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Keywords:

  • proepicardium;
  • photoablation;
  • rose bengal;
  • chick embryo;
  • heart development

Abstract

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

The proepicardium (PE) is a primarily extracardiac progenitor cell population that colonizes the embryonic heart and delivers the epicardium, the subepicardial and intramyocardial fibroblasts, and the coronary vessels. Recent data show that PE-derived cells additionally play important regulatory roles in myocardial development and possibly in the normal morphogenesis of the heart. Developmental Dynamics 233, 2005. Research on the latter topics profits from the fact that loss-of-PE-function can be experimentally induced in chick embryos. So far, two microsurgical techniques were used to produce such embryos: (1) blocking of PE cell transfer with pieces of the eggshell membrane, and (2) mechanical excision of PE. Both of these techniques, however, have their shortcomings. We have searched, therefore, for new techniques to eliminate the PE. Here, we show that loss-of-PE-function can be induced by photoablation of the PE. Chick embryos were treated in ovo by means of a window in the eggshell at Hamburger and Hamilton (HH) stage 16 (iday 3). The pericardial coelom was opened, and the PE was externally stained with a 1% solution of Rose Bengal by means of a micropipette. Photoactivation of the dye was accomplished by illumination of the operation field with visible light. Examination on postoperative day 1 (iday 4, HH stages 19/20) disclosed complete removal of PE in every experimental embryo. On iday 9 (HH stages 33/34), the survival rate of experimental embryos was 35.7% (15 of 42). Development of the PE-derivatives was compromised in the heart of every survivor. The abnormalities encompassed hydro- or hemopericardium, epicardium-free areas with aneurysmatic outward bulging of the ventricular wall, thin myocardium, defects of the coronary vasculature, and abnormal tissue bridges between the ventricles and the pericardial wall. Our results show that photoablation of the PE is a powerful technique to induce long-lasting loss-of-PE-function in chick embryos. We have additionally obtained new data that suggest that the embryonic epicardium may make important contributions to the passive mechanics of the developing heart. Developmental Dynamics 233:1454–1463, 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

The early embryonic heart is a relatively simple, tube-like organ built up only of endothelial and myocardial cell lineages. Endothelial cells form an inner endothelial tube in contact with the blood—the primitive endocardium—whereas the developing myocardial cells form an outer epithelial tube in contact with the pericardial fluid—the primitive myocardium. The majority of the other cell lineages present in the mature heart do not derive from the embryonic heart tube but originate from progenitor cell populations that are primarily located outside the developing heart (Poelmann et al., 2002; Gittenberger-de Groot, 2003). One of these cell populations is the proepicardium (PE). The PE is a cauliflower-like accumulation of villous or bleb-like protrusions of the pericardial mesothelium that forms at a circumscribed region of the embryonic pericardial wall near the venous pole of the embryonic heart tube (Virágh and Challice, 1981; Männer, 1992b; Virágh et al., 1993; Männer et al., 2001).

In avian embryos, the villous protrusions of the PE attach to the opposite dorsal surface of the heart loop and establish a secondary tissue bridge between the ventral wall of the sinus venosus and the dorsal wall of the developing ventricles. This “secondary dorsal mesocardium” normally serves as the main route for the transfer of cells from the PE to the developing heart of avian embryos (Männer, 1992a, b, 1993, 1999). The first tissue to be formed by PE-derived cells is the epicardium, hence the name proepicardium (Virágh et al., 1993; Kálmán et al., 1995). Formation of the epicardium starts from the point of attachment of the secondary dorsal mesocardium to the heart from where PE-derived mesothelium spreads as a continuous epithelial sheet over the originally naked myocardial surface (Shimada and Ho, 1980; Hiruma and Hirakow, 1989; Männer, 1992b). Subsequent to formation of the epicardium, a subset of mesothelial cells delaminates from the epicardium and transforms into mesenchymal cells (Pérez-Pomares et al., 1997, 1998, 2002a; Dettman et al., 1998) that colonize the subepicardial, myocardial, and subendocardial layers of the cardiac wall and the atrioventricular (AV) endocardial cushions (Gittenberger-de Groot et al., 1998; Männer, 1999). The PE-derived mesenchymal cells deliver nearly all of the cellular elements of the subepicardial and intermyocardial connective tissue and of the coronary vasculature (Mikawa and Fischman, 1992; Mikawa and Gourdie, 1996; Dettman et al., 1998; Männer, 1999; Landerholm et al., 1999; Wada et al., 2001; Pérez-Pomares et al., 2002a).

