N1ICD Expression During Epicardial Formation
An N1ICD-specific antibody against the conserved N-terminal V1744 amino acid of N1ICD was used for this study (Del Monte et al.,2007). By Western blot analysis, we detected a specific N1ICD band (110-kDa, arrow) in chicken and quail embryonic (Hamburger and Hamilton stage [HH] 30) hearts (Fig. 1A) and an additional lower molecular weight band (asterisk) in adult rat epicardial/mesothelial cells (ARMECs; Eid et al.,1992; Wada et al.,2003a) (Fig. 1B,C). Western blotting analysis showed that N1ICD levels were cell density dependent (Fig. 1C) and decreased in ARMECs treated with γ-secretase inhibitor (L685,485 at 10 μm, for 48 hr) compared with dimethyl sulfoxide (DMSO) vehicle exposed negative controls (Fig. 1B). The secretase inhibitor prevents Notch cleavage and therefore inhibits the formation of N1ICD.
Figure 1. Immunoblot analysis of Notch1 intracellular domain (N1ICD) levels in adult rat epicardial/mesothelial cells (ARMECs), quail (Hamburger and Hamilton stage [HH] 30) and chicken (HH30) heart tissue. A: The N1ICD antibody recognized a 110-kDa protein band (arrow) in quail (HH30) and chicken (HH30) whole heart lysates. B: In whole cell lysates of ARMECs, a 110-kDa band (arrow) and a smaller molecular weight band (asterisk, *) was detected by N1ICD antibody. Both protein bands decreased in intensity after treatment with γ-secretase inhibitor L-685,458 (10 μm, 48 hr) compared with that treated by dimethyl sulfoxide (DMSO) vehicle control (DMSO). C: N1ICD levels were elevated with increased cell density (25%, 50% and 100% confluence of ARMECs). D: Hypoxia (1% O2 incubation for 24 hr) and ectopic HIF1 expression [infection with adenovirus encoding constitutively active HIF1α (100MOI)) increased Notch1ICD (lower panel) levels correlated with the induction of HIF1α expression showed in upper panel. The 50-μg proteins per lane were loaded and β-actin was used as the loading control.
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At a stage when the tubular chicken heart is looped and beginning chamber differentiation, N1ICD was not expressed in any cell types in the PEO (Fig. 2B), while intense staining was present in endothelial cells of the endocardium of the atrial ventricular canal (AVC; Fig. 2B arrow) in a pattern similar to that described for E8.5–E10.5 mouse hearts (Schroeter et al.,1998; Serneels et al.,2005; Del Monte et al.,2007). The mesothelial cells on the surface of PEO were identified as epicardial cells by immunostaining for the epicardial marker, Wilms tumor suppressor gene1 (Wt1) protein, in the immediately adjacent section (Fig. 2C arrow). After the PEO cells attached to the myocardium and during epicardial monolayer formation at HH21, N1ICD immunostaining appeared in clusters of mesothelial cells that were covering the myocardium from the dorsal to the ventral surfaces (Fig. 2E, arrows). These cells were Wt1-positive in the immediately adjacent section (Fig. 2F, arrow). At this time point, the elongated mesothelial protrusions extending from the PEO at the sinus venosus and adhering to the myocardium was detected by Wt1 immunostaining (Fig. 2I). Some mesothelial cells on the PEO extension showed N1ICD staining (Fig. 2G,H, arrows) that was comparable in intensity to the signal in the endocardium (Fig. 2E, arrowhead). Lateral induction of Notch signaling was reported in cardiac valve development (Gittenberger-de Groot et al.,1998) and in other systems such as wing margin boundary formation in flies (Timmerman et al.,2004), induction of proneural domains in the ear in vertebrates (Panin et al.,1997), limb bud margin formation (Daudet and Lewis,2005), and somite boundary formation (Irvine and Vogt,1997). The formation of a contiguous cell layer may stimulate the lateral induction mechanism among mesothelial cells of the epicardium and induce the N1ICD expression.
