Experimental cardiac morphogenesis: I. Development of the ventricular septum in the chick

The ventricular septum in the chick embryo is first noted approximately at four days of incubation as a loose meshwork of trabeculae. Between day 4 and 8 of incubation, this meshwork of trabeculae solidifies into the muscle mass that is the muscular ventricular septum in the mature heart. A portion of the heart loop is thus separated into areas that will later become the definitive right and left ventricles.

A number of studies has been reported to resolve the origin of the ventricular septum in both human and experimental animals. These studies represent descriptive embryology based upon information from histologic sections of embryonic hearts. They have given rise to a number of different views on how the septum arises (Flack, 1909; Tandler, 1912; Murray, 1919; Waterston, 1919; Takahashi, 1923; Frazer, 1932; Kramer, 1942; Streeter, 1948; Grant, 1962; De Vries & Saunders, 1962; Van Mierop, Alley, Kausel & Stranahan, 1963; Patten, 1964; Van Mierop & Netter, 1969; Goor, Edwards & Lillehei, 1970). Much of this controversy is based upon limitations in the techniques utilized.

We have been able to label specific cells of the heart by inserting microfibers coated with tritiated thymidine in the chick embryo heart during the period in which ventricular septation is taking place. Labeling specific locations of the embryo with a durable, non-diffusible marker circumvents many of the criticisms raised against the earlier investigations.

White Leghorn eggs were used throughout this study. Fertile eggs were incubated at 38 ± 0·5 °C for appropriate periods and all embryos were staged according to the criteria of Hamburger & Hamilton (1951). After candling each egg, a window was made in the shell and the vitelline and amniotic membranes were torn with forceps, exposing the pericardium of the embryo heart. The operation on the embryonic heart was made in the manner described below. The shell opening was then sealed with parafilm and melted paraffin and the egg was returned to the incubator.

Sterile nylon fibers (black braided suture, Dektanel) approximately 12 μm in diameter were implanted into the embryonic myocardium perpendicular to the surface of the ventricle, using ultrafine forceps under a dissecting microscope (× 4–25). The distal end of each fiber was barely intruded into the ventricular cavity; the proximal end, which protruded from the surface, was trimmed even with the surface so that the final length of each fiber was approximately 200 μm. Particular care was taken to minimize intrusion of the fibers into the ventricular cavity since it is possible for such intrusion to distort the embryonic bloodstreams (Harh et al. 1973).

A pair of fibers was placed in each embryo in one of the following three ways: group I, one fiber in each primitive ventricle to each side of the future site of the ventricular septum; group 11, both fibers in the primitive right ventricle; group III, both fibers in the primitive left ventricle. The distance between the two fibers in each embryo was measured with a micrometer under direct visualization, and this measurement was repeated daily. To reduce error in measuring the distance between fibers, the embryos were lifted up with an instrument to stop the pulsation of the heart for a second or so. Embryos that survived were autopsied at 7 days of embryonic age and those that died less than 48 h post-operatively or those in which one or both fibers had been extruded, were excluded from the final analysis. Following gross external examination of all the embryos, they were fixed in Bodian’s solution (80% ethanol, 90 parts; glacial acetic acid, 5 parts; formaldehyde, 5 parts), and final examination was made under a dissecting microscope.

Sterile nylon fibers approximately 12 μm in thickness were dipped in concentrated thymidine-methyl H³, 20 Ci/ mmol/c.c. (New England Nuclear); dehydrated using an air dryer, then left at room temperature overnight. The fibers were implanted into the embryonic myocardium in the manner described above for unlabeled fibers and the embryos were reincubated for 6 h (36 embryos), 24 h (27 embryos), 48 h (37 embryos), 72 h (49 embryos), and 84 h (57 embryos) so that the resulting total incubation period was ⁠, 5, 6, 7, and days respectively. The sacrificed embryos were fixed in Bodian’s solution and prepared for serial section at 10 μm thickness. Deparaffinized slides were dipped in Kodak type 3 NTB liquid emulsion. After 30–45 days’ exposure, the sections were then developed in Kodak D-19 developer, fixed in Kodak fixer, stained in 0·1% nuclear fast red and 1% picric acid and examined for labeled silver granules under the microscope (Belanger & Leblond, 1946; Gross, Bogoroch, Nadler & Leblond, 1951; Messier & Leblond, 1957; Sissman, 1966; Rosenquist & DeHaan, 1966; Feitelberg & Gross, 1970).

