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Cold-induced protein RBM3 orchestrates neurogenesis via modulating Yap mRNA stability in cold stress

Rockefeller University Press
Journal of Cell Biology (JCB)
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In mammals, a constant body temperature is an important basis for maintaining life activities. Here, we show that when pregnant mice are subjected to cold stress, the expression of RBM3, a cold-induced protein, is increased in the embryonic brain. When RBM3 is knocked down or knocked out in cold stress, embryonic brain development is more seriously affected, exhibiting abnormal neuronal differentiation. By detecting the change in mRNA expression during maternal cold stress, we demonstrate that Yap and its downstream molecules are altered at the RNA level. By analyzing RNA-binding motif of RBM3, we find that there are seven binding sites in 3′UTR region of Yap1 mRNA. Mechanistically, RBM3 binds to Yap1-3′UTR, regulates its stability, and affects the expression of YAP1. RBM3 and YAP1 overexpression can partially rescue the brain development defect caused by RBM3 knockout in cold stress. Collectively, our data demonstrate that cold temperature affects brain development, and RBM3 acts as a key protective regulator in cold stress.
Cooling affects neurogenesis. (A–C) Cold stress was induced at E14, and brains were collected at E16. Embryonic brain sections from rostral to caudal in different temperature conditions were costained with anti-TUJ1 and anti-Nestin antibodies. The quantification shows the relative density of fluorescence. The sketch on the left shows the rostral, central, and caudal sections, and the area selected for the analysis of the phenotype. (n = 3 brains). (D) Cold stress was induced at E14, and brains were collected at E16. BrdU was injected 2 h before brain collection. Embryonic brain sections in different temperature conditions were costained with anti-PAX6 and anti-BrdU antibodies. The bar graph shows the number of BrdU+PAX6+ cells in the VZ/SVZ per 40,000 µm² (n = 3 brains). (E) Cold stress was induced at E14, and brains were collected at E16. BrdU was injected 2 h before brain collection. Embryonic brain sections in different temperature conditions were costained with anti-Tbr2 and anti-BrdU antibodies. The bar graph shows the number of BrdU+Tbr2+ cells in the VZ/SVZ per 40,000 µm² (n = 3 brains). (F) Cold stress was induced at E14, and brains were collected at E16. Embryonic brain sections in different temperature conditions were stained with anti-SOX2 antibody. The bar graph shows the number of SOX2 cells in the VZ/SVZ per 40,000 µm² (n = 3 brains). (G) Cold stress was induced at E14, and brains were collected at E16. Embryonic brain sections in different temperature conditions were costained with anti-SATB2 and anti-CTIP2 antibodies. The bar graph shows the number of SATB2+ (or CTIP2+) cells per 250,000 µm² (n = 3 brains). (H) Cold stress was induced at E14, and brains were collected at E16. BrdU was injected 24 h before brain collection. Embryonic brain sections in different temperature conditions were costained with anti-Ki67 and anti-BrdU antibodies. The arrows indicate the cells that are BrdU+Ki67−. The bar graph shows the ratio of BrdU+Ki67−/BrdU+ cells (or BrdU+Ki67+/BrdU+ cells) in the VZ/SVZ (n = 3 brains). (I) NSCs were isolated at E12 and then cultured at 37°C or 32°C for 2 d. BrdU was added into the culture medium 1 h before the cells were fixed. In addition, the cells at different temperatures were costained with anti-TUJ1 and anti-BrdU antibodies. The bar graphs represent the ratio of TUJ1+/Dapi+ cells (or BrdU+/Dapi+ cells; n = 3 independent experiments). (J) NSCs were isolated from the E12 brain and then cultured at 37°C or 32°C for 2 d. The cells in different temperature conditions were costained with anti-Ki67 and anti-Map2 antibodies. The bar graphs represent the ratio of Map2+/Dapi+ cells (or Ki67+/Dapi+ cells). (n = 3 independent experiments). Bars: 30 µm (A–C); 20 µm (D, E, and H); 15 µm (F); 50 µm (G); 10 µm (I and J). *, P < 0.05; **, P < 0.01; ***, P < 0.001. Error bars represent the mean ± SEM.
… 
Maternal cold stress can induce the expression of RBM3 and affect the development of the cerebral cortex. (A and B) The results of IUE showed that the induction of maternal cold stress decreased the GFP+ cells in CP and increased the GFP+ cells in the VZ/SVZ region. The GFP-expression plasmid was electroporated into E13 embryonic mouse brains; cold stress was induced at E14, and the phenotype was analyzed at E16. The percentage of GFP-positive cells in each region is shown (n = 3 brains). (C and D) The GFP-expression plasmid was electroporated into E13 embryonic mouse brains, and cold stress was induced at E14; brain sections at the different temperatures were stained by anti-MAP2 antibody at E16. The percentage of GFP and MAP2 double-positive cells in the CP layer is shown (n = 3 brains). (E and F) The GFP-expression plasmid was electroporated into E13 embryonic mouse brains, and cold stress was induced at E14; brain sections at the different temperatures were stained by anti-PAX6 antibody at E16. The percentage of GFP and PAX6 double-positive cells in the VZ/SVZ is shown (n = 3 brains). (G) Cold stress was induced at E14, and the cortex was isolated for mRNA collection at E16. The indicated genes were detected by real-time PCR (n = 3 independent experiments). (H) The indicated cold-related genes’ mRNA levels in the NSCs at different development periods were examined. (I and J) The protein samples from cold stress–induced and normal body temperature embryonic brains were analyzed by Western blotting. The bar graph shows the change in RBM3 under different temperature conditions (n = 6 brains). (K and L) Western-blot analysis showing the neurogenesis-related markers’ changes in embryonic brains from the cold stress–induced and normal body temperature groups. The bar graph shows the changes at the different temperature conditions (n = 6 brains). Bars: 100 µm (A–D); 50 µm (E and F). *, P < 0.05; **, P < 0.01; ***, P < 0.001. Error bars represent the mean ± SEM.
… 
Maternal cold stress induces a global change in the transcriptome and promotes high expression of Yap1 in NSCs. (A) Schematic explaining the steps of the RNA-Seq experiment. (B and C) The volcano plot and heat map analysis showed the gene profiling expression. Gene analysis revealed that the transcript levels of 2,702 genes were up-regulated (455 genes) or down-regulated (2,247 genes) in the RBM3-knockdown and cold stress–induced group. (D) The indicated genes associated with neurogenesis were detected by real-time PCR in the cold stress–induced and normal body temperature groups. The cold stress was induced at E14, and then total mRNA from the embryonic brain was collected for quantitative analysis at E16 (n = 5 independent experiments). (E) The expression of Yap1 downstream genes, including ctgf, cyr61, and ccnd1, were increased in the cold stress–induced brains (n = 5 independent experiments). (F and G) IUE was performed at E13, and then cold stress was induced at E14. Next, GFP+ cells were isolated from the embryonic brains. Real-time PCR (F) and Western blotting (G) were used to detect the expression of YAP (n = 3 independent experiments). (H and I) The protein level of Yap1 was increased when cold stress was induced. Cold stress was induced at E14, and the embryonic cortex was collected at E16 for Western blotting (n = 6 brain samples). (J) Yap1 mRNA was degraded faster in NSCs cultured at 37°C versus 32°C, as measured by an mRNA semiquantitative experiment. Actinomycin D (5 µg/ml) was added into the culture medium to inhibit the transcription. The same amount of mRNA in the indicated time phase was reverse transcribed, and PCR was performed (n = 3 independent experiments). (K) Under the 32°C culture conditions, Yap increased faster at the mRNA level in the NSCs. The NSCs isolated from the E12 brains were cultured in the proliferation medium at 37°C for 24 h and then placed at the indicated temperature for analysis at different time phases (n = 3 independent experiments). *, P < 0.05; **, P < 0.01; ***, P < 0.001. Error bars represent the mean ± SEM.
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ARTICLE
Rockefeller University Press https://doi.org/10.1083/jcb.201801143 3464
J. Cell Biol. 2018 Vol. 217 No. 103464–3479
Rockefeller University Press
In mammals, a constant body temperature is an important basis for maintaining life activities. Here, we show that when
pregnant mice are subjected to cold stress, the expression of RBM3, a cold-induced protein, is increased in the embryonic
brain. When RBM3 is knocked down or knocked out in cold stress, embryonic brain development is more seriously aected,
exhibiting abnormal neuronal dierentiation. By detecting the change in mRNA expression during maternal cold stress,
we demonstrate that Yap and its downstream molecules are altered at the RNA level. By analyzing RNA-binding motif of
RBM3, we nd that there are seven binding sites in 3′UTR region of Yap1 mRNA. Mechanistically, RBM3 binds to Yap1-3′UTR,
regulates its stability, and aects the expression of YAP1. RBM3 and YAP1 overexpression can partially rescue the brain
development defect caused by RBM3 knockout in cold stress. Collectively, our data demonstrate that cold temperature
aects brain development, and RBM3 acts as a key protective regulator in cold stress.
Cold-induced protein RBM3 orchestrates neurogenesis
via modulating Yap mRNA stability in cold stress
WenlongXia1,2,3, LiboSu1,2, and JianweiJiao1,2
Introduction
Stress affects neuronal development of the fetus during maternal
pregnancy. For example, nutritional status (Steenweg-de Graaff
et al., 2017), emotions (Dworkin and Losick, 2001), alcoholism
(Tyler and Allan, 2014), and infection (Toyama et al., 2015; Tang
et al., 2016) in the progeny have been reported to be associated
with the development of the fetal brain.
