Embed Size (px)
Citation preview
Article
The Primate-Specific Gen
e TMEM14B Marks OuterRadial Glia Cells and Promotes Cortical Expansionand FoldingGraphical Abstract
Highlights
d TMEM14B is a primate-specific gene expressed in oRG cells
in human neocortex
d TMEM14B expression in mice induces IP and oRG expansion
and induces gyrification
d TMEM14B promotes phosphorylation and nuclear
translocation of IQGAP1
d TMEM14B/IQGAP1 regulate progenitor proliferation via
promoting G1/S transitions
Liu et al., 2017, Cell Stem Cell 21, 635–649November 2, 2017 ª 2017 Elsevier Inc.https://doi.org/10.1016/j.stem.2017.08.013
Authors
Jing Liu, Wensu Liu, Lu Yang, ...,
Jun Zhang, Fuchou Tang,
Xiaoqun Wang
[email protected] (J.Z.),[email protected] (F.T.),[email protected] (X.W.)
In Brief
Wang and colleagues show that the
primate-specific gene TMEM14B marks a
subset of human neural progenitors and
induces cortical folding, providing
insights into human brain evolution.
Expressing TMEM14B in the fetal mouse
brain increases proliferation of progenitor
subsets and cortical thickening through
nuclear shuttling of IQGAP1, which
promotes G1/S transitions.
Data Resources
GSE90734
Cell Stem Cell
Article
The Primate-Specific Gene TMEM14BMarks Outer Radial Glia Cells and PromotesCortical Expansion and FoldingJing Liu,1,4,6 Wensu Liu,1,4,6 Lu Yang,2,6 Qian Wu,1,6 Haofeng Zhang,3 Ai Fang,1,4 Long Li,1,4 Xiaohui Xu,3 Le Sun,1
Jun Zhang,3,* Fuchou Tang,2,* and Xiaoqun Wang1,4,5,7,*1State Key Laboratory of Brain and Cognitive Science, CAS Center for Excellence in Brain Science and Intelligence Technology (Shanghai),
Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China2BIOPIC, ICG, School of Life Sciences, Peking University, Beijing 100871, China3Obstetrics and Gynecology Medical Center of Severe Cardiovascular Disease of Beijing Anzhen Hospital, Capital Medical University,
Beijing 100029, China4University of Chinese Academy of Sciences, Beijing 100049, China5Beijing Institute for Brain Disorders, Beijing 100069, China6These authors contributed equally7Lead Contact
*Correspondence: [email protected] (J.Z.), [email protected] (F.T.), [email protected] (X.W.)https://doi.org/10.1016/j.stem.2017.08.013
SUMMARY
Human brain evolution is associated with expansionand folding of the neocortex. Increased diversity inneural progenitor (NP) populations (such as basallylocated radial glia [RG], which reside in an enlargedouter subventricular zone [OSVZ]) likely contributesto this evolutionary expansion, although their char-acteristics and relative contributions are onlypartially understood. Through single-cell transcrip-tional profiling of sorted human NP subpopulations,we identified the primate-specific TMEM14B geneas a marker of basal RG. Expression of TMEM14Bin embryonic NPs induces cortical thickening andgyrification in postnatal mice. This is accompaniedby SVZ expansion, the appearance of outer RG-likecells, and the proliferation of multiple NP subsets,with proportional increases in all cortical layers andnormal lamination. TMEM14B drives NP proliferationby increasing the phosphorylation and nuclear trans-location of IQGAP1, which in turn promotes G1/S cellcycle transitions. These data show that a single pri-mate-specific gene can drive neurodevelopmentalchanges that contribute to brain evolution.
INTRODUCTION
Expansion and gyrification in the neocortex are associated with
unique cognitive abilities that distinguish Homo sapiens from
other mammalian species (Dehay et al., 2015; Florio and Huttner,
2014; Garcıa-Moreno et al., 2012; Lui et al., 2011; Rakic, 1995,
2003). These evolutionary features reflect increasing cortical
neurons founded by various neural stem and progenitor cell sub-
types and their neurogenic divisions (Betizeau et al., 2013; Bor-
rell and Gotz, 2014; Fietz et al., 2010; Garcıa-Moreno et al., 2012;
Cell S
Hansen et al., 2010; Martınez-Cerdeno et al., 2012; Miyata et al.,
2004; Noctor et al., 2001; Reillo et al., 2011). The outer subven-
tricular zone (OSVZ) has recently been suggested to account for
evolutionary expansion of the human neocortex and humans’
unique cognitive abilities (Borrell and Gotz, 2014; Dehay et al.,
2015; Florio and Huttner, 2014; LaMonica et al., 2012; Lui
et al., 2011; Smart et al., 2002). The extraordinary diversity and
complexity of the OSVZ arise from various neural progenitor
(NP) subtypes, such as the outer radial glia (oRG or basal RG
[bRG]) and intermediate progenitor (IP) cells (Fietz et al., 2010;
Hansen et al., 2010; Reillo et al., 2011; Wang et al., 2011). De-
fects in OSVZ progenitors likely underlie some neurodevelop-
mental disorders affecting cerebral cortex structure and func-
tion. However, the cellular and molecular mechanisms
regulating the development of oRG cells and the OSVZ, as well
as their potential capacity for driving neocortical formation and
folding, are not fully studied.
To understand molecular aspects of oRG cells, various tran-
scriptional profiling strategies have been used to identify genes
uniquely expressed in oRG cells, such as transcriptome analyses
of microdissected VZ and SVZ tissues from rodents, primates,
and humans (Fietz et al., 2012; Hawrylycz et al., 2012; Johnson
et al., 2009, 2015; Kang et al., 2011; Lui et al., 2014; Miller
et al., 2014; Pollen et al., 2014, 2015; Stahl et al., 2013). Compar-
isons between purified cell populations isolated with specific
surface molecular markers and bioinformatic analyses using sin-
gle-cell in silico assays have provided additional insightful mo-
lecular information about oRG cells (Florio et al., 2015; Johnson
et al., 2009, 2015; Pollen et al., 2015; Thomsen et al., 2016).
However, profiling oRG-specific gene expression is challenging
due to the threshold of high-throughput single-cell RNA
sequencing (RNA-seq) and difficulties in isolating bona fide
oRG cells.
Here we used flow cytometry with cell-surface markers to
isolate individual ventricular RG (vRG), oRG, and IP cells from
the VZ and the OSVZ and maturating neurons from the cortical
plate (CP) of developing human fetal brains. Then we identified
specific markers for these populations by single-cell RNA-seq.
tem Cell 21, 635–649, November 2, 2017 ª 2017 Elsevier Inc. 635
Of those, transmembrane protein 14B (TMEM14B) was selected
for further studies. The TMEM14B sequence evolutionarily exists
only in primates, including humans. Our data showed that
TMEM14B was specifically localized in the OSVZ in the human
fetal neocortex. Ectopic expression of TMEM14B in embryonic
NP cells during mouse development increased the proportion
of IP and oRG cells and their subventricular dispersion, resulting
in gyrification of mouse brains. In addition, our results revealed
that TMEM14B interacts with and contributes to the phosphory-
lation of IQGAP1, resulting in its nuclear translocation and activa-
tion of cell proliferation. These observations suggest that
TMEM14B specifically marks oRG and contributes to cortical
expansion and folding in the developing human neocortex.
RESULTS
Distinct Molecular Markers for Different Subtype CellsTo create detailed transcriptome datasets of vRG, oRG, and IP
cells and neurons, three human fetal brains from 16 to 17 weeks
gestation (GW16–17) were carefully microdissected to isolate
VZ, OSVZ, and CP tissues, which were then dissociated to sin-
gle-cell suspensions for flow cytometry (Figure 1A and S1A).
CD15 and CD184 surface markers were used to purify NPs,
and we removed neuroblasts, mature neurons, and oligoden-
drocytes by CD56 and CD140a/PDGFaR (Pruszak et al.,
2009; Wang et al., 2013; Yuan et al., 2011). CD15+/CD184+/
CD56�/CD140a� NPs from the VZ and OSVZ and CD56+/
CD184� neurons from the CP were obtained (Figure 1B). In to-
tal, �400 single cells were collected from these sorted
populations.
To validate the molecular identity of these vRG, oRG, IP cells,
and neurons, we used real-time qPCR to examine the expression
of three marker genes, TBR2, SOX2, and TUBB3. GAPDH was
used as an internal control (Figure 1C). As internal quality con-
trols and technical replicates, for some of the single-cell samples
we divided the single-cell lysate into two aliquots to generate
separate libraries for deep sequencing (Tang et al., 2009,
2011). We removed genes with low expression in all cell sub-
types in the library and calculated the Pearson’s correlation
value for all genes to evaluate data quality (Figure 1D).We subse-
quently assessed differential expression of the remaining genes
(p < 0.05 in any two cell types) across the four cell types. A total of
562 differentially expressed genes were identified, and mRNA
expression patterns reflecting the specificity to cell subtype
were analyzed (Figure 1E). The vRG and oRG cells were charac-
terized by high expression of radial glial cell markers, including
SOX2, PAX6, and recently identified human oRG marker HOPX
(Figure 1E, samples were arranged by Pearson’s correlation co-
efficient), while CCNB1 and EOMES were found mainly ex-
pressed in IP cells (Figure 1E). The mRNA levels of known
neuronal markers, such as NEUROD6 and STMN2, were high
in neurons (Figure 1E).
We next examined genes highly specific to each cell type by
comparing the expression of a gene in one cell type with the
other three cell types. Those genes with p < 0.05 in all compar-
isons were selected, and they all showed at least 2-fold enrich-
ment in expression levels in one specific cell type over any other.
Nine oRG cell-, 17 vRG cell-, two IP cell-, and 21 neuron-specific
candidate genes were identified among these 562 genes (Fig-
636 Cell Stem Cell 21, 635–649, November 2, 2017
ure S1B). Then we analyzed the remaining 513 genes with
Venn analysis contrasting neuron (Figure S1C), principal-
component analysis (PCA) (Figure S1D), and weighted gene
co-expression network analysis (WGCNA) (Figure S1E) (Lang-
felder and Horvath, 2008). The combined method effectively
simulated both linear and non-linear information in gene expres-
sion and, thus, significantly reduced the rate of false discovery
(Figure S1F).
TMEM14B Is an oRG-Specific Molecular MarkerTMEM14B,KCNK10,DAG1, andHP1BP3, identified through our
analysis as oRG-specific genes, have not previously been stud-
ied in this context (Figure 2A), while PTPRZ1, HOPX, TNC,
FAM107A, and MOXD1 were previously reported as oRG
markers (Pollen et al., 2015; Thomsen et al., 2016). We then per-
formed in situ hybridization in GW16–17 cortical tissue sections
to further investigate these four oRG candidate genes. We found
that TMEM14B, KCNK10, DAG1, and HP1BP3 were mainly ex-
pressed in the OSVZ of the fetal brains (Figure 2B), consistent
with our single-cell RNA-seq data. Thus, these genesmay be po-
tential markers for oRG cells. TMEM14B encodes a membrane
protein and arose in the primate lineage, including Old World
and New World monkeys and apes according to phylogenetic
analysis, suggesting that this gene might contribute to OSVZ
expansion in primates or human beings (Figure S2A). Interest-
ingly, the TMEM14B sequences among apes and Old World
monkeys were more conserved than in New World monkeys
(Figure S2B).
To explore the function of TMEM14B in corticogenesis,
TMEM14B was fused with EGFP and 33 FLAG tag and was ex-
pressed in the lateral ventricle of mouse neocortex by in utero
electroporation on embryonic day 13.5 (E13.5). Although surface
perimeter and area measured in intact half brains were not
different (Figures S2C and S2D), TMEM14B expression in the lis-
sencephalic mouse brain resulted in regional thickening of
neocortex and gyrus-like structures after 2 days (Figure 2C, as-
terisks and arrows; Figures S2E–S2H). PAX6+/EGFP+ NP cells
increased, leading to an expanded NP-enriched area above
the traditional SVZ in mice at E15.5 (Figures 2C and 2D) that
resembled an expanded SVZ. There were increased PAX6+/
EGFP+ cells with basal, but no apical, processes in the expanded
SVZ, typical of oRG cells (Figures 2C and 2E). Consistent with the
increase in PAX6+ cells, SOX2+ cells increased similarly 2 days
after TMEM14B electroporation (Figures 2F–2H). Interestingly,
we found that expression of TMEM14B also significantly
elevated the percentage of TBR2+ IP cells (Figures 2F and 2I).
Additionally, we observed a few oRG-like cells (3.1%) were
TBR2+ (Figure 2J), suggesting TMEM14B may increase different
progenitor subtypes.
We then looked at the effects of TMEM14B expression on pro-
liferation. Following a 2-hr bromodeoxyuridine (BrdU) pulse and
immunolabeling with Ki67, we found that expression of
TMEM14B increased cellular proliferation with approximately
21.2% more cells entering S phase (Figures 2K–2M). We
observed enlarged brains, with a corresponding increase in
both cortical thickness and surface area at 5 days post-electro-
poration (Figures S2I–S2O). TMEM14B-expressing cells were
mostly PAX6+ progenitors in the expanded VZ/SVZ (Figures
S2K and S2L), suggesting that TMEM14B overexpression
Figure 1. Single-Cell Transcriptome Profiling in Human Fetal Brain
(A) Schematic diagram for isolation single neural progenitors from the VZ, OSVZ, and neurons from CP in human fetal brain.
