J. Embryo/, exp. Morph. Vol. 65, pp. 235-256, 1981
Printed in Great Britain © Company of Biologists Limited 1981
235
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.
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 1 0 - 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 14C-labelled
methylated acid-soluble calf-skin collagen or methyl-methaemoglobin (New
England Nuclear) as substrates respectively. 14C-labelled collagen was dissolved
in the assay buffer (final reaction condition; 0-36 mM-CaCl2; 0-05 MTris-HCl,
pH 7-5; unlabelled acid-soluble calf-skin collagen, 100/tg/ml; 14C-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
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 ([14C]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 [3H]
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 0-001 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 0-05-0-01 /A. The uncertainty
resulted from both back pressure and leakage from the heart.
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
20-25 min at room temperature. Embryos were post fixed with 1 % OsO4 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
The structural role of cardiac jelly
239
15 -
30
40
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
22-37 indicating that the native glycosaminoglycans are sensitive to the enzyme.
240
A. NAKAMURA AND F. J. MANASEK
'V
c
_
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 22-37 indicate that the enzyme does degrade the native
glycosaminoglycans.
The injection of hyaluronidase directly into the cardiac jelly of embryos
ranging from stage 1 0 - 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
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.
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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.
The structural role of cardiac jelly
243
B"
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
244
A. NAKAMURA AND F. J. MANASEK
30C -
.5
E
200
100
10
20
30
40
50
Fraction number
60
70
80
Fig. 6. Effect of purified collagenase on [14C]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
2000.
300
r
S 200
-
20
30
40
50
60
70
80
Fraction number
Fig. 7. Test for non-specific proteolytic activity of purified collagenase. [14C]methylMet-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
The structural role of cardiac jelly
245
0 min
60 min
300 r
70
80
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 [14C]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 [14C]methyl met-haemoglobin and [14C]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
246
A. NAKAMURA AND F. J. MANASEK
B
\
D
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.
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.
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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.
The structural role of cardiac jelly
249
Histological observations
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.
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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.
251
The structural role of cardiac jelly
Table 1. Effects of enzyme injection on the shape of early developing chick
embryonic hearts*
Enzyme
Concentration
Testicular hyaluronidase
2 mg/ml, 5 mg/ml, 10
mg/ml
Pure collagenase
1 mg/ml, 2 mg/ml,
5 mg/ml
2 mg/ml, 5 mg/ml,
10 mg/ml
Crude collagenase
Pronase
0-1 mg/ml
Trypsin
0-0! mg/ml, 0-02 mg/ml
0-05 mg/ml, 0-1 mg/ml
2 mg/ml + 1 mg/ml
Pure collagenase+
testicular hyaluronidase
Trypsin + testicular
0-05 mg/ml + 1 mg/ml
hyaluronidase
Pure collagenase + trypsin 2-5 mg/ml+1 mg/ml
Effectsf
Shrinkage and flabbiness but the
shape was maintained. Age
dependent (younger-less
sensitive)
No clearly noticeable effect
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 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 15-20 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
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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
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
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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 13831 from the National Heart, Lung and Blood
Institute, National Institutes of Health.
The structural role of cardiac jelly
255
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{Received 25 March 1981, Revised 30 April 1981)