Among the potential subpopulations vulnerable to
temperature changes, pregnant women have received less
attention. During the process of pregnancy, the temperature of
pregnant mammals remains stable. Compared with exposure to
room temperature (24.4°C), weekly exposures during the last 4
wk of pregnancy to extreme cold was found to be associated with
a 17.9% increased risk of preterm birth (He et al., 2016).
Cumulative and acute exposures to extremely low temperatures
may induce maternal stress during pregnancy (Lin et al., 2017b).
There is increasing evidence that temperature plays a role as a
trigger of adverse birth outcomes, such as preterm birth, low birth
weight, and stillbirth (Ha et al., 2017; Zhang et al., 2017). Howeve r,
the relationship between maternal temperature and fetal brain
development remains unknown. Cold stress is an important
stimulus to the mother and fetus during pregnancy (Kali et al.,
2016). Maternal cold stress can lead to abnormal fetal development
and may even cause miscarriage. However, whether maternal cold
stress affects the brain development is largely unclear.
Neocortical development is a spatially and temporally
regulated process that is defined by an early expansion of
proliferative neural stem cells (NSCs) that reside in the
ventricular zone (VZ) of the embryonic cortical epithelium (Fang
et al., 2013). In the development process, any external stimuli are
likely to affect the fate of NSCs and then affect the structure or
function of the brain (Durak et al., 2016).
The biological function of RBM3 during embryonic brain
development is little known. RBM3, which was initially defined
as an RNA-binding protein (Derry et al., 1995; Dresios et al.,
2005), is induced to be expressed at low temperatures (Danno
et al., 1997). It is associated with the structural plasticity and
protective effects of cooling in neurodegeneration (Peretti et
al., 2015). RBM3 is also related to changes in the expression
of different RNAs during the circadian rhythm of body
temperature (Liu et al., 2013) and regulates the expression of
temperature-sensitive miRNAs (Wong et al., 2016). However,
whether RBM3 is involved in the temperature-associated
regulation of brain development and neural stem cell
development is also unknown.
The activity and expression of Yap1 can be rapidly regulated
by a variety of life activities (Lin et al., 2015, 2017a). For example,
Yap1 has been associated with energy homeostasis (Wang et al.,
2015), mechanical pressure (Aragona et al., 2013), G-protein
coupled receptor signaling, and oxidative stress (Lehtinen
et al., 2006). However, whether YAP1 is able to participate in
certain biological processes under a low temperature followed
by prolonged cold stress is still unknown.
© 2018 Xia et al. is article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the rst six months aer the
publication date (see http:// www .rupress .org/ terms/ ). Aer six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0
International license, as described at https:// creativecommons .org/ licenses/ by -nc -sa/ 4 .0/ ).
1State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China; 2University of Chinese Academy of
Sciences, Beijing, China; 3School of Life Sciences, University of Science and Technology of China, Hefei, Anhui, China.
Correspondence to Jianwei Jiao: jwjiao@ ioz .ac .cn. 
Journal of Cell Biology
https://doi.org/10.1083/jcb.201801143
Xia et al.
RBM3 regulates brain development
3465
Figure 1.Cooling aects neurogenesis. (A–C) Cold stress was induced at E14, and brains were collected at E16. Embryonic brain sections from rostral to
caudal in dierent temperature conditions were costained with anti-TUJ1 and anti-Nestin antibodies. e quantication shows the relative density of uores-
cence. e sketch on the le shows the rostral, central, and caudal sections, and the area selected for the analysis of the phenotype. (
n
= 3 brains). (D) Cold
stress was induced at E14, and brains were collected at E16. BrdU was injected 2h before brain collection. Embryonic brain sections in dierent temperature
conditions were costained with anti-PAX6 and anti-BrdU antibodies. e bar graph shows the number of BrdU+PAX6+ cells in the VZ/SVZ per 40,000 m2
(
n
= 3 brains). (E) Cold stress was induced at E14, and brains were collected at E16. BrdU was injected 2h before brain collection. Embryonic brain sections in
dierent temperature conditions were costained with anti-Tbr2 and anti-BrdU antibodies. e bar graph shows the number of BrdU+Tbr2+ cells in the VZ/SVZ
per 40,000 m2 (
n
= 3 brains). (F) Cold stress was induced at E14, and brains were collected at E16. Embryonic brain sections in dierent temperature condi-
tions were stained with anti-SOX2 antibody. e bar graph shows the number of SOX2 cells in the VZ/SVZ per 40,000 m2 (
n
= 3 brains). (G) Cold stress was
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RBM3 regulates brain development
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Here, we examined the in vivo effects of RBM3 disruption
on embryonic mammalian brain development under differ-
ent maternal body temperature environments. We found that
knockdown of RBM3 in cold stress, but not in the normal body
temperature condition, results in reduced cortical neural pro-
genitor proliferation and altered neurogenesis. In particular, the
expression of Yap1 in the sample of the embryonic cerebral cor-
tex in cold stress was increased compared with that at normal
temperature. During cold stress, the expression of Yap1 in the
sample of the RBM3 knockout embryonic cerebral cortex was
decreased compared with that in the littermate control. Fur-
thermore, our results showed that RBM3 regulated the stability
of Yap1 mRNA by binding to the 3UTR region of Yap1 mRNA.
We confirmed that knockout of RBM3 led to brain development
defects in cold stress.
Together, these observations provide new insights into the
roles of cold stress during brain development. Our results reveal
the function of RBM3 during neocortical development in prena-
tal cold stress. The data suggest that RBM3 and Yap1 act as poten-
tial cold shock proteins in brain development.
Results
Cold stress affects neurogenesis and induces the
expression of RBM3
It has been reported that many external environmental factors
affect the development of the fetal brain. Whether a change in
constant body temperature, an important feature of mammals,
affects the development of the cerebral cortex is unknown. To
examine the effect of maternal body temperature changes on em-
bryonic brain development, we injected 5-AMP (0.7 mg/g; Tao
et al., 2011; Peretti et al., 2015) into pregnant mice to induce cold
stress, and the rectal temperatures were recorded at different
time points to determine the reduction in body temperature in
these pregnant mice (Fig. S1 A). The immunofluorescence stain-
ing results showed that TUJ1+ neuronal cells were decreased after
the induction of cold stress in pregnant mice, and the proportion
of NES TIN+ NSCs was increased (Fig.1, A–C). The immunoflu-
orescence staining results also showed that the proportion of
PAX6+BrdU+ (or TBR2+BrdU+ or SOX2+) NSCs was increased
(Fig.1, D–F), and SATB2 (or CTIP2)-positive neuronal cells were
decreased after the induction of cold stress in pregnant mice
(Fig.1G). The result of the cell cycle exit experiment showed that,
after the induction of maternal cold stress, the proliferation of
NSCs was increased, and the percentage of NSCs exiting the cell
cycle was decreased (Fig.1H). The results of in vitro culture of
NSCs also showed that cooling inhibited the differentiation into
neurons and promoted the proliferation of NSCs (Fig.1, I and J).
We also found that after induction of cold stress, fetal and neo-
natal mice had a reduced brain size at embryonic day 16 (E16)
and postnatal day 2 (P2) compared with that of the control group
(Fig. S1, B–E). Together, these results suggest that maternal body
temperature as an important physiological indicator plays an im-
portant role in regulating embryonic brain development.
To study the effects of cold stress on embryonic cortical devel-
opment, in utero electroporation (IUE) was used to introduce a
GFP-expression plasmid into the cold stress–induced embryonic
cerebral cortex and into the cortex of the normal body tempera-
ture group. The results of IUE showed that the induction of ma-
ternal cold stress decreased the GFP+ cells in the cortical plate
(CP) and increased the GFP+ cells in the VZ/subventricular zone
(SVZ) region compare with the control group. (Fig.2, A and B).
The ratio of GFP+MAP2+ neuronal cells in the CP layer decreased,
and the ratio of PAX6+GFP+ NSCs increased in the VZ/SVZ re-
gion when the maternal cold stress was induced (Fig.2, C–F).
To detect which changes occurred in the gene expression level
in the embryonic brain leading to the abnormal development
during maternal cold stress, according to the previous studies,
the indicated genes clusters were tested, including genes asso-
ciated with low temperature sensitivity (RBM3 [Danno et al.,
1997, Cirbp [Nishiyama et al., 1997], trpm8 [Palkar et al., 2018],
and trpa1 [Moore et al., 2018]), metabolic sensitivity (HIF1a [Luo
et al., 2018] and Ucp1 and Ucp2 [Margaryan et al., 2017]), and
high temperature sensitivity (trpm2, trpv1, and trpv3 [Tan and
McNaughton, 2016]; Fig.2G). The results showed that the mRNA
of the cold-induced protein RBM3 was significantly increased.
We also examined the expression of these low temperature sen-
sitive genes (RBM3, Cirbp, trpm8, and trpa1) at different develop-
ment periods (E10, E12, E15, and E18) and found that RBM3 was
the most highly expressed gene in the embryonic brain among
the genes analyzed here (Fig.2H). Western blotting analysis and
immunofluorescence staining of RBM3 showed that the expres-
sion of RBM3 (Fig.2, I and J; and Fig. S1, F and G), NSC markers
(Pax6 and Tbr2), and proliferation markers (PCNA and pH3)
were increased, but the neuronal marker Tuj1 was decreased
(Fig.2, K and L), when the cold stress was induced. These results
indicate that RBM3 is involved in the brain development process
under cold stimulation conditions.
