SEARCH

SEARCH BY CITATION

Keywords:

  • chicken embryo;
  • heart development;
  • coronary development;
  • PDGF;
  • epicardium-derived cells;
  • pro-epicardial organ

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Platelet-derived growth factors (PDGFs) are important in embryonic development. To elucidate their role in avian heart and coronary development, we investigated protein expression patterns of PDGF-A, PDGF-B, and the receptors PDGFR-α and PDGFR-β using immunohistochemistry on sections of pro-epicardial quail–chicken chimeras of Hamburger and Hamilton (HH) 28–HH35. PDGF-A and PDGFR-α were expressed in the atrial septum, sinus venosus, and throughout the myocardium, with PDGFR-α retreating to the trabeculae at later stages. Additionally, PDGF-A and PDGFR-α were present in outflow tract cushion mesenchyme and myocardium, respectively. Small cardiac nerves and (sub)epicardial cells expressed PDGF-B and PDGFR-β. Furthermore, endothelial cells expressed PDGF-B, while vascular smooth muscle cells and interstitial epicardium-derived cells expressed PDGFR-β, indicating a role in coronary maturation. PDGF-B is also present in ventricular septal development, in the absence of any PDGFR. Epicardium-derived cells in the atrioventricular cushions expressed PDGFR-β. We conclude that all four proteins are involved in myocardial development, whereas PDGF-B and PDGFR-β are specifically important in coronary maturation. Developmental Dynamics 233:1579–1588, 2005. © 2005 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Normal avian and mammalian coronary development is dependent on correct outgrowth of the epicardium, the outer cell layer of the heart (Poelmann et al., 1993; Virágh et al., 1993; Mikawa and Gourdie, 1996; Gittenberger-de Groot et al., 1998; Vrancken Peeters et al., 1999). This layer is not derived from cells of the primitive heart itself but from an extracardiac, cauliflower-like structure, called the pro-epicardial organ (PEO; Virágh et al., 1993). The PEO is located at the dorsal wall of the embryo near the sinus venosus and the liver. It protrudes in the direction of the heart and after contacting the heart, cells of the PEO start to cover the entire myocardium, thereby forming the epicardium (Vrancken Peeters et al., 1995). A subepicardial mesenchyme of epicardium-derived cells (EPDCs; Gittenberger-de Groot et al., 1998) is formed from the epicardial sheet by epithelial–mesenchymal transformation (EMT; Vrancken Peeters et al., 1999; Morabito et al., 2001). EPDCs do not only form the subepicardial layer but are also involved in the formation of the atrioventricular valves (Gittenberger-de Groot et al., 1998; Manner et al., 2001). Furthermore, they migrate into the myocardium to form the interstitial fibroblasts (Gittenberger-de Groot et al., 1998) and the smooth muscle cells of the coronary vasculature (Poelmann et al., 1993; Mikawa and Gourdie, 1996; Dettman et al., 1998; Gittenberger-de Groot et al., 1998; Vrancken Peeters et al., 1999).

The development of the coronary vasculature starts with the formation of a primitive endothelial network (Poelmann et al., 1993; Lie-Venema et al., 2005), which only stabilizes after recruitment of supporting mural cells, such as vascular smooth muscle cells (vSMCs; Vrancken Peeters et al., 1999; Lu et al., 2001). The major cellular source for the developing coronary arterial and venous walls is the EPDC population. Although the processes involved in EMT, migration and differentiation of the epicardium and the EPDCs have been investigated and described (Tomanek, 1996; Gittenberger-de Groot et al., 1998; Morabito et al., 2002; Munoz-Chapuli et al., 2002; Perez-Pomares et al., 2002; Reese et al., 2002; Luttun and Carmeliet, 2003; Wessels and Perez-Pomares, 2004), many aspects are still unresolved, one of them being the role of platelet-derived growth factor (PDGF) -signaling molecules.

The PDGF family is presently thought to consist of four different members, which can form five different dimers, namely PDGF-AA, -AB, -BB, -CC, and -DD. These ligands are secreted into the extracellular matrix to exert their function. The distance of action depends on the presence of heparin-binding retention motifs in their C-terminus, which is regulated through alternative mRNA splicing in the case of PDGF-A and is thought to be regulated through proteolytical processing in PDGF-B. When such motifs are present, the ligand will not be able to diffuse over a long distance, as it will be caught in the extracellular matrix, due to binding to heparin. When the motifs are not present, the ligand is able to diffuse further from the site of production, thereby increasing the distance of action. Although a developmental role seems to be present for these different variants, their exact role and distribution patterns are not clear (Betsholtz, 2003). Two receptors, PDGF receptor-α (PDGFR-α) and PDGFR-β, are known. They can form homo- and heterodimers upon ligand binding (Heldin et al., 2002). In vitro, PDGFR-α binds PDGF-A, -B, and -C and PDGFR-β binds PDGF-B and -D (Heldin et al., 2002). In vivo, however, not all of these combinations seem to play a biological role. A role for PDGF-B signaling through PDGFR-α, for example, seems not to exist in mouse embryonic development, because the expression patterns of the ligand and the receptor do not colocalize (Betsholtz, 2003). PDGF-B is described to be expressed solely by endothelial cells and megakaryocytes during mouse embryonic development (Betsholtz et al., 2001), whereas PDGFR-α is expressed in non-neuronal neural crest (Morrison-Graham et al., 1992) and paraxial mesoderm-derived tissues like the mesenchyme around the vertebrae, the kidneys, the cardiac valves, and the pericardium (Takakura et al., 1997). Similarly, nonoverlapping expression patterns have been found for PDGF-A, present in different epithelia (Orr-Urtreger and Lonai, 1992) and PDGFR-β, present in vSMC or pericyte precursors (Lindahl et al., 1997a; Hellström et al., 1999; Betsholtz et al., 2001). Less is known about the expression and function of the two ligands PDGF-C and PDGF-D (Zhuo et al., 2003; Fang et al., 2004), but PDGF-C does not seem to play a role in heart development as it has been described not to be expressed at all in the developing mouse heart (Ding et al., 2000).

