Murine Jagged1/Notch signaling in the second heart field orchestrates Fgf8 expression and tissue-tissue interactions during outflow tract development (original) (raw)
Inhibition of Notch in the second heart field results in cardiovascular defects. To elucidate the role of Notch in the second heart field, we took advantage of the fact that all 4 mammalian Notch receptors (Notch1–4) interact with mastermind-like proteins after activation and translocation to the nucleus. A dominant-negative truncated form of mastermind-like (DNMAML) is able to interact with intracellular Notch but cannot recruit transcriptional coactivators. This construct has been shown to be an effective and specific inhibitor of Notch signaling (16, 22). We used mice in which a fusion of DNMAML and GFP has been inserted into the Rosa26 locus downstream of a floxed PGK Neo cassette such that Notch can be inhibited in a tissue-specific manner after activation by Cre recombinase (22). DNMAML mice were crossed with 2 different Cre recombinase lines that each express in the second heart field. The first of these lines contains Cre knocked into the Islet1 locus (Islet1Cre) (23). The second is a transgenic line in which Cre is under control of a promoter fragment from the Mef2c gene, which has been shown to drive expression specifically in the anterior/second heart field (Mef2c-AHF-Cre) (1). Activation of DNMAML with either of these Cre lines resulted in fully penetrant neonatal lethality associated with severe cardiac defects (Table 1 and Supplemental Table 1; supplemental material available online with this article; doi:10.1172/JCI38922DS1). At late gestation, mutant embryos were found at approximately the expected Mendelian ratios. However, no mutant offspring survived after 1 day of age from either cross (Supplemental Table 1).
Summary of phenotypes in second heart field mutants (E17.5 to P0)
Analysis of the hearts of late-gestation and neonatal Islet1Cre/+;DNMAML mutants demonstrated a number of severe cardiac abnormalities (Table 1). All of the mutants had OFT defects, including persistent truncus arteriosus or double outlet right ventricle (Figure 1, B and C). In addition, nearly all mutants had aortic arch defects including right-sided aortic arch, hypoplastic aortic arch, or retroesophageal subclavian arteries (Table 1 and Figure 1, B and I). We also observed several vascular rings, a potentially lethal congenital malformation in which vascular structures surround and constrict the trachea and esophagus (Figure 1C). Sectioning through the Islet1Cre/+;DNMAML hearts also revealed multiple intracardiac defects. Three of 10 hearts demonstrated severe right ventricular hypoplasia and tricuspid valve atresia (Figure 1M). All 3 of these mutants had atrial septal defects allowing passage of blood from the right to left atrium (Figure 1N). All Islet1Cre/+;DNMAML mutants displayed ventricular septal defects (Figure 1O). In addition to cardiovascular defects, the mutants also showed disruption of other pharyngeal structures, including the thymus (Table 1). These results demonstrate a critical role for Notch in the development of the cardiac OFT and right ventricle, the cardiac inflow tract, and pharyngeal structures including the aortic arch and the thymus. Islet1Cre/+ mice had no discernible cardiac defects (refs. 23, 24; data not shown), indicating neither Islet1 haploinsufficiency nor Cre expression could account for the phenotype in the Islet1Cre/+;DNMAML mutants.
