Anat Embryol (1992) 186:379 385
Anatomy and Embryology 9 Springer-Verlag 1992
The development of pericardial villi in the chick embryo Ji~rg M~inner Abteilung Embryologie, Universitfit G6ttingen, Kreuzbergring 36, W-3400 G6ttingen, Federal Republic of Germany Accepted June 17, 1992
Summary. The development of pericardial villi and their relation to the development of the cardiac surface was studied in chick embryos from the 3rd to 10th day of incubation by scanning electron microscopy. During the 3rd day of incubation (stage 14-17 HH) the coelomic epithelium covering the ventral wall of the sinus venosus forms villous protrusions. By the end of the 3rd day (stage 17 HH) these protrusions contact the dorsal wall of the heart, so that a secondary dorsal mesocardium is formed. This bridges the pericardial cavity between the ventral wall of the sinus venosus and the dorsal base of the ventricles. This sinu-ventricular mesocardium exists only temporarily, as on the 8th day of incubation it becomes a part of the cardiac wall due to fusion with the epicardium of the coronary sulcus. During the 4th and 5th day of incubation (stage 1 7 - 25 HH), the formation of the epicardium proceeds from the point of attachment of the sinu-ventricular mesocardium. Although these findings suggest that the epithelium of the villous protrusions spreads over the surface of the embryonic heart, one cannot exclude other hypotheses on epicardial origin. The impression of a spreading epicardium could also be created if epicardial cells were to delaminate from a local epithelium in a temporally and spatially organized pattern. Key words: Chick embryo Pericardial villi - Secondary mesocardium - Formation of the epicardium
Introduction The wall of the early embryonic heart tube consists of two distinct cell layers, an inner endothelial tube (the anlage of the endocardium), and an outer epithelium which is derived from the coelomic epithelium. The two cell layers are connected with each other by a layer of a cell-free extracellular matrix called "cardiac jelly" (Davis 1924).
Since the last century, the coelomic epithelium of the early embryonic heart has generally been regarded as the anlage of both the myocardium and the epicardium (K611iker 1879; De Haan 1965). Correspondingly this epithelium is called the myoepicardium. His (1885) and Kurkiewicz (1909), having studied the development of the cardiac wall in human and chick embryos, reported that the myoepicardium could not be the source of the epicardium, because it consists solely of myoblastic cells. Their findings did not receive sufficient recognition until Manasek (1968) published his TEM observations on the development of the myocardium in the early embryonic chick. According to his findings, the myoepicardium is composed of a more or less uniform population of myoblasts. He concluded that the epicardium must be derived either from an extramyoepicardial source, or that a process of dedifferentiation occurs within the superficial layer of the myoepicardium. To clarify the origin of epicardial cells, the formation of the epicardium was investigated in the years following 1968 (Manasek 1969; Viragh and Challice 1973, 1981; Shimada and Ho 1980; Shimada et al. 1981; Komiyama et al. 1987; Kuhn and Liebherr 1988; Hiruma and Hirakow 1989). These studies support Kurkiewicz (1909), who suggested that epicardial cells are derived from pericardial villi facing the dorsal wall of the early embryonic heart. The formation of the epicardium is thought to be a process of secondary covering of the developing heart. However, by what means the epithelium of the pericardial villi is transferred to the surface of the beating heart is still not clarified. Some findings suggest that the pericardial villi make contact with the heart, forming continuous connections between the pericardial wall and the dorsal surface of the developing heart (Kurkiewicz 1909; Shimada and Ho 1980; Shimada et al. 1981; Viragh and Challice 1981). Other findings suggest that villi form free vesicles, which are released into the fluid of the pericardial cavity where they float to the surface of the heart (Komiyama et al. 1987; Kuhn and Liebherr 1988).
380 It is the aim of this study to discover how cells of pericardial villi are transferred to the surface of the embryonic heart. For this purpose, the development of the pericardial villi and their involvement in the development of the cardiac surface were investigated in chick embryos with an SEM.
Materials and methods Fertilized eggs (White Leghorn) were incubated at 38~ C and 75% humidity for 3-10 days. Embryos ranged from stage 14 to 36 according to Hamburger and Hamilton (1951). For investigation with the scanning electron microscope, 17 embryos were used from the 3rd, 15 from the 4th, 9 from the 5th, 4 from the 6th, 4 from the 7th, 4 from the 8th, 4 from the 9th, and 2 from the 10th day of incubation. The embryos were removed fi'om the eggs and dissected in a dish in Locke's solution prior to fixation. After opening the pericardial cavity, the surfaces of the heart and the pericardial wall were rinsed with Locke's solution. Because it is advantageous to fix hearts in a standardized state of contraction, the beating hearts were then externally rinsed with a calcium-free solution of 20 retool/1 manganese chloride (Asami 1979). MgClz causes a cardiac arrest in a general dilatation by biocking the calcium channels. After cardiac arrest, the hearts were shifted craniad by forceps to expose the dorsal wall of the heart and the dorsal wall of the pericardial cavity. This situs was preserved by rinsing the pericardial cavity with glutaraldehyde (25%) for a short time (1-2 rain). Short fixation with 25% glutaraldehyde did not produce visible coagulations of the tissues. The final fixation was carried out in a 2% solution of glutaraldehyde in 0.2 tool/1 cacody!ate buffer for 3-12 h. After dehydration in graded ethanols, the embryos were critical point dried using ethanol as the transitional and CO2 as the exchange fluid (CPD apparatus E 3000, Polaron Equipment). The dried specimens were mounted with conducting silver, and sputtered with gold-palladium to a layer of about 40 nm (SEM coating unit E 5100, Polaron Equipment). A Zeiss Novascan 30 scanning electron microscope was used to observe and photograph the specimens.
