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First published online April 25, 2008
doi: 10.1242/10.1242/dev.016147
1 Department of Cell Biology,, Duke University Medical Center, Durham, NC,
USA.
2 Department of Pediatrics, Duke University Medical Center, Durham, NC,
USA.
3 Center for In Vivo Microscopy, Duke University Medical Center, Durham, NC,
USA.
Author for correspondence (e-mail:
kling{at}cellbio.duke.edu)
Accepted 19 March 2008
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Sonic hedgehog, Atrioventricular septal defect, Intracardiac septation, Heart development, Morphogenesis, Mouse
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). AV
septation defects (AVSDs) occur in over 1 in 2800 live births, with highly
variable presentation (Hoffman,
1995; Hoffman and Kaplan,
2002). The three defects most common to AVSD are atrial septum
primum defect, membranous ventricular septum defect, and abnormal or missing
AV valve septal leaflets. The combined effect is juncture among all four
chambers of the heart, resulting in mixing of freshly oxygenated and
oxygen-depleted blood.
Our understanding of the process of AV septation comes in part from study
of the mouse embryo. At embryonic day (E) 9.0, the interface between the atria
and ventricles becomes specified as the AV canal, and formation of endocardial
cushions begins. Two AV cushions form as a result of epithelial to mesenchymal
transition and subsequent proliferation. The cushions themselves undergo
extensive remodeling to contribute to the septal leaflets of the mitral and
tricuspid valves, as well as to a large region of the membranous ventricular
septum. In addition to AV cushions, the AV septal complex also includes
outflow tract (OFT) endocardial cushions, the muscular ventricular septum and
components of the atrial septa (reviewed by
Lamers and Moorman, 2002).
The complicated process of atrial septation and its impact on AV septation
is an emergent body of research. Recent work has focused on the extracardiac
contribution to atrial septation by the dorsal mesocardium (DM), the region of
the splanchnic mesoderm ventral to the endodermal tube and dorsal to the heart
that maintains contact with the venous pole of the heart tube throughout
development. Starting at E10.5, mesenchymal cells from the DM enter the atria
at the site of pulmonary vein formation
(Webb et al., 1998). The
primary atrial septum, a myocardial extension of the dorsal atrial wall that
becomes capped by mesenchyme, is already present. Webb et al.
(Webb et al., 1998)
re-examined via histology the contribution of DM to an atrial structure
identified originally by Wilhelm His as the spina vestibuli
(His, 1880), and suggested
that this extracardiac tissue has a significant contribution to atrial
septation. Wessels and colleagues found a similar `dorsal mesenchymal
protrusion' (DMP) in human embryos, composed of mesenchyme entering the heart
between the pulmonary ridges at the site of future pulmonary vein formation
(Wessels et al., 2000). These
authors suggested that this tissue contributes to the mesenchyme capping the
primary atrial septum, and helps to close the primary atrial foramen by
filling the space between the primary atrial septum and AV cushions. Studies
of transgenic markers of gene expression in mesenchymal cells have provided
further support for a discrete contribution from the DM to the septal complex
(Mommersteeg et al., 2006;
Snarr et al., 2007a). Here we
refer to this structure descriptively as the DMP.
The identification of the DMP as a source of cells contributing to atrial septation raises many questions. For example, what is the structural contribution of the DMP to atrial septation, and more broadly to AV septation? Does the DMP functionally interact with other components of the AV complex? What genetic pathways are important for DMP development? Can defective DMP development result in AVSD? To address these questions, we use a multi-technique approach. We report a genetic marker for the DM, and use novel imaging techniques to address its morphogenesis and ultimate contribution to AV septation. We then use this knowledge to address the functional relevance of the DMP to the pathogenesis of an uncharacterized mouse model of AVSD, demonstrating a crucial role for hedgehog signaling in this process.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Descriptions of remaining mouse lines used here can be found in Goddeeris et
al. (Goddeeris et al., 2007).
