Importance of Flectin in the Dorsal Mesocardium and Splanchnic Mesoderm for Looping
Reported genetic misexpression studies in the chick usually end with a read-out of heart looping direction. We decided to take the analyses further downstream to address whether the expression of the specific matrix molecule flectin, that appears associated with the looping process, is changed by misexpression of upstream laterality genes during randomization of looping direction. Taking together (1) the asymmetric extracellular matrix characteristics of the chick heart that we have reported earlier (Tsuda et al., 1996; Linask et al., 2002), (2) the lack of a role for the cardiac jelly in looping in the rat embryo (Baldwin and Solursh, 1989), (3) biomechanical considerations (Taber et al., 1995), and (4) perturbation studies of flectin resulting in randomization of looping direction (Linask et al., 2002), these findings suggest that flectin in the dorsal mesocardial folds at the cardiac midline is important for establishing looping direction. To establish the validity of flectin within the dorsal region as being important to heart looping, we carried out two sets of experiments: (1) We treated chick embryos with hyaluronidase to remove extracellular matrix from the cardiac jelly and from the basal lamina of the myocardium, followed by flectin immunolocalization. (2) We treated chick embryos with flectin antibody that we previously had shown randomizes heart looping (Linask et al., 2002) and immunolocalized hLAMP-1, an extracellular matrix protein that has been shown to localize to the cardiac ECM during the looping stages (Sinning, 1997). Expression of hLAMP1 in both right- and left-looping hearts was then characterized and compared with that of flectin in the CFC misexpression experiments that resulted also in right- and left-looping hearts.
Hyaluronidase experiments using chick embryos.
Previous experiments in rat embryos demonstrated that even though degradation of hyaluronic acid in the ECM of the rat heart essentially removes the extracellular matrix of the cardiac jelly, the directionality and normal looping of the heart is maintained (Nakamura and Manasek, 1978; Baldwin and Solursh, 1989). We used hyaluronidase degradation here with avian embryos, as a means to remove the extensive ECM in the chick cardiac jelly and in the basal lamina of the myocardium to address whether flectin still remains localized anywhere within the heart ECM and, if so, where. The rationale was that any remaining localization of flectin in the ECM will provide information as to the region that may be important for the observed normal looping. Stage 5 chick embryos were incubated in the presence of hyaluronidase and after a 20- to 22-hr incubation period, the embryos had reached stages 12 to 14, a time period in which heart looping is well under way. As previously reported for the rat embryo, the rightward looping heart tube has collapsed into a flat structure. Despite the extensive removal of the cardiac jelly and the ECM of the myocardial basal lamina, flectin remains expressed in the left dorsal mesocardial fold and in the left splanchnic mesoderm that is closely apposed to the ventral floor of the foregut (Fig. 1). Although some flectin is also expressed within the right dorsal mesocardial fold, it is not as extensive as in the left. Only small areas of flectin are apparent in the myocardial wall. The accompanying diagram is to aid in understanding the dorsal mesocardial (DM) folds/splanchnic mesoderm (SM) areas of the collapsed heart. In summary, this experiment documents that flectin remains localized in the left dorsal mesocardium and in the left splanchnic mesoderm adjacent to the ventral floor of the foregut, after extensive removal of matrix molecules in a heart that continues to loop in the normal direction. Hence, this region was highlighted as an important area for heart looping and to be analyzed further in our CFC misexpression studies.
Figure 1. Hyaluronidase-treated embryo immunostained for flectin. A: The heart has noticeably collapsed and is flattened. Although the cardiac jelly has been degraded, flectin continues to show asymmetric localization in the myocardial wall (MYO), primarily on the left side (L) in the left dorsal mesocardium (DM) and splanchnic mesoderm (SM; large arrows) now flattened against the floor of the foregut (FG). The small arrow points to limited flectin localization within right fold of the dorsal mesocardium. Little, if any, flectin is apparent in splanchnic mesoderm on right. B: Lower magnification for orientation of boxed-in region shown at higher magnification in A. The diagram depicts the relationships and foldings of the various tissues seen. NT, neural tube; EN, endocardium. Scale bars = 60 μm in A, 100 μm in B.
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Localization of hLAMP-1 extracellular matrix protein in right- and left-looping hearts.
