1 Department of Cell Biology, Duke University Medical Center, Durham, NC 27710,
USA
2 Laboratory of Reproductive and Developmental Toxicology, National Institute of
Environmental Health Sciences, Research Triangle Park, NC 27709, USA
3 Department of Pediatrics, Duke University Medical Center, Durham, NC 27710,
USA
* Author for correspondence (e-mail: kling{at}cellbio.duke.edu)
Accepted 22 January 2004
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SUMMARY |
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Key words: BMPRIA, Neural crest, Heart, Outflow tract, Myocardium, Epicardium
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Introduction |
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In conjunction with several other developmental signaling molecules, bone
morphogenetic proteins (BMPs), particularly BMP2 and BMP4, have been
implicated in promoting NCC induction, maintenance, migration and
differentiation in several different model organisms
(Aybar and Mayor, 2002;
Christiansen et al., 2000
;
Knecht and Bronner-Fraser,
2002
). Most evidence has come from gain-of-function manipulations,
such as application of recombinant BMP proteins, or overexpression of
transgenes that modulate BMP signaling. However, zebrafish embryos with
reduced BMP2 show deficient NCCs (Nguyen
et al., 1998
). Mouse embryos null for BMP2 show no evidence of
migratory NCCs, but die while neural crest is forming, obscuring the
requirement for BMP2 in this process
(Kanzler et al., 2000
). The
involvement of other BMPs and signaling pathway components are masked by
earlier roles or, possibly, by functional redundancy. Thus, the requirements
for BMP activity in various aspects of mammalian neural crest development
remain unclear.
We have used a conditional gene ablation approach to address the
requirements for BMP signaling in mammalian NCC biology. BMP ligands activate
a bipartite receptor complex composed of a type I and type II receptor
(Kawabata et al., 1998). Mouse
has three known type I receptors for BMP2 and BMP4, BMPRIA (ALK3), BMPRIB
(ALK6) and possibly ActRIA (ALK2)
(Miyazono et al., 2001
).
Neither Bmpr1b nor Actr1a is expressed in the neural tube
until well after NCC migration has begun
(Gu et al., 1999
;
Panchision et al., 2001
;
Yoshikawa et al., 2000
). By
contrast, Bmpr1a is expressed throughout the early embryo, including
the dorsal neural folds, but null mutants fail to gastrulate
(Mishina et al., 1995
).
Fortunately, a conditional null allele of Bmpr1a allows the deletion
of this gene in tissues expressing Cre recombinase
(Mishina et al., 2002
). We
used a Wnt1-Cre transgene, which triggers recombination exclusively
in forming NCCs (Brault et al.,
2001
; Chai et al.,
2000
; Danielian et al.,
1998
; Jiang et al.,
2000
), to delete Bmpr1a specifically in the neural crest
from the outset of migration. Embryos lacking Bmpr1a in NCCs die
abruptly at midgestation with no defects in the induction, maintenance,
delamination or initial migration of NCCs. Instead, we observed severe defects
in the cardiac outflow tract and ventricular myocardium.
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Materials and methods |
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Gene expression assays and histology
RNA probes were labeled with digoxigenin (Roche) and used for whole-mount
in situ hybridization according to standard protocols
(Belo et al., 1997). All
riboprobes were synthesized from published plasmids: Tcfap2a
(Mitchell et al., 1991
),
Crabp1 (Stoner and Gudas,
1989
), cadherin 6 (Inoue et
al., 1997
), Tcf21
(Quaggin et al., 1998
).
lacZ transgenes were visualized by fixing embryos for 15 minutes at
room temperature with 4% paraformaldehyde, rinsed and stained overnight with
X-gal stain. Tie2-lacZ embryos were cleared to benzyl alcohol: benzyl
benzoate (BABB). Histological analysis was conducted on 8 µm paraffin
wax-embedded sections using established protocols
(Hogan et al., 1994
). Section
immunohistochemistry with antibodies for smooth muscle actin (Sigma clone 1A4)
and PECAM1 (BD Pharmingen) used colorimetric substrates (NBT/BCIP). The WT1
antibody (Santa Cruz) was used on cryosections at 1:500 and visualized with
AlexaFluor 594 Goat anti-Rabbit IgG (Molecular Probes) at 1:500. Whole-mount
immunohistochemistry was carried out with 2H3 supernatant from the
Developmental Studies Hybridoma Bank at the University of Iowa. Proliferating
cells were marked with an antibody against phosphorylated histone H3 (Upstate
Biotechnologies) at 1:1000 overnight at 4°C; Goat anti-Mouse Cy3 (Jackson)
overnight at 4°C at 1:1000. Confocal microscopy utilized a Zeiss LSM 510.
Mitotic indices were calculated as percent of labeled cells among the total
number of DAPI counterstained cells (staining for 5 minutes at room
temperature).
Ink injections
To analyze blood flow, dissected embryos were injected with India ink into
the ventricle using a pulled capillary tube and mouth pipette. Embryos were
subsequently fixed in paraformaldehyde and cleared in BABB
(Hogan et al., 1994).
RT-PCR and PCR
To test for the presence of Cre, tissues were microdissected from embryos.
RNA was purified using Trizol reagent and standard procedures, including DNAse
treatment to prevent genomic DNA contamination
(Sambrook and Russell, 2001).
Total RNA was used for in vitro cDNA synthesis with MMLV reverse transcriptase
(MMLV-RT; Gibco). Cre was amplified with the same primers as for genotyping.
HPRT primers were used as published
(Stottmann et al., 2001
).
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Results |
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Recombination at the dorsal neural folds of the midbrain/hindbrain junction
was visible by 4 s (Fig. 1A,B),
reflecting a domain of Wnt1 expression required for midbrain
development (McMahon and Bradley,
1990). The first distinct NCCs were seen along the dorsal neural
folds of the hindbrain at the 5 s stage; these are more numerous by 6 s
(Fig. 1C,D). Recombination
extended into the rostral spinal cord by 8 s
(Fig. 1E,F). Transverse
sections of Wnt-1Cre; R26R embryos at
10 s (E8.5) revealed
migration from the neural tube to the pharyngeal arches and other target
tissues (Fig. 1G and data not
shown). Recombination then extended caudally along the dorsal neural tube to
include the entire neural crest and its descendents
(Fig. 1H,I). Wnt1-Cre
activity in the neural crest at 5 s coincides with the time at which the first
NCCs have been detected by cell labeling studies
(Serbedzija et al., 1992
).
