|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online March 9, 2006
doi: 10.1242/10.1242/dev.02292
1 Department of Cell and Developmental Biology and Anatomy, School of Medicine,
University of South Carolina, Columbia, SC 29208, USA.
2 Program in Women's Studies, College of Arts and Sciences, University of South
Carolina, Columbia, SC 29208, USA.
3 Department of Cell Biology and Anatomy and Cardiovascular Developmental
Biology Center, Medical University of South Carolina, Charleston, SC 29425,
USA.
* Author for correspondence (e-mail: ramsdell{at}musc.edu)
Accepted 18 January 2006
 |
SUMMARY |
|---|
 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Key words: ActRIB, ALK4, Cardiac development, Cardiomyocyte, Congenital heart defect, Heterotaxy, Laterality, Left-right asymmetry, Situs inversus, TGFß
|
INTRODUCTION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
). Deviations in
normal LR pattern during embryogenesis result in a wide spectrum of abnormal
laterality phenotypes that are broadly classified as either heterotaxy or
situs inversus (reviewed by Bowers et al.,
1996). Heterotaxy is a condition of discordant LR organ
asymmetries, such that some organs are normal in their LR asymmetry, and
others are not. The most severe type of heterotaxy is a condition called
isomerism, in which normally asymmetric organs instead develop left or right
symmetry. In contrast to heterotaxy, situs inversus is a condition in which
all organs are LR reversed, resulting in a complete mirror-image asymmetry of
the heart and viscera.
Many of the most severe and life-threatening complex congenital heart
defects (CHDs) occur in individuals who are afflicted by laterality disease.
Whereas the overall incidence of CHDs in the general population is estimated
to occur in 0.08% of live births (Ferencz
et al., 1985), this incidence dramatically rises to 90% or higher
for individuals exhibiting heterotaxic phenotypes
(Nugent et al., 1994). CHDs in
individuals with heterotaxy commonly include atrial septal defects,
ventricular septal defects, transposition (or corrected transposition) of the
great arteries, a double outlet right ventricle, anomalous venous return, a
single ventricle and aortic arch anomalies (reviewed by
Bowers et al., 1996;
Bartram et al., 2005). CHDs
commonly associated with isomerism include formation of two morphologically
`leftish' or `rightish' atria, an absent or deficient atrial septum, anomalous
venous return and loss of the coronary sinus (reviewed by
Bowers et al., 1996;
Bartram et al., 2005). Despite
the concordance of organ systems in situs inversus, the incidence of CHDs in
situs inversus individuals is nevertheless elevated (3% versus 0.08%) compared
with that in individuals exhibiting normal body situs (situs solitus)
(Ferencz et al., 1985;
Nugent et al., 1994;
Sternick et al., 2004). In
addition, the risk for developing heterotaxy, and hence CHDs, is greatly
increased for progeny of individuals with situs inversus
(Burn, 1991;
Gebbia et al., 1997).
The prevalence of CHDs that is associated with laterality disease indicates
that heart development is greatly affected by the mechanisms driving LR body
axis determination. This association is not surprising, given the many LR
asymmetries that the heart must develop during its formation (reviewed by
Ramsdell, 2005). During
vertebrate cardiogenesis, cells located in the paired left and right
splanchnic mesodermal heart fields (called the primary heart fields) coalesce
to form a relatively straight tube-shaped structure containing an outer
myocardium and an inner endocardium (reviewed by
Brand, 2003;
Eisenberg and Markwald, 2004).
As the cardiac tube elongates through continued fusion of the primary heart
fields and addition of cells from the secondary/anterior heart field, it
initiates dextral (rightward and dorsal) looping morphogenesis that brings
together regions of the heart tube that were initially non-adjacent to one
another (reviewed by Manner,
2000). Concomitant with looping, additional cardiomyocytes are
contributed from diverse sources such as the neural crest, transformation of
intracardiac and extracardiac mesenchyme cells, and perhaps even the
recruitment of circulating stem cells (reviewed by
Eisenberg and Markwald, 2004).
The rearrangement of the heart that results from looping morphogenesis is a
necessary prelude to chamber formation, septation and differentiation of the
inflow and outflow tracts – all processes that establish LR differences
necessary for maintaining separation of systemic and pulmonary blood flow in
the fully developed heart.
