© 2015. Published by The Company of Biologists Ltd | Development (2015) 142, 1616-1627 doi:10.1242/dev.120543
RESEARCH ARTICLE
STEM CELLS AND REGENERATION
Lin28 promotes the proliferative capacity of neural progenitor cells
in brain development
ABSTRACT
Neural progenitor cells (NPCs) have distinct proliferation capacities at
different stages of brain development. Lin28 is an RNA-binding
protein with two homologs in mice: Lin28a and Lin28b. Here we show
that Lin28a/b are enriched in early NPCs and their expression
declines during neural differentiation. Lin28a single-knockout mice
show reduced NPC proliferation, enhanced cell cycle exit and a
smaller brain, whereas mice lacking both Lin28a alleles and one
Lin28b allele display similar but more severe phenotypes. Ectopic
expression of Lin28a in mice results in increased NPC proliferation,
NPC numbers and brain size. Mechanistically, Lin28a physically and
functionally interacts with Imp1 (Igf2bp1) and regulates Igf2-mTOR
signaling. The function of Lin28a/b in NPCs could be attributed,
at least in part, to the regulation of their mRNA targets that encode
Igf1r and Hmga2. Thus, Lin28a and Lin28b have overlapping
functions in temporally regulating NPC proliferation during early
brain development.
KEY WORDS: Lin28a, Lin28b, Neural progenitor cells, Proliferation,
Brain development, Mouse
INTRODUCTION
Neural progenitor cells (NPCs) use distinct self-renewal and
differentiation programs for brain development and homeostasis.
At different stages, NPCs exhibit unique morphologies,
proliferation capacities and differentiation potentials. Early
embryonic NPCs replicate rapidly to expand their population, and
their proliferation rate declines later in development to coordinate
with the neuronal differentiation programs (He et al., 2009;
Salomoni and Calegari, 2010). This temporal change of NPC
properties has been partially attributed to intricate gene expression
programs. For example, unique sets of transcription factors are
sequentially expressed in different populations of NPCs during
brain development, including Pax6, Tbr2 (also known as Eomes)
and Sox2 (Englund et al., 2005; Graham et al., 2003; Sessa et al.,
2008). Epigenetic regulators modulate chromatin states to control
NPC self-renewal and neurogenic potentials in a developmental
stage-dependent manner (Kishi et al., 2012; Lim et al., 2009;
Nishino et al., 2008). Although these studies have provided
1
Department of Genetics, Biochemistry & Molecular Biology, University of Georgia,
2
Athens, GA 30602, USA. Department of Biochemistry & Molecular Genetics,
3
University of Colorado Denver, Aurora, CO 80045, USA. Lunenfeld-Tanenbaum
Research Institute, Mount Sinai Hospital, Toronto, ON M5T 3L9, Canada.
4
Department of Pediatrics, University of Colorado Anschutz Medical Campus,
5
Children’s Hospital Colorado, Aurora, CO 80045, USA. Department of Molecular
Biology, Rowan University, Stratford, NJ 08084, USA.
*These authors contributed equally to this work
‡
Author for correspondence ([email protected])
Received 1 December 2014; Accepted 10 March 2015
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significant insights into the highly coordinated gene expression
program at transcriptional levels for NPC behavior control, little is
known about gene regulation at post-transcriptional levels for NPC
self-renewal and brain development.
Signals from local and extrinsic sources regulate NPC proliferation
and differentiation during brain development (Johansson et al., 2010),
including insulin-like growth factor 1/2 (Igf1/2) signaling (Lehtinen
et al., 2011; O’Kusky and Ye, 2012). The action of Igf1/2 is mainly
mediated by insulin-like growth factor receptor 1 (Igf1r) followed by
Akt activation and downstream signaling events, including mTOR
pathway activation (Laplante and Sabatini, 2012; O’Kusky and Ye,
2012). In humans, mutations in IGF1 or IGF1R are associated with
severe growth retardation and microcephaly (Abuzzahab et al., 2003;
Woods et al., 1997). Knockout of Igf1, Igf2 or Igf1r in mice
dysregulates progenitor cell division leading to deficiencies in
embryonic and postnatal growth, and ultimately results in reduced
brain size (Baker et al., 1993; Liu et al., 1993). Although the
importance in NPC behaviors is established, how Igf1/2 signaling is
temporally regulated to contend with the changes in NPC
proliferation that occur during brain development remains largely
unknown.
Lin28 is an RNA-binding protein with a cold-shock domain (CSD)
and retroviral-type CCHC zinc knuckle RNA-binding domain.
Mammals have two Lin28 homologs: Lin28a and Lin28b. Since
our original identification of LIN28 as a developmental timing
regulator in C. elegans (Moss et al., 1997), substantial efforts have
been put into understanding Lin28a/b functions in mammals. Studies
from recent years suggest that Lin28a/b function in a wide spectrum
of biological processes and diseases, including embryonic stem cell
(ESC) self-renewal, induced pluripotent stem cell (iPSC) generation,
cancers and diabetes (Shyh-Chang and Daley, 2013; Thornton and
Gregory, 2012). Our previous studies, together with those by other
groups, suggest that Lin28a regulates cell proliferation and
neurogenesis in vitro (Balzer et al., 2010; Cimadamore et al.,
2013). However, the physiological functions of Lin28a/b in somatic
stem cells remain largely unknown, and their in vivo roles in NPC
self-renewal and brain development have not been determined.
Whereas previous research focused on the microRNA (miRNA)
let-7 as the major target mediating Lin28 functions (Shyh-Chang
and Daley, 2013; Thornton and Gregory, 2012), our recent studies
suggest that Lin28a could function in a let-7-independent manner
(Balzer et al., 2010). Moreover, recent genome-wide studies suggest
that mRNAs are the major targets of Lin28a, whereas miRNA loci
represent only 0.07% of Lin28a binding sequence reads (Cho et al.,
2012; Hafner et al., 2013). Overall, these studies raise questions as
to the identity of mRNA targets of Lin28 and their importance in
mediating Lin28 functions during brain development.
Here, we use both gain- and loss-of-function genetic approaches
to reveal that Lin28a and Lin28b have overlapping functions in
promoting NPC proliferation and brain development in mouse.
DEVELOPMENT
Mei Yang1, *, Si-Lu Yang1, *, Stephanie Herrlinger1, Chen Liang1, Monika Dzieciatkowska2, Kirk C. Hansen2,
Ridham Desai3, Andras Nagy3, Lee Niswander4, Eric G. Moss5 and Jian-Fu Chen1,‡
Lin28a/b function, at least in part, through modulation of Igf2mTOR signaling activities and the protein expression of the
chromatin regulator Hmga2.
