1
Division of Neonatology, Department of Pediatrics, PO Box 3179, Duke
University Medical Center, Durham, NC 27710, USA
2
Departments of Cellular Biology and Anatomy, and Pharmacology, Medical College
of Georgia, Augusta, GA 30912, USA
*
Author for correspondence
(e-mail:kirby013{at}mc.duke.edu
)
Accepted 4 May 2001
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SUMMARY |
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Key words: Chick-quail chimeras, Myocardium, FGF-8, BMP-2, Heart development, Secondary heart field, Outflow tract, Conotruncus
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INTRODUCTION |
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Mapping studies of these primary heart fields have traditionally relied on
whole embryo culture of chick embryos where viability of the explanted embryos
is limited to early straight heart tube stages. In the earliest mapping
studies, it was assumed that the components of the straight heart tube
represented all of the endocardial and myocardial progenitors of the adult
heart endo/myocardium (Rosenquist and DeHaan,
1966). However, marking
experiments performed in ovo at later, looping stages of heart development
suggested that the atrioventricular canal, atria and conotruncus are added
secondarily to the straight heart tube during looping (Arguello et al,
1975
; de la Cruz et al.,
1977
; de la Cruz et al.,
1987
; de la Cruz et al.,
1989
; de la Cruz et al.,
1991
). According to de la
Cruz, the inflow segments are added first, followed by the outflow segments.
Recent studies in mice support the idea that the myocardium of the primary
heart tube does not represent all of the myocardium of the septated heart
(Christoffels et al.,
2000
).
In support of the data of de la Cruz, we have identified a previously unrecognized `secondary' heart field in the splanchnic mesoderm that underlies the caudal pharynx. The field provides myocardium to the outflow tract during looping. In contrast, the inflow myocardium appears to be accreted from the bilateral primary heart fields. The secondary heart field expresses Nkx2.5 and Gata-4 prior to differentiation as myocardium, and cells are induced to become myocardium in a manner similar to that which occurs in the primary heart fields.
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MATERIALS AND METHODS |
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For injections, Mitotracker (50 µg, Molecular Probes) was dissolved in 1 ml DMSO and diluted 1:10 with phosphate buffered saline (PBS). Pulled capillaries were used to inject approximately 1-2 nl of the diluted dye into the splanchnic mesoderm just caudal to the cardiac outflow tract. The embryos were reincubated for 48 hours or until they reached stage 22.
To produce chimeras, quail embryos at a comparable stage were dissected from the yolk and placed in PBS. The splanchnic mesoderm just behind the cardiac outflow tract was removed and transferred to the same site in an explanted chick embryo or into the lateral wall of the pharynx. The chick embryos were reincubated for 48 hours or until they reached stage 22.
Embryos for immunohistochemistry or in situ hybridization were collected in DEPC-treated phosphate buffered saline (PBS) and immersion fixed in 4% paraformaldehyde in PBS.
Tissue culture
Conditioned medium was made by culturing ventral pharynx, myocardium or
secondary outflow heart field from chick embryos at stage 14-15 overnight in a
small volume of Dulbecco's minimal eagles medium (DMEM) with 6% fetal bovine
serum. Secondary outflow heart field was removed from embryos at stages 14-15
and cultured overnight in 20 µl of conditioned medium.
5-Bromo-2'deoxyuridine (BrdU, 20 µl of a 40 mM solution, Boehringer)
was added to the cultured heart fields for 1 hour before fixation in 4%
paraformaldehyde in phosphate-buffered saline as reported by Brand-Saberi et
al. (Brand-Saberi et al.,
1995). Fixation was carried out
for 10 minutes followed by double immunohistochemistry as described above.
