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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Dev Biol. Author manuscript; available in PMC Nov 15, 2007.
Published in final edited form as:
PMCID: PMC1866362

N-cadherin is required for neural crest remodeling of the cardiac outflow tract


Cardiac neural crest cells undergo extensive cell rearrangements during the formation of the aorticopulmonary septum in the outflow tract. However, the morphogenetic mechanisms involved in this fundamental process remain poorly understood. To determine the function of the Ca2+-dependent cell adhesion molecule, N-cadherin, in murine neural crest, we applied the Cre/loxP system and created mouse embryos genetically mosaic for N-cadherin. Specifically, deletion of N-cadherin in neural crest cells led to embryonic lethality with distinct cardiovascular defects. Neural crest cell migration and homing to the cardiac outflow tract niche were unaffected by loss of N-cadherin. However, N-cadherin-deficient neural crest cells were unable to undergo the normal morphogenetic changes associated with outflow tract remodeling, resulting in persistent truncus arteriosus in the majority of mutant embryos. Other mutant embryos initiated aorticopulmonary septum formation, however the neural crest cells were unable to elongate and align properly along the midline, and remained rounded with limited contact with their neighbors. Interestingly, rotation of the outflow tract was incomplete in these mutants suggesting that alignment of the channels is dependent on N-cadherin-generated cytoskeletal forces. A second cardiac phenotype was observed where loss of N-cadherin in the epicardium led to disruption of heterotypic cell interactions between the epicardium and myocardium resulting in a thinned ventricular myocardium. Thus, we conclude that in addition to its role in myocardial cell adhesion, N-cadherin is required for neural crest cell rearrangements critical for patterning of the cardiac outflow tract and in the maintenance of epicardial – myocardial cell interactions.

Keywords: cell adhesion, cellular rearrrangement, epicardium, myocardium


Neural crest ontogeny is a complex cell biological process involving an epithelial – mesenchymal transition and a concomitant delamination from the neural tube, emigration, prolific migration, homing, remodeling and differentiation into a bewildering array of phenotypes along the entire rostrocaudal axis (Knecht and Bronner-Fraser, 2002; Le Douarin and Kalcheim, 1999). A greater understanding of the molecular mechanisms involved in neural crest development is critical not only to revealing the congenital or environmental factors that disproportionately affect neural crest development but also to the prevention and clinical treatment of neural crest-related congenital defects such as DiGeorge syndrome in humans. Cardiac neural crest cells (NCC), a neural crest subpopulation derived from the postotic hindbrain neural fold, play an essential role in outflow tract (OFT) septation and tissue remodeling in the conotruncal region of the heart (Kirby et al., 1983; Kirby and Waldo, 1995). The structural determinants that regulate this complex morphological process are unknown.

Classical cadherins comprise a family of cell surface adhesion molecules that are anchored to the actin cytoskeleton via catenins, specifically β- and α-, or γ- and α-catenin (Wheelock and Johnson, 2003). Cadherins are single pass transmembrane proteins that bind calcium extracellularly, a prerequisite for their adhesive function. In most cell types, cadherins are concentrated at cell-cell contact sites called adherens junctions. The strength of adhesion is modulated by p120ctn binding to the juxtamembrane region of the cadherin cytoplasmic domain. Many of the morphogenetic processes cadherins are thought to regulate, such as adhesion and motility, also require dynamic rearrangement of the actin cytoskeleton.

