An experimental study of the relation of cardiac jelly to the shape of the early chick embryonic heart

Transcription

1 J. Embryo/, exp. Morph. Vol. 65, pp , Printed in Great Britain Company of Biologists Limited 1981 An experimental study of the relation of cardiac jelly to the shape of the early chick embryonic heart By ATSUYO NAKAMURA 1 AND FRANCIS J. MANASEK From the Department of Anatomy, The University of Chicago SUMMARY The structural roles of cardiac jelly components were examined in the early developing chick embryonic heart. Cardiac jelly matrix components were enzymically removed in situ by injecting nanogram quantities of enzymes directly into the cardiac jelly. Injection of ovine testicular hyaluronidase caused shrinkage and the heart became flaccid, but overall heart shape did not change. These responses were the result of enzymatic removal of glycosaminoglycan sugar moieties and were not due to lumenal collapse. Although purified collagenase did not cause any noticeable change, enzymes with non-specific proteolytic activity induced marked cardiac shape changes. In such hearts the dorsal mesocardium opened completely, and the myocardium as well as splanchnic mesoderm of foregut detached from their substrata and the entire heart region swelled. Consequently the shape of the heart was altered completely. These results suggested that in the normal condition the myocardial envelope was under an internal pressure due to the presence of glycosaminoglycans in the cardiac jelly space, and that some matrical non-collagenous protein components were essential to control the internal pressure. Therefore it is suggested that the internal pressure of cardiac jelly may be the direct driving force for the looping process and protein components of cardiac jelly may be important in directing the force for the morphogenetic process. INTRODUCTION One of the earliest critical morphogenetic events in vertebrate embryonic heart development is the process known as 'looping'. Tn this process the roughly symmetrical tubular heart, which is formed by fusion of bilateral precardiac splanchnic mesoderm, simultaneously bends and rotates toward the right side of the body axis (D-loop). Bending and rotation are separate events as shown by the fact that the bending heart can rotate to the right (normal direction); to the left (L-loop) or not rotate at all (Okamoto, Satow, Hidaka & Akimoto, 1980). It is clear that the forces initiating bending originate largely from within the cardiac rudiment (Butler, 1952) and not from other embryonic structures, and the mechanisms controlling these events are gradually being elucidated (for reviews see Manasek, 1976; Manasek, 1981). 1 Author's address: Department of Anatomy, The University of Chicago, 1025 East 57th Street, Chicago, Illinois 60637, U.S.A.

2 236 A. NAKAMURA AND F.J. MANASEK The structure of the developing heart at these early stages is simple. There is an outer layer of differentiating myocytes (myocardium), a single layer of flat cells (endocardium), and an interposing proteoglycan-rich connective tissue, the cardiac jelly (Davis, 1924; Manasek, 1968; Manasek et al. 1973). The cardiac jelly has a unique set of properties that may be instrumental in mediating looping. It was shown that chemically isolated native cardiac jelly was able to undergo hydrostatic behaviour in vitro (Nakamura & Manasek, 1978a). Also, cardiac jelly contains a uniquely organized microfibrillar network which may permit rapid bending without extensive remodelling of its structure (Nakamura & Manasek, 19786; Nakamura, Kulikowski, Lacktis & Manasek, 1980; Hurle, Icardo & Ojeda, 1980). These properties, as well as its presence in large quantities in the developing heart wall, suggest significant physical involvement of cardiac jelly in the looping process. Nonetheless, the idea that there is direct involvement of cardiac jelly in heart morphogenesis remains largely inferential. The present study was designed to experimentally analyse the possible relationship of this compartment to the shape of the heart. Our rationale was to disrupt enzymically various components of the cardiac jelly and determine the effect on resulting heart shape. These experiments were done using microinjection techniques to introduce enzymes directly into the cardiac jelly of hearts in situ. The results are consistent with our hypothesis that glycosaminoglycans produce an internal pressure that provides turgor but that the utilization of this force in the morphogenetic process may be mediated by the proteinaceous components. Such components may include the fibrillar elements of cardiac jelly and the material which is involved in the interaction between the myocardium and its substrate. Collagen by itself, however, does not seem to be directly involved in the morphogenesis of the early chick embryonic heart. MATERIALS AND MBTHODS Hearts from stage 10- to stage 13 (Hamburger & Hamilton, 1951) chick embryos were used throughout this study. Enzyme assays Collagenase. Column-purified collagenase (Calbiochem, A-grade) and crude collagenase (Worthington Biochemical Co, CLS) were assayed qualitatively both for collagenolytic and non-specific proteolytic activity by using 14 C-labelled methylated acid-soluble calf-skin collagen or methyl-methaemoglobin (New England Nuclear) as substrates respectively. 14 C-labelled collagen was dissolved in the assay buffer (final reaction condition; 0-36 mm-cacl 2 ; 0-05 MTris-HCl, ph 7-5; unlabelled acid-soluble calf-skin collagen, 100/tg/ml; 14 C-labelled collagen, 0-1 /*Ci/10 /ig/m\), warmed to 37 C and the reaction begun by adding enzyme solution (final concentrations; pure collagenase, 10/^g/ml; or crude