In this set of experiments, we determined whether the localization of another ECM molecule that is present during heart looping would be altered upon randomization of heart looping. Heart specific lectin-associated matrix protein-1 (hLAMP-1; Sinning, 1997) has been localized to the extracellular matrix of the looping chick heart and is present also in the extensive basal lamina of the chick embryo (Fig. 2). We incubated stage 5 chick embryos in the presence of flectin monoclonal antibody that we demonstrated previously results in both normally right-looping and abnormally left-looping hearts (Linask et al., 2002). Control untreated hearts (Fig. 2A), and flectin antibody-perturbed right- (Fig. 2B) and left- looping (Fig. 2C) hearts were then immunostained with an antibody for hLAMP-1. As seen in the control embryo, hLAMP-1 localizes to the cardiac jelly ECM and the ECM of the basal lamina and to an attachment region of the endocardium to the midline of the ventral foregut floor (shown in Fig. 2A). hLAMP-1 is not expressed, however, within the myocardium, in the dorsal mesocardium, or splanchnic mesoderm adjacent to the foregut wall. In the flectin perturbed right-looping (Fig. 2B) and left-looping (Fig. 2C) hearts, the ECM appears somewhat diminished. However, the hLAMP-1 expression pattern does not change and continues to be expressed in the same manner in the basal lamina and cardiac jelly of the heart, regardless of sidedness of looping. This finding is in contrast to the modulation of the specific asymmetric expression pattern of flectin within the dorsal mesocardium/splanchnic mesoderm that correlates closely with the change in looping direction reported previously (Tsuda et al., 1996; Linask et al., 2002). Note also in the left-looping heart, the foregut is displaced well to the left of the embryo midline, as defined by the position of the notochord. The heart is positioned relative to the midline of the ventral floor of the foregut and not relative to the embryo midline.
Figure 2. Immunolocalization of hLAMP-1 in the control, untreated, heart of a stage 12 chick embryo, after a 22-hr incubation period in culture (A) and in flectin antibody perturbed right- (B) and left-looping (C) hearts. A: hLAMP-1 localizes primarily to the basal lamina of most developing tissues. In the heart, it is detectable also in the cardiac jelly, in association with the endocardium, in the basal lamina of the myocardium, and in the extracellular matrix (ECM) between the splanchnic mesoderm and the apposing wall of the foregut (FG). Little, if any, hLAMP-1 is detectable within the myocardium, in the dorsal mesocardium (see arrowheads), or splanchnic mesoderm. In flectin antibody perturbed hearts, less cardiac jelly was apparent in both right-looping (B) and left-looping (C) hearts. There was no major change relative to the controls in localization of hLAMP-1, as it continues to be expressed in the basal laminae, endocardium, and cardiac jelly. No expression of hLAMP is observed in the dorsal mesocardial folds (arrowheads) or in the splanchnic mesoderm as was seen with flectin. Note in C of the left-looping heart that the FG is displaced laterally far to the left of the embryo midline as defined by the notochord (N). hLAMP-1 is also highly expressed in the ECM in association with endocardial cells seen in this region adjoining the ventral foregut midline (white arrow points to the hLAMP-1 in this midregion near foregut floor). NT, neural tube. Scale bars = 50 μm in A (applies to A,C), 100 μm in B.
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CFC misexpression was carried out in the present study because it has two principal effects: it transiently represses Lefty-1 expression at the midline and leads downstream to bilateral expression of Pitx2 (Schlange et al., 2001). Results of chick CFC misexpression using antisense oligonucleotides on heart looping, Pitx2 expression, and flectin localization are shown in Figure 3A–O, Figure 4A,B, and Figure 5A–D, in Tables 1 and 2, and a control heart in Figure 6. Forty-three anterior halves of the antisense-oligonucleotide–treated embryos were immunostained for flectin as a cardiac extracellular matrix asymmetry marker, and 31 of these embryos were sectioned anteriorly to posteriorly through the heart (Fig. 3). Posterior part of each embryo cut just below the heart was analyzed for Pitx2 mRNA expression by whole-mount in situ hybridization. The Pitx2c in situ hybridization and flectin protein patterns are indicated for all the different classes of embryos examined (see Tables 1, 2). The relationship of the heart to the mid-region of the ventral floor of the foregut and to the embryo midline was noted also in the sectioned embryos. Representative embryos are shown in the figures.