Using embryos of the frog Xenopus laevis, we recently identified a
pivotal role for a type I TGFß serine-threonine kinase receptor,
activin-like kinase receptor 4 (ALK4), in modulating LR axis determination and
cardiac LR development. Misregulation of ALK4 signaling, through either
left-side attenuation or right-side ectopic activation, causes LR reversals in
heart and gut asymmetries, suggesting that ALK4 ordinarily functions on the
left side of the Xenopus embryo to establish normal body situs
(Chen et al., 2004). ALK4
signaling also regulates the classically defined
nodal
Pitx2c pathway in the lateral plate mesoderm,
indicating that ALK4 functions upstream of heart and visceral organ formation
in modulating LR development (Chen et al.,
2004). How cells of the heart use LR axis information, including
that imparted by the ALK4 pathway, to generate anatomical asymmetries during
organogenesis is not known.
Because the heart derives from multiple bilaterally paired sources of cells that are located to the left and right sides of the embryonic midline, we hypothesized that the regulated allocation of these left and right side lineages is an important aspect of normal cardiac LR development. To test this, we first determined the left versus right side lineage origins of all myocytes, regardless of heart field ancestry, that are present in the post-septated, looped heart. To then determine whether LR cardiomyocyte lineages are regulated by the LR body axis, results were compared with those obtained in embryos with various laterality defects, including abnormal Pitx2c expression, induced via ectopic activation or attenuation of ALK4 signaling. Our results define a comprehensive map of the LR myocyte composition of the vertebrate heart and show that LR lineage composition is abnormal in hearts of embryos with heterotaxy and situs inversus phenotypes. Defects in LR cardiomyocyte composition are almost always associated with cardiac malformations, demonstrating that allocation of cardiac LR lineages is an important target of LR axial patterning. We propose that the ability of cells to become differentiated into various cardiac structures is related to LR lineage origin and that when LR cardiomyocyte lineages are altered, different types of CHDs will occur depending on which region(s) of the heart are affected. In addition, because cardiac malformations, altered Pitx2c expression and abnormal LR cardiomyocyte compositions also are present in many of the embryos that fail to develop heterotaxy or situs inversus following experimental manipulation of ALK4 signaling, this suggests that some `isolated' CHDs can nevertheless arise from subtle errors in LR patterning processes, even in the absence of overt body situs defects.
|
MATERIALS AND METHODS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
).
Using cell size and pigmentation patterns to distinguish left and right
blastomeres, Oregon Green-conjugated dextran (Mr 10,000,
lysine-fixable) and Alexa 647-conjugated dextran (10,000
Mr) (Molecular Probes) were pressure injected in the
marginal zone of single cells of stage 3 embryos as described
(Ramsdell et al., 2005).
Induction of laterality defects
GpppG-capped RNA was transcribed with the mMessage mMachine kit (Ambion)
using 300 ng of linearized CA-ALK4 or tALK4 cDNAs
(Chang et al., 1997) as
templates. RNA was pressure-injected into left or right ventrolateral vegetal
cells of 16-cell stage embryos as described
(Chen et al., 2004). After
reaching stages 45-46, embryos were anesthetized with 0.01% benzocaine and
scored for dorsoanterior phenotype using the dorsoanterior index (DAI)
(Kao and Elinson, 1988) and LR
phenotype using orientation of the heart, gut and gallbladder as indicators of
body situs. Embryos exhibiting an abnormal DAI or other gross morphological
defects were not used for lineage analysis.
Confocal imaging
Embryos were fixed in MEMFA (Sive et
al., 2000) and processed for paraffin wax-embedded sectioning as
described (Ramsdell et al.,
2005). Prior to imaging the heart, embryos were prescreened at low
(5-10x) magnification to ascertain that the dextrans were appropriately
targeted only to one side of the embryo. Embryos not showing distinct LR
hemi-labeling were discarded with a total of 34 prescreened embryos (control
and experimental) used for lineage analyses.
Images of embryo sections were collected using a Leica TCS SP2 AOBS Confocal System mounted onto a Leica DM RE-7 upright microscope. Samples typically were viewed using a 20x Plan APO objective, and the dextran fluorophores were excited using either 488 nm (Argon laser) for Oregon Green or 633 nm (HeNe laser) for Alexa 647. Each tissue section was imaged at a depth giving maximum emission signal in both emission channels, and pseudo-colored green for Oregon Green and red for Alexa 647. Images were imported into Adobe Photoshop for adjustment of contrast and brightness.