3 The structural role of cardiac jelly 237 collagenase, 100/^g/ml) and incubating at 37 C with continuous agitation. The non-specific proteolytic activity assays of enzymes were performed in the similar manner as described above except the final concentration of substrates and enzymes ([ 14 C]methyl methaemoglobin, 0-1 /ici/4/tg/ml; unlabelled methaemoglobin, 96/*g/ml; pure collagenase, 20/tg/ml; or crude collagenase, 1 mg/ml). Reactions were stopped at 0 min, 15, 30 and 60 min by boiling for 5 min with an equal volume of 10 mm tris-hcl, ph 7-5; 2 % SDS, 0016 % beta mercaptoethanol, 0-2 mm EDTA. Digestion products were analysed on a 0-9 x 15 cm column of Bio Rad P-100 equilibrated with 5 mm Tris-HCl, ph 7.5; 1 % SDS, 0-008% beta mercaptoethanol, 0-1 mm EDTA and calibrated with blue dextran and 3 H 2 O. Hundredmicrolitre aliquots were loaded on the column and eluted with the same buffer at a rate of 5 ml/h. Radioactivity was determined in a Searle, mark III scintillation counter. Hyaluronidase. Hyaluronidase activity was determined qualitatively using newly synthesized radioactive glycosaminoglycans produced by embryonic hearts. Six hearts from embryos, stages 11 to 13, were dissected out and incubated for 3 h at 37 C in Tyrode's medium containing 500/^Ci/ml [ 3 H] glucosamine. Following incubation unincorporated glucosamine was washed out. Individual hearts were placed in serological tubes containing 50/tl of Tyrode's and incubated with 50/d of hyaluronidase (Ovine testicular, Type V, Sigma) solution (1 mg/ml in Tyrodes solution) for 30 min, 1 h and 2 h at 37 C. The reaction was stopped and hearts were solubilized as above except using 0-01 M phosphate buffer, ph 7-4, in place of 5 mm Tris-HCl. Digests were developed through a Bio-Rad P-4 column (0-9 x 60 cm) and radioactivity was measured as above. Injection of enzymes. Enzyme solutions were prepared as follows: Hyaluronidase and collagenases were dissolved in Tyrode's solution at concentrations of 1, 2, 5 or 10 mg/ml. Trypsin (pancreatic trypsin, A-grade, Calbiochem) was dissolved in N-HC1 as 1 mg/ml stock solution and then diluted with Tyrode's to a final concentration of 0-01 mg/ml, 0-02, mg/ml, 0-05 mg/ml or 0-1 mg/ml. Pronase (B-grade, Calbiochem) was dissolved in Tyrode's solution at 0-1 mg/ml. Mixtures of enzymes contained the following concentrations: hyaluronidase + collagenase, 1 mg/ml and 2 mg/ml respectively; hyaluronidase and trypsin, 1 mg/ml and 50 /*g/ml respectively. Enzyme solutions were loaded into a microinjection apparatus employing a glass microneedle. Explanted (New, 1955) embryos were placed under a dissecting microscope and the microneedle inserted into the cardiac jelly using a micromanipulator. Direct visualization permitted precise localization of the tip of the microneedle and it was possible to see the injected enzyme solution enter the cardiac jelly. The actual volume injected into a heart could not be precisely determined, but ranged from about /A. The uncertainty resulted from both back pressure and leakage from the heart.

4 238 A. NAKAMURA AND F. J. MANASEK Controls. Injection of Tyrode's medium without any enzymes was made. Sham injections were also made. A microneedle was inserted into the cardiac jelly but no fluid injected. Enzymes were also injected into the pericardial space. Isolated hearts were incubated in Tyrode's medium; Tyrode's + hyaluronidase (1 mg/ml) or Tyrode's containing bovine serum albumin (1 mg/ml). Recording. All cultured embryos were treated in a similar manner. Embryos were maintained at 37 C except during injection which was done at room temperature. The developing hearts were photographed before and after injection and at suitable times thereafter. Microscopy. After termination of the experiments embryos were processed for histological and cytological examination. After making two cuts on the splanchnopleure, one on each side of the heart, embryos were fixed by drop-wise application of full-strength Karnovsky's (1965) fixative. Then the entire embryo was dissected out and placed in fresh fixative. The total fixation time was min at room temperature. Embryos were post fixed with 1 % OsO 4 in 0-1 M cacodylate buffer, ph 7-4 for 1 h at room temperature and treated with tannic acid (Simionescu & Simionescu, 1976). The samples were dehydrated and embedded in Epon 812. Serial sections 1 or 2/<m thick, were cut and stained with toluidine blue for light microscopy. Thin sections were cut with a diamond knife and stained with uranyl acetate and lead citrate for electron microscopy. Sample preparation for scanning electron microscopy is the same as in our previous publication (Nakamura & Manasek, 19786). An Hitachi HSS-2 scanning electron microscope was used. RESULTS Control experiments In order to assess the morphogenetic consequences of enzymic degradation of the cardiac jelly, we did an extensive series of control procedures. Since, in all injection experiments, the microneedle has to penetrate both the pericardial membranes and the myocardial wall to gain access to the cardiac jelly, it is essential to determine what the effects of such an insult are on morphogenesis. Sham injections were performed, where the microneedle penetrated to the cardiac jelly and was withdrawn. In order to test for effects of injection per se, Tyrode's medium was injected into the cardiac jelly. In both cases heart shape was unaltered and development proceeded normally, indicating that the mechanical procedure of injection did not interfere with development. We next tested the possibility that the enzymes used might interfere with development or cause shape changes by acting directly on the myocardial cells. To test this possibility we injected enzymes into the pericardial cavity. Thus, the bare myocardial surface was in direct contact with enzymes and any direct effect on morphogenesis would have been apparent. These hearts developed

