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Expression of bone morphogenetic protein-10 mRNA during chicken heart development.

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Expression of Bone Morphogenetic
Protein-10 mRNA During Chicken
Heart Development
Experimental and Molecular Cardiology Group, Cardiovascular Research Institute
Amsterdam, Academic Medical Center, Amsterdam, The Netherlands
In this communication we describe the expression pattern of BMP10 mRNA during
cardiac development in chickens. BMP10 is considered an important factor in the regulation
of cardiac growth and trabeculation in the murine embryo. We identified chicken Ests, which
are similar to mouse and human BMP10 in the UMIST database. The cDNA clone that
contained most sequences was obtained, verified by sequence analysis, and used to determine
the spatiotemporal pattern of gene expression. BMP10 mRNA is initially expressed at HH10
in the myocardium of the arterial pole of the heart tube, anterior to the interventricular
groove. Between HH14 and HH22, BMP10 mRNA becomes broadly expressed in the outflow
tract, the distal part of the inflow tract, and the trabeculated part of the developing ventricles
and atria. From HH31 onward, BMP10 mRNA expression decreases in the ventricular
myocardium by first disappearing from the compact myocardium and then from the tips of the
trabecules. At HH44, BMP10 mRNA is expressed only in the trabeculated myocardium of the
atria and the endocardium of the ventricles. The observed expression pattern of BMP10
mRNA suggests that it may play a role in regulating the formation of the ventricular wall and
trabecules. © 2004 Wiley-Liss, Inc.
Key words: BMP10; TGF␤-superfamily; heart
Congenital heart malformations affect almost eight of
1,000 live births (Šamánek, 2000). Bone morphogenetic
proteins (BMPs) are considered to be of significant importance in the regulation of multiple aspects of cardiogenesis (for review see Brand, 2003). However, genetic
evidence is limited because functional impairment of
BMP signaling in mice results in embryo lethality prior
to heart development, or in mice without cardiac defects
(for reviews see Chang et al., 2002; Zhao, 2003). To gain
insight into the potential roles of BMPs during cardiac
development, we obtained a comprehensive description
of the spatiotemporal expression pattern of BMP2, -4,
-5, -6, and -7 mRNA during chicken heart development
(Somi et al., in press). To identify known and novel
BMPs that are expressed in the developing heart, we
used a polymerase chain reaction (PCR) approach (Somi
et al., 2003) and in silico cloning using the chicken Est
databases. From these databases, we identified BMP10,
determined its spatiotemporal expression pattern, and
compared this to the initial description, using wholemount in situ hybridization (Teichmann and Kessel,
In mice, BMP10 mRNA is expressed only in the developing heart. In the heart, BMP10 mRNA is expressed
in the trabeculated part of the ventricles and the proximal outflow tract at days 9.0 –14.5 of development. In
the developing atria, BMP10 mRNA expression is observed concomitantly with the appearance of trabecules
at 12.5 days of development (Neuhaus et al., 1999). An
initial report on the functional disruption of BMP10
revealed embryo lethality due to severe cardiac defects,
including hypoplastic ventricular wall, absence of ventricular trabeculation, and impaired contractile function (Chen et al., 2003).
Grant sponsor: The Netherlands Heart Foundation; Grant
number: M96.002.
*Correspondence to: Maurice van den Hoff, Ph.D., Academic
Medical Center, Department of Anatomy and Embryology,
Meibergdreef 15, Rm. K2-264, 1105 AZ Amsterdam, The Netherlands. Fax: ⫹31 20 697 6177.
Received 20 February 2004; Accepted 2 March 2004
DOI 10.1002/ar.a.20052
Published online 3 June 2004 in Wiley InterScience
Fertilized chicken eggs were obtained from a local
hatchery (Drost BV, Nieuw Loosdrecht, The Netherlands),
incubated at 38.5°C in a moist atmosphere, and automatically turned every hour. After the appropriate incubation
times, the embryos were isolated and staged according to
Hamburger and Hamilton (HH) (Hamburger and Hamilton, 1951). The embryos were fixed in 4% paraformaldehyde dissolved in phosphate-buffered saline (PBS) for 4
hr. Nonradioactive in situ hybridization was performed on
whole chicken embryos ranging from HH4 to HH18, and
on serial sections ranging from HH8 to HH44, as recently
described (Moorman et al., 2001).