Results On the 3rd day of incubation (stage 14-18 H H ) , the sinus venosus is still incorporated in the dorsal wall of the pericardial cavity. Thus its ventral surface, covered by the coelomic epithelium, is a part of the primary pericardium. During this period, the epithelium covering the ventral wall of the sinus venosus forms villous protrusions. At stage 14, there are some small vesicular protrusions present which are directed into the pericardial cavity (Fig. 1). These protrusions quickly increase in n u m b e r and grow in length, forming an accumulation of pericardial villi which is best described as resembling a cauliflower (Fig. 2). Due to the f o r m a t i o n of the cervical flexure during stages 14 to 17, the dorsal surface o f the heart approaches the ventral wall of the sinus venosus. In consequence, the pericardial villi begin to establish contact with the dorsal wall of the heart. A touching of the cardiac surface is followed by the establishment of firm contacts between the villi and the heart. Initially, filipodia, extending f r o m the tips of some villi, adhere to the opposing surface of the heart. Later on, the apical surfaces of the villi come into intimate contact with the cardiac surface (Figs. 3, 4).
At the end of the 3rd day of incubation (stage 1 7 + ) most villi are in firm contact with the heart. A secondary dorsal mesocardium is then formed, bridging the pericardial cavity from the ventral wall of the sinus venosus to the dorsal wall of the heart (Fig. 5 b). The m o r p h o l o g y of this mesocardium is best described as resembling a tree with a short trunk and multiple ramifications. At the mesocardium, new villi are formed which we call accessory villi. During the 4th and 5th day of incubation (stages 18-25), an epithelium different from the original epithelium of the heart, but similar to the epithelium of the villi, seems to spread over the cardiac surface. " S p r e a d i n g " of this epithelium proceeds from the point of attachment of the secondary dorsal mesocardium (Figs. 5a, b, 6a, b). During the same period the m o r p h o l o g y of the secondary dorsal mesocardium changes. The trunk grows and elongates, whereas its branches and most of the accessory villi fuse with the cardiac wall. As a result, the secondary dorsal mesocardium is found as a free cord bridging the pericardial cavity between the ventral wall of the sinus venosus and the dorsal base of the ventricles (Fig. 7). F r o m the 4th to the 9th day of incubation, the sinus venosus is separated f r o m the pericardial wall and incorporated into the right atrium. Due to this process, the origin of the secondary dorsal mesocardium shifts from the pericardial to the cardiac wall. During the 6th and 7th day of incubation its free cord fuses with the epicardium covering the dorsal wall of the coronary sulcus. As a result, the secondary dorsal mesocardium becomes a part of the cardiac wall. On the 8th day of incubation (stage 31), the remnant of the secondary dorsal mesocardium is found as a sagittal fold (Fig. 8), which flattens further (Fig. 9). During the dissection of the embryos in Locke's solution, inside the fold a vessel was found which opened into the sinus venosus.
Discussion Our discussion on the origin of the epicardium concentrates on observations which support the view that the myoepicardium is the source of myocardial cells only. To our knowledge, His (1885) was the first to report on such findings in h u m a n embryos. He stated, " V o n der ursprfinglichen Herzanlage liefert der /iul3ere Schlauch die Muskulatur und zwar nur die Muskulat u r . " ( " T h e m y o c a r d i u m and only the m y o c a r d i u m is derived f r o m the outer tube of the primitive cardiac anlage." His 1885 p. 171). He supposed that the ceils of the epicardium and the connective tissue of the heart were derived from endocardial cells, which colonize the cardiac wall f r o m the inside to the outside. It seems that this publication from His (1885) did not receive sufficient recognition. Consequently, the first discussion on a nonmyoepicardial origin of the epicardium is c o m m o n l y attributed to Kurkiewicz (1909).
381
Fig. 1. Embryo stage 14. Heart loop seen from caudal direction. The coelomic epithelium covering the ventral wall of the sinus venosus shows vesicular protrusions. The sinus venosus is still a part of the pericardial wall, x 125 1, common atrium, 2, anlage of the liver
Fig. 3. Embryo stage 17. Same view as in Fig. 1. Some of the villi adhere to the cardiac surface, x 100 Fig. 4. Embryo stage 17. Higher magnification showing a pericardial villus adherent to the cardiac surface. The tip of this villus is branching at the cardiac surface, x 1750
Fig. 2. Embryo stage 16. Same view as in Fig. 1. The vesicular protrusions of the coelomic epithelium have developed into an accumulation of pericardial villi, x 125
Although Kurkiewicz (1909) was the first to recognize the possible relation between the formation of pericardial villi and the f o r m a t i o n of the epicardium, he was not the first to describe these villi (" villi of Kurkiewicz" Kuhn and Liebherr 1988). The first and best descriptions of the villi in chick embryos had already been made by R e m a k (1843, 1855), but he failed to show their developmental significances. Later, pericardial villi were described in m a m m a l i a n embryos by Lieberkfihn (1876), K611iker (1879), His (1881), U s k o w (1883a, b), and Born (1889). Because the villi are found to be in close proximity to the developing
liver, a possible contribution to the formation of hepatic blood vessels was discussed. A good review on this discussion was given by U s k o w (1883 b). Current discussions about the role of the pericardial villi have focused on two items, the development of the epicardium (Kurkiewicz 1909; Shimada and H o 1980; Shimada et al. 1981 ; Viragh and Challice 1981 ; Komiyam a et al. 1987; K u h n and Liebherr 1988) and the develo p m e n t of the cardiac blood vessels (Grant 1926; Voboril and Schiebler 1969; Steinhoff 1971 ; Los and Verwoerd 1972; Langford et al. 1990; Viragh et al. 1990). With regard to the possible relationship between the
382
Fig. 5a, b. Embryos stages 17 (a) and 18 (b). Same views as in Fig. 1. Due to the attachment of the villi to the heart a secondary dorsal mesocardium is formed. From the point of its attachment, an epithelium different from the original epithelium of the heart
but similar to the epithelium of the villi seems to spread over the cardiac surface. Arrows mark the borderline between the two epithelia, a • 150; b • 125
Fig. 6a, b. Embryos stages 16 (a) and 20 (b). a Surface ofa pericardial villus, b Surface of a developing heart showing original (1), and ~ spreading" (2) epithelium. The surface morphology of the
"spreading" epithelium resembles the surface morphology of the villous epithelium, x 1500
formation of pericardial villi and the formation of the epicardium, three findings are of major interest: 1. In all species investigated, pericardial villi are found in the region of the cardiac inflow tract, where they face the dorsal wall of the developing heart (Remak 1843, 1855; Lieberkiihn 1876; K611iker 1879; His 1881; Uskow 1883 a, b; Born 1889; Kurkiewicz 1909; Shimada and Ho 1980; Shimada et al. 1981; Viragh and Challice 1981; Komiyama et al. 1987; Kuhn and Liebherr 1988; Hiruma and Hirakow 1989). 2. The formation of the epicardium commonly begins at the dorsal wall of the embryonic heart (Greil 1903; Kurkiewicz 1909; Steinhoff 1971; Viragh and Challice 1973, 1981 ; Shimada and Ho 1980; Shimada et al. 1981 ; Kuhn and Liebherr 1988; Hiruma and Hirakow 1989).
