C-looping represents the first easily visible manifestation of morphological L–R asymmetries in the originally bilaterally symmetric embryo. The biophysical mechanisms driving it are now becoming better understood (for a review see Taber, 2006) as are the molecular mechanisms (for reviews, see Linask and VanAuker, 2007; Brand, 2003; Mercola and Levin, 2001). A three-dimensional map of cell-proliferation and cell-growth is also available for the c-looping stages (Soufan et al., 2006).
By contrast, little is known about the forces involved in s-looping and the process has attracted scant attention (Manner, 2000). Because s-looping in the chick embryo covers a roughly 20-hr time span from stage 12 to stage 18, we restricted our attention to a 6-hr span from stage 12 to stage 15. We refer to this period as “early s-looping.” During early s-looping, the ventricle moves inferior to the common atrium and distance between the conotruncus and the common atrium decreases (see Heart Morphology During Early S-Looping section, Fig. 1).
Most of our knowledge on the biophysical mechanisms leading to s-looping come from a series of studies by Jorg Manner (Manner et al., 1993, 1995a, 1995b; Manner, 2000, 2004). These prior studies posit a strong causal role for external forces, particularly those due to cervical flexure, in s-looping.
Our results support and significantly extend Manner's results (please see Results section). In this study, we considered some of the major players in the mechanics of c-looping and studied their effect on early s-looping. The following is a summary of our major new findings.
First, it is known that the SPL plays an important role in c-looping (Voronov et al., 2004; Nerurkar et al., 2006; Ramasubramanian et al., 2008); our experiments show that its role continues into early s-looping (see Effect of Removing the Splanchnopleure [SPL] section). Second, while it is accepted that cardiac jelly inflation is not necessary for c-looping, our experiments show that the matter is not so black and white for early s-looping (see Effect of Dissolving the Cardiac Jelly section). Finally, while isolated straight hearts have the capability to bend on their own during c-looping, we found that isolated c-looped hearts do not have the capability to form an s-loop (see Effect of Culturing Hearts in Isolation section). In summary, we tested the role of intrinsic and extrinsic forces in early cardiac s-looping and our results show that the latter are much more important than the former.
As far as we know, we are the first group to develop a three-dimensional computer model for early cardiac s-looping that includes both the myocardium and the cardiac jelly. The model uses stage-12 topologies and measured nonlinear material properties for the myocardium and the cardiac jelly. We used the model to propose and test the following hypothesis for the mechanics of early cardiac s-looping.
Remarks on the Link Between Heart and Head Morphogenesis
Two of the most visible developmental changes in vertebrate embryos are the flexure and rotation of the embryonic axis. These morphological changes transform the initially straight embryonic axis into a curved axis leading to the familiar “fetal position.” The curvature of the embryonic axis is seen in embryos of reptiles, birds, and mammals possibly due to the space limitations inside an egg or uterine cavity; it is not seen in fish and amphibian embryos which develop in open water without any space constraints (Patten, 1951). Since reptilian, avian, and mammalian embryos normally develop with the ventral side facing the yolk, flexing is prevented by the presence of the yolk. Hence, the embryo must first rotate so that its lateral side now faces the yolk so that flexing, which brings the anterior and posterior ends together, can now commence (Patten, 1951).
In the chick embryo, flexion and rotation occur simultaneously, starting at about stage 11. By stage 20, the embryo is fully rotated on its side. By about stage 13, rotation has progressed to the regions containing the heart and two prominent bends, the cranial flexure and the cervical flexure can now be seen (Fig. 1). The location of cranial flexure is remote from the looping heart and its influence in early s-looping has been ruled out (Manner et al., 1995b).
Cervical flexure, on the other hand, occurs in close proximity to the heart and leads to a prominent bend in the cervical region (Fig. 1A–E). Recall that during early s-looping, the two ends of the heart, the conotruncus and the common atrium, come into close proximity to each other (Fig. 1A'–E'). It is clear from looking at the morphology that either of these processes can lead to the other; shortening of the distance between the ends of the heart can pull the cervical portion into a curved configuration or cervical flexure can lead to early s-looping in the heart. Flynn et al. (1991) and Waddington (1937) support the former idea; these researchers severed the conotruncus and noted that these embryos, where the heart can no longer exert any forces in the neck region, lacked cervical flexure.