(B) Representative fluorescence-activated cell sorting (FACS) plots showing gating strategy sorting of vRG, oRG, and IP cells and neurons.
(C) Different cell subtype identification by real-time PCR.
(D) Pearson’s correlation map of all expressed genes based on value of all samples.
(E) Differential expression genes with known markers in different subtypes of cells. See also Figure S1.
increases the proportion of PAX6+ progenitors and thus pro-
motes the potential for increased generation of TUJ1+ neurons
(Figures S2K and S2M). Most TMEM14B+ neurons were
SATB2+ (Figures S2N and S2O), indicating that TMEM14B pref-
erentially induces upper layer neuron formation. Thus, high levels
of TMEM14B promote the formation of an expanded SVZ with
proliferation of both oRG-like and IP cells in the developing
mouse neocortex.
Cell Stem Cell 21, 635–649, November 2, 2017 637
Figure 2. Overexpression of Primate-Specific Gene TMEM14B Promotes Neural Progenitor Cell Generation in Mouse
(A) Relative expression of the 16 most oRG-specific genes in each cell subtype.
(B) In situ hybridization for representative genes belonging to the oRG cluster, including TMEM14B, KCNK10, DAG1, and HP1BP3, in GW16 human cortical
sections. Inset shows higher magnification of positively stained region. Scale bar, 200 mm.
(legend continued on next page)
638 Cell Stem Cell 21, 635–649, November 2, 2017
TMEM14B Expands the SVZ and Causes Brain FoldingIn VivoTo further examine TMEM14B function in cerebral cortex devel-
opment, we generated conditional knockin mice in which human
TMEM14B expression is driven by Nestin-Cre (Figure S3A; here-
after referred to as TMEM14BCKI mouse). Nestin-dependent
expression of TMEM14B was examined in the mouse neocortex
at E15.5 (Figure S3B). During mouse development, oRG cells are
rare and there is no distinct OSVZ (Wang et al., 2011). However,
conditional expression of TMEM14B in mouse NPs resulted in
thickening of the neocortex with increased PAX6+ progenitors
(Figures 3A–3C, S3C, and S3D), consistent with transient
expression. Although the PAX6+ progenitor population in
TMEM14BCKI mice was elevated from 15.1% to 29.5% in the
expanded SVZ, no differences were observed in the VZ/SVZ
(Figures 3B, 3D, and S3C). We also observed that the PAX6+
area expanded radially toward the intermediate zone (IZ), which
could be considered as an OSVZ-like region in the developing
TMEM14BCKI mouse neocortex, where we observed a signifi-
cant increase in PAX6+ oRG cells visualized by phospho-vimen-
tin staining (Figures 3B, 3E, and S3E).
HOPX, a recently published human oRG marker (Pollen et al.,
2015; Thomsen et al., 2016), was expressed broadly in the VZ
and SVZ in control mice neocortex (Figure 3F). In the
TMEM14BCKI mouse neocortex, HOPX-expressing cells were
more numerous andextended into the expandedSVZ (Figure 3F).
To further confirm the identities of these cells, we examined the
shape of HOPX+ cells by phospho-vimentin staining. As ex-
pected, some HOPX+ cells in the expanded SVZ exhibited
oRG cell-like morphology in the developing TMEM14BCKI mouse
neocortex (Figure 3G), similar to human oRG cells in the OSVZ at
GW16 neocortex (Figure S3F). To further investigate the molec-
ular characteristics of TMEM14B+ oRG cells, we performed a
combination of in situ hybridization and immunostaining on
GW16–17 human cortices, and we found 74.9% of the
TMEM14B+ cells in the OSVZ expressed HOPX (Figures S3G
and S3H). In addition, SOX2+ and TBR2+ progenitors were
increased in the expanded SVZ of E15.5 mice (Figures 3H–3J),
reminiscent of the expanded diffuse TBR2+ band described in
the OSVZ of ferrets, macaques, and humans (Bayatti et al.,
2008; Martınez-Cerdeno et al., 2012; Reillo and Borrell, 2012).
We noticed that PAX6+, SOX2+, and TBR2+ cells in the VZ/
SVZ/expanded SVZ and total protein expression in the
(C) Overexpression of TMEM14B promotes progenitor generation and gyrus-like s
structures, respectively. Control and TMEM14B images (left panel) are composi
images show that the PAX6+ cell (arrows) has a long basal process (arrowhe
20 mm (right).
(D) Quantification for the percentage of PAX6+ cells among EGFP+ cells (n = 3 ea
(E) Quantification for the percentage of PAX6+ oRG cells among EGFP+ cells (n =
(F) Overexpression of TMEM14B expands oRG and IP cells. Higher magnifica
(arrowheads), but no apical process. Scale bars, 50 mm (left) and 20 mm (right).
(G) Quantification for the percentage of SOX2+ cells among EGFP+ cells (n = 6 e
(H) Quantification for the percentage of SOX2+ TBR2� oRG cells among EGFP+
(I) Quantification for the percentage of TBR2+ cells among EGFP+ cells (n = 6 ea
(J) Quantification for the percentage of TBR2+ cells with basal process among to
(K) The experimental scheme and E15.5 cortices labeled for Ki67 and BrdU. Sca
(L) Quantification for the percentage of BrdU+ cells among EGFP+ cells (n = 4 ea
(M) Quantification for the percentage of Ki67+ cells among EGFP+ cells (n = 4 ea
Data are presented as mean ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p
neocortex were increased in the developing TMEM14BCKI mice
(Figures S3I–S3L), suggesting an overall increase of NP popula-
tion. In summary, PAX6+/pVIM+ progenitors in the expanded
SVZ, HOPX+ progenitors, and TBR2+ IP cells were significantly
enriched in TMEM14BCKI mice, indicating that TMEM14B
expression in mice primarily increased oRG and IP cells during
cortical development.
We next explored the basis of progenitor cell number expan-
sion. BrdU-pulsing analysis and Ki67/PH3 staining showed
more actively cycling and mitotic cells (Figures 3K–3M, S3M,
and S3N), suggesting that TMEM14B increases proliferative po-
tential of NPs. However, the increase in proliferating cells was
significant in the SVZ and the expanded SVZ, but not in the VZ
(Figures 3K and 3M). We then examined cell cycle re-entry by
performing dual-pulse labeling. An EdU pulse was administered
to pregnant mice 8 hr after E13.5, and 14 hr later a 2-hr BrdU
pulse was given (Figure 3N). We observed 85.4% EdU+ cells in
TMEM14BCKI mice re-entered S phase within 16 hr compared
to controls, suggesting that TMEM14B may shorten the cell cy-
cle of NPs. To further investigate the mechanism by which
TMEM14B induced oRG generation and since oRG cells are
known to arise from vRG cells (Wang et al., 2011), we investi-
gated the cleavage angles of vRG cells (Figure S3O). Previous re-
ports suggested oblique divisions of vRGprimarily generate oRG
cells in mice (LaMonica et al., 2013; Shitamukai et al., 2011). We
observed that oblique divisions increased from 13.7% to 27.5%
in TMEM14B-expressing vRG cells compared to controls, sug-
gesting that TMEM14B may modulate cleavage angle of vRG
cells to promote oRG production (Figure 3O).
We next investigated whether TMEM14B expression can
induce cortical gyrification. We found conditional expression of
TMEM14B in NPs consistently led to the development of
enlarged brains with featured sulci and gyri in newborn mice
(post-natal day [P]0) (Figures 4A and 4B). To test whether in-
creases in oRG and IP cells in the gyrated brains of P0
TMEM14BCKImice contribute to upper layer or deep layer forma-
tion, we examined the structure of sulci and gyri by staining for
SATB2 (mainly expressed in layer II/III) and CUX1 (layer II/III).
We observed that the gyrated CP in TMEM14BCKI animals was
enlarged when compared with the smooth control neocortex
that exhibited proper cortical lamination (Figures 4C, 4D, and
S4A). The average thicknesses of the neocortex and SATB2+ up-
per layer neurons were elevated (Figures 4E–4G) in TMEM14BCKI
tructure formation in mouse. Asterisk and arrowhead indicate gyrus and sulcus
te images of three and two separate fields, respectively. Higher magnification
ads), but no apical process. Scale bars, 200 mm (left), 50 mm (middle), and
ch group).
3 each group).
tion images show the SOX2+ TBR2� cell (arrow) has a long basal process
ach group).
cells (n = 6 each group).
ch group).
tal EGFP+ cells (n = 3 each group).
le bars, 50 mm.
ch group).
ch group).
< 0.0001, Student’s t test). See also Figure S2.
Cell Stem Cell 21, 635–649, November 2, 2017 639
Figure 3. Neural Progenitor Cell Increases in TMEM14B-Conditioning Knockin Mouse Cortex
(A) Staining for PAX6 in E15.5 NesCre and TMEM14BCKI mice. Scale bar, 200 mm.
(B) Staining for PAX6 and pospho-VIMENTIN in E15.5 NesCre and TMEM14BCKI mice. Note the cells double positive for p-VIM and PAX6 (arrows) with the basal
process (arrowheads) in expanded SVZ. Scale bars, 100 mm (left) and 20 mm (right).
(C) The thickness of the cortical wall of TMEM14BCKI increases during early corticogenesis (nNesCre = 3 and nTMEM14BCKI = 4).
(D) Quantification for the percentage of PAX6+ cells in the expanded SVZ (nNesCre = 3 and nTMEM14BCKI = 4).
(legend continued on next page)
640 Cell Stem Cell 21, 635–649, November 2, 2017
animals. To further explore the effects of TMEM14B on deep
layer formation, we used CTIP2 (layer V) and FOXP2 (layer VI)
as markers, and we observed an increase in CTIP2+ and
FOXP2+ deep layer neurons following TMEM14B expression
(Figures 4D, 4F, 4H–4J, and S4B). Thus, TMEM14B expression
in mouse CNS leads to expansion of the SVZ and the appear-
ance and increase in oRG and IP cells, thus ultimately increasing
both upper and deep layer neurons to form a folded cortex with
proper lamination.
Identification of TMEM14B-Interacting ProteinsTo identify TMEM14B-interacting proteins, we transfected either
a FLAG-tagged-TMEM14B expression construct or an empty
FLAG-tag vector into HEK293T cells (Figure S5A). The cells
were then lysed, immunoprecipitated, and analyzed by SDS-
PAGE (Figure S5B). TMEM14B-specific bands were excised
from the gels (Figure 5A, arrowheads) and then analyzed by
liquid chromatography-tandem mass spectrometry (LC-MS/
MS). We identified 283 proteins (Table S1), and gene ontology
analysis indicated that the proteins had roles in cell cycle regula-
tion and development (Figure 5B). Among the putative
TMEM14B-interacting proteins, we found Ras GTPase-acti-
vating-like protein IQGAP1 (Figure S5C). The interaction be-
tween TMEM14B and IQGAP1 was verified by co-immunopre-
cipitation (coIP) and western blot analyses (Figure 5C).
Next, we asked whether overexpression of IQGAP1 could
phenocopy the effects of TMEM14B expression. To test this,
human IQGAP1 was expressed in E13.5 mouse neocortex by
in utero electroporation. We observed concentrated oRG-like
SOX2+ cells in the SVZ and expanded SVZ region (Figure 5D).
IQGAP1 expression induced an increase in the number of
SOX2+ progenitors, which displayed oRG-like morphologies
with cortical extensions (Figures 5D–5F, S5D, and S5E). We
also noted increases in TBR2+ IP cells and TBR2+ oRG-like
cells (Figures S5F–S5H). oRG cells undergoing self-renewal
were also observed (Figure 5D), suggesting that IQGAP1 may
promote progenitor proliferation similarly to TMEM14B. Consis-
tently, cell cycle analyses showed fewer IQGAP1+ cells in
G0/G1 phase and more in S phase, suggesting that IQGAP1
promotes G1/S transition (Figures 5G–5I). This is consistent
with the observed dramatic increase in S-phase cells in the
developing neocortex of the TMEM14BCKI mice (Figures 3N
and S3M).
(E) Quantification for the percentage of PAX6+ p-VIM+ cells in the expanded SVZ
(F) Immunostaining andquantificationofHOPXcells (arrows) in theexpandedVZof
Scale bars, 50 mm (left) and 10 mm (right).
(G) Staining for HOPX and phospho-VIMENTIN in E15.5 NesCre and TMEM14BC
basal process (arrowheads) in the expanded SVZ. Scale bars, 50 mm (left) and 2
(H) Staining for TBR2 and SOX2 in E15.5 NesCre and TMEM14BCKI mice. Scale
(I) Quantification for the percentage of SOX2+ cells in the expanded SVZ (nNesCre
(J) Quantification for the percentage of TBR2+ cells in the expanded SVZ (nNesCr
(K) Staining for Ki67 and PH3 in E15.5 NesCre and TMEM14BCKI mice. Scale ba
(L) Quantification for the percentage of Ki67+ cells in the VZ/SVZ and expanded
(M) Quantification for the percentage of PH3+ cells in the VZ and SVZ/expande
nNesCre = 3 and nTMEM14BCKI = 4.