3. The period during which most pericardial villi are found coincides with the period in which the formation of the epicardium takes place (Kurkiewicz 1909; Komiyama et al. 1987; Kuhn and Liebherr 1988). According to Kurkiewicz (1909), these findings reflect the fact that the epicardium is derived from the epithelium of the pericardial villi. However, how epithelium from the pericardial wall was transferred to the cardiac surface remained obscure. Some authors stated only that cells from this epithelium migrated to the heart (Viragh and Challice 1973); other authors were more definite and reported that cells from pericardial villi migrated to the cardiac surface after villi had become attached to the dorsal wall of the heart (Kurkiewicz 1909; Shimada and Ho 1980; Viragh and Challice 1981).
383
Fig. 7. Embryo stage 27. Same view as in Fig. 1. In order to expose the dorsal wall of the heart, the apex of the heart was shifted craniad by forceps prior to fixation. The secondary dorsal mesocardium is found as a free cord bridging the pericardial cavity between the ventral wall of the sinus venosus and the dorsal base of the ventricles. The incorporation of the sinus venosus into the heart is nearly completed. Due to the development of the subepicardial connective tissue, the surface of the heart, especially its ventricular surface, shows fissures and folds, l, left horn of the sinus venosus; 2, left atrium; 3, right atrium; 4, ventricles, x 60 Fig. 8. Embryo stage 31. Same view as in Fig. 7. The mesocardium has become a part of the cardiac wall; its remnant is found as a sagittal fold. Accessory villi are found as appendages of the atrial wall. x 50 Fig. 9. Embryo stage 34. Same view as in Fig. 7. The sagittal fold, which is the remnant of the secondary dorsal mesocardium, has flattened. The incorporation of the sinus venosus is completed. x 60
384 This would suggest that pericardial villi form continuous connections between the pericardial wall and the heart, bridging the pericardial cavity. However, all these authors failed to show such connections. Consequently, Kuhn and Liebherr (1988) stated that they could not imagine that villi were able to establish continuous connections to the rhythmically moving surface of the ventricular part of the heart. They supposed that epithelium from the pericardial wall was transferred across the pericardiat cavity to the cardiac surface in the form o f free vesicles, which had been detached from the pericardial villi (Komiyama et al. 1987; Kuhn and Liebherr 1988). However, secondary connections between the pericardial wall and the ventricular part of the heart are not unknown in vertebrates, although it seems that previous descriptions have not found their way into textbooks of embryology. The first description of such a connection was made by Greil (1903). In Lacerta viridis, the apex of the mature heart is connected with the pericardial wall by a cord. According to Greil (1903) this "apical c o r d " is formed by fusion between epicardial villi at the cardiac apex, and villi at the opposing side o f the pericardial wall. Hochstetter (1906) and Patten (1922) described a connection between the ventral wall of the sinus venosus and the dorsal wall of the heart in chick embryos. Although they stated that it must have been formed secondarily, they could not show how this happened. Regarding the possible role of this " f u s i o n " they supposed that it served as a pathway along which one of the main coronary veins reached the sinus venosus during development. The present findings show that this connection, which has frequently been misinterpreted as being a remnant of the primary dorsal mesocardium (Hamilton 1952, p. 263, Fig. 125; Steinhoff 1971 ; Los and Verwoerd 1972; Langford et al. 1990), is a secondary dorsal mesocardium. It is formed by the attachment of the pericardial villi to the dorsal surface of the heart (Figs. 2, 3, 5a, b), and bridges the pericardial cavity between the ventral wall of the sinus venosus and dorsal wall of the ventricular part of the heart (sinu-ventricular mesocardium) (Fig. 7). Due to its formation, epithelium from the pericardial wall reaches the dorsal surface of the heart. We then have to consider whether this epithelium is the source of the epicardium, and how the formation of the epicardium takes place. Most authors state that cells from the pericardial villi migrate over the cardiac surface forming the epicardium (Kurkiewicz 1909; Shimada and Ho 1980; Shimada et al. 1981; Viragh and Challice 1981). The observations presented here seem to support this view. After the sinuventricular mesocardium has formed, local differences in the surface morphology of the cardiac epithelium are found. An epithelium, similar to t h e epithelium of the pericardial villi, but different from the original epithelium of the heart, seems to spread over the cardiac surface. " S p r e a d i n g " of this epithelium proceeds from the point of attachment of the sinu-ventricular mesocardium (Figs. 5 a, b, 6 a, b). These findings correspond to previous SEM observations which have been
interpreted as " s p r e a d i n g " epicardium (Shimada and Ho 1980; Shimada et al. 1981). Although these observations suggest that the epithelium o f the pericardial villi is spreading, one cannot exclude other hypotheses of epicardial origin. The impression of a spreading epicardium could also be created if epicardial cells were to delaminate from local myoepicardium in a temporally and spatially organized pattern. In this case, the border between the epicardium and myoepicardium would appear as if it were shifting over the cardiac surface without real substantial displacement of the cells. Because neither the existence of undifferentiated precursor cells, nor dedifferentiating myoblasts, can be fully excluded by descriptive studies alone, such a process, which is best described as resembling a travelling wave, cannot be excluded morphologically. Therefore, experimental investigations are needed to clarify the origin of the epicardium.