Several cadherin family members exhibit a dynamic expression pattern during NCC development including N-cadherin (Akitaya and Bronner-Fraser, 1992; Duband et al., 1988; Hatta et al., 1987; Nakagawa and Takeichi, 1995; Pla et al., 2001). Following neural tube closure, N-cadherin and cadherin-6B are both expressed in the dorsal region where the NCC will emerge. These cadherins are downregulated as epithelial-mesenchymal transition occurs and the NCC migrate either dorso-ventral or dorso-lateral toward their ultimate destinations. Cadherin-7 and -11 are upregulated in migrating NCC, and downregulated after homing and during differentiation. Proper cadherin-mediated cell-cell adhesion is important for NCC emigration from the neural tube and their subsequent migration, in which transient cadherin-mediated cell-cell contacts are thought to be required for NCC to migrate as a stream. Injection of N-cadherin function-blocking antibodies into the neural tube resulted in ectopic NCC in chick embryos (Bronner-Fraser et al., 1992). Conversely, overexpression of N-cadherin or cadherin-7 inhibits NCC emigration from the chick neural tube (Nakagawa and Takeichi, 1998). Transplantation experiments in Xenopus embryos demonstrated that misexpression of cadherin-11 blocks migration and differentiation of NCC (Borchers et al., 2001). These previous studies examined the effect of misexpression or dominant negative cadherin on NCC delamination and initial migration away from the dorsal neural tube, but they did not address the role of cadherin-mediated adhesion in NCC re-aggregation.

Using ex vivo neural tube cultures, we previously demonstrated that nascent N-cadherin-deficient NCC migrate faster than wild-type accompanied with a loss of directionality, resulting in a decrease in overall migration distance (Xu et al., 2001). The early embryonic lethality of N-cadherin mutant embryos (Radice et al., 1997) precluded later and more extensive analysis of N-cadherin function in the NCC lineage. In this study, we employed a conditional knockout to examine NCC behavior in the absence of N-cadherin. Here we show that N-cadherin-deficient NCC reach the heart, however they are incapable of remodeling the OFT. Furthermore, we found that N-cadherin-mediated adhesion is required for maintaining heterotypic epicardial-myocardial cell – cell interactions in the developing heart.

Materials and methods

Generation of N-cad/Wnt1-Cre embryos

To generate NCC-specific deletion of N-cadherin, N-cad null/+, Wnt1-Cre mice were mated with N-cad flox/flox, R26R/R26R mice and embryos were recovered from timed pregnancies. The N-cad null/flox, Wnt1-Cre, R26R genotype served as the CKO embryos and all other genotypes as controls. All embryos inherited a mixed genetic background. Wnt1-Cre mice were provided by A. McMahon (Harvard University) and R26R reporter mice were obtained from the Jackson Laboratories.

Morphological and molecular analysis of N-cad/Wnt1-Cre embryos

Whole-mount X-gal staining on embryos was performed as previously described (Luo et al., 2005). TUNEL reactions were performed using the In Situ Cell Death Kit, Fluorescein (Roche). Transmission electron microscopy was performed on frontal sections through the thoracic region of E12.5 embryos according to standard protocol. Indirect immunofluorescence was performed on paraffin sections of embryos as previously described (Luo et al., 2001). Sections were incubated with the following primary antibodies: α-smooth muscle actin (Sigma), N-cadherin (Zymed), β-catenin (Zymed) and β-galactosidase (Santa Cruz), followed by incubation with secondary antibody either Cy3- or FITC-conjugated goat anti-mouse (Jackson ImmunoResearch Laboratories) and visualized with a confocal microscope.

Intracardiac ink injections

For gross examination of the cardiovascular system, Indian ink was injected into the ventricle of E10.5 and E11.75 embryos. After injection, embryos were fixed in 4% paraformaldehyde for 12 hours, dehydrated and cleared in benzyl benzoate:benzyl alcohol (2:1). Embryos were digitally photographed under a dissecting microscope.

Epicardial cell culture

To derive primary epicardial cells, E10.5 hearts were dissected and atria and OFT were removed and the remaining cardiac tissue was cultured as previously described (Chen et al., 2002). Epicardial cells migrate away and form a monolayer surrounding the cardiac tissue. The cardiac tissue was removed after 3 days and the epicardial monolayer was immunostained for N-cadherin.


