© 2015. Published by The Company of Biologists Ltd | Development (2015) 142, 732-742 doi:10.1242/dev.119016
RESEARCH ARTICLE
Akt1 signaling coordinates BMP signaling and β-catenin activity to
regulate second heart field progenitor development
Wen Luo1, Xia Zhao1, Hengwei Jin1, Lichan Tao1, Jingai Zhu1, Huijuan Wang1, Brian A. Hemmings2 and
Zhongzhou Yang1,3,*
Second heart field (SHF) progenitors exhibit continued proliferation
and delayed differentiation, which are modulated by FGF4/8/10, BMP
and canonical Wnt/β-catenin signaling. PTEN-Akt signaling regulates
the stem cell/progenitor cell homeostasis in several systems, such as
hematopoietic stem cells, intestinal stem cells and neural progenitor
cells. To address whether PTEN-Akt signaling is involved in
regulating cardiac progenitors, we deleted Pten in SHF progenitors.
Deletion of Pten caused SHF expansion and increased the size of
the SHF derivatives, the right ventricle and the outflow tract. Cell
proliferation of cardiac progenitors was enhanced, whereas cardiac
differentiation was unaffected by Pten deletion. Removal of Akt1
rescued the phenotype and early lethality of Pten deletion mice,
suggesting that Akt1 was the key downstream target that was
negatively regulated by PTEN in cardiac progenitors. Furthermore,
we found that inhibition of FOXO by Akt1 suppressed the expression
of the gene encoding the BMP ligand (BMP7), leading to dampened
BMP signaling in the hearts of Pten deletion mice. Cardiac activation
of Akt also increased the Ser552 phosphorylation of β-catenin, thus
enhancing its activity. Reducing β-catenin levels could partially
rescue heart defects of Pten deletion mice. We conclude that Akt
signaling regulates the cell proliferation of SHF progenitors through
coordination of BMP signaling and β-catenin activity.
KEY WORDS: Second heart field, Akt, BMP, β-catenin, Mouse
INTRODUCTION
Lineage tracing and retrospective clonal analysis have identified two
populations of cardiac progenitors during early mouse heart
development (Kelly et al., 2001; Vincent and Buckingham, 2010;
Watanabe et al., 2012). These two pools of cardiac progenitors are
localized in the first heart field (FHF, or primary heart field) and the
second heart field (SHF, or anterior heart field) (Bruneau, 2008;
Harvey, 2002; Olson, 2006). Although the FHF contributes mainly
to the left ventricle (LV), the SHF develops into the right ventricle
(RV), the inflow tract and the outflow tract (OFT) (Black, 2007).
Starting at embryonic day (E) 8.5, the migration of SHF
progenitors from the splanchnic mesoderm (SM) and pharyngeal
mesoderm (PM) into the linear heart tube is essential for heart
development in mice (Watanabe et al., 2012). Genetic studies in
mice have revealed that disruption of SHF formation and
1
MOE Key Laboratory of Model Animal for Disease Study, Model Animal Research
Center, Nanjing Biomedical Research Institute, Nanjing University, Nanjing
2
210061, China. Friedrich Miescher Institute for Biomedical Research, 4058 Basel,
3
Switzerland. Collaborative Innovation Center for Genetics and Development,
Institute of Plant Biology, School of Life Sciences, Fudan University, Shanghai
200438, China.
*Author for correspondence ([email protected])
Received 23 October 2014; Accepted 12 December 2014
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migration severely impaired heart development. For instance,
each deletion of Isl1, Tbx5, Mef2c and Nkx2.5 (Nkx2-5 – Mouse
Genome Informatics) affects the SHF development, resulting in
developmental heart defects, with a single ventricle (the left
ventricle) and the absence of SHF derivatives, including the RV and
the OFT, being observed (Dodou et al., 2004; Hu et al., 2004; Lin
et al., 1997; Prall et al., 2007; Xu et al., 2004).
