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. Author manuscript; available in PMC: 2006 Sep 14.
Published in final edited form as: Mech Dev. 2006 Jul 14;123(8):626–640. doi: 10.1016/j.mod.2006.06.003

Notch signaling plays a key role in cardiac cell differentiation

Mary DL Chau a, Richard Tuft b, Kevin Fogarty b, Zheng-Zheng Bao a,*
PMCID: PMC1567976  NIHMSID: NIHMS11948  PMID: 16843648

Abstract

Results from lineage tracing studies indicate that precursor cells in the ventricles give rise to both cardiac muscle and conduction cells. Cardiac conduction cells are specialized cells responsible for orchestrating the rhythmic contractions of the heart. Here, we show that Notch signaling plays an important role in the differentiation of cardiac muscle and conduction cell lineages in the ventricles. Notch1 expression coincides with a conduction marker, HNK-1, at early stages. Misexpression of constitutively active Notch1 (NIC) in early heart tubes in chick exhibited multiple effects on cardiac cell differentiation. Cells expressing NIC had a significant decrease in expression of cardiac muscle markers, but an increase in expression of conduction cell markers, HNK-1, and SNAP-25. However, the expression of the conduction marker connexin 40 was inhibited. Loss-of-function study, using a dominant-negative form of Suppressor-of-Hairless, further supports that Notch1 signaling is important for the differentiation of these cardiac cell types. Functional studies show that the expression of constitutively active Notch1 resulted in abnormalities in ventricular conduction pathway patterns.

Keywords: Cell differentiation, Notch pathway, Cardiomyocyte, Ventricles, Molecular mechanism

1. Introduction

The cardiac conduction system is a specialized tissue responsible for setting, maintaining, and coordinating the rhythmic contractions of the heart (Gourdie et al., 1999; Moorman et al., 1998; Myers and Fishman, 2003). Precisely timed electrical impulses are generated at the sinoatrial node, spread through the atrial myocytes, and are received at the atrioventricular node. This impulse is then rapidly propagated along the His bundles and its branches, spreading into the ventricular muscle via the Purkinje fiber network. Although much progress has been made in the understanding of heart development (Eisenberg and Markwald, 2004; Srivastava and Olson, 2000), the mechanism underlying the development of the cardiac conduction system is only partially understood.

Cardiac conduction cells are distinguished by their unique gene expression pattern. Antibodies to Leu-7 (HNK-1) have been used widely to delineate the developing conduction system in mammals and in chicken (Chuck and Watanabe, 1997; Gorza et al., 1988; Moorman et al., 1998; Nakagawa et al., 1993; Verberne et al., 2000). The HNK-1 antibody recognizes a complex sulfate-3-glucuronyl carbohydrate moiety, which is present on a series of molecules involved in cell adhesion and extracellular matrix interaction. The antibody to SNAP-25 protein, a component of the SNARE complex, has also been used to mark the elements of the ventricular conduction system in chick (Verberne et al., 2000). Among the gap junction protein connexins, Connexin40 (Cx40) or the chicken homolog Cx42, has been used as a marker for the conduction system in many species (Bastide et al., 1993; Delorme et al., 1997; Gourdie et al., 1993b; Gros et al., 1994).

Retroviral lineage analyses have provided compelling evidence that conduction cells are derived from precursor cells in the heart, sharing a common lineage with working cardiomyocytes (Cheng et al., 1999; Gourdie et al., 1995). Clonally related cells are found in both cardiac muscle cells and in the conduction system. Following microinjection of replication-defective retrovirus into the cardiac neural crest, however, no virally tagged cells could be traced into the Purkinje fiber lineage, excluding contribution from the neural crest. It has been demonstrated that the selection of conduction cells within myocardial clones occurs as a result of paracrine signals from endocardial cells and endothelial cells from coronary arteries, including endothelin-1 (ET-1) in chick and Neuregulin-1 in mouse (Hall et al., 2004; Kanzawa et al., 2002; Rentschler et al., 2002; Takebayashi-Suzuki et al., 2000). Coexpression of preproendothelin (preproET-1) and endothelin converting enzyme (ECE-1) in the embryonic myocardium induced myocytes to express Purkinje fiber markers (Hall et al., 2004; Takebayashi-Suzuki et al., 2000). Addition of neuregulin-1 to embryo cultures of CCS-lacZ mice, in which lacZ delineates the cardiac conduction system, increased lacZ expression (Rentschler et al., 2002). Several transcription factors, including Nkx2.5, Tbx5, and HF-1b have also been shown to play an important role in the development of the cardiac conduction system (Jay et al., 2004; Kondo et al., 2003; Kupershmidt et al., 1999; Moskowitz et al., 2004; Nguyen-Tran et al., 2000; Pashmforoush et al., 2004; Thomas et al., 2001). Mouse mutants deficient in either of the transcription factors HF-1 b, Tbx5 or Nkx2.5 exhibit defects in the development and function of the conduction system.

The Notch signaling pathway is an evolutionarily conserved mechanism used by metazoans to control cell fate decisions through local cell interactions (Artavanis-Tsakonas et al., 1999). The notch gene encodes a single-pass transmembrane protein receptor that interacts with its ligands, Delta and Serrate/Jagged. Upon binding of the ligand, the intracellular domain of Notch (NIC) undergoes proteolytic cleavage, and is translocated to the nucleus. In the nucleus, NIC binds to its major downstream effector, Suppressor-of-Hairless [Su(H)]. Su(H) binds to the regulatory sequences of the Enhancer-of-Split [E(spl)] locus, upregulating the expression of basic helix-loop-helix (bHLH) proteins, which in turn regulate the expression of downstream target genes. Signals transmitted through the Notch receptor, in combination with other cellular factors, influence differentiation of various cell types, in the nervous system, immune system and pancreas (Artavanis-Tsakonas et al., 1999).