Figure 2. Expression of Notch1 intracellular domain (N1ICD), Wilms tumor suppressor gene1 (Wt1), and vascular endothelial growth factor A (VEGFA) in Hamburger and Hamilton stage (HH) 17 and HH21 chicken hearts during epicardial formation. A: A semi-sagittal section of HH17 chicken heart. B,C: Higher magnifications of the boxed area in A. B: Nuclear staining for N1ICD (green) was absent in the cells of the pro-epicardial organ (PEO) at HH17 but was intense in endothelial cells of the endocardium at the atrial ventricular canal (AVC; solid arrowhead). C: An immediately adjacent 3-μm serial section was stained with Wt1, a marker for epicardial cells in this setting (red). Cardiomyocytes did not express Notch1ICD in the myocardial wall (empty arrowhead). D: A sagittal section of a HH21 heart. E,F: Higher magnifications of the boxed area in D. A few epicardial mesothelial cells covering the ventricular myocardium had nuclear staining for N1ICD (E, arrows) colocalized with Wt1 in a consecutive sections (F, arrows). G–I: Higher magnifications of the boxed area in E and F showed the N1ICD (G, arrows) colocalized with Wt1 in a consecutive sections (I, arrows). E and F were immediately adjacent sections. Nuclei were visualized by 4′,6-diamidine-2-phenylidole-dihydrochloride (DAPI) staining (blue; G,H). Scale bars = 500 μm in A (applies to D), 50 μm in B (applies to C,E,F), 5 μm in G (applies to H).
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N1ICD Expression During Epicardial EMT and EPDC Differentiation
To identify endothelial cell type specific N1ICD expression, the quail endothelial cell marker, QH1, was used for co-staining in embryonic quail hearts. At HH17 and HH21, N1ICD was not colocalized with QH1-positive cells in the PEO (data not shown). From HH17 (the earliest stage we studied), the expression of N1ICD was always present in the endothelial cells lining the endocardium of the ventricles, atria, and AV cushion, but it was never observed in cardiomyocytes in the compact myocardium. By HH25 when the epicardial coverage over the ventricular myocardium was complete, a subset of Wt1-positive epicardial mesothelial cells (Fig. 3B, empty arrowheads) and epicardial-derived mesenchymal cells were N1ICD-positive (Fig. 3C, arrowheads). Some QH1-positive endothelial cells at the AVJ (Fig. 3C, arrows) and some endothelial cells in myocardial vessels (Fig. 3B, arrows) showed intense N1ICD expression. The N1ICD immunostaining in mesothelial cells at the ventricular epicardium (Fig. 3B, empty arrowheads) was less intense compared with N1ICD immunostaining of endothelial cells suggesting an increase in N1ICD levels with EMT. Similarly, at the AVJ: endothelial cells and subepicardial mesenchymal cells (Fig. 3C, arrowhead) adjacent to the endothelial cells (Fig. 3C, arrows) showed more intense staining than mesothelial cells (Fig. 3C, empty arrowhead).
Figure 3. Expression of Notch1 intracellular domain (N1ICD), QH1 (quail endothelial cell marker), and vascular endothelial growth factor A (VEGFA) in sections of Hamburger and Hamilton stage (HH) 25 quail heart. A: A semi-sagittal section of a Hamburger and Hamilton stage (HH) 25 quail heart. N1ICD immunostaining was intensely positive in the endothelial cells lining the endocardium of the ventricles, atria, and AV cushions as well as on some mesenchymal cells within the atrioventricular (AV) cushions. B,C: Higher magnifications of the interventricular sulcus (B) and atrial–ventricular junction (AVJ; C) regions indicated by boxed areas in A. QH1-positive endothelial cells in endocardium were N1ICD-positive (B, solid arrowheads). Notch1ICD was expressed in epicardial mesothelial (B,C, empty arrowheads) and mesenchymal cells (C, solid arrowhead) and colocalized with Wt1 at the AVJ. Some QH1-positive (red) endothelial cells in the myocardium (B arrows) and nascent vessels (C arrows) within the epicardium at the AVJ showed intense N1ICD staining. D: Alternate sections immunostained for N1ICD, Wilms tumor suppressor gene1 (Wt1), and VEGFA revealed staining for all three in epicardial mesothelial cells and subepicardial mesenchymal cells at the AVJ. The dotted line indicates the boundary of the epicardium (left) and myocardium (right). Nuclei were labeled by 4′,6-diamidine-2-phenylidole-dihydrochloride (DAPI) staining (dark blue). Scale bars = 500 μm in A; 50 μm in B (applies to C–F).
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At HH25, staining of alternative sections showed that VEGFA immunostaining coincided with N1ICD expression in a subset of the epicardial mesothelial cells and subepicardial mesenchymal cells at the AVJ and less intense VEGFA staining was also observed in the myocardium adjacent to the epicardium (Fig. 3F, arrowheads). Notch1 was proposed as a downstream gene of VEGF in arterial endothelial cells (Timmerman et al.,2004). VEGF stimulation was shown to up-regulate Notch1 and Dll4 expression exclusively in arterial endothelial cells (Lawson et al.,2002). Whereas VEGFA could not rescue arterial marker gene expression in Notch deficient zebrafish embryos, the expression of an activated Notch1 in VEGFA-deficient embryos could rescue the expression of arterial markers. Furthermore, the spatiotemporal VEGF-distribution as well as specificity of the isoforms are important for proper coronary vascular development (van den Akker et al.,2007a). It is likely that epicardium-derived and myocardium-derived VEGFA may have an additive effect to induce N1ICD expression in epicardial and subepicardial mesenchymal cells during epicardial EMT and endothelial cell differentiation.