Although it is possible for the fibers to cause mechanical distortion of the embryonic bloodstreams during the early developmental stages of the ventricular system, the fibers do not appear to disturb normal development of the heart in our experiments. Both the hearts in which the fibers were extruded from the ventricles immediately following operation and the hearts in which the fibers remained for additional periods of incubation developed normal and normally related great vessels. This is not surprising since the fiber is only 12 μm in diameter, which is less than the diameter of a single trabecula of the ventricle of the 4-day-old embryo.

In group I embryos (42), when the fibers were placed no further than 200–250 μmi to each side of the center of the future site of the ventricular septum (Table 1), the distance between the two fibers was shorter on each subsequent examination until at 7 days both fibers were found within the ventricular septum. In those hearts in which the fibers were not visible on the surface at 7 days of incubation, they were found within the septal myocardium when it was dissected under the dissecting microscope (Figs. 1 and 2). The closer the fibers were placed to the site of the future ventricular septum, the earlier they were embedded in the septum (Table 2). As a result, they were found at the septum most centrally. All the fibers found within the septum pointed toward the site of the interventricular foramen. However, when the fibers were placed further than 250 μm to each side of the site of the future ventricular septum, the distance between them increased by the following day. Subsequently the distance between them decreased, but these fibers remained in the free wall of the ventricular myocardium and never became incorporated into the septum (Table 3). In some embryos in which the fibers were placed within 250 μmi of the center of the future ventricular septum, the distance between the two fibers remained unchanged or increased after 24 h of incubation, and subsequently decreased. Interestingly in these hearts, the ventricular septum was either not formed at all at 5 days of incubation, or was barely noticeable externally, but then resumed normal morphogenesis. In group 11 embryos (28), in which two fibers were placed in the primitive right ventricle, the distance between them always increased on each subsequent examination (Fig. 3, Table 4). Jhis also occurred in group III embryos (26), in which two fibers were placed in the primitive left ventricle, as illustrated in Table 5.

Six h after fiber placement (⁠-day-old embryos), a major portion of the myocytes of the trabeculae in contact with and adjacent to the fibers were labeled with silver granules (Fig. 4). The myo-cardium of the free wall of the ventricle was more heavily labeled than the bodies of the trabeculae, as was the epicardium and pericardium around the site of the fiber implantation. This was perhaps due to tritiated thymidine scraping off and diffusing into the neighboring area as the fiber entered.

When the fibers were placed anywhere within 200–250 μm. to each side of the future ventricular septum, the distance between the labeled cells associated with the two fibers became shorter 24 h after fiber placement (5-day-old embryos, Fig. 5). The trabeculae within 100–125 μm of the septum had made contact with each other. Those to either side of this 100–125 μm wide zone had made loose contact with septum at their ends projecting into the ventricular cavity, while their bases were still separate from each other. The bases of the trabeculae were more heavily labeled at this stage than the projecting portions of the trabeculae. 48 h after fiber placement (6-day-old embryo), the labeled trabeculae were aggregated densely near the region that would become the crest of the smooth septum and upper portion of the trabeculated septum (Fig. 6). The labeled, cells of the trabeculae originally within 100–125 μm to each side of the future septum were amassed in the central portion of the septum. The labeled cells, on the other hand, in the zone from 125 to 250 μm to each side of the future septum were superficially located within the septum, and the bases of the trabeculae were still more heavily labeled. 72–84 h after fiber placement (7-and -day-old embryos), the entire length of all these trabeculae was incorporated into the septum (Fig. 7). The labeled myocytes were aligned from the basal portion of the smooth septum to the most apical portion of the trabeculated septum, although the silver granules were sparse where the muscular septum and myocardium of the free wall met. The labeled portions of the trabeculae and myocardium of the ventricular free wall in -day-old embryos were progressively shifted into the trabeculae with growth of the ventricles in the 5-, 6-, 7-, and -day-old embryos as demonstrated in Fig. 8 and newly formed ventricular myocytes were added to below as the ventricles expanded apically. The trabeculae which were immediately outside the zone 400–500 μm wide just described contributed to the ventricular septum at their projecting ends only, and the bases of these trabeculae still remained in the free wall of the ventricle.

