Cardiac dextral looping (C-looping) is a morphogenetic process that transforms the straight primitive heart tube into a C-shaped tube that is bent toward the right side of the embryo (Fig. 1A,B). Looping is the first obvious morphologic manifestation of left-right asymmetry in vertebrate embryos. Abnormal looping can lead to major congenital heart defects.
The chick embryo is a popular experimental model to study the mechanisms of C-looping due to its advantages for experimental procedures. Development of the chick heart parallels that of the human heart. This study, therefore, focuses on C-looping in the chick embryo.
Normal C-looping consists of two deformation components: ventral bending and rightward rotation (Manner, 2000). The bending component likely is a process intrinsic to the heart (Butler, 1952; Manning and McLachlan, 1990). The rightward rotation also may have an intrinsic component (Itasaki et al., 1991), but there are at least two possible sources of external forces that can influence the rotational component. First, the heart is restricted ventrally by an extraembryonic membrane, the splanchnopleura, which can push against the heart (Patten, 1922). Second, in the “traditional” culture methods used to study C-looping, the chick embryo is placed ventral side up on the surface of the culture medium (New, 1955; Stern and Bachvarova, 1997; Darnell and Schoenwolf, 2000; Chapman et al., 2001). Such a position of the embryo is useful to facilitate its oxygen supply. However, in this case, the ventral side of the embryo with the developing heart can be subjected to the influence of surface tension, a force that does not exist during normal development within the egg. Therefore, a question arises: can surface tension interfere with the mechanics of C-looping under experimental conditions? The purpose of this study is to investigate this question.
To study the influence of surface tension, we have developed a culture method that allows the embryo to be immersed in a liquid medium so that surface tension is not a factor. By using this method, we have shown that surface tension can interfere with C-looping in some experiments for which traditional cultivation methods are used (Lepori, 1967; Stalsberg and DeHaan, 1969; Castro-Quezada et al., 1972; Manasek et al., 1972; De la Cruz et al., 1977). In some cases, surface tension can affect results dramatically.
To eliminate the effect of surface tension on looping, we modified traditional culture methods to enable culturing of the chick embryo under a thin layer of liquid media. In the following, we first show that the embryo develops normally in our new preparation. Then, we estimate the forces exerted on the embryo by tension in the splanchnopleura and by surface tension when embryos are exposed to air. Next, we illustrate the influence that these forces can have on the morphology of the looped heart tube. Finally, we demonstrate how surface tension affects the results in one particular classic experiment.
Development of the Embryo
In our method, chick embryos were cultivated while submerged under a thin layer (approximately 0.5 mm) of Dulbecco's modified Eagle's medium (DME), with the gaseous phase in the incubator replaced by a mixture of 95% oxygen and 5% carbon dioxide (see Experimental Procedures section). All intact embryos developed normally from Hamburger and Hamilton (HH) stage 9 (Hamburger and Hamilton, 1951) up to completion of C-looping at HH stage 11–12 (Fig. 1B, 93 embryos). Some embryos were cultivated for 2 days from HH stage 8 to HH stage 18, and they developed normally and established an extensive extraembryonic vascular system.
In intact embryos submerged in DME, no abnormal left looping was observed. In contrast, left looping was observed in 5 of 77 intact embryos cultivated from HH stage 9 on the surface of the medium. The difference in the occurrence of left looping between embryos cultivated in DME and on the surface is significant at the level P < 0.05 (Fisher exact test). Therefore, cultivation of intact chicken embryos on the surface can increase the occurrence of spontaneous left-looping.
Replacement of air by pure oxygen in our method is critical. The development of 16 of 16 embryos incubated in DME under a mixture of atmospheric air and 5% carbon dioxide was abnormal. They demonstrated multiple abnormalities: slow development and growth, progressive degradation of somites, inhibition of body rotation and cervical flexure, and abnormal heart formation (Fig. 1E,F). Similar abnormalities occurred in six of six embryos cultured in the oxygen-enriched atmosphere but under a thicker (approximately 4 mm instead of 0.5 mm) layer of DME.
Estimation of External Forces
External forces due to surface tension and extraembryonic membranes were estimated by comparing the deformation of the embryo due to these forces with deformations of the same embryo compressed by thin glass strips of known weights (Fig. 2). Lacktis and Manasek (1978) used a similar approach to study the mechanical properties of the developing chick heart. As a measure of deformation, we measured the change in the maximal diameter of the anterior part of the embryo (“head”) (see Fig. 3A).
