An essential role for Notch in neural crest during cardiovascular development and smooth muscle differentiation (original) (raw)
Multiple Notch receptors and ligands are expressed in the developing cardiac outflow tract. In order to determine which Notch family members are likely to play a role in cardiac outflow tract development, we performed in situ hybridizations to determine the expression patterns of all known Notch receptors and ligands in the developing heart. We examined embryos at E12.5, during a critical time period for remodeling of the outflow tract and the aortic arch arteries. Notch1 and Notch4 were expressed in a thin layer lining the heart and the aortic arch arteries, consistent with expression restricted to the endothelium (Figure 1, A and D). Notch1 was also expressed in the region of the aortic valve (Figure 1A), consistent with its role in development of this valve (7). Notch2 and Notch3 transcripts were detected in the mesenchymal cells surrounding the aortic arch arteries, consistent with expression within neural crest (Figure 1, B and C).
Multiple Notch receptors and ligands are expressed in the developing cardiac outflow tract. In situ hybridization analysis of cross-sections through the outflow tract and aortic arch arteries of E12.5 embryos is shown. (A) Notch1 is expressed by endothelial cells lining the aortic arch arteries (AAA) and by endocardial cushion tissue of the developing aortic valve (AV). (B) Notch2 is expressed broadly throughout the pharyngeal mesenchyme and notably by the neural crest–derived cells surrounding the aortic arch arteries. (C) Notch3 is expressed by neural crest–derived cells surrounding the aortic arch arteries. (D) Notch4 is expressed by endothelial cells of the aortic arch arteries. (E) Jagged1 is expressed by neural crest–derived cells surrounding the aortic arch arteries. (F) Jagged2 is expressed broadly in the pharyngeal region, including cells surrounding the aortic arch arteries. (G) Delta-like1 is expressed by endocardium but is absent from the aortic arch arteries. (H) Delta-like4 is expressed by endothelium of the aortic arch arteries. Scale bars: 100 μm.
The Notch ligand Jagged1 was expressed in cells surrounding the aortic arch arteries, consistent with expression by neural crest–derived tissues including vascular smooth muscle (Figure 1E). Jagged2 was expressed diffusely throughout the pharyngeal region, including neural crest–derived tissue surrounding the aortic arch arteries (Figure 1F). Delta-like1 transcripts were detectable in the endocardium, but there was no detectable signal in the aortic arch arteries at this stage (Figure 1G). The expression pattern for Delta-like4 was consistent with other reports describing endothelium-specific expression (Figure 1H) (23). Delta-like3 transcripts were not detectable in the developing cardiac outflow tract at this time point (data not shown).
Neural crest–specific expression of dominant-negative MAML results in cardiac outflow tract defects. The expression of multiple Notch family members suggests possible redundancy among different Notch receptors in the developing cardiac outflow tract. Therefore, we used a dominant-negative approach that would inhibit the activity of all 4 Notch receptors. MAML proteins are important components of the Notch transcriptional complex and are critical for recruitment of other coactivators (24). The dominant-negative version of MAML1 (DNMAML) consists of a 62-amino-acid peptide at the N terminus of MAML1 that has been shown in crystal structures to bind specifically to the Notch-CSL transcriptional complex (25). This construct effectively inhibits canonical (CSL-dependent) signaling by all 4 mammalian Notch receptors (26, 27). We used a mouse model in which DNMAML fused to GFP (DNMAML-GFP) was targeted to the ubiquitously expressed ROSA26 locus. In this model, DNMAML-GFP is placed downstream of a neomycin cassette, which is flanked by loxP sites. In the presence of Cre recombinase, neomycin is excised and DNMAML-GFP comes under control of the ubiquitous ROSA26 promoter. This provides a system in which DNMAML-GFP can be activated in a tissue-specific manner by crossing DNMAML-GFP mice with a mouse line that expresses a tissue-specific Cre recombinase (27).