NCC Migrate to the OFT without N-cadherin

To investigate the role of N-cadherin in NCC behavior, mice inheriting a floxed N-cadherin gene (Kostetskii et al., 2005) were bred to Wnt1-Cre transgenic mice (Danielian et al., 1998) to delete N-cadherin specifically from the NCC lineage. In addition, the R26R Cre reporter (Soriano, 1999) was introduced into the N-cadherin floxed background. In this way, histochemical staining for β-galactosidase activity with X-gal could easily monitor the fate of Wnt1-Cre deleted cells. Ablation of N-cadherin using Wnt1-Cre resulted in embryonic lethality by E13 associated with severe cardiovascular defects. The N-cadherinnull/flox, Wnt1-Cre, R26R (herein referred to as N-cad/Wnt1-Cre) embryos appeared grossly normal along the anterior-posterior axis between E10.5 and E12.5 with the exception of abnormal brain and craniofacial development (Fig. 1). The protrusion of neural tissue from the hindbrain is consistent with N-cadherin’s role in closure of the neuropore along the midline (Luo et al., 2001). This study will focus on the cardiovascular phenotypes and the description of other defects in the N-cad/Wnt1-Cre embryos will be presented elsewhere. By E12.5, N-cad/Wnt1-Cre embryos were pale and apparently lacked any blood circulation (Fig. 1C).

Figure 1
Loss of N-cadherin in NCCs leads to embryonic lethality. Whole-mount images of E10.5 (A), E11.5 (B) and E12.5 (C) wild-type (WT) and N-cadherin CKO (CKO) embryos. Note the pale appearance of the CKO compared to WT at E12.5.

To determine if N-cadherin affects NCC migration and homing to the OFT of the developing heart, we examined the distribution of NCC in N-cad/Wnt1-Cre embryos. The overall pattern of X-gal staining appeared remarkably similar between N-cad/Wnt1-Cre and wild-type embryos (Fig. 2A,B and Supplementary Fig. 1 online) demonstrating that neither NCC genesis nor migration were significantly affected by the loss of N-cadherin in the nascent NCC lineage. There was no apparent deficiency of NCC in the OFT region of N-cad/Wnt1-Cre compared to wild-type embryos at E10.5 (Fig. 2C–F) and E11.5 (see Supplementary Fig. 2 online).

Figure 2
Normal distribution pattern of NCC in E10.5 embryos after deletion of N-cadherin. Wnt1-Cre recombination in NCC is marked by the expression of β-galactosidase in wild-type (WT) and N-cadherin CKO (CKO) R26R embryos (A,B). Higher magnification ...

N-cadherin is Required for Remodeling of the OFT

Cardiac NCCs are the primary cellular component of the aorticopulmonary septation complex responsible for remodeling the embryonic heart. To examine development of the OFT, serial frontal sections of E11.75 embryos were examined histologically (Fig. 3). N-cad/Wnt1-Cre embryos exhibited two types of OFT abnormalities (Table I); lack of septation (undivided) or septation initiated (divided), but not normal. All other genotypes exhibited normal septation of the OFT. The majority of embryos exhibited persistent truncus arteriosus as illustrated by the single channel in the mutant (n=13) compared to the presence of the aorta and pulmonary artery in the wild-type (Fig. 3). To further examine OFT and pharyngeal arch artery development, intracardiac India ink injections were performed on N-cad/Wnt1-Cre and wild-type embryos (Fig. 4). Initially, at E10.5 the OFT and pharyngeal arch arteries appeared morphologically similar in mutant (n=2) and wild-type embryos (Fig. 4A,B). By E11.75, aorta and pulmonary artery were observed in wild-type, in contrast, only one ink-stained channel was connected to the arch arteries in the mutant embryos (n=3). NCC populate the pharyngeal arch arteries and differentiate into smooth muscle. To determine if this process occurs normally in the N-cad/Wnt1-Cre embryos, frontal sections of E11.5 embryos were immunostained using an α-smooth muscle actin antibody, a marker of differentiated smooth muscle cells (Fig. 5). The staining pattern around the arch arteries appeared similar between the wild-type and mutant embryos (n=3) indicating that N-cadherin-deficient NCC reached their destination and differentiated into smooth muscle cells normally.