Chicken cDNA (604156553F1), which is similar to human and mouse BMP10, was identified in the UMIST Est
database (Boardman et al., 2002) and obtained from the
MRC geneservice (Cambridge, UK). Cardiac Troponin I
(cTnI) was used to visualize the cardiomyocytes (Houweling et al., 2002). Digoxigenine-labeled antisense RNA
(Roche, Almere, The Netherlands) was produced by in
vitro transcription according to the manufacturer’s instructions.
Images were taken with a digital Nikon Coolpix 950 or
an Olympus DP12 camera coupled to a Leica MZFLIII
stereomicroscope or a Zeiss Axiophot microscope equipped
with differential interference contrast (DIC) optics. A flatfield correction was applied to all images with the use of a
user-written macro in PMIS 4.1 ( Contrast and color saturation were adjusted by means of the
Levels and Hue/Saturation functions of Adobe Photoshop
The BMP10 cDNA clone was sequence-verified, and
comprised 1,641 bp. The cDNA clone encodes the mature
part of BMP10 protein and 1,294 bp of the 3⬘ untranslated
region of the mRNA. The TGF-␤ family ligands are translated as prepropeptide precursors with an N-terminal signal peptide followed by the prodomain and the mature
domain (for review see Chang et al., 2002). The mature
domain of the chicken BMP10 protein showed 89% identity (92% homology) and 87% identity (92% homology),
respectively, with mouse and human BMP10 mature protein. A nucleotide comparison revealed an 80% sequence
identity within this region of mouse and human BMP10.
Blasting the protein sequence within Genbank showed
that chicken Dorsalin-1 is the closest relative within the
TGF-␤ superfamily showing 72% identity (85% homology).
In a previous study (Chang et al., 2002), a progressive
cluster multiple-sequence alignment of known mouse and
human mature BMPs showed that BMP10 clusters with
BMP9. A comparison of chicken BMP10 with human
BMP9 revealed only 68% similarity (80% homology).
Taken together, these results indicate that the identified
chicken cDNA clone most probably encodes chicken
In contrast to a previous report by Teichmann and Kessel (2004), BMP10 mRNA can be detected 12 hr earlier
than that described by Techmann and Kessel (2004), during chicken development at HH10, when the linear heart
tube is formed (Fig. 1A and B). BMP10 mRNA is not
expressed throughout the myocardium of the entire heart,
but is restricted to the myocardium of the arterial pole,
i.e., anterior to the interventricular groove (Fig. 1C and
D). At HH14, BMP10 mRNA is expressed in the entire
heart, with the exception of the atrioventricular canal
(Fig. 1E). Serial sections of HH16 hearts show that
BMP10 mRNA is expressed throughout the entire myocardial wall of the heart (Fig. 1F and G). The observed
expression pattern is much broader than that described by
Teichmann and Kessel (2004), who observed it only in the
inner myocardial layer of the developing ventricles. At
this stage, BMP10 mRNA expression is not observed in
the atrioventricular canal and pro-epicardium (Fig. 1F
and G). At HH22, BMP10 mRNA expression starts to
decrease in the outflow tract and inflow tract myocardium,
and becomes confined to the trabeculated myocardium of
the ventricles and atria. From HH26 onward, a transmural gradient is observed. This gradient is highest at the
luminal side of the ventricles, and decreases toward the
compact myocardium of the ventricular free wall and the
interventricular septum, in which BMP10 mRNA is barely
visible (Fig. 1H–K). Subsequently, BMP10 mRNA expression also disappears from the trabecules, being longest
expressed in the most distal myocardium of the tips of the
trabecules (Fig. 1L and M). Furthermore, while the ventricular expression of BMP10 mRNA decreases to nondetectable levels, intense staining remains in the trabeculated myocardium of the atria, the interatrial septum (Fig.
