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/. Embryol. exp. Morph. Vol. 43, pp. 167-183, 1978
Printed in Great Britain © Company of Biologists Limited 1978
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Experimental studies of the shape and structure
of isolated cardiac jelly
By A T S U Y O N A K A M U R A 1 AND F R A N C I S J. M A N A S E K 1
From the Department of Anatomy, The University of Chicago
SUMMARY
The properties of the early chick embryonic heart cardiac jelly were studied. The cells of
the heart were removed by sequential treatments with calcium magnesium-free medium; the
same medium containing 5 mM EDTA; and aqueous 0 1 % deoxycholate. The transparent,
naked cardiac jelly retained the original shape and size of the untreated original heart when
immersed in physiological ionic strength medium. Its size and shape responded to changes
in the ionic strength of the surrounding media. Alcian blue, cetylpyridinium chloride and
testicular hyaluronidase abolished the ability of the jelly to respond to ionic strength changes.
Electron microscope examination of the negatively stained spread cardiac jelly revealed an
extensive network of collagenous fibrils and fine filaments with some amorphous adhering
material. Treatment with testicular hyaluronidase removed much of the amorphous material
and improved the details of the filaments. These results suggest that glycosaminoglycans play
an important part in the hydration of the cardiac jelly and that the stability of the cardiac
jelly shape is mainly due to the filamentous network and their possible interactions with
macromolecules of the cardiac jelly matrix. It is suggested that the factors that control the
deposition of the connective tissue macromolecules and the assembly of the filamentous
network are significant factors which influence the morphogenesis of the early embryonic
heart.
INTRODUCTION
During early developmental stages, the heart has an extensive connective
tissue layer situated between the myocardium and endocardium. This extracellular material is called 'cardiac jelly' (Davis, 1924), and is particularly
prominent at the time the heart rudiment bends to the right, a process called
looping (for discussion see Manasek, 1976«).
The cardiac jelly contains glycosaminoglycans as shown histochemically
(Barry, 1951 ; Markwald & Adams Smith, 1972; Ortiz, 1958). Later biochemical
and ultrastructural studies revealed that the cardiac jelly contains three major
components, glycosaminoglycans, glycoproteins and collagen (Gessner &
Bostrom, 1965; Gessner, Lorincz & Boström, 1965; Johnson, Manasek,
Vinson & Seyer, 1974; Manasek et al. 1973; Manasek, 1976&). The presence of
these components suggested various possible physiological roles of the cardiac
jelly such as the formation of a microenvironment controlling the passages of
1
Authors' address: Department of Anatomy, The University of Chicago, 1025 East
57th Street, Chicago, Illinois 60637, U.S.A.
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A. N A K A M U R A A N D F. J. M A N A S E K
substances (Manasek, 1975). Earlier, Barry (1948) and Patten, Kramer &
Barry (1948) had suggested a functional significance of cardiac jelly as a viscoelastic element effecting diastolic rebound.
Recent studies (Manasek, 1916b; Markwald, Fitzharris & Adams Smith,
1975 a) have suggested that cardiac jelly is not simply a solution of macromolecules. Different solute molecules such as glycosaminoglycans and fucosylated glycoproteins interact to form exceedingly large complexes (Manasek,
1977). This observation suggested that the large components of cardiac jelly
might not be able to translocate freely relative to each other. If this is true then
one would expect to be able to demonstrate that the cardiac jelly compartment
has an intrinsic shape that is not disrupted readily.
The experiments we report in this paper explore these properties of cardiac
jelly. We have succeeded in isolating the cardiac jelly from single isolated
embryonic hearts and have shown that it retains its shape even in the absence
of the myocardial cell layer. Further, we examined the effects of differing ionic
strengths on the shape of the cardiac jelly and found that it can be made to swell
reversibly, without losing its shape by alternating between a low and physiological ionic strength environment. These findings have enabled us to propose
a structural morphogenetic role for cardiac jelly during early heart development.
MATERIALS AND METHODS
Isolation of embryonic heart
Hearts were collected from stages (Hamburger & Hamilton, 1951) stage 10 +
to 17 - chick embryos (11-30 pairs of somites). After carefully removing the pericardial splanchnopleure with fine glass needles, the heart was removed by
cutting the cephalic end of the bulbus arteriosus and, caudally, the omphalomesenteric veins. Some of the early embryonic stage hearts often included
a small part of the foregut. The isolated hearts were washed several times with
Tyrode's medium.