Functional roles in embryogenesis for PDGF-A, PDGF-B, and the two receptors have been investigated in several null-mutant mouse models. The defects seen in the Pdgf-b and Pdgfr-β knockout mice are comparable, again suggesting that PDGF-B only has a role in development by signaling through PDGFR-β. The embryos die perinatally (Battegay et al., 1994; Lindahl et al., 1997a; Hellström et al., 1999) and present with heart malformations (ventricular septal defect, thin ventricular myocardium), reduction of the number of pericytes in the vessel wall, and overall edema, probably because of microvascular leakage. Based on these observations, PDGF-B and PDGFR-β seem to be involved in maturation and stabilization of several different vascular networks. This idea is further supported by the fact that loss of the heparin-binding domain in the PDGF-B protein leads to structurally abnormal vessels (Lindblom et al., 2003). Knockout mouse embryos for Pdgf-a and Pdgfr-α show different phenotypes. Pdgf-a knockouts either die before embryonic day (E) 10 of an unknown cause or die later (several weeks after birth) as a result of lung emphysema (Betsholtz et al., 2001). They present with several other malformations, such as intestinal, testicular, and hair follicle defects. However, no heart defects have been described (Betsholtz et al., 2001). Pdgfr-α knockouts, as well as Patch mutants (a mutation that involves the Pdgfr-α gene), show quite severe heart malformations, including atrial and ventricular septal defects, outflow tract (OFT) malformations, malformed valves, and thin myocardium (Schatteman et al., 1995). They die between E8 and E16 (Betsholtz et al., 2001). Also, other malformations such as facial cleft, spina bifida, and skeletal, lung, and gut defects have been found. The OFT and facial abnormalities have been related to abnormalities in neural crest development (Morrison-Graham et al., 1992; Schatteman et al., 1995; Soriano, 1997). The large differences between Pdgf-a and Pdgfr-α knockouts might be explained by functional redundancy of PDGF-B or -C, the other ligands for PDGFR-α. Thus, PDGF-A and PDGFR-α seem to be involved predominantly in the morphogenesis of several organs.

We, therefore, hypothesize that PDGF-B and PDGFR-β, rather than PDGF-A and PDGFR-α, are involved in maturation and stabilization of the coronary vasculature and, thus, are expressed in cells participating in coronary development, such as coronary endothelial cells and EPDCs. To investigate this hypothesis, we describe the protein expression patterns of PDGF-A, PDGF-B, and their receptors PDGFR-α and PDGFR-β in the hearts of normal chicken and quail embryos. To elucidate whether EPDCs specifically express these factors, we analyzed expression in quail–chicken chimeras, in which the PEO of the quail was transplanted into the chicken embryo.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Cardiac Protein Expression Patterns of PDGF-A and PDGFR-α

In the time-span we investigated, PDGF-A was present in several areas of the avian heart during development (Table 1), namely in the myocardial wall of the cardinal veins of the sinus venosus, in the atrial and atrial septal myocardium, in both the compact and the trabecular ventricular myocardium (Fig. 1e,f), and in the OFT cushion mesenchyme. The presence of PDGF-A decreased in time in the myocardial wall of the cardinal veins, in the atrial septal myocardium, and in the OFT cushion mesenchyme, but stayed constant in the atrial myocardium and in the compact and trabecular ventricular myocardium.

thumbnail image

Figure 1. Expression of platelet-derived growth factor (PDGF)-A and PDGF receptor-α (PDGFR-α) in proepicardial quail–chicken chimeras of HH28–HH35. Photomicrographs of representative transverse sections examined by light (a–d), fluorescence (e,g), or confocal fluorescence microscopy (f,h,i). ad: PDGFR-α is present in the atrial wall and atrial septal myocardium (AS) at HH28 (a,b) and myocardial wall of the left and right cardinal veins (LCaV and RCaV, respectively) at HH28 (c,d). b and d are magnifications of the boxed areas in a and c, respectively. e: PDGF-A (green) and PDGFR-α (red) colocalize (yellow) throughout the trabecular myocardium (TM) at HH35. f: Enlarged view of double-staining (yellow) of PDGF-A (green) and PDGFR-α (red) in the TM at HH35. g: Quail-derived epicardium-derived cells (EPDCs) (QCPN-positive, green) do not express PDGFR-α (red) at HH35. h: Enlarged view of PDGFR-α (red) negative EPDCs (green) in a consecutive section of g. i: Double-positive (yellow) staining of cardiomyocytes expressing PDGFR-α (red) and α/γ muscle actin (green) at HH35. HH, Hamburger and Hamilton stage; CM, compact myocardium; LA, left atrium; RA, right atrium. Scale bar = 250 μm in a,c, 50 μm in b,d,e,g, 10 μm in f,h,i.

Download figure to PowerPoint

Table 1. Summary of the Spatio-temporal Protein Expression Patterns of PDGF-A, PDGFR-α, PDGF-B and PDGFR-β in the Avian Embryonic Heart
  1. a

    This table reflects alterations in total protein expression per area; for the receptor staining this relates to the number of positive cells per area, whereas for the ligands the overall amount of protein per area is indicated. PDGF-A and PDGFR-α have been observed mainly in myocardial tissues, while PDGF-B and PDGFR-β are mainly expressed in cells related to the developing coronary system. A, atrial; AS, atrial septal; AV, atrioventricular; CA-ECs, coronary arterial endothelial cells; CaV, cardinal vein; C-vSMCs, coronary vascular smooth muscle cells; OFT, outflow tract; VC, ventricular compact; VCM, ventricular compact myocardium; VS, ventricular septal; VT, ventricular trabecular.

inline image

PDGFR-α expression colocalized with PDGF-A staining (Table 1), both in location and in time, in the myocardial wall of the cardinal vein (Fig. 1c,d), the atrial myocardium, and the atrial septal myocardium (Fig. 1a,b). In the ventricular myocardium, PDGFR-α expression decreased in time in the compact myocardium, while increasing in the trabecular myocardium (Fig. 1e–i). Furthermore, PDGFR-α was not present in the OFT cushion mesenchyme but in the adjacent OFT myocardium, where the expression decreased in time.

