The Interleukin 3 Gene (IL3) Contributes to Human Brain
Volume Variation by Regulating Proliferation and
Survival of Neural Progenitors
Xiong-jian Luo1,2., Ming Li1., Liang Huang3,2, Kwangsik Nho4,5,6,7, Min Deng2, Qiang Chen8,
Daniel R. Weinberger8, Alejandro Arias Vasquez9,10, Mark Rijpkema11, Venkata S. Mattay8,
Andrew J. Saykin4,6,7, Li Shen4,6,7, Guille ´n Ferna ´ndez11,12, Barbara Franke9,10, Jing-chun Chen13, Xiang-
ning Chen13, Jin-kai Wang1, Xiao Xiao1, Xue-bin Qi1, Kun Xiang1, Ying-Mei Peng1, Xiang-yu Cao1, Yi Li14,
Xiao-dong Shi14, for the Alzheimer’s Disease Neuroimaging Initiative, Lin Gan2,15*, Bing Su1*
1State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan, China, 2University of
Rochester Flaum Eye Institute, University of Rochester, Rochester, New York, United States of America, 3Gannan Medical University, Ganzhou, Jiangxi, China, 4Center for
Neuroimaging, Department of Radiology and Imaging Sciences, Indiana University School of Medicine, Indianapolis, Indiana, United States of America, 5Center for
Computational Biology and Bioinformatics, Indiana University School of Medicine, Indianapolis, Indiana, United States of America, 6Regenstrief Institute, Indianapolis,
Indiana, United States of America, 7Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, Indiana, United States of America,
8Genes, Cognition and Psychosis Program, National Institute of Mental Health, the National Institutes of Health, Bethesda, Maryland, United States of America,
9Department of Genetics, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands, 10Department of Psychiatry, Donders Institute for Brain, Cognition
and Behaviour, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands, 11Donders Institute for Brain, Cognition and Behaviour, Donders Centre for
Cognitive Neuroimaging, Radboud University Nijmegen, Nijmegen, The Netherlands, 12Department of Cognitive Neuroscience, Donders Institute for Brain, Cognition
and Behaviour, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands, 13Virginia Institute for Psychiatric and Behavioral Genetics, and Department of
Psychiatry, Virginia Commonwealth University, Richmond, Virginia, United States of America, 14Biological Resources and Environmental Science College, Qujing Normal
University, Qujing, Yunnan, China, 15College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou, Zhejiang, China
One of the most significant evolutionary changes underlying the highly developed cognitive abilities of humans is the
greatly enlarged brain volume. In addition to being far greater than in most other species, the volume of the human brain
exhibits extensive variation and distinct sexual dimorphism in the general population. However, little is known about the
genetic mechanisms underlying normal variation as well as the observed sex difference in human brain volume. Here we
show that interleukin-3 (IL3) is strongly associated with brain volume variation in four genetically divergent populations. We
identified a sequence polymorphism (rs31480) in the IL3 promoter which alters the expression of IL3 by affecting the
binding affinity of transcription factor SP1. Further analysis indicated that IL3 and its receptors are continuously expressed in
the developing mouse brain, reaching highest levels at postnatal day 1–4. Furthermore, we found IL3 receptor alpha (IL3RA)
was mainly expressed in neural progenitors and neurons, and IL3 could promote proliferation and survival of the neural
progenitors. The expression level of IL3 thus played pivotal roles in the expansion and maintenance of the neural progenitor
pool and the number of surviving neurons. Moreover, we found that IL3 activated both estrogen receptors, but estrogen
didn’t directly regulate the expression of IL3. Our results demonstrate that genetic variation in the IL3 promoter regulates
human brain volume and reveals novel roles of IL3 in regulating brain development.
Citation: Luo X-j, Li M, Huang L, Nho K, Deng M, et al. (2012) The Interleukin 3 Gene (IL3) Contributes to Human Brain Volume Variation by Regulating
Proliferation and Survival of Neural Progenitors. PLoS ONE 7(11): e50375. doi:10.1371/journal.pone.0050375
Editor: Xiao-Jiang Li, Emory University, United States of America
Received September 5, 2012; Accepted October 18, 2012; Published November 30, 2012
Copyright: ? 2012 Luo et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was supported by grants from the National 973 project of China (2011CBA00401), the National Natural Science Foundation of China
(31130051 and 31071101), and the United States National Institute of Health (EY013426 to L.G.). The Brain Imaging Genetics study is supported by the
Hersenstichting Nederland and the Biobanking and Biomolecular Resources Research Infrastructure. The funders had no role in study design, data collection and
analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org (BS); Lin_Gan@urmc.rochester.edu (LG)
. These authors contributed equally to this work.
The greatly expanded brain size and highly developed cognitive
abilities are the most significant features that set humans apart from
thegeneralpopulation,rangingfrom981 mlto1,795 ml(1,462 mlin
males and 1,266 ml in females, on average) . Recent imaging
0.87) of brain volume and its correlation with general intelligence
[2,3], working memory, perceptual organization and processing
volume has been reported in several brain diseases such as
schizophrenia  and Attention-Deficit/Hyperactivity Disorder
PLOS ONE | www.plosone.org1November 2012 | Volume 7 | Issue 11 | e50375
(ADHD) . As a complex quantitative trait with high heritability,
brain volume is likely regulated by many genes. But so far only a
handful of such genes have been reported by studying patients with
reported microcephalin genes are important in explaining the
enlarged human brain during evolution , recent studies have
indicated that they only account for a small part of brain volume
variation in the general population [9,10]. Fortunately, recent
genome wide association studies have identified several promising
loci significantly associated with intracranial volume and head
circumference [11–13]. Nevertheless, all of these studies were
performed only in populations of European ancestry and some of
the variants (e.g., rs7890687 and rs9915547) identified in these
Additionally, the genetic dissection of schizophrenia (SCZ), a
common mental disorder with high heritability provides opportu-
nities to identify genes associated with brain volume variation since
SCZ patients have decreased total brain volume compared to
normal controls [5,14,15]. This is consistent with the hypothesis
that the pathogenesis of SCZ is related to abnormal brain
development . Though numerous linkage and association
studies, especially recent genome wide association studies have
identified many loci significantly associated with schizophrenia
[17–22], the etiology of schizophrenia remains poorly understood.
Among the hypotheses that explain the etiology of schizophrenia,
the neurodevelopmental hypothesis  has been supported by
the majority of the published data. This hypothesis predicts that a
disruption of brain development during early life underlies the
later emergence of psychosis during adolescence or early
adulthood. These evidences indicate that schizophrenia suscepti-
bility genes may regulate the unique features of human brain
development and dysfunction of these genes likely disrupted the
normal development of brain, which eventually lead to schizo-
phrenia susceptibility. In fact, recent studies demonstrated that
some schizophrenia susceptibility genes do regulate brain devel-
opment [24,25]. In light of these findings, we hypothesize that
schizophrenia susceptibility genes may regulate brain development
and affect the total brain volume.
To detect the relationship between schizophrenia susceptibility
genes and brain volume, we earlier systematically studied the
genetic association between schizophrenia susceptibility genes and
brain volume variation in a large cohort of healthy subjects. This
led to identification of a highly significant chromosomal region,
5q23.2–33.1, a region that has been well studied and shown strong
association with SCZ in multiple world populations [26–32].
Recently, Chen et al. systematically studied this region by using a
large sample (N=3,422, including case-control and family-based
samples) and dense SNP markers. They found haplotypes
spanning SPEC2, PDZ-GEF2, LOC728637, and ACSL6 were
significantly associated with schizophrenia in five independent
samples [33,34]. We further replicated the associations in a
Chinese sample . Collectively, these consistent results strongly
suggested genetic variants near these four genes (SPEC2, PDZ-
GEF2, LOC728637, and ACSL6) may contribute to schizophre-
nia susceptibility and brain development.
