Loss of RhoA in neural progenitor cells causes the
disruption of adherens junctions and hyperproliferation
Kei-ichi Katayamaa, Jaime Melendezb, Jessica M. Baumanna, Jennifer R. Lesliea, Bharesh K. Chauhanc, Niza Nemkula,
Richard A. Langc, Chia-Yi Kuana, Yi Zhengb, and Yutaka Yoshidaa,1
Divisions ofaDevelopmental Biology,bExperimental Hematology and Cancer Biology, andcOphthalmology, Cincinnati Children’s Hospital Medical Center,
Cincinnati, OH 45229
Edited* by Pasko Rakic, Yale University, New Haven, CT, and approved March 31, 2011 (received for review January 25, 2011)
The organization of neural progenitors in the developing mamma-
lian neuroepithelium is marked by cadherin-based adherens junc-
tions. Whereas RhoA, a founding member of the small Rho GTPase
family, has been shown to play important roles in epithelial adhe-
rens junctions, its physiological roles in neural development re-
main uncertain due to the lack of specific loss-of-function studies.
Here, we show that RhoA protein accumulates at adherens junc-
tions in the developing mouse brain and colocalizes to the cad-
herin–catenin complex. Conditional deletion of RhoA in midbrain
and forebrain neural progenitors using Wnt1-Cre and Foxg1-Cre
mice, respectively, disrupts apical adherens junctions and causes
massive dysplasia of the brain. Furthermore, RhoA-deficient neural
progenitor cells exhibit accelerated proliferation, reduction of cell-
cycle exit, and increased expression of downstream target genes of
the hedgehog pathway. Consequently, both lines of conditional
RhoA-deficient embryos exhibit expansion of neural progenitor
cells and exencephaly-like protrusions. These results demonstrate
a critical role of RhoA in the maintenance of apical adherens junc-
tions and the regulation of neural progenitor proliferation in the
developing mammalian brain.
CNS development|cell adhesion
produce the organ of predetermined size (1–3). Apart from
maintaining intercellular adhesion, adherens junctions that are
preferentially located at the ventricular surface of the neuro-
epithelium are linked to several major signaling pathways to
regulate the rate and mode of cell division in neural progenitors
(4, 5). Classic cadherins are core components of adherens
junctions. The extracellular regions of classic cadherins mediate
adhesive recognition through homophilic interactions, whereas
cytoplasmic domains associate with a set of cytoplasmic proteins
collectively called catenins. Deletion of αE-catenin in the de-
veloping nervous system results in dysregulated proliferation of
neural progenitor cells as well as disruption of adherens junc-
tions (6). Furthermore, Lgl1-knockout mice and compound
knockout mice for both Numb and Numbl also show disruption
of adherens junctions together with abnormal proliferation of
neural progenitor cells (7–9). These findings indicate that the
cadherin–catenin complex has broader functions beyond cell–cell
adhesion in mammalian neural progenitors.
Cadherin–catenin complexes interact with the actin cytoskele-
ton through α-catenin. Formation of adherens junctions is ac-
companied by profound changes in the actin cytoskeleton and
accumulation of polymerized actin at the contact area, and both
the formation and the maintenance of adherens junctions depend
on the actin cytoskeleton (10). The Rho family of small GTPases,
including RhoA, Cdc42, and Rac1, are key regulators of the actin
cytoskeleton and coordinate junction assembly, stability, and
function (11, 12). However, these functions are mainly deduced
or constitutively active small Rho GTPases. However, the con-
sequences of GTPase signaling alteration depend profoundly on
uring brain development, proliferation, differentiation, and
cell death of neural progenitor cells are tightly controlled to
cellular context and the specificity of the mutant proteins. The
recent development of a conditional gene-targeting strategy has
provided many new insights into the physiological functions of
small Rho GTPases (13). For example, although Cdc42 and Rac1
are both implicated in epithelial apical junctions, conditional
gene deletion in the telencephalon revealed that Cdc42, but not
Rac1, is indispensable for the formation of apical adherens junc-
tions in the developing brain (14–16). These findings underlie the
importance of using conditional gene deletion to ascertain the
biological functions of small Rho GTPases.