Thus, Wnt1-Cre allows recombination of Bmpr1a in the neural
crest from very early stages of its development.
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Thin ventricular wall in Bmpr1a neural crest mutants
To elucidate the cause of the cardiovascular dysfunction and lethality in
embryos lacking Bmpr1a in NCCs, we examined whether there might be
defects in cardiac tissues not known to involve NCC contributions. Ventricular
structure at E10.5 and E11.0 looked normal in mutant embryos
(Fig. 6A-D) with formation of
both the compact myocardium on the periphery of the ventricle, and the
trabecular myocardium adjacent to the ventricular cavity. At E11.5, however,
both layers were significantly reduced in mutants, showing a severely thinned
compact myocardial wall and little expansion of the trabeculated myocardium
relative to wild type (Fig.
6E-H). These defects were seen prior to the onset of embryonic
necrosis (Fig. 6F).
|
A population of neural crest cell derivatives may colonize the epicardium
The mechanism by which the lack of a gene specifically in NCCs could lead
to a deficient ventricular myocardium is unclear. To determine whether there
might be a previously unknown NCC contribution to the mouse heart, we used
Wnt1-Cre; R26R embryos to assess the distribution of NCC derivatives
in and around the ventricles. Embryos at E9.5 showed a few
ß-galactosidase-positive cells immediately ventral to the heart and in
the epicardium (Fig. 7A), a
thin mesothelial tissue surrounding the heart. The epicardium derives
primarily from cells on the posterior side of the septum transversum, which
lies just caudal to the primitive ventricles. The epicardium expands to cover
the ventricular surface in an epithelial sheet by E10.0
(Komiyama et al., 1987). Some
of the epicardial cells undergo an epithelial-mesenchymal transition and
contribute to several lineages within the heart, including connective tissue
and coronary vascular elements
(Munoz-Chapuli et al., 2002
).
In E10.5 and E11.5 Wnt1-Cre; R26R embryos, progressively more marked
cells were seen in the epicardium, and also in the outer ventricular
myocardium (Fig. 7B,C). No
labeled cells were ever seen in the epicardium (or elsewhere) of R26R
embryos lacking Cre; furthermore, the same distribution of stained cells is
seen in Wnt1-Cre; R26R embryos regardless of whether they were
processed for ß-galactosidase activity and then sectioned, or sectioned
and then processed for activity (Fig.
7F). Together, these data eliminate the possibility that the
labeled cells are artifacts of ß-galactosidase staining. Although an
epicardial NCC lineage has not been documented previously using this
transgene, all other Wnt1-Cre lineages detected in our embryos are
identical to those shown in previous studies
(Jiang et al., 2000
).
|
Like other epicardial cells, a few of these lineage-labeled cells then invade the ventricular myocardium (Fig. 7F). Because cells of the epicardium populate the coronary arteries, we examined these vessels in late-gestation Wnt1-Cre; R26R embryos for the presence of labeled cells. We observed labeled cells in the coronary arteries in both whole-mount (Fig. 7D) and sectioned hearts (Fig. 7E). Collectively, this distribution of marked cells suggests that a small population of NCCs migrates to the epicardium, where it integrates into the nascent epicardial epithelium and has similar cell fates as neighboring epicardial cells from other origins.
We conducted additional tests to further evaluate the hypothesis that the
labeled epicardial cells derive from the neural crest. First, we assessed the
distribution of cells labeled by an independent NCC genetic lineage marker,
P0-Cre (Yamauchi et al.,
1999). P0-Cre; R26R embryos showed a similar contribution
of NCCs to the epicardium and myocardium
(Fig. 7G,H). We then considered
whether these cells might reflect coincidental activity of Wnt1-Cre
and P0-Cre transgenes in some novel domain outside the neural crest,
such as the proepicardial organ or epicardium itself. To determine whether
these cells could result from ectopic Cre expression outside the
neural crest, we assayed tissue ventral to the neural tube, including the
heart and all other thoracic tissues, for Wnt1-Cre expression by
reverse transcription coupled to the polymerase chain reaction (RT-PCR). We
found no Cre expression in ventral tissues collected from E9.5 and E10.5
Wnt1-Cre embryos, covering a wide range of somite numbers (0/37;
Fig. 7I). We cannot exclude the
possibility that there may be a brief burst of short-lived Cre expression in a
temporal window not represented in our samples, although this seems unlikely.
Instead, these data taken together strongly suggest that a small population of
labeled epicardial and myocardial cells derive from the neural crest.
Among several BMPs expressed in the developing heart, BMP2 and BMP4 are
known to be ligands for BMPRIA. BMP2 is expressed in the atrioventricular
canal from E9 to E11 (Zhang and Bradley,
1996). Using a Bmp4-lacZ transgene
(Lawson et al., 1999
), we
observed that Bmp4 is expressed at high levels in the parietal
pericardium of the thoracic body wall from E9.5 through E11.5
(Fig. 7J-L; data not shown).
This suggests that at least one BMPRIA ligand, BMP4, is present in a cell
population adjacent to potential receiving cells in the epicardium.
