Cellular Biology
Bmp Signaling Exerts Opposite Effects on
Cardiac Differentiation
Emma de Pater, Metamia Ciampricotti, Florian Priller, Justus Veerkamp, Ina Strate, Kelly Smith,
Anne Karine Lagendijk, Thomas F. Schilling, Wiebke Herzog, Salim Abdelilah-Seyfried,
Matthias Hammerschmidt, Jeroen Bakkers
Rationale: The importance for Bmp signaling during embryonic stem cell differentiation into myocardial cells has
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
been recognized. The question when and where Bmp signaling in vivo regulates myocardial differentiation has
remained largely unanswered.
Objective: To identify when and where Bmp signaling regulates cardiogenic differentiation.
Methods and Results: Here we have observed that in zebrafish embryos, Bmp signaling is active in cardiac
progenitor cells prior to their differentiation into cardiomyocytes. Bmp signaling is continuously required during
somitogenesis within the anterior lateral plate mesoderm to induce myocardial differentiation. Surprisingly, Bmp
signaling is actively repressed in differentiating myocardial cells. We identified the inhibitory Smad6a, which is
expressed in the cardiac tissue, to be required to inhibit Bmp signaling and thereby promote expansion of the
ventricular myocardium.
Conclusion: Bmp signaling exerts opposing effects on myocardial differentiation in the embryo by promoting as
well as inhibiting cardiac growth. (Circ Res. 2012;110:578-587.)
Key Words: BMP 䡲 Smad6 䡲 Heart 䡲 Zebrafish 䡲 Bmpr1a
T
he complex multichambered vertebrate heart is formed
from a rapidly expanding primary heart tube due to the
combined processes of cardiac differentiation and tissue
morphogenesis.
heart tube by the addition of cardiomyocytes to both the arterial
and venous pole of the heart tube.7 At the arterial pole, Fgf
signaling is required for the growth of the heart.7 At the venous
pole we described that the transcription factor Isl1 is present in
the differentiating cardiomyocytes, and embryos lacking Isl1
have a small reduction in the number of atrial cardiomyocytes.
Bmp ligands have been implicated in regulating cardiac
differentiation in invertebrates as well as vertebrates.8 –14 Bmp
proteins are synthesized as large preproteins, which are
cleaved to release the dimeric C-terminal mature and active
region.15 Bmp2 and Bmp4 are closely related, and they can
bind to their Bmp type 1 receptors (alk2/8, alk3a, alk3b, and
alk6) as homo- or heterodimers.16 –18 Interaction of the type 1
receptor with the Bmp type 2 receptor results in the phosphorylation of Smad transcriptional activators. In case of
Bmp signaling there are 3 receptor-Smad proteins (Smad1,
-5, and -8) that can be phosphorylated and interact with the
co-Smad, Smad4. This phosphorylated Smad-protein complex
is translocated to the nucleus where it activates gene expression.
Secreted antagonists such as Noggin can block the interaction
between Bmp ligand and the receptor and thereby prevent Bmp
In This Issue, see p 523
In zebrafish, cardiac progenitor cells are located in the lateral
marginal domain at both sides of the organizer of blastula stage
embryos.1,2 During gastrulation, the bilateral pools of cardiac
progenitor cells migrate dorsal and anterior to form part of the
anterior lateral plate mesoderm (ALPM). In the ALPM, several
transcription factors (nkx2.5, gata5, and hand2) are expressed
that promote cardiac differentiation. The first progenitor cells
that are determined to become cardiomyocyte in the ALPM can
be identified at the 14-somite stage by expression of the cardiac
differentiation marker cardiac myosin, light polypeptide 7
(myl7).3 Around 19 hours postfertilization (hpf) (20-somite
stage), these 2 bilateral pools of cardiomyocytes have fused at
the midline to form a cardiac disk.4,5 This cardiac disk rotates
and extends in an anterior and leftward direction to form a linear
heart tube.6 We previously described a continuous growth of the
Original received November 18, 2011; revision received December 29, 2011; accepted January 5, 2012. In December 2011, the average time from
submission to first decision for all original research papers submitted to Circulation Research was 14.29 days.
From the Hubrecht Institute—KNAW & University Medical Centre Utrecht (E.P., M.C., I.S., K.S., A.K.L., J.B.); Interuniversity Cardiology Institute
of the Netherlands, The Netherlands (J.B.); Max Delbrück Centrum for Molecular Medicine Berlin, Berlin, Germany (F.P., J.V., S.A.S.); Department of
Developmental and Cell Biology, University of California, Irvine (T.F.S.); Max Planck Institute for Molecular Biomedicine, Münster, Germany (W.H.);
Biocenter Cologne, Institute of Developmental Biology, Cologne University, Cologne, Germany (M.H.).
The online-only Data Supplement is available with this article at http://circres.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA.111.261172/-/DC1.
Correspondence to Jeroen Bakkers, PhD, Associate Professor, Cardiac development and genetics group, Hubrecht Institute for Developmental Biology
and Stem Cell Research, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands. E-mail [email protected]
© 2012 American Heart Association, Inc.
Circulation Research is available at http://circres.ahajournals.org
DOI: 10.1161/CIRCRESAHA.111.261172
578
de Pater et al
A
B
Bmp
579
Non-standard Abbreviations and Acronyms
Type I: Alk2/3/8
Type II: BmpRII
cytoplasm
P
P-Smad1/5/8
nucleus
gene
expression
alk3 +/-
Bmp and Myocardial Differentiation
ALPM
AVC
Bmp
BrdU
BRE
hpf
MO
RT-PCR
anterior lateral plate mesoderm
atrioventricular canal
bone morphogenetic protein
5-bromo-2⬘-deoxyuridine
Bmp responsive element
hours postfertilization
Morpholino oligo
reverse transcriptase, polymerase chain reaction
alk3 -/-
E
F
G
H
hand2
nkx2.5
D
tbx1
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
C
Figure 1. Alk3a/Bmpr1a is required for cardiac specification.
A, Illustration representing a dorsal view on a zebrafish embryo
at the 12-somite stage (15 hours postfertilization) with bilateral
cardiac fields colored in blue. B, Schematic diagram of Bmp
signaling. C through H, Expression of nkx2.5 (C and D), hand2
(E and F), and tbx1 (G and H) in wild-type siblings or alk3a/
bmpr1a mutant embryos. Embryos at 12-somite stage are
shown as dorsal views with anterior to the top.
signaling, while the inhibitory Smad protein Smad6 attenuates
Bmp signaling in the cytoplasm. Smad6 has been shown to
specifically inhibit Bmp signaling but only weakly Tgf-␤/activin
signaling. Several mechanisms have been proposed for the
inhibitory effect of Smad6 on Bmp signaling. Smad6 can bind to
Smad1 and interfere with complex formation between Smad1
and Smad4.19 Smad6 can also form a stable complex with the
Bmp type I receptors. This interaction can inhibit phosphorylation of Bmp-specific R-Smads and result in inhibition of Bmp
signaling.20 Furthermore, Smad6 recruits the E3 ubiquitin ligase
Smurf1 (Smad ubiquitin regulatory factor 1) to the signaling
receptor complex and enhances the down-regulation of type I
receptors through proteasome-dependent degradation.21
In this study, we investigated the role of Bmp signaling in
cardiomyocyte differentiation. We observed that in zebrafish
embryos, Bmp signaling is active in the ALPM but is absent
from the myocardial cells at similar stages. These results raise
2 questions: (i) when is Bmp signaling required for cardiac
differentiation and (ii) is the observed inhibition of Bmp
signaling in differentiating cardiomyocytes relevant for heart
formation? As an answer to the first question, we identified a
previously unrecognized continuous requirement for Bmp
signaling during cardiac differentiation in the ALPM. As an
answer to the second question, we found that Smad6 is
required to inhibit Bmp signaling and that the downregulation of Bmp signaling is required for proper formation
of the ventricle and preventing expression of atrial myosin in
regions outside the atrium.
Methods
All transgenic strains were analyzed as heterozygotes in our studies, and all
embryos were grown at 28.5°C. Mutants used in this study were lin/
bmpr1aahu4087, bmpr1absa0028, and laf/acvr1ltm110b. Transgenic lines used
were Tg(hsp70:alk8), Tg(hsp70I:Nog3)fr14, Tg(hsp70:Bmp2b)fr13, and
Tg(Bre:GFP)p77.
An expanded Methods section is available in the online-only Data
Supplement.