Figure 3
Deletion of N-cadherin in neural crest cells leads to truncus arteriosus. Histological analysis of serial frontal sections from WT (A,C,E) and N-cadherin CKO (B,D,F) E11.75 embryos. The OFT (arrow) is undivided in the CKO compared to the WT heart. Ao, ...
Figure 4
Undivided OFT in the N-cadherin/Wnt1-Cre mutant embryos. Left lateral view after intracardiac ink injection to visualize OFT and pharyngeal arch arteries at E10.5 (A,B) and E11.75 (C–F). The pharyngeal arch arteries are numbered. At E10.5, the ...
Figure 5
Normal pattern of smooth muscle cells surrounding the pharyngeal arch arteries in N-cad/Wnt1-Cre embryos. Histological (A,C) and immunofluorescence (B,D) analysis was performed on adjacent frontal sections from E11.5 wild-type (A,B) and CKO (C,D) embryos ...
Table I
Summary of OFT Defect and Survival in N-cad/Wnt1-Cre Embryos

A small number of N-cad/Wnt1-Cre embryos initiated the septation process, however the resulting OFT was abnormal. NCC normally elongate and align with their neighbors eventually aggregating at the midline of the OFT to septate the lumen into aortic and pulmonary channels (Fig. 6A–D). The diameter of the OFT was smaller in the E12.5 N-cad/Wnt1-Cre embryo with rounded, less condensed NCC compared to wild-type. In addition, the lumens of the developing aortic and pulmonary channels were collapsed in the mutant embryos. To examine NCC at the ultrastructural level, transmission electron microscopy (TEM) was performed on the OFT region of E12.5 embryos (Fig. 6E,F). Wild-type NCC were elongated and aligned with their neighbors, in contrast mutant NCC were rounded with fewer cell-cell contact sites compared to wild-type. Furthermore, expression of α-smooth muscle actin was significantly reduced in these loosely aggregated NCC compared to wild-type suggesting that differentiation of NCC in the OFT may be dependent on N-cadherin-mediated cell-cell contact (Fig. 6G,H). Interestingly, the orientation of the OFT in the N-cad/Wnt1-Cre embryo did not change significantly from a day earlier, E11.5 (see Supplementary Fig. 3 online), indicating that rotation of the aorticopulmonary septation complex is dependent on N-cadherin-mediated NCC rearrangement and differentiation. By E12.75, the mutant NCC began to undergo programmed cell death as determined by TUNEL (n=3) and TEM analysis (see Supplementary Fig. 4 online). The requirement for N-cadherin-mediated adhesion in NCC survival is consistent with the increased apoptosis observed previously in N-cadherin-deficient neuroepithelium and somites at earlier stages in embryonic development (Luo et al., 2001).

Figure 6
Abnormal outflow tract development in N-cadherin CKO embryos. Histological sections of outflow tract of wild-type (WT) and N-cadherin CKO (CKO) E12.5 embryos (A–D). Note the smaller, less developed outflow tract in the CKO compared to WT embryo. ...

N-cadherin Expression during OFT Development

To determine if the dynamic cellular rearrangements of the NCC correlated with changes in N-cadherin, we examined N-cadherin expression in the OFT from E10.5 – E12.5. At E10.5, when NCC arrive at the OFT N-cadherin was weakly expressed and restricted to limited regions of cell-cell contact (Fig. 7A). As expected, N-cadherin was absent from the NCC in the N-cad/Wnt1-Cre embryo whereas it remained expressed in the myocardium (Fig. 7B). There was a dramatic increase in N-cadherin levels as NCC elongated and condensed in the OFT between E11.5 and E12.5 (Fig. 7C,D). To determine if the cadherin/catenin complex was present in the cardiac NCC after deletion of N-cadherin, OFT was immunostained for the cadherin binding partner, β-catenin (Fig. 7E,F). Beta-catenin was still present at the cell membrane in N-cadherin-deficient NCC, albeit at low levels, compared to wild-type indicating that at least one other classical cadherin was present in these cells. However, the remaining cadherin/catenin complex in the mutant NCC was evidently insufficient to mediate OFT remodeling.