Intact epicardium in Bmpr1a neural crest mutants
We then assessed whether the epicardium might show abnormalities in
Bmpr1a neural crest mutants. Examination of Wnt1-Cre;
Bmpr1aflox; R26R embryos revealed an indistinguishable
distribution of labeled cells in the epicardium between mutant and wild-type
embryos (Fig. 8A,B compared
with Fig. 7A,B). This is in
contrast to the truncus arteriosus, where there is a striking reduction in NCC
population in mutants. Although mutant NCC derivatives populate the
epicardium, it is conceivable nonetheless that the epicardium is structurally
abnormal in the mutants. Physical delay of migration of cells from the
proepicardial organ results in a less extensive epicardial covering of the
ventricles; this in turn is associated with reduced myocardial proliferation
in underlying tissue (Perez-Pomares et
al., 2002). To analyze the structure of the epicardium, we
compared ventricular epicardium in sectioned mutant hearts and control
littermates, and found no evidence for a disrupted or patchy epicardium
(Fig. 8C-F). Moreover, the
epicardial cell density in sections was not significantly different
(P>0.100) between stage-matched mutant and wild-type embryos.
|
Taken together, these data indicate that cells may migrate from the neural crest to the epicardium, where they are potentially exposed to several BMPs, including BMP4. Loss of Bmpr1a in NCCs results in midgestational defects in the underlying ventricular myocardium similar to those seen in epicardial ablations. The epicardial population of NCCs is distributed normally in mutant embryos, and the epicardium is structurally intact. These results therefore suggest a specific compromise in the proliferative influence of epicardium on ventricular myocardium upon loss of BMPRIA in neural crest derivatives.
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Discussion |
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Neural crest develops in the absence of BMPRIA
Although Bmpr1a is expressed throughout the neural plate, this
receptor is dispensable for many aspects of NCC development. Definitive neural
crest is first detectable at 5 s, beginning in the rostral hindbrain
(Nichols, 1981), and
production of NCCs is ongoing for
9 hours at any given axial location,
ending first in the hindbrain at 11 s to 14 s
(Serbedzija et al., 1992
).
Meanwhile, Wnt1-Cre is expressed from 5 s in the hindbrain, and has
been strongly expressed there for several hours by 11 s. Even if there were
perdurance of BMPRIA protein long after the gene has been ablated (not
suggested by previous studies with this allele), it seems unlikely that
significant numbers of putative NCCs would retain BMPRIA protein during the
whole window of NCC production. If the receptor were required for early NCC
development but were not ablated soon enough, one would have expected that the
initial NCCs would form and migrate away, but that continued production would
decline and stop. Instead, we observed continuous production of NCCs
indistinguishable from wild-type controls. Moreover, the BMPRIA protein would
have been absent for many hours as the NCCs migrate to target tissues and
differentiate. It may be that the only crucial period for BMPRIA signaling
occurs prior to 5-6 s, when Wnt1-Cre recombination becomes prevalent
in the forming neural crest; our current experiment is unable to assess the
necessity for this receptor during the earliest phases of NCC specification.
Taken together, these considerations strongly suggest that BMPRIA signaling is
unnecessary in vivo for neural crest delamination, migration and
differentiation into most derivative cell types.
A potential implication of our results is that BMP signaling in general may
not have an essential role in many aspects of mammalian NCC development.
Neither of the other known BMP type I receptors, BMPRIB and ActRIa, is known
to be expressed in the neural tube during early NCC development
(Gu et al., 1999;
Panchision et al., 2001
;
Yoshikawa et al., 2000
).
However, it remains possible that ectopic expression of Bmpr1b or
Actr1a, or perhaps an unknown TGFß family type I receptor, can
compensate for the absence of Bmpr1a in this context.
BMP signaling in neural crest cells is required for outflow tract development
Our study reveals a critical requirement for BMPRIA signaling in proper
formation and septation of the cardiac OFT. Although NCCs successfully migrate
to the OFT in mutant embryos, we observed severe OFT defects, including a
failure of septation and reduced conotruncal length. These phenotypes, which
result from the ablation of a single gene product, closely resemble those
resulting from physical ablation of the entire cardiac neural crest in chick
embryos, roughly extending from the otic vesicle to the fourth somite
(Kirby and Waldo, 1995).
Further studies into the mechanism of the reduced OFT in cardiac NCC ablations
reveal an accompanying defective migration of the outflow tract myocardium
from an anterior heart field (Yelbuz et
al., 2003
). We see no evidence for a change in cell number through
cell death or cell proliferation changes in the OFT of our mutant embryos.
Therefore, we suggest that NCCs require BMPRIA for proper colonization of the
OFT. Furthermore, we speculate that the NCCs colonizing the OFT in wild-type
embryos may secrete a factor downstream of BMPRIA signal transduction, lacking
in our mutants, that stimulates migration of future outflow tract myocardial
cells from the secondary heart field to form a fully mature OFT and right
ventricle (Kelly et al., 2001
;
Mjaatvedt et al., 2001
;
Waldo et al., 2001
).
Although other organogenesis defects also result from cardiac neural crest
ablations, such as deficient thymus and parathyroid, the Bmpr1a
mutant embryos studied here died too soon (prior to E12.5) to address whether
BMPR1A in NCCs is required for development of these organs. Nevertheless, our
results complement earlier genetic evidence that BMP signaling is necessary
for OFT septation. A hypomorphic allele of the gene encoding BMPRII, the
binding partner of BMPRIA, also results in OFT defects
(Delot et al., 2003).
Overexpression of the BMP antagonist Noggin in chick heart and great vessels,
expected to decrease effective BMP ligand levels in these tissues, results in
OFT septation anomalies as well (Allen et
al., 2001
). In both the chick Noggin cardiac misexpression
experiments and the mouse NCC Bmpr1a ablation studies, NCCs entered
the OFT but failed to migrate as far towards the ventricles as in control
embryos. Several BMP ligands are expressed in the early OFT, including BMP2,
BMP4, BMP6 and BMP7 (Bitgood and McMahon,
1995
; Jones et al.,
1991
; Kim et al.,
2001
; Lyons et al.,
1990
). Together, these considerations suggest that local BMPs in
and around the conotruncus signal through BMPRIA in the incoming NCCs to
promote myocardial morphogenesis and NCC colonization of the septating truncus
arteriosus.
The acute heart failure observed just before death of the
Wnt1-Cre;Bmpr1aflox/null embryos was not due to a failure
of blood to flow through the OFT to the periphery. Persistent (unseptated)
truncus arteriosus is not necessarily lethal until birth, when the fetus
requires distinct pulmonary and systemic circulations. Although the aortic
arch arteries were occasionally somewhat hypoplastic in the mutants and may
have reduced flow, the lumens of neither these arteries nor the truncus
arteriosus were occluded. Indeed, other mutants with hypoplastic aortic arch
arteries survive beyond E12.5 (Abu-Issa et
al., 2002; Lindsay and
Baldini, 2001
; Yanagisawa et
al., 2003
), by which time the Bmpr1a NCC mutants have
died.