Immunohistochemistry
The embryos were embedded in paraffin and sectioned at 8 µm. The
sections were incubated as described previously (Waldo et al.,
1998). HNK-1 (obtained from
American Type Culture Collection, Rockville, MD) was visualized with Alexa 488
goat anti-mouse IgG conjugate (Molecular Probes, Eugene, OR) as reported
previously (Waldo et al.,
1996
). MF20 (red) and QH-1
(green) were visualized with Alexa 568 goat anti-mouse IgG conjugate
(Molecular Probes). The MF20, developed by Donald A. Fischman, and QH-1 were
obtained from the Developmental Studies Hybridoma Bank developed under the
auspices of the NICHD and maintained by The University of Iowa, Department of
Biological Sciences, Iowa City, IA. The mouse ABC Elite kit (Vector) was used
for the secondary reaction with DAB visualization. Details of the protocol
have been described previously (Waldo et al.,
1996
).
Quail cells in the quail-chick chimeras were visualized with QCPN, a
quail-specific antibody, as described previously (Waldo et al.,
1998). Chick Slug antibody was
used at 1:50 with the ABC Elite kit for the secondary reaction. The Slug
antibody, developed by Thomas M. Jessell, was obtained from the Developmental
Studies Hybridoma Bank. BrdU was visualized using the Zymed Kit with DAB as
the secondary antibody. The preparations were counterstained with hematoxylin
(Anatech, Battle Creek, MI).
Isolation and sequence analysis of cDNA
The GATA-4 probe was made from a GATA-4 PCR fragment cloned by Todd Evans
(clone 7) and sent via Katherine Yutzey. A 221 bp fragment of chick Nkx2.5
(Accession Number X91838) was made using RT-PCR with primers and protocol as
described previously (Schultheiss et al.,
1995). A 225 bp fragment of
FGF-2 was cloned using the primers as described by
Désiré et al.
(Désiré et
al., 1998
) and cDNA from stage
18 whole embryos. A 239 bp fragment of FGF-4 (Accession Number U14654) was
made using primers spanning the region of 321-559bp and cDNA from stage 18
whole embryos. A 393 bp fragment of FGF-8 (Accession Number U55189) was made
using primers spanning the region of 17-409 bp and cDNA from hearts from stage
14-18 embryos. A 337 bp fragment of BMP-2 (accession number X75914) was made
using primers spanning the region of 239-575 bp and cDNA from stage 14 and 18
embryonic hearts. A 283 bp fragment of BMP-4 (accession number X75915) was
made using primers spanning the region of 11-293bp and cDNA from stage 18
whole chick embryos. The fragments were cloned into the vector PCRII
(Invitrogen) and sequenced to ensure their identity. PCR fragments containing
one of the promoter primers and one of the gene specific primers were
generated and labeled with digoxigenin.
In situ hybridization and sectioning of stained embryos
The in situ hybridization was carried out with digoxigenin-labeled
riboprobes generated from the cDNA fragment cloned into pCRII (Invitrogen)
using one promoter primer and one of the gene specific primers. The protocol
followed that described by Wilkinson (Wilkinson,
1992). After examination and
documentation of whole-mount staining, the embryos were embedded in paraffin,
sectioned transversely at 12 µm and mounted.
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RESULTS |
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HNK-1 but not SLUG is expressed by cells in the secondary heart
field
HNK-1 was expressed in the Nkx2.5/Gata-4-positive splanchnic mesoderm
underlying the pharyngeal endoderm. We have previously noted the expression of
HNK-1 in this mesenchyme and determined through quail-chick chimeric embryos
that it is not associated with neural crest (K. L. W. and M. L. K.,
unpublished). Because of its appearance in other tissues and, during
development, at the cardiac inflow and outflow tracts, it appears to be
expressed by cells undergoing translocation or differentiation (Vincent and
Thiery, 1984; Tucker et al.,
1984
; Canning and Stern,
1988
). From stage 14 onwards,
the myocardium of the caudal part of the distal outflow tract was particularly
HNK-1-positive, as was the splanchnic mesoderm below the floor of the pharynx
immediately caudal to the outflow tract
(Fig. 2A-D). The timing of
HNK-1 expression in these regions correlated with the period when the
secondary or definitive outflow segments are added to the heart tube as shown
in the marking experiments of de la Cruz and colleagues (Arguello et al.,
1975
; de la Cruz et al.,
1977
; de la Cruz et al.,
1987
; de la Cruz et al.,
1989
; de la Cruz et al.,
1991
).