RBM3 regulates neurogenesis during the maternal cold stress
We used IUE to introduce RBM3 shRNA into neural progenitor
cells in E13 mouse embryos in pregnant mice under different
induced at E14, and brains were collected at E16. Embryonic brain sections in dierent temperature conditions were costained with anti-SATB2 and anti-CTIP2
antibodies. e bar graph shows the number of SATB2+ (or CTIP2+) cells per 250,000 m2 (
n
= 3 brains). (H) Cold stress was induced at E14, and brains were
collected at E16. BrdU was injected 24h before brain collection. Embryonic brain sections in dierent temperature conditions were costained with anti-Ki67
and anti-BrdU antibodies. e arrows indicate the cells that are BrdU+Ki67−. e bar graph shows the ratio of BrdU+Ki67−/BrdU+ cells (or BrdU+Ki67+/BrdU+
cells) in the VZ/SVZ (
n
= 3 brains). (I) NSCs were isolated at E12 and then cultured at 37°C or 32°C for 2 d. BrdU was added into the culture medium 1h before
the cells were xed. In addition, the cells at dierent temperatures were costained with anti-TUJ1 and anti-BrdU antibodies. e bar graphs represent the ratio
of TUJ1+/Dapi+ cells (or BrdU+/Dapi+ cells;
n
= 3 independent experiments). (J) NSCs were isolated from the E12 brain and then cultured at 37°C or 32°C for
2 d. e cells in dierent temperature conditions were costained with anti-Ki67 and anti-Map2 antibodies. e bar graphs represent the ratio of Map2+/Dapi+
cells (or Ki67+/Dapi+ cells). (
n
= 3 independent experiments). Bars: 30 m (A–C); 20 m (D, E, and H); 15 m (F); 50 m (G); 10 m (I and J). *, P < 0.05; **,
P < 0.01; ***, P < 0.001. Error bars represent the mean ± SEM.
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Figure 2.Maternal cold stress can induce the expression of RBM3 and aect the development of the cerebral cortex. (A and B) e results of IUE
showed that the induction of maternal cold stress decreased the GFP+ cells in CP and increased the GFP+ cells in the VZ/SVZ region. e GFP-expression plas-
mid was electroporated into E13 embryonic mouse brains; cold stress was induced at E14, and the phenotype was analyzed at E16. e percentage of GFP-pos-
itive cells in each region is shown (
n
= 3 brains). (C and D) e GFP-expression plasmid was electroporated into E13 embryonic mouse brains, and cold stress
was induced at E14; brain sections at the dierent temperatures were stained by anti-MAP2 antibody at E16. e percentage of GFP and MAP2 double-positive
cells in the CP layer is shown (
n
= 3 brains). (E and F) e GFP-expression plasmid was electroporated into E13 embryonic mouse brains, and cold stress was
induced at E14; brain sections at the dierent temperatures were stained by anti-PAX6 antibody at E16. e percentage of GFP and PAX6 double-positive cells
in the VZ/SVZ is shown (
n
= 3 brains). (G) Cold stress was induced at E14, and the cortex was isolated for mRNA collection at E16. e indicated genes were
detected by real-time PCR (
n
= 3 independent experiments). (H) e indicated cold-related genes’ mRNA levels in the NSCs at dierent development periods
were examined. (I and J) e protein samples from cold stress–induced and normal body temperature embryonic brains were analyzed by Western blotting. e
bar graph shows the change in RBM3 under dierent temperature conditions (
n
= 6 brains). (K and L) Western-blot analysis showing the neurogenesis-related
markers’ changes in embryonic brains from the cold stress–induced and normal body temperature groups. e bar graph shows the changes at the dierent
temperature conditions (
n
= 6 brains). Bars: 100 m (A–D); 50 m (E and F). *, P < 0.05; **, P < 0.01; ***, P < 0.001. Error bars represent the mean ± SEM.
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Figure 3.RBM3 regulates NSC proliferation and neuronal dierentiation during the maternal cold stress. (A) RBM3 knockdown causes GFP-positive
cell positioning changes in the embryonic brain in the cold stress–induced mice. An empty shRNA control or RBM3-KD plasmid was electroporated into E13
embryonic mouse brains, and the phenotype was analyzed at E16. e pregnant mice were cooled using intraperitoneally injected 5-AMP (0.7 mg/g). e per-
centage of GFP-positive cells in each region is shown (
n
= 3 brains). (B) RBM3 knockdown decreases neuronal dierentiation. E16 brain sections were stained
for TUJ1 aer the electroporation of the control or RBM3 knockdown plasmid into the brain at E13; the cold stress was induced at E14, and then the mice were
maintained at low temperature or room temperature for 48h. e percentage of GFP and TUJ1 double-positive cells relative to the total GFP-positive cells is
shown as a bar graph (
n
= 3 brains). Bar: 100 m. (C) e percentage of GFP and PAX6 double-positive progenitor cells is decreased by RBM3 knockdown in
cold stress. e mouse brain was electroporated at E13; the cold stress was induced at E14, and then the mice were maintained at low temperature or room
temperature for 48h. e percentage of GFP+PAX6+ cells relative to the total GFP-positive cells in the VZ/SVZ is shown. (
n
= 3 brains). Bars: 100 m (A and
B); 50 m (C). *, P < 0.05; **, P < 0.01; ***, P < 0.001. Error bars represent the mean ± SEM.
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RBM3 regulates brain development
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body temperatures (normal body temperature or induced cold
stress). We used immunostaining and Western methods to detect
the knockdown efficiency (Fig. S2, A–D). Interestingly, we found
that in the induction of maternal cold stress conditions, knock-
down of RBM3 led to significant changes in the GFP-positive cell
distribution. There was a significant loss of GFP-positive cells in
the proliferation VZ/SVZ, and GFP-positive cells in the cortical
plate were also reduced (Fig.3A). These results indicated that
RBM3 was involved in the development of the fetal cerebral cor-
tex in the maternal cold stress state. When the cold stimulation
occurred during development, the embryonic cortical develop-
ment was more seriously affected without RBM3 participation.
To confirm whether RBM3 knockdown leads to decreased
neuronal differentiation when cold stress is induced, we im-
munostained electroporated brain sections with the neuronal
markers TUJ1 and MAP2. The results revealed that knockdown
of RBM3 during the maternal cold stress led to a significant de-
crease in GFP-TUJ1 double-positive cells in CP layer (Fig.3B). The
quantification of GFP-MAP2 double-positive cells revealed that
knockdown of RBM3 led to an obvious decrease in the percentage
of MAP2+ neurons (Fig. S2 E). These results demonstrated that
RBM3 knockdown in the induced maternal cold stress resulted in
a decrease in neuronal differentiation. The result of PAX6 immu-
nofluorescence staining showed that knockdown of RBM3 in cold
stress resulted in a decreased proportion of radial glial cells in
the VZ/SVZ (Fig.3C). To test neural progenitor cell proliferation,
pregnant dams were injected with BrdU 2h before brain collec-
tion at E16. The result showed that knockdown of RBM3 during
the maternal cold stress caused a reduction of BrdU and GFP dou-
ble-positive cells versus the control (Fig. S2 F). The result of TBR2
immunofluorescence staining showed that knockdown of RBM3
in cold stress resulted in a decreased proportion of BPs in the VZ/
SVZ (Fig. S2 G). IUE under the shorter cold exposure time was
preformed to ensure that the abnormal brain development was
Figure 4.RBM3 knockdown aects the neu-
ronal development in cold stress–induced
mice. (A and B) RBM3 knockdown caused
GFP-positive cell positioning changes in the
cold stress–induced embryonic brain in a long-
term experiment. e empty shRNA control or
RBM3-KD plasmid was electroporated into E13
embryonic mouse brains, and the phenotype was
analyzed at P0. e pregnant mice were cooled at
E14. e percentage of GFP-positive cells in each
region is shown (
n
= 3 brains). (C and D) RBM3
knockdown causes abnormal development of
GFP-positive cells, which were isolated from the
cold stress–induced group. IUE was performed
at E13, and then cold stress was induced at E14.
Subsequently, the GFP+ cells were isolated from
the embryonic brains and cultured for 2 d. e
length of the process in each indicated condition
is shown (
n
= 3 independent experiments). Bars:
100 m. ***, P < 0.001. Error bars represent
the mean ± SEM.
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Figure 5.Maternal cold stress induces a global change in the transcriptome and promotes high expression of Yap1 in NSCs. (A) Schematic explaining
the steps of the RNA-Seq experiment. (B and C) e volcano plot and heat map analysis showed the gene proling expression. Gene analysis revealed
that the transcript levels of 2,702 genes were up-regulated (455 genes) or down-regulated (2,247 genes) in the RBM3-knockdown and cold stress–induced
group. (D) e indicated genes associated with neurogenesis were detected by real-time PCR in the cold stress–induced and normal body temperature
groups. e cold stress was induced at E14, and then total mRNA from the embryonic brain was collected for quantitative analysis at E16 (
n
= 5 independent
experiments). (E) e expression of Yap1 downstream genes, including ctgf, cyr61, and ccnd1, were increased in the cold stress–induced brains (
n
= 5
independent experiments). (F and G) IUE was performed at E13, and then cold stress was induced at E14. Next, GFP+ cells were isolated from the embryonic
brains. Real-time PCR (F) and Western blotting (G) were used to detect the expression of YAP (
n
= 3 independent experiments). (H and I) e protein level
of Yap1 was increased when cold stress was induced. Cold stress was induced at E14, and the embryonic cortex was collected at E16 for Western blotting
(
n
= 6 brain samples). (J) Yap1 mRNA was degraded faster in NSCs cultured at 37°C versus 32°C, as measured by an mRNA semiquantitative experiment.