Acknowledgements. I should like to thank Mr. Hannes Sydow for technical assistance, Ms. Kirsten Falk for photographical assistance, and Ms. Cyrilla Maelicke for correcting the English manuscript. References Asami I (1979) Development of the outflow tract of the rat embryonic heart. 5th European Anatomical Congress, Prag, Argumenta Communicationum : 14 Born G (I 889) Beitr/ige zur Entwicklungsgeschichte des S/iugetierherzens. Arch Mikrosk Anat 33:284~378 Davis CL (1924) The cardiac jelly of the chick embryo. Anat Rec 27 : 201-202 De Haan RL (1965) Morphogenesis of the vertebrate heart. In: De Haan RL, Ursprung H (eds) Organogenesis. Holt, Reinhardt and Winston, New York, pp 377419 Grant RT (1926) Development of the cardiac coronary vessels in the rabbits. Heart 13:261-271 Greil A (1903) Beitr/ige zur vergleichenden Anatomic und Entwicklungsgeschichte des Herzens und des Truncus arteriosus der Wirbelthiere. Gegenbaurs Morphol JB 31:123-310 Hamburger V, Hamilton HL (1951) A series of normal stages in the development of the chick embryo. J MorphoI 88:49-92 Hamilton HL (1952) Lillie's Development of the chick. 3rd edn. Holt, New York Hiruma T, Hirakow R (1989) Epicardial formation in embryonic chick heart: computer-aided reconstruction, scanning and transmission electron microscopic studies. Am J Anat 184:129 138 His W (1881) Mittheilungen zur Embryologie der S/iugethiere und des Menschen. Arch Anat Entwickl Gesch Jg 1881:303-329 His W (1885) Anatomic menschlicher Embryonen. Teil III Zur Geschichte der Organe. Vogel, Leipzig Hochstetter F (1906) Beitr/ige zur Anatomic und Entwicklungsgeschichte des Blutgeffil3systemsder Krokodile. In: Voeltzkow A (ed) Reise in Ostafrika. Wissenschaftliche Ergebnisse. Anatomic und Entwicklungsgeschichte. Vol 4, pp 1-133 K611iker A (1879) Entwicklungsgeschichte des Menschen und der Thiere, 2. Aufl. Engelmann, Leipzig Komiyama M, Ito K, Shimada Y (1987) Origin and development of the epicardium in the mouse embryo. Anat Embryol 176:183-189 Kuhn HJ, Liebherr G (1988) The early development of the epicardimn in Tupaja beIangerie. Anat Embryol 177:225 234 Kurkiewicz T (1909) O histogenezie miesnia sercowego zwierzat kregowych - Zur Histogenese des Herzmuskels der Wirbeltiere. Bull Internat Acad Sci Cracovie: 148 191
385 Langford JK, Hay DA, Bolender DL (1990) Fine structural features of coronary vasculogenesis in collagen lattices. In: Bockman DA, Kirby ML (eds) Embryonic origins of defective heart development. Ann NY Acad Sci 588:404408 Lieberkfihn N (1876) Ober die AIlantois und die Nieren von S/iugethierembryonen. Sitzungsberichte d Ges z Bef6rderung der gesamten Naturwiss z Marburg 1 : 1 11 Los JA, Verwoerd CDA (1972) The development of a primary venous system from epicardial villi in the cardiac wall of the chicken and the mouse embryo, and the relationship between this venous system and the arterial vascularisation in the mouse. Acta Morphol Neerl Scand 8 : 233 Manasek FJ (1968) Embryonic development of the heart I. A light and electron microscopic study of myocardial development in the early chick embryo. J Morphol 125:329 366 Manasek FJ (1969) Embryonic development of the heart II. Formation of the epicardium. J Embryol Exp Morphol 22:333 348 Patten BM (1922) The formation of the cardiac loop in the chick. Am J Anat 30:373-397 Remak R (1843) Ober die Entwicklung des Hfihnchens im El. Arch Anat Physiol Wiss Med Jg 1843:478484 Remak R (1855) Untersuchungen fiber die Entwicklung der Wirbelthiere. Berlin Shimada Y, Ho E (1980) Scanning electron microscopy of the embryonic chick heart: formation of the epicardium and surface
structure of the four heterotypic cells that contribute to the embryonic heart. In: van Praagh R, Takao A (eds) Etiology and morphogenesis of congenital heart disease. Futura, New York, pp 63-80 Shimada Y, Ho E, Toyota N (1981) Epicardial covering over myocardial wall in the chicken embryo as seen with the scanning electron microscope. Scanning Electron Microsc 11:275-280 Steinhoff W (1971) Zur Entwicklung der terminalen Strombahn im Hfihnerherzen. Anat Entwickl Gesch 134: 255-277 Uskow N (1883 a) Ober die Entwicklung des Zwerchfells, des Pericardiums und des Coeloms. Arch Mikrosk Anat 22:143 219 Uskow N (1883b) Bemerkungen zur Entwicklungsgeschichte der Leber und der Lungen. Arch Mikrosk Anat 22:219 227 Viragh S, Challice CE (1973) Origin and differentiation of cardiac muscle cells in the mouse. J Ultrastruct Res 42 : 1-24 Viragh S, Challice CE (1981) The origin of the epicardium and the embryonic myocardial circulation in the mouse. Anat Rec 201:157-168 Viragh S, Kalman F, Gittenberger de Groot AC, Poelmann RE, Moorman AFM (1990) Angiogenesis and hematopoesis in the epicardium of the vertebrate embryo heart. In: Bockman DE, Kirby ML (eds) Embryonic origins of defective heart development. Ann NY Acad Sci 588:455458 Voboril Z, Schiebler TH (1969) Ober die Entwicklung der Geffil3versorgung des Rattenherzens. Anat Entwickl Gesch 129 : 2440
Anatomy and Embryology 9 Springer-Verlag 1992
The development of pericardial villi in the chick embryo Ji~rg M~inner Abteilung Embryologie, Universitfit G6ttingen, Kreuzbergring 36, W-3400 G6ttingen, Federal Republic of Germany Accepted June 17, 1992