|
To examine whether these cells were undergoing a classical
epithelial-to-mesenchymal transformation, we used an antibody to Slug, which
is expressed in cells undergoing epithelial-to-mesenchymal transformation
(Nieto et al., 1994; Romano
and Runyan, 1999
; Carmona et
al., 2000
). Slug was expressed
in the myocardium of the looping heart as previously reported (Carmona et al.,
2000
), but not at its poles or
in the HNK-1-positive splanchnic mesenchyme caudal to the outflow tract
(Fig. 2E).
To determine whether the HNK-1-positive cells at the ends of the cardiac
tube expressed myocardial contractile proteins we used double
immunohistochemistry for HNK-1 and MF20, a standard myocardial cell marker
(Han et al., 1992). In both
poles of the heart we found that HNK-1 was expressed in the Nkx2.5-positive
mesenchyme below the pharyngeal endoderm. The HNK-1-positive cells in the
mesenchyme most distant from the myocardium were negative for MF20
(Fig. 3A,C, green). However,
some of the HNK-1-positive cells that were closer to the myocardium were also
positive for MF20 (yellow cells in 3A,C). Myocardium more centrally located in
the tubular heart, that is, more distant from the attachments of the inflow
and outflow to the pharynx, was HNK-1-negative, MF20-positive
(Fig. 3A,C). Caudal to the
outflow tract, the midline splanchnic mesoderm ventral to the pharynx was
strongly HNK-1-positive (Fig.
3B, arrow).
|
Proximity to the cardiac outflow tract initiates myocardial
differentiation
To demonstrate directly that the outflow tract lengthens by accretion of
cells from these secondary centers, mitotracker, a fluorescent dye, was
injected into the splanchnic mesoderm behind the outflow tract at stage 14
(Fig. 4A). The hearts were
examined using confocal microscopy at stage 22. The myocardium of the most
proximal segment, the conus, was fluorescent while that of the distal segment,
the truncus arteriosus, did not fluoresce
(Fig. 4B), indicating that two
new segments were added to the outflow tract between stages 14 and 22. As the
truncal myocardium was not fluorescent, it was most probably generated after
the time of the injection and arose from an uninjected region of the
splanchnic mesoderm. Significantly, after the injection, the splanchnic
mesenchyme caudal to the outflow tract at stage 22 was not fluorescent, but a
tiny spot of fluorescence, signifying the original site of injection, could be
seen cranial to the attachment of the outflow tract to the body wall
(Fig. 4B, arrow). An
examination of the outflow attachment to the ventral pharynx showed that the
attachment point has a dynamic relationship with the pharynx and is located
progressively more caudally. Thus, at stage 12 the outflow joins the pharynx
ventral to pharyngeal arch/artery 1, and by stage 24 is located ventral to
pharyngeal arches/arteries 4-6 (Fig.
5).
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|
Quail-chick chimeras were generated to examine the potential of the ventral pharyngeal mesoderm for differentiating as myocardium. Donor tissue from quail ventral pharyngeal mesenchyme was transplanted into the corresponding region in chick embryos at stage 14 (Fig. 4A). The embryos were collected at stage 22 and double-labeled for an anti-quail antibody (QCPN) and MF-20. The quail implants formed rounded bodies comprised of quail cells, which were encircled by MF-20-positive myocardium derived from the quail graft (Fig. 4C,D). When similar grafts were implanted in the lateral wall of the pharynx, not in proximity to the cardiac outflow tract, they formed vesicles that did not contain MF-20-expressing cells (data not shown).
