Loss of Tsc2 in radial glia models the brain
pathology of tuberous sclerosis complex
in the mouse
Sharon W. Way, James McKenna III, Ulrike Mietzsch, R. Michelle Reith, Henry Cheng-ju Wu
and Michael J. Gambello?
Division of Medical Genetics, Department of Pediatrics, University of Texas Health Science Center, 6431 Fannin
Street, MSB 3.144, Houston, TX 77030, USA
Received December 15, 2008; Revised December 15, 2008; Accepted January 12, 2009
Tuberous sclerosis complex (TSC) is an autosomal dominant, tumor predisposition disorder characterized by
significant neurodevelopmental brain lesions, such as tubers and subependymal nodules. The neuropatho-
logy of TSC is often associated with seizures and intellectual disability. To learn about the developmental per-
turbations that lead to these brain lesions, we created a mouse model that selectively deletes the Tsc2 gene
from radial glial progenitor cells in the developing cerebral cortex and hippocampus. These Tsc2 mutant mice
were severely runted, developed post-natal megalencephaly and died between 3 and 4 weeks of age. Analysis
of brain pathology demonstrated cortical and hippocampal lamination defects, hippocampal heterotopias,
enlarged dysplastic neurons and glia, abnormal myelination and an astrocytosis. These histologic abnorm-
alities were accompanied by activation of the mTORC1 pathway as assessed by increased phosphorylated S6
in brain lysates and tissue sections. Developmental analysis demonstrated that loss of Tsc2 increased the
subventricular Tbr2-positive basal cell progenitor pool at the expense of early born Tbr1-positive post-mitotic
neurons. These results establish the novel concept that loss of function of Tsc2 in radial glial progenitors is
one initiating event in the development of TSC brain lesions as well as underscore the importance of Tsc2 in
the regulation of neural progenitor pools. Given the similarities between the mouse and the human TSC
lesions, this model will be useful in further understanding TSC brain pathophysiology, testing potential thera-
pies and identifying other genetic pathways that are altered in TSC.
The autosomal dominant tumor suppressor disorder, tuberous
sclerosis complex (TSC), bears its name from the tuber-like
brain lesions described by Desire-Magloire Bourneville in
the late 1800s (1). Most affected patients have a germline inac-
tivating mutation of either TSC1 or TSC2, encoding the pro-
teins hamartin and tuberin, respectively (2,3). Loss of the
second allele, leading to loss of heterozygosity (LOH), has
been shown to predispose to tumor formation in many
organs (4,5). Although any organ can be affected, the brain,
skin, kidney, lung and heart are principal sites of pathology
(6). The effects of TSC on the brain are particularly debilitat-
ing, encompassing a variety of lesions such as cortical tubers,
subependymal nodules, white matter linear migration lines,
corpus callosum abnormalities and transmantal cortical dys-
plasia associated with hemimegalencephaly (7). The histo-
pathology of tubers is characterized by generalized cellular
disorganization, astrocytosis and characteristic giant cells.
The neuropathology of TSC is associated with epilepsy, devel-
opmental delay, behavioral abnormalities and autism spectrum
disorders (8–10). Because the neurologic sequelae of TSC
cause significant morbidity and mortality, there is intense
interest in understanding and treating the brain manifestations
of this disease.
Hamartin and tuberin form a biochemical TSC complex that
inhibits the insulin- and nutrient-induced mTOR/S6K/4E-BP1
signal transduction pathwaythatcontrolstranslation,
?To whom correspondence should be addressed. Tel: þ1 713 500 5760; Fax: þ1 713 500 5689; Email: firstname.lastname@example.org
# 2009 The Author(s)
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Human Molecular Genetics, 2009, Vol. 18, No. 7
Advance Access published on January 15, 2009
proliferation and cell growth (11–13). This inhibition is
mediated primarily through the GTPase activity of the TSC
complex toward Rheb-GTP, a small Ras-like protein (14,15).
Rheb-GTP normally activates the rapamycin-sensitive mTOR
ated with the proteins raptor and GbL (16,17). Loss of either
TSC1 or TSC2 in TSC abolishes Rheb-GTPase activity, result-
ing in constitutively activated mTORC1. Activated mTORC1
kinase leads to increased levels of several phosphorylated pro-
teins of the translational apparatus, such as S6 kinase and ribo-
somal protein S6. The end result is increased translation, cell
size and proliferation. The giant cells of tubers stain intensely
for both phosphorylated S6 kinase and phosphorylated S6, indi-
cating that mTORC1 activation is an important mechanism for
TSC neuropathology (5,18). The identification of the TSC1–
TSC2 complex as an inhibitor of mTORC1 has paved the way
for a novel medical treatment of TSC. The fungal metabolite
rapamycin, which is widely used to suppress the immune
system in organ transplant patients, inhibits mTORC1 activity
(19). The mTORC1-inhibitory function of rapamycin led to
the hypothesis that rapamycin might be able to restore the inhi-
bition lost in TSC. Subsequent cell culture and animal studies
confirmedthathypothesis (20–23).Ithasrecently beendemon-
strated that rapamycin can rescue the seizure phenotype in an
astrocyte-specific Tsc1 mouse model (24). Rapamycin also
improved survival and rescued cell size and myelination
abnormalities in a neuronal-specific Tsc1 model of TSC (25).
Indeed, rapamycin appears to be very promising for the treat-
ment of TSC, and several human trials have already yielded
encouraging results (26,27). In spite of the recent discoveries
regarding the functions of hamartin and tuberin, the pathophy-
siology of TSC brain lesions remains poorly understood. Most
brain lesions are present at birth, suggesting that the neuro-
pathology of TSC represents developmental defects (1,7).