Interleukin-3 is Strongly Associated with Brain Volume
Variation in Chinese
For the initial analyses in Chinese population, we performed a
genetic screening to detect the association of cranial volume (the
approximate of brain volume, which is highly correlated with
brain volume [36,37]) with sequence variations located in the
5q23.2–33.1 region. The cranial volumes of 1,013 healthy
individuals (460 males and 553 females) were measured (see
methods), followed by genotyping of 20 single nucleotide
polymorphisms (SNPs) in the 5q23.2–33.1 region spanning about
809 kb. To test whether schizophrenia susceptibility variants in
5q23.2–33.1 are associated with brain volume, we initially
genotyped 8 tagging SNPs covering the four genes (SPEC2,
PDZ-GEF2, LOC728637, and ACSL6). The single SNP associ-
ation was conducted using linear regression under an additive
model and the p-values were obtained by the Wald test as
implemented in PLINK . The results showed that six of these
8 SNPs were significantly associated with cranial volume in
females, but not in males (Table S1). For fine-scale mapping, we
genotyped another 12 SNPs and identified a sharp signal in the
region containing IL3, showing a strong female-specific association
with cranial volume (Fig. 1a and Table S1). Among the 7 highly
significant SNPs (-logP.3.3) covering IL3, one was located in
exon 1 (rs40401, Ser to Pro), one in intron 2 (rs31481), one in the
promoter (rs31480), and four in the upstream region (rs3914025,
rs3916441, rs31400 and rs3846726) (Fig. 1a), clearly implicating
IL3 as the responsible gene. The associations between these 7
SNPs and brain volume were still highly significant (corrected
p,0.01) even using the most stringent Bonferroni correction for
multiple testing (Table S1). Further haplotype analysis combining
the 7 SNPs indicated strong linkage disequilibrium (LD) among
the SNPs (Fig. S1a) with only two major haplotypes, one showing
positive association (P=461025), the other showing negative
association (P=861024) with cranial volume in females (Table
S2). None of the described associations in females were observed
in males (Table S3), implying that the association of IL3 with
brain volume is sex-specific. To capture missing common SNPs,
we re-sequenced the IL3 gene region (4 kb) in 150 randomly
selected Chinese individuals and found no additional SNPs.
Replication of the Association between IL3 and Brain
Volume Variation in Europeans
To confirm our initial findings from the Chinese population, we
conducted a replication analysis in three independent samples of
European ancestry, for which total brain volume had been
determined based on magnetic resonance imaging (MRI). For the
seven SNPs showing strong association in Chinese sample, two
SNPs in different LD regions in Europeans (rs3916441 and
rs40401, Fig. S1) were included in the replication analysis. Only
the healthy controls of these samples were used. We found that the
most significant SNP in Chinese, rs3916441, was also significantly
associated with total brain volume in the BIG (Dutch Brain
(Table 1). In CBDB/NIMH (Clinical Brain Disorders Branch/
National Institute of Mental Health Sibling Study) sample
(p=0.0516) (Table 1). We noticed the female specific association
of rs3916441 with brain volume in Chinese was not the situation
in European samples. Interestingly, rs3916441 was also signifi-
cantly associated with total gray matter volume in the CBDB/
NIMH and BIG samples (Table 2), which may point towards a
mechanism explaining the effects of IL3 on brain structure.
Impacts of rs31480 on Transcription Factor Binding and
To capture the causal variants of IL3 in Chinese population, we
performed bioinformatics analysis for the 7 highly significant SNPs
IL3 Contributes to Human Brain Development
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Figure 1. Genetic association of the 5q23.2–33 SNPs with brain volume and impacts of promoter SNP (rs31480) on the expression
of IL3. (a) The distribution of the –logP of the 20 SNPs tested across the 5q23.3–33.1 region (middle panel). The locations of the six known coding
genes are displayed. (b) The brain volume distributions of the three genotypes at rs31480, on average, TT genotype carriers have a brain volume of
1257 ml and CC genotypes have 1216 ml (***P,0.001, two tailed Student’s t-test). (c) C allele of rs31480 is completely conserved across a variety of
species. (d) The oligonucleotides for testing the binding activity of SP1. The predicted binding sequence is underlined containing the rs31480
variation site (red). (e) The result of electrophoretic mobility shift assay, showing that the probe containing T allele can bind SP1 (Lane 2) but the C
allele cannot (Lane 3). Similar results were obtained using HeLa or MCF7 nuclear extracts (Lane 6 and 7, 10 and 11). Competition experiments using a
100-fold excess of unlabeled probe (Lane 4 and 5, 8 and 9) confirm the specificity of the probe. Binding to the unknown protein/complex was also
IL3 Contributes to Human Brain Development
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according to their genomic locations and allelic differences in
transcription factor binding affinities, and we found SNP rs31480
showing potential functional effects. rs31480 is located in the IL3
promoter (216 bp upstream of the transcription start site (TSS),
Fig. S2), within a putative binding site of the transcription factor
SP1. Interestingly, there is a significant difference of 41 ml in the
average cranial volume between individuals carrying the two
homozygotes at rs31480 (1,257 ml for TT carriers and 1,216 ml
for CC carriers, p=4.7 61024, two tailed student t-test) (Fig. 1b
and Table S4), suggesting that rs31480 could regulate brain
volume variation. In addition, we noticed the C allele (ancestral
allele, determined by comparison with the chimpanzee homolo-
gous sequence) of rs31480 is completely conserved across a wide
variety of species (Fig. 1c and Fig. S2), also suggesting a
functional conservation of rs31480. The T allele (derived allele) is
prevalent in East Asian populations (0.556 in Chinese and 0.568 in
Japanese), but relatively rare in Europeans (0.198) and Africans
The C to T change at rs31480 could change the binding affinity
of SP1 and influence the expression of IL3. Electrophoretic
mobility shift assay (EMSA) with purified recombinant human SP1
protein (Fig. 1d) showed that SP1 binds to the sequence
containing the T allele (Fig. 1e, lane 2) but not the C allele
(Fig. 1e, lane 3). Similar results were observed when using HeLa
(Fig. 1e, lane 6 and 7) and MCF-7 (Fig. 1e, lane 10 and 11) cell
nuclear extracts as the source of SP1 protein (Fig. 1e). Finally,
competition experiments using unlabeled oligonucleotides corrob-
orated the SP1 binding specificity to the T allele (Fig. 1e, lane 4
and 5, lane 8 and 9). These data suggest that rs31480 has an ‘‘on’’
or ‘‘off’’ effect on SP1 binding to the IL3 promoter.
To test whether rs31480 also influences IL3 promoter activity,
we performed transactivation assays using the luciferase reporter
gene. The promoter region encompassing nucleotides 2436 to
+164 (relative to the ATG start codon at +1) of IL3 was amplified
by PCR from genomic DNA of two individuals homozygous with
respect to the corresponding genotypes (TT and CC) for rs31480.
Sequencing analysis of the amplified promoter fragments did not
detect other sequence differences except for rs31480. As shown in
Fig. 1f and Fig. S3a–c, the transcriptional activity of the IL3
promoter containing the T allele was indeed significantly higher
than that of the C allele in all cell lines tested (Hela, CHO, SK-N-
SH, and COS-7). The T allele of rs31480 thus enhances the IL3
promoter activity through the binding of transcription factor SP1.
For the most significant SNP rs3916441, since our functional
prediction analysis did not give any hint for the functional role of
this SNP, whether it plays any functional role for IL3 is yet to be
It should be noted that, rs31480 was not significantly associated
with brain volume in the BIG sample of Europeans, and it was not
available in the ANDI and CBDB/NIMH samples. Another SNP
rs40401 in high linkage with rs31480 was also not significant in
these samples. The differences in association for this SNP
(rs31480) are likely due to the genetic heterogeneity between
Chinese and Europeans as shown in Fig. S1.
IL3 and its Receptors are Mainly Expressed in Neural
Progenitors and Mature Neurons
IL3 exerts its biological effects through a receptor that is
composed of a ligand-specific a (IL3RA) subunit and a signal
transducing b subunit (IL3RB) common to IL3/IL5/GM-CSF.
The mouse IL3 receptor has two distinct b subunits, one that
functions only in IL3-mediated cell signaling (bIL3) and a second
that is shared with IL5 and GM-CSF (IL3RB or CSF2RB). We
studied the expression of IL3 and its receptors in the developing
mouse brain and found that IL3 and its receptors were
continuously expressed in mouse brain from embryonic day (E)
12.5 to adult life as revealed by RT-PCR (Fig. 2a), with a peak
expression level at postnatal day (P) 1 to 4 (Fig. 2b), a stage with
active neural proliferation and neurogenesis. We also noticed that
bIL3 is only expressed from E14.5 to P7 (Fig. 2a), a stage
accompanied by the dramatic increase of the neocortex volume
. Immunostaining revealed that IL3 and its receptors were
mainly expressed in the neocortex region of the mouse brain
(Fig. 2c–n and Fig. S4 and S5). We also detected weak
expression of IL3RA in the CA1 and CA3 regions of hippocampus
(Fig. S6), hilus of the dentate gyrus (Fig. S7), and lateral septal
nucleus, dorsal part (LSD) (Fig. S8). Compared to IL3RA, IL3RB
showed higher expression in the mouse brain (Fig. 2a, b). It was
extensively expressed in mouse brain including neocortex (Fig. 2i–
k) and hippocampus (Fig. S6). Since all IL3RA positive cells also
expressed IL3RB (Fig. S6 and S9), we focused on IL3RA
hereinafter. To characterize IL3RA-positive cells, we performed
co-immunostaining and found IL3RA was expressed in SOX2-
and nestin-positive neural progenitors at early developmental stage
(Fig. 3a–e, Fig. S4 and S10). As development continues, the
observed (Figure 1e, arrow C and D), again, the probe containing T allele showed stronger binding than C allele. (f) Assays of promoter activities by
relative luciferase expression in HeLa, construct with T allele has significant higher expression activity than C allele. Values of relative luciferase activity
are expressed as mean 6 s.d. (results of three independent experiments, each containing three replicates). ***P,0.001 (one tailed Student’s t-test).