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caused by a primary response to maternal cold stress. Abnormal
NSC proliferation and differentiation were also observed with
short time cold stress treatment (Fig. S3, A–E).
Long-term experimental results (from E13 to P0) showed that
the RBM3 knockdown defect phenotype was persistent under the
maternal cold stress condition, but there was no obvious phe-
notype in the normal body temperature group (Fig.4, A and B).
Our results also showed that RBM3 knockdown affected neuro-
nal development in vitro when cells were isolated from the cold
stress–induced group, but there was no obvious phenotype for
the cells isolated under the normal body temperature condition
(Fig.4, C and D).
Maternal cold stress induces a global change in
the transcriptome
To gain a deeper insight into how RBM3 regulates neural progen-
itor proliferation and differentiation in cold stress, we examined
the transcriptome of dividing cells following RBM3 knockdown.
We performed IUE using either control or RBM3 shRNA in the
cold stress. The GFP+ dividing cells were isolated through flu-
orescence-activated cell sorting 48h after the IUE. Following
total RNA extraction, transcriptional sequencing was performed
to identify differentially expressed genes between control and
RBM3-knockdown cells (Fig.5A).
Our results revealed that 2,702 genes (including 2,247
down-regulated genes and 455 up-regulated genes; fold change
>2) in the RBM3 knockdown samples were differentially ex-
pressed compared with control samples when cold stress was
induced (Fig.5, B and C). Together, these data suggest that RBM3
has an essential role in regulating the prenatal genes necessary
for temperature-related embryonic development. In addition,
cold stress did not change the expression of certain important
neuronal migration-related genes, and further study revealed
that the abnormal phenotype induced by RBM3 knockdown was
also not caused by neuronal migration (Fig. S3, F and G).
Maternal cold stress promotes high expression
of Yap1 in NSCs
To determine which genes were changed in the induction
of maternal cold stress that was associated with the NSC
proliferation and differentiation in our experiment, we detected
some of the NSC proliferation- and differentiation-related
genes. The results showed that Yap1 and its downstream genes
(ctgf, cyr61, and ccnd1) were significantly up-regulated when
cold stress was induced (Fig.5, D and E). After checking the
RNA-Seq data, we found that there was 60% decrease for Yap1
expression in RBM3 knockdown sample under the cold stress–
induced condition. Real-time PCR and Western blotting were
used for further confirmation of YAP reduction (Fig.5, F and G).
Western blot analysis showed that the expression of YAP1
was increased in the cold stress embryonic cortex (Fig.5, H and
I). To investigate whether low temperature stress affects the
stability of Yap1 mRNA and thus affects the expression of YAP1,
we checked the half-life of Yap1 mRNA at 37°C and 32°C in NSCs
by adding actinomycin D, which is an intercalating transcription
inhibitor. The result of the mRNA semiquantitative experiment
showed that the half-life of Yap1 mRNA was extended at the 32°C
condition relative to the 37°C condition (Fig.5 J). In addition,
under the 32°C culture condition, Yap1 increased faster at the
mRNA levels in the NSCs (Fig.5K). These results indicate that
Yap1 may respond to cold stimuli and act as a potential cold-
induced protein.
RBM3 promotes YAP expression by binding to the Yap1 mRNA
3′UTR and increasing its stability
Knowing that the expression of Yap1 and RBM3 may have a cer-
tain correlation at low temperatures, we wanted to determine
the intrinsic molecular mechanism. The RBM3 binding motif has
been reported in the previous study (Liu et al., 2013; Fig.6A),
and we found that RBM3 could bind to the 3UTR to increase
the stability of some mRNAs. After analyzing the 3UTR region
of the mouse Yap1 mRNA, we found that there are seven RBM3
binding sequences in the 3UTR of Yap1 (Fig.6B). When RBM3
was knocked down in the NSCs, the half-life of Yap1 mRNA was
reduced (Fig.6C), but the half-life was extended when the RBM3
was overexpressed (Fig.6D) in the NSCs maintained at 32°C. To
further verify that RBM3 stabilizes the Yap1 mRNA though bind-
ing to the Yap1-3UTR region, we constructed a luciferase plasmid
containing the full-length Yap1-3UTR region. At 32°C, there was
a sixfold increase in the translation of the message containing the
Yap1-3UTR, compared with the empty vector control (Fig.6E’).
Further, the expression of RBM3 was increased by culturing
cells at 32°C, which resulted in a 1.4-fold increase in translation
of the message compared with that of cells that were cultured
at 37°C (Fig.6E”). To further prove that RBM3 mediates the sta-
bility of the Yap1 mRNA, knockdown of RBM3 was performed
in N2A cells, which were cultured at 32°C. The result showed
that the activity of the Yap1-3UTR–containing luciferase was
decreased (Fig.6F). RBM3 overexpression increased the activity
of the Yap1-3UTR–containing luciferase (Fig.6G). To show the
specificity of the YAP pathway activation, a cell model of cooling
using an 8×GTI IC-luciferase reporter was used to show specific
YAP activity. The result showed that the relative activity of the
8×GTI IC-luciferase was increased in cells that were cultured at
32°C versus the cells cultured at 37°C (Fig.6H). The overexpres-
sion of RBM3 induced the increase in the relative activity of the
8×GTI IC-luciferase (Fig.6I). These results suggest that the 3UTR
region of the Yap1 mRNA and the low temperature–induced high
expression of RBM3 improved the stability of the RNA. Collec-
tively, the data support a role for RBM3 in controlling the Yap1 ex-
pression through binding to its 3UTR, and Yap1 acts downstream
of RBM3 in regulating embryonic cortical development.
Actinomycin D (5 g/ml) was added into the culture medium to inhibit the transcription. e same amount of mRNA in the indicated time phase was reverse
transcribed, and PCR was performed (
n
= 3 independent experiments). (K) Under the 32°C culture conditions, Yap increased faster at the mRNA level in the
NSCs. e NSCs isolated from the E12 brains were cultured in the proliferation medium at 37°C for 24h and then placed at the indicated temperature for
analysis at dierent time phases (
n
= 3 independent experiments). *, P < 0.05; **, P < 0.01; ***, P < 0.001. Error bars represent the mean ± SEM.
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RBM3 knockout affects embryonic neurogenesis
under cold stress
To confirm the phenotype of the RBM3 knockdown, we con-
structed RBM3 knockout mice through a CRI SPR-Cas9 system.
No expression of RBM3 was detected in knockout mice (Fig.
S4, A–D). In the cold stress–induced embryonic mice cortex,
histological analysis showed the changes between the RBM3
knockout and the WT (Fig.7A). Further Western blot analysis
showed that some neurogenesis associated markers changed
between the RBM3 knockout and the WT when cold stress was
induced (Fig.7B).
Our results showed that the TUJ1-positive cells in the RBM3
knockout mice at E16 was decreased compared with the WT mice
(Fig.7 C). The immunostaining results showed that the prolif-
eration markers of Ki67, SOX2, PAX6, and TBR2 were decreased
(Fig.7, D–G), and neuronal markers of STAB2 and CTIP2 were also
decreased (Fig.7H). In addition, apoptosis was not significantly
affected by RBM3 knockout (Fig. S4 E).
In the cold stress–induced RBM3 knockout mice, we also found
that the neuronal differentiation was decreased at P2 (Fig.7, I–K);
RBM3 knockout affected the neuronal development in vivo when
maternal cold stress was induced (Fig.7L). However, there was
no significant difference between WT and knockout in the nor-
mal body temperature group (Fig. S4, F–H).
To confirm the phenotype, IUE was used in the E13 knockout
embryo, and the phenotype was analyzed 72h later. At normal
body temperature, the RBM3 knockout mice showed no differ-
ence in GFP distribution compared with the WT mice. However,
the RBM3 knockout phenotype was similar to that of the RBM3
knockdown in the cold stress–induced condition. In the cold
Figure 6.RBM3 promotes the stability of Yap1 mRNA by binding to the 3′UTR of Yap1 mRNA. (A) Schematic showing the motif that has been published
for the mouse RBM3 mRNA-recognition motif. (B) Schematic showing there were seven sequences located in the Yap1-3UTR region matching the mouse RBM3
recognition motif by sequence alignment. (C and D) e half-life of Yap1 mRNA was prolonged by RBM3. e results showed that when RBM3 was knocked
down in the NSCs, the half-life of Yap1 mRNA was shortened (C). Aer overexpression of RBM3, the Yap1 half-life was extended (D). e quantication shows the
relative Yap1 mRNA at each time point, compared with the level of Yap1 mRNA at time zero (taken as 1). In addition, the half-life of Yap1 mRNA was calculated
and is shown as the
t
1/2 value (
n
= 3 independent experiments). (E) e full-length Yap1-3UTR was cloned into a pisCheck2-vector. HEK293 cells were trans-
fected with a plasmid containing the Yap1-3UTR or the control empty vector and then cultured at 32°C. e relative luciferase activity was detected 48h aer
the transfection and is presented as a bar graph (E’). HEK293 cells were transfected with a plasmid containing the Yap1-3UTR or the control vector and then
were cultured at 32°C or 37°C (E”). e relative luciferase activity was detected 48h aer the transfection and is presented as a bar graph (
n
= 3 independent
experiments). (F) N2A cells were transfected with vector and Yap1-3UTR luciferase plasmids or (RBM3-sh1 and Yap1-3UTR luciferase plasmids), and cultured at
32°C. e relative luciferase activity was detected 48h aer the transfection and is presented as a bar graph (
n
= 3). (G) N2A cells were transfected with vector
and Yap1-3UTR luciferase plasmids (or RBM3-OE and Yap1-3UTR luciferase plasmids) and then cultured at 32°C. e relative luciferase activity was detected
48h aer the transfection and is presented as a bar graph (
n
= 3). (H) HEK293 cells were transfected with a plasmid containing the 8XGT IIC luciferase reporter
and then cultured at 32°C or 37°C. e relative luciferase activity was detected 48h aer the transfection and is presented as a bar graph (
n
= 4). (I) HEK293
cells were transfected with the indicated plasmid combinations. e relative luciferase activity was detected 48h aer the transfection and is presented as a
bar graph (
n
= 4). **, P < 0.01; ***, P < 0.001. Error bars represent the mean ± SEM.