Among all small Rho GTPases, RhoA is one of the last
remaining members whose in vivo gene-deletion consequences in
the mammalian central nervous system are yet to be reported
(13). In addition to RhoA, there are two other Rho isoforms in
mammals, RhoB, and RhoC, which are highly homologous, and
all three members induce stress fiber formation when overex-
pressed in fibroblasts (13). However, in knockout studies, RhoB-
null and RhoC-null mice are viable and have normal de-
velopment (17, 18). Recently, conditional deletion of RhoA in
the developing skin was reported, but the mutant mice did not
show any obvious phenotypes in vivo (19). Therefore, RhoA and
its close isoforms, RhoB and RhoC, may have redundant func-
tions in skin development. In mammalian epithelial cells, RhoA
induces actomysin contractility to expand cell–cell adhesion (20).
Furthermore, loss-of-function experiments using Drosophila em-
bryos demonstrated that the Drosophila RhoA homolog, Rho1,
is required for the organization of cadherin-based adherens
junctions (21, 22). These studies indicate that mammalian RhoA
may play a role in adherens junctions, but the physiological role
of RhoA in adherens junctions in mammalian nervous system
In this study, we examined the role of RhoA in the developing
mouse brain using a conditional gene-targeting strategy with two
lines of Cre drivers (Wnt1-Cre for the midbrain and Foxg1-Cre
for the forebrain mutation). These conditional gene deletions
lead to similar phenotypes including the disruption of adherens
junctions, massive expansion of neural progenitors, and disor-
ganization of the brain. These findings uncover an essential and
nonredundant role of RhoA in neural progenitor cells in the
mouse central nervous system.
Localization of RhoA Protein in the Developing Brain. We first ex-
amined the spatial distribution of RhoA protein in the developing
brain. RhoA was detected throughout the neuroepithelium and
Author contributions: K.-i.K., R.A.L., C.-Y.K., Y.Z., and Y.Y. designed research; K.-i.K., J.M.,
J.M.B., J.R.L., B.K.C., and N.N. performed research; K.-i.K., J.M., C.-Y.K., Y.Z., and Y.Y.
analyzed data; and K.-i.K., C.-Y.K., Y.Z., and Y.Y. wrote the paper.
The authors declare no conflict of interest.
*This Direct Submission article had a prearranged editor.
1To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| May 3, 2011
| vol. 108
| no. 18
day 12.5 (E12.5) (Fig. 1A). Because the apical surface of the
ventricular zone is also enriched with cadherin–catenin proteins,
we compared the localization of RhoA with those of the adherens
that RhoA is extensively colocalized with αE-catenin, β-catenin,
iments further revealed the association of RhoA with β-catenin
interaction was supported by the reciprocal use of anti-RhoA
and anti–β-catenin antibodies in precipitation immunoblot but
yielded the same result, as well as the loss of the interaction signal
in conditional RhoA-null embryos (Fig. 1G; see below for condi-
tional deletion of RhoA). Together, these data suggest that RhoA
Deletion of RhoA in Mesencephalon Causes Exencephaly. To de-
termine the in vivo functions of RhoA in neural progenitors, we
generated RhoA-floxed mice (Fig. S1) and crossed them with
Wnt1-Cre mice that express Cre recombinase in the mesen-
cephalon as well as in neural crest derivatives (23). By crossing
with stop-floxed EGFP reporter mice, we detected the Wnt1-
Cre–mediated Cre/loxP recombination in the mesencephalon at
E9.5 (Fig. S2). In Wnt1-Cre; RhoAflox/flox(hereafter referred to as
RhoA-CKO) embryos, we validated the absence of RhoA ex-
pression in the mesencephalon by immunostaining, as well as
immunoblotting analysis (Fig. S3). Deletion of RhoA did not
markedly affect the expression of other small Rho GTPase
members (Fig. S3). At E13.5, RhoA-CKO embryos showed en-
largement of the mesencephalon, compared with their litter-
mates (Fig. 2 A and B, arrowheads), and at E15.5, they exhibited
exencephaly-like protrusions (Fig. 2 C and D). About 80% of
RhoA-CKO embryos exhibited exencephaly-like abnormalities
from E15.5 to E16.5 (Table S1). Histologically, the protrusion
consisted of a neuronal mass originating from the midbrain,
which not only exposed dorsally but also pushed forebrain
structures to the ventral side (Fig. S4). Because RhoA-CKO
embryos did not exhibit exencephaly-like abnormalities before
E12.5 (Table S1), we concluded that this anomaly was not caused
by the defects of neural tube closure. Rather, it is more likely to
be caused by abnormal expansion of neuronal cells.