Experimental manipulation of isolated hearts
Isolated hearts were placed individually in glass troughs measuring 55 x 10 x
1 mm containing Tyrode's medium. A cover-glass (22 x 30 mm) was laid over
the trough. There was ample space for the heart to float between the cover-glass
and the bottom of the trough. Solutions were introduced into one open end of
the trough and withdrawn from the other end with a piece of paper. All of the
operations were carried out under a dark-field light microscope and recorded
with Polaroid film. During the exchanges of solutions great care was necessary
to prevent loss of the sample.
Structure of isolated cardiac jelly
169
Experimental manipulation of intact hearts
The Tyrode's medium in the trough was replaced with distilled water (DW)
photographs were taken at various time intervals. Then DW was exchanged
with 0-1 M-NaCl, 5 m M sodium phosphate buffer, pH 7-4, and pictures were
taken. After repeating this sequence with 0-1 M-NaCl solution or with Tyrode's,
1 % cetylpyridinium chloride (CPC) was added to the trough.
Removal of cell layers
(a) 0-1 % Deoxycholate {DOC)
0-1% DOC (Na-salt, Sigma Chemical Co., St Louis) was prepared from
10% stock aqueous solution. Although it is not necessary, the isolated heart
was washed with 10 mM phosphate buffer, pH 8-0, before being placed in the
trough. 0-1 % DOC was introduced into the trough and the effects were photographed. After 0-5-2 h of treatment, DOC was replaced sequentially with DW,
0-1-0-15 M-NaCl, DW, 0-1-0-15 M-NaCl. Some of the DOC treated samples
were further treated with 0-8 % Alcian blue, 1 % CPC or testicular hyaluronidase
(THyase; 1 mg/ml in 0-15 M-NaCl, 0 1 M sodium phosphate buffer, pH 5-3) for
1 h at 37 °C.
(b) Calcium magnesium-free medium (CMF) - CMF.EDTA
In this procedure the intention was to reduce the adhesion between cells and
between cells and connective tissue. Isolated hearts were washed with CMF
(NaCl 8 g, KCl 0-2 g, NaH 2 P0 4 -H 2 0 0-05 g, NaHC0 3 1 g, glucose 2 g, per
liter; Moscona, 1952) and incubated in the same solution for 25 min at 37 °C.
This was followed by CMF.EDTA (5 mM) for 25 min at 37 °C. After a brief
rinse with DW, the heart was treated with 0-1% DOC at room temperature
while mechanically shaking the trough. This treatment was continued for
25 min to 1-5 h. Then the sample was bathed sequentially with DW and
0-1-0-15 M-NaCl, 5 mM sodium phosphate, pH 7-4. Some of the samples were
treated with 0-8 % Alcian blue, or 1 % CPC, or THyase.
The initial CMF treatment can be omitted without any noticeable effect on
the results. The concentration of EDTA in CMF. EDTA should be above 1 mM
since the removal of cell layers is more difficult at or below 2 mM EDTA. The
transparent, barely visible cardiac jelly was negatively contrasted by adding
India ink to the medium and photographed with a bright-field light microscope.
Electron microscopy
DOC-treated (0-1 % DOC) samples and also the bare cardiac jelly were fixed
with full strength Karnovsky's (1965) fixative for 5 min at 4 °C and then with
the same fixative containing 0-8% Alcian blue. The total fixation time was
20 min. Samples were washed with 0-1 M cacodylate buffer pH 7-4 for 30 min
at 4 °C and were post-fixed with 1 % Os0 4 in the same buffer for 1 h at 4 °C.
170
A. N A K A M U R A A N D F. J. M A N A S E K
Dehydration was carried out through graded ethyl alcohol and samples were
embedded in Araldite 502. Sections were cut on a Sorval MT-2 microtome with
a diamond knife. Sections were mounted on copper grids, double stained with
alcoholic uranyl acetate and lead citrate.