In situ hybridization
Non-injected and experimental embryos were collected at stages 25-26 and
processed for whole-mount in situ hybridization using a digoxigenin-labeled
antisense Pitx2c RNA probe synthesized with the Maxiscript kit
(Ambion) as described (Chen et al.,
2004). Sibling embryos from control and experimental groups were
maintained to monitor LR phenotypes. Images were acquired with a Spot RT
camera and imported into Adobe Photoshop for adjustment of contrast and
brightness.
|
RESULTS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
5
days of development), when the heart has undergone septation and looping
morphogenesis. As shown in Fig.
1, the dextrans were not only detectable during these stages, but
were also clearly restricted to the left and right sides of the embryonic
midline, confirming fidelity of the lineage marker targeting. This approach
differs from previous attempts to generate a LR fate map of the heart
(Gormley and Nascone-Yoder,
2003; Stalsberg,
1969), in which only the primary heart fields were labeled.
Because the heart comprises cells derived from multiple locations within the
embryo (e.g. primary heart field, anterior/secondary heart field, neural
crest, etc), labeling only the primary heart fields cannot give a
comprehensive map. However, the strategy used in this study is sufficient to
trace left versus right side origins of all cardiomyocytes, regardless of
initial sources.
|
To determine whether cardiac LR cell lineage composition is regulated by
the LR body axis, dextran-labeled embryos were microinjected with RNA encoding
constitutively active ALK4 (CA-ALK4) as described
(Chen et al., 2004). ALK4 is a
type I serine-threonine kinase TGFß receptor that modulates LR axis
determination in Xenopus. As previously shown
(Chen et al., 2004), the
majority of embryos with right-side CA-ALK4 injection had body situs
defects (Table 1). Left-side
expression of CA-ALK4 caused only a minority of embryos to develop
body situs defects (Table 1),
consistent with its role in specifying left cell lineages
(Chen et al., 2004).
|
|
|
From the second group of embryos with heterotaxy (l-looped hearts), three hearts are shown in Fig. 5. The first heart contained an unseptated common atrium that comprised green cells in its left region and red cells in the right region (Fig. 5A). No difference in size between the left and right halves was evident. The left and right cardinal veins (Fig. 5B) and left and right aortic arches (not shown) showed normal lineage derivations. The myocardium of the AVC and ventricle comprised green and red halves; these two lineages appeared inverted because of the reversed loop of the heart (Fig. 5C,D). The OFT also comprised green and red halves (Fig. 5C,D). The second heart in this subgroup contained an unseptated common atrium that comprised green and red cell regions (not shown). The remaining regions appeared anatomically normal, except for the orientation of the heart loop, and no LR lineage anomalies were observed (Fig. 5E-I). By contrast, the third heart in this subgroup contained distinct left and right atria, separated by an interatrial septum; however, the anatomical positions of the atria were inverted and both atrial chambers and the interatrial septum comprised exclusively green cells (Fig. 5J). The cardinal veins (Fig. 5J) and aortic arches (Fig. 5L) in this heart had normal unilateral lineage compositions, and the myocardium of the AVC and the ventricle were normal in morphology and lineage composition (Fig. 5K and other sections not shown). However, the OFT region was shortened and incompletely looped; the proximal OFT comprised nearly exclusively green cells, with red cardiomyocytes present only in the distal-most region just adjacent to and including the aortic sac (Fig. 5L and other sections not shown). Endocardial cushion tissue formation appeared reduced in the OFT region (Fig. 5L) but not in the AVC.
|
Unlike ectopic right-side CA-ALK4 expression, left-side
CA-ALK4 expression does not cause body situs defects because this is
the side of the embryo on which ALK4 ordinarily functions
(Chen et al., 2004). As
expected, the majority of embryos exhibited normal body situs following
left-side CA-ALK4 expression
(Table 1). However, close
examination of hearts from situs solitus embryos in this group revealed that
almost all contained some combination of morphological and cell lineage
abnormalities (Table 2). One
heart in this group contained an unseptated common atrium that comprised
exclusively green cells (Fig.
7A). The AVC and ventricle comprised nearly all green cells except
for some red cells mingled with green cells in the ventricular trabeculae
(Fig. 7B,C). The OFT
predominantly comprised green cells in the proximal region (not shown), which
appeared shortened relative to the proximal OFT in normal hearts
(Fig. 7B,C). In the distal OFT
and the aortic sac, green and red cells, which did not mix across the midline
of the heart, were present (Fig.
7B,C). The OFT endocardial cushions were formed; however,
endocardial cushion tissue formation was detected only in the left-derived
region of the AVC region (i.e. only the `inferior' cushion was present)
(Fig. 7B). The left and right
cardinal veins and aortic arches had normal unilateral lineage compositions.