RESULTS
Lin28a deletion results in reduced body and brain size in
mice
Mammals possess two Lin28 genes, encoding the Lin28a and
Lin28b proteins (supplementary material Fig. S1A), which may
have arisen by duplication of an ancestral gene (Guo et al., 2006;
Moss and Tang, 2003). We and others have previously observed
Lin28a expression in early neural development (Balzer et al.,
2010; Yokoyama et al., 2008). The purpose of this study was to
investigate the in vivo roles of Lin28 with a particular focus on the
developing mammalian nervous system. To do so we created
Lin28a null mice, which harbor an exon 2 deletion. Heterozygous
Lin28a mice were viable and fertile and of normal size, whereas
homozygous mutant mice exhibited reduced body size at birth
(Fig. 1A). Western blot analysis verified the complete elimination
of Lin28a (Fig. 1B, top panel) and not the homolog Lin28b
(Fig. 1B, middle panel) protein in the brain of homozygous
mutant embryos. Mutant organ mass was proportionally reduced
with total body weight at embryonic day (E) 18.5 and postnatal day
(P) 1 compared with wild type (Fig. 1C; supplementary material
Fig. S1C), including a reduction in brain size (Fig. 1C,D;
supplementary material Fig. S1C). No significant difference in
morphology or organ size was observed at E15.5 between
homozygous mutant, heterozygous and wild-type embryos
(supplementary material Fig. S1B). Histological staining and
statistical analysis revealed a ∼10% reduction in cerebral cortex
Development (2015) 142, 1616-1627 doi:10.1242/dev.120543
thickness in Lin28a mutant brains compared with littermate
controls at E17.5 and P1 (Fig. 1E,F). These findings suggest that
Lin28a is crucial for murine growth control, including brain
development.
To guide our mechanistic studies of Lin28a in brain development,
we assessed the distribution of Lin28a protein in the cerebral cortex
at different stages. These experiments showed that Lin28a is
expressed in the NPCs, as indicated by its localization in early
neural tube and at the ventricular/subventricular zone (VZ/SVZ) of
the developing cerebral cortex (Fig. 1G-I; supplementary material
Fig. S1D). At subcellular levels, Lin28a is localized in the
cytoplasm of NPCs labeled by Pax6 or nestin (Fig. 1H;
supplementary material Fig. S1D). Immunoblots of whole-cell
extracts of cortices from different embryonic stages showed that
Lin28a/b are predominant at early stages when the neuroepithelium
is primarily composed of NPCs and that protein levels decrease as
neural differentiation proceeds (supplementary material Fig. S1E).
These data, together with our previous studies (Balzer et al., 2010),
suggest that Lin28a/b are highly expressed in NPCs in the early
developing brain.
Lin28a deletion leads to reduced NPC numbers due to
decreased proliferation and enhanced cell cycle exit
The enriched expression of Lin28a in NPCs (Fig. 1G-I) and reduced
brain size of Lin28a mutants (Fig. 1D-F) prompted us to test whether
there is a decreased number of NPCs in the mutant brains. We used
Pax6 and Tbr2, respectively, to mark apical progenitors (APs) and
intermediate neural progenitors (INPs) in the developing cerebral
cortex. We focused our analyses on the VZ/SVZ of the cerebral
cortex (the area below the white lines in Fig. 2). Examination of
Fig. 1. Loss of Lin28a results in
reduced body and brain size.
(A) Lin28a mutant and its wild-type
(WT) littermate on P1. (B) Western blot
analyses of Lin28a and Lin28b
expression in the cerebral cortex of
E10.5 wild-type, heterozygous and
homozygous mutant embryos. β-actin
serves as a loading control. (C) Relative
weights of different organs isolated
from P1 wild-type and mutant mice.
Error bars indicate s.e.m. of
measurements from three independent
experiments. *P<0.05 for each WT
versus mutant comparison (Student’s
t-test). (D) Dorsal views of P1 wild-type
and mutant mouse brains. (E) Coronal
sections of P1 cerebral cortex stained
with H&E. (F) Lin28a-deficient cortex
shows decreased radial thickness.
Error bars indicate s.e.m. of nine
sections at comparable positions along
the rostral/caudal axis between
wild-type and mutant cerebral cortex
from three independent experiments.
*P<0.05 (Student’s t-test).
(G-I) Confocal microscopy images of
coronal sections of the developing
brain at different stages using the
antibodies indicated. Hoechst stains
nuclei (blue). Overlap refers to merge.
Scale bars: 2 mm in D; 1 mm in E;
50 µm in G; 100 µm in H, except 5 µm
bottom right; 100 µm top row and 20 µm
bottom row in I.
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Development (2015) 142, 1616-1627 doi:10.1242/dev.120543
comparable cerebral cortical sections from wild-type and mutant
littermates showed that the numbers of Pax6+ APs and Tbr2+ INPs
were significantly decreased in the mutants at E17.5 and P1, whereas
no difference was detected at E15.5 (Fig. 2A-C). Together, these data
indicate that Lin28a is required for the maintenance of AP and INP
populations during normal development.
Programed cell death occurs during normal cerebral cortex
development (Blaschke et al., 1998), and reduced brain size could
be due to increased cell death in Lin28a mutant mice. TUNEL
staining of comparable coronal sections in wild-type and mutant
brains showed no significant increase in TUNEL+ cells in mutants
compared with wild-type controls, either prior to or during stages of
observable brain size reduction (supplementary material Fig. S2A,B).
Because TUNEL detects the final step of the apoptosis process, we
also examined an early event of apoptosis: the activation of caspase
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3. Again, we did not find any significant difference in the expression
of activated caspase 3 in the cerebral cortex of the wild type and
Lin28a mutants (supplementary material Fig. S2D,E).