To inhibit Notch in derivatives of the neural crest, we crossed the DNMAML-GFP mouse with 2 different neural crest–specific Cre recombinase lines. First, we used a mouse in which Cre is knocked into the Pax3 locus (Pax3Cre/+) (28). Pax3 is a paired-type transcription factor that is expressed in premigratory neural crest cells and presomitic mesoderm. Previous fate-mapping studies using the Pax3Cre/+ mouse have shown efficient recombination in derivates of the cardiac neural crest, including the developing aortic arch vessels and the conotruncal septum. In addition to neural crest derivatives, the Pax3Cre/+ mouse also induces recombination in derivatives of the somites, including skeletal muscle of the diaphragm and limb muscles (28).
Examination of the hearts of late-gestation Pax3Cre/+ DNMAML embryos revealed multiple significant cardiac defects that mimicked those seen in human congenital heart disease. A detailed list of the defects observed in these mice is presented in Table 1. Twenty-seven of 36 (75%) of the embryos examined at E17.5 or E18.5 showed at least 1 cardiac anomaly. The most common defect observed was stenosis of the pulmonary artery in 15 of 36 (42%) of the embryos (Figure 2, A and B). Some of these embryos also demonstrated membranous ventricular septal defects (Figure 2, C and D) and a malalignment of the outflow tract such that the aorta was located lateral to, rather than posterior to, the pulmonary artery. This suggests a phenotype similar to tetralogy of Fallot in a subset of the embryos.
Neural crest–specific expression of DNMAML results in cardiac outflow tract defects. (A and B) Lateral view of the left side of hearts removed from E17.5 control (A) and Pax3Cre/+ DNMAML (B) embryos. Compared with the control pulmonary artery, the mutant pulmonary artery and ductus arteriosus are significantly narrowed (arrowheads in A and B). (C and D) Hematoxylin and eosin stained cross-sections through the hearts of E17.5 control (C) or Pax3Cre/+ DNMAML (D) embryos demonstrate a membranous ventricular septal defect in the mutant (arrow in D). (E–J) Photographs and drawings depicting the aortic arch phenotypes in several E17.5 embryos. A control embryo (E) shows the normal branching pattern of the great vessels. (F) Pax3Cre/+ DNMAML mutant showing an abnormal arch structure similar to the human defect known as persistent fifth arch. In addition, the ductus arteriosus is absent, and there is a retroesophageal right subclavian artery. (G) Pax3Cre/+ DNMAML mutant showing a right-sided aortic arch and isolated left subclavian artery arising from the pulmonary artery (arrowhead). (H) Pax3Cre/+ DNMAML mutant with an atretic ductus arteriosus (arrow) and isolated right subclavian artery (arrowhead). (I) Wnt1-Cre DNMAML mutant showing duplication of the left common carotid artery (arrows). (J) Wnt1-Cre DNMAML mutant with an absent ductus arteriosus. The asterisk indicates ventricular septal defects that were observed upon sectioning the hearts. rv, right ventricle; lv, left ventricle; ao, aorta; da, ductus arteriosus; rsa, right subclavian artery; rca, right common carotid artery; lca, left common carotid artery; lsa, left subclavian artery. Scale bars: 200 μm.
Distribution of cardiac defects observed in DNMAML crosses (E17.5–P0)
Twenty-three of 36 (64%) of these embryos displayed aortic arch patterning defects, with a spectrum of severity (Figure 2, E–H, and Table 1). All of these aortic arch defects could be attributed to improper persistence or regression of the aortic arch arteries during development. Defects in the development of the sixth arch artery were most common, including defects in the pulmonary artery and ductus arteriosus (Figure 2, F–I). However, third and fourth arch artery defects were also apparent. Anomalous origin of the subclavian arteries was observed, either as a retroesophageal right subclavian artery arising from the descending aorta (Figure 2F) or as an isolated subclavian artery arising from the main pulmonary artery (Figure 2, G and H). We also observed other arch anomalies, including right-sided aortic arch (Figure 2G), duplication of the common carotid arteries (Figure 2I), and a defect similar to a rare congenital malformation known as persistent fifth arch (Figure 2F).