Cardiovascular defects in Notch second heart field mutants. E17.5–P0 control (A), Islet1Cre/+;DNMAML (B and C), and Mef2c-AHF-Cre;DNMAML (D–F) mutants. (A) Arrows indicate pulmonary artery and aorta. (B) A single OFT vessel (arrow) and retroesophageal right subclavian artery. (C) Double outlet right ventricle (arrows) with a right aortic arch and left ductus arteriosus producing a vascular ring. (D) Cervical right aortic arch (arrow). (E) Interruption of the aortic arch, type B (IAA-B, arrow), and retroesophageal right subclavian artery. (F) Double aortic arch (arrows). (G–P) H&E sections of E17.5–E18.5 hearts. (G) Control with separate aorta and pulmonary artery. (H) Islet1Cre/+;DNMAML mutant with single OFT arising from right ventricle. (I) Islet1Cre/+;DNMAML mutant with retroesophageal right subclavian artery dorsal to the trachea. (J and K) Sections from a Mef2c-AHF-Cre+;DNMAML mutant showing a vascular ring. A right-sided circumflex arch (J) runs posterior to the trachea and esophagus en route to a left-sided descending aorta and left ductus arteriosus (K). (L) Control with normal tricuspid valve (arrow). (M and N) Islet1Cre/+;DNMAML with tricuspid atresia (arrow, M), right ventricular hypoplasia, and atrial septal defect (arrow in N). (O and P) Islet1Cre/+;DNMAML (O) and Mef2c-AHF-Cre+;DNMAML (P) hearts showed ventricular septal defects (arrows). ao, aorta; da, ductus arteriosus; e, esophagus; IAA-B, interruption of the aortic arch, type B; la, left atrium; lca, left carotid artery; lsa, left subclavian artery; lv, left ventricle; pa, pulmonary artery; pv, pulmonic valve; ra, right atrium; rca, right carotid artery; rersa, retroesophageal right subclavian artery; rsa, right subclavian artery; rv, right ventricle; ta, truncus arteriosus; tr, trachea; tv, tricuspid valve. Original magnification: ×30 (A–F). Scale bars: 250 μm (G–K), 500 μm (L–P).
The hearts of Mef2c-AHF-Cre+;DNMAML mutants demonstrated similar defects to those seen in the Islet1Cre/+;DNMAML mice (Table 1 and Figure 1, D–F), though the OFT defects were slightly less severe. All of the mutants analyzed had aortic arch defects. The most common defects seen in the Mef2c-AHF-Cre+;DNMAML mutants were right-sided aortic arch and cervical aortic arches, extending high in the neck (Figure 1D). Complete interruption of the aortic arch (Figure 1E), double aortic arch (Figure 1F), aberrant subclavian arteries (Figure 1, D, E, J, and K) and vascular rings (Figure 1, J and K) were also observed. Sectioning through the hearts of Mef2c-AHF-Cre+;DNMAML mutants revealed ventricular septal defects in 100% of mutants (Figure 1P and Table 1).
In order to determine which tissues were expressing DNMAML in Islet1Cre/+;DNMAML and Mef2c-AHF-Cre;DNMAML mutants, we performed immunostaining for GFP to detect the DNMAML-GFP fusion protein (Supplemental Figure 1). Mid-gestation Islet1Cre/+;DNMAML embryos showed DNMAML-GFP expression in the pharyngeal mesenchyme and the pharyngeal endoderm, while Mef2c-AHF-Cre;DNMAML embryos showed DNMAML-GFP expression in the mesenchyme only (Supplemental Figure 1, A and B). At late gestation, Islet1Cre/+;DNMAML embryos expressed DNMAML-GFP in derivatives of the second heart field, including the base of the truncus arteriosus, myocardium of the OFT and right ventricle, a small subset of cells in the left ventricle, the inflow tract, and both atria, as well as endoderm derivatives (Supplemental Figure 1, C and D). A similar pattern was seen when Islet1Cre and Mef2c-AHF-Cre mice were crossed with R26RLacZ to generate a fate map (Supplemental Figure 1, E and F). In summary, Islet1Cre resulted in Cre-mediated recombination broadly throughout the heart including both the OFT and inflow tract, as well as in the pharyngeal endoderm derivatives. Mef2c-AHF-Cre mice had more restricted Cre-mediated recombination in the right ventricle and proximal OFT. Therefore, it is possible that the atrial septal defects in Islet1Cre/+;DNMAML mice are, in part, due to defects of inflow tract formation. Importantly, neither Cre line results in significant recombination in the aortic arch or its major branches. These results are consistent with other reports using these mouse lines (1, 25). The broader expression pattern of the Islet1Cre/+ line, as well as the fact that Mef2c is downstream of Islet1 (26) and therefore likely activated later, may explain the more severe cardiac phenotypes observed in the Islet1Cre/+;DNMAML mutants when compared with the Mef2c-AHF-Cre;DNMAML embryos (Table 1).
