© 1997 American Heart Association, Inc.
Articles |
Expression Pattern of Connexin Gene Products at the Early Developmental Stages of the Mouse Cardiovascular System
From the Laboratoire de Génétique et Physiologie du Développement (B.D., T.J-G., D.G., M.T-R.), UMR C9943, Institut de Biologie du Développement de Marseille (France), Faculté des Sciences de Luminyce; GSF Forschungszentrum (E.D.), Institute for Mammalian Genetics, Oberschleisshem, Germany; Abt. Molekulargenetik (K.W.), Institut für Genetik, Universität Bonn (Germany); and Laboratoire d'Immunochimie des Peptides et Virus (J.-P.B.), UPR 9021, Strasbourg, France.
Correspondence to Dr Magali Théveniau-Ruissy, LGPD-IBDM, Campus de Luminy, Case 907, 13288 Marseille Cédex 09, France. E-mail thevenia{at}lgpd.univ-mrs.fr
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Abstract The synchronized contraction of myocytes in cardiac muscle requires the structural and functional integrity of the gap junctions present between these cells. Gap junctions are clusters of intercellular channels formed by transmembrane proteins of the connexin (Cx) family. Products of several Cx genes have been identified in the mammalian heart (eg, Cx45, Cx43, Cx40, and Cx37), and their expression was shown to be regulated during the development of the myocardium. Cx43, Cx40, and Cx45 are components of myocyte gap junctions, and it has also been demonstrated that Cx40 was expressed in the endothelial cells of the blood vessels. The aim of the present work was to investigate the expression and regulation of Cx40, Cx43, and Cx37 during the early stages of mouse heart maturation, between 8.5 days post coitum (dpc), when the first rhythmic contractions appear, and 14.5 dpc, when the four-chambered heart is almost completed. At 8.5 dpc, only the reverse-transcriptase polymerase chain reaction technique has allowed identification of Cx43, Cx40, and Cx37 gene transcripts in mouse heart, suggesting a very low activity level of these genes. From 9.5 dpc, all three transcripts became detectable in whole-mount in situ–hybridized embryos, and the most obvious result was the labeling of the vascular system with Cx40 and Cx37 anti-sense riboprobes. Cx40 and Cx37 gene products (transcript and/or protein) were demonstrated to be expressed in the vascular endothelial cells at all stages examined. By contrast, only Cx37 gene products were found in the endothelial cells of the endocardium. In heart, Cx37 was expressed exclusively in these cells, which rules out any direct involvement of this Cx in the propagation of electrical activity between myocytes and the synchronization of contractions. Between 9.5 and 11.5 dpc, Cx40 gene activation in myocytes was demonstrated to proceed according to a caudorostral gradient involving first the primitive atrium and the common ventricular chamber (9.5 dpc) and then the right ventricle (11.5 dpc). During this period of heart morphogenesis, there is clearly a temporary and asymmetrical regionalization of the Cx40 gene expression that is superimposed on the functional regionalization. In addition, comparison of Cx40 and Cx43 distribution at the above developmental stages has shown that these Cxs have overlapping (left ventricle) or complementary (atrial tissue and right ventricle) expression patterns.
Key Words: connexin • gene expression • heart
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Introduction |
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The gap junctions are clusters of membrane channels that allow the direct cell-to-cell passage of small cytoplasmic components, such as ions, metabolic intermediates, and second messengers, and that are thus responsible for intercellular metabolic and electrical coupling. The proteins that form these channels are encoded by a multigene family, the Cx family. The structure and assembly of Cxs into gap junctions have recently been reviewed by several authors.1 2 3 4
Since the elegant experiments of Barr et al,5 it has been known that the propagation of electrical activity in the myocardium and, accordingly, the coordination of the contraction of the atrial and ventricular myocytes require the structural integrity of the gap junctions between myocytes. The physiological importance of these structures in the myocardium has given rise to numerous investigations dealing with their distribution in the different cardiac tissues, their functioning mechanism, and the regulation of their permeability.6 7 8 The identification of the Cxs that form the intermyocyte gap junctions is a first step toward a molecular understanding of the conduction phenomenon in adult and embryonic hearts.
The products of six Cx genes (Cx50, Cx46, Cx45, Cx43, Cx40, and Cx37) have been detected in the mammalian heart, and the first task that arises is to determine with which cell types, among those found in the heart (myocytes, fibroblasts, pericytes, endothelial cells, smooth muscle cells, and nerve cells), these Cxs are associated. The cardiac cells that express Cx46, a Cx whose mRNA has been detected in low amount in rat heart,9 have not yet been identified, but these cells are likely not myocytes. Cx50 was localized within the atrioventricular valves of rat heart during the postnatal period and is probably associated with interstitial cells.10 The abundance of the transcripts of the other four Cx genes (Cx45, Cx43, Cx40, and Cx37) is regulated during heart development,11 12 13 and each of these Cxs forms homomeric gap junction channels with unique functional properties.14 15 Cx43 is the major gap junction protein in the adult mammalian heart.16 17 18 From the prenatal stages on, it has been shown to be abundantly expressed in heart, where it is associated with myocytes.11 12 19 20 However, neither Cx43 nor its mRNA has been detected in the proximal regions of the conduction system in rats,10 20 21 22 and the expression of the Cx43 gene in the nodal cells of mammalian hearts is still a matter of debate.14 23 Homozygous embryos of mice lacking Cx43 survive until birth but die by asphyxiation shortly after delivery.24 Death is the consequence of an obstruction of the outflow tract, which prevents the blood flow from reaching the lungs. These findings demonstrate that, unexpectedly, Cx43 plays an essential role in cardiac morphogenesis and that at least one, or possibly several, Cxs have been able to replace Cx43 in its current-carrying capacity. Cx45 mRNA was detected in very low abundance in adult and embryonic hearts, including mouse hearts.13 25 26 In dog hearts, Cx45 has been shown to be a component of the gap junctions of all types of myocytes (working, conductive, and nodal myocytes).25 26 It was also reported to be expressed in human ventricular myocytes27 28 and in cultured neonatal rat heart myocytes.29 Cx40 gene expression has been the subject of numerous studies in different mammalian species, and a feature consistently emerging from these investigations is that Cx40 is expressed in the atrial myocytes and the conductive myocytes (which form the His bundle, the two bundle branches, and the Purkinje fibers).14 21 30 In adult mouse ventricular muscle, the distribution of Cx40 is restricted to the conduction system, and this has been demonstrated to be the consequence of the strong spatiotemporal downregulation of its expression, which occurs during development.13 A similar pattern of regulation has also been described in rat hearts.22 In addition to the atrial and conductive myocytes, Cx40 in the heart has also been detected in the endothelial cells of the coronary vessels.13 31 32
In a previous study,13 we demonstrated that the downregulation of Cx40 gene expression, which occurs in mouse embryonic hearts from 14 dpc, correlated with the differentiation of the conduction system. Here, we have investigated the distribution of Cx40 gene products at the early stages of heart maturation, between 8.5 dpc, when the first cardiac contractions appear, and 14.5 dpc, when the formation of the four-chambered heart is almost completed. The spatiotemporal distribution of Cx40 has been compared with that of Cx43 at the same stages, and each Cx was found to be expressed with a unique pattern of distribution. We have also shown that Cx37 in heart is expressed exclusively in the endothelial cells of the endocardium and the coronary vessels, which rules out any direct involvement of this Cx in the propagation of electrical activity between myocytes.
