/ . Embryol. exp. Morph. Vol. 56, pp. 211-223, 1980
Printed in Great Britain © Company of Biologists Limited 1980
211
Compositional and structural
heterogenicity of the cardiac jelly of the chick
embryo tubular heart: a TEM, SEM and
histochemical study
By J. M. HURLE, 1 J. M. ICARDO AND J. L. OJEDA
From the Department of Anatomy, Faculty of Medicine, University
of Santander, Spain
SUMMARY
The hearts of chick embryos of stages 9-13 were subjected to SEM, TEM and histochemical studies to ascertain possible regional differences in the structure and composition
of the cardiac jelly. Two distinct regions, the cardiac jelly filling the space located between the
myocardium and the endocardium (MECJ) and the cardiac jelly filling the dorsal mesocardium (EECJ), were distinguished by their structural and histochemical properties. MECJ
is formed by amorphous and fibrillar material arranged between the endocardial and myocardial layer. The amount of its components increases when cetylpyridinium chloride is
introduced into the fixative, and it appears intensely stained by ruthenium red and alcian
blue at low concentrations of MgCl2. The amount and arrangement of its components
increase during the beginning of the looping process of the heart tube. The EECJ is very
rich in ruthenium-red-positive basal-lamina-like material and the addition of cetylpyridinium
chloride to the fixative does not modify its appearance. It also appears poorly stained by
alcian blue at low concentrations of MgCl2 and its arrangement undergoes modifications
closely associated with the events of endocardial fusion. The possible significance of these
results in the early morphogenesis of the heart is discussed.
INTRODUCTION
Numerous recent papers show that the embryonic extracellular matrix plays
an important role in morphogenesis and differentiation of tissues and organs
(Bernfield, Cohn & Banerjee, 1973; Manasek, 1975; Overton & Collins, 1976;
Toole, 1973). The morphogenetic role of the extracellular matrix is dependent
on the kind, amount and distribution of its components.
During the early stages of heart development the extracellular matrix,
'cardiac jelly' (CJ) (Davis, 1924), constitutes the major component of this
organ. Among the different components of the CJ, collagen (Johnson, Manasek,
Vinson & Seyer, 1974; Hurle & Ojeda, 1977), glycoproteins (Manasek, 19766,
1977), and cellular detritus (Ojeda & Hurle, 1975) and mucopolysaccharides
1
Author's address: Departamento de Anatomia, Facultad de Medicina (Poligono de
Cazona), Santander, Spain.
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J. M. HURLE, J. M. ICARDO AND J. L. OJEDA
(glycosaminoglycans) are very abundant (Manasek, 1970; Markwald & Adams
Smith, 1972; Ortiz, 1958) and abnormal development of the heart occurs when
the synthesis of mucopolysaccharides is altered (Overman & Beaudoin, 1971;
Satow & Manasek, 1977). Biochemical analysis of the chick embryo CJ showed
the presence of the following mucopolysaccharides: hyaluronate and chondroitin (Manasek et al. 1973; Orkin & Toole, 1978); undersulphated chondroitin
sulphate (Manasek et al., 1973) chondroitin 4- or 6-sulphate (Gessner, Lorincz
& Bostrom, 1965; Manasek et al. 1973) and heparin/heparan sulphate (Gessner
et al. 1965; Satow & Manasek, 1977). However, it must be noted, as Manasek
pointed out (1976a), that more components could be present since most of the
studies are synthetic analyses rather than total compositional analyses.
Temporal and regional differences in the composition and arrangement of
the CJ would explain some of the proposed functions for the CJ (see Manasek,
1976 c, and Hurle & Ojeda, 1977). However, most of the studies focused on that
problem have been made in the period in which the CJ is invaded by mesenchymal cells (Markwald, Fitzharris & Manasek, 1977; Markwald, Fitzharris,
Bank & Bernake, 1978) while earlier stages have received less attention although
during these stages important morphogenetic events occur, such as the fusion
of the paired cardiac primordia and the beginning of the looping process of the
tubular heart.