SHF progenitors exhibit continued proliferation and a delay in
differentiation (Srivastava, 2006; Watanabe et al., 2012). Fgf8 and
Fgf10, the first molecular marker of the murine SHF, are the two
important regulators that promote SHF proliferation (Ilagan et al.,
2006; Kelly et al., 2001; Park et al., 2006; Watanabe et al., 2010,
2012). Canonical Wnt/β-catenin signaling also drives SHF
progenitor cell proliferation (Klaus and Birchmeier, 2009; Klaus
et al., 2007; Kwon et al., 2007). Bone morphogenetic protein
(BMP) signaling is required to induce the SHF formation and to
subsequently inhibit cardiac cell proliferation (Klaus et al., 2007;
McCulley et al., 2008; Prall et al., 2007).
PTEN-Akt signaling regulates the stem cell/progenitor cell
homeostasis (Cully et al., 2006; Oudit and Penninger, 2009;
Sussman et al., 2011; Walsh, 2006). In several stem/progenitor cell
systems, such as hematopoietic stem cells, intestinal stem cells
(ISCs) and neural progenitor cells, deletion of Pten causes increased
cell proliferation through Akt activation (Groszer et al., 2001; He
et al., 2007; Li and Clevers, 2010).
To determine whether PTEN-Akt signaling is involved in the
SHF regulation, we deleted Pten in cardiac progenitor cells. We
found that enhanced Akt signaling promoted SHF progenitor cell
proliferation through the coordination of BMP signaling and
β-catenin activity.
RESULTS
Deletion of Pten in the SHF progenitors results in SHF
expansion and enlarged size of SHF derivatives
Mef2c-AHF-Cre (hereafter referred to as Mef2c-Cre) mice express
the Cre recombinase in the anterior heart field (AHF), a subset of the
SHF which gives rise to OFT/RV (Verzi et al., 2005). This Cre line
has been extensively utilized to delete genes for investigation of the
SHF development in mice (Ai et al., 2007; Bai et al., 2013; Engleka
et al., 2012; Xie et al., 2012). Using Mef2c-Cre, Pten was deleted in
the SHF. Staining for β-galactosidase clearly labeled the SHF and its
derivatives, i.e. the OFT and the RV (Fig. 1A,B). Deletion of Pten
resulted in a substantial expansion of SHF in the PM (Fig. 1A,B).
The OFT and the RV in Pten deletion mice also showed
significantly increased sizes compared with those of the control
tissue (Fig. 1A,B). Quantitative analysis revealed that the SHF area
(in the PM) and the length of the OFT in Pten-deletion mice were
significantly greater than in control mice (supplementary material
Fig. S1A,B). Anatomical and histological analysis of embryos at
later stages displayed significantly enlarged RV and arteries in Pten
DEVELOPMENT
ABSTRACT
RESEARCH ARTICLE
Development (2015) 142, 732-742 doi:10.1242/dev.119016
deletion mice (Fig. 1C-H). The heart function of these mice showed
signs of impairment in the forms of edema (Fig. 1I). The majority of
the mice died within 10 days after birth (Fig. 1S). Dead newborns
were not presented with milk in the stomach or with hypertrophic
heart (Fig. 1J-L). In addition, WGA staining showed no significant
difference in cardiomyocyte size between Pten deletion and control
mice by mid-gestational stage (E14.5) (Fig. 1M,N,Q), although at
this stage cardiomyocyte proliferation was profoundly higher in
Pten deletion mice than that of control mice (supplementary
material Fig. S1C-K). Furthermore, we observed enlarged sizes
of cardiomyocytes in Pten deletion newborns compared with
those of control mice (Fig. 1O-Q). Accordingly, expression of
hypertrophic markers, such as Anp and Bnp (Nppa and Nppb,
respectively – Mouse Genome Informatics), was not changed in the
heart of Pten deletion mice at E14.5, but their levels were
significantly increased in Pten deletion mice at birth (Fig. 1R).
Taken together, these results indicate that PTEN inactivation
affects SHF progenitors and SHF development.
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DEVELOPMENT
Fig. 1. Deletion of Pten in SHF
progenitors resulted in SHF
expansion. (A-D) β-galactosidase
staining to visualize the SHF and its
derivatives, the OFT and the RV, at E9.5
and E14.5, respectively. The SHF
progenitors in the PM are circled by
dashed lines in E9.5 mice. Deletion of
Pten caused SHF expansion and
enlarged size of the OFT and RV.
(E-H) Histological analysis of the hearts
at E12.5 and E14.5, respectively (H&E
staining). (I) Gross analysis of mouse
embryos. Arrow indicates edema.