Noon of the day of vaginal plug detection was defined as embryonic day (E)
0.5. Embryos were genotyped for Cre
(Meyers et al., 1998) and for
the Smo wild-type and flox alleles
(Zhang et al., 2001). In all
figures, wild-type (WT) refers to littermates that are either Cre(+) and
flox/+, or flox/flox but Cre(-), unless otherwise specified.
Immunohistochemistry and β-galactosidase detection
Histological preparation of embryos followed standard procedures
(Goddeeris et al., 2007;
Hogan et al., 1994). Section
immunohistochemistry was conducted on sagittal paraffin sections (8 µm)
incubated with rat anti-PECAM-1 (1:250; PharMingen, CN557355) or rabbit
anti-phosphorylated histone H3 antibody (1:250; Upstate Biotechnology).
Anti-phosphorylated histone H3 signal was amplified with biotinylated
anti-rabbit (Vector Labs). Detection was with Cy3 conjugated streptavidin
(Vector Labs, 1:250) and Cy5 conjugated anti-rat (Jackson Immuno, 1:250).
Nucleus detection was with Syto13 (Invitrogen, 1:1000) in PBT. Undiluted MF-20
supernatant (Developmental Studies Hybridoma Bank) was used with
methanol-fixed embryos and detected with rhodamine red-conjugated goat
anti-mouse IgG, Fc
subclass 2b specific secondary (Jackson
Immunoresearch) followed by paraffin sectioning and nuclei detection with
Syto13. Lysotracker Red cell death analysis was as described
(Abu-Issa et al., 2002;
Zucker et al., 1999).
Whole-mount in situ hybridization with digoxigenin-labeled riboprobes was as
described (Neubuser et al.,
1997). The Shh riboprobe has been previously reported
(Echelard et al., 1993).
Confocal microscopy was performed on a Zeiss LSM 510 META. Images were prepared in Adobe Photoshop 7.0.1. Control and mutant images were treated identically and represent the data set as a whole. Manual mesenchyme cell counts were analyzed by Student's t-test. Volume was measured using ImageJ Freeware (1.37v, NIH).
Transwell explant cultures
Dorsal mesocardium was dissected from E10.5 ICR embryos and cultured on rat
type 1 collagen for 24 hours at 37°C in DMEM with 10% fetal calf serum
(FCS). Pooled explants were dissociated with trypsin, washed and resuspended
in 500 µL OPTI-MEM/1% FCS. 15,000 cells in 100 µL OPTI-MEM/1% FCS were
added to each transwell insert (Costar 3422) coated with type 1 collagen and
placed in a 24-well plate. Each bottom well was filled with 600 µL DMEM/10%
FCS. In some wells, 10 µm cyclopamine in DMSO was added. Control cells were
treated with DMSO, or cultured in media alone. Cells were incubated at
37°C for 48 hours. Cells on the underside of the transwell insert membrane
were fixed and stained with Gills #1 Hematoxylin and Eosin.