(N) EdU/BrdU double pulse in E13.5 NesCre and TMEM14BCKI mice. Note the c
20 mm (right). Quantification for the percentage of EdU+ BrdU+ cells among EdU
(O) Quantification for the plane of division at ventricle surface (**p (0–30) < 0.005,
Data are presented as mean ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p
We then performed loss-of-function analyses of IQGAP1 by in
utero electroporation of a specific short hairpin RNA to suppress
its expression (shIQGAP1; Figure S6A). IQGAP1 short hairpin
RNA (shRNA), but not scrambled shRNA, disrupted increases
in SOX2+ RG and TBR2+ IP cell numbers induced by transient
expression of TMEM14B (Figures 6A–6D and S6B). Knockdown
of IQGAP1 also reduced RG and IP cells in TMEM14BCKI devel-
oping neocortex (Figures 6E–6H). In addition, we noted that
TMEM14B expression in HEK293T cells reduced G0/G1 cells
and proportionally increased S-phase cells (Figures 6I, 6J, and
6L). This could be partially rescued by IQGAP1 knockdown (Fig-
ures 6I–6L), providing further evidence that TMEM14B interacts
with IQGAP1 and modulates the cell cycle.
TMEM14B Regulates IQGAP1 Phosphorylation andNuclear TranslocationIQGAP1 is concerved across different species and its main
localization is at plasma membrane and cytoplasm (Figures S7A
and S7B).Given IQGAP1 is known to accumulate in the nucleus
at the G1/S phase of the cell cycle to regulate DNA replication
and transcription (Johnson et al., 2011), we asked whether the
subcellular localization of IQGAP1 in cells depends on TMEM14B
expression. IQGAP1-tdTomato and TMEM14B-EGFP were
co-transfected into NIH 3T3 cells. We observed an increase in
IQGAP1 nuclear localization when co-expressed with TMEM14B
compared to controls (Figures 7A and 7B). Biochemical fraction-
ation of HEK293Tcells confirmed thatmore endogenous IQGAP1
accumulated in nuclei after TMEM14B expression (Figure 7C).
Since phosphorylated IQGAP1 has been reported to regulate
cell proliferation (Wanget al., 2009),we askedwhether TMEM14B
could affect phosphorylation of endogenous IQGAP1. HEK293T
cells were transfected with TMEM14B or empty vector and
analyzed by immunoblotting. The upper band detected by
IQGAP1antibodieswasalso recognizedbyapSer (PKC-substrate)
antibody (Figure 7D), suggesting it represents a phosphorylated
form of endogenous IQGAP1. Together these results suggest
that TMEM14B increases IQGAP1 phosphorylation and induces
its nuclear translocation, thereby promoting G1/S transition.
DISCUSSION
The development of single-cell transcriptome analyses can
enhance the identification of cell-type-specific markers, which
(nNesCre = 3 and nTMEM14BCKI = 4).
E15.5NesCreandTMEM14BCKImicecortices (nNesCre =3andnTMEM14BCKI = 4).
KI mice. Note the cells double positive for HOPX and p-VIM (arrows) with the
0 mm (right).
bar, 50 mm.
= 3 and nTMEM14BCKI = 4).e = 3 and nTMEM14BCKI = 4).
r, 100 mm.
SVZ (nNesCre = 3 and nTMEM14BCKI = 4).
d SVZ. For VZ, nNesCre = 3 and nTMEM14BCKI = 4; for SVZ and expanded SVZ,
ells only positive for EdU are indicated by arrows. Scale bars, 50 mm (left) and+ cells (nNesCre = 4 and nTMEM14BCKI = 3).
**p (30–60) < 0.005, and *p (60–90) < 0.05; nNesCre = 3 and nTMEM14BCKI = 4).
< 0.0001, Student’s t test). See also Figure S3.
Cell Stem Cell 21, 635–649, November 2, 2017 641
(legend on next page)
642 Cell Stem Cell 21, 635–649, November 2, 2017
could be useful for understanding the molecular changes asso-
ciated with increased numbers, density, and complexity of
neuronal subtypes and progenitor lineages in the human brain.
In this study, we identified four oRG-specific markers, including
the primate-specific gene TMEM14B. Genetic expression of
TMEM14B in mice caused an increase in all NP subtypes, espe-
cially IP cells, and an expansion of the VZ/SVZ, leading to
increased generation of upper and deep layer neurons and
cortical thickening and folding. In addition, our data revealed
that TMEM14B associates with IQGAP1 and facilitated its phos-
phorylation, thereby promoting its nuclear translocation to drive
G1/S transition (Figure 7E). Thus, TMEM14B-dependent control
of proliferation may represent an important mechanism that con-
tributes to neocortical thickening with proper lamination, surface
area expansion, and folding. Furthermore, the TMEM14BCKI
mice provide a model for studying cellular and molecular mech-
anisms underlying these processes.
The mammalian cerebral cortex varies tremendously across
species in size and shape, from the lissencephalic mouse cortex
to the large and complicated gyrencephalic primate cortex (Lui
et al., 2011; Rakic, 1995; Wilsch-Br€auninger et al., 2016). Evolu-
tionary expansion of cortical thickness and surface area, as well
as increased diversity of neural subtypes, particularly in human,
reflects the importance of progenitor subtypes and their prolifer-
ative potential (Geschwind and Rakic, 2013). The radial unit hy-
pothesis is an important model of cortical expansion, which sug-
gests that radial size (thickness) is determined by the number of
neurons generated per radial unit (ontogenetic column) whereas
tangential size (surface area) depends on the number of radial
units established by the vRG (Malatesta et al., 2000; Miyata
et al., 2001; Noctor et al., 2001; Rakic, 1995). Evidence indicates
that over-proliferation of vRG, caused either by constitutive
expression of b-catenin (Chenn and Walsh, 2002, 2003) or by
deletion of caspase-9 (Kuida et al., 1998), leads to cortical gyri-
fication with thinner and disrupted lamination in mice. Those re-
sults suggest that increased vRG numbers are necessary, but
not sufficient, for cortical thickening and folding.
With the discovery of IP cells/basal progenitors and neuro-
genic divisions outside of the VZ, it was proposed that IP cells
undergo symmetric division to amplify neuronal production and
are likely important in determining brain size. In mice, however,
IP cells are derived from RG with limited proliferative capacity
(Englund et al., 2005; Kawaguchi et al., 2008; Kowalczyk et al.,
2009; Martınez-Cerdeno et al., 2012; Noctor et al., 2004; Pon-
Figure 4. TMEM14B Expression in Mouse Progenitor Cells Induces Co
(A) Dorsal view of NesCre and TMEM14BCKI brains at P0. Scale bars, 1 mm.
(B) Quantification for the volumes of P0 cortices due to augmented anterior-po
percentage control (n = 6).
(C) Staining for CTIP2 in P0 TMEM14BCKI sagittal brain section. Asterisks and a
separate fields. Scale bar, 500 mm.
(D) Layer-specific markers CTIP2 and SATB2 in P0 NesCre and TMEM14BCKI g
fields. Images 1 and 2 are high-magnification images of (C). Scale bar, 100 mm.
(E) Quantification for the thickness of the cortical wall at P0 (n = 5 each group).
(F) Layer-specific markers CTIP2 and SATB2 in P0 NesCre and TMEM14BCKI ne
Each panel is a composite image of three separate fields. Scale bar, 100 mm.
(G) Quantification for the SATB2+ cells radially at P0 (nNesCre = 4 and nTMEM14BCK
(H) Quantification for the CTIP2+ cells radially at P0 (nNesCre = 4 and nTMEM14BCKI
(I) Layer-specific marker FOXP2 in P0 NesCre and TMEM14BCKI neocortex. Sca
(J) Quantification for the FOXP2+ cells radially at P0 (n = 3 each group).
Data are presented as mean ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p
tious et al., 2008). Previous experimental attempts to expand
basal progenitor pools via Cdk4 and Cyclin D1 overexpression
found that brain surface area cannot be expanded in lissence-
phalic mice, but, remarkably, neocortical folding can be
observed earlier in gyrencephalic ferrets (Nonaka-Kinoshita
et al., 2013). This suggests that an increase of basal progenitors
alone is not sufficient to explain gyrification and evolutionary
cortical expansion.
oRG cells, located in the OSVZ, display a greater capacity for
transit amplification in gyrencephalic animals as they typically
undergo multiple rounds of cell division before generating the
majority of upper layer neurons (Fietz et al., 2010; Hansen
et al., 2010; Lui et al., 2011; Reillo et al., 2011; Wilsch-Br€auninger
et al., 2016). However, recent observations in marmoset (lissen-
cephalic) and agouti (a rodent with moderate gyrencephalic cor-
tex) cortices indicate that oRG proliferation and OSVZ expansion
are present in diverse mammalian orders and may not correlate
with gyrencephaly (Garcıa-Moreno et al., 2012; Kelava et al.,
2012). We found that overexpression of TMEM14B increases
the number of TBR2+ IPcells, causes detachment and moderate
increases in the OSVZ HOPX+ cells, and increases both deep
layer CTIP2+ neurons and upper layer SATB2+ neurons. These
findings suggest that increases in all progenitor subtypes, espe-
cially IPs, drive cortical folding in the mouse (Figures 3F–3H).
Thus, TMEM14B-induced expansion of neocortical surface
area and folding are a consequence of increases in progenitor
subtypes, numbers, and proliferative potentials, consistent
with the radial unit hypothesis that predicts increases in prolifer-
ative zones and apical and bRG are the basis for the evolutionary
growth of the cortex (Geschwind and Rakic, 2013; Hevner and
Haydar, 2012; Molnar and Clowry, 2012).
Although we cannot conclude whether increased IP cells arise
from vRG, oRG, or self-division, our results suggest that the
transit amplification of multiple progenitors in a short period of
time is critical for brain enlargement and cortical folding. Further
progenitor subtype-specific labeling and lineage-tracing experi-
ments will provide deeper insights into the cellular mechanisms
underlying cortical expansion and folding. We also observed
expansion of upper and deep layer neurons (V and VI) upon over-
expression of TMEM14B, indicating that a larger cortex might be
determined early by neural stem cells in the VZ and that the
neocortex cannot expand in surface area and folding without
increased production of VZ/SVZ-dependent deep layer neurons
prior to OSVZ formation. Considering that increased neuronal
rtical Folding with Proper Cortical Lamination
sterior (AP), mediolateral (ML), perimeter, and area. Values are expressed as
rrows indicate gyrus and sulcus structures. This is a composite image of 21
yrus and sulcus structures. Each panel is a composite image of two separate
ocortex.
I = 6).
= 6).
le bar, 100 mm.
< 0.0001, Student’s t test). See also Figure S4.
Cell Stem Cell 21, 635–649, November 2, 2017 643
Figure 5. TMEM14B Regulates Cell Cycle by Associating with IQGAP1
(A) Sliver-stained SDS-PAGE of IP lysate. Black arrows, special expression proteins in TMEM14B complex; asterisks, bait proteins.
(B) Gene ontology (GO) analysis of proteins binding with TMEM14B.
(C) CoIP of TMEM14B and IQGAP1 in HEK293T cells. IP, immunoprecipitation; IB, immunoblotting.
(D) Overexpression hIQGAP1 in mice cortex induces oRG generation. Note the SOX2+ oRG-like cells (arrows) with long basal process (arrowheads), but no apical
process. Asterisk, dividing oRG cell. Scale bars, 50 mm (left) and 20 mm (right).
(E) Quantification for the percentage of SOX2+ cells among EGFP+ cells (nControl = 4 and nIQGAP1 = 5).
(F) Quantification for the percentage of SOX2+ TBR2� EGPF+ cells with basal process among EGFP+ cells (nControl = 4 and nIQGAP1 = 5).
(G and H) FACS analysis of HEK293T cells transfected with (G) PCMV-FLAG-EGFP and (H) PCMV-FLAG-hIQGAP1-EGFP (n = 3 for each group). Data are
presented as mean ± SEM.
(I) Quantification and comparison of data presented in (G) and (H)
Data are presented as mean ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001, Student’s t test). See also Figure S5.
diversity is another feature of primate cortices (Briggs et al.,
2002; Chenn and Walsh, 2002; Goto et al., 2013), whether
TMEM14B expression leads to the generation of more neuronal
subtypes or other non-neural cells needs future study.
TMEM14B interacts with and facilitates phosphorylation of
IQGAP1, which shuttles into the nucleus to promote the G1/S
transition. Previous studies indicate that shortening the cell cy-
cle by reducing G1 phase increases OSVZ progenitor prolifera-
tion and reduces their propensity for exiting the cell cycle,
644 Cell Stem Cell 21, 635–649, November 2, 2017
thereby resulting in rapid expansion of progenitor pools and ac-
celeration of neurogenesis in primates (Betizeau et al., 2013;
Lange et al., 2009; Nonaka-Kinoshita et al., 2013; Pilaz et al.,
2009). Consistently, we also observed that TMEM14B or
IQGAP1 expression promotes G1/S transition and shortens
the cell cycle, suggesting that TMEM14B/IQGAP1 may in-
crease the progenitor pool in mice in an analogous manner.