5 The structural role of cardiac jelly Fraction number Fig. 1. Effects of ovine testicular hyaluronidase on newly synthesized embryonic cardiac glycosaminoglycans in situ. Hearts (stages 11-13) were incubated with ph]glucosamine to label glycosaminoglycans. After washing away unincorporated label hearts were either incubated in control medium (A) or with testicular hyaluronidase (B). Incubation was stopped after 30 min and the hearts were solubilized and passed through Bio-Rad P-4. The control (A) shows four prominent peaks; the hyaluronidase digested specimen (B) shows additional radioactivity in fractions indicating that the native glycosaminoglycans are sensitive to the enzyme.

6 240 A. NAKAMURA AND F. J. MANASEK c 'V Fig. 2. The effects of testicular hyaluronidase microinjection into the cardiac jelly of an embryonic heart from a stage-12 embryo. The injected volume was in the range of 1 x 10~ 5 to 5 x 10" 5 ml of enzyme solution (2 mg/ml). (A) Pre-injection. (B) Post-injection. (C) 35 min post-injection. The heart shrank and became flaccid, but the general shape was maintained. Heart was beating but not forcefully. Arrow heads indicate margin of the heart. Bar indicates 0-1 mm. _ normally, indicating that direct enzymic action on the myocardial surface does not alter cardiac morphogenesis significantly. Injection experiments: hyaluronidase We first ascertained if native embryonic cardiac glycosaminoglycans could be digested in situ by our hyaluronidase preparation under physiological conditions. Radioactive, newly synthesized GAG from isolated hearts eluted from P-4 in four major well-defined peaks (Fig. 1 A) and 30 min digestion by hyaluronidase resulted in the elution profile shown in Fig. IB. The additional intermediate peaks in fractions indicate that the enzyme does degrade the native glycosaminoglycans. The injection of hyaluronidase directly into the cardiac jelly of embryos ranging from stage 10- to stage 13 (9-19 pairs of somites) resulted generally in shrinkage of the heart, a slight retardation of development and a general flaccid appearance. However, the degree of response varied widely, from no noticeable change to severe shrinkage. Younger hearts, such as these from stage-10 embryos, tended to show less shrinkage, but their subsequent looping process seemed somewhat retarded. In some cases after injection of hyaluronidase the heart shrinkage was extreme, with the myocardium becoming rough and dense. Although such hearts continued to beat for hours, the beat was not forceful and the hearts appeared flaccid. Their overall shape remained unchanged. Hearts with these severe shrinkages and flaccidity never recovered even during prolonged culture (Fig. 2). In contrast, hearts which responded mildly showed only transient, slight

7 The structural role of cardiac jelly 241 r Fig. 3. Light micrographs of cross sections of hearts. (A) Normal heart: myocardium and endocardium are separated by a thick acellular cardiac jelly layer (stage 12 + ). (B) Hyaluronidase-injected heart: the heart is characterized by the extremely narrow cardiac jelly layer and thick myocardium. The lumen is patent (stage 12 ). These features suggest that the shrinkage of the heart is due to the removal of cardiac jelly material and not lumenal collapse. M, Myocardium; E, endocardium; L, lumen; Cj, cardiac jelly. 2/*m thick, plastic sections, stained with 1% toluidine blue. Bar indicates 0-1 mm.

8 242 A. NAKAMURA AND F. J. MANASEK Fig. 4. Electron micrographs of hyaluronidase-injected hearts. (A) There are only small amounts of cardiac jelly matrix (arrows) remaining between the myocardium and endocardium. (B) There are numerous fibrous structures with different diameters within the narrow cardiac jelly. The distribution density of these materials is much higher than in the normal cardiac jelly. M, myocardium; E, endocardium; Cj, cardiac jelly. Stained with uranylacetate and lead citrate. Bars indicate 1 /an.