MRM
Embryos were imaged at E14. Contrast procedures and imaging were as
described (Petiet et al.,
2007), using a vertical bore 9.4T magnet interfaced to a GE EXCITE
console, modified for MRM through an intermediate stage in the radiofrequency
(rf) chain. Images were acquired using 3D rf refocused spin warp encoding with
extended dynamic range (Johnson et al.,
2007) yielding 3D image arrays of 512x512x1024 with
isotropic spatial resolution of 19.5 µm3. Acquisition time was 4
hours. Hearts were color-labeled and segmented using VoxPort Software by
MRPath (Durham, NC) and a Matlab routine. Volume-rendered images were
generated using VGStudio Max by Volume Graphics GmbH (Germany).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The AHF includes a splanchnic mesodermal region that
contributes to the arterial pole of the primary heart tube to form much of the
OFT and right ventricle (Kelly et al.,
2001; Mjaatvedt et al.,
2001; Waldo et al.,
2001). Intriguingly, expression of AHF markers also has been
described in or near the developing atria, supporting the view that this
domain contributes to both the arterial and venous poles of the primary heart
tube (Cai et al., 2003;
Huynh et al., 2007;
Kelly et al., 2001;
Mommersteeg et al., 2006;
Snarr et al., 2007b). In the
course of our previous studies, we noted similar atrial expression with the
Cre transgenic line Mef2C-AHF-Cre
(Verzi et al., 2005) using the
Cre recombination reporter R26R
(Soriano, 1999), which
indicates where recombination has occurred via β-galactosidase
(β-gal) expression. The atrial expression pattern includes regions of the
atrial septum thought to be of extracardiac mesenchymal origin, specifically
the DMP. As little is understood about the nature of this tissue, we sought to
address its origin and contributions to atrial septation using
Mef2C-AHF-Cre as a genetic tool. We crossed Mef2C-AHF-Cre with R26R in a series of pseudo-lineage trace experiments to determine the extent to which this cell population contributes to AV septation in later stages of heart development. By E10.5, this Cre line leads to β-gal expression in the splanchnic mesoderm, which is continuous with the DM (Fig. 1A,B). Mef2C-AHF-Cre was also observed in the medial dorsal myocardial wall of the atria continuous with the splanchnic mesoderm. By E11.5, R26R expressing mesenchymal cells continuous with the DM are seen to protrude into the atria at the future point of pulmonary vein attachment. This DMP expression domain extends to the inferior AV cushion, and comprises part of the mesenchymal cap that extends to the superior AV cushion (Fig. 1C). This pattern indicates that Mef2C-AHF-Cre is either expressed in the DMP and mesenchymal cap, or that these tissues are derived from tissue that once expressed Cre, such as the DM.
In embryos from E12.5-E16.5, cells derived from the Mef2C-AHF-Cre expression domain constitute much of the tissue continuous with the AV cushion-derived central mesenchymal mass, as well as much of the primary atrial septum (Fig. 1D-F). Cre expression is specific to this midline tissue and largely excludes AV cushion-derived mesenchyme. At E14.5, labeled cells were observed continuous with the primary atrial septum, forming a wedge over the fused AV cushion-derived tissue. By this stage, the AV cushion tissue has redistributed to form the septal leaflets of the mitral and tricuspid valves. In summary, histological analysis suggests that the primary atrial septum is partially derived from Mef2C-AHF-Cre-positive cells.
Using magnetic resonance microscopy (MRM) of E14.5 embryos in comparison to
Mef2C-AHF-Cre; R26R embryos, we generated three-dimensional (3D)
renderings of the Mef2C-AHF-Cre lineage contribution to the atrial
septum. Heart structures were color-labeled from the MRM slices according to
the pattern observed in closely matched Mef2C-AHF-Cre; R26R sections
(Fig. 1G). These heart
structures were isolated and recompiled to generate a 3D structure
(Petiet et al., 2007).
Multiple views suggest that these labeled cells contribute much of the atrial
septal complex and present a large interface with the remodeling AV cushions
(Fig. 1H). Near the dorsal
atrial wall, cells derived from the Mef2C-AHF-Cre domain occupy a
greater volume than the remaining component of the primary atrial septum.
Together, these data confirm that the DMP is derived from the DM, and show
that the DMP in turn gives rise to much of the tissue mass in the atrial
septal complex.
Hh responsive DMP is deficient in Shh-/- mutant embryos
As the pathogenic mechanisms of many AVSDs are unclear, we wished to
determine whether abnormal DMP development could be a causal defect. Besides
OFT phenotypes, Shh-/- mutant embryos have ventricular and
atrial septal defects (Washington Smoak et
al., 2005), as well as incompletely-penetrant AVSD with a single
AV valve (Fig. 2A,B). Most
Shh-/- mutants have rudimentary septal AV valve leaflets,
though some lack even these. The causes of the large atrial septation defect
observed in Shh-/- mutants have not been addressed, but
the phenotype is consistent with the absence of tissue from the AV septal
complex. Recent data indicate that the ventral pharyngeal endoderm (PE) is the
source of Shh required for normal OFT development
(Goddeeris et al., 2007), but
there are no overt domains of Shh expression within the heart to
explain a role in AV septation. We therefore investigated this apparent
contradiction.