(J-L) Gross and microscopic analysis of
embryos and their hearts. Arrows in J
indicate the stomach. (M-P) WGA
staining to display the cell size of
cardiomyocytes at E14.5 and P0
(newborn). (Q) Quantification of cell size
in M-P. (R) RT-qPCR analysis of gene
expressions in heart tissue. (S) Survival
curve. Scale bars: 50 µm.
Pten deletion promotes the proliferation of SHF progenitor
cells
The enlarged SHF resulting from Pten loss suggests enhanced cell
proliferation of cardiac progenitors. Therefore, we assessed cell
proliferation in the SHF. Islet1 (Isl1)-positive cells in the PM and
proximal OFT indicate cardiac progenitor identity at E9.5. Thus,
the proliferation of Isl1-positive cardiac progenitors in these
regions was analyzed via immunofluorescence (IF) staining
Development (2015) 142, 732-742 doi:10.1242/dev.119016
(Cai et al., 2003). The results indicated that there were
significantly more cells expressing Isl1 and the mitotic marker
Ki67 (Mki67 – Mouse Genome Informatics) in Pten deletion
embryos than in control embryos (Fig. 2A-I′,N). Co-staining of
phosphorylated histone H3 ( pHH3) and Isl1 further confirmed
these results (Fig. 2J-M′,O). In addition, loss of Pten led to more
β-gal- and Isl1-double-positive cells than in control animals
(supplementary material Fig. S1L).
Fig. 2. Pten deletion promotes the proliferation of SHF progenitors. (A) Schematic to display the horizontal section of E9.5 embryo. Boxed area marks
the SHF for investigation (in red). (B-I) IF staining against Ki67/Isl1 of sections from E9.5 mouse embryos. Ki67/Isl1 double-positive nuclei are shown in yellow.
B′-I′ are higher magnification views of the boxed areas in B-I. (J-M) IF staining against PHH3/Isl1 of sections from E9.5 mouse embryos. PHH3/Isl1 doublepositive nuclei are shown in yellow (indicated by arrows). K′ and M′ are higher magnification views of the boxed areas in K and M. (N) Quantification of Ki67/
Isl1 double-positive nuclei in B-I′. (O) Quantification of PHH3/Isl1 double-positive nuclei in J-M,K′ and M′. (P) Gene expression levels examined by RT-qPCR.
(Q-T) IF staining. Q′-T′ are higher magnification views of the boxed areas in Q-T.
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DEVELOPMENT
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Development (2015) 142, 732-742 doi:10.1242/dev.119016
Next, we performed real-time quantitative PCR (RT-qPCR) to
examine gene expression levels in the SHF region (Fig. 2A) of
control and Pten deletion embryos. As expected, the expression
levels of Pten were significantly decreased in Pten deletion mice
compared with control mice at E9.5 (Fig. 2P). However, we
observed profoundly enhanced Isl1 expression in Pten deletion
mice compared with control mice, which was consistent with
increased cell proliferation of SHF progenitors (Fig. 2P). The
expression levels of the key cardiac transcription factors, including
Nkx2.5, Hand2, Mef2c, Gata4, Tbx5 and Tbx1, were comparable
between control and Pten deletion mice (Fig. 2P). FGF8 and FGF10
are crucial signaling molecules promoting SHF progenitor
proliferation, and their expression levels were not altered in Pten
deletion mice (Fig. 2P). In addition, we investigated early cardiac
muscle-specific genes, such as tropomyosin (Tpm) and α-smooth
muscle actin (αSMA; Acta2 – Mouse Genome Informatics) by IF
staining and observed similar patterns in control and Pten deletion
mice (Fig. 2Q-T′). These results suggest that cardiac differentiation
was nearly normal in Pten deletion mice.