9 The structural role of cardiac jelly 243 Fig. 5. The effect of testicular hyaluronidase on isolated hearts. (A) Control: this heart of an embryo of stage 11 was incubated in Tyrode's medium and developed normally. A, O', A', 25', A", 135'. (B) Hyaluronidase treatment. This heart of a stage 11 embryo was incubated in testicular hyaluronidase solution (1 mg/ml in Tyrode's). Heart quickly shrank and became flaccid, but the overall shape was retained. B, O', B', 10', B", 115'. The orientation of isolated hearts could not be controlled since the hearts were suspended in the incubation medium. Bar indicates 0-1 mm. shrinkage and flaccidity, but during further incubation they recovered and proceeded with morphogenesis normally. Histological observations Cross-sections of normal hearts show the cardiac jelly as a wide clear space between the myocardium and endocardium (Fig. 3 A). The dorsal mesocardium is still relatively wide and filled with cardiac jelly. However, hearts severely shrunken as a result of hyaluronidase are markedly different. The most prominent difference is the narrowness of the cardiac jelly layer. In some cases the B"

10 244 A. NAKAMURA AND F. J. MANASEK 30C -.5 E Fraction number Fig. 6. Effect of purified collagenase on [ 14 C]methylated collagen substrate. The substrate alone elutes from P-100 in a single peak of large molecular weight in the void volume similar to 0' digestion. Essentially, all of the large molecular weight material has been digested by 60 min incubation. Digestion with crude collagenase produced the similar shift of the substrate peak (not shown). BD, Blue dextran r S Fraction number Fig. 7. Test for non-specific proteolytic activity of purified collagenase. [ 14 C]methyl- Met-Hb was incubated with enzyme. No appreciable degradation, as detected by elution from P-100, could be seen after 60 min incubation compared to 0 min. endocardium came very close to the myocardium as a result of loss of cardiac jelly (Fig. 3B). The lumen of the injected heart is usually large. Both endocardial and myocardial cells are bulging out into the lumen of the heart or into the pericardiac space respectively. These features suggested that the shrinkage of the heart was due primarily to the degradation and removal of cardiac jelly material, probably glycosaminoglycans. Electron-microscope examinations

11 The structural role of cardiac jelly r 0 min 60 min Fraction number Fig. 8. Test for non-specific proteolytic activity of crude collagenase. The preparation of crude collagenase used in our study contains a high level of non-specific proteolytic activity. After 60 min incubation [ 14 C]methyl-Met-Hb substrate was substantially degraded as determined by elution from P-100. Virtually all of the large-molecular-weight substrate has been degraded. confirmed this. The cardiac jelly layer is seen to be extremely narrow and more fibrous than normal (Fig. 4A, B). Another prominent structure, other than fibrils, in the narrow cardiac jelly space is electron-dense material of various sizes and shapes. The distribution density of these fibrous and electron-dense structures appeared much higher than in the normal heart. This suggests that these structures are compacted as the glycosaminoglycans are digested away by injected hyaluronidase and the cardiac jelly space collapses. Experiments with isolated hearts were also carried out. The results with isolated hearts were similar to those where hyaluronidase was injected into hearts in situ. Isolated hearts, incubated in Tyrode's medium with hyaluronidase (1 mg/ml), shrank rapidly and became flaccid, but shapes did not change (Fig. 5B). Incubation of isolated hearts in Tyrode's alone or in Tyrode's with 1 mg/ml Bovine serum albumin did not produce any effect after several hours of incubation and the hearts developed normally (Fig. 5 A). Therefore, the observed shrinkage and flaccid appearance of isolated hearts were enzymatic effects and not simple osmotic ones due to the presence of hyaluronidase. Collagenase injections Two different enzyme preparations were used, and each was tested for both collagenase activity and non-specific proteolytic activity. Enzyme solutions were incubated separately with [ 14 C]methyl met-haemoglobin and [ 14 C]methyl acid-soluble calf-skin collagen. Enzyme activity was indicated by production of smaller fragments which were displayed on Biogel P-100 elution pattern. Both crude and purified collagenase degraded radioactive collagen (Fig. 6). Purified

12 246 A. NAKAMURA AND F. J. MANASEK B \ Fig. 9. Effect of crude collagenase injection on heart shape. Enzyme was injected directly into the cardiac jelly of a cultured stage 11 embryo. The pre-injection heart shows normal heart morphology (A) as seen from the ventral surface. Immediately post injection (B) the morphology remains virtually identical. The arrowhead indicates the site of injection. After 10 min (C) the left side (arrows) has ballooned outward and the discrete left margin is no longer visible. By 30 min (D) the entire heart region has ballooned and the shape of the heart has been completely lost except around the anterior intestinal portal (A1P). Arrows outline the margin of the splanchnic mesoderm of foregut. Bar indicates 0-1 mm. D

13 The structural role of cardiac jelly 247 B Fig. 10. Cross section of embryos at the level of the heart. (A) Normal heart (stage 11). The heart has already rotated toward the right side of the body. (B) Crude-collagenase-injected heart (stage 11+). Splanchnic mesoderm of foregut and myocardium has detached from their substrata. The dorsal mesocardium is completely open and the myocardium has unfolded. Note the complete loss of heart shape and the myocardium is now an essentially flat sheet of cells. The effect of the enzyme reached beyond the heart region and even the paraxial mesenchyme is affected. Cj, Cardiac jelly; DM, dorsal mesocardium; ED, endoderm of foregut; E, endocardium; M, myocardium. Bar indicates 0-1 mm.