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Figure 7.RBM3 knockout decreases the dierentiation and proliferation of NSCs under maternal cold stress. (A) H&E staining showed the dierence
between the RBM3 knockout mouse brain and the WT mouse brain at E16 and P2. Maternal cold stress was induced at E14, and then the brain was collected at
E16 or P2 for H&E staining. e bar graph shows the height or width of the indicated brains at dierent stages (
n
= 3 brains). (B) Western blot analysis showed
that the indicated proliferation and dierentiation markers were reduced in cold stress–induced RBM3 knockout embryos. Maternal cold stress was induced at
E14 and then maintained at the low temperature until E16 for Western analysis (
n
= 3 experiments). (C) e ratio of Tuj1-positive cells was reduced in the RBM3
knockout in cold stress. Maternal cold stress was induced at E14, and then the embryonic brain was collected at E16. e bar graph shows the uorescence
intensity of Tuj1 (
n
= 3 brains). (D) e number of Ki67-positive NSCs was reduced in the cold stress–induced and RBM3 knockout brain sections. e bar graph
shows the number of Ki67+ cells in the VZ/SVZ per 250,000 m2 (
n
= 3 brains). (E) e number of SOX2-positive NSCs was reduced in the cold stress–induced
and RBM3 knockout brain sections. e bar graph shows the number of SOX2+ cells in the VZ/SVZ per 40,000 m2 (
n
= 3 brains). (F) e number of BrdU and
PAX6 double-positive intermediate progenitors was decreased in cold stress–induced RBM3 knockout brains. e cold stress was induced at E14 and then
maintained at the cold temperature until E16. e bar graph shows the number of BrdU+PAX6+ cells in the VZ/SVZ per 40,000 m2 (
n
= 3 brains). (G) e
number of BrdU and TBR2 double-positive intermediate progenitors was decreased in the cold stress–induced RBM3 knockout brains. e cold stress was
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stress–induced RBM3 knockout mice, the number of GFP cells
was decreased in the VZ/SVZ and CP. Analysis of neuronal dif-
ferentiation showed that the MAP2+ (or TUJ1+) cells in the CP of
the RBM3 knockout embryonic mice were also decreased in cold
stress. The PAX6 (or Tbr2) immunofluorescence results showed
that the proliferation of the NSCs was decreased in the RBM3
knockout mice during cold stress. When RBM3-OE or YAP1-OE
was electroporated into the RBM3 knockout mice, the pheno-
type of the GFP+ cell distribution and the ratio of GFP+MAP2+
(or GFP+TUJ1+, GFP+PAX6+, and GFP+Tbr2+) cells could be partly
rescued (Fig.8, A–F). To gain a deeper insight into how RBM3
regulates neural progenitor proliferation and differentiation in
cold stress, we examined the transcriptome of the embryonic
cortex following RBM3 knockout in the maternal cold stress–
induced condition. These data also suggested that RBM3 had an
essential role in regulating gene expression during embryonic
development (Fig. S4, I and J). RBM3 overexpression could rescue
the expression of Yap and its downstream molecules (cyr61 and
ctgf ) in the RBM3 knockout NSCs, which were isolated at E13 and
cultured at 32°C (Fig.8G). The number of TUJ1+ (or MAP2+) neu-
rons in CP layer were also decreased at P30 in the RBM3 knockout
and maternal cold stress–induced mice cortex (Fig. S5, A and B).
In addition, YAP1 knockdown in the maternal cold stress–induced
mice had the same phenotype as the RBM3 knockout (Fig. S5, C
and D). These results further confirm that RBM3 is involved in
the regulation of the brain development process when maternal
cold stress was induced (Fig.8H).
Discussion
Cold stress is a common physiological phenomenon (Byard and
Bright, 2017; Moler et al., 2017), and it has been reported that
deep cold stress causes neurological dysfunction (Gatti et al.,
2017). However, its roles in pregnancy are little studied. Our
results show that induction of maternal cold stress can lead to
abnormal development of embryonic mice, especially with re-
spect to NSC development. Here, we report that RBM3 is one of
molecules that mediates the effects of cold stress on brain devel-
opment. Our results show that cold stress affects the expression
of RBM3 in the embryonic cerebral cortex.
During the cold stress, RBM3 is induced. RBM3 has been
reported to be associated with neuroprotective functions, and
RBM3 regulates protein expression by selectively binding to dif-
ferent poly(A)-tail sites during the process of circadian rhythms
(Liu et al., 2013). However, the role of RBM3 in embryonic brain
neurogenesis was unclear. Our results showed that RBM3 in the
embryo may regulate the proliferation and differentiation of
NSCs during maternal cold stress by binding to the Yap1-3UTR,
promoting Yap1 expression by stabilizing its mRNA. A maternal
hypothermic environment will lead to fetal cerebral cortex de-
velopment abnormalities. In addition, when RBM3 was knocked
down in the cold stress–induced embryonic cortex, the neuronal
differentiation and proliferation of NSCs were reduced. We ob-
tained the same phenotype in the RBM3 knockout mice. These
results confirm that RBM3 is involved in the development of the
cerebral cortex at cold stress.
To explain the intrinsic causes of these results, we
screened molecules and found that Yap1 may be the down-
stream target of RBM3 under cold stress conditions. We
observed that the half-life of the Yap1 mRNA in NSCs was
longer at 32°C versus 37°C. When RBM3 was knocked down
in cultured NSCs at 32°C, the half-life of the Yap1 mRNA was
shortened. In contrast, when RBM3 was overexpressed in the
NSCs cultured at 32°C, the half-life of the Yap1 mRNA was
longer. Our results showed that a low temperature induced a
high expression of RBM3, which could enhance the expres-
sion of Yap1 by stabilizing its 3UTR region. In addition, the
results of our RNA sequencing analysis showed that the in-
duction of cold stress can affect the development of the fetal
cerebral cortex. Interestingly, our results show that Yap1 may
also be a potential cold shock protein, and the abundance of
Yap1 mRNA and protein was changed at low temperature.
These results may enhance our understanding of Yap1.
However, RBM3 knockdown or knockout did not affect
the neurogenesis process when the body temperature was
normal. These differences may be caused by cooling-induced
transcriptome reprogramming. Under the condition of cold
stimulation, RBM3 was highly expressed, which may have
increased the stability of intracellular protein transcription.
It may be explained that RBM3, as an RNA-binding protein,
could bind to different regions of mRNAs, which may affect
the normal development process, including the development
of the cerebral cortex.
RBM3-overexpression and YAP-overexpression partially res-
cue the RBM3 knockout phenotype when the maternal cold stress
was induced. The RBM3 pathway is likely to be one of the effector
pathways, which are important for responses to cold stress. It is
possible that there are other pathways, which plays function in
maternal cold stress condition.
In the IUE experiment, the neurons are still stuck in the inter-
mediate zone and do not properly differentiate or migrate in the
absence of RBM3 when the maternal cold stress was induced. We
detected some migration related genes (
lis1
,
rac1
,
dcx
,
cdh2
, and
cdk5
) expression and did not find significant expression change
of these genes. However, we cannot exclude the possibility that
cold stress affects migration.
induced at E14 and then maintained at the cold temperature until E16. e bar graph shows the number of BrdU+TBR2+ cells in the VZ/SVZ per 40,000 m2 (
n
= 3 brains). (H) e number of SATB2- (or CTIP2-) positive neurons was reduced in the cold stress–induced and RBM3 knockout brain sections. e bar graph
shows the number of SATB2- (or CTIP2-) positive cells in the VZ/SVZ per 250,000 m2 (
n
= 3 brains). (I–K) e number of CUX1-, SATB2-, and CTIP2-positive
cells was reduced in the RBM3 knockout in cold stress at P2. e maternal cold stress was induced at E14, and then the brain was collected at P2. e bar
graph shows the number of CUX1-, SATB2-, and CTIP2-positive cells (
n
= 3 brains). (L) RBM3 knockout caused abnormal development of GFP-positive cells at
P2 when maternal cold stress was induced. IUE was performed at E13, and then cold stress was induced at E14. Aer that, the brains were collected at P2. e
length of the leading process in each indicated condition is shown (
n
= 3 independent experiments). Bars: 500 m (A, E16); 750 m (A, P2); 50 m (C, H, and
L) 20 m (D–G); 100 m (I–K). *, P < 0.05; **, P < 0.01; ***, P < 0.001. Error bars represent the mean ± SEM.
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In conclusion, we report that, following the induction of
maternal cold stress, there are changes in the expression of
specific cold-inducible proteins, including the reported cold
shock protein, RBM3, and the never-reported protein Yap1.
Interestingly, we show that Yap1 is a downstream target of RBM3.
Additionally, our results suggest that RBM3 and Yap1 are involved
in the brain development under cold stress.