BMPRIA functions outside the ventricular myocardium to promote its proliferation
The primary cause of death in the Wnt1-Cre; Bmpr1a mutants is
ventricular dysfunction and a consequent failure to pump an adequate supply of
blood. The lethal phase is very narrow: mutants occur at Mendelian levels
(25%) at E11, but are entirely absent by E13. The ventricular myocardium
appears reduced shortly before death, particularly the outer, compact layer.
Although the ventricles of mutant embryos look normal through E11.0, the
ventricular myocardium shows significantly reduced cell proliferation by
E10.5.
The thin-walled ventricular myocardial phenotype that we observe resembles
phenotypes reported for loss-of-function mutations in several other mouse
loci. These include the genes encoding WT1
(Kreidberg et al., 1993),
RAR
(Kastner et al.,
1997
), RXR
(Sucov et
al., 1994
),
4 integrin
(Yang et al., 1995
), VCAM1
(Kwee et al., 1995
), FOG2
(Tevosian et al., 2000
),
erythropoietin and the erythropoietin receptor
(Wu et al., 1999
), and others.
These mutants are all general `knockout' mutations, in which the entire embryo
has deficient gene activity. By contrast, our ablation of Bmpr1a was
restricted to a very limited extracardiac population, with Bmpr1a
activity intact in myocardial cells. Thus, the myocardial phenotype is caused
indirectly, specifically as a result of deficient BMP signaling in an
extracardiac cell population that must function in a tissue interaction that
regulates myocardial proliferation in the ventricles.
A small cell population in the epicardium that signals through BMPRIA may promote ventricular proliferation
We observed a novel population of labeled cells contributing to the
epicardium from E9.5, and propose that deficient BMPRIA activity in this
lineage is responsible for the ventricular defects. Considerable genetic
evidence suggests that a defective epicardium can lead to reduced myocardial
proliferation. For example, Wt1 is expressed in epicardium, but not
in myocardium, yet has a very similar thin compact myocardium phenotype;
expression of Wt1 specifically in the epicardium rescued the
thin-walled phenotype and death (Moore et
al., 1999). Interference with retinoic acid signaling in the
epicardium mimicked the thin-walled myocardium phenotype of Rxra
mutant embryos (Chen et al.,
2002
). In the VCAM1, FOG2 and
-4integrin mutants, the tight
juxtaposition of epicardium with ventricular myocardium was abnormal, with
corresponding thinning of the myocardium
(Kwee et al., 1995
;
Tevosian et al., 2000
;
Yang et al., 1995
).
Embryological studies complement these results. Ablation of the proepicardial
organ has a similar phenotype, and reduced migration to the heart results in
patchy epicardium with reduced ventricular myocardium thickness underlying the
deficient areas of the epicardium
(Gittenberger-de Groot et al.,
2000
; Perez-Pomares et al.,
2002
; Poelmann et al.,
2002
). This reduction in ventricular wall thickness resulting from
interference with the epicardium is caused primarily by reduced cell
proliferation (Pennisi et al.,
2002
).
Thus, the thin-walled ventricular phenotype we observe in Wnt1-Cre;Bmpr1a mutants is very similar to those resulting from molecular or structural epicardial defects. Our analysis indicated that the epicardium in the mutants has no detectable structural problems, and that the expression of at least some epicardial markers was normal. Moreover, we found that the epicardial population of recombined (labeled) cells was indistinguishable between mutants and wild-type littermates. These data suggest that the relevant defect in the mutants is the specific lack of functional BMPRIA in this population of epicardial cells.
Two recent studies have demonstrated directly that the epicardium produces
secreted factors that promote proliferation of the underlying ventricular
myocardium. Chen and colleagues (Chen et
al., 2002) found that cultured mouse epicardial cells produce
trophic factors for myocardial cell proliferation, and production of this
trophic activity depends on retinoid signal transduction in the epicardial
cells. Stuckmann et al. (Stuckmann et al.,
2003
) also found that blocking retinoid signaling in the
epicardium blocks production of a secreted myocardial proliferation factor, as
does blocking erythropoietin signaling. Neither study identified the
proliferation factor(s) produced by epicardial cells.
Our data lead us to propose a model for an essential role of BMPRIA
signaling in an epicardial cell population in promoting ventricular
development (Fig. 9A-C). An
extracardiac lineage of cells (reflecting Wnt1-Cre recombination)
migrates to the epicardium, and via BMPRIA transduces a BMP signal. We found
that BMP4 (a known ligand) is expressed at high levels in cells adjoining the
epicardium, though other work has demonstrated expression of Bmp5, Bmp6,
Bmp7 and Bmp10 in the myocardium at many stages
(Dudley and Robertson, 1997;
Kim et al., 2001
;
Neuhaus et al., 1999
;
Solloway and Robertson, 1999
).
Transduction of BMP signals by BMPRIA in these epicardial cells would in turn
stimulate production within the epicardium of an unidentified proliferation
signal for the underlying ventricular myocardium. As we can detect only a
limited contribution of recombined cells to the epicardium, this population
probably acts upon other cells of the epicardium to stimulate production of
this myocardial proliferation signal(s). Thus, a limited number of recombined
cells may lead to a defect in signaling from a much larger region of the
epicardium. As a result, the Wnt1-Cre; Bmpr1aflox/null
embryos would produce reduced proliferation signal(s) and consequently
experience a critical underproliferation of the ventricular myocardium.
Reduced compact and trabeculated myocardium precludes sufficient blood flow
for the metabolic demands of the rapidly growing embryo. Embryonic lethality
then results primarily from ventricular failure, caused by deficient
myocardial maturation. The ventricular deficiency of the mutants analyzed here
may be compounded by restricted flow through the unseptated outflow tract and
hypoplastic aortic arch arteries. Within the ventricular myocardium itself,
BMPRIA has been proposed to transduce a myocyte growth signal
(Gaussin et al., 2002
), though
in our mutants the myocardium per se is wild type.
|
Is there a neural crest contribution to the epicardium?