The head diameter was measured for the following conditions: (1) embryo under fluid layer with splanchnopleura intact (Fig. 3A), (2) embryo under fluid layer with splanchnopleura removed (Fig. 3B), (3) embryo exposed to air with splanchnopleura removed (Fig. 3C), and (4) dissected head under no load or various applied loads (Fig. 3D–I).
The reliability of the method was estimated by using repeated measurements of three embryos. Each embryo was loaded and unloaded three times with each glass strip. Differences between the measured diameters at a given load were found to be within the range of error of the measurement technique (approximately 2%).
When the head was dissected, its diameter did not change due to, for example, a possible relief of inner pressure (compare Figs. 3B,D). Moreover, the embryo maintained its elasticity during the loading procedure, as the head returned to its original diameter (Fig. 3D) when the loads were removed (Fig. 3I).
The mean results from 10 HH stage 9+ embryos (9 somites, the stage immediately before C-looping) are given in the Figure 4. The circles in Figure 4 correspond to the glass strip compression test, the triangle represents the diameter of the head in embryos with the membrane intact and under a fluid layer (Fig. 3A), and the square represents the diameter of the head in embryos without the membrane on the surface of medium (Fig. 3C). Linear interpolation of the compression test curve yielded the compressive forces exerted by the splanchnopleura alone (triangle) and the surface tension (square). The estimated values of the compressive force developed by the splanchnopleura varied between 0.33 and 0.86 dynes, with a mean value being 0.6 ± 0.2 dynes (± SD). The force exerted by surface tension varied between 3.0 and 7.7 dynes with a mean value 5.3 ± 1.6 dynes. The difference between these forces is significant at the level P < 0.0001 (Mann-Whitney rank sum test, used here for data that failed to pass normality test).
For the same 10 embryos, the mean heart diameter (see Fig. 3A) in extra solution was 0.29 ± 0.03 mm for the embryo without membranes and 0.31 ± 0.03 mm for the embryo with intact membranes. The difference between raw values of these heart diameters is not significant (t-test, P = 0.08). However, after normalization of these values by dividing by the unloaded heart diameter (Fig. 3B), the difference between hearts with and without membranes is significant at the level P < 0.0001 (Mann-Whitney rank sum test). This normalization eliminates the effects of individual heart size. For the embryos without membranes or extra fluid (Fig. 3C), the heart diameter was 0.45 ± 0.05 mm. The difference between the heart diameter with or without extra fluid is significant at the level P < 0.0001. So, the heart diameter is increased by approximately 5% due to the influence of the splanchnopleura and by 50% due to the influence of surface tension.
For eight embryos placed on the semisolid medium, to assess the influence of gravity, we measured the head diameters before and after turning the Petri dishes containing them upside down. For all of these embryos, the head diameter did not change (Fig. 5).
Effects of External Forces on Looping
To examine effects of extraembryonic forces, we removed the splanchnopleura in 24 HH stage 9–10 embryos and cultured them for several hours in DME. C-looping was abnormal in all experiments, as the heart bent ventrally with a slight tilt toward the right side and little rotation (Fig. 6A–D). Carbon particles placed along the ventral midline of the heart helped in visualizing rotation. However, when the fluid layer was removed, all of the hearts immediately rotated toward the right side and normal C-looping then was observed (Fig. 6E). This rotation was reversible, as when the fluid layer was restored, the hearts returned to their shape with ventral bending and only slight rotation (Fig. 6F).
The splanchnopleura also was removed in 8 HH stage 11 embryos that had well developed C-looping. Although the hearts did not return to the sagittal plane immediately, they returned after a short (20–30 min) re-incubation in DME under the oxygen enriched atmosphere (Fig. 6G–I).
Longitudinal Cut of Right Splanchnopleura
In an often-cited experiment, Lepori (1967) found that cutting the splanchnopleura longitudinally can influence the direction of looping in cultured chick embryos. In his experiments, longitudinally cutting the membrane on the left side of the embryo resulted in normal looping to the right, whereas cutting the membrane on the right side produced looping to the left.
We repeated this experiment without or with the extra layer of fluid medium. The splanchnopleura was cut on the right side in HH stage 8–9 embryos, which were then cultured for 6 hr. Under conditions similar to those used by Lepori (1967), i.e., without the extra fluid layer, we obtained similar results, namely, left looping in all 35 experimental embryos. In contrast, when the embryos were cultured after the cut under the medium, 35 of 39 hearts looped normally to the right and only 4 to the left (Fig. 7). According to Fisher's exact test, the differences between these results are significant at the level P < 0.0001.
In all cases, after cutting the membrane to the right of the embryo, the left part of the membrane moves leftward. This movement was visualized by labeling the membrane with carbon particles (Fig. 7).