Summary. The development of pericardial villi and their relation to the development of the cardiac surface was studied in chick embryos from the 3rd to 10th day of incubation by scanning electron microscopy. During the 3rd day of incubation (stage 14-17 HH) the coelomic epithelium covering the ventral wall of the sinus venosus forms villous protrusions. By the end of the 3rd day (stage 17 HH) these protrusions contact the dorsal wall of the heart, so that a secondary dorsal mesocardium is formed. This bridges the pericardial cavity between the ventral wall of the sinus venosus and the dorsal base of the ventricles. This sinu-ventricular mesocardium exists only temporarily, as on the 8th day of incubation it becomes a part of the cardiac wall due to fusion with the epicardium of the coronary sulcus. During the 4th and 5th day of incubation (stage 1 7 - 25 HH), the formation of the epicardium proceeds from the point of attachment of the sinu-ventricular mesocardium. Although these findings suggest that the epithelium of the villous protrusions spreads over the surface of the embryonic heart, one cannot exclude other hypotheses on epicardial origin. The impression of a spreading epicardium could also be created if epicardial cells were to delaminate from a local epithelium in a temporally and spatially organized pattern. Key words: Chick embryo Pericardial villi - Secondary mesocardium - Formation of the epicardium
Introduction The wall of the early embryonic heart tube consists of two distinct cell layers, an inner endothelial tube (the anlage of the endocardium), and an outer epithelium which is derived from the coelomic epithelium. The two cell layers are connected with each other by a layer of a cell-free extracellular matrix called "cardiac jelly" (Davis 1924).
Since the last century, the coelomic epithelium of the early embryonic heart has generally been regarded as the anlage of both the myocardium and the epicardium (K611iker 1879; De Haan 1965). Correspondingly this epithelium is called the myoepicardium. His (1885) and Kurkiewicz (1909), having studied the development of the cardiac wall in human and chick embryos, reported that the myoepicardium could not be the source of the epicardium, because it consists solely of myoblastic cells. Their findings did not receive sufficient recognition until Manasek (1968) published his TEM observations on the development of the myocardium in the early embryonic chick. According to his findings, the myoepicardium is composed of a more or less uniform population of myoblasts. He concluded that the epicardium must be derived either from an extramyoepicardial source, or that a process of dedifferentiation occurs within the superficial layer of the myoepicardium. To clarify the origin of epicardial cells, the formation of the epicardium was investigated in the years following 1968 (Manasek 1969; Viragh and Challice 1973, 1981; Shimada and Ho 1980; Shimada et al. 1981; Komiyama et al. 1987; Kuhn and Liebherr 1988; Hiruma and Hirakow 1989). These studies support Kurkiewicz (1909), who suggested that epicardial cells are derived from pericardial villi facing the dorsal wall of the early embryonic heart. The formation of the epicardium is thought to be a process of secondary covering of the developing heart. However, by what means the epithelium of the pericardial villi is transferred to the surface of the beating heart is still not clarified. Some findings suggest that the pericardial villi make contact with the heart, forming continuous connections between the pericardial wall and the dorsal surface of the developing heart (Kurkiewicz 1909; Shimada and Ho 1980; Shimada et al. 1981; Viragh and Challice 1981). Other findings suggest that villi form free vesicles, which are released into the fluid of the pericardial cavity where they float to the surface of the heart (Komiyama et al. 1987; Kuhn and Liebherr 1988).
380 It is the aim of this study to discover how cells of pericardial villi are transferred to the surface of the embryonic heart. For this purpose, the development of the pericardial villi and their involvement in the development of the cardiac surface were investigated in chick embryos with an SEM.
Materials and methods Fertilized eggs (White Leghorn) were incubated at 38~ C and 75% humidity for 3-10 days. Embryos ranged from stage 14 to 36 according to Hamburger and Hamilton (1951). For investigation with the scanning electron microscope, 17 embryos were used from the 3rd, 15 from the 4th, 9 from the 5th, 4 from the 6th, 4 from the 7th, 4 from the 8th, 4 from the 9th, and 2 from the 10th day of incubation. The embryos were removed fi'om the eggs and dissected in a dish in Locke's solution prior to fixation. After opening the pericardial cavity, the surfaces of the heart and the pericardial wall were rinsed with Locke's solution. Because it is advantageous to fix hearts in a standardized state of contraction, the beating hearts were then externally rinsed with a calcium-free solution of 20 retool/1 manganese chloride (Asami 1979). MgClz causes a cardiac arrest in a general dilatation by biocking the calcium channels. After cardiac arrest, the hearts were shifted craniad by forceps to expose the dorsal wall of the heart and the dorsal wall of the pericardial cavity. This situs was preserved by rinsing the pericardial cavity with glutaraldehyde (25%) for a short time (1-2 rain). Short fixation with 25% glutaraldehyde did not produce visible coagulations of the tissues. The final fixation was carried out in a 2% solution of glutaraldehyde in 0.2 tool/1 cacody!ate buffer for 3-12 h. After dehydration in graded ethanols, the embryos were critical point dried using ethanol as the transitional and CO2 as the exchange fluid (CPD apparatus E 3000, Polaron Equipment). The dried specimens were mounted with conducting silver, and sputtered with gold-palladium to a layer of about 40 nm (SEM coating unit E 5100, Polaron Equipment). A Zeiss Novascan 30 scanning electron microscope was used to observe and photograph the specimens.
