© 1998 American Heart Association, Inc.
Original Contributions |
Versican Expression Is Associated With Chamber Specification, Septation, and Valvulogenesis in the Developing Mouse Heart
From the Neural Development Unit, Institute of Child Health, University College London, London, UK.
Correspondence to Dr Deborah Henderson, Neural Development Unit, Institute of Child Health, 30 Guilford St, London, WC1N 1EH, UK. E-mail dhender{at}ich.ucl.ac.uk
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Abstract |
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Abstract—The versican (PG-M) gene encodes a chondroitin sulfate proteoglycan that is nonpermissive for cell migration and appears in association with slow cell proliferation and cytodifferentiation. Using the techniques of in situ hybridization and immunocytochemistry on sectioned mouse embryos, we found that the mRNA and protein for versican show similar distributions and are expressed in a dynamic pattern during development of the heart. Versican exhibits generalized expression in the tubular heart but becomes rapidly downregulated in the atrium and exhibits higher transcript levels on the right side of the ventricular chamber than the left, before the onset of ventricular septation. Versican is expressed strongly in the trabeculated ventricular myocardium, whereas the compact proliferative zone has lower transcript abundance. It is expressed in the outer layers and on the crest of the ventricular septum and is prominent on the mesenchymal cap of the primary atrial septum. Versican is particularly strongly expressed in the endocardial cushions of the atrioventricular and outflow tract regions and in the atrioventricular, semilunar, and venous valves. This study raises the possibility that versican may be involved in specification of the ventricular chambers, in growth and fusion of the atrial and ventricular septa, and in the transformation from epithelium to mesenchyme that characterizes development of the endocardial cushions. Versican may be a key participant in cardiogenesis, responding to the many diffusible signals that mediate interactions between the developing endocardium and myocardium.
Key Words: mouse • proteoglycan • embryo • endocardial cushion • gene expression
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Introduction |
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Recent years have seen a dramatic increase in the number of genes known to be expressed in the developing heart. Moreover, the advent of gene-targeting technology1 2 3 has provided a wide range of mutant mice that develop heart defects, thereby identifying model systems with which to probe the origins of human congenital cardiac disease. Gene targeting has revealed key roles for MEF2c and dHAND in chamber specification4 5 and has pointed to the importance of neuregulin, N-myc, neurofibromatosis-1 (NF-1), and retinoid X receptor-
in the development of septa and endocardial
cushions.6 7 8 9 10 11 These advances in the
identification of some of the key genes of cardiac development open the
way toward understanding the cascades of gene expression that underlie
events such as chamber specification and the formation of valves and
septa. It is striking, however, that although a number of transcription
factors and signaling molecules have been shown to play a functional
role in heart development, much less is known about the genes
downstream from these regulatory molecules, which are likely to mediate
the cellular interactions that constitute heart development. The
present study describes the expression of one such gene,
versican. Versican (or PG-M) is a chondroitin sulfate proteoglycan expressed in the pathways of neural crest cell migration and in prechondrogenic areas of the developing chick and mouse.12 13 This molecule has been proposed to be nonpermissive for cell movements and is expressed in barrier tissues for neural crest migration and neurite outgrowth.12 13 14 Versican is also associated with regions of epithelium to mesenchyme transformation within the developing mouse embryo (D.J. Henderson, M. Kolatsi-Joannou, and A.J. Copp, unpublished data, 1998). To study the role of versican in cardiac development, we examined the expression of the mRNA and protein by in situ hybridization and immunocytochemistry, respectively, in developing mouse hearts from embryonic day (E) 8.5, before cardiac looping has begun, until E14.5, when septation is complete and the cardiac valves have formed. Versican gene expression occurs at high levels in specific regions of the developing heart throughout this period of embryogenesis. Specifically, versican is expressed in a chamber-specific manner, with downregulation in the atrium as the heart loops. It is expressed at much higher levels in the trabeculations of the right than the left ventricle, before formation of the ventricular septum is apparent, and is found in the crest of the developing atrial and ventricular septa and throughout the development of the endocardial cushions and valves. Our findings suggest that versican may play multiple roles in cardiac development, including regulation of chamber specification, growth and fusion of septa, and modeling of the endocardial cushions.
