(Circulation. 1995;92:3289-3296.)
© 1995 American Heart Association, Inc.
Articles |
Vascular Smooth Muscle Cells of H-2Kb-tsA58 Transgenic Mice
Characterization of Cell Lines With Distinct Properties
From the Institute of Molecular Biology (E.E., A.D.), Austrian Academy of Sciences, Salzburg, Austria; Ludwig Institute for Cancer Research (P.S.J., M.D.N.), Riding House Street, London, UK; and Department of Biology (S.C.), University of Padova, Padova, Italy.
Correspondence to Dr Annette Draeger, Institute of Anatomy, University of Berne, Bühlstr 26, 3000 Berne 9, Switzerland.
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Abstract |
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Background The vascular wall is composed of at least two different populations of smooth muscle cells that are distinct in their structure and protein composition. According to the developmental stage of tissue taken for culture, the ratio between cells of epithelioid phenotype and spindle-shaped cells is variable. In particular, the epithelioid cells display characteristic features associated with immaturity. Because their increased appearance can be observed in endothelial denudation, they represent a dedifferentiated, proliferative smooth muscle cell type with a repair function in vascular injury.
Methods and Results To investigate this cellular heterogeneity, we established vascular smooth muscle cell lines from H-2Kb-tsA58 transgenic mice. Due to temperature-sensitive expression of the SV 40 large T-antigen in cells derived from this mouse strain, our smooth muscle lines were conditionally immortalized from the onset of their life in culture. Thus, we were able to clone cell lines representing the two different phenotypes described so far. Epithelioid cells derived from newborn animals are characterized by their expression of cytokeratins and the development of tight junctional complexes. Spindle-shaped cells, which could be isolated from newborn or adult animals, corresponded in phenotype and protein expression to smooth muscle cell lines established previously.
Conclusions The special properties of vascular smooth muscle cells of the epithelioid phenotype suggest an endothelial replacement function in the course of injury to the vascular wall.
Key Words: muscle, smooth • arteries • differentiation • genetics
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Introduction |
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Due to their association with vascular disease, smooth muscle cells have attracted increasing attention during the past decade. As a result, it is clear that the cells forming the medial wall are not a homogeneous population but rather show important differences in their composition of structural and contractile proteins.1 2 3 These variations are as much species specific4 as dependent on the blood vessel site.5 6 In particular, within the intimal layer of the avian aorta, a stellate cell type with sparse myofilaments can be found that only loosely resembles medial smooth muscle.7 Probably due to its low abundance, this special class of cells is rarely represented in primary cultures derived from healthy adult aorta. The pattern of cell types obtained from developing vessels or those undergoing regenerative responses to injury show, however, more pronounced phenotypic diversity. Smooth muscle cells isolated from fetal or neonatal blood vessels8 9 10 11 12 as well as cultures of neointimal cells after balloon-catheter injury contain cells of epithelioid shape that contrast with spindle-shaped "adult" smooth muscle cells.13 14
In an attempt to investigate distinct lineages of vascular smooth muscle, we established cell lines from newborn and adult aortas. The source of our cultures was a recently described transgenic mouse strain (H-2Kb-tsA58) harboring a temperature-sensitive mutant of the SV40 large T-antigen.15 Cells derived from this mouse are conditionally immortalized from the onset of their life in culture. Thus, our approach obviated the need for manipulations in vitro, known to interfere with cellular morphology and gene expression, to establish permanent cell lines.15 As the ratio of spindle-shaped to epithelioid cells increases during vascular development, newborn as well as adult mice were selected for the present study.
The cell lines we obtained are representative of the two divergent phenotypes described thus far. Their distinct structural features are discussed with regard to their possible role in endothelial injury.
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Methods |
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Cell Culture
Smooth muscle cells were obtained by digestion of freshly isolated aortae of H-2Kb-tsA58 transgenic mice15 (at 7 days postpartum and 3 months) for 30 minutes in collagenase (1 mg/mL, type 1, Sigma Chemical Co) in DMEM at 37°C. After trituration and centrifugation, the cells were seeded in tissue culture flasks (Falcon) and cultivated in DMEM (GIBCO Life Technologies) supplemented with 10% heat-inactivated FCS (GIBCO), 50 IU/mL penicillin (GIBCO), and 20 U/mL murine recombinant IFN-
(GIBCO). Permissive
conditions were defined as proliferation at 33°C, 7%
CO2, and nonpermissive conditions were defined as
proliferation at 39.5°C, 7% CO2. Activity of large
T-antigen was monitored by immunofluorescence of
cultured cells under permissive and nonpermissive conditions with a
specific antibody16 (Santa Cruz Biotechnology). Cell lines
were generated by single cell cloning two times by limiting dilution.