In the atrioventricular sulcus, some of the epicardially derived mesenchymal cells coalesced to form channels within the extracellular matrix and formed the endothelium of the coronary vessels at HH30 quail heart (Fig. 4B,F). N1ICD was present in endothelial cells in these nascent vessels in the AVJ epicardium (Fig. 4,F, arrows) and within the compact ventricular myocardium (Fig. 4D–G). At HH30, no smooth muscle actin-positive cells were detected (data not shown). At this stage, there is no blood flow and, hence, no blood pressure because the coronary arteries do not connect to the aorta until HH32. This is consistent with previous findings that the timing of coronary smooth muscle cell differentiation occurs after the formation of ostia when the blood flow may stimulate SMC formation (Lawson et al.,2002).
Figure 4. Notch1 intracellular domain (N1ICD) immunostaining in the Hamburger and Hamilton stage (HH) 30 quail heart. A: Double immunofluorescence staining for N1ICD (green) and Qh1 (red; a quail endothelial cell marker) at lower magnification of a frontal section of HH30 quail heart. B: At the atrioventricular junction (AVJ), endothelial cells in nascent capillary vessels are Notch1ICD-positive. C: An immediately adjacent section to that shown in B, was exposed to N1ICD antibody blocked with N1ICD peptide (1:4) for the negative control (Fig. 4 C). D,E: Higher magnification of regions from A showed N1ICD stained vessels in the myocardium of the right ventricle and IVS. F: Higher magnification of the boxed region in B showed that N1ICD and Qh1 were colocalized in endothelial cells of the nascent coronary plexus (arrows). G: N1ICD colocalized with Qh1 in endothelial cells of myocardial vessels (arrows), G is an enlargement of the region within the white box in D. Nuclei were stained with 4′,6-diamidine-2-phenylidole-dihydrochloride (DAPI; blue). Scale bars = 500 μm in A, 50 μm in B (applies to C,D,E), 10 μm in F (applies to G).
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Two sources of coronary smooth muscle cells were proposed, the neural crest derivatives and epicardial cells. It was determined that the proximal coronary stems contain some neural crest-derived SMCs for a short distance from their origin at the aortic root, using a neural crest-specific Wnt1-Cre recombinase system for lineage mapping (van den Akker et al.,2007b). As was described for aortic arch arteries in chick–quail chimeras (Hood et al.,1992; Gittenberger-de Groot et al.,1998), mouse neural crest cells in the coronary stems exhibit sharp boundaries and little or no intermixing with PEO-derived SMCs (Jiang et al.,2000). SMCs of coronary arteries originate mainly from the epicardium in avian systems. Fate mapping studies using retroviral labeling (Mikawa and Gourdie,1996) provided evidence that proepicardial cells may be already committed to distinct endothelial or SMC fates before contact with the heart. We chose HH35 to study the role of N1ICD in SMC recruitment to the nascent plexus. Double immunofluorescence staining of N1ICD showed clear colocalization with SMA. Notch 1ICD was expressed in SMCs lining coronary vessels at the AVJ in HH35 quail heart (Fig. 5B arrows). The endothelial cells lining the vessel lumen were N1ICD-positive (Fig. 5E, arrowheads) as were SMCs surrounding vessels within the myocardial compact layer (Fig. 5D box). Of interest, the N1ICD staining of epicardial mesothelial cells and subepicardial mesenchyme around the vessels was absent by stage HH35. These cells appeared to have lost N1ICD expression after epicardial EMT and differentiation after the initiation of coronary circulation. Notch-mediated lateral inhibition of N1ICD was demonstrated in several developmental events such as neuroblast segregation in Drosophila (Daudet and Lewis,2005), vertebrate early neurogenesis (Skeath and Thor,2003); and sensory hair cell formation in the vertebrate inner ear (Chitnis,1995) and may be playing a role in down-regulating N1ICD expression at this late stage. Alternatively, hypoxia and VEGF levels may be lower at these sites because of the initiation of coronary circulation.