RhoA Deficiency Disrupts Adherens Junctions at the Ventricular
Surface. We next examined Nissl-stained sections from RhoA-
CKO embryos and control littermates. The initial abnormality in
the RhoA-deleted mesencephalon was detected as multiple nod-
ules protruding from the apical surface around E11.5. This
anomaly intensified by E12.5, and the cellular organization in the
ventricular zone of the mesencephalon became highly irregular in
RhoA-CKO embryos (Fig. 3 A and B). After E12.5, the dysplasia
was observed in all of the RhoA-CKO embryos we examined. The
apical F-actin staining was lost in dysplastic regions whereas the
cellular organization at the basal side, including accumulation of
raising the possibility that RhoA deficiency may lead to disruption
of the cadherin-based apical cell–cell adhesion. Consistent with
this notion, we found that the localization of components of the
cadherin–catenin complex, including αE-catenin, β-catenin, and
N-cadherin, was all disrupted in the dysplastic region (Fig. 3 E–J).
These results suggest that RhoA deletion leads to disruption of
adherens junctions in the ventricular surface of the developing
Loss of RhoA Causes Massive Dysplasia in the Mesencephalon. At
E13.5, the dysplasia expanded throughout the mesencephalon
and the cellular organization was markedly disorganized in
RhoA-CKO embryos (Fig. S5 A and B). Nestin-positive neural
progenitor/radial glial processes were also disorganized in
RhoA-CKO embryos (Fig. S5 E and F). In addition, we fre-
quently found neuroblast rosette-like structures that were also
observed in αE-catenin and N-cadherin null mutants (6, 24) (Fig.
S5 C and D). In the midbrain of control embryos, Ki67+pro-
liferative cells and βIII-tubulin+postmitotic neurons were sep-
arated in the apical and basal compartments, respectively (Fig.
4A). However, these two cell populations were intermingled and
the ratio of postmitotic neurons decreased in RhoA-CKO em-
bryos [Fig. 4 B and C; control, 23.8 ± 3.4% (n = 3); RhoA-CKO,
16.5 ± 1.5% (n = 4); mean ± SD; P < 0.05 by Student’s t test].
These results suggest that RhoA deficiency leads to massive
interacts with β-catenin. (A) Immunohistochemistry for RhoA revealed that
RhoA protein is enriched at the apical portion of the ventricular zone of the
mesencephalon at E12.5. RhoA-positive signals colocalize with positive sig-
nals for αE-catenin (B), β-catenin (C), and N-cadherin (D–F) at E13.5. Nuclei
are visualized by ToPro3. (Scale bars: A, 50 μm; B, 10 μm.) (G) Immunopre-
cipitation revealed the interaction of RhoA with β-catenin, and the in-
teraction was not observed in conditional RhoA-null (RhoA-CKO) brain.
RhoA accumulates at adherens junctions in the ventricular zone and
subsequently leads to exencephaly-like protrusion formation. General ap-
pearance of control (A and C) and RhoA-CKO (B and D) embryos at E13.5 (A
and B) and E15.5 (C and D).
Deletion of RhoA induces enlargement of the mesencephalon and
| www.pnas.org/cgi/doi/10.1073/pnas.1101347108 Katayama et al.
dysplasia and expansion of neural progenitor cells in the de-
Accelerated Proliferation and Decreased Cell-Cycle Exit in RhoA-
Deleted Neural Progenitor Cells. To assess whether the loss of
RhoA leads to changes in cell proliferation, we performed
immunostaining with anti–phospho-histone H3 (p-H3) antibody
to identify mitotic cells. In control embryos, p-H3–positive nuclei
were localized close to the ventricular surface of the neuro-
epithelium (Fig. 4D). In contrast, in RhoA-CKO embryos, the p-
H3–positive nuclei were dispersed throughout the brain (Fig.
4E). Quantitative analysis of the p-H3 staining demonstrated
about a twofold increase in the number of mitotic cells among
total neuronal cells in the mesencephalon in RhoA-CKO em-
bryos [Fig. 4F; control, 2.8 ± 0.3% (n = 4); RhoA-CKO, 5.1 ±
0.8% (n = 4); P < 0.01].