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Biological Material
Parental HeLa cells and HeLa cells transfected with mouse Cx37 cDNA were cultured at 37°C under 10% CO2 atmosphere in DMEM supplemented with 10% fetal calf serum and puromycin (0.5 mg/mL) as described previously.33
Female Swiss mice, 6 to 8 weeks of age, were mated overnight with males of the same strain. The day the vaginal plug was found was considered to be day 0.5 of pregnancy.34 The hearts were collected from 8.5- to 14.5-dpc embryos under sterile conditions. Great care was taken to ensure that only the heart was removed. The developmental stage of the embryos was checked by determining the number of somites and analyzing the morphology of the heart.35
RNA was extracted from mouse embryo hearts collected at different developmental stages using the guanidium thiocyanate method.36 Bluescript SK+ plasmids containing the complete coding regions of mouse Cx37, Cx40, or Cx4337 38 were propagated in Escherichia coli XL1 BLUE and purified according to standard protocols.
Preparation and Purification of Site-Directed Antibodies
The peptide (Y)GGRKSPSRPNSSASKKQ, corresponding to amino acids
315 to 331 of mouse Cx37,38 was synthesized in solid phase
and conjugated to keyhole limpet hemocyanin as described
previously.32 The immunization of rabbits and the affinity
purification of antibodies (antibodies 315 to 331) from immune sera
have been described in detail by Dupont et al.39 The
preimmune sera were subjected to the same purification steps as the
immune sera. The collected fractions, used in control experiments, will
be referred to as preimmune fractions.
Immunoblotting Analyses
Parental HeLa cells and HeLa-Cx37 transfectants, cultured at
confluence in 75-cm2 flasks, were rinsed twice with
ice-cold PBS solution (pH 7.2) and harvested in 300 µL of RIPA buffer
(50 mmol/L Tris-HCl, pH 7.5, 150 mmol/L NaCl,
1% NP-40, 1% sodium deoxycholate, and 0.1% SDS) containing protease
inhibitors (leupeptin, pepstatin, antipain, and
benzamidine, at a final concentration of 1 mmol/L). The
cell suspensions were frozen at -20°C, thawed, kept for 30 minutes
at room temperature, and centrifuged for 10 minutes at
13 000g. The supernatants, which will be referred to as
homogenates, were diluted 1:1 with the sample buffer
(125 mmol/L Tris-HCl, pH 6.8, 4% SDS, 10%
2-mercaptoethanol, 20 mmol/L EDTA, and 10% glycerol), and
aliquots of the mixtures were fractionated by electrophoresis and
electrotransferred onto nitrocellulose membranes as described
previously.32 Immunoreplicas were successively incubated
with rabbit antibodies 315 to 331 (10 µg/mL), biotinylated
goat anti-rabbit antibodies, and peroxidase-labeled streptavidin
(Jackson Immunoresearch Laboratories) as described
previously.32 Peroxidase activity was revealed with
H2O2 and 4-chloronaphthol according to standard
procedures. The specificity of the labelings detected in the
immunoreplicas was checked by (1) the analysis of
nontransfected parental cell homogenates, (2) the omission
of the primary antibodies (antibodies 315 to 331), (3) the replacement
of the primary antibodies by a preimmune fraction, and (4) the
replacement of primary antibodies by a solution of primary antibodies
preincubated overnight at 4°C with the immunogenic peptide (50
µg/mL). Protein content of the cell homogenates
was determined using the bicinchoninic acid assay40 (Sigma
Aldrich Chimie).
Hearts collected from 14.5-dpc mouse embryos were freeze-dried and stored at -80°C until use. Samples were sonicated in a minimal volume of RIPA buffer, kept for 30 minutes at room temperature, and centrifuged for 10 minutes at 13 000g. The supernatants, referred to as homogenates, were fractionated by electrophoresis, electrotransferred as described above, and then analyzed by immunoblotting with antibodies 315 to 331 (1 µg/mL) using a chemiluminescent detection reagent kit (Boehringer-Mannheim) as previously described.13
Immunofluorescence Analyses
Antibodies
Anti-desmin mouse monoclonal antibodies (clone DE-U-10, Sigma
Aldrich Chimie), diluted to 1:50, and anti-mouse CD31/PECAM-1 rat
monoclonal antibodies (clone MEC 13.3; Pharmingen, Inc), diluted to
1:50, were used as markers of myocytes and endothelial
cells, respectively.41 42 43 The anti-mouse CD 31/PECAM-1 rat
antibodies will be referred to hereafter as anti-PECAM antibodies.
Anti-Cx43 antibodies were rabbit or hen anti-peptide antibodies,
previously characterized,32 44 and they were used at 3 and
8 µg/mL, respectively. The characterization of the rabbit
anti-Cx40 antibodies has also been previously published.32
The rabbit anti-Cx37 antibodies were those characterized in this
article (antibodies 315 to 321). The anti-Cx40 and anti-Cx37 antibodies
were used at 5 and 7 to 8 µg/mL, respectively. The above
primary antibodies were detected with appropriate fluorescent
secondary antibodies (see below). All antibodies were diluted in a
saturation solution made with PBS containing 2% (wt/vol) BSA and
0.05% (wt/vol) saponin.