Table 1. Replication of the most significantly associated SNPs in genetically divergent populations.
BIG sample (3.0 T)
P value (Total brain volume)P value (Total brain volume)P value (Total brain volume)
rs3916441C/T 0.6970.0467 0.164 0.05160.04160.462 3.561024
rs40401G/A 0.147 0.2610.467 0.7030.580 NANANANA
NA: Not available.
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expression of IL3RA was down-regulated in neural progenitors
(Fig. 3f–h). After birth, expression of IL3RA was found in some
Tuj1-positive neurons (Fig. S11). However, we noticed that many
IL3RA positive cells were Tuj1-negative (Fig. 3i–k, Fig. S11a–i).
Co-labeling with GFAP excluded their identity as glial cells
(Fig. 3l–n). Further double immunostaining showed many IL3RA
positive cells also weakly expressed Tbr2, a marker for interme-
diate progenitor cells (IPCs) (Fig. 3o–q and Fig. S12). In
contrast, in addition to co-expression with IL3RA, IL3RB was
expressed in neurons and glial cells (Fig. S13). Taken together,
these results demonstrate that IL3 and its receptors are mainly
expressed in neural progenitors and neurons in the developing
IL3 Promotes the Proliferation of Neural Progenitors
IL3 is known to activate three signaling pathways, the JAK/
STAT, the MAPK, and the PI3K/AKT pathways, among which
the MAPK signal pathway regulates cell proliferation . Since
we found IL3RA co-expressed with the cell proliferation markers
Ki67 and pH3 (Fig. S14), IL3 could play a role in promoting the
proliferation of neural progenitors. We thus examined the effect of
IL3 treatment on the proliferation of neural progenitors isolated
from E13.5 cortex. We first verified the expression of IL3RA and
IL3RB in cultured neural progenitors (Fig. S15a). Anti-pH3
immunolabeling showed that the number of proliferating cells was
significantly increased in IL3-treated samples compared to the
controls (Fig. 4a,b), which is consistent with published observa-
tions . Western blotting confirmed that IL3 could activate
MAPK pathway in both MEG01 cells (Fig. 4f) and cultured
neural progenitors (Fig. 4g). The phosphorylation of MAPK1/2
was significantly increased after IL3 treatment, indicating that IL3
can activate proliferation pathway in neural progenitors. We also
investigated another proliferation related pathway, the JAK/
STAT pathway. We found that JAK2 phosphorylation was
increased after IL3 treatment (Fig. 4g), further supporting the
involvement of IL3 in the proliferation of neural progenitors.
We next tested whether IL3 could drive neuronal differentiation
in vitro. The progenitor cells were cultured under differentiation
condition, treated with IL3, and the expression level of cell type-
specific markers was measured by quantitative PCR. We found
that IL3 had no effect on neuronal differentiation in vitro. After IL3
(10 ng/ml) treatment, the expression of all tested genes was not
changed significantly (Fig. S16). Collectively, these data suggest
that IL3 promotes the proliferation of neural progenitors through
the activation of MAPK and JAK/STAT pathways, but has no
effects on neural differentiation.
Neurotrophic Effects of IL3 on Neural Progenitors and
IL3 is reported to have trophic effects on neurons . It
promotes the survival of sensory neurons and protects against
neuronal death induced by FeSO4 and Ab [43,44]. We speculated
that IL3 might also have similar trophic effects on cortical neural
progenitors. To test this, we determined cell viability of cultured
neural progenitors and neurons in media with different growth
factors and supplements. The results suggest that when the
nutrition is deficient, the IL3 pathway could be activated to protect
against cell death induced by starvation (Fig. S15b,c, Fig. 4c,d).
Similar results were obtained on neurons when cultured using the
previously reported culture method  (Fig. 4e).
The Bcl-xL was reported has a role in neuronal survival
mediated by IL3 pathway , so we studied the expression of
Bcl-xLin neural progenitors and found there was no significant
change after treated with IL3 (Fig. S15e). The signaling through
the PI3K/AKT pathway is one of the most potent intracellular
mechanisms to promote cell survival. It is well established that IL3
can activate the PI3K/AKT pathway . To further study the
mechanism of neural progenitor survival mediated by IL3 and to
test if the PI3K/AKT pathway participates in IL3-mediated
survival of neural progenitors, we studied the interactions between
IL3 and AKT1 in neural progenitors by western blotting. In
untreated progenitors, the level of phosphorylated AKT (the active
form) is low (Fig. 4g). However, the level of phosphorylated AKT
was dramatically increased after IL3 treatment (Fig. 4g). In
addition, analysis of three AKT1 SNPs indicated significant
association with brain volume (Table S5). Taken together, these
results indicate that IL3 promotes the survival of neural
progenitors by activating the PI3K/AKT pathway.
IL3 Activates Estrogen Receptor a and b in vitro
As shown above, the genetic association of the IL3 SNPs with
brain volume was female-specific in Chinese. To test whether
estrogen could regulate the expression of IL3, we treated the K562
cell line (which expresses both IL3 and estrogen receptor (Fig.
S17a)) with estrogen and found no overt change of IL3 expression
(Fig. S17c–e), while the expression of TFF1 as a control was
significantly increased after estrogen treatment (P,0.001) (Fig.
S17b–d). These results suggest that the transcription of IL3 could
not be directly regulated by estrogen.
It was reported that the MAPK and PI3K/AKT pathways
could activate the estrogen receptor (ER) [45,46]. To investigate
whether IL3 could activate ER genes through the two regulated
pathways, we constructed three vectors, 3ERE-PGL3 (contains
three repeats of estrogen response element (ERE)), ERaand ERb
Table 2. Association of rs3916441 with gray matter and white matter in genetically divergent populations.
SNP PolymorphismReplication samples
(Healthy controls)BIG sample (3.0 T) (Healthy controls)
P value (Total gray matter volume)P value (Total gray matter volume)P value (Total white matter volume)
rs3916441 C/T 0.0228 0.128 0.1100.001 0.0010.178 0.0020.004 0.138
rs40401 G/A0.1710.128NA NANA NANANANA
NA: Not available.
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Figure 2. Spatiotemporal expression profiling of IL3 and its receptor in the developing mouse brain. (a) RT-PCR revealed the expression
of IL3 and its receptor in developing mouse brain from E12.5 to adult. (b) Quantitative PCR showed that the expression of IL3 and its receptors peaks
at P1–P4, a period with active neural proliferation and neurogenesis. Data are expressed as mean 6 s.e.m. (n=3). (c–n) Immunohistochemistry
analysis indicated that IL3 and its receptor were expressed in the mouse brain. Co-expression of IL3 and IL3RA were detected (arrows in l–n),
indicating the activation of IL3-mediated signaling pathways in the developing mouse brain. (o–p) IL3RA is expressed in radial glia (resides in
ventricular zone and characterized by long radial processes, arrowhead in o) and migratory neurons (arrowheads in p). Ctx, cortex; VZ, ventricular
zone. Scale bars, (c, d, e) 25 mm; O, 10 mm.
Figure 3. IL3RA is mainly expressed in neural progenitors and neurons. (a, b) Expression of IL3RA was detected in SVZ and IZ regions. Co-
labeling with SOX2 showed IL3RA expression cells in SVZ are SOX2 positive, indicating these cells are neural progenitors. However, IL3RA positive
cells in IZ are SOX2 negative, indicating these cells are not neural progenitors. (c–e) In the early stages of brain development, co-expression of IL3RA
and SOX2 was found in neocortex region. With the development of the central nervous system, expression of SOX2 was down-regulated or
disappeared in IL3RA positive cells (f–h). We also found many IL3RA positive cells were not mature neurons (i–k) or glial cells (l–n). Co-labeling with
TBR2 demonstrated IL3RA positive cells are intermediate progenitor cells (IPCs) (o–q). VZ, ventricular zone; SVZ, subventricular zone; IZ, intermediate
zone; CP, cortex plate. Scale bars, 25 mm.