Figure 3. Misexpression of CFC and resultant flectin localization patterns. Ventral view of hearts is shown in whole-mounts. Right (R) and left (L) sides are the same for all panels. In all rows of panels corresponding to different embryos, the left panel shows the in situ hybridization pattern for Pitx2 mRNA expression, the center panel shows flectin localization in the whole-mount, and the right panel shows a section through the same heart shown in the middle panel. A–C: Heart is looping to the right as seen in embryo in B. In this embryo, Pitx2 mRNA expression was on the left (green arrows, A). This heart when sectioned displays predominant localization of flectin in left dorsal mesocardial fold and in adjacent myocardial wall (in C, large green arrow). D–F: Heart is looping to the right (E) within an embryo showing bilateral Pitx2 localization (D, green arrows). When sectioned, the heart shows slightly more flectin on left side (F). Note in F and in O that the midline of the foregut (FG) is displaced more to the right or left of the embryo midline, as defined by the notochord's (N) position than is normally seen (compare with foregut localization in C, I, and L). The heart develops, however, in relation to the midline of the ventral floor of the foregut in all embryos analyzed. G–I: Heart shows a definite leftward loop. This embryo also showed a bilateral Pitx2 localization (G, green arrows). A predominant right-sided flectin localization pattern is now apparent (I, larger green arrows. Small arrows show comparable region on other side of tube with little flectin expression detectable). J–L: In this whole-mount, the heart appears unlooped (K), while Pitx2 pattern was left-sided (J) as is normally observed for Pitx2 expression. The heart sectioned displayed a symmetric expression of flectin in the dorsal mesocardial folds and adjacent myocardial wall. This finding is consistent with earlier observations of hearts that do not loop. M–O: Heart in the whole-mount appeared to be not looping (seen in N). The Pitx2 expression pattern was bilateral (see green arrows in M). This heart sectioned showed a relatively low level of intensity for flectin protein expression, as well as a symmetrical pattern of flectin within the mesocardial fold regions and adjacent myocardium (O, arrows). Scale bars = 100 μm in C (applies to C,F,I,L), 50 μm in O, 200 μm in E (applies to B,E,H,K,N).
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Figure 4. This CFC-treated embryo displayed an abnormally, leftward, looping heart with a normal, left-sided Pitx2 mRNA expression pattern. A: As was expected with leftward looping hearts, the sectioned heart showed a predominant right-sided flectin expression pattern (“painted” yellow). The rectangle demarcates the region in which the area painted was measured. B: A similar area was defined in the red on the left side, and the painted region was measured. The areas were expressed as left–right ratios and were defined as asymmetry quotients (AQ) to obtain relative measurements of flectin localization. FG, foregut; R, right; HT, heart.
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Figure 5. Some hearts were described as S-shaped (A shows the whole-mount of one such heart). These embryos showed normal, left-sided Pitx2 expression. Heart in A is shown sectioned in B–D. The anterior region has normal rightward bending with predominant left-sided flectin localization. The flectin localization area defined in red relative to yellow was measured anteriorly (B,C), as well as posteriorly (D). The more posterior heart regions of this same heart express little flectin and show no extracellular matrix predominance, and, as a result, this part of the tube does not show bending and appears in whole-mounts to be S-shaped (A). L, left; R, right; Ant, anterior; AIP, anterior intestinal portal; N, notochord; FG, foregut; MYO, myocardial wall. Scale bar in D = 100 μm in A, 50 μm in D (applies to B,C).
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Table 1. CFC-Treated Embryos
|Situs||Pitx2 expression||Number (n)||Embryos sectioned|
Table 2. Asymmetry Quotients (AQ) of CFC Antisense-Oligonucleotide–Treated Embryos
|Situs||Flectin AQa||Figure no.||Pitx2 expression|
|Right||4.6b; 3.3; 2.3||3C||Left-sided|
|Right||1.1; 1.4; 1.1||3F||Bilateral|
|Left||0.2; 0.8; 0.7||3I||Bilateral|
|Unlooped||0.9; 1.0; 0.9||3O||Bilateral|
|S-shaped|| || || |
| R-loop/Antc||1.7; 1.2||5B,C||Left-sided|
| No loop/Posc||1.0; 0.8||5D||Left-sided|
Figure 6. A control embryo treated with scrambled oligonucleotides displays rightward looping. The area defined in yellow (A) relative to the area defined in red (B) was determined to have an asymmetry quotient of 2.2, consistent with rightward looping and more flectin in the left dorsal mesocardial fold and adjacent splanchnic mesoderm than on the right. FG, foregut; En, endocardium. Scale bar = 50 μm in B (applies to A–B).
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In Table 2, the first asymmetry quotient (AQ) given depicts the asymmetry quotient that was calculated for the embryo shown in the figures. The additional AQs represent quotients that were calculated for flectin localization in hearts of embryos within the same class but are not shown separately in the figures. However, they are the same with regard to Pitx2 expression and direction of heart looping. A statistical analysis was not attempted due to reasons given in Experimental Procedures section.