The Notch pathway has been previously shown to influence cardiogenesis. In Xenopus, it is suggested that the interaction of Notch1 with its ligand Serrate1 apportions myogenic and non-myogenic cell fates within the early heart field (Rones et al., 2000). In mouse, null mutations in both notch1 and RBP-J, the mammalian homolog of Suppressor-of-Hairless, leads to embryonic lethality and pericardial edema (Oka et al., 1995; Swiatek et al., 1994). The absence of RBP-J in mouse ES cells causes an increase in cardiac muscle development suggesting that Notch/RBP-J signaling is required for the specification of cell fates within the heart field by suppressing cardiomyogenesis (Schroeder et al., 2003). Recently, mutations in Notch1 in humans have been shown to cause aortic valve defects and activation of Notch1 in mouse leads to abnormal cardiogenesis characterized by deformities of the ventricles and atrioventricular canal (Garg et al., 2005; Watanabe et al., 2006). Additionally, mutations in various Notch signaling pathway genes, including Jagged1, mind bomb 1, Hesr1/Hey1, and Hesr2/Hey2, result in cardiac defects, such as pericardial edema, atrial and ventricular septal defects, cardiac cushion, and valve defects (Donovan et al., 2002; Fischer et al., 2004; Gessler et al., 2002; Kokubo et al., 2005; Kokubo et al., 2004; Koo et al., 2005; McCright et al., 2002; Sakata et al., 2002).

Here, we demonstrate a role for Notch1 in the differentiation of cardiac cell types in the ventricles. notch1 mRNA transcripts are expressed in the ventricular conduction cell lineage at early stages. Forced expression of constitutively active Notch1 in progenitor cells inhibits muscle marker expression but promotes expression of conduction marker HNK-1 and SNAP-25. Cells expressing constitutively active Notch were localized predominantly in the trabeculae where conduction cells are concentrated, and not in the future myocardial compact zone. Loss-of-function study further demonstrate the requirement for Notch in this lineage decision. By optical mapping, we have further shown that expression of constitutively active Notch1 resulted in abnormal conduction patterns in the heart consistent with a defect in cardiac cell differentiation.

2. Results

To study the mechanism of cardiac cell differentiation, we analyzed the expression of the notch1 gene by in situ hybridization. At embryonic days 6 (E6), in situ hybridization on heart sections showed that notch1 was expressed in the ventricles and the atria, concentrated in a subset of cells in the trabecular myocardium, and atrioventricular canal (Figs. 1A, B, and data not shown). Some very weak signals were also detected in the endocardium (Fig. 1B). To determine which cardiac cell type in the myocardium expressed notch1 mRNA, we performed in situ hybridization on heart sections using the chick notch1 probe, followed by immunostaining with markers for different cardiac cell types. From E3 to E6, the notch1 in situ signals in the ventricles were largely associated with staining by HNK-1 (Fig. 1C). HNK-1 antibody recognizes a complex carbohydrate moiety on the cell surface and has been used extensively as a marker for the ventricular conduction system in many species including chick, rat, rabbit and human (Aoyama et al., 1993; Aoyama et al., 1995; Chuck and Watanabe, 1997; Gorza et al., 1988; Ikeda et al., 1990; Luider et al., 1993; Nakagawa et al., 1993; Nakamura et al., 1994; Sakai et al., 1994; Verberne et al., 2000), and overlaps with another conduction system marker, Cx40 (Supplementary Fig. 1S). As in situ signals are localized in the cytoplasm whereas HNK-1 staining is found on the plasma membrane, the association of the expression patterns suggests that these cells may be expressing HNK-1 and notch1. By E9, however, notch1 in situ signals in the ventricles appeared to be concentrated in the grooves between the trabeculae, with limited association to HNK-1 staining (Fig. 1C). This result suggests that notch1 is expressed in the ventricular conduction cell lineage at early stages.

Fig. 1.

Fig. 1

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Expression patterns of notch1 mRNA in the embryonic chick heart, by in situ hybridizations on heart sections at embryonic days 3 (HH19), 6 (HH29), and 9 (HH35) (E3, E6, E9). (A) notch1 expression at E6 is shown at a low magnification in the ventricles. (B) The boxed area in A is shown at a higher magnification. Note the expression of notch1 in the myocardium (arrows), in addition to very weak signals in the endocardium (arrowhead). (C) To identify the cells expressing notch1, we immunostained the heart section with the antibody HNK-1 (red), after the completion of in situ hybridization. Note that notch1 in situ signals appear to be closely associated with cells positive for HNK-1 staining on the plasma membrane at E3 and E6 (arrows). The HNK-1 staining appears subendocardial. The sections shown are of oblique angles. At E9, many notch1-positive cells are no longer associated with HNK-1 staining. V, ventricles; t, trabecula. Scale bars in B, C = 20 μm; in A = 200 μm.

To define the role of notch1 in heart development, we first took a gain-of-function approach by expressing a constitutively active form of Notch1 in a replication-competent avian retrovirus (RCAS-NIC) (Fig. 2A). The truncated protein consisting only of the intracellular domain of Notch1 is known to localize largely in the nucleus, and elicits a constitutively active phenotype (Kopan et al., 1994). RCAS-NIC, or a control virus RCAS-GFP, encoding green fluorescent protein (GFP), was injected into the early heart tube at HH 9. As shown in Fig. 2B, by co-staining with the anti-myc and anti-viral GAG antibodies, NIC protein was observed in the nuclei of the infected cardiac cells. However, not all infected cells appeared positive for the anti-myc antibody staining, possibly due to low detection sensitivity with a single copy of myc-tag present on the NIC protein. Because most of the embryos injected with RCAS-NIC died around E5, we analyzed some embryos at E4.5. For later analyses at E6 or E10, we injected diluted viral stocks to improve survival. Because some molecular markers are more specific in the ventricles, we focused our analysis within the ventricles.

Fig. 2.

Fig. 2

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Expression of constitutively active Notch1 (NIC) via a retroviral construct in embryonic chick heart. (A) Retroviral constructs, RCAS-GFP and RCAS-NIC. LTR, long terminal repeat; gag, gene encoding the viral capsid proteins; env, gene encoding viral envelope protein; pol, gene encoding viral reverse transcriptase. (B) RCAS-NIC injected chick hearts were harvested at E6, sectioned and immunostained with p27 (anti-viral GAG protein, green) to identify the infected cells, and an antibody against the myc tag, 9E10 (red). DAPI (blue) was used to stain for the nuclei. Note that the myc-tag staining coincides with the nuclear stain. Scale bar, 10 μm.