Next, we investigated whether the decrease in NPC numbers in
Lin28a−/− mutants is due to decreased cell proliferation and/or
premature differentiation of NPCs. We addressed this using
comparable cerebral cortex sections from wild-type and mutant
embryos at E15.5, when no significant morphological or cellular
differences were observed (Fig. 2B,C; supplementary material
Fig. S1B), examining cells in S phase by performing 1-h
bromodeoxyuridine (BrdU) labeling experiments. There was a
significant reduction of BrdU+ cells in the VZ/SVZ of Lin28a−/−
cerebral cortex compared with wild type (Fig. 2D,E). Since a BrdU
pulse experiment only labels cells undergoing S phase at a given
time, we used a phospho-histone H3 ( p-H3) antibody to label
DEVELOPMENT
Fig. 2. Lin28a deficiency results in reduced proliferation, enhanced cell cycle exit and depletion of cortical NPCs. (A) Confocal micrographs of coronal
sections from E17.5 or P1 wild-type and mutant cortex stained with antibodies against Pax6 (red) or Tbr2 (green). Hoechst stains nuclei (blue). The ventricular/
subventricular zone (VZ/SVZ) is the area below the white line. (B,C) Percentage of Pax6+ (B) or Tbr2+ (C) cells among total cells within the VZ/SVZ in the
experiment in A at the stages indicated. n.s., not significant (P>0.05) at E15.5; P<0.05 at E17.5 and P1 (Student’s t-test). (D) Confocal micrographs of coronal
sections from E15.5 wild-type and mutant cortex stained with antibodies against p-H3 (green) and BrdU (red) 1 h after a single pulse label of BrdU. The VZ/SVZ
regions (below the white line) are enlarged to the right. (E,F) Percentage of BrdU+ cells (E) and p-H3+ cells (F) among total cells within the VZ/SVZ (below the white
line) in the experiment in D. P<0.05 in E and F (Student’s t-test). (G) Confocal micrographs of coronal sections from E16.5 mice 24 h after a single pulse label of
BrdU; sections were stained with antibodies against BrdU (green) and Ki67 (red). Arrowheads indicate cells that exited the cell cycle (BrdU+/Ki67–). (H,I) The
percentage of BrdU+/Ki67− cells (H) or BrdU+/Ki67+ cells (I) among total BrdU+ cells within the VZ/SVZ (below the white line in the experiment in G). n.s., not
significant (P>0.05) at E15.5; P<0.05 at E16.5 (Student’s t-test). (B,C,E,F,H,I) Error bars indicate s.e.m. of nine sections from three independent experiments.
Scale bars: 20 µm.
Development (2015) 142, 1616-1627 doi:10.1242/dev.120543
Table 1. Genotypes of 91 embryos from E14.5 to E18.5
Observed
Expected
Genotype
No.
%
No.
%
Lin28a+/+; Lin28b+/+
Lin28a+/–; Lin28b+/+
Lin28a–/–; Lin28b+/+
Lin28a+/+; Lin28b+/–
Lin28a+/+; Lin28b–/–
Lin28a+/–; Lin28b+/–
Lin28a+/–; Lin28b–/–
Lin28a–/–; Lin28b+/–
Lin28a–/–; Lin28b–/–
4
13
7
12
5
26
10
14
0
4.4
14.3
7.7
13.2
5.5
28.6
11.0
15.4
0.0
5.7
11.4
5.7
11.4
5.7
22.8
11.4
11.4
5.7
6.3
12.5
6.3
12.5
6.3
25
12.5
12.5
6.3
Shown are the numbers of wild-type and compound mutant embryos
recovered alive from litters dissected at the indicated developmental stages.
Parents of these embryos were Lin28a+/–; Lin28b+/–.
mitotic cells. In E15.5 wild-type cerebral cortex, p-H3+ NPCs were
mainly localized at the VZ, with some scattered cells in the SVZ
(Fig. 2D, upper panel), whereas the Lin28a−/− brains exhibited a
reduction of p-H3+ cells in the VZ/SVZ (Fig. 2D,F). Thus, Lin28a is
required to maintain the proper NPC proliferation rate during
cerebral cortex development.
To determine whether Lin28a−/− NPCs prematurely exit the cell
cycle in addition to reducing their proliferation rate, we performed
24-h BrdU pulse labeling experiments at different stages of
cortical development, including E15.5 and E16.5. Using Ki67 to
mark cells in all phases of the cell cycle except G0, we assessed
the fraction of BrdU+ cells that had exited the cell cycle within
24 h after the incorporation of BrdU (referred to as BrdU+/Ki67−
cells) (Fig. 2G). Although no difference was detected at E15.5, we
found a significant increase in the proportion of mutant NPCs that
had exited the cell cycle compared with the wild-type control at
E16.5 (Fig. 2G,H). We also quantified the number of actively
proliferating cells within 24 h after BrdU labeling (referred to as
BrdU+/Ki67+), and found that actively proliferating cells are
slightly reduced in the Lin28a mutants compared with the wildtype control at E16.5 (Fig. 2I). Together, these data indicate that
Lin28a is required for NPCs to maintain an appropriate
proliferation rate and prevent premature cell cycle exit during
cerebral cortex development.
Lin28a and Lin28b are both required for normal NPC
proliferation and brain development
The high protein sequence similarity between Lin28a and Lin28b
(supplementary material Fig. S1A) and their similar protein expression
patterns during cerebral cortex development (supplementary material
Fig. S1E) prompted us to examine whether they play redundant roles in
brain development. It has been reported that Lin28b null mice
exhibit no detectable developmental phenotypes (Shinoda et al.,
2013). We crossed Lin28a+/− with Lin28b+/− mice to produce
compound heterozygotes, which were intercrossed to generate
various genotypes including double-homozygous knockout (DKO)
offspring. No DKO mice were found postnatally or between E14.5
and E18.5 (Table 1), suggesting early lethality of DKO embryos.
By contrast, Lin28a−/−; Lin28b+/− (referred to as Lin28a−/−b+/−)
compound heterozygotes were recovered at normal Mendelian
ratios (12.5%) before birth (Table 1), suggesting that they can
survive through development. Intriguingly, Lin28a−/−b+/−
compound heterozygotes had visibly smaller brains than littermate
controls at E14.5 with more than 90% penetrance (Fig. 3A), and
brain size reduction was more severe than with Lin28a−/− embryos,
which showed a normal brain size at least a day later in
development. Therefore, we focused our NPC behavior analysis
on the cerebral cortex of Lin28a−/−b+/− compound mutants.
The defect in NPC numbers and proliferation was also more severe
in Lin28a−/−b+/− compound mutants than in Lin28a−/− embryos.