Recent data have shown that PE-derived cells and tissues play important regulatory roles in the development of the working myocardium (Eid et al., 1992; Chen et al., 2002; Stuckmann et al., 2003) and the differentiation of the Purkinje fibers of the cardiac conduction system (Harris et al., 2002). Further but partially inconsistent data suggest that the presence of PE-derived cells or tissues possibly might be necessary for proper morphogenesis of the AV endocardial cushions (Gittenberger-de Groot et al., 2000; Pérez-Pomares et al., 2002b; Poelmann et al., 2002), proper formation of the interventricular septum (Gittenberger-de Groot et al., 2000; Pérez-Pomares et al., 2002b), and proper remodeling of the cardiac outflow tract (Gittenberger-de Groot et al., 2000; Rothenberg et al., 2002; Schaefer et al., 2004).

So far, two microsurgical techniques were used to produce a loss-of-PE-function in chick embryos: (1) The implantation of a piece of eggshell membrane between the PE and the heart to block the normal cell transfer from the PE to the heart (Männer, 1993; Gittenberger-de Groot et al., 2000, 2004; Pérez-Pomares et al., 2002b; Poelmann et al., 2002; Rothenberg et al., 2002; Pennesi et al., 2003; Eralp et al., 2005) and (2) the mechanical excision of the PE villi with fine tungsten needles (Pérez-Pomares et al., 2002b). Both of these techniques, however, have their shortcomings. One major drawback, for example, is that they cannot permanently prevent the colonization of the developing heart with PE-derived cells but rather cause a delay in the formation of some PE-derived tissues. In both kinds of experiments, the formation of a compensatory epicardium was observed that derived, first, from regenerating remnants of incompletely removed PE (Männer, 1993; Pérez-Pomares et al., 2002b) and, second, from pericardial mesothelium at the arterial pole of the heart (Männer, 1993; Gittenberger-de Groot et al., 2000).

The complete mechanical excision of the highly proliferating PE is practically difficult, because the PE of chick embryos occupies the thin and fragile wall of the sinus venosus, so that any mechanical damage to the PE might cause lethal ruptures of the vessel wall. We have searched, therefore, for new microsurgical techniques that might facilitate an almost complete and long-lasting elimination of the PE without significant damage to non-PE tissues. In medicine, photodynamic therapy (PDT) is a promising therapeutic approach for selective elimination of pathological tissues or cell populations (Dolmans et al., 2003; Moan and Peng, 2003). PDT is the combined application of two individually nontoxic components to induce tissue damage in an oxygen-dependent manner. These two components are (1) a photoactivatable substance—the photosensitizer—and (2) light of a specific wavelength that excites the photosensitizer. In the presence of oxygen, the excitation of photosensitizers induces the generation of reactive oxygen species, which in turn can damage cells and tissues. Reactive oxygen species have a high reactivity and a short half-life so that the direct phototoxic effects are confined to cells and tissues that have been labeled with the photosensitizer. Photoablation has been used successfully for selective killing of cells in experimental neurobiology (Miller, 1979; Picaud et al., 1990; Nirenberg and Cepko, 1993) and in experiments on invertebrate embryos (Shankland, 1984; Nishida and Satoh, 1989; Ettensohn, 1990). We hoped that photoablation might be a promising new approach that might facilitate the complete and selective elimination of the PE in chick embryos. Here, we show that photoablation of the PE indeed is a powerful technique to induce long-lasting loss-of-PE-function in chick embryos. Our results give new insights into the functional roles of PE-derived tissues in normal development of the embryonic heart.

RESULTS

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

Photoablation of the PE was performed by staining the villous protrusions of the PE with the reddish colored photosensitizer Rose bengal (RB; Fig. 1) on incubation day 3 at Hamburger and Hamilton (HH) stage 16 (Hamburger and Hamilton, 1951). After 24 hr of re-incubation (incubation day 4, HH stages 19–21), all experimental embryos (n = 46) were examined in ovo under a stereomicroscope. At this time point, all embryos were alive and normally developed. Upon examination of their pericardial cavity, no visible trace of RB and neither PE villi nor a secondary dorsal mesocardium were found in any of the experimental embryos. The ventral surface of the sinus venosus was smooth, and its wall appeared opaque. In the group of sham-operated embryos (n = 5), the secondary dorsal mesocardium and PE villi were present in every embryo.

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Figure 1. A–F: A series of microphotographs showing the technique of application of Rose bengal (RB) to the PE (arrow) of a Hamburger and Hamilton (HH) stage16 embryo in ovo. A–D: A piece of egg shell membrane is temporarily interposed between the heart and the PE to protect the heart from damage caused by unintended staining of the myocardium. C,D: Subsequent to the placement of the protecting membrane, small boluses of a 1% solution of RB were injected into the pericardial fluid surrounding the PE by means of a micropipette. To prevent unintentional staining of non-PE tissues, any trace of RB that did not become adherent to the surface of the PE was immediately sucked away from the pericardial fluid by means of a second micropipette (not shown). The application of small boluses of RB solution to the PE was repeated until the entire population of PE villi showed an intense red staining. E,F: Subsequent to complete staining of the PE with RB, the protecting membrane was removed from the pericardial coelom. During the whole operation procedure, the embryos were illuminated with visible light, which led to the photoactivation of RB. v, ventricular loop; o, outflow tract.