Results
Bmp Signaling Is Required and Precedes
Cardiac Differentiation
We recently showed that Bmp receptor 1a (alk3a;alk3b)
double-mutant embryos (further referred to as alk3 mutants)
lack differentiated cardiomyocytes.22 Further examination of
the alk3 mutant embryos revealed that the precardiac marker
hand2 and the cardiac marker nkx2.5 were lost in alk3 mutant
embryos (Figure 1A through 1F), while tbx1 was still expressed in the ALPM (Figure 1G and 1H). Although these
results demonstrate that Bmp signaling is required for the
induction of cardiac mesoderm, they do not address when and
where this takes place. To visualize active Bmp signaling, we
used an antibody recognizing phosphorylated Smad1, Smad5,
and Smad8 protein (referred to as P-Smad), in combination
with the transgene Tg(myl7:GFP) labeling differentiated
cardiomyocytes. In agreement with our previous findings,7
we observed that in the ALPM the number of cardiomyocytes
expressing myl7:GFP progressively increases from 15 to 20
580
Circulation Research
A
ectoderm
LPM
myocardium
neural
tube
February 17, 2012
B
myocardium
endoderm
yolk
12-somite
C
myl7:GFP P-Smad
D
dorsal
myocardium
ventral
myl7:GFP P-Smad
endocardium
E
20-somite
myl7:GFP P-Smad
G
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
F
dorsal
myocardium
ventral
Tpm
endocardium
Bre:GFP
H
20-somite
Tpm
Bre:GFP
Figure 2. Restricted activation of Bmp signaling in ALPM. A,
C, and F, Illustrations representing transverse sections through
the anterior region of a zebrafish embryo at the 12- or
20-somite stage. B, A single transverse confocal image of a
Tg(myl7:eGFP) embryo at 15 hours postfertilization (hpf) (12somite stage) stained for phosphorylated-Smad1,5,8 (P-Smad).
Arrow points to an myl7:eGFP-expressing cell, which is negative for P-Smad. Bar indicates region containing several
P-Smad positive cells. D, Single confocal scan of
Tg(myl7:eGFP) embryos at 19 hpf (20-somite stage) stained for
phosphorylated-Smad1,5,8 (P-Smad). Arrow points to the dorsal
part of the neural tube. E, Higher magnification of the myocardium
shown in D. G, A single confocal scan of a Tg(bre:GFP) embryo
stained for tropomyosin (Tpm) to indicate the myocardium. H,
Higher magnification of the myocardium from images shown in G.
Corresponding gray-scale images of single-color channels are
provided in Online Figure I.
hpf (Figure 2A through 2E). Interestingly, at the 12 somite
stage (15 hpf) a nuclear P-Smad protein was detected in cells
near the myl7:GFP-expressing cells, while most myl7:GFPexpressing cells were negative for P-Smad (Figure 2A, B and
Online Figure I). This was even more apparent at the 20
somite stage (20 hpf) (Figure 2C through 2E and Online Figure
I). At this stage a majority (84%, n⫽6 embryos) of the
myl7:GFP-expressing myocardial cells lacked a nuclear
P-Smad1 signal, while nuclear P-Smad signal was clearly
present in cells near the myl7:GFP-expressing myocardial
cells and in the dorsal part of the neural tube (Figure 2D).
Together, these results demonstrate that while Bmp signaling
is active in the ALPM, there is very little Bmp signaling
activity in the differentiating cardiomyocytes. Since Bmp
signaling is required for cardiomyocyte differentiation, we
addressed whether Bmp signaling was active in cardiac
progenitor cells prior to their differentiation into cardiomyocytes. Therefore we analyzed Tg(Bre:eGFP) embryos in
which GFP expression is induced by Bmp signaling due to
the presence of a conserved Bmp responsive element (BRE)
upstream of the coding sequence for eGFP.23 Since GFP has
a half-life time of 24 hours, it is possible to trace cells after
the Bmp signal has been turned off. When analyzing Tg(Bre:eGFP) embryos, we observed that differentiating cardiomyocytes, marked by tropomyosin expression, were positive for Bre:GFP, albeit at lower levels in comparison with
cells neighboring the myocardial tissue (Figure 2F through
2H and Online Figure I). Together, these results demonstrate
that while cardiomyocytes are exposed to Bmp signaling
prior to their differentiation and that the signaling activity is
required for proper cardiomyocyte differentiation, Bmp signaling is no longer active in differentiating cardiomyocytes.
Bmp Signaling Is Required for Cardiomyocyte
Differentiation Until Midsegmentation
Independent of Mesoderm Specification
To address when Bmp signaling is required to induce cardiac
differentiation, we studied the zebrafish lost-a-fin (laf) mutant, encoding a Bmp receptor type I (acvr1l/alk8). While
alk3 mutant embryos display severe dorsoventral patterning
defects resulting in early embryo lethality, zygotic (Z) laf/
alk8 mutant embryos have only mild dorsoventral defects due
to the presence of maternal alk8 mRNA in the oocyte during
gastrulation.16,17,22 Maternal zygotic (MZ) alk8 mutants, lacking all maternal and zygotic alk8 mRNA, display very severe
mesodermal patterning defects and as a consequence die
between 16 and 24 hpf.17 As reported previously, zygotic (Z)
laf/alk8 mutant embryos have a reduced ventral tailfin together with a smaller atrium in comparison with their wildtype siblings at 48 hours postfertilization (hpf) (Online Figure
IIA).12,16,17,24 To investigate when zygotic alk8 mRNA is
required to allow atrial cardiomyocytes to form correctly, we
re-expressed a functional alk8 gene in Zlaf/alk8 mutant
embryos at various developmental time points. For this
purpose we used a previously described heat-shock inducible
transgenic line driving alk8 expression Tg(hsp70:alk8).25 In
agreement with an earlier report, inducing alk8 expression
during gastrulation (6 hpf) completely rescued the Zlaf/alk8
phenotypes, including the small atrium (Shin et al25 and data
not shown). Interestingly, re-expressing alk8 after gastrulation at 16 hpf (14 somites stage) in Zlaf/alk8 mutant embryos
still restored the number of atrial cardiomyocytes to wild-type
numbers (Figure 3A and 3B) (wild-type control; 85.6⫾5
atrial cells versus laf/alk8 mutant; 51.8⫾4 atrial cells versus
laf/alk8 mutant/ Tg(hsp70:alk8); 82.6⫾6 atrial cells,
P⬍0.01), although it no longer rescued the loss of the ventral
tailfin (Online Figure IIA). These results suggest that even
after gastrulation, Bmp signaling is still required for the
correct differentiation of cardiomyocytes. Re-expressing alk8
at 16 hpf (14 somites stage) in Zlaf/alk8 mutant embryos did
not, however, restore cardiac looping (Online Figure IIA),
indicating that cardiac looping and cardiomyocyte differentiation are 2 distinct processes both requiring Bmp signaling
but at different time points during embryo development.
To address when Bmp signaling is still required to stimulate
cardiac differentiation after gastrulation, we blocked Bmp signaling
at different postgastrula stages using a previously described heat-
de Pater et al
A
Bmp and Myocardial Differentiation
B
temporal rescue of Bmp signaling
alk8 +/alk8 -/alk8 -/-;
Tg(hsp70:alk8)
Zlaf/alk8 -/-
0
10 hpf
24 hpf
16 hpf
analysis: 48 hpf
hsp70:alk8
rescue
atrial CM
HS
C
cardiomyocytes
alk8
zygotic
alk8
maternal
581
140
120
100
80
60
40
20
0
**
ventricle
atrium
temporal reduction of Bmp signaling
hsp70:Noggin3
10 hpf
analysis: 48 hpf
HS
normal CM
reduced CM (atr.)
reduced CM
(atr.&ven.)
HS
HS
control
140
E
HS @ 10 hpf
140
120
100
**
80
60
**
40
20
0
ventricle
atrium
cardiomyocytes
D
cardiomyocytes
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
24 hpf
16 hpf
Tg(hsp70:Noggin3)
120
100
**
80
HS @ 24 hpf
F
HS @ 16 hpf
60
40
20
140
cardiomyocytes
0
120
100
80
60
40
20
0
0
ventricle
atrium
ventricle
atrium
Figure 3. Bmp signaling is required during mid- and late-somite stages for myocardial differentiation. A, Experimental set-up for
temporal rescue of myocardial defect in laf/alk8 mutant embryos. B, Quantification of the number of ventricular and atrial cells in laf/
alk8⫾, laf/alk8⫺/⫺, and laf/alk8⫺/⫺ Tg(hsp70:alk8) embryos heat-shocked at 16 hours postfertilization (hpf). Bars represent mean ⫾ SEM.