Figure 7
Cadherin/catenin expression in the NCC of the outflow tract. Immunofluorescence was performed on E10.5 (A,B), E11.5 (C,E,F), E12.5 (D) embryos for N-cadherin or β-catenin (E,F). N-cadherin was present at a low level in WT (arrow, inset) and absent ...

Disruption of Epicardial-Myocardial Cell-Cell Interactions in N-cad/Wnt1-Cre Embryos

N-cadherin deletion in NCC leads to embryonic lethality between E12.5–E13.5, which is unexpected since OFT defect alone is not sufficient to cause embryonic death at this stage. Therefore, we examined the heart for additional abnormalities. Histological analysis showed a thinned ventricular myocardium accompanied by a detached epicardium in N-cad/Wnt1-Cre embryos at E12.5 (Fig. 8). The epicardium is critical for providing proliferative signals to the underlying ventricular myocardium (Chen et al., 2002; Stuckmann et al., 2003). Both the compact myocardium on the periphery of the ventricle and the trabecular myocardium adjacent to the ventricular cavity were significantly reduced in mutant embryos compared to wild-type (Fig. 8A,B). The epicardium appeared structurally abnormal in the N-cad/Wnt1-Cre embryos having detached from the underlying myocardium (Fig. 8D) resulting in a bubbling appearance (Fig. 8F) compared to the normal adherent epicardium in the wild-type embryos. Indeed, we observed loss of N-cadherin in the epicardium in the N-cadherin CKO embryo (Fig. 8H). Due to the strong expression of N-cadherin in the myocardium, it was difficult to appreciate the weaker staining in the epicardium, therefore epicardial cells were isolated from E10.5 embryos, cultured 2 days, and immunostained for N-cadherin. Consistent with our in vivo results, N-cadherin was significantly decreased in the epicardial cells derived from the N-cad/Wnt1-Cre embryo compared to wild-type (Fig. 8I,J). Thus, the thinned ventricular myocardium may be explained by the disruption of epicardial – myocardial cell-cell interactions leading to loss of proliferative signals from the epicardium.

Figure 8
Loss of epicardial – myocardial cell interactions in the N-cadherin CKO embryos. Histological sections of wild-type (WT) and N-cadherin CKO (CKO) E12.5 hearts show a thinned myocardial wall associated with a detached epicardium (arrow) in the ...


Recent studies have begun to elucidate the molecular mechanisms involved in NCC genesis and migration. However, little is known about the regulation of NCC rearrangement and re-aggregation once they arrive at their final destinations. N-cadherin exhibits a dynamic expression pattern during cardiac NCC ontogeny. N-cadherin must be down-regulated in order for the NCC to emigrate from the dorsal neural tube and migrate to their ultimate destinations. In migrating NCC in vitro, weak N-cadherin immunostaining is apparent at the tip of cellular processes in regions of intercellular contact (Monier-Gavelle and Duband, 1995; Xu et al., 2001). We conclude that N-cadherin is dispensable in migratory cardiac NCC as no deficit in NCC was observed in the OFT of N-cad/Wnt1-Cre embryos. Cardiac NCC migrate to the OFT and contribute to the formation of aorticopulmonary channels over a two day period thus providing a unique model to identify the structural determinants in this complex morphogenetic process. Coincident with the morphological changes of the cardiac NCC, N-cadherin expression dramatically increases between E10.5 – E12.5. Our results demonstrated that N-cadherin is the dominant cadherin in cardiac NCC and its up-regulation is required for the remodeling of the OFT in the developing heart. Persistent truncus arteriosus was observed in the majority of the embryos, however some of the mutants undergo septation with abnormal aorticopulmonary channel development. In contrast, the pharyngeal arch arteries developed normally in the N-cad/Wnt1-Cre embryos with NCC-derived smooth muscle surrounding the arteries.

