The Development of the Arterial Outflow Tract in the Chick Embryo Heart OSCAR CHARLES JAFFEE 1 Department of Biology,z State University of New YoTk at Buffalo ABSTRACT Bloodstream flow patterns have been outlined in the arterial outflow tract (ventricular outflow tract and bulbus arteriosus) of the chick embryo heart during the period in which septation takes place. Hemodynamic factors underlying flow changes during this period are discussed. The mapping of flow patterns did not support the concept of a conoventricular flange reported previously. Septation was found to take place between two separate and discrete bloodstreams. The cellular nature of the aorticopulmonary septum has been described. The spiral ridges that farm this septum expand by cellular growth, explaining the ability of this septum to develop against the direction of blood flow. The aorticopulmonary septum divides about two-thirds of the arterial outflow tract; the h a l partitioning of the most proximal portion of the outflow tract was found to take place by means of the apposition of endocardia1 cushion tissue masses. Failure of aorticopulmonary septum development (truncus arteriosus communis persistens) was found to follow fusion of the bloodstreams in experimental studies. In experimental aortic stenosis the appearance of a small left stream was found to be followed by the development of a stenotic aorta. Thus in the first instance the septum apparently cannot develop unless the streams remain separate and in the second case the size of the primordial bloodstreams appears to determine the diameter of the vessel. The development of the arterial outflow tract is described in the present report. Although a number of hemodynamic interpretations of outflow tract development have been presented (cf. Spitzer, '23; Bremer, '32; Goerttler, '58; de Vries and Saunders, '62) none of these are based upon descriptions of blood flow in Living hearts during the period in which septation takes place. Descriptions of blood flow patterns for this period are presented; these cover the third to seventh days of incubation in the chick embryo (Jaffee, '65a). The development of blood flow patterns and the rheological basis of this development have been described (Jaff ee, '65a; '66a). The developmental morphology of the arterial outflow tract has been the subject of a number of studies (Tonge, 1869; Odgers, '38; Kramer, '42; de Vries and Saunders, '62) but some disputes remain. The cono-ventricular flange (Kramer, '42) is disputed by de Vries and Saunders ('62); since both studies are based primarily upon histological material the outlining of flow patterns in this region should resolve ANAT. REC., 158: 35-42. this dispute since the pathways of blood flow is the issue at hand. The cellular structure of the aorticopulmonary septum has not been described in detail. This is considered of special interest since it has long been known (cf. Tonge, 1869) that this septum arises from a spur between the fourth and sixth aortic arches and develops toward the heart, against the direction of blood flow, while other cardiac septae develop in the direction of blood flow (Patten, '60; Jaffee, '63a). Analyses of blood flow patterns in experimentally produced cardiac malformations (Rychter, '62; Jaffee, '65a) have provided another approach to the study of the dynamics of cardiac development. Analyses of experimental aortic stenosis (Jaffee, '64; '66b) and common truncus arteriosus (Jaffee, '65b; 66b) have been presented in preliminary form and will be discussed with relation to the findings of the present study. IAided by a grant from the National Foundation. 2 Present address: Department of Biology, University of Dayton, Dayton, Ohio 45409. 35 36 OSCAR CHARLES JAFFEE MATERIALS AND METHODS White Leghorn eggs were incubated in forced draft incubators at 38.5"C. Several hundred embryos were observed both in this study and as controls in a study of experimental cardiac defects in progress. Living embryos were examined and photographed at intervals (stated in the text) from 3 to 7 days incubation. The methods for observing blood flow and cinephotomicrography have been described (Jaffee, '63a; '65a). Histological preparations of embryos were made utilizing the paraffin method and hematoxylin and eosin staining. Methyl green pyronin staining (Brachet, ' 5 3 ) was also utilized with ribonuclease treated sections as a control (also cf. Jaffee, '63b). RESULTS Two well defined bloodstreams are found in the heart on the third day. Each is composed of a core of blood cells surrounded by plasma; thus each column of blood cells