Cardiac neural crest cell migration is altered by Notch inhibition in the second heart field. The spectrum of aortic arch defects in both the Islet1Cre/+;DNMAML and Mef2c-AHF-Cre+;DNMAML mice suggests a defect in the formation or remodeling of the aortic arch arteries earlier in development. To investigate this, we analyzed the aortic arch arteries of Islet1Cre/+;DNMAML mutants at E10.5. To visualize the endothelium of the aortic arch arteries, we performed immunostaining for the endothelial marker PECAM on sections through the pharyngeal arches of control and mutant embryos (Figure 2, A–D). In control embryos the lumens of the third, fourth, and sixth aortic arch arteries were large and clearly patent (Figure 2, A and C). However, Islet1Cre/+;DNMAML mutants showed generalized hypoplasia of the fourth pharyngeal arch and severe narrowing or complete absence of the fourth and sometimes the sixth aortic arch arteries (Figure 2, B and D). Similar findings were also present in the Mef2c-AHF-Cre;DNMAML mutants (data not shown). The fourth embryonic aortic arch arteries are the precursors of the main segment of aortic arch and the proximal region of the right subclavian artery. Therefore, defective formation of the fourth arch artery is consistent with the types of aortic arch defects that were observed in the late-gestation embryos, such as interrupted or right-sided aortic arches and aberrant subclavian arteries.
Notch inhibition in the second heart field results in abnormal patterning of the pharyngeal arches and impaired cardiac neural crest cell migration. (A–D) PECAM immunostaining of coronal sections through E10.5 embryos to visualize the endothelium of the aortic arch arteries (numbered). Note the hypoplasia of the fourth pharyngeal arch and narrowing of the fourth and sixth aortic arch arteries in the mutant (B). (C and D) Higher-magnification views of the fourth aortic arch artery in control (C) and Islet1Cre/+;DNMAML (D) embryos show severe hypoplasia in the mutant. (E–H) In situ hybridization for the neural crest markers Sema3C (E and F) and PlexinA2 (G and H) of coronal sections through the OFT of E11.5 control (E and G) and mutant (F and H) embryos. Dotted lines in G highlight neural crest cells within the outflow endocardial cushions expressing PlexinA2. Control genotype was Islet1+/+;DNMAML. Scale bars: 50 μm (A–D), 100 μm (E–H).
Cardiac neural crest cells populate the aortic arches and cardiac OFT and play critical roles in patterning of the aortic arch arteries and in septation of the truncus arteriosus. We examined the expression of markers of cardiac neural crest by in situ hybridization in the developing OFTs of wild-type and mutant embryos. PlexinA2 and Sema3C were both expressed by post-migratory cardiac neural crest. In wild-type embryos, expression is evident in 2 clusters of cells within the outflow endocardial cushions that represent the leading edge of the 2 prongs of cardiac neural crest invading the OFT (27). However, in Islet1Cre/+;DNMAML mutants, expression in this region was lost, though myocardial expression of Sema3C was preserved (Figure 2, F and H). We examined indexes of apoptosis and proliferation at E9.5–E10. No significant difference in the percentage of phospho–histone H3–positive neural crest cells was apparent in the ventral pharynx between E9.5 Islet1Cre/+;DNMAML (3.91% ± 1.19%, n = 6) and Islet1+/+;DNMAML (2.90% ± 2.46%, n = 6) embryos (P = 0.39). In addition, we did not detect a significant difference in apoptosis (as assayed by TUNEL staining) of neural crest cells populating the ventral pharynx (Islet1Cre/+;DNMAML: 1.05% ± 0.90%, n = 6, versus Islet1+/+;DNMAML: 1.57% ± 1.28%, n = 4; P = 0.47) (Supplemental Figure 2). These results suggest that inhibition of Notch in the secondary heart field affects migration of cardiac neural crest cells into the OFT, suggesting that a Notch-dependent signal from the second heart field can modulate cardiac neural crest behavior.
The Notch ligand Jag1 is required in the second heart field. The expression pattern of Jag1 suggests that it is a candidate to act as an important Notch ligand in the second heart field. At E10.5 and E11.5, Jag1 is expressed in the pharyngeal endoderm, pharyngeal mesenchyme, and the OFT of the heart (Figure 3, A and B). In addition, it is expressed in the endothelium and smooth muscle of the aortic arch arteries (Figure 3B). To understand the role of Jag1 in the second heart field, we crossed Islet1Cre mice with mice harboring a conditional allele of Jag1 (Jag1flox). Immunostaining for Jag1 at E11.5 revealed efficient deletion in pharyngeal mesenchyme and the OFT of Islet1Cre/+;Jag1flox/flox, while expression was maintained in the region of the aortic arch arteries (Figure 3, C and D).