Tubers have been detected in utero using high-resolution ultra-
sound and fetal MRI (18,28). Neuroglial progenitor markers,
vimentin and nestin, have been demonstrated in the cells of
tubers (29,30). These observations have led to a prevailing
hypothesis that neuroglial precursor cells might be the cells of
origin of the brain lesions of TSC (18,31). This hypothesis
Initial attempts to model TSC in the mouse did not result
in significant TSC-like brain pathology. Mice with hetero-
zygous disruptions of Tsc1 or Tsc2 developed kidney and
liver lesions, but limited brain pathology (22,32–35). Homo-
zygous deletion of either gene caused midgestation lethality,
possibly related to liver hypoplasia, though some embryos
had exencephaly. Recent models using a conditional allele
of the Tsc1 have been more fruitful. Conditional disruption
of Tsc1 in astrocytes, using a GFAP-Cre driver, produced
mice that demonstrated enlarged brains, an astrocytosis and
mTORC1 activation (36). Tsc1flox/flox;GFAP-Cre mice had
normal cortical organization, mild hippocampal abnormalities
and died from seizures by 3 months of life. Generation of a
neuron-specific deletion of Tsc1 using a synapsin-Cre driver
also produced severely compromised animals that died within
a few months of life from seizures (37,38). There is some
discrepancy as to whether these animals exhibit histologic
activated mTORC1 and myelination defects. Although these
Tsc1-based models do not demonstrate tubers or subependy-
mal nodules per se, they exhibit significant features of the
human disease and will be useful to dissect out the functions
of the TSC complex in different neuronal populations. None-
theless, neither model has tested the hypothesis that loss of
Tsc1 or Tsc2 in a neuroprogenitor cell initiates TSC neuro-
pathology. Moreover, these studies are all based upon the
Tsc1 gene. Although patients with TSC can have mutations
in either gene, TSC2-based disease appears to be more
severe and causes more profound central nervous system
(CNS) dysfunction (39,40). Therefore, in an effort to
develop a Tsc2-based model for TSC neuropathology, we
have created a conditional allele of Tsc2 in the mouse
(41). Our goal was to use this floxed allele to delete Tsc2
in a neuroglial progenitor population to recapitulate the
brain manifestations of TSC.
Over the past several years, the traditional function of radial
glial cells as a mere scaffold for migrating and developing
neurons has been expanded to that of neuroglial precursor
cells (42). Multiple lineage tracing experiments using Cre
transgenic mice and real-time imaging have demonstrated
that radial glial cells likely give rise to a majority of cells in
the cerebral cortex (43,44). At midneurogenesis, radial glia
can divide symmetrically to generate other radial glia, or
asymmetrically to self-renew and produce post-mitotic
neurons (45). Some radial glia can also generate a second pro-
genitor pool in the subventricular zone containing neurogenic
basal progenitor cells (46,47). At later stages of development,
radial glia are primarily gliogenic. Given these neuroglial
progenitor properties, we speculated that loss of Tsc2 in
radial glial might recapitulate the brain pathology of TSC.
We used an extensively characterized hGFAP-Cre mouse
(48–50) to remove the Tsc2 gene from radial glial progenitor
cells. The hGFAP-Cre transgenic is notably very different
from the GFAP-Cre used to create the astrocyte-specific
Tsc1 knockout model (36). In the hGFAP-Cre mouse,
Cre recombinase is expressed in the radial glia of the
hippocampal anlage at E12 and in the cortical radial glia at
E13.5–14 (48–50). Using the hGFAP-Cre mouse allowed
us to remove Tsc2 from these progenitor cells and their
neuronal and glial progeny, creating a model more similar to
the human disease. In the Tsc1flox/flox;GFAP-Cre mouse,
leaving normal Tsc1 function in all neurons. We generated
Tsc2flox/ko;hGFAP-Cre mice to mimic TSC patients with het-
erozygous loss of TSC2 in all cells and selective loss of the
remaining copy of Tsc2 only in radial glial cells. This strategy
allowed us to bypass the lethality of homozygous knockouts
and create a novel Tsc2-based model of TSC that recapitulates
many features of the human disease in the brain. Our data
demonstrate new roles of the TSC complex in radial glial
biology and cerebral cortical development.
Generation of Tsc2flox/ko;hGFAP-Cre mice
We crossed male Tsc2þ/ko;hGFAP-Cre mice with female
Tsc2flox/floxmice to generate the desired progeny, Tsc2flox/ko;
Human Molecular Genetics, 2009, Vol. 18, No. 71253
hGFAP-Cre. These mutant mice represented TSC patients with
one inactivated Tsc2 allele in all cells. Cre expression only
occurred in radial glial cells; hence, the second floxed allele
was only lost in radial glial cells and their neuronal and glial
descendents. This scheme also tested whether the two hit
hypothesis is operative in TSC neuropathology, a somewhat
controversial issue (51,52). Tsc2flox/ko;hGFAP-Cre mice were
born in the expected Mendelian ratio and appeared healthy until
Figure 1. Generation of Tsc2flox/ko;hGFAP-Cre mice. (A) Weight curves of mutant (Tsc2flox/ko;hGFAP-Cre) mice (red, n ¼ 17) compared with the control
(Tsc2þ/flox) mice (blue, n ¼ 15) demonstrating the retarded growth of the mutant animals. (B) A runted mutant, 21-day-old (P21) mouse compared with a
control littermate. Note the domed head and splayed feet in the mutant animal. (C) The brain of the mutant mouse (right) was noticeably larger than the
control (left). (D) Ventricles in the mutant mouse were dilated (right). (E) Immunoblot analyses of cortical and hippocampal lysates from mutant (‘Creþ’)
mice demonstrated a marked decrease of tuberin, slightly decreased levels of hamartin and large increase in pS6 levels compared with the control (‘Cre-’).
a-Tubulin was used as the loading control. (F and G) Pax6 and pS6 immunohistochemistry in E15.5 control (F) and mutant (G) mice showed increased activation
of mTORC1 in the radial glial cells of the developing cortex. Scale bars, C, 1 mm; F and G, 10 mm.