NPCs were significantly decreased at E14.5 in Lin28a−/−b+/−
cerebral cortex, as evidenced by reduced numbers of Pax6+ APs and
Tbr2+ INPs compared with controls (Fig. 3B-D), whereas Lin28a
knockout mice start to exhibit reduced NPC numbers at E17.5
(Fig. 2A-C). Furthermore, 1-h BrdU labeling and p-H3 staining
experiments suggest that there is a significant reduction of S-phase
and M-phase NPCs in Lin28a−/−b+/− compared with control E14.5
cerebral cortex (Fig. 3E-G). TUNEL and activated caspase 3 assays
did not show any significant difference in cell death between wildtype and Lin28a−/−b+/− cerebral cortex at different stages, including
E12.5, E13.5 and E14.5 (supplementary material Fig. S2C,F).
Together, these results suggest that Lin28a and Lin28b have
overlapping functions in regulating NPC proliferation and brain
development.
Ectopic expression of Lin28a in NPCs results in NPC
expansion and an enlarged brain
The loss-of-function studies above suggest that Lin28a/b are
required for NPC proliferation and brain development. We next
performed Lin28a gain-of-function studies. We created an inducible
Lin28a transgenic mouse model (Lin28a Tg), in which a STOP
codon flanked with loxP sequences is placed downstream of a
constitutive promoter and upstream of a copy of the Lin28a gene
(supplementary material Fig. S1F); Lin28a expression is induced
upon the spatiotemporal expression of Cre. In the absence of Cre,
the floxed Lin28a transgenic mice do not overexpress Lin28a, as
indicated by our immunohistochemical staining studies at different
developmental stages (supplementary material Fig. S1G,H), and
there are no noticeable morphological or behavioral abnormalities.
The Nestin-Cre driver induces recombination at ∼E11.5 in NPCs of
the cerebral cortex (Kim et al., 2009; Tronche et al., 1999). In
Lin28a Tg; Nestin-Cre embryos, Lin28a expression is significantly
increased in E14.5 NPCs (Fig. 4B); brain size and weight are
increased at E18.5 (Fig. 4A,C) and Hematoxylin and Eosin (H&E)
staining and statistical analysis revealed slightly increased thickness
of the cerebral cortex compared with the control at E18.5, although
no difference was detected at E16.5 (Fig. 4D,E). We found no
evidence of altered cell death in Lin28a-overexpression cerebral
cortex compared with control (supplementary material Fig. S2G-J).
These results suggest that Lin28a could play instructive roles in
brain development.
The enlarged brain size of Lin28a Tg; Nestin-Cre mice prompted
us to examine whether there is increased proliferation and numbers
of NPCs in the brain. Indeed, Pax6+ APs were significantly
increased in Lin28a Tg; Nestin-Cre cerebral cortex compared with
the control (Fig. 4F,G) and this was detected as early as E15.5, when
the cerebral cortex is similar in size in wild-type and Lin28aoverexpression mice, suggesting that an increased NPC population
could be the cause of the enlarged brain. Correspondingly, there is a
robust increase in the cell proliferation rate, as indicated by
increased numbers of EdU+ cells as well as p-H3+ cells in the
VZ/SVZ zone of Lin28a Tg; Nestin-Cre mice compared with the
control (Fig. 4F,H,K,L). Interestingly, the numbers of Tbr2+ INPs
were significantly reduced in Lin28a Tg; Nestin-Cre cerebral cortex
compared with the control (Fig. 4I,J). Together, these data suggest
that Lin28a overexpression results in the amplification of Pax6+ APs
and in an imbalance in the conversion to INPs, and this could
preferentially generate neurons at the expense of INPs.
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We assayed whether Lin28a overexpression delays NPC cell
cycle exit in the VZ/SVZ by applying a single BrdU pulse 24 h prior
to sacrifice and Ki67 labeling. Consistent with the observation that
loss of Lin28a results in premature cell cycle exit (Fig. 2G-I), we
found that Lin28a overexpression significantly inhibits the cell
cycle exit of NPCs compared with the control (Fig. 4M,N).
Together, these results indicate that Lin28a plays regulatory roles in
NPC proliferation and brain development.
Lin28a interacts with Imp1 to regulate NPC proliferation
Lin28 could function in NPCs through miRNA let-7-dependent
and/or -independent mechanisms (Balzer et al., 2010; Thornton and
Gregory, 2012; Zhu et al., 2010). We examined let-7 (let7a-d)
expression in NPCs isolated from the cerebral cortex of E14.5 wildtype and Lin28a−/−b+/− embryos, when NPC proliferation, numbers
and brain size are reduced (Fig. 3). However, we did not detect any
significant difference in let-7 expression in NPCs between wildtype and Lin28a−/−b+/− NPCs (Fig. 5A).
To broaden our viewpoint of Lin28a/b functional
mechanisms, we sought to identify Lin28a-associated proteins
in NE-4C cells, a cell line established from the cerebral vesicle
of E9.0 mouse embryos and which mimics NPC proliferation
and differentiation in vitro (Varga et al., 2008). We generated a
genetically modified Lin28a bacterial artificial chromosome
(BAC) vector that encodes a Lin28a fusion protein containing
two N-terminal tags: EGFP and Flag (supplementary material
Fig. S3A). An NE-4C stable cell line with the genetically
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modified Lin28a BAC was used for affinity purification
(supplementary material Fig. S3B). Of ∼40 proteins pulled
down with Lin28a (supplementary material Fig. S3C,D), Imp1
(also known as Igf2bp1) was of particular interest because our
previous studies have shown that Lin28a is associated with
Imp1-3 in skeletal muscle (Polesskaya et al., 2007). In addition,
recent studies suggest that Imp1 plays essential roles in NPC
self-renewal during brain development (Nishino et al., 2013).
We confirmed that Lin28a specifically interacts with Imp1, and
not its homolog Imp2 (Igf2bp2), in NE-4C cells (Fig. 5B).
Furthermore, Lin28a colocalizes with Imp1 in the cytoplasm of
NE-4C cells (Fig. 5C). Deletion analysis indicates that the
interaction between Lin28a and Imp1 is specific and requires the
region between the RNA-binding CSD and CCHC domains of
Lin28a (Fig. 5D-F). These data suggest that Lin28a is associated
with Imp1 in NE-4C cells.
To examine the functional importance of the domain that interacts
with Imp1, we used in utero electroporation to label individual
NPCs and to introduce full-length Lin28a or the Lin28a deletion
mutant (Lin28aΔ113-138) into NPCs of Lin28a−/−b+/− compound
mutants. BrdU labeling experiments show that wild-type Lin28a
almost fully rescues the proliferation deficiency of NPCs seen in
Lin28a−/−b+/− mutants (Fig. 5G-K). By contrast, Lin28aΔ113-138
exhibits significantly reduced rescue effects (Fig. 5G-K). These
studies suggest that the region of Lin28a that interacts with Imp1 is
functionally important in regulating NPC proliferation during brain
development.