The trough is not convenient for preparing a number of naked cardiac jelly
preparations simultaneously. Multiwell tissue culture plates (Falcon, no. 3008)
are more suitable for this purpose. The cell layers of the heart were removed by
the same treatment as before, but in this case samples were transferred from
one well to another with a Pasteur pipette while they were being observed with
a dark-field dissecting microscope. The naked cardiac jelly in DW was picked
up with a Pasteur pipette with a minimal amount of fluid and was mixed with
a drop of 0-3-0-5% bovine serum albumin (BSA) in a Microtest Terasaki tissue
culture plate (Falcon, no. 3034) to improve the hydrophilicity of supporting
film. The sample was picked up with a small loop of copper wire and mounted
on a carbon enforced formvar or collodion film coated copper grid. Some of the
samples mounted without BSA were digested with THyase (1 mg/ml) for 1 h
at 37 °C prior to negative staining. Negative staining was done with 2 %
aqueous uranyl acetate. All samples were examined in RCA-EMU 4 electron
microscope operated at 50 kV.
Planimetry
Images of cardiac jelly were carefully cut out of photographic enlargements,
desiccated under vacuum and weighed.
RESULTS
Studies on the intact heart
The freshly dissected heart maintained its shape throughout the initial
handling period, during which time it was removed from the embryo and
placed in the trough. Under the dark-field microscope the untreated freshly
isolated embryonic heart appeared to be covered with numerous brightly
contrasting, tightly packed granules. When the Tyrode's medium was exchanged
with distilled water, the heart swelled rapidly (Fig. 1). Maximum size was
reached within 5-10 min, after which the heart gradually became somewhat
smaller again. The rapid swelling was accompanied by the loss from the heart
of many of the granular surface particles, probably as a result of cell lysis.
Replacement of the DW with 0-1-0-15 M-NaCl solution restored the heart to
nearly its normal size. Alternation of DW and salt solution caused the heart to
undergo swelling and shrinking cycles that could be repeated at least twice. The
ability to swell and shrink in response to medium changes was abolished by the
addition of a few drops of 1 % CPC. This was accompanied by marked permanent shrinkage of the specimen.
Structure of isolated cardiac jelly
Fig. 1. Effects of ionic strength on the isolated early chick embryonic heart (Jl
somites). (A) The dissected heart. Distilled water 0 min. (B) Distilled water 5 min.
Heart swelled extensively with simultaneous removal of granular material. (C) Distilled water 30 min. Note the increased loss of granular material and concomitant
increase in cardiac jelly transparency. (D) 01 M-NaCl, 5 rriM sodium phosphate,
pH 7-4. The heart shrank to its nearly normal size and shape, but some cellular
materials remained. (E) Distilled water 30 min. The heart swelled again as salt was
washed out. (F) Tyrode's solution 5 min. The heart shrank again to approximately
the normal size and shape. Dark-field light micrographs. Scale 1 mm. x 35.
171
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A. NAKAMURA AND F. J. MANASEK
Studies on cardiac jelly
(a) Effects of 0-1% deoxycholate (DOC)
Within a few minutes after replacement of the Tyrode's solution with 0-1 %
DOC, the myocardial layer became more transparent and started swelling
(Fig. 2). The entire heart became very soft. Simultaneously, a number of
particles began to fall off from the myocardial layer. The size of the heart
increased, reaching about twice that of the original heart. After 30 min in
0-1 % DOC solution, a large number of particles had been shed by the myocardium into the medium. Prolonged 0-1 % DOC treatment (up to 1 h), and
subsequent shaking, DW and salt solution treatment (0-1-0-15 M-NaCl)
removed most of the granular material from the myocardial layer, although
some material still remained.
Electron microscopic examination of the 0-1 % DOC-treated heart showed
clearly that there were still many myofibrillar and nuclear remnants in the
myocardial and endocardial layers (Fig. 3). The myofibrils (Fig. 3, M), although
grossly disorganized, were still recognizable. The intercalated discs (Fig. 3)
were relatively intact, despite the loss of large amounts of membranes.
Replacement of 0-1 % DOC with DW caused further swelling of the heart
(Fig. 2C). Although the general shape of the original heart still could be
detected, some parts of the swollen structure appeared deformed. Salt solution
had the same effect on the DOC-treated heart as it did on the intact heart, and
caused shrinkage. Cyclic swelling and shrinking could also be repeated at least
twice by alternating DW and 0-1 M-NaCl (Fig. 2C-E). It is important to point
out that the shape of the heart after shrinking was very similar to that of the
freshly dissected heart (Fig. 2A, D).