This study has shown that external forces due to membrane and surface tension can significantly influence cardiac looping morphology in the chick embryo. The force applied by the splanchnopleura may be necessary for normal cardiac development. Our results suggest, however, that surface tension present in most traditional culture preparations can alter looping behavior. Hence, results from experiments in which the surface of the embryo is exposed to air should be interpreted with caution.
In our experiments, all embryos developed normally in DME up to HH stage 18, similar to traditional methods of culture on the surface of the medium. Because C-looping occurs in less than a day, our method can be considered as satisfactory in studies of this process. The method is useful for various experimental interventions such as microsurgery and microinjections.
For successful cultivation in DME, the use of oxygen-enriched atmosphere is important, and although the medium must cover the embryo, the fluid layer must be as thin as possible (3 ml of medium is enough for a 6-cm Petri dish). Incubation in conventional incubators with a mixture of atmospheric air and 5% carbon dioxide or with a thick layer of fluid does not support normal development and produces a variety of embryonic defects, probably due to hypoxia (Fig. 1E,F). The influence of hypoxia has been examined previously in chick embryos older than those used in the present study. Nevertheless, the described abnormalities are similar to those of embryos developed in DME under an air mixture in our experiments, e.g., slow development, impaired growth, and abnormal heart formation (Grabowski and Schroeder, 1968; Altimiras and Phu, 2000). It also has been shown that cervical flexure depends on oxygen supply to the embryo (Manner et al., 1995).
It is known that, for massive explants such as whole embryos or organs, a slow rate of oxygen diffusion is a critical problem (Freshney, 1994). There are at least three ways to solve this problem: (1) culture on the surface of the medium to facilitate gas exchange, (2) stirring of the medium, and (3) increase oxygen concentration up to pure oxygen or use high pressure (Freshney, 1994). Traditional methods of chick embryo cultivation use the first method (Spratt, 1947; New, 1955; Stern and Bachvarova, 1997; Chapman et al., 2001), but in this case, surface tension is an inevitable factor. Cultivation in roller tubes (Connolly et al., 1995) uses the second method, but it can hardly be adopted to study mechanisms, as the embryo is sealed within extraembryonic membranes and, therefore, is invisible and inaccessible for experimental interventions. Our method uses the third possibility, and at the same time, it is convenient for experiments and eliminates surface tension.
According to our measurements, HH stage 9+ chick embryos placed on the surface of the medium are compressed by a mean force of 5.3 dynes. Constancy of head diameter after turning these embryos upside down (Fig. 5) suggests that this compressive force can be attributed mainly to surface tension rather than gravity. For the same embryos submerged in DME, the mean force exerted by the splanchnopleura is 0.6 dynes. The difference between these two forces is almost 1 order of magnitude and is statistically significant. Due to these forces, the diameter of the heart is increased 5% by the splanchnopleura alone and approximately 50% by surface tension (or by surface tension and the splanchnopleura). Actually, in traditional preparations, surface tension essentially transforms the tubular embryonic heart into a considerably flat structure. Therefore, it is not surprising that surface tension can interfere with the results of looping experiments.
To estimate the value of the surface tension on the heart, we use Laplace's law. Considering the embryonic heart as a cylinder, this law can be written as P = T/R, where T is surface tension per unit length, P is the pressure developed by the surface tension, and R is the radius of the cylinder. We do not know the precise value of T for DME, but for a wide range of body fluids, it is at least 5.5 dynes/mm (Hrncir and Rosina, 1997; Hjelde and Brubakk, 2000). Substituting this value for T in the above formula and taking the heart radius as 0.25 mm gives the pressure 22 dynes/mm2. With the projected surface area of the heart being approximately 0.5 mm2 (1 mm long and 0.5 mm wide), the total force due to this pressure is 11 dynes. Previously, Lacktis and Manasek (1978) showed that, under a compressive load of this magnitude, the diameter of HH stage 11–12 chick hearts increased approximately 50%, which is consistent with our observations.
It is known that C-looping involves a combination of two morphogenetic processes, namely, bending of the heart tube in the ventral direction and its simultaneous rotation (torsion) to the right around the craniocaudal axis (Patten, 1922; Manasek et al., 1984; Icardo, 1996; Manner, 2000). Patten (1922) proposed that C-looping can be explained by external spatial restrictions: bending is inevitable due to elongation of the heart tube while fixed on both ends, and rotation is forced by ventral constraint due to splanchnopleura. In later experiments with isolated hearts, Butler (1952) and Manning and McLachlan (1990) found that the bending is likely intrinsic to the heart tube. Moreover, as heart rotation has been observed in labeling experiments after the splanchnopleura is removed (Stalsberg and DeHaan, 1969; Castro-Quezada et al., 1972; Manasek et al., 1972; De la Cruz et al., 1977), Manasek et al. (1984) postulated that this process also is intrinsic to the heart. However, in all these experiments in which the splanchnopleura was removed, the influence of surface tension on the process of C-looping was not excluded.