DEVELOPMENT
Fig. 3. Lin28a and Lin28b have overlapping functions in NPC proliferation and brain development. (A) Reduced brain size in Lin28a−/−b+/− compound
mutants compared with wild type and Lin28a/b double heterozygotes at E14.5. (B) Confocal micrographs of coronal sections from E14.5 wild-type and
Lin28a−/−b+/− cortex stained with antibodies against Pax6 (red) or Tbr2 (green). Hoechst stains nuclei (blue). The VZ/SVZ regions (below the white line) are
enlarged to the right. (C,D) Percentage of Pax6+ (C) or Tbr2+ (D) cells among total cells within the VZ/SVZ (below the white line in the experiment in B).
(E) Confocal micrographs of coronal sections from E14.5 wild-type and Lin28a−/−b+/− compound mutant cortex stained with antibodies against p-H3 (green) and
BrdU (red) 1 h after a single pulse label of BrdU. The VZ/SVZ regions (below the white line) are enlarged to the right. (F,G) Percentage of BrdU+ cells (F) and p-H3+
cells (G) among total cells within the VZ/SVZ (below the white line in the experiment in E). (C,D,F,G) Error bars indicate s.e.m. of nine sections from three
independent experiments; P<0.05 (Student’s t-test). Scale bars: 1 mm in A; 20 µm in B,E.
Development (2015) 142, 1616-1627 doi:10.1242/dev.120543
Fig. 4. Ectopic expression of Lin28a results in NPC expansion and enlarged brains. (A) Dorsal views of E18.5 wild-type and Nestin-Cre-directed
Lin28a-overexpression mouse brains. (B) Western blot analyses of Lin28a protein expression in NPCs isolated from E14.5 wild-type and Lin28a-overexpression
cerebral cortex. β-actin serves as a loading control. (C) Analysis of brain weights of E18.5 wild-type and Lin28a-overexpression brains. Error bars indicate s.e.m.
from four independent experiments. P=0.022 (Student’s t-test). (D) Coronal sections of E18.5 cerebral cortex stained with H&E. (E) The Lin28a-overexpression
cortex shows increased radial thickness. n.s., not significant at E16.5; P=0.034 at E18.5 (Student’s t-test). (F) Confocal micrographs of coronal sections from
E17.5 wild-type and Lin28a-overexpression mouse cortex labeled for Pax6 (green) and with EdU (red) 1 h after a single pulse label of EdU. The VZ/SVZ is the
area below the white line. (G) Percentage of Pax6+ cells among total cells within the VZ/SVZ at E15.5 and E17.5. P<0.05 (Student’s t-test). (H) Percentage of
EdU+ cells among total cells within the VZ/SVZ at E17.5 in the experiment in F. P<0.01 (Student’s t-test). (I) E17.5 cerebral cortex samples from wild-type
and Lin28a transgenic mice were immunostained with an antibody against Tbr2 (red). Tbr2+ cells from the boxed area are shown in the right-hand panel.
(J) Percentage of Tbr2+ cells among total cells in the experiment in I. P=0.024 (Student’s t-test). (K) Confocal micrographs of coronal sections from E17.5 wild-type
and Lin28a transgenic mouse cortex stained with antibodies against Pax6 (green) and p-H3 (red). (L) Percentage of p-H3/Pax6 double-positive cells among
Pax6+ cells in K. P=0.03 (Student’s t-test). (M) Confocal micrographs of coronal sections from E17.5 mice 24 h after a single pulse label of BrdU; sections were
stained with antibodies against BrdU (green) and Ki67 (red). Arrowheads indicate cells that exited the cell cycle (BrdU+/Ki67–). (N) Percentage of BrdU+/Ki67–
cells among total BrdU+ cells in the experiment in M. P<0.05 (Student’s t-test). (E,G,H,J,L,N) Error bars indicate s.e.m. of nine sections from three independent
experiments. Scale bars: 1 cm in A; 0.5 mm in D; 50 µm in F,I,K; 20 µm in M.
Lin28 regulates Igf2-mTOR signaling in NPCs
Recent genome-wide studies have identified Lin28a and Imp1 targets
in HEK 293T cells (Hafner et al., 2013, 2010). Our finding that Lin28a
and Imp1 interact in NE-4C cells prompted us to examine their
common targets following RNA immunoprecipitation (RIP) to isolate
Lin28a-associated RNAs in NE-4C cells. This showed that Lin28a
is associated with mRNAs encoding Hmga2, a regulator of NPC
self-renewal (Nishino et al., 2008), and several components of the
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Development (2015) 142, 1616-1627 doi:10.1242/dev.120543
Igf2-mTOR signaling pathway, including Igf2, Igf1r, Akt1/3 and
Imp1 (Fig. 6A). Therefore, we next tested the hypothesis that Lin28a/b
regulates Igf2-mTOR signaling by examining the responses of wildtype and Lin28a−/− cerebral cortex tissues to Igf2 stimulation. Cortex
tissues were dissected from the lateral pallium of mouse brains at
E12.5, when more than 85% of the cells are NPCs, as evidenced by
Pax6 expression (data not shown), and cultured the tissue for 30 min
with Igf2. We used phosphorylation of Erk1/2 (Mapk3/1) at Thr202/
204 (p-Erk1/2) and of Akt at Ser473 (p-Akt) as readouts of Igf2
signaling activity (Lehtinen et al., 2011). Western blot results showed
that Igf2 treatment significantly increased p-Erk1/2 and p-Akt levels in
wild-type cortex tissue, whereas this response was significantly
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blunted in Lin28a mutant tissue (Fig. 6B-D). These studies indicate
that the response of Lin28a mutant cerebral cortex to Igf2 stimulation
is compromised.