is separated from the other and from the heart wall by plasma (cf. Jaffee, '66a). The rheological basis for this structure has been discussed (Jaffee, '66a). The configuration of the bloodstreams in the arterial outflow tract on the third day is illustrated in figure 1. The left stream emerges from the left side of the undivided ventricle, flows dorsally and to the right into the right side of the bulbus arteriosus turning very slightly posteriorly and exiting from the bulbus, contributing to the second and third aortic arches (fig. 1 ) . The right stream flows into the bulbus ventrad to the left stream, turning cephd a d at the same time and into the left side of the bulbus; the right stream can be traced through an arc which goes dorsal and posteriorly and exits from the bulbus in a posterior direction, contributing to the third and fourth aortic arches (fig. 1). With the continued development of the cardiac loop the bulbus becomes pointed more in a posterior direction. This is especially marked from the third to the fourth day (cf. Patten, '22; '51). During the course of the third day a lessening amount of blood is seen flowing into the second arch as compared to the fourth, and the second arch disappears by the end of the third day (cf. Romanoff, '60). The posterior rerouting of the bulbar outflow is probably a factor in the vascularization of the sixth arch. A small volume of blood was seen flowing into the sixth arch early on the fourth day. During the course of the fourth day the volume of blood flowing into the sixth arch becomes increased so that by the end of the fourth day the sixth arch is well vascularized. An increase in the volume of blood directed into the right stream on the fourth day (Jaffee, '65a) also appears to contribute to the development of the sixth arch. The fifth aortic arch is small and transitory and has been considered a branch of the sixth; this arch has no bearing on the development of the cardioaortic complex according to Romanoff ('60) and Rychter ('62). The stage of development of the heart in the fourth day embryo (fig. 3, also cf. Patten, '51) may be compared to that of a human embryo illustrated by Kramer ('42, fig. 6); this is the stage in which the problem of the cono-ventricular flange arises. According to Kramer ('42, p. 259) : "The location of this flange makes it necessary for blood from the left ventricle to negotiate a sharp reverse turn through the interventricular foramen into the right ventricle before i t can leave the heart by way of the truncus." De Vries and Saunders ('62), on the other hand, stated that their reconstructions showed no Obstruction to the egress of blood from the left ventricle into the bulbus; this is confirmed in observations of blood flow in the arterial outflow tract of the four-day chick embryo heart (fig. 3). The present study has further established that at no time during normal development does an obstruction from the left ventricle into the bulbus exist and thus septation in the arterial outflow tract takes place between two bloodstreams. Development of the aorticopulmonary septum was noted on the fourth day (also cf. Tonge, 1869). At this time the bloodstreams flowing into the aortic arches flow into the fourth (left stream) and sixth (right stream) arches. Flow of either stream into more than one arch (cf. fig. 1 ) was not seen at this time. The third arch ARTERIAL OUTFLOW TRACT DEVELOPMENT 37 Fig. 1 Arterial outflow tract of a three day embryo illustrating the bloodstream flow pattern. Abbreviations: LS, left stream; RS, right stream. The aortic arches are numbered. Abbreviations d, dorsal cellular spiral ridge; ec, endocardial cushion tissue; 1, left bloodstream; la, left atrium; r, right bloodstream; ra, right atrium; s, aorticopulmonary septum; v, ventral cellular spiral ridge. 38 OSCAR CHARLES JAFFEE Fig. 2 Flow patterns in the arterial outflow tract of a five day embryo. Abbreviations: LA, left atrium; LS, left stream; RA, right atrium; RS, right stream. ARTERIAL OUTFLOW TRACT DEVELOPMENT Fig. 3 Print from 1 6 m m motion picture film illustrating the complete separation of the left ventricular outflow (LVO) tract and the right ventricular outflow (RVO) tract in a four day embryo heart. Fig. 4 Flow pattern in the arterial outflow tract of a seven day embryo heart. The pulmonary artery (PA) is ventral to the aorta (AO). has become a branch of the fourth (cf. fig. 2 ) probably because of the posterior routing of the entire bulbar outflow noted above. The aorticopulmonary septum forms as a fusion of two groups of cells. This is first found at the point of exit