Another heart in this group contained two atria of similar size separated by
an interatrial septum; the atrial chambers and the interatrial septum
comprised green cells, with only a few red cells present in the right-side
wall of the right-side atrial chamber (Fig.
7D). The left and right cardinal veins (not shown) and aortic
arches (Fig. 7F) appeared
normal in this heart. The lineage composition of the AVC myocardium was normal
and cushion tissues were present in this region
(Fig. 7E-G). However, despite
normal morphological appearance of the ventricular myocardium, it comprised
primarily green cells in its left half and a mix of mostly green cells with
some red cells in its right half (Fig.
7E-G). The OFT and the aortic sac, like the AVC, comprised green
and red cells that did not cross the midline of the heart, and endocardial
cushion tissue was present (Fig.
7E-G). Yet another heart from this group contained an unseptated
common atrium and comprised green cells in its left region and a mixture of
green and red cells in its right side region
(Fig. 7H). The region of the
common atrium containing the green cells was larger than that containing the
red cells, suggesting an inversion of the rudimentary atrial chamber
components. A normal contribution of green and red cells to each half of the
AVC and OFT myocardium was present (Fig.
7I,J), but the ventricular myocardium comprised exclusively green
cells in its left half with a mixture of mostly green cells with some red
cells in its right half (Fig.
7I). Despite this abnormality in ventricular lineage composition,
no gross morphological defects could be discerned in this region. Yet another
heart in this group differed from all others in that it appeared normal in
every respect, including cell lineage compositions that were identical to
those observed in control hearts (not shown).
|
|
|
;
Ramsdell, 2005)
(n=10; Fig. 8A).
Consistent with previous work (Chen et al.,
2004) and with the mix of heterotaxy and situs inversus phenotypes
caused by right-side CA-ALK4 targeting, the location of
Pitx2c expression was bilateral (38%) or right-side only (54%)
(n=13; Fig. 8B,C). In
addition, the intensity and area of staining also were elevated compared with
controls. By contrast, the majority of embryos (85%) with left-side
CA-ALK4 targeting had Pitx2c expression exclusively on the
left side of the embryo (n=13;
Fig. 8D). However, as observed
for the right-side CA-ALK4-injected embryos, the intensity and extent
of staining were markedly increased, with a dorsal and/or posterior expansion
of staining (Fig. 8D). This
subtle alteration in the amount and extent of Pitx2c expression in
CA-ALK4-injected embryos was not appreciated in our previous study
and suggests that the duration and/or dose, in addition to asymmetry, of ALK4
signaling are important for normal LR development.
|
|
). Similar to
hearts from heterotaxic embryos caused by CA-ALK4 expression, no two
hearts obtained from embryos in the DN-ALK4-induced heterotaxy group
were exactly alike. The atrium was the most frequently affected region, with
abnormal lineage composition present in five out of six embryos examined. One
embryo from this group is shown in which the left and right atria were
inverted (Fig. 9A). The
morphological left atrium and the interatrial septum comprised a mixture of
red and green cells, and the morphological right atrium comprised green cells
only (Fig. 9A). The left and
right cardinal veins comprised correct unilateral lineages, and the AVC
comprised green and red lineages that segregated with respect to the cardiac
midline (Fig. 9A,B). The
ventricle also comprised green and red lineages; however, the two lineages
mixed across the cardiac midline and abnormal trabeculation was present
(Fig. 9C). The OFT of this
heart also showed mixing of green and red cells across the cardiac midline
(Fig. 9C), although the two
lineages remained segregated at the level of the aortic sac (not shown). No
abnormalities were noted for the aortic arches. Defects in left-right myocyte
lineage allocation and/or cardiac anatomy present in other heterotaxic embryos
in this group are summarized in Table
3.
|
|
DISCUSSION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Ramsdell,
2005). Our results demonstrate that allocation of LR cell lineages
to the heart is an important target of LR axial patterning. When LR axis
determination is compromised, as occurred in the heterotaxic and situs
inversus embryos generated in this study, LR cell lineage composition of the
heart is always abnormal. Moreover, even subtle perturbations of the LR
developmental pathway can cause LR cell lineage abnormalities, as occurred in
many of the experimental embryos that exhibited situs solitus following
activated or attenuated ALK4 signaling. Close examination of the cardiac
anatomy of the laterality defective and experimental situs solitus embryos
showed that cardiac LR lineage defects almost always are associated with the
presence of CHDs in the affected region(s), suggesting that proper allocation
of LR cell lineages is an important step in normal heart morphogenesis. Despite the symmetric appearance of the AVC, ventricle, OFT and the paired cardinal veins and aortic arches, these regions exhibit distinct LR differences in cell lineage compositions. Regions of the heart that are morphologically asymmetric, such as the left and right atria, also exhibit differences in LR lineage compositions. Thus, with respect to the anteroposterior axis of the heart, each region exhibits a particular cellular laterality, with some regions comprised unilateral lineages and others comprised dual lineages (summarized in Fig. 10).
