Brain size is precisely regulated during development and involves coordination of neural progenitor cell proliferation, differentiation,
sion is high during embryonic development but diminishes as cells differentiate and switches to ShcB/Sck/Sli and ShcC/N-Shc/Rai. To
controls throughout postnatal and adult life. The cerebrum appeared most severely affected, but the gross architecture of the brain is
normal. Body weight was mildly affected with a delay in reaching mature weight. At a mechanistic level, the ShcFFF microencephaly
which declined by E14.5. Apoptosis remained at normal basal levels throughout postnatal development. Proliferation indices were not
nestin-Cre transgene, conditional deletion of ShcA in mice with a homozygous floxed shc1 locus also showed a similar microencephaly
balance between cell proliferation and apoptosis. Mutations af-
fecting either of these processes have dramatic effects on brain
et al., 1996, 1998; Pompeiano et al., 2000) cause an enlarged
forebrain because of the lack of normal neural progenitor apo-
ptosis during development. Conversely, null mutations in the
proto-oncogene Ski (Berk et al., 1997), the transcription factor
AP-2 (Schorle et al., 1996), as well as the double null mutant
combination of Jnk1 and Jnk2 cause an increase in apoptosis in
the forebrain accompanied by a severely reduced embryonic
brain size (Sabapathy et al., 1999).
survival of neuronal progenitors. ShcA transmits extracellular
signals from transmembrane surface receptors to mitogen-
activated protein kinase (MAPK)/extracellular signal-regulated
kinase (ERK) and phosphatidylinositol 3-kinase (PI3K)/Akt ki-
nase signaling pathways (Luzi et al., 2000; Ravichandran, 2001).
The importance of ShcA signaling during development is dem-
onstrated by the homozygous knock-out mouse which dies by
embryonic day 11.5 (E11.5) (Lai and Pawson, 2000). ShcA is
expressed in three isoforms, p66, p52, and p46, which differ only
at the N terminus; however, all three isoforms contain the dis-
tinctive modular organization of phosphotyrosine binding
(PTB), proline-rich (CH1), and Src homology 2 (SH2) domains
(Pelicci et al., 1992). On binding of the PTB domain of ShcA to
phosphotyrosine residues on receptor tyrosine kinases, tyrosine
1996; van der Geer et al., 1996), are phosphorylated, providing a
docking site for the Grb2/Sos nucleotide exchange factor com-
plex, which then initiates downstream kinase signaling cascades
(Rozakis-Adcock et al., 1992).
blotting, is elevated during early embryonic neural development
but then decreases during later stages, and ShcA mRNA is found
in proliferative areas in both embryonic and adult brains (Conti et
al., 1997). In contrast, other Shc family members, ShcB/Sck/Sli and
led to the hypothesis that ShcA is important for proliferation of
neural progenitors, whereas ShcB/Sck/Sli and ShcC/N-Shc/Rai are
In this study, we sought to directly determine brain-specific
TheJournalofNeuroscience,July26,2006 • 26(30):7885–7897 • 7885
functions for ShcA. Transgenic mice harboring a STOP cassette
flanked by loxP sites downstream of the elongation factor-1?
(EF-1?) promoter and upstream of a dominant-negative ShcA
(ShcFFF) construct (Zhang et al., 2002) were crossed with mice
expressing Cre recombinase under the control of the nestin pro-
moter and a neural specific enhancer (Tronche et al., 1999). Our
proliferation of neural progenitors but rather their survival dur-
in a C57BL/6 background. Experimental animals were obtained from
matings of nestin-Cre males to STOPfloxShcFFF females with the day of
birth designated as postnatal day 0 (P0). Embryos were obtained from
timed matings with the morning of vaginal plug detection defined as
E0.5. All animals were handled in compliance with the University of
Virginia’s Animal Care and Use Committee guidelines.
DNA extraction and PCR. Tail biopsies from transgenic animals were
obtained at the time of weaning or experiment, and genomic DNA was
PCR. Transgenes were detected with the following primers under stan-
dard PCR conditions: cre (?), gcggtctggcagtaaaaactatc; cre (?), gtgaaa-
cagcattgctgtcactt; ShcFFF (?), tcctaagcttgatggaattgga; ShcFFF (?), cgg-
gaagtcattaaagaactgatggt; ShcFlox (?), cagccggccaactctaag; and ShcFlox
(?), gccctcggacagagcaatcatgtc. For control PCRs, the native IL-2 gene
gatct; and IL-2 (?), gtaggtggaaattctagcatcatcc.
For DNA isolation from fixed embryonic tissue, 16- to 20-?m-thick
hol series into 1? PBS. Using a dissection microscope for easy visualiza-
tion, all non-neural tissue was carefully removed from the slide in 1?
PBS using a pipette tip and moved into a Microfuge tube. The slide with
the remaining neural tissue was rinsed gently in PBS to remove residual
non-neural tissue. The neural tissue was then scraped from the slide in a
similar manner and placed into a separate Microfuge tube. Both neural
and non-neural tissues were pelleted by gentle centrifugation, and the
(150 mM NaCl, 25 mM EDTA, 0.5% Tween 20, 0.5 ?g/?l Proteinase K)
overnight in a 55°C water bath, and then heat denatured at 80°C for 1 h
with the resulting lysate used directly in PCRs. To detect the presence of
the STOP cassette in ShcFFF transgene-containing mice, the following
primers were used under standard PCR conditions: STOP (?), gcctcat-
native IL-2 gene was detected as described above.
5-Bromo-2?-deoxyuridine injections. Postnatal animals were injected
with 5-bromo-2?-deoxyuridine (BrdU) (Sigma, St. Louis, MO) intra-
intraperitoneally with a single dose of BrdU at 100 mg/kg and killed 6 h
later. Animals were killed with a lethal dose of nembutol and perfused
ethanol. Embryos and postnatal brains were dissected and postfixed in
70% ethanol, embedded in paraffin, and then sectioned in the sagittal or
coronal planes. Six-micrometer sections were cut on a microtome for
tissue staining and 16–20 ?m sections were cut for DNA isolation. For
Human brain samples were procured from archival autopsy tissue at
protocol. Brain tissue was chosen from autopsy patients with non-
neurological causes of death.