Enlargement of the progenitor pool in RhoA-deficient mid-
brain may result from accelerated proliferation, decreased cell
death, failure in cell-cycle exit, or any combination of these
factors. First, to reveal potential changes in the proliferation, we
counted the proportion of neural progenitor cells labeled by
a 30-min pulse of BrdU and found a significant increase in the
fraction of BrdU-labeled nuclei among all Ki67+neural pro-
genitor cells in RhoA-CKO embryos [Fig. 4 G–I; control, 52.9 ±
3.1% (n = 5); RhoA-CKO, 66.5 ± 4.6% (n = 3); P < 0.01]. The
increase of BrdU+fractions among Ki67+cells suggests accel-
erated proliferation of neural progenitor cells. In addition,
BrdU-positive nuclei of control embryos localized at the basal
side of the ventricular zone (Fig. 4G), whereas those of RhoA-
CKO embryos localized not only at the basal side but also at the
apical side (Fig. 4H).
To examine whether loss of RhoA causes changes in cell-cycle
withdrawal, we counted the proportion of cells that exit the cell
cycle after 24 h labeling with BrdU, which was calculated as the
ratio of BrdU+/Ki67−cells among all BrdU-labeled cells. This
analysis revealed a significant decrease of the cell-cycle exit index
in RhoA-CKO embryos, compared with control embryos [Fig. 4
J–L; control, 34.0 ± 2.1% (n = 6); RhoA-CKO, 22.9 ± 3.3%
(n = 3); P < 0.01].
Finally, we compared the extent of apoptosis using the terminal
deoxynucleotidyl transferase-mediated dUTP nick-end labeling
(TUNEL) method and found that the number of apoptotic cells
was not decreased but rather increased in RhoA-CKO embryos
(Fig. S6). Thus, our results suggest that expansion of neural pro-
genitor cells in the RhoA-deleted midbrain is caused by both ac-
celerated proliferation and failure to exit the cell cycle.
Similar Defects in Adherens Junctions and Expansion of the Neural
Progenitor Pool in Forebrain-Specific RhoA Deletion. To examine
whether RhoA has important neurodevelopmental functions
outside the mesencephalon, we crossed RhoA-floxed mice with
another Cre-driver line, Foxg1-Cre. In Foxg1-Cre mice, the Cre
recombinase was expressed by telencephalic progenitors from
E9.5 (25). Genotyping by PCR detected near-complete re-
combination of the floxed RhoA allele in the forebrain, but not
the tail, of Foxg1-Cretg; RhoAflox/flox(hereafter referred to as
Foxg1-RhoA-CKO) embryos at E13.5 (Fig. 5B). From E13.5 to
E14.5, Foxg1-RhoA-CKO embryos exhibited an increasing in-
cidence of the exencephaly phenotype (55% at E13.5 and 80% at
E14.5, Fig. 5A and Table S1), similar to the Wnt1-Cre–mediated
RhoA-CKO embryos. Histologically, the mass consisted of
Pax6+neural progenitors, which were greatly expanded com-
pared with control embryos (Fig. 5 C and D). The ventricular
surface was disorganized and the apical expression of αE-catenin
became absent in Foxg1-RhoA-CKO embryos, Instead, there
were rings of intense αE-catenin expression inside the brain mass
in mutant embryos (Fig. 5 E and F). Ki67+and Pax6+progen-
itors either were extensively intermingled with βIII-tubulin+
postmitotic neurons (Fig. 5 G and H) or formed rosette-like
clusters in E14.5 Foxg1-RhoA-CKO embryos, similar to those
detected in Wnt1-Cre–derived embryos (Fig. S5 G and H). To-
gether, the similar phenotypes in midbrain and forebrain suggest
that RhoA may have important functions in neuroepithelial
progenitors throughout the developing nervous system.
Downstream Targets of the Hedgehog Signaling Pathway. Finally,
we turned to the issues of signaling pathways responsible for the
abnormality of neural progenitors in RhoA-CKO embryos. In this
context, previous studies of mutants with a disrupted cadherin–
catenin complex reported that αE-catenin null mice exhibit abnor-
mal activation of the hedgehog pathway (6) and that deletion of
Lgl1 causes loss of cell polarity and up-regulation of the Notch
known to induce hyperproliferation in neural progenitor cells (26).