Single-Labeling Experiments
Parental and Cx37-transfected HeLa cells were cultured on
gelatin-coated coverslips, fixed, and permeabilized
according to a previously described procedure.45 The cells
were incubated overnight at 4°C with antibodies 315 to 331, washed
with PBS, and incubated for 1 hour at room temperature with
TRITC-labeled goat anti-rabbit IgGs (Sigma Aldrich Chimie), diluted
to 1:200. The cells were carefully washed and mounted on slides with a
Citifluor antifading solution (Citifluor Products). They
were examined with a Zeiss III light microscope equipped for
epifluorescence. The specificity of the immunolabelings in
HeLa-Cx37 transfectants was checked by carrying out control experiments
similar to those described above for the immunoblotting
analyses.
Double-Labeling Experiments
Mouse embryos (8.5 to 9.5 dpc), fixed for 15 minutes in 2%
formaldehyde solution in PBS, and isolated hearts, unfixed, collected
from late embryos or adult mouse were embedded in Tissue-Tek OCT
compound (Miles Laboratories) and frozen in isopentane precooled at
-30°C. Frozen serial sections (5 to 10 µm thick) were mounted
on gelatin-coated slides and stored at -20°C until use. The sections
were incubated overnight at 4°C with rabbit anti-Cx antibodies,
washed with PBS, and incubated for 1 hour at room temperature with
either anti-desmin mouse monoclonal antibody or anti-PECAM rat
monoclonal antibody. The sections were then washed with PBS and
incubated for 1 hour with TRITC-labeled goat anti-rabbit IgGs (Sigma
Aldrich Chimie) mixed with either FITC-labeled sheep anti-mouse
IgGs (Sigma Aldrich Chimie) or DTAF-conjugated donkey anti-rat IgGs
(Jackson Immunoresearch Laboratories). The secondary antibodies used
were diluted to 1:200. The sections were processed and analyzed
as described above.
Photobleach Triple-Labeling Experiments
A standard double-immunofluorescence
labeling for one of the Cxs and for desmin was carried out as described
above. Immunopositive cells were analyzed and imaged, and then
the FITC and TRITC signals were photobleached using the light of the
microscope.46 After photobleaching, a single-labeling
experiment was performed using the anti-PECAM rat monoclonal antibody.
The sections were then washed with PBS and incubated for 1 hour with
DTAF-conjugated donkey anti-rat IgGs (Jackson Immunoresearch
Laboratories). The sections were processed and analyzed as
described above.
Confocal Microscopy Analysis
Frozen sections of 10.5-, 12.5-, and 14.5-dpc mouse embryonic
hearts were processed for double-labeling
immunofluorescence experiments with rabbit
anti-Cx40 antibodies and hen anti-Cx43 antibodies as described
previously.32 The primary antibodies were detected with
Texas red–labeled goat anti-rabbit IgGs (Jackson Immunoresearch
Laboratories) and FITC-labeled goat anti-chicken IgGs (Kirkegaard and
Perry Laboratories) diluted to 1:200. The sections were
analyzed by confocal laser scanning microscopy using a Leica
TCS 4D instrument (Leica Lasertechnik) based on an Leitz DMRBE
microscope interfaced with an argon-krypton laser adjusted to 488 and
568 nm for excitation of FITC and Texas red, respectively, and equipped
with filters and detectors designed for the simultaneous
recording of FITC and Texas red. The emitted signals were
digitized, and pictures from screen images were printed with an NP 1600
Kodonics CS video print.
Whole-Mount In Situ Hybridization
The anti-sense and sense riboprobes were synthesized from
linearized plasmids (see "Biological Material") in the presence
of UTP-digoxygenin according to the manufacturer's instructions
(Boehringer-Mannheim). The 1.06-kb mouse Cx37 anti-sense probe
was synthesized with T7 polymerase from the Xba
I–linearized plasmid. Mouse Cx40 and Cx43 anti-sense probes (1.35 and
0.7 kb, respectively) were both synthesized with T3 polymerase from
Xho I–linearized plasmids. The Cx37 and Cx40 probes matched
the complete coding regions of their respective
genes37 38 ; the Cx43 probe matched 400 bp of the
C-terminal coding region of the mouse Cx43 gene plus 300 bp of the 3'
untranslated region.37
Whole-mount in situ hybridizations were essentially performed as described previously,47 with the following modifications: (1) The embryos were bleached with 6% H2O2 in 80% methanol for 1 hour after fixation. (2) The hybridization mix was prepared with 50% formamide, 1.3x SSC, 5 mmol/L EDTA, 0.2% Tween 20, 0.5% CHAPS, 100 µg/mL heparin, and 50 µg/mL yeast RNA. Briefly, embryos of 9.5- to 12.5-dpc stages were dissected from their decidua in PBS containing 2 mmol/L EGTA and fixed overnight at 4°C with 4% formaldehyde. Permeabilization, prehybridization, and hybridization were as described previously.47 Hybridization signals were detected by processing the embryos with alkaline phosphatase–coupled anti-digoxygenin antibodies (Boehringer-Mannheim), followed by reaction with nitro blue tetrazolium and 5-bromo-4-chloro-3-indolylphosphate. When the color was developed to the required degree, the embryos were washed three times for 5 minutes each at room temperature with TBST (140 mmol/L NaCl, 2.7 mmol/L KCl, 0.1% Tween 20, and 25 mmol/L Tris-HCl, pH 7.5), dehydrated through a methanol/TBST series, and then rehydrated by incubating the embryos through this methanol/TBST series in reverse. Before microscope analysis, the embryos were cleared for 1 hour with a 1:1 (vol/vol) glycerol/PBS containing 1 mmol/L EDTA and 0.05% Tween 20.
In some experiments, the whole-mount hybridized embryos were sectioned after color development. Stained embryos were washed for 1 hour in PBS, incubated 1 hour in 0.86% NaCl, and subjected to a series of incubations in 50%, 75%, 90%, and 100% isopropanol for 2, 3, and 6 hours and overnight, respectively. The embryos were then incubated overnight at 60°C in a mixture consisting of 1:1 (vol/vol) isopropanol/paraffin, followed by a 2-day incubation at 60°C in paraffin. Finally, the embryos were embedded in fresh paraffin, and 10-µm sections were prepared with Leica or Leitz microtomes.
Analyses of control experiments carried out with either sense probes or anti-digoxygenin antibodies did not reveal any labeling.