In the present paper we report the results of a structural and histochemical
study of the CJ in the early stages of heart morphogenesis. The structure of the
CJ was studied by scanning electron microscopy which gives three-dimensional
information on the arrangement of the CJ. In addition, to assess possible
regional differences in the composition of the CJ, ruthenium-red staining was
employed in transmission electron microscopy and alcian blue staining at the
'critical electrolyte concentration' (Scott & Dorling, 1965) in light microscopy.
This technique gives an approximate analysis of the composition in sulphated
mucopolysaccharides by-passing the difficulty of the biochemical studies
derived from the small amount of material available. In addition it allows
detection of the regional distribution of the sulphated mucopolysaccharides
impossible in biochemical analysis.
MATERIAL AND METHODS
Fertile White Leghorn eggs were incubated at 38 °C to yield normal embryos
ranging between stage 9 and 13 (Hamburger & Hamilton, 1951) and the structure of the CJ was studied by the following techniques:
Scanning electron microscopy (SEM)
The embryos were fixed for 3-5 h in 4 % cacodylate-buffered glutaraldehyde
with and without 1 % cetylpyridinium chloride and then transferred into
cacodylate buffer in which the specimens were transversely sectioned throughout
Heterogenicity of the cardiac jelly
213
the heart. The fragments were dehydrated through a series of acetones dried
by the critical-point method (Anderson, 1951) and gold sputter-coated. The
specimens were viewed using a Philips SEM 501.
Although the addition of cetylpyridinium chloride to the fixative produces
alterations of the cell membranes (Morris & Solursh, 1978) it was employed
here because it has also been reported that it retains abundant extracellular
components of the CJ which are extracted by conventional fixative fluids
(Markwald et al. 1978; Pratt, Larsen & Johnson, 1975).
Transmission electron microscopy (TEM)
Ruthenium-red staining was carried out according to the Luft (1971) procedure. The embryos were immersed in 3 % glutaraldehyde buffered in 0-1 M
sodium cacodylate pH 7-3 containing 0-1% ruthenium red, and quickly
transversely sectioned through the heart for better penetration of the dye.
After 4-10 h of fixation, the specimens were washed in cacodylate buffer,
postfixed for 2 h in 2 % osmium tetroxide containing 0-1 % ruthenium red and
processed for electron microscopy according to the usual procedure.
Ultrathin sections either unstained or counterstained with uranyl acetate
and lead citrate were examined with a Philips EM 201 electron microscope.
Histochemistry
Embryos were fixed in Carnoy's fluid or Newcomer's solution, embedded in
paraplast and serial sectioned at 8 or 30 jum. Sections were processed to detect
sulphated mucopolysaccharides as follows:
(a) Differential sulphated mucopolysaccharide staining by the l critical electrolyte concentration'' method of Scott & Dorling (1965). Sections were stained
in solutions of 0 1 % alcian blue 8GX (G. T. Gurr Ltd) in 0-025 M acetate buffer
(pH 5-8) plus MgCl2 in concentrations ranging from 0-1 to 1 M. Sections were
rinsed in MgCl2 solutions of the appropriate molarity before and after staining
and examined microscopically after dehydration, clearing and mounting in
DPX. For a better evaluation of the staining intensity, in addition to subjective
evaluation, the sections were analyzed with the help of the grey scale of a
Micro-Videomat (Zeiss, Oberkochen). This technique allows an approximate
differentiation of the sulphated mucopolysaccharides owing to their different
critical electrolyte concentrations (at which the anionic polymers change from
binding dye to binding Mg 2+ ).
(b) Hyaluronidase digestion. To distinguish chondroitin sulfate A/C from
chondroitin sulphate B (dermatan sulphate), heparin and Keratan sulphate,
deparaffinized sections were incubated overnight with bovine testicular hyaluronidase at 37 °C in pH 6 citrate buffer prior to staining (Leppi & Soward, 1965).