To determine the effect of constitutively active Notch on cardiac muscle differentiation, we co-immunostained the samples with anti-viral GAG p27 antibody to visualize the infected areas, and an antibody that recognizes the sarcomeric myosin heavy chain band, MF20. We did not use myc staining for quantitative analyses because it is difficult to ascertain whether the cell type markers are expressed in the same cells positive for myc in the nuclei, among the densely packed cells. Control RCAS-GFP-infected cells appeared to be mostly positive for MF20 staining, similar to wild type uninjected hearts (Fig. 3A upper panels, and B). The majority of the RCAS-NIC-infected cells, however, appeared to have much decreased MF20 staining (Fig. 3A, middle and bottom panels). Loss of MF20 staining was more apparent in samples with wide spread infection, nearly 100% of the ventricular cells in some cases (Fig. 3A, bottom panels). At higher magnification by using confocal microscopy, the control RCAS-GFP-infected cells showed a strong sarcomeric banding pattern with MF20 staining, and exhibited a typical rod-shaped cardiomyocyte morphology (Fig. 3C). However, RCAS-NIC-infected cells lacked this muscle cell morphology, appeared more rounded, and the majority of the cells lacked MF20 staining (Fig. 3C). In the hearts with limited infection, only RCAS-NIC-infected cells appeared negative for MF20 staining, while neighboring uninfected cells were positive for MF20, suggesting that the effect of the NIC protein is likely cell-autonomous (Fig. 3C).

Fig. 3.

Fig. 3

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Constitutively active Notch1 (NIC) inhibits cardiac muscle cell differentiation. RCAS-NIC or control RCAS-GFP injected hearts were harvested at E4.5 (A) and E6 (C), sectioned and immunostained with p27 (green) and the anti-sarcomeric myosin heavy chain marker, MF20 (red). Hoechst dye 34580 (blue) was used to stain for the nuclei. (A) E4.5 stained heart sections were analyzed at low magnification on an epifluorescence microscope. Note that, in the RCAS-GFP-infected sample (top panel), MF20 staining is widely distributed throughout the heart. RCAS-NIC-infected patches, however, show a substantial decrease in MF20 staining (arrowheads). In some heavily infected hearts, little MF-20 staining was seen in the ventricles (bottom panel). (B) Staining pattern of MF20 on a wild type E6 heart is shown. (C) To quantify the results, E6 heart samples were analyzed on confocal microscopy. Note that the control RCAS-GFP infected cells are mostly positive for MF20, showing characteristic banding patterns, whereas many RCAS-NIC infected cells are negative for MF20 staining. (D) Quantification of the results by scoring for the percent of infected cells stained with MF20 at E6 and E10, respectively. Scale bar in A common to B = 500 μm, and C = 10μm.

The results of partially infected E6 samples were quantified by scoring randomly chosen infected cells on the confocal microscope. Heart sections infected with the RCAS-NIC virus showed a marked decrease in the percentage of MF20-positive cells, compared to the control RCAS-GFP-injected samples (mean ± std: 41.9 ± 4.0% and 89.4 ± 3.7%, respectively; p < 0.001, Student's t-test) (Fig. 3D). To determine whether the decrease of MF20 staining is due to a delay in cardiomyocyte differentiation by constitutive Notch1 activity, we examined RCAS-NIC-infected hearts at a later stage, E10. We observed similar results at E10: RCAS-NIC-infected hearts displayed a similar decrease in the number of MF20 positive cells (38.6 ± 8.4 vs. 95.2 ± 1.5% in control cells, p < 0.001) (Fig. 3D). Furthermore, we analyzed the expression of another muscle marker α-actin in the RCAS-NIC infected cells and a similar inhibitory effect was observed (data not shown). These results suggest that persistent Notch activity inhibits cardiomyocyte differentiation.

We next examined the effects of constitutively active Notch1 on the differentiation of conduction cells. In the control RCAS-GFP injected samples similar to uninjected wild type samples, conduction marker HNK-1 staining was observed in the areas of the trabeculae and interventricular septum (Fig. 4A and B). At higher magnification, HNK-1 staining appeared to line the trabeculae with limited staining inside the trabeculae (Fig. 4C). However, in hearts heavily infected with RCAS-NIC, the expression of HNK-1 was found in nearly all of the ventricular myocardium (Fig. 4A). Instead of a normal pattern lining the trabeculae, HNK-1 staining appeared to surround every cell in the RCAS-NIC injected samples (Fig. 4C). Similar increase in HNK-1 staining was observed in the E6 samples, despite the fact that these samples were not well-infected by injection with diluted viral stocks. By scoring randomly chosen infected areas on the confocal microscope, a significant increase was observed in the proportion of RCAS-NIC-infected cells at E6 expressing HNK-1, compared to RCAS-GFP-infected cells (53.8 ± 6.6 and 15.7 ± 4.0%, respectively; p < 0.001) (Fig. 4D and E). These results support our notion that constitutively active Notch1 increases the expression of HNK-1.

Fig. 4.

Fig. 4

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RCAS-NIC increases expression of the conduction system marker, HNK-1. RCAS-GFP or RCAS-NIC injected hearts were harvested at E4.5(A, C) or E6 (D), sectioned and immunostained with p27 (green), to identify infected cells, and the conduction lineage marker HNK-1 (red). Hoechst dye 34580 or DAPI (blue) were used to stain for the nuclei. (A) E4.5 stained heart sections were analyzed at low magnification on an epifluorescence microscope. Note that the RCAS-NIC infected sample shows a significant increase in HNK-1 staining as compared to the RCAS-GFP infected heart. (B) The staining patterns of HNK-1 in wild type E4.5 and E6 hearts are shown. Note that HNK-1 staining is concentrated around the trabeculae. (C) The areas of the hearts marked by arrows in (A) are shown in a higher magnification. The RCAS-NIC infected sample shows HNK-1 staining around almost every cell within the trabeculae, while HNK-1 staining mostly outlines the trabeculae in the RCAS-GFP infected heart. (D) The overlay images of the infected hearts at E6 are shown (red, HNK-1; green, P27; blue, Hoechst dye). Note that the trabeculae partially infected with RCAS-NIC has an increase in HNK-1 expression, especially within the trabeculae. (E) Quantification of the percentage of the cells infected with RCAS-GFP or RCAS-NIC expressing HNK-1 at E6. Scale bar in A common to B = 500 μm; C = 100 μm; D = 100 μm.