Immunohistochemistry. Paraffin sections were dewaxed, rehydrated,
and blocked in either 2% normal goat or horse serum in PBS plus 0.1%
Triton X-100. Primary antibodies were incubated overnight at 4°C and
401, 1:50; Developmental Studies Hybridoma Bank (DSHB), University
of Iowa, Iowa City, IA], cleaved caspase-3 (CC3) (1:100; Cell Signaling
Technology, Beverly, MA), Cre (1:20,000; Novagen, San Diego, CA),
neurofilament (2H3, 1:50; DSHB), TuJ1 (1:500; Covance, Princeton,
NJ), NeuN (1:1000; Chemicon, Temecula, CA), Brn-1 (1:50; Santa Cruz
Biotechnology, Santa Cruz, CA), calretinin (1:500; Chemicon), reelin
(1:500; Chemicon), and Cux-1 (1:500; Santa Cruz Biotechnology).
GABAAreceptor subunit ?2 was recognized by a rabbit antibody (1:200;
Alomone Labs, Jerusalem, Israel). GABAAreceptor subunit ?5 was rec-
ognized by a rabbit antibody (amino acids 337–388; 8 ?g/ml) (gift from
Werner Sieghart, Brain Research Institute, Vienna, Austria).
For fluorescent studies, Oregon Green-conjugated goat anti-mouse
and rhodamine-conjugated goat anti-rabbit secondary antibodies (In-
vitrogen, Eugene, OR) were used at a 1:200 dilution in the blocking
solution. Counterstaining of the tissue was performed using 4?,6?-
itate reactions, goat anti-rabbit and horse anti-mouse biotin-conjugated
secondary antibodies (Sigma) were used at a 1:200 dilution in the block-
ing solution. Antibody detection was performed using the ABC (avidin–
(DakoCytomation, Carpinteria, CA). Tissue sections were counter-
stained with hematoxylin. “No primary” antibody staining controls did
not show any color precipitate.
Cytochrome oxidase staining was performed as described previously
Sections of control littermates were chosen using embryonic (Scham-
common morphological landmarks. Images of stained tissue were cap-
tured using Adobe Photoshop 7.0. Confocal images were captured using
a PerkinElmer (Wellesley, MA) UltraviewRS system attached to a Zeiss
nipulations were performed identically for both experimental and con-
Morphometric measurements of brain structures and cortical layers.
Equivalent sections of ShcFFF and control littermate brains were chosen
using prenatal (Schambra et al., 1992) or postnatal (Paxinos and Frank-
lin, 2001) brain atlases as described above. Structures including the stri-
images were captured using a 4? objective, and their area was measured
using NIH ImageJ 1.32j. For each animal, structures from each one-half
of the brain were measured separately, and then averaged together. The
area of the subventricular zone (SVZ) was calculated while performing
BrdU counts as described below. For cortical layers, each layer was iden-
tified based on morphology of the neurons in each layer, and the transi-
tion between layers was noted. ImageJ was then used to measure the
distance between each layer. Total thickness of the cortical wall was cal-
culated by measuring from the top of layer 1 to the bottom of layer 6
using ImageJ 1.32j. Gross area analyses of whole brains were performed
using ImageJ 1.32j to measure each indicated dimension.
Cell counts in tissue sections. At embryonic time points, equivalent
sections of the forebrain and hindbrain were chosen as described above.
Images of each area were recorded, and positive BrdU or cleaved
caspase-3 cells were counted in a manner similar to that described pre-
viously (Gambello et al., 2003). Briefly, five random, nonoverlapping
counting boxes that were 200 ?m in width by the thickness of the cortex
were chosen for each image. To examine cortical layering, the thickness
of the cortex for each of the five random counting boxes was divided
equally into five sections and labeled one through five from the ventric-
ular to the pial surface. Positive BrdU or cleaved caspase-3 cells (brown)
and the total number of nuclei (blue) were counted in each subdivided
area. Because cell counting data can be confounded by systematic differ-
ences in cell size, a correction factor known as Abercrombie’s formula
can be applied (Abercrombie, 1946). However, because our nuclear and
soma measurements of ShcFFF and control mice revealed no differences
between control and ShcFFF samples in either embryonic or postnatal
brain, we did not need to apply this correction factor. Proliferation and
7886 • J.Neurosci.,July26,2006 • 26(30):7885–7897McFarlandetal.•ShcAandSurvivalSignalinginNeuralProgenitors
apoptotic indices for each cortical bin were calculated by dividing the
thickness of the cortex and multiplying by 100%. NeuN-positive cells
were counted in a similar manner with no divisions in the cortical thick-
ness. Total proliferation and apoptotic indices were calculated by divid-
ing the total number of positive cells throughout the thickness of the
cortex by the total number of nuclei in the cortex and multiplying by
For BrdU counts at postnatal time points, equivalent sections of the
SVZ in the lateral wall of the lateral ventricle were chosen as described
above. In the SVZ, cell counts for positively stained nuclei and total
nuclei number or total area were performed using ImageJ 1.32j. In post-
Graphs were generated using KaleidaGraph, version 4.02. Graphs and
data are expressed as the mean ? SEM. Data were statistically compared
and two-tailed p values were obtained using a nonpaired t test with
QuickCalc online software (GraphPad, San Diego, CA). Littermate con-
trol data were combined from the animals genotyped as containing the
ShcFFF transgene alone, the nestin-Cre transgene alone, and those com-
pletely wild type.
To test the role of ShcA in the developing brain, we used the
conditional transgenic expression of a FLAG-tagged, dominant-
negative p52ShcA transgene termed ShcFFF (Zhang et al., 2002).
This ShcFFF transgene has the three critical tyrosine residues,
Y239, Y240, and Y317, mutated to phenylalanine and affects the
activation of endogenous ShcA (Zhang et al., 2002). To prevent
basal expression of the dominant-negative protein, ShcFFF is lo-
are specifically recognized by Cre recombinase. Cre-mediated
excision of the upstream STOP cassette allows expression of the
transgene from the EF-1? promoter (supplemental Fig. 1A,B,
available at www.jneurosci.org as supplemental material). Using
the nestin promoter and neural specific enhancer located in the
and glial precursors (Zimmerman et al., 1994; Tronche et al.,
1999; Mignone et al., 2004), thereby allowing removal of the
STOP cassette specifically in these cells. This approach using the
nestin-Cre transgene has been used successfully to conditionally
shown to occur throughout the majority of the CNS during em-
bryonic development (Jiang et al., 2005).