(c) Digestion in methanol-ClH. Control of specificity of the alcian-blue
staining for sulphated mucosubstances was made by treating the sections with
214
J. M. HURLE, J. M. ICARDO AND J. L. OJEDA
Heterogenicity of the cardiac jelly
215
0 1 N-C1H in absolute methanol at 60 °C for 4h. This treatment abolishes
sulphate-specific staining (Quintarelli, Scott & Dellovo, 1964).
Sections of human umbilical cord were included in all staining tests to ensure
validity of dye reactions and enzyme digestion.
OBSERVATIONS
Scanning electron microscopy
During the stages studied in the paper the formation of the tubular heart
takes place by the fusion in the midline of two lateral primordia (for details see
Ojeda & Hurle, 1975). During this fusion process, the heart is a very simple
organ consisting of three concentric tissue layers. It has an outer wall, termed the
myocardial layer (after Manasek, 1969; and Ho & Shimada, 1978), which is
open at the dorsal side forming the dorsal mesocardium. The innermost structure is the endocardium. Between the endocardium and the myocardium there is
a large extracellular compartment filled by an acellular matrix, the cardiac jelly
(CJ). This extracellular matrix is also observed in the dorsal mesocardium arranged between the ventral foregut endoderm and the endocardium. As can be
seen in Fig. 1 two morphologically different regions can be distinguished within
the CJ: the CJ located between the myocardium and the endocardium (MECJ)
and the CJ located between the endoderm and the endocardium (EECJ).
The MECJ appears as a mesh work of extracellular material arranged between
the basal surfaces of the endocardium and the myocardium. A progressive
modification in the structure of this zone of the CJ can be observed, Initially
(stages 9-10) it appears to be formed by radially arranged fibrillar material
associated with abundant fine granular material (Fig. 2). The total amount of
material at these stages is small, and numerous spaces which lack material
are observed. In the next stages the amount of material increases and the
amorphous material appears as rounded masses, 0-5-1 fim in diameter (in the
coated material) closely associated with the fibrillar material (Fig. 3). By stage
12-13 the endocardium and the myocardium are closer to each other, diminishing the space filled by MECJ. In these stages the MECJ is very rich in material
consisting mainly of thick strands of fibrillar material with a clear radial arrange-
Fig. 1. Transverse fracture through the level of fusion of the endocardial tubes (E) of
a stage-10 embryo showing the location of EECJ (arrow) and MECJ (M).
Fig. 2. High magnification SEM view of the MECJ of the chick embryo shown in
Fig. 1. The MECJ appears to be formed by fine granular material and fibrillar
material between the mycardium (M) and the endocardium (E).
Fig. 3. MECJ of a stage-11 embryo. Note the abundant fibrillar material intertwined with granular masses of amorphous material (arrows).
Fig. 4. MECJ of a stage-13 embryo. Thefibrillarmaterial is the major component of
MECJ at this stage and shows a clear radial arrangement.
216
J. M. HURLE, J. M. ICARDO AND J. L. OJEDA
Fig. 5. SEM view of the endocardial wall of a stage-13 embryo showing the dense
fibrillar sheet located under the endocardial surface.
Fig. 6. Panoramic view of the MECJ of a stage-10 embryo fixed in glutaraldehydecetylpyridinium chloride. Note the abundant fine granular material retained and
compare with Fig. 2. Endocardium (E). Myocardium (M).
Fig. 7. EECJ of a chick embryo heart after fusion of the endocardial tubes. This
CJ consist of amorphous and fibrillar material and contains cell fragments (C).
Ventral foregut endoderm (F). Myocardium (M). Endocardial tube (E).
Fig. 8. EECJ of a chick embryo heart during fusion of the endocardial tubes (E).
This CJ appears to be formed by two sheets (arrows) that are continuous with the
basal lamina of the ventral foregut endoderm (F).
Heterogenicity of the cardiac jelly
111
ment, joining one another by delicate strands of fibrils (Fig. 4). Amorphous
material is not abundant at these stages and on the basal surfaces of the myocardium and endocardium a sheet of close-packed fibrils is observed (Fig. 5).