PTEN is a negative genetic regulator of Akt1 signaling
PTEN is a lipid phosphatase antagonizing the PI3K signaling, which
mediates several signal transduction pathways, including the
mitogen-activated protein kinase (MAPK) signaling pathway and
the PDK1 signaling pathway (Cantley, 2002). PDK1 phosphorylates
and subsequently activates a panel of kinases, such as Akt,
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DEVELOPMENT
Fig. 3. PTEN is a negative genetic regulator of Akt1 signaling. (A) Schematic to display the horizontal section of E9.5 embryo. Boxed areas mark the
SHF progenitor pool for investigation (in red). (B-G) IF staining against phospho-Akt S473 and PTEN. (H-O) Microscopic and histological analysis of mouse
embryos and hearts. (P-U) Microscopic and histological analysis of mouse postnatal hearts. (V-X) WGA staining to display the cardiomyocyte size in mice of
3 months age. (Y) Quantification of cell size in V-X. (Z) Survival curve. Survival rate is identical between control and Pten f/f; Akt1 f/f; Mef2c-Cre mice. Note that the
data for the control and Pten f/f; Mef2c-Cre mice are from the same experiment as those shown in Fig. 1S.
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Development (2015) 142, 732-742 doi:10.1242/dev.119016
Fig. 4. PTEN-Akt signaling suppresses BMP-SMAD signaling. (A-H) IF staining against phospho-SMAD1/5 in the SHF at E9.5. (A′-H′) Higher magnification views
of the boxed areas in A-H. (I) Quantification of p-SMAD1/5 nuclei in the SHF of A-H′. (J) Western blot analysis. (K,L) RT-qPCR analysis of Bmp ligands and receptors.
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lethality beginning at E9.5, and complete lethality by E12.5. However,
the deletion of Akt1 from Pten deletion mice generated the Pten/Akt1
double-deletion mice (Ptenf/f; Akt1f/f; Mesp1-Cre), which were able to
develop to full term, with some progeny surviving for 3-4 months
(supplementary material Fig. S2A,D-G). Furthermore, we found that
deletion of Akt2 or Akt3 also delayed the embryonic lethality of Pten
deletion mice (supplementary material Fig. S2B,C). Histological
analysis revealed nearly normal heart development in Pten/Akt1
double-deletion mice, although a slightly enlarged heart was observed
(supplementary material Fig. S2A).
Taken together, these results indicate that Akt1 is the key
downstream target of PTEN and is negatively regulated by PTEN
during heart development. The increased size of the SHF and the
defective SHF development found in Pten deletion mice are a
consequence of enhanced Akt activity.
PTEN-Akt signaling suppresses BMP-SMAD signaling
through FOXO
BMP signaling is required for cardiac progenitor specification,
proliferation and differentiation (Brand, 2003; Klaus et al., 2012;
Mercola et al., 2011; Olson and Schneider, 2003; van Wijk et al.,
2007). To understand how enhanced Akt activity gives rise to SHF
expansion, we studied BMP signaling in the SHF of Pten deletion
mice. IF staining of phospho-SMAD1/5, an indicator of the
activation of BMP signaling, showed a markedly reduced signal
in the SHF of Pten deletion hearts (Fig. 4A-I).
DEVELOPMENT
PKC (Prkcg – Mouse Genome Informatics), SGK (Sgk1 – Mouse
Genome Informatics) and S6K (Rps6k – Mouse Genome
Informatics) (Pearce et al., 2010). Although Akt is a key
downstream target that is negatively regulated by PTEN, genetic
evidence is lacking regarding the relationship between PTEN and
Akt during mouse embryogenesis and organogenesis.
To examine whether Akt activity is promoted in the heart tissue of
Pten deletion mice, we performed IF staining in E9.5 embryonic
hearts. Phosphorylation of Akt at serine 473 (S473) should indicate
Akt activation, and IF staining of Akt phospho-S473 revealed a
stronger signal in Pten deletion SHF than in that of controls
(Fig. 3A-G).
Next, we tested the genetic relationship between PTEN and Akt
during heart development. The deletion of Akt1 from Pten deletion
mice generated Pten/Akt1 double-deletion mice (Ptenf/f; Akt1f/f;
Mef2c-Cre). Anatomic and histological analysis revealed comparable
heart sizes between Pten/Akt1 double-deletion mice and control mice,
indicating that the increased heart size of Pten deletion mice was
rescued by abrogation of Akt1 (Fig. 3H-O). The majority of Pten
deletion mice died within 10 days after birth and showed
hypertrophic hearts. However, the Pten/Akt1 double-deletion mice
were able to survive for more than 3 months and showed significantly
reduced cardiomyocyte size compared with Pten deletion mice, as
evidenced by histological analysis (Fig. 3P-Z).