Cardiac Protein Expression of PDGF-B and PDGFR-β

Presence of PDGF-B was observed in completely different areas in the heart compared with PDGF-A (Table 1). PDGF-B was present in the ventricular septal myocardium (Fig. 2a), in the endothelial cells (Fig. 2b) and vSMCs of the coronary arteries, in the subepicardium, and in small cardiac nerves (Fig. 2c). In the ventricular septal myocardium, PDGF-B was only present during early development of the septum. At Hamburger and Hamilton stage (HH) 30, when the ventricular septum was almost completed, the presence of PDGF-B was already strongly diminished compared with HH28, and at HH35 PDGF-B was completely absent from the ventricular septum. Furthermore, while the endothelial cells of the coronary arteries already stained with the α-PDGF-B antibody at HH28, the vSMCs only showed PDGF-B staining from HH30 onward. In addition, the presence of PDGF-B in the subepicardium decreased and the presence in the cardiac nerves stayed constant in time.

thumbnail image

Figure 2. Expression of platelet-derived growth factor (PDGF) -B and PDGF receptor-β (PDGFR-β) in pro-epicardial quail–chicken chimeras of HH28–HH35. Photomicrographs of representative transverse sections examined by light (a–c,g), fluorescence (h), or confocal fluorescence microscopy (d,e,f,i). ac: PDGF-B is present in the developing ventricular septum (VS) at HH28 (a), in coronary arterial (CA) endothelial cells (b), and in cardiac nerves (c; arrow) at HH30. df: Quail-derived epicardium-derived cells (EPDCs; QCPN-positive, green nuclear staining) express PDGFR-β (red membranous staining) located in the compact myocardium (CM) at HH30 (d), in subendocardial cells of the AV cushions (Cu; e), and in cells around a CA (f) at HH35. g: α-smooth muscle actin (α-SMA) expression around a CA at HH35. h: Consecutive section of g in which PDGFR-β expression (red) is seen in QCPN-positive EPDCs (green) around a CA. i: Microvascular strands of quail-derived endothelial cells (QH-1, green) do not express PDGFR-β (red) at HH35. AM, atrial myocardium; AVC, atrioventricular canal; CV, coronary vein; LV, left ventricle; RV, right ventricle; VM, ventricular myocardium. Scale bar = 100 μm in a, 50 μm in b,c,g,h, 10 μm in d–f,i.

Download figure to PowerPoint

With regard to PDGFR-β (Table 1), expression was observed in the atrioventricular valves (Fig. 2e), in vSMCs of the coronary arteries (Fig. 2f–h), in the epicardium, in the subepicardium, in interstitial cells located in the myocardium (Fig. 2d) and in small cardiac nerves. In the atrioventricular valves and in the vSMCs of the coronary arteries, expression was only seen from HH30 onward. However, no expression of either PDGFR-α or PDGFR-β in the interventricular septum was observed. The expression of PDGFR-β in the epicardium and subepicardium decreased in time, whereas the expression stayed constant in the interstitial cells in the myocardium and in the small cardiac nerves.

Expression of the PDGFRs in the EPDCs

The population of PDGFR-β–positive cells in the heart closely resembled the spatiotemporal distribution of EPDCs (Poelmann et al., 1993; Dettman et al., 1998; Gittenberger-de Groot et al., 1998; Vrancken Peeters et al., 1999; Manner et al., 2001). To analyze the identity of the PDGFR-β–positive cells, we used pro-epicardial quail–chicken chimeras, in which quail-derived EPDCs were detected by the QCPN antibody (pan-quail nuclear staining). Double-staining of the QCPN antibody with an antibody against PDGFR-α showed that EPDCs did not express PDGFR-α between HH28 and HH35 (Table 1; Fig. 1g,h). PDGFR-β–positive EPDCs were present (i.e., in a QCPN/PDGFR-β double-staining, these cells showed green nuclear and red membranous staining) in all areas that showed PDGFR-β–positive cells (Table 1; Fig. 2d–h). The small PDGFR-β–positive cardiac nerves are not derived from EPDCs but from neural crest cells (Verberne et al., 2000). These results are compiled in the schematic figure (Fig. 3) that represents frontal views of the hearts of quail–chicken chimeras at HH28 and HH35.

thumbnail image

Figure 3. Compilation of platelet-derived growth factor receptor (PDGFR) expression in epicardium-derived cells (EPDCs). ad: Drawing of a frontal view of a chicken embryonic heart of HH28 (a,c) and HH35 (b,d) in which the white circles represent coronary arteries. Green staining reflects the nuclei of the EPDCs, the red staining reflects PDGFR-α staining in a and b, and PDGFR-β staining in c and d. In a and b, the EPDCs do not express PDGFR-α; therefore, no cells with both green and red staining are visible. In c and d, the green cells represent the PDGFR-β–negative EPDCs and the cells with green nuclear and red membranous staining represent PDGFR-β–positive EPDCs. These expression patterns suggest that PDGFR-α signaling plays a role in embryonic myocardial development, whereas PDGFR-β signaling is important in the maturation of the coronary vasculature.

Download figure to PowerPoint

PDGFRs in Cardiomyocytes, Endothelial Cells, and Smooth Muscle Cells Between HH28 and HH35

To further identify the type of cells that express PDGFR-α or PDGFR-β, double-stainings were performed with either α/γ muscle actin (HHF35; cardiomyocytes and smooth muscle cells) or QH-1 (quail endothelial cells). Consecutive sections were stained for α-smooth muscle actin (1A4; smooth muscle cells), because no double-staining with α–PDGFR-β antibody could be performed due to identical IgG subunits of the antibodies. Double-staining for PDGFR-α and α/γ muscle actin showed that all ventricular PDGFR-α–positive cells from HH28 to HH35 also expressed α/γ muscle actin, implicating that these were cardiomyocytes (Fig. 1i). Staining for PDGFR-β and α-smooth muscle actin at HH35 showed overlapping expression patterns around the coronary vessels, indicating that the epicardium-derived vSMCs in the medium of the coronary vessels express PDGFR-β (Fig. 2g,h). Furthermore, QH-1–positive endothelial cells were never PDGFR-β–positive between HH28 and HH35 (Fig. 2i).