1254 Human Molecular Genetics, 2009, Vol. 18, No. 7
about post-natal day 8, when their weight gain slowed compared
with littermate controls (Fig. 1A). By weaning, Tsc2flox/
ko;hGFAP-Cre mice were severely runted (Fig. 1B) and died
between 3 and 4 weeks, likely from seizures, as we observed
several mice seizing (n ¼ 6) and the majority of dead mice
were found in extensor posturing. The Tsc2flox/ko;hGFAP-Cre
mice had splayed feet, domed heads, and were often tremulous
and generally less active than littermate controls. Because
mutant animals were often quite sick at weaning and died
during the week after, all analyses on adult mice were conducted
at post-natal day 21 (P21). The brains of the Tsc2flox/
ko;hGFAP-Cre mice were substantially larger than control
brains and demonstrated dilated lateral ventricles (Fig. 1C and
D). In the Tsc2flox/ko;hGFAP-Cre mice, Cre recombinase
deleted the floxed Tsc2 allele in the majority of radial glia in the
hippocampal anlage at E12.5 and in the cortical ventricular
zone at E13.5–14 (49,50). Consequently, all the neurons and
astrocytes derived from Tsc2-null radial glia should have had
complete loss of tuberin antigen. To demonstrate extensive loss
of tuberin, western analysis of cell lysates from P21 cortex and
hippocampus of the Tsc2flox/ko; hGFAP-Cre and control mice
was performed and demonstrated significant loss of tuberin
antigen (Fig. 1E). Hamartin levels were also slightly decreased
in experimental lysates, reflecting the dependence of its stability
on the presence of its binding partner tuberin (53). Loss of
tuberin antigen was accompanied by an expected activation of
the mTORC1 pathway based on increased levels of phosphory-
lated (Ser 240/244) S6 (pS6). We then demonstrated that
mTORC1was activated in radial glia by performing immunohis-
tochemistry on E15.5 brains using antibodies against the radial
glial marker Pax6 and pS6 (Fig. 1F and G) (54). In the mutant
embryonic brains, more intensely red pS6 staining was seen sur-
rounding the green Pax6-labeled nuclei of radial glial cells in the
ventricular zone compared with the control. This increased pS6
expression in radial glial cells demonstrated activation of
mTORC1 caused by loss of Tsc2.
Loss of Tsc2 in radial glia causes cortical and hippocampal
enlargement, enlarged cells, lamination defects,
heterotopias and activated mTORC1 in neurons and glia
Histologic analysis of P21 mice revealed a thicker cerebral
cortex from rostral to caudal telencephalon, a blurring of the
gray–white junction, a smaller marginal zone (Layer I) and
apparent lamination defects (Fig. 2A and B) in the mutant.
Higher magnification demonstrated many cells with enlarged
somata and nuclei, and more extracellular matrix in the
mutant compared with the control (Fig. 2C and D). The loss
of Tsc2 in radial glia should have activated mTORC1 in all
their neuronal and glial progeny, similar to what has been
observed in human TSC lesions (18,55). To assess cortical
neuronal and astrocyte mTORC1 activation, we performed
double immunohistochemistry against NeuN, a neuronal
marker, or S100, an astrocyte marker, and pS6. In the rostral
cerebral cortex, almost all NeuN-positive cells in the mutant
demonstrated increased pS6 antigen (Fig. 2F and G). Note
the increased size (P , 0.005) of almost all the mutant
neurons compared with the control (Fig. 2E). Similarly, the
S100-positive astrocytes in the rostral cortex of mutant mice
were larger and expressed more pS6 than those of control
animals (Fig. 2H and I).
The hippocampus of mutant Tsc2flox/ko;hGFAP-Cre mice
also demonstrated defects in organization. There were lami-
nation defects throughout the pyramidal layer, most severe
in regions CA1 and CA3 (Fig. 3B), with many ectopic,
enlarged neurons in the stratum oriens (SO) (Fig. 3F and G).
These defects were in stark contrast to the tight, ordered pyr-
amidal layer of the control animals, with a relatively cell
sparse SO (Fig. 3A and E). The dentate gyrus (DG) retained
its overall structure and contained approximately equal
numbers of granule cells. However, in the mutant hippo-
campus, there were large, ectopic granule cells lining the
outer limit of this region (Fig. 3C and D). Another striking
defect was the presence of ring heterotopias throughout the
stratum lacunosum moleculare (SLM) (Fig. 3B and H).
These heterotopias were mainly composed of NeuN-positive
neurons with a few enlarged S100-positive astrocytes
(Fig. 3I and J). There were, however, several DAPI-stained
nuclei that were neither NeuN nor S100 positive, suggesting
that differentiation into a neuronal or glial lineage was abnor-
mal. Hippocampal NeuN-positive cells and S100-positive cells
also demonstrated increased pS6 (Fig. 3K, L, N and O).
However, the DG of both mutant and control hippocampus
did not express a similar increase in the intensity of pS6
antigen compared with respective pyramidal layers (PL). Pre-
vious studies have verified that Cre is expressed in DG granule
cell precursors in the hGFAP-Cre transgeneic mouse (49,50).
Therefore, we postulated that increased mTORC1 activity in
granule cells of the DG might not result in elevated pS6
levels comparable with the pyramidal cell layer. Nonetheless,
mTORC1 activation should result in an increase in cell size, so
we compared the areas of DG granule cells between mutant
and control animals (Fig. 3M). Granule cells from the
mutant DG were on average 10% larger (P , 0.0005) than
those in the control DG, suggesting that Tsc2 deletion and
mTORC1 activation occurred in the DG of the mutant.