Jag1 is an essential Notch ligand in the second heart field. (A and B) In situ hybridizations for Jag1 in control embryos. (A) Jag1 expression in pharyngeal endoderm (arrows, E10.5 embryo). (B) Jag1 expression in pharyngeal mesenchyme (arrow), OFT (arrowheads), and aortic arch arteries (asterisks, E11.5 embryo). (C and D) Jag1 immunostaining through E11.5 control (C) and mutant (D) embryos demonstrated loss of Jag1 expression in pharyngeal mesenchyme (arrow) and OFT (black arrowheads) of the mutant. Expression was maintained in the aortic arch arteries (white arrowheads). (E–G) Photographs and diagrams of hearts from E18.5 control (E) and mutant (F and G) embryos. (E) Arrows indicate pulmonary artery and aorta. (F) Double outlet right ventricle (arrows). (G) Double outlet right ventricle (arrows) and an interrupted aortic arch. (H–K) H&E-stained sections of E18.5 control (H and J) and mutant (I and K) hearts, demonstrating a double outlet right ventricle and a ventricular septal defect (arrow). (L–O) In situ hybridization of E10.5 control (L and N) and mutant (M and O) embryos. Control sections (L and N) demonstrated normal neural crest expression of Sema3C (L) and PlexinA2 (N) in the 2 clusters of cells in the center of the developing OFT cushions (dotted circles in N), while mutants (M and O) demonstrated reduced expression. Original magnification, ×30 (E–G). Scale bars: 250 μm (A, C, D, H, and I), 500 μm (B, J, and K), 100 μm (L–O).
Islet1Cre/+;Jag1flox/flox mutants died in the neonatal period (Supplemental Table 1). Analysis of late-gestation embryos revealed severe cardiac and aortic arch defects in Islet1Cre/+;Jag1flox/flox mutants similar to those seen in the second heart field DNMAML mutants, and included persistent truncus arteriosus, double outlet right ventricle, ventricular septal defects, atrial septal defects, and aortic arch defects such as right-sided aortic arch and interrupted aortic arch (Figure 3, F–K, and Table 1). Similar cardiovascular defects were seen when Mef2c-AHF-Cre was used to delete Jag1 (Table 1). Expression of the neural crest markers Sema3C and PlexinA2 is deficient in the OFT cushions of Islet1Cre/+;Jag1flox/flox embryos (Figure 3, L–O). Thus, second heart field–specific Jag1 deletion phenocopies the second heart field DNMAML mutants, suggesting that Jag1 functions as an essential Notch ligand in the second heart field.
Notch inhibition diminishes Fgf8 expression in the developing OFT. We examined the hearts of Isl1Cre/+;DNMAML embryos at E9.5, a relatively early stage, when the second heart field contributes a substantial number of progenitors to the developing right ventricle and OFT. At this time, the mutants showed significant hypoplasia of the right ventricle and OFT when compared with littermate controls (Figure 4, A and B). These results show that Notch plays a critical role in the early patterning of second heart field–derived structures. H&E staining of sections through the OFT at E10.5 demonstrated a statistically significant reduction in the cellularity of the endocardial cushions in the mutant embryos (Figure 4, C and D, and Supplemental Figure 3). The clusters of condensed mesenchyme of neural crest cells within the endocardial cushions, which can be discerned with H&E staining, were not evident in the mutant when compared with wild-type control (Figure 4, C and D).