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Materials and Methods |
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Mouse Strains and Embryos
Randomly bred CD1 mice were used throughout the study. Females were placed with males overnight and checked for copulation plugs the following morning. Noon on the day of finding a plug was designated 0.5 days of gestation (E0.5). Pregnant females were killed by cervical dislocation, and the embryos were placed in DMEM (GIBCO) containing 10% FCS. The yolk sac and amnion were opened, and the umbilical cord was cut. For in situ hybridization, embryos were washed in PBS and then fixed in 4% paraformaldehyde in PBS by immersion overnight at 4°C, followed by dehydration and embedding in paraffin wax. For immunocytochemistry, embryos were washed in PBS and then immersed in St Marie's fixative (1% acetic acid and 95% ethanol) overnight at room temperature. The embryos were washed twice in 100% ethanol and processed for embedding in paraffin wax.
Radioactive In Situ Hybridization on Paraffin Wax–Embedded
Sections
Oligonucleotide primers were designed in order
to amplify 2 regions of the sequence encoding the versican core protein
as described by Ito et al.15 The regions
amplified corresponded to base pairs 1469 to 2029 and 6919 to 7357 of
the published sequence. These regions correspond to the and ß
domains of the core protein, respectively, and show <15% homology at
the amino acid level to any known genes, including the other
chondroitin sulfate proteoglycans (aggrecan, neurocan, and brevican).
This makes cross hybridization with other chondroitin sulfate
proteoglycans very unlikely. The corresponding DNA fragments were
amplified by polymerase chain reaction, cloned into a plasmid vector,
and sequenced, as described previously.13 The
plasmids were used to produce 35S-labeled
riboprobes, and in situ hybridization was carried out according to a
standard protocol,16 with modification as
follows: Briefly, 8-µm paraffin sections were dewaxed and rehydrated,
treated with 20 µg/mL proteinase K for 8 minutes, washed in PBS,
dipped in 0.5% acetic anhydride, dehydrated, and air-dried. Labeled
probe was applied to the sections at a final concentration of
5x104 cpm/µL at 55°C overnight in a humid
environment. The sections were washed at high stringency to 0.1x SSC
at 50°C, dipped in Ilford K5 emulsion, and exposed in the dark for up
to a week at 4°C. After development, the sections were counterstained
with toluidine blue, mounted, and photographed using a Zeiss Axiophot
microscope. Probes to both the and ß domains showed identical
expression patterns in initial experiments, so the probe for the ß
domain alone was used in all further experiments. In no case did sense
controls show hybridization above background.
Immunocytochemistry for Versican on Paraffin Wax–Embedded
Sections
A polyclonal antiserum to the ß domain of mouse versican was
obtained as a gift from Dr T. Shinomura (Nagakute, Japan). The antibody
is an affinity-purified preparation of antiserum, raised from a fusion
protein of the ß domain of versican expressed in Escherichia
coli (T. Shinomura and K. Kimata, unpublished data, 1998).
Briefly, 8-µm sections were dewaxed, rehydrated, and treated with 3%
hydrogen peroxide in deionized water for 5 minutes to block
endogenous peroxidase. The sections were subsequently
blocked for 15 minutes with serum-free block (Dako). The versican
antibody was diluted 1:20 in 2% rat serum in Tris-buffered saline and
incubated with the sections for 2 to 3 hours at room temperature. The
sections were washed in Tris-buffered saline and then overlaid with a
diluted (1:600 with 2% rat serum in Tris-buffered saline) biotinylated
goat anti-rabbit secondary antibody (Dako) for 1 hour at room
temperature. This was followed by incubation with horseradish
peroxidase– linked avidin (Dako) to amplify the signal. Positive
signal was detected by incubation with metal-enhanced diaminobenzidine
tetrahydrochloride (Sigma Chemical Co) as a substrate for horseradish
peroxidase. Rabbit serum was used in place of the primary antibody as a
negative control. In no case was signal above background seen in these
sections. The sections were counterstained with toluidine blue,
dehydrated, mounted, and photographed using a Zeiss Axiophot
microscope.