From these results and double immunohistochemistry with HNK-1 and MF-20, it appeared that proximity to the primary heart tube influenced the conversion of splanchnic mesoderm cells to myocardium.
Expression of FGF-8 and BMP-2
Because FGF and BMP cytokine family members are essential in induction of
myocardial cells from the primary heart fields, we examined the expression of
FGF-2, FGF-4 and FGF-8, and BMP-2 and BMP-4 in the pharynx at stages 14 and
18. FGF-2 and FGF-4 and BMP-4 were not expressed in or near the secondary
heart field at either stage. At stage 14, FGF-8 was expressed in the lateral
pharynx in both the ectoderm and endoderm
(Fig. 6A,B). Expression was
continuous craniocaudally from arch 1 to the arch 6 region in the ectoderm,
but expressed in the endoderm only where it was close or in contact with the
ectoderm. Expression in arches 1 and 2 was less than in arches 3-6. By stage
18, the expression of FGF-8 was much reduced so that it was only between the
maxillary and mandibular prominences of arch 1 and in the cleft/pouch of arch
2. Expression continued at a reduced level in the lateral ectoderm of arches
3-6 (Fig. 6C,D).
|
At stage 14, BMP-2 was expressed most strongly in the splanchnic mesoderm just caudal to the outflow myocardium. The myocardium also showed positive expression although less intense than that in the splanchnic mesoderm. The dorsal mesocardium and myocardium of the inflow tract was also positive (Fig. 6E,F,H). It was not expressed in the splanchnic mesenchyme underlying the pharyngeal endoderm between the cardiac outflow and inflow tracts (Fig. 6G). By stage 18, BMP-2 expression was notable in the endocardium but was completely absent from the secondary heart field (Fig. 6I).
Secondary heart field induces myocardial differentiation
To determine whether the lateral endoderm/ectoderm or secondary heart field
acted as a signaling center, we explanted various tissues to produce
conditioned medium, which was then used in secondary heart field cultures. The
secondary heart field was tested for its ability to differentiate as
myocardium using MF-20 as a marker. Proliferation was also assessed using
BrdU. Conditioned medium was made using explanted lateral pharyngeal
endoderm/ectoderm, outflow myocardium or secondary outflow field.
Unconditioned medium, or medium conditioned by secondary heart field, distal
outflow myocardium or endoderm/ectoderm, induced myocardial differentiation,
although MF20 staining was poor or absent in the endoderm/ectoderm conditioned
medium (Fig. 7A,B,D). However,
in every case exposure to endoderm/ectoderm caused a robust proliferative
response in the secondary heart field (Fig.
7C). Proliferation was completely absent in the secondary heart
field cultured in secondary heart field-conditioned medium.
|
Because myocardial differentiation from the primary heart field is dependent on BMP-2 induction, we examined whether BMP-2, which is expressed in both the secondary heart field and outflow myocardium might be necessary for secondary myocardial differentiation. Noggin (15 µg/ml, gift from Dr Richard Harland) was added to a duplicate set of secondary heart field cultures in the various conditioned media. In most cases, myocardial differentiation was less robust in the presence of noggin and proliferation was increased (Fig. 7E-H). However, myocardial differentiation appeared unaffected in the secondary heart field cultured in secondary heart field-conditioned medium, which could perhaps be explained by the combined presence of abundant BMP-2 from the conditioned medium and the cultured secondary heart field itself overriding the noggin inhibition. This also might explain the paucity of proliferation in these cultures with or without noggin. Proliferation in the endoderm/ectoderm-conditioned medium with noggin was remarkable for its abundance although it would be difficult to make the case that noggin altered proliferation in these cultures.