Murine vascular endothelial cells were a gift of Dr
H.C. Bauer (Salzburg) and were maintained in medium 199 with Earle's
salts and L-glutamine supplemented with 10% FCS, 50 IU/mL
penicillin, 20 µg/mL endothelial cell–derived
growth substance (Collaborative Research), and 100 µg/mL heparin
(Sigma) at 37°C, 5% CO2, as described
previously.17
Proliferation Assays
Cells were seeded at a density of
1.5x103 cells per
well into a 96-well plate (Falcon) and synchronized for 36 hours in
medium containing 0.5% FCS before subjecting them to the different
culture conditions for 12 to 24 hours. Their rate of proliferation was
determined with the BrdU labeling and detection kit III
(Boehringer-Mannheim) according to the manufacturer's
instructions. In brief, cells were incubated for 3 hours with 110
µmol/L BrdU, washed with DMEM containing 10% FCS, and fixed with 0.5
mol/L HCl in 70% ethanol for 30 minutes at -20°C. After being
washed with 10% FCS/PBS and nuclease digestion to improve the
accessibility of BrdU for the antibody reaction, the cells were
incubated with Fab fragments of a peroxidase-conjugated, monoclonal
antibody against BrdU. The bound conjugate was visualized with the
soluble chromogenic substrate ABTS, and its absorbance was
measured in a microtiter plate reader (SLT) at 450 nm. Each graph
represents the mean value of three identical experiments.
Immunofluorescence
For immunofluorescence, cells were
cultivated on glass coverslips (12 mm), fixed in 4%
paraformaldehyde in PBS for 10 minutes at room
temperature, followed by brief (30 seconds) permeabilization with 0.5%
Triton X-100. Immunofluorescent staining was performed as
described previously.18 Microscopy was carried out with a
Zeiss Axioskop equipped with epifluorescence microscope
optics (Carl Zeiss Inc) and photographs taken with Agfa Pan
black-and-white 400 film.
The monoclonal antibodies against desmin
(clone DE-U-10)19
and vimentin (clone V9)20 were a gift of Prof M. Osborn
(Göttingen); the monoclonal antibodies against 
-smooth
muscle actin (clone 1A4)21 and caldesmon (clone
C21)22 were from Sigma. A polyclonal antibody against
cytokeratin (wide spectrum)23 was from Dako Corp. A
monoclonal anticytokeratin 18 antibody (clone Ks 18.04)24
was obtained from Progen. The polyclonal antibody against calponin/sm
2225 was a gift from Dr M. Gimona (Cold Spring Harbor). A
polyclonal antibody to von Willebrand factor was obtained from
Sigma. Cell contacts were visualized with monoclonal antibodies against
vinculin (clone hVIN-1)26 (Sigma), plakoglobin (clone PG
5.1)27 from (Progen), and ZO-1 (clone R
40.76)28 (Chemicon), and polyclonal antibodies against
desmoplakin29 were a gift from Dr A. Magee (London) and
cingulin.30 Secondary antibodies were obtained from Vector
Laboratories Inc (Texas Red conjugates) and Dako Corp (FITC
conjugates). Negative controls were performed by incubating fixed cells
with fluorophore-conjugated antibodies and omitting first layers or
substituting first antibodies for irrelevant ones (data not shown).
SDS-PAGE and Western Blot Analysis
SDS-PAGE was performed
according to the procedure of Matsudaira
and Burgess31 on a linear acrylamide gradient
of 8% to 22% concentration in the buffer system of
Laemmli32 of SDS samples of fully confluent cell cultures
containing equal amounts of protein. Blotting of gels onto
nitrocellulose sheets (Schleicher and Schuell) was carried out
according to Towbin et al.33 Immunoreactions were detected
with the silver-enhanced immunogold method as described by
Moeremans et al34 with a secondary antibody conjugated to
a colloidal gold marker (Amersham). For chemiluminescence, a secondary
antibody conjugated to peroxidase was used, and the reaction was
performed according to the manufacturer's instructions (Amersham). The
reaction was visualized on Kodak-X-OMAT AR film.