To localize ShcA protein expression during embryonic develop-
ment, we performed immunohistochemistry at various develop-
mental time points. We find that ShcA immunoreactivity is
strongly expressed throughout the neuroepithelium at E10.5
(Fig. 1A) but decreases and is diffuse throughout the developing
nervous system by E12 (Fig. 1B) and with even lower expression
at E14.5 (Fig. 1C). During postnatal development, some expres-
sion persists in the proliferating SVZ (Fig. 1F) as well as in blood
vessels (Fig. 1E). No immunoreactivity was seen in mature neu-
ropil or white matter (Fig. 1E), determined by staining adjacent
sections with neuronal and glial markers (data not shown). This
expression pattern agrees with the previously described mRNA
localization and the time course agrees with Western blotting
data (Conti et al., 1997).
In the nestin-Cre transgenic mouse, Cre protein expression is
more, nestin expression (Fig. 1G) in the subventricular zone at
early postnatal stages is similar to that seen for endogenous ShcA
protein (Fig. 1F). These observations for nestin protein and
tions of nestin promoter activity (Lendahl et al., 1990; Zimmer-
man et al., 1994; Tronche et al., 1999).
To compare its expression in human tissues, we stained fetal
and adult brain tissue for ShcA. In human brain tissue from a 23
week fetus, ShcA immunoreactivity is found in proliferating re-
the adult human brain, ShcA protein is observed only in the
endothelium of blood vessels and is not found in neurons or glia
of the adult cortex as determined by neuronal morphology and
staining adjacent sections with the astroglial marker GFAP (Fig.
1I) (data not shown).
Cre transgenic mouse at E10.5 (D). Expression of nestin in the SVZ at P2 (G). ShcA protein
ShcA and nestin promoter-driven Cre proteins are expressed in the proliferative
McFarlandetal.•ShcAandSurvivalSignalinginNeuralProgenitorsJ.Neurosci.,July26,2006 • 26(30):7885–7897 • 7887
Mice that were doubly positive for the
nestin-Cre and STOPFloxShcFFF trans-
genes were viable and observed at the ex-
pected mendelian ratio of 25% when sire
Cre transgene and the STOPFloxShcFFF
transgene, respectively. However, we were
unable to detect expression of the ShcFFF
transgene using immunohistochemical
Therefore, we designed primers to the
STOP cassette of the ShcFFF transgene to
cassette in various tissues (supplemental
Fig. 1, available at www.jneurosci.org as
a PCR product from these primers de-
pends on the presence of the STOP cas-
sette, such as in non-neural tissues of em-
bryos genotyped with both nestin-cre and
STOPfloxShcFFF transgenes or in both
neural and non-neural tissues of embryos
genotyped with only the STOPfloxShcFFF
transgene (supplemental Fig. 1A,C, avail-
material). A lack of a PCR product from
these primers occurs when the STOP cas-
sette is absent, such as in embryos that do
not carry the STOPfloxShcFFF transgene
recombinase in neural tissue of embryos genotyped with both
nestin-Cre and STOPfloxShcFFF transgenes (supplemental Fig.
1B; C, asterisks; available at www.jneurosci.org as supplemental
E12 and E14.5 embryos and tested with the STOP cassette prim-
ers and with primers to the native IL-2 gene as a control (supple-
mental Fig. 1C, available at www.jneurosci.org as supplemental
material). We obtained the results described above indicating
that at the DNA level the STOP cassette is removed from the
the STOPfloxShcFFF transgenes (henceforth referred to as
ShcFFF animals) allowing for transcription of the ShcFFF trans-
gene. The inability to detect the protein product of the ShcFFF
transgene is most likely attributable to a low level of expression.
Using the same STOPFloxShcFFF mouse, Zhang et al. (2002)
detected very low levels of ShcFFF expression compared with
endogenous ShcA, yet observed a very strong phenotype in thy-
At P2, ShcFFF mice demonstrated microencephaly with brain
weights reduced to one-half that of littermate controls (Fig.
ment (Fig. 2B,B?) and into maturity at all time points assayed
including 1 year of age (Fig. 2C). Furthermore, at birth and dur-
ing early postnatal development, there was a slight body weight
difference between ShcFFF animals and their control littermates
but ShcFFF animals subsequently reached weights near those of
their control littermates during maturity (Fig. 2E,E?,F). How-
ever, at these later time points, the difference in body size was
more noticeable in males than in females (data not shown) (fe-
males shown in Fig. 2E,E?).
Because the dominant-negative ShcFFF transgene could, in
theory, perturb signaling through other Shc family members, we
undertook an independent approach using the conditional dis-
ruption of the shc1 locus. To this end, we created animals in
which ShcA was conditionally removed from nestin-expressing
neural progenitors by crossing a nestin-Cre; ShcAloxP/?female to
phenotype in animals that are nestin-Cre; ShcAloxP/loxP(Fig. 2C).
the ShcFFF animals. Intriguingly, the brain size phenotype ob-
served for the expression of ShcFFF in neural tissue was more
severe than that of deleting the ShcA gene using the same nestin-
of ShcFFF once it is transcribed after the STOP cassette deletion
versus the potentially long half-life of the extant ShcA protein
even after disruption of the shc1 locus via the nestin-Cre trans-
ShcFFF and nestin-Cre; ShcAloxP/loxPanimals provides strong ev-
idence for a ShcA-specific role in the regulation of brain size.
Histological analysis of brains from ShcFFF animals revealed
that, on a gross anatomic level, overall architecture and organi-
However, on closer inspection and quantification, certain struc-
tures of these brains are more severely affected than others.
Quantitative comparisons revealed differences that were statisti-
cally significant including the thickness of the cerebral cortex
(Fig. 3J), the area of the subventricular zone (Fig. 3I), and the
area of the striatum (Fig. 3K). However, other structures such as
7888 • J.Neurosci.,July26,2006 • 26(30):7885–7897McFarlandetal.•ShcAandSurvivalSignalinginNeuralProgenitors
the area of the hippocampus at P2 (Fig.
3M), dentate gyrus (Fig. 3N), and area of
the lateral ventricles (Fig. 3L) did not have
statistically significant differences. Addi-
tionally, a gross area analysis of the whole
brain at P2 and postnatal year 1 revealed
size of control at P2) was affected more
severely in ShcFFF mice than the olfactory
bulb (82% the size of control at P2) (Fig.
3O–Q). Moreover, ShcFFF animals have
no obvious abnormalities in their normal
home cage behavior and are fertile and are
able to raise healthy pups (data not
To determine whether the reduction in
cortical thickness was layer specific or a
generalized reduction in total cortical
cal layer-specific markers were examined.