**P⬍0.01. C, Experimental set-up for temporal inhibition of Bmp signaling by heat shock–induced Noggin3 expression. D and F, Quantification of the number of ventricular and atrial cardiomyocytes in wild-type sibling or Tg(hsp70:Noggin3) embryo heat-shocked at 10
hpf (D), 16 hpf (E), and 24 hpf (F), respectively. Bars represent mean ⫾ SEM. **P⬍0.01.
shock inducible Noggin3 transgenic line.24 The Tg(hsp70:Noggin3)
carriers were crossed with Tg(myl7:dsRed-nuc) carriers, and the
embryos derived from this cross were heat-shocked at various
stages and fixed at 48 hpf to quantify the number of cardiomyocytes in the ventricle and atrium (Figure 3C). After an early
block in Bmp signaling from 10 hpf (bud stage), both cardiac
chambers were observed to contain fewer cardiomyocytes (wildtype siblings; 122.7⫾4 ventricular and 84.4⫾6 atrial cells versus
Tg(hsp70:Noggin3) embryos; 84.1⫾11 ventricular and 24.8⫾6
atrial cells; n⫽11, P⬍0.01) (Figure 3D and Online Figure IIB).
In contrast, Tg(hsp70:Noggin3)/Tg(myl7: dsRed-nuc) embryos
heat-shocked at 16 hpf (14 somite stage) and fixed at 48 hpf
contained normal numbers of ventricular cells (111.8⫾4 in
wild-type siblings versus 105.5⫾6 ventricular cells in
Tg(hsp70:Noggin3)). However, the number of atrial cells
was significantly reduced in the Tg(hsp70:Noggin3) embryos heat-shocked at 16 hpf (wild-type siblings; 97.6⫾5
versus Tg(hsp70:Noggin3); 78.8⫾2 atrial cells, n⫽11,
P⬍0.01) (Figure 3E and Online Figure IIB). Finally,
Tg(hsp70:Noggin3)/Tg(myl7: dsRed-nuc) embryos heatshocked at 24 hpf and fixed at 48 hpf contained normal
numbers of both ventricular and atrial cardiomyocytes
(109.1⫾3 versus 107.8⫾9 ventricular cardiomyocytes in
wild-type siblingsversus Tg(hsp70:Noggin3); 82.5⫾4 versus 73.6⫾3 atrial cardiomyocytes in wild-type siblings
versus Tg(hsp70:Noggin3)) (Figure 3F and Online Figure
IIB). However, looping morphogenesis was affected in
these embryos (Online Figure IIB).
In conclusion, these data demonstrate that in the embryo
proper, there is a continuous requirement for Bmp signaling
activity to induce cardiomyocyte differentiation. However,
atrial and ventricle cardiomyocytes require a Bmp signal at
different stages. While progenitor cells that form the myocardium of the ventricle require activation of Bmp signaling
from gastrulation until early segmentation stages (bud stage),
those that form the myocardium of the atrium require activation of Bmp signaling from early through late segmentation
stages (12–15 somites). This observation is consistent with
nkx2.5
myocardium
B
C
nkx2.5
E
sections
alk8 +/?
D
alk8-/-
BrdU
alk8+/?
wild-type
Tg(hsp70:noggin3)
A
B
C
D
tbx20
ALPM
February 17, 2012
alk8-/-
E
our previous observation that cardiomyocyte differentiation is
initiated in the ventricle and continues in the atrium.7
Bmp Signaling via Alk8 Is Dispensable for Cell
Proliferation in the ALPM
Previous studies in chick and mouse embryos showed that
inhibiting Bmp signaling stimulates cell proliferation in the
ALPM.26 –29 To analyze whether cell proliferation was affected in the ALPM of Zlaf/alk8 mutant embryos, we pulselabeled the embryos from 16 to 19 hpf with BrdU and
quantified the number of BrdU positive cells. Expression of
nkx2.5 was used to mark the cardiac mesoderm. By counting
all BrdU positive cells in ALPM of serial sections (from
anterior nkx2.5 positive cells until the notochord appeared in
the sections), no difference in the number of BrdU positive
cells was observed between laf/alk8 mutant and their wildtype sibling embryos (wild-type sibling 412⫾6 versus laf/
alk8 mutant 420⫾63) (Figure 4A through 4E). Similar results
were obtained with the Tg(hsp70:Noggin3) embryos in which
Bmp signaling was inhibited at 16 hpf (data not shown).
Bmp Signaling Induces tbx2b and
tbx20 Expression
Since we observed no effect on cell proliferation in the
ALPM upon reducing Bmp signaling, we wondered whether
Bmp signaling could affect cardiac differentiation by regulating the expression of cardiogenic factors. The T-box
factors tbx20 and tbx2 play independent roles during cardiomyocyte differentiation in mouse embryos.30 In addition, both
tbx20 and tbx2 are direct transcriptional targets of Bmp
signaling.30,31 To address when BMP-dependent regulation of
tbx2b and tbx20 occurs during cardiac differentiation, we
reduced Bmp signaling at the mid- to late-somite stage. We
observed a reduced expression of both tbx20 and tbx2b in the
cardiac region as a consequence of noggin3 expression at the
15-somite stage (Figure 5A through 5D). Quantitative
RT-PCR analysis on cardiac explants revealed that tbx20 and
Tweezers
F
fold-change
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
Figure 4. Cell proliferation in ALPM is unaffected in laf/alk8
mutant embryos. A and B, Illustrations indicating the location and
orientation of the transverse sections shown in C and D. C and D,
Transverse sections of wild-type sibling (⫹/⫹ and ⫹/⫺) (C) and
laf/alk8 mutant (⫺/⫺) (D) embryo stained for nkx2.5 expression
(blue staining) and BrdU (brown), counterstained with hematoxilin.
Region of ALPM is indicated by red dotted line, and arrows point
to BrdU positive nuclei. E, Quantification of BrdU positive nuclei in
serial sections of the ALPM (indicated by presence of nkx2.5 positive cells and absence of notochord) of wild-type siblings and laf/
alk8 mutant embryos (n⫽3). Bars represent ⫾ SEM.
tbx2b
A
Circulation Research
BrdU+ cells
582
WT control
Tg(hsp:nog3)
1
0.75
0.5
**
0.25
***
0
tbx20
tbx2b
Figure 5. Overexpression of noggin3 reduces expression of
cardiac differentiation markers. A through D, In situ hybridization for tbx20 and tbx2b at 24 hours postfertilization in wildtype siblings and Tg(hsp70:noggin3) embryos heat-shocked at
15-somite stage. White dotted line in C outlines the linear heart
tube. E, Schematic representation of manual dissection of the
cardiac region from 25-somite stage embryos subjected to
RT-qPCR. F, Quantification of RT-qPCR results showing a
down-regulation of the cardiac transcription factors tbx2b and
tbx20 in 25-somite hearts of Tg(hsp70:noggin3) embryos heatshocked at 18-somite stage. **P⬎0.01. ***P⬎0.001.
tbx2b were significantly down-regulated, 3.5- fold and 5-fold,
respectively, as a response to Noggin3 expression during late
segmentation stages (Figure 5E and 5F). These results demonstrate that Bmp signaling at midsomite stages is required to
induce tbx20 and tbx2b expression, 2 transcription factors that
regulate myocardial differentiation.