Identification of Cx Transcripts by RT-PCR
Primer Design and Synthesis
For the PCR and RT-PCR experiments, the primers were designed
using GeneWorks software (IntelliGenetics Inc) from the murine
sequences of Cx37, Cx40, Cx43, and desmin.37 38 48 49 Each
pair of upstream and downstream primers had closely similar Tm values
(Table 1
), and they were checked for
minimal self-priming and upper/lower dimer formation.
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RT-PCR Experiments
RNA samples (2 µg per experiment), extracted from mouse
embryonic heart at different developmental stages or prepared from
wild-type HeLa cells, were reverse-transcripted using the murine RT
(included in the First Strand cDNA Synthesis Kit, Pharmacia Biotech)
with 50 pmol of each specific primer (Table 1
). Reactions (33 µL)
were incubated for 1 hour at 37°C. Eight microliters of the
first-strand reaction of each stage investigated was amplified using
primer pairs specific for each of the three Cx genes or the desmin gene
(Table 1) in a typical 100-µL reaction containing 200
µmol/L dNTPs, 2 mmol/L MgCl2, 50
mmol/L KCl, 10 mmol/L Tris-HCl (pH 9.0 at 25°C),
0.1% Triton X-100, 100 ng of each primer, and 2.5 U Taq DNA polymerase
(Promega Corp). The PCR reactions included 30 cycles with three
temperature steps (94°C, 1 minute; 53°C, 1 minute; and 72°C, 1
minute) and were performed with a Perkin-Elmer-Cetus DNA thermal cycler
(Roche Molecular System, Inc). One third of the amplification reaction
was run in parallel with a known amount of a molecular weight marker
(PM VI, Boehringer-Mannheim) on a 1.5% agarose gel stained
with ethidium bromide.
All the cardiac RNA samples (2 µg) were tested for DNA contamination in reactions carried out in the absence of RT. Blank reactions were carried out without RNA in the presence of RT. RT-PCR reactions also included blank reactions in which the Taq polymerase activity was tested in the absence of cDNA preparations.
Southern Blot Analysis of Amplification Products
To check that the amplification products were those
predicted, they were analyzed by Southern blot with specific
radioactive probes.
DNAs, used to probe the amplicons derived from Cx37 and Cx40 mRNAs,
were PCR products generated from cloned cDNAs containing the
complete coding regions of mouse Cx37 and Cx40 (see "Biological
Material"). The experimental conditions and the primer pairs used
were similar to those described above. The amplification reactions were
carried out with 100 ng of plasmid DNA. The control experiments
included amplification cycles with the vectors alone, without the
cloned cDNAs. The PCR products were analyzed by
electrophoresis as above, and the DNA fragments were recovered from the
agarose gels using the Geneclean II kit (BIO 101, Inc). These fragments
of 244 bp (Cx37 probe) and 255 bp (Cx40 probe) were radiolabeled with
[
-32P]dCTP by random priming using the Multiprime DNA
labeling kit (Amersham International).
DNAs, used to probe the amplification products derived from Cx43
and desmin mRNAs, were synthesized oligonucleotides
whose sequences, included in the amplification products, were as
follows: 5'-GCCTACTCCACGGCCGGAGG-3' (CX43 probe, positions 45 to 65;
Tm, 70°C)48 and 5'-TCAACTTCCGAGAAACCAGC-3' (desmin
probe, positions 1273 to 1293; Tm, 60°C).49 These
oligonucleotides were radiolabeled at their 5' ends
with [
-32P]ATP using T4 polynucleotide
kinase (Boehringer-Mannheim).
The DNA fragments amplified by RT-PCR from the RNAs, as described in the previous paragraph, were analyzed by electrophoresis in agarose gels and then transferred onto nylon membranes (Hybond N+) (Amersham International). The membranes were hybridized overnight at 65°C with the Cx37 and Cx40 probes or at 55°C with the Cx43 and desmin probes in 0.2 mol/L NaPO4 (pH 7.2), 1 mmol/L EDTA, 1% BSA, 7% SDS, and 15% formamide. The membranes were then washed at 65°C or 55°C in 40 mmol/L NaPO4 (pH 7.2), 1 mmol/L EDTA, and 1% SDS and exposed to Kodak XAR5 film (Eastman Kodak Co) for 1 hour or overnight.
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Detection of Cx37, Cx40, and Cx43 Transcripts by RT-PCR at the Early Stages of Heart Maturation (8.5 dpc)
At 8.5 dpc, the mouse heart, which is starting to beat,34 35 is an S-shaped tube. The slow peristaltic contractions that run through it suggest that the primitive myocytes express one or more Cxs assembled into gap junction channels. In order to verify this hypothesis, the double-labeling immunofluorescence technique was applied on serial sections of 8.5-dpc embryos using antibodies specific for Cx40 or Cx43 in combination with anti-desmin or anti-PECAM antibodies, allowing identification of the myocytes or the endothelial cells, respectively.41 42 43 Although the myocytes and the endothelial cells were strongly labeled, no immunoreactivity due to anti-Cx antibodies was observed in the heart or the vascular system. Nor were the transcripts of the three Cxs (Cx37, Cx40, and Cx43) detected in the myocardium using the in situ hybridization technique. A moderate signal was seen, however, in the paired dorsal aortas of the embryos probed for the Cx40 transcript (not shown). These results were at least partly in agreement with the previous observations of Ruangvoravat and Lo50 and Yancey et al,51 who reported having detected no Cx43 gene expression by in situ hybridization or immunofluorescence in the heart of 8.5-dpc mouse embryos. The results obtained were not altogether surprising, because the slow propagation of action potentials between embryonic myocytes52 and the synchronization of their contraction require only very few gap junction channels.53 54 55 On the assumption that the techniques used were perhaps not sensitive enough to detect Cx gene products in the 8.5-dpc hearts, RT-PCR was applied to RNA extracted from these hearts.