Accretion of myocardium by the cardiac inflow tract is from the
primary heart fields
The inflow does not lengthen much after stage 12, which means that it is
finished by the time secondary myocardium is being added to the outflow tract
at stage 14. We examined the possibility that accretion of myocardium to the
inflow tract is also from a secondary heart field. At stage 11, the inflow end
of the heart and its attached dorsal mesocardium was HNK-1 positive, while the
remaining heart tube was negative (Fig.
8A). Subsequently, the mesenchyme adjacent to the inflow lost its
HNK-1 reactivity while the mesocardium just behind the outflow myocardium
became positive (Fig. 2C).
NKX2.5 and GATA-4 were expressed by the inflow myocardium, dorsal mesocardium
and extended slightly into the splanchnic mesoderm underlying the pharynx
(Fig. 8B,D), while myocardial
differentiation occurred slightly into the dorsal mesocardium but not into the
splanchnic mesoderm (Fig. 8C). Neither BMP-2 nor FGF-8 was expressed in a pattern that suggested induction in
the same manner as that observed for the cardiac outflow tract
(Fig. 8E,F). Because of the
continuity of the cardiac inflow tract with the sinus venosus and systemic
veins, all of which are invested in myocardium (Franco et al.,
2000), it appeared that
accretion of myocardium that occurs close to the time that the single heart
tube is fusing could be a continuation of this process of fusion. In this case
the myocardium would be added from the primary heart fields rather than from a
special secondary heart field, as appears to happen in building the definitive
outflow tract.
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DISCUSSION |
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The outflow myocardium has been recognized for a long time as being
molecularly distinct from ventricular myocardium (Ruzicka and Schwartz,
1988). The primary myocardium
begins to regionalize and proceeds to maturation through a set program of
contractile isoform expression, while the outflow myocardium retains
characteristics of myocardium in the primary heart tube (Christoffels et al.,
2000
). For example, expression
of
smooth muscle actin, the first myocardial contractile gene to be
expressed in myocardial cells derived from the primary heart fields (Colas et
al., 2000
), continues long
after the ventricular myocardium has stopped expressing the smooth muscle
isoform in favor of cardiac
actin (Ruzicka and Schwartz,
1988
). During septation, the
outflow myocardium is incorporated into the right ventricular outflow tract,
although some of the myocardial cells undergo apoptosis during this process
(Watanabe et al., 1998
). The
fact that the definitive outflow myocardium is incorporated from a separate
heart field well after the primary heart tube has formed helps to explain why
it is different from the primary myocardium.
The transformation of cells from the secondary heart field into myocardium
appears to require many of the same steps undertaken by cells in the primary
heart fields (Fig. 9). The
primary heart fields are separated, paired heart fields in the lateral plate
mesoderm (Rosenquist and DeHaan, 1976; Colas et al.,
2000). Differentiation of
myocardial phenotype from these fields requires at least three separate
inductions. The earliest induction of myocardial potential occurs at
gastrulation by activin signaling from the organizer (Antin et al.,
1994
; Nascone and Mercola,
1995
; Yatskievych et al.,
1997
). Because activin induces
both mesoderm and endoderm, and interactions between the two may be
responsible for heart induction, the role of activin may be indirect (Logan
and Mohun, 1993
). In any case,
after this initial induction, BMP signaling is necessary to maintain the
myocardial lineage potential (Schultheiss et al.,
1997
; Andree et al.,
1998
; Yatskievych et al.,
1997
). Signaling from the
anterior endoderm appears to be important in a number of species, including
mouse (Arai et al., 1997
),
chick (Schultheiss et al.,
1995
), newt (Sater and
Jacobson, 1990
) and
Xenopus (Nascone and Mercola,
1995
). The endoderm induces
Nkx-2.5 and Gata-4 expression, the first molecular indication of myocardial
cell commitment, in these fields. Expression of both Nkx-2.5 and Gata-4
appears to be required prior to myocardial differentiation (Schultheiss et
al., 1997
; Jiang et al.,
1998
; Reecy et al.,
1999
; Schlange et al.,
2000
). Finally myocardial
differentiation of the Nkx-2.5/Gata-4-positive cells depends on FGF-2 and
BMP-4 signaling from the anterior endoderm (Sugi and Lough,
1994
) as treatment with either
factor alone does not induce Nkx-2.5 expression in explants of primary heart
field mesoderm (Lough et al.,
1996
; Yatskievych et al.,
1997
; Barron et al.,
2000
; Lough and Sugi,
2000
). Exposure to FGF-2 for
30 minutes with continuous exposure to BMP-4 elicits myocardial
differentiation from mesoderm explanted from the primary heart fields (Barron
et al., 2000
). Most of these
studies have been carried out in chick, but BMP signaling is also required for
expression of myocardial markers by the primary myocardium in Xenopus
embryos (Shi et al., 2000
).