Bmp Signaling in the Cardiac Field Is Inhibited by
the Inhibitory Smad6
We observed that cardiomyocytes were exposed to Bmp signaling prior to differentiation and that Bmp signaling was lost in
differentiating cardiomyocytes (Figure 1). This raised the question of whether the reduced Bmp signaling observed in the
differentiating myocardium is relevant for normal heart formation and, if so, how this is regulated. A possible explanation for
the low Bmp signaling activity in cardiomyocytes would be that
in cardiomyocytes, Bmp ligands are a limiting factor. To test this
de Pater et al
Control
A
myl7:GFP
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
Tg(hsp70:bmp2b) Tg(hsp70:bmp2b) Control
A’
myl7:GFP
E
myl7:GFP
E’
myl7:GFP
B
P-Smad
B’
P-Smad
F
P-Smad
F’
P-Smad
Bmp and Myocardial Differentiation
C
D
myl7:GFP P-Smad
myl7:GFP P-Smad dapi
C’
D’
myl7:GFP P-Smad
myl7:GFP P-Smad dapi
G
H
myl7:GFP P-Smad
myl7:GFP P-Smad dapi
G’
H’
myl7:GFP P-Smad
myl7:GFP P-Smad dapi
possibility we overexpressed bmp2b in the entire embryo including the myl7-expressing cardiac cells using the heat-shock
inducible Tg(hsp70:bmp2b) transgene. We observed that cardiomyocytes located medially in the cardiac field, which in wildtype embryos lack P-Smad (Figure 6A through 6D⬘), were
positive for P-Smad in heat-shocked Tg(hsp70:Bmp2b) embryos
(Figure 6E through 6H⬘, indicated by the white arrows). P-Smad
levels in these cardiomyocytes, however, were still low when
compared with other cell types such as those in the ventral neural
tube (Figure 6E through 6H⬘, indicated by the red arrowhead).
This suggested that either medial cardiomyocytes lack the
appropriate receptors to respond to the Bmp signal or they may
contain an endogenous inhibitory factor. Since expression of
alk3a, alk3b, and alk8 was reported to be ubiquitous at the
relevant stages,16,17,22 we addressed the involvement of an
inhibitor. The inhibitory Smad6 protein can repress Bmp signaling by competing with Smad1 for Smad4 binding or by
preventing Smad1,5,8 phosphorylation.19 –21 In zebrafish, smad6
is duplicated, and we observed that smad6a was expressed in 2
bilateral populations of cells at the 15-somite stage (Figure 7A
and 7B). At later stages when the cardiac fields have fused at the
midline, we observed smad6a expression in the cardiac disk
(Figure 7C). When the heart tube has formed at 24 hpf, we
observed smad6a expression in the linear heart tube predominantly in the future ventricle and in a region posterior of the
arterial pole (Figure 7D). To test the function of Smad6a during
cardiac differentiation, we generated 2 nonoverlapping antisense
morpholino oligos (MOs) complementary to the 5⬘UTR and
translation start site of the smad6a transcript. Injection of these
smad6a MOs did not affect overall embryo morphology, but we
observed ectopic phospho-Smad1,5,8 signals specifically in the
583
Figure 6. Bmp signaling is inhibited in
the cardiac field. A through Hⴕ, Single
confocal scan of Tg(myl7:eGFP) embryos
stained for eGFP in green and P-Smad in
red at 25-somite stage. A through D,
Wild-type embryo with enlargement of the
myocardium in Aⴕ through Dⴕ. E through
H, Representative Tg(hsp70:bmp2b)
embryo heat-shocked at 18-somite stage
with enlargement of the myocardium
shown in Eⴕ through Hⴕ. Red arrowhead
points to strong P-Smad signal in neural
tube. White arrows point to myocardial
cells with intermediate level of P-Smad
staining. Scale bars represent 50 ␮m.
Scale bars in enlargements represent
10 ␮m.
medial differentiating cardiomyocytes (Figure 7 E through 7H⬘
for ATG MO and data not shown) in comparison with embryos
injected with a control MO (Figure 7A through 7D). In wild-type
embryos, only 16% of myl7:GFP expressing cardiomyocytes
were P-Smad positive, whereas in smad6a morphants (MO
injected), this percentage was increased to 62% (n⫽6 for
control, n⫽5 for MO injected). Together, these results demonstrate that the inhibitory Smad6a is expressed during myocardial
differentiation, during which it is required to prevent Smad
phosphorylation and thereby activation of the Bmp signaling
pathway.
Ectopic Bmp Signaling Results in a Hypoplastic
Ventricle and Ectopic Expression of Atrial Myosin
To address the biological relevance of Bmp inhibition in
the cardiac field during cardiac differentiation, we analyzed the
heart tubes of Tg(hsp70:bmp2b) embryos heat-shocked at the
14- or 18-somite stages as well the heart tubes of smad6a
morphant embryos at 48 hpf (Figure 8A through 8C). In both the
Tg(hsp70:bmp2b) and the smad6a morphant embryos, we observed that cardiac looping was compromised and the size of the
ventricle was reduced. To further investigate the effect of ectopic
Bmp signaling on cardiac growth, we quantified the number of
atrial and ventricle myocytes in those hearts. Surprisingly, we
observed that the number of ventricular cardiomyocytes in
heat-shocked Tg(hsp70:bmp2b) embryos as well as in smad6a
morphant embryos was significantly reduced (wild-type
150.0⫾9; Tg(hsp70:bmp2b) 122.3⫾9; smad6a morphants
89.9⫾4 ventricular cells; P⬍0.05 and P⬍0.01) (Figure 8E),
while the number of atrial cardiomyocytes was not significantly
different from wild-type siblings on ectopic Bmp signaling in
584
Circulation Research
15 som B
A
February 17, 2012
15 som C
e
23 som D
Control MO
E
myl7:GFP
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
Smad6 MO atg Smad6 MO atg Control MO
E’
myl7:GFP
I
myl7:GFP
I’
myl7:GFP
e
h
e
smad6a
24 hpf
e
e
smad6a
F
P-Smad
F’
P-Smad
J
P-Smad
J’
P-Smad
smad6a
smad6a
G
H
myl7:GFP P-Smad
myl7:GFP P-Smad dapi
G’
H’
myl7:GFP P-Smad
myl7:GFP P-Smad dapi
K
L
myl7:GFP P-Smad
myl7:GFP P-Smad dapi
K’
L’
myl7:GFP P-Smad
myl7:GFP P-Smad dapi
Tg(hsp70:bmp2b) embryos or smad6a morphants (wild-type
123.8⫾6; Tg(hsp70:bmp2b) 111.0⫾8; smad6a morphants
107.6⫾6) (Figure 8E). The reduction in the number of ventricle
myocytes was most prominent in the smad6a morphant embryos. To address whether Bmp ligands are limiting Bmp
signaling in the absence of the inhibitory Smad6, we combined
heat shock–induced bmp2b expression with the knock-down of
smad6a. However, when we induced ectopic bmp2b expression
at the 14-somite stage in smad6a morphants, we did not observe
an additional reduction of the number of cells in the ventricle
(smad6a morphants; 89.9⫾4 versus Tg(hsp70:bmp2b)/smad6a
morphants; 84.2⫾7) (Figure 8D and 8E). Interestingly, we did
observe the appearance of cells located within the ventricle or
arterial pole that expressed the atrial myosin Myh6 (wild-type
0.0⫾0 cells in n⫽5 embryos; Tg(hsp70:bmp2b) 1.8⫾0.5 cells in
n⫽5 of 6 embryos; smad6a morphants 1⫾0.3 cells in n⫽5 of 7
embryos; Tg(hsp70:bmp2b)/smad6a MO 8.2⫾1.8 cells in n⫽5
of 6 embryos) (Figure 8D through D⬘⬘). Since we had observed
that Bmp signaling was required to induce tbx20 and tbx2b
expression in the differentiating myocardium, we next analyzed
whether Bmp activity was also sufficient to induce the expression of these cardiac transcription factors. Indeed, we observed
ectopic expression of tbx20 in the region posterior to the arterial
pole and enhanced expression of tbx2b at places where tbx2b is
Figure 7. The smad6a expression in the
cardiac field is required to inhibit
Smad1,5,8 phosphorylation. A through
D, In situ hybridization for Smad6a at
15-somite stage in lateral (A) and dorsal
(B through D) views, 23-somite stage and
24 hours postfertilization; h, linear heart
tube; e, eye. E through Lⴕ, Single confocal scan of Tg(myl7:eGFP) embryos
stained for eGFP in green and P-Smad in
red at 25-somite stage. One half of the
ALPM is shown with the midline to the
left. The tissue is counterstained with
DAPI indicating nuclei. E through H,
Embryo injected with control morpholino
(MO) with enlargement of myocardium
shown in Eⴕ through Hⴕ. I through L,
Representative Smad6a atg MO injected
embryo with enlargement of the myocardium shown in Iⴕ through Lⴕ. White
arrows point to P-Smad positive myocardial cells. Scale bars represent 50 ␮m.
Scale bars in enlargements represent
10 ␮m.
normally already expressed, including the myocardium. Together, these results demonstrate that inhibition of Bmp signaling by Smad6 is required to allow efficient myocardial
differentiation.