Our results suggest that, although bending is likely intrinsic to the heart, rotation may not be. In our experiments with the splanchnopleura removed, we eliminated surface tension by cultivation of the embryo in DME, and the heart could develop without any spatial restrictions on the ventral side of embryo. In all these experiments, the hearts bent ventrally with only slight torsion to the right side (Fig. 6D). However, they rotated much more to the right after removing the liquid medium, as surface tension pushed the hearts dorsally (Fig. 6E). This rotation was reversible, as the hearts restored their shape after the fluid layer was restored (Fig. 6F). Similar behavior was observed in experiments with normal HH stage 11 embryos, after significant C-looping had occurred. After the splanchnopleura was removed, the heart rotated slowly back toward the sagittal plane (Fig. 6G–I). This process likely was slow due to viscous forces. These results suggest that surface tension was a main cause of heart rotation in previous labeling experiments with the splanchnopleura removed (Stalsberg and DeHaan, 1969; Castro-Quezada et al., 1972; Manasek et al., 1972; De la Cruz et al., 1977).
Surface tension does not exist in ovo, as in the embryos cultured in DME. We speculate, however, that in ovo or in DME the splanchnopleura constrains ventral bending of the heart and, therefore, compels the heart to rotate left or right, depending on the initial asymmetry of the heart tube (Fig. 8). Normally, there is a slight initial bias toward the right (Fig. 6A) and the heart loops to the right (Fig. 8A–C). On the other hand, if the heart tube forms with a slight bias to the left, the splanchnopleura forces the heart to loop abnormally to the left (Fig. 8A,D,E). In other words, a passive rotation of the heart due to external constraints is an important component of normal C-looping, as Patten (1922) hypothesized. We speculate that chemical or genetic perturbations that result in left looping interfere with the normal initial rightward bias.
In embryos cultured on the surface of the medium, longitudinal cutting of the splanchnopleura on the right side of the cardiac tube leads to abnormal looping to the left (Fig. 7A,B), whereas cutting it on the left side does not change the direction of normal looping (Lepori, 1967; Castro-Quezada et al., 1972). The results of these experiments are extremely uniform, as abnormal left C-looping was observed in all experiments that have been reported: 24 by Lepori (1967), 25 by Castro-Quezada et al. (1972), and in 35 of our experiments. However, in almost all embryos submerged in DME with right-side splanchnopleura dissection, normal right C-looping was observed (Fig. 7C,D).
At least two hypotheses have been proposed for the cause of left C-looping after cutting the right splanchnopleura. Lepori (1967) and Castro-Quezada et al. (1972), although disagreeing on details, proposed that the splanchnopleura cut results in a shift of the anterior intestinal portal to the opposite side of the cut, and the same thing happens later with the looped heart. On the other hand, Itasaki et al. (1991) proposed that after right-side dissection, the splanchnopleura contracts to the left and pulls the heart toward the left side through its attachment to the ventral mesocardium at HH stage 9.
Our experiments support the last hypothesis, but with a correction. We speculate that in the experiments of Lepori (1967) and Castro-Quezada et al. (1972), the splanchnopleura dragged the heart to the left through friction forces. Friction is likely very high in embryos cultivated on the medium surface, as surface tension pushes the splanchnopleura strongly and tightly against the heart. In embryos cultured in DME, the link between the splanchnopleura and heart tube is considerably weaker, and the splanchnopleura, although moving leftward after dissection on the right, can slide over the heart tube, as visualized by labeling (Fig. 7). As a result, in almost all of these experiments normal right C-looping was observed, regardless of the right-side cut of the splanchnopleura. Therefore, in normal conditions (under the medium or in ovo) a possible asymmetrical splanchnopleura contraction probably cannot regulate effectively the direction of cardiac looping. However, the splanchnopleura is important for increasing of the rotation, pushing the heart farther dorsally and to the right.
Consequently, the heart in traditionally cultivated chick embryos is under considerable force due to surface tension. This force significantly exceeds the force developed by extraembryonic membranes and can affect results of looping experiments. Moreover, it can be a source of considerably high occurrence of spontaneous abnormal left-looping in intact embryos developed on the surface. Therefore, to study mechanisms of C-looping, cultivation under liquid medium is recommended. Our method of cultivation in DME provides a way to eliminate surface tension and to study C-looping without this potential source of artifacts.