Materials and methods
Mice
ICR pregnant mice were purchased from Vital River. The mice
were maintained in standard conditions (Mice were housed
in a 12h:12h light: dark cycle with lights on at 7 a.m.). All mice
were taken care according to the Guide for the Care and Use of
Laboratory Animals. All animal experiments were approved
by the Animal Committee of the Institute of Zoology, Chinese
Academy of Sciences. The RBM3 knockout mice were generated
by using CRI SPR-Cas9 system.
RBM3 knockout mice generation
RBM3 knockout mice were generated using the CRI SPR-Cas9
system. In brief, upstream-gRNA1: 5-CGG GCG TGC TGC ATC GGA
ACG GG-3, upstream-gRNA2: 5-5GCG GGC GTG CTG CAT CGG AAC
GG-3; downstream-gRNA1: 5-GTA TTC TTG AGA TTG GTA CAT
GG-3; and downstream-gRNA2: 5-ATG TGG CTT GGA TAT ATC
AAA GG-3 were cloned into pUC57-kan-T7-gRNA vector. After
transcription and purification, the gRNAs and Cas9 expressing
vector were microinjected into fertilized one-cell embryos. Then
the injected zygotes were implanted into the host dams. When
the offspring were born, the genotyping PCR experiments were
done, and the PCR production were sequenced to identify the
mutations. Following genetic testing, mice with 1,980-bp deletion
were screened and bred for the sequential experiments. The
genotyping primers used for RBM3 knockout mice were RBM3-
269F: 5-CTC GTT CCC CAC CTC ACT T C-3 and RBM3-2575R: 5-GAG
TAG CGG TCA TAG CCA CC-3. The PCR products were 2,000 bp
for WT and 200 bp for RBM3 knockout mice.
Plasmid constructs
The sequences for small hairpin RNA are as follows: RBM3-
shRNA1, 5-GTT GAT CAT GCA GGA AAG TCT-3; RBM3-shRNA2,
5-GTC GTC CTG GAG GAT ATG GAT-3; YAP-shRNA1, 5-CGG TTG
AAA CAA CAG GAA TTA-3.
The shRNAs were subcloned into the pSicoR-GFP vector.
RBM3 and YAP cDNA was amplifed by PCR and subcloned into
pCDH-CMV-GFP vector.
Induction of cold stress
The pregnant mice were cooled using 5-AMP as described.
Freshly prepared 5-AMP (Sigma) was injected intraperitoneally
(0.7 mg/g). Control mice were injected with saline. Mice were
maintained at room temperature 1h until core body tempera-
ture decreased to 33°C. Subsequently, mice were transferred to a
refrigerator (5° C) and core body temperature lowered to 30°C
(Peretti et al., 2015), and then the pregnant mice were housed
24h at 10°C and 24h at 5°C (Fischer et al., 2017). For short-term
cold experiment, after the maternal body temperature dropped
to 30°C, the pregnant mice were housed 4h at 10°C, and then they
were housed in the room temperature. During the experiment we
observe the physiological status of pregnant mice every hour to
ensure the normal survival of pregnant mice.
IUE
The pregnant mice were anesthetized by given an injection of
pentobarbital sodium, and the uterine horns were exposed. Re-
combinant plasmid mixed with Venus-GFP at a 3:1 molar ratio,
and fast green (2 mg/ml; Sigma) was microinjected into the fetal
brain ventricles. And the mixed plasmid was electroporated into
the brain ventricle cells. After electroporation, the pregnant mice
were sacrificed at different time point for phenotype analysis.
The fetal brains were fixed in 4% PFA overnight and then de-
hydrated in 30% sucrose at 4°C. For knockdown tissue RNA-se-
quence, IUE of dividing cells with either control or RBM3 shRNA
was used. GFP-containing tissue from three to five embryos
were collected under the microscope, followed by FACS sorting
of GFP-positive cells. The total RNA of these cells was collected
by using trace RNA extraction kit for RNA-seq.
Cell culture
293FT (or N2A) cells were cultured in DMEM that contained 10%
FBS, non-essential amino acid, and penicillin/streptomycin.
Lentiviral package DNA and the core DNA was transfected
into HEK293FT by GenEscort (Wisegen). The supernatant con-
taining the virus was collected at 24, 48, and 72h after trans-
fection, and the cell debris was removed by centrifugation at
3,000 rpm for 5 min.
NSCs were isolated from E12 cortex. In brief, E12 brain cor-
tex tissue around the ventricle was dissected out and digested in
papain (20 U/mg; Worthington) for 5 min at 37°C to acquire cells
suspension. Cells suspension was washed three times by NSCs
culture medium (NeuroBasal/DMEM/F12 with penicillin-strep-
tomycin, GlutaMAX, nonessential amino acids, B27 supplement
[2%], bFGF [5 ng/ml], and EGF [5 ng/ml]) to remove the papain.
Cell suspensions were then filtered through a 40-m strainer.
Figure 8.e abnormal neurogenesis process caused by RBM3 knockout in the maternal cold stress group could be rescued. (A–F) RBM3 knockout
caused GFP-positive cell positioning changes in the cold stress–induced embryonic brain, and the RBM3 or Yap1 overexpression could partially rescue this
phenotype. e indicated plasmid was electroporated at E13, and the phenotype was analyzed at E16. e pregnant mice were cooled by using 5-AMP injection.
e percentage of GFP-positive cells in each region is shown (B), and the GFP+Map2+ cells in the CP (C), GFP+TUJ1+ cells in the CP (D), GFP+PAX6+ neural
progenitors in VZ/SVZ (E), or GFP+Tbr2+ neural progenitors in VZ/SVZ (F) were calculated (
n
= 3 brains). Bars: 50 m (A). (G) e expression of Yap and its
downstream molecules (cyr61 and ctgf) could be rescued by RBM3 overexpression in the RBM3 knockout NSCs. e WT and RBM3 knockout NSCs were iso-
lated at E13, and the RBM3-overexpression virus was used to rescue the level of RBM3. e indicated gene expression was detected by real-time PCR (
n
= 3
independent experiments). (H) Schematic diagram: cold stress aects brain development, and RBM3 deciency reduces neuronal dierentiation. *, P < 0.05;
**, P < 0.01; ***, P < 0.001. Error bars represent the mean ± SEM.
Journal of Cell Biology
https://doi.org/10.1083/jcb.201801143
Xia et al.
RBM3 regulates brain development
3477
Acquired cells were seeded in plates coated with Poly--Lysine
and Laminin before cell culture. 1.5 million cells per well were for
a 6-well plate, while 5,000 cells per well were for a 24-well plate.
Cells were infected with lentivirus by the addition of 2 g/ml
polybrene to improve the efficacy of infection. 8h later, the pro-
liferation medium was changed into a differentiation medium,
which consisted of low glucose DMEM (Gibco), 2% B27, and 1%
FBS (Invitrogen).
BrdU labeling
For NPC proliferation analysis, BrdU (50 mg/kg) was adminis-
tered to pregnant female mice for 2h before harvesting the em-
bryonic brains. For the birth dating experiment, BrdU (50 mg/kg)
was administered to pregnant mice 24h after electroporation,
and the embryonic brains were collected 3 d later for phenotype
analysis. For the cell-cycle exit experiment, BrdU (50 mg/kg) was
administered to pregnant mice for 24h before the mouse brains
were separated and processed. Then, the brains were costained
with anti-Ki67 and anti-BrdU antibodies for analysis.
Western blotting
Cells were lysed with radioimmunoprecipitation assay in
combination protease inhibitor on ice for 5 min, and the cell
debris was eliminate by centrifuged at 4°C for 5 min. The
BCA method was used for measuring the concentration of
protein. The protein samples were loaded onto SDS-PAGE
gel for electrophoresis, and the bands were transferred to
nitrocellulose or polyvinylidene fluoride membranes. The
membranes were blocked in 5% nonfat milk in PBST (PBS with
0.1% Tween-20) for 1 h at room temperature, and incubated
with primary antibody at 4°C. The different secondary
antibodies were used to visualize the bands.
Immunostaining
Immunostaining for cultured cells or brain slices was performed
according to the following procedure: the samples were washed
with PBST (1% Triton X-100 in 1M PBS), fixed in 4% PFA, blocked
by 5% BSA (in 1% PBST) for 1h, incubated with primary antibod-
ies overnight at 4°C, and visualized using fluorescence-labeling
secondary antibodies.
RNA half-life test
The freshly prepared Actinomycin D (5 g/ml, Sigma; Jiang et
al., 2016) was added in cultured cells to stop transcription. After,
cells were collect at different time point (0, 1, 2, and 4h). RNA
was extracted using TRIzol reagent (Invitrogen Life Technolo-
gies) according to the manufacturer's instructions. Total mRNA
was reversed into cDNA using RNA reverse-transcription kit.
Real-time reverse transcription (RT) PCR was then performed as
previously described; mouse Yap1 mRNA levels were determined
by qRT-PCR and normalized by β-Actin. The expression of the
Yap mRNA at the starting point was taken as 1.
t
1/2 was calculated
from RNA level trend plot.