To examine whether Igf2-mTOR signaling is disrupted in vivo, we
used phosphorylation of the S6 ribosome protein (Ser240/244 or
Ser235/236; p-S6) as a downstream readout of Igf2-mTOR signaling
activity (Ferrari et al., 1991; Lehtinen et al., 2011; Magri et al., 2011).
mTOR activity was reduced in NPCs (labeled by Pax6) of the Lin28a
knockout, as evidenced by a significant decrease of p-S6 levels
compared with controls at P1 (Fig. 6E,F; supplementary material
Fig. S4A,B) and at E14.5 in Lin28a−/−b+/− brains (Fig. 6I,J), at a time
when let-7 expression levels are unchanged in the mutant NPCs
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Fig. 5. Lin28a interacts with Imp1. (A) miRNA RT-PCR analysis of let-7 expression levels in NPCs isolated from E14.5 cerebral cortex of wild type and
Lin28a−/−b+/− compound mutants. n.s., not significant (P>0.05) (Student’s t-test). Error bars indicate s.e.m. from three independent experiments. (B) Lin28a
BAC with Flag tag was used to generate the NE-4C stable cell line. Lin28a protein was immunoprecipitated (IP) with m2 beads (anti-Flag) followed by western
blot (WB) analysis using an antibody against Imp1 or Imp2. K.D. indicates kDa. (C) Confocal micrographs of Flag-Lin28a stable NE-4C cells stained with antibodies
against Flag (Lin28a, green) and Imp1 (red). Hoechst stains nuclei (blue). Scale bar: 10 µm. (D) Schematic representation of Lin28a full-length and deletion
constructs. The interval between the RNA-binding CSD and CCHC domains of Lin28a is required for its interaction with Imp1. (E,F) Total lysates from HEK 293T
cells transfected with Flag-Lin28a or various deletion constructs were immunoprecipitated with anti-Flag antibody and western blot analysis was performed using
the antibodies indicated. (G-J) Twenty-four hours after in utero electroporation of the indicated plasmids, together with pCAG-H2BGFP constructs, into E14.5
brains, the animals were administered a single pulse label of BrdU for 1 h followed by immunostaining of cerebral cortex for GFP (green) and BrdU (red).
Arrowheads indicate GFP/BrdU double-positive cells. (K) Quantification of GFP/BrdU double-positive cells among total GFP+ cells from the experiment in G-J. Data
are expressed as s.e.m. of six sections from two independent experiments. *P<0.05 (Student’s t-test).
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Development (2015) 142, 1616-1627 doi:10.1242/dev.120543
(Fig. 5A). These data suggest that the integrity of Igf2-mTOR
signaling is compromised in NPCs of Lin28 loss-of-function mutants.
Consistent with the idea that Lin28a positively regulates Igf2-mTOR
signaling, the overexpression of Lin28a significantly increased p-S6
staining in the Lin28a Tg; Nestin-Cre mouse NPCs compared with
controls (Fig. 6G,H; supplementary material Fig. S4C,D) and
specifically in the VZ/SVZ of the cerebral cortex as early as E14.5
(Fig. 6K,L). Together, these data suggest that Lin28a regulates Igf2mTOR signaling in NPCs in vivo.
Igf1r and Hmga2 are functionally important targets of Lin28
in regulating NPC proliferation
To further understand the molecular mechanisms by which Lin28
regulates Igf2-mTOR signaling and NPC proliferation, we examined
the expression of proteins encoded by Lin28a/b-associated mRNAs
as confirmed in our RIP studies in NE-4C cells (Fig. 6A). The protein
levels of Igf1r, Akt3 and Hmga2 were significantly reduced in
NPCs isolated from Lin28a−/−b+/− cerebral cortex (Fig. 7A,B;
supplementary material Fig. 4G,H). By contrast, Akt1, Akt2 and
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Fig. 6. Lin28a/b regulate Igf2-mTOR signaling in NPCs. (A) Using the NE-4C stable cell line with Flag-tagged Lin28a, mRNAs associated with Lin28a were
pulled down using m2 (Lin28a) antibodies followed by RT-PCR analyses with the probes indicated. Error bars indicate s.e.m. of three independent experiments.
*P<0.05; n.s., not significant (P>0.05) (Student’s t-test). (B) Western blot analyses of the levels of p-Erk1/2 (Thr202/204) and p-Akt (Ser473) using
embryonic cortex tissues dissected from the lateral pallium of E12.5 mouse brains. The embryonic cortices were grown in DMEM in the presence or absence of
20 ng/ml Igf2 for 30 min. Total Erk1/2 and total Akt serve as controls. (C,D) Quantification of western blot data in experiment B using three independent blots.
Densitometry of blot signals was quantified with ImageJ software (NIH); levels of p-Erk1/2 or p-Akt in samples with Igf2 treatment were normalized to respective
controls without Igf2 treatment. Error bars indicate s.e.m. from three independent experiments. P<0.01 (C) and P=0.016 (D) (Student’s t-test). (E,G) Confocal
micrographs of coronal sections of cerebral cortex stained with antibodies against Pax6 (red) and p-S6 (Ser240/244) (green). Hoechst stains nuclei (blue).
The erebral cortex tissues are from P1 wild-type and Lin28a−/− mutant mice (E) or from E17.5 wild-type and Nestin-Cre-directed Lin28a-overexpression mice (G).
(F,H) Statistical analysis of fluorescence intensity of p-S6 (Ser240/244) in the VZ/SVZ of the cerebral cortex. P<0.01 (F) and P<0.001 (H) (Student’s t-test).
(I,K) Confocal micrographs of coronal sections from E14.5 Lin28a−/−b+/− compound mutant (I) or Lin28a-overexpression (K) cortex stained with antibodies
against p-S6 (Ser240/244). Hoechst stains nuclei (blue). The VZ/SVZ is the area below the white line. (J,L) Statistical analysis of fluorescence intensity of p-S6
(Ser240/244) at the VZ/SVZ in experiments in I or K. P<0.05 (Student’s t-test). (F,H,J,L) Error bars indicate s.e.m. of nine sections from three independent
experiments. Scale bars: 20 µm in E,G and I left; 50 µm in K left; 10 µm in I,K right.
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Development (2015) 142, 1616-1627 doi:10.1242/dev.120543
Fig. 7. Igf1r and Hmga2 are functionally important targets of Lin28a/b. (A) Western blot analysis of Igf1r and Hmga2 expression using lysates from NPCs of
wild-type and Lin28a−/−b+/− compound mutant cerebral cortex. (B) Quantification of western blot data in experiment A using three independent blots. n.s., not
significant; *P<0.05 (Student’s t-test). Error bars indicate s.e.m. from three independent experiments. (C,D) Confocal micrographs of coronal sections from
E14.5 wild-type and Lin28a−/−b+/− cerebral cortex stained with antibodies against Igf1r (red in C) and Hmga2 (red in D). Hoechst stains nuclei (blue).