IQGAP1 is a key integrator of cellular signaling pathways that
regulate cell adhesion, migration, proliferation, and gene
Figure 6. IQGAP1 Is the Downstream Molecule of TMEM14B
(A and B) Knockdown of IQGAP1 rescues the neuronal progenitor increase induced by TMEM14B expression in mice. Co-expression of RFP-shIQGAP1 and
EGFP-TMEM14B by electroporation on E13.5 is shown. Sections of the E15.5 cerebral cortex are stained by (A) SOX2 and (B) TBR2. Scale bar, 50 mm.
(C) Quantification for the percentage of SOX2+ cells among electroporated EGFP+ RFP+ cells (Control + shScramble n = 4, TMEM14B + shScramble n = 6, and
TMEM14B + shIQGAP1 n = 6; one-way ANOVA).
(D) Quantification for the percentage of TBR2+ cells among EGFP+ RFP+ cells (Control + shScramble n = 4, TMEM14B + shScramble n = 5, TMEM14B +
shIQGAP1 n = 5; one-way ANOVA).
(E and F) Expression of RFP-shIQGAP1 reduces the neuronal progenitor-increasing phenotype in TMEM14BCKI neocortex. Sections of the cerebral cortex 2 days
after electroporation on E13.5 stained by (E) SOX2 and (F) TBR2 are shown. Scale bar, 50 mm.
(G) Quantification for the percentage of SOX2+ cells among RFP+ cells (NesCre + shScramble n = 4, TMEM14BCKI + shScramble n = 4, and TMEM14BCKI +
shIQGAP1 n = 3; one-way ANOVA).
(H) Quantification for the percentage of TBR2+ cells among RFP+ cells (NesCre + shScramble n = 4, TMEM14BCKI + shScramble n = 4, and TMEM14BCKI +
shIQGAP1 n = 3; one-way ANOVA).
(legend continued on next page)
Cell Stem Cell 21, 635–649, November 2, 2017 645
Figure 7. Phosphorylation of IQGAP1 by TMEM14B Induces Nuclear Localization
(A) Immunofluorescencemicroscopy and typical cell images of NIH 3T3 co-transfected with TMEM14B-EGFP and IQGAP1-tdTomato for 36 hr. Scale bar, 10 mm.
(B) Quantification for fluorescence density of nuclear IQGAP1 versus cytoplasm IQGAP1 in each cell (nControl = 61 and nTMEM14B = 60; ***p < 0.0005, Student’s t
test). Data are presented as mean ± SEM.
(C) Nuclear and membrane extraction followed by western blot to indicate nuclear IQGAP1 increased by TMEM14B overexpression. Endogenous Histone3 and
Na+/K+ ATPase were detected as loading controls for nuclear and membrane fraction, respectively.
(D) Blotting with antibodies for IQGAP1 and pSer. g-tubulin was detected as a loading control.
(E) Proposed model for the role of primate-specific gene TMEM14B in cortical expansion and folding. TMEM14B expression increases nuclear localization of
IQGAP1 through upregulating IQGAP1 phosphorylation. This process promotes neural progenitor generation by regulating G1/S progression in human cortical
development.
See also Figure S7.
transcription (Smith et al., 2015). Overexpression of IQGAP1
enhances b-catenin nuclear import, accumulation, and tran-
scriptional co-activation, which contributes to Wnt-signaling
activation (Briggs et al., 2002; Chenn and Walsh, 2002; Goto
et al., 2013). Interestingly, mice expressing a constitutively
active version of b-catenin have enlarged brains and gyrifica-
tion with horizontal expansion of progenitors due to decreased
cell cycle exit (Chenn and Walsh, 2002). This suggests that
TMEM14B-induced mouse brain enlargement and cortical
folding may be due to increased Wnt signaling via IQGAP1
and b-catenin nuclear localization.
In summary, TMEM14B can regulate cortical expansion and
folding. Our results suggest that a single gene is sufficient to pro-
mote the appearance and proliferation of all progenitor subtypes
required for gyrus formation in the mouse cerebral cortex, along
with proportional increases in neurons across all cortical layers.
Thus, brain size and gyrification may be experimentally manipu-
lated by changing the number of progenitor cells or the
neurogenic divisions via overexpression of a specific gene,
similar to increases in progenitors induced by overexpression
(I–K) FACS analysis of HEK293T cells co-transfected with (I) PCMV-FLAG-E
shScramble, and (K) PCMV-FLAG-hTMEM14B-EGFP and RFP-shIQGAP1-3 (n =
(L) Quantification and comparison of data presented in (I)–(K).
Data are presented as mean ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p
646 Cell Stem Cell 21, 635–649, November 2, 2017
of b-catenin, Trnp1, ARHGAP11B, TBC1D3, or a combination
of multiple genes (Chenn and Walsh, 2002; Florio et al., 2015;
Ju et al., 2016; Martınez-Martınez et al., 2016; Stahl et al.,
2013). The TMEM14BCKI mouse model therefore provides a
powerful tool for examining cellular and molecular mechanisms
behind cortical expansion and folding.
STAR+METHODS
Detailed methods are provided in the online version of this paper
and include the following:
d KEY RESOURCES TABLE
d CONTACT FOR REAGENT AND RESOURCE SHARING
d EXPERIMENTAL MODEL AND SUBJECT DETAILS
GFP a
3 for e
< 0.00
B Mouse Maintenance
B Human Tissue Samples
B Isolation of NPCs from Fetal Neocortex
B Preparation of Human Embryonic Fibroblast
B Cells lines Culture
nd RFP-shScramble, (J) PCMV-FLAG-hTMEM14B-EGFP and RFP-
ach group). Data are presented as mean ± SEM.
01). See also Figure S6.
d METHOD DETAILS
B Single-cell RNA-seq
B Read Trimming and Mapping
B Filter Data for Potential Marker Genes
B Principal Component Analysis
B Venn Analysis in the Categorizing Genes
B WGCNA Analysis in Categorizing Genes
B Comparison of TMEM14B among Species
B cRNA Probe Synthesis and ISH
B SDS-PAGE Separation and in-gel Digestion
B LC-MS/MS Analysis and Proteins Identification
B Plasmid Construction and IUE
B BrdU Labeling, EdU/BrdU Double Pulsing
B Concentrations of Antibodies
B Immunostaining of Brain Sections
B IP, Co-IP and Subcellular Fractionation
B Western Blotting
B FACS for Neural Progenitor Cells Screening
B FACS for PI Staining
B Cortical Expansion index Measurement
d QUANTIFICATION AND STATISTICAL ANALYSIS
B Imaging and Quantification on Brain Slices
B Quantification of PI Cell Cycle
B Quantification of Cortical Expansion
B Statistics
d DATA AND SOFTWARE AVAILABILITY
B Data Resources
SUPPLEMENTAL INFORMATION
Supplemental Information includes seven figures and two tables and can be
found with this article online at https://doi.org/10.1016/j.stem.2017.08.013.
AUTHOR CONTRIBUTIONS
Q.W., J.L., J.Z., F.T., and X.W. conceived the project, designed the experi-
ments, and wrote the manuscript. J.L., W.L., L.Y., Q.W., H.Z., and J.Z. con-
ducted the experiments, including animal surgery, tissue preparation, immu-
nostaining, imaging, cell culture, RNA-seq data collection, analysis, and
interpretation. A.F. prepared animals, L.L. analyzed Mass Spectrometry (MS)
data, and X.X. performed flow cytometry experiments. L.S. prepared the sam-
ples. F.T. participated in RNA-seq and data interpretation. All the authors edi-
ted and approved the manuscript.
ACKNOWLEDGMENTS
We thank Yanbing Zhu, Shasha Wei, Xiaoming Ma, Junjing Zhang, and
Shiyang Chang for suggestions and technical assistance and the staff at the
Anzhen Hospital for providing access to donated tissue samples. We also
thank Dr. Guohong Li for suggestions and members of the Wang laboratory
for discussion. This work was supported by National Basic Research Program
of China (2014CB964600 and 2015CB964800), the Strategic Priority Research
Program of the Chinese Academy of Sciences (XDA01020309), the National
Natural Science Foundation of China (NSFC) (31371100 and 91332105 to
X.W.; 31400937 and 31671072 to Q.W.), the Grants of Shanghai Brain-Intelli-
gence Project from STCSM (16JC1420500 to X.W.), Newton Advanced
Fellowship (NA140416 to X.W.), and Youth Innovation Promotion Association
CAS to Q.W.
Received: December 12, 2016
Revised: April 6, 2017
Accepted: April 16, 2017
Published: October 12, 2017
REFERENCES
Bayatti, N., Moss, J.A., Sun, L., Ambrose, P., Ward, J.F., Lindsay, S., and
Clowry, G.J. (2008). A molecular neuroanatomical study of the developing hu-
man neocortex from 8 to 17 postconceptional weeks revealing the early differ-
entiation of the subplate and subventricular zone. Cereb. Cortex 18,
1536–1548.
Betizeau, M., Cortay, V., Patti, D., Pfister, S., Gautier, E., Bellemin-Menard, A.,
Afanassieff, M., Huissoud, C., Douglas, R.J., Kennedy, H., and Dehay, C.
(2013). Precursor diversity and complexity of lineage relationships in the outer
subventricular zone of the primate. Neuron 80, 442–457.
Borrell, V., and Gotz, M. (2014). Role of radial glial cells in cerebral cortex
folding. Curr. Opin. Neurobiol. 27, 39–46.
Briggs, M.W., Li, Z., and Sacks, D.B. (2002). IQGAP1-mediated stimulation of
transcriptional co-activation by beta-catenin is modulated by calmodulin.
J. Biol. Chem. 277, 7453–7465.
Chen, H., and Boutros, P.C. (2011). VennDiagram: a package for the genera-
tion of highly-customizable Venn and Euler diagrams in R. BMC
Bioinformatics 12, 35.
Chenn, A., and Walsh, C.A. (2002). Regulation of cerebral cortical size by con-
trol of cell cycle exit in neural precursors. Science 297, 365–369.
Chenn, A., and Walsh, C.A. (2003). Increased neuronal production, enlarged
forebrains and cytoarchitectural distortions in b-catenin overexpressing trans-
genic mice. Cereb. Cortex 13, 599–606.
Dehay, C., Kennedy, H., and Kosik, K.S. (2015). The outer subventricular zone
and primate-specific cortical complexification. Neuron 85, 683–694.
Englund, C., Fink, A., Lau, C., Pham, D., Daza, R.A., Bulfone, A., Kowalczyk, T.,
and Hevner, R.F. (2005). Pax6, Tbr2, and Tbr1 are expressed sequentially by
radial glia, intermediate progenitor cells, and postmitotic neurons in devel-
oping neocortex. J. Neurosci. 25, 247–251.
Fietz, S.A., Kelava, I., Vogt, J., Wilsch-Br€auninger, M., Stenzel, D., Fish, J.L.,
Corbeil, D., Riehn, A., Distler, W., Nitsch, R., and Huttner, W.B. (2010).
OSVZ progenitors of human and ferret neocortex are epithelial-like and
expand by integrin signaling. Nat. Neurosci. 13, 690–699.
Fietz, S.A., Lachmann, R., Brandl, H., Kircher, M., Samusik, N., Schroder, R.,
Lakshmanaperumal, N., Henry, I., Vogt, J., Riehn, A., et al. (2012).
Transcriptomes of germinal zones of human and mouse fetal neocortex sug-
gest a role of extracellular matrix in progenitor self-renewal. Proc. Natl.
Acad. Sci. USA 109, 11836–11841.
Florio, M., and Huttner, W.B. (2014). Neural progenitors, neurogenesis and the
evolution of the neocortex. Development 141, 2182–2194.
Florio, M., Albert, M., Taverna, E., Namba, T., Brandl, H., Lewitus, E., Haffner,
C., Sykes, A., Wong, F.K., Peters, J., et al. (2015). Human-specific gene
ARHGAP11B promotes basal progenitor amplification and neocortex expan-
sion. Science 347, 1465–1470.
Garcıa-Moreno, F., Vasistha, N.A., Trevia, N., Bourne, J.A., and Molnar, Z.
(2012). Compartmentalization of cerebral cortical germinal zones in a lissence-
phalic primate and gyrencephalic rodent. Cereb. Cortex 22, 482–492.
Geschwind, D.H., and Rakic, P. (2013). Cortical evolution: judge the brain by its
cover. Neuron 80, 633–647.
Goto, T., Sato, A., Adachi, S., Iemura, S., Natsume, T., and Shibuya, H. (2013).
IQGAP1 protein regulates nuclear localization of b-catenin via importin-b5 pro-
tein in Wnt signaling. J. Biol. Chem. 288, 36351–36360.
Hansen, D.V., Lui, J.H., Parker, P.R.L., and Kriegstein, A.R. (2010). Neurogenic
radial glia in the outer subventricular zone of human neocortex. Nature 464,
554–561.
Hawrylycz, M.J., Lein, E.S., Guillozet-Bongaarts, A.L., Shen, E.H., Ng, L.,
Miller, J.A., van de Lagemaat, L.N., Smith, K.A., Ebbert, A., Riley, Z.L., et al.