Pax3Cre/+ DNMAML embryos were observed at expected Mendelian ratios throughout gestation. However, no offspring survived after birth (Table 2). This neonatal mortality could not be explained by cardiac defects alone, as several dead Pax3Cre/+ DNMAML neonates had no obvious cardiac phenotype. On the other hand, all mutant neonates failed to show signs of lung inflation, and examination revealed a very thin diaphragm with severe reduction in skeletal muscle mass (data not shown). Therefore, we conclude that the major cause of death in these mice was respiratory failure. This finding is consistent with the fact that Pax3 is expressed in developing somites and that there is a well-established role for Notch signaling in both somite and skeletal muscle development (29, 30).
Survival of DNMAML offspringA
To confirm that the cardiac phenotype seen in the Pax3Cre/+ DNMAML mice was specific to the neural crest, we also crossed DNMAML mice with _Wnt1-Cre_–transgenic mice (13). Wnt1 is also expressed in premigratory neural crest cells, but it is not expressed in skeletal muscle precursors. Wnt1-Cre DNMAML mice also demonstrated cardiac outflow tract and aortic arch abnormalities that were similar to those seen in the Pax3Cre/+ DNMAML mice (Table 1 and Figure 2, I and J). These results confirm a critical cell-autonomous role for Notch signaling in the cardiac neural crest. Unlike the Pax3Cre/+ DNMAML mice, many of the Wnt1-Cre DNMAML mice survived after birth. As Wnt1-Cre does not induce recombination in skeletal muscle precursors, these mice did not display diaphragm defects. However, we did observe significant mortality during the first 2 weeks of life (Table 2). This can be attributed to the cardiac phenotype, as several dead pups had severe cardiac malformations.
Two different control mouse crosses were also generated. First, we examined the Cre-negative littermates from the Pax3Cre/+ DNMAML cross. Second, to control for the presence of Pax3Cre/+, as well as for any possible effects of GFP overexpression, we crossed the Pax3Cre/+ mice with R26RGFP mice (31). The R26RGFP mice contain GFP knocked into the ROSA26 locus, allowing for activation of GFP using Cre recombinase. This acts as an ideal control for the DNMAML-GFP mouse line. Importantly, we did not observe any cardiac defects in either of these control groups (Table 1).
DNMAML is activated by Pax3Cre specifically in derivatives of the neural crest and somites and does not affect neural crest migration. One possible explanation for the cardiac phenotype in these mice is that inhibition of Notch signaling causes a defect in neural crest cell number or migration from the neural tube into the cardiac outflow tract. To follow the fate of neural crest cells in the Pax3Cre/+ DNMAML mice and to ensure that DNMAML was activated specifically within neural crest cells and their derivatives, we made use of the GFP tag on the DNMAML molecule. We compared these embryos with control embryos in which Pax3Cre/+ was crossed with Z/EG mice, which contain a Cre-inducible GFP reporter (32). Pax3Cre/+ DNMAML embryos at E10.5 demonstrated DNMAML-GFP expression as expected in tissues derived from Pax3-expressing precursors, including neural tube, pharyngeal arches, and somites. This pattern was identical to that of Pax3Cre/+ Z/EG controls (Figure 3, A and B).
DNMAML-GFP is activated specifically in neural crest and somites by Pax3-Cre and does not affect neural crest cell number or migration. (A and B) E10.5 Pax3Cre/+ Z/EG control (A) and Pax3Cre/+ DNMAML mutant (B) embryos demonstrating expression of DNMAML-GFP in pharyngeal arches (arrows) and somites (arrowheads). (C and D) Immunostaining for GFP with Hoechst nuclear counterstain on frontal sections through the pharyngeal arches of E10.5 Pax3Cre/+ DNMAML embryos. (C) Low-magnification view showing GFP-positive cells investing the third, fourth, and sixth aortic arch arteries. (D) Higher magnification showing GFP expression specifically in the neural crest–derived mesenchyme of the pharyngeal arch (nc), but not in pharyngeal epithelium (ep) or endothelial cells (ec). (E and F) Immunostaining for GFP on frontal sections through the conotruncus of E11.5 embryos, showing an equivalent number of GFP-positive cells in the conotruncal cushions (arrows) of control (E) and mutant (F) embryos. (G–J) Immunostaining for GFP and α-SMA on frontal sections through the aortic arch arteries of E11.5 embryos. (G and H) Low-magnification view showing equivalent numbers of GFP-positive cells in the pharyngeal region surrounding the 6 major aortic arch arteries (arrows) in control (G) and mutant (H) embryos. (I and J) High-magnification views of the left sixth aortic arch arteries shown in G and H. (K–N) In situ hybridizations for the neural crest cell marker PlexinA2 on frontal sections through the conotruncus (K and L) and the aortic arch arteries (M and N) of E11.5 embryos, showing equivalent expression in control (K and M) and mutant (L and N) embryos. (O and P) GFP expression in the mature aortic arch of control Pax3Cre/+R26RGFP (O) and mutant Pax3Cre/+ DNMAML (P) mice. Scale bars: 100 μm (C, E–H, and K–N), 20 μm (D).