Discussion
Our genetic analysis demonstrates that Bmp signaling activity
is strictly controlled during cardiomyocyte differentiation,
since it is required in the cardiac progenitor cells to induce
cardiac differentiation but dispensable and even deleterious
once differentiation is initiated. While Bmp signaling is
active in cardiac progenitor cells to induce their differentiation, Smad6a actively inhibits Bmp signaling once differentiation is initiated. Inhibition of Bmp signaling in the myocardium is required to allow restricted levels and localization
of tbx20 and tbx2b expression and proper differentiation of
the cardiac chambers (Online Figure III).
The temporal distinct effects of Bmp signaling on cardiomyocyte number provide insights into the cellular mechanism by
which the size of the vertebrate heart is controlled. Our analysis
of mutant embryos deficient for Bmpr1a demonstrated that Bmp
signaling is required to induce expression of nkx2.5 and hand2 in
the ALPM, corroborating previous data from mouse mutants in
which Bmpr1a was deleted in the lateral mesoderm by using
de Pater et al
Bmp and Myocardial Differentiation
585
Figure 8. Ectopic activation of Bmp signaling reduces cardiomyocyte numbers. A through Dⴕⴕ, Reconstruction of
myl7:DsRed B
A
confocal z-stacks of the hearts of a repreMyh6
sentative uninjected wild-type sibling (A),
v
v
a Tg(hsp70:bmp2b) heat-shocked at 18
a
somites (B), a Smad6a MO injected
a
embryo (C), and a Tg(hsp70:bmp2b)
embryo injected with Smad6a MO (D
through Dⴕⴕ). All embryos shown are
ventricle atrium
Tg(myI7:DsRed2-Nuc) and are stained
myl7:DsRed
D’
D’’
C
D
with ␣-DsRed antibody (red) and an
D’
Myh6
␣-S46/Myh6 antibody (green). Scale bars
v
D’’
in A represent 50 ␮m. E, Quantification of
v
a
the total number of cardiomyocytes in
ventricle or atrium of control embryos,
a
v
Tg(hsp70:bmp2b) heat-shocked at 18
somites, smad6a MO injected embryos,
and a Tg(hsp70:bmp2b)/smad6a MO
injected embryos at 48 hours postfertilizahs:bmp2b G
hs:bmp2b H
control I
hs:bmp2b
F
tion (hpf), heat-shocked at 18 hpf. Bars
e
e
represent mean ⫾ SEM *P⬍0.05.
h
h
**P⬍0.01 in comparison with control
e
embryos. F and G, Dorsal views of in situ
e
hybridization for tbx20 and tbx2b expression in Tg(hsp70:bmp2b) embryos heattbx20
tbx2b
tbx2b
tbx2b
shocked at 15 somites and analyzed at
24 hpf. Wild-type controls for panels F
and G are presented in Figure 5A (tbx20)
and 5C (tbx2b). Arrow in F points to ectopic posterior expression domain. H and I, Lateral views of in situ hybridization for tbx2b
expression in wild-type and Tg(hsp70:bmp2b) embryos heat-shocked at 15 somites and analyzed at 36 hpf. Arrows indicate position
of the heart; h, heart; e, eye.
E
# cardiomyocytes
Tg(hsp70:bmp2b)
wild-type
smad6a
MO
*
**
**
Tg(hs:bmp2)
Tg(hs:bmp2)
+ smad6a MO
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
ISH
Smad6a MO
Control MO
Control
MesP1-cre.8 During gastrulation a gradient of Bmp signaling
along the dorsoventral axis patterns the mesoderm. In both
Drosophila and zebrafish, Bmp signaling during gastrulation is
required to specify the initial pool of cardiac progenitor cells that
express nkx2.5.9,10,12 Our data demonstrate that beyond gastrulation, Bmp signaling is still required to promote cardiac differentiation. This conclusion differs from a previous report suggesting that Bmp signaling after gastrulation is dispensable for
cardiac differentiation.12 In the latter study, the authors used
different methods to perturb Bmp signaling than were used in
our study, which could have been less potent in inhibiting Bmp
signaling. Indeed, it was reported that Tg(Hsp70l:dnBmpr1beGFP) line and the chemical Bmp inhibitor Dorsomorphin are
both less potent than Tg(hsp70:noggin3) line used in this study
in recapitulating a full loss of Bmp signaling.24,32,33 We therefore
believe that this difference in experimental setup used to perturb
Bmp signaling provides a likely explanation for the different
outcome of the 2 studies.
Our results are consistent with a model in which initial
Bmp signaling activity in the ALPM is required to induce
cardiac differentiation by the induction of transcription factors such as nkx2.5, hand2, tbx20, and tbx2, while during
myocardial differentiation a more restricted and confined
activation of the Bmp pathway is required to allow compartmentalization of the myocardium into distinct chamber and
primitive myocardium (Online Figure III). We observed that
inhibiting Bmp signaling by ectopic Noggin expression at
mid- to late-somite stages resulted in the down-regulation of
the cardiac transcription factors tbx20 and tbx2b. Bmp signaling strictly regulates the expression of both tbx20 and tbx2
during myocardial differentiation, and it was suggested that
these transcription factors are direct targets of this path-
way.30,31 Our results from the Tg(Bre:GFP) embryo experiment are consistent with such a cell-autonomous role for Bmp
signaling during myocardial differentiation. Tbx20 and
tbx2 regulate the compartmentalization of the myocardium
into primitive and chamber myocardium. Mice homozygous mutant for tbx20 establish a small heart tube that fails
to undergo looping morphogenesis and to initiate chamber
formation.34 –37 The transcriptional repressor tbx2 was
shown to regulate AVC formation by preventing chamber
differentiation.38,39
We and others previously showed that Bmp signaling is
required for normal looping morphogenesis.11,22,24,40,41 Here we
observed that while cardiomyocyte differentiation was rescued,
the looping defect observed in alk8 mutant hearts was not
rescued by re-expressing wild-type alk8 at mid-somite stages. In
addition we observed abnormal looping morphogenesis in embryos in which Noggin was expressed at 24 hpf, at which time
cardiomyocyte differentiation was unaffected. These results
suggest that Bmp signaling independent from its role during
myocardial differentiation controls looping morphogenesis. Corroborating such a model is the observation that Bmp2/4 is
expressed in the looped heart tube, where it is required to confine
tbx2 expression to the AVC.42,43
Inhibition of Bmp Signaling by Smad6 Is Required
for Myocardial Differentiation
Our results demonstrate that Smad6a is expressed in the
cardiac field and linear heart tube to inhibit Bmp signaling.
Interestingly, Smad6 is also expressed in the myocardium of
the chick heart at stages 10 and 15, where it is regulated by
Bmp signaling.28 In addition, in human embryonic stem cells,
Smad6 expression is up-regulated during cardiac differentiation (personal communication, S. Braam, R. Passier, and C.
586
Circulation Research
February 17, 2012
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
Mummery), suggesting that the role for Smad6 during cardiac
differentiation has been conserved in higher vertebrates.
Smad6 is not the only negative regulator of Bmp signaling
in the myocardium since other mechanisms have been identified. First, a detailed analysis of the mouse nkx2.5 mutant
demonstrated that nkx2.5 negatively regulates phosphSmad1,5,8 levels in the cardiac field during myocardial
expansion, possibly by repressing Bmp2 expression.27 Second, it was reported that Bmp signaling in the myocardium is
negatively regulated by a direct interaction of tbx20 with
phosphorylated Smad1 and Smad5.30 Since Bmp signaling
regulates expression of nkx2.5, tbx20, and Smad6 in the
myocardium, they may be part of a concerted feedback
inhibition loop to control and fine-tune Bmp signaling activity. While Bmp signaling is initially activated broadly in the
cardiac mesoderm, it becomes confined to AVC, where it
induces the transcriptional repressor tbx2, which is required
to maintain a primitive myocardium. Ectopic expression of
tbx2 in the entire myocardium of the early mouse heart results
in small and unlooped hearts,44 illuminating the necessity to
prevent premature activation of tbx2 by Bmp signaling in the
heart tube and prospective chambers.
Our results potentially clarify some of the seemingly contradictory results obtained from in vitro experiments. In cardiac
differentiation assays using embryonic and induced pluripotent
stem cells, both stimulating and inhibitory effects of Bmp
growth factors have been reported.45– 48 Interestingly, recent
results were reported showing that myocardial differentiation is
most efficient when embryoid bodies are incubated with a
combination of Bmp and activin for 2 days, after which they are
cultured in the presence of dorsomorphin and SB431542 to
inhibit Bmp and Nodal signaling, respectively.48 Thus, our
observations described here contribute to a better understanding
of how cardiogenic differentiation is regulated in vivo. A good
understanding of the in vivo regulation will allow optimizing
this process in vitro in the future.