The addition of cetylpyridinium chloride to the fixative results in the preservation of abundant fine granular material within the MECJ which makes it
difficult to recognize the fibrillar component of this zone of the jelly (Fig. 6).
The EECJ fills the extracellular space of the dorsal mesocardium and in
addition to fibrillar material the amorphous material is a major component
of this zone of the CJ. Rounded cell fragments are frequently found associated
with the extracellular material (Fig. 7). The arrangement of this zone of the CJ
also shows differences between the studied stages but, as has been previously
described by us (Hurle & Ojeda, 1977), these modifications appear to be closely
related with the fusion of the endocardial tubes. Before and especially after the
fusion of the endocardial tubes EECJ appears as thick chains of closely packed
extracellular material joining the endoderm with the endocardium (Fig. 7).
During the process of fusion both endocardial tubes are located lateral to each
other and the EECJ appears as two sheets of dense basal lamina-like material
occupying the limited space between the fusing tubes (Fig. 8). These sheets are
continuous dorsally with the basal lamina of the endoderm (Fig. 8). The addition of cetylpyridinium chloride to the fixative does not produce gross differences in the appearance of this zone of the CJ.
Ruthenium-red staining
Ruthenium red (RR) penetrates well into the heart tissues and gives a strong
positive reaction both with the cell surfaces of all the heart tissues and with the
extracellular material of the CJ (Figs. 9, 10). As can be seen in Fig. 9, the
MECJ appears in the sections as a loose meshwork of RR-positive fibrils. This
fibrillar material is more abundant in the proximity of the myocardium where
the fibrils are often seen to contact the RR-positive coat of the developing
myocardial cells (Fig. 9). The EECJ appears composed mainly of an amorphous
RR-stained mass associated with RR-positive fibrils among which crossbanded
collagen fibrils can be recognized (Fig. 10). Large amounts of EECJ are often
observed filling invaginations of the endocardial cells of the midline in the
stages in which the endocardial tubes undergo fusion (fig. 11). This location of
the extracellular matrix seems particulary interesting since most of these cells are
detached towards the bloodstream and it could be a mechanism of elimination
of the EECJ when it is no longer necessary.
No gross modifications in the staining pattern of the CJ were observed during
the studied stages.
Alcian-blue staining
In all the studied embryos CJ appears strongly stained with alcian blue (AB)
when MgCl2 is added at concentrations lower than 0-2 M (Fig. 12a, b). However,
218
J. M. HURLE, J. M. ICARDO AND J. L. OJEDA
Fig. 9. Panoramic view of the MECJ stained with RR. Note the relation of the extracellularfibrilswith the RR-positive coat of the myocardial cell basal surface (arrows).
Fig. 10. Electron micrograph showing the EECJ stained with RR. This zone of the
CJ consist of strongly RR-positive amorphous material and fibrillar material.
Endocardium (E).
Fig. 11. Electron micrograph showing abundant RR-positive extracellular material
filling a large invagination of a midline endocardial cell.
the AB-positive material does not appear homogeneously distributed within the
CJ sleeve. The MECJ is richer in stained extracellular material than EECJ.
Furthermore the distribution of the AB-positive material does not appear
homogeneous within the MECJ itself. Most of the stained material appears
arranged as two laminae associated with the basal surface of the myocardial
and the endocardial layers (Fig. 12a). A less-intense stained material appears
radially arranged between the endocardium and the myocardium.
When the concentration of MgCl2 is increased at 0-5 M the staining pattern is
similar to that described above but the intensity of staining is drastically
reduced. At concentrations of 0-6 M MgCl2 a small amount of AB-positive
material is retained which is mainly associated with the basal surface of the
myocardium (Fig. 12 c, d). The staining practically disappears at concentrations
Heterogenicity of the cardiac jelly
219
Fig. 12. Two consecutive sections of a stage-11 embryo stained with AB plus MgCl2,
(a) Staining with 0-2 M MgCl2. Note that most of the stained material is associated
with the basal surfaces of the myocardium and endocardium and very little with
EECJ (arrow), (b) Same section observed in the micro-videomat at the appropriate
grey level to discriminate the AB-staining. (c) Staining with 0-6 M MgCl2. Note that
some dye can be recognized at the basal surface of the myocardium, (d) same section
observed in the micro-videomat at the same grey level as that of {b). (Note the same
level in both grey scales -G-.)
over 0-8 M MgCl2. The amount of stained material at concentrations of MgCl2
greater than 0-5 M increases in the older stages.