(E-H) Twenty-four hours after in utero electroporation of the indicated plasmids, together with pCAG-H2BGFP constructs, into E14.5 brains, the animals were
administered with a single pulse label of BrdU for 1 h followed by immunostaining of cerebral cortex for GFP (green) and BrdU (red). Arrowheads indicate
GFP/BrdU double-positive cells. (I) Quantification of GFP/BrdU double-positive cells among total GFP+ cells from the experiment in E-H. Data are expressed as
s.e.m. of six sections from two independent experiments. *P<0.05 (Student’s t-test). (J) In the working model, Lin28a interacts with Imp1 to regulate the levels
of Igf1r and Hmga2 protein, therefore providing early NPCs with higher self-renewal capacity as compared with late NPCs in the developing brain. Scale
bars: 20 µm in C,D left; 10 µm in C,D right; 50 µm in E-H.
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Lin28a−/−b+/− NPCs (Fig. 7E-I). Together, these data suggest that
Lin28a/b regulate NPC proliferation, at least in part, through the
regulation of Igf1r and Hmga2 protein expression in the developing
brain.
DISCUSSION
Lin28a/b play essential roles in a variety of biological processes and
diseases, including ESC self-renewal, iPSC formation, cancers and
type II diabetes (Peng et al., 2011; Shyh-Chang and Daley, 2013;
Thornton and Gregory, 2012; Yu et al., 2007). However, the roles of
Lin28a/b in somatic progenitor cells remain largely unknown, and
their potential in vivo functions in neural stem/progenitor cells and
brain development have not been examined. Here, our gain- and
loss-of-function studies have identified Lin28a/b as crucial
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Imp1 protein levels were not significantly changed in Lin28a−/− or
Lin28a−/−b+/− NPCs compared with the control (supplementary
material Fig. 4E-H). Immunohistochemical staining confirmed the
reduction in Igf1r and Hmga2 in the VZ of the Lin28a−/−b+/−
cerebral cortex (Fig. 7C,D). These studies suggest that Lin28a/b
regulate the protein expression of Igf1r and Hmga2 in vivo.
Previous studies suggest that Igf2-mTOR signaling and Hmga2
are crucial regulators of NPC self-renewal and brain development
(Lehtinen et al., 2011; Magri et al., 2011; Nishino et al., 2008). To
examine the functional importance of Igf1r/Hmga2 downregulation
in mediating Lin28a/b functions in NPC proliferation, we
performed rescue experiments using the in utero electroporation
method. BrdU labeling studies showed that ectopic expression of
Hmga2 or Igf1r significantly rescued the proliferation deficiency of
regulators for the temporal control of NPC proliferation during
mouse brain development (Fig. 7J).
Our previous studies suggest that Lin28 regulates neurogliogenesis
in vitro (Vadla et al., 2012). Recent studies of human ESCdifferentiated NPCs suggest that LIN28 is capable of rescuing NPC
proliferation and neurogenic deficits in the absence of SOX2
(Cimadamore et al., 2013). Our current studies provide substantial
new information about Lin28a/b functions in vivo. First, we found that
Lin28a is not only necessary but also sufficient for promoting NPC
proliferation in mice. Second, this Lin28a-mediated NPC regulation is
functionally important for brain development because Lin28a lossand gain-of-function mice exhibit smaller and enlarged brains,
respectively. Third, whereas it is unclear if Lin28a and Lin28b play
distinct or redundant roles, our studies suggest that they have
overlapping functions in regulating NPC proliferation and brain
development. The RNA-binding proteins Lin28 and Lin41 are
founding members of the heterochronic pathway, which was
originally characterized in C. elegans, and are required for the
progression of specific developmental stages in the seam cells
(Ambros, 2011; Blaschke et al., 1998; Nimmo and Slack, 2009;
Rougvie and Moss, 2013). Previously, we showed that mammalian
Lin41 (Trim71 in mouse) regulates the balance of NPC self-renewal
and differentiation during neural development, and that loss of Lin41
results in precocious NPC differentiation (Chen et al., 2012); our
current study suggests that Lin28a/b promote the temporal
proliferation capacities of NPCs and brain development in mice.
Thus, these heterochronic genes play evolutionarily conserved roles to
regulate progenitor cell behaviors across different species.
Lin28a/b are highly expressed in early NPCs and their expression
is almost absent in the cerebral cortex after E14.5, based on western
blot analysis (supplementary material Fig. S1E), whereas E15.5 is
the earliest stage at which the cellular phenotypes can be detected in
the Lin28a−/− cerebral cortex (Fig. 2D-F). There are several possible
explanations for these observations. First, the early molecular
changes, such as the compromised Igf2 signaling in Lin28a−/−
NPCs at E12.5 (Fig. 6B-D), are not sufficient to cause detectable
cellular/morphological phenotypes until later developmental stages,
such as E15.5. Second, Lin28a/b have redundant functions, as
suggested by our analysis of Lin28a−/−b+/− compound mutants
(Fig. 3), which might lead to the late manifestation of cellular
phenotypes in the Lin28a single-knockout cerebral cortex. Lastly,
Lin28a is locally restricted to the VZ/SVZ of the cerebral cortex at
late developmental stages, based on immunohistochemical staining
(Fig. 1I). The near absence of Lin28a in western blot assays
(supplementary material Fig. S1E) could be due to the fact that the
protein lysate is from a mixture of NPCs and differentiated neurons.
Mechanistic studies of Lin28a/b functions have mainly focused
on inhibition of miRNA let-7 biogenesis (Shyh-Chang and Daley,
2013; Thornton and Gregory, 2012). Our previous in vitro studies
suggest that Lin28a regulates neurogliogenesis through let-7independent mechanisms (Balzer et al., 2010). Here, our in vivo
evidence further suggests that Lin28a/b may function through let7-independent mechanisms to regulate NPC proliferation. First,
let-7 expression levels remain unchanged in NPCs isolated from
E14.5 Lin28a−/−b+/− compound mutants (Fig. 5A), which already
exhibit smaller brains and reduced NPC numbers compared with
controls (Fig. 3A-D). Second, Lin28a is associated with mRNAs
encoding Hmga2 and components of the Igf2-mTOR pathway,
including Igf2, Igf1r, Akt1/3 and Imp1 (Fig. 6A). In the E14.5
NPCs of Lin28a−/−b+/− mutants, the protein levels of Hmga2
and Igf1r are reduced compared with controls (Fig. 7A-D).