of the bulbus into the aortic arches. In the specimen from which figure 5 was taken the aorticopulmonary septum extends through five 39 cross sections, cut at 10 LI,but it is difficult to determine exactly where this septum and the arterial walls of the fourth and sixth arches end since these structures are continuous. The groups of cells forming the aorticopulmony septum form cellular ridges extending into the lumen of the bulbus as seen in figure 5 which is taken 100 c1 proximal to the formed septum. One of these ridges is dorsal and to the right and the other ventral and to the left at this level (fig. 5). These cells are continuous with the endocardium of the bulbus (fig. 5) and also continuous with the cells forming the walls of the aortic arches. A rapid increase in the rate of growth of the cells comprising the aortic arches has been reported in the four day chick embryo by Hughes ('43) who stated that these cells were derived from the surrounding mesenchyme. The increase of artery wall forming cells was found to coincide with the development of the aorticopulmonary septum in the present study and the suggestion is made that these events are related. The histological appearance of the cells forming both structures was found to be similar. With regard to the cells forming the septum, however, the possibility that these are derived from the endocardium of the bulbus seems very probable; this may take place in the manner that the cells invading the cardiac jelly to transform this substance into endocardial cushion tissue was observed by Patten, Kramer and Barry ('48). A marked uptake of hematoxylin by the septum forming cells suggested a basophilia to the present author. This was confirmed with toluidine blue and pyronin staining. Since the greater part of the pyronin stain was found extractable with ribonuclease, the appearance of ribonucleic acid in the cytoplasm of these cells was indicated. High ribonucleic acid levels has been found associated with cellular differentiation in other embryonic tissues (Jaffee, '63b). High ribonucleic acid levels were demonstrated in the walls of the forming aortic arches in the same manner. The forming septum (fig. 5) extends over the distal third of the bulbus at four days. When sections comprising the middle third of the bulbus were examined at this time columns of cells continuous with 40 OSCAR CHARLES JAFFEE the forming septum (fig. 5) were found in the midst of the endocardial cushion tissue (fig. 6). These are no longer in contact with the endocardium but appear to invade the endocardial cushion tissue (fig. 6). In most cases such columns of cells extend throughout both the dorsal and ventral aspects of the bulbus, but in some cases, such as illustrated in figure 6, one of these columns may extend somewhat further proximally. The media of the most proximal portion of the bulbus was found to be comprised entirely of endocardial cushion tissue in the four day embryo; the development of this tissue has been described by Patten, Kramer and Barry ('48). In five day embryos the formed septum, which appeared circular in cross section (fig. 7) was found at the level of the forming valves (fig. 7). At this time the region distal to the forming valves will be designated as the truncus and that proximal to the valves as the conus, as suggested by Kramer ('42). In the conus the aorticopulmonary septum again forms in the same manner as in the four day embryo, i.e. beginning with two columns of cells which invade the endocardial cushion tissue (fig. 8). The pyronin staining of these cells is illustrated in figure 8. Final closure of the arterial outflow tract does not involve the aorticopulmonary septum, as pointed out by Odgers ('38). Confirmation of Odgers work is made on a histological basis. The cellular aorticopulmonary septum was not found to extend throughout the arterial outflow tract and the closing off of the interventricular foramen was found to take place by means of the apposition of endocardial cushions. No evidence was noted that these cushions expanded through cellular division. Marked changes in blood flow patterns were noted between the third and seventh days, the latter time corresponding to the completion of outflow tract septation (Rychter, '62; Jaffee, '65a). Between three and four days an increase in the degree of spiralling was seen but the basic patterns remained unchanged. Beginning with the fifth day a lessening degree in the amount of spiralling, sometimes referred to as unspiralling, was found (cf. figs. 2 and 1). By the seventh day (fig. 4 ) the definitive prehatching flow patterns