Northern Blot Analysis
Total RNA was isolated from
1x107 cultured cells
per cell line according to the procedure of Chirgwin et
al35 and separated by gel electrophoresis on a 1% agarose
gel containing 6% formaldehyde. The gels were blotted overnight onto
Hybond-N (Amersham) membranes as described previously.36
The transferred RNA was cross-linked by exposing the filter to UV
light (312 nm) and hybridized with a probe to total
actin37 or smooth muscle myosin38 (RAMHC-15;
a gift from J.P. Babij, Wyeth-Ayerst Research, Princeton)
radioactively labeled with a Random-Priming kit
(Boehringer-Mannheim) according to the manufacturer's
instructions. The filters were exposed to radiographic film
(Kodak X-OMAT).
Electron Microscopy
Cells grown on glass coverslips (12 mm)
were fixed in 1%
glutaraldehyde in PBS for 30 minutes at room
temperature and postfixed in 0.5% osmium tetroxide in PBS on ice,
washed in PBS, and dehydrated. After infiltration with propylenoxide,
propylenoxide-araldite (1:1 mixture), and pure araldite
(Gröpl), the coverslips were mounted cell side down onto a drop
of araldite. After polymerization at 60°C for 24 hours, the coverslip
was dissolved with hydrofluoric acid, and areas of interest were cut
out under a dissecting microscope and mounted, parallel to the cell's
dorsal surface, onto an araldite stub. Ultrathin sections were cut on a
Reichert microtome, and the sections were retrieved on grids,
contrasted with 1% uranyl acetate and Reynold's lead citrate, and
examined with a Zeiss EM 10A electron microscope.
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Results |
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Morphology and Growth Properties
Isolation and cultivation of cells derived from the aorta of normal newborn mice result in a mixed population, displaying an epithelioid, polygonal shape or an elongated, spindle shape (Fig 1A
). The same result is obtained in primary cultures of
neonatal aortic cells recovered from H-2Kb-tsA58 transgenic
mice. Because cells derived from this mouse strain are conditionally
immortalized, cell lines of the different populations could be
established from very early passages. After cloning by limiting
dilution, distinct morphologies were retained, and several stable lines
of each phenotype were established (Fig 1B and
1C). Juvenile,
epithelioid cells (Fig 1B) formed monolayers, whereas juvenile,
spindle-shaped cells (Fig 1C) overlapped at confluence to
produce a
hill-and-valley pattern typical for smooth muscle cells in
culture. Aorta from adult mice of the same strain gave rise to lines of
spindle-shaped cells only (Fig 1D). No reactivity with an
antibody
to von Willebrand factor, applied to identify
endothelial cells, was observed in either cell type
(not shown). All lines have shown phenotypic stability over more than
100 passages.
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Because both cell types have been described to derive from adult as well as fetal tissue, we use terms indicating their morphological appearance ("epithelioid" and "spindle-shaped") rather than referring to an assumed degree of maturity.
Like other cells
derived from this new strain of transgenic
mice,15 39 40 our lines harbor the
temperature-sensitive mutant of the SV 40 large T-antigen.
Therefore, cultivation at a condition defined as "permissive"
(33°C) activates the oncogene, and the cells grow with a
doubling time of 42 hours for juvenile cells and 36 hours for adult
cells. At a nonpermissive (39.5°C) temperature, proliferation ceased
(Fig 2A and 2B), and after 3 to 5 days, the
cells began
to detach from the culture dish. Activation of large T-antigen could be
visualized through nuclear labeling of cells grown at permissive
temperature by reaction with a specific antibody (not shown). We found
that the temperature during cultivation determined the proliferation
rate and addition of IFN- as described by others15 had
little or no effect on cell growth (not shown). In general, juvenile
cells replicated more slowly than adult cells.
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Expression of Smooth Muscle Actin and Myosin Isoforms
Northern
blot analysis detected transcripts for
-smooth muscle actin and cytoplasmic actins in primary cultures
of smooth muscle as well as in every smooth muscle–derived cell
line (Fig 3). Expression levels of the -smooth
muscle actin isoform that were equivalent to primary cultures (lane 4)
could be observed only in juvenile spindle-shaped cells (lane 2),
whereas transcription of this gene was noticeably reduced in
epithelioid (lane 1) and adult (lane 3) lines. An
endothelial cell sample showed very weak expression of
cytoplasmic actin only (lane 5).