Notch regulates Fgf8 expression in the second heart field. (A and B) Light microscopy of E9.5 hearts of control (A) and Islet1Cre/+;DNMAML mutants (B). The mutant OFT (arrow) and right ventricle (arrowhead) were hypoplastic. (C and D) H&E-stained sections of E10.5 OFT cushions in control (C) and Islet1Cre/+;DNMAML mutant (D). The mutant OFT cushions (arrowheads) were hypocellular (D). (E and F) Quantitative RT-PCR of DAPT-treated cultured pharyngeal explants (E) or tissue directly isolated from E10.5 embryos (F), expressed relative to controls. (E) The data represent an average of 4 independent experiments, with error bars indicating 1 standard deviation. Asterisks indicate gene products that showed statistically significant changes in all 4 experiments (P < 0.02). (F) Fgf8 expression levels in the anterior pharynx and OFT of E10.5 control and mutant embryos. The data represent the average of 3 pools of 3–4 embryos per genotype. (G–J) In situ hybridizations showing Fgf8 expression in the OFT of E9.5 control (G and I) and mutant embryos (H and J). Arrowheads point to developing OFT myocardium. (K and L) In situ hybridization for Bmp4 at E11.5 in control (K) and mutant (L) embryos. (M and N) Immunohistochemistry for phospho-Smad in the OFT at E10.5 of control (M and O) and mutant (N and P) embryos showed robust phospho-Smad expression in the OFT cushions (circled) and endothelium (arrowheads) of controls with decreased expression in mutants. Control genotype was Islet1+/+;DNMAML. Scale bars: 100 μm (C–H, K–P), 250 μm (A, B, I, and J).
In order to identify downstream molecules involved in Notch signaling in the second heart field, we utilized a pharyngeal explant assay. In this assay, a region of the pharynx and the distal OFT was dissected from E9.5 mouse embryos and cultured in vitro. This region of the embryo is enriched for Islet1-positive progenitor cells that reside in the anterior pharynx and cardiac OFT and also includes surrounding tissues including the pharyngeal endoderm, pharyngeal ectoderm, and neural crest cells (28). Wild-type pharyngeal explants were treated with the γ-secretase inhibitor DAPT to inhibit Notch signaling. The explants were then analyzed by quantitative RT-PCR for the expression of a number of candidate genes involved in OFT development. Of several candidate genes examined, Fgf8 was notable for being altered in 4 of 4 independent experiments (Figure 4E). We also examined the Ets family transcription factors Pea3, Erm, and Er81, which have been implicated downstream of Fgf8. Pea3 was significantly downregulated in the DAPT-treated explants. Erm and Er81 also trended downward, but the decrease was not statistically significant. We also observed decreased expression of the Notch target genes Hrt1 and Hrt3, indicating that DAPT treatment effectively inhibited Notch signaling. Interestingly, Hrt2 expression was unchanged, consistent with several reports suggesting that regulation of this gene in the heart may not be strictly Notch dependent (29–31). Additional candidates that were tested using the explant assay but that did not reveal any statistically significant differences included Islet1, Mef2c, Tbx1, and Fgf10 (Figure 4E). The relative preservation of expression of these and other markers of cardiac OFT development suggests that the downregulation of Fgf8 is a specific effect of γ-secretase inhibition, rather than a nonspecific result of gross perturbations in patterning of the cardiac mesoderm.
Based upon the results of the explant assays, we examined Fgf8 mRNA expression by quantitative RT-PCR on tissue directly dissected from the pharyngeal region and cardiac OFT of wild-type and Islet1Cre/+;DNMAML mutant embryos at E10.5. These experiments revealed a statistically significant reduction in Fgf8 expression in mutants when compared with littermate controls (Figure 4F). Furthermore, in situ hybridization on sections through the OFT of E9.5 embryos demonstrated a clear reduction in Fgf8 mRNA in the developing OFT myocardium in the Islet1Cre/+;DNMAML mutants (Figure 4, G–J). In order to determine whether this reduction was due to a change in the number of Islet1-expressing cells in the OFT of mutant embryos, we performed immunohistochemistry and counted positively staining cells on serial sections of E10.5 Islet1Cre/+;DNMAML embryos compared with Islet1Cre/+ embryos, but we did not detect a significant difference (Islet1Cre/+;DNMAML: 325.4 ± 110.3 cells, n = 5, versus Islet1Cre/+: 347.3 ± 33.5 cells, n = 4; P = 0.72). Similar results were found when comparing E10.0 Islet1Cre/+;Jag1flox/+ (226.4 ± 26.8 cells, n = 6) with Islet1Cre/+;Jag1flox/flox littermates (257.0 ± 72.2 cells, n = 5) littermates (P = 0.503) (Supplemental Figure 4).