were found es- tablished. Changes in flow patterns were noted in comparing the relative positions of the semilunar valves; at five days these are lateral to each other (fig. 7) while at seven days the pulmonary valve is anterior to the aortic valve in the definitive positions of the valves (also cf. Tonge, 1869; Kramer, '42). The changes in flow patterns appear to be greatly influenced by circulatory changes in the atrial region including the establishment of interatrial flow (Jaffee, '65a). One of the most important effects of the cardiac inflow changes was considered to be the equalization of the sizes of the bloodstreams so that the arterial outflow tract divides between two bloodstreams of relatively equal diameter (fig. 4). From three to five days the right stream appears to be larger (Jaffee, '65a, also figs. 7, 8). Rising ventricular pressures and a greater degree of competency of the semilunar valves (Paff et al., '65), along with the equalization of the bloodstreams, are considered dynamic factors in the unspiralling process. DISCUSSION The mechanism whereby the aorticopulmonary septum develops against the direction of blood flow does not appear to have been explained up to the present. The finding that this septum develops as an active cellular growth has provided an answer to this question. The finding that the aorticopulmonary septum develops at a time of rapid aortic arch development indicates that these events are related and suggests that this relationship is worthy of further inquiry . Review of studies of arterial outflow tract development in the human in the light of the present study indicate that the mechanisms involved are similar. De Vries and Saunders ('62) have described the reticular layer in the truncus as being more cellular than the corresponding region of the infundibulum while the aorticopulmonary septum is developing. The developing aorticopulmonary septum, as described in figure 7 of this study, appears to be present in figure 4B of Kramer's ('42) study. Failure of aorticopulmonary septa1 development (truncus arteriosus communis persistens, Lev and Saphir, '42) has been ARTERIAL OUTFLOW TRACT DEVELOPMENT 41 Fig. 5 Cross section proximal to the formed septum i n the bulbus of a four day embryo (cf. text). The cellular masses comprising the spiral ridges that form the aorticopulmonary septum may be noted. Hematoxylin and eosin staining. Fig. 6 Section from specimen shown i n figure 5 taken two-thirds the distance from the distal end of the bulbus. The mass of cells in the endocardial cushion tissue is the first sign of the development of the aorticopulmonary septum. Hematoxylin and eosin staining. Fig. 7 Forming semilunar valves of a five day embryo heart. The formed aorticopulmonary septum appears circular in cross section and is found between the forming valves. Hematoxylin and eosin staining. Fig. 8 Conus of a five day embryo illustrating a high level of ribonucleic acid in the forming aorticopulmonary septum. Methyl green pyronin stain. The association of ventricular septa1 deproduced experimentally (Le Douarin, '60; Jaffee, '65b) and later shown to follow a fects with truncus arteriosus communis fusion of the bloodstreams in the arterial persistens (Lev and Saphir, '42) also beoutflow tract (Jaffee, '66b), providing evi- comes clarified with the present study dence that the aorticopulmonary septum since the aorticopulmonary septum has cannot develop unless a pathway between been found to extend well into the region two discrete bloodstreams is present. Such of the forming valves. Septation of the arterial outflow tract a fusion of the streams might have an anatomical (Le Douarin, '60) or physio- has been shown to take place between two bloodstreams of equal size. The finding logical (Jaffee, '66a) basis. 42 OSCAR CHARLES JAFFEE that a smaller left stream is followed by 1965b The effects of cytosine arabinoside on cardiac development. Fifth Annual the development of a stenotic aorta (Jaffee, Meeting or the Teratology Society, San Fran'64; '66b) has indicated that the positioncisco. ing of the septae dividing the outflow tract - 1966a Rheological aspects of the cieis determined by the diameters of the velopment of blood flow patterns in the chick embryo heart. Biorheology, 3: 59-62. bloodstreams. ACKNOWLEDGMENT The author wishes to thank David Bellucci for the drawings and Milda Spindler for the preparation of the plates. LITERATURE CITED Brachet, J. 1953 The use of basic dyes and ribonuclease for the cytochemical detection of RNA. Q. J. Mic. Sci., 94: 1-10. 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