenic
brains. TUNEL? cells were confirmed by ver-
ifying condensed nuclei labeled with the DNA
binding dye Hoechst 33342. Counts of total
numbers of labeled cells revealed that cell death
in transgenic brains was not substantially less
than found in wild type (Fig. 5B); in fact, there
appeared to be greater than twofold increased
rates of apoptosis in transgenic brains [F(4,11)?
26.00, P ? 0.0002). Taken together, the BrdU-
labeling studies and TUNEL studies suggest
that the progenitor cell population expansion
cannot be explained by a simple mitogenic ef-
fect of ?-catenin or by decreased apoptotic cell
Progenitor divisions that give rise to addi-
tional progenitors can expand the progenitor
pool exponentially. Consequently, small alter-
ations in the fraction of cell divisions that ex-
pand the progenitor pool can result in large
changes in the final size of the brain (5, 34). To
examine whether the increase in the progenitor
pool results from a shift in the fraction of pro-
genitors that choose to remain progenitors in-
stead of differentiating, we examined cell cycle
exit and re-entry by examining the fraction of
cells dividing after pulse labeling with BrdU 24
cell cycle as BrdU? and Ki67–, and we identi-
fied cells that remained in the cell cycle as
BrdU? and Ki67?. At E15.5, we found an
?twofold increase in the proportion of trans-
genic precursors that re-enter the cell cycle
when compared with wild-type neural precur-
sors [F(4, 15)? 11.00, P ? 0.0009] (Fig. 5C).
Together, these studies suggest that ?-catenin
activation functions in neural precursors to in-
fluence the decision to re-enter the cell cycle
instead of differentiate.
Our results support recent findings suggest-
ing that epithelial architecture and adherens
junctions regulate growth control and cell pro-
liferation (35). Because ?-catenin is an integral
component of adherens junctions (12), disrup-
tions of adherens junctions may cause misregu-
Fig. 4. Neuronal differentiation in transgenic brains. In situ hybridization for Hes5 labels cortical
precursors (adjacent to lumen of ventricle), but not differentiated neurons in both E15.5 wild-type
and transgenic brains. In situ hybridization for Tbr-1 in adjacent sections indicate that Tbr-1 is
expressed in the cortical plate and intermediate zone, but not in the precursor zone of both control
and transgenic brains. In situ hybridization of adjacent sections show strong Reln expression in the
outermost layer of neurons of both control and transgenic brains. Sections stained with the TuJ1
antibody reveal the location of newly postmitotic neurons in the intermediate zone and developing
cortical plate, but not in the ventricular zone in both wild-type and transgenic animals. The relative
position of Hes-5, Tbr-1, Reln, and TuJ1 staining is maintained in wild-type versus transgenic
animals. The boxed portion in the upper panels is enlarged in the lower panels. The ventricular
surface is outlined to aid visualization. Bar, 1mm (top) and 200 ?m (bottom).
Fig. 5. Cell cycle re-entry increased
in transgenic precursors. (A) The
(Ki67?, red) labeled with BrdU
(green) after a 30-min pulse label is
not altered in transgenic animals.
DNA stain (blue) reveals that wild
type developing cortex is thicker
outside the progenitor population,
containing relatively more postmi-
totic cells (Ki67–), as compared with
transgenic brains [F(6,18)? 0.970,
P ? 0.471]. (B) Normalized for area,
transgenic brains have more apo-
ptotic cells labeled by TUNEL (red).
DNA is counterstained (blue) with
Hoechst 33342 [F(4,11)? 26.00, P ?
0.0002]. (C) Animals were exposed
to a single-pulse label of BrdU 24
hours before being killed; sections
were stained with antibodies to BrdU (green) and Ki67 (red). The fraction of cells labeled only with
BrdU (BrdU?/Ki67–, no longer dividing) 24 hours after pulse label, as compared with BrdU?/
Ki67? cells (yellow, re-entered cell cycle). Approximately twice as many wild-type precursors leave
the cell cycle, as compared with transgenic precursors [F(4, 15)? 11.00, P ? 0.0009].
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19 JULY 2002 VOL 297SCIENCEwww.sciencemag.org
lation and accumulation of cytoplasmic ?-cate-
nin. Our findings that ?-catenin signaling can
regulate the decisions of neural precursors to
re-enter or exit the cell cycle lend support to the
possibility that ?-catenin signaling may mediate
the loss of growth control when adherens junc-
tions are disrupted.
It has been hypothesized that mutations in
regulatory genes that control the decision of
neural precursors to divide or differentiate can
underlie the expansion of the precursor popula-
(5, 11). Here, we find that ?-catenin activation
by influencing the decision to divide or differ-
entiate, without increasing cell cycle rate, de-
creasing cell death, or grossly altering neuronal
differentiation. Larger brains can be generated
in different ways as well. For example, reduc-
tion of programmed cell death by targeted mu-
tation of Caspase 9 causes severe brain malfor-
mations characterized by cerebral enlargement,
zone (36, 37). In contrast, mice with targeted
deletions of the cell cycle regulator p27kip1have
increased body size and uniformly enlarged
brains with virtually no anatomic abnormalities
other than increased cell number and cell den-
sity (38–40). Notably, cortical surface area was
not disproportionately increased (38). In con-
trast, our findings suggest that subtle changes in
the expansion or maintenance of the neural pre-
cursor population result in horizontal expansion
of the surface area of the developing cerebral
cortex without increases in cortical thickness
(41). Further understanding of how the decision
to divide or differentiate is regulated by ?-cate-
nin will lend valuable insight into the mecha-
nisms that underlie the disproportionate
growth of the cerebral cortex in higher
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Supporting Online Material
Materials and Methods
References and Notes
21 May 2002; accepted 25 June 2002
R E P O R T S
Nonresonant Multiple Spin
Thilo M. Brill,* Seungoh Ryu, Richard Gaylor, Jacques Jundt,
Douglas D. Griffin, Yi-Qiao Song, Pabitra N. Sen, Martin D. Hu ¨rlimann
Nonresonant manipulation of nuclear spins can probe large volumes of sample
situated in inhomogeneous fields outside a magnet, a geometry suitable for
However, the interference by Earth’s magnetic field causes rapid decay of the
signal within a few milliseconds for protons and is detrimental to this method.
Here we describe a technique to suppress the effects of Earth’s field by using
adiabatic rotations and sudden switching of the applied fields. We observed
hundreds of spin echo signals lasting for more than 600 milliseconds and
accurately measured the relaxation times of a liquid sample.
Conventional nuclear magnetic resonance
(NMR) experiments are almost always car-
ried out by manipulating nuclear spins using
radio frequency (rf) pulses at the spin Larmor
frequency ? ? ?B, where ? is the gyromag-
netic ratio and B is the magnitude of the
magnetic field. Such resonant NMR experi-
ments allow the imaging of spins in materials
and the characterization of spin interactions,
enabling applications extending to materials
such as soft condensed matter (1), plants (2),
food products (3), cement and concrete (4),
and geological materials (5, 6). The field
applications are the motivation for several
recent developments in ex situ NMR (7–10),
where a mobile NMR detector is used to
examine the sample outside the NMR mag-
net. However, as a result of the geometry of
such mobile tools, the applied magnetic fields
exhibit large inhomogeneities, and all reso-
nant techniques will result in small sensitive
volumes where the resonance condition is
satisfied. Composite (11) and adiabatic (12)
pulses may be used to expand the excitation
bandwidth to a limited extent at the expense
of higher irradiation power.
Alternatively, spins can be manipulated
R E S E A R C H A R T I C L E S
www.sciencemag.org SCIENCEVOL 29719 JULY 2002