To determine the effect of Notch signaling on other known conduction cell lineage markers, we examined the expression of SNAP-25 and connexin 40 in the RCAS-NIC injected hearts. SNAP-25 is a component of the SNARE complex that is involved in exocytosis on synaptic terminals. The antibody of SNAP-25 has been shown to label components of the ventricular conduction system in the chick embryo (Verberne et al., 2000). At low magnification on an epifluorescence microscope, SNAP-25 expression in E6 wild type hearts was found to be concentrated in the trabeculae, in areas similar to HNK-1 staining. But unlike HNK-1 staining which lines the trabeculae, SNAP-25 staining was found inside the trabeculae (Fig. 5A and B). At higher magnification, SNAP-25 staining was visible in the cytoplasm and plasma membrane of the conduction cells, in addition to punctate staining amidst the muscle cell fibers (Fig. 5C). Because SNAP-25 is not expressed in the ventricles until E6, we could not analyze the effect of NIC on SNAP-25 in heavily infected E4.5 hearts. When we scored randomly selected RCAS-NIC-infected cells at E6 on a confocal microscope, a significant increase in the percentage of SNAP-25-positive cells was observed, compared to the control samples (35.8 ± 4.2 and 18.8 ± 7.6% of the infected cells, respectively; p < 0.01) (Fig. 5D). At lower magnification, these NIC-expressing cells were found to localize to the trabeculae region, which is normally enriched with SNAP-25 positive cells (Fig. 5A, bottom panels). In contrast, the control RCAS-GFP-positive cells were largely distributed in the myocardium destined for the future compact zone (Fig. 5A, top panels).

Fig. 5.

Fig. 5

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Increase in proportion of the RCAS-NIC infected cells expressing SNAP-25. RCAS-GFP or RCAS-NIC injected hearts were harvested at E6, sectioned and immunostained with anti-GAG antibody p27 (green), and the conduction marker SNAP-25 (red). Hoechst dye 34,580 (blue) was used to stain for the nuclei. (A) At low magnification, RCAS-NIC-infected cells are found to be largely localized to the trabeculae region where SNAP-25 expression is enriched. In contrast, the control RCAS-GFP-positive cells were largely distributed in the myocardium destined for the future compact zone. (B) The staining pattern of SNAP-25 antibody is shown on wild type E6 heart section. Note that the SNAP-25 antibody stains the tips of the trabeculae. (C) Stained heart sections were analyzed at higher magnification by confocal microscopy. Many RCAS-NIC-infected cells are SNAP-25 positive. High levels of expression of SNAP-25 were observed on the membrane and the cytoplasm of the RCAS-NIC expressing cells with some punctate staining among the myocardial fibers (top panels). (D) Quantification of the percentage of the cells infected with RCAS-GFP or RCAS-NIC expressing SNAP-25. Scale bar in A common to B = 500 μm and in C = 10 μm.

Another conduction system marker, connexin 40 (also-called Cx42 in chick), encodes a gap junction protein (Gourdie et al., 1993a; Moorman et al., 1998; Takebayashi-Suzuki et al., 2001). By in situ hybridization, we found low levels of Cx40 expression in cardiomyocytes in addition to intense staining in conduction system cells, in uninjected samples or the control samples injected with RCAS-GFP (Fig. 6A). This pattern has been reported previously (Becker et al., 1998; Minkoff et al., 1993). By in situ hybridization on heart sections infected with RCAS-GFP or RCAS-NIC virus using the chick Cx40 probe, followed by immunostaining with the anti-GAG antibody, we found that RCAS-NIC-infected areas appeared to have decreased Cx40 in situ signals (Fig. 6C and D). Few strongly stained cells were observed and the overall weak staining in the cardiomyocytes was further reduced. This result demonstrates that constitutively active Notch1 decreases the expression of Cx40.

Fig. 6.

Fig. 6

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The expression of the conduction system marker connexin 40 (Cx40) is decreased by constitutively active Notch. RCAS-GFP (A, B) or RCAS-NIC (C, D) infected heart sections were analyzed by in situ hybridization with the Cx40 probe. Trabeculae areas were shown for both samples. Note the dark subendocardial Cx40 signals were not decreased by RCAS-GFP expression (arrows). The entire area shown was infected by RCAS-GFP. Because the dark precipitates of the in situ signals quench the fluorescence, small non-fluorescent areas overlapping exactly with the in situ signals are likely infected. In contrast, the RCAS-NIC-infected areas correlated with decreased Cx40 in situ signals, both in the subendocardial cells (arrowheads) and myocardial cells. Scale bar, 20 μm.

We next took a loss-of-function approach by expressing a dominant-negative form of the Suppressor-of-Hairless through retroviral infection, RCAS-Su(H)DN. The dominant-negative form of Suppressor-of-Hairless has been shown to interfere with transcriptional activation of target genes by the Notch1 protein, thereby inhibiting Notch1 function (Morrison et al., 2000; Wettstein et al., 1997). The injected hearts were harvested at E4 and E6 and processed similarly as in our gain-of-function study. In contrast to the control hearts which show high levels of Cx40 expression in the subendocardial cells in addition to low level expression in the myocardial cells (Fig. 6A and B), expression of Su(H)DN resulted in loss of cells expressing high levels of Cx40 (Fig. 7A). However, low-level of Cx40 expression in myocardial cells was unaltered.

Fig. 7.