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Results |
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To investigate the expression pattern of versican mRNA in the developing mouse heart, we hybridized 35S-labeled riboprobes specific for the versican core protein to serial sections of mouse embryos harvested at intervals between E8.5 and E14.5. This was complemented with immunocytochemistry for versican protein.
Versican mRNA Expression is Chamber Specific
Versican transcripts can be seen in the heart of E8.5 embryos with
5 or 6 somites (Figure 1A
and 1B). At
this stage, the heart consists of a double-layered tube, in which
myocardium surrounds the inner endocardium, with
extracellular cardiac jelly in between. Versican mRNA is present
solely in the myocardium (small arrowheads in Figure 1B)
and is absent from the endocardium (arrows in Figure 1B).
Analysis of serial sections shows that versican transcripts are
found throughout the length of the heart tube at this stage (data not
shown). Versican is also expressed in the splanchnic lining of the
dorsal mesocardium, where the heart tube is attached to the body wall
(large arrowheads in Figure 1B). As the heart tube begins to loop at
E9.0 (11 to 13 somites), versican mRNA continues to be expressed in the
myocardium of the outflow tract and ventricle, whereas it
is rapidly downregulated in the presumptive atrial region (Figure 1C).
This regional difference in versican expression occurs before
endocardial cushions have formed in the
atrioventricular canal and before any morphological
differences can be discerned between atrial and ventricular
chambers. Versican transcripts are not seen in the atrial
myocardium at any subsequent stage of development.
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Expression differences between the presumptive right and left
ventricles can be seen at E9.0 (arrowheads indicate more intense
expression in the right ventricle in Figure 1C
). This is before any
evidence of trabecular formation and well before the onset
of ventricular septation. At E10.0 to E10.5, the site at
which the ventricular septum will form can be distinguished
(arrows in Figure 1D and 1E). Although versican mRNA continues to be
expressed in both right and left sides of the ventricle, transcripts
are more abundant in the presumptive right ventricle and outflow tract
than in the left ventricle (Figure 1D to 1F). This difference in
expression between the right and left sides of the heart becomes even
more obvious at E11.5, when a sharp boundary of expression can be seen
between the trabeculations of the presumptive right and
left ventricles (Figure 1H). This boundary coincides precisely with the
site of the developing ventricular septum, which, in some
preparations, appears to be "bisected" by the boundary between
high- and low-expression domains of versican. By E12.5, versican
expression is declining in both ventricles (Figure 2A and 2B), and at E13.5, transcript
levels are almost indistinguishable between the 2 ventricles (Figure 2D). Versican mRNA expression is completely absent from the ventricle
by birth, remaining only in the valves (Figure 2F). Thus, asymmetry of
versican expression precedes the morphological division of the common
ventricle into right and left chambers.
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Versican Is Expressed in the Trabeculations of the
Developing Myocardium
Between E9.5 and E11.5, the ventricle develops from a simple
thin-walled sac (closely resembling the atrial chamber) into a complex
structure with trabeculations and a myocardial wall several
cell layers thick. Trabeculations are first visible at
E10.0 (23 to 25 somites), at which time the myocardial wall is still
thin, with only 1 or 2 cell layers. At this stage, versican is
expressed at a high level in the developing trabeculations
but at a much lower level in the wall of the myocardium
(Figure 3A and 3B). As
trabeculation proceeds, expression of versican reaches a
peak of intensity, with transcripts abundant in both
trabeculations and myocardial wall (Figure 3C and 3D). At
E12.5 to E13.5, the ventricular wall differentiates to form
a compact outer layer and an inner spongy layer. Versican expression
becomes downregulated in both the compact and spongy layers of the
myocardial wall but is maintained in the trabeculated
myocardium (Figure 3E and 3F). Later, as the
trabeculations differentiate to form the papillary muscles
and the tendinous cords that connect the muscles to the
atrioventricular septum, the expression of versican
becomes less intense, so that by the time of birth, versican is no
longer expressed in the ventricle (data not shown).