Together, these results indicated that mTORC1 remained acti-
vated in the neuronsand
Tsc2-deficient radial glia. This mTORC1 activation resulted
in increased cell size, both in the cortex and in the hippo-
campus, as previously reported in several model systems
Developmental analysis of Tsc2flox/ko;hGFAP-Cre mice
reveals post-natal cerebral enlargement and possible
To analyze the development of the cerebral abnormalities seen
at P21, we examined hematoxylin and eosin (H&E) stained
sections from E16.5–P15 (Fig. 4). At E16.5, we observed
no notable difference in thickness of the developing cortex
between mutant and control (data not shown). At P0, the
difference in thickness of the cerebral cortex was also
minimal, though the ventricular zone appeared thicker in the
mutant (Fig. 4A and B). In the hippocampus, defects in organ-
ization of the CA3 layer of the mutant were already apparent
(Fig. 4C and D). By P5, the difference in cortical thickness
became more pronounced while the lamination abnormalities
in CA1 and CA3 and ectopic neurons in the SO were
Human Molecular Genetics, 2009, Vol. 18, No. 7 1255
evident. In the hippocampal fissure, there were several ectopic
clusters of cells in the mutant brains (Fig. 4E–H). These
clusters appeared to be the site of origin of the future ring
heterotopias. By P10, the mutant cortex was noticeably
becoming more obvious (Fig. 4I–M). By P15, the mutant
brains were similar histologically to brains analyzed at P21
than control and the ringheterotopias were
Figure 2. Cerebral cortical defects and up-regulation of mTORC1 in cortical neurons and astrocytes in Tsc2flox/ko;hGFAP-Cre mice. All sections were taken from
P21 mice. (A and B) The cortex of the mutant (B) was thicker than the control (A) and displayed lamination defects, blurring between the gray–white junction,
and a much less-defined molecular layer (Layer I). (C and D) Higher magnification revealed enlarged cells in the cortex of the mutant (D) and more extracellular
matrix between the cells compared with the control (C). (E) Comparison of the areas of NeuN-labeled neurons from mutant and control cortex revealed that
mutant neurons are significantly larger (??P , 0.005, n ¼ 6) than control neurons. (F and G) NeuN-labeled neurons in the mutant cortex (G) displayed substan-
tial increase in pS6 expression compared with the control (F), indicating elevated mTORC1 activation. (H and I) S100-labeled astrocytes in the mutant cortex (I)
also showed notable increase in pS6 expression compared with the control (H). Scale bars, A and B, 100 mm; C and D, 20 mm; F and G, 50 mm; H and I, 40 mm,
inset 10 mm.
1256 Human Molecular Genetics, 2009, Vol. 18, No. 7
Loss of Tsc2 in radial glia causes perturbations in neuronal
subsets and an astrogliosis
To gain further insight into the enlarged brains of the mutant
animals, we assessed cortical lamination with layer-specific
immunohistochemistry. Antibody to Cux-1 labels neurons in
Layers II–IV of the mammalian cerebral cortex (Fig. 5A)
(56). In the Tsc2flox/ko;hGFAP-Cre cortex, labeled Cux1 cells
were not restricted to Layers II–IV, but detected throughout
the entire cortex. Moreover, though the difference was not
found to be significant (Fig. 5D), there was a slight increase
in Cux-1 positive neurons in the mutant cerebral cortex com-
pared with the control. The integrity of Layer VI was assessed
using antibody against the transcription factor FoxP2 (Fig. 5B)
(57). In the mutant cortex, there was a well-demarcated
FoxP2-positive Layer VI; however, there were ?30% less
FoxP2-positive cells (P , 0.05) in the mutant Layer VI than
the control. TUNEL analysis at E16.6 and P10 suggested
that the fewer FoxP2 cells were not due to increased cell
death (data not shown). Since there were significantly
fewer FoxP2-labeled neurons, and because the macrocephaly
occurred post-natally, we asked whether the total number of
NeuN-positive neurons or S100-positive glia in the cerebral
cortex of the mutant animals were different. Quantitation of
both cell populations did not reveal a significant difference
(Fig. 5C) or of S100-positive glia (Fig. 5H) in the cortex of
the control versus mutant animals. Further analysis with
PCNA, a proliferation marker, showed no significant differ-
ence in PCNA-positive cells in the cortex at P10 (data not
shown), in accordance with the total cell counts.
An astrogliosis is often present in TSC tubers (55). Astro-
gliosis usually occurs when insult to the brain is sustained,
yet tubers display alterations in astrocytes, even though no
physical damage has occurred. In the synapsin-Cre-based
Tsc1 mouse model, no evidence of astrogliosis was observed
(37). GFAP-Cre-driven Tsc1 deletion in astrocytes did yield
an astrogliosis throughout the cortex and the hippocampus
(36). To determine whether our conditional deletion of Tsc2
immunohistochemistryfor GFAP on
normal mouse cerebral cortex expresses very weak GFAP
Figure 3. Hippocampal defects and up-regulation of mTORC1 in hippocampal neurons and astrocytes in Tsc2flox/ko;hGFAP-Cre mice. All sections were taken
from P21 mice. (A and B) Severe lamination defects in the mutant mouse (B) were apparent in H&E-stained sections of the hippocampus, especially in the CA1
and CA3 regions. White arrows represent ring heterotopias that were evident throughout the SLM. (C and D) The DG was lined with large, ectopic granule cells
in the mutant (D). (E–G) Numerous ectopic neurons (F, black arrows) and enlarged cells (G, black arrowhead) were also present in the SO of the mutant hip-
pocampus (F), in contrast to the tight, well-organized pyramidal layer in the control (E). (H–J) High power magnification of a ring heterotopia demonstrated that
they were mainly composed of NeuN-positive neurons (green cells) with some S100-positive astrocytes (I, J, red cells, white arrowheads), though some cells do
not stain for either marker (I, J, black arrowheads). (K and L) NeuN-labeled neurons in the hippocampus of the mutant (L) displayed greater pS6 expression than
the control (K). However, expression of pS6 in the DG of both the mutant and the control is reduced compared with their respective PL. (M) Despite low immu-
nohistochemical pS6 expression in the mutant DG, average cell area of cells in the DG of the mutant was significantly larger (???P , 0.0005) than the control
(n ¼ 6). (N and O) S100-labeled astrocytes in the hippocampus of the mutant (O) had increased pS6 expression. The SLM region of the hippocampus is shown.