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

The present study describes the cardiac expression patterns of PDGF-A, PDGF-B, PDGFR-α, and PDGFR-β in the avian embryo, highlighting the later stages of coronary maturation. Here, their possible roles in the development of the avian heart in general and in that of the coronary system in particular will be discussed.

PDGF-A was found in the atrial and ventricular myocardium, with at HH28 an additional signal in the OFT cushion mesenchyme, while expression of PDGFR-α was seen in the OFT myocardium. It is likely that PDGF-A attracts PDGFR-α–positive cells from the myocardium of the outflow tract into the cushion mesenchyme, where these cells lose their PDGFR-α expression. These expression patterns correlate well with OFT malformations observed in Patch mice, harboring a mutated Pdgfr-α gene (Schatteman et al., 1995). At HH28, PDGFR-α is also strongly expressed in the developing atrial septum. A role for PDGFR-α in septal development was also suggested by similar protein expression patterns in mice (Takakura et al., 1997) and by the finding that atrial and ventricular septal defects are often encountered in Patch mice (Schatteman et al., 1995). The presence of PDGF-A and PDGFR-α in the myocardial wall of the cardinal veins of the sinus venosus at HH28 might relate to the still active process of incorporation of the sinus venosus into the atrium. In the literature, expression patterns of PDGF-A and PDGFR-α in the sinus venosus have never been described. In the ventricular myocardium, the presence of PDGF-A and PDGFR-α was initially observed at low levels in both the compact and the trabecular myocardium. At later stages, PDGF-A remained present in the compact myocardium, but PDGFR-α became restricted to the trabeculae. This finding is in agreement with mRNA expression data of Pdgfr-α in mouse embryos (Schatteman et al., 1992). In the trabeculae, PDGF-A colocalized with, and thus seemed bound to, PDGFR-α, which was expressed by cardiomyocytes, especially in the older embryos (HH30–HH35). The role of PDGF-A/PDGFR-α signaling in trabecular formation is unknown, although PDGFR-α stimulation by PDGF-A has a role in epithelial–mesenchymal interactions (Betsholtz, 2003). It has been proposed that this signaling is responsible for the formation of a smooth muscle cell layer in newly formed bronchial branches by promoting proliferation and migration of PDGFR-α–positive precursors of alveolar SMCs (Betsholtz, 2003; Lindahl et al., 1997b). Trabecular formation might be a process similar to branching, so it can be hypothesized that PDGF-A might be produced and secreted by the endocardium and promotes the ingrowth of PDGFR-α–expressing cardiomyocytes or cardiomyocyte precursors into the newly formed trabeculae, in a similar way as has been suggested for the bronchi. PDGF-A might have diffused into the myocardium, due to the possible lack of heparin-binding domains (Betsholtz, 2003), thereby explaining the lack of protein expression in the endocardium.

PDGF-B expression previously has only been described in endothelial cells and megakaryocytes during mouse embryonic development (Lindahl et al., 1997a; Betsholtz et al., 2001; Betsholtz, 2003). However, in our avian model, the PDGF-B protein was highly present in the region of the developing ventricular septum, where neither of these cell types is present at that time, suggesting an additional PDGF-B–producing cell type such as the cardiomyocytes. Combined with observations in Pdgf-b and Pdgfr-β knockout mouse embryos, which have a higher incidence of ventricular septal defects compared with wild-type mice, these data suggest a role for PDGF-B signaling in the formation of the ventricular septum. The exact pathways through which PDGF-B exerts its effect during ventricular septal development, however, are unclear, as neither PDGFR-α nor PDGFR-β are expressed in the ventricular septum at that time (data not shown). Furthermore, PDGF-B might be involved in embryonic nervous development as well, since staining was observed in (cardiac) nerves. It has been described in rats that PDGF-B might be involved in peripheral nerve regeneration (Oya et al., 2002). With regard to PDGFR-β, its function has mainly been analyzed in vSMCs (Lindahl et al., 1997a; Betsholtz, 2003). Therefore, the presence of PDGFR-β–positive EPDCs in AV-cushions, with unknown differentiation characteristics and function is remarkable. These cells might play a role in regulation of endocardial EMT in cushion tissue (Gittenberger-de Groot et al., 2003), perhaps by means of activation of extracellular matrix degradation. In vitro blockade of PDGFR-β signaling with antibodies decreases matrix metalloproteinase-2 (MMP-2) levels (Kenagy et al., 1997). MMP-2 is an extracellular matrix degrading protein shown to be necessary for EMT in the atrioventricular cushions by facilitating migration (Alexander et al., 1997; Song et al., 2000; Enciso et al., 2003). As PDGF-B staining is lacking at this site, PDGF-D might be responsible for activating PDGFR-β in this situation.

In the cells that contribute to the coronary system (epicardial cells, EPDCs, vSMCs, and endothelial cells), PDGF-A or PDGFR-α staining was not observed between HH28–HH35. This finding indicates that PDGF-A signaling through PDGFR-α does not play a crucial role in avian coronary maturation. This explanation is in agreement with data concerning Pdgf-a knockout and Patch (Pdgfr-α mutated) mice, in which no structural coronary malformations were described (Betsholtz et al., 2001; Schatteman et al., 1995). However, an indirect role for PDGFR-α in coronary angiogenesis cannot be excluded, because, first, Patch mice show diminished numbers of blood vessels in the heart (Schatteman et al., 1992) and second, epicardial PDGFR-α positivity has been described in embryonic day 12.5 mouse embryos (comparable to HH25 in avian embryos; Takakura et al., 1997).

Conversely, PDGF-B and PDGFR-β are present in all cell types of the developing coronary system, at all stages that we investigated. This finding, and the fact that Pdgf-b and Pdgfr-β knockout mice have severe coronary defects (Lindahl et al., 1997a), confirm an essential role for PDGF-B and PDGFR-β in coronary maturation. Correct formation of a coronary vasculature with mature and stable vessel walls, depends largely on the properly timed presence and differentiation of EPDCs (Poelmann et al., 1993; Gittenberger-de Groot et al., 1998, 2000; Vrancken Peeters et al., 1999; Lie-Venema et al., 2003; Eralp et al., 2005). We hypothesize that the PDGF-B/Rβ pathway plays a prominent role at all stages of EPDC formation and differentiation.