Antibodies
Antibodies used were as follows: anti-β III Tubulin (MAB1637;
Millipore and T2200; Sigma), anti-PCNA (SC7907; Santa
Table1.Primers used for RT-PCR
Primer Forward or reverse Sequence (5′–3′)
RBM Forward ATG CAG GAA AGT CTG CCA GG
Reverse TCT CTA GAC CGC CCA TAC CC
CIR BP Forward TCT CCG AAG TGG TGG TGG TA
Reverse CAT CAT GGC GTC CTT AGC GT
trpm Forward TAT GAG ACC CGA GCA GTG GA
Reverse GGC TGA GCG ATG AAA TGC TG
trpa Forward GGC AAT GTG GAG CAA TAG CG
Reverse TGA GGC CAA AAG CCA GTA GG
Hifa Forward AGG ATG AGT TCT GAA CGT CGA AA
Reverse GGG GAA GTG GCA ACT GAT GA
ucp Forward CGT CCC CTG CCA TTT ACT GT
Reverse CCC TTT GAA AAA GGC CGT CG
ucp Forward GGC CTC TGG AAA GGG ACT TC
Reverse GAC CAC ATC AAC AGG GGA GG
trpv Forward TCA CCG TCA GCT CTG TTG TC
Reverse GAT CAT AGA GCC TTG GGG GC
trpv Forward GAC CCA TAA GAC GGA CAG CA
Reverse ACC GAC GTT TCT GGG AAT TCAT
trpm Forward CTG ACT GTC ACC CTG CTC TG
Reverse TCT GTG TGT TCC TGC ACC TC
Yap Forward TTC GGC AGG CAA TAC GGA AT
Reverse GCA TTC GGA GTC CCT CCA TC
ctgf Forward AAG GAC CGC ACA GCA GTTG
Reverse GGC TCG CAT CAT AGT TGG GT
cyr Forward CTT TTC AAC CCT CTG CAC GC
Reverse CTC GTG TGG AGA TGC CAG TT
il Forward CCA GCT GGA CAA CAT ACT GC
Reverse TTC TGG GCC ATG CTT CTC TG
ngn Forward CTC ACC ATC CAA GTG TCC CC
Reverse AGT CAC CCA CTT CTG CTT CG
Ascl Forward GGA ACT GAT GCG CTG CAA AC
Reverse GTG GCA AAA CCC AGG TTG AC
Brn Forward GTT TGC TCT ATT CGC AGC CG
Reverse TCT GCA TGG TGT GGC TCA TC
Bdnf Forward CCG GTA TCC AAA GGC CAA CT
Reverse CTG CAG CCT TCC TTG GTG TA
Ngf Forward ACA GCC ACA GAC ATC AAG GG
Reverse TGA CGA AGG TGT GAG TCG TG
Fgf Forward CGC GAG AAA TCC AAT GCC TG
Reverse TTC TGC GCC TCT TCT TGG AG
Axin Forward CCC GGA GCT ATT CCG AGA AC
Reverse TCT CAG CGT CCT CTG TGG TA
actb Forward GCA AGT GCT TCT AGG CGG AC
Reverse AAG AAA GGG TGT AAA ACG CAGC
ccnd Forward CCC TGG AGC CCT TGA AGA AG
Reverse AGA TGC ACA ACT TCT CGG CA
cdkna Forward GTC GCA GGT TCT TGG TCA CT
Reverse CAT GTT CAC GAA AGC CAG AGC
Journal of Cell Biology
https://doi.org/10.1083/jcb.201801143
Xia et al.
RBM3 regulates brain development
3478
Cruz), anti-PAX6 (AB2237; Millipore), anti-TBR2(AB23345;
Abcam), anti-Histone H3 (4499; Cell Signaling Technology),
anti–β-ACT IN (20536-1-Ap; Proteintech), anti-NES TIN
(MAB353; Millipore), anti-Flag (F1804; Sigma), anti–phospho-
Histone H3(Ser10) (3377; Cell Signaling Technology), anti-
BrdU (AB6326; Abcam), anti-RBM3 (14363-1-AP; Proteintech),
anti-Map2 (MAB3418; Millipore), anti-Ki67 (ab15580;
Abcam), anti-TUJ1(MAB1637; Millipore), anti-YAP (4912;
Cell Signaling Technology), anti-SOX2 (3728; Cell Signaling
Technology), anti-CTIP2 (ab18465; abcam), and anti-SATB2
(ab51502; Abcam).
Real-time PCR
The total RNA was extracted by using the TRIzol (Invitrogen) ac-
cording to the instructions. FastQuant RT kit (with DNase [TIA
NGEN]) was used to get first-strand cDNA. Real-time PCR was
performed on the real-time PCR machine (ABI).
Confocal imaging and statistical analysis
All confocal images were acquired with the Zeiss LSM 780
microscope in room temperature and analyzed with Photoshop
CS4 (Adobe); Detector is PMT; Plan-Apochromat 10/0.45 or
Plan-Apochromat 20/0.8 was used; 50% Glycerin was applied
as imaging medium; The software for image acquisition and
processing was ZEN 2010.
The fluorescence density is calculated using the ImageJ and
Zeiss ZEN2012 blue software; for fluorescence density detecting,
we use the same concentration of antibodies for different
samples, the same incubation time, the same confocal microscope
fluorescence excitation light intensity, and the fluorescence
density of the control group is taken as one.
Statistical analyses were performed using Student’s
t
test
(*, P < 0.05; **, P < 0.01; ***, P < 0.001). All bar graphs in the
figures are shown as the means ± SEM.
For transcriptome analysis, the IUE was used to introduce the
RBM3 knockdown GFP plasmid or control GFP plasmids into the
E13 embryonic cortex. Then, GFP-positive NSCs were isolated
from E15 mice by FACS. RNA used for global transcriptome
analysis were extracted from these NSC and sequenced by
Hiseq 2500. Significantly differentially expressed genes were
identified when we compared normalized reads count between
groups with P < 0.05 and log2 fold change > 1.
Primers for real-time PCR
Primers for RT-PCR are listed in Table1.
Luciferase assays
Luciferase assays were performed in HEK293 or N2A with the
Yap1-3UTR insert psi-Check2 luciferase reporter in different
conditions as previously reported (Jiang et al., 2016).
8×GTI IC-lux Luciferase assays were performed in HEK293
cells with the YAP/TAZ-responsive reporter 8×GTI IC-Lux accord-
ing to previous study (Dupont et al., 2011).
The indicated gene expression vectors were transfected into
the cells, and the sample was collected 48h later; the Dual-Lucif-
erase Reporter Assay System (Promega) was used to detect the
expression of the luciferase.
The GEO accession numbers
The GEO accession nos. for the cold stress–induced control and
RBM3-knockdown transcriptome sequencing data reported in
this paper is GSE104300. The GEO accession nos. for the cold
stress–induced WT and RBM3 knockout transcriptome sequenc-
ing data reported in this paper is GSE109362.
Online supplemental material
Fig. S1 shows that induction of maternal cold stress can affect the
development of offspring. Fig. S2 shows that RBM3 regulates NSC
proliferation and neuronal differentiation during the maternal
cold stress. Fig. S3 shows that RBM3 regulates NSC proliferation
and neuronal differentiation during short-term cold stress (4-h
exposure). Fig. S4 shows that RBM3 knockout does not affect the
neuronal differentiation in the normal body temperature group.
Fig. S5 shows that RBM3 knockout decreases the differentiation
under maternal cold stress conditions at P30.
Acknowledgments
This work was supported by grants from Chinese Academy of
Science Strategic Priority Research Program (XDA16020602),
the National Natural Science Foundation of China (31730033
and 31621004), the National Key Basic Research Program of
China (2015CB964501 and 2014CB964903), and the K.C. Wong
Education Foundation.
The authors declare no competing financial interests.
Author contributions: W. Xia and J. Jiao designed the research;
W. Xia and L. Su performed the research, analyzed the data, and
wrote the manuscript; J. Jiao supervised the project and ob-
tained funding support.
Submitted: 22 January 2018
Revised: 6 May 2018
Accepted: 2 July 2018
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... Exposure to cold stress increases YAP expression in certain tissues, such as the brain and adipose tissue, where it plays a crucial role in maintaining cellular homeostasis. In the context of neural development, YAP has been implicated in the cold shock response by stabilizing its expression through interactions with RNA-binding proteins like RBM3 [49]. This upregulation promotes the proliferation and differentiation of neural stem cells [50], which is essential for maintaining brain health during cold stress. ...
... Thermal therapies, including hyperthermia and cold treatments, provide additional avenues for modulating YAP activity. The heat-induced activation of YAP can enhance tissue repair by promoting stem cell differentiation towards osteogenesis, while cold therapy's ability to reduce inflammation through YAP modulation highlights its role in creating a favorable environment for healing [46,[46][47][48][49][50][51]. The dynamic application of heat and cold therapies, tailored to the specific needs of the patient, can provide synergistic benefits by leveraging the YAP pathway to optimize tissue repair, reduce pain, and improve overall recovery [46,[46][47][48][49][50][51]. ...
... The heat-induced activation of YAP can enhance tissue repair by promoting stem cell differentiation towards osteogenesis, while cold therapy's ability to reduce inflammation through YAP modulation highlights its role in creating a favorable environment for healing [46,[46][47][48][49][50][51]. The dynamic application of heat and cold therapies, tailored to the specific needs of the patient, can provide synergistic benefits by leveraging the YAP pathway to optimize tissue repair, reduce pain, and improve overall recovery [46,[46][47][48][49][50][51]. ...
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Cellular behavior is strongly influenced by mechanical signals in the surrounding microenvironment, along with external factors such as temperature fluctuations, changes in blood flow, and muscle activity, etc. These factors are key in shaping cellular states and can contribute to the development of various diseases. In the realm of rehabilitation physical therapies, therapeutic exercise and manual treatments, etc., are frequently employed, not just for pain relief but also to support recovery from diverse health conditions. However, the detailed molecular pathways through which these therapies interact with tissues and influence gene expression are not yet fully understood. The identification of YAP has been instrumental in closing this knowledge gap. YAP is known for its capacity to perceive and translate mechanical signals into specific transcriptional programs within cells. This insight has opened up new perspectives on how physical and rehabilitation medicine may exert its beneficial effects. The review investigates the involvement of the Hippo/YAP signaling pathway in various diseases and considers how different rehabilitation techniques leverage this pathway to aid in healing. Additionally, it examines the therapeutic potential of modulating the Hippo/YAP pathway within the context of rehabilitation, while also addressing the challenges and controversies that surround its use in physical and rehabilitation medicine.