The developing cardiac outflow tract and aortic arch arteries were observed more closely by sectioning and immunostaining using anti-GFP antibodies. Frontal sections through the pharyngeal arches at E10.5 revealed DNMAML-GFP expression specifically in the neural crest–derived mesenchyme, while it was absent in the pharyngeal endoderm and the endothelium of the aortic arch arteries (Figure 3, C and D). At E11.5, Pax3 derivatives expressing DNMAML-GFP were readily apparent in the cushions of the developing outflow tract and were found in numbers equivalent to those in control Pax3Cre/+ Z/EG embryos (Figure 3, E and F). Sections through the aortic arch arteries at E11.5 also revealed DNMAML-GFP expression throughout the pharyngeal region at levels equivalent to those in controls (Figure 3, G and H). High-magnification views showing costaining for GFP and α-SMA demonstrated that DNMAML-GFP–expressing neural crest cells were present surrounding the aortic arch arteries at the onset of smooth muscle differentiation (Figure 3, I and J).
As an additional method to track these cells, we performed in situ hybridizations for PlexinA2, a marker of migratory and postmigratory cardiac neural crest cells (33). When compared with Cre-negative controls, Pax3Cre/+ DNMAML embryos at E11.5 showed equivalent levels of PlexinA2 in the conotruncal cushions (Figure 3, K and L) and in the mesenchyme surrounding the aortic arch arteries (Figure 3, M and N).
Previous studies have shown that neural crest–derived vascular smooth muscle contributes to the mature aortic arch, ductus arteriosus, and the proximal regions of the major aortic arch branches (13, 28). Pax3Cre/+ DNMAML embryos at late gestation (E17.5–E18.5) showed DNMAML-GFP expression in these regions of the aortic arch, similar to the pattern seen in Pax3Cre/+R26RGFP control embryos (Figure 3, O and P).
Given these results, we conclude that expression of DNMAML within neural crest cells does not affect their overall numbers, their migration into the cardiac outflow tract, or their contribution to the mature aortic arch. This suggests that the cardiac defects in Pax3Cre/+ DNMAML mice are likely due to defective function of postmigratory neural crest cells.
Notch target genes are activated in the developing vascular smooth muscle layer of the aortic arch arteries, and their expression is inhibited by DNMAML. In order to analyze the spatial and temporal patterns of Notch activity in cardiac neural crest cells, we examined the expression of known Notch target genes in both wild-type and Pax3Cre/+ DNMAML embryos. We focused on members of the HRT gene family, as they have been shown to be important targets of Notch in the cardiovascular system (2).
At E10.0, prior to the onset of smooth muscle differentiation, HRT1 was expressed in the mesenchyme of the pharyngeal arches and in a thin layer lining the aortic arch arteries, consistent with endothelium-specific expression (data not shown). Neither HRT2 nor HRT3 showed significant expression around the aortic arch arteries at this time point (data not shown). By E11.5, HRT1, HRT2, and HRT3 showed distinct expression patterns in the cells surrounding the aortic arch arteries in control embryos (Figure 4, A–C). Immunostaining of adjacent sections for α-SMA showed that the activation of HRT genes coincides with the differentiation of neural crest cells into smooth muscle (Figure 4D). Importantly, the expression of all 3 HRT genes was significantly downregulated in the aortic arch arteries of Pax3Cre/+ DNMAML embryos. A small amount of signal was still detected in a thin layer lining the aortic arch arteries. This is consistent with remaining expression of the HRT genes in endothelial cells, where they have also been reported to play a critical role (2). HRT1 and HRT3 expression was also maintained in the non–neural crest–derived pharyngeal epithelium of the Pax3Cre/+ DNMAML mutants (Figure 4, E–G).