(2012). An anatomically comprehensive atlas of the adult human brain tran-
scriptome. Nature 489, 391–399.
Hevner, R.F., and Haydar, T.F. (2012). The (not necessarily) convoluted role of
basal radial glia in cortical neurogenesis. Cereb. Cortex 22, 465–468.
Insolera, R., Bazzi, H., Shao,W., Anderson, K.V., and Shi, S.-H. (2014). Cortical
neurogenesis in the absence of centrioles. Nat. Neurosci. 17, 1528–1535.
Cell Stem Cell 21, 635–649, November 2, 2017 647
Johnson, M.B., Kawasawa, Y.I., Mason, C.E., Krsnik, Z., Coppola, G.,
Bogdanovi�c, D., Geschwind, D.H., Mane, S.M., State, M.W., and Sestan, N.
(2009). Functional and evolutionary insights into human brain development
through global transcriptome analysis. Neuron 62, 494–509.
Johnson, M., Sharma, M., Brocardo, M.G., and Henderson, B.R. (2011).
IQGAP1 translocates to the nucleus in early S-phase and contributes to cell cy-
cle progression after DNA replication arrest. Int. J. Biochem. Cell Biol.
43, 65–73.
Johnson, M.B., Wang, P.P., Atabay, K.D., Murphy, E.A., Doan, R.N., Hecht,
J.L., andWalsh, C.A. (2015). Single-cell analysis reveals transcriptional hetero-
geneity of neural progenitors in human cortex. Nat. Neurosci. 18, 637–646.
Ju, X.-C., Hou, Q.-Q., Sheng, A.-L., Wu, K.-Y., Zhou, Y., Jin, Y., Wen, T., Yang,
Z., Wang, X., and Luo, Z.-G. (2016). The hominoid-specific gene TBC1D3 pro-
motes generation of basal neural progenitors and induces cortical folding in
mice. eLife 5, e18197.
Kang, H.J., Kawasawa, Y.I., Cheng, F., Zhu, Y., Xu, X., Li, M., Sousa, A.M.,
Pletikos, M., Meyer, K.A., Sedmak, G., et al. (2011). Spatio-temporal transcrip-
tome of the human brain. Nature 478, 483–489.
Kawaguchi, A., Ikawa, T., Kasukawa, T., Ueda, H.R., Kurimoto, K., Saitou, M.,
and Matsuzaki, F. (2008). Single-cell gene profiling defines differential progen-
itor subclasses in mammalian neurogenesis. Development 135, 3113–3124.
Kelava, I., Reillo, I., Murayama, A.Y., Kalinka, A.T., Stenzel, D., Tomancak, P.,
Matsuzaki, F., Lebrand, C., Sasaki, E., Schwamborn, J.C., et al. (2012).
Abundant occurrence of basal radial glia in the subventricular zone of embry-
onic neocortex of a lissencephalic primate, the common marmoset Callithrix
jacchus. Cereb. Cortex 22, 469–481.
Kowalczyk, T., Pontious, A., Englund, C., Daza, R.A., Bedogni, F., Hodge, R.,
Attardo, A., Bell, C., Huttner, W.B., and Hevner, R.F. (2009). Intermediate
neuronal progenitors (basal progenitors) produce pyramidal-projection neu-
rons for all layers of cerebral cortex. Cereb. Cortex 19, 2439–2450.
Kuida, K., Haydar, T.F., Kuan, C.Y., Gu, Y., Taya, C., Karasuyama, H., Su,M.S.,
Rakic, P., and Flavell, R.A. (1998). Reduced apoptosis and cytochrome
c-mediated caspase activation in mice lacking caspase 9. Cell 94, 325–337.
LaMonica, B.E., Lui, J.H., Wang, X., and Kriegstein, A.R. (2012). OSVZ progen-
itors in the human cortex: an updated perspective on neurodevelopmental dis-
ease. Curr. Opin. Neurobiol. 22, 747–753.
LaMonica, B.E., Lui, J.H., Hansen, D.V., and Kriegstein, A.R. (2013). Mitotic
spindle orientation predicts outer radial glial cell generation in human
neocortex. Nat. Commun. 4, 1665.
Lange, C., Huttner, W.B., and Calegari, F. (2009). Cdk4/cyclinD1 overexpres-
sion in neural stem cells shortens G1, delays neurogenesis, and promotes the
generation and expansion of basal progenitors. Cell Stem Cell 5, 320–331.
Langfelder, P., and Horvath, S. (2008). WGCNA: an R package for weighted
correlation network analysis. BMC Bioinformatics 9, 559.
Langfelder, P., and Horvath, S. (2012). Fast R Functions for Robust
Correlations and Hierarchical Clustering. J. Stat. Softw. 46, i11.
Li, L., Dong, J., Yan, L., Yong, J., Liu, X., Hu, Y., Fan, X., Wu, X., Guo, H., Wang,
X., et al. (2017). Single-Cell RNA-Seq Analysis Maps Development of Human
Germline Cells andGonadal Niche Interactions. Cell StemCell 20, 858–873.e4.
Lui, J.H., Hansen, D.V., and Kriegstein, A.R. (2011). Development and evolu-
tion of the human neocortex. Cell 146, 18–36.
Lui, J.H., Nowakowski, T.J., Pollen, A.A., Javaherian, A., Kriegstein, A.R., and
Oldham, M.C. (2014). Radial glia require PDGFD-PDGFRb signalling in human
but not mouse neocortex. Nature 515, 264–268.
Malatesta, P., Hartfuss, E., and Gotz, M. (2000). Isolation of radial glial cells by
fluorescent-activated cell sorting reveals a neuronal lineage. Development
127, 5253–5263.
Martınez-Cerdeno, V., Cunningham, C.L., Camacho, J., Antczak, J.L.,
Prakash, A.N., Cziep, M.E., Walker, A.I., and Noctor, S.C. (2012).
Comparative analysis of the subventricular zone in rat, ferret and macaque:
evidence for an outer subventricular zone in rodents. PLoS ONE 7, e30178.
Martınez-Martınez, M.A., De Juan Romero, C., Fernandez, V., Cardenas, A.,
Gotz, M., and Borrell, V. (2016). A restricted period for formation of outer sub-
ventricular zone defined by Cdh1 and Trnp1 levels. Nat. Commun. 7, 11812.
648 Cell Stem Cell 21, 635–649, November 2, 2017
Miller, J.A., Ding, S.L., Sunkin, S.M., Smith, K.A., Ng, L., Szafer, A., Ebbert, A.,
Riley, Z.L., Royall, J.J., Aiona, K., et al. (2014). Transcriptional landscape of the
prenatal human brain. Nature 508, 199–206.
Miyata, T., Kawaguchi, A., Okano, H., and Ogawa, M. (2001). Asymmetric in-
heritance of radial glial fibers by cortical neurons. Neuron 31, 727–741.
Miyata, T., Kawaguchi, A., Saito, K., Kawano, M., Muto, T., and Ogawa, M.
(2004). Asymmetric production of surface-dividing and non-surface-dividing
cortical progenitor cells. Development. 131, 3133–3145.
Molnar, Z., and Clowry, G. (2012). Cerebral cortical development in rodents
and primates. Prog. Brain Res. 195, 45–70.
Noctor, S.C., Flint, A.C., Weissman, T.A., Dammerman, R.S., and Kriegstein,
A.R. (2001). Neurons derived from radial glial cells establish radial units in
neocortex. Nature 409, 714–720.
Noctor, S.C., Martınez-Cerdeno, V., Ivic, L., and Kriegstein, A.R. (2004).
Cortical neurons arise in symmetric and asymmetric division zones and
migrate through specific phases. Nat. Neurosci. 7, 136–144.
Nonaka-Kinoshita, M., Reillo, I., Artegiani, B., Martınez-Martınez, M.A.,
Nelson, M., Borrell, V., and Calegari, F. (2013). Regulation of cerebral
cortex size and folding by expansion of basal progenitors. EMBO J. 32,
1817–1828.
Palop, J.J., Roberson, E.D., and Cobos, I. (2011). Step-by-Step In Situ
Hybridization Method for Localizing Gene Expression Changes in the Brain.
In Alzheimer’s Disease and Frontotemporal Dementia: Methods and
Protocols, E.D. Roberson, ed. (Totowa, NJ: Humana Press), pp. 207–230.
Pilaz, L.J., Patti, D., Marcy, G., Ollier, E., Pfister, S., Douglas, R.J., Betizeau,
M., Gautier, E., Cortay, V., Doerflinger, N., et al. (2009). Forced G1-phase
reduction alters mode of division, neuron number, and laminar phenotype in
the cerebral cortex. Proc. Natl. Acad. Sci. USA 106, 21924–21929.
Pollen, A.A., Nowakowski, T.J., Shuga, J., Wang, X., Leyrat, A.A., Lui, J.H., Li,
N., Szpankowski, L., Fowler, B., Chen, P., et al. (2014). Low-coverage single-
cell mRNA sequencing reveals cellular heterogeneity and activated signaling
pathways in developing cerebral cortex. Nat. Biotechnol. 32, 1053–1058.
Pollen, A.A., Nowakowski, T.J., Chen, J., Retallack, H., Sandoval-Espinosa,
C., Nicholas, C.R., Shuga, J., Liu, S.J., Oldham, M.C., Diaz, A., et al. (2015).
Molecular identity of human outer radial glia during cortical development.
Cell 163, 55–67.
Pontious, A., Kowalczyk, T., Englund, C., and Hevner, R.F. (2008). Role of in-
termediate progenitor cells in cerebral cortex development. Dev. Neurosci.
30, 24–32.
Pruszak, J., Ludwig, W., Blak, A., Alavian, K., and Isacson, O. (2009). CD15,
CD24, and CD29 define a surface biomarker code for neural lineage differen-
tiation of stem cells. Stem Cells 27, 2928–2940.
Rakic, P. (1995). A small step for the cell, a giant leap for mankind: a hypothesis
of neocortical expansion during evolution. Trends Neurosci. 18, 383–388.
Rakic, P. (2003). Developmental and evolutionary adaptations of cortical radial
glia. Cereb Cortex. 13, 541–549.
Reillo, I., and Borrell, V. (2012). Germinal zones in the developing cerebral cor-
tex of ferret: ontogeny, cell cycle kinetics, and diversity of progenitors. Cereb.
Cortex 22, 2039–2054.
Reillo, I., de Juan Romero, C., Garcıa-Cabezas, M.A., and Borrell, V. (2011). A
role for intermediate radial glia in the tangential expansion of the mammalian
cerebral cortex. Cereb. Cortex 21, 1674–1694.
Shitamukai, A., Konno, D., and Matsuzaki, F. (2011). Oblique radial glial divi-
sions in the developing mouse neocortex induce self-renewing progenitors
outside the germinal zone that resemble primate outer subventricular zone
progenitors. J. Neurosci. 31, 3683–3695.
Smart, I.H., Dehay, C., Giroud, P., Berland, M., and Kennedy, H. (2002). Unique
morphological features of the proliferative zones and postmitotic compart-
ments of the neural epithelium giving rise to striate and extrastriate cortex in
the monkey. Cereb. Cortex 12, 37–53.
Smith, J.M., Hedman, A.C., and Sacks, D.B. (2015). IQGAPs choreograph
cellular signaling from the membrane to the nucleus. Trends Cell Biol. 25,
171–184.
Stahl, R., Walcher, T., De Juan Romero, C., Pilz, G.A., Cappello, S., Irmler, M.,
Sanz-Aquela, J.M., Beckers, J., Blum, R., Borrell, V., and Gotz, M. (2013).
Trnp1 regulates expansion and folding of the mammalian cerebral cortex by
control of radial glial fate. Cell 153, 535–549.
Tang, F., Barbacioru, C., Wang, Y., Nordman, E., Lee, C., Xu, N., Wang, X.,
Bodeau, J., Tuch, B.B., Siddiqui, A., et al. (2009). mRNA-Seq whole-transcrip-
tome analysis of a single cell. Nat. Methods 6, 377–382.
Tang, F., Lao, K., and Surani, M.A. (2011). Development and applications of
single-cell transcriptome analysis. Nat. Methods 8 (4, Suppl), S6–S11.
Thomsen, E.R., Mich, J.K., Yao, Z., Hodge, R.D., Doyle, A.M., Jang, S.,
Shehata, S.I., Nelson, A.M., Shapovalova, N.V., Levi, B.P., and Ramanathan,
S. (2016). Fixed single-cell transcriptomic characterization of human radial glial
diversity. Nat. Methods 13, 87–93.
Wang, J.B., Sonn, R., Tekletsadik, Y.K., Samorodnitsky, D., and Osman, M.A.
(2009). IQGAP1 regulates cell proliferation through a novel CDC42-mTOR
pathway. J. Cell Sci. 122, 2024–2033.
Wang, X., Tsai, J.-W., LaMonica, B., and Kriegstein, A.R. (2011). A new sub-
type of progenitor cell in the mouse embryonic neocortex. Nat. Neurosci. 14,
555–561.
Wang, J., O’Bara, M.A., Pol, S.U., and Sim, F.J. (2013). CD133/CD140a-based
isolation of distinct human multipotent neural progenitor cells and oligoden-
drocyte progenitor cells. Stem Cells Dev. 22, 2121–2131.