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In four experimental embryos, the second and final examination of their phenotype was carried out on incubation day 6 (HH stages 25/26) when the formation of the primitive epicardium normally is completed in the chick embryo (Hiruma and Hirakow, 1989; Männer, 1992b). Two of these embryos (50%) were alive, and their hearts were examined for the presence of epicardium using an anti–retinoic acid-synthesizing aldehyde dehydrogenase (anti-RALDH2) antibody (Xavier-Neto et al., 2000). The hearts of normal HH stages 25/26 control embryos were completely covered with a layer of RALDH2-positive cells (Fig. 2A,C). In the hearts of the two experimental embryos, epicardial formation was severely compromised. One heart was almost completely devoid of epicardium. RALDH2-positive cells were found only at the distal portion of the cardiac outflow tract (Fig. 2B,D). In the second heart, only the dorsal wall of the ventricles was covered with RALDH2-positive cells.

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Figure 2. A–D: Normal (A,C) and experimental Hamburger and Hamilton (HH) stages 25/26 chick embryo heart (B,D) subsequent to immunohistochemical staining with an anti–retinoic acid-synthesizing aldehyde dehydrogenase (anti-RALDH2) antibody to detect the epicardium. A,B: Frontal views (stereomicroscopic pictures) of the whole heart before sectioning. C,D: Sagittal sections through the primitive atrium (a), primitive left ventricle (lv), and outflow tract (oft). A–D: The normal heart is covered with a layer of RALDH2-positive cells (A,C), whereas the experimental heart almost completely lacks such a cell layer (B,D). RALDH2-positive cells are only found at the distal portion of the outflow tract (arrow). rv, primitive right ventricle.

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In the remaining 42 experimental embryos, the final examination of their phenotype was carried out on incubation day 9 (HH stage 33/34) when the hearts of chick embryos normally have acquired the mature four-chambered phenotype and the main stems of the coronary arteries normally are connected to the aorta. From these 42 embryos, 27 (64.3%) were dead and 15 (35.7%) were still alive. Upon external examination of the embryos, an abnormal hydropic enlargement of the pericardial cavity (hydropericardium) was noted in 9 of the 15 survivors, which, in its turn, was associated with a pericardial bleeding (hemopericardium) in 7 of them. The majority of embryos with a hemopericardium showed hearts with incomplete epicardial covering (6× grade IV defects, 1× grade II defects; see below). After opening of the pericardial cavity, abnormal tissue bridges between the heart and the pericardial wall were noted in four embryos (Fig. 3B). Except for the presence of “macroscopically” visible defects in development of PE derivatives (see below), the outer morphology of the hearts (dimensions and positional relationships of the cardiac chambers and great vessels) was found to be normal in 13 embryos. Two embryos had hearts with an abnormal outer morphology. One of these hearts showed a side-by-side position of the great arterial trunks and an abnormal herniation of the apical portions of its ventricles through an open defect in the pericardial wall. The other heart had a prominent hypoplasia of the right ventricle.

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Figure 3. A–F: Normal (A,C,E) and experimental hearts (B,D,F) from incubation day 9 (Hamburger and Hamilton [HH] stages 33/34). A,B: Views of the cardiac apex (stereomicroscopic pictures of vital hearts before perfusion and fixation). C,D: Views of the cardiac apex (scanning electron microscopy pictures). E,F: Transverse histological sections through the ventricles (hematoxylin and eosin staining). Normal hearts are completely covered with epicardium and show a thick, translucent layer of subepicardial mesenchyme (marked by arrows in A), which shrinks during fixation and dehydration for histological examinations. In the experimental hearts, only the dorsal and lateral walls of the ventricles are covered with compensatory epicardium. The boundary between the compensatory epicardium and the naked myocardial surface is marked by arrowheads. D,F: Epicardium-free areas show an aneurysmatic outward bulging of the ventricular wall which is especially prominent at a dilated state. Note the absence of the subepicardial space in B and F, and the presence of a tissue bridge (asterisk) between the ventricles and the ventral wall of the pericardial cavity. e, epicardium; ra, right atrium; rv, right ventricle; vm, naked ventricular myocardium.