Hyaluronidase digestion drastically reduces the AB-staining at concentration
of 0-1-0-2 M MgCl2 but it does not significantly affect the staining at concentrations over 0-5 M MgCl2. Treatment of the sections with methanol/ClH
resulted in complete inhibition of the staining at all the concentrations of MgCl2.
DISCUSSION
Structure of the CJ
Since the early description of Davis (1924) an intriguing question about the
CJ has been the divergence between its physical properties in vivo and the
amount of materials detectable by light and electron microscopy. Our SEM
observations show that the CJ is very rich in solid materials supporting the
hypothesis which gives important mechanical functions to this structure (Barry,
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J. M. HURLE, J. M. ICARDO AND J. L. OJEDA
1948; Patten, Kramer & Barry, 1948). The general appearance of the chick CJ
under the SEM appears similar to that observed in rat and chick embryos of
older stages (Markwald, Fitzharris & Adams Smith, 1975; Markwald et al 1977;
Markwald et al. 1978) and to other embryonic extracellular matrices (Overton
& Collins, 1976; Tosney, 1978).
The present observations confirm our previous ultrastructural study (Hurle
& Ojeda, 1977) showing that two distinct regions can be distinguished within the
CJ. The MECJ located between the myocardium and the endocardium appears
as the major component of the CJ and comprises mainly RR-positive unbanded
fibrillar material. The fact that the amount of matrical components increases
when cetylpyridinium chloride is introduced into the fixative suggests that
hyaluronate is an important component of this zone of the CJ (Pratt et al. 1975).
The presence of hyaluronate in this zone of the CJ can be related to the invasion
of the CJ by mesenchymal cells as has been pointed out by other authors
(Markwald et al. 1978; Orkin & Toole, 1978). Our observations show that the
fibrillar components of the MECJ tend to appear radially arranged between the
endocardium and the myocardium. This is accentuated in the latest studied
stages in which the looping process of the heart begins. This observations
supports the hypothesis of Nakamura and Manasek (1978), who have suggested
that CJ has an intrinsic shape which could be responsible for the looping process
of the heart tube. Contrary to our observations, it has been reported (Markwald, Fitzharris, Bolender & Bernake, 1979) that the CJ of the atrio-ventricular
cushions does not display a radial arrangement until it is invaded by mesenchymal cells. This discrepancy can be explained by the difference in the stages
studied, or by the fact that the SEM observations of those authors are based on
material fixed in glutaraldehyde plus cetylpyridinium chloride. In these conditions the large amount of fine granular material retained makes it difficult to
recognize the arrangement of the fibrillar material. An alternative explanation
is that the differences can be due to artefacts produced either by the omission
or by the addition of cetylpyridinium chloride. Recently Morris & Solursh (1978)
have reported gross ultrastructural artefacts due to the addition of cetylpyridinium chloride to the fixative.
The EECJ fills the dorsal mesocardium and it can only be observed during
the stages in which this structure is present in the heart. Recent studies of
Manasek (1976b) show that this zone of the jelly is particularly rich in
fucose-containing glycoproteins probably elaborated by the endoderm. In our
study this zone appears very rich in RR-positive amorphous material similar
to the basal lamina material, and hyaluronate does not seem to be very abundant
since the addition of cetylpyridinium chloride to the fixative does not produce
gross modifications in its appearance. On the other hand the fact that the
arrangement of this zone of the CJ is modified during the fusion of the endocardial tubes suggests that it may be involved in that process (see Hurle &
Ojeda, 1977, for a detailed discussion).