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).
However, the ventral PE expresses Shh and is juxtaposed to the dorsal
mesocardium during DMP formation (Fig.
2D). Consistent with this observation, we have previously
demonstrated that loss of Shh from the PE results in AVSD
(Goddeeris et al., 2007). The
lack of overt Shh expression within the heart itself and the presence
of AVSD in embryos lacking endodermal Shh led us to hypothesize that
Shh produced in the PE acts on the DM, which in turn forms the DMP and
contributes to atrial septation.
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).
Ptch1lacZ activity appears to be restricted to the
developing atria and distal OFT myocardium
(Fig. 2E,F)
(Washington Smoak et al.,
2005). Atrial expression is first observed at E10.5 in the DM near
the pulmonary pit (data not shown). At later stages (E11.5-E13.5), weak
expression is observed in the DMP within the atria
(Fig. 2E,F). This
Ptch1 domain is Shh dependent, as Shh-/-
embryos lack Ptch1lacZ expression within the atria
(Fig. 2G,H; see Fig. S1 in the
supplementary material). Thus, the DMP is the only component of the AV
septation complex with detectable, Shh-dependent,
Ptch1lacZ expression.
Early development of the AV cushions does not require Shh signaling
Classical models for the etiology of AVSD have focused on early
abnormalities in AV cushion development. To see whether these pertain to
Shh-/- mutants, we conducted histological analysis at
early stages in AV formation (E9.5-E11.5), a hallmark of which is epithelial
to mesenchymal transition (EMT) of endocardial cells to populate the cardiac
jelly.
The AV cushions appeared normal in size and morphology at E9.5. Appropriate numbers of mesenchymal cells were present in the cushion cardiac jelly (data not shown), suggesting that AV cushion specification and the initial events of EMT proceed normally in the AV canal of Shh-/- embryos. To assess the subsequent extent of EMT, we performed section immunohistochemistry on Shh-/- mutants and wild-type littermates at E10.5 and E11.5 (Fig. 3A,B). Cushion mesenchymal cells were assessed by location and lack of the endothelial marker PECAM1, and counted in serial sections through a given embryo. No significant difference in total cell number was found in either cushion at E10.5 (Fig. 3C) or E11.5 (data not shown). Cell proliferation was assayed by immunodetection of phosphorylated histone H3. No difference was observed in E10.5 AV cushion proliferation (data not shown) or total AV cushion volume (Fig. 3D) between wild type and Shh-/- mutants. Together, these data indicate early stages of AV cushion formation and growth are normal despite loss of Shh.
By E14.5, AV cushion structure is abnormal in Shh mutants (Fig. 2B); thus, it is possible that disrupted Hh signaling during later stages of AV cushion development might play an important role in generating AVSD. On the other hand, our phenotypic analysis, combined with the observation that the DM and DMP are Shh responsive, suggests that loss of the DMP may be part of the etiology of Shh-/- AVSD. Such considerations suggested three hypotheses: (1) that defective Hh signaling in the AV cushions causes defects not only in this tissue but in AV septation in general; (2) loss of Hh signaling in the developing DMP alone is sufficient to recapitulate the full AVSD; or (3) defective signaling in both DMP and AV cushion development lead to the Shh-/- AVSD phenotype. To distinguish between these possibilities, we used tissue-specific gene ablation to disrupt Hh signaling.
Neither endocardium nor myocardium requires direct Hh signaling for AV septation
To test whether direct Hh signaling to the developing AV cushions is
required for normal AV septation, we ablated the Smo receptor, and
thus Hh receptiveness, from the two primary tissues of the AV cushions: the
myocardium, the endocardium, or both, in combination, then analyzed mutant
embryos for AVSD. To test necessity of Hh signaling in the myocardium, we
generated mutants of the genotype TnT-Cre; Smoflox/-.
TnT-Cre is expressed throughout the differentiated myocardium of the
heart but not in the endocardium (Jiao et
al., 2003) (Fig.