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Cellular behavior is regulated by mechanical signals within the cellular microenvironment. Additionally, changes of temperature, blood flow, and muscle contraction also affect cellular state and the development of diseases. In clinical practice, physical therapy techniques such as ultrasound, vibration, exercise, cold therapy, and hyperthermia are commonly employed to alleviate pain and treat diseases. However, the molecular mechanism about how these physiotherapy methods stimulate local tissues and control gene expression remains unknow. Fortunately, the discovery of YAP filled this gap, which has been reported has the ability to sense and convert a wide variety of mechanical signals into cell-specific programs for transcription, thereby offering a fresh perspective on the mechanisms by which physiotherapy treat different diseases. This review examines the involvement of Hippo/YAP signaling pathway in various diseases and its role in different physical therapy approaches on diseases. Furthermore, we explore the potential therapeutic implications of the Hippo/YAP signaling pathway and address the limitations and controversies surrounding its application in physiotherapy.
... hypothermia-mediated structural derangements, which explains its temporary abundance at birth in the normothermic brain. 33 Similarly, CIRBP KO mice have increased hippocampal and BBB damage in a hypothermia model of cardiopulmonary bypass. 34 Finally, RTN3 promotes axonal growth and contributes to coolinginduced neuroprotection. ...
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Cooling or the application of mentholated liniments to the skin has been used to treat itch for centuries, yet remarkably little is known of how counter-stimuli such as these induce itch relief. Indeed, there is no clear consensus in the scientific literature as to whether or not cooling does in fact block the transduction of itch signals or if it is simply a placebo effect. This gap in our understanding led us to hypothesize that cooling is antipruritic and, like cooling analgesia, requires function of the cold-gated ion channel TRPM8, a receptor for menthol expressed on peripheral afferent nerve endings. Using a combination of pharmacological, genetic, and mouse behavioral assays, we find that cooling inhibits both histaminergic and non-histaminergic itch pathways, and that inhibition of itch by cooling requires TRPM8 channels or intact and functional TRPM8-expressing afferent neurons. The cold mimetic menthol is also effective in ameliorating itch in a TRPM8-dependent manner. Moreover, we find that chronic itch can be ameliorated by cooling, demonstrating that this counter-stimulus activates a specific neural circuit that leads to broad itch relief and a potential cellular mechanism for treatment of chronic itch.
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Nociception is an important physiological process that detects harmful signals and results in pain perception. In this review, we discuss important experimental evidence involving some TRP ion channels as molecular sensors of chemical, thermal, and mechanical noxious stimuli to evoke the pain and itch sensations. Among them are the TRPA1 channel, members of the vanilloid subfamily (TRPV1, TRPV3, and TRPV4), and finally members of the melastatin group (TRPM2, TRPM3, and TRPM8). Given that pain and itch are pro-survival, evolutionarily-honed protective mechanisms, care has to be exercised when developing inhibitory/modulatory compounds targeting specific pain/itch-TRPs so that physiological protective mechanisms are not disabled to a degree that stimulus-mediated injury can occur. Such events have impeded the development of safe and effective TRPV1-modulating compounds and have diverted substantial resources. A beneficial outcome can be readily accomplished via simple dosing strategies, and also by incorporating medicinal chemistry design features during compound design and synthesis. Beyond clinical use, where compounds that target more than one channel might have a place and possibly have advantageous features, highly specific and high-potency compounds will be helpful in mechanistic discovery at the structure-function level.
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Introduction: Type 2 diabetes mellitus (T2DM) and obesity are metabolic disorders whose major hallmark is insulin resistance. Impaired mitochondrial activity, such as reduced ratio of energy production to respiration, has been implicated in the development of insulin resistance. Uncoupling proteins (UCPs) are proton carriers, expressed in the mitochondrial inner membrane, that uncouple oxygen consumption by the respiratory chain from ATP synthesis. Aim: The aim of the study was to determine transcriptional levels of UCP1 and UCP2 in peripheral blood mononuclear cells (PBMCs) from patients with metabolic disorders: T2DM, obesity and from healthy individuals. Material/Methods: The mRNA levels of UCP1, UCP2 were determined by Real-Time PCR method using Applied Biosystems assays. Results: The UCP1 mRNA expression level was not detectable in the majority of studied samples, while very low expression was found in PBMCs from 3 obese persons. UCP2 mRNA expression level was detectable in all samples. The median mRNA expression of UCP2 was lower in all patients with metabolic disorders as compared to the controls (0.20+0.14 vs. 0.010+0.009, p=0.05). When compared separately, the differences of medians UCP2 mRNA expression level between the obese individuals and the controls as well as between the T2DM patients and the controls did not reach statistical significance. Conclusions: Decreased UCP2 gene expression in mononuclear cells from obese and diabetic patients might contribute to the immunological abnormalities in these metabolic disorders and suggests its role as a candidate gene in future studies of obesity and diabetes.
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The Hippo pathway controls organ size and tissue homeostasis, with deregulation leading to cancer. The core Hippo components in mammals are composed of the upstream serine/threonine kinases Mst1/2, MAPK4Ks and Lats1/2. Inactivation of these upstream kinases leads to dephosphorylation, stabilization, nuclear translocation and thus activation of the major functional transducers of the Hippo pathway, YAP and its paralogue TAZ. YAP/TAZ are transcription co-activators that regulate gene expression primarily through interaction with the TEA domain DNA-binding family of transcription factors (TEAD). The current paradigm for regulation of this pathway centres on phosphorylation-dependent nucleocytoplasmic shuttling of YAP/TAZ through a complex network of upstream components. However, unlike other transcription factors, such as SMAD, NF-κB, NFAT and STAT, the regulation of TEAD nucleocytoplasmic shuttling has been largely overlooked. In the present study, we show that environmental stress promotes TEAD cytoplasmic translocation via p38 MAPK in a Hippo-independent manner. Importantly, stress-induced TEAD inhibition predominates YAP-activating signals and selectively suppresses YAP-driven cancer cell growth. Our data reveal a mechanism governing TEAD nucleocytoplasmic shuttling and show that TEAD localization is a critical determinant of Hippo signalling output. © 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
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Background: Maternal psychological stress during pregnancy has essentially been conceptualized as a teratogen. However, little is known about the effect of temperature on maternal stress during pregnancy. The aim of this study is to investigate the relationship between temperature and maternal stress during pregnancy. Methods: In 2010, a total of 1931 eligible pregnant women were enrolled across Shanghai from four prenatal-care clinics during their mid-to-late pregnancy. Maternal life-event stress and emotional stress levels during pregnancy were assessed by the "Life Event Scale for Pregnant Women" (LESPW) and "Symptom Checklist-90-Revised Scale" (SCL-90-R), respectively. Exposure to ambient temperature was evaluated based on daily regional average in different moving average and lag days. The generalized estimating equations were used to evaluate the relationship between daily average temperature/temperature difference and maternal stress. Results: After adjusting for relevant confounders, an U-shaped relationship was observed between daily average temperature and maternal Global-Severity-Index (GSI) of the SCL-90-R. Cumulative exposures to extremely low temperatures (< P5, 1.4-10.5℃, lag 0-1 days, 0-2 days and 0-5 days) and extremely high temperatures (≥ P95, 31.2-34.1℃, lag 0-1 days and 0-2 days), and acute exposures to extremely low (lag day 0, 1, 2 and 3) and high (lag day 0, 1) temperatures, all induced higher risks of high GSI (the highest tertile), compared to the risk induced by exposed to an optimal temperature range (20-25℃) (P< 0.05). Increased temperature difference was associated with high maternal GSI (P< 0.05). However, non-significant associations were observed between daily average temperatures/temperature differences and maternal log-transferred LESPW scores. Conclusions: Cumulative and acute exposures to extremely low/high temperatures may both induce emotional stress during pregnancy.
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Adaptive thermogenesis is the process of heat generation in response to cold stimulation. It is under the control of the sympathetic nervous system, whose chief effector is the catecholamine norepinephrine (NE). NE enhances thermogenesis through β3-adrenergic receptors to activate brown adipose tissue and by 'browning' white adipose tissue. Recent studies have reported that alternative activation of macrophages in response to interleukin (IL)-4 stimulation induces the expression of tyrosine hydroxylase (TH), a key enzyme in the catecholamine synthesis pathway, and that this activation provides an alternative source of locally produced catecholamines during the thermogenic process. Here we report that the deletion of Th in hematopoietic cells of adult mice neither alters energy expenditure upon cold exposure nor reduces browning in inguinal adipose tissue. Bone marrow–derived macrophages did not release NE in response to stimulation with IL-4, and conditioned media from IL-4-stimulated macrophages failed to induce expression of thermogenic genes, such as uncoupling protein 1 (Ucp1), in adipocytes cultured with the conditioned media. Furthermore, chronic treatment with IL-4 failed to increase energy expenditure in wild-type, Ucp1−/− and interleukin-4 receptor-α double-negative (Il4ra−/−) mice. In agreement with these findings, adipose-tissue-resident macrophages did not express TH. Thus, we conclude that alternatively activated macrophages do not synthesize relevant amounts of catecholamines, and hence, are not likely to have a direct role in adipocyte metabolism or adaptive thermogenesis.