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Development of the PE derivatives was compromised in the heart of every survivor. Depending on the grade of deficient development of PE-derivatives (epicardium, subepicardial mesenchyme, subepicardial coronary vessels), hearts could be categorized as follows.

Grade I

These hearts had a relatively normal appearance. They were completely covered with epicardium and subepicardial mesenchyme and showed subepicardial coronary vessels. Compared with normal controls, however, they showed a general reduction in thickness of the subepicardial mesenchyme.

Grade II

These hearts were completely covered with epicardium but incompletely covered by subepicardial mesenchyme. The presence of subepicardial mesenchyme and coronary vessels was locally confined to the atrioventricular sulcus and the base of the ventricles only.

Grade III

These hearts were completely covered with epicardium but were almost completely devoid of subepicardial mesenchyme and coronary vessels.

Grade IV

These hearts were only incompletely covered with epicardium. They showed large epicardium-free areas on the ventral wall of the right and left ventricles. These areas could already be identified in the beating hearts of living embryos under a dissecting microscope because they were characterized by an aneurysma-like outward bulging of their naked myocardial wall (Fig. 3B). The aneurysma-like outward bulging of the epicardium-free areas of the ventricular wall was especially prominent in dilated ventricles (Figs. 3C–F, 4), e.g., at the end of ventricular diastole. Histological analyses showed that the epicardium-free areas additionally were characterized by a severe reduction in thickness of their myocardial walls (Figs. 3E,F, 4A,B). The epicardium-covered areas of grade IV hearts were almost completely devoid of subepicardial mesenchyme and coronary vessels. The exact status of the coronary vessels was analyzed in two specimens fixed for histology. In one of these hearts only a tiny left coronary artery branched off from the aorta (Fig. 5), whereas the right coronary artery was missed completely. In the other specimen, neither the left nor the right coronary artery was present (data not shown). Grade IV defects regularly occurred in association with a pericardial bleeding (see above), resulting from small ruptures of their thinned myocardial wall. From the hearts of the 15 survivors, two had grade I defects, four had grade II defects, two had grade III defects, and seven had grade IV defects.

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Figure 4. A–C: Details from the right ventricular wall of normal (A) and experimental hearts (B,C) from incubation day 9 (Hamburger and Hamilton [HH] stages 33/34). A,B: Transverse histological sections (hematoxylin and eosin staining). C: Scanning electron micrograph of the lateral surface of the right ventricle. In the experimental hearts, only the dorsal and lateral walls of the ventricles are covered with compensatory epicardium. The boundary between the epicardium and the naked myocardial surface is marked by arrows/arrowheads. B: Note the absence of the subepicardial space in the experimental heart. e, compensatory epicardium; rv, lumen of the right ventricle; se, subepicardial mesenchyme; vm, ventricular myocardium.

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Figure 5. A–D: Normal (A,C) and experimental grade IV heart (B,D) from incubation day 9 (Hamburger and Hamilton [HH] stages 33/34). Transverse histological sections (hematoxylin and eosin staining) through the heart base at the level of the origin of the left coronary artery (marked by arrow). A,B: Views of whole sections. C,D: Higher magnification views from A,B. In the experimental heart, only a tiny left coronary artery branches off from the aorta (B,D). Note also that the experimental heart shows a marked reduction in thickness of the atrial myocardial wall compared with the normal heart. ao, aorta; la, left atrium; p, pulmonary trunk; ra, right atrium.

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Examination of the internal cardiac morphology disclosed the normal HH stages 33/34 phenotype in eight hearts, the presence of an isolated large ventricular septal defect (VSD) in four hearts, and the presence of a large VSD combined with double-outlet right ventricle in three hearts. Large VSDs affected the avian equivalent of the mammalian membranous septum and the subvalvular portion of the outflow septum. The primarily muscular portion of the interventricular septum was not affected. Isolated large VSDs were found in hearts with high grades in deficiency of PE derivatives (3× grade IV, 1× grade III). A double-outlet right ventricle was found in one grade II heart, one grade III heart, and one grade IV heart. Two of these hearts had additional abnormalities (herniation of the apical portions of its ventricles through a pericardial defect; tricuspid atresia). Except for the one heart with tricuspid atresia, further abnormalities of the AV valves were not found in the present study.

In the sham-operated group, all embryos (n = 5) had survived until incubation day 9 (HH stage 33/34). No abnormalities were recorded upon examination of the external and internal morphology of their hearts.