1J–O), and the venous valves (Fig. 1L and M), which was
not previously reported (Teichmann and Kessel, 2004). At
HH44, BMP10 mRNA is no longer detectable in the ventricular myocardium, but it becomes apparent in the endocardium of the ventricles (Fig. 1N and O).
In contrast to the mouse, BMP10 mRNA in chicken is
not restricted to the heart, but is also expressed in the
endothelium of the sinusoids of the liver from HH22 onward (data not shown). Since 9.0 days of mouse development is comparable to HH16 in the chicken (Pexieder,
1978), the onset of BMP10 mRNA expression occurs later
in the mouse heart than in the chicken heart (Neuhaus et
al., 1999). Nevertheless, the expression of BMP10 mRNA
in the developing ventricle and outflow tract is similar in
mouse and chicken. The expression of BMP10 mRNA in
the atria is observed from the start of its development in
chicken (Fig. 1E), whereas in mouse the expression of
BMP10 mRNA is first observed at 12.5 days of development (comparable to HH27 in chicken). Because the
mouse expression pattern is only described up to 14.5 days
of development, being comparable to HH30, the disappearance of BMP10 mRNA from the mouse ventricular
myocardium has not been observed (Neuhaus et al., 1999)
and remains to be determined.
The observed expression pattern of BMP10 mRNA during heart development in mouse and chicken supports the
notion that it plays a role in the development of the ventricular wall and trabecules, as previously observed in
BMP10 knockout mice (Chen et al., 2003). Cardiomyocytespecific deletion of the BMP receptor 1A (Alk3) has been
reported to lead to underdevelopment of the ventricular
compact myocardium and trabecules (Gaussin et al.,
2002), which suggests that the myocardially produced
BMP10 supports chamber development in an autocrine
loop via Alk3. Furthermore, the idea that BMP10 plays a
role in the initiation of chamber formation in chicken
(Teichmann and Kessel, 2004) is not supported by its
expression pattern, because the formation of chamber
myocardium becomes apparent as early as HH9 – when
Fig. 1. Whole-mount and serial sections of HH8 –HH44 chicken
hearts showing BMP10 (A, B, D, E, G, I, K, M, and O) and cardiac
Troponin I (C, F, H, J, L, and N) mRNA expression. No expression of
BMP10 mRNA is observed prior to HH10 (A). BMP10 mRNA is initially
expressed in the myocardium of the arterial pole anterior to the interventricular groove (ivg) at HH10 (B–D). At HH14, BMP10 mRNA is
expressed throughout the entire heart, with the exception of the atrioventricular canal (avc) (E). At HH16 (F and G), BMP10 mRNA is located
in the developing embryonic ventricles (ev) and atria (ea), the outflow
tract (oft), and inflow tract (ift). Arrowheads in G indicate BMP10 mRNA
expression in the oft and ift. During subsequent development, BMP10
mRNA becomes gradually confined to the trabeculated part of the
ventricles and atria (H and I). At HH31, BMP10 mRNA becomes re-
stricted to the ventricular trabecules and the trabeculated myocardium
of the atria (J and K). At HH39, its expression in the ventricles is
restricted to the tip of the trabecules (arrowheads), while atrial expression remains intense (L and M2). BMP10 mRNA is also observed in the
venous valves (vv), and is absent from the myocardial sleeves of the
caval vein (cv) and pulmonary vein (pv) (L1 and M1). At HH44, BMP10
mRNA is only observed in the atrial myocardium and the ventricular
endocardium (endo; arrowheads) beneath the atrioventricular valves (N
and O2). Abbreviations: aip, anterior intestinal portal; ao, aorta; cm,
cardiac mesoderm; Hn, Hensen’s node; ias, interatrial septum; ivs, interventricular septum; LA, left atrium; li, liver; LV, left ventricle; mv, mitral
valve; pe, pro-epicardium; RA, right atrium; RV, right ventricle; tv, tricuspid valve. Scale bar: 100 ␮m.
atrial natriuretic factor mRNA is used as a marker for the
developing chamber myocardium (Houweling et al., 2002).
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development, expressions, protein, morphogenetic, heart, chickens, bones, mrna
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