Notch target genes are expressed in the developing smooth muscle layer of the aortic arch arteries and are suppressed by DNMAML. (A–H) Frontal sections through the aortic arch arteries of E11.5 control (A–D) and Pax3Cre/+ DNMAML (E–H) embryos showing the aortic arch arteries (arrows) and pharyngeal epithelium (ep). Adjacent sections were used for in situ hybridizations for the Notch target genes HRT1, HRT2, and HRT3 (A–C and E–G) and immunostaining for α-SMA (D and H). (A–D) In control embryos, HRT1, HRT2, and HRT3 are expressed in the cells surrounding the aortic arch arteries, consistent with expression in the developing smooth muscle layer. (E–H) Pax3Cre/+ DNMAML embryo shows a loss of HRT1, HRT2, and HRT3 expression surrounding the aortic arch arteries, while HRT1 and HRT3 are maintained in the pharyngeal epithelium (ep), and a thin layer of HRT2-positive cells persists in the endothelium of the aortic arch arteries. (I) Semiquantitative RT-PCR from primary smooth muscle cells derived from the aortic arches of late-gestation control and Pax3Cre/+ DNMAML embryos. Cells were stimulated with immobilized control-Fc (ctrl) or Jagged1-Fc (Jag1). Jagged1 stimulation induces HRT1, HRT2, and HRT3 expression in control cells (lane 2) but not in DNMAML-expressing cells (lane 4). In contrast, expression of the Mef2 target genes histidine-rich calcium-binding protein (HRC) and Mef2c is unchanged in cells expressing DNMAML compared with controls. Scale bars: 100 μm.
It was recently reported that, in addition to its role in the Notch transcriptional complex, MAML1 also acts as a coactivator for Mef2c, a member of the MEF2 (myocyte enhancer family 2) family of transcription factors that can influence muscle-specific gene activation (34). We therefore determined whether DNMAML could inhibit Mef2c in addition to Notch. Using luciferase reporter assays, we show that even a low dose of DNMAML robustly inhibited the activity of a Notch-responsive promoter. On the other hand, similar levels of DNMAML had no effect on an artificial promoter containing 4 Mef2 binding sites (Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/JCI30070DS1). Furthermore, a reporter construct driven by an endogenous Mef2c target promoter (histidine-rich calcium-binding protein–luciferase [HRC-luciferase]) was not inhibited over a broad range of both Mef2c and DNMAML doses (Supplemental Figure 2).
The reporter assays suggest that DNMAML acts as a much more potent inhibitor of Notch than Mef2c, but they do not rule out the possibility that DNMAML may inhibit Mef2c in some contexts. Therefore, we also addressed the effect of DNMAML on endogenous Notch and Mef2c targets in our system. We utilized primary smooth muscle cell cultures generated from the aortic arches of Pax3Cre/+ DNMAML and Cre-negative DNMAML (control) embryos. When stimulated with the Notch ligand Jagged 1, control smooth muscle cells demonstrated significant increase in expression of HRT1, HRT2, and HRT3. This increase in expression was completely blocked in DNMAML-expressing smooth muscle cells generated from the Pax3Cre/+ DNMAML embryos (Figure 4I). Importantly, we were unable to detect differences in the expression levels of the Mef2 target genes HRC (35) or Mef2c (Figure 4I). Therefore, in the context of our experiments, DNMAML appears to act as a potent and specific inhibitor of Notch-dependent, but not Mef2c-dependent, transcriptional activation.
Together, these results show that Notch signaling is active in postmigratory neural crest cells, specifically in the developing smooth muscle layer of the aortic arch arteries, and that DNMAML can effectively and specifically block this activity. It also suggests that HRT1, HRT2, and HRT3 are critical Notch targets in neural crest–derived vascular smooth muscle.