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Smooth muscle myosin transcripts could not be found by Northern blot analysis with a probe encoding the carboxyl terminus of myosin heavy chain38 (not shown).
Expression of Other Smooth Muscle–Associated
Proteins
Because the presence of -smooth muscle actin has been
described in a multitude of cells and tissues unrelated to smooth
muscle,41 42 43 44 we
investigated the expression of additional
structural and regulatory proteins, considered to be smooth muscle
associated.
Samples of density growth–arrested juvenile and adult
cells were
immunoblotted with monoclonal antibodies against vinculin
(cross-reacting with metavinculin), caldesmon, desmin, and a
polyvalent antibody against calponin and SM 22. In addition, we
monitored the expression of cytokeratin 18, known to be developmentally
regulated in smooth muscle, and plakoglobin, a component of adherens
junctions (Fig 4).
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Mouse myometrium (lane 1) or mouse aorta (not shown) contained all differentiation markers. Epithelioid (lane 3), spindle-shaped adult (lane 4), and juvenile (not shown) cells all express h-caldesmon, calponin, and SM 22, whereas metavinculin and desmin could not be observed or induced by raising the temperature to nonpermissive levels. Endothelial cells (lane 2) were negative for every smooth muscle–associated protein investigated, with the exception of SM 22.
The expression of cytokeratin 18 was restricted to smooth
muscle cells
of the epithelioid phenotype (lane 3). A monoclonal antibody to
plakoglobin gave a strong reaction with endothelial
cells (lane 2) and cells of epithelioid lineage (lane 3). In smooth
muscle control (lane 1) and spindle-shaped (lane 4) cells, this
protein is only scantily expressed (see also Fig 7).
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In all cell lines, the smooth muscle–associated proteins could still be detected after multiple passages (80 to 100) (not shown). However, these proteins were expressed only in samples of densely confluent cultures.
Epithelioid Cells Express Intermediate Filaments of the
Cytokeratin Type
During embryogenesis, vascular smooth muscle cells
transiently
express cytokeratins, which are subsequently
downregulated.45 46 In primary cultures of murine
neonatal
vascular cells, epithelioid cells were consistently reactive
with a polyclonal pancytokeratin antibody and a monoclonal antibody to
cytokeratin 18 (not shown). Cytokeratin expression was restricted to
cells of epithelioid phenotype and was retained after
subcloning (Fig 5A). Spindle-shaped cells did not
give a positive reaction with this antibody under any culture condition
(Fig 5D). We found that the expression of cytokeratin was
unrelated to
the number of population doublings that the epithelioid cells had
undergone. In primary cultures, the intermediate filament proteins
vimentin and, dependent on the topography of the aorta used for
isolation,47 also desmin could be detected in either cell
type (not shown). After stable cell lines had been established
(passages 5 to 8), desmin reactivity could no longer be observed,
whereas all cells were labeled with a monoclonal antibody to vimentin
(Fig 5B and 5C).
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Cell Contacts
The striking morphological similarity with
endothelial cells prompted the ultrastructural
investigation of epithelioid cells. Thin sections, cut parallel to the
cell's plane of attachment, revealed tight junctional complexes and a
band of actin filaments closely apposed (Fig 6A)
alternating with areas of membranous interdigitations (Fig 6B).
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Antibodies to focal contacts, adherens, and tight junction components
disclosed a complex pattern of cell-cell contacts. As expected
after Western blotting (Fig 4
), vinculin was expressed in focal
contacts in adult and juvenile cell lines of both phenotypes
(Fig 7A, 7D, and 7G). An antibody
to plakoglobin exposed
a filamentous web around the membrane of epithelioid cells (Fig
7B) and
outlined cell-cell contacts in spindle-shaped juvenile (Fig 7E)
and, to a lesser degree, in adult (Fig 7H, arrows) cells.
However,
neither spindle-shaped juvenile nor adult cells were reactive with
antibodies to cingulin (Fig 7F and 7I) or ZO-1
(not shown); both of
them were present in epithelioid cells at all passages examined (up
to 105) (Fig 7C, ZO-1 not shown). This cell type was connected
by a
belt of tight junctions to all of its neighbors as ultrastructural and
immunocytochemical observations clearly demonstrated. Using electron
microscopy, we did not observe desmosomes in the epithelioid cell type,
neither could a reaction with a polyclonal antibody to desmoplakin be
elicited (not shown).