RNA extracted from 8.5-, 9.5-, and 14.5-dpc embryonic hearts or from
wild-type HeLa cells (in which no Cx mRNA has yet been
identified)33 was reverse-transcribed using primers unique
to the three Cx genes (Cx37, Cx40, and Cx43) and to the mouse desmin
gene (Table 1). Primer pairs unique to the above genes (Table 1) were
used to amplify the cDNAs generated by reverse transcription, and the
resulting amplicons were analyzed by electrophoresis (Fig 1). RNA samples of 14.5-dpc embryonic
hearts were included in the experiments, because it had previously been
demonstrated that the transcripts of Cx43, Cx40, and Cx37 were
present in mouse heart at this stage of development (authors'
unpublished data, 1995). In addition, the desmin transcript was used as
a positive control in the cardiac samples. Amplicons generated from
8.5-dpc embryonic heart cDNA preparations were of the predicted size
(Table 1) for the three Cxs and desmin (Fig 1A). Amplicons of identical
size were also generated from 9.5- and 14.5-dpc heart cDNAs (Fig 1B and 1C). The identity of each of these amplicons was confirmed by Southern
blotting (Fig 1D) at the three developmental stages investigated. No
DNA fragment was amplified from wild-type HeLa cell cDNA preparations
(not shown). The analyses of the final reactions of the control
experiments (see "Materials and Methods") did not reveal any
signal. These results indicated that Cx37, Cx40, and Cx43 gene
transcripts were present in very low amount in 8.5- and 9.5-dpc
hearts. The probability of the contamination of analyzed
samples by noncardiac cells was extremely low, but it could not be
completely ruled out.
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Cx37 Is Expressed in Both Vascular and Endocardial Endothelial
Cells
From 9.5 dpc, the three Cx gene transcripts became detectable by
in situ hybridization in embryos (see below). To further characterize
the expression of these genes, previously characterized anti-Cx40 and
anti-Cx43 antibodies were used in combination with anti-Cx37
antibodies. Characterization of the latter antibodies (also called
antibodies 315 to 331) is described in the paragraph below.
Characterization experiments were carried out with Cx37-transfected
HeLa cells and mouse embryonic hearts. In addition to the specificity
of the antibodies, these experiments demonstrated for the first time
that Cx37 was expressed in the endocardial endothelial
cells.
Parental HeLa cells were shown to have a very low level of gap
junctional communication,56 and the above experiments
demonstrated that the Cx37 gene transcript was not detectable in these
cells. Analysis of parental HeLa cells by
immunofluorescence and Western blotting with
antibodies 315 to 331 revealed no immunoreactivity (not shown). The
expression in these cells of DNA coding for murine Cx37 was shown to
induce high electrical and dye coupling, thus providing evidence of the
formation of functional gap junction channels upon
transfection.33 In the immunoreplicas of HeLa-Cx37
transfectant homogenates, probed with antibodies 315 to
331, Cx37 was detected as a single-band protein with an
Mr of 37 000 (Fig 2A, lane b). This was similar to the
Mr deduced from immunoprecipitation experiments
carried out with in vitro translation products of rat Cx37
mRNA.57 No labeling was detected in immunoreplicas probed
with either preimmune fractions (Fig 2A, lane c) or antibodies 315 to
331 preincubated with the immunogenic peptide (Fig 2A, lane d).
Analysis of HeLa-Cx37 transfectants by fluorescence
microscopy showed that immunoreactivity due to antibodies 315 to 331 is
localized both in the intercellular contact sites, where the gap
junctions usually occur, and in cytoplasmic vesicles, which accumulate
at one pole of the nucleus (Fig 2B and 2C). Recent investigations have
shown that the overexpression of Cxs induces the aberrant formation of
gap junctions in various cell compartments, including the nuclear
envelope and the endoplasmic reticulum.58 Thus, the
presence of immunoreactive sites in cytoplasmic vesicles of transfected
HeLa cells probably reflects the overexpression of the Cx.
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In the mouse heart, the abundance of Cx37 mRNA is much higher during
fetal life than at the adult stage,13 and accordingly, the
protein should be more abundant at the embryonic than at the adult
stages. Thus, the characterization of antibodies 315 to 331 was
continued using mouse embryonic heart. In the immunoreplicas of
14.5-dpc embryonic heart homogenates probed with antibodies
315 to 331, a single protein was detected (Fig 2A, lane e). Its
electrophoretic mobility matched that of Cx37 expressed in HeLa
transfectants (Fig 2A, lane b). No protein was revealed in replicas
incubated with either preimmune fractions (Fig 2A, lane f) or
antibodies preincubated with the immunogenic peptide (not shown).
Double-labeling immunofluorescence experiments,
carried out with 14.5-dpc embryonic heart sections, using antibodies
315 to 331 and anti-PECAM antibody, showed that immunoreactivity due to
antibodies 315 to 331 was exclusively associated with intercellular
domains of the endothelial cells of both the blood
vessels and the endocardium (Fig 2D through 2F; also see Fig 10C).
Specific labelings of vascular and endocardial
endothelial cells were also observed in younger embryos
(9.5 dpc, vascular endothelial cells, Fig 4A and 4D;
10.5 dpc, endocardial endothelial cells, Fig 8D through
8F; and 13.5 dpc, endocardial endothelial cells, Fig 6G
through 6H). No immunofluorescence signal was ever
observed in embryo sections or in Cx37-transfected HeLa cells using
preimmune fractions or antibodies 315 to 331 preincubated with the
immunogenic peptide (not shown).
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The results described above demonstrate that antibodies 315 to 331 are specific for Cx37, and they will hereafter be referred to as anti-Cx37 antibodies. They were used in the work described below to investigate the distribution of Cx37 in the cardiovascular system.
As reported above, the Cx gene products became detectable by in situ hybridization in the embryos from 9.5 dpc. From this stage until the 12.5-dpc stage, distribution of transcripts was investigated by in situ hybridization. Protein expression was studied until 14.5 dpc by double- and triple-labeling immunofluorescence techniques using anti-Cx antibodies (anti-Cx37, -Cx40, and -Cx43) in combination with anti-desmin and anti-PECAM antibodies. For the sake of clarity, gene expression will be described first in the vascular system (next paragraph) and then in the heart, where atrial and ventricular tissues will be examined separately.