Neural crest–specific DNMAML expression is associated with loss of vascular smooth muscle cell markers. We propose that Notch functions in the postmigratory neural crest to mediate proper development of the cardiac outflow tract and remodeling of the aortic arch arteries. As a major fate of cardiac neural crest cells is to form the vascular smooth muscle cells of the aortic arch, we carefully examined the effect of DNMAML on vascular smooth muscle marker gene expression in vivo. First, we crossed Pax3Cre/+ DNMAML with mice in which LacZ is knocked into the _SM22_α locus (SM22_α_LacZ/+ mice) (36). At E11.5, all control SM22_α_LacZ/+ embryos showed strong LacZ activity in the 6 main aortic arch arteries (left and right third, fourth, and sixth arch arteries) (Figure 5, A and B). However, all Pax3Cre/+ DNMAML, SM22_α_LacZ/+ embryos showed diminished or absent LacZ activity in 1 or more of the 6 aortic arch arteries (Figure 5, E and F). The left sixth aortic arch artery, the embryonic precursor of the pulmonary artery and ductus arteriosus, was disproportionately affected. However, diminished staining was also observed in the third and fourth aortic arch arteries in some of the embryos.
Neural crest–specific inhibition of Notch results in loss of smooth muscle markers in the developing aortic arch arteries. (A, B, E, and F) Lateral views of the aortic arch arteries as visualized by whole mount staining for β-galactosidase activity in E11.5 SM22_α_LacZ/+ embryos. Control embryo shows strong staining in all 3 major aortic arch arteries on both the right (A) and left (B). In contrast, a Pax3Cre/+ DNMAML embryo shows absence of β-galactosidase activity in the right sixth aortic arch artery (E) and diminished β-galactosidase activity in the left sixth aortic arch artery (F). (C, D, G, and H) Immunohistochemistry for α-SMA on frontal sections through the developing aortic arch arteries of E12.5 embryos. At low magnification, the smooth muscle layer surrounding all aortic arch arteries is robust and uniform in control embryos (C). However, a Pax3Cre/+ DNMAML embryo shows diminished α-SMA staining in the sixth aortic arch arteries but normal staining in the other vessels (G). (D and H) High-magnification views of the left sixth aortic arch artery shown in C and G. Compared with control (D), the mutant artery (H) shows diminished α-SMA staining and a general disruption in the architecture of the smooth muscle layer. Scale bars: 100 μm (C and G), 20 μm (D and H).
In addition, we performed immunohistochemistry for α-SMA, another marker of vascular smooth muscle cells. Frontal sections through wild-type embryos at E12.5 showed a thick layer of α-SMA–positive cells surrounding all 6 aortic arch arteries (Figure 5C). However, Pax3Cre/+ DNMAML embryos showed localized downregulation of α-SMA staining in 1 or more of the aortic arch arteries (Figure 5G). As was observed with the SM22αLacZ marker, the sixth aortic arch arteries were disproportionately affected. High-magnification views of sections through the left sixth aortic arch artery of wild-type embryos showed strongly staining smooth muscle cells arranged in an organized, concentric pattern around the vessel (Figure 5D). On the other hand, the left sixth aortic arch arteries of Pax3Cre/+ DNMAML embryos often showed a disorganized smooth muscle cell layer with diminished α-SMA staining (Figure 5H).
These results demonstrate a highly reproducible defect in neural crest–derived vascular smooth muscle cells during the time period critical for aortic arch artery development. The disproportionate effect on smooth muscle markers in the sixth aortic arch artery is consistent with the high frequency of pulmonary artery and ductus arteriosus defects observed in the late-gestation embryos.
Activation of DNMAML after the onset of smooth muscle differentiation has no effect on aortic arch artery remodeling. A number of in vitro studies indicate that Notch can play a role in the modulation of vascular smooth muscle cell proliferation, apoptosis, migration, and contractility (15–18). One model to explain the aortic arch defects in these mice is that defective vascular smooth muscle cell proliferation or survival may lead to loss of aortic arch artery integrity and subsequent defects in aortic arch remodeling. However, we failed to see any differences in apoptosis or cell division between Pax3Cre/+ DNMAML and control embryos using TUNEL assays and staining for proliferation markers at E11.5 and E12.5 (data not shown).