14 248 A. NAKAMURA AND F. J. MANASEK * *. Fig. 11. Electron micrograph of a portion of two adjacent myocytes of a crude collagenase injected heart similar to that of Fig. 10B. Developing intercalated disc (ICD) and myofibril (M) look normal even though the heart lost its shape. N, nucleus. Bar indicates 0-1 fim. collagenase had no detectable non-specific proteolytic activity (Fig. 7) but the crude enzyme did (Fig. 8). Injection of pure collagenase directly into cardiac jelly was without noticeable effect upon the shape of the heart and injected hearts developed normally. Crude collagenase, on the other hand, had dramatic effects upon the shape of the heart rudiment when injected. Injected hearts of stage-10 to stage-ll + embryos enlarged rapidly and the outlines of the heart rudiment became indistinct (Fig. 9). Ten to fifteen minutes after injection the entire pericardial region was swollen considerably except for the area very close to the anterior intestinal portal. The degree of swelling and of vagueness of the heart shape is agedependent. The response is more rapid and extreme in younger hearts. During the early stages of swelling, heartbeat was normal, but as the swelling proceeded the beating became irregular, especially where the swelling was extensive. In those extensively swollen areas fibrillation, or twitching was seen and subsequently the heart stopped beating completely.

15 Histological observations The structural role of cardiac jelly 249 Histological cross sections of embryos injected with crude collagenase (Fig. 10) show that the myocardium is still intact but has, in essence, unrolled. The crude collagenase detached part of the splanchnic rnesoderm as well as the myocardium from their substrata and simultaneously opened up the dorsal mesocardium. As a consequence there is now a continuous sheet of splanchnic mesoderm stretched across the ventral side of the embryo (Fig. 10B). The morphological distinction between myocardium, dorsal mesocardium and splanchnic mesoderm has been lost. The endocardium was left behind as a collapsed cell cord. Electron-microscopic observation: Myocardium The ultrastructure of the myocardium appeared largely normal. Cells were bound to each other by means of desmosomes and developing intercalated discs (Fig. 11), providing additional evidence that disaggregation had not occurred Electron-microscope observation of the cardiac jelly near the myocardium revealed very few recognizable structures. It appeared largely 'empty'. Near the endocardium we observed a few microfibrils. Normally the cardiac jelly would contain many microfibrils, especially in the dorsal mesocardium where they are associated with lamina-like material. The general impression of the cardiac jelly of crude collagenase-injected hearts is that there are much fewer visible inclusions and there is a complete disruption of the normal filament alignment (Fig. 12). Injection of other enzymes We injected other proteolytic enzymes. Pronase injection elicited responses virtually identical to those of crude collagenase but which were more rapid and extensive. The myocardium and splanchnic mesoderm detached from their substrata and heart shape was lost within 20 min. Trypsin injection did not cause noticeable changes and the heart continued normal morphogenesis. A combination of trypsin and pure collagenase did cause abnormal heart shapes in some embryos but this appeared to be the result of actual cell loss and not the result of detachment of the myocardium and splanchnic mesoderm from the substrate. The dorsal mesocardium appeared normal. Other combinations were tried and the results of the injection experiments are tabulated in Table 1.

16 250 A. NAKAMURA AND F. J. MANASEK Fig. 12. Scanning electron micrographs of freeze-fractured embryos at the level of the heart (views from rostral end). (A) Normal embryo (stage 11 ). Heart rotated toward right side and the tubular structure of the heart is clear. There are numerous radially oriented microfibrils within the cardiac jelly space. (B) Crude-collagenaseinjected embryo (stage 11). The myocardium and splanchnic mesoderm have detached from their substrata and unfolded completely. Endocardium is collapsed and appears as a cell cord on the endoderm of foregut. The microfibrillar network of cardiac jelly is completely destroyed. Compare these views to Fig. 10. Cj, Cardiac jelly; D, dorsal mesocardium; E, endocardium; ED, endoderm of foregut; F, foregut M, myocardium; Sp, splanchnic mesoderm. Bar indicates 0-1 mm.