The converse idea, i.e., cervical flexure leads to early s-looping, is not new and was proposed by early researchers such as Patten (1951) and Romanoff (1960). A classic experiment performed by Manner and his colleagues lends strong support to this theory: they inserted a human hair into the neural tube of a stage-12 (completed c-looping) embryo; the hair, being stiffer than the developing cephalic region, prevents cervical flexure (Manner et al., 1993). It was noted that preventing cervical flexure in this manner led to an arrest of cardiac morphogenesis in the c-looped state. Separately, Manner also showed that the lack of cervical flexure in Flynn et al. (1991) is due to oxygen deprivation and that heart-deprived embryos can still form normal flexures when cultured in an oxygen-rich environment (Manner et al., 1995a).
New experiments performed in this study support the results of Manner et al. (1993) . Two of our perturbations, SPL removal and ablation of the cardiac jelly, led to irregularities in the looping process, but did not have any effect on cervical flexure or head rotation (Figs. 3 and 7). This implies that cardiac morphogenesis is not a necessary cause for cervical flexure. Also, our experiments with isolated heart cultures indicate that external forces are necessary for s-looping (Fig. 8).
Material property measurements also support the hypothesis that cervical flexure causes s-looping and not vice-versa. Results from the Taber group indicate that the developing neural tissue is about 10 times stiffer compared to developing cardiac tissue at stage 12 (Zamir and Taber, 2004; Xu et al., 2010). It is of course easier for a stiffer tissue (i.e., developing brain) to deform a softer tissue (i.e., developing heart) than vice versa.
There is, thus, strong evidence that the formation of the cervical flexure is crucial for early cardiac development. As pointed to in Manner et al. (1993), this could be the reason why some congenital malformations of the neck are accompanied by congenital cardiac malformations. The actual mechanism of cervical flexure formation is not currently known although differential cell proliferation in the axial structures (neural tube, notochord, etc.) and cell-shape changes caused by actin filaments have been proposed as possible mechanisms (Goodrum and Jacobson, 1981; Schoenwolf and Smith, 1990).
Although simulations strongly suggest a role for head rotation (our results; see A Hypothesis for Early S-Looping section and Fig. 9; the idea is also mentioned in Manner, 2004), so far no study has done experiments on the effect of head rotation on cardiac s-looping. Our hypothesis does not say anything about the relative importance of head rotation and SPL pressure during Action 1. This warrants further study.
Remarks on SPL Removal
The splanchnopleure (SPL) is a membrane that lies on the side of the pre-looped heart (Fig. 6A') and applies pressure on it. Experiments performed by other researchers indicate a strong role for the SPL in c-looping (Voronov et al., 2004). New results from this study indicate a similarly important role for it in early s-looping. We first consider the short-term effects of SPL removal.
Immediately following SPL removal, the heart swings forward (i.e., toward the observer in a left lateral view). This effect has been observed in a previous study (Filas et al., 2007) and suggests that the SPL applies a compressive force, the removal of which allows the heart to be free to swing in an arc (Fig. 3C',C''). The heart tube also becomes markedly thinner (Figs. 3B',D' and 4A,B) and the heart wall becomes considerably thicker (Fig. 4A',B'). These effects are first noticed at t=2 hr following SPL removal and become established at t=5 hr. There is some recovery in both effects when SPL-lacking hearts are incubated in the presence of blebbistatin, i.e., the heart diameter increases (Fig. 4B,C) and the heart wall becomes thinner again (Fig. 4B',C'). These results indicate potential activation of cytoskeletal contraction when the SPL is removed. Since circumferential contraction results in a smaller-diameter tube, there must be some thickening in the radial direction due to material incompressibility.
The short-term effects of SPL removal on s-looping have similarities to those observed during SPL removal on c-looping. Nerurkar et al. (2006) and Ramasubramanian et al. (2008) also report a decrease in heart diameter following SPL removal during c-looping. And as mentioned earlier, Filas et al. (2007) also report a forward swing (i.e, toward the observer in a left lateral view) of the heart tube immediately following SPL removal in the c-looping stages.
The long-term (10 hr of SPL-free culture) effects of SPL removal are also significant: the ventricle still has the potency to move caudad, but not to the extent seen in control hearts (Fig. 3B',D'). Our hypothesis for early s-looping posits that SPL pressure together with head rotation is necessary to displace the cranial portion of the heart tube (see previous text, Fig. 9). This sets the stage for cervical flexure to complete the process. Removal of the SPL pressure possibly results in an inadequately displaced cranial portion, which when acted on by cervical flexure, results in an immature heart loop. The model with SPL pressure removed, but head rotation retained, results in topologies that are remarkably similar to experiment (Fig. 3D',E). The importance of the SPL to early s-looping is also demonstrated by experiments in which the SPL manages to regrow during culture (Fig. 6). These embryos tended to loop normally.