To compare the consequences of RhoA deletion with the
previous studies, we examined the expression of transcriptional
targets of hedgehog, Notch, and Wnt/β-catenin pathways by re-
verse transcription–quantitative PCR analysis. We found that the
expression of Gli1 and Fgf15 was significantly elevated in the
RhoA-deficient midbrain at E13.5 (Fig. 6A). The in situ hybrid-
ization analysis showed that Gli1 and Fgf15 transcripts were
cells. (A and B) Nissl-stained sections through the developing mesencephalon
from control (A) and RhoA-CKO (B) embryos at E12.5. The apical structure is
partially disrupted in RhoA-CKO embryos, and neural progenitor cells are
irregularly arranged and protruded to the ventricle in this region (B). (C and
D) Double immunostaining for F-actin and βIII-tubulin of control (C) and
RhoA-CKO (D) embryos. Apical F-actin staining is disrupted in the dysplastic
region, whereas basal βIII-tubulin (TuJ1) staining remains unaffected (D). (E–
I) Immunostaining for αE-catenin (E and F), β-catenin (G and H), and N-
cadherin (I and J) of control (E, G, and I) and RhoA-CKO (F, H, and J) embryos
at E12.5. Apical staining of these molecules is also disrupted in the dysplastic
region. (Scale bars, 50 μm.)
Disruption of adherens junctions in RhoA-deleted neural progenitor
Katayama et al.PNAS
| May 3, 2011
| vol. 108
| no. 18
markedly up-regulated in neural progenitor cells of the RhoA-
deficient midbrain at E13.5 (Fig. 6 B–E). We also found a mild
but significant increase in the expression of Notch pathway target
genes (Hes5 and Hes1) and a Wnt/β-catenin target gene
(CyclinD1) (Fig. 6A). However, because these three genes are
highly expressed in neural progenitor cells (27, 28) (Fig. 6 F and
G), the slight increase in the expression of these genes may re-
flect the expansion of neural progenitor cells in RhoA-CKO
embryos (Fig. 4 A–C). Indeed, the expression of Sox2, a marker
for neural progenitor cells, was increased in RhoA-CKO em-
bryos similar to that of Hes5, Hes1, and CyclinD1 (29) (Fig. 6A).
In addition, because there were no significant changes in the
expression of Axin2 and c-myc (Fig. 6 A, H, and I), the Wnt/
β-catenin pathway is unlikely to be involved in the hyper-
proliferation of neural progenitor cells in RhoA-CKO embryos.
Together, these results suggest that the hedgehog pathway is
activated in RhoA-deficient neural progenitor cells.
RhoA, a founding member of the small Rho GTPase family, has
been implicated in a host of cellular processes, but its physio-
logical functions remain uncertain due to the lack of specific loss-
of-function studies (13). In the present study using two lines of
Cre drivers to delete RhoA in the midbrain or forebrain early in
development, we showed that RhoA deficiency resulted in dis-
ruption of adherens junctions, hyperproliferation of neural pro-
genitors, and brain dysplasia. These results demonstrated that
RhoA has important and indispensable functions in mammalian
RhoA Is Indispensable for the Adherens Junction Formation in the
Developing Brain. The neuroepithelium of developing mammalian
brains has a string of adherens junctions that connects neural
progenitors at the ventricular surface, similar to apical epithelial
junctions (4). The main building blocks of adherens junctions are
cadherins and catenins, and in the developing mammalian
brains, the critical components are N-cadherin, β-catenin, and
αE-catenin, according to their unique gene-expression patterns
and null-mutation phenotypes (5). In addition, cell-culture stud-
ies suggest that small Rho GTPases—especially Rac1, Cdc42, and
RhoA—are important components of adherens junctions, regu-
lating the interactions between catenins and the cytoskeleton
C) Immunohistochemistry for Ki67 and βIII-tubulin revealed increased Ki67+neural progenitor cells and decreased βIII-tubulin+postmitotic neurons in RhoA-
CKO embryos. The graph represents ratios of βIII-tubulin+postmitotic neurons to the total neuronal cell number. (D–F) Immunostaining for p-H3 revealed
increased mitosis in the RhoA-deficient mesencephalon. The graph shows ratios of mitotic cells to the total neuronal cell number. (G–I) Accelerated pro-
liferation in RhoA-deficient neural progenitors. A higher percentage of RhoA-deficient progenitors (Ki67+) are labeled with BrdU after a 30-min pulse. The
percentages of K67+cells that incorporated BrdU are shown. (J–L) Cell-cycle withdrawal is compromised in RhoA-deficient neural progenitors. Pregnant mice
were injected with BrdU 24 h before being killed. Cells reentering the cell cycle are BrdU+/Ki67+, whereas cells withdrawn from the cell cycle are BrdU+/Ki67−.