Wilsch-Br€auninger, M., Florio, M., and Huttner, W.B. (2016). Neocortex expan-
sion in development and evolution - from cell biology to single genes. Curr.
Opin. Neurobiol. 39, 122–132.
Yang, H., Wang, H., and Jaenisch, R. (2014). Generating genetically modified
mice using CRISPR/Cas-mediated genome engineering. Nat. Protoc. 9,
1956–1968.
Yuan, S.H., Martin, J., Elia, J., Flippin, J., Paramban, R.I., Hefferan, M.P., Vidal,
J.G., Mu, Y., Killian, R.L., Israel, M.A., et al. (2011). Cell-surface marker signa-
tures for the isolation of neural stem cells, glia and neurons derived from hu-
man pluripotent stem cells. PLoS ONE 6, e17540.
Cell Stem Cell 21, 635–649, November 2, 2017 649
STAR+METHODS
KEY RESOURCES TABLE
REAGENT or RESOURCE SOURCE IDENTIFIER
Antibodies
Mouse monoclonal anti-CD184 BD Biosciences Cat# 560670; RRID: AB_1727436
Mouse monoclonal anti-CD15 eBioscience Cat# 50-8813-42; RRID: AB_11219681
Rat monoclonal anti-CD140a BD Biosciences Cat# 558774; RRID: AB_397117
Mouse monoclonal anti-CD56 BD Biosciences Cat# 560916; RRID: AB_2033963
Rat monoclonal anti-GFP Nacalai Tesque Cat#GF090R;RRID: AB_2314545
Goat polyclonal anti-Sox2 Santa Cruz Cat# sc17319;RRID: AB_661259
Rabbit polyclonal anti-Tbr2 Abcam Cat# ab23345;RRID: AB_778267
Rabbit polyclonal anti-Pax6 BioLegend Cat# 901301;RRID: AB_2565003
Rat monoclonal anti-BrdU Abcam Cat# ab6326; RRID: AB_305426
Rabbit polyclonal anti-Ki67 Millipore Cat#AB9260;RRID: AB_2142366
Mouse monoclonal anti- Phospho-Vimentin (Ser55) MBL International Cat# D076-3; RRID: AB_592963
Mouse monoclonal anti- Phospho-Vimentin (Ser82) MBL International Cat# D095-3; RRID: AB_592969
Mouse monoclonal anti-PH3 Abcam Cat# ab14955;RRID: AB_443110
Rabbit polyclonal anti-HOPX Sigma-Aldrich Cat#HPA030180;RRID: AB_10603770
Rabbit polyclonal anti-Ctip2 Novus Cat# NB100-79809; RRID: AB_1108022
Rabbit polyclonal anti-Satb2 Abcam Cat# ab34735; RRID: AB_2301417
Rabbit polyclonal anti-Cux1 Santa Cruz Cat# sc-13024; RRID: AB_2261231
Rabbit polyclonal anti-Foxp2 Abcam Cat# ab16046; RRID: AB_2107107
Mouse monoclonal anti-Sox2 Millipore Cat# MAB4343; RRID: AB_827493
Mouse monoclonal anti-Flag (M2) Sigma-Aldrich Cat# F1804; RRID: AB_262044
Mouse monoclonal anti-IQGAP1 BD Biosciences Cat# 610611; RRID: AB_397945
Rabbit polyclonal anti-IQGAP1 Santa Cruz Cat# sc-10792; RRID: AB_2249072
Rabbit polyclonal anti-Phospho-(Ser) PKC Substrate Cell Signaling Technology Cat# 2261L; RRID: AB_330311
Rabbit polyclonal anti-Histone3 Cell Signaling Technology Cat# 9715L; RRID: AB_823526
Rabbit polyclonal anti-Na+-K+ ATPase Santa Cruz Cat# sc-28800; RRID: AB_2290063
Mouse monoclonal anti-g-Tubulin Sigma-Aldrich Cat# T5326; RRID: AB_532292
Alexa Fluor 647Conjuated Affinipure Donkey
anti-Rabbit IgG(H+L)
Invitrogen Cat# A-31573; RRID: AB_2536183
Alexa Fluor 647Conjuated Affinipure Donkey
anti-Goat IgG(H+L)
Invitrogen Cat# A21447; RRID: AB_10584487
Alexa Fluor 647Conjuated Affinipure Donkey
anti-Mouse IgG(H+L)
Invitrogen Cat# A-31571; RRID: AB_162542
Alexa Fluor 594 Conjuated Affinipure Donkey
anti-Rabbit IgG(H+L)
Invitrogen Cat# A21207; RRID: AB_141637
Alexa Fluor 546 Conjuated Affinipure Donkey
anti-Goat IgG(H+L)
Invitrogen Cat# A11056; RRID: AB_10584485
Alexa Fluor 546 Conjuated Affinipure Donkey
anti-Mouse IgG(H+L)
Invitrogen Cat# A10036; RRID: AB_2534012
Alexa Fluor 488 Conjuated Affinipure Donkey
anti-Rat IgG(H+L)
Invitrogen Cat# A-21208; RRID: AB_2535794
Goat anti-Rabbit HRP Abcam Cat# ab136817
Goat anti-Mouse HRP Abcam Cat# ab136815
(Continued on next page)
e1 Cell Stem Cell 21, 635–649.e1–e8, November 2, 2017
Continued
REAGENT or RESOURCE SOURCE IDENTIFIER
Biological Samples
Human fetal brains at gestation16 weeks Anzhen Hospital of Capital Medical
University
N/A, materials available on request
from lead contact
Human fetal brains at gestation17 weeks Anzhen Hospital of Capital
Medical University
N/A, materials available on request
from lead contact
Human fetal brains at gestation 16.5 weeks Anzhen Hospital of Capital
Medical University
N/A, materials available on request
from lead contact
Chemicals, Peptides, and Recombinant Proteins
5-Bromo-2-deoxyuridine (BrdU) Sigma-Aldrich Cat# B5002
Propidium iodide (PI) Sigma-Aldrich Cat# P4170
4’,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI) Thermo Fisher Cat# D1306; RRID: AB_2629482
5-ethynyl-20-deoxyuridine (EdU) Thermo Scientific Cat# C10639
SDS-RIPA lysis buffer Bioteke Cat# PP1901
RNase inhibitor Takara Cat# 2313A
Triton X-100 Sigma-Aldrich Cat# T9284
dNTP Fermentas Cat# R0192
ERCC RNA Spike-In Mix Life Technologies Cat#4456740
SuperScript II reverse transcriptase Invitrogen Cat#18064-014
Betaine Sigma-Aldrich Cat# 61962
HotStart ReadyMix KAPA Biosystems Cat# KK2601
Agencourt AMPure XP beads Beckman Cat# A63881
Dynabeads MyOne Streptavidin C1 beads Thermo Fisher Cat# 65501
Critical Commercial Assays
DIG RNA Labeling Kit (SP6/T7) Roche Cat# 11175025910
Click-iT Plus EdU Alexa Fluor 594 Imaging Kit Thermo Scientific Cat# C10639
Plasma Membrane Protein Isolation Kit Invent Biotechnologies Cat# SM-005
NE-PER Nuclear and Cytoplasmic Extraction Reagent Thermo Scientific Cat# 78833
KAPA Hyper Prep Kit Kapa Biosystems Cat# KK8505
NEBNext Ultra DNA Library Prep Kit for Illumina NEB Cat# E7370L
EndoFree Plasmid Maxi Kit QIAGEN Cat# 12362
PrimeScript II 1st strand cDNA Synthesis Kit Takara Cat# 6210A
Deposited Data
Single-cell RNA sequencing Accession# GEO: GSE90734
Experimental Models: Cell Lines
NIH 3T3 ATCC Cat# CRL-6442
HEK293T ATCC Cat# CRL-11268
Human Embryonic Fibroblast This paper; derived from
fetal skin
N/A
Experimental Models: Organisms/Strains
Mouse: CD-1 Charles River laboratory Stock number: 201
Mouse: hTMEM14Bf/+ C57BL/6 This paper N/A
Mouse: B6.Cg-Tg(Nes-cre)1Kln/J Jackson lab Stock number: 003771
Mouse: NestinCre:: TMEM14B+/� This paper N/A
Recombinant DNA
pSPT18 Roche Cat# 11175025910
pSPT18-hTMEM14B This paper For primers, see Table S2; NM_030969.4
pSPT18-hHP1BP3 This paper For primers, see Table S2; NM_016287.1
pSPT18-hKCNK10 This paper For primers, see Table S2; NM_138318.2
pSPT18-hDAG1 This paper For primers, see Table S2; NM_004393.1
(Continued on next page)
Cell Stem Cell 21, 635–649.e1–e8, November 2, 2017 e2
Continued
REAGENT or RESOURCE SOURCE IDENTIFIER
pCMV-3Flag-EGFP Genechem Cat# GOSE50280
pCMV-3Falg-TMEM14B-EGFP This paper For primers, see Table S2;NM_030969.4
pCMV-3Falg-IQGAP1-EGFP This paper For primers, see Table S2; NM_003870.3
pTdtomato-N1 Addgene Cat#54642
pTdtomato-N1-IQGAP1 This paper For primers, see Table S2; NM_003870.3
PLL3.7-Tdtomato This paper N/A
pLL3.7-shIQGAP1-Tdtomato This paper For primers, see Table S2
Sequence-Based Reagents
See Table S2 for the list of oligos This paper Table S2
Software and Algorithms
GraphPad Prism 5.0 GraphPad Software http://www.graphpad.com/scientific-
software/prism/
Origin 8.0 OriginLab http://originlab.com/
ImageJ NIH https://imagej.nih.gov/ij/
Adobe photoshop CS6 Adobe Systems http://www.adobe.com
DNAMAN Lynnon Biosoft http://en.bio-soft.net/format/
DNAMAN.html
R(3.1.3) R-project https://www.r-project.org/
TopHat(2.0.10) Johns Hopkins University
Center for Computational
Biology
http://ccb.jhu.edu/software/tophat/
index.shtml
Cufflinks(2.1.1) Cole Trapnell’s lab http://cole-trapnell-lab.github.io/cufflinks/
Scatterplot3d(0.3.35) R-project https://cran.r-project.org/src/contrib/
Archive/scatterplot3d/
Plotrix(3.5.12) R-project https://cran.r-project.org/src/contrib/
Archive/plotrix/
VennDiagram(1.6.9) R-project https://cran.r-project.org/src/contrib/
Archive/
RColorBrewer(1.1.2) R-project https://cran.r-project.org/web/packages/
RColorBrewer/index.html
WGCNA(1.47) R-project https://cran.r-project.org/src/contrib/
Archive/WGCNA/
CONTACT FOR REAGENT AND RESOURCE SHARING
Further information and requests for reagents should be directed to and will be fulfilled by the Lead Contact, Xiaoqun Wang
EXPERIMENTAL MODEL AND SUBJECT DETAILS
Mouse MaintenanceAnimal housing and all experimental procedures in this study were in compliance with the guidelines of the Institutional Animal Care
and Use Committee of the Institute of Biophysics, Chinese Academy of Sciences. All mice had free access to food and water, were
housed in the institutional animal care facility (SPF) with a 12h light-dark schedule. In this study, presence of a vaginal plug deter-
mined the embryonic day (E) 0.5 and the day of birth is designated as postnatal day (P) 0. All mice used in these studies had never
been involved in previous procedures (drugs or other tests).
CD-1 mice purchased from Charles River Laboratories in China (Vital river, Bejing, China) were used for all in utero electroporation
experiments. The following transgenic mouse lines were on a pure C57BL/6 genetic background. Nestin-Cre mice (B6.Cg-Tg(Nes-
cre)1Kln/J No: 003771) was purchased from Jackson laboratory.
TMEM14B conditional knockin mouse model (TMEM14Bflox/+) was generated with CRISPR/Cas9 technique according to previous
report (Yang et al., 2014). Briefly, sgRNAs targeting Rosa26 insertion (between exon1 and exon2) site were selected using CRISPR
design by the Feng Zhang lab (crispr.mit.edu). CAGpr-loxP-Stop-loxp-3XFlaghTMEM14B-IRES-EGFP-WPRE-pA was cloned into
the targeting vector (Figure S3A) flanked with 2kb 50 and 30 homologous arms as donor DNA. An injection mix of Cas9 (100 ng/ml),
e3 Cell Stem Cell 21, 635–649.e1–e8, November 2, 2017
sgRNA (50 ng/ml) and DNA donor (200 ng/ml) were injected into �150–250 one-cell embryos from C57BL/6 mice each time. In total,
854 zygotes were implanted into C57BL/6 surrogate mothers, and 149 pups were delivered. 5 mice were identified as founders (F0)
by collecting genomic DNA from tails and PCR. F0 mice were bred with wild-type C57BL/6 mice and F1 pups were examined by tail
genotyping and Southern blotting to confirm the complete targeting DNA was inserted at the correct location. These mice were used
as TMEM14Bflox/+ mice. TMEM14BCKI mice were generated by crossing TMEM14Bflox/+ with Nestin-Cre mice. TMEM14BCKI mice
(TMEM14Bflox/+/Nestin-Cre) were identified by genotyping (genotyping primers see Table S2) and confirmed by EGFP expression
in brain. The TMEM14BCKI mice were randomly selected for experiments with littermates used as control (TMEM14B+/+/Nestin-Cre).
