* Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville 22908; Department of Cell
Biology and the Cardiovascular Developmental Biology Center, Medical University of South Carolina, Charleston 29425; § Invitrogen Corporation, San Diego, California 92121;
Division of Neurosciences, Beckman Research Institute of the City of
Hope, Duarte, California 91010; ¶ Department of Chemistry, University of Virginia, Charlottesville 22901; and ** Department of
Pathology, University of Virginia, Charlottesville 22908
The assembly of the vessel wall from its cellular and extracellular matrix components is an essential event in embryogenesis. Recently, we used the descending aorta of the embryonic quail to define the
morphological events that initiate the formation of a
multilayered vessel wall from a nascent endothelial cell tube (Hungerford, J.E., G.K. Owens, W.S. Argraves,
and C.D. Little. 1996. Dev. Biol. 178:375-392). We generated an mAb, 1E12, that specifically labels smooth
muscle cells from the early stages of development to
adulthood. The goal of our present study was to characterize further the 1E12 antigen using both cytological and biochemical methods. The 1E12 antigen colocalizes
with the actin cytoskeleton in smooth muscle cells
grown on planar substrates in vitro; in contrast, embryonic vascular smooth muscle cells in situ contain 1E12
antigen that is distributed in threadlike filaments and in
cytoplasmic rosette-like patterns. Initial biochemical analysis shows that the 1E12 mAb recognizes a protein,
Mr = 100,000, in lysates of adult avian gizzard. An additional polypeptide band, Mr = 40,000, is also recognized in preparations of lysate, when stronger extraction conditions are used. We have identified the 100-kD
polypeptide as smooth muscle -actinin by tandem
mass spectroscopy analysis. The 1E12 antibody is an
IgM isotype. To prepare a more convenient 1E12 immunoreagent, we constructed a single chain antibody (sFv) using recombinant protein technology. The sFv
recognizes a single 100-kD protein in gizzard lysates.
Additionally, the recombinant antibody recognizes purified smooth muscle
-actinin. Our results suggest that
the 1E12 antigen is a member of the
-actinin family of
cytoskeletal proteins; furthermore, the onset of its expression defines a primordial cell restricted to the
smooth muscle lineage.
The proper recruitment and subsequent differentiation of primordial vascular smooth muscle cells
(VSMCs)1 is essential for formation of a functionally mature vessel wall. The regulation of these processes
is not well understood, due in part to the very nature of
the VSMC. Unlike most other cell types, VSMCs do not
terminally differentiate into a single phenotype. Instead,
VSMCs exist along a continuum of phenotypes during embryogenesis and subsequent maturity. At one extreme is
a synthetic, fibroblastic cell, and at the other is a physiologically mature, contractile cell (Chamley-Campbell et al.,
1981 Skeletal muscle maturation is perhaps the best example
of a paradigm for the regulation of differentiation (for reviews see Olson, 1993a Not only has there been a paucity of specific developmental markers, but the molecular mechanisms that regulate smooth muscle cell differentiation remain undefined.
Studies from several labs have identified regions within
the promoters of smooth muscle genes that account for
regulated tissue-specific transcription. These include the
SM To study the early events of VSMC development and
differentiation during the formation of a multilayered vessel wall, we generated mAbs to embryonic vessel wall antigens (Hungerford et al., 1996 Embryonic Aortic Explants
Dorsal aortae were dissected from 10-d-old embryonic quail, placed in
cold Hank's solution, minced, and then transferred to a solution of 1×
trypsin-EDTA (GIBCO BRL, Gaithersburg, MD) at 37°C for ~10 min.
DME (GIBCO BRL), supplemented with 10% chicken serum (Sigma
Chemical Co., St. Louis, MO), glutamine, penicillin, and streptomycin (Irvine Scientific, Irvine, CA), was added to the aortae/trypsin-EDTA mixture and centrifuged at 1,000 rpm for 10 min. The pellet of small aortic
pieces was reconstituted in a small amount of complete media, pipetted
onto sterile No. 1 coverslips, and allowed to attach. The resultant explants
were cultured ~3 d (5% CO2 at 37°C). After washing in PBS, cells were fixed and permeabilized in ice-cold methanol for 5 min, followed by dipping in ice cold acetone for several seconds. The fixed, permeabilized cells
were then immunolabeled (see below).
Contractile Amniotic Smooth Muscle Cells
Amniotic SMCs were isolated and cultured in serum-free media using a
previously described protocol (Bowers and Dahm, 1993 Immunofluorescence Labeling of Coverslips
Fixed and permeabilized aortic smooth muscle cells, amniotic smooth
muscle cells, and chicken embryo fibroblasts, grown on glass coverslips,
were rehydrated in PBS and blocked (15 min) in 3% BSA/PBS. The cells
were then incubated with primary antibody for 30 min to 1 h, washed
three times in PBS, and incubated with a fluorochrome-conjugated secondary antibody. Finally, the cells were washed three times in PBS, and
the coverslips were mounted on standard slides with Gel Mount (Biomeda
Corp., Foster City, CA).
