Sema3D, Sema3F, and Sema5A Are Expressed in Overlapping and Distinct Patterns in Chick Embryonic Heart

Abstract

INTRODUCTION

As the first organ to form in the embryo, the four-chambered heart is generated through a series of complex morphogenetic processes (Markwald et al., 1996; Srivastava and Olson, 2000; Bruneau, 2002; Moorman and Christoffels, 2003; Armstrong and Bischoff, 2004; Eisenberg and Markwald, 2004). The cardiogenic mesoderm arises from paired regions of dorsolateral mesoderm, which subsequently fuse along the midline to assemble the linear heart tube. The heart tube undergoes rightward looping, which leads to positioning of the cardiac segments at proper positions of the four chambers. After looping, some of the endocardial cells of the outflow tract and atrioventricular segments delaminate and migrate into the adjacent extracellular matrix to form cardiac cushion tissues, a process characterized as “epithelial–mesenchymal transformation.” Cardiac cushion tissues undergo further remodeling to give rise to the atrioventricular valves and cardiac septa (Markwald et al., 1996; Eisenberg and Markwald, 2004; Lincoln et al., 2004; Person et al., 2005). For the ventricular septum, there is also a muscular component, which has been shown to form largely through fusion and coalescence of ventricular trabeculae (Harh and Paul, 1975; Ben-Shachar et al., 1985). Abnormal development of the endocardial cushion contributes to the pathogenesis of various types of congenital heart defects, including transposition of the great arteries, double outlet right ventricle, tetralogy of Fallot, and atrioventricular septal defect (Nakajima et al., 2000; Anderson et al., 2003; Armstrong and Bischoff, 2004).

Although originally characterized as guidance factors for axons in the nervous systems, many proteins in the Semaphorin, Netrin, Slit, and Ephrin families have now been implicated to play a role in the development of a variety of non-neural tissues (Hinck, 2004). ephrinB2 and one of the receptors, EphB4, mRNA are exclusive markers for arteries and veins, respectively (Wang et al., 1998). Major defects were found in remodeling of the capillary network in ephrinB2 and EphB4 homozygous null mice (Gerety et al., 1999). Moreover, Slit/Robo signaling has been shown to be important for kidney development (Grieshammer et al., 2004).

Semaphorins comprise one of the largest conserved families of axon guidance cues (Raper, 2000; Pasterkamp and Kolodkin, 2003). All Semaphorin family proteins are secreted or membrane-bound and have a conserved SEMA domain containing approximately 500 amino acids. Eight subclasses were characterized based on sequence similarity and structural features. Semaphorins in class I and II are found in invertebrate species and class V in the genomes of certain DNA viruses. Class III–VII contain vertebrate Semaphorins: secreted proteins in class III, glycosylphosphatidylinositol (GPI)-anchored proteins in class VII, and transmembrane proteins in classes IV to VI. Two receptor families, plexins and neuropilins, have been characterized in mediating Semaphorin functions.

The roles of some Semaphorin proteins in heart development have also been demonstrated, mainly by studies of targeted deletion in mice. Targeted mutation of Sema3C in mice exhibit severe cardiac defects, consisting of interruption of the aortic arch and improper septation of the cardiac outflow tract (Feiner et al., 2001). Sema3A−/− mutant mice have postnatal hypertrophy of the right ventricle and ventricular septal defect (Behar et al., 1996). Deletion of one of the plexin receptors, plexinD1, results in cardiovascular defects involving the outflow tract of the heart and derivatives of the aortic arch arteries (Gitler et al., 2004). In addition, Sema6D has been shown to play dual roles in cardiac morphogenesis, promoting migration of cells from the conotruncal segment, and inhibiting migration of ventricular cells (Toyofuku et al., 2004a). Reverse signaling by Sema6D enhances the migration of myocardial cells from the compact zone into the trabeculae (Toyofuku et al., 2004b).

Although the expression patterns of Semaphorins have been well documented in the nervous system (Shepherd et al., 1996; Chilton and Guthrie, 2003), the expression analyses of semaphorin genes in the heart are relatively limited. Here, we describe the expression patterns of three semaphorin genes in chick heart development: Sema3D, Sema3F, and Sema5A. These genes exhibit overlapping but distinct expression patterns in the embryonic chick heart. All three genes are expressed in the cardiac cushion regions during the stages of cardiac remodeling. However, these genes additionally display their own unique patterns in the heart. Sema3D is expressed in the tips of the trabeculae, whereas Sema3F is expressed in scattered cells within the ventricles, and Sema5A is expressed in the newly formed atrioventricular valves.