4A,A'). All mutants sectioned (7/7) had normal AV septation
(Fig. 4B). Thus, loss of Hh
signaling to the myocardium fails to recapitulate the
Shh-/- AVSD phenotype. Interestingly, R26R
analysis of TnT-Cre expression at E11.5 and later stages revealed
TnT-Cre expression at the leading edge of the DMP (data not shown),
consistent with observations that this mesenchyme becomes myocardial after
entry into the atria (Soufan et al.,
2004). As TnT-Cre; Smoflox/- mutants have no
atrial septation defects, we conclude that Hh signaling to the DMP is not
required after differentiation into myocardium.
We next tested whether the endocardium requires a direct Hh signal. We
ablated Smo from the endocardium using Tie2-Cre
(Fig. 4C,C')
(Koni et al., 2001). As the AV
cushion mesenchyme is derived from the endocardium, this tissue also lacks
Smo. Histological analysis at late stages revealed no intracardiac
defects in these mutants (9/9, Fig.
4D), indicating that the endocardium and its derivatives do not
require Hh signaling for normal AV septation.
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Mef2C-AHF-Cre; Smoflox/- mutants recapitulate Shh-/- intracardiac septation defects
To test whether Hh signaling is required within the DM for heart septation,
we generated Mef2C-AHF-Cre; Smoflox/- embryos.
This strategy removed the ability of the DM to respond to Shh from the
pharyngeal endoderm. These embryos survive to term with a failure in OFT
septation (Goddeeris et al.,
2007). In addition to this phenotype, section analysis of mutant
and wild-type embryos between E14.5 and E18.5 revealed intracardiac defects
consistent with those found in Shh-/- mutants
(Fig. 5A,B). All
Mef2C-AHF-Cre; Smoflox/- mutants observed
(15/15) had a large atrial septation defect as well as a pronounced VSD at the
level of the OFT-ventricle connection. The atrial septation defect is
consistent with loss of the DMP (Fig.
5A-D). Typically, the muscular primary atrial septum was observed
along the dorsal-most atrial wall (data not shown). In addition to atrial
septation defects, most Mef2C-AHF-Cre;
Smoflox/- mutants (11/15) have abnormally short,
rounded septal AV valve leaflets. In most cases, the central mass of
mesenchyme affixed to the muscular ventricular septum appeared much larger and
more rounded than in control embryos (Fig.
5B,D). This mass is probably comprised entirely of AV
cushion-derived tissue, as it is not β-gal positive in Mef2C-AHF-Cre;
Smoflox/-; R26R embryos
(Fig. 5B). The most severe
forms of AVSD found at low penetrance in Shh-/- mutants,
i.e. lack of all septal leaflets of the AV valves and the central mesenchymal
mass, were not observed in Mef2C-AHF-Cre,
Smoflox/- mutants. These results indicate that the
DM requires Hh signaling for intracardiac septation and that loss of
receptiveness within this domain can largely recapitulate the AV septation
defects observed in Shh-/- embryos.
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Taken together, our tissue-specific ablations indicate that the DMP is essential for intracardiac septation. Hh signaling is necessary within the DM for DMP morphogenesis and contribution to the septal complex. Moreover, absence of the DMP contribution has an indirect effect on the morphology of AV cushions.
Loss of Hh signaling results in abnormal DM differentiation and migration
To understand the early etiology of the AVSD in Mef2C-AHF-Cre;
Smoflox/- embryos, we characterized the
distribution of β-gal positive cells in Mef2C-AHF-Cre;
Smoflox/-;R26R and control embryos. At
E10.5, no differences in the total amount or location of β-gal positive
cells were observed (Fig.
6A,B). However, far fewer mesenchymal cells were observed within
the atria of mutant embryos at E11.5 relative to wild-type littermates
(Fig. 6C,D). At E10.5 and E11.5
there appeared to be an increase in midline β-gal positive atrial
myocardial cells. However, increased Mef2C-AHF-Cre contribution to
the atrial myocardium was not detectable at E14.5 and later stages
(Fig. 5A,B and data not shown).