DISCUSSION

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

Induction of loss-of-function is an established experimental approach to gain evidence for the functional roles of genes, molecules, single cells, or cell populations in embryonic development (Gilbert, 2003). Loss-of-function of embryonic cell populations can be induced by two kinds of microsurgical experiments: (1) blocking of their normal behavior by means of the implantation of physical barriers that, for example, block the migration of cells to their normal local destinations within the embryo; or (2) the physical elimination of cell populations before the onset of their presumed function. During the past decade, several loss-of-function experiments were carried out on avian embryos to clarify the functional significance of the PE (Männer, 1992a, 1993; Gittenberger-de Groot et al., 2000, 2004; Poelmann et al., 2002; Pérez-Pomares et al., 2002b; Pennisi et al., 2003; Eralp et al., 2005). All these studies used a blocking technique originally developed by one of us (Männer, 1992a, 1993). Only a single study used this blocking technique as well as a mechanical excision technique (Pérez-Pomares et al., 2002b).

One major drawback of the PE-blocking and PE-excision techniques is that they cannot permanently prevent the colonization of the developing heart with PE-derived cells but rather cause a delay in the formation of some PE-derived tissues. In both kinds of experiments, the formation of a compensatory epicardium was observed which derived, first, from regenerating remnants of incompletely removed PE (Männer, 1993; Pérez-Pomares et al., 2002b) and, second, from pericardial mesothelium at the arterial pole of the heart (Männer, 1993; Gittenberger-de Groot et al., 2000). The formation of the compensatory epicardium led to the establishment of a complete mesothelial covering of the hearts in every embryo surviving up to the ninth incubation day (Männer, 1993; Pérez-Pomares et al., 2002b), which was a 3-day delay compared with normal hearts. PE-blocking or excision experiments, therefore, can deliver information on early effects of the loss-of-PE-function but seems to be of limited use if one wants to gain information on the effects of a long-lasting loss-of-PE-function. In the present study, therefore, we have used a photoablation technique to eliminate the PE. We hoped that photoablation might facilitate the complete elimination of the PE, so that, under ideal conditions, the formation of a compensatory epicardium might be prevented. Our results show that photoablation indeed facilitates the complete removal of the villous protrusions of the PE without damaging adjacent structures. Elimination of the PE was sufficient to prevent visible regeneration of PE villi in every treated embryo for at least 24 hr and led to the development of an abnormal cardiac phenotype that corresponded to the loss-of-PE-function phenotype observed subsequent to PE-blocking/ excision experiments. Our expectation, however, that photoablation of the PE might prevent the formation of compensatory epicardium could not be realized in the present study. Compared with the previously used blocking or excision techniques, photoablation of the PE could delay the initiation of the formation of the compensatory epicardium so that nearly 50% of the surviving experimental embryos (7 of 15) still showed hearts with large epicardium-free areas on the ninth incubation day, when the hearts of PE-blocked or PE-excised embryos were regularly completely covered with compensatory epicardium (Figs. 3B,D,F, 4B,C). These results show that photoablation facilitates the induction of a longer-lasting loss-of-PE-function compared with the previously used blocking or excision techniques.

Loss-of-PE-Function Phenotype

Defective formation of the epicardium and subepicardial mesenchyme, myocardial growth defects (“thin myocardium syndrome”), and coronary vessel defects are features of the loss-of-PE-function phenotype that have been reported consistently in previous as well as in the present study. The pathogenesis of these defects has been discussed extensively elsewhere (Männer, 1992a, 1993; Gittenberger-de Groot et al., 2000; Poelmann et al., 2002; Pérez-Pomares et al., 2002b; Pennisi et al., 2003; Eralp et al., 2005). We, therefore, will not discuss them here again. Instead, we will focus on abnormal features that previously have received only minor attention and on abnormalities that have been recognized after photoablation of the PE, for the first time.

Abnormalities of the AV Endocardial Cushions/Valves

Quail–chick chimera studies have shown that a subset of PE-derived mesenchymal cells invades the endocardium-derived mesenchyme of the AV cushions (Gittenberger-de Groot et al., 1998; Männer, 1999). It has been postulated, therefore, that PE-derived cells might play an important role in the morphogenesis of the AV valves (Gittenberger-de Groot et al., 1998). In accord with this idea, abnormalities of the AV cushions were indeed recorded in some of the PE-blocking/ excision experiments (Gittenberger-de Groot et al., 2000; Poelmann et al., 2002; Pérez-Pomares et al., 2002b; Eralp et al., 2005). The reported data, however, did not match with each other. In one laboratory, it was found that the AV cushions were regularly nearly absent and did not fuse so that all mature experimental hearts showed common AV canals (Gittenberger-de Groot et al., 2000; Poelmann et al., 2002; Eralp et al., 2005). In another laboratory, the AV cushions of experimental hearts were found to be abnormally large and fusion defects were not encountered (Pérez-Pomares et al., 2002b). In the present study, only 1 of 15 surviving embryos had a malformed AV valve (tricuspid atresia), whereas the remaining 14 did not show any overt anomaly of the AV valves. We do not know the reason(s) for this divergence of results. It should be noted, however, that hypoplasia of the AV cushions/valves was observed subsequent to PE-blocking in quail embryos (Gittenberger-de Groot et al., 2000; Poelmann et al., 2002; Eralp et al., 2005), whereas normal or hyperplastic AV valves were observed subsequent to PE-blocking/-photoablation in chick embryos (Pérez-Pomares et al., 2002b; present study). We, therefore, speculate that the above-mentioned divergence in the development of the AV cushions subsequent to PE-blocking/photoablation experiments might be best explained by species- and stock-specific differences in the susceptibility of the developing AV cushions to perturbations in epicardial development. The elucidation of the role of PE-derived cells in AV cushion development obviously awaits future studies. At the present time, we only know that the subpopulation of PE-derived cells invading the mesenchyme of the AV cushions does not contribute any substantial number of cells to the mature AV valves (de Lange et al., 2004).