Cx40 and Cx37 Are Coexpressed in the Vascular
Endothelial Cells From 9.5 dpc
At 9.5 dpc, the most patent result of in situ hybridization
experiments was the labeling of the vascular system with Cx40 and Cx37
anti-sense riboprobes (Fig 3A and 3B). By
contrast, Cx43 transcript was never detected in vessels whatever the
developmental stage investigated (Fig 3C). Cx40 transcript was highly
expressed in the vasculature and was observed in the paired dorsal
aortas, the intersegmental arteries sprouting from the aortas, the
brachial arch arteries, the primary head arteries and branching
vessels, the atrium, and the common ventricular chamber
(Fig 3A, 3D, 3E, and 3F). More caudally, Cx40 transcript was detected
in the common aorta and the vitelline, umbilical, and caudal vessels
(Fig 3A and 3E). During development, between 9.5 and 12.5 dpc, the
vascular expression pattern of Cx40 transcript was modified after the
rearrangement of the vasculature. Analyses of sections prepared
from 9.5- to 12.5-dpc embryos hybridized with anti-sense Cx40 riboprobe
(Fig 3G and 3I) and sections incubated with anti-Cx40 antibodies (Fig 4C and 4F) demonstrated that Cx40 gene
products were expressed in the endothelial cells of
the vessels. At 9.5 dpc, Cx40 transcript was also
seen to be expressed, with a periodic pattern, in the lateral myoblasts
of the anterior region of embryos (Fig 3A), as previously described by
Dahl et al59 in the mouse and Van Kempen et
al22 in the rat, at later developmental stages. The
presence of this transcript in myoblasts was observed until the last
stage analyzed by in situ hybridization, ie, 12.5 dpc (E. Dahl
and K. Willecke, unpublished data, 1997).
|
Cx37 transcript was detected both in the vessels and in the heart, but
hybridization signals were weaker than for Cx40 transcript (Fig 3B).
Its distribution in the vascular system was similar to that described
above for Cx40 transcript. Analyses of vessels present in
sections prepared from embryos hybridized with Cx37 anti-sense
riboprobe indicated that the transcript was expressed in
endothelial cells (Fig 3H through 3J). Expression of
the protein in the same cells was demonstrated by
immunofluorescence experiments (Fig 4A and 4D).
Cx37 was never observed in myocytes whatever the stage investigated,
including the adult stage.
Cx40 Gene Is Activated Earlier Than Cx43 Gene in
Atrial Myocytes
At 9 dpc, the looping process of the primitive heart is completed,
placing the cardiac chambers near their final relative position. By 9.5
dpc, the primitive atrium is a single chamber not yet divided into
right and left parts but separated from the common
ventricular chamber by the constriction of the
atrioventricular canal. At this stage, in situ
hybridization experiments showed that Cx40 transcript was clearly
detectable in the thin atrial wall formed by two or three cell layers
(Fig 3A and Fig 5A).
Immunofluorescence experiments carried out at the
same stage demonstrated that the Cx40 protein was associated with the
myocytes (Fig 6A through 6E). Cx40 was
never detected in the endocardium, either at 9.5 dpc (compare panels E
and F in Fig 6) or at later developmental stages or in the adult heart.
This held true for both atrial and ventricular endocardium.
Between 9.5 dpc and 12.5 dpc, the atrial segmentation develops, and by
the end of the 12.5 dpc stage, the atrium is completely partitioned off
by the septum primum, leaving the left and right atria separated from
each other, except for the interatrial foramen secundum. From 10.5 dpc
onward, Cx40 was much more highly expressed in the atrial myocytes than
at the previous stages (Fig 5C, 5D, and 5E), and this expression level
was seen to be maintained at the later developmental
stages.13 No expression of the Cx40 gene was detected in
the atrioventricular canal (Table 2).
|
|
The Cx37 gene transcript was detected in the primitive atrium at 9.5
dpc (Fig 3B). At all stages investigated, hybridization and
immunofluorescence signals specific for Cx37 gene
products were seen in the atrial endocardium, associated with the
endothelial cells (Fig 5G and 5H and Fig 6H). In the
endocardium lining the atrioventricular canal, Cx37 was
detectable in the endothelial cells at the 10.5-dpc
stage (Table 2).
Expression of the Cx43 gene in the mouse embryo, and in
particular in the heart, has previously been the subject of several
detailed investigations.11 19 50 51 59 Our results are in
complete agreement with these investigations, and only the more
pertinent points will be mentioned as a basis for comparison with Cx40
expression. No hybridization or immunoreactivity signal was observed in
the atrium of 9.5- to 11.5-dpc embryos treated with the Cx43 riboprobe
(Fig 3C and Fig 5J and 5K) or anti-Cx43 antibodies. Cx43 gene
expression in the atrium was detected as a weak hybridization signal
from the 12.5-dpc stage only (not shown, but see References 50 and 5150 51 ).
At the later developmental stages, Cx43 was shown to be expressed at a
much higher level (Fig 6I). Cx43 gene expression was never
detected in the atrioventricular canal, whatever
the stage investigated (Table 2).
Activation of the Cx40 Gene Is Delayed in the Myocytes of the
Future Right Ventricle
At 9.5 dpc, the common ventricular chamber (ie, the
primitive ventricle), formerly situated cephalically to the primitive
atrium, is brought into its characteristic adult position, and it is
demarcated from the bulbus cordis35 60 by the
interventricular constriction. The common
ventricular chamber and the bulbus cordis will participate
later in the formation of the left and right ventricles,
respectively.35 At 9.5 dpc, the Cx43 gene transcript was
undetectable or barely detectable in the primitive ventricle (Fig 3C).
However, evidence for activation of the Cx43 gene was provided by the
immunoreactivity of ventricular outermost myocytes to
anti-Cx43 antibodies (Fig 7). Cx43
expression was limited to these cells; myocytes that formed the
primordial trabecular network remained free of labeling
(Fig 7). Hybridization or immunofluorescence
signals reflecting a very low activation of the Cx40 gene, dispersed in
the primordial trabecular network of the
ventricular chamber, were detected as early as 9.5 dpc (Fig 3A and Fig 5B). The expression level of Cx37 gene transcript and
protein in the endocardium of the ventricular regions was
similar to that seen in the atrium (Fig 3B and Table 2).
|
From 10.5 dpc, the ventricular free wall thickens, the
trabecular network develops, and the anlage of the septum
forms at the level of the interventricular constriction. A
striking feature at this stage was the strong activation of the Cx40
gene in the left ventricle, which was expressed by an accumulation of
transcript in this region (Fig 5C, 5D, and 5F). By contrast, no
hybridization signal was detected in the right ventricle (Fig 5C and 5D). As expected, immunofluorescence experiments
revealed a prominent expression of Cx40 in the myocytes of the
trabecular network and the compact free wall of the left
ventricle (Fig 8A and 8B), but no
immunoreactivity was detected in the right ventricle at 10.5 dpc (Table 2). At this stage, Cx43 gene expression, which was limited to the thin
and compact ventricular free wall at 9.5 dpc, was seen to
have spread, although very irregularly, to the myocytes of the
trabecular network of both ventricles (Fig 5L and Fig 8A and 8C). In the ventricular regions, where both Cx40 and
Cx43 were expressed, they were colocalized in most of the gap
junctions, as seen by conventional fluorescence microscopy (Fig 8B and 8C) and confirmed by confocal microscopy analyses (not
shown). In the endocardium, signals of hybridization and
immunoreactivity of Cx37 gene products were of the same intensity
as at the previous stage (9.5 dpc) (Fig 5G and 5I and Fig 8E and 8F).