If, following incubation with 0-1 % DOC, the specimens were exposed to
Alcian blue (0-8 %), CPC (less than 1 %) or testicular hyaluronidase they
shrank markedly and no longer changed their dimensions in response to
changes in salt concentration (Fig. 2F).
(b) CMF-CMF.EDTA-0 1 % DOC treatment
An attempt was made to effect complete myocardial removal. Since it is
common procedure to use CMF medium with or without EDTA in loosening
up the intercellular junctions (Moscona, 1952; Muir, 1966), the dissected heart
was incubated with CMF and CMF.EDTA (above 2mM-EDTA) prior to
DOC treatment.
The effects of CMF-CMF.EDTA treatment became apparent during the
subsequent 0-1 % DOC treatment (Fig. 4). Small patches of flocculent material
with granules came off the heart and left transparent areas on the surface. The
removal of this material from the heart was facilitated by vigorous shaking of
the trough or by flushing out the DOC with DW and then with 0-1-0-15 M-NaCl
while shaking the trough. In the latter case the swollen heart shrank to almost
Structure of isolated cardiac jelly
173
Fig. 2. Effects of the 01 % deoxycholate treatment on the isolated early chick
embryonic heart (14 somites). (A) The dissected heart. Deoxycholate (DOC)
0 min. (B) DOC 60 min. The heart swelled and most of the granular materials were
more effectively removed than they were by distilled water. (C) Washed with distilled
water. Some cellular materials were released. (D) 0-15 M-NaCl, 5 mM sodium
phosphate, pH 7-4, 5 min. The swollen heart quickly shrank and returned to its
original shape and size. (E) Distilled water 10 min. The heart swelled again and
more cellular materials were released. The degree of swelling decreased gradually
with repeated cycles. (F) 1 % cetylpyridinium chloride. The heart shrank extremely
and irreversibly. Dark field light micrographs. Scale 1 mm. x 35.
EMB 43
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A. NAKAMURA AND F. J. MANASEK
Fig. 3. Electron micrograph of the DOC-treated heart. 01 % DOC treatment
followed by distilled water and salt solution caused the disruption of myocardial
cells. Although a large part of membranous structures are removed, disorganized
myofibrils (M) and intercalated discs (arrow) are still present. Scale 1 /im. x 28000.
physiological shape and size (Fig. 4E). This shrinkage forced out the endocardial
cell debris also, which was otherwise trapped in the lumen of the heart. Even
after overnight exposure to DW, the swollen heart was able to shrink when
placed in salt solution. During the course of the removal of the myocardial
layer, the cells along the dorsal mesocardium came oif most easily (Fig. 4C, D).
Conventional electron microscopic examination of the resulting transparent
material revealed that the myocardium, as well as endocardium, was almost
completely removed from the cardiac jelly (Fig. 5). Thus, the transparent
material remaining after the complete removal of the myocardial flocculent
materials was undoubtedly the naked cardiac, jelly itself (Fig. 5, inset). The
cardiac jelly contained electron-dense materials of various sizes (up to 111 nm
thick). Most of this material appeared amorphous, but sometimes some thin
filaments (6-10 nm) were seen (Fig. 5, arrowheads).
Spread and negatively stained cardiac jelly showed a completely different
picture. The negatively stained cardiac jelly preparations were often too thick
to reveal details. However, structure could be discerned in well spread peripheral
regions of the preparations (Fig. 6, 7). There were numerous fibrils running in
various directions. The fibrils were extremely long and relatively straight, but
Structure of isolated cardiac jelly
175
Fig. 4. Removal of the cell layers from the isolated chick embryonic heart (16 somites)
by calcium magnesium-free medium (CMF)-CMF. EDTA-01 % DOC treatment.
(A) Dissected heart. (B) CMF.EDTA 10 min at 37 °C. No appreciable change is
seen. (C) 01 % DOC 30 min. Heart swelled and cells along the dorsal mesocardium
were removed. (D) 0-1 % DOC 1-5 h. Heart swelled further and patches of myocardial cells began to come cff. (E) India ink. Naked cardiac jelly is virtually invisible
using either bright- or dark-field illumination and can be visualized best by means
of negative contrast, in this case using dilute India ink. Note that the cardiac jelly
has the shape and size of the original heart (A). A small remnant of cellular material
is stained dark by india ink. Dark field light micrographs except (F), which is bright
field. Scale 1 mm. x 36.