Figure 5. Expression of Notch1 intracellular domain (N1ICD) and smooth muscle actin (SMA) in Hamburger and Hamilton stage (HH) 35 quail heart. A: Co-immunofluorescence staining for N1ICD (nuclear, green) and SMA (cytoplasmic, red) of a frontal section of the HH35 quail heart. B: At the right atrioventricular junction (AVJ), several smooth muscle cells (red) enwrapping coronary vessels had Notch1ICD-positive nuclei (green). C: The coronary vessels at the left AVJ were also covered by N1ICD-positive smooth muscle cells (SMCs; arrows). D: The SMCs (red) adjacent to vessels within the myocardial compact layer of the ventricles were stained by N1ICD (green). E: Higher magnification of the boxed region in C showed nuclear staining of N1ICD (green) colocalized (arrows) with SMA staining (red) around a vessel containing a nucleated (blue) red blood cell (negative for N1ICD, asterisk). A few endothelial cells lining the vessel lumen were N1ICD-positive (arrowheads). F: A higher magnification of a region from D showed N1ICD staining (green) within the left ventricular wall. N1ICD (green) colocalized with SMA (red) in SMCs of myocardial vessels (arrows). Nuclei were stained by 4′,6-diamidine-2-phenylidole-dihydrochloride (DAPI; blue). Scale bars = 500 μm in A, 50 μm in B (applies to C,D), 10 μm in E (applies to F).
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As summarized in Figure 6, N1ICD appears in mesothelial cells of the pro-epicardium during epicardial formation at looped heart stages. After epicardial EMT, a subset of N1ICD-positive EPDCs differentiate and link to form a nascent plexus that induces “local” N1ICD-positive mesenchymal cells to become smooth muscle cells. And the “local” mesenchymal cells were most likely the EPDCs migrating with the endothelial cells. Once these EPDCs commit to the endothelial cell and SMC/pericyte lineage, NICD expression is lost in the neighboring EPDCs and mesothelial cells. We propose that Notch1 signaling may play essential roles in coronary progenitor cell fate determination. Recently, evidence was obtained that epicardial cells may also serve as precursors for a subset of cardiomyocytes at sulcus regions (Cai et al.,2008; Zhou et al.,2008). It will be interesting to look at the role of Notch 1 during development of this special set of cardiomyocytes.
Figure 6. Notch1 intracellular domain (N1ICD) expression pattern during coronary vascular development. A: The N1ICD is not present in cells of the pro-epicardial organ (PEO) before the PEO attaches to the myocardium. B: N1ICD (red nuclei) appears in mesothelial cells extending from the pro-epicardium on the looping heart. C,D: After epicardial epithelial/mesenchymal transition (EMT), a subset of N1ICD-positive mesenchymal cells (red nuclei) differentiate into endothelial cells (D, pink cells with red nuclei) and form a nascent vessel plexus within the subepicardial extracellular matrix (SEM) of the atrioventricular junction (AVJ) and within the compact layer of myocardium (Myo) (Hamburger and Hamilton stage [HH] 25–HH30). E: The endothelial cells expressing N1ICD in the vessels recruit local N1ICD-positive mesenchymal cells and the latter commit to the smooth muscle cell fate (yellow cells with red nuclei; HH32–HH35). There is a loss of NICD expression in the neighboring and mesothelial cells (with light blue nuclei) surrounding the mature coronary vessels.
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HIF1 and Notch
The regulation of Notch signaling has recently been linked to hypoxia and HIF-1. Hypoxia or ectopically expressed HIF-1 α elevates Notch1 ICD protein levels and increases the Notch downstream response in vitro (Eddison et al.,2000). The crosstalk between hypoxia with the Notch signaling pathway has been shown to be required for hypoxia-mediated reduction of progenitor cell differentiation in neuronal and myogenic differentiation (Gustafsson et al.,2005). The Notch intracellular domain (ICD) interacted with HIF-1α, and HIF-1α was recruited to Notch-responsive promoters upon Notch activation during hypoxic conditions. Hypoxia induced Dll4, Hey1, and Hey2 in various cell types including embryonic endothelial progenitor cells (eEPCs), was mediated by activation of HIF-1α and Notch signaling. Hypoxia might also play essential roles in arterial cell fate determination by activation of the Dll4-Notch-Hey2 signalling cascade with subsequent repression of COUPTFII (Sainson and Harris,2006). In ARMECs, we also found that hypoxia (1% O2 incubation for 24 hr) and ectopic stabilized HIF1 expression [infection by constitutively active HIF1α described previously (Kelly et al.,2003)] increased Notch1ICD levels correlated with the induction of HIF1α expression. (Fig. 1D.) This observation is consistent with a similar observation in C2C12 cells (Gustafsson et al.,2005). We hypothesize that N1ICD may be also be stabilized in adult rat epicardial cell EMT in some circumstance, such as under ischemic conditions. Our findings suggest that the hypoxia/HIF1-VEGF-Notch pathway plays a role in epicardial cell interactions that promote epicardial EMT and determines coronary progenitor cell fate and differentiation during epicardial development and coronary vasculogenesis in particularly hypoxic microenvironments at sulcus regions.