The single midline primary heart tube forms from the two heart fields during
body wall closure. In the chick embryo this occurs at about stages 8-9.
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After body wall closure, the secondary heart field lies just underneath the
same endoderm that induces Nkx 2.5 and Gata-4 expression in the primary heart
fields. Sater and Jacobson (Sater and Jacobson,
1990) showed that the ventral
pharyngeal endoderm is still capable of inducing heart development in
non-cardiac mesoderm until after the third pharyngeal pouch has formed, which
is well into the looping stage of heart development. The ventral pharyngeal
endoderm retains the same potency in chick embryos until about stage 16 (M. J.
Farrell and M. L. K., unpublished).
The decision of the Nkx-2.5/Gata-4-positive cells in the secondary heart
field to translocate into the outflow tract coincides with the onset of HNK-1
expression at about stage 14. The HNK-1 carbohydrate epitope is part of a
cell-surface glycoprotein that mediates cell-cell or cell-substrate
interactions. It is widely accepted in avian embryology as a marker of
migrating neural crest cells (Bronner-Fraser,
1986), but is it expressed in a
number of non-neural crest cell types. Canning and Stern (Canning and Stern,
1988
) have noted the onset of
HNK-1 expression in epiblast cells as they traversed the primitive streak to
become mesodermal.
While it is possible that HNK-1 is expressed by cells in the secondary heart fields in association with their differentiation as myocardium, the time of its appearance is more consistent with the onset of migration or translocation into the outflow tract. We were not able to see any morphological indications of myofibrils in the splanchnic mesoderm using either confocal or transmission electron microscopy (K. L. W and M. L. K., unpublished). It is only as the HNK-1-positive cells from the secondary heart field approach and incorporate into the existing primary myocardium that they commence expression of a myocardial phenotype, as shown here by the onset of myosin heavy chain expression. Once the cells have completed their incorporation into the myocardium, they lose their HNK-1 reactivity.
While FGF-2 and FGF-4 are not expressed in the splanchnic mesoderm or
outflow myocardium, FGF-8 is expressed strongly by ectoderm and endoderm of
the lateral walls of the pharynx. FGF-8 expression is extinguished in the
lateral pharynx by about stage 18, when most of the secondary myocardium has
been added to the outflow tract. In the zebrafish, FGF-8 is required for
expression of Nkx-2.5 and Gata-4 in cardiac precursors (Reifers et al.,
2000).
Because the HNK-1-positive cells begin to express MF-20 only when they are
near the outflow myocardium/endocardium, the final signal needed for
expression of a myocardial phenotype may emanate from the outflow tract
itself. Interestingly, BMP-2 has been shown in previous studies to be
expressed by the primary outflow myocardium during the stages that the
secondary myocardium is incorporated (Yamada et al.,
1999). Smad6, an inhibitory
SMAD, is expressed throughout the outflow myocardium at the same time (Yamada
et al., 1999
), which might
protect the newly incorporated myocardium from BMP signaling.