Results On the 3rd day of incubation (stage 14-18 H H ) , the sinus venosus is still incorporated in the dorsal wall of the pericardial cavity. Thus its ventral surface, covered by the coelomic epithelium, is a part of the primary pericardium. During this period, the epithelium covering the ventral wall of the sinus venosus forms villous protrusions. At stage 14, there are some small vesicular protrusions present which are directed into the pericardial cavity (Fig. 1). These protrusions quickly increase in n u m b e r and grow in length, forming an accumulation of pericardial villi which is best described as resembling a cauliflower (Fig. 2). Due to the f o r m a t i o n of the cervical flexure during stages 14 to 17, the dorsal surface o f the heart approaches the ventral wall of the sinus venosus. In consequence, the pericardial villi begin to establish contact with the dorsal wall of the heart. A touching of the cardiac surface is followed by the establishment of firm contacts between the villi and the heart. Initially, filipodia, extending f r o m the tips of some villi, adhere to the opposing surface of the heart. Later on, the apical surfaces of the villi come into intimate contact with the cardiac surface (Figs. 3, 4).
At the end of the 3rd day of incubation (stage 1 7 + ) most villi are in firm contact with the heart. A secondary dorsal mesocardium is then formed, bridging the pericardial cavity from the ventral wall of the sinus venosus to the dorsal wall of the heart (Fig. 5 b). The m o r p h o l o g y of this mesocardium is best described as resembling a tree with a short trunk and multiple ramifications. At the mesocardium, new villi are formed which we call accessory villi. During the 4th and 5th day of incubation (stages 18-25), an epithelium different from the original epithelium of the heart, but similar to the epithelium of the villi, seems to spread over the cardiac surface. " S p r e a d i n g " of this epithelium proceeds from the point of attachment of the secondary dorsal mesocardium (Figs. 5a, b, 6a, b). During the same period the m o r p h o l o g y of the secondary dorsal mesocardium changes. The trunk grows and elongates, whereas its branches and most of the accessory villi fuse with the cardiac wall. As a result, the secondary dorsal mesocardium is found as a free cord bridging the pericardial cavity between the ventral wall of the sinus venosus and the dorsal base of the ventricles (Fig. 7). F r o m the 4th to the 9th day of incubation, the sinus venosus is separated f r o m the pericardial wall and incorporated into the right atrium. Due to this process, the origin of the secondary dorsal mesocardium shifts from the pericardial to the cardiac wall. During the 6th and 7th day of incubation its free cord fuses with the epicardium covering the dorsal wall of the coronary sulcus. As a result, the secondary dorsal mesocardium becomes a part of the cardiac wall. On the 8th day of incubation (stage 31), the remnant of the secondary dorsal mesocardium is found as a sagittal fold (Fig. 8), which flattens further (Fig. 9). During the dissection of the embryos in Locke's solution, inside the fold a vessel was found which opened into the sinus venosus.
Discussion Our discussion on the origin of the epicardium concentrates on observations which support the view that the myoepicardium is the source of myocardial cells only. To our knowledge, His (1885) was the first to report on such findings in h u m a n embryos. He stated, " V o n der ursprfinglichen Herzanlage liefert der /iul3ere Schlauch die Muskulatur und zwar nur die Muskulat u r . " ( " T h e m y o c a r d i u m and only the m y o c a r d i u m is derived f r o m the outer tube of the primitive cardiac anlage." His 1885 p. 171). He supposed that the ceils of the epicardium and the connective tissue of the heart were derived from endocardial cells, which colonize the cardiac wall f r o m the inside to the outside. It seems that this publication from His (1885) did not receive sufficient recognition. Consequently, the first discussion on a nonmyoepicardial origin of the epicardium is c o m m o n l y attributed to Kurkiewicz (1909).
381
Fig. 1. Embryo stage 14. Heart loop seen from caudal direction. The coelomic epithelium covering the ventral wall of the sinus venosus shows vesicular protrusions. The sinus venosus is still a part of the pericardial wall, x 125 1, common atrium, 2, anlage of the liver
Fig. 3. Embryo stage 17. Same view as in Fig. 1. Some of the villi adhere to the cardiac surface, x 100 Fig. 4. Embryo stage 17. Higher magnification showing a pericardial villus adherent to the cardiac surface. The tip of this villus is branching at the cardiac surface, x 1750
Fig. 2. Embryo stage 16. Same view as in Fig. 1. The vesicular protrusions of the coelomic epithelium have developed into an accumulation of pericardial villi, x 125
Although Kurkiewicz (1909) was the first to recognize the possible relation between the formation of pericardial villi and the f o r m a t i o n of the epicardium, he was not the first to describe these villi (" villi of Kurkiewicz" Kuhn and Liebherr 1988). The first and best descriptions of the villi in chick embryos had already been made by R e m a k (1843, 1855), but he failed to show their developmental significances. Later, pericardial villi were described in m a m m a l i a n embryos by Lieberkfihn (1876), K611iker (1879), His (1881), U s k o w (1883a, b), and Born (1889). Because the villi are found to be in close proximity to the developing
liver, a possible contribution to the formation of hepatic blood vessels was discussed. A good review on this discussion was given by U s k o w (1883 b). Current discussions about the role of the pericardial villi have focused on two items, the development of the epicardium (Kurkiewicz 1909; Shimada and H o 1980; Shimada et al. 1981 ; Viragh and Challice 1981 ; Komiyam a et al. 1987; K u h n and Liebherr 1988) and the develo p m e n t of the cardiac blood vessels (Grant 1926; Voboril and Schiebler 1969; Steinhoff 1971 ; Los and Verwoerd 1972; Langford et al. 1990; Viragh et al. 1990). With regard to the possible relationship between the
382
Fig. 5a, b. Embryos stages 17 (a) and 18 (b). Same views as in Fig. 1. Due to the attachment of the villi to the heart a secondary dorsal mesocardium is formed. From the point of its attachment, an epithelium different from the original epithelium of the heart
but similar to the epithelium of the villi seems to spread over the cardiac surface. Arrows mark the borderline between the two epithelia, a • 150; b • 125
Fig. 6a, b. Embryos stages 16 (a) and 20 (b). a Surface ofa pericardial villus, b Surface of a developing heart showing original (1), and ~ spreading" (2) epithelium. The surface morphology of the
"spreading" epithelium resembles the surface morphology of the villous epithelium, x 1500
formation of pericardial villi and the formation of the epicardium, three findings are of major interest: 1. In all species investigated, pericardial villi are found in the region of the cardiac inflow tract, where they face the dorsal wall of the developing heart (Remak 1843, 1855; Lieberkiihn 1876; K611iker 1879; His 1881; Uskow 1883 a, b; Born 1889; Kurkiewicz 1909; Shimada and Ho 1980; Shimada et al. 1981; Viragh and Challice 1981; Komiyama et al. 1987; Kuhn and Liebherr 1988; Hiruma and Hirakow 1989). 2. The formation of the epicardium commonly begins at the dorsal wall of the embryonic heart (Greil 1903; Kurkiewicz 1909; Steinhoff 1971; Viragh and Challice 1973, 1981 ; Shimada and Ho 1980; Shimada et al. 1981 ; Kuhn and Liebherr 1988; Hiruma and Hirakow 1989).