Heterogenicity of the cardiac jelly
221
Histochemistry
AB-staining of the CJ reveals the presence of abundant sulphated mucopolysaccharides as previously reported by numerous authors (Gessner &
Bostrom, 1965; Gessner, Lorincz & Bostrom, 1965; Manasek, 1970; Markwald
& Adams Smith, 1972; Ortiz, 1958). Manasek et al. (1973) in a synthetic
analysis made in cultured chick embryos, showed that the synthesis of sulphated
mucopolysaccharides undergoes changes related to myocardial differentiation;
before stage 11, only undersulphated chondroitin sulphate is produced in the
heart, while during the stages 11 to 13~, chondroitin 4- or 6-sulphate is also
produced. In our observations the disappearance of most of the AB-staining
at concentrations over 0-5 M MgCl2 and the hyaluronidase lability of this
staining indicate that most of the stained material is chondroitin sulphate
(Leppi & Stoward, 1965; Scott & Dorling, 1965) but it can not be ascertained
whether it corresponds to undersulphated or to 4- or 6-sulphate. However, it
should be noted that a weak staining, labile to methylation and resistant to
hyaluronidase digestion, remains at concentrations of 0-6 and even at 0-8
M MgCl2. This suggests that other strongly anionic mucopolysaccharides such
as heparin or dermatan sulphate and keratan sulphate might be present. These
results are in agreement with similar studies made in rat embryos (Markwald &
Adams Smith, 1972). Heparin has been detected in the CJ of older embryos
(Gessner et al. 1965; Satow & Manasek, 1977) but other sulphated mucopolysaccharides have never been detected in synthetic biochemical studies. These
observations suggest that the turnover of those mucopolysaccharides, if present,
must be very slow.
One of the principal purposes of this study was to find out possible regional
differences in the distribution of sulphated mucopolysaccharides which could
explain some of the proposed functions for the cardiac jelly. In this regard we
found that the EEGJ, despite being very rich in ultrastructural components,
appears very poor when stained with AB, suggesting that sulphated mucopolysaccharides are not the main components of this zone of the jelly. This observation supports the view that the two zones of the CJ have different origins and
functions (Manasek, 19766; Hurle & Ojeda, 1977). On the other hand the
MECJ is very rich in sulphated mucopolysaccharides and most of these are
located in the basal surfaces of the myocardium and endocardium. This arrangement can represent a gradient in the diffusion of those materials since they are
produced both by the myocardium (Kosher & Searls, 1973; Manasek, 1970)
and by the endocardium (Gross, Challice & Schrevel, 1974; Johnstone & Comar,
1957; Markwald et al. 1975) and it is consistent with an involvement of this
extracellular matrix in the cytodifferentiation of both tissues as suggested by
other authors (Manasek, 19766; Markwald et al. 1975).
We conclude from this study that the CJ is not a homogeneous material
either in its ultrastructure, its tridimensional arrangement or its histochem15
EMB 56
222
J. M. HURLE, J. M. ICARDO AND J. L. OJEDA
istry. On the contrary, our observations suggest that CJ has a precise structure
and composition possibly related to the morphogenetic events taking place in
the heart during these early stages (fusion and looping). It seems therefore
erroneous to us to consider this cardiac component as a passive structure
playing only unspecific mechanical functions. The recent experimental study
of Nakamura & Manasek (1978) constitutes a nice demonstration of this hypothesis.
This work was supported by a grant of the Fondo Nacional para el Desarrollo de la
Investigation Cientifica from the Spanish government.
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MANASEK,
(Received 23 June 1979, revised 1 October 1979)
Note added in proof
Nakamura & Manasek (1978 in Morphogenesis and Malformation of the Cardiovascular
System, G. C. Rosenquist and D. Bergsma (ed.), pp. 229-250. Alan R. Liss: New York)
have recently published a SEM study of the EECJ of chick embryos of the same stages
studied here and their results are in agreement with our present observations.
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