To determine whether allocation of cardiac cell lineages is regulated by
the LR body axis, LR lineage composition was compared between hearts from
control embryos and hearts from laterality defective embryos. Hearts from all
laterality defective embryos induced by misregulated ALK4 signaling contained
LR cell lineage defects that were preceded by altered Pitx2c
expression in the lateral plate mesoderm. Pitx2c is a
well-characterized laterality gene that is a crucial downstream target of
symmetry-breaking events during vertebrate LR axis determination (reviewed by
Levin, 2005;
Ramsdell, 2005).
Pitx2c ordinarily is expressed in the left, but not right, lateral
plate mesoderm just prior to cardiogenesis. The asymmetric expression pattern
of Pitx2c was inverted in situs inversus embryos, and in all but one
situs inversus embryo, hearts had completely inverted LR lineage composition
that corresponded with mirror-image reversed cardiac anatomy. In heterotaxic
embryos, Pitx2c expression was typically inverted or bilateral,
reflecting the heterogeneity of LR phenotypes that is characteristic of this
condition. With respect to morphology and cell lineage composition, no two
hearts from heterotaxic embryos had exactly the same phenotype, indicating
that some regions of the heart were mis-specified for cellular laterality
without other regions being affected. This suggests not only that each region
of the heart can become independently LR specified, but also that, in
instances of heterotaxy, specification of the different cardiac regions is
random, as occurs on the gross anatomical level when organs develop
asymmetries that are stochastic in their LR orientation. One important
implication of this finding is that the pleiotropic CHDs typically found in
individuals with heterotaxy (especially those with the same genetic etiology)
could be attributed to developmental errors that cause discordance in this LR
specification process.
Abnormal LR cardiomyocyte compositions were frequently associated with
CHDs, which suggests that the ability of different regions of the heart to
become differentiated into particular structures is linked to cardiac cell
lineage. For example, the left and right atria derive from a common
progenitor, the common atrium, which must become divided into two chambers
with distinct LR differences (Anderson,
1992; Markwald et al.,
1998; Min et al.,
2000). LR labeling of the primary heart fields previously
suggested that this division is related to the LR origin of cells that
contribute to this region (Gormley and
Nascone-Yoder, 2003). Our results not only confirm this
interpretation by showing that each atrium is derived from a unilateral
lineage, but also provide direct evidence that atrial chamber formation rarely
proceeds normally if cell lineage composition in the common atrium is altered.
The two morphological defects that most often accompanied lineage defects in
this region were atrial inversion, which was most commonly seen in situs
inversus hearts, and atrial chamber isomerism, which was most commonly seen in
hearts from heterotaxic embryos.
Studies of the interatrial septum suggest that its formation also is
related to differences in the ability of left versus right-side-derived cells
to become differentiated. Myocardial cells of the interatrial septum (IAS)
share common gene expression with cells in the left but not right atrial wall,
suggesting that the IAS and left atrial chamber both form from a cell
population specified for `leftness'
(Franco and Campione, 2003;
Liu et al., 2002;
Wessels et al., 2000). In
support of this possibility, these two cardiac components ordinarily comprised
exclusively left-derived cells and the cell lineage compositions of the
morphological left atrium and interatrial septum are concordantly reversed in
situs inversus hearts (i.e. they are both right-side derived). Moreover, in
instances where left-side signaling is impaired (i.e. some of the heterotaxic
embryos), the IAS did not form. The latter experimental result correlates with
clinical observations that the IAS is oftentimes either deficient or
completely absent in hearts of individuals with heterotaxy (reviewed by
Bowers et al., 1996;
Bartram et al., 2005).