Although Wnt1-Cre expression has been associated exclusively with
the neural crest and its derivatives
(Brault et al., 2001;
Chai et al., 2000
;
Jiang et al., 2000
), the
epicardium of Wnt1-Cre; R26R embryos contained a small proportion of
recombined cells. This indicates that they are derived from a Cre-expressing
lineage. Although we were unable to detect such cells in every specimen
(probably for technical reasons), control experiments indicated that the
labeling was not a staining artifact. These labeled cells later populated the
same tissues as epicardial derivatives (coronary arteries and scattered cells
in the ventricular wall). Because these epicardial cells clearly underwent
epithelial-mesenchymal transition (EMT) to clonally populate these structures,
it is possible that this population represents a fraction of the epicardium
that is competent to undergo EMT rather than remaining an obligate part of the
epicardial mesothelium.
We observed a Wnt1-Cre lineage of cells in the septum transversum adjacent to the ventricles at E9.5. This is the location of the proepicardial organ, the source of epicardial cells. We therefore suspect that the labeled cells of the epicardium came from this tissue, though it is also possible that they have migrated in from a known neural crest domain elsewhere. We are uncertain whether these labeled cells are of neural crest origin, or if they represent a hitherto undetected independent expression domain of Wnt1-Cre. However, we saw exactly the same distribution of labeled cells when we used PO-Cre as an independent neural crest lineage marker. Moreover, using the sensitive assay of RT-PCR, we never detected Cre expression in non-dorsal tissues dissected from Wnt1-Cre embryos from E9.5-10.5 (during which time the epicardium forms). These considerations lead us to suspect that the labeled epicardial cells are of neural crest origin, though they probably arrive via the proepicardial organ.
Although an epicardial NCC lineage has not been otherwise observed in mice
or chicks, recent evidence suggests NCCs contribute to ventricular myocardium
in zebrafish (Li et al., 2003;
Sato and Yost, 2003
). In mice
and chickens with NCC defects, myocardial defects are seen before NCCs are
known to populate the outflow tract, suggesting that NCCs produce factor(s)
involved in myocardial development
(Creazzo et al., 1998
).
Moreover, Pax3 expression in NCCs rescues the reduced myocardial
tissue phenotype of Pax3-null mutants
(Li et al., 1999
). We suggest
that lesions in an early population of NCC derivatives, migrating into the
epicardium and/or outflow tract, account for previously observed myocardial
defects associated with neural crest perturbations. Overall, our work
demonstrates that BMPRIA is dispensable for most aspects of early neural crest
development, and identifies a novel, crucial role for BMPRIA signal
transduction in extra-cardiac cells during the development of the outflow
tract and ventricular myocardium of the heart.
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ACKNOWLEDGMENTS |
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Abu-Issa, R., Smyth, G., Smoak, I., Yamamura, K. and Meyers, E.
N. (2002). Fgf8 is required for pharyngeal arch and
cardiovascular development in the mouse. Development
129,4613
-4625.
Ahn, K., Mishina, Y., Hanks, M. C., Behringer, R. R. and Crenshaw, E. B., 3rd (2001). BMPR-IA signaling is required for the formation of the apical ectodermal ridge and dorsal-ventral patterning of the limb. Development 128,4449 -4461.[Medline]
Allen, S. P., Bogardi, J. P., Barlow, A. J., Mir, S. A., Qayyum, S. R., Verbeek, F. J., Anderson, R. H., Francis-West, P. H., Brown, N. A. and Richardson, M. K. (2001). Misexpression of noggin leads to septal defects in the outflow tract of the chick heart. Dev. Biol. 235,98 -109.[CrossRef][Medline]
Aybar, M. J. and Mayor, R. (2002). Early induction of neural crest cells: lessons learned from frog, fish and chick. Curr. Opin. Genet. Dev. 12,452 -458.[CrossRef][Medline]
Belo, J. A., Bouwmeester, T., Leyns, L., Kertesz, N., Gallo, M., Follettie, M. and de Robertis, E. M. (1997). Cerberus-like is a secreted factor with neutralizing activity expressed in the anterior primitive endoderm of the mouse gastrula. Mech. Dev. 68, 45-57.[CrossRef][Medline]
Bitgood, M. J. and McMahon, A. P. (1995). Hedgehog and Bmp genes are coexpressed at many diverse sites of cell-cell interaction in the mouse embryo. Dev. Biol. 172,126 -138.[CrossRef][Medline]
Brault, V., Moore, R., Kutsch, S., Ishibashi, M., Rowitch, D.
H., McMahon, A. P., Sommer, L., Boussadia, O. and Kemler, R.
(2001). Inactivation of the beta-catenin gene by
Wnt1-Cre-mediated deletion results in dramatic brain malformation and failure
of craniofacial development. Development
128,1253
-1264.
Chai, Y., Jiang, X., Ito, Y., Bringas, P., Jr, Han, J., Rowitch,
D. H., Soriano, P., McMahon, A. P. and Sucov, H. M. (2000).
Fate of the mammalian cranial neural crest during tooth and mandibular
morphogenesis. Development
127,1671
-1679.
Chen, T. H., Chang, T. C., Kang, J. O., Choudhary, B., Makita, T., Tran, C. M., Burch, J. B., Eid, H. and Sucov, H. M. (2002). Epicardial induction of fetal cardiomyocyte proliferation via a retinoic acid-inducible trophic factor. Dev. Biol. 250,198 -207.[CrossRef][Medline]
Christiansen, J. H., Coles, E. G. and Wilkinson, D. G. (2000). Molecular control of neural crest formation, migration and differentiation. Curr. Opin. Cell Biol. 12,719 -724.[CrossRef][Medline]
Conway, S. J., Godt, R. E., Hatcher, C. J., Leatherbury, L., Zolotouchnikov, V. V., Brotto, M. A., Copp, A. J., Kirby, M. L. and Creazzo, T. L. (1997). Neural crest is involved in development of abnormal myocardial function. J. Mol. Cell Cardiol. 29,2675 -2685.[CrossRef][Medline]
Creazzo, T. L., Godt, R. E., Leatherbury, L., Conway, S. J. and Kirby, M. L. (1998). Role of cardiac neural crest cells in cardiovascular development. Annu. Rev. Physiol. 60,267 -286.[CrossRef][Medline]
Danielian, P. S., Muccino, D., Rowitch, D. H., Michael, S. K. and McMahon, A. P. (1998). Modification of gene activity in mouse embryos in utero by a tamoxifen-inducible form of Cre recombinase. Curr. Biol. 8,1323 -1326.[Medline]
Delot, E. C., Bahamonde, M. E., Zhao, M. and Lyons, K. M.