RESULTS

Using cDNAs prepared from chick embryonic day (E) 6 heart tissues as templates, we performed RT-PCR with degenerate primers, designed based on the conserved sequences in the SEMA domain (see Experimental Procedures section). After sequencing approximately 60 clones, we found that the nucleotide sequences of some of our clones are identical to the previously reported Sema3D and Sema3F genes (Luo et al., 1995; Chilton and Guthrie, 2003; Watanabe et al., 2004) and the newly annotated Sema5A gene from the chick genomic sequence (Supplementary Table S1, which can be viewed at http://www.interscience.wiley.com/jpages/1058-8388/suppmat). Further analysis with the bl2seq program provided by National Center for Biotechnology Information (NCBI) shows that the Sema3D and Sema3F clones are 59% identical at amino acid level, but much less homology is detected between Sema5A and Sema3 subfamily clones. Despite conservation in amino acid sequences, the nucleotide sequences of these clones are divergent enough that no signifi-cant homology can be found. We, therefore, have isolated partial cDNA clones for the chick Sema3D, Sema3F, and Sema5A genes.

To determine the expression patterns of these semaphorin genes in heart development, we performed in situ hybridization on cryosections of the heart with digoxigenin (DIG) -labeled probes. As shown in Figure 1, Sema3D transcripts are expressed inside the heart at E6. Sema3D transcripts were found in the atrioventricular junction, and the interventricular septum. At higher magnification, the expression can be clearly seen in the cardiac cushion regions, in both the mesenchymal cells inside the cardiac jelly and the epithelial cells in the endocardial layer (Fig. 1B,D). The expression in the interventricular septum appears to be concentrated at the leading edge, and absent from the more basal regions of the septum (Fig. 1A,C,E). Judging by the staining patterns, Sema3D transcripts appear to be both in the endocardial and myocardial layers (Fig. 1C,E). No signal was detected in the control samples hybridized with the Sema3D sense probe (Fig. 1F).

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Fig. 1

Expression of Sema3D transcripts in embryonic chick heart at embryonic day (E) 6. In situ hybridization experiments were carried out on cryosections of chick heart at E6. Results from the sections of two different hearts are shown. A: Expression of Sema3D was found at the atrioventricular junction (avj) and the tip of the forming interventricular septum (ivs). Higher magnification images of the avj and ivs areas are shown in B and D, and C and E, respectively. B,D: At the atrioventricular junction regions, expression of Sema3D was found both in the epithelial cells in the endocardial layer (arrows) and in the mesenchymal cells inside the cardiac jelly (arrowheads). C,E: In the interventricular septum regions, Sema3D expression was detected at the tip of the interventricular septum, both in the endocardial (arrows) and myocardial layers (arrowheads). F: A Sema3D sense probe was used for the control experiments. Note that no signal was detected with the sense probe. Scale bars = 250 μm in A, 50 μm in F (applies to B–F).

Expression patterns of Sema3D in the hearts at E9 were observed at the cardiac cushion tissues and the leading edge of the interventricular septum, similar to that at E6 (Fig. 2A). In addition, Sema3D transcripts are also present at the periphery of the inter-ventricular septum and the leading edges of some trabeculae in the ventricles (Fig. 2A-C). At higher magnification, high levels of Sema3D transcript expression were clearly visible in the cells at the leading edges of the trabeculae, including both the endocardial and myocardial layers, while the cells at more trailing positions expressed a low amount or not at all (Fig. 2C). At an even later stage, E12, Sema3D is still expressed in the cells at the very periphery of the interventricular septum and the tips of a small number of trabeculae, but no longer expressed in the atrioventricular junction regions or the tip of the interventricular septum (Fig. 2E-G). No signal was observed with the control sense probe in either the E9 or E12 heart sections (Fig. 2D,H).

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Fig. 2

A–C,E–G: Expression of Sema3D transcripts in embryonic chick hearts at embryonic day (E) 9 (A–C) and E12 (E–G). In situ hybridizations were carried out on cryosections of chick hearts. Higher magnification images of the boxed areas in A are shown in B and C. Note that the cells at the tip and peripheral margins of the interventricular septum (ivs) and the leading edges of some trabeculae in the ventricles are positive for the expression of Sema3D transcripts (arrows). D: E9 heart sections hybridized with a control Sema3D sense probe did not show any signal. Similar patterns at the peripheral margins of the interventricular septum were found at E12 (E–G). However, most trabeculae and the tip of the interventricular septum are negative for Sema3D expression at this stage. Higher magnification image of the boxed area in E is shown in F. G: The ivs area of another heart sample hybridized with the antisense Sema3D probe is shown. H: E12 heart section hybridized with a control Sema3D sense probe did not show any signal. a, atrium; v, ventricle; ivs, interventricular septum. Scale bars = 500 μm in A,E, 50 μm in H (applies to B–D,F–H).