Cx37 was detected in the endocardium from the subsequent developmental
stages until adult stage.
Cx40 was detected in the right ventricle from the 11.5-dpc stage. At
this stage, and at the later stage of 12.5 dpc, Cx40 was seen
colocalized with Cx43 in numerous gap junctions of the
trabecular network and the free ventricular
wall (Fig 9A through 9C). From 12.5 dpc,
a gradient of expression of Cx40 appeared. Cx40 became barely
detectable in the compact subepicardial layers, whereas its expression
was maintained, if not increased, in the trabecular
network, as illustrated in Fig 10A and 10B for the 14.5-dpc stage. A similar
gradient was also observed for Cx43 (not shown, but see Reference
1919 ).
|
Between 10.5 and 14.5 dpc, the interventricular septum
develops, and by 14.5 dpc, the septation of the ventricles is almost
complete. The heart has achieved the definitive arrangement that will
persist until birth. Cx40 expression was never observed in the myocytes
of the interventricular septum (Fig 10D through 10F and
Table 2). In contrast, from 13.5 dpc, the peripheral
myocytes located at the very top of the septum and lying on its flanks,
ie, the myocytes forming the primitive conduction system, were seen to
highly express Cx40 (Fig 10D through 10F). Remarkably, Cx43 was neither
detected in the myocytes of the primitive His bundle nor in those of
the anterior regions of the primitive bundle branches (Fig 10E and 10F). This differential expression of Cx40 and Cx43 in the anterior
region of the conduction system persisted at the subsequent
developmental stages and was also observed in the adult heart (not
shown). The latter feature has previously been described in adult and
embryonic rat hearts.10 20 21 22 32 Beyond 14.5 dpc, the
progressive spatiotemporal restriction of the Cx40 expression and, in
parallel, the upregulation of Cx43 have been described by Delorme et
al13 and Fromaget et al,19 respectively.
Finally, it should be pointed out that neither the Cx40 gene
products nor those of the Cx43 gene were ever observed in the
outflow tract myocardium (as recently
defined),60 whatever the developmental stages, in
agreement with the results reported for the rat heart20 22
(Table 2).
During heart maturation, the coronary vessels differentiate in
the compact subepicardial layers from precursor cells migrating from
the liver region.61 In the 10.5-dpc mouse heart,
Virágh and Challice62 have described the appearance
of vascular buds, which were the earliest signs of coronary
vessel formation. From 13.5 to 14.5 dpc, when the gradient of Cx40
expression has been established, discrete, cordlike anti-Cx40
immunoreactive sites were observed in the subepicardial layers (Fig 10A and 10B). The topology of these sites, parallel to the epicardium,
suggested the presence of vascular channels, precursors of
coronary vessels formed by endothelial cells.
This was confirmed by the immunoreactivity of channel-forming cells to
anti-Cx37 (Fig 10C) and anti-PECAM antibodies (not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The present work combined RT-PCR, in situ hybridization, and immunofluorescence techniques to investigate the expression and regulation of three Cxs (Cx37, Cx40, and Cx43) during the early stages of mouse heart maturation. We have demonstrated that Cx37 was expressed in endothelial cells of both blood vessels and endocardium and that expression of the Cx40 gene in myocytes depends on a unique activation program. Three points will be discussed below regarding (1) the Cxs in the endothelial cells, (2) the regionalization of Cx40 gene expression in heart, and (3) the redundancy and complementarity of Cx40 and Cx43 gene expression in myocytes of different cardiac regions.
Cxs in the Endothelial Cells
Expression of Cx37 in vascular endothelial cells
is consistent with the presence of gap junctions between these
cells,63 64 and this finding is in agreement with various
results reporting the detection of Cx37 mRNA and/or Cx37 protein in
cultured or in situ vascular endothelial
cells.65 66 67 In contrast, the expression of Cx37 in the
endocardium has not yet been reported, and it is so far the only Cx
detected in this thin tissue layer. Thus, Cx37 would appear to be
involved either alone or with other as-yet-unidentified Cxs in the
formation of gap junctions seen between endocardial
endothelial cells.68 69 Besides Cx37, a
second Cx, Cx40, was found to be expressed in vascular
endothelial cells of the mouse embryo in general and of
myocardium in particular (coronary vessels). These
results are in agreement with previously published reports that
described in various species Cx40 expression in the
endothelial cells of the vascular
tree.31 32 59 67 70 71 It is not yet known, however,
whether Cx37 and Cx40, which are compatible Cxs,3 33 are
expressed in the same endothelial cells and, if they
are, whether they are colocalized in the same gap junctions. Cx40
was found not to be expressed in the atrial and ventricular
endocardium. This Cx could be considered as a marker of the vascular
endothelial cells. Consequently, a detailed
investigation (which would go well beyond the scope of the present
study) of the expression of Cx40 and Cx37 in the
endothelial cells of the outflow tract area and the
heart venous pole would perhaps make it possible to answer the question
that is posed by embryologists concerning the boundary between vascular
endothelium and endocardial
endothelium.
Our results demonstrate the early expression of Cxs in endothelial cells of mouse embryos, and of the three Cx genes investigated, the Cx40 gene was shown to be the first gene activated in the vascular system: its transcript was detectable, weakly, by in situ hybridization as early as the 8.5-dpc stage (data not shown). From 9.5 dpc onward, both Cx40 and Cx37 were detected in endothelial cells of developing and remodeling large vessels, and when the myocardial vascularization was initiated, they were also both expressed in the coronary vessels. Thus, early in development, Cx37 and Cx40 were demonstrated to be closely associated with the vascularization process. Both Cxs were also seen in the vascular endothelial cells in the adult, but they were never detected in heart capillaries, which perhaps indicated a very low Cx expression level in these structures. No hybridization or immunoreactivity signal suggesting the expression of Cx43 in vascular endothelial cells was ever detected in mouse embryos or in adult hearts. Cx43 expression has, however, been described in endothelial cells of certain regions of the vascular tree in other species, such as rats72 and humans.67
The physiological role of gap junctions in the coordinated contraction of uterine smooth muscle cells73 and cardiac myocytes14 is well documented. Their role is much less clear in vascular endothelium. Vascular endothelial cells communicate via electrical and dye coupling,65 66 67 74 and it has recently been demonstrated that their gap junctions are permeable to inositol 1,4,5-trisphosphate.66 Junctional communication between these cells is thought to be involved in their migration capacity during wound repair, but our results suggest they also have a potential role in vasculogenesis and angiogenesis. Another aspect that has to be considered regarding the vessels is the junctional communication between vascular endothelial cells and subjacent smooth muscle cells of the tunica media. This heterotypic communication, demonstrated by several authors, would appear to be implicated in the coordination of vasomotor responses (for reviews, see References 75 and 7675 76 ). To our knowledge, the physiological role of gap junctions in endocardial endothelial cells has not yet been addressed, but if it is confirmed that gap junctions between these cells are constituted only of Cx37, then endocardium endothelial cells would be a suitable model for the investigation of Cx37 endogenous channels.