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respectively. We first studied the expression of IL3 receptors and
estrogen receptors in MEG-01 and HEK293T cell lines and we
found IL3 receptors were expressed in both cell lines (Fig. 5a,b).
The vectors (3ERE-PGL3 and ERa,or 3ERE-PGL3 and ERb)
were then co-transfected into the IL3 receptor-expressing MEG-
01 cell line, followed by IL3 or E2 treatments. As expected, IL3
Figure 4. IL3 promotes proliferation and survival of neural progenitors. (a) IL3 promotes neural progenitor’s proliferation. (b) Quantification
of proliferating cells (pH3+) after IL3 treatment.#P,0.05 (n=4, one-tailed Student’s t-test). (c) Trophic effect of IL3 on neural progenitors. Neural
progenitors from E12.5 mice were grown in the absence of any factor and in the presence of different concentrations of IL3 for 36 hours, then cell
viability were determined. Note that 3.0 ng/ml IL3 could promote survival of neural progenitors significantly (n=8 for each condition). (d–e)
Neurotrophic effects of IL3 on neural progenitors and neurons. Neural progenitors were first cultured in neurobasal medium with B27 supplement for
about 24 hours, then the medium was replaced with neurobasal medium containing N2 supplement and different concentrations of IL3 were added.
The cultures were maintained for 3 days and cell viability was determined. IL3 has significant effects on this culture condition on progenitors (d) and
neurons (e) (n=8 for control group and 20 ng/ml group, n=16 for other groups). y-axis, cell viability (normalized to control), x-axis, concentration of
IL3 (ng/ml). Data are expressed as mean 6 s.e.m. *P,0.05, **P,0.01 (two-tailed Student’s t-test). (f) IL3 activates PI3K-AKT, MAPK1/2 and Gsk3b signal
pathways in MEG01 cell line. (g) IL3 activate MAPK, JAK/STAT, and PI3K/AKT pathways in primary cultured neural progenitors. The phosphorylation
level of AKT, MAPK1/2, JAK2 and GSK3b was increased after IL3 treatment.
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could activate both ER genes, especially ERb (Fig. 5c,d). In
addition, the activation of ERbinduced by E2 was enhanced by
IL3. We confirmed these results in HEK293T cells (Fig. 5e,f).
Hence, the activation of ER genes by IL3 and estrogen and the
interaction between them may explain the sex-specific functional
effects of the sequence polymorphism at rs31480 on brain volume
in females. The reported association studies on SCZ patients is
consistent with our observation, in which IL3 showed a significant
association with schizophrenia only in females. The hypothesized
sex-specific regulation of brain volume is illustrated in Fig. 6 and
Brain volume is an important quantitative trait that underlies
our most complex cognitive abilities and human evolution is
characterized by a dramatic expansion in brain size and
complexity. Undoubtedly, our unique genetic makeup played
a decisive role in our enlarged brain and human brain size must
be highly regulated during development. The hypothesis that
SCZ is a brain disease unique to humans  suggests that
SCZ susceptibility genes may regulate the unique features of
human brain development and dysfunction of these genes may
disrupt the normal development of the human brain. In this
study, we provide evidence that IL3 may regulate human brain
volume variation. First, our genetic association results strongly
suggest the association between IL3 and brain volume. IL3 is
located in 5q31.1, one of the most successfully replicated regions
that may harbor SCZ susceptibility genes [26–35]. In fact,
5q23–31 was ranked number 2 of all chromosomal regions
implicated to harbor SCZ susceptibility genes in a genome-wide
meta-analysis of SCZ . As accumulating data support that
SCZ is a neurodevelopmental disorder, it’s likely that there are
potential genes in 5q23.2–33.1 that regulate brain development.
However, though studies repeatedly found association between
genes located in 5q23.2–33.1 and SCZ, the detailed expression
pattern ofthesegenes and
development is not known. Here, for the first time, we detailed
the spatiotemporal expression pattern of IL3 and its receptors in
developing mouse brain and we found IL3RA is mainly
expressed in neural progenitors and neurons, which also support
the importance of IL3 signaling pathway in brain development.
Also, our in vitro proliferation and survival assays further validate
the pivotal roles of IL3 in the development of central nervous
system. Collectively, these results provide novel insights to the
involvement of IL3 in brain development, supporting the
neurodevelopmental hypothesis of schizophrenia.
It should be noted that during the initial screening in the
Chinese sample, we used cranial volume as proxy of brain volume.
Though cranial volume is not exactly equal to brain volume, the
correlation between these two variables is very high . More
importantly, we have successfully identified the association
between cranial volume and MCPH1 gene by applying this
method in our previous study . In addition, the successful
replication of our initial findings in genetically divergent popula-
tions further support the reliability of our method.
We realized that the association data and the functional data
did not refer to the same SNP, which could be explained by
several possible reasons. First, though rs3916441 has the
smallest p value in our screening sample, it is located about
27 kb upstream of IL3, and the likelihood of rs3916441’s direct
regulation of IL3 expression is relatively small. Second, the p
value of rs3916441 and rs31480 is very close in our screening
sample, and they are highly linked (r2=0.89) in Chinese.
Hence, the functional data suggests rs31480 is probably the
causal SNP in brain volume regulation. Nevertheless, we have
successfully replicated the significant associations of IL3 variants
with brain volume in BIG sample (rs3916441), and we also
observed a marginal significant association in CBDB/NIMH
sample (rs3916441). Although the association of these SNPs did
not reach genome-wide significance, considering the non-overlap
of the studied samples and different genetic backgrounds of
Chinese and Europeans, IL3 is likely an authentic gene
contributing to brain volume variation in general populations.
To date, no genes have been shown significantly associated with
brain volume and only a few genes were associated with
intracranial volume in recent genome-wide association studies
[11–13], suggesting an extremely complicated genetic regulation
of brain volume.
between immune and nervous systems may play an important
role in the pathogenesis of schizophrenia . The immune and
nervous systems interact with each other through cytokines, a
family of proteins that are secreted by a specific group of cells
of the immune system and have pleiotropic effects on many cell
types, including proliferation, differentiation, and survival. IL-3
is a cytokine that induces growth and differentiation of
hematopoietic stem cells and a variety of cell types originating
in the bone marrow. Recent studies have demonstrated the
important role of IL3 in the central nervous system (CNS). It is
expressed in the hippocampus and cortices of normal mouse
brain , and it stimulates the growth and proliferation of
microglial cells [51,52]. Studies also found that IL3 facilitates
the survival of sensory neurons significantly and stimulates the
formation of the neural network . In addition, IL-3 has
been found to be able to promote the process extension of
cultured cholinergic  and prevent delayed neuronal death in
the hippocampus . In fact, rat interleukin 3 receptor b-
subunit was cloned from cultured microglia , and disruption
of IL3 production in brain led to neurologic dysfunction .
All of these studies strongly suggest that IL3 is a pivotal
protective factor for CNS. More importantly, Chen et al.