Scale bars, A–F, 50 mm; G–J, 10 mm; K and L, 100 mm; M, 50 mm; N and O, 20 mm.
Human Molecular Genetics, 2009, Vol. 18, No. 71257
immunostaining, whereas the Tsc2flox/ko;hGFAP-Cre mutant
cortex revealed a marked increase in GFAP expression
throughout the entire cortical thickness (Fig. 5F). There was
also increased GFAP staining around the mutant ventricular
zone surrounding the lateral ventricles. Increased GFAP
expression was also seen throughout the hippocampus,
where astrocytes in all hippocampal regions, especially the
SLM, displayed more intense GFAP expression. The cell
bodies of the astrocytes in the mutant were also larger, with
shorter and thicker processes than those of the control cells,
a characteristic of activated astrocytes (Fig. 5G, inset). Quan-
titation of GFAP-labeled cells within the hippocampus showed
significantly more astrocytes in the mutant (Fig. 5I). The
increased number of GFAP-positive cells in the hippocampus
was also accompanied by an increase in PCNA-positive cells
(data not shown). These results verified that Tsc2 deletion in
radial glia caused an astrogliosis in the cortex mainly by astro-
cyte activation, but in the hippocampus by both activation and
Loss of Tsc2 in radial glia alters progenitor cell
populations with minimal effect on proliferation
S100-positive glia between the mutant and the control cerebral
cortex suggested that loss of Tsc2 had a minimal effect on pro-
liferation of radial glial cells. To address this, we pulse-labeled
E14.5 embryos with BrdU for 30 min before sacrifice to label
cells in S phase. Quantitation of BrdU-positive cells revealed
no significant difference between control and mutant animals
(Fig. 6A–C), suggesting that loss of Tsc2 had a small if any
effect on proliferation at this time point. Since we did
observe notably fewer FoxP2-labeled early born neurons in
the mutant cortex, we reasoned that loss of Tsc2 in radial
glia might affect their differentiation into post-mitotic
neurons versus subventricular basal progenitor cells. Immuno-
histochemistry for Tbr2, a marker of basal progenitor cells,
demonstrated a significant increase in this progenitor popu-
lation compared with wild-type (P , 0.0005, Fig. 6D–F)
equal numbersof NeuN-positiveneurons and
Figure 4. Post-natal developmental analysis of Tsc2flox/ko;hGFAP-Cre mice. H&E-stained sections from P0 to P15. (A–D) At P0, the size of the cerebral cortex
was comparable between the control and the mutant. The ventricular zone (arrowheads) appeared thicker in the mutant and lamination abnormalities in CA1 and
CA3 (arrows) were beginning to develop. (E–H) At P5, an increase in the cortical thickness of the mutant (F) was more apparent. The layering abnormalities in
CA3 (arrow) were more pronounced. There were many ectopic cells in the SO of the mutant (H, white arrowheads) compared with the control. There was also the
appearance of clusters of cells in the hippocampal fissure (black arrowhead). (I–M) By P10, the cerebral cortex of the mutant had continued to enlarge, and the
layering was indistinct compared with the control. The ring heterotopias in the SLM were evident (inset J). The corpus callosum was also noticeably thicker.
Sectioning through a heterotopia (K–M) demonstrated that they represent a nodule of ectopic cells. (N–Q) By P15, the same abnormalities were present as those
seen at weaning. Scale bars, A, B, E–J, N, O, 100 mm; C, D, K–M, P, Q, 50 mm.
1258Human Molecular Genetics, 2009, Vol. 18, No. 7
Tbr2-positive basal progenitor cells might be accompanied
by a decrease in the number of Tbr1-positive post-mitotic
neurons at E14.5 that might explain the lower number of
FoxP2-labeled Layer VI neurons. Indeed, Tbr1-positive cells
were significantly increased (P , 0.05) in the control com-
pared with the mutant (Fig. 6G–I). To determine whether
this effect was a result of increased radial glial progenitors,
we counted pax6-labeledradial
significant difference between the control and the mutant
glia and foundno
Loss of Tsc2 in radial glia caused causes myelin formation
defects and increases oligodendrocytes
Advances in MRI imaging techniques have identified a variety
of white matter defects in the brains of TSC patients (58,59).
Diffusion tension MRI, which detects the direction and magni-
tude of water diffusion in tissues, has even identified micro-
structrural defects in normal-appearing white matter from
TSC patients (59). Some of the most common white matter
lesions identified in the brains of TSC patients were gliosis
and hypomyelination. The neuron-specific Tsc1 mouse
Figure 5. Lamination defects and astrogliosis in the Tsc2flox/ko;hGFAP-Cre mice. All sections and counts conducted in P21 mice. (A and b) Cux1 (A), a Layer
II–IV marker, and FoxP2 (B), a Layer VI marker, were used to assess lamination defects (n ¼ 6 and 4, respectively). The Cux1-labeled cells of the control
resided mostly in their designated layers, whereas the labeled cells of the mutant were scattered throughout the cortex. FoxP2-labeled cells were generally
in Layer VI for both control and mutant. (C–E) Although NeuN-labeled neurons (C) and Cux1-labeled cells (D) were not found to be significantly different
in the cortex, there were significantly less FoxP2-labeled cells in the mutant (E,?P , 0.05). (F and H) Immunostaining of GFAP in the cortex (F) revealed
substantial increase of GFAP-expressing astrocytes in the cortex compared with the control. Intense GFAP expression was also seen in the mutant ventricular
zone of the lateral ventricle (white arrows). As quantification of GFAPþ cells in the cortex was difficult in the control, S100 (H) was used to label cortical
astrocytes. No significant difference was found in S100þ cells between the mutant and the control. (G and I) GFAP expression was notably higher in the
mutant hippocampus where astrocytes had larger cell bodies but shorter, thicker processes compared with the control. Significantly more GFAP-labeled cells
(I) were present in the mutant hippocampus.