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Versican Is Expressed in the Septa Dividing the
Atria, Ventricles, and Outflow Tract
The common atrium of the mouse heart begins to divide at E9.5 as a
result of the formation, modification, and eventual fusion of the
primary and secondary atrial septa. During this process, the primary
septum grows out from the roof of the atrium, and its mesenchymal cap
fuses with the atrioventricular endocardial cushions.
Perforations appear in the septum, allowing flow of blood between the
right and left atria. In embryos harvested at E10.0, versican mRNA can
be detected in the region of the atrial wall where a thickening marks
the site of formation of the primary septum (Figure 1D). This septum
begins to grow out at E10.5 and initially expresses versican throughout
its length (Figure 1E). By E11.5, the septum has grown down from the
roof of the atrium, forming a thin muscular structure with a
mesenchymal cap.17 Versican mRNA can clearly be
detected in the crest of the septum (small arrowhead in Figure 1I),
whereas the remainder of the septum is now negative for versican mRNA
(arrow in Figure 1I). By E12.5, the mesenchymal cap of the primary
septum has fused with the atrioventricular cushions.
Versican appears to be completely downregulated in the primary septum
once fusion with the atrioventricular cushion tissue
has taken place (Figure 2A [arrow], 2C, and 2D [arrows]). At
E12.5 to E13.5 of development, the atrioventricular
cushion tissue expresses versican intensely, and there is a clear
boundary of expression between the negative septum and the positive
cushion tissue (Figure 2C and 2D). The secondary atrial septum does not
appear to express versican mRNA at any stage of its development (data
not shown).
The ventricular septum first appears at E10.0 in the floor
of the versican-positive ventricular chamber. At this
stage, versican expression in the rudimentary septum is
indistinguishable from that in the rest of the
trabeculations (arrow in Figure 1D). At E11.5, the growing
septum exhibits heterogeneity of versican mRNA
expression, with the right side expressing at a higher level than the
left side (data not shown), reflecting the differences in expression of
versican mRNA between the right and left ventricles (Figure 1H). By
E12.5, the septum has fused with the atrioventricular
cushions (Figure 2A) but has not yet fused with the outflow tract
septum (Figure 2B). At this stage, the core of the septum expresses
versican only at a low level, in a manner similar to that in the
compact zone of the ventricular wall, whereas the outer
layers of the septum continue to express versican more intensely, with
the leading edge exhibiting particularly strong expression (arrowheads
in Figure 2B). At E13.5, after the septum has fused with the
atrioventricular cushions and with the septum of the
outflow tract, the former crest of the ventricular septum
retains high expression of versican transcripts, whereas the core of
the septum continues to express versican at a lower level than the
outer layers (Figure 2D).
Intense Expression of Versican in the Endocardial Cushions and
Developing Valves
Endocardial cushions first appear in the looping heart tube at
about E9.5. They consist of swellings within the
atrioventricular canal and outflow tract in which
endocardial cells transform from an epithelial to mesenchymal
morphology, and they migrate throughout the cardiac jelly toward the
myocardium. Versican transcripts can be detected at E10.0
in the early atrioventricular endocardial cushions
(Figure 1D). This expression is detected as the cells deepithelialize
and migrate into the cardiac jelly. No versican mRNA is detected in the
presumptive cushions before the transformation process. By E10.5,
versican is strongly expressed in the endocardial cushions of the
atrioventricular canal (Figure 1E) and in the newly
developing truncal ridges of the outflow tract (Figure 1F). As the
loose early endocardial cushion develops into the cell-dense structure
characteristic of the more mature valvular tissue, versican
mRNA expression increases in both the atrioventricular
and outflow tract cushions (Figure 1G and 1I). The
atrioventricular (Figure 2D) and semilunar (large
arrowheads in Figure 2E) valves remain strongly positive for versican
mRNA throughout the remainder of fetal development. However, in the
more distal region of the outflow tract, mRNA transcript expression is
downregulated throughout the walls of the aorta and the
pulmonary trunk after their separation (Figure 2E).