recently reported that IL3 was significantly associated with
brain disease such as schizophrenia in three independent Irish
samples [34,56,57].IL3 receptors,
CSF2RB (or IL3RB), were also found significantly associated
with schizophrenia in three different populations [58–60]. In
addition, decreased IL-3 levels in the first-episode and drug-
naı ¨ve patients with schizophrenia was also reported . These
convergent evidences strongly indicate the involvement of the
IL3 pathway in schizophrenia. Interestingly, we noticed that
rs3916441, which is most significantly associated with brain
volume, was also significantly associated with schizophrenia in
females , implying the interaction between IL3 and gender
may play vital roles in normal brain development and
schizophrenia susceptibility. Though many investigations support
the involvement of IL3 in brain function and schizophrenia, the
precise expression pattern of IL3 and its receptors in developing
brain is not well characterized and it’s not clear how genetic
variation within IL3 affect brain development and schizophrenia
In summary, we have demonstrated that IL3 plays crucial roles
in the development of the central nervous system. We identified a
genetic variant (rs31480) in the promoter of IL3 that is
significantly associated with brain volume in the general popula-
tion. This polymorphism influences the expression of IL3 and the
differential IL3 expression of the two alleles at rs31480 can
influence neural progenitor pool expansion and maintenance
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Figure 5. IL-3 activates estrogen receptors in MEG01 and HEK293T cells. There are two estrogen receptors, ERaand ERb. MEG01 and
HEK293T cell lines were used to test whether IL3 can activate estrogen receptors. Expression of IL-3 receptors (IL3RA, CSF2RB) were verified by RT-PCR
in MEG01(a) and HEK293T (b) cell lines. In HEK293T cell line, we also detected the expression of estrogen receptors (ERaand ERb) (b). Constructs
containing three repeats of estrogen response element (3ERE) and estrogen receptor (ERaor ERb) were co-transfected into MEG01 and HEK293T cell
lines prior to IL-3 or estrogen (E2) treatment. IL-3 can activate ERaand ERbin both cell lines (c-d for MEG01 and e-f for HEK293T), and this effect was
enhanced by the estrogen, indicating there were interactions between IL-3 and estrogen activation. Data are expressed as mean 6 s.e.m. (three
IL3 Contributes to Human Brain Development
PLOS ONE | www.plosone.org 10November 2012 | Volume 7 | Issue 11 | e50375
during neurodevelopment. Furthermore, our findings that IL3 can
promote proliferation and survival of neural progenitors further
support the proposed novel role of IL3 in the central nervous
Materials and Methods
Our screening samples are from Yunnan province of south-
western China (n=1,013). Replication samples included samples
from the Alzheimer’s Disease Neuroimaging Initiative (ADNI;
n=204), samples from the US National Institute of Health
(CBDB/NIMH; n=188) and samples from the Dutch Brain
Imaging Genetics study (BIG; n=486).
Screening Samples: Chinese Samples
The detailed information of the sample screening was described
in our previous study . Briefly, a total of 1,013 unrelated healthy
individuals including 460 males and 553 females were included.
The identities of the subjects were self-declared and confirmed by
their written ID profiles. All the sampled individuals are from
Yunnan province of southwestern China. Written informed
consents for this study were obtained from all the subjects, and
the research protocol was approved by the internal review board
of Kunming Institute of Zoology, Chinese Academy of Sciences.
The ages of the 1,013 individuals range from 19 to 28 years with
98% of them being 21–26 years old.
Replication Samples: ADNI Sample
The MRI and genotyping data in this replication sample were
obtained from the Alzheimer’s Disease Neuroimaging Initiative
(ADNI) database (adni.loni.ucla.edu). One goal of ADNI has been
to test whether serial magnetic resonance imaging (MRI), positron
emission tomography (PET), other biological markers, and clinical
and neuropsychological assessment can be combined to measure
the progression of mild cognitive impairment (MCI) and early
Alzheimer’s disease (AD). For up-to-date information, see www.
adni-info.org. The ADNI participants consist of patients with AD,
patients with MCI, and elderly healthy individuals. They were
aged 55–90 years and recruited from 59 sites across the U.S. and
Canada. Written informed consent was obtained from all 822
participants and the study was conducted with prior Institutional
Review Board approval. Of 822 participants, 204 unrelated non-
Hispanic Caucasian healthy controls were used in this study .
Replication Samples: CBDB/NIMH Sample
All subjects are the healthy control participants of Clinical Brain
Disorders Branch/National Institute of Mental Health Sibling
Study, a study aimed at identifying schizophrenia susceptibility
genes and related intermediate biologic phenotypes . Subjects
with good quality of structural data and genotyping were included
Replication Samples: BIG Sample
In this study, a total of 486 healthy control subjects aged 18–35
years from the Brain Imaging Genetics (BIG) study at the Donders
Institute for Brain, Cognition and Behaviour of the Radboud
University Nijmegen (Medical Centre) were included. The BIG
study is a study of self-reported healthy individuals included into
earlier imaging studies at the Donders Centre for Cognitive
Neuroimaging. Subjects are of European Caucasian descent and
generally highly educated . The study was approved by the
regional medical ethics committee (CMO regio Arnhem/Nijme-
gen) and all participants provided written informed consent prior
independent assays, each containing 3 replicates).
Figure 6. Model for regulation of brain volume by IL3 genetic variation and expression level. Individuals with different genotypes at
rs31480 have differential expression level of IL3 (individuals with TT genotype have higher IL-3 expression than CC carriers), which lead to differential
activation of signaling pathways mediated by IL3. The differential activation of signaling pathways further influence the proliferation and survival of
neural progenitors, eventually lead to brain volume variation for individuals with different genotypes at rs31480.
IL3 Contributes to Human Brain Development
PLOS ONE | www.plosone.org11 November 2012 | Volume 7 | Issue 11 | e50375
Measurement of Cranial Volume: Chinese Screening
The cranial volume was measured and calculated as described
in our previous study . Three principal dimensions of the
cranium were measured including 1) Maximum antero-posterior
length (L, measured between glabella and the inion). 2) Maximum
breadth (B, biparietal diameter; measured between two parietal
eminences). 3) Cranial height (H, basi-bregmatic height, measured
between the internal acoustic meatus to the highest point of the
vertex). Then the cranial volumes were computed using the
following formula [9,65]: Male, 0.337 (L-1.1) (B-1.1) (H-1.1)
+406.01 cc; Female, 0.400 (L-1.1) (B-1.1) (H-1.1) +206.60 cc.
Measurement of Brain Volume: Replication Samples
3D T1-weighted brain MRI scans were acquired using a sagittal
3D MP-RAGE sequence following the ADNI MRI protocol .
Baseline 1.5T MRI scans from 204 participants were downloaded
from the ADNI public website (http://www.loni.ucla.edu/ADNI/
) onto local servers at Indiana University School of Medicine. As
detailed in previous studies , FreeSurfer V4 software (http://
surfer.nmr.mgh.harvard.edu/), a widely employed brain segmen-
tation and cortical parcellation tool, was used to label cortical and
subcortical tissue classes using an atlas-based Bayesian segmenta-
tion procedure and to extract the measure of brain volume.
Measurement of Brain Volume: Replication Samples
All structural MRI were acquired on a 1.5 Tesla GE scanner
(GE Medical Systems, Milwaukee, Wisconsin) using a T1-
weighted spoiled gradient recalled (SPGR) sequence (repetition
time, 24 ms; echo time, 5 ms; number of excitations, 1; flip angle,
45u; matrix size 2566256; FOV 24624 cm2), with 124 sagittal
slices (0.9460.9461.5 mm3resolution). Images were processed
using the FreeSurfer  toolbox (version 5). Total Brain Volume
(TBV) and Total Grey Matter volume (TGM) measurements were
calculated as previously described [63,69]. TGM was defined as
sum of tissue probabilities for the grey matter region. TBV was
defined as the sum of total gray matter volume, total white matter
volume, and cerebrospinal fluid.
Measurement of Brain Volume: Replication Samples (BIG
Subjects were scanned at 3 Tesla (n=486) MRI scanners and
T1-weighted structural magnetic resonance imaging data (3D
MPRAGE) were acquired (more information on the image
acquisition can be found in our previous study ). All scans
covered the entire brain and had a voxel-size of 16161 mm3. To
calculate total brain volume, raw DICOM MR imaging data were
converted to NIFTI format using the conversion as implemented
in SPM5 (http://www.fil.ion.ucl.ac.uk/spm/software/spm5/).
Normalizing, bias-correcting, and segmenting into gray matter,
white matter, and cerebrospinal fluid was performed using the
VBM toolbox (VBM5.1 Toolbox version 1.19) in SPM using
priors (default settings). This method uses an optimized VBM
Protocol [71,72] as well as a model based on Hidden Markov
Random Fields (HMRF) developed to increase signal-to-noise
ratio . Total volume of gray matter, white matter, and
cerebrospinal fluid was calculated by adding the resulting tissue
probabilities. Total brain volume was defined as the sum of white
matter and gray matter volume.
SNP Selection, SNP Tagging, Genotyping and
SNP selection was based on the previous association studies
including our recent data [33–35]. We focused on the four genes
that identified by Chen et al. recently using systematically mapping
in large independent samples. In addition, our recent data and LD
in Chinese were also considered. We selected 8 SNPs for the initial
screening (rs3756295, rs40396, rs1291602, rs31251, rs1355095,
rs2240525, rs3914025, rs31400), additional SNPs were included
according to the association significance and whether they are
tagging SNPs. Totally, we selected 20 SNPs for fine mapping. The
20 SNPs were genotyped using the SNaPshot method (Applied
Biosystems). We sequenced the IL3 gene (including the 500 bp
upstream and downstream, respectively) in 150 randomly selected
individuals through direct sequencing. The conservation analyses
were performed by using UCSC genome browser . (http://
Prediction of DNA-binding Motifs
We used Dragon ERE finder , a web-based program for
identification and interactive analyses of estrogen response
elements (EREs) to predict EREs in the upstream region of IL3.
alibaba2/index.html) was used to predict and compare DNA-
binding motifs in the promoter region with alternative alleles.