Human Tissue SamplesThe de-identified fetal cortical tissue samples were collected with patient informed consent in strict observance of the legal and insti-
tutional ethical regulation at Anzhen Hospital of Capital Medical University. Protocols were approved by the institutional review board
(ethics committee) at the Institute of Biophysics, Chinese Academy of Sciences.
The donors in this study were pregnant women who cannot continue pregnancy because of their own heart disease. All patients
signed informed consents and voluntarily donated the fetus for this research. The replicates in this study derived from 3 independent
samples (Brain 1 - GW16 female, Brain 2 - GW16.5 male, Brain 3 - GW17male) were obtained at autopsy within 3h after abortion with
recorded of sex and age. All these 3 samples were normal by karyotyping analysis (Figure S1A). All samples used in these studies had
never been involved in previous procedures (drugs or other tests). The stages of human embryos in this study were calculated from
last menstruation bleeding time. The clinicians who obtained the samples also confirmed the development stages.
Isolation of NPCs from Fetal NeocortexBrains were dissected in ice-cold artificial cerebrospinal fluid (ACSF) containing 125 mMNaCl, 5 mM KCl, 1.25 mM NaH2PO4, 1 mM
MgSO4, 2 mM CaCl2, 25 mM NaHCO3 and 20 mM glucose; pH 7.4, 310 mOsm/Kg, bubbled with carbogen (95% O2 5% CO2). The
right hemisphere was coronally divided into four slabs; the first slab was embedded in 4% low-melting-temperature agarose in ACSF
and sectioned at 300 mm using a vibratome (Leica Microsystems). Under visual guidance, the VZ, OSVZ and CP cellular zones were
carefully microdissected in HBSS from the brain slices using stereoscope (Olympus SZ2-LGB), and subsequently the tissues were
dissociated using 1mg/ml papain at 37�C for 10 min and passed through cell-strainer caps. Cells were harvested and then single cell
suspension was collected by centrifugation and resuspension.
Preparation of Human Embryonic FibroblastFetal skin tissues from fetus were dissected in a clean Petri dish by using a sterile scissor. Added 2 mL of 0.05% trypsin/EDTA
(GIBCO, Invitrogen) and incubated for 15 min at 37�C. Inactivated the trypsin by adding about 2 volume of FBS. Centrifuged the cells
with 1000rpm for 5 min, carefully removed the supernatant and resuspended cell pellet in warm medium. Then human embryonic
fibroblast cells were planted on a new 10cm Petri dish. Cells were maintained at 37�C with 5% CO2.
Cells lines CultureCell lines including NIH 3T3 and HEK293T cells were used for transfection (Lipofectamine 2000, Life Tech). Cells were maintained in
Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, US) supplemented with 10% fetal bovine serum (FBS) and penicillin and
streptomycin at 37�C with 5% CO2.
METHOD DETAILS
Single-cell RNA-seqWe prepared the single-cell RNA-seq cDNA libraries according to the protocol described previously (Li et al., 2017). After isolation of
single NPCs from fetal neocortex, the single cells were picked into 2.55 mL lysis buffer by mouth pipette. Each single cell was lysed
with 2.55 mL solution containing: 0.78 U/ml RNase inhibitor, 0.37% Triton X-100, 1.96 mMdNTP, 0.59 mMbarcoded primer (25nt oligo
dT primer anchored with an 8nt cell-specific barcode and 8nt unique molecular identifiers) and about 30,000 molecular number of
ERCC. After incubation at 72�C for 3 min, the first- and second- strand cDNAs were synthesized by adding 2.85 mL solution contain-
ing: 17.54 U/ml SuperScript II reverse transcriptase, 1.75 U/ml RNase inhibitor, 1.75 3 Superscript II first-stranded buffer, 8.78 mM
DTT, 1.75 M Betaine, 10.52 mM MgCl2 and 1.75 mM TSO primer, and incubated at 25�C for 5 min, 42�C for 1 hr, 50�C for 30 min,
72�C for 10 min and held at 4�C. The double stranded cDNAs were amplified with solution containing: 1.67 3 KAPA HiFi HotStart
ReadyMix, 0.17 mM IS PCR primers and 0.83 mM 30P2 primers with the PCR program of 95�C for 3 min, 4 cycles of 98�C for 20 s,
65�C for 30 s and 72�C for 5 min, then 16 cycles of 98�C for 20 s, 67�C for 15 s and 72�C for 5 min, 72�C for 5 min, and held at
4�C. Pull 6 mL the amplified cDNAs together, purify with 0.8 3 XP beads (Beckman) twice, and amplify about 30 ng pulled cDNAs
by 50 mL solution containing: 1 3 KAPA HiFi HotStart ReadyMix, 0.4 mM IS PCR primers and 0.4 mM Biotin index primer, using
the PCR program of 95�C for 3 min; 4 cycles of 98�C for 20 s, 67�C for 15 s, 72�C for 5 min; 72�C for 5 min and held at 4�C. About300ng biotinylated cDNAs were purified with 0.83 XP beads once and sheared into 300 bp fragments with Covaris instrument. After
purified with 0.83 XP beads once, the sheared fragments were enriched with Dynabeads�MyOne Streptavidin C1 beads. And the
cDNA libraries were constructed by KAPA Hyper Prep Kit and sequenced on Illumina HiSeq 4000 platform with 150 bp paired-end
(sequenced by Novogene).
Cell Stem Cell 21, 635–649.e1–e8, November 2, 2017 e4
Read Trimming and MappingFor the single-cell cDNA libraries prepared using the protocol described previously (Li et al., 2017), the raw reads with adaptor con-
taminations and low-quality bases (N > 10%)were removed andwere split by the cell specific barcode sequences located in read 2 of
the pair-end reads to get single-cell specific data. Unique molecular identifier (UMI) sequences were aligned to the corresponding
read 1, which were then trimmed to remove the TSO sequences and poly-A tail sequences, and the single-cell clean data were gener-
ated. Then the single-cell clean data were aligned to the hg19 human transcriptome (UCSC) using TopHat (http://ccb.jhu.edu/
software/tophat/index.shtml; version 2.0.12) using the default parameters. Only the unique mapped reads were counted by
htseq-count (HTSeq package). FPKM values were calculated by Cufflinks (version 2.2.1) and were converted to TPM value. The
duplicated transcripts with the same UMI sequences were discarded. Finally, for a given individual cell, only distinct UMIs of each
gene were counted as the copy number of the transcript.
Filter Data for Potential Marker GenesR codes (https://www.r-project.org/; R version 3.1.3) were specially written to filter single cell RNA-seq data for identifying potential
marker genes. Low quality single cell samples were removed with the cutoff of gene numbers < 1000. Lowly expressed genes were
removed with the cutoff of averaged TPM% 1. Pearson’s correlation coefficient for all genes based on all samples was calculated to
evaluate the sample quality. 562 Geneswere filtered on the standard at least one Student’s t test p values of TPM for all combinations
of two cell subtypes was < 0.05 for downstream analysis. Reported markers within the 562 genes were extracted for further evalu-
ation. Within the 562 genes, 26 genes were filtered with all Student’s t test p values of TPM < 0.05 for vRG or oRG cells against all
other three cell subtypes and with at least 2-fold changes for vRG or oRG cells compared with all other three cell subtypes. We per-
formed combined categorizing analysis on the remained 536 genes (see Figure S1F), including principal component analysis (PCA),
Venn analysis contrasting neurons and weighted gene co-expression network analysis (WGCNA). The three categorizing methods
separated genes into cell subtype specific categories in distinct standards (methods were described in detail in the following three
sections). The combined categorizing method selected 49 genes which were in the same cell subtype specific category with all three
categorizing methods. The 49 selected genes and the 26 previous picked genes were considered as potential marker genes.
Principal Component AnalysisR codes were specially written to perform PCA analysis (https://www.r-project.org/; R version 3.1.3) and decompose the data into
primary components and projections. PC1 projection represented the sum of all four cell subtypes and was not cell subtype specific.
PC2 projection (positive direction) was vRG and oRG cells specific, PC3 projection (positive direction) was neural cells specific, and
PC4 projection (negative direction) was IPC cells specific. Categories were separated based on the standard tomaximize the number
within the absolute value of PCA projection of PC2 (vRG and oRG), PC3 (Neuron) and PC4 (IPC) (see Figure S1D). 190 genes were
classified as vRG and oRG category, 55 genes were classified as neuron category and 69 genes were classified as IPC category.
Venn Analysis in the Categorizing GenesVenn analysis was performed by R package ‘‘VennDiagram’’ (Chen andBoutros, 2011)(R version 3.1.3, https://cran.r-project.org/src/
contrib/Archive/; package version 1.6.9) and the neurons were selected as reference for Venn analysis. Venn analysis was performed
using Student’s t test p value filtering by comparing vRG, ORG and IPC cells with neuron respectively (p < 0.05) and separated genes
into vRG, oRG and IPC categories (n1, n2 and n3) . Genes within intersection of the three categories (n123) were considered as neural
cell specific genes. Intersection genes with more than one cell subtypes (n13, n12 and n23) were excluded. Genes only within group
of one cell subtype (34 vRG specific genes, 21 oRG specific genes, 57 IPC specific genes and 21 neural specific genes) and with no
intersection with other groups (n1-n12-n13-n123 for vRG, etc.) were considered as vRG, oRG and neural cell subtype specific genes
respectively (see Figure S1C).
WGCNA Analysis in Categorizing GenesWGCNA analysis was performed by R package ‘‘WGCNA’’ (Langfelder and Horvath, 2008, 2012) (R version 3.1.3, https://cran.
r-project.org/src/contrib/Archive/WGCNA; package version 1.47). The WGCNA softpower value was determined by navigating
the soft-threshold-mean-connectivity curve. The minimum WGCNA module size was 30 genes (see Figure S1E). Modules
with < 0.25 similarity were merged. Modules correlated with specific cell subtype were considered as standard modules for catego-
rizing genes into certain cell subtypes. 8 modules were selected and 4 of these modules were respectively related to vRG, oRG, IPC,
and neural cells. 54 genes were separated into vRG category, 33 genes were separated into oRG category, 129 genes were sepa-
rated into neuron category and 46 genes were separated into IPC category.
Comparison of TMEM14B among SpeciesPhylogenetic evolutionary tree analysis on TMEM14B was performed by ClustalX (version 2.1, http://www.softpedia.com/get/
Science-CAD/Clustal-X.shtml) and Mega (version 7.0.14, http://www.megasoftware.net/download_form).TMEM14B sequence for
all available species was downloaded from NCBI database. Phylogenetic relationship was calculated with UPGMA algorithm in
ClustalX and phylogenic trees were generated by MEGA. Divergence time was calculated comparing the divergence time between
e5 Cell Stem Cell 21, 635–649.e1–e8, November 2, 2017
Homo sapiens and pan troglodytes against UPGMA phylogenic relation. Conservative analysis was performed by UCSC genome
browser (https://genome.ucsc.edu/cgi-bin/hgGateway) selectingmodel species expressing TMEM14B including old worldmonkeys
and new world monkeys.
cRNA Probe Synthesis and ISHFor in situ hybridization, all operations were carried out under RNase-free conditions. Related primer sequences were listed in Table
S2. Fetal brain sections of 40mm thickness were hybridized with cRNA probes at a final concentration of 400 ng/ml overnight at 62�Cin hybridization solution (50% formamide, 10% dextran sulfate, 0.2% tRNA (Invitrogen), 1 x Denhardts solution (from a 50x stock;
Sigma), 1x salt solution (containing 0.2 M NaCl, 0.01 M Tris, 5 mM NaH2PO4, 5 mM Na2HPO4, 5 mM EDTA pH 7.5)). After the sec-
tions were washed, alkaline phosphatase-coupled anti-digoxigenin Fab fragments were applied. For visualization of the labeled
cRNAs, the sections were incubated in the dark in nitrobluetetrazolium/5-bromo-4-chloro-3-indolyl phosphate solution (Roche).
The detailed procedure for in situ hybridization was performed as described previously (Palop et al., 2011). Images were taken by
Leica SCN400 (Leica Microsystems). 3 sections of each embryo were analyzed.
For subsequent immunolabelling with antibody, slides were subjected to antigen retrieval, and then follow the procedure of immu-
nostaining. For in situ hybridization and immunostaining co-labeling slice, images were taken by Nikon-EclipseTi, 3 sections from
GW16-17 embryo were analyzed, 351 cells were counted.
SDS-PAGE Separation and in-gel DigestionTwo sets of equi-volume elution from immunoprecipitation were separated on a 10%SDS-PAGE gel with a 5%stacking gel andwere
stained with silver nitrate solution. Compared with control, the differential bands in the TMEM14B-overexpressed lanewere cut into 6
slices, the proteins in the individual gel sliceswere separately reduced in-gel with dithiothreitol and alkylatedwith iodoacetamide. The
proteins were subsequently digested at 37�Cwith trypsin (Promega, Madison, WI) overnight. Digested peptides were extracted from
gels with 0.1% FA and then with 50% ACN/0.1% FA. The resulting peptide mixtures were dried and stored at�80�C until further LC-
MS/MS analysis.