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A. NAKAMURA AND F. J. MANASEK
Fig. 5. Electron micrograph of the naked cardiac jelly (27 somites). No cellular components are discernible. The jelly appeared to be a mixture of varied sized cross or
obliquely cut electron-dense rods and some granular material. Fine filamentous
materials often interconnect these rods (arrows). Frequently filamentous substructures
can be seen in the rods (arrowheads). Scale 1 /tm. x 37200. Inset: dark field light
micrograph of the naked cardiac jelly. Scale 01 mm. x 48. The naked cardiac jelly
was prepared by the CMF-CMF. EDTA method, and was fixed with Karnovsky's
fixative for 5 min followed with Karnovsky-0-8 % Alcian blue fixative.
they did not appear to be entangled extensively in spite of their length (Figs. 6 A,
7 A). They could be roughly classified into two groups based on the presence or
absence of cross-banding pattern. Cross-banded fibrils were uniformly about
30 nm thick throughout their length. They often showed some adhering
materials which tended to obscure the details of the cross-banded pattern
(Fig. 6B). Testicular hyaluronidase treatment of the mounted sample prior to
negative staining removed most of the adhering materials (Fig. 7) and greatly
enhanced detail. The periodicity of the cross-banding pattern was about 65 nm,
and the alternating light and dark band pattern was characteristic of fibrillar
collagen (Fig. 7B). The fibrils frequently appeared side by side in a parallel
fashion, but their cross-banding pattern was not necessarily in register. The
apparent number of thesefibrilsincreased with embryo age. Filaments without
cross striations were thinner and had varying diameters of up to 10 nm. Some
of these thin filaments had globular clumps of material associated with them,
spaced about 90-100 nm along the filament (Fig. 7B). The regularity of the
Structure of isolated cardiac jelly
Fig. 6. Electron micrographs of a negatively stained spread cardiac jelly. Spread
cardiac jelly shows numerous filaments of varying diameters. They are very long
and show some adhering substances which tend to obscure their detail. Negatively
stained with 2 % aqueous uranyl acetate. Scale 1 [im. (A) x 6160; (B) x 18100.
111
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A. NAKAMURA AND F. J. MANASEK
Fig. 7. Electron micrographs of testicular Hyaluronidase treated spread cardiac
jelly. Much of the adhering substance is removed, revealing filament detail (7 A).
There are two major types of filaments (7B), a thin type (up to 10 nm) showing
globular material at regular intervals (90-100 nm) and another which is typical
fibrillar collagen with characteristic cross-banding (7B). Negatively stained with
2 % aqueous uranyl acetate. Scale 1 /tm (A) and 0-1 /*m (B). (A) x 8800; (B) x 66000.
periodic association of the globules with filaments suggests that this association
is not an artifact.
After complete removal of the myocardial layer, the remaining material was
transparent. It could not be seen by transmitted light microscopy and its
outlines could only barely be perceived by dark field microscopy. The only way
it could be studied, visually or photographically, was to outline it negatively
by flooding the trough with dilute India ink and examining the negative image
(Fig. 8). Despite removal of the cell layers, the shape of the remaining material
was very similar to that of the originally dissected heart. The naked cardiac
jelly retained the ability to swell and shrink in response to changes in salt
concentration. Although this response was much less than that exhibited by
specimens containing either complete (Fig. 1) or partial (Fig. 2) cell layers, it
was still clearly demonstrable (Fig. 8). Because of the smaller response, the
changes shown by the naked cardiac jelly were measured by planimetry.
Distilled water (Fig. 8B) resulted in an increase in image area to 1-6 times that
of the original heart (Fig. 8 A). The swollen size was restored to the original size
Structure of isolated cardiac jelly
t*
Fig. 8. Cyclic response of the naked cardiac jelly (20 somites) to changes in ionic
strength. (A) Calcium magnesium-free medium 0 min. (B) Distilled water-India
ink. Just after removal of the myocardium. Note the swollen size of the jelly.
(C) 0-1 M-NaCl, 5 mM Na-phosphate, pH 7-4 India ink. The cardiac jelly shrank to
its original size and shape. (D) Distilled water-India ink 20 min. The cardiac jelly is
somewhat swollen. (E) Distilled water 15 min. The cardiac jelly is swollen further
and the size is very similar to that of (B). (F) 0-8 % Alcian blue. 0-8 % Alcian blue
was diffused into the trough. The cardiac jelly shrank irreversibly. Bright field light
micrographs except (A) and (E), which are dark field. Scale 1 mm. x 27.