Fig. 7

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Expression of the dominant-negative form of Suppressor-of-Hairless alters the expression of cardiac cell type markers. Chick embryos were injected with the control virus, RCAS-GFP, or with the RCAS-Su(H)DN virus. (A) Hearts were harvested at E6 and E4 (not shown), sectioned and immunostained with anti-viral GAG antibody, and various cell type specific markers. Note that most of the RCAS-Su(H)DN infected are positive for MF20, but negative for HNK-1. Expression of Cx40 was also analyzed by in situ hybridization on the infected heart sections, followed by staining with anti-GAG antibody. In the RCAS-Su(H)DN infected areas, the Cx40 expression appears to be at a low level uniformly, unlike the control hearts which show relatively high levels of Cx40 in some subendocardial cells (Fig. 6A, B). Scale bars, 20 μm. (B) Quantification of the results by scoring the percentage of the RCAS-Su(H)DN infected cells that express various markers. Note that, compared to the GFP control (black bar), expression of Su(H)DN (gray bar) increased the percentage of cells positive for MF20, and decreased the percentage of cells positive for HNK-1 or SNAP-25. Asterisks indicate statistical significance.

For MF20, HNK-1 and SNAP-25 markers, we found reversed phenotypes with the RCAS-Su(H)DN-expressing cells as compared to those expressing constitutively active Notch1 (RCAS-NIC). At E4, we found a small but significant increase of RCAS-Su(H)DN-infected cells showing MF20 staining, compared to RCAS-GFP infected cells (98.4 ± 0.8 vs. 90.9 ± 1.2% in control, p < 0.001) (Fig. 7A and B). Additionally, expression of Su(H)DN also appeared to significantly decrease the percentage of HNK-1 expressing cells (4.7 ± 1.1 vs. 13.5 ± 1.9% in controls, p < 0.001) (Fig. 7A and B). We found similar results at E6 as those at E4; a significant increase of RCAS-Su(H)DN-infected cells showing MF20 staining, compared to RCAS-GFP-infected cells, and a significant decrease in the percentage of RCAS-Su(H)DN-infected cells positive for HNK-1 and SNAP-25 (Fig. 7B).

To determine the effect of constitutive Notch signaling on the functional development of the ventricular conduction system, we utilized an optical mapping technique to visualize the propagation pathway of action potentials across the ventricular myocardium. High-speed imaging was performed on uninjected control, RCAS-GFP-injected and RCAS-NIC-injected hearts. The hearts were dissected, stained with a voltage sensitive fluorescent dye, di-4-ANEPPS, and recorded for both the dorsal and ventral sides. After recording, hearts were fixed, sectioned, and the degree of infection was determined by anti-GAG staining. As the fluorescent signal decreases with an increase in membrane voltage, the most rapid decrease in fluorescent signal corresponds to an action potential. A custom software was developed to compute absolute values of rates of fluorescent change at each 5 × 5 pixel area of the entire ventricular surface and the maximum slope of the action potential was displayed as red in the color scale. Five beat series were analyzed for each heart and the patterns appeared consistent from beat to beat.

Because a great majority of RCAS-NIC injected embryos died by E5 and hearts younger than E4.5 were difficult to handle because of their small size and fragility, we imaged live hearts at E4.5-5. Seven out of 12 uninjected control hearts at this stage exhibited an immature, unidirectional propagation pattern (Fig. 8A, upper sequence). After activation of the atrium, the impulse travels along the myocardial wall in a unidirectional fashion towards the outflow tract. The action potential travels across the heart within 8–10 ms. The rest of the uninjected hearts (5 out of 12) displayed a mature, apex to base sequence of activation within a similar time frame (Fig. 8A, lower sequence), suggesting that the transition from an immature to a mature activation pattern occurs at around E4.5-5. These results are consistent with previous optical mapping studies in chick showing similar mature and immature activation patterns (Chuck and Watanabe, 1997; Hall et al., 2004; Reckova et al., 2003).

Fig. 8.

Fig. 8

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Constitutively active Notch1 alters the conduction propagation pattern in embryonic chick hearts. (A) Optical mapping was performed on uninjected control hearts at E4.5-5. Images were collected at 2 ms/frame, and processed using a custom software. The first derivative was computed and the maximum upstroke velocity was defined as dF/dt max and depicted as red in the color scale. Note, in the top panel, the impulse propagates along the myocardial wall from the atrium towards the outflow tract in the immature propagation pattern (red arrows) in about 8–10 ms. Also note, in the bottom panel, the impulse travels from the apex to the base (red arrows) within 8 ms in the mature activation sequence. (B) Optical mapping was similarly performed on RCAS-NIC-injected hearts at E4.5-5. After imaging, the hearts were fixed, sectioned and stained with anti-P27 to visualize the extent of infection (shown in green fluorescence). Note, the impulse fails to advance towards the base and dissipates within 4ms in the top two panels, and a diffuse activation pattern in the bottom panel. The heart in the bottom panel was more extensively infected with the RCAS-NIC virus.

In 5 out of 24 hearts injected with RCAS-NIC, we observed an altered apex-to-base pattern in which the breakthrough impulse at the apex failed to advance towards the base. Instead of a normal 8–10 ms propagation time from apex to base, the activation sequence dissipated within 4 ms (Fig. 8B, upper and middle sequence). Upon analysis of the samples after imaging, we found that these hearts expressed NIC in a relatively wide spread area (approx. 20–50%) (Fig. 8B, green fluorescence images in top and middle panels). Another abnormal pattern found in the hearts injected with RCAS-NIC was a relatively diffuse activation pattern where the impulse at the apex of the heart traveled across the majority of the ventricular surface to the base (5 out of 24) (Fig. 8B, bottom sequence). These hearts appeared to be infected most broadly in the myocardium but not in the epicardium (approx. 70–95%) (Fig. 8B, green fluorescence image in bottom panel). The rest of the RCAS-NIC injected hearts had largely normal conduction patterns in the ventricle. These hearts were not as well infected, with only small patches of infection (data not shown). We also examined the activation patterns of RCAS-GFP-injected hearts (n = 14). These hearts displayed normal activation patterns within the same time frame as the control uninjected hearts (14 of 14), suggesting that viral infection alone did not affect the conduction patterns. Taken together, these data suggest that expression of a constitutively active Notch resulted in an abnormality in the functional development of the cardiac conduction system.