Other aspects of cardiac morphogenesis also may be dependent upon proper
allocation of LR cardiomyocyte lineages. When LR cardiomyocyte lineage
abnormalities were present in the AVC, endocardial cushion tissue formation
frequently was absent or greatly diminished. Because endocardial cushions
ultimately become remodeled into valve and septal tissues, deficiencies in
endocardial cushion tissue formation are a leading cause of valvuloseptal
defects (reviewed by Person et al.,
2005). A previous study of the endocardial cushions in the AVC of
the chick embryo demonstrated that consistent with their initial left- and
right-side origins, the inferior and superior cushions exhibit distinct
properties throughout septation morphogenesis, including differences in
proliferative rates, spatial distribution and amount of endocardial tissue
formed (Moreno-Rodriguez et al.,
1997). As the AVC myocardium is the primary source of signals that
induce endocardial cushion tissue formation (reviewed by
Person et al., 2005), it is
possible that LR cardiomyocyte composition in the AVC is causatively related
to the asymmetries reported for inferior versus superior cushion formation and
differentiation.
|
|
). As was found in
hearts with defective AVC cushion formation, correlation between abnormal LR
cell lineage composition and absent/reduced cushion tissue also was present in
the OFT region of several hearts from heterotaxic embryos. In some (but not
all) of these hearts, the cell lineage aberrancies were correlated with
formation of a shortened, abnormally looped OFT. Double-outlet right
ventricle, transposition of the great arteries and ventricular septal defects
are types of CHDs that can result from errors in length and/or rotation of the
OFT during its formation (reviewed by
Hutson and Kirby, 2003).
Relevant to these findings, Pitx2 loss of function in
Xenopus causes anomalous shifting of the OFT during looping
morphogenesis (Dagle et al.,
2003), suggesting that normal formation and positioning of the OFT
is indeed affected by LR patterning events during vertebrate
cardiogenesis.
Other regions of the heart that were affected in the laterality defective
embryos included the ventricle, aortic sac and aortic arches. Disorganized
and/or decreased trabeculation were present in the ventricles of some hearts
that had either unilateral (left-side) lineage composition or abnormally
`mixed' left and right lineages that each were distributed on both sides of
the midline of the heart. However, in other hearts, abnormal LR lineage
composition was present in the ventricle, but the size and shape of the
ventricle as well as ventricular trabeculation appeared grossly normal. In
these hearts, as well as the few others showing lineage defects without
obvious malformations, it is possible that subtle abnormalities were present
but not readily detectable by histological examination (e.g. conduction or
other physiological defects). Alternatively, it is possible that the cell
lineage defects were not sufficiently abnormal to cause detrimental
development. In addition to the ventricle, the aortic sac and the aortic
arches were two other regions in which lineage defects were observed in a
small minority of laterality defective embryos. Again, obvious morphological
defects were not present in these cases. It is possible that either the
abnormalities in cell lineage composition were too minor to cause significant
structural defects, or alternatively, that with continued development, CHDs
reflective of dysmorphogenesis in these regions (e.g. aortic arch anomalies)
might have been found. Because dextran lineage markers lose stability after
approximately stage 46 due to lysosomal degradation
(Sive et al., 2000), it was
not possible to distinguish between these possibilities.
The other type of embryos that were examined for cardiac lineage
composition were experimental embryos that failed to develop heterotaxy or
situs inversus phenotypes in response to misregulated ALK4 signaling. Much to
our surprise, the majority of embryos in this group exhibited LR cardiac
lineage defects, despite a situs solitus phenotype. LR lineage defects were
prevalent in the atrial regions, and were associated with abnormal atrial
chamber development and absent interatrial septation. In addition, the
ventricles of many of these embryos were altered in LR cardiomyocyte lineage
composition, although obvious morphological defects were not found in this
region for all hearts examined. Two hearts additionally showed shortened OFTs
that corresponded with unilateral or mixed lineage compositions in the
proximal regions. The variable defects in LR cell lineage composition were
preceded by increased Pitx2c expression in the lateral plate
mesoderm, suggesting that dosage and/or duration of laterality gene expression
may be just as important as unilateral restriction of expression in modulating
LR development. Consistent with this interpretation, an increased
susceptibility of the atria to altered Pitx2 expression has been
noted in Pitx2-null mice in which hypomorphic alleles can rescue all
defects typical of the knockout with the exception of right atrial isomerism,
which requires a higher dose of Pitx2c expression/activity
(Liu et al., 2001). These
findings, coupled with results obtained in our experimental situs solitus
embryos, indicate that subtle laterality defects can occur in embryos with
otherwise normal body situs. This suggests that some incidences of seemingly
isolated CHDs, particularly those that occur in individuals whose families are
afflicted with some form of heritable laterality disease
(Morelli et al., 2001), may in
fact represent less obvious manifestations of a LR asymmetry defect. Although
important details of the molecular pathway linking LR lineage allocation,
Pitx2c expression and cardiogenesis remain to be defined, the ability
of the LR axis to regulate cell lineage allocation reveals a fundamental and
previously unappreciated mechanism by which organ laterality becomes patterned
during vertebrate embryogenesis.
|
ACKNOWLEDGMENTS |
|---|
|
REFERENCES |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Anderson, R. H. (1992). Understanding the nature of congenital division of the atrial chambers. Br. Heart J. 68,1 -3.