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Discussion |
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TopAbstractIntroductionMethods Results Discussion References |
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Smooth Muscle Cell Lines
Clonal cell lines derived from vascular smooth muscle have been established after spontaneous immortalization,48 49 retroviral infection, or transfection with oncogenes.50 51 52 53 Their largely uniform, fibroblast-like phenotype has been generally taken to reflect a homogeneous cell population within the vascular wall. Recently, however, cloned aortic smooth muscle cells from rat pups have revealed a strikingly complex pattern with respect to morphology, proliferation characteristics, and gene expression.12 To identify and characterize distinct lineages of smooth muscle cells without the need for immortalization procedures, we established cell lines of vascular smooth muscle cells from the aorta of H-2Kb-tsA58 transgenic mice.
Vascular Smooth Muscle Lineage Diversity
Several distinct
classes of smooth muscle cells have been
described within the vascular wall. In vivo, they have been identified
by their ultrastructural features7 54 as well as
their
divergent pattern of protein
expression.3 10 55 56 57 58 59
In
tissue culture, their structural and biochemical differences become
even more pronounced: cells displaying an epithelioid morphology, more
reminiscent of endothelial cells, contrast sharply with
elongated, spindle-shaped smooth muscle cells. In cultures derived
from fetal or neonatal blood
vessels,9 60 61 epithelioid
cells predominate, whereas this cell type is scarce in cultures of
adult vascular tissue,8 in which the majority of cells are
spindle shaped. Our work suggests that the structural features of
vascular smooth muscle cells in primary culture are to a large extent
dependent on the developmental stage of the tissue of origin.
Detailed analysis of proliferation characteristics and gene expression13 indicates that the observed phenotypic diversity is not a tissue culture artifact but rather a reflection of functional differences within the vascular wall. Judging by their independence of platelet-derived mitogens11 12 62 and their increased appearance in cultivated neointima, epithelioid cells have been ascribed a role in repair processes after endothelial injury.13 14 Finally, in view of their strong expression of developmentally regulated genes, their potential stem cell properties have been discussed repeatedly.10 12
Smooth Muscle Characterization
Due to extensive modulation of
their phenotype, cultivated
smooth muscle cells are notoriously difficult to characterize. A number
of structural and regulatory proteins associated with smooth muscle
allow the discrimination of fibroblasts, macrophages, or
endothelial cells from fully differentiated smooth
muscle cells.57 63 64 65
However, smooth
muscle–associated proteins have also been found to be expressed in
striated and cardiac muscle (desmin)19 ; in cardiac muscle
(metavinculin)63 ; in striated muscle, fibroblasts, and
astroglia (-smooth muscle
actin)44 66 67 ; and in
fibroblasts (SM 22).68 Immunoreactive forms of calponin
have been reported in fibroblasts,41 and this reactivity
has been ascribed to an acidic calponin isoform widely present in
nonmuscle cells.69 Our antibody recognized only the smooth
muscle calponin isoform.25
We have found several smooth muscle–associated proteins in spindle-shaped and epithelioid cell lines, whereas smooth muscle myosin, the most specific marker for cells of the myogenic lineage known,70 could not be demonstrated. Transcripts of myosin heavy chain in cultured smooth muscle cells beyond the first passage have not been observed previously70 ; therefore, no reaction was expected by Northern blot analysis of epithelioid and spindle-shaped cells with specific cDNA probes, and none was observed. Smooth muscle–associated proteins are downregulated in tissue culture,71 and our cell lines are no exception to this rule. However, in contrast to the endothelial controls, both spindle-shaped and epithelioid cell lines express a number of specific proteins that allow their classification as smooth muscle derived, despite their divergent phenotype.
Biochemical and
immunohistochemical studies3 58 59 have
established an intimal-adventitial gradient in human
arterial differentiation, with the luminal or
subendothelial cells displaying fewer smooth
muscle–associated proteins. In our murine vascular lines, we did
not observe a difference between epithelioid and spindle-shaped
cells in the expression of smooth muscle–associated proteins.