Cell Culture, Treatment, and RNA Extraction
K562 cells were routinely cultured in DMEM (Gibco)
supplemented with 10% FBS (Hyclone), 100 u/ml penicillin and
100 ug/ml streptomycin. Before treatments, the cells were
maintained in phenol red-free DMEM containing 10% dextran-
coated charcoal-stripped fetal bovine serum (DCC-FBS) (Hyclone) for
a minimum of 3 days with the media changed every day. Cells
were treated with 10 nM 17-beta-estradiol (E2) (Sigma) for 2 to 24
hours. Total RNA was harvested and prepared using TRIzol
(Invitrogen) following the manufacturer’s instructions.
Quantitative Real-time PCR
Reverse transcription PCR (RT-PCR) was performed using the
Omniscript RT Kit (Qiagen) following the manufacturer’s
instructions. We carried out real-time quantitative PCR using
gene specific primers, and the fold change in expression was
calculated using the DDCt(threshold cycle) method. The GAPDH
was used as the internal control.
EMSAs were performed with a LightshiftTMchemilumescent
EMSA kit (Pierce). The single-strand oligonucleotides were
biotinylated with Biotin 39 End DNA labeling Kit (Pierce) and
then annealed to form double strands. The nuclear extracts of
MCF-7 and U2OS were prepared by CelLyticTMNuCLEARTM
Extraction kit (Sigma). HeLa nuclear extracts were purchased
from Santa Cruz Biotech. The binding reactions were performed
for 20 mins at room temperature in 10 mM Tris-HCl (PH 7.5),
1 mM MgCl2, 0.5 mM EDTA, 0.5 mM DTT, 50 mM NaCl,
50 ug/ml poly (dI-dC)(dI-dC) and 4% glycerol, 35 fmol biotin 39-
end -labeled double-stranded oligonucleotides, and purified
recombinant SP1 protein (Alexis) or nuclear extracts. After
incubation, samples were separated on a native 6% polyacryl-
amide gel and then transferred to a nylon membrane. The
positions of biotin end-labeled oligonucleotides were detected by a
chemilumescent reaction with streptavidin-horseradish peroxidase
according to the manufacturer’s instructions and visualized by
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autoradiography. For competition assays, we pre-incubated 100-
fold excess of unlabeled oligonucleotide probe with SP1 or nuclear
extracts before adding the biotin-labeled probe. The nucleotide
sequences of the double-stranded oligonucledtides with either T or
C allele are:
Promoter Cloning and Reporter Gene Assays
To construct IL3 promoter, we amplified fragments encom-
passing nucleotides -436 to +164 (relative to the ATG start codon
at +1) of IL3 by PCR from genomic DNA of two individuals
homozygous with respect to the corresponding genotypes (TT and
CC) for rs31480, using primers tailed with Xhol and HindIII
restriction sites, and directionally subcloned them into the Xhol and
HindIII sites of the pGL3-Basic expression vector (Promega). We
verified all recombinant clones by bi-directional DNA sequencing.
HeLa, COS-7, CHO, and SK cells were routinely cultured in
DMEM supplemented with 10% FBS with antibiotics. The cells
were plated at 2.56105cells per well in a 24-well plate the day
before transfection and incubated overnight at 37uC in 5% CO2.
Transient transfection assays were conducted in these cells using
the Lipofectamine 2000 transfection reagent (Invitrogen), all assays
were performed in at least three independent experiments with
minimum of three replicates. The reporters containing either T
allele or C allele were transfected into these cells together with a
Renilla luciferase control vector. After 24h incubation, we collected
the cells and measured luciferase activity using the Dual-Luciferase
Reporter Assay System (Promega).
3ERE Cloning and IL3 Activation Assays
Three repeats of Estrogen Response Elements (EREs) tailed
with Xhol and HindIII restriction sites were synthesized and
annealed to form double-stranded nucleotides. The sequence is 59-
CCG CTCGAG TA GGTCA GCG TGACC TA TA GGTCA
GCG TGACC TA TA GGTCA GCG TGACC TA AAGCTT
GGG-39. We directionally cloned it into the pGL3-Basic vector
after restriction enzyme digestion. We confirmed the construct by
sequencing. The estrogen receptor alpha and beta vectors were
kindly provided by professor Sylvie Mader (Faculte ´ de Me ´decine,
Universite ´ de Montre ´al) and Leigh C. Murphy (University of
Manitoba). MEG-01 and HEK293T, IL3 receptor expression
positive cell lines were cultured in PRMI 1640 and DMEM
respectively supplemented with 10% FBS, 2 mM L-glutamine,
1 mM Sodium pyruvate and 1% antibiotics. Co-transfection of
3ERE-pGL3 (2 ug), ERa(1 ug) or ERb(1 ug) were performed by
using a Nucleofector Device from Amaxa Biosystems (Lonza
Cologne AG, Germany) in MEG01 cell line, while co-transfection
in HEK293T cell line were used Lipofectamine 2000 transfection
reagent (Invitrogen) as previously described, and with pRL-TK as
the internal control. After six hours incubation, 17-beta-estradiol
and recombinant human IL3 protein (Invitrogen) were added into
the medium with a final concentration of 10 nM. Cells were
harvested and their luciferase activity was measured after
additional 24 h incubation. All assays were performed in at least
three independent experiments with a minimum of triplications.
Hardy-Weinberg equilibrium of each SNP was assessed by
using GENEPOP (v 4.0) . Association of single SNP with total
brain volume (TBV) and their additive effects on this quantitative
trait were tested by utilizing PLINK or SAS statistical software
using the linear regression option, with age, sex and IQ (optional)
as covariates; for the analyses on total gray/white matter, TBV
was also considered as a covariate . To account for sex-specific
effects, we used a statistical model where the mean effect of SNP
dose on the phenotype was allowed to differ for the two sexes. The
p-value was adjusted by the conservative Bonferroni correction
according to the number of independent SNPs and the divided
internal samples separated by sex. We used the Haplo Stats  to
infer the haplotype frequency and to perform the haplotype
association test. We used the Haploview  to calculate pairwise
LD indices r2and D’, to define LD blocks and to select the tag
SNPs. Haplotypes were inferred with the PHASE program by the
Bayesian statistical methods based on the genotype data .
Sequence alignment and assembly were conducted by DNASTAR
software package. The analysis of quantitative PCR data was
based on the DCtvalues.
The C57BL/6J mice were used in this paper and all animal
procedures described herein were approved by the University
Committee of Animal Resources at the University of Rochester.
For the purposes of staging embryos, noon of the day a vaginal
plug was detected was taken to be embryonic day 0.5 (E0.5). Mice
were deeply anesthetized, perfused transcardially with PBS,
followed by 4% PFA in PBS, pH 7.3. Then the brains were
dissected, postfixed in 4% PFA in PBS at 4uC overnight, washed
three times in PBS, and cryoprotected in 30% sucrose in PBS
before rapid freezing in OCT compound (TissueTek). For antigen
retrieval, cryosections (20 mm) were heated in 10 mM citrate
buffer (pH 6.0) at 95uC for 10 min. The sections were permea-
bilized and blocked in PBS plus 0.1% Tween-20, 5% horse serum,
and incubated with primary antibody overnight at 4uC, washed in
PBS three times and incubated with fluorescently labeled
secondary antibody for 1 h at room temperature. The primary
antibodies used were mouse monoclonal anti-IL3RA (Santa Cruz,
1:100), rabbit polyclonal anti-IL3RA (Santa Cruz, 1:100), rabbit
polyclonal anti-IL3RB (Santa Cruz, 1:100), goat polyclonal anti-
IL3 (Santa Cruz, 1:100), rabbit monoclonal anti-b-III tubulin
(Tuj1) (Covance, 1:1000), mouse anti-NeuN (Chemicon, 1:300),
goat anti-SOX2 (Santa Cruz, 1:500), rabbit anti-Nestin (Abcam,
1:300), rabbit anti-TBR2 (Millipore, 1:200), rabbit anti-pH3
(Santa Cruz, 1:200), rabbit anti-PROX1 (Covance, 1:1000),
anti-estrogen receptor b (Santa Cruz, 1:100), rabbit anti-Ki67
(Novocastra, 1:1000), rabbit anti-GFAP (1:2000). Secondary
antibodies used were Alexa Fluor 488 (Invitrogen, 1:500), 546
(Invitrogen, 1:500) conjugated to donkey anti-mouse, rabbit or
anti-goat (Invitrogen). DNA was stained with 49,6-diamidino-2-
phenylindole (DAPI; Molecular Probes). Images were acquired
with a Zeiss laser confocal microscope and analysed with LSM 510
software (Carl Zeiss).