Similarly, we also observed that Mesp1-Cre-mediated Pten
deletion (Ptenf/f; Mesp1-Cre) caused heart defects and embryonic
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Development (2015) 142, 732-742 doi:10.1242/dev.119016
Western blot analysis also revealed a consistent change in
phospho-SMAD1/5 (Fig. 4J; supplementary material Fig. S3A,B).
Among the ∼20 Bmp ligands, Bmp2, 4, 7 and 10 are expressed in
the SHF (Liu et al., 2004; Wang et al., 2010; Yuasa and Fukuda,
2008). We examined the expression levels of these Bmp ligands and
their receptors in the SHF via RT-qPCR, and the results revealed
significantly reduced levels of Bmp7 in Pten deletion mice
compared with those of the controls (Fig. 4K). However, the
expression levels of BMP receptors, including Alk1-3, Alk6 and
Bmpr2, were comparable between control and Pten deletion mice
(Fig. 4L).
Collectively, these data indicate that BMP signaling is dampened
in the SHF of Pten deletion embryos.
Akt1 signaling was hyperactivated upon Pten deletion, and we
observed nuclear localization of active Akt. Therefore, we
hypothesized that the expression of Bmp 7 was suppressed by Akt
signaling at the transcriptional level. The forkhead box O (FOXO)
family of transcription factors are well-established Akt substrates,
and Akt signaling might repress the expression of BMP signaling
components through inhibition of FOXO (Evans-Anderson
et al., 2008). To test this hypothesis, we first investigated the
phosphorylation status of FOXO in heart tissue via IF staining. The
levels of FOXO phosphorylation in the Pten deletion hearts were
profoundly enhanced compared with control hearts (Fig. 5A-G).
Phosphorylation of FOXO by Akt translocates FOXO from the
nucleus to the cytosol. Accordingly, we observed more cytosolic
phospho-FOXO in the Pten deletion hearts than in control hearts
(Fig. 5A-G; and supplementary material Fig. S4). Furthermore,
western blot analysis revealed significantly enhanced FOXO
phosphorylation levels in the heart of Pten deletion mice compared
with control mice (Fig. 5H).
To test whether FOXO activates the transcription of Bmp7, we
ligated a luciferase reporter into the upstream cis-elements of the
Bmp7 gene, which contains a FOXO-binding consensus sequence
(Fig. 5I). This luciferase reporter assay showed that constitutively
active FOXO1 (FOXO1-AAA) strongly activated the transcription
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DEVELOPMENT
Fig. 5. Akt regulates Bmp7 expression through the FOXO transcription factor. (A-F) IF staining against phospho-FOXO1/3 in the SHF at E9.5. More
pFOXO1/3 is localized in the cytosol of Pten deletion mice. C′,F′ are higher magnification views of the boxed areas in C,F. (G) Quantification of pFOXO1-positive
nuclei. (H) Western blot analysis. (I) Bmp7-luciferase construct. There is a FOXO binding site at the −637 bp position in the Bmp7 5′-upstream region.
(J) Luciferase reporter assay. In the Bmp7 mutant, the FOXO binding motif of GTAAATAT was changed to GTACCTAT (highlighted in red).
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Development (2015) 142, 732-742 doi:10.1242/dev.119016
of the luciferase reporter, whereas mutation of the FOXO-binding
consensus sequence blocked this activation (Fig. 5I,J).
Taken together, these results demonstrate that Akt signaling
suppresses BMP signaling through phosphorylation and inhibition
of FOXO.
PTEN-Akt signaling regulates β-catenin activity
Wnt signaling regulates early cardiogenesis and is involved in SHF
development. Deletion of the Wnt effector β-catenin in the SHF
caused impaired RV formation (Kwon et al., 2007). It was
previously reported that phosphorylation of β-catenin at the
C-terminal S552 by Akt promotes β-catenin nuclear localization
and activation in ISCs (He et al., 2007). We first investigated
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β-catenin phosphorylation levels in heart tissues through western
blot analysis and found much higher phospho-β-catenin levels in
the hearts of Ptenf/f; Mef2c-Cre mice than in control hearts
(Fig. 6A). Ptenf/f; Akt1f/+; Mesp1-Cre mice also showed strong
phosphorylation of β-catenin in the heart (Fig. 6B).