3. The period during which most pericardial villi are found coincides with the period in which the formation of the epicardium takes place (Kurkiewicz 1909; Komiyama et al. 1987; Kuhn and Liebherr 1988). According to Kurkiewicz (1909), these findings reflect the fact that the epicardium is derived from the epithelium of the pericardial villi. However, how epithelium from the pericardial wall was transferred to the cardiac surface remained obscure. Some authors stated only that cells from this epithelium migrated to the heart (Viragh and Challice 1973); other authors were more definite and reported that cells from pericardial villi migrated to the cardiac surface after villi had become attached to the dorsal wall of the heart (Kurkiewicz 1909; Shimada and Ho 1980; Viragh and Challice 1981).
383
Fig. 7. Embryo stage 27. Same view as in Fig. 1. In order to expose the dorsal wall of the heart, the apex of the heart was shifted craniad by forceps prior to fixation. The secondary dorsal mesocardium is found as a free cord bridging the pericardial cavity between the ventral wall of the sinus venosus and the dorsal base of the ventricles. The incorporation of the sinus venosus into the heart is nearly completed. Due to the development of the subepicardial connective tissue, the surface of the heart, especially its ventricular surface, shows fissures and folds, l, left horn of the sinus venosus; 2, left atrium; 3, right atrium; 4, ventricles, x 60 Fig. 8. Embryo stage 31. Same view as in Fig. 7. The mesocardium has become a part of the cardiac wall; its remnant is found as a sagittal fold. Accessory villi are found as appendages of the atrial wall. x 50 Fig. 9. Embryo stage 34. Same view as in Fig. 7. The sagittal fold, which is the remnant of the secondary dorsal mesocardium, has flattened. The incorporation of the sinus venosus is completed. x 60
384 This would suggest that pericardial villi form continuous connections between the pericardial wall and the heart, bridging the pericardial cavity. However, all these authors failed to show such connections. Consequently, Kuhn and Liebherr (1988) stated that they could not imagine that villi were able to establish continuous connections to the rhythmically moving surface of the ventricular part of the heart. They supposed that epithelium from the pericardial wall was transferred across the pericardiat cavity to the cardiac surface in the form o f free vesicles, which had been detached from the pericardial villi (Komiyama et al. 1987; Kuhn and Liebherr 1988). However, secondary connections between the pericardial wall and the ventricular part of the heart are not unknown in vertebrates, although it seems that previous descriptions have not found their way into textbooks of embryology. The first description of such a connection was made by Greil (1903). In Lacerta viridis, the apex of the mature heart is connected with the pericardial wall by a cord. According to Greil (1903) this "apical c o r d " is formed by fusion between epicardial villi at the cardiac apex, and villi at the opposing side o f the pericardial wall. Hochstetter (1906) and Patten (1922) described a connection between the ventral wall of the sinus venosus and the dorsal wall of the heart in chick embryos. Although they stated that it must have been formed secondarily, they could not show how this happened. Regarding the possible role of this " f u s i o n " they supposed that it served as a pathway along which one of the main coronary veins reached the sinus venosus during development. The present findings show that this connection, which has frequently been misinterpreted as being a remnant of the primary dorsal mesocardium (Hamilton 1952, p. 263, Fig. 125; Steinhoff 1971 ; Los and Verwoerd 1972; Langford et al. 1990), is a secondary dorsal mesocardium. It is formed by the attachment of the pericardial villi to the dorsal surface of the heart (Figs. 2, 3, 5a, b), and bridges the pericardial cavity between the ventral wall of the sinus venosus and dorsal wall of the ventricular part of the heart (sinu-ventricular mesocardium) (Fig. 7). Due to its formation, epithelium from the pericardial wall reaches the dorsal surface of the heart. We then have to consider whether this epithelium is the source of the epicardium, and how the formation of the epicardium takes place. Most authors state that cells from the pericardial villi migrate over the cardiac surface forming the epicardium (Kurkiewicz 1909; Shimada and Ho 1980; Shimada et al. 1981; Viragh and Challice 1981). The observations presented here seem to support this view. After the sinuventricular mesocardium has formed, local differences in the surface morphology of the cardiac epithelium are found. An epithelium, similar to t h e epithelium of the pericardial villi, but different from the original epithelium of the heart, seems to spread over the cardiac surface. " S p r e a d i n g " of this epithelium proceeds from the point of attachment of the sinu-ventricular mesocardium (Figs. 5 a, b, 6 a, b). These findings correspond to previous SEM observations which have been
interpreted as " s p r e a d i n g " epicardium (Shimada and Ho 1980; Shimada et al. 1981). Although these observations suggest that the epithelium o f the pericardial villi is spreading, one cannot exclude other hypotheses of epicardial origin. The impression of a spreading epicardium could also be created if epicardial cells were to delaminate from local myoepicardium in a temporally and spatially organized pattern. In this case, the border between the epicardium and myoepicardium would appear as if it were shifting over the cardiac surface without real substantial displacement of the cells. Because neither the existence of undifferentiated precursor cells, nor dedifferentiating myoblasts, can be fully excluded by descriptive studies alone, such a process, which is best described as resembling a travelling wave, cannot be excluded morphologically. Therefore, experimental investigations are needed to clarify the origin of the epicardium.