Ventricular Septal Defects

Defects or absence of the interventricular septum occurred in quail and chick embryonic hearts subsequent to PE-blocking or PE-excision experiments (Gittenberger-de Groot et al., 2000; Pérez-Pomares et al., 2002b; Eralp et al., 2005). Large VSDs were also found in most of our HH stages 33/34 hearts with large epicardial defects. It, therefore, might be possible that PE-derived cells normally play a critical role in the formation of the interventricular septum. In this respect, we should note that the VSDs found in our experimental hearts affected the outflow septum and the “membranous” septum. This finding suggests that the role of PE-derived cells in the formation of the interventricular septum might be confined to its primarily mesenchymal anlagen. Further studies are needed to unravel the mechanism(s) by which PE-derived cells might regulate the formation of the interventricular septum.

Complex Cardiac Malformations

Complex cardiac malformations with double-inlet left ventricle/double-outlet right ventricle connections occurred frequently in quail embryos subsequent to PE-blocking experiments (Gittenberger-de Groot et al., 2000; Eralp et al., 2005). Information on the occurrence of such malformations is missed in previous PE-blocking/-excision studies carried out on chick embryos (Männer, 1993; Pérez-Pomares et al., 2002b; Rothenberg et al., 2002; Pennesi et al., 2003). In the present study, however, we found malformed hearts with double-outlet right ventricle connection in 20% of the survivors. Differences in the incidence of complex heart defects between studies carried out on chick versus quail embryos might be explained by species-specific differences in the susceptibility of the developing heart to perturbations in epicardial development. The pathogenesis of complex heart defects subsequent to PE-blocking/-ablation experiments has not been clarified to date. Recent findings, however, suggest that these malformations might result from alterations in the normal spatiotemporal patterning of apoptosis in the outflow tract myocardium (Rothenberg et al., 2002, 2003; Schaefer et al., 2004).

Aberrant Secondary Tissue Bridges Between the Heart and the Pericardial Wall

The PE-derived secondary dorsal mesocardium normally is the only secondary tissue bridge that forms between the heart and the pericardial wall of chick embryos (Männer, 1992b). In the present study, we found that, in the absence of the PE, some hearts became connected with the ventral pericardial wall or with the bottom of the pericardial cavity by means of slender secondary tissue bridges (Fig. 3B). The occurrence of such aberrant secondary tissue bridges might be of interest because it suggests that the potential of the pericardial wall to form PE-like tissue may not be confined to the region of the PE, only. It will be a challenge for future studies to identify the factor(s) that are normally needed for the induction of the PE as well as for the induction of aberrant PE-like tissues.

Hemopericardium

Bleeding into the pericardial cavity (hemopericardium) was observed previously in transgenic mice with defective formation of the epicardium (Kwee et al., 1995; Yang et al., 1995; Moore et al., 1999) but was not observed in avian embryos subsequent to PE-blocking/-excision experiments (Männer, 1993; Gittenberger-de Groot et al., 2000, 2004; Pérez-Pomares et al., 2002b; Poelmann et al., 2002; Rothenberg et al., 2002; Pennesi et al., 2003; Eralp et al., 2005). The present study is the first to report on this anomaly in avian embryos with defective formation of the epicardium. Photoablation of the PE obviously seems to produce a loss-of-PE-function phenotype that is closer to the phenotype found in mouse models with epicardium-deficient hearts than the phenotype produce by PE-blocking/-excision.