This study shows that PDGF-B and its β-receptor are present in the epicardium and subepicardium during the first step in this process: the formation of the subepicardial mesenchyme by EMT. In vitro studies show that this process can be induced by several factors, among which are PDGF-B (Lu et al., 2001), fibroblast growth factor (FGF)-1, and epidemal growth factor (EGF) (Morabito et al., 2001). The expression patterns observed in this study indicate that PDGF-B also contributes to epicardial EMT in vivo. Probably, the role of PDGF-B herein is by means of the up-regulation of the expression of the transcription factor Ets-1 (Naito et al., 1998), an intermediate in the PDGF-B signaling cascade that has been shown to be important for the formation of the subepicardial mesenchyme (Lie-Venema et al., 2003). Furthermore, stimulation of PDGFR-β might facilitate EMT by increasing MMP-2 levels locally, as has been postulated for endocardial cushion EMT (see earlier).

After EMT, EPDCs start to migrate from the subepicardial mesenchyme into the underlying myocardium. We observed PDGFR-β–positive, interstitial EPDCs in the myocardium at HH28, interstitial and vessel-related PDGFR-β–positive EPDCs at HH30, and solely vessel-related PDGFR-β–positive EPDCs at HH35. This finding might reflect the migration of PDGFR-β–positive EPDCs toward the developing coronary system, guided by chemokines such as PDGF-B produced by the endothelial cells. However, because with immunohistochemistry no PDGF-B gradient but only strong signal in close proximity to the endothelial cells is seen, this signaling pathway might be specifically responsible for the last part of the guided migration of the EPDCs, whereas other chemokines like PDGF-D or heparin binding EGF-like growth factor (HB-EGF; Iivanainen et al., 2003) might be involved in its initiation. PDGF-D is another ligand for PDGFR-β, but its embryonic expression patterns and, thus, its role in development are yet to be described. HB-EGF recently has been described to be involved in the attraction of vSMCs toward endothelial cells. Therefore, the distribution pattern of PDGFR-β–positive cells might not solely be reflecting PDGF-B dependent migration. Another function of PDGF-B produced by coronary endothelial cells might be stabilization of the structural integrity of the vascular wall (Lindblom et al., 2003), explaining the nondiffuse localization and the increase in expression in the vascular wall in time.

EPDCs have been shown earlier to be the source of the coronary vSMCs (Dettman et al., 1998; Vrancken Peeters et al., 1999; Munoz-Chapuli et al., 2002). At stage HH35, the PDGFR-β–positive EPDCs around the coronary vessels colocalize with cells expressing α-smooth muscle actin, indicating that these EPDCs have differentiated into vSMCs. Since PDGF-B has been shown to induce migration rather than differentiation (Hellström et al., 1999), the differentiation of the PDGFR-β–positive cells into vSMCs is probably directed by other factors, such as transforming growth factor-β (TGFβ; Hirschi et al., 1998; Nishishita and Lin, 2004) and/or serum response factor (SRF; Landerholm et al., 1999). The role for PDGFR-β–positive cells in the development and stabilization of the coronary vessel wall was demonstrated earlier in vitro (Zerwes and Risau, 1987; Hirschi et al., 1998) and in many other vascular beds (Lindahl et al., 1997a; Hellström et al., 1999; Hoch and Soriano, 2003).

In conclusion, the spatiotemporal localization of PDGF-A and -B and their α- and β-receptors suggests that PDGF-A signaling through PDGFR-α is important in the remodeling of the myocardium (sinus venosus, OFT, septa, trabeculae). PDGF-B might play a role in ventricular septum formation, however, not through interaction with PDGFR-α or PDGFR-β as expression of these receptors is not observed. Furthermore, PDGF-B/PDGFR-β signaling seems to be predominantly involved in maturation of EPDCs contributing to the coronary system and atrioventricular valves, both processes that start with EMT and are followed by the maturation of epicardium-derived cells.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Embryos

Fertilized eggs of the White Leghorn chicken (Gallus domesticus) and of the Japanese quail (Coturnix coturnix japonica) were incubated at 37°C (80% humidity) and staged according to the criteria of Hamburger and Hamilton (Hamburger and Hamilton, 1951). For normal chicken and quail controls, eggs were incubated for 6–9 days, harvested, and staged (HH28–HH35). The thoraxes were fixed in 20% dimethylsulfoxide and 80% methanol or in 4% paraformaldehyde in phosphate buffered saline (PBS) for 24 hr. The thoraxes were embedded in paraffin and subsequently sectioned transversely (5 μm).

Chimerization Technique

Chimerization was performed as described before (Poelmann et al., 1993). In short, after 3 days of incubation, the quail embryos were removed from the eggs, staged (HH15–HH18), and the PEO was microsurgically removed. In the shell of the chicken eggs, a small window was made. The embryos were staged (HH15–HH18), and a quail PEO was inserted into the pericardial cavity. The eggs were closed with Scotch tape and re-incubated. At 3 to 6 days later (6–9 days of incubation), the chimeric embryos were harvested, staged (HH28–HH35), and processed as the controls. A QCPN (pan-quail nuclear) staining was used to determine the extent of chimerization. A total of 14 chimeras were included in this study.

Western Blotting

To confirm the specificity of the polyclonal antibodies against PDGF-A, PDGF-B, PDGFR-α, and PDGFR-β in chicken embryonic material, we performed Western blots. In short, total protein was extracted from hearts of normal chicken embryos in ice-cold RIPA buffer (PBS with 1% [v/v] Igepal-CA630, 0.5% [wt/vol] sodium deoxycholate, and 0.1% sodium dodecyl sulfate) containing phenylmethyl sulfonyl fluoride serine protease inhibitor at a concentration of 1 mmol/L. The total protein content of the extracts was determined using the bicinchronic acid method (BCA Kit, Pierce). Western blot analysis of total protein was performed on extracts from three independent experiments using the ECL Advance Western Blotting Detection Kit (Amersham). The results of these experiments are that all antibodies, viz. α-PDGF-A, α-PDGF-B, α-PDGFR-α, and α-PDGFR-β, specifically recognized the expected protein. From this finding, we conclude that the antibodies recognize the chicken PDGF-ligands and receptors with adequate specificity.