Interestingly, the valves guarding the entry of the superior caval vein
are also versican-positive throughout their development, even though
these structures do not arise from endocardial cushions (large
arrowheads in Figures 1I, 2A, and 2D). Versican mRNA continues to be
expressed in the mitral and tricuspid valves of the neonatal mouse
heart (Figure 2F), suggesting that versican may be involved in the
continued remodeling of the valve leaflets after birth.
Versican Protein Mimics the Pattern of the mRNA Expression
Throughout Heart Development
Localization of versican protein during heart development in the
mouse embryo reveals a pattern broadly similar to versican mRNA
expression. At E8.5 to E9.5, protein is found surrounding both the
endocardial and myocardial cells in the presumptive atrial and
ventricular regions (Figure 4A). This suggests that the restriction
of versican protein to the ventricle occurs slightly later in
development than the restriction of the mRNA. However, by E10.5
versican is downregulated in the atrium (Figure 4B and 4C) but is found
surrounding the endocardium of the common ventricle (Figure 4B).
Versican is still found surrounding the endocardial cells until E11.5
but is also found in the myocardium at this stage of
development (Figure 4C). Versican expression in the developing
ventricle is downregulated by E12.5 (Figure 4F and 4G).
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Versican is also abundant in the developing
atrioventricular and outflow tract cushions and in the
atrial septum as it begins to form, mirroring the mRNA pattern (Figure 4B to 4G). Moreover, at E11.5, versican staining is noticeably more
intense in the right ventricle than in the left ventricle (Figure 4C),
as was also seen in the mRNA study (Figure 1H). Although protein levels
are declining in the ventricular myocardium at
E12.5, expression can still be seen in the developing valves (Figure 4F and 4G [arrows]). Interestingly, staining can be seen
throughout the development of the venous valves, supporting a role for
versican in the development of these structures (arrowheads in Figure 4F and 4G).
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Discussion |
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We have studied the cardiac expression, at both mRNA and protein levels, of the chondroitin sulfate proteoglycan versican (PG-M) from the earliest stages of mouse heart tube formation until the neonatal period. We find it to be associated with all aspects of valve and septum formation. In addition, versican is expressed in the myocardium in a chamber-specific manner before any morphological indications of septation have occurred. This myocardial expression is restricted to the developing trabeculations, showing rapid downregulation in the myocardial wall. These differences may reflect a role in specification of the ventricles and/or functional differences between the 2 ventricles.
Versican May Be Involved in Chamber Specification
Versican mRNA is expressed initially in the myocardium
throughout the heart tube, but this generalized expression is quickly
downregulated in the presumptive atrium as the heart loops at E9.0 (11
to 13 somites). At the same time, versican becomes differentially
expressed between the presumptive right and left ventricles, and by
E10.5, it is clearly expressed in a right ventricle–specific manner.
Versican protein is expressed in a similar pattern, with the right
ventricle showing more abundant staining than the left. This right
ventricle– specific expression pattern mirrors the expression of the
basic helix-loop-helix gene dHAND,5 raising the
possibility that versican may be regulated by dHAND. MEF2c, a known
regulator of skeletal, smooth, and cardiac
myogenesis,18 19 is expressed throughout the
myocardium of the developing heart. It was proposed
recently that the MEF2c protein might promote right ventricle–specific
expression by binding to a regulatory region within several genes,
including myosin light chain-2 and desmin.20 21
This raises the possibility that MEF2c is upstream from dHAND in a
cascade to specify the right ventricle.4 It is
possible, therefore, that the versican gene promoter might also contain
an MEF2c binding site or that MEF2c might regulate versican via dHAND.