Brain-1 (Brn-1) (Fig. 4A,B) and Cux-1
pressed in cortical layers II through IV
(McEvilly et al., 2002; Ferrere et al., 2006)
as well as reelin (Fig. 4E,F) and calretinin
(Fig. 4C,D) staining in cortical layer I (Del
Rio et al., 1995) were analyzed. Addition-
ally, cytochrome oxidase (Fig. 4G,H; sup-
plemental Fig. 2E,F, available at www.
jneurosci.org as supplemental material)
rel region of the somatosensory cortex in
ShcFFF and controls. GABAAreceptor ?2
jneurosci.org as supplemental material)
and GABAAreceptor ?5 (supplemental
Fig. 2C,D, available at www.jneurosci.org
ined in the somatosensory cortex.
ness of the Cux-1-positive area (layers II–
resenting layer I and layers V–VI) were
whisker barrels in layer IV of the somato-
sensory cortex were identified by staining
supplemental Fig. 2E,F, available at www.
jneurosci.org as supplemental material).
The barrels in layer IV in the somatosen-
resenting layers I through III) and below
(representing layers V through VI) were
identified and the thickness of each was
measured (Fig. 4L).
These data reveal that the entire thick-
ness of the somatosensory cortex is re-
of controls). However, the upper cortical
Gross area analysis of P2 brains indicating the measured parameters. a, cerebellum height; b, cerebellum width; c, cerebrum
McFarlandetal.•ShcAandSurvivalSignalinginNeuralProgenitorsJ.Neurosci.,July26,2006 • 26(30):7885–7897 • 7889
layers (layers II through IV) appear to be more dramatically af-
fected (reduced by ?60% of controls in layer IV) than layers
V–VI (reduced by 92% of controls). A narrower swath of Brn-1
staining in layers II through IV (Fig. 4A,B) and GABAAreceptor
staining (supplemental Fig. 2A–D, available at www.jneurosci.
finding. Moreover, the reduction in cortical thickness in ShcFFF
out the cortex (supplemental Fig. 2G, available at www.
jneurosci.org as supplemental material).
as evidenced by reelin (Fig. 4E,F) and calretinin (Fig. 4C,D)
determine whether the reduced brain size in ShcFFF-expressing
animals was attributable to a reduction in the proliferation of
neural progenitors, we performed BrdU injections to mark pro-
liferating cells at embryonic stages E10.5, E12, and E14.5 (Fig. 5)
and at P2 and P21 (Fig. 6). During the embryonic stages, prolif-
5A,B,E,F,I,J) and in the pons surrounding the fourth ventricle
five equally sized bins, labeled 1 (nearest ventricle) through 5
(nearest pial surface), and BrdU?cells and the total number of
cells were counted in each. This was done to determine whether
there was any difference in cortical layering in ShcFFF embryos
during embryogenesis. Graphed data (Fig. 5A?–L?) represents
this proliferation index as the number of BrdU?nuclei in the
cortical thickness. Additionally, the total proliferation index was
calculated by counting the total number of BrdU?nuclei
the total number of nuclei in the cortical thickness.
At E10.5, there were no significant differences in the prolifer-
in any of the cortical bins in both the frontal cortex (Fig.
5A,A?,B,B?) and the pons (Fig. 5C,C?,D,D?). Additionally, total
proliferation indices throughout the entire cortical thickness
were unchanged between ShcFFF and control littermates in the
2.57%, n ? 3, p ? 0.7414) and in the pons (ShcFFF, 38.32 ?
3.59%, n ? 2, vs control, 34.71 ? 10.47%, n ? 3, p ? 0.8149).
Similar results were obtained between ShcFFF and control
littermates at E12 for both the frontal cortex (Fig. 5E,E?,F,F?)
and the pons (Fig. 5G,G?,H,H?). Total proliferation indices
throughout the thickness of the entire cortex at E12 were also
unchanged between ShcFFF and control animals in the frontal
cortex (ShcFFF, 48.64 ? 1.74%, n ? 2, vs control, 52.93 ? 4.93,
2, vs control, 22.42 ? 2.49%, n ? 3, p ? 0.4190).
maintained in the pons (Fig. 5K,K?,L,L?). However, in the fron-
rate in cortical bin 4 (Fig. 5J,J?) compared with littermate con-
and littermate controls remains unchanged in both the frontal
control, 3.97 ? 1.21%, n ? 3, p ? 0.3942).
During postnatal development, proliferation in the subven-
tricular zone, a source of postnatal and adult neural progenitors
in ShcFFF animals and compared with littermate controls at P2
and P21 (Fig. 6). The proliferation index (calculated as the num-
and ShcFFF (F) animals. Total number of animals examined for calretinin, reelin, and Brn-1
of the somatosensory cortex (L). Total number of animals for cytochrome oxidase and Cux-1
staining was as follows: ShcFFF, n ? 4; control, n ? 5. Error bars indicate SEM. cc, Corpus
7890 • J.Neurosci.,July26,2006 • 26(30):7885–7897 McFarlandetal.•ShcAandSurvivalSignalinginNeuralProgenitors
ber of BrdU?nuclei divided by the total number of nuclei or the
also observed that the SVZ in ShcFFF animals was smaller when
compared with littermate controls at both time points (Fig. 3I).
To determine whether microencephaly in
ShcFFF animals is the result of premature
neuronal differentiation, we examined
such markers via immunohistochemical
ronal differentiation were occurring, we
would observe aberrant staining patterns
for markers of early and late neuronal dif-
ferentiation including NeuN (Fig. 7A–D),
microtubule-associated protein 2 (MAP2)
(Fig. 7E–H), neurofilament (Fig. 7I–L),
ber of animals observed: control, n ? 3;
ShcFFF, n ? 2) and E12 (total number of
animals observed: control, n ? 5; ShcFFF,
n ? 3) in ShcFFF and control littermate
brains. We observed these staining patterns
to have a normal intensity and distribution
in ShcFFF brains. Additionally, quantifica-
tion of the NeuN staining pattern indicates
there are no differences in the percentage of
NeuN-positive nuclei between ShcFFF and
control brains in either location at E10.5
(frontal cortex: ShcFFF, 0.00 ? 0.00%, n ?
2, vs control, 0.14 ? 0.08%, n ? 3, p ?
0.2546; pons: ShcFFF, 0.52 ? 0.16%, n ? 2,
and E12 (frontal cortex: ShcFFF, 0.15 ?