LC-MS/MS Analysis and Proteins IdentificationThe nanoLC-MS/MS analysis of peptide mixtures was performed on a Q Exactive mass spectrometer with a nanoelectrospray ioni-
zation source (Thermo Scientific) coupled with Easy n-LC 1000 HPLC system (Thermo Scientific). The peptides were loaded onto a
100 mm id 3 2 cm fused silica trap column packed in-house with reversed phase silica (Reprosil-Pur C18 AQ, 5 mm, Dr. Maisch
GmbH) and then separated on an a 75 mm id 3 20 cm C18 column packed with reversed phase silica (Reprosil-Pur C18AQ,
3 mm, Dr. Maisch GmbH). The peptides bounded on the column were eluted with a78 min linear gradient. The solvent A consisted
of 0.1% formic acid in water solution and the solvent B consisted of 0.1% formic acid in acetonitrile solution. The segmented gradient
was 5%–8% B, 8 min; 8%–22% B, 50 min; 22%–32% B, 12 min; 32%–95% B, 1 min; 95% B, 7 min at a flow rate of 280 nL/min. The
MS analysis was performed with Q Exactive mass spectrometer (Thermo Scientific). With the data-dependent acquisition mode, the
MS data were acquired at a high resolution 70,000 (m/z 200) across the mass range of 300-1600m/z. The top 20 precursor ions were
selected from each MS full scan with isolation width of 2 m/z for fragmentation in the HCD collision cell with normalized collision en-
ergy of 27%. Subsequently, MS/MS spectra were acquired at resolution 17,500 atm/z 200. The dynamic exclusion timewas 40 s. For
nano electrospray ion source setting, the spray voltage was 2.0 kV; no sheath gas flow; the heated capillary temperature was 320�C.Proteome Discovery (version 1.4, Thermo Scientific) was used as data processing interface. Data processing was performed using
Sequest HT search engine for protein identification and Percolator for FDR (false discovery rate) against the human Uniprot database
(25/10/2015 release). The carbamidomethylation of cysteine was set as a fixed modification and oxidized methionine was variable
modifications; Trypsin was selected as the proteolytic enzyme, with a maximum of two potential missed cleavages; the mass toler-
ance of precursor was set as 10 ppm and the product ions tolerance was 0.02 Da. FDR analysis was performed with Percolator and
FDR < 1% was set for protein identification. The peptides confidence was set as high for peptides filter.
Plasmid Construction and IUEFetal human brain RNA was reverse transcribed with PrimeScript II 1st strand cDNA Synthesis Kit using oligo (dT) primers. For the
generation of ISH template plasmid, TMEM14B cDNA and KCNK10 cDNA were acquired by PCR and subcloned into the XbaI/SacI
site of pSPT18; HP1BP3 cDNA and DAG1 cDNA were acquired by PCR and subcloned into the BamHI/EcoRI site of pSPT18. To
construct the fluorescent protein fusion protein plasmid, TMEM14B cDNA was subcloned into the XhoI/KpnI site of pCMV-3Flag-
EGFP; IQGAP1 cDNA was subcloned into the XhoI/SacII site of pCMV-3Flag-EGFP; IQGAP1 cDNA was subcloned into the XhoI/
SacII site of pTdtomato-N1. For the generation of vectors encoding small interference RNA targeting IQGAP1: shIQGAP1, annealed
oligos were inserted into HpaI/XhoI digested pLL3.7-Tdtomato. For pLL3.7-tdtomato plasmid generation, tdtomato full length was
subcloned into Pll3.7 (addgene #11795) from tdTomato-C1 (addgene #54653) with EcoRI and NHeI.
For in-utero electroporation, a timed pregnant CD-1 mouse at E13.5 was anaesthetized, the uterine horns were exposed and 1 mL
of plasmid DNA (2mg/ml) mixed with 0.1 mg/ml Fast Green (Sigma) was manually microinjected through the uterus into the lateral
ventricle, using a beveled and calibrated glass micropipette (Drummond Scientific). For electroporation, five 50-ms pulses of 40–
50V with a 950-ms interval were delivered across the uterus with two 9-mm electrode paddles positioned on either side of the
head (ECM830, BTX). After the procedure, the uterus was placed back in the abdominal cavity and the woundwas surgically sutured.
Cell Stem Cell 21, 635–649.e1–e8, November 2, 2017 e6
The mouse was then placed in a 28�C recovery incubator under close monitoring until it recovered and resumed normal activity
(Wang et al., 2011). Related primer sequences were listed in Table S2.
BrdU Labeling, EdU/BrdU Double PulsingIn utero electroporation was conducted at E13.5 of pregnant CD-1 mouse according to the method described above. The pregnant
female mousewas injected with 50mg/kg BrdU (Sigma) at E15.5. After 2h, embryonic brains were dissected and fixed immediately in
cold 4% PFA in PBS at 4�C overnight, followed by sequentially dehydration in 20% and 30% sucrose in PBS at 4�C. For transgenicmice, BrdU were injected at E15.5 and sacrificed two hours later.
For EdU/BrdU double labeling, procedures were performed mainly as previously described with some modifications (Insolera
et al., 2014). Pregnant female TMEM14BCKI mice were injected with 50 mg/kg EdU (Sigma) at E13.5 and 50 mg/kg BrdU (Thermo
Scientific) 14 hr later. After 2h, pregnant female sacrificed and brains were dissected.
Concentrations of AntibodiesFor the source and identifier of the following antibodies, see Key Resources Table. For flow cytometry analysis: CD184 (1/1000);
CD15 (1/300); CD140a (1/1000), and CD56 (1:1000). Immunofluorescence: GFP (1/1000); Sox2 (1/200); Tbr2 (1/300); Pax6(1/300);
BrdU (1/500); Ki67 (1/300);P-Vim(Ser55) (1/500); P-Vim (MO82) (1/500); PH3(1/500); HOPX (1/1000); Ctip2 (1/300); Satb2 (1/500);
Cux1(1/300); Foxp2 (1/500). Secondary antibodies usedwere as follows: donkey anti-rat Alexa 488, donkey anti-mouse or anti-rabbit
or anti-goat Alexa-546-, and Alexa-647-conjugated antibodies (1/500). DNA was stained with DAPI (1/10,000). Western blot: Sox2
(1/4000); Tbr2 (1/4000); Pax6 (1/4000); Flag M2 (1/10000); g-Tubulin (1/5000); IQGAP1 (1/4000); IQGAP1 (1/500); Phosphoserine
(pSer) PKC-substrate (1/1000); Histone 3(1/5000); Na+-K+ ATPase (1/5000).
Immunostaining of Brain SectionsBrains were removed and transcardially perfused with ice-cold PBS (pH 7.4) followed by 4%paraformaldehyde in PBS (pH 7.4), then
the brain was dissected out and post-fixed into cold 4% PFA in PBS at 4�C overnight. The fixed brain was dehydrated in 20% and
30% sucrose in PBS at 4�C sequentially. For immunohistochemistry, brain slides were subjected to antigen retrieval, followed by
permeabilization (0.1% Triton X-100 in PBS), blocking (10% donkey serum in PBS) and antibody incubation.
IP, Co-IP and Subcellular FractionationCells after 36h transfection were washed in ice-cold phosphate-buffered saline, (PBS), scraped into lysis buffer (150mMNaCl, 50nM
Tris7.5, 1% NP40, 0.25% Na-deoxycholate, 0.5mM EDTA and protease inhibitor cocktail) for 30min on ice, and then centrifuged at
12,000x rpm for 15min. For all experiments, protein concentrations were determined (BCA kit, Pierce) and equal amount precleared
with 15 mL PBS-equilibrated protein G or A beads for 2h at 4�C. Antibody were added and incubated with rotation overnight at 4�C,and 30 mL beads were added and incubated with rotation for 4h at 4�C. The beads were washed five times with 1 mL lysis buffer,
boiled for 10 min in 25 mL 1.5x SDS loading buffer, resolved on SDS-PAGE and immunoblotted. Nuclear-cytoplasmic fractionation
was performed by Plasma Membrane Protein Isolation Kit and NE-PER Nuclear and Cytoplasmic Extraction Reagent.
Western BlottingFor western blotting analysis, cortical cells were lysed in SDS-RIPA lysis buffer and protease inhibitor cocktail. Proteins were sepa-
rated by SDS–PAGE and transferred onto a PVDF membrane. After membranes were blocked with 5% milk for 1h at room temper-
ature, blocking buffer was changed to be 3%BSA for phosphorylation, they were probedwith various primary antibodies overnight at
4�C, followed by incubation with secondary antibodies for 1h at room temperature and visualized with enhanced chemiluminescence
reagent (Thermo Scientific).
FACS for Neural Progenitor Cells ScreeningCells for flow cytometry analysis were resuspended in PBSwith 1%BSA; the cell suspension was incubated for 30min with antibody
on ice. All cell populations were isolated in a single sort using Becton Dickinson FACS Aria II. Cells were collected in PBS with 1%
BSA. Gates were set manually by using control samples. Data were analyzed with FlowJo 9.3 data analysis software.
FACS for PI StainingCells were pelleted by centrifugation at 1500 rpm for 5min andwashed in PBS and then centrifuged again at 1500 rpm for 5min. Cells
were resuspended in 1 mL of ice-cold 70% ethanol overnight at 4�C. Next, the cells were pelleted by centrifugation at 1500 rpm for
5 min and wash in PBS for three times, then suspended in 600 mL of PBS containing RNase A (50mg/ml) and propidium iodide
(50mg/ml). Cell cycle profiles were determined using a BD Biosciences FACScalibur flow cytometer. The percentage of cells in
each cell cycle phase was determined with CellQuest software.
Cortical Expansion index MeasurementCortical expansion index were measured on the image taken by stereoscope by ImageJ. AP (anterior-posterior) extension was from
olfactory bulb/frontal cortex boundary to caudal hemisphere boundary. ML (Medial-lateral) extension was measured from ventral
e7 Cell Stem Cell 21, 635–649.e1–e8, November 2, 2017
longitudinal fissure to lateral hemisphere boundary. IUE brains were measured by the electroporated hemispheres, TMEM14B trans-
genic mice were measured by two hemispheres. The graph showed the ratio of TMEM14B/Control *100.
QUANTIFICATION AND STATISTICAL ANALYSIS
Imaging and Quantification on Brain SlicesImmunostaining brain slices were imaged by Olympus confocal microscope FV1000 (Olympus) and images were analysed by
FluoView (Olympus), Imaris (Bitplane) and Photoshop (Adobe Systems). Composite images of more than one separate field were
stitched by Photoshop (Adobe Systems) and noted in figure legends.
Quantifications of the double positive immunostained cells from in utero electroporated mice or TMEM14BCKI mice were per-
formed on brain coronal or sagittal sections from at least n R 3 mice. Cells were considered as double positive when the signal
of the fluorescent protein co-localized with the marker present in the nucleus (Sox2, Pax6, Tbr2, BrdU, EdU, PH3, Ki67, Ctip2,
Satb2, Foxp2, Cux1), or cytosol (perinuclear and processes) (P-Vimentin, HOPX), or membrane (IQGAP1) on a single optical section.
DAPI was used to visualize the cell nuclei. For the quantification of marker expression, at least total 120 cells were analyzed, from at
least 3 different mice. All the WTmice (including Control and NesCre) and TMEM14B mice (including TMEM14B overexpression and
TMEM14BCKI) were pups of the same litter with randomization.
For electroporated cells, a mean of n = 235.2 ± 29.32 cells were counted for each marker for each animal analyzed (total cells
counted = 6820). For the embryonic TMEM14BCKI mice analysis, a mean of n = 516.7 ± 36.93 cells were counted for each marker
for each animal analyzed (total cells counted = 16533). For the postnatal TMEM14BCKI mice analysis, a mean of n = 2822.7 ±
215.2 cells were counted for each marker for each animal analyzed (total cells counted = 16936).
Quantification of PI Cell CycleFor the PI FACS analysis of cell cycle, total 60000 cells transfected with control /TMEM14B/IQGAP1were analyzed for a single exper-
iment. Data were collected from 3 independent experiments.
Quantification of Cortical ExpansionFor the quantification of cortical expansion, datas were collected from at least 3 different mice. All theWTmice (including Control and
NesCre) and mice (including TMEM14B/IQGAP1 overexpression and TMEM14BCKI) were were randomly selected for experiments
with littermates used as control. A mean of n = 5.5 ± 0.38 brains for each group analyzed (total brains counted = 44).
StatisticsAll data were represented as the mean ± the SEM. Comparisons between two groups were made using t tests. Comparisons among
three ormore groups weremade using one-way ANOVA analyses followed by Newman–Keuls tests. . The quantification graphs were
analysed by using GraphPad Prism (GraphPad Software). Statistical details are described in Figure legends.
DATA AND SOFTWARE AVAILABILITY
Data ResourcesThe accession number for the RNA sequencing data reported in this paper is GEO: GSE90734.
Cell Stem Cell 21, 635–649.e1–e8, November 2, 2017 e8