Aneurysma-Like Outward Bulging of Epicardium-Deficient Areas of the Ventricular Wall

Aneurysma-like outward bulging of the epicardium-deficient areas of the ventricular wall is a prominent pathomorphological feature of the loss-of-PE-function phenotype (Figs. 3B,D,F, 4B,C) that, surprisingly, has been recognized in the present study for the first time. It shows that epicardium-deficient areas of the cardiac wall develop a lower passive stiffness than epicardium-covered areas. Failure to recognize this feature in previous studies might be explained by the fact that it might become apparent only at advanced stages of development when the hearts of PE-blocked or PE-excised embryos were completely engulfed with compensatory epicardium (Männer, 1993; Pérez-Pomares et al., 2002b). The outward bulging of epicardium-deficient areas of the ventricular wall might be caused by (1) mechanical weakness of its thinned myocardial component, (2) the absence of a passive girdling effect of the epicardium/subepicardial connective tissue, or (3) a combination of both factors. In the past, several studies have focused on the myocardial growth defect induced by loss-of-PE-function (Gittenberger-de Groot et al., 2000; Pérez-Pomares et al., 2002b; Poelmann et al., 2002; Rothenberg et al., 2002; Pennesi et al., 2003) but only one study has focused on the mechanical properties of the embryonic subepicardial connective tissue (Tidball, 1992). The few available data suggest that, in the chick embryo, the subepicardial connective tissue might start to make an important contribution to cardiac mechanics only in the latter half of development (Tidball, 1992). It might be tempting, therefore, to attribute the outward bulging of the epicardium-deficient areas of the ventricular wall preferentially to mechanical weakness of its thinned myocardial component. The fact that these areas are surrounded by a sharp waist-like boundary that correlates with the free border of the compensatory epicardium (Figs. 3D,F, 4B,C), however, strongly suggests the presence of a passive girdling effect of the epicardium in the epicardium-covered portions of the cardiac wall. We, therefore, speculate (1) that the absence of a passive girdling effect of the epicardium/subepicardium has significantly contributed to the abnormally low passive stiffness of the epicardium-deficient portions of the ventricular wall and (2) that the embryonic epicardium and subepicardial connective tissue might start to play a significant role in the passive mechanics of the heart already with the beginning of epicardium formation. These ideas are supported by morphological and physiological data obtained from normal chick embryo hearts during the phase of formation of the epicardium. Morphological analyses of hearts fixed in general dilation show that the naked areas of their outer myocardial wall are slightly more dilated than the areas already covered with epicardium (Fig. 6). Physiological measurements of the intramyocardial pressures suggest that, in HH stage 18 chick embryo hearts, the passive stiffness of the dorsal ventricular wall is greater than that of the ventral wall (Chabert and Taber, 2002). At this developmental stage, the dorsal wall of the cardiac ventricles is just covered with epicardium, whereas the ventral wall is still free of epicardium (Männer, 1992b; Männer et al., 2001). The possibility that the epicardium/subepicardium might make important contributions to passive mechanics of the embryonic heart did not receive any attention in the past. This, however, is a pity, because studies on adult hearts have shown that the expression patterns of myocardial genes can be changed by external alterations of the passive mechanics of the heart (Sabbah et al., 2003). It, therefore, might be possible that the embryonic epicardium modulates myocardial development not only by the secretion of a hitherto unknown mitogenic factor (Eid et al., 1992; Chen et al., 2002; Stuckmann et al., 2003) but also by changing the passive mechanical properties of the cardiac wall. Our present observations have opened the door for future studies on an exciting new aspect of epicardial development.

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Figure 6. A–D: Scanning electron micrographs of a normal chick embryo heart during the phase of formation of the epicardium (fourth incubation day, Hamburger and Hamilton [HH] stage 22). A: Frontal view. B–D: Left lateral views. During normal formation of the epicardium (borders marked by arrowheads), epicardium-free areas of the cardiac wall show a slightly stronger dilation than the areas already covered with epicardium. This finding is especially prominent at the primitive atrium. am, naked surface of the atrial myocardium; e, epicardium; om, naked surface of the outflow tract myocardium; pe, proepicardial villi; rv, lumen of the right ventricle; se, subepicardial mesenchyme; vm, naked surface of the ventricular myocardium.

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EXPERIMENTAL PROCEDURES

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

Chick Embryos

Fertilized chicken eggs (White Leghorn, Gallus gallus) were obtained from the Georg-August-University research farm. Eggs were incubated at 38°C and 75% relative humidity. Staging of the embryos was performed according to Hamburger and Hamilton (1951).

Photosensitizer

We used the reddish colored fluorescein derivative RB (4,5,6,7-tetrachloro-2′,4′,5′,7′-tetraiodofluorescein sodium salt; Sigma-Aldrich R 3877) as the photosensitizer. Use of a colored photosensitizers facilitates selective and complete application of the photosensitizer to the target tissue under visual control. The phototoxic effects of RB have been documented in numerous in vitro and in vivo experiments (e.g., Manev et al., 1995; Menon et al., 1989). Solutions of RB were freshly prepared (1% RB dissolved in physiological saline) and kept in the dark at room temperature until use.