To determine whether the reduction in
brain size may be a result of an increase in
apoptosis, we used antibodies to CC3, the
activated form of an effector molecule of
the apoptotic pathway. In the developing
CC3 correlates well with terminal deoxy-
ated UTP nick end labeling (TUNEL)-
positive cells with the majority of CC3?
cells being TUNEL?(Urase et al., 2003).
sis; yet, in these special cases, the CC3?
cells do not have any other characteristics
some condensation, DNA fragmentation,
or TUNEL staining (Yan et al., 2001; Lossi et al., 2004; Oomman
et al., 2004).
We noted the presence of condensed and pyknotic nuclei in
the nervous tissue of ShcFFF embryos but not in their control
littermates (Fig. 8E–H, DAPI-stained insets; Fig. 7C?,G?,O?, ar-
pons (C, D) in control littermates (A, C) and ShcFFF animals (B, D) at E10.5. Associated average binned proliferation indices
locations. In all panels, the ventricle is located to the top of the panel and a dashed line indicates the pial surface. Arrow,
McFarlandetal.•ShcAandSurvivalSignalinginNeuralProgenitorsJ.Neurosci.,July26,2006 • 26(30):7885–7897 • 7891
www.jneurosci.org as supplemental mate-
CC3 (supplemental Fig. 3C–J, arrows;
available at www.jneurosci.org as supple-
mental material). Thus, we interpret these
data as CC3?cells in ShcFFF prenatal
brains being apoptotic. There are occa-
sional pyknotic nuclei that are CC3-
negative (supplemental Fig. 3E–J, arrow-
heads; available at www.jneurosci.org as
supplemental material), indicating that
CC3 staining does not necessarily mark all
apoptotic cells in ShcFFF embryos. We
quantified both the binned apoptotic in-
dex (Fig. 8A?–L?) and the total apoptotic
At E10.5, cells in the frontal cortex of
ShcFFF animals (Fig. 8B) showed a slight,
of CC3?cells in each cortical bin (Fig. 8B?) and in the total
0.15%, n ? 3, p ? 0.0741) compared with littermate controls
(Fig. 8A,A?). However, in the pons surrounding the fourth ven-
tricle, there was an even larger increase in the number of CC3?
cells in ShcFFF animals (Fig. 8D). Although these increases were
not significant for all cortical bins (Fig. 8D?), the total apoptotic
index showed a significant increase in ShcFFF animals (ShcFFF,
9.42 ? 3.84%, n ? 2, vs control, 0.00 ? 0.00%, n ? 3, p ?
At E12, the apoptotic index appeared to reach its peak. In the
frontal cortex of ShcFFF animals (Fig. 8F), the apoptotic index
(Fig. 8F?) dramatically increased in all cortical bins, with the
majority of apoptotic cells located from the middle of the cortex
to the pial surface of the brain. In the pons of ShcFFF animals
with the majority of apoptotic cells located in the periventricular
area. Total apoptotic indices were increased significantly in both
the frontal cortex (ShcFFF, 11.44 ? 2.71%, n ? 3, vs control,
4.78%, n ? 3, vs control, 0.06 ? 0.06%, n ? 3, p ? 0.0488).
to levels seen in control littermates. In the frontal cortex, the
able from that of control littermates (Fig. 8I,I?) in both the cor-
tical bins and the total apoptotic index (ShcFFF, 0.54 ? 0.30%,
n ? 4, vs control, 0.08 ? 0.03%, n ? 3, p ? 0.1720). Similar
compared with their littermate controls (Fig. 8K,K?) (total apo-
ptotic index: ShcFFF, 0.51 ? 0.32%, n ? 4, vs control, 0.56 ?
0.15%, n ? 4, p ? 0.8804).
To determine the identity and differentiated state of these apo-
ptotic cells, double immunofluorescent staining was performed
for mature, differentiated neurons, such as NeuN and neurofila-
ment (data not shown). Additionally, CC3?cells did not stain
with the early neuronal marker, TuJ1 (Fig. 9A,B). However, the
majority of CC3?cells are nestin-positive (Fig. 9C–F). High-
power confocal images (Fig. 9E,F) and stacks throughout the
entire volume of these cells (supplemental movies 1 and 2, avail-
able at www.jneurosci.org as supplemental material) reveal radi-
staining. Thus, apoptotic cells in ShcFFF embryos are composed
primarily of nestin-positive neural progenitors.
in the survival of neural progenitors during development of the
nervous system. This conclusion differs from previous hypothe-
of neural precursors (Pelicci et al., 1992; Conti et al., 1997; Cat-
taneo and Pelicci, 1998; Conti et al., 2001).
Dominant-negative molecules can function in a nonspecific
members. However, several lines of evidence lead us to believe
that disruption of the p46 and/or p52 isoforms and not the p66
of our observed phenotypes.
During embryonic brain development, p66Shc expression is
(Conti et al., 1997). Additionally, mice lacking only the p66ShcA
isoform, which is involved in oxidative stress-induced apoptosis
fat diet (Napoli et al., 2003). Therefore, if the expression of
the developing brain to be resistant to apoptosis, which may lead
to the opposite phenotype of what we have observed.
tions of other Shc family members, ShcB and ShcC. In the brain,
development and are expressed in mature neurons throughout
al., 1996; Conti et al., 1997; Nakamura et al., 1998; Ponti et al.,
2005). Our data show that the increase in apoptosis in ShcFFF
neurons. Additionally, ShcB and double ShcB/ShcC knock-out
7892 • J.Neurosci.,July26,2006 • 26(30):7885–7897McFarlandetal.•ShcAandSurvivalSignalinginNeuralProgenitors
mice have very subtle defects in the brain
that do not affect its size (Sakai et al.,
would be mild and would not lead to
Moreover, the spatial and temporal
pattern of apoptosis in ShcFFF mice is
consistent with the expression of endoge-
nous ShcA protein. Additionally, the mi-
croencephaly observed in conditional
of the ShcFFF transgene can phenocopy
removal of brain-specific ShcA function.
Together, these data suggest that the phe-
notype seen by expressing ShcFFF in neu-
ral tissue results from interfering specifi-
cally with ShcA function.