17 The structural role of cardiac jelly 251 Table 1. Effects of enzyme injection on the shape of early developing chick embryonic hearts* Enzyme Concentration Effectsf Testicular hyaluronidase Pure collagenase Crude collagenase Pronase 2 mg/ml, 5 mg/ml, 10 mg/ml 1 mg/ml, 2 mg/ml, 5 mg/ml 2 mg/ml, 5 mg/ml, 10 mg/ml 0-1 mg/ml Trypsin 0-0! mg/ml, 0-02 mg/ml 0-05 mg/ml, 0-1 mg/ml Pure collagenase+ 2 mg/ml + 1 mg/ml testicular hyaluronidase Trypsin + testicular hyaluronidase Pure collagenase + trypsin 2-5 mg/ml+1 mg/ml Shrinkage and flabbiness but the shape was maintained. Age dependent (younger-less sensitive) No clearly noticeable effect 0-05 mg/ml + 1 mg/ml Some shrinkage Rapid swelling. Loss of heart shape Age dependent (younger - more sensitive) Rapid swelling. Loss of heart shape shape. Age dependent (Younger - more sensitive) No noticeable effect except a slight shrinkage Some shrinkage Some showed cell release * Nanogram quantity enzymes were injected into the cardiac jelly of stages 10 to 12 + chick embryonic hearts. t These effects were observed within an hour (mostly within min) after injection to avoid secondary effects. DISCUSSION In this study we considered the morphogenetic role of two general classes of matrix components, proteins and the carbohydrate moiety of glycosaminoglycans, in the early development of heart shape. The effects of disrupting either class of matrix molecules are dramatically different and these differences imply different functional roles. The experiments all involve the introduction of enzymes directly into embryonic extracellular matrix enclosed within an epithelial layer (the myocardium). In any such experiment it is essential to distinguish between two possible effects of the enzyme. The first is a direct effect of enzyme upon endogenous matrix substrate which in turn results in an alteration of shape. Secondly we must consider a possible direct effect of the enzyme upon cells, in this case myocardial cells. Tn such a case a morphogenetic response to injection might reflect a cellular response to enzyme and not a response to an altered matrix. Our control experiments indicate that the responses we detect are largely results of enzyme action on the matrix, since injecting enzymes into the pericardial cavity where it comes into direct contact with the developing myocytes but not

18 252 A. NAKAMURA AND F. J. MANASEK the underlying matrix has no detectable effect on the heart. Additionally, microscopic examination reveals that the changes of the heart do not result from collapse of the lumen, disruption of cytoarchitecture or pathological changes within the cells. Although we have ruled out direct effects via the apical membrane we cannot rule out a possible direct effect via the basal surface which cannot be tested. Hyaluronidase does not change the overall general shape of the heart but does cause shrinkage and flaccidity. The observed variability in response to hyaluronidase injection is probably due to the differences in the final amount of injected enzyme relative to glycosaminoglycans in the cardiac jelly space. In younger hearts especially, the volume of enzyme solution that can be injected without exploding the heart wall is limited by the small size of the heart. Furthermore, the wide dorsal mesocardium of early stages may permit extensive leakage of enzyme. In all stages the effect of enzymic degradation may be lessened further by the relatively high content of glycosaminoglycans and their continued rapid synthesis. The retention of heart shape after hyaluronidase injection shows that the myocardial layer has the ability to sustain its shape but not its size even if cardiac jelly glycosaminoglycans are disrupted. Earlier studies have shown that removal of myocardium leaves behind native cardiac jelly that also retains its original heart shape (Nakamura & Manasek, 1978a). Since in the present experiment we have, in effect, done the reverse, we can conclude that both acellular and cellular compartments with hyaluronidase-resistant matrix material have an intrinsic shape. This is not to say that these shapes are independent of each other since newly synthesized matrix probably conforms to the general shape of the myocardium and the myocardium is dependent also upon the matrix. The heart normally has an internal pressure that is high relative to the outside. This is demonstrated by the flaccidity and loss of turgor that follows hyaluronidase injection. Specifically, this suggests that the internal pressure is a function of the glycosaminoglycan (GAG) carbohydrate moieties. The flaccidity and loss of turgor are not the result of lumenal collapse because the lumen remains wide. These observations suggest that the normal tubular heart is a structure supported by the internal pressure of cardiac jelly. It may be expected that the internal pressure resulting from hydrated GAGs would increase continuously with development since GAGs are synthesized throughout this period (Manasek et al. 1973) and endogenous hyaluronidase activity is at undetectable levels (Nakamura, 1980). This, continued accumulation of GAG occurs. The presence of such an internal pressure in a developing system may be of profound importance in morphogenesis. The myocardial wall must contain this pressure but, as in any physical system under internal pressure, there is stress. This stress, if it is sufficient to exceed the elastic modulus of its container (in this case the myocardium), will result in a shape change. An analysis of the effects of this