Next, we tested whether reducing β-catenin activity could
reverse the phenotype of Pten deletion mice. We found that
partial deletion of β-catenin partially reduced the heart size of
Pten deletion mice (Fig. 6C-L) and also decreased cell
proliferation of the SHF progenitors of Pten deletion mice
(Fig. 6M-P).
Collectively, these results showed that β-catenin activity was
elevated in Pten deletion mice, which disrupted heart development.
DEVELOPMENT
Fig. 6. PTEN-Akt signaling regulates
β-catenin activity. (A,B) Western blot
analysis of β-catenin S552 phosphorylation
in heart tissues. (C-L) Microscopic and
histological analysis of embryos and their
hearts. (M-O) IF staining against Ki67/Isl1 at
E9.5. M′-O′ and M″-O″ are higher
magnification views of the boxed areas in
M-O. (P) Quantification of Isl1/Ki67 doublepositive nuclei in M-O″.
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Development (2015) 142, 732-742 doi:10.1242/dev.119016
Reducing Akt signaling in SHF progenitors
PDK1 is indispensable for Akt activation during heart development
(Feng et al., 2010). We deleted Pdk1 in the SHF and observed
impaired development of the RV and the OFT (Fig. 7A-J). IF staining
of phospho-SMAD1/5 revealed an increased signal in the hearts of
Pdk1 deletion mice compared with the controls (Fig. 7K-S).
DISCUSSION
Our findings provide new insights into SHF development, which is a
fundamental, but poorly understood, developmental event that
governs OFT formation and ventricular expansion in mammals.
We found that enhancing Akt signaling through Pten deletion caused
expansion of the SHF and overgrowth of SHF derivatives, the OFT
and the RV, whereas the inhibition of Akt signaling upon PDK1 and
Akt inactivation results in a smaller OFT and RV. Thus, our findings
demonstrate a crucial role of Akt signaling in SHF development.
Furthermore, we uncovered a mechanism underlying the
coordination of BMP and Wnt-β-catenin signaling to regulate SHF
proliferation and differentiation (supplementary material Fig. S5A).
Genetic relationship between PTEN and Akt1 in SHF
development
One property of SHF progenitors is continued cell proliferation
(Watanabe et al., 2012). FGF signaling drives the cell proliferation
of SHF progenitors. Among the numerous FGF ligands, FGF8 and
10 are important regulators involved in SHF development. FGF10
was the first identified molecular marker of the murine SHF, and
mouse genetic studies have revealed that both FGF8 and FGF10
promote SHF progenitor proliferation through activation of the
MAPK/ERK pathway (Watanabe et al., 2010). In a variety of
mouse and human tissues, FGFs exert their functions by
modulating two pivotal signaling pathways: the MAPK/ERK
signaling pathway and the PI3K-Akt signaling pathway.
Therefore, FGF ligands, including FGF4, 8 and 10, are most
likely the upstream intercellular growth factors that activate PI3KAkt signaling in the SHF.
There are three Akt proteins in mice and humans, Akt1, 2 and 3,
and previous studies by our group have demonstrated that all of
these proteins are expressed in embryonic heart tissues (Chang et al.,
2010). Although deletion of Akt1 is sufficient to suppress tumor
development in Pten +/− mice, genetic evidence of the relationship
between PTEN and Akt1 during mouse embryonic development is
lacking. In this study, we demonstrated that Akt1 is the key
downstream target that is negatively regulated by PTEN, and we
established the first genetic relationship between PTEN and Akt1 in
the SHF and heart development.
Pten is expressed throughout embryogenesis, and germline
deletion of Pten causes embryonic lethality by E9.5, indicating
that PTEN is essential for mouse embryonic development (Suzuki
et al., 1998). Thus, it is possible that modulation of Pten expression
or PTEN protein levels by signals other than FGF signaling impacts
the SHF development.
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DEVELOPMENT
Fig. 7. Reducing Akt signaling in SHF progenitors. (A-J) Microscopic analysis of E10.5 and E14.5 hearts and embryos. RVs are marked by dashed lines.
(K-R) IF staining against phospho-SMAD1/5 in the RV at E14.5. (S) Quantification of p-SMAD1/5-positive nuclei in the RV.