Bartram, U., Wirbelauer, J. and Speer, C. P. (2005). Heterotaxy syndrome – asplenia and polysplenia as indicators of visceral malposition and complex congenital heart disease. Biol. Neonate 88,278 -290.[CrossRef][Medline]
Bowers, P. N., Brueckner, M. and Yost, H. J. (1996). Laterality disturbances. Prog. Pediatr. Cardiol. 6,53 -62.
Brand, T. (2003). Heart development: molecular insights into cardiac specification and early morphogenesis. Dev. Biol. 258,1 -19.[CrossRef][Medline]
Burn, J. (1991). Disturbance of morphological laterality in humans. In Ciba Foundation Symposium, Vol.162 (ed. G. R. Bock and J. Marsh), pp.282 -296. Chichester: John Wiley & Sons Ltd.[Medline]
Chang, C., Wilson, P. A., Mathews, L. S. and Hemmati-Brivanlou, A. (1997). A Xenopus type I activin receptor mediates mesodermal but not neural specification during embryogenesis. Development 124,827 -837.[Abstract]
Chen, Y., Mironova, E., Whitaker, L. L., Edwards, L., Yost, H. J. and Ramsdell, A. F. (2004). ALK4 functions as a receptor for multiple TGF beta-related ligands to regulate left-right axis determination and mesoderm induction in Xenopus. Dev. Biol. 268,280 -294.[CrossRef][Medline]
Dagle, J. M., Sabel, J. L., Littig, J. L., Sutherland, L. B., Kolker, S. J. and Weeks, D. L. (2003). Pitx2c attenuation results in cardiac defects and abnormalities of intestinal orientation in developing Xenopus laevis. Dev. Biol. 262,268 -281.[CrossRef][Medline]
Delot, E. C. (2003). Control of endocardial cushion and cardiac valve maturation by BMP signaling pathways. Mol. Genet. Metab. 80,27 .[CrossRef][Medline]
Eisenberg, L. M. and Markwald, R. R. (2004). Cellular recruitment and the development of the myocardium. Dev. Biol. 274,225 -232.[CrossRef][Medline]
Ferencz, C., Rubin, J. D., McCarter, R. J., Brenner, J. I.,
Neill, C. A., Perry, L. W., Hepner, S. I. and Downing, J. W.
(1985). Congenital heart disease: prevalence at livebirth. The
Baltimore-Washington infant study. Am. J. Epidemiol.
121, 31-36.
Franco, D. and Campione, M. (2003). The role of Pitx2 during cardiac development. Linking left-right signaling and congenital heart diseases. Trends Cardiovasc. Med. 13,157 -163.[CrossRef][Medline]
Gebbia, M., Ferrero, G. B., Pilia, G., Bassi, M. T., Aylsworth, A., Penman-Splitt, M., Bird, L. M., Bamforth, J. S., Burn, J., Schlessinger, D. et al. (1997). X-linked situs abnormalities result from mutations in ZIC3. Nat. Genet. 17,305 -308.[CrossRef][Medline]
Gormley, J. P. and Nascone-Yoder, N. M. (2003). Left and right contributions to the Xenopus heart: implications for asymmetric morphogenesis. Dev. Genes Evol. 213,390 -398.[CrossRef][Medline]
Hutson, M. R. and Kirby, M. L. (2003). Neural crest and cardiovascular development: a 20-year perspective. Birth Defects Res. C Embryo Today 69,2 -13.[CrossRef][Medline]
Kao, K. R. and Elinson, R. P. (1988). The entire mesodermal mantle behaves as Spemann's organizer in dorsoanterior enhanced Xenopus laevis embryos. Dev. Biol. 127, 64-77.[CrossRef][Medline]
Levin, M. (2005). Left-right asymmetry in embryonic development: a comprehensive review. Mech. Dev. 122,3 -25.[CrossRef][Medline]
Liu, C., Liu, W., Lu, M. F., Brown, N. A. and Martin, J. F.
(2001). Regulation of left-right asymmetry by thresholds of
Pitx2c activity. Development
128,2039
-2048.