Beta-catenin is thought to play a critical role in neural crest ontogeny due to its dual function in cell adhesion, as a link between cadherin and the cytoskeleton, and in cell signaling, as a key mediator of the canonical Wnt-signaling pathway. In migrating NCC, β-catenin is primarily found associated with N-cadherin at sites of cell-cell contact. In early migrating NCC located near the dorsal neural tube, β-catenin is detectable in the nuclei suggesting that β-catenin signaling is required transiently at the onset of migration, but that sustained signaling is not required later during migration (de Melker et al., 2004). Interestingly, β-catenin/Wnt1-Cre embryos (Brault et al., 2001) have defects in brain and craniofacial development and survive longer (i.e. E18.5) than N-cad/Wnt1-Cre embryos presumably due to the ability of plakoglobin to substitute for β-catenin in the cadherin/catenin complex. The inability of the OFT to develop normally in N-cad/Wnt1-Cre embryos, including its incomplete rotation, suggests N-cadherin plays a critical role in the overall morphogenesis of the cardiac OFT. In N-cad/Wnt1-Cre embryos, NCC reached the OFT indicating loss of N-cadherin does not have a significant affect on NCC migration and homing. In contrast, a reduced number of NCC reached the OFT in β-catenin/Wnt1-Cre embryos (Lee et al., 2004). In another study (Kioussi et al., 2002), BrdU pulse labeling of β-catenin/Wnt1-Cre embryos at E10.5 demonstrated a decrease in cellular proliferation in the OFT. In β-catenin/Wnt1-Cre embryos, all cadherins expressed in NCC will be affected by loss of β-catenin from the cadherin/catenin adhesion complex. In our model, specific loss of the N-cadherin/β-catenin complex was not sufficient to prevent cardiac NCC from reaching the OFT allowing comparison between the two models. In the N-cad/Wnt1-Cre model, other cadherins (e.g. cadherin-7 or –11) and/or other migration machinery was sufficient for NCC migration to the OFT. In the β-catenin/Wnt1-Cre model, perturbation of the canonical Wnt signaling pathway may lead to a decreased number of cardiac NCC due to stem cell fate decisions as previously proposed (Lee et al., 2004).

The ability of the mutant neural crest cells in N-cad/Wnt1-Cre embryos to emigrate normally from the neural tube is somewhat surprising given previous results (Bronner-Fraser et al., 1992); however there may be a simple explanation. N-cadherin function-blocking antibodies injected into the cranial neural tube of chick embryos cause misshapened neural tubes and ectopic aggregates of neural crest cells located dorsally of the neural tube. Interestingly, this phenotype was observed when the embryos were injected at the 3 – 9 somite stage, however few defects were observed when embryos of 9 or more somites were injected with antibody (Bronner-Fraser et al., 1992). Consistent with this latter result, a dominant negative N-cadherin molecule virally transduced into chick embryos at the 20 somite stage did not affect dorsolateral migration of neural crest cells (Nakagawa and Takeichi, 1998). In the mouse model, Wnt1-Cre is active in the dorsal neural tube around the 15 somite stage, therefore the differences between the initial chick study and ours may be due to inhibition of N-cadherin function at an earlier stage of neural crest development.

