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
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