(2003). BMP signaling is required for septation of the outflow
tract of the mammalian heart. Development
130,209
-220.
Dudley, A. T. and Robertson, E. J. (1997). Overlapping expression domains of bone morphogenetic protein family members potentially account for limited tissue defects in BMP7 deficient embryos. Dev. Dyn. 208,349 -362.[CrossRef][Medline]
Etchevers, H. C., Vincent, C., le Douarin, N. M. and Couly, G.
F. (2001). The cephalic neural crest provides pericytes and
smooth muscle cells to all blood vessels of the face and forebrain.
Development 128,1059
-1068.
Farrell, M., Waldo, K., Li, Y. X. and Kirby, M. L. (1999). A novel role for cardiac neural crest in heart development. Trends Cardiovasc. Med. 9, 214-220.[CrossRef][Medline]
Gaussin, V., van de Putte, T., Mishina, Y., Hanks, M. C.,
Zwijsen, A., Huylebroeck, D., Behringer, R. R. and Schneider, M. D.
(2002). Endocardial cushion and myocardial defects after cardiac
myocyte-specific conditional deletion of the bone morphogenetic protein
receptor ALK3. Proc. Natl. Acad. Sci. USA
99,2878
-2883.
Gittenberger-de Groot, A. C., Vrancken Peeters, M. P.,
Bergwerff, M., Mentink, M. M. and Poelmann, R. E. (2000).
Epicardial outgrowth inhibition leads to compensatory mesothelial outflow
tract collar and abnormal cardiac septation and coronary formation.
Circ. Res. 87,969
-971.
Gu, Z., Reynolds, E. M., Song, J., Lei, H., Feijen, A., Yu, L.,
He, W., MacLaughlin, D. T., van den Eijnden-van Raaij, J., Donahoe, P. K. et
al. (1999). The type I serine/threonine kinase receptor
ActRIA (ALK2) is required for gastrulation of the mouse embryo.
Development 126,2551
-2561.
Hebert, J. M., Mishina, Y. and McConnell, S. K. (2002). BMP signaling is required locally to pattern the dorsal telencephalic midline. Neuron 35,1029 -1041.[Medline]
Hogan, B., Beddington, R., Costantini, F. and Lacy, E. (1994). Manipulating the Mouse Embryo. New York: Cold Spring Harbor Press.
Inoue, T., Chisaka, O., Matsunami, H. and Takeichi, M. (1997). Cadherin-6 expression transiently delineates specific rhombomeres, other neural tube subdivisions, and neural crest subpopulations in mouse embryos. Dev. Biol. 183,183 -194.[CrossRef][Medline]
Jamin, S. P., Arango, N. A., Mishina, Y., Hanks, M. C. and Behringer, R. R. (2002). Requirement of Bmpr1a for Mullerian duct regression during male sexual development. Nat. Genet. 32,408 -410.[CrossRef][Medline]
Jiang, X., Rowitch, D. H., Soriano, P., McMahon, A. P. and
Sucov, H. M. (2000). Fate of the mammalian cardiac neural
crest. Development 127,1607
-1616.
Jones, C. M., Lyons, K. M. and Hogan, B. L. (1991). Involvement of Bone Morphogenetic Protein-4 (BMP-4) and Vgr-1 in morphogenesis and neurogenesis in the mouse. Development 111,531 -542.[Abstract]
Kanzler, B., Foreman, R. K., Labosky, P. A. and Mallo, M.
(2000). BMP signaling is essential for development of
skeletogenic and neurogenic cranial neural crest.
Development 127,1095
-1104.
Kastner, P., Messaddeq, N., Mark, M., Wendling, O., Grondona, J.
M., Ward, S., Ghyselinck, N. and Chambon, P. (1997). Vitamin
A deficiency and mutations of RXRalpha, RXRbeta and RARalpha lead to early
differentiation of embryonic ventricular cardiomyocytes.
Development 124,4749
-4758.
Kaufman, M. H. (1992). The Atlas of Mouse Development. San Diego: Academic Press.
Kawabata, M., Imamura, T. and Miyazono, K. (1998). Signal transduction by bone morphogenetic proteins. Cytokine Growth Factor Rev. 9, 49-61.[CrossRef][Medline]
Kelly, R. G., Brown, N. A. and Buckingham, M. E. (2001). The arterial pole of the mouse heart forms from Fgf10-expressing cells in pharyngeal mesoderm. Dev. Cell 1,435 -440.[Medline]
Kim, R. Y., Robertson, E. J. and Solloway, M. J. (2001). Bmp6 and Bmp7 are required for cushion formation and septation in the developing mouse heart. Dev. Biol. 235,449 -466.[CrossRef][Medline]
Kirby, M. L. and Waldo, K. L. (1995). Neural
crest and cardiovascular patterning. Circ. Res.
77,211
-215.
Knecht, A. K. and Bronner-Fraser, M. (2002). Induction of the neural crest: a multigene process. Nat. Rev. Genet. 3,453 -461.[Medline]
Komiyama, M., Ito, K. and Shimada, Y. (1987). Origin and development of the epicardium in the mouse embryo. Anat. Embryol. 176,183 -189.[Medline]
Kreidberg, J. A., Sariola, H., Loring, J. M., Maeda, M., Pelletier, J., Housman, D. and Jaenisch, R. (1993). WT-1 is required for early kidney development. Cell 74,679 -691.[Medline]
Kwee, L., Burns, D. K., Rumberger, J. M., Norton, C., Wolitzky, B., Terry, R., Lombard-Gillooly, K. M., Shuster, D. J., Kontgen, F., Stewart, C. et al. (1995). Creation and characterization of E-selectin-and VCAM-1-deficient mice. Ciba Found. Symp. 189,17 -28.[Medline]
LaBonne, C. and Bronner-Fraser, M. (1999). Molecular mechanisms of neural crest formation. Annu. Rev. Cell Dev. Biol. 15,81 -112.[CrossRef][Medline]
Lawson, K. A., Dunn, N. R., Roelen, B. A., Zeinstra, L. M.,
Davis, A. M., Wright, C. V., Korving, J. P. and Hogan, B. L.