Human Molecular Genetics, 2009, Vol. 18, No. 7 1259
model demonstrated significant hypomyelination (37). To
investigate white matter defects in our mutant mice, we per-
formed immunohistochemistry for myelin basic protein
(MBP). Myelination is progressing in control and mutant
animals in the corpus callosum at P10 (Fig. 7A). In the
mutant, myelination appeared to be discontinuous at several
places. By P15, myelination had progressed, though there
was a paucity of myelinated fibers in the more superficial
layers of the cortex, as well as in the hippocampus
(Fig. 7B). There was no significant difference between
myelin patterns between P15 and P21. To assess whether
defects in myelin-producing oligodendrocytes might have con-
tributed to this phenotype, we performed immunohistochemis-
try to CC1, an oligodendrocyte marker. Interestingly, mutant
mice had more oligodendrocytes than control (665 cells/
mm2+46 versus 532 cell/mm2+35, P ¼ 0.05). Double
labeling with CC1 and pS6 revealed that a larger number of
oligodendrocytes in the mutant have up-regulated mTORC1
activity (Fig. 7C and D). In spite of an increased number of
activated oligidendrocytes, myelination does not proceed nor-
mally in mutant animals.
We have created a novel Tsc2-based brain-specific model of
TSC by Cre-mediated ablation in radial glial progenitor
cells. Consequently, all neurons and glia derived from
Tsc2-null radial glia were devoid of Tsc2 and demonstrated
mTORC1 activation, similar to the cells of human TSC
brain lesions (18). The brains of the Tsc2flox/ko;hGFAP-Cre
mice exhibited many neuropathologic features of human
TSC, such as cortical thickening, enlarged cells, lamination
defects, heterotopias and abnormal myelination. Although
there were no focal tubers per se, there were focal lesions
in the hippocampus and the entire cerebral cortex had many
characteristics of tubers. Our results support the neuropro-
genitor hypothesis of TSC brain lesions (18,31). TSC is
also considered a tumor suppressor disorder; however, LOH
has been difficult to demonstrate for some human brain
lesions (51,52). Our data demonstrate that LOH of Tsc2 in
radial glia model TSC neuropathology and support the
two-hit hypothesis for the formation of human TSC brain
The Tsc2flox/ko;hGFAP-Cre model differs significantly from
the neuronal-specific Tsc1flox/ko;SynICre mouse and the glial-
specific Tsc1flox/flox;GFAP-Cre mouse models of TSC in two
important ways (36,37). First, the mouse homolog of the
TSC2 gene, associated with more severe human neurologic
defects, was conditionally inactivated in this model. Secondly,
since the Tsc2 gene was deleted in radial glial progenitor cells,
all their resulting neuronal and glial progeny were also lacking
in Tsc2 activity as demonstrated by mTORC1 activation. The
survival of these mice was more severely compromised than
Figure 6. Cortical neural progenitor pool analysis at embryonic day E14.5. (A–C) BrdU pulse-labeling failed to detect any significant proliferative differences
along the ventricles of the control and mutant embryos at E14.5 (n ¼ 6). (D–F) Significantly more (F,???P , 0.0005) Tbr2-labeled basal progenitor cells were
found in the subventricular zone of the mutant (E) compared with the control (D) (n ¼ 6). (G–I) The number of post-mitotic Tbr1-labeled neurons was signifi-
cantly decreased (I,?P , 0.05) in the control (G) (n ¼ 6). (J–L) Radial glia numbers (L), as labeled by pax6, were not found to be significantly different
between the control and the mutant (J) (n ¼ 4). CP, cortical plate, SVZ, subventricular zone, VZ, ventricular zone. A, B, G and H taken at 20? magnification,
D, E, J and K taken at 40?.
1260Human Molecular Genetics, 2009, Vol. 18, No. 7
the other neuronal and astrocyte-specific Tsc1 brain-specific
knockouts, dying between 3 and 4 weeks rather than 2–3
months of age (36,37). Although we have not performed
EEG recordings of epileptiform activity, we have observed
generalized seizures in several animals. When animals were
found dead, they typically were in extensor posturing
suggesting that they died from a tonic seizure. Future electro-
physiologic studies will be required to understand the epilepsy
that these animals have.
TSC has been considered a disorder of neuronal migration
(6). The defects observed in the cerebral cortex and hippo-
campus provide evidence to support a neuronal migration
defect. The lamination defects of the hippocampal pyramidal
layer were similar to defects seen in mice with targeted disrup-
tions of neuronal migration genes such as Reelin, Lis1 and
Doublecortin (60–62). The ring heterotopias also appeared
to represent abnormal migration of neurons, glia and other uni-
dentified cells. The presence of more Cux-1-expressing cells
Figure 7. Post-natal developmental analysis of hypomyelination and oligodendrocytes in the mutant. (A and B) Immunostaining of MBP at P10 (A) and P15 (B)
demonstrated a defect in myelin formation in the mutant cortex (arrows). (C and D) Oligodendrocyte analysis using CC1 revealed a 1.25-fold increase in
CC1-labeled cells in the mutant (n ¼ 4, data not shown) compared with the control. Colocalization of pS6 and CC1 (arrowheads) showed that most oligoden-
drocytes in the mutant had up-regulated mTORC1 activity.