Acknowledgments
We gratefully acknowledge Dr. Mullins for providing the
Tg(hsp70:alk8) line, and Bilge San and the Hubrecht Imaging Center
for technical assistance. We gratefully thank Robert Kelly and
members of the Bakkers laboratory for valuable discussions during
the preparation of this manuscript.
Sources of Funding
Work in Jeroen Bakkers’ laboratory was supported by EU FP7
FP7-NMP-2007-214539 (BioScent). Salim Abdelilah-Seyfried
was supported by a Heisenberg fellowship by the Deutsche
Forschungsgemeinschaft.
Disclosures
None.
References
1. Stainier DY, Lee RK, Fishman MC. Cardiovascular development
in the zebrafish: I. Myocardial fate map and heart tube formation.
Development. 1993;119:31– 40.
2. Keegan BR, Meyer D, Yelon D. Organization of cardiac chamber progenitors in the zebrafish blastula. Development. 2004;131:3081–3091.
3. Yelon D, Horne SA, Stainier DY. Restricted expression of cardiac myosin
genes reveals regulated aspects of heart tube assembly in zebrafish. Dev
Biol. 1999;214:23–37.
4. Stainier DY. Zebrafish genetics and vertebrate heart formation. Nat Rev
Genet. 2001;2:39 – 48.
5. Glickman NS, Yelon D. Cardiac development in zebrafish: coordination
of form and function. Semin Cell Dev Biol. 2002;13:507–513.
6. Smith KA, Chocron S, von der Hardt S, de Pater E, Soufan A, Bussmann
J, Schulte-Merker S, Hammerschmidt M, Bakkers J. Rotation and asymmetric development of the zebrafish heart requires directed migration of
cardiac progenitor cells. Dev Cell. 2008;14:287–297.
7. De Pater E, Clijsters L, Marques SR, Lin Y-F, Garavito-Aguilar ZV, Yelon
D, Bakkers J. Distinct phases of cardiomyocyte differentiation regulate
growth of the zebrafish heart. Development. 2009;136:1633–1641.
8. Klaus A, Saga Y, Taketo MM, Tzahor E, Birchmeier W. Distinct roles of
wnt/beta-catenin and bmp signaling during early cardiogenesis. Proc Natl
Acad Sci U S A. 2007;104:18531–18536.
9. Frasch M. Induction of visceral and cardiac mesoderm by ectodermal dpp
in the early drosophila embryo. Nature. 1995;374:464 – 467.
10. Kishimoto Y, Lee KH, Zon L, Hammerschmidt M, Schulte-Merker S.
The molecular nature of zebrafish swirl: bmp2 function is essential during
early dorsoventral patterning. Development. 1997;124:4457– 4466.
11. Zhang H, Bradley A. Mice deficient for bmp2 are nonviable and have
defects in amnion/chorion and cardiac development. Development. 1996;
122:2977–2986.
12. Marques SR, Yelon D. Differential requirement for bmp signaling in
atrial and ventricular lineages establishes cardiac chamber proportionality. Dev Biol. 2009;328:472– 482.
13. Schultheiss TM, Burch JB, Lassar AB. A role for bone morphogenetic proteins
in the induction of cardiac myogenesis. Genes Dev. 1997;11:451–462.
14. Schlange T, Andree B, Arnold HH, Brand T. Bmp2 is required for early heart
development during a distinct time period. Mech Dev. 2000;91:259–270.
15. Nakayama T, Cui Y, Christian JL. Regulation of bmp/dpp signaling
during embryonic development. Cell Mol Life Sci. 2000;57:943–956.
16. Bauer H, Lele Z, Rauch GJ, Geisler R, Hammerschmidt M. The type i
serine/threonine kinase receptor alk8/lost-a-fin is required for bmp2b/7
signal transduction during dorsoventral patterning of the zebrafish
embryo. Development. 2001;128:849 – 858.
17. Mintzer KA, Lee MA, Runke G, Trout J, Whitman M, Mullins MC. Lost-a-fin
encodes a type i bmp receptor, alk8, acting maternally and zygotically in dorsoventral pattern formation. Development. 2001;128:859–869.
18. Little SC, Mullins MC. Bone morphogenetic protein heterodimers
assemble heteromeric type i receptor complexes to pattern the dorsoventral axis. Nat Cell Biol. 2009;11:637– 643.
19. Hata A, Lagna G, Massague J, Hemmati-Brivanlou A. Smad6 inhibits
bmp/smad1 signaling by specifically competing with the smad4 tumor
suppressor. Genes Dev. 1998;12:186 –197.
20. Goto K, Kamiya Y, Imamura T, Miyazono K, Miyazawa K. Selective
inhibitory effects of smad6 on bone morphogenetic protein type i
receptors. JBiol Chem. 2007;282:20603–20611.
21. Murakami G, Watabe T, Takaoka K, Miyazono K, Imamura T. Cooperative inhibition of bone morphogenetic protein signaling by smurf1 and
inhibitory smads. Mol Biol Cell. 2003;14:2809 –2817.
22. Smith KA, Noel E, Thurlings I, Rehmann H, Chocron S, Bakkers J. Bmp
and nodal independently regulate lefty1 expression to maintain unilateral
nodal activity during left–right axis specification in zebrafish. PLoS
Genet. 2011;7:e1002289.
23. Alexander C, Zuniga E, Blitz IL, Wada N, Le Pabic P, Javidan Y, Zhang
T, Cho KW, Crump JG, Schilling TF. Combinatorial roles for bmps and
endothelin 1 in patterning the dorsal–ventral axis of the craniofacial
skeleton. Development. 2011;138:5135–5146.
24. Chocron S, Verhoeven MC, Rentzsch F, Hammerschmidt M, Bakkers J.
Zebrafish bmp4 regulates left–right asymmetry at two distinct developmental time points. Dev Biol. 2007;305:577–588.
25. Shin D, Shin CH, Tucker J, Ober EA, Rentzsch F, Poss KD, Hammerschmidt M, Mullins MC, Stainier DY. Bmp and fgf signaling are essential
for liver specification in zebrafish. Development. 2007;134:2041–2050.
26. Waldo KL, Kumiski DH, Wallis KT, Stadt HA, Hutson MR, Platt DH,
Kirby ML. Conotruncal myocardium arises from a secondary heart field.
Development. 2001;128:3179 –3188.
27. Prall OW, Menon MK, Solloway MJ, Watanabe Y, Zaffran S, Bajolle F,
Biben C, McBride JJ, Robertson BR, Chaulet H, Stennard FA, Wise N,
Schaft D, Wolstein O, Furtado MB, Shiratori H, Chien KR, Hamada H,
Black BL, Saga Y, Robertson EJ, Buckingham ME, Harvey RP. An
nkx2–5/bmp2/smad1 negative feedback loop controls heart progenitor
specification and proliferation. Cell. 2007;128:947–959.
28. Tirosh-Finkel L, Zeisel A, Brodt-Ivenshitz M, Shamai A, Yao Z, Seger
R, Domany E, Tzahor E. Bmp-mediated inhibition of fgf signaling
de Pater et al
promotes cardiomyocyte differentiation of anterior heart field progenitors. Development. 2010;137:2989 –3000.
Hutson MR, Zeng XL, Kim AJ, Antoon E, Harward S, Kirby ML.
Arterial pole progenitors interpret opposing fgf/bmp signals to proliferate
or differentiate. Development. 2010;137:3001–3011.
Singh R, Horsthuis T, Farin HF, Grieskamp T, Norden J, Petry M,
Wakker V, Moorman AFM, Christoffels VM, Kispert A. Tbx20 interacts
with smads to confine tbx2 expression to the atrioventricular canal. Circ
Res. 2009;105:442– 452.
Mandel EM, Kaltenbrun E, Callis TE, Zeng X-XI, Marques SR, Yelon D,
Wang D-Z, Conlon FL. The bmp pathway acts to directly regulate tbx20
in the developing heart. Development. 2010;137:1919 –1929.
Pyati UJ, Webb AE, Kimelman D. Transgenic zebrafish reveal stagespecific roles for bmp signaling in ventral and posterior mesoderm development. Development. 2005;132:2333–2343.