(1999). Bmp4 is required for the generation of primordial germ
cells in the mouse embryo. Genes Dev.
13,424
-436.
Le Douarin, N. M. and Kalcheim, C. (1999). The Neural Crest. New York: Cambridge University Press.
Li, J., Liu, K. C., Jin, F., Lu, M. M. and Epstein, J. A.
(1999). Transgenic rescue of congenital heart disease and spina
bifida in Splotch mice. Development
126,2495
-2503.
Li, Y. X., Zdanowicz, M., Young, L., Kumiski, D., Leatherbury, L. and Kirby, M. L. (2003). Cardiac neural crest in zebrafish embryos contributes to myocardial cell lineage and early heart function. Dev. Dyn. 226,540 -550.[CrossRef][Medline]
Lindsay, E. A. and Baldini, A. (2001). Recovery
from arterial growth delay reduces penetrance of cardiovascular defects in
mice deleted for the DiGeorge syndrome region. Hum. Mol.
Genet. 10,997
-1002.
Lyons, K. M., Pelton, R. W. and Hogan, B. L. (1990). Organogenesis and pattern formation in the mouse: RNA distribution patterns suggest a role for bone morphogenetic protein-2A (BMP-2A). Development 109,833 -844.[Abstract]
Mair, D., Edwards, W., Fuster, V., Seward, J. and Danielson, G. (1992). Truncus arteriosus and aortopulmonary window. In Paediatric Cardiology, Vol. 2, pp. 913-929. Edinburgh: Churchill Livingstone.
McMahon, A. P. and Bradley, A. (1990). The Wnt-1 (int-1) proto-oncogene is required for development of a large region of the mouse brain. Cell 62,1073 -1085.[Medline]
Mishina, Y., Hanks, M. C., Miura, S., Tallquist, M. D. and Behringer, R. R. (2002). Generation of Bmpr/Alk3 conditional knockout mice. Genesis 32, 69-72.[CrossRef][Medline]
Mishina, Y., Suzuki, A., Ueno, N. and Behringer, R. R. (1995). Bmpr encodes a type I bone morphogenetic protein receptor that is essential for gastrulation during mouse embryogenesis. Genes Dev. 9,3027 -3037.[Abstract]
Mitchell, P. J., Timmons, P. M., Hebert, J. M., Rigby, P. W. and Tjian, R. (1991). Transcription factor AP-2 is expressed in neural crest cell lineages during mouse embryogenesis. Genes Dev. 5,105 -119.[Abstract]
Miyazono, K., Kusanagi, K. and Inoue, H. (2001). Divergence and convergence of TGF-beta/BMP signaling. J. Cell Physiol. 187,265 -276.[CrossRef][Medline]
Mjaatvedt, C. H., Nakaoka, T., Moreno-Rodriguez, R., Norris, R. A., Kern, M. J., Eisenberg, C. A., Turner, D. and Markwald, R. R. (2001). The outflow tract of the heart is recruited from a novel heart-forming field. Dev. Biol. 238,97 -109.[CrossRef][Medline]
Moore, A. W., McInnes, L., Kreidberg, J., Hastie, N. D. and
Schedl, A. (1999). YAC complementation shows a requirement
for Wt1 in the development of epicardium, adrenal gland and throughout
nephrogenesis. Development
126,1845
-1857.
Munoz-Chapuli, R., Macias, D., Gonzalez-Iriarte, M., Carmona, R., Atencia, G. and Perez-Pomares, J. M. (2002). The epicardium and epicardial-derived cells: multiple functions in cardiac development. Rev. Esp. Cardiol. 55,1070 -1082.[Medline]
Neuhaus, H., Rosen, V. and Thies, R. S. (1999). Heart specific expression of mouse BMP-10 a novel member of the TGF-beta superfamily. Mech. Dev. 80,181 -184.[CrossRef][Medline]
Nguyen, V. H., Schmid, B., Trout, J., Connors, S. A., Ekker, M. and Mullins, M. C. (1998). Ventral and lateral regions of the zebrafish gastrula, including the neural crest progenitors, are established by a bmp2b/swirl pathway of genes. Dev. Biol. 199,93 -110.[CrossRef][Medline]
Nichols, D. H. (1981). Neural crest formation in the head of the mouse embryo as observed using a new histological technique. J. Embryol. Exp. Morphol. 64,105 -120.[Medline]
Panchision, D. M., Pickel, J. M., Studer, L., Lee, S. H.,
Turner, P. A., Hazel, T. G. and McKay, R. D. (2001).
Sequential actions of BMP receptors control neural precursor cell production
and fate. Genes Dev. 15,2094
-2110.
Pennisi, D. J., Rentschler, S., Gourdie, R. G., Fishman, G. I. and Mikawa, T. (2002). Induction and patterning of the cardiac conduction system. Int. J. Dev. Biol. 46,765 -775.[Medline]
Perez-Pomares, J. M., Phelps, A., Sedmerova, M., Carmona, R., Gonzalez-Iriarte, M., Munoz-Chapuli, R. and Wessels, A. (2002). Experimental studies on the spatiotemporal expression of WT1 and RALDH2 in the embryonic avian heart: a model for the regulation of myocardial and valvuloseptal development by epicardially derived cells (EPDCs). Dev. Biol. 247,307 -326.[CrossRef][Medline]
Poelmann, R. E., Lie-Venema, H. and Gittenberger-de Groot, A. C. (2002). The role of the epicardium and neural crest as extracardiac contributors to coronary vascular development. Tex. Heart Inst. J. 29,255 -261.[Medline]
Quaggin, S. E., Vanden Heuvel, G. B. and Igarashi, P. (1998). Pod-1, a mesoderm-specific basic-helix-loop-helix protein expressed in mesenchymal and glomerular epithelial cells in the developing kidney. Mech. Dev. 71,37 -48.[CrossRef][Medline]
Robb, L., Mifsud, L., Hartley, L., Biben, C., Copeland, N. G., Gilbert, D. J., Jenkins, N. A. and Harvey, R. P. (1998). epicardin: A novel basic helix-loop-helix transcription factor gene expressed in epicardium, branchial arch myoblasts, and mesenchyme of developing lung, gut, kidney, and gonads. Dev. Dyn. 213,105 -113.[CrossRef][Medline]
Sambrook, J. and Russell, D. (2001). Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press.