We also used in situ hybridization to examine the expression of Bmp4, a downstream effector of Fgf8 signaling in the cardiac OFT (24, 32, 33). Bmp4 expression was dramatically reduced in the OFT cushions and surrounding myocardium of Islet1Cre/+;DNMAML mutants compared with wild-type embryos (Figure 4, K and L). Bmp signaling results in Smad phosphorylation, and immunohistochemistry revealed decreased phospho-Smad1/5/8 levels in the cardiac mesenchyme populating the OFT cushions and the endothelium lining the OFT of Islet1Cre/+;DNMAML mutants compared with wild-type (Figure 4, M–P). Similar abnormalities of endocardial cushion development, and reductions in Fgf8, Bmp4, and phospho-Smad1/5/8 expression, were observed in the Islet1Cre/+;Jag1flox/flox embryos (Supplemental Figure 5). Thus, Notch inhibition or Jag1 deletion in the second heart field results in abnormal endocardial cushions, reduced myocardial Fgf8 in the OFT, and reduced Bmp signaling.
Notch signaling in the second heart field mediates Fgf8-dependent EMT in the OFT cushions. Endocardial cushions form through a process of EMT in which endothelial cells lining the anlagen of the cardiac valves respond to signals from overlying myocardium to invade the extracellular matrix of the cardiac jelly (34). The hypocellularity of the endocardial cushions in Islet1Cre/+;DNMAML mutants suggested that EMT might have been altered. Therefore, we used an established explant assay system to investigate EMT in vitro (35). Microdissected E10.5 OFT explants, including endothelium and myocardium derived from the second heart field in the region of the forming endocardial cushions, were placed on collagen gels and cultured for 24–72 hours. At all time points examined (24, 48, and 72 hours), wild-type explants exhibited elongated cells migrating away from the beating myocardium and invasion of the collagen gel (Figure 5, A and E data not shown). Many fewer cells were evident in explants derived from Islet1Cre/+;DNMAML mutants at all time points examined (Figure 5, C and G; data not shown). Moreover, the cells that did migrate away from the explant were circular and of abnormal morphology (Figure 5, C and G). Quantification of explants pooled from 3 independent experiments resulted in a nearly 50% reduction in average radial migration at 24, 48, and 72 hours (Figure 5, I–K).
Defective EMT in Islet1Cre/+;DNMAML mutants is rescued by rFgf8. (A–D) Representative images of outflow explants taken 48 hours (24 hours after treatment with rFgf8 or vehicle) after plating. Control explants treated without (A) or with (B) rFgf8 underwent EMT and invasion into the collagen gel. (C) Islet1Cre/+;DNMAML explants displayed deficient EMT. (D) Addition of rFgf8 to mutant explants rescued EMT. (E–H) Similar results were seen at 72 hours. (I–K) Quantitative analysis was performed on samples pooled from 3 independent experiments at 24, 48, and 72 hours. Error bars represent 1 standard deviation. Control genotype was Islet1+/+;DNMAML. Scale bars: 100 μm.
These results demonstrate a tissue-specific requirement for Notch in second heart field–derived myocardium during EMT, suggesting that Notch may regulate a soluble signal from the myocardium that modulates EMT in the adjacent endothelial cells. Our finding of reduced Fgf8 expression in Islet1Cre/+;DNMAML mutants led us to test whether soluble Fgf8 could rescue EMT in mutant explants. Twenty-four hours after initial plating, we added recombinant Fgf8 (rFgf8) (50 ng/ml) or vehicle to wild-type and mutant explant cultures, and we analyzed the ability of explants to undergo EMT. While rFgf8 had little effect on wild-type explants (Figure 5, B and F), mutant explants showed a restoration of cellular morphology (Figure 5, D and H) and ability to invade the collagen matrix at both 48 and 72 hours (Figure 5, J and K). These results suggest that decreased Fgf8 observed in second heart field–derived myocardium of Islet1Cre/+;DNMAML mutants is at least partially responsible for defective EMT and hypocellular outflow endocardial cushions in these embryos, though changes in proliferation cannot be excluded.