19 777? structural role of cardiac jelly 253 stress and the regulation of the ensuing strain will be presented in another publication (Manasek et al. 1981). Non-specific proteolytic enzymes cause dramatic loss of heart shape, indicating that extracellular proteins are essential in maintaining the shape of the heart rudiment and in modulating its development. In particular, Pronase and crude collagenase caused the developing dorsal mesocardium to widen to the extent that the tubular form was completely lost. This widening of the dorsal mesocardium and the flattening out of the entire myocardium coincident with the loss of extracellular protein suggest the myocardium is normally under tension (as also suggested by the hyaluronidase experiment) and that protein moieties in the matrix assist in counteracting and in directing these forces. It is most likely that the system of matrix radial fibres (Nakamura & Manasek, 19786; Hurle, Icardo & Ojeda, 1980) are important in this process. These fibres which extend across the width of the cardiac jelly from endocardium to myocardium appear to be in tension. Previous studies of experimental deformation of the tubular heart (Lacktis & Manasek, 1978) showed that the heart can be deformed readily to 150% of its size and then becomes relatively resistant to further deformation. This levelling of the stress-strain curve was attributed to the presence of the radial fibre system which, when straightened, would resist further deformation. We propose that the in situ enzymic disruption of matrix fibre system permits a major shape change in response to internal pressure. The myocardial basal lamina is not yet complete at this early developmental stage (Manasek, 1968) and it is difficult to assess the extent of enzyme action on it. However, it seems that the basal lamina is not obligatory to the process of active shape change during looping at least in its early stage, because it is not yet fully developed and also because pure collagenase does not have any noticeable effect on the shape of the heart. These results are somewhat different from the case of the branching morphogenesis of mouse salivary gland (Banerjee, Cohn & Bernfield, 1977; Bernfield, Banerjee & Cohn, 1972; Bernfield & Banerjee, 1972), where the epithelial basal lamina, especially the glycosaminoglycans associated with it, is essential for the active epithelial shape change, presumably by providing an extracellular scaffolding in the presence of mesenchyme (Cohn, Banerjee & Bernfield, 1977). The difference may be related to the fact that in the early developing heart cardiac jelly acts as an active force generator for morphogenesis and the epithelium (myocardium) deforms rather passively in the complete absence of mesenchymal cells. In this system the poorly developed myocardial basal lamina may be advantageous since the myocardium, being less rigid, is more deformable. Collagen appears to play a minor morphogenetic role in the early embryonic heart. Purified collagenase, with no detectable non-specific proteolytic activity did not alter the heart shape. Although collagen is being synthesized during this period (Johnson, Manasek, Vinson & Seyer, 1974), cross-banded fibrils are extremely scarce. Evidence has been accumulating in other systems, notably 9 EMB 65

20 254 A. NAKAMURA AND F. J. MANASEK developing salivary gland, that collagen deposition is necessary for morphogenesis (Spooner & Faubion, 1980). In this context it is important to point out that the events of cardiac morphogenesis that we are investigating in the present study occur much earlier in embryonic development when there are not yet significant amounts of collagen. It is difficult to assess the negative results obtained when trypsin is injected alone. Either trypsin is too selective and does not degrade structurally important matrix components or it is inactivated in situ. The latter is unlikely since when injected in combination with purified collagenase the myocardium begins to dissociate into individual cells. Since pure collagenase alone does not do this the trypsin must be active. It is also possible that, in situ, trypsin activity is reduced to the level where its effects are too slight to alter morphogenetic events. Certainly one would expect a morphogenetic consequence to the proteolysis of the fibronectin present in the heart (Waterman & Balian, 1980) especially in light of its known sensitivity to trypsin (Hynes, 1973). Early cardiac shape changes are deformative changes (Manasek, Burnside & Waterman 1972; Lacktis & Manasek, 1978; Nakamura et al. 1980). This means that physical forces are responsible, but to date the origin and regulation of these forces have remained elusive. The present study addresses this problem and the results implicate the extracellular matrix. Cardiac jelly is a product largely of the myocardium (Manasek, 1976; Johnson et al. 1974; Manasek et al. 1973). Thus, biochemical events such as matrix synthesis, turnover and matrix filament formation must be viewed from a biomechanical perspective. It is tempting to propose that the deformations that characterize looping are the results of mechanical forces organizing within the matrix and directed by both matrix anisotropy and the myocardium itself. Collectively the data suggest that glycosaminoglycan carbohydrate moieties function to provide filler or 'stuffing' but spatial relations are maintained by protein components. Scanning electron microscopy as well as classical staining techniques (Nakamura & Manasek, 1978a, b; Markwald, Fitzharris, Bank & Bernanke, 1978; Hurle et al. 1980) has demonstrated the flbrillar form of some of the cardiac matrix proteins. We can view the system as a set of guy wires holding cells and tissues in their relative positions against an expansive force of glycosaminoglycans. This dynamic equilibrium can be altered, as we have done, by exogenous factors or by some endogenous means such as selective proteolysis of synthesis of matrix components in an ordered manner to effect morphogenesis. Such a model, where epithelial deformation depends upon force derived from matrix hopefully could be generalized to the young embryo as a whole. This work was supported by grant HL from the National Heart, Lung and Blood Institute, National Institutes of Health.