BMP-induced cardiomyocyte differentiation is mediated by
TGF-ß-activating kinase 1 (TAK1), a novel member of the mitogen-activated
protein kinase kinase kinase superfamily (Monzen et al.,
1999). TAK1 inhibits cyclin D1
promoter activity and proliferation (Terada et al.,
1999
). In the present study,
secondary heart field cultured with pharyngeal endoderm-conditioned medium
showed elevated proliferation. Both secondary heart field- and
myocardium-conditioned medium induced cardiomyocyte differentiation and both
are sources of BMP-2 during the time that secondary myocardium is incorporated
into the looping heart. Thus, signaling from the endoderm/ectoderm may require
local signaling by BMP-2 released from the distal myocardium and secondary
heart field to initiate myocardial contractile protein expression. If this is
the case, proliferation appears to be inhibited concurrent with myocardial
differentiation. Because of the robust expression of BMP-2 by the secondary
myocardium, it was difficult to completely inhibit myocardial differentiation,
even in the presence of the endoderm/ectoderm if this is indeed one of the
functions of BMP-2. The addition of noggin, a BMP inhibitor, suppressed
myocardial differentiation to some extent but only in a few cases did
myocardium not appear in the cultures.
A recent study has shown that FGF-8 in the ventral pharynx caused a
decrease in the myocardial transient, concomitant with increased proliferation
after neural crest ablation (Farrell et al.,
2001). Neural crest cells
migrating into the caudal pharynx are believed to block the detrimental effect
of the pharyngeal FGF-8.
Failure of the quail cells to be incorporated into the myocardium of the
outflow tract in chimeras of the secondary heart field is somewhat puzzling.
However, the architecture of the ventral pharynx is established during
formation of the foregut pocket by closure of the body wall (Patten,
1971). It is during this
process that the bilateral primary heart fields are brought to the ventral
midline to form the primary heart tube. Because the cells in the dorsal
mesocardium and splanchnic mesoderm are laid down at this time, it is not
surprising that grafted cells would not be in an appropriate position to be
incorporated into the myocardium of the outflow tract. Grafts in the ventral
pharynx are difficult because of the movement of the outflow tract relative to
the pharynx and movement of the region because of the heartbeat.
It remains to be determined whether the mammalian myocardium also arises
from primary and secondary heart fields; however, recently acquired
information suggests that the myocardium of the primary heart tube in mouse
embryos does not represent all of the myocardial cells that will build the
four-chambered heart (Christoffels et al.,
2000). If normal
differentiation of the chambers of the heart is dependent on addition of the
definitive outflow and inflow, then these chambers may not differentiate
normally if the secondary heart fields fail to be induced or the cells do not
become incorporated (Christoffels et al.,
2000
). Embryonic mice with
Mef2c or dHAND (Hand2 Mouse Genome Informatics) null mutations fail to
express molecular markers specific to the right ventricle (Srivastava et al.,
1997
; Lin et al.,
1997
). If right ventricular
chamber specification requires the addition of the secondary myocardium, then
the defect in the case of these two mutations may be lack of addition of the
secondary or definitive outflow tract to the primary myocardium.
Another mouse with a similar phenotype provides a potential mechanism for
movement of cells from a secondary heart field. The hdf mouse mutant, which
has a transgenic insertional mutation in the Cspg2 (versican) gene located on
chromosome 13, also fails to differentiate right ventricular chamber markers
or an outflow tract (Mjaatvedt et al.,
1998). Versican expression is
highest in the outflow tract during looping (Capehart et al.,
1999
). The fact that the hdf
mouse appears to lack a definitive outflow tract suggests that versican may
provide a migratory matrix for incorporation of the HNK-1-positive cells from
the secondary heart field. It has recently been found that neural crest cells
will not migrate through a three-dimensional collagen-aggrecan gel, but will
move through a collagen/versican substrate via engagement of HNK-1
antigen-bearing cell-surface components (Perissinotto et al.,
2000
).
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ACKNOWLEDGMENTS |
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