We also analyzed the expression of Sema3F, another gene in the same subfamily, by in situ hybridization on chick embryonic heart sections. At E6, Sema3F is also expressed in the cardiac cushion in the atrioventricular junction, and at the tip of the growing interventricular septum (Fig. 3A,B). In the cardiac cushion region, Sema3F transcripts are present in both the epithelial and mesenchymal cells, similar to Sema3D. At E9, Sema3F expression remains in the cardiac cushion regions and the tip of the interventricular septum (Fig. 3C,D). In addition, Sema3F is also expressed in a subset of cells scattered inside the myocardium of the heart at E9, more in the myocardium below the trabeculae. Although this pattern is reminiscent of the coronary vascular cells that have migrated from outside of the heart into the myocardium, these cells are not grouped in a pattern resembling typical vessels. At E12, the expression of Sema3F is no longer present in the cardiac cushion and interventricular septum (Fig. 3E,F). The expression in the scattered cells remains, although the Sema3F-positive cells are now localized more in the trabeculae than in the compact zone.

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Fig. 3

A–F: Expression of Sema3F transcripts in the embryonic chick heart at embryonic day (E) 6 (A,B), E9 (C,D), and E12 (E,F). In situ hybridizations were performed on cryosections of the hearts. A: At E6, Sema3F expression was detected in the cardiac cushion (marked with an asterisk) at the atrioventricular junction (avj) and at the leading edge of the growing interventricular septum (ivs). B: The boxed area in A is shown at higher magnification. Note both the epithelial (arrowheads) and mesenchymal cells (arrow) in the cardiac cushion (cc) express Sema3F transcripts. C: At E9, Sema3F is similarly expressed at the atrioventricular junction and at the tip of the interventricular septum. D: A higher magnification image of the boxed area in C; cells positive for Sema3F are visible throughout the heart, scattered inside the myocardium (arrows in D), but are more concentrated in the area outside of the trabeculae. E,F: At E12, scattered Sema3F-positive cells (arrows) are still present inside the heart, now more in the trabeculae than in the compact zone (arrows). Scale bars = 250 μm in A,C, 50 μm in F (applies to B,D–F).

In addition, we found that Sema5A is also expressed in the embryonic chick heart. At E6, Sema5A expression was detected inside the cardiac cushion regions, in both the epithelial and mesenchymal cell populations (Fig. 4A,B). At E9, high levels of Sema5A transcripts were observed in the cardiac cushion region inside the outflow tract. In addition, Sema5A transcripts were found in the cells in the cardiac cushions from both sides of the atrioventricular canal, apposed to each other (Fig. 4C,D). It has been shown previously that cardiac cushion cell proliferation decreases and valve primordial maturation occurs around E10 in chick (Lincoln et al., 2004). These Sema5A-positive cells, thus, correspond to the atrioventricular valve primordial cells in the process of maturation. The expression of Sema5A becomes restricted to the atrioventricular valves at E12, including the newly formed mitral and tricuspid valves (Fig. 4E,F).

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Fig. 4

Expression of Sema5A transcripts in embryonic chick heart at embryonic day (E) 6 (A,B), E9 (C,D), and E12 (E,F). A,B: At E6, Sema5A expression was only detected in the cardiac cushion region (cc), shown at lower (A) and higher (B) magnifications. Note that the expression of Sema5A was detected both in the epithelial (arrow) and mesenchymal cells (arrowhead) in the cardiac cushion. C,D: At E9, Sema5A transcripts are expressed in the conotruncus cardiac cushion (ccc), the forming tricuspid valve (tcv), and mitral valve (mv). E,F: Similar expression patterns were observed in the E12 heart. rv, right ventricle; la, left atrium; lv, left ventricle. Scale bar = 250 μm in A, 50 μm in B (applies to C–F).

DISCUSSION

Although initially characterized in axon pathfinding, several axon guidance factors have been shown to play important roles in embryonic development. Several semaphorin genes, including Sema3A, Sema3C, and Sema6D, have been demonstrated to be essential for cardiac morphogenesis (Behar et al., 1996; Feiner et al., 2001; Gitler et al., 2004; Toyofuku et al., 2004a,b). However, the expression of other semaphorin genes has not been characterized in heart development in great detail. Here, we report the expression patterns of three semaphorin genes during chick heart development: Sema3D, Sema3F, and Sema5A. All three genes are expressed in the cardiac cushion regions during the period of cardiac remodeling to form valve primordia. However, they also show distinct patterns in the embryonic heart. Sema3D exhibits an interesting expression pattern at the tip of the trabeculae; Sema3F is expressed in scattered cells in the myocardium; Sema5A is expressed in the newly formed atrioventricular valves. These results suggest that more semaphorin genes are expressed in the heart in addition to those that have been analyzed functionally, and they may play overlapping yet distinct roles in heart development.