Delta1 is a well-characterized ligand known to bind to the Notch receptor and activate the Notch signaling pathway. We analyzed the expression of Delta1 by performing in situ hybridization on chick heart sections. As shown in Fig. 9A, Delta1 transcripts were detected widely in myocardial cells in the ventricles at E3 and E6, but the signal was reduced at E9. It has been previously shown that Delta1 expression can be negatively regulated by Notch signaling through downstream basic helix-loop-helix (bHLH) transcription factors (Heitzler et al., 1996; Heitzler and Simpson, 1991; Lutolf et al., 2002). To test whether a similar feedback loop is also at work in this system, expression of Delta1 transcripts was analyzed in control RCAS-GFP or RCAS-NIC-infected hearts. As shown in Fig. 9B, the expression of Delta1 was decreased in areas infected with RCAS-NIC, but not in areas infected with control RCAS-GFP. These results suggest that Delta1 may act as a ligand for the Notch receptor and a negative feedback loop may also be present during cardiac differentiation.

Fig. 9.

Fig. 9

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Expression of Delta1 is decreased in the cells infected with RCAS-NIC. (A) Expression of the chick Delta1 transcripts in the developing heart. In situ hybridizations were performed on cardiac sections of E3, E6, and E9 chick embryos. Note that Delta1 is widely expressed in the myocardium. (B) Expression of Delta1 was reduced in the areas infected with the RCAS-NIC virus. Heart tubes were injected with control RCAS-GFP or RCAS-NIC virus at HH9-10 and the infected hearts were harvested at E6. In situ hybridizations were performed on the infected tissues with the Delta1 probe, followed by anti-viral GAG staining. Note that Delta1 transcript expression was inhibited by the expression of NIC (arrows), but not GFP. Scale bars, 50 μm.

3. Discussion

In this paper, we show that Notch signaling plays an important role in cardiac cell differentiation. Notch1 transcripts are expressed in the early conduction lineage but not in cardiomyocytes in the ventricles. Expression of constitutively active Notch inhibits the expression of cardiac muscle proteins including sarcomeric myosin heavy chain and α-actin. The effects of constitutively active Notch on the conduction cell markers are more complex: there was an increase in the expression of conduction lineage markers, HNK-1 and SNAP-25, but a decrease in the expression of Cx40. By using a Su(H)DN construct, we found that reducing Notch signaling resulted in an increase of MF20 expression and a decrease of conduction markers including HNK-1, SNAP-25, and a high level of Cx40 expression. These results suggest that Notch signaling plays a role, along with the inductive signals, in the genetic network regulating cardiac cell type specification and determination.

Multiple functions have been reported for the Notch pathway in heart development, in specification of the cardiogenic field in Xenopus and mouse (Rones et al., 2000; Schroeder et al., 2003). Mutations in the genes in the Notch signaling pathway result in various cardiac defects including pericardial edema, defects in formation of valves, atrial and ventricular septa, and in endocardial cushions (Donovan et al., 2002; Fischer et al., 2004; Gessler et al., 2002; Kokubo et al., 2005; Kokubo et al., 2004; Koo et al., 2005; McCright et al., 2002; Sakata et al., 2002; Timmerman et al., 2004). We injected retroviruses after the completion of early cardiogenesis. Thus, initial cardiogenic processes in the RCAS-NIC injected samples were unaffected and the hearts retained largely normal morphology. Our study thus provides evidence for an additional, later role of Notch1 in heart development, in the differentiation of ventricular cell types. The following evidence supports the notion that inhibition of cardiac muscle marker expression by RCAS-NIC is due to specific effects of Notch signaling, and not nonspecific effects of the virus. First, samples infected with a control virus expressing GFP displayed normal expression of myocardial and conduction cell markers, and have a normal conduction pathway, suggesting that the virus itself does not cause non-specific effects on cardiac differentiation. This type of virus has been widely used to study the development of many organ systems, and no significant adverse effects have been reported. Second, a significantly higher proportion of the RCAS-NIC-infected cells expressed the conduction cell markers, HNK-1 and SNAP-25. Third, the retrovirally expressed dominant negative Su(H) gave rise to the opposite effects of those with RCAS-NIC, suggesting that these effects on marker expression are likely due to specific effects of the transgenes, not nonspecific effects of viral infection. Fourth, the effects of constitutively activated Notch1 on cell differentiation are also consistent with its endogenous expression pattern; Notch1 is expressed in the conduction cell lineage at early stages, but not in myocardial cells.

Notch activity has been shown to influence various cell differentiation processes, by selecting a subset of cells from an initially homogenous precursor population (Artavanis-Tsakonas et al., 1999). This is mainly achieved through a process termed “lateral inhibition”, in which a small difference in signaling among the cells is amplified through a feedback mechanism. A key element of this mechanism is that the expression of Delta is repressed by Notch signaling through downstream basic helix-loop-helix (bHLH) transcription factors (Heitzler et al., 1996; Heitzler and Simpson, 1991). We have shown that Delta1 is similarly downregulated by constitutively activated Notch, suggesting that a feedback loop is possibly at work in cardiac differentiation.