Importantly, ectopic expression of Hmga2 or Igf1r can partially
Development (2015) 142, 1616-1627 doi:10.1242/dev.120543
rescue the proliferation defects of Lin28a−/−b+/− NPCs,
suggesting that these downstream targets are functionally
important in mediating Lin28a/b regulation of NPCs. Together,
these results suggest that Lin28a/b regulate NPC proliferation, at
least in part, through modulation of Igf2-mTOR signaling
activities and the production of Hmga2 protein (Fig. 7J).
A recent study showed that Imp1 temporally regulates NPC selfrenewal in the developing brain (Nishino et al., 2013). Interestingly,
we found that Lin28a specifically associates with Imp1 and not its
homolog Imp2 (Fig. 5B), and that the Imp1 protein level is
unchanged in Lin28a−/−b+/− NPCs. Thus, epistasis analysis will be
informative in defining the genetic interactions between Lin28a and
Imp1 in NPC regulation. It has been shown that Imp1 increases
protein production by enhancing the stability of its target mRNA
Hmga2 (Nishino et al., 2013), whereas Lin28a regulates mRNA
translation, based on studies in cultured cells (Cho et al., 2012;
Hafner et al., 2013). Therefore, examining the mRNA expression
levels of Lin28 candidate targets, including Hmga2 and Igf1r, in
Lin28a/b genetically modified mouse brains will further advance
our understanding of Lin28a/b functional mechanisms.
MATERIALS AND METHODS
Lin28a/b mutant and Lin28a transgenic mice
The generation of Lin28a knockout and inducible Lin28a transgenic mice is
described in the supplementary Materials and Methods. Lin28b knockout
mice were kindly provided by the Dr George Daley laboratory, and have
been described elsewhere (Shinoda et al., 2013).
Mutant phenotypic and BrdU/EdU labeling analyses
Histological processing, TUNEL assays, BrdU/EdU labeling and
immunohistochemical labeling of cryosections were performed as described
previously (Chen et al., 2014); details are provided in the supplementary
Materials and Methods and antibodies are listed in Table S1.
RNA immunoprecipitation (RIP)
To examine Lin28a-associated RNAs, we grew Flag-tagged Lin28a stable
NE-4C cells in 6×100 mm plates to 100% confluence. Cells were then crosslinked for 4 min using 245 nm UV, and collected using 1 ml of lysis buffer
[50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.5% NP40, and
one tablet of protease inhibitor (Roche) per 10 ml]. Cell lysate was
incubated on ice for 15 min, vortexed three times during incubation, and
centrifuged at 13,000 rpm (15,700 g) for 15 min. Supernatant was collected
in fresh tubes, and myc beads as a control (50 µl for three plates; Sigma) and
Flag beads (50 µl for three plates; Sigma) were added. The lysate was rotated
with beads at 4°C for 3 h followed by centrifugation at 6000 rpm (3300 g)
for 2 min. Supernatant was removed and the beads washed four times with
1 ml wash buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA,
0.5% NP40) for 10 min each. Trizol (Invitrogen) was used to extract RNAs
followed by RT-PCR analyses.
Affinity purification of Lin28a-associated proteins
Lin28a-interacting proteins were purified from genetically modified Lin28a
BAC-transfected NE-4C mouse cells as detailed in the supplementary
Materials and Methods.
Quantitative RT-PCR
For the mRNA RT-PCR analyses in Fig. 6A, RNA samples were from RIP
experiments as described above and the probes listed were obtained from
Life Technologies. For the miRNA let-7 RT-PCR in Fig. 5A, E14.5 wildtype and Lin28a−/−b+/− dorsal telencephalon tissue was microdissected, and
isolated NPCs were cultured in adherent culture medium overnight or in
self-renewal medium for 5 days before RNA collection (for NPC isolation
and culture see the supplementary Materials and Methods). RNAs were
extracted using Trizol and converted into cDNA using the TaqMan
MicroRNA Reverse Transcription Kit (Applied Biosystems). Stem-loop
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RESEARCH ARTICLE
reverse transcription primers for individual let-7 members were used for the
reverse transcription reaction, followed by the real-time PCR analysis,
which provides quantitation of mature let-7 miRNA expression.
In utero electroporation
Mouse cortical NPCs were electroporated with various plasmids as
described previously (Chen et al., 2014). Briefly, DNA constructs were
prepared using the EndoFree Plasmid Maxi Kit (Qiagen), including
pCAG-H2BGFP, pCAG-Lin28a, pCAG-Lin28aΔ113-138, pCAG-Igf1r
and pCAG-Hmga2. Individual plasmids or combinations, as indicated
(Fig. 5G-J and Fig. 7E-H), plus 0.5% Fast Green (Sigma) were injected
into the lateral ventricle of E14.5 brains followed by electroporation
using an ECM 830 electroporator (BTX) with four 100 ms pulses
separated by 100 ms intervals at 32 V. To improve retention of the
pregnancy, we avoided plasmid injection and electroporation of the two
embryos next to the upper vagina. In utero development was allowed to
continue for 24 h (or as indicated) followed by intraperitoneal injection
of BrdU at 100 mg/kg body weight for 1 h before embryo dissection.
E15.5 brains were dissected and processed for immunohistochemical
staining analysis as described above.
Acknowledgements
We thank Lori Bulwith for technical assistance and our laboratory colleagues for
stimulating discussions; Dr F. C. Nielsen for Imp1, Imp2 and Imp3 antibodies;
Dr Anthony A. Hyman for the R6Kamp-hNGFP plasmid used for the generation of
tagged Lin28a BAC; and the Dr George Daley laboratory for sharing Lin28b
knockout mice.
Competing interests
The authors declare no competing or financial interests.
Author contributions
M.Y., S.-L.Y., C.L. and J.-F.C. conceived and performed all experiments. S.H.,
E.G.M. and L.N. helped with the manuscript writing. R.D. and A.N. generated Lin28a
transgenic mice. M.D. and K.C.H. performed mass spectrophotometry. J.-F.C.
designed and interpreted the experiments and wrote the manuscript.
Funding
We are grateful for the support of The National Institutes of Health (NIH)/NICHD
[K99HD073269 to J.-F.C.] and the work was supported in part by the University of
Georgia start-up fund, NIH [HD081562 and NS085749 to L.N.], NIH/NCATS Colorado
CTSI [UL1 TR000154 to L.N.], the Neuroscience Program [NS48154 to L.N.], the
Colorado IDDRC (Animal Models Core; to L.N.) and the National Science Foundation
(NSF) [IOS-0924497 to E.G.M.]. Deposited in PMC for release after 12 months.
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.120543/-/DC1
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