To directly test the effect of Notch inhibition after the onset of smooth muscle differentiation, we also crossed the DNMAML mice with SM22_α_-Cre mice (37). Unlike the Pax3Cre/+ DNMAML embryos, which showed strong expression of DNMAML at E10.5, prior to the onset of smooth muscle differentiation, the SM22_α_-Cre DNMAML embryos did not express DNMAML until after the activation of smooth muscle markers surrounding the aortic arch arteries (Supplemental Figure 3). These mice failed to show defects in the smooth muscle layer of the developing aortic arch arteries (Supplemental Figure 3) and also did not display any cardiac outflow tract or aortic arch defects at later time points (Table 1). These studies indicate that the critical time period for Notch activity in cardiac neural crest–derived tissues is prior to E11.5. Therefore, we do not believe that an effect of Notch on smooth muscle proliferation or survival could account for the defects seen in the neural crest–specific DNMAML mice.
Notch promotes the differentiation of cardiac neural crest precursors into smooth muscle ex vivo. Another explanation for the smooth muscle defects in these mice is the possibility that Notch plays a critical role in the differentiation of neural crest into smooth muscle. We used neural tube explant assays to address this possibility. Neural tubes from the cardiac neural crest region were isolated from E8.5 embryos and grown in culture medium containing serum for 48 hours. Under these conditions, neural crest cells from control embryos readily migrated out of the explanted neural tube and differentiated robustly into smooth muscle cells, as determined by immunofluorescence staining for α-SMA or SM22α (Figure 6, A and D). On the other hand, neural tube explants from Pax3Cre/+ DNMAML embryos showed a dramatic reduction in the number of α-SMA–positive cells (Figure 6B). Some of the explants we examined showed inefficient Cre-mediated recombination, as determined by costaining for GFP. In these explants, the majority of DNMAML-GFP–positive cells were α-SMA negative, while adjacent DNMAML-GFP–negative cells in the same explant were usually α-SMA positive. This demonstrates that the inhibition of smooth muscle differentiation by DNMAML was cell autonomous (Figure 6C).
Inhibition of Notch activity blocks the differentiation of neural crest precursors into smooth muscle ex vivo. (A–C) Neural tube explants from E8.5 control (A) and Pax3Cre/+ DNMAML (B and C) embryos, immunostained for α-SMA (green) and GFP (red). The majority of cells in the control explant express α-SMA, as do the DNMAML-GFP–negative cells in the Pax3Cre/+ DNMAML explants (B and C). Conversely, most of the DNMAML-GFP–positive cells in B and C are α-SMA negative. (D–F) Wild-type neural tube explants treated with the γ-secretase inhibitor DAPT, immunostained for α-SMA (green) and SM22α (red). Compared with DMSO-treated controls (D), explants treated with 1 μM DAPT (E) or 5 μM DAPT (F) show a decrease in the number of cells expressing smooth muscle markers. (G) Quantification of neural tube explant assays from Pax3Cre/+R26RGFP (control) and Pax3Cre/+ DNMAML embryos. (H) Quantification of assays on neural tube explants treated with the γ-secretase inhibitor DAPT. Error bars indicate 1 SD. P values, indicated by brackets, were determined by Student’s t test. Scale bars: 100 μm.
We quantified this effect by counting cells from the explants. To control for any nonspecific effects of GFP, we also performed neural tube explant assays on Pax3Cre/+R26RGFP embryos. In these control embryos, there was no significant difference in α-SMA expression between GFP-negative and GFP-positive cells (Figure 6G, first and second bars). On the other hand, in Pax3Cre/+ DNMAML explants, an average of 29% of DNMAML-GFP–positive cells coexpressed α-SMA, compared with 71% of DNMAML-GFP–negative cells (Figure 6G, third and fourth bars).
As an additional method of inhibiting Notch, we treated wild-type neural tube explants with the γ-secretase inhibitor DAPT. Both 1 μM and 5 μM DAPT resulted in a statistically significant reduction in the number of cells expressing either α-SMA or SM22α when compared with DMSO-treated controls (Figure 6, D–F and H). Therefore, DAPT mimics the effect of DNMAML, further supporting the specificity of DNMAML as an inhibitor of Notch signaling. Together, these results demonstrate that Notch promotes the differentiation of cardiac neural crest precursors into smooth muscle.