Because our cell type markers are on the plasma membrane (HNK-1) or sarcomeric (MF-20), and the cells are large and densely packed, we used cytoplasmic staining of GAG rather than the nuclear myc staining for scoring. Although not all GAG-positive cells are positive for myc due to low sensitivity of a single copy myc, we think NIC is expressed by most of the GAG-positive cells. This is evident in the well-infected samples such as those shown in Figs. 4A and 3A, that nearly all the ventricular cells are positive for HNK-1 and negative for MF-20, respectively. Our results of constitutively active Notch on the expression of myocardial markers, MF20 and α-actin, and conduction markers, HNK-1 and SNAP-25, support that Notch1 is involved in cardiac differentiation by inhibiting cardiomyocyte but promoting early conduction cell differentiation. This is reminiscent of the role of Notch1 in the nervous and immune systems, where Notch inhibits neural and B cell fates, and promotes glial and T cell differentiation, respectively. However, the effect of Notch signaling on another conduction marker, Cx40, is more complex. Decreasing Notch signaling by using a dominant negative Su(H) construct shows that the high level of Cx40 expression is diminished but the low level of Cx40 expression in the myocardium remains unchanged. Because conduction cells express high levels of Cx40 whereas the myocardial cells express low levels of Cx40, this result is consistent with our model that reduction of Notch signaling increases myocardial but decreases conduction cell differentiation. However, because Cx40 is a relatively later marker, Notch signaling may need to be turned down before high levels of Cx40 can be expressed in the conduction cells. Prolonged expression of constitutive active Notch may inhibit the expression of Cx40 in both the myocardial and conduction cells. This is consistent with our observation that Notch1 is only transiently expressed in the conduction cells (Fig. 1 and data not shown). Additional signals may also be required with the Notch signaling to turn on the maturation program of the conduction cells including high levels of Cx40 expression. Previous works have shown that paracrine factors released by the endocardium and endothelial cells of the coronary arteries, endothelin in chick and Neuregulin-1 in mouse, can increase the expression of conduction markers and cause a change in conduction pathway that is consistent with excess recruitment of functional Purkinje cells (Hall et al., 2004; Kanzawa et al., 2002; Rentschler et al., 2002; Takebayashi-Suzuki et al., 2000). Therefore, our current model is that transient Notch activity may be required for the initial separation of myocardial and conduction lineages by inhibiting cardiomyocyte differentiation and promoting early conduction cell differentiation, possibly through regulating the responsiveness of the cells to the paracrine factors.

Although HNK-1 has been shown as a conduction cell marker in chick and other species, it has also been used as a neural crest marker. However, we believe that the HNK-1 expression induced by NIC indicates an increase in the conduction lineage cells rather than the neural crest cells for the following reasons. First, NIC increases the expression of another conduction marker SNAP-25 in addition to HNK-1. SNAP-25 has not been shown as a neural crest cell maker, which argues against an increase in neural crest cells. Second, expression of NIC through a non-viral vector by electroporation resulted in a similar increase of HNK-1 expression (data not shown). Because this vector cannot replicate, cells entering the heart after the initial electroporation, such as neural crest cells, will not be infected. Third, in some heavily infected hearts, nearly 100% of the ventricular cells are positive for HNK-1, but negative for MF-20. This is unlikely to be an exclusive effect on neural crest cells.

Our optical mapping studies show that approximately 41% of the RCAS-NIC injected mutants had obvious abnormality in the conduction pathway. Because optical mapping analyzes the pattern of action potential in epicardial cells, it is less likely to be affected by factors which may affect heart rate or contractility. For each heart, we analyzed at least five beat series to confirm that the abnormality is present consistently in each beat. In addition, the degree of abnormality correlated well with the degree of infection by the RCAS-NIC virus. The altered conduction system function revealed by optical mapping is consistent with our model that cells expressing constitutively active Notch are not fully differentiated functional conduction cells. These results are different from the phenotype observed in chick hearts with excess production of endothelin or mouse hearts treated with neuregulin-1, which induced alterations in activation patterns consistent with additional recruitment of Purkinje cells (Hall et al., 2004; Rentschler et al., 2002). Optical mapping studies of the (Cx40) knockout mice indicated some delay or block in conduction velocities in the right bundle branch, and more diffuse breakthrough sites in the left ventricle (Tamaddon et al., 2000; van Rijen et al., 2001). Chimeric mice generated from stem cells deficient for connexin 43, a gap junction predominantly expressed in the ventricular myocardium, displayed conduction delay (Gutstein et al., 2001). Thus, reduced expression of gap junction proteins can lead to conduction abnormalities. Because constitutively active Notch down-regulates the expression of Cx40, we speculate that the conduction abnormalities of the RCAS-NIC injected hearts may be in part due to decreased expression of Cx40. In the hearts moderately infected with RCAS-NIC, a general correlation of blocked pathway with the area of infection was observed, suggesting a block in the infected areas. In the hearts highly infected by RCAS-NIC, Cx40 expression may be downregulated throughout the myocardium, causing the electrical impulse to disperse across the epicardial surface. Because our optical mapping protocol detects electrical propagation across the epicardial surface, we therefore observed a diffuse activation pattern.

Retroviral lineage analyses have shown that the central and peripheral conduction systems may arise separately although they both share lineages with cardiomyocytes (Cheng et al., 1999; Gourdie et al., 1995). In chick, two different conduction cell localizations have been described: subendocardial and periarterial (Gourdie et al., 1999). Because most of the well-infected RCAS-NIC embryos died around E5, prior to the formation of periarterial conduction cells, our current study has been focused on subendocardial conduction cells in the ventricles. However, at early stages such as E3 and E4.5, cells expressing Notch1 and HNK-1 appear not always associated closely with the endocardium. This possibly represents an early pattern prior to the establishment of more defined subendocardial localization, as we have observed this pattern in multiple samples in multiple experiments. It is also interesting to note, that Notch1 appears to be expressed in vascular endothelial cells in the coronary vessels after E9.

While our results support a role for Notch1 in cardiac cell differentiation in the ventricles, we currently do not have evidence whether it is involved in atrial cell differentiation. We focused our study on ventricles because some of the cell type markers we used are not as specific in the atria as they are in the ventricles. Although Notch1 expression was reported in the outflow tract, the atrioventricular canal, the trabeculae of the ventricles, the epicardium, in the aorta (Loomes et al., 2002), and in the endocardium (Del Amo et al., 1992; Reaume et al., 1992; Timmerman et al., 2004), the expression of Notch1 has yet to be reported in myocardium in mouse. The differences in expression patterns of Notch1 reported in the heart likely reflect dynamic and transient nature of the expression patterns of Notch1. Mutant mice with a targeted deletion of the Notch1 gene die before E11. Although severe pericardial edema was reported for these mutant mice, these mice have a beating heart at E10.5 (Swiatek et al., 1994). It is likely, therefore, that the central conduction system is differentiated to a certain extent by this stage. It is possible that Notch1 function is not required for the differentiation of the central conduction system, or other Notch receptors expressed in the heart may compensate for the loss of the Notch1 receptor. It has been reported that the Notch2 and Notch3 genes are also expressed in the developing heart (Krebs et al., 2003; McCright et al., 2001). With the characterization of Notch function in chick conduction system development, further study on the role of Notch in the murine conduction system development is warranted.