We considered several possibilities for the
cause of the ShcFFF microencephaly
phenotype. First, the disruption of ShcA
could interrupt proliferation, as previous
hypotheses have suggested. Second, dis-
turbing ShcA function could cause a pre-
mature neuronal differentiation. Addi-
tionally, interference with ShcA function
creased apoptosis. Alternatively, the in-
crease in apoptotic cells in ShcFFF embry-
onic brains could be caused by an
dead cells, leading to an accumulation of
Based on our data, there are no ob-
differentiation between ShcFFF animals
and their littermate controls. In addition,
delaying the phagocytic clearance of dead
cells would not account for the major re-
duction in brain size. Indeed, the most
likely cause for the smaller brains in
apoptosis of neural progenitors during
move these cells from the neural progeni-
ferentiating further but would further
reduce the pool of transiently amplifying
progenitor cells. The ShcFFF phenotype is
of animals observed was as follows: E10.5, control, n ? 3;
tative animals are shown. The arrows indicate pyknotic
and/or fragmented nuclei. In all panels, the ventricle is lo-
McFarlandetal.•ShcAandSurvivalSignalinginNeuralProgenitorsJ.Neurosci.,July26,2006 • 26(30):7885–7897 • 7893
mice irradiated during specific stages of
embryonic development (Kitamura et al.,
2001). These mice were microencephalic,
with increased levels of CC3-positive cells
in their brains, in addition to a moderate
decrease in body weight.
Our data therefore suggest that ShcA
prosurvival pathway. Such a pathway may
use neurotrophin receptors, such as the
Trk family, which regulate survival signal-
(Barnabe-Heider and Miller, 2003) and
signal through the mitogen-activated pro-
tein kinase kinase (MEK)/ERK or PI3K/
Akt pathways to elicit a prosurvival re-
sponse. Additionally, in various cell types,
ShcA is a part of the activated ERK path-
way via EGFR (epidermal growth factor
receptor) and Grb2/Sos in response to re-
active oxygen stress (Huang et al., 1996;
proapoptotic protein Bad via p90Rsk, a
target kinase of ERK (Bonni et al., 1999;
Eisenmann et al., 2003; Chen et al., 2005).
Thus, in the absence of a survival signal
through ShcA, the apoptotic pathway may
proceed unrestrained in neural progeni-
tors, thereby leading to the widespread ac-
tivation of caspase proteins and hence
Additional evidence that the Ras3Erk
pathway is critical for brain development
comes from the recent discovery that
cardio-facio-cutaneous syndrome, char-
macrocephaly (Kavamura et al., 2002), is
caused by mutations in either B-Raf,
2006). These mutations lead to increased
when transfected in vitro. Thus, precise
ing appears to be critical for normal brain
period of neurogenesis predicts subse-
quent consequences on cortical lamina-
tion during later development. The mam-
malian cortex forms “inside-out” with the
earliest born neurons occupying deeper
layers in the brain and later born neurons
in more superficial layers (Kubo and Na-
progenitor apoptosis at E12 would result in a reduction of later
born neurons impacting the thickness of cortical layers II
through IV more so than earlier born, deeper neurons. This is
exactly what we observed in ShcFFF animals.
7894 • J.Neurosci.,July26,2006 • 26(30):7885–7897 McFarlandetal.•ShcAandSurvivalSignalinginNeuralProgenitors
as well. In the frontal cortex, apoptotic cells are located from the
the pons are located in the periventricular zone. Because neuro-
nal cells undergo differentiation as they migrate away from the
ventricular layer toward the pial surface and neurogenesis pro-
within each brain region.
The ShcFFF microencephaly phenotype is also reminiscent of
some human microencephaly disorders. Human primary mi-
ence greater than three SDs from the age- and sex-matched pop-
ulation mean, is caused by a reduction of brain growth in utero
and is thought to result from insufficient production of neurons
during neurogenesis (Woods, 2004).
The apoptotic phenotype in ShcFFF
mice suggests an alternative mechanism
To date, all six of the nonsyndromic pri-
mary microencephaly disorders in hu-
location of human Shc1 at 1q21 (Huebner
et al., 1994; Yulug et al., 1995; Jamieson et
al., 2000; Jackson et al., 2002; Bond et al.,
2003; Leal et al., 2003). Although four of
these disorders are associated with muta-
tions in genes (MCPH1/Microcephalin;
MCPH6/CENPJ) encoding proteins that
are thought to affect the normal progres-
sion of the cell cycle in neural progenitors
(Jackson et al., 2002; Bond et al., 2003,
2005; Kumar et al., 2004; Trimborn et al.,
lecular identity of the remaining two pri-
mary disorders and of the numerous sec-
disorders are unknown.
In conclusion, our work establishes
in the survival of neural precursors during
embryogenesis. Uncontrolled neuropro-
genitor apoptosis may provide an alterna-
tive mechanism for some forms of human
ShcA or other members of its signaling
Abercrombie M (1946) Estimation of nuclear
population from microtome sections. Anat
Alvarez-Buylla A, Herrera D, Wichterle H (2000)
The subventricular zone: source of neuronal
precursors for brain repair. Prog Brain Res
enously produced neurotrophins regulate sur-
via distinct signaling pathways. J Neurosci
F (2003) Endog-
Berk M, Desai S, Heyman H, Colmenares C (1997) Mice lacking the ski
proto-oncogene have defects in neurulation, craniofacial patterning, and
skeletal muscle development. Genes Dev 11:2029–2039.
A, Walsh C, Roberts E, Woods C (2003) Protein-truncating mutations
in ASPM cause variable reduction in brain size. Am J Hum Genet
Bond J, Roberts E, Springell K, Lizarraga S, Scott S, Higgins J, Hampshire D,
Morrison E, Leal G, Silva E, Costa S, Baralle D, Raponi M, Karbani G,
Rashid Y, Jafri H, Bennett C, Corry P, Walsh C, Woods C (2005) A
centrosomal mechanism involving CDK5RAP2 and CENPJ controls
brain size. Nat Genet 37:353–355.
Bonni A, Brunet A, West A, Datta S, Takasu M, Greenberg M (1999) Cell
dependent and -independent mechanisms. Science 286:1358–1362.
Cattaneo E, Pelicci P (1998) Emerging roles for SH2/PTB-containing Shc
adaptor protein in the developing mammalian brain. Trends Neurosci
tional fluorescent microscopic image of CC3 (red) and nestin (green) staining with DAPI counterstaining (blue). D, Confocal
McFarlandetal.•ShcAandSurvivalSignalinginNeuralProgenitors J.Neurosci.,July26,2006 • 26(30):7885–7897 • 7895
Chen X, Lau L, Fung Y, Yu A (2005) Inactivation of bad by site-specific
phosphorylation: the checkpoint for ischemic astrocytes to initiate or
resist apoptosis. J Neurosci Res 79:798–808.