Immunohistochemistry

Immunohistochemical stainings were performed as described before (Bergwerff et al., 1998). Sections were stained for QCPN, QH-1, α-smooth muscle actin (1A4), PDGF-A, PDGF-B, and PDGFR-α. Microwave antigen retrieval was applied, except for the QH-1 and the α-smooth muscle actin staining. Endogenous peroxidase activity was quenched by incubation for 15 min in 0.3% H2O2 in PBS. Sections were incubated overnight with the primary antibody (α-QCPN, 1:4; α-QH-1, 1:500; α-α-smooth muscle actin, 1:3,000; α-PDGF-A, 1:100; α-PDGF-B, 1:50; α-PDGFR-α, 1:50) and incubated with a secondary peroxidase-labeled antibody (QCPN, QH-1, and α-smooth muscle actin staining) or a biotin-labeled (PDGF-A, PDGF-B, and PDGFR-α staining) antibody for 1 hr. Sections incubated with a biotin-labeled antibody were then incubated with Vectastain ABC staining kit for 1 hr. Slides were rinsed with PBS and Tris/Maleate (pH 7.6). 3-3′ diaminobenzidine tetrahydrochloride (DAB) was used as chromogen and Mayer's hematoxylin as counterstaining.

Immunofluorescence

The following double-stainings were performed: PDGFR-α/QCPN, PDGFR-α/PDGF-A, PDGFR-α/HHF-35, PDGFR-β/QCPN, PDGFR-β/QH-1, PDGFR-β/PDGF-B, and PDGFR-α/PDGF-B. Sections were pretreated as described above and incubated overnight with the first primary antibody (α-PDGFR-α, 1:50; α-PDGFR-β, 1:25). Subsequently, they were incubated with a biotin-labeled secondary antibody for 1 hr, followed by incubation with avidin-labeled with fluorescein isothiocyanate (FITC; PDGFR-α) or tetrarhodamine isothiocyanate (TRITC; PDGFR-β) for 1 hr. Sections were incubated for 2 hr with second primary antibodies (QCPN, 1:4; PDGF-A,1:100; HHF-35, 1:500; QH-1, 1:500; or PDGF-B, 1:50) and incubated for 1 hr with secondary TRITC- or FITC-labeled antibodies. Finally, sections were mounted with Vectashield containing 4′,6-diamidine-2-phenylidole-dihydrochloride to stain the nuclei.

Materials

Primary antibodies used were rabbit-α-human PDGF-A (SC-128, Santa Cruz), rabbit–α-human PDGF-B (SC-7878, Santa Cruz), goat–α-human PDGFR-α (P2110, Sigma-Aldrich), mouse–α-human α-smooth muscle actin (clone 1A4, A2547, Sigma-Aldrich), mouse–α-human PDGFR-β (610114, Falcon), mouse–α-quail QCPN, mouse–α-quail QH-1 (both Hybridoma Bank, Iowa City), and mouse–α-human α/γ-muscle actin (HHF35; M0635, DAKO). Secondary antibodies used were biotin-labeled goat–α-rabbit (BA-1000, Vector Labs), biotin-labeled horse–α-mouse (BA-2000, Vector Labs), biotin-labeled horse–α-goat (BA-9500, Vector Labs), FITC-labeled avidin (A-2001, Vector Labs), TRITC-labeled avidin (A-2002, Vector Labs), TRITC-labeled rabbit–α-mouse (R270, DAKO), FITC-labeled goat–α-mouse IgG1 (1075-02, Southern Biotechnology Associates), FITC-labeled donkey–α-rabbit (SC-2090, Santa Cruz), and peroxidase-labeled rabbit–α-mouse (P0260, DAKO). Furthermore, normal goat serum (S-1000, Vector Labs), normal horse serum (S-2000, Vector Labs), the Vectastain ABC staining kit (PK-6100, Vector Labs), Vectashield (H-1200, Vector Labs), DAB (D5637, Sigma-Aldrich) and Entellan (Merck) were used in the immunohistochemistry and immunofluorescence. Photographs were made using an Olympus AX70 light microscope equipped with an Olympus DP12 digital camera (Olympus), a Leica IRBE fluorescence microscope equipped with a Leica DC350F digital camera (Leica Microsystems) or a Bio-Rad confocal laser scanning microscope. Fluorescent images were analyzed using Leica FW4000 software and confocal images were processed with Image J.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