These data indicate that although an adequate population of DM cells is
present at E10.5 in mutants, it is not capable of DMP formation.
Next we analyzed whether loss of Hh signaling in the DM affects proliferation, cell death, differentiation or migration. Analysis of proliferation in Mef2C-AHF-Cre; Smoflox/- embryos was assessed via anti-phosphorylated histone H3 antibody detection; no differences were observed in the DM (data not shown). Cell death was assayed using the Lysotracker Red fluorescent probe, a lysosomal marker, at E10.5 and E11.5. No differences in cell death within the DM or atria were observed comparing Mef2C-AHF-Cre; Smoflox/- embryos with control littermates (Fig. 6E-H). Together, these data suggest that loss of DMP tissue in Mef2C-AHF-Cre; Smoflox/- embryos is not the result of decreased proliferation or increased cell death within the DM at these stages.
As discussed here and previously
(Soufan et al., 2004;
Snarr et al., 2007b), a region
of the DMP undergoes differentiation into myocardium after its formation. One
possible explanation for abnormal DMP development is abnormal control of
differentiation. We addressed this possibility by testing for the presence of
striated muscle specific myosin using the myofilament-20 (MF-20) antibody in
E11.5 embryos (Bader et al.,
1982). We observed the presence of MF-20 positive cells within the
splanchnic mesoderm and DM of Mef2C-AHF-Cre;
Smoflox/- mutants
(Fig. 6I-J'). MF-20
positive cells were never observed outside of the heart in the body wall in
wild-type controls (Fig.
6I'). Therefore, inappropriate differentiation may
contribute to the loss of DMP formation in these mutants.
Another mechanism for the DMP defect might be a reduction in the ability of
DM cells to move into the atria, for example by compromised migration. To
address this possibility, we employed a transwell migration assay (see
Materials and methods). Explanted DM (and closely adherent tissues) from E10.5
wild-type embryos was assayed for migratory properties. Three migration
culture conditions were tested using the same starting population of DM cells:
media only, 10 µm cyclopamine [a small molecule Smo inhibitor
(Chen et al., 2002)] and a DMSO
(cyclopamine carrier) control. We observed a 37% reduction in migration with
cyclopamine treatment compared with the DMSO control treatment
(P=0.001, n=3, Fig.
6K). Therefore, in addition to inappropriate differentiation of DM
cells, another cellular cause of the defect may be a reduction in their
migratory capability.
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), causing general disrupted heart curvature. Consistent with
this, Mef2C-AHF-Cre; Smoflox/- embryos (which
have relatively normal OFT and right ventricle length) were not observed to
have abnormal AV cushion shape or distribution (data not shown), suggesting
normal OFT lengthening is required for proper AV cushion rightward expansion.
Together, these data imply that a combination of defective AV cushion
rightward expansion and DMP development culminates in the most severe forms of
AVSD in Shh-/- mutants. |
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Snarr et al., 2007b;
Wirrig et al., 2007) and human
(Blom et al., 2003) fetuses
with AVSD. As such individuals exhibit malformations in other septal
structures, the specific role of DMP defects in generating these malformations
has been unclear. Here, we show that the DMP is crucial for atrial and AV
valve development. We demonstrate that AVSD can arise from a primary defect in
the DMP, rather than in the AV cushions. These studies further support a new
paradigm for understanding the pathogenic mechanisms of AVSD
(Snarr et al., 2007b).
Mef2C-AHF-Cre activity marks extracardiac contributions to the primary atrial septum
Recently, several groups have recognized the late extra-cardiac
contribution of the DM to the atria
(Mommersteeg et al., 2006;
Webb et al., 1998;
Wessels et al., 2000;
Snarr et al., 2007a;
Snarr et al., 2007b). These
studies found that continuous with the DM, the DMP expands into the atria and
wedges to close the gap formed between the primary atrial septum and the AV
cushion mesenchyme. However, owing to a lack of adequate markers for this
population, direct confirmation of these observations and assessment of DMP
contribution to later atrial development has been difficult. We demonstrate
that Mef2C-AHF-Cre is a faithful marker for the DM and its
contribution to atrial septation.