Yu PB, Hong CC, Sachidanandan C, Babitt JL, Deng DY, Hoyng SA, Lin
HY, Bloch KD, Peterson RT. Dorsomorphin inhibits bmp signals required
for embryogenesis and iron metabolism. Nat Chem Biol. 2008;4:33– 41.
Singh MK, Christoffels VM, Dias JM, Trowe MO, Petry M, SchusterGossler K, Burger A, Ericson J, Kispert A. Tbx20 is essential for cardiac
chamber differentiation and repression of tbx2. Development. 2005;132:
2697–2707.
Stennard FA, Costa MW, Lai D, Biben C, Furtado MB, Solloway MJ,
McCulley DJ, Leimena C, Preis JI, Dunwoodie SL, Elliott DE, Prall OW,
Black BL, Fatkin D, Harvey RP. Murine t-box transcription factor tbx20 acts
as a repressor during heart development, and is essential for adult heart
integrity, function and adaptation. Development. 2005;132:2451–2462.
Takeuchi JK, Mileikovskaia M, Koshiba-Takeuchi K, Heidt AB, Mori AD,
Arruda EP, Gertsenstein M, Georges R, Davidson L, Mo R, Hui CC, Henkelman RM, Nemer M, Black BL, Nagy A, Bruneau BG. Tbx20 dosedependently regulates transcription factor networks required for mouse heart
and motoneuron development. Development. 2005;132:2463–2474.
Cai CL, Zhou W, Yang L, Bu L, Qyang Y, Zhang X, Li X, Rosenfeld
MG, Chen J, Evans S. T-box genes coordinate regional rates of proliferation and regional specification during cardiogenesis. Development.
2005;132:2475–2487.
Habets PE, Moorman AF, Clout DE, van Roon MA, Lingbeek M, van
Lohuizen M, Campione M, Christoffels VM. Cooperative action of tbx2
29.
30.
31.
32.
33.
34.
35.
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
Bmp and Myocardial Differentiation
587
and nkx2.5 inhibits anf expression in the atrioventricular canal: implications for cardiac chamber formation. Genes Dev. 2002;16:1234 –1246.
Harrelson Z, Kelly RG, Goldin SN, Gibson-Brown JJ, Bollag RJ, Silver
LM, Papaioannou VE. Tbx2 is essential for patterning the atrioventricular
canal and for morphogenesis of the outflow tract during heart development. Development. 2004;131:5041–5052.
Chen JN, van Eeden FJ, Warren KS, Chin A, Nusslein-Volhard C, Haffter
P, Fishman MC. Left–right pattern of cardiac bmp4 may drive asymmetry
of the heart in zebrafish. Development. 1997;124:4373– 4382.
Schilling TF, Concordet JP, Ingham PW. Regulation of left–right asymmetries in the zebrafish by shh and bmp4. Dev Biol. 1999;210:277–287.
Ma L, Lu MF, Schwartz RJ, Martin JF. Bmp2 is essential for cardiac
cushion epithelial–mesenchymal transition and myocardial patterning.
Development. 2005;132:5601–5611.
Verhoeven MC, Haase C, Christoffels VM, Weidinger G, Bakkers J. Wnt
signaling regulates atrioventricular canal formation upstream of bmp and
tbx2. Birth Defects Res A Clin Mol Teratol. 2011;91:435– 440.
Christoffels VM, Hoogaars WMH, Tessari A, Clout DEW, Moorman
AFM, Campione M. T-box transcription factor tbx2 represses differentiation and formation of the cardiac chambers. Dev Dyn. 2004;229:
763–770.
Yuasa S, Itabashi Y, Koshimizu U, Tanaka T, Sugimura K, Kinoshita M, Hattori
F, Fukami S-I, Shimazaki T, Okano H, Ogawa S, Fukuda K. Transient inhibition
of bmp signaling by noggin induces cardiomyocyte differentiation of mouse
embryonic stem cells. Nature Biotechnology. 2005;23:607–611.
Hao J, Daleo MA, Murphy CK, Yu PB, Ho JN, Hu J, Peterson RT,
Hatzopoulos AK, Hong CC. Dorsomorphin, a selective small molecule
inhibitor of bmp signaling, promotes cardiomyogenesis in embryonic
stem cells. PloS One. 2008;3:e2904.
Takei S, Ichikawa H, Johkura K, Mogi A, No H, Yoshie S, Tomotsune D,
Sasaki K. Bone morphogenetic protein-4 promotes induction of cardiomyocytes from human embryonic stem cells in serum-based embryoid body
development. AJP: Heart and Circulatory Physiology. 2009;296:
H1793–H1803.
Kattman SJ, Witty AD, Gagliardi M, Dubois NC, Niapour M, Hotta A,
Ellis J, Keller G. Stage-specific optimization of activin/nodal and bmp
signaling promotes cardiac differentiation of mouse and human pluripotent stem cell lines. Cell Stem Cell. 2011;8:228 –240.
Novelty and Significance
What Is Known?
●
●
In embryos in which bone morphogenetic protein (BMP) signaling
activity is compromised, cardiomyocyte specification is reduced.
Both stimulation and inhibition of BMP signaling activity can enhance
cardiomyocyte differentiation of embryonic stem cells.
What New Information Does This Article Contribute?
●
●
●
Cardiac progenitor cells located in the lateral plate mesoderm of the
embryo are exposed to and require a BMP signal to initiate
cardiomyocyte differentiation.
Once cardiomyocyte differentiation is initiated, BMP signaling is
inhibited due to the expression of Smad6 in the cardiomyocytes.
Ectopic BMP signaling activity induces the expression of Tbx2 and
Tbx20 and prevents cardiac chamber formation.
After the first report that in Drosophila embryos, Dpp signaling
(related to vertebrate BMP and TGF-␤) is required to induce
cardiac mesoderm, it was recognized that in vertebrates also,
BMP signaling induces cardiomyocyte specification and differentiation. However, it is unclear when and where in the
vertebrate embryo BMP signaling is active during myocardial
differentiation. Here we demonstrate that, cardiomyocyte specification in the lateral plate mesoderm is completely absent in
zebrafish mutant embryos that lack BMP signaling. We show
that cardiac progenitor cells are exposed to and require BMP
signaling to initiate differentiation; however, once differentiation
is intiated, BMP signaling is actively repressed by the expression
of Smad6, which is required to allow cardiac chamber differentiation. We also show that activating BMP signaling in the
myocardium results in the ectopic expression of Tbx2 and Tbx20
and prevents cardiac chamber formation. From these results we
conclude that Bmp signaling activity is strictly controlled during
cardiomyocyte differentiation. It is required in the cardiac
progenitor cells to induce cardiac differentiation, but it is
dispensable or even deleterious once differentiation is initiated.
Understanding the signals that are required for cardiomyocyte
differentiation in the embryo proper will facilitate optimization of
in vitro protocols for cardiomyocyte differentiation of multipotent, embryonic stem cells.
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
Bmp Signaling Exerts Opposite Effects on Cardiac Differentiation
Emma de Pater, Metamia Ciampricotti, Florian Priller, Justus Veerkamp, Ina Strate, Kelly
Smith, Anne Karine Lagendijk, Thomas F. Schilling, Wiebke Herzog, Salim
Abdelilah-Seyfried, Matthias Hammerschmidt and Jeroen Bakkers
Circ Res. 2012;110:578-587; originally published online January 12, 2012;
doi: 10.1161/CIRCRESAHA.111.261172
Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 2012 American Heart Association, Inc. All rights reserved.
Print ISSN: 0009-7330. Online ISSN: 1524-4571
The online version of this article, along with updated information and services, is located on the
World Wide Web at:
http://circres.ahajournals.org/content/110/4/578
Data Supplement (unedited) at:
http://circres.ahajournals.org/content/suppl/2012/01/12/CIRCRESAHA.111.261172.DC1
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published
in Circulation Research can be obtained via RightsLink, a service of the Copyright Clearance Center, not the
Editorial Office. Once the online version of the published article for which permission is being requested is
located, click Request Permissions in the middle column of the Web page under Services. Further information
about this process is available in the Permissions and Rights Question and Answer document.
Reprints: Information about reprints can be found online at:
http://www.lww.com/reprints
Subscriptions: Information about subscribing to Circulation Research is online at:
http://circres.ahajournals.org//subscriptions/
Online Methods
Zebrafish lines:
To identify cardiomyocytes we used the Tg(myl7:EGFP)twu26
5.1myl7:nDsRed2)f2
2
lines.