Human Molecular Genetics, 2009, Vol. 18, No. 71261
in the deep layers of the mutant cortex, and the thickened ven-
tricular zone of P0 mutant mice also suggested a neuronal
migration defect. Further investigation into the role of the
TSC complex in neuronal migration will be required,
but this model should make in vivo developmental studies
The initial, striking feature of the brains of the TSC mice
was the megalencephaly. The enlarged brains of these
animals are reminiscent of the enlarged eye phenotype seen
in the seminal studies in Drosophila that established the
roles of the Tsc1 and Tsc2 genes in controlling cell and
organ size (63,64). Hemimegalencephaly has also been
reported as a rare manifestation of TSC (65,66). Cortical
size is controlled by a delicate balance of proliferation, differ-
entiation and apoptosis (67–69). The TSC complex has been
demonstrated to regulate all these processes in different
model systems. The developmental analysis indicated that
the enlarged brain phenotype was predominantly a post-natal
event. Minimal proliferation abnormalities were found in the
cortex, whereas significantly increased PCNA-positive cells
were found in the hippocampus. It appears that increased
cell size and matrix are the principle determinants for the cor-
tical enlargement, whereas hippocampal enlargement was
dependent on both increased proliferation and increased cell
size and matrix.
The differences in neuronal composition of the adult
Tsc2flox/ko;hGFAP-Cre cortex suggested that loss of Tsc2
might alter cell fate. Embryonic studies indicated that although
total proliferation and radial glial cell numbers were unaf-
fected at E14.5, there were notably more basal progenitor
cells and fewer post-mitotic neurons in the mutant embryos.
These findings indicate that loss of Tsc2 perturbs progenitor
differentiation. Given the fewer early born post-mitotic
neurons in the mutant embryo, it follows that there were
fewer Layer VI FoxP2-positive neurons in the adult mutant
cortex. However, although the large increase in basal progeni-
tor cells in the mutant indicated that a similarly large increase
in superficial-layer neurons should be present in the adult
cortex, we were unable to find a significant increase in Cux-1-
positive cells in Layers II–IV in the mice we have analyzed in
the P21 cortex (n ¼ 6). Recent studies have demonstrated that
Tbr2-positive basal progenitor cells not only contribute to
superficial cortical layers, but also deeper layers (70). Conse-
quently, we hypothesize that loss of Tsc2 may increase pools
of basal progenitor progeny in regions we have not studied,
such as Layer V. Though we have not been able to detect
apoptosis at E16.5, P5 or P10, we cannot completely
exclude cell death of basal progenitors or their progeny as
factors that contribute to the final P21 mutant brain.
Astrocytes can become reactive in response to a variety of
CNS pathologies such as infection, ischemia or neurodegen-
erative disease (71,72). This reactive gliosis is accompanied
by morphologic and cell physiologic changes, most notably,
the up-regulation of intermediate filament proteins such as
vimentin and GFAP. Cortical tubers contain characteristic
giant cells surrounded by a severe astrogliosis. The cause of
this TSC-associated astrogliosis is unknown, but suggests
that dysfunctional, reactive astrocytes may be important in
TSC neuropathology (55). The Tsc2flox/ko;hGFAP-Cre mice
displayed an extensive astrogliosis throughout the cortex and
hippocampus, similar to the human disease. Though the
giant cell is a hallmark feature of tubers, human and animal
studies suggest that dysfunctional astrocytes may be a major
site of neuropathology of TSC (36,55). This new mouse
model will allow further assessment of the role of activated
astrocytes in TSC pathology.
Brain myelination is a process whereby the membranes of
oligodendrocytes wrap around and extend along neuronal
axons (73). Normal myelination appears to rely not solely
on oligodendrocytes, but also upon the correct signals from
target axons (74). Abnormal myelination has recently been
found to be another pathologic feature in the brains of TSC
showed abnormalities in the progression of myelination that
resulted in an undermyelinated cerebral cortex and hippo-
campus. Abnormalities in neurons and/or oligodendrocytes
may have caused these defects as both cell populations demon-
strated mTORC1 activation. Interestingly, the neuronal Tsc1
model mouse, in which post-mitotic neurons are deleted for
Tsc1, also demonstrated very similar defects in myelination
(37). Those data suggested that loss of Tsc1 disrupted neuronal
signals that are crucial for the correct progression of myelina-
tion. Since both models demonstrate similar myelination
defects, it is likely that the neuronal absence of Tsc2 is a
major cause for the aberrant myelination. Future co-culture
studies will be required to assess the effects of the TSC
complex on the interplay between oligodendrocytes and
neurons and the regulation of myelination.
The Tsc2flox/ko;hGFAP-Cre model of TSC suggests that the
radial glial cell might be a cellular site for LOH in human TSC
brain lesions. Although our results do not preclude LOH in
other progenitor cells such as neuroepithelial or basal progeni-
tor cells, the resultant phenotype suggests a crucial role for
tight regulation of the mTORC1 pathway in radial glial pro-
genitor differentiation and neuronal migration. Loss of
mTORC1 regulation plays a role in other developmental
brain disorders such as neurofibromatosis type 1 and
PTEN-associated macrocephaly (75,76). Further study of the
developmental defects in radial glia caused by loss of
TSC-regulated mTORC1 should provide insight not only
into TSC, but also many other neurodevelopmental defects.