In addition to cell-cell interactions, NCC behavior is dependent on cell-extracellular matrix (ECM) interactions (Perris and Perissinotto, 2000). Based on the lack of a migration defect in the N-cad/Wnt1-Cre embryos, it is possible that cell-ECM interactions are more important for cardiac NCC migration to the OFT than cadherin-mediated cell-cell interactions. In vitro studies have demonstrated that N-cadherin and β1-integrin are coordinately regulated in NCC (Monier-Gavelle and Duband, 1997). Blocking NCC interactions with the ECM results in rapid restoration of intercellular contacts mediated by N-cadherin. Interestingly, we have observed upregulation of β1-integrin in cardiomyocytes (Kostetskii et al., 2005) and endothelial cells (Luo and Radice, 2005) after depletion of N-cadherin. If cadherin and integrin compensate for one another, it may be necessary to remove both adhesion systems (i.e. double mutants) to interfere with NCC migration. Loss of β1-integrin in NCC after initiation of migration using the Ht-PA-Cre transgene (Pietri et al., 2003) results in aberrant development of the peripheral nervous system (Pietri et al., 2004). Earlier deletion of β1-integrin from NCC using Wnt1-Cre results in embryonic lethality at E12.5, however the phenotype has not been reported (Pietri et al., 2004). It will be interesting to directly compare the N-cad/Wnt1-Cre and the β1-integrin/Wnt1-Cre phenotypes to determine how cell-cell and cell-ECM interactions regulate cardiac NCC behavior.

A second distinct and unexpected cardiovascular phenotype was observed in the N-cad/Wnt1-Cre embryos where the epicardial cell layer detached from the underlying myocardium. In support of our observations, it was recently demonstrated using R26R reporter that Wnt1-Cre is active in epicardial cells (Stottmann et al., 2004). We also observed Wnt1-Cre activity in epicardial explant cultures consistent with the in vivo results (see Supplementary Fig. 5 online). Interestingly, the epicardium remained intact in the N-cad/Wnt1-Cre embryos whereas the epicardial – myocardial cell interactions were disrupted indicating that other cell adhesion molecule(s) is responsible for epicardial cell-cell interaction. E-cadherin or Bves (Wada et al., 2001), a novel cell adhesion molecule expressed in epicardial cells, may be involved in maintaining the structural integrity of the epicardium whereas N-cadherin appears more important for heterotypic cell interactions between the epicardium and myocardium. The functional relevance of this low level of N-cadherin expression in the epicardium compared to the N-cadherin rich myocardium was not previously appreciated. N-cadherin-deficient epicardium caused myocardial cell hypoplasia or thinned ventricular myocardium in the N-cad/Wnt1-Cre embryos possibly due to loss of proliferative signals (e.g. BMP and FGF) originating from the epicardium. The retinoic acid inducible factor, FGF-9, expressed in the epicardium is critical for myocardial cell proliferation via signaling through FGFR1 and FGFR2 found in the myocardium (Lavine et al., 2005). Disruption of epicardial-myocardial cell interactions in the N-cad/Wnt1-Cre embryos may interfere with this ligand-receptor signaling pathway. Alternatively, the myocardial defect may be secondary to the NCC defect in the N-cad/Wnt1-Cre embryos. For example, Pax3-deficient Splotch mice have a defect in NCC development causing persistent truncus arteriosus as well as a thinned ventricular myocardium (Conway et al., 1997; Creazzo et al., 1998). Although we can’t rule out a secondary affect from the mutant NCC, we believe the myocardial cell hypoplasia in the N-cad/Wnt1-Cre embryos can be explained by disruption of epicardial – myocardial cell interactions. The separation of the epicardium from the underlying myocardium is very similar to the structural defect observed in the α4-integrin and V-CAM knockout mice (Kwee et al., 1995; Yang et al., 1995). The explanation for the adhesion defect in these animals is that epicardium expresses α4-integrin whereas V-CAM, the ligand for α4β1-integrin, is found in the myocardium. The majority of embryos null for either α4-integrin or V-CAM die earlier than the N-cad/Wnt1-Cre embryos, however some of the V-CAM KO embryos succumb at E12.5 with a thinned ventricular myocardium (Kwee et al., 1995). In our model, N-cadherin likely acts homophilically to maintain the heterotypic cell-cell interactions between the N-cadherin-expressing myocardium and the epicardium.