Liu, C., Liu, W., Palie, J., Lu, M. F., Brown, N. A. and Martin,
J. F. (2002). Pitx2c patterns anterior myocardium and aortic
arch vessels and is required for local cell movement into atrioventricular
cushions. Development
129,5081
-5091.
Manner, J. (2000). Cardiac looping in the chick embryo: a morphological review with special reference to terminological and biomechanical aspects of the looping process. Anat. Rec. 259,248 -262.[CrossRef][Medline]
Markwald, R. R., Trusk, T. and Moreno-Rodriguez, R. (1998). Formation and septation of the tubular heart: integrating the dynamics of morphology with emerging molecular concepts. In Living Morphogenesis of the Heart (ed. M. V. De la Cruz and R. R. Markwald). Heidelberg: Springer-Verlag.
Min, J. Y., Kim, C. Y., Oh, M. H., Chun, Y. K., Suh, Y. L., Kang, I. S., Lee, H. J. and Seo, J. W. (2000). Arrangement of the systemic and pulmonary venous components of the atrial chambers in hearts with isomeric atrial appendages. Cardiol. Young 10,396 -404.[Medline]
Morelli, S. H., Young, L., Reid, B., Ruttenberg, H. and Bamshad, M. J. (2001). Clinical analysis of families with heart, midline, and laterality defects. Am. J. Med. Genet. 101,388 -392.[CrossRef][Medline]
Moreno-Rodriguez, R. A., De la Cruz, M. V. and Krug, E. L. (1997). Temporal and spatial asymmetries in the initial distribution of mesenchyme cells in the atrioventricular canal cushons of the developing chick heart. Anat. Rec. 248, 84-92.[CrossRef][Medline]
Nieuwkoop, P. D. and Faber, J. (1967).Normal Table of Xenopus laevis (Daudin) . Amsterdam: North-Holland Publishing.
Nugent, E. W., Plauth, W. H., Edwards, J. E. and Williams, W. H. (1994). The pathology, pathophysiology, recognition and treatment of congenital heart disease. In Hurst's The Heart (ed. R. C. Schlant and R. Alexander), pp.1773 -1777. New York: McGraw-Hill.
Person, A. D., Klewer, S. E. and Runyan, R. B. (2005). Cell biology of cardiac cushion development. Int. Rev. Cytol. 243,287 -335.[CrossRef][Medline]
Ramsdell, A. F. (2005). Left-right asymmetry and congenital cardiac defects: getting to the heart of the matter in vertebrate left-right axis determination. Dev. Biol. 288, 1-20.[CrossRef][Medline]
Ramsdell, A. F. and Yost, H. J. (1999). Cardiac looping and the left-right axis: antagonism of left-sided Vg1 activity by a right-sided ALK2-dependent BMP pathway. Development 126,5195 -5205.[Abstract]
Ramsdell, A. F., Bernanke, J. M., Johnson, J. and Trusk, T. C. (2005). Left-right lineage analysis of AV cushion tissue in normal and laterality defective Xenopus hearts. Anat. Rec. 287,1176 -1182.[CrossRef]
Sive, H. L., Grainger, R. M. and Harland, R. M. (2000). Early Development of Xenopus laevis. A Laboratory Manual. New York: Cold Spring Harbor Laboratory Press.
Stalsberg, H. (1969). The origin of heart asymmetry: right and left contributions to the early chick embryo heart. Dev. Biol. 19,109 -127.[CrossRef][Medline]
Sternick, E. B., Marcio Gerken, L., Max, R. and Osvaldo Vrandecic, M. (2004). Radiofrequency catheter ablation of an accessory pathway in a patient with Wolff-Parkinson-White and Kartagener's Syndrome. PACE 27,401 -404.
Wessels, A., Anderson, R. H., Markwald, R. R., Webb, S., Brown, N. A., Viragh, S., Moorman, A. F. and Lamers, W. H. (2000). Atrial development in the human heart: an immunohistochemical study with emphasis on the role of mesenchymal tissues. Anat. Rec. 259,288 -300.[CrossRef][Medline]
Related articles in Development:
This article has been cited by other articles:
|
|
 |
|
  E. N. Hilton, F. D.C. Manson, J. E. Urquhart, J. J. Johnston, A. M. Slavotinek, P. Hedera, E.-L. Stattin, A. Nordgren, L. G. Biesecker, and G. C.M. Black Left-sided embryonic expression of the BCL-6 corepressor, BCOR, is required for vertebrate laterality determination Hum. Mol. Genet., July 15, 2007; 16(14): 1773 - 1782. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||