Figure 3A–C shows anterior and posterior halves of a CFC antisense-oligonucleotide–treated embryo with a rightward looping heart. This embryo displayed a left-sided Pitx2 mRNA expression pattern (Fig. 3A, green arrows). The anterior half of the embryo shown in Figure 3A was immunostained for flectin (ventral view shown of whole-mount in Fig. 3B: heart wall is outlined in yellow) and the same heart sectioned (Fig. 3C). A dominant, left-sided, flectin localization is apparent in the left dorsal mesocardial fold (Fig. 3C, large green arrow) and in the outer curvature of the heart. The same pattern is seen also in control and untreated embryos (not shown). The L/R asymmetry quotient equals 4.6, which is consistent with a predominant left-sided flectin expression (see Experimental Procedures section and Fig. 4).
Figure 3D–F shows an embryo in which the heart looped to the right, but Pitx2 expression was bilateral (Fig. 3D, green arrows). In sections through the heart, greater localization of flectin is seen in left, outer curvature of the myocardial wall (Fig. 3F, large green arrows) than in comparison to the same regions of the right myocardial wall (smaller green arrows). Although flectin is present in the dorsal mesocardial fold on the right side, it is almost absent where the bend actually occurs. In contrast to the heart in the Figure 3A, there is now Pitx2 present also on the right side. Although the detectable flectin matrix difference is small in this section of an anterior region of the heart, there is more flectin on left (AQ = 1.1) and a rightward bend has occurred. The higher flectin expression seen on the dorsal side of the foregut is often, but not always, observed. Flectin is also observed in the extraembryonic splanchnic membrane covering the heart and also is seen at high levels in this embryo. It should be noted in this embryo that the midline of the ventral floor of the foregut (FG) has shifted more than is normally observed to the right of the embryo midline (as defined by notochord, N, location). A similar shift was also seen earlier, when Pitx2c was directly misexpressed (Linask et al., 2002). The heart develops, however, in relation to the ventral foregut midline, not embryo midline, as defined by the position of the notochord.
In an abnormally, leftward looping heart shown in Figure 3G–I, bilateral Pitx2 expression was also observed in this embryo. When this heart was sectioned, a predominance of flectin was observed in the right dorsal mesocardial fold and right outer curvature of the heart in comparison to the left (AQ = 0.2). The asymmetry within the dorsal mesocardial area of this abnormally looping heart is seen clearly in the section in Figure 3I.
In hearts that were classified as “unlooped” based on whole-mount observation (Fig. 3K,N), the Pitx2 mRNA expression differed: in embryo 3K, Pitx2 was left-sided only (in situ hybridization pattern for Pitx2 mRNA expression shown in Fig. 3J). This heart, when sectioned (Fig. 3L), showed relatively equal intensity for flectin in the left and right myocardium and dorsal mesocardium (AQ = 1.0). The heart tube did not show any distinct looping direction. The unlooped heart (shown in Fig. 3N) displayed bilateral Pitx2 expression (in situ hybridization in Fig. 3M). This heart also displayed relatively symmetric, although in contrast to heart in Figure 3L, little flectin expression in the two sides of the tubular heart (Fig. 3O) and in the dorsal mesocardial folds (AQ = 0.9). Thus, these results provide additional evidence that regardless of Pitx2 expression being left only or bilateral, the asymmetric predominance of flectin, or sometimes symmetric expression, in the dorsal mesocardial folds and splanchnic mesoderm, as well as in the myocardial wall, is associated directly with the direction of heart looping, or no looping, respectively. Note also in these embryos that the midline of the ventral floor of the foregut (FG) may be slightly to the left or right of the embryo midline, as defined by the location of the notochord (N). As seen with embryonic hearts in all panels, the heart and dorsal mesocardial folds develop in relation to the midline of the ventral floor of the foregut, not the embryo midline. Thus, the positioning of the foregut, and specifically the ventral floor, defines the position of the cardiac midline. For all 31 embryos analyzed, the results were similar to these shown.
Among the CFC antisense-oligonucleotide–treated embryos one embryo only showed abnormal leftward looping, but a normal, left-sided Pitx2 expression. Based upon the above results, one would predict that this heart when sectioned would have a predominant right-sided flectin expression. This was found to be the case (Fig. 4A,B). This figure also shows how we obtained the AQ indicated above. In Figure 4A, brighter intensity values were painted in yellow for the right side and red on the left. The areas “painted” within the same fixed region defined by the same-sized rectangular box for each side were then calculated in numbers of pixels (3 × 3) and are expressed as an asymmetry quotient L/R obtained from dividing the means calculated for the two sides. The AQ for the heart region in this embryo equals 0.6, consistent with a leftward looping direction taken.