For neural progenitor’s culture, cerebral cortices from C57BL/6
mice embryos (E12.5–14.5) were dissected in HBSS solution
(Invitorgen), the meninges and other parts were removed under
dissecting microscope and only the cortices were retained. After
several washes with HBSS, the cortices were minced and
dissociated mechanically with trituration, filtered through a
70 mm cell strainer (BD Falcon). Then, the cells were culture in
neurobasal medium (Invitrogen) or DMEM/F12 (Millipore) with
different supplements and growth factors according to experimen-
tal requirements. For neurons culture, the procedures are same
with neural progenitors except the age of mice embryos (E14.5–
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For generation of neurospheres, the dissociated cells from
E14.5–17.5 mice cortices were cultured for 2–5d in the medium
containing neurobasal medium, B27 (Invotrogen) and N2 (In-
vitrogen) supplements, 100 U/mL penicillin, 100 mg/mL strepto-
mycin, FGF2 (10 ng/ml), and EGF (10 ng/ml). For monolayer
cultures, the generated neurospheres were collected and spun
down (200 g for 5 minutes), then triturated with a Pasteur pipette
to obtain single cells. The single cell suspensions were replated
onto poly-L-lysine (50 mg/ml)/laminin (10 mg/ml)–coated Lab-
Tek Chamber Slide (Thermo Scientific). Cells were treated with
different concentrations of recombinant mouse IL3 (10 and
100 ng/ml) and cultured in above medium. Untreated cells served
as control. After 24 or 48 hours culture, cells were fixed with 4%
PFA and subjected to mmunohistochemistry. Immunostaining and
morphometry were carried out to assess the numbers of
proliferating progenitors (Ki67 and pH3 positive). More than 4
random microscopic fields (206) were analyzed and about 8000
cells were counted for each condition.
Assessment of Differentiation Markers in vitro
To investigate whether IL3 can drive neural differentiation, we
used quantitative real-time PCR method described previously 
to evaluate the effects of IL3 on neural differentiation. First, neural
progenitors were cultured in neurobasal medium (containing B27
supplements, N2 supplements,10 ng/ml FGF2 and 10 ng/ml
EGF) for 2 days, then FGF2 and EGF was removed and replaced
by neurobasal medium with 1% serum (vol/vol), B27 supplements,
N2 supplements and IL3 (10, 100, and 200 ng/ml). Four days
after addition of recombinant mouse IL3 (Invitrogen), cells were
harvested for the RNA extraction. Untreated cells served as
control. RNA was isolated using Trizol reagent (Invitrogen)
according to the manufacturer’s instructions and treated with
DNase I (Fermentas). cDNA was synthesized from 3 mg total RNA
using oligo-dT primers and Superscript III Reverse Transcriptase
(Invitrogen). Quantitative PCR was performed by using the Bio-
Rad iCycler & iQ Real-Time PCR Systems. The following primer
TACTTATCCTT; mouse Nestin-F,
GCCTCAGACATAGGTGGGATG; mouse b-III-tub-F, TA-
CCCTGGCTCGTGTGGATTT, mouse GFAP-R: GACCGA-
GTCCCTGGCCGTGTGTAAG, mouse ENO2-R: CATCCC-
GAAAGCTCTCAGC; mouse nestin-F: CCCTGAAGTCGAG-
GAGCTG, mouse nestin-R: CTGCTGCACCTCTAAGCGA;
mouse PLP1-F: TGAGCGCAACGGTAACAGG, mouse PLP1-
R: CCCACAAACTTGTCGGGATG. The iCycler PCR analysis
was performed using the SYBR Green master mix, according to
the manufacturer’s recommendations (BioRad). The specificity of
product was ensured by melting curve analysis and agrose gel
electrophoresis. cDNA content of samples was normalized to the
expression of GAPDH.
Cell Viability Assays
To investigate whether IL3 has protective or trophic effects on
neural progenitor’s survival, cerebral cortices from E12.5 or E13.5
mice embryos were dissociated and the isolated cells (56104) were
plated onto poly-L-lysine coated 96-well plate (Corning). The
survival of neural progenitors was investigated by three culture
conditions. Firstly, we studied the neurotrophic effects of IL3 on
neural progenitors through culturing the progenitors in the
absence of any factor (with neurobasal medium only) and in the
presence of IL3 (0.02–50 ng/ml). Recombinant mouse IL3 was
added into the medium after the culture initiated two hours. After
36 hours incubation, cell viability was determined by measurement
of cellular ATP levels (CellTiter-Glo Luminescent Cell Viability
Assay, Promega). Secondly, neural progenitors were first cultured
in neurobasal medium supplemented with B27 and Glutamax
(Invitrogen). On day 2 of culture, the medium was replaced with
serum-free neurobasal medium containing N2 supplement and
different concentrations of mouse IL3 (0.1–20 ng/ml). The
cultures were maintained for 3 days and cell viability was
measured. Thirdly, neural progenitors were first cultured in
neurobasal medium supplemented with B27 and Glutamax. On
day 2 of culture, IL3 was added into the medium and the cultures
were maintained for 3 days and cell viability was measured. For
studying the effects of IL3 on neurons, cerebral cortices from
E16.5–E18.5 mice embryos were dissociated and cultured in
DMEM/F12 medium with 5% FBS. On day 2 of culture, the
medium was replaced with serum-free DMEM/F12 containing
N2 supplement and different concentrations of IL3 (0.1–20 ng/
ml). The cultures were maintained for 2 or 3 days and cell viability
was determined as described above.
Neural progenitors from E13.5 mice were first cultured in
neurobasal medium under proliferating condition (containing B27
supplement, Glutamax, 10 ng/ml FGF2 and EGF) for one week.
To exclude the interference of other factors, the supplements,
FGF2 and EGF were removed from the medium for about 16
hours prior to IL3 treatment (3 ng/ml). Proteins from MEG01
cells (IL3 treated) and neural progenitors were homogenized in
RIPA lyses buffer (Cell signaling) containing a cocktail of protease
inhibitor (Sigma Chemical, MO, USA) and phosphatase inhibitor
(Cell Signaling). Proteins were quantified by BCA method (Pierce).
Extracted protein (40 mg) was separated by SDS-polyacrylamide
gel electrophoresis and transferred to PVDF or Nitrocellulose
membrane by electrophoretic transfer. The membrane was
blocked, incubated with primary antibodies for overnight at 4uC,
washed three times with TBST, and then incubated with
secondary antibody for 1 hour at room temperature. Antibodies
used in western blot are as follows: Rabbit anti-phospho-AKT
(Thr308) (Cell Signaling), Rabbit anti-AKT1 (Cell Signaling),
Rabbit anti-phospho-JAK2 (Tyr 1007/1008) (Cell Signaling),
Rabbit anti-JAK2 (Cell Signaling), Rabbit anti-phospho-GSK3b
(Ser9) (Cell Signaling), Rabbit anti-phospho-ERK1/2 (Cell
Signaling), Rabbit anti-ERK1/2 (Cell Signaling), Rabbit anti-
GSK3b (BD), and Rabbit anti-actin (Abcam). Immunoreactivity
was detected with an enhanced chemiluminescence system (Pierce,
IL, USA) with colored markers (Fermentas) as molecular size
studies SNPs in Chinese (CHB) and Europeans (CEU). (a)
In screening sample (CHB), they are four haplotype blocks and all
the 7 highly significant association SNPs are located in block 2. (b)
In CEU, they are five haplotype blocks and the 7 highly linked
SNPs in CHB are disrupted. LD values (r2) for each pair of
markers were calculated by Haploview (v4.2). Haplotype blocks
were defined according to the criteria of Gabriel et al.
Linkage disequilibrium (LD) pattern of the
IL3 Contributes to Human Brain Development
PLOS ONE | www.plosone.org14 November 2012 | Volume 7 | Issue 11 | e50375
vertebrate. rs31480 (box in red) is locates 216 bp upstream of
the IL3 promoter, and only 10 bp downstream of the highly
conserved TATA binding site (box in blue). The C allele (ancestral
allele) is completely conserved in all of the listed species, implying
functional importance of rs31480. Note that rs31480 is lies in a
primates conserved region (up panel), also suggesting the
importance of rs31480 in primates. TSS, transcription start site.