The cardiac cushion is a developmental structure important for cardiac morphogenesis (Nakajima et al., 2000; Anderson et al., 2003; Armstrong and Bischoff, 2004; Person et al., 2005). The formation of the cardiac cushions is a complex event initiated by epithelial–mesenchymal transition (EMT) of a subset of endocardial cells that are specified in the cushion-forming regions to delaminate and invade the underlying extracellular matrix. Inside the extracellular matrix, the cardiac jelly, these cells proliferate and complete their differentiation into mesenchymal cells. The cardiac cushion tissues then undergo extensive remodeling to form the atrioventricular valves. Interestingly, all three semaphorin genes are expressed in the cardiac cushion regions during the period of cardiac remodeling, in both the epithelial and mesenchymal cells. Because the three semaphorin genes also have additional distinct expression patterns, the overlapping expression patterns are not likely due to cross-hybridization of the probes used for in situ hybridization. In addition, sequence comparison using the bl2seq program provided by NCBI has confirmed that the nucleotide sequences of these clones are divergent and that no significant homology is detected (Supplementary Table S1). Two previously reported semaphorin genes, Sema6D and Sema3C, have different expression patterns. Sema6D transcripts were reported to be expressed broadly throughout the entire heart, including the conotruncal segment, the atrioventricular segment, and the ventricular myocardium (Toyofuku et al., 2004a). Sema3C expression is shown only to be in the myocardium of the outflow tract (Feiner et al., 2001; Gitler et al., 2004).

Sema3D is expressed additionally at the tips of the trabeculae. It has been documented that, in chick, the muscular component of the ventricular septum is formed largely through fusion and coalescence of ventricular trabeculae (Harh and Paul, 1975; Ben-Shachar et al., 1985). Therefore, the tips of the trabeculae are composed of highly motile cells inside the ventricles. In addition to the staining in the cardiac cushion, Sema3F is also expressed in a subset of cells scattered throughout the ventricles. This pattern is reminiscent of the vascular precursor cells of coronary vessels (Pennisi and Mikawa, 2005). It is well established that endothelial and smooth muscle cells of the coronary vessel system are derived from a structure called the proepicardial organ (PEO) outside of the heart (Mikawa and Fischman, 1992; Dettman et al., 1998; Reese et al., 2002). In the first wave of migration, the PEO cells migrate over the myocardium to envelope the heart. A subset of the PEO cells then undergo EMT and subsequently move through the myocardium in a second wave. These cells populate throughout the myocardium and establish blood vessels through a vasculogenic process. Due to a lack of a suitable antibody to the endothelial cells in chick, we cannot confirm whether Sema3F is expressed in these vascular precursor cells. The identity of the Sema3F-expressing cells remains to be determined.

In summary, all three semaphorin genes appear to be expressed in regions of the heart that undergo active remodeling. It has been shown previously that the Semaphorin signaling receptor plexinA1 is expressed in the cardiac cushion and ventricular myocardium, whereas plexinD1 is expressed in the endothelial cells (Gitler et al., 2004; Toyofuku et al., 2004a). As Sema3D and Sema3F both encode secreted proteins, the role of these genes in cardiac remodeling currently is unclear. It has been demonstrated that SemaA3 controls vascular morphogenesis by inhibiting integrin function in an autocrine manner (Serini et al., 2003). Disruption of SemaA3 signaling stimulates integrin-mediated adhesion and migration to extracellular matrices. Unlike the class III semaphorin genes, Sema5A encodes a transmembrane protein. Another transmembrane Semaphorin, Sema6D, has been shown to mediate both forward and reverse signaling in the myocardium (Toyofuku et al., 2004a,b). Forward signaling of Sema6D exerts a region-specific effect on the migration of cardiomyocytes. Constitutive activation of Sema6D reverse signaling enhances migration of myocardial cells. Unlike Sema3D and Sema3F, Sema5A transcripts remain expressed in the atrioventricular valves after the valves are formed. Sema5A has been shown to interact with the glycosaminoglycan portion of chondroitin sulfate proteoglycans and heparan sulfate proteoglycans (Kantor et al., 2004). Inactivation of the Sema5A gene results in early embryonic lethality at E11.5 to 12.5 due to defective remodeling of the cranial vascular system (Fiore et al., 2005). It awaits further study to uncover how these semaphorin genes are involved in cardiac morphogenesis and remodeling.

EXPERIMENTAL PROCEDURES

ACKNOWLEDGMENT

Footnotes

REFERENCES