Conti L, De Fraja C, Gulisano M, Migliaccio E, Govoni S, Cattaneo E (1997)
Expression and activation of SH2/PTB-containing ShcA adaptor protein
reflects the pattern of neurogenesis in the mammalian brain. Proc Natl
Acad Sci USA 94:8185–8190.
Conti L, Sipione S, Magrassi L, Bonfanti L, Rigamonti D, Pettirossi V,
Peschanski M, Hadded B, Pelicci P, Milanesi G, Pelicci G, Cattaneo E
(2001) Shc signaling in differentiating neural progenitor cells. Nat Neu-
DelRioJ,MartinezA,FonsecaM,AuladellC,SorianoE (1995) Glutamate-
as identified with calretinin antibody. Cereb Cortex 5:13–21.
Eisenmann K, VanBrocklin M, Staffend N, Kitchen S, Koo H-M (2003)
Mitogen-activated protein kinase pathway-dependent tumor-specific
survival signaling in melanoma cells through inactivation of the proapo-
totic protein Bad. Cancer Res 63:8330–8337.
Ferrere A, Vitalis T, Gingras H, Gaspar P, Cases O (2006) Expression of
Cux-1 and Cux-2 in the developing somatosensory cortex of normal and
Gambello M, Darling D, Yingling J, Tanaka T, Gleeson J, Wynshaw-Boris A
(2003) Multiple dose-dependent effects of Lis1 on cerebral cortical de-
velopment. J Neurosci 23:1719–1729.
Gao Q, Wolfgang M, Neschen S, Morino K, Harvath T, Shulman G, Fu X
Natl Acad Sci USA 101:4661–4666.
GotohN,TojoA,ShibuyaM (1996) Anovelpathwayfromphosphorylation
IL-3. EMBO J 15:6197–6204.
Haigh J, Morelli P, Gerhardt H, Haigh K, Tsien J, Damert A, Miquerol L,
Muhlner U, Klein R, Ferrara N, Wagner E, Betsholtz C, Nagy A (2003)
Cortical and retinal defects caused by dosage-dependent reductions in
VEGF-A paracrine signaling. Dev Biol 262:225–241.
Huang R, Wu J, Fan Y, Adamson E (1996) UV activates growth factor re-
ceptors via reactive oxygen intermediates. J Cell Biol 133:211–220.
Huebner K, Kastury K, Druck T, Salcini A, Lanfrancone L, Pelicci G, Lowen-
stein E, Li W, Park S, Cannizzaro L, Pelicci P, Schlessinger J (1994)
Chromosome locations of genes encoding human signal transduction
adapter proteins, Nck (NCK), Shc (Shc1) and Grb2 (GRB2). Genomics
Jackson A, Eastwood H, Bell S, Adu J, Toomes C, Carr I, Roberts E, Hamp-
shire D, Crow Y, Mighell A, Karbani G, Jafri H, Rashid Y, Mueller R,
Markhan A, Woods C (2002) Identification of microcephalin, a protein
implicated in determining the size of the human brain. Am J Hum Genet
Jamieson C, Govaerts C, Abramovicz M (1999) Primary autosomal reces-
sive microcephaly: homozygosity mapping of MCPH4 to chromosome
15. Am J Hum Genet 65:1465–1469.
Jiang Y, de Bruin A, Caldas H, Fangusaro J, Hayes J, Conway E, Robinson M,
Altura R (2005) Essential role for survivin in early brain development.
J Neurosci 25:6962–6970.
Kavamura M, Peres C, Alchorne M, Brunoni D (2002) CFC index for the
diagnosis of cardiofaciocutaneous syndrome. Am J Hum Genet
Kitamura M, Itoh K, Matsumoto A, Hayashi Y, Sasaki R, Imai Y, Itoh H
(2001) Prenatal ionizing radiation-induced apoptosis of the developing
murine brain with special references to the expression of some proteins.
Kobe J Med Sci 47:59–73.
Kubo K, Nakajima K (2003) Cell and molecular mechanisms that control
cortical layer formation in the brain. Keio J Med 52:8–20.
Kuida K, Zheng T, Na S, Kuan C-Y, Yang D, Karasuyama H, Rakic P, Flavell
R (1996) Decreased apoptosis in the brain and premature lethality in
CPP32-deficient mice. Nature 384:368–372.
P, Flavell R (1998) Reduced apoptosis and cytochrome c-mediated
caspase activation in mice lacking caspase-9. Cell 94:325–337.
Kumar A, Blanton S, Babu M, Markandaya M, Girimaji S (2004) Genetic
analysis of primary microcephaly in Indian families: novel ASPM muta-
tions. Clin Genet 66:341–348.
Lai K, Pawson T (2000) The ShcA phosphotyrosine docking protein sensi-
tizes cardiovascular signaling in the mouse embryo. Genes Dev
Leal G, Roberts E, Silva E, Costa S, Hampshire D, Woods C (2003) A novel
locus for autosomal recessive primary microcephaly (MCPH6) maps to
13q12.2. J Med Genet 40:540–542.
Lendahl U, Zimmerman L, McKay R (1990) CNS stem cells express a new
class of intermediate filament protein. Cell 60:585–595.
Lossi L, Tamagno I, Merighi A (2004) Molecular morphology of neuronal
apoptosis: analysis of caspase 3 activation during postnatal development
of mouse cerebellar cortex. J Mol Histol 35:621–629.
Luzi L, Confalonieri S, Di Fiore P, Pelicci P (2000) Evolution of Shc func-
tions from nematode to human. Curr Opin Genet Dev 10:668–674.
Transcriptional regulation of cortical neuron migration by POU domain
factors. Science 295:1528–1532.
Migliaccio E, Giorgio M, Mele S, Pelicci G, Reboldi P, Pandolfi P, Lanfran-
cone L, Pelicci P (1999) The p66shc adaptor protein controls oxidative
stress response and life span in mammals. Nature 402:309–313.
MignoneJ,KukekovV,ChiangA-S,SteindlerD,EnikolopovG (2004) Neu-
ral stem and progenitor cells in Nestin-GFP transgenic mice. J Comp
Moynihan L, Jackson A, Roberts E, Karbani G, Lewis I, Corry P, Turner G,
Mueller R, Lench N, Woods C (2000) A third novel locus for primary
Nakamura T, Muraoka S, Sanokawa R, Mori N (1998) N-Shc and Sck, two
neuronally expressed Shc adapter homologs. Their differential regional
Napoli C, Martin-Padura I, de Nigris F, Giorgio M, Mansueto G, Somma P,
Condorelli M, Sica G, De Rosa G, Pelicci P (2003) Deletion of the
Natl Acad Sci USA 100:2112–2116.