Application and Photoactivation of Rose Bengal

RB was applied to the PE of normal chick embryos from HH stage 16 (third incubation day). Embryos were treated in ovo under stereomicroscopic control. Eggs were windowed, and the embryos were prepared for operation according to Hara (1971). The pericardial cavity was opened as described previously (Männer, 1993). The PE was inspected to ensure that the villous protrusions of the PE had not made any firm contacts to the dorsal wall of the heart loop. Only embryos lacking these contacts were used. We found that unintentional staining of the surface of the heart loop with RB caused cardiac arrest. The embryonic hearts, therefore, had to be protected from staining with RB during the application of the dye to the PE. For this purpose, a rectangular piece of eggshell membrane was placed between the PE and the dorsal wall of the heart before the application of RB (Fig. 1A,B). Subsequent to the placement of the protecting membrane, a 1% solution of RB was applied to the PE by means of a micropipette (Fig. 1C,D). We found that embryos did not survive the direct injection of RB solutions into the mesenchyme of the PE. The PE, therefore, was externally stained with RB. Small boluses of the dye solution were injected into the pericardial fluid surrounding the PE, where most of the dye was instantly bound to the surface of the PE. To prevent unintentional staining of non-PE tissues, any trace of RB that did not become adherent to the surface of the PE, but remained in solution, was immediately sucked away from the pericardial fluid by means of a second micropipette. The application of small boluses of RB solution to the PE was repeated until the entire population of PE villi showed an intense red staining. Subsequent to complete staining of the PE with RB, the protecting membrane was removed from the pericardial coelom (Fig. 1E,F). During the entire operation procedure, the embryos were illuminated with visible light as described by Hara (1971). Because the photochemical reaction of RB is induced by irradiation with visible light (Menon et al., 1989), photoactivation of RB occurred concomitantly with the application of the dye to the PE. The eggs were closed again with adhesive tape and the embryos were re-incubated under the conditions described above.

Sham Operations

Embryos undergoing sham operations (n = 5) underwent the same treatment as the experimental embryos (windowing of the eggs, preparation for operation according to Hara [1971], opening of the pericardial coelom, transient implantation of a protecting membrane, illumination by visible light) except that RB was not applied to the PE. Instead, a small bolus of RB solution was injected into the free pericardial cavity where the dye was immediately sucked away by means of a second micropipette.

Immunohistochemistry

For immunohistochemical detection of epicardial cells, we used an anti-RALDH2 antibody (Xavier-Neto et al., 2000) kindly provided by Peter McCaffrey and Ursula Dräger. Embryos were washed three times in phosphate buffered saline (PBS) and fixed in a methanol/DMSO mixture (4:1) at 4°C overnight. Endogenous peroxidase was blocked by incubating the embryos with methanol/DMSO/30% H2O2 mixture (4:1:1) for 2 hr at room temperature. Unspecific binding sites were blocked by incubating the embryos 2× 1 hr in 2% bovine serum albumin (BSA), 0.1% Triton X-100 in PBS at room temperature. Embryos were incubated overnight in a 1:400 dilution of the RALDH2 antibody, which had been preincubated with chick embryo powder. After 5 washes for 1 hr with TBST (0.8% (w/v) NaCl, 0.02% (w/v) KCl, 25 mM Tris-Cl, pH7.5, 1% Tween-20), the embryos were incubated with a 1:200 dilution of a horseradish peroxidase–conjugated horse anti-mouse antibody (Vector Laboratories). After 5 washes for 1 hr with TBST, the immunoreaction was color developed using diaminobenzidine. For sectioning, the embryos were infiltrated overnight at 50°C with a 7.5% gelatin/15% sucrose solution and snap-frozen in dry ice cooled isopentane. Sections (20 μm) were obtained in a cryostat.

Histology and Scanning Electron Microscopy

To analyze the long-term effects of loss-of-PE-function, the majority of experimental embryos were re-incubated until incubation day 9 (HH stages 33/34). Test experiments had shown that, due to spontaneous ruptures of their thin myocardial walls, chick embryos rarely survive photoablation of the PE beyond incubation day 9. Embryos were fixed and prepared for examination either by conventional histology or scanning electron microscopy according to established protocols (Männer et al., 1996).

Acknowledgements

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

We thank Drs. Peter McCaffrey and Ursula Dräger for their kind gift of the RALDH2 antibody. We also thank Mrs. Kirsten Falk-Stietenroth and Mr. Hannes Sydow for technical and photographical assistance and Mrs. Cyrilla Maelicke for correcting the English manuscript.

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  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
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