From our pseudo-lineage trace and MRM analysis, we confirm that DM enters
the developing atria at the site of pulmonary vein formation. The resulting
mesenchymal mass, the DMP, elongates to fuse with the inferior AV cushion.
Mef2C-AHF-Cre cells make up a region of the `mesenchymal cap' of the
primary atrial septum, which directly fuses with the superior AV cushion
(Fig. 7A). These findings are
consistent with previous models (Lamers
and Moorman, 2002; Wessels et
al., 2000). Loss of Hh signaling affects both the DMP and
mesenchymal cap of the primary atrial septum, consistent with a common origin
of these tissues.
The specificity of Mef2C-AHF-Cre allowed for the analysis of DMP contributions to later stages of atrial development. As visualized by MRM, the DMP maintains a large and clearly demarcated interface with the tissue derived from the AV cushions. We observed Mef2C-AHF-Cre; R26R expressing cells in the dorsal-medial myocardium of the atria. We suggest that splanchnic mesodermal derivatives distinct from the DMP add to the atria during the same time frame as the DMP. However, in contrast to the DMP, these cells do not directly require Hh signaling, as they are still present in Mef2C-AHF-Cre; Smoflox/- mutants. Alternatively, these cells may independently express the Mef2C-AHF-Cre transgene at this point in development.
The DMP is an outgrowth into the atrial lumen from the DM, in turn composed
largely of mesenchyme ventral to the foregut. Given that
Mef2C-AHF-Cre activity results in recombined cells in both the DMP
and splanchnic mesoderm, it is conceivable that these tissues arise from a
common precursor. We note that DMP labeling by cells from the
Mef2C-AHF-Cre expression domain is probably not unique to this
particular AHF-Cre driver, as similar atrial expression has been reported in
studies of other mouse genes expressed in the AHF, such as Fgf10,
Isl1 and Tbx1 (Cai et al.,
2003; Huynh et al.,
2007; Kelly et al.,
2001; Mommersteeg et al.,
2006; Snarr et al.,
2007b).
AVSD can result directly from DMP defects
To address whether the DMP is necessary for atrial septation and to further
explore its role in AV septation, we identified a mouse mutant in which this
structure is defective. Shh-/- mutants suffer from a
severe atrial septal defect and AVSD, and Hh responsiveness occurs
endogenously in the DM (Fig.
7B). Results reported here and previously
(Goddeeris et al., 2007) imply
that Shh signaling from the foregut endoderm is required for normal AV
septation, as it is the only relevant domain of Shh expression. This
relatively distant ligand source elicits its effect on AV septation by being
juxtaposed to the DM prior to its contribution to the AV complex. We
demonstrate that Ptch1lacZ is expressed within the DM and
that Cre-mediated loss of Hh receptiveness within the Mef2C-AHF-Cre
expression domain is sufficient to recapitulate the consequences of absent Shh
ligand on septal development (Fig.
7D).
Little is understood concerning the mechanism by which the DM enters the atria, and several possibilities might underlie the mechanism underlying the DMP defect. First, Shh produced by foregut endoderm may signal directly to the DM to elevate levels of proliferation, thus providing a sufficient population of cells to form the DMP. Although we were unable to observe proliferation differences in Mef2C-AHF-Cre; Smoflox/- mutant DM, such differences may occur at time points not studied here. Alternatively, Shh may promote cell shape changes or the coordinated migration of DM cells into the atria, resulting in an actual `protrusion' into the lumen. Equally plausible is that a combination of both migration and proliferation is responsible. In tissue culture assays, we found a reduction of over 35% in migrating DM cells after 24 hours of Hh inhibition. Perhaps a prolonged reduction of Hh signaling (as in the mutants in vivo) would increase the level of migration inhibition, accounting for the severe DMP deficiency. Finally, we detected DM cells that express the myofibril marker MF-20 within the body wall of E11.5 Mef2C-AHF-Cre; Smoflox/- mutants. Normally, these cells should only express such markers after they have entered the atria. Based on this surprising result, we suggest two possible explanations. DM cells may require Hh signaling to repress actively the expression of myocardial differentiation genes, and loss of this signaling results in premature differentiation of cells en route to the atrial compartment. Alternatively, the DM we observed in mutants may be differentiating in the normal time frame, but in an inappropriate location owing to a cell-motility defect.