Until recently it was thought that the primary source of myocardial progenitors was the bilaterally symmetrical precardiac mesoderm in the gastrulating embryo. However, there is a new appreciation for extracardiac contribution to the heart from a region called the anterior heart field or secondary heart field (Kelly and Buckingham, 2002; Mjaatvedt et al., 2001; Waldo et al., 2001). The myocardial cells arise from the pharyngeal mesoderm and contribute to the developing OFT. Growth factors such as FGF8 and BMP2 are involved in the induction of these mesodermal cells into myocardial cells. In a similar manner, it is possible that the N-cadherin-deficient cardiac NCC don’t respond appropriately to signals from the secondary heart field leading to a septation defect.

We previously showed that N-cadherin is required in the myocardial (Luo et al., 2001) and endocardial (Luo and Radice, 2005) cell compartments of the developing tubular heart. In the present study, we demonstrated that N-cadherin-mediated adhesion is required for NCC remodeling of the cardiac OFT and maintenance of epicardial – myocardial cell-cell interactions in the nascent four-chamber heart. In conclusion, N-cadherin plays an essential role in multiple cardiac cell lineages and at different times during cardiac morphogenesis.

Supplementary Material


Suppl. 1. Normal distribution pattern of NCC in E9.5 N-cad/Wnt1-Cre embryo. Wnt1-Cre recombination in NCC is marked by the expression of β-galactosidase in wild-type (WT) and N-cadherin CKO (CKO) R26R embryos (A,B). Note at E8.5 (A), Wnt1-Cre activity is primarily restricted to the midbrain. Cross-section through the postotic region of the X-gal stained E9.5 embryos (C,D). No apparent deficit of NCC was observed in the CKO compared to WT embryo.


Suppl. 2. Normal distribution pattern of NCC in E11.5 N-cad/Wnt1-Cre embryo. Wnt1-Cre recombination in NCC is marked by the expression of β-galactosidase in wild-type (A) and N-cadherin CKO (B) R26R embryos. Cross-section through the outflow tract of the X-gal stained embryos (C,D). A similar number of NCC appeared in the outflow tract of WT and CKO embryos. Scale bars: C,D 50 μm.


Suppl. 3. N-cadherin-deficient NCC were poorly organized in the outflow tract at E11.5. Histological sections of outflow tract of wild-type (A,C) and N-cadherin CKO (B,D) embryos. Higher magnification (inset) shows the rounded, less compacted morphology of the NCC in the CKO compared to WT embryo. Scale bars: A,B 25 μm; C,D 50 μm.


Suppl. 4. Loss of N-cadherin-mediated adhesion leads to NCC programmed cell death. Beta-galactosidase immunostaining (A,C) and TUNEL analysis (B,D) of adjacent frontal sections from wild-type (A,B) and N-cadherin CKO (C,D) outflow tract at E11.5. Arrows indicate TUNEL positive NCC. Note the nonspecific fluorescence of blood cells in the lumen of the wild-type embryo (A,B). Transmission electron microscopy of NCC in the outflow tract of E12.75 embryos. Nuclear condensation in the CKO NCC confirmed the TUNEL analysis. Scale bars: A-D 25 μm; E,F 2 μm.


Suppl. 5. Wnt1-Cre activity in epicardial cells in culture. Wnt1-Cre recombination in epicardial cells is marked by the expression of β-galactosidase in N-cadherin CKO (B) R26R culture. Epicardial culture without Wnt1-Cre (A) served as a negative control. X-gal staining demonstrated that approximately 25% of the epicardial cells migrating away from the explant were positive.


We thank Karen Knudsen, Robert Moore and John Burch for their comments on the manuscript, Yanming Xiong for technical assistance, Neelima Shah and the University of Pennsylvania Biomedical Imaging Core Facility for performing the electron microscopy analysis. This study was supported by the American Heart Association (G.R.) and the NIH P01 HL075215, RO1 HL61475 (J.E.). Y.L. was supported in part by a postdoctoral fellowship from the PA/DE Affiliate of the American Heart Association. G.R. is an Established Investigator of the American Heart Association.


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