Looping could also be inhibited because of the observed reduction in heart size. It is possible that the crowding effect of cervical flexure formation on the embryonic heart tube may be less strong in an abnormally small heart loop within a normal-sized pericardial cavity as compared to a normal-sized heart loop within a normal-sized pericardial cavity.
Long-term effects of SPL removal are quite different between c and s-looping. Normal c-looping is only delayed following SPL removal. As reported in Nerurkar et al. (2006), a secondary mechanism involving asymmetric cytoskeletal contraction manifests itself about 5 hr following the perturbation and normal c-looping is restored 10 hr following the perturbation. In contrast, we found that, following SPL removal, normal s-looping was not restored in longer-term (> 10 hr) cultures.
While prior studies do not mention anything about the effects of SPL removal on heart rate during c-looping, our results indicate a jump in heart rate following the same perturbation. It is possible that the more rapid pumping is a compensatory mechanism since the heart tube is now thinner and more frequent beats are needed to achieve the same volume of blood flow. Another possible reason for the increase in heart rate is the change in fluid pressure in the coelomic cavity brought about by SPL removal. Experiments with elasmobranch fish (sharks and rays) and sturgeon show that altering the fluid pressure in the various body cavities produces significant alterations in cardiac function (Shabetai et al., 1985; Gregory et al., 2004).
We note here that head flexure and rotation are not impaired by SPL removal even though heart looping is. This lends support to the study of Manner et al. (1993), which claims that cardiac s-looping is caused by head flexure and not vice versa as claimed by other studies (Flynn et al., 1991; Waddington, 1937).
Remarks on Cardiac Jelly Inflation
The cardiac extracellular matrix, the cardiac jelly (CJ), is a significant component of the looping heart, occupying a large volume between the myocardium (MY) and the endocardium (EN) (Fig. 6). The CJ is not a passive substrate and plays an active role in many aspects of cardiac morphogenesis (for a review, see Bowers and Baudino, 2010). Recent research indicates that the CJ is also implicated in cell convection leading to endocardial morphogenesis (Aleksandrova et al., 2012). Despite its name, the CJ is structurally sound with a modulus that is roughly 25% of that of the MY (Zamir and Taber, 2004). Furthermore, when the MY is removed, the isolated CJ can hold its shape (Nakamura and Manasek, 1978).
There was considerable debate regarding the role of CJ in c-looping. Taking inspiration from tubular worms whose shape is determined by internal turgor pressure (Wainwright et al., 1982), Manasek and coworkers (1984) proposed that the c-loop in the tubular heart is also formed by turgor pressure due to hydration-driven expansion of the CJ. This hypothesis was contradicted by experiments of Baldwin and Solursh (1989), who found that hearts whose CJ was ablated by the enzyme hyaluronidase looped normally. Later experiments by Linask et al. (2003) also indicated that CJ inflation was not necessary for c-looping. This view is now widely accepted (Taber, 2006).
Our results from s-looping show many similarities to these previous studies on c-looping. Like the results of Manasek et al. (1984) and Baldwin and Solursh (1989), our hearts also became flaccid when treated with hyaluronidase. These previous studies report that enzyme treatment resulted in a greatly expanded lumen with the MY and EN almost directly apposed. OCT imaging confirmed the same finding in our results (Fig. 6). Our OCT data also show an unevenly thick CJ layer with the original (i.e., pre c-looped) left and right sides having the greatest thickness (Fig. 6A'). These results are in agreement with previous studies (Manner et al., 2008, 2010).
New results from our study shed light on the role of CJ inflation in s-looping morphogenesis; we found that looping was negatively affected (delayed or inhibited) in 79% of the cases (see Effect of Dissolving the Cardiac Jelly section). S-looping was normal in the remaining 21% of the cases. It is curious that CJ removal has no influence on c-looping (Baldwin and Solursh, 1989; Linask et al., 2003), but delays development in the morphogenetic phase immediately following it (our results). We offer three explanations.
First, similar to the effect of SPL removal, the decrease in heart size could make looping more difficult to accomplish by the application of head forces, i.e., the crowding effect of cervical flexure formation is less strong in an abnormally small heart loop within a normal-sized pericardial cavity compared to a normal-sized heart loop within a normal-sized pericardial cavity.
Second, looping could be affected because blood flow is interrupted. Assuming circular cross-sections for the MY and EN and a 20% shortening of the MY during systole, Barry (1948) showed that the tubular heart at the stages considered in this study cannot effectively pump blood unless a layer of CJ is present between the MY and the EN. By culturing rat embryos in the presence of hyaluronidase, Baldwin et al. (1994) showed that ablation of cardiac jelly during looping leads to “substantial hemodynamic alterations” compared to control embryos. A more recent OCT study found that the CJ layer is not perfectly circular in cross-section. The latter study also suggests that the uneven thickness of the CJ leads to an elliptical endocardial tube during diastole with a higher pumping efficiency (Manner et al., 2008).