Sato, M. and Yost, H. J. (2003). Cardiac neural crest contributes to cardiomyogenesis in zebrafish. Dev. Biol. 257,127 -139.[CrossRef][Medline]
Schlaeger, T. M., Bartunkova, S., Lawitts, J. A., Teichmann, G.,
Risau, W., Deutsch, U. and Sato, T. N. (1997). Uniform
vascular-endothelial-cell-specific gene expression in both embryonic and adult
transgenic mice. Proc. Natl. Acad. Sci. USA
94,3058
-3063.
Serbedzija, G. N., Bronner-Fraser, M. and Fraser, S. E.
(1992). Vital dye analysis of cranial neural crest cell migration
in the mouse embryo. Development
116,297
-307.
Solloway, M. J. and Robertson, E. J. (1999).
Early embryonic lethality in Bmp5; Bmp7 double mutant mice suggests
functional redundancy within the 60A subgroup.
Development 126,1753
-1768.
Soriano, P. (1999). Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat. Genet. 21, 70-71.[CrossRef][Medline]
Stoner, C. M. and Gudas, L. J. (1989). Mouse cellular retinoic acid binding protein: cloning, complementary DNA sequence, and messenger RNA expression during the retinoic acid-induced differentiation of F9 wild type and RA-3-10 mutant teratocarcinoma cells. Cancer Res. 49,1497 -1504.[Abstract]
Stottmann, R. W., Anderson, R. M. and Klingensmith, J. (2001). The BMP antagonists Chordin and Noggin have essential but redundant roles in mouse mandibular outgrowth. Dev. Biol. 240,457 -473.[CrossRef][Medline]
Stuckmann, I., Evans, S. and Lassar, D. B. (2003). Erythropoietin and retinoic acid, secreted from the epicardium, are required for cardiac myocyte proliferation. Dev. Biol. 255,334 -349.[CrossRef][Medline]
Sucov, H. M., Dyson, E., Gumeringer, C. L., Price, J., Chien, K. R. and Evans, R. M. (1994). RXR alpha mutant mice establish a genetic basis for vitamin A signaling in heart morphogenesis. Genes Dev. 8,1007 -1018.[Abstract]
Tevosian, S. G., Deconinck, A. E., Tanaka, M., Schinke, M., Litovsky, S. H., Izumo, S., Fujiwara, Y. and Orkin, S. H. (2000). FOG-2, a cofactor for GATA transcription factors, is essential for heart morphogenesis and development of coronary vessels from epicardium. Cell 101,729 -739.[Medline]
Waldo, K. L., Kumiski, D. and Kirby, M. L. (1996). Cardiac neural crest is essential for the persistence rather than the formation of an arch artery. Dev. Dyn. 205,281 -292.[CrossRef][Medline]
Waldo, K., Zdanowicz, M., Burch, J., Kumiski, D. H., Stadt, H.
A., Godt, R. E., Creazzo, T. L. and Kirby, M. L. (1999). A
novel role for cardiac neural crest in heart development. J. Clin.
Invest. 103,1499
-1507.
Waldo, K. L., Kumiski, D. H., Wallis, K. T., Stadt, H. A., Hutson, M. R., Platt, D. H. and Kirby, M. L. (2001). Conotruncal myocardium arises from a secondary heart field. Development 128,3179 -3188.[Medline]
Willette, R. N., Gu, J. L., Lysko, P. G., Anderson, K. M., Minehart, H. and Yue, T. (1999). BMP-2 gene expression and effects on human vascular smooth muscle cells. J. Vasc. Res. 36,120 -125.[CrossRef][Medline]
Wu, H., Lee, S. H., Gao, J., Liu, X. and Iruela-Arispe, M.
L. (1999). Inactivation of erythropoietin leads to defects in
cardiac morphogenesis. Development
126,3597
-3605.
Yamauchi, Y., Abe, K., Mantani, A., Hitoshi, Y., Suzuki, M., Osuzu, F., Kuratani, S. and Yamamura, K. (1999). A novel transgenic technique that allows specific marking of the neural crest cell lineage in mice. Dev. Biol. 212,191 -203.[CrossRef][Medline]
Yanagisawa, H., Clouthier, D. E., Richardson, J. A., Charite, J.
and Olson, E. N. (2003). Targeted deletion of a branchial
arch-specific enhancer reveals a role of dHAND in craniofacial development.
Development 130,1069
-1078.
Yang, J. T., Rayburn, H. and Hynes, R. O.
(1995). Cell adhesion events mediated by alpha 4 integrins are
essential in placental and cardiac development.
Development 121,549
-560.
Yelbuz, T. M., Waldo, K. L., Zhang, X., Zdanowicz, M., Parker, J., Creazzo, T. L., Johnson, G. A. and Kirby, M. L. (2003). Myocardial volume and organization are changed by failure of addition of secondary heart field myocardium to the cardiac outflow tract. Dev. Dyn. 228,152 -160.[CrossRef][Medline]
Yoshikawa, S. I., Aota, S., Shirayoshi, Y. and Okazaki, K. (2000). The ActR-I activin receptor protein is expressed in notochord, lens placode and pituitary primordium cells in the mouse embryo. Mech. Dev. 91,439 -444.[CrossRef][Medline]
Zhang, H. and Bradley, A. (1996). Mice
deficient for BMP2 are nonviable and have defects in amnion/chorion and
cardiac development. Development
122,2977
-2986.