The C allele of rs31480 is highly conserved in
CHO, SK-N-SH and COS-7 cell lines. Promoter activity of
the construct with T allele is significantly higher than C allele in
CHO (a) and COS-7 (b) cells. In SK-N-SH cells (c), the trend is
same as CHO and COS-7 though the differences were not
reached significant level. Values of relative luciferase activity are
expressed as mean 6 s.d. (results of a triplicate assay).#P=0.07,
**P,0.005 (Student’s t-test).
Impacts of rs31480 on promoter activity in
brain (E12.5). IL3RA is expressed in SOX2 positive cells in
Frontal cortex and hippocampus, two regions that associated with
higher level cognitive functions. Scale bar, 50 mm.
Expression of IL3RA in embryonic mouse
region of mouse brain. (a–f) Co-expression of IL3RA and
IL3RB in agranular retrosplenial cortex (RSA), barrel field of the
primary somatosensory cortex (S1BF). (g–l) Expression of IL3RA
was also found in perirhinal cortex (Prh), primary Motor Cortex
(M1) and secondary Motor Cortex (M2). Note that IL3RA positive
cells were also expressed Tbr2 weakly, indicating they were not
mature neurons. Scale bar, 25 mm.
IL3RA is mainly expressed in the neocortex
campus. (a–c) Co-expression of IL3RA and IL3RB in CA3
region of hippocampus. (d–i) IL3RA is expressed in CA1 and CA3
of hippocampus, some of IL3RA positive cells were Tuj1 positive,
indicating they were mature neurons, whereas others were Tuj1
negative. Scale bar, 25 mm.
Expression of IL3RA and IL3RB in hippo-
Prox1 was used to label the granule cell layer (GCL) of the dentate
gyrus, note IL3RA positive cells in hilus were prox1 negative.
Scale bar, 50 mm.
IL3RA is expressed in hilus of dentate gyrus.
revealed co-localization of IL3RA and SOX2 in developing
mouse brain. (a–c) IL3RA expression cells were SOX2 positive in
lateral septal nucleus, dorsal part (LSD), indicating they were neural
progenitors. (d–i) In cortex, some IL3RA positive cells still express
SOX2, but for many of IL3RA positive cells, expression of SOX2
was down-regulated or turned-off, demonstrating they were
converted into immediate progenitors or neurons. Scale bar, 25 mm.
Double immunostaining of IL3RA and SOX2
brain. All IL3RA positive cells were also expression IL3RB. Scale
bar, 25 mm.
Co-expression of IL3RA and IL3RB in mouse
genitors at early embryonic stage. (a–c) At E12.5, IL3RA is
expressed in sox2 positive progenitors in frontal cortex. (b–e) Co-
IL3RA is mainly expressed in neural pro-
expression of IL3RA and nestin, a marker for neural progenitors.
(f–h) At E14.5, IL3RA is expressed in some sox2 positive
progenitors in cingulate cortex. Scale bar, 25 mm.
and Tuj1 in the developing mouse brain. At early stage of
brain development (From E14.5-P1), IL3RA is not expressed in
mature neurons (a–i). However, at P2 stage, a proportion of
IL3RA positive cells are mature neurons as revealed by co-
localization with tuj1 (j–l), a marker for mature neurons. Scale bar,
Double immunostaining analysis of IL3RA
cells (IPCs). IL3RA positive cells also express Tbr2 weakly,
indicating these cells were immediate progenitors. Scale bar,
IL3RA is expressed in immediate progenitor
(a–c) Co-immunofluorescence of IL3RB and NeuN revealed
expression of IL3RB in mature neurons. (d–f) IL3RB also
expressed in some glia cells (GFAP positive). Scale bar, 25 mm.
IL3RB is expressed in neurons and glia cells.
progenitors. Some IL3RA positive cells also expressed Ki67 and
pH3, marker for proliferation cells, indicating they were active
proliferation. Scale bar, 25 mm.
Expression of IL3RA in proliferating neural
IL3RB) in cultured neural progenitors and trophic
effects of IL-3 on neural progenitors and neurons. (a)
IL3 receptors (IL3RA and IL3RB) were expressed in cultured
neural progenitors (from E13.5 mice) as revealed by RT-PCR,
however, bIL3 was not detected. (b) When there were 5% FBS in
neurobasal medium, IL-3 has no trophic effects on neural
progenitors. Neural progenitors from E12.5 mice were cultured in
neurobasal medium (supplemented with 5% FBS and IL-3) for 36
hoursthen cellviabilitywas measured.(c) IL-3(1 ng/ml)significantly
promotes survival of neurons when there were no any factors in
neurobasal medium. Neurons from E17.5 mice were cultured in
neurobasal medium (supplemented with B27 and Glutamax) for 12
days, then B27 and Glutamax were removed from medium and
different concentrations of IL-3 were added. Cell viability was
determined after IL-3 treatment for 2 days. (d) However, when there
were B27 supplement in neurobasal medium, trophic effects of IL-3
on neurons was disappeared. Neurons from E18 mice were first
cultured in neurobasal medium (supplemented with B27 and
different concentrations of IL-3) for 24 hours, cell viability was then
measured. Y-axis, cell viability (normalized to control); X-axis,
concentration of IL-3 (ng/ml). Data are expressed as mean 6 s.e.m.
BCL-xL was not regulated by IL3 in neural progenitors.
Expression of IL3 receptors (IL3RA and
Neural progenitors were first cultured in neurobasal medium
under proliferation condition (containing 10 ng/ml FGF2 and
EGF), after 4 day’s culture, FGF2 and EGF were removed. Then
2% FBS and different concentrations of IL3 (10, 100, 200 ng/ml)
were added. The cultures were maintained for 4 days and RNA was
isolated for quantification. Relative gene expression was not
changed for all of the tested genes, indicating IL3 has no effect on
neural differentiation. Real-time PCR analysis of beta-III Tubulin
IL3 has no effects on neural differentiation.
IL3 Contributes to Human Brain Development
PLOS ONE | www.plosone.org15November 2012 | Volume 7 | Issue 11 | e50375
(Tubb3) (a) and Enolase 2 (ENO2) (b), two neuron specific markers,
GFAP (glia cells marker) (c), sox2 (neural stem cells marker) (d),
Tbr2 (immediate progenitors marker) (e), and DCX (new born
neurons marker) (f). None of these cell specific markers showed
significant change after different concentration IL-3 treatment.
confirmed that K562 cell line expressed IL3 and estrogen
receptors (ESR1 and ESR2) by RT-PCR (a). (b–c) Expression of
TFF1 and IL3 was not changed after treated by vehicle (DMSO)
for different times (0 h–24 h). TFF1, an estrogen response gene,
showed significantly elevated after treated by estrogen (10 nM) (d),
however, expression of IL-3 was not changed after estrogen
treatment (e), indicating IL-3 is not regulated by estrogen. Data
are expressed as mean 6 s.e.m. (three independent assays, each
containing 3 replicates).**P,0.01.
IL-3 is not regulated by estrogen. We first
and brain volume. Different genotypes at rs31480 (TT vs. CC)
influence IL-39s expression in both males and females. However,
since the estrogen receptors (ER) level is low in males, therefore,
even the signaling pathways mediated by IL-3 were different in
TT and CC carriers, the total activation level of ERs was not
significant changed. But in females, the activation level of ERs was
different between TT and CC carriers due to high level of ERs. In
addition, estrogen can further enhance ER activity in females. As a
result, signaling pathways mediated by ER were greatly activated
in TT carriers than in CC carriers, which may influence brain
development, eventually lead to difference of brain volume in TT
and CC carriers at rs31480.
Model for sex-specific association of IL-3
nificance in females.
Marker characteristics and association sig-
Core haplotype association analysis in fe-
nificance in males.
Marker characteristics and association sig-
with three different genotypes at each of the seven SNPs
Average cranial volumes of female individuals
Marker characteristics of AKT1 and associa-
We are grateful to all the voluntary donors of DNA samples in this study.
We thank Wei Zhang, Hui Zhang, Xiaoling Xie and Yan-jiao Li for their
Conceived and designed the experiments: XJL ML LG BS. Performed the
experiments: XJL ML LH MD. Analyzed the data: XJL ML KN QC
DRW AAV MR VSM AJS LS GF BF JCC XNC. Contributed reagents/
materials/analysis tools: XX JKW XBQ KX YMP XYC YL XDS. Wrote
the paper: XJL ML LG BS.
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