Although gene knockouts exist for dHAND and MEF2c, both types of null
mutant embryos fail to develop a right
ventricle,4 5 precluding a study to determine
whether the right ventricle–specific expression of versican is
dependent on expression of these transcription factors.
Apart from the HAND genes, versican is the only characterized gene to be expressed in a ventricular chamber– specific manner before chamber septation, raising the possibility that it may have a functional role in the specification of the chambers. The ventricular septum develops precisely at the junction between the domains of high and low intensity of versican expression. Thus, it is possible that the boundary of versican expression may be involved in specifying the site at which the ventricular septum forms. Versican expression has been associated with changes in cell fate and the proliferative rate12 22 (D.J. Henderson, M. Kolatsi-Joannou, and A.J. Copp, unpublished data, 1998), both of which might play a part in chamber specification. A random transgene insertion has located another gene, named hdf, which also has right ventricle–specific expression before chamber septation.23 Mice homozygous for this insertional mutation die at E10.5 of gestation, with abnormalities of the endocardial cushions and the ventricular trabeculations, both of which exhibit strong versican expression. Moreover, the hdf integration site is mapped close to the versican locus on mouse chromosome 13,23 24 25 raising the possibility that the hdf insertion is within the versican gene. Indeed, recent unpublished mapping data support versican as a strong candidate for the gene mutated by the hdf mutation (C. Mjaatvedt, written communication, 1998). A number of other structural genes, including SM22, have also been shown to be differentially expressed between the ventricles. These differences appear after chamber septation, however, and seem likely to reflect differences in maturation rate of the myocardium in the 2 chambers rather than a role in chamber specification.26
Versican May Be Involved in Signaling Between the Endocardium
and Myocardium
Versican transcripts are seen initially throughout the
myocardium, but expression becomes upregulated in the
developing ventricular trabeculations. This
high level of expression is maintained until the
trabeculations form the papillary muscles and tendinous
cords that contribute to the mature cardiac skeleton. Versican protein
is most abundant surrounding the endocardial cells, with lower levels
surrounding the myocardial cells in the preseptation heart. Versican is
an extracellular matrix protein that is secreted from the cell and is
thought to play a role in cell-cell or cell-substratum adhesion. It
appears therefore that versican is synthesized in the
myocardium and is secreted to surround the endocardium.
This suggests that versican may have a role in the interaction between
myocardial and endocardial cells. A variety of other molecules appear
to be involved in this signaling between the myocardium and
endocardium, including neuregulin, angiopoietin-1, and their
receptors6 27 and the chemokine
PBSF/SDF-1.28 Gene targeting has revealed the
importance of these molecules for trabeculation and
septation of the ventricles.6 27 28 If the
neuregulin or angiopoietin-1 signaling pathways are disrupted, the
ventricle remains untrabeculated, and the
ventricular septum does not form, supporting a role for
signaling between the endocardium and myocardium in the
development of trabeculae. Versican is expressed at sites
of cell-cell interaction throughout the developing mouse embryo
(D.J. Henderson, M. Kolatsi-Joannou, and A.J. Copp, unpublished
data, 1998), and this supports a general role for versican in mediating
interactions between different cell types during development.
Versican Is Regulated According to the Proliferative and
Differentiated State of Myocardial Cells
As the myocardial wall develops, versican becomes downregulated in
the outer compact layer but is maintained in the spongy inner layer.
Similarly, versican is abundant in the outer layer and crest of the
ventricular septum but is downregulated in the inner core.