In embryos in which looping is described as being S-shaped (see also Schlange et al., 2001), the anterior region is beginning to loop to the right, but the posterior region is seemingly bending to the left or is unlooped, thus giving an S-shape to the heart. (See whole-mount of S-shaped heart in Fig. 5A. AIP designates the anterior intestinal portal. Arrow shows plane of anteroposterior axis). In one such heart, the anterior region shows normal rightward bending with predominant left-sided flectin localization (AQ = 1.7; Fig. 5B,C). The more posterior heart regions of the same heart tube, however, express little flectin as yet and show no ECM predominance. This finding correlates with the part of the cardiac tube that does not show bending (AQ = 1; Fig. 5D). Thus, the heart appears in whole-mounts to be S-shaped. In some embryos in the S-shaped heart group, a slight left predominance of expression was apparent. One would expect these hearts to continue bending in the rightward direction as development continues.
Figure 6A,B shows a control heart of an embryo treated with a scrambled sequence of oligonucleotides. This embryo had developed to a slightly older stage than seen in previous figures. Flectin AQ based on measurements in the dorsal mesocardial folds was 2.2, consistent with hearts that loop to the right and display left-sided Pitx2 expression.
Relationship to Secondary Heart Field and Pharyngeal Region
Once looping direction is specified in anterior regions within chick stages 9/10 and becomes detectable at stage 11, heart morphogenesis proceeds directly into the next stage with the addition of new segments to the outflow region from the secondary heart field, as well as to the caudal inflow region beginning with stage 12 (de la Cruz et al., 1991; Waldo et al., 2001). The secondary heart field encompasses the splanchnic mesoderm that is adjacent to the ventral floor of the gut tube (caudal pharyngeal region) and is continuous with the dorsal mesocardial folds. At stage 12, flectin continues to be asymmetrically expressed in the secondary heart field, primarily in the left splanchnic mesoderm adjacent to the lateral wall of the pharyngeal foregut and in the left dorsal mesocardial fold (see arrows in Fig. 7A and in Fig. 7B of same region shown at higher magnification). The dorsal mesocardial region, as we specified above, continues to be closely associated with the endoderm of the ventral floor of the foregut, specifically encompassing this mid-region of the foregut floor. A strand of endocardial cells pass through the sleeve of the dorsal mesocardium to associate with the gut tube cells at the midline of the ventral pharynx.
Figure 7. Stage 12 normal embryo immunostained for flectin. A: For orientation, A shows a section at lower magnification through the heart and the secondary heart field. Note the left (L) and right (R) dorsal mesocardial areas and the splanchnic mesoderm adjacent to ventral foregut (FG) endoderm continue to show asymmetry in that more flectin is seen in left side than in the right (compare regions indicated by arrows). B: At higher magnification showing flectin immunolocalization, as well as the alignment of embryo midline as designated by the notochord (N) with the ventral floor of the foregut endoderm with which the endocardial cells (EN) associate as a cord of cells in between the dorsal mesocardial folds. NT, neural tube. Scale bars = 100 μm in A, 50 μm in B.
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From our previous studies on N-cadherin/β-catenin localization during heart development, we noted that catenins are involved in compartment formation in the early embryo (Linask et al., 1992, 1997). We now document that β-catenin–like plakoglobin defines a compartment of cells within the foregut floor endoderm at stages 11 and 12. A distinct compartment of cells within the foregut endoderm is delineated by plakoglobin-mediated cell to cell junctions (see arrows in Fig. 8A, showing a stage 11 embryo and Fig. 8B, a stage 12 embryo). The arrowheads in Figure 8B designate the lateral boundaries of the compartment. It is this ventral endoderm compartment with which the endocardial cells (EN) associate and that the dorsal mesocardium/splanchnic mesoderm encompasses at the cardiac midline. If Pitx2 or flectin is misexpressed, often the position of the foregut is seen to be more lateral to the left or right than normal (see Fig. 2; also Linask et al., 2002). The position of the heart is defined in all of our analyses by its association with the foregut endoderm compartment.
Figure 8. Localization of plakoglobin in stage 11 (A) and stage 12 (B) embryos. Similar region as shown above. Expression of plakoglobin is detectable within cell–cell junctions (see arrows) of a compartment of endoderm cells at the mid-region of the ventral foregut floor. Arrowheads in B designate the lateral boundary of pharyngeal endoderm compartment at stage 12. This compartment may be a signaling component during development of the secondary heart field into additional cardiac segments. This addition of segments occurs during subsequent looping, right after looping direction has been specified. DM, dorsal mesocardial fold; EN, endocardial cells; FG, foregut; N, notochord; NT, neural tube. Scale bar = 50 μm in A (applies to A,B).
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