The authors thank Jan Lens for photographic assistance, Bas Blankenvoort for the graphics in Figure 3, and Marleen Hessel (Department of Cardiology, LUMC, Leiden, the Netherlands) for technical assistance with the Western blots. N.M.S.v.d.A. and H.L.-V. were funded by the Netherlands Heart Foundation.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  • Alexander SM, Jackson KJ, Bushnell KM, McGuire PG. 1997. Spatial and temporal expression of the 72-kDa type IV collagenase (MMP-2) correlates with development and differentiation of valves in the embryonic avian heart. Dev Dyn 209: 261268.
  • Battegay EJ, Rupp J, Iruela-Arispe L, Sage H, Pech M. 1994. PDGF-BB modulates endothelial proliferation and angiogenesis in vitro via PDGF β-receptors. J Cell Biol 125: 917928.
  • Bergwerff M, Gittenberger-de Groot AC, DeRuiter MC, van Iperen L, Meijlink F, Poelmann RE. 1998. Patterns of paired-related homeobox genes PRX1 and PRX2 suggest involvement in matrix modulation in the developing chick vascular system. Dev Dyn 213: 5970.
  • Betsholtz C. 2003. Biology of platelet-derived growth factors in development. Birth Defects Res 69: 272285.
  • Betsholtz C, Karlsson L, Lindahl P. 2001. Developmental roles of platelet-derived growth factors. Bioessays 23: 494507.
  • Dettman RW, Denetclaw W, Ordahl CP, Bristow J. 1998. Common epicardial origin of coronary vascular smooth muscle, perivascular fibroblasts, and intermyocardial fibroblasts in the avian heart. Dev Biol 193: 169181.
  • Ding H, Wu XL, Kim I, Tam PPL, Koh GY, Nagy A. 2000. The mouse Pdgfc gene: dynamic expression in embryonic tissues during organogenesis. Mech Dev 96: 209213.
  • Enciso JM, Gratzinger D, Camenisch TD, Canosa S, Pinter E, Madri JA. 2003. Elevated glucose inhibits VEGF-A-mediated endocardial cushion formation: modulation by PECAM-1 and MMP-2. J Cell Biol 160: 605615.
  • Eralp I, Lie-Venema H, DeRuiter MC, Van Den Akker NM, Bogers AJ, Mentink MM, Poelmann RE, Gittenberger-de Groot AC. 2005. Coronary artery and orifice development is associated with proper timing of epicardial outgrowth and correlated Fas ligand associated apoptosis patterns. Circ Res 96: 526534.
  • Fang L, Yan YB, Komuves LG, Yonkovich S, Sullivan CM, Stringer B, Galbraith S, Lokker NA, Hwang SS, Nurden P, Phillips DR, Giese NA. 2004. PDGF C is a selective alpha platelet-derived growth factor receptor agonist that is highly expressed in platelet alpha granules and vascular smooth muscle. Arterioscler Thromb Vasc Biol 24: 787792.
  • Gittenberger-de Groot AC, Bartram U, Oosthoek PW, Bartelings MM, Hogers B, Poelmann RE, Jongewaard IN, Klewer SE. 2003. Collagen type VI expression during cardiac development and in human fetuses with trisomy 21. Anat Rec 275A: 11091116.
  • Gittenberger-de Groot AC, Vrancken Peeters M-PFM, Bergwerff M, Mentink MMT, Poelmann RE. 2000. Epicardial outgrowth inhibition leads to compensatory mesothelial outflow tract collar and abnormal cardiac septation and coronary formation. Circ Res 87: 969971.
  • Gittenberger-de Groot AC, Vrancken Peeters M-PFM, Mentink MMT, Gourdie RG, Poelmann RE. 1998. Epicardial derived cells, EPDCs, contribute a novel population to the myocardial wall and the atrioventricular cushions. Circ Res 82: 10431052.
  • Hamburger V, Hamilton HL. 1951. A series of normal stages in the development of the chick embryo. J Morphol 88: 4992.
  • Heldin CH, Eriksson U, Ostman A. 2002. New members of the platelet-derived growth factor family of mitogens. Arch Biochem Biophys 398: 284290.
  • Hellström M, Kalén M, Lindahl P, Abramsson A, Betsholtz C. 1999. Role of PDGF-B and PDGFR-β in recruitment of vascular smooth muscle cells and pericytes during embryonic blood vessel formation in the mouse. Development 126: 30473055.
  • Hirschi KK, Rohovsky SA, D'Amore PA. 1998. PDGF, TGF-β, and heterotypic cell-cell interactions mediate endothelial cell-induced recruitment of 10T1/2 cells and their differentiation to a smooth muscle fate. J Cell Biol 141: 805814.
  • Hoch RV, Soriano P. 2003. Roles of PDGF in animal development. Development 130: 47694784.
  • Iivanainen E, Nelimarkka L, Elenius V, Heikkinen SM, Junttila TT, Sihombing L, Sundvall M, Maatta JA, Jukka V, Laine O, Yla-Herttuala S, Higashiyama S, Alitalo K, Elenius K. 2003. Angiopoietin-regulated recruitment of vascular smooth muscle cells by endothelial-derived heparin binding EGF-like growth factor. FASEB J 17: 16091621.
  • Kenagy RD, Hart CE, Stetler-Stevenson WG, Clowes AW. 1997. Primate smooth muscle cell migration from aortic explants is mediated by endogenous platelet-derived growth factor and basic fibroblast growth factor acting through matrix metalloproteinases 2 and 9. Circulation 96: 35553560.
  • Landerholm TE, Dong X-R, Lu J, Belaguli NS, Schwartz RJ, Majesky MW. 1999. A role for serum response factor in coronary smooth muscle differentiation from proepicardial cells. Development 126: 20532062.
  • Lie-Venema H, Eralp I, Maas S, Gittenberger-de Groot AC, Poelmann RE, DeRuiter MC. 2005. Myocardial heterogeneity in permissiveness for epicardium-derived cells and endothelial precursor cells along the developing heart tube at the onset of coronary vascularization. Anat Rec A Discov Mol Cell Evol Biol 282: 120129.
  • Lie-Venema H, Gittenberger-de Groot AC, van Empel LJP, Boot MJ, Kerkdijk H, de Kant E, DeRuiter MC. 2003. Ets-1 and Ets-2 transcription factors are essential for normal coronary and myocardial development in chicken embryos. Circ Res 92: 749756.
  • Lindahl P, Johansson BR, Levéen P, Betsholtz C. 1997a. Pericyte loss and microaneurysm formation in PDGF-B-deficient mice. Science 277: 242245.