Acknowledgements. I should like to thank Mr. Hannes Sydow for technical assistance, Ms. Kirsten Falk for photographical assistance, and Ms. Cyrilla Maelicke for correcting the English manuscript. References Asami I (1979) Development of the outflow tract of the rat embryonic heart. 5th European Anatomical Congress, Prag, Argumenta Communicationum : 14 Born G (I 889) Beitr/ige zur Entwicklungsgeschichte des S/iugetierherzens. Arch Mikrosk Anat 33:284~378 Davis CL (1924) The cardiac jelly of the chick embryo. Anat Rec 27 : 201-202 De Haan RL (1965) Morphogenesis of the vertebrate heart. In: De Haan RL, Ursprung H (eds) Organogenesis. Holt, Reinhardt and Winston, New York, pp 377419 Grant RT (1926) Development of the cardiac coronary vessels in the rabbits. Heart 13:261-271 Greil A (1903) Beitr/ige zur vergleichenden Anatomic und Entwicklungsgeschichte des Herzens und des Truncus arteriosus der Wirbelthiere. Gegenbaurs Morphol JB 31:123-310 Hamburger V, Hamilton HL (1951) A series of normal stages in the development of the chick embryo. J MorphoI 88:49-92 Hamilton HL (1952) Lillie's Development of the chick. 3rd edn. Holt, New York Hiruma T, Hirakow R (1989) Epicardial formation in embryonic chick heart: computer-aided reconstruction, scanning and transmission electron microscopic studies. Am J Anat 184:129 138 His W (1881) Mittheilungen zur Embryologie der S/iugethiere und des Menschen. Arch Anat Entwickl Gesch Jg 1881:303-329 His W (1885) Anatomic menschlicher Embryonen. Teil III Zur Geschichte der Organe. Vogel, Leipzig Hochstetter F (1906) Beitr/ige zur Anatomic und Entwicklungsgeschichte des Blutgeffil3systemsder Krokodile. In: Voeltzkow A (ed) Reise in Ostafrika. Wissenschaftliche Ergebnisse. Anatomic und Entwicklungsgeschichte. Vol 4, pp 1-133 K611iker A (1879) Entwicklungsgeschichte des Menschen und der Thiere, 2. Aufl. Engelmann, Leipzig Komiyama M, Ito K, Shimada Y (1987) Origin and development of the epicardium in the mouse embryo. Anat Embryol 176:183-189 Kuhn HJ, Liebherr G (1988) The early development of the epicardimn in Tupaja beIangerie. Anat Embryol 177:225 234 Kurkiewicz T (1909) O histogenezie miesnia sercowego zwierzat kregowych - Zur Histogenese des Herzmuskels der Wirbeltiere. Bull Internat Acad Sci Cracovie: 148 191
385 Langford JK, Hay DA, Bolender DL (1990) Fine structural features of coronary vasculogenesis in collagen lattices. In: Bockman DA, Kirby ML (eds) Embryonic origins of defective heart development. Ann NY Acad Sci 588:404408 Lieberkfihn N (1876) Ober die AIlantois und die Nieren von S/iugethierembryonen. Sitzungsberichte d Ges z Bef6rderung der gesamten Naturwiss z Marburg 1 : 1 11 Los JA, Verwoerd CDA (1972) The development of a primary venous system from epicardial villi in the cardiac wall of the chicken and the mouse embryo, and the relationship between this venous system and the arterial vascularisation in the mouse. Acta Morphol Neerl Scand 8 : 233 Manasek FJ (1968) Embryonic development of the heart I. A light and electron microscopic study of myocardial development in the early chick embryo. J Morphol 125:329 366 Manasek FJ (1969) Embryonic development of the heart II. Formation of the epicardium. J Embryol Exp Morphol 22:333 348 Patten BM (1922) The formation of the cardiac loop in the chick. Am J Anat 30:373-397 Remak R (1843) Ober die Entwicklung des Hfihnchens im El. Arch Anat Physiol Wiss Med Jg 1843:478484 Remak R (1855) Untersuchungen fiber die Entwicklung der Wirbelthiere. Berlin Shimada Y, Ho E (1980) Scanning electron microscopy of the embryonic chick heart: formation of the epicardium and surface
structure of the four heterotypic cells that contribute to the embryonic heart. In: van Praagh R, Takao A (eds) Etiology and morphogenesis of congenital heart disease. Futura, New York, pp 63-80 Shimada Y, Ho E, Toyota N (1981) Epicardial covering over myocardial wall in the chicken embryo as seen with the scanning electron microscope. Scanning Electron Microsc 11:275-280 Steinhoff W (1971) Zur Entwicklung der terminalen Strombahn im Hfihnerherzen. Anat Entwickl Gesch 134: 255-277 Uskow N (1883 a) Ober die Entwicklung des Zwerchfells, des Pericardiums und des Coeloms. Arch Mikrosk Anat 22:143 219 Uskow N (1883b) Bemerkungen zur Entwicklungsgeschichte der Leber und der Lungen. Arch Mikrosk Anat 22:219 227 Viragh S, Challice CE (1973) Origin and differentiation of cardiac muscle cells in the mouse. J Ultrastruct Res 42 : 1-24 Viragh S, Challice CE (1981) The origin of the epicardium and the embryonic myocardial circulation in the mouse. Anat Rec 201:157-168 Viragh S, Kalman F, Gittenberger de Groot AC, Poelmann RE, Moorman AFM (1990) Angiogenesis and hematopoesis in the epicardium of the vertebrate embryo heart. In: Bockman DE, Kirby ML (eds) Embryonic origins of defective heart development. Ann NY Acad Sci 588:455458 Voboril Z, Schiebler TH (1969) Ober die Entwicklung der Geffil3versorgung des Rattenherzens. Anat Entwickl Gesch 129 : 2440