There is thus considerable evidence that the CJ layer is needed for heart function. Treatment with hyaluronidase results in a lumen that is open all the time (Fig. 6), disrupting normal blood flow and possibly leading to the pooling of blood visible in 50% of our enzyme-treated hearts (Fig. 7D'). Our results also show that the hyaluronidase-treated hearts have MY and EN layers that are almost directly apposed. The lumen is no longer closed shut during systole and the heart ceases to be an effective pump. Although blood flow and heart beat are not necessary for c-looping (Manasek and Monroe, 1972), their effects on s-looping are not known. It is, therefore, possible that morphogenesis is interrupted because normal blood flow is interrupted.
Third, enzyme-treated hearts are flaccid and we found it somewhat challenging to determine if they had looped or not. By our criteria, a heart is not considered normal unless the loop had the same morphology as control at the same time point. It is possible that Baldwin and Solursh (1989) and Linask et al. (2003) found similar distortions due to flaccidity, but nonetheless classified looping as normal based purely on whether the heart acquired a c-loop at the end of culturing.
Effect of hyaluronidase on head morphogenesis
Previous studies have found that the developing head region has a high concentration of glycosaminoglycans (Toole, 1976). Hence, it is possible that shrinkage in the head region, which we observed in many of our enzyme-treated embryos (Fig. 7), is due to transport of the enzyme to the head region. A previous study also found that rat embryos cultured in hyaluronidase showed a slight reduction in neuroepithelial cell number (Morriss-Kay et al., 1986).
Remarks on Isolated Heart Cultures
Isolated straight stage-10 hearts have the potency to bend and form a c-shape (Latacha et al., 2005; Butler, 1952; Manning and McLachlan, 1990; Flynn et al., 1991). New results from the current study show that isolated c-looped hearts do not have the potency to form an s-loop (see Effect of Culturing Hearts in Isolation section).
However, when c-looped hearts are cultured in isolation, they bend into a shape where the two ends of the heart come together (please see Fig. 8). These findings are consistent with the results of Flynn et al. (1991) who did a similar experiment. However, our conclusions are quite different. The earlier study posits that bending of a c-shaped heart tube into an o-shaped one is the cause for cervical flexure. Our conclusions are more in agreement with those of Manner et al. (1993) and are the exact opposite, i.e., cervical flexure is responsible for heart looping.
Since forces due to cervical flexure are not found in isolated heart cultures, the question arises as to what causes these hearts to continue bending. Latacha et al. (2005) showed that cell shape changes driven by actin polymerization cause the straight heart tube to bend into a c-shaped one. In the absence of external constraints, it is possible that these forces cause continued bending into an o-shaped tube. Indeed, some of the straight hearts in Latacha et al. (2005) were cultured for 24 hr (c-looping only takes 12 hr to complete) and these were found to bend into an o-shaped tube with the arterial and venous poles almost in contact.
We found that many of our isolated hearts had out-of-plane deformations, an effect that diminished over time. This suggests a slow relieving of stresses that were present when the heart was still attached to the embryo. Ramasubramanian and Taber (2008) suggest some feedback mechanisms by which this can be achieved.
While the model-predicted topologies match the experiment, there is some difference between their respective shapes (Fig. 1). There are several possible reasons. First, our hypothesis focuses on the role of external forces and internal loads are not included. Previous studies have shown that cell-shape changes brought out by actin polymerization drive the bending component of c-looping (Latacha et al., 2005) while cytoskeletal contraction plays an important role in the restoration of the rotational component of c-looping when the SPL is removed (Nerurkar et al., 2006). The role of these forces in s-looping is currently under investigation (Chu-LaGraff et al., unpublished data) and they are not included in our hypothesis. CJ inflation is also not included in the model. Second, the model dimensions are chosen to demonstrate s-loop formation in a tubular heart of fixed length and diameter; no attempt has been made to accurately capture the changing length and diameter (Fig. 1A'–E') of the heart tube as it undergoes s-looping. Finally, there are stark differences in the shape of the normal s-loop (see A Hypothesis for Early S-Looping section) due to natural biological variability, and the model does not attempt to capture the heart shape of any particular embryo. Rather, it only captures the general topology that is present in all control hearts (as described in Heart Morphology During Early S-Looping section).
The earliest hypothesis for c-looping was put forth in 1922 and even now there is debate regarding the forces driving it. While we have demonstrated a strong role for external forces in early s-looping, we realize that our hypothesis is but a starting point. It will need to be modified when further data become available.