RESEARCH ARTICLE
Previously, Li and colleagues reported that BMP suppresses PTEN
phosphorylation that inactivates PTEN, resulting in enhanced
PTEN activity in ISCs (He et al., 2004). Their results indicate that
BMP suppresses Akt activity through PTEN in ISCs. In this study,
we found that Akt signaling represses the expression of Bmp ligand
(Bmp7) in SHF progenitors. As a result, BMP signaling is reduced
by Akt activation. There might be a reciprocal antagonizing effect
between Akt and BMP signaling in SHF development. Whether
BMP inhibits Akt signaling in the SHF needs further investigation.
Regulation of the stem cell/progenitor cell switch from
quiescent to active state
Based on their own and others’ research on stem cells, Clevers and
Li proposed a model for the switch of stem cells from a quiescent
state to an active state (Li and Clevers, 2010). In the quiescent state,
BMP signaling is active, and Wnt signaling is turned off in stem
cells. Once stem cells are activated for proliferation (the active state),
BMP signaling is shut off and Wnt signaling is switched on.
Nevertheless, how the switch from a quiescent/BMP-on state to an
active/Wnt-on state is regulated remains elusive. Our findings
suggest that Akt signaling is a mediator coordinating both BMP and
Wnt signaling during the transition state of stem/progenitor cells
(supplementary material Fig. S5B). In the future, it will be
intriguing to test this hypothesis in adult stem cells, such as hair
follicle, gut and bone marrow stem cells.
We observed enlarged LVs of Pten deletion mice from late
gestational stage. Lineage tracing showed that Pten-deletion
cardiomyocytes moved from the RV to the LV, suggesting that
the mutant cardiomyocytes have gained migratory ability or
dominated the RV due to increased proliferation. This hypothesis
deserves further investigation.
MATERIALS AND METHODS
Mice
The previously described mouse strains used in this study included Pten
conditional mice (Suzuki et al., 2001), Mesp1-Cre and Mef2c-AHF-Cre mice
(Klaus and Birchmeier, 2009; Saga et al., 1999; Verzi et al., 2005), β-cateninfloxed mice (Huelsken et al., 2001) and Akt1 and Pdk1 conditional mice
(Di et al., 2012; Feng et al., 2010; Zhao et al., 2014). All mouse lines were
maintained in a B6 genetic background. The experimental animal facility has
been accredited by the Association for Assessment and Accreditation of
Laboratory Animal Care International (AAALAC), and the Institutional
Animal Care and Use Committee (IACUC) of Model Animal Research
Center of Nanjing University, China, approved all animal protocols used in this
study.
Histological analysis and β-galactosidase staining
Mouse embryos were collected and fixed in 4% paraformaldehyde, then
dehydrated, embedded in paraffin and sectioned. Histological sections
were stained with hematoxylin and eosin (H&E) or used for other
analyses, such as immunohistochemistry (IHC), immunofluorescence
(IF) staining and terminal deoxynucleotidyl transferase dUTP nick end
labeling (TUNEL) assays. For β-galactosidase staining, embryos were
harvested and pre-fixed for 2 h in 4% paraformaldehyde on ice, and
β-galactosidase staining was subsequently performed as previously
described (Prall et al., 2007).
incubated with appropriately diluted primary antibodies at 4°C overnight.
Afterwards, the sections were washed three times in PBS for 5 min each and
then incubated with secondary antibodies or other dyes at room temperature
for 2 h. The fixed and stained sections were mounted with nail polishing oil
for analysis. IHC staining was carried out according to manufacturer’s
recommendations and detected with 3,3-diaminobenzidine (DAB) (MaixinBio). The following antibodies were used for IF staining: anti-p-Akt (Cell
Signaling Technology, CST 4060; 1:200), anti-p-Smad1/5 (Cell Signaling
Technology, CST 9516; 1:200), anti-p-β-catenin S552 (Cell Signaling
Technology, CST 5651S; 1:200), anti-Ki67 (DakoCytomation, M724901;
1:200), anti-p-Histone H3 (Cell Signaling Technology, CST 9701S; 1:200),
anti-αSMA (Sigma, c6198; 1:200), anti-p-FOXO (Cell Signaling
Technology, CST 2880; 1:200), and anti-Isl1 (1:200) and antiTropomyosin (1:200) from the Developmental Studies Hybridoma Bank
(DSHB; University of Iowa, Iowa City, USA).