4. Experimental procedures

4.1. Whole-mount and section in situ hybridization

Standard specific pathogen-free white Leghorn chick embryos from closed flocks were provided fertilized by Charles River Laboratories (North Franklin, Connecticut). Eggs were incubated inside a moisturized 38 °C incubator. The embryos were staged according to Hamburger and Hamilton (Hamburger and Hamilton, 1992). Because our analyses were focused on embryos at relatively late stages, we chose to describe the ages of embryos by embryonic days rather than Hamburger–Hamilton stages. E3, E4.5, E6, E9 are equivalent to Hamburger and Hamilton stages 20, 25, 29, 35, respectively.

The hearts were dissected and fixed in 4% paraformaldehyde at 4 °C for 12–24 h. Cryosections of 20 μm thickness were prepared from tissue OCT blocks on a cryostat (Leica, Deerfield, IL) and collected on Super-frost Plus slides (Fisher Scientific, Pittsburgh, PA). Whole-mount and section in situ hybridization were performed as previously described in Bao et al., 1999; and Jin et al., 2006. cDNA plasmids used for generating the Digoxigenin-labeled Notch1 and Delta1 probes were provided by Dr. D. Henrique. Full-length chicken connexin 42 cDNA was obtained from the chicken EST database (MRC Geneservice).

4.2. Immunofluorescence staining and data analysis

Immunofluorescence staining was carried out on cryosections of the heart. Sections were fixed in 4% paraformaldehyde, and blocked in 10% calf serum DME with 0.2% Triton X-100. Primary and secondary antibodies were diluted in block, and incubated for 1 h at room temperature or overnight at 4 °C. Viral infection was confirmed by using the mouse polyclonal anti-gag antibody, p27 (SPAFAS, Norwich, CT). The mouse monoclonal antibody of the muscle marker, MF20, was obtained from the Developmental Studies Hybridoma Bank (University of Iowa, Iowa City, IA). HNK-1 was obtained from ATCC (Manassas, VA), α-actin antibody was obtained from Dako Cytomation (Carpinteria, CA), and SNAP-25 was obtained from Sternberger Monoclonals, Inc. (Lutherville, MD).

The nuclear stain, DAPI and Hoechst Dye 34580 were obtained from Roche (Indianapolis, IN), and Molecular Probes (Eugene, OR), respectively.

Stained sections were analyzed and scored using the Leica TBS SP2 confocal microscope and software. A total of 500 randomly selected cells were scored from each heart, and a total of four different hearts were examined for each marker. The percentage of infected cells expressing the observed marker was used for statistical analysis by unpaired Student's t-test.

4.3. Viral constructs

The mouse Notch intracellular domain (NIC) construct was obtained from Dr. Jeffrey Nye. NIC insert was released from the plasmid and cloned into the avian replication-competent retrovirus, RCAS. G-coat viruses were prepared by transient transfection as previously described in Bao et al., 1999. Early heart tubes of HH stage 9 embryos were injected with the viral stocks. Because the embryos injected with the undiluted viral stock had high rate of mortality, we diluted the viral stocks 1:2 to increase the chances of survival. The dominant-negative form of Suppressor-of-Hairless was a gift from Dr. Nathan Lawson (UMass Medical School, Worcester, MA). The 2.5 kb fragment was cloned into the ClaI site of RCAS. RCAS virus was similarly prepared and used for injection into the heart tubes of HH 9 embryos.

4.4. Optical mapping

Optical mapping technique was modified from previously published procedures (Reckova et al., 2003). Hearts were dissected from uninjected control, RCAS-GFP-injected, and RCAS-NIC-injected E4.5 embryos, and stained by submerging in a 0.002% solution of voltage sensitive fluorescent dye, di-4-ANEPPS (Molecular Probes), in Tyrodes-HEPES buffer, pH 7.4, for 4 min at room temperature. The hearts were then transferred to oxygenated 37 °C Tyrode's solution imaged on a custom-built upright wide-field epifluorescence microscope equipped with a 128 × 128 pixel, high-speed, 100% imaging duty cycle electron multiplying CCD camera (Cascade 128+, Photometrics, Tucson, AZ). The laser shutter, camera control and image storage were managed by Metaview software (Universal Imaging, Philadelphia, PA). Hearts were imaged using an Olympus 2× objective lens, with an overall magnification of 10 μ/pixel. As the contraction of the heart at this stage did not appear to interfere with the imaging, no motion inhibitors were used. The dye was excited with the 514 nm line of an argon laser, and the emitted fluorescence was imaged onto the camera through a 580 nm long pass emission filter. A simple image streaming protocol was set up in Metaview and used for all image acquisitions. For each sequence, the laser shutter was opened and 4000 images were streamed directly to system memory at 500 frames/sp (2 ms exposure per image frame). The data were processed using a custom software program as follows. Images were first smoothed using a 5 × 5 box filter, and the first time derivative was computed by subtracting successive images (dFi[unk]dt≈|Fi − Fi−1|[unk]2 ms, where F is the average fluorescence measured in each smoothed picture element). The maximum upstroke velocity was defined as dF/dt max and depicted as red in the color scale accompanying the sequence of difference images. For most of the hearts, the difference images were analyzed for five beat sequences to confirm the results. In all cases, we found that the activation patterns are consistent from beat to beat, despite some minor differences.

Supplementary Material

Supplementary Figure 1

Acknowledgements

We are grateful to Dr. Gregory Morley for helpful advice on optical mapping technique, Drs. N. Lawson, J. Nye, and D. Henrique for reagents, M. Grabowski, S. Heilman, T. Cronin, Dr. R. Mozell for critical reading of the manuscript. This work is funded by the American Heart Association and the Worcester Foundation for Biomedical Research.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.mod.2006.06.003.

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Supplementary Figure 1
NIHMS11948-supplement-Figure1S.pdf (103.5KB, pdf)

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