Regionalization of Cx40 Gene Expression in Heart
During the developmental period covered by the present
investigation (8.5 to 14.5 dpc), Cx40 gene expression in mouse heart
myocytes is subordinated to strong spatiotemporal regulation. The Cx40
gene was shown to be transcribed at 8.5 dpc, but its mRNA was
present in very low amounts, and it was detectable only by RT-PCR.
At 9.5 dpc, the gene was found to be activated: its transcript
and the protein were both detected by in situ hybridization and
immunofluorescence in the primitive atrium and, to
a lesser extent, in the common ventricular chamber.
Activation of the Cx40 gene in the embryonic right ventricle was seen
from 11.5 dpc only. Beyond this stage and until 14 to 14.5 dpc, atria
and ventricles equally expressed Cx40, except in the
interventricular septum, where Cx40 gene expression was
never detected (Table 2). Thus, at the early stages of mouse heart
maturation, Cx40 gene activation proceeds according to a caudorostral
gradient involving first the primitive atrium and the left
ventricular region and then the right
ventricular region, while avoiding, however, the
atrioventricular canal and the
interventricular septum. During this period, there is
clearly a temporary regionalization of the Cx40 gene expression. The
regionalization of this Cx is in fact superimposed on a morphological
and functional regionalization of the myocardium, which (in
chickens but probably also in rodents, although this has yet to be
formally demonstrated) sees the alternating of segments with slow- and
fast-conducting properties.77 The slow-conducting inflow
tract, the fast-conducting atrium, the slow-conducting
atrioventricular canal, the fast-conducting ventricle,
and, finally, the slow-conducting outflow tract follow each other, in
the heart, in the caudorostral direction.77 During the
heart morphogenesis, as shown in Table 2 for stages 9.5 to 14.5 dpc,
expression of Cx40, and also of Cx43, is associated with the
fast-conducting regions. From 14 to 14.5 dpc, the Cx40 gene expression
is modified in space and time, as previously described,13
leading to a further regionalization of Cx40 in the adult heart. This
regionalization, which has been demonstrated in the adult
myocardium of other mammal species, again associates the
expression of Cx40 with domains of fast conduction.21
Redundancy and Complementarity of Cx Gene Expression in
Heart
Homozygous Cx43-/- mouse embryos die shortly after
birth because of their inability to establish autonomous
pulmonary circulation. Death is the consequence of an
obstruction of the outflow tract due to a proliferation of myocytes,
producing interconnected septa and blind-ended cavities, in the right
ventricular chamber.24 Before birth, the heart
contracts rhythmically,78 which implies that one or
several Cxs substitute for Cx43 in its current-carrying capacity. Among
the known cardiac Cxs, two are candidates for this substitution role:
Cx40 and Cx45. The distribution of Cx40 in embryonic, newborn, and
adult mouse heart is known (the present study and Delorme et
al13 ), and this Cx, highly expressed in embryonic heart,
could replace Cx43 functionally. However, whatever the developmental
stage considered, the expressions of Cx40 and Cx43 are never
overlapping in all cardiac tissues.
Immunofluorescence investigations have shown, for
example, that in the late fetal stages and at birth, Cx40 is not
expressed in the interventricular septum and the
ventricular free walls. In the light of these results, it
is difficult to explain the propagation of electrical activity in the
ventricular walls of the homozygous neonates for the Cx43
null mutation,79 especially since Cx40 regulation in heart
of mutant embryos is identical to that of wild-type embryos (J. Ya,
A.F.M. Moorman, W.H. Lamers, unpublished data, 1997). The simplest
hypothesis to explain the persistence of the conduction phenomenon is
to postulate that a Cx other than Cx40 is involved in the electrical
coupling between myocytes, at least in heart regions lacking Cx40.
Cx45, whose expression is not upregulated in the myocardium
of Cx43 knockout mouse embryos (J. Ya, A.F.M. Moorman, W.H. Lamers,
unpublished data, 1997), could be this Cx, and consequently, its
distribution in the developing heart deserves particular consideration.
Finally, the possibility that another Cx, which is not normally
expressed in the myocardium, could be upregulated in the
heart of Cx43-/- mice cannot be excluded.
Looping of the heart to the right is the first indication of left-right asymmetry in the embryo. Several signaling molecules and extracellular matrix molecules that may play a role in cardiac looping have been identified recently (for reviews, see References 80 and 8180 81 ). Within this context, it is interesting to observe that at the early stages of heart maturation, ie, between 9.5 and 11.5 dpc, Cx40 is expressed only in one of the two compartments (the common ventricular chamber) that will participate in the formation of the ventricles. In contrast, at the same stages, Cx43 is expressed in both ventricles. Whether this asymmetrical expression of Cx40 may play a role in the morphogenesis of the heart is not known.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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These investigations were supported by grants from the Ministère de l'Education Nationale, de l'Enseignement Supérieur et de la Recherche (grant ACC SV No. 95-09003, Dr Gros), the Association Française contre les Myopathies (Dr Gros), the European Economic Community (grant BMH4-CT 96-1427, Drs Gros and Willecke), and the Deutsche Forschungsgemeinschaft SFB 284 C1 (Dr Willecke). We thank C. Moretti, P. Weber, G. Turini, and N. Barrois for their valuable assistance in image analysis and photography. We thank also Drs W. Lamers, H.J. Jongsma, and P. Durbec for the critical reading of the manuscript and their suggestions.
Received January 17, 1997; accepted June 3, 1997.
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