Fertilized White Leghorn chicken eggs were incubated in a humidified atmosphere at 38°C to yield embryos at stages 8–10 of Hamburger and Hamilton (1951).
To extract the embryos, the eggs were opened and emptied into a 15-cm Petri dish, and the albumen was removed by blunt-ended forceps. Then, the embryos were extracted by using filter paper (Whatman #2) rings of outer diameter 30 mm and inner diameter 15 mm (Flynn et al., 1991; Chapman et al., 2001) and washed in chick Ringer's solution (0.7% NaCl, 0.037% KCl, 0.018% CaCl2 in distilled water) to remove the yolk.
Submerged embryos were cultured in a fluid medium prepared by mixing 89% DME (Sigma, D1152), 10% chicken serum (Sigma, C5405), and 1% antibiotics (penicillin-streptomycin-neomycin, Gibco BRL #15640). An embryo attached to the filter paper ring was placed ventral side up in an empty 6-cm Petri dish, and then a second paper ring was put on the first one to obtain a sandwich-like structure with the yolk membrane fixed between these two paper rings (Fig. 9B). To avoid floating of the embryo, a stainless steel ring (diameter, 20–22 mm; wire cross-section, 1 mm) was placed on the upper paper ring, and then 3 ml of medium was added. The steel ring was not completely closed to allow medium exchange between the inner (where the embryo is) and outer volumes (Fig. 9A). The depth of the layer of medium above the heart was approximately 0.5 mm. Several embryos were cultured in Petri dishes with 10 ml of medium, and in this case, the layer of medium above the heart was approximately 4 mm deep. Petri dishes containing the embryos were placed in 12 × 20-cm plastic bags filled with a mixture of 95% O2 and 5% CO2. Then, the sealed bags were put into the incubator. To increase humidity, several drops of water were put into the bags.
Other embryos were cultivated on a semisolid medium (Fig. 9C) that was prepared by mixing equal volumes of warm egg albumen and 0.3% melted agar in chick Ringer's solution, with 1% antibiotics added (Flynn et al., 1991; Chapman et al., 2001). Five milliliters of medium were put into a 6-cm Petri dish. The embryos on the paper rings were placed on the agar surface ventral side up. They developed in an incubator (Queue Systems, Inc., model QMI300SABA) with a 5% CO2 atmosphere at 38°C.
Quantitative Estimation of External Forces
Embryos were prepared as for cultivation in the medium, with the yolk membrane held between two paper rings. Measurements were carried out in PBS (Dulbecco's phosphate-buffered saline, Gibco BRL #14040) and on the surface of the semisolid medium described above. To estimate the deformation of the embryo due to the splanchnopleura and surface tension, we measured the diameter of the head before and after removing the membrane, on the surface or under the medium (Fig. 3A–C). Then, compression tests were conducted on the head after it was dissected from the embryo and immersed in PBS (Figs. 2, 3D–I). Loads were applied by using rectangular glass strips that allowed us to record images. One narrow side of the strip was placed on the head and the other side on the bottom of the Petri dish (Fig. 2). Simple equilibrium considerations give the force compressing the head as F = 0.6W(Lab/Lbc)/2, where W is the weight of the strip (in dynes), Lab is the length of the strip, Lbc is the distance between the center of the head and the remote side of the strip, and the multiplier 0.6 is needed to take into account the loss of the glass strip weight due to hydrostatic Archimedes' force, as the density of glass is equal to 2.5 g/cm3, and of PBS is approximately 1 g/cm3. The center of the head was used as the approximate location of the contact point c (Fig. 2B).
Dissections were performed by using fine glass needles. Experiments of two types were carried out: (1) Removing of the splanchnopleura around the heart at HH stage 10-, to allow it to develop freely. To visualize heart rotation, we placed small charcoal particles along the ventral midline of the heart (see Fig. 6) (Stalsberg and DeHaan, 1969; Castro-Quezada et al., 1972; Manasek et al., 1972; De la Cruz et al., 1977); (2) Longitudinal dissection of the right splanchnopleura along a line parallel to the embryo at HH stage 8–9, as Lepori (1967) described (Fig. 7). After the operations, the embryos were cultivated below or on the surface of medium until C-looping developed.
Pictures were taken by using a dissecting microscope (Leica MZ8) and a CCD camera (COHU, model 4915-2000/0000) and electronically processed by using a PC with a frame-grabbing board (FlashPoint 128 4M).
For the statistical analysis, we used SigmaStat software (SPSS, Chicago, IL).