Cell-cycle exit was determined as the ratio of cells that exited the cell cycle (BrdU+/Ki67−) to all cells that incorporated BrdU. All these analyses were carried out
using E13.5 embryos. The graphs represent mean + SD. n = 3–6, *P < 0.05, **P < 0.01, Student’s t test. (Scale bars, 50 μm.)
Accelerated proliferation and failure in cell-cycle withdrawal result in expansion of neural progenitor cells in the RhoA-deficient mesencephalon. (A–
| www.pnas.org/cgi/doi/10.1073/pnas.1101347108 Katayama et al.
(11, 12). In our study, we showed that RhoA is concentrated at
the apical surface of developing brains and colocalized with
cadherin–catenin proteins. RhoA-deleted embryonic brain showed
disruption of adherens junctions and severe dysplasia, similar to
the phenotypes of αE-catenin, β-catenin, and N-cadherin mutant
mice (6, 24, 30). These results together with the finding that
RhoA physically interacts with β-catenin strongly suggest that
RhoA is an integral and indispensable component of adherens
junctions in the developing mammalian brain.
Differential Roles of RhoA and Cdc42 in Adherens Junctions of Neural
Progenitors. Although Cdc42 deletions in previous reports also
showed their essential roles in adherens junctions in the nervous
system (14, 15), there are also significant differences in the
phenotypes of RhoA and Cdc42 deletions, even when the same
Cre driver (i.e., Foxg1-Cre mice) was used. In Foxg1-Cdc42 null
embryos, the disappearance of apical cadherin–catenin proteins
started as early as E9.5–E10.5, causing holoprosencephaly due to
failure to expand the telencephalon (15). In contrast, RhoA de-
letion by Foxg1-Cre or Wnt1-Cre drivers caused a gradual dis-
ruption of apical cadherin–catenin protein localization between
E11.5 and E14.5. Although we cannot exclude the possibility that
the distinct phenotypes may be in part due to different amounts
of residual RhoA and Cdc42 protein after Cre/loxP recombi-
nation, it is likely that Cdc42 has essential functions for the initial
establishment of apical adherens junctions, whereas RhoA is
essential for their maintenance in the developing mammalian
brain. This interpretation is in keeping with the notion that dif-
ferent small Rho GTPases are involved in different steps of the
adherens junction formation, expansion, and plasticity (11, 12).
Dysregulated Proliferation of RhoA-Deficient Neural Progenitor Cells.
During brain development, proliferation, differentiation, and cell
death of neural progenitor cells are tightly regulated to produce
the organ of predetermined size (1–3). Recent studies using
mutant mice have shown the coincidence of defects in adherens
junctions and hyperproliferation of neural progenitor cells, in-
dicating the involvement of adherens junctions in this process
(5). For example, deletion of αE-catenin in the developing ce-
rebral cortex leads to defects in adherens junctions, massive
disorganization of the neuroepithelium, and hyperproliferation
of neural progenitor cells (6). Although the molecular mecha-
tions and expansion of neural progenitor cells, leading to exencephaly-like
protrusion formation. (A) Gross appearance of control (Left) and Foxg1-
RhoA-CKO (Right) embryos at E14.5. (B) PCR analysis showed near-complete
recombination of the floxed RhoA allele in the forebrain, but not the tail, of
Foxg1-RhoA-CKO embryos at E13.5. (C and D) At E13.5, Foxg1-RhoA-CKO
embryos exhibited expansion of the Pax6+cell population, which formed
masses in the developing cerebral cortex. (E and F) At E14.5, the apical/
ventricular wall became disrupted and the localization of αE-catenin was lost
in Foxg1-RhoA-CKO embryos. (G and H) Whereas Ki67+neural progenitor
cells and βIII-tubulin+postmitotic neurons were completely segregated in
control embryos (G), these two components were extensively intermingled
and neural progenitor cells were expanded in Foxg1-RhoA-CKO embryos (H).
(Scale bars: C and D, 100 μm; E–H, 50 μm.)
Forebrain RhoA deletion also causes disruption of adherens junc-
hog signaling pathway in the RhoA-deficient mesencephalon. (A) Reverse
transcription–quantitative PCR analysis of Gli1, Fgf15, Hes5, Hes1, Axin2,
c-myc, CyclinD1, and Sox2 in control and RhoA-deficient midbrains at E13.5.