  • Lindahl P, Karlsson L, Hellström M, Gebre-Medhin S, Willetts K, Heath JK, Betsholtz C. 1997b. Alveogenesis failure in PDGF-A-deficient mice is coupled to lack of distal spreading of alveolar smooth muscle cell progenitors during lung development. Development 124: 39433953.
  • Lindblom P, Gerhardt H, Liebner S, Abramsson A, Enge M, Hellstrom M, Backstrom G, Fredriksson S, Landegren U, Nystrom HC, Bergstrom G, Dejana E, Ostman A, Lindahl P, Betsholtz C. 2003. Endothelial PDGF-B retention is required for proper investment of pericytes in the microvessel wall. Genes Dev 17: 18351840.
  • Lu J, Landerholm TE, Wei JS, Dong XR, Wu SP, Liu XS, Nagata K, Inagaki M, Majesky MW. 2001. Coronary smooth muscle differentiation from proepicardial cells requires RhoA-mediated actin reorganization and p160 rho-kinase activity. Dev Biol 240: 404418.
  • Luttun A, Carmeliet P. 2003. De novo vasculogenesis in the heart. Cardiovasc Res 58: 378389.
  • Manner J, Perez-Pomares JM, Macias D, Munoz-Chapuli R. 2001. The origin, formation and developmental significance of the epicardium: a review. Cells Tissues Organs 169: 89103.
  • Mikawa T, Gourdie RG. 1996. Pericardial mesoderm generates a population of coronary smooth muscle cells migrating into the heart along with ingrowth of the epicardial organ. Dev Biol 174: 221232.
  • Morabito CJ, Dettman RW, Kattan J, Collier JM, Bristow J. 2001. Positive and negative regulation of epicardial-mesenchymal transformation during avian heart development. Dev Biol 234: 204215.
  • Morabito CJ, Kattan J, Bristow J. 2002. Mechanisms of embryonic coronary artery development. Curr Opin Cardiol 17: 235241.
  • Morrison-Graham K, Schatteman GC, Bork T, Bowen-Pope DF, Weston JA. 1992. A PDGF receptor mutation in the mouse (Patch) perturbs the development of a non-neuronal subset of neural crest-derived cells. Development 115: 133142.
  • Munoz-Chapuli R, Macias D, Gonzalez-Iriarte M, Carmona R, Atencia G, Perez-Pomares JM. 2002. The epicardium and epicardial-derived cells: multiple functions in cardiac development. Rev Esp Cardiol 55: 10701082.
  • Naito S, Shimizu S, Maeda S, Wang JW, Paul R, Fagin JA. 1998. Ets-1 is an early response gene activated by ET-1 and PDGF-BB in vascular smooth muscle cells. Am J Physiol Cell Physiol 43: C472C480.
  • Nishishita T, Lin PC. 2004. Angiopoietin 1, PDGF-B, and TGF-beta gene regulation in endothelial cell and smooth muscle cell interaction. J Cell Biochem 91: 584593.
  • Orr-Urtreger A, Lonai P. 1992. Platelet-derived growth factor-A and its receptor are expressed in separate, but adjacent cell layers of the mouse embryo. Development 115: 10451058.
  • Oya T, Zhao YL, Takagawa K, Kawaguchi M, Shirakawa K, Yamauchi T, Sasahara M. 2002. Platelet-derived growth factor-B expression induced after rat peripheral nerve injuries. Glia 38: 303312.
  • Perez-Pomares JM, Carmona R, Gonzalez-Iriarte M, Atencia G, Wessels A, Munoz-Chapuli R. 2002. Origin of coronary endothelial cells from epicardial mesothelium in avian embryos. Int J Dev Biol 46: 10051013.
  • Poelmann RE, Gittenberger-de Groot AC, Mentink MMT, Bökenkamp R, Hogers B. 1993. Development of the cardiac coronary vascular endothelium, studied with antiendothelial antibodies, in chicken-quail chimeras. Circ Res 73: 559568.
  • Reese DE, Mikawa T, Bader DM. 2002. Development of the coronary vessel system. Circ Res 91: 761768.
  • Schatteman GC, Morrison-Graham K, van Koppen A, Weston JA, Bowen-Pope DF. 1992. Regulation and role of PDGF receptor α-subunit expression during embryogenesis. Development 115: 123131.
  • Schatteman GC, Motley ST, Effmann EL, Bowen-Pope DF. 1995. Platelet-derived growth factor receptor alpha subunit deleted patch mouse exhibits severe cardiovascular dysmorphogenesis. Teratology 51: 351366.
  • Song W, Jackson K, McGuire PG. 2000. Degradation of type IV collagen by matrix metalloproteinases is an important step in the epithelial-mesenchymal transformation of the endocardial cushions. Dev Biol 227: 606617.
  • Soriano P. 1997. The PDGFα receptor is required for neural crest cell development and for normal patterning of the somites. Development 124: 26912700.
  • Takakura N, Yoshida H, Ogura Y, Kataoka H, Nishikawa S, Nishikawa S-I. 1997. PDGFRα expression during mouse embryogenesis: immunolocalization analyzed by whole-mount immunostaining using the monoclonal anti-mouse PDGFRα antibody APA5. J Histochem Cytochem 45: 883893.
  • Tomanek RJ. 1996. Formation of the coronary vasculature: a brief review. Cardiovasc Res 31: E46E51.
  • Verberne ME, Gittenberger-de Groot AC, VanIperen L, Poelmann RE. 2000. Distribution of different regions of cardiac neural crest in the extrinsic and the intrinsic cardiac nervous system. Dev Dyn 217: 191204.
  • Virágh Sz, Gittenberger-de Groot AC, Poelmann RE, Kálmán F. 1993. Early development of quail heart epicardium and associated vascular and glandular structures. Anat Embryol (Berl) 188: 381393.
  • Vrancken Peeters M-PFM, Gittenberger-de Groot AC, Mentink MMT, Poelmann RE. 1999. Smooth muscle cells and fibroblasts of the coronary arteries derive from epithelial-mesenchymal transformation of the epicardium. Anat Embryol (Berl) 199: 367378.
  • Vrancken Peeters M-PFM, Mentink MMT, Poelmann RE, Gittenberger-de Groot AC. 1995. Cytokeratins as a marker for epicardial formation in the quail embryo. Anat Embryol (Berl) 191: 503508.
  • Wessels A, Perez-Pomares JM. 2004. The epicardium and epicardially derived cells (EPDCs) as cardiac stem cells. Anat Rec 276A: 4357.
  • Zerwes HG, Risau W. 1987. Polarized secretion of A platelet-derived growth-factor like chemotactic factor by endothelial-cells in vitro. J Cell Biol 105: 20372041.
  • Zhuo Y, Hoyle GW, Zhang J, Morris G, Lasky JA. 2003. A novel murine PDGF-D splicing variant results in significant differences in peptide expression and function. Biochem Biophys Res Commun 308: 126132.