Western blot analysis
Embryonic hearts were harvested, snap-frozen in liquid nitrogen and stored
until use. Tissue lysates were prepared in lysis buffer as previously described
(Chang et al., 2010). Approximately 50 µg of protein per sample was
separated via electrophoresis and transferred to a polyvinylidene difluoride
(PVDF) membrane (Millipore). The membranes were blocked with TBST
(50 mM Tris, 150 mM NaCl, 0.5 mM Tween 20, pH 7.5) and incubated with
primary antibodies overnight. The samples were analyzed via immunoblotting
with antibodies against t-Akt (Bioworld, BS1810; 1:1000), p-Akt (Cell
Signaling Technology, CST 4060; 1:1000), Smad1 (Cell Signaling
Technology, CST 6944; 1:500), p-Smad1/5 (Cell Signaling Technology,
CST 9516; 1:500), p-β-catenin552 (Cell Signaling Technology, CST 5651S;
1:500) and anti-GAPDH (Bioworld, AP0063; 1:20,000). The membranes
were then incubated with a secondary anti-rabbit or anti-mouse HRPconjugated antibody, and the resulting signals were detected through
enhanced chemiluminescence (ECL).
RT-qPCR
Total RNA from embryonic hearts or pharyngeal arches was isolated with
TRIzol reagent (Invitrogen), and 1 μg of RNA was reverse-transcribed using
a reverse transcription system kit (Promega). RT-qPCR was performed
using an ABI7900 thermocycler with the Roche SYBR Green RT-qPCR
reagent. The expression of selected genes involved in cardiac development
was examined, and the results were normalized to the expression of
GAPDH.
Plasmid construction and luciferase reporter assay
A ∼3.4 kb fragment, corresponding to the Bmp7 5′ flanking sequence, was
amplified via PCR from mouse genomic DNA and cloned into the pGL3Basic Vector (Promega) to generate the Bmp7-luc vector. The Bmp7 mut-luc
vector, which contains a 2-bp mutation in the mouse Bmp7 FHRE box (AA
to CC), was generated using the MutaBest kit (Takara). Luciferase assays
were performed using the Dual Luciferase Assay System (Promega),
following a standard protocol. At least ten independent transfections were
conducted, and all assays were performed three times.
Statistical analysis
All results are presented as mean±s.e.m. Statistical analyses were performed
using Student’s t-test with GraphPad Prism 4.03 software. Multiple groups
were tested via one-way ANOVA, and comparisons between two groups
were performed using Student’s t-test. A P-value of <0.05 was considered
significant (*), and P-values of <0.01 (**) or <0.005 (***) were considered
highly significant.
IF and IHC staining
Acknowledgements
Embryos were fixed in 4% paraformaldehyde for no more than 2 h. After
fixation, the embryos were rinsed in PBS and incubated in 30% sucrose at
4°C for at least 8 h. The embryos were then embedded in OCT medium in
the required direction. The embedded embryos were snap-frozen in liquid
nitrogen and stored at −80°C. Sections were cut at a thickness of 7-10 μm.
Subsequently, the sections were blocked with 10% goat serum and
We thank Dario Alessi for Pdk1 floxed mice, Brian Black for Mef2c-AHF-Cre mice
and Yeguang Chen for discussions. We thank Teng Teng for English editing and
Heng Wang for drawing schematics.
740
Competing interests
The authors declare no competing or financial interests.
DEVELOPMENT
Crosstalk between PTEN-Akt signaling and BMP signaling
Development (2015) 142, 732-742 doi:10.1242/dev.119016
Author contributions
W.L. and Z.Y. conceived and designed the experiments; W.L., X.Z., L.T., J.Z., H.W.
and H.J. performed experiments and analyzed the data; W.L. and Z.Y. wrote the
manuscript; W.L., X.Z., L.T., J.Z., H.W., H.J. and Z.Y. discussed and reviewed the
manuscript.
Funding
This work was supported by the National Natural Science Foundation of China
[31130037, 91019002 and 31071282 to Z.Y.] and the National Key Basic Research
Program of China [2011CB943904 and 2012CB966602 to Z.Y.]. The Friedrich
Miescher Institute for Biomedical Research is part of the Novartis Research
Foundation.
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.119016/-/DC1
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