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A. NAKAMURA AND F. J. MANASEK
when the distilled water was replaced by 0-1 M-NaCl (Fig. 8C). The ability to
respond to changes in salt concentration was lost rapidly by the naked cardiac
jelly preparations. However, even after no* visible swelling was elicited by
distilled water, the addition of 0-8 % Alcian blue or a few drops of 1 % CPC
resulted in shrinkage of the cardiac jelly (Fig. 8 F).
DISCUSSION
The present study demonstrates a major property of the cardiac jelly. The
cardiac jelly has a shape that reflects the shape of the heart itself, and this shape
can be manipulated experimentally by altering the ionic environment of the
cardiac jelly. This finding is important to furthering our understanding of the
regulation of embryonic morphogenesis.
The cardiac jelly is not simply a viscous solution. Complete removal of the
myocardial layer by a variety of procedures did not cause the jelly to lose the
shape it had while it was still confined by the myocardial investment. There was
no notable slumping, or time-dependent shape change of the bare cardiac jelly
in 0-1-0-15 M-NaCl. Such intrinsic stability might, of course, simply reflect an
extremely high viscosity. This possibility is not. consistent with the results from
our experimental manipulation of cardiac jelly shape. Furthermore, it is
unlikely that our procedures to remove the myocardium resulted in this shape
stability since all the procedures were extractive. Hence, any putative action on
the cardiac jelly itself would have tended to remove material, making the
structure less viscous and less stable rather than increasing its structural
stability.
The shape and volume of the cardiac jelly can be altered reversibly by altering
the ionic strength of the surrounding solution. This ability to expand in a low
ionic medium (distilled water) is lost if the jelly is treated with testicular,
hyaluronidase, Alcian blue or CPC. All of these agents are known to act on the
glycosaminoglycans which are known to be in the cardiac jelly (Gessner &
Boström, 1965; Gessner et al. 1965; Manasek et al. 1973). Thus, THyase
hydrolyses chondroitin sulfate and hyaluronic acid; CPC precipitates these
molecules and Alcian blue will bind to them. The increase in jelly size (swelling)
in low ionic strength therefore appears to result from hydration of the resident
glycosaminoglycans, a situation analogous to that shown by Fessier (1960) in
Wharton's jelly.
The naked cardiac jelly does not swell in response to distilled water as
dramatically as do those specimens that have either a complete or partial
myocardial covering. This suggests that some of the increase in size shown by
the latter specimens may represent swelling of the cells or their remnants in
addition to swelling of the jelly. However, the response of the specimens devoid
of myocardium clearly establishes that the naked cardiac jelly has an intrinsic
ability to respond to changes in ionic strength. The rapidity with which this is
Structure of isolated cardiac jelly
181
lost in naked specimens, as a function of repeated cycling, suggests that extraction
is occurring more rapidly in the absence of a myocardial investment.
The observation that the grossly deformed and swollen jelly returns to its
normal shape with increasing ionic strength suggests that the jelly has intrinsic
structure that serves to regulate or stabilize its overall shape. Without such
intrinsic regulation it would not be expected to resume its morphology after
being distorted grossly in distilled water.
There are two lines of evidence that provide clues to the nature of the intrinsic
shape of the cardiac jelly. Careful light and electron microscope observation
(Johnson et al. 1974; Markwald et al. 1975 a; Patten et al. 1948) have demonstrated the presence of fibrils within the cardiac jelly. The present electron
microscope study of negatively stained preparations of cardiac jelly supports
these earlier observations. The cardiac jelly is therefore a multiphasic system.
It is interesting to note that the fibrous components, particularly fibrillar
collagen, appear to increase during the time that the heart undergoes looping.
We therefore propose that presence of matrical fibrils increases the morphological stability of the cardiac jelly. The recent observation that matrical
glycoproteins, which may also be filamentous (Markwald, Fitzharris & Bank,
1975 b), interact with glycosaminoglycans (Manasek, 1977) is also noted as
a mechanism whereby the molecular composition of the cardiac jelly is capable
of stabilizing its morphology.