The Tsc2flox/ko;hGFAP-Cre mouse demonstrated activation
of mTORC1, so rapamycin treatment should rescue some of
the neuropathology. Since neurons and glia are affected in
this model, it will be interesting to assess whether there is a
differential cellular response to rapamycin treatment. The cre-
ation of a Tsc1flox/ko;hGFAP-Cre is in progress and will allow
a comparison with the present model to provide a possible bio-
logic basis for the differences in severity between patients with
TSC1 versus TSC2 mutations. This Tsc2-based model also
broadens the availability of models for the preclinical testing
of established and novel drugs for the treatment of TSC and
MATERIALS AND METHODS
Mice and genotyping
All animal experimentation was approved by the UTHSC
Animal Welfare Committee. Mice were in a mixed 129 and
1262 Human Molecular Genetics, 2009, Vol. 18, No. 7
C57/Bl6 background. Tsc2þ/flox, Tsc2þ/koand hGFAP-Cre
mice have been previously described (41,48,49). Mice were
genotyped for Tsc2 alleles using three primers in one PCR
reaction: KO1: 50-GCAGCAGGTCTGCAGTGAAT-30, KO2:
50-CCTCCTGCATGGAGTTGAGT-30; WT2: 50-CAGGCAT
Band sizes were wild-type (390 bp), Tsc2flox(434 bp) and
Tsc2ko(547 bp). Cre primers: CreF: 50-GGACATGTTCA
AACTCCCAGACAC-30, RapB: 50-AGCTTCTCATTGCTGC
GCGCCAGGTTCAGG-30. Band sizes were Cre (219 bp)
and Rap (590 bp) as a positive control band. Cre and Rap
primers were generously provided by the laboratory of
Adult mice were deeply anesthetized before undergoing trans-
cardiac perfusion with PBS and then with 4% paraformalde-
hyde (PFA). Mouse brains were post-fixed in PFA overnight
and stored in 70% ethanol prior to embedding in paraffin. Par-
affin blocks were sectioned at 5 mm. For embryo analysis, the
day of the vaginal plug was 0.5. Dams were anesthetized with
2.5% avertin and killed by cervical dislocation before embryos
were dissected into cold PBS and staged. A small piece of the
embryo was used for genotyping. Embryos were fixed in PFA
4–6 h and washed in 1? PBS before being stored in 70%
ethanol and embedded in paraffin. Slides were rehydrated,
stained with routine H&E, and coverslipped.
Immunofluorescence was performed by blocking in 10%
serum from animal in which secondary antibody was raised
and 0.05% Triton X-100 in 1? PBS for 1 h. Primary antibody
was allowed to incubate overnight at 48C. Sections were
washed in 1? PBS followed by secondary antibody incubation
for 1 h. Tissue was directly visualized for fluorescence-
using anOlympusBX51 or IX81 microscope and captured with
a Qimaging RETIGA-2000RV digital camera or a Bio-Rad
1024 MP confocal microscope. Digital images were then pro-
cessed using Adobe Photoshop (San Jose, CA, USA).
The antibodies used for immunohistochemistry were as
follows: Pax6 (1:2000, Developmental Studies Hybridoma
Bank, Iowa City, IA, USA), phosphorylated (Ser 240/244)
S6 (1:100, Cell Signaling Technology, Bedford, MA, USA),
Cux-1 (1:50, Santa Cruz Biotechnology, Santa Cruz, CA,
USA), FoxP2 (1:4000, Abcam, Cambridge, MA, USA),
BrdU (1:50, Becton Dickinson, San Jose, CA, USA), GFAP
(1:400, Sigma), CC1 (1:5, Calbiochem, Gibbstown, NJ,
USA), S100 (1:100, DakoCytomation, Denmark and 1:500,
Abcam), PCNA (1:50, Santa Cruz) and NeuN (1:100), Tbr2
(1:2000), Tbr1 (1:500) and MBP (1:200) from Millipore,
Billerica, MA, USA. Prolong Gold antifade reagent with
DAPI (Invitrogen, Eugene, OR, USA) was used for DAPI
staining and coverslipping of post-natal tissue. Embryonic
tissue was stained with Hoechst 33258 (Invitrogen) after
removal of the secondary antibody and coverslipped using
Fluoromount-G (SouthernBiotech, Birmingha, AL, USA).
The antibodies used for immunoblotting were: tuberin
(1:1000), hamartin (1:1000), pS6 (1:2000) and a-tubulin
(1:1000), from Cell Signaling.
Two or three serial sections from each mouse were used for
analysis unless otherwise noted. For post-natal cell counts,
equal-sized images spanning the thickness of the somatosen-
sory cortex were taken at the same lateral distance from the
midline. Count results were corrected to represent a percen-
tage of cells in the cortex in order to account for differences
in total cortex size. For embryo counts, equal-sized images
spanning most of the length of the lateral ventricles were
used. Marker-labeled cells with visible nuclei were manually
counted using Photoshop and ImageJ v1.38x (W. Rasband,
National Institutes of Health, Bethesda, MD, USA). For
neuron cell size determination, the cortex was divided into
five equal bins from the bottom of Layer I to the bottom of
Layer VI. In one section each from three pairs of control
and mutant mice, 50 NeuN-labeled neurons from Bin 3 were
outlined and filled using Photoshop and area was analyzed
in micrometers using ImageJ. For DG area calculation, one
section each from three pairs of mice were stained with
H&E. DG cell images were captured using a 60? objective
and cells near the midline of the DG were outlined for area
calculation in the same manner. Data were analyzed using
repeated-measures ANOVA in SPSS (Chicago, IL, USA)
and Microsoft Excel (Seattle, WA, USA).
Whole cell lysates were made from P21 cerebral cortex and
hippocampus that were quick-frozen in liquid nitrogen.
Samples were homogenized in a dounce homogenizer with
10 volumes of Ripa buffer with protease inhibitor cocktail
and phosphatase inhibitor cocktail (Sigma). Lysates were cen-
trifuged at 48C, sonicated and frozen until use. Protein concen-
trations were determined with a BCA reagent kit (Pierce,
amounts of protein were separated on a denaturing 4–12%
gradient gel (Invitrogen) and transferred to nitrocellulose.
The membrane was cut into sections and each section
probed with different antibodies using a stripping procedure
after each experiment if necessary. Secondary antibodies
were horseradish peroxidase conjugated. Visualization was
conducted with an ECL kit (Amersham, Piscataway, NJ,
We would like to thank Drs. Seonhee Kim, Seo-Hee Cho and
S. Shahrukh Hashmi for their helpful suggestions on this
Conflict of Interest statement. None declared.
Human Molecular Genetics, 2009, Vol. 18, No. 7 1263
Funding to Pay the Open Access Charge was provided by
the National Institutes of Health.
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