A surprising finding is the role of the DMP in AV cushion maturation. The
AV valves are completely derived from tissues unrelated to the DMP and
theoretically should not be affected (de
Lange et al., 2004). Nevertheless, discrete loss of the DMP
results in severe defects in AV valve formation. Shh-/-
and Mef2C-AHF-Cre; Smoflox/- mutant embryos
each had abnormal AV valve development, for which several possibilities exist.
The DMP may provide remodeling signals to the AV cushion mesenchyme that are
crucial to AV valve formation. Alternatively, the DMP may provide a physical
force, pressing the AV mesenchyme into its proper conformation. We observed
the central AV mesenchymal mass to be abnormally large and rounded atop the
muscular septum in multiple Mef2C-AHF-Cre;
Smoflox/- mutants, consistent with both models. If
loss of the DMP results in loss of a secondary signal from the DMP to the AV
cushions, this secondary signal is unlikely to be a Hh ligand, as we were
unable to detect any PtchlacZ activity within the AV
cushions. Furthermore, myocardial and endocardial loss of Smo does
not affect AV septation (Fig.
7C).
OFT defects can increase the severity of AVSDs
Mef2C-AHF-Cre; Smoflox/- mutant AVSDs are
often less severe than those of Shh-/- embryos. This may
be due to the AV cushion expansion defect we observed in some
Shh-/- embryos at E11.5. In these embryos the AV cushions
do not expand correctly or evenly to the right. As schematized in
Fig. 7, the major cardiac
phenotypic difference between these two classes of embryos is that
Shh-/- embryos have a much shorter OFT and smaller right
ventricle (Goddeeris et al.,
2007; Washington Smoak et al.,
2005). Webb et al. (Webb et
al., 1999) linked a similar AV cushion expansion defect in Ts16
trisomic mouse mutants with abnormalities in the curvature of the heart tube.
Shh-/- mutants have significant shortening of the right
ventricle and OFT, which subsequently results in decreased outer curvature.
Thus, we propose that more severe aspects of AVSD in
Shh-/- embryos are the combined result of DMP
developmental failure and abnormal AV cushion rightward expansion, in turn
related to severe OFT and right ventricle shortening. Consistent with this
connection, another class of mutants with severe AVSD,
Nkx2.5Cre; Shhflox/-, has both
abnormal AV cushion shape and OFT shortening (data not shown)
(Goddeeris et al., 2007).
Mef2C-AHF-Cre; Smoflox/- mutants, which possess
relatively normal OFT and right ventricle lengthening, do not have these more
severe defects.
Mef2C-AHF-Cre; Smoflox/- mutant intracardiac defects resemble human AVSD
Mouse models that directly impact AV cushion development typically result
in embryonic lethality, limiting their clinical relevance to human AVSD. In
our study of Mef2C-AHF-Cre; Smoflox/- and
Shh-/- mutant AV septation defects, we found that the
range of severity and constellation of AVSDs observed are consistent with
those found in humans. For example, individuals with Down syndrome (Trisomy
21) have remarkably similar types of AVSD that are associated with defects in
DMP expansion (Blom et al.,
2003). AVSD is also observed in the Ts16 Down syndrome mouse
model, in which the DMP is deficient (Webb
et al., 1999; Snarr et al., 2007). Here, we demonstrate that AVSD
can be the direct result of defective DMP development in mice. A broader
analysis of defective DMP development in humans with AVSD is necessary to
better understand the basis of these serious birth defects.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/10/1887/DC1
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ACKNOWLEDGMENTS |
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Footnotes |
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Present address: St Luke's Children's Hospital, Boise, ID, USA
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