The graphs represent mean + SD. n = 5 for control and n = 7 for RhoA-CKO
embryos, *P < 0.05, **P < 0.01, Student’s t test. (B–I) In situ hybridization for
Gli1 (B and C), Fgf15 (D and E), Hes5 (F and G), and Axin2 (H and I) of the
mesencephalon from control (B, D, F, and H) and RhoA-CKO (C, E, G, and I)
embryos at E13.5. (Scale bar, 50 μm.)
Increased expression of downstream target genes of the hedge-
Katayama et al.PNAS
| May 3, 2011
| vol. 108
| no. 18
nisms that link adherens junctions and proliferation control re-
main unclear, recent evidence suggests the involvement of major
developmental signaling pathways as a cause of hyperprolif-
eration (5). In αE-catenin mutant mice, the expression of Gli1
and Fgf15, targets of the hedgehog pathway, was up-regulated,
and the activation of the hedgehog signaling pathway is likely
to explain the hyperproliferation of neural progenitors (6). We
found that the expression of Gli1 and Fgf15 was markedly in-
creased in RhoA-CKO embryos, similar to that in the αE-catenin
null mutants. Importantly, the hedgehog pathway plays a critical
role in brain development and cancer, and Gli1 promotes pro-
liferation of neural progenitor cells in the developing brain (31,
32). Thus, the hyperproliferation of neural progenitor cells in
RhoA-CKO embryos may be caused, at least in part, by hyper-
activation of the hedgehog pathway.
In conclusion, the present study identifies RhoA as a critical
protein for the maintenance of adherens junctions and con-
strained proliferation of neural progenitors in the developing
mammalian brain (Fig. S7).
Materials and Methods
Animals. Mice harboring conditional RhoA alleles, in which exon 3 is flanked
by loxP sites, were generated as described in Fig. S1.
Histological Analysis. Nissl staining, in situ hybridization, and immunohisto-
4% paraformaldehyde, cryoprotected in 30% sucrose, embedded in OCT
compound, and sectioned at 16 μm. Antibodies used in this study are listed in
Table S2. To detect apoptotic cells, we carried out the TUNEL method using
the ApopTag fluorescein in situ apoptosis detection kit (Millipore). We also
used phalloidin-tetramethylrhodamine B isothiocyanate (Sigma) and ToPro3
or DAPI (Molecular Probes) to visualize F-actin and nuclei, respectively.
Images were taken by an Axio Imager Z1 microscope and an LSM510 con-
focal microscope (Carl Zeiss).
Quantitation of Cell Differentiation, Mitosis, Proliferation, Cell-Cycle Exit, and
Cell Death. BrdU labeling and analysis of cell-cycle exit were performed as
described (6, 8, 26). For quantitation of the data, immunohistochemical
images were taken by an Axio Imager Z1 microscope (Carl Zeiss) and the
total numbers of cells and cells stained with indicated antibodies were
counted using AxioVision software (Carl Zeiss).
Immunoprecipitation. Midbrains from E13.5 embryos were lysed in tissue lysis
buffer [20 mM Tris-HCl (pH 7.6), 100 mM NaCl, 10 mM MgCl2, 1% Triton-
X-100, 0.2% sodium deoxycholate, 2 mM phenylmethylsulphonyl fluoride,
100 nM okadaic acid, and a mixture of protease inhibitors]. Supernatants
were exposed to the antibodies. Then, the immunocomplexes were captured
by adding Protein A/G-agarose beads (Santa Cruz Biotechnology).
RNA Extraction and Reverse Transcription–Quantitative PCR. Midbrains were
dissected out from E13.5 embryos and total RNA was extracted using the
RNeasy Micro Kit (Qiagen). Reverse transcription was performed with Su-
perScript III reverse transcriptase (Invitrogen). Real-time PCR was performed
with QuantiTect (Qiagen) by the CFX96 Real-Time System (Bio-Rad). The
intensity relative to β-actin was calculated, and the fold change relative to
the relative intensity in control embryos is presented. Primers were designed
by Primer3 (http://frodo.wi.mit.edu/primer3/).
ACKNOWLEDGMENTS. The authors are grateful to Dr. Masato Nakafuku
and Dr. Akira Nagafuchi for providing us with antibodies. The authors thank
Dr. Fumiyasu Imai for critical comments on the manuscript. K.-i.K. is
supported by Japan Society for the Promotion of Science Postdoctoral
Fellowships for Research Abroad.
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| www.pnas.org/cgi/doi/10.1073/pnas.1101347108 Katayama et al.