21 The structural role of cardiac jelly 255 REFERENCES BANERJEE, S. D., COHN, R. H. & BERNFIELD, M. R. (1977). Basal lamina of embryonic salivary epithelia: production by the epithelium and role in maintaining lobular morphology. J. Cell Biol. 73, BERNFIELD, M. R. & BANERJEE, S. D. (1972). Acid mucopolysaccharide (glycosaminoglycan) at the epithelial-mesenchymal interface of mouse embryo salivary glands. J. Cell Biol. 52, BERNFIELD, M. R., BANERJEE, S. D. & COHN, R. (1972). Dependence of salivary epithelial morphology and branching morphogenesis upon acid mucopolysaccharide-protein (proteoglycan) at the epithelial surface. J. Cell Biol. 52, BUTLER, J. K. (1952). An experimental analysis of cardiac loop formation in the chick. Master's Thesis, University of Texas. COHN, R. H., BANERJEE, S. D. & BERNFIELD, M. R. (1977). Basal lamina of embryonic salivary epithelia: Nature of glycosaminoglycans and organization of extracellular materials. J. Cell Biol. 73, DAvrs, C. L. (1924). The cardiac jelly of the chick embryo. Anat. Ree. 27, HAMBURGER, V. & HAMILTON, H. (1951). A series of normal stages in the development of the chick embryo. /. Morph. 88, HURLE, J. M., ICARDO, J. M. & OJEDA, J. L. (1980). Compositional and structural heterogenicity of the cardiac jelly of the chick embryo tubular heart: a TEM, SEM and histochcmical study. J. Embryol. exp. Morph. 56, HYNES, R. O. (1973). Alteration of cell-surface proteins by viral transformation and by proteolysis. Proc. nutn. Acad. Sci. U.S.A. 70, JOHNSON, R. C, MANASEK, F. J., VINSON, W. C. & SEVER, J. M. (1974). The biochemical and ultrastructural demonstration of collagen during early heart development. Devi Biol. 36, KARNOVSKY, M. J. (1965). Formaldehyde-glutaraldehyde fixative of high osmolality. J. Cell Biol. 27, 137a. LACKTIS, J. W. & MANASEK, F. J. (1978). An analysis of deformation during a normal morphogenetic event. In Morphogenesis and Malformation of the Cardiovascular System (ed. G. C. Rosenquist and D. Bergsma), pp New York: Alan R. Liss. MANASEK, F. J. (1968). Embryonic development of the heart. I. A light and electron microscopic study of myocardial development in the early chick embryo. J. Morph. 125, MANASEK, F. J. (1976). Heart development: interactions involved in cardiac morphogenesis. In The Cell Surface in Animal Embryogenesis and Development (ed. G. Post and G. Nicholson), pp Amsterdam: North Holland. MANASEK, F. J. (1981). Determinants of heart shape in early embryos. Fedn. Proc. (in the Press). MANASEK, F. J., KULIKOWSKI, R. R., NAKAMURA, A., NGUYENPHUC, Q. & LACKTIS, J. W. (1981). The mechanisms causing and regulating bending and rotation of the embryonic heart. (In preparation.) MANASEK, F. J., REID, M. VINSON, W., SEYER, J. & JOHNSON, R. (1973). Glycosaminoglycans synthesis by the early embryonic chick heart. Devi Biol. 3f>, MANASEK, F. J., BURNSIDE, M. B. & WATERMAN, R. L. (1972). Myocardial cell shape changes as a mechanism of embryonic heart looping. Devi Biol MARKWALD, R. R., FITZHARRIS, T. P., BANK, H. & BERNANKE, D. H. (1978). Structural analysis on the matrical organization of glycosaminoglycans in developing endocardial cushions. Devi Biol. 62, NAKAMURA, A. (1980). Cardiac hyaluronidase activity of chick embryos at the time of endocardial cushion formation. J. molec. cell. Cardiol. 12, NAKAMURA, A. & MANASEK, F. J. (1978a). Experimental studies of the shape and structure of isolated cardiac jelly. J. Embryol. exp. Morph. 43, NAKAMURA, A. & MANASEK, F. J. (1978/)). Cardiac jelly fibrils: Their distribution and organization. In Morphogenesis and Malformation of the Cardiovascular System (ed. G. C. Rosenquist and D. Bergsma), pp New York: Alan R. Liss. 9-2

22 256 A. NAKAMURA AND F. J. MANASEK NAKAMURA, A., KULIKOWSKI, R. R., LACKTIS, J. W. &MANASEK, F. J. (1980). Heart looping: A regulated response to deforming forces. In Etiology and Morphogenesis of Congenital Heart Disease (ed. R. van Praagh and A. Takao), pp New York: Futura. NEW, D. A. T. (1955). A new technique for the cultivation of the chick embryos in vitro. J. Embryol. exp. Morph. 3, OKAMOTO, N., SATOW, Y., HIDAKA, N. & AKIMOTO, N. (1980). Anomalous development of the conotruncus in Neutron-Irradiated rats. In Etiology and Morphogenesis of Congenital Heart Disease (ed. R. van Praagh and A. Takao), pp New York: Futura. SIMIONESCU, N. & SIMIONESCU, M. (1976). Galloylglucose of low molecular weight as mordant in electron microscopy. I. Procedure and evidence for mordanting effect. J. Cell Biol. 70, SPOONER, B. S. & FAUBION, J. M. (1980). Collagen involvement in branching morphogenesis of embryonic lung and salivary gland. Devi Biol. 11, WATERMAN, R. E. & BALIAN, G. (1980). Indirect immunofluorescent staining of fibronectin associated with the floor of the foregut during formation and rupture of the oral membrane in the chick embryo. Am. J. Anat. (in the Press). {Received 25 March 1981, Revised 30 April 1981)