Mutants
used
in
this
1
and Tg(-
study
were
lin/bmpr1aahu4087, bmpr1absa0028 and laf/acvr1ltm110b. To re-express alk8 we
3
used Tg(hsp70:alk8)
Tg(hsp70I:Nog3)fr14
4
Tg(hsp70:Bmp2b)fr13
4
.
, to down regulate bmp signaling we used
and
To
to
detect
overexpress
Bmp
Bmp2b
signaling
activity
we
we
used
used
Tg(Bre:GFP)p77 5.
Cell counts:
To count cardiomyocytes at various stages we used the Tg(myl7:DsRed2nuc) line stained using an α-DsRed antibody (Clontech, 1:100) and an α-Myh6
antibody (s46 from the Hybridoma bank 1:10) and a nuclear counter stain with
DAPI. Images were acquired by confocal microscopy with a Leica CLSM
confocal microscope. 3-D reconstruction and cell counting were performed
using Volocity software (Improvision).
Histological methods:
Whole mount immunofluorescence was performed according to 6. Vibratome
sections were used to visualize pSmad in the lateral plate mesoderm.
Embryos were embedded in 3% agarose in PBS and 100 µm sections were
stained O/N with primary antibody and for 4 h with secondary antibody.
Agarose slices were subsequently counterstained O/N with DAPI (Invitrogen,
1:2500). Antibodies used in this study: α-DsRed (Clontech, 1:200), α-Myh6
antibody (Hybridoma bank, s46, 1:10), α-eGFP (Chemokine, 1:200) and αphospho-Smad1, 5, 8 (Cell Signaling, 1:200).
Heat-shock experiments:
Heat-shock experiments were performed according to 4. Embryos collected
from crosses between heterozygous transgenic carriers and wild-type fish
were heat-shocked by transferring the embryos to E3 medium preheated to
37 °C and incubation at 37 °C for 30 min.
BrdU incorporation:
BrdU labeling was performed by soaking the embryos in embryo medium
containing 5 mg/ml BrdU (Roche) for 3 hours. α-BrdU (Roche) antibody
labeling was performed on 10-µm thick paraffin sections which were then
stained with 3,3’- Diaminobenzidine (DAB).
Recovery of cardiac tissue and RNA extraction:
Whole
intact
hearts
from
homozygous
Tg(myl7:EGFP)twu34
and
Tg(myl7:EGFP)twu34/ Tg(hsp70l:nog3)fr14 embryos were separated from the
body proper by fluorescence guided manual dissection. About 300 hearts
were retrieved per genotype and stored in RNAlater (Ambion) for further
processing. Total RNA was extracted with the RNeasy Micro Kit (Qiagen)
according to manufacturer’s instructions. Obtained RNA was assessed for
yield and quality with a ND-1000 spectrophotometer (Nanodrop Technologies)
and a 2100 Bioanalyzer RNA 6000 Nano chip (Agilent Technologies),
respectively. RNA was stored at -80°C
RT-qPCR:
For each sample, 100ng of total RNA were reverse transcribed with the
Omniscript RT Kit (Qiagen) according to manufacturer’s instructions.
Quantitative real-time PCR was carried out on the iQ5 detection system
(BioRad) in iQ 96-Well PCR Plates (BioRad). Efficiencies were calculated with
REST2009 software (Qiagen). Cqs were determined automatically by the iQ5
software v2.0.148.60623 (BioRad) via the PCR Base Line Subtracted Curve
Fit mode. gnb2l1 was chosen as a stable reference gene determined by the
geNorm algorithm. Differential gene expression was determined by an
efficiency corrected ΔΔCq method. Statistical significance of gene regulation
compared to the wild-type control was assessed using Student’s t-test.
References:
1.
Huang CJ, Tu CT, Hsiao CD, Hsieh FJ, Tsai HJ. Germ-line
transmission of a myocardium-specific gfp transgene reveals critical
regulatory elements in the cardiac myosin light chain 2 promoter of
zebrafish. Dev Dyn. 2003;228:30-40
2.
Mably JD, Mohideen MA, Burns CG, Chen JN, Fishman MC. Heart of
glass regulates the concentric growth of the heart in zebrafish. Curr
Biol. 2003;13:2138-2147
3.
Shin D, Shin CH, Tucker J, Ober EA, Rentzsch F, Poss KD,
Hammerschmidt M, Mullins MC, Stainier DY. Bmp and fgf signaling are
essential
for
liver
specification
in
zebrafish.
Development.
2007;134:2041-2050
4.
Chocron S, Verhoeven MC, Rentzsch F, Hammerschmidt M, Bakkers
J. Zebrafish bmp4 regulates left-right asymmetry at two distinct
developmental time points. Dev Biol. 2007;305:577-588
5.
Alexander C, Zuniga E, Blitz IL, Wada N, Le Pabic P, Javidan Y, Zhang
T, Cho KW, Crump JG, Schilling TF. Combinatorial roles for bmps and
endothelin 1 in patterning the dorsal-ventral axis of the craniofacial
skeleton. Development. 2011;138:5135-5146
6.
Smith KA, Chocron S, von der Hardt S, de Pater E, Soufan A,
Bussmann J, Schulte-Merker S, Hammerschmidt M, Bakkers J.
Rotation and asymmetric development of the zebrafish heart requires
directed migration of cardiac progenitor cells. Dev Cell. 2008;14:287297
Online Figure I: Restricted activation of Bmp signaling in ALPM. (A,C,F) Cartoons
representing transverse sections through the anterior region of a zebrafish embryo at
the 12 or 20 somite stage. (B-B’’) A single transverse confocal image of a
Tg(myl7:eGFP) embryo at 15 hpf (12-somite stage) stained for phosphorylatedSmad1,5,8 (P-Smad) and DAPI. Arrow points to a myl7:eGFP expressing cell which
is negative for P-Smad. Bar indicates region containing several P-Smad positive cells.
(D-D’’) Single confocal scan of Tg(myl7:eGFP) embryos at 19 hpf (20-somite stage)
stained for phosphorylated-Smad1,5,8 (P-Smad). Arrow points to the dorsal part of
the neural tube. (E-E’’) Higher magnification of the myocardium shown in D. (G-G’’)
A single confocal scan of a Tg(bre:GFP) embryo stained for tropomyosin (Tpm) to
indicate the myocardium. (H-H’’) Higher magnification of the myocardium from
images shown in G.
Online Figure II: Bmp signaling is required during mid- and late- somite stages for
myocardial differentiation.
(A) Live images of tails and reconstruction of confocal z-stacks of hearts of laf/alk8+/, laf/alk8-/- and laf/alk8-/- ; Tg(hsp70:alk8) embryos, heat-shocked at 16 hpf. All
embryos carry Tg(myl7:DsRed2-Nuc) and were stained for DsRed in red and
S46/Myh6 in green.
(B) Reconstruction of confocal z-stacks of a heart of a representative wild-type
sibling or Tg(hsp70:Noggin3) embryo heat-shocked at 10 hpf, 16 hpf and 24 hpf
respectively. All embryos express Tg(cmlc2:DsRed2-Nuc) and are stained for DsRed
in red and S46/Myh6 in green. Scale bar represents 50 µm.
A
10-­24 hpf
Nkx2.5
Hand2
Tbx20
Tbx2b
Smad6a
Bmp
myocardial differentiation
B
24-­48 hpf
F
K
D
P
E
H
U
Nkx2.5
Hand2
Tbx20
Smad6a
Bmp
v
avc
a
Tbx2b
Online Figure III; Model for Bmp function in myocardial differentiation to promote
and inhibit cardiac growth.
(A) At stages prior to heart tube formation, Bmp signaling promotes myocardial
differentiation in the ALPM by inducing expression of nkx2.5, hand2, tbx20 and
tbx2b. Ventricle progenitor cells require Bmp signaling at gastrula and early somite
stages while atrial progenitor cells require Bmp signaling at late segmentation stages
corroborating the observed temporal difference in cardiomyocyte differentiation. In
differentiating cardiomyocytes Smad6a inhibits Bmp signaling by preventing
phosphorylation of Smad1/Smad5 and their translocation to the nucleus. (B) During
heart tube formation high Bmp signaling activity in the AVC maintains tbx2b
expression. Tbx2b prevents chamber formation and may promote primitive
myocardium formation in the AVC. Tbx20 and Smad6a prevent expression of tbx2b
in the ventricle by inhibiting Bmp signaling activity.