O’Bryan J, Songyang Z, Cantley L, Der C, Pawson T (1996) A mammalian
adaptor protein with conserved Src homology 2 and phosphotyrosine-
Proc Natl Acad Sci USA 93:2729–2734.
Oomman S, Finckbone V, Dertien J, Attridge J, Henne W, Medina M, Man-
souriB,SinghH,StrahlendorfH,StrahlendorfJ (2004) Activecaspase-3
atically or consistently associated with apoptosis. J Comp Neurol
Paxinos G, Franklin K (2001) The mouse brain in stereotaxic coordinates,
Ed 2. San Diego: Academic.
I, Grignani F, Pawson T, Pelicci P (1992) A novel transforming protein
(SHC) with an SH2 domain is implicated in mitogenic signal transduc-
tion. Cell 70:93–104.
Pelicci G, Dente L, De Giuseppe A, Verducci-Galletti B, Giuli S, Mele S,
VetrianiC,GiorgioM,PandolfiP,CesareniG,PelicciP (1996) Afamily
of Shc related proteins with conserved PTB, CH1 and SH2 regions. On-
Pellegrini M, Pacini S, Baldari C (2005) p66SHC: the apoptotic side of Shc
proteins. Apoptosis 10:13–18.
Ponti G, Conti L, Cataudella T, Zuccato C, Magrassi L, Rossi F, Bonfanti L,
Cattaneo E (2005) Comparative expression profiles of ShcB and ShcC
phosphotyrosine adapter molecules in the adult brain. Neuroscience
PompeianoM,BlaschkeA,FlavellR,SrinivasanA,ChunJ (2000) Decreased
apoptosis in proliferative and postmitotic regions of the caspase-3 defi-
cient embryonic central nervous system. J Comp Neurol 423:1–12.
Rao G (1996) Hydrogen peroxide induces complex formation of SHC-
Grb2-SOS with receptor tyrosine kinase and activates Ras and extracellu-
lar signal-regulated protein kinases group of mitogen-activated protein
kinases. Oncogene 13:713–719.
Ravichandran K (2001) Signaling via Shc family adapter proteins. Onco-
Roberts E, Jackson A, Carradice A, Deeble V, Mannan J, Rashid Y, Jafri H,
McHaleD,MarkhamA,LenchN,WoodsC (1999) Thesecondlocusfor
autosomal recessive primary microcephaly (MCPH2) maps to chromo-
some 19q13.1–13.2. Eur J Hum Genet 7:815–820.
7896 • J.Neurosci.,July26,2006 • 26(30):7885–7897McFarlandetal.•ShcAandSurvivalSignalinginNeuralProgenitors
McCormick F, Rauen K (2006) Germline mutations in genes within the
MAPK pathway cause cardio-facio-cutaneous syndrome. Science
Romanko M, Rola R, Fike J, Szele F, Dizon M, Felling R, Brazel C, Levison S
(2004) Roles of the mammalian subventricular zone in cell replacement
after brain injury. Prog Neurobiol 74:77–99.
Thomas S, Brugge J, Pelicci P, Schlessinger J, Pawson T (1992) Associa-
tion of the Shc and Grb2/Sem5 SH2-containing proteins is implicated in
activation of the Ras pathway by tyrosine kinases. Nature 360:689–692.
Sabapathy K, Jochum W, Hochedlinger K, Chang L, Karin M, Wagner E
absence of both JNK1 and JNK2. Mech Dev 89:115–124.
Sakai R, Henderson J, O’Bryan J, Elia A, Saxton T, Pawson T (2000) The
mammalian ShcB and ShcC phosphotyrosine docking proteins function in
SalgiaR,GriffinJ (2000) TheBCR/ABLtyrosinekinaseinducesproduc-
tion of reactive oxygen species in hematopoietic cells. J Biol Chem
Schambra U, Lauder J, Silver J (1992) Atlas of the prenatal mouse brain, Ed
1. San Diego: Academic.
SchorleH,MeierP,BuchertM,JaenischR,MitchellP (1996) Transcription
factor AP-2 essential for cranial closure and craniofacial development.
ReisA,SperlingK,NeitzelH,JacksonA (2004) Mutationsinmicrocephalin
cause aberrant regulation of chromosome condensation. Am J Hum Genet
Schutz G (1999) Disruption of the glucocorticoid receptor gene in the
nervous system results in reduced anxiety. Nat Genet 23:99–103.
Urase K, Kouroku Y, Fujita E, Momoi T (2003) Region of caspase-3 activa-
tion and programmed cell death in the early development of the mouse
forebrain. Dev Brain Res 145:241–248.
van der Geer P, Wiley S, Gish G, Pawson T (1996) The Shc adaptor protein
is highly phosphorylated at conserved, twin tyrosine residues (Y239/
Y240) that mediate protein-protein interactions. Curr Biol 6:1435–1444.
Wong-RileyM (1979) Changesinthevisualsystemofmonocularlysutured
or enucleated cats demonstrable with cytochrome oxidase histochemis-
try. Brain Res 171:11–28.
Woods C (2004) Human microcephaly. Curr Opin Neurobiol 14:112–117.
Xu X, Lee J, Stern D (2004) Microcephalin is a DNA damage response pro-
tein involved in regulation of CHK1 and BRCA1. J Biol Chem
Yan X, Najbauer J, Woo C, Dashtipour K, Ribak C, Leon M (2001) Expres-
sion of active caspase-3 in mitotic and postmitotic cells of the rat fore-
brain. J Comp Neurol 433:4–22.
Yulug I, Egan S, See C, Fisher E (1995) A human SHC-related sequence
maps to chromosome 17, the SHC gene maps to chromosome 1. Hum
Zhang L, Camerini V, Bender T, Ravichandran K (2002) A nonredundant
J, Vassileva G, McMahon A (1994) Independent regulatory elements in
the nestin gene direct transgene expression to neural stem cells or muscle
precursors. Neuron 12:11–24.
McFarlandetal.•ShcAandSurvivalSignalinginNeuralProgenitorsJ.Neurosci.,July26,2006 • 26(30):7885–7897 • 7897