In the present report we have drawn different conclusions about the morphogenesis of the ventricular septum than others have suggested. Tandler (1912), Waterston (1919), Murray (1919), Takahashi (1923), Kramer (1942), and Patten (1964) suggested that the muscular septum is formed by upward polarized growth of a muscle ridge from the caudal floor of the primary cardiac tube. As Grant (1962) and Goor et al. (1970) have stated, the crest of the septum is stationary and the interventricular foramen is not invaginated by the septum. They demonstrated this by measuring the diameter of the interventricular foramen at each stage of fetal growth. There was no change during the first few stages, a period of time during which the ventricle had increased several fold in length. Currently this view has been largely disregarded. On the other hand, others have considered more favorably that the septum forms passively by expansion of the two trabeculated pouches from the primary heart tube on each side of the interventricular foramen, and that as these pouches become larger and deeper their medial walls coalesce into a common wall. The ventricular septum, then, is formed by the folding of the two ventricular myocardial walls (Flack, 1909; Frazer, 1932; Streeter, 1948; Grant, 1962; Van Mierop et al. 1963; Van Mierop & Netter, 1969). Since the septum is formed passively by a folding of the two myocardial walls, the epicardium and the outermost layers of the ventricular myocardium should be aligned most centrally along the longitudinal axes of the ventricular septum. Therefore, one would not expect to see any communication between the two ventricular chambers at the septum. We have failed to confirm this finding. Instead we found multiple interventricular communications from the projecting ends to the basal portions of the trabeculae during the period of septal morphogenesis. These intertrabecular spaces disappeared only at the final stage of trabecular coaptation. More importantly, we found the morphogenesis of the septum resulted from an active downward growth rather than upward polarized growth. Newly formed ventricular myocytes were added to the bases of the trabeculae as the ventricles further expanded apically (Fig. 8). De Vries & Saunders (1962) believed that the ventromedial rotation of the right ventricular limb increases the height of the myocardial crest internally, and this becomes the septum as a result of continued ventral infolding and coaptation of the ventricular walls, as well as fusion of the trabeculae. This view is essentially a slight modification of Streeter’s view, and does not agree with our findings as stated above. Goor et al. (1970) postulated dual origins of the muscular ventricular septum from the appearance of its two morphologic structures: a smooth wall above and a trabeculated septum below. They then hypothesized that the smooth septum originates by active growth of the crest of the smooth wall, which is a process of competition between outward expansion and inward proliferation. It is clearly indicated in the present study that the septum is of a single developmental origin; the smooth wall of the muscular septum is formed by the projecting ends of the trabeculae while the trabeculated septum is formed by the basal portions of the trabeculae. This was indicated by the fact that the labeled myocytes are originally located throughout the length of the trabeculated septum.

The long dimensions of the developing myocytes in the free wall of the ventricular myocardium are parallel to the surface, in a fashion reminiscent of pavement epithelium. The cells in the trabeculae are oriented along the long axes of the trabeculae. The increased cell multiplications in the ventricular myocardial wall result in distribution of part of their cells into the trabeculae. This was shown in this report by the fact that the labeled cells of the common wall where the septum and myocardium of the free wall met were frequently replaced by cells which were unlabeled (Fig. 8). It is of interest to note that these differences in cell multiplication rate between the septum and myocardial wall can be seen in early chick embryo studies published earlier (Grohmann, 1961).

The trabecular aggregations in septal morphogenesis are evidenced by the shortening of the distance between the two fibers and between the labeled cells from the site of initial fiber implantation.

The trabeculation of the muscular septum is a form of incomplete coaptation of the trabeculae located near the margins of the 400–500 μm belt-like zone in the 4-day-old embryo. The trabeculae located immediately outside this zone become the trabeculation of the muscular septum by the fusion of projecting ends of the trabeculae into the cavity and their bases remain in the myocardium of the ventricular free wall.

The authors are indebted to Dr Gerald Odell for his support in this work and to Drs Glenn Rosenquist and Alan Cohen for their laboratory equipment and their interest in our work. The Ms Soame Christianson and Lauren Sweeney are acknowledged for their assistance in the preparation of the manuscript. This study was supported by USPHS Grants HD 00091 and HL 10191, and Park Ridge (Illinois) United Fund, Inc.