Expression levels of smooth muscle–associated proteins are
generally reduced in cultured cells.70 71 Therefore,
the
relative amounts of these proteins in the different cell lines do not
necessarily reflect the situation in vivo within the vascular wall. It
is conceivable that specific genes are downregulated to a low, basal
level that is identical in cell lines of both phenotypes. A
notable exception is the expression of -smooth muscle actin,
which has been shown to be elevated in cultured aortic cells from
newborn compared with adult animals.72
In agreement with the findings by Campbell et al73 and Shirinsky et al74 that the presence of smooth muscle–associated proteins in culture is dependent on the degree of confluency of the cells, we noted an increased reactivity with our antibodies in SDS samples taken from overconfluent, epithelioid, or spindle-shaped cells.
Epithelioid Cells Express Cytokeratins
Although cytokeratins
and vimentin can be found coexpressed in
myoepithelial cells of adult salivary and breast
glands,75 76 the concept of several cell populations
displaying various stages of maturity within the vascular wall
correlates well with the previously
reported13 14 77 focal
reappearance of developmentally regulated proteins in vascular disease.
Immature precursors to extracellular matrix proteins have been
identified in fetal vascular smooth muscle cells in
rat.59 79 During angiogenesis, cytokeratins are
transiently expressed within the vascular wall,45 and
their reappearance has been noted in cultured neointima.
The expression of cytokeratins appears to be lineage specific because
no reaction was observed in spindle-shaped cells regardless of
their developmental stage.
In our cultures, vimentin was always synthesized in preference to desmin. This is in agreement with a study by Jahn et al,79 who could show that cytokeratin-positive cells coexpress vimentin rather than desmin.
Intercellular Junctions
Although smooth muscle cells of
epithelioid phenotype have
been isolated previously, the nature of their cell contacts has not
been investigated. Unlike spindle-shaped cells, epithelioid cells
possess tight junctions, expressing characteristic proteins such as
cingulin29 80 81 and
ZO-1.82 83 Their
proliferation in a monolayer as well as their reactivity with a
cytokeratin antibody suggest a relation with epithelial cells, but they
do not show desmosomal structures in the electron microscope, neither
do they have desmoplakin (not shown).
Actin attachment sites in both neonatal and adult cell lines contain vinculin. Areas of cell-cell contacts in all smooth muscle lines also react with an antibody to plakoglobin, an adherens junction protein not observed in smooth muscle cells in vivo.84 However, in vitro, the de novo expression of plakoglobin in primary cultures (not shown) and in vascular cell lines may simply indicate a lack of extracellular matrix that in vivo separates smooth muscle layers and thus prevents direct cellular contact.
Separating plasma membrane domains, the presence of tight junctions indicates structural and functional polarization of a cell.85 The reason for the existence of a polarized class of smooth muscle cells is not immediately obvious. After experimental endothelial denudation, a cell population emerges in the vascular intima that differs in gene expression and protein synthesis from the cells showing "conventional" spindle-shaped morphology,12 14 and in vivo, the development of focal tight junctions in the subendothelial layer has been noted previously.86
These cells resemble endothelial cells that are also linked by tight junctional complexes,30 suggesting the possibility that epithelioid cells constitute a reserve cell population within the intima to protect and seal off the underlying layers of spindle-shaped smooth muscle cells in the event of damage to the endothelium.
Our findings confirm and extend previous observations of separate smooth muscle lineages within the vascular wall, the epithelioid phenotype probably corresponding to the "stellate" cell type found by Moss and Benditt7 in the intimal layer of the avian aorta. Although epithelioid cells display certain stem cell characteristics, we have been unable to unequivocally identify transitions between cells belonging to either phenotype. However, cells that have been credited with a role in the formation of atherosclerotic plaques87 are now available in clonal lines of unlimited supply. Closer investigation of their properties may lead to new insight into angiogenesis and vascular pathology.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This work was supported by grants from the Austrian Science Research Council and the Austrian Ministry for Science and an EMBO short-term fellowship (Dr Draeger). We thank Dr H.C. Bauer for the donation of murine vascular endothelial cells and for help and encouragement throughout the study and Prof J.V. Small for critical comments on the manuscript. The gift of antibodies and probes by Prof M. Osborn and Drs M. Gimona, A. Magee, and P. Babij is gratefully acknowledged. M. Schmittner provided excellent technical assistance, and A. Weber provided excellent photographic assistance.
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Footnotes |
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Dr Ehler's present address is The Randall Institute, King's College, Drury Lane, London, UK.
Dr Draeger's present address is Institute of Anatomy, University of Berne, Bühlstr 26, 3000 Berne 9, Switzerland.
Received November 15, 1994; revision received May 22, 1995; accepted July 7, 1995.
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References |
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