Proliferative differences are known to exist in the
ventricular myocardium, with a rapid rate of
proliferation in the compact zone of the myocardial wall and the core
of the ventricular septum and a slower proliferative rate
in the trabeculations and outer layers of the
ventricular septum.29 30 The
N-myc transcription factor is associated with cell
proliferation7; consistent with this
role, N-myc is expressed in the compact zone of the
ventricular myocardium and in the core of the
ventricular septum but not in the more slowly dividing
trabeculations and outer layers of the
septum.31 The expression of N-myc is
therefore reciprocal to versican, reinforcing the idea that versican is
expressed in differentiating rather than rapidly proliferating cells. A
number of mouse mutants have defects in
trabeculation and the formation of the
ventricular septum. These include N-myc and
NF-1, both of which are thought to be involved in the control of cell
proliferation.7 9 NF-1 is thought to negatively
regulate proliferation, whereas N-myc stimulates
proliferation, suggesting that these processes are tightly coupled in
the myocardium. It will be interesting to determine whether
downregulation of versican expression is necessary for differentiation
of the myocardium and/or growth of the core of the
ventricular septum. Versican expression has also been shown
to be related to the proliferative/differentiated state in the dermis,
where versican is abundant at low cell densities but is downregulated
when cell density is high or when cells undergo terminal
differentiation.22 The authors of that
study22 suggest that versican may serve to
modulate cell proliferation and it may be that, in the developing
heart, versican may also play a regulatory role, perhaps directing the
switch from rapid proliferation to differentiation of the
myocardium. Cardiomyocytes undergo terminal differentiation
during the neonatal period, when versican is completely switched off in
the ventricle. This suggests that versican may need to be downregulated
before terminal differentiation can take place.
A Role for Versican in Epithelium to Mesenchyme
Transformation
Before E9.5 in the mouse, the endocardial cushions constitute an
acellular accumulation of extracellular matrix ("cardiac jelly")
enclosed by endocardium and myocardium. From E9.5,
endocardial cells overlying these sites undergo an epithelium to
mesenchyme transformation, under the influence of the
myocardium, and migrate into the cardiac jelly to form the
loose mesenchymal structure of the cushion
tissue.32 33 34 The endocardial cushions then act
as primitive valves within the looping heart. We have shown that
versican is expressed in cushion tissue as cells delaminate from the
endocardium and migrate into the cardiac jelly and that it continues to
be expressed in the developing valves into the neonatal period.
Endocardial cells do not express versican mRNA transcripts, although
the protein is found surrounding these cells up to around E11.5.
Versican is also expressed throughout the development of the primary atrial septum. It is found initially in the roof of the atrium as the septum is forming, but as the septum grows toward the endocardial cushions, it is expressed solely in the mesenchymal crest. Neural cell adhesion molecule, which is expressed by the endothelial cells of the developing septum, becomes polysialylated coincident with this cellular change, possibly mediating a reduction in cell-cell adhesion and thereby facilitating cell migration.35 The expression of versican may well be involved in this same cellular process, providing an antiadhesive influence to facilitate cell migration as the atrial septum grows toward the endocardial cushions. Interestingly, versican is also found in the developing venous valves, which are not known to arise from endocardial cushion tissue or to involve an epithelial to mesenchyme transformation. It is possible that the venous valves do in fact develop by a process of epithelial-mesenchymal transformation or that versican has a role in the sculpting of the valves after they have formed.
Versican is well known for its antiadhesive properties,36 37 and its expression at sites of epithelial-mesenchymal transformation may mediate the detachment and migration of mesenchymal cells rather than play a role in the transformation process itself. Versican binds to hyaluronan38 and colocalizes with tenascin at numerous sites during development and in the adult.39 Hyaluronan and tenascin are expressed in the developing endocardial cushions,40 41 and it is possible that versican interacts with these molecules to mediate downstream events. Versican is also expressed at sites of epithelial-mesenchymal transformation outside the heart in developing chick and mouse embryos12 (D.J. Henderson, M. Kolatsi-Joannou, and A.J. Copp, unpublished data, 1998), providing support for a generalized role of versican in these cellular transformations.
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Acknowledgments |
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We are grateful to Dr T. Shinomura and Professor K. Kimata for the kind gift of the versican antibody, to Professor Robert Anderson for critically reading the manuscript, and to the British Heart Foundation for financial support (grant PG/96034).
Received January 7, 1998; accepted June 16, 1998.
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