Up to now we have considered factors that stabilize the shape of the cardiac
jelly. The cardiac jelly however is a dynamic structure that undergoes ontogenic
remodelling as the organ changes its shape. For example, in a time period of
about 5 h, the heart transforms itself from a midline to a ' c ' shaped structure.
This process is called 'looping'. Obviously, the cardiac jelly must be able to
change its shape too. Therefore, when we speak of 'morphological stability' of
the jelly we do so in a restricted sense, involving a single developmental time
point such as that utilized, of necessity, in our experiments. The factors that
induce such stability must also be capable of physiological modification to
permit normal organ morphogenesis. We therefore note another required
property of cardiac jelly; it must be capable of undergoing time dependent
shape change. Obviously, the most simple mechanism would be for the
enveloping myocardium to simply deform the jelly, and hold it in its new
shape. This, however, is not consistent with our demonstration that the shape of
the jelly is maintained without myocardium. If morphogenetic movements
exceed the elastic limit of the jelly one could imagine that such a bend could
become permanent. However, one cannot experimentally 'bend' the jelly into
a new or different shape suggesting that this is not a normal morphogenetic
mechanism. Indeed, with each systole the jelly is deformed far more than it is
during morphogenetic changes, yet such contractile deformations are also
transient.
There are a number of possible mechanisms by which the shape of the cardiac
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A. NAKAMURA AND F. J. MANASEK
jelly can be altered. There are three concomitant events that occur during
looping: (i) continuous synthesis of matrix macromolecules (Manasek, 1973)
which interact with each other to form supramolecular aggregates (Manasek,
1977); (ii) increased fibrillar structures within the cardiac jelly (Johnson et al.
1974); (iii) a coordinated change in shape of myocardial cells which results in
localized changes in myocardial surface area (Manasek, Burnside & Waterman,
1972). Taken collectively, these observations as well as the present results permit
us to propose the following model: myocardial cell shape changes, occurring
in small continuous increments, result in small incremental deformational
changes in organ shape. While the cells are changing shape they are also
secreting matrix macromolecules which continuously and rapidly interact with
each other in the extracellular compartment. These interactions (possibly
covalent) serve to stabilize continuously the shape of the newly deposited
matrix, which conforms to the limits of the myocardial mantle. Thus, the shape
change of the myocardium does not have to deform the entire jelly continuously
or at any given time; rather it needs to provide a mold only for those newly
elaborated matrix components not yet fully stabilized within the jelly. Once
they are incorporated into the matrix no additional work on the part of the
myocardium is required to maintain their relative positions, hence shape of the
jelly. In this model, we view the morphogenetic force as being myocardial in
origin (cell shape change) and the jelly as a filler that assumes the shape dictated
by the myocardium, and retains this shape by means of cross links. We therefore
expect the jelly to demonstrate a continuum that ranges from older, 'fixed'
molecules to those newly synthesized ones that are relatively mobile and conform
to the new shape prior to being 'fixed'.
There is an additional mechanism that could be operational. Since we have
demonstrated that the cardiac jelly can swell and shrink in response to ionic
environment it is possible that a similar mechanism operates in situ. Thus, by
regulating the local ionic environment in different regions of cardiac jelly local
volume changes could be effected. These local volume changes would be
manifested as changes in the shape of the organ. Again, such changes would have
to be stabilized (since the cardiac jelly does indeed retain its shape as shown by
our experiments), most likely by matrix molecule interaction. This hydration
model requires that the myocardium regulates the ionic strength of its substrate.
The role of the myocardium is predictably different in these two models. In
the first, the myocardium itself exerts a deforming force directly by changing
the shape of its cells. In the second, it produces local shape changes in the
cardiac jelly by ionic means and, it would be assumed, conforms to the resulting
shape. The shape changes detected in the myocardial cells (Manasek et al.
1972) would then not be primary determinative morphogenetic events, but
secondary ones. We cannot decide which of these models is correct. Both give
us predictions which are testable experimentally and which should clarify the
mechanisms involved in morphogenesis.
Structure of isolated cardiac jelly
183
This work was supported by grant HL 13831 from the National Heart, Lung and Blood
Institute, National Institutes of Health.
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BARRY, A. (1948). The functional significance of the cardiac jelly in the tubular heart of the
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DAVIS, C. L. (1924). The cardiac jelly of the chick embryo. Anat. Rec. 27, 201-202.
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(Received 2 May 1977, revised 11 August 1977)