Whole-Mount Immunofluorescent Labeling
Embryos were removed from the yolk, washed in PBS, staged (according
to the criteria of Hamburger and Hamilton, 1951 Acrylamide Embedding and Vibrotome Sectioning
Whole-mount immunolabeled embryos were postfixed in 3% paraformaldehyde in PBS for 30 min at room temperature, and then embedded in a
15% acrylamide mixture using a modification of Germroth et al. (1995) Acrylamide-embedded, whole-mount, immunolabeled embryos were
sectioned on a Lancer Vibratome (Series 1000; Sherwood Medical, St.
Louis, MO) at ~200 µm thickness. Sections were mounted on clean glass
slides in antibleaching medium (5% n-propylgalate, 0.25% 1-4 diaza-bicyclo-(2,2,2)octane, and 0.0025% paraphenylenediamine in glycerol) under
a No.1 coverslip.
Immunological Reagents
SM Confocal Microscopy and Image Analysis
Immunofluorescently labeled acrylamide sections and cultured amniotic
SMCs were viewed on an MCR-1000 Bio-RadTM laser scanning confocal
microscope (LSCM; Bio Rad Laboratories, Hercules, CA). Sequential optical planes were acquired in 1-µm steps along the z axis through the wall
of the descending aorta or through the cultured amniotic cells. The stored
graphics files were, in some cases, collapsed to a single virtual image (referred to as a "z-series projection") using the manufacturer's proprietary software (Bio Rad Laboratories).
Graphics files obtained from confocal microscopy were imported into
Adobe PhotoshopTM (Adobe Systems, Inc., Mountain View, CA) for further
image processing and pseudocoloration. Biochemical data was scanned on
a UMAX Power Look Scanner (UMAX Data Systems, Taiwan, R.D.C.)
and imported into Adobe PhotoshopTM for further image processing.
Chicken Gizzard Tissue Extracts
Adult chicken gizzard smooth muscle was dissected from associated connective tissue and fat. Muscle tissue was then minced finely with a sharp
razor. Small preparations were made by homogenization of ~0.25 g of tissue in 1-1.5 ml of TBS with a disposable Kontes (Vineland, NJ) micropestle. Homogenized tissue was stirred overnight at 4°C, and insoluble material was pelleted in a microfuge. The insoluble pellet was reextracted in
either TBS or 6 M urea/TBS. Homogenates were stirred overnight at 4°C
and repelleted, after which the supernatant was decanted for further use.
A standard protease inhibitor cocktail (i.e., EDTA, leupeptin, sodium
vanadate, and PMSF) was included in all preparations.
Perfusion Flow Chromatography and ELISA Analysis
Perfusion flow chromatography was performed on a BioCad Sprint system (Perspective Biosystems, Cambridge, MA). Weak anion exchange
chromatography, using an HQ column (Perspective Biosystems), was performed on adult chicken gizzard extracts. 50-100 µl of extract was injected
for each fractionation. A pH of 8.0 demonstrated the best separation of
protein with a salt gradient of 0.0-1.0 M NaCl. Resultant fractions were
tested by ELISA for 1E12 mAb immunoreactivity.
ELISA assays were performed by the standard method described by
Chapman et al. (1984) Mass Spectrometry Analysis
Samples analyzed by mass spectrometry were subjected to SDS-PAGE
(see below) and transferred to polyvinyldifluoride (PVDF; Millipore
Corp., Bedford, MA) membranes, using a 10 mM CAPS (3-[cyclohexylamino]-1-propanesulfonic acid) buffer, which included 10% methanol.
The membrane was rinsed in distilled water and stained with 0.1% Ponceau S dissolved in 1% acetic acid for 5 min. The putative 1E12-reactive
band was cut from the PVDF membrane and digested in situ with trypsin, following the method of Aebersold et al. (1987) The peptide sequences obtained from the above procedures were analyzed for potential matches, using the MOWSE Peptide Mass Fingerprint
Database.
Recombinant Antibody Production
A single chain antibody (sFv), consisting of an antibody light chain variable domain (VL) and heavy chain variable domain (VH) connected by a
short linker peptide, [(Gly)4Ser]3, was generated from 1E12 hybridoma
cells. Briefly, mRNA was isolated from the 1E12 hybridoma cell line and
used as a template for synthesis of a single strand cDNA. The first strand
cDNA used to generate the light chain product was primed with oligo dT.
The mRNA used to generate the VH cDNA was primed with a constant
region-specific primer from the Ig-Prime kit (Novagen, Madison, WI), as
the heavy chain message is larger and it is essential that the 5 The 5 To produce soluble 1E12 sFvs, single ampicillin-resistant colonies of infected E. coli HB2151, a non-supE strain, were inoculated into 150 µl of
LB-amp-glu in 96-well plates and grown with shaking until an OD600 nm of
0.8-1.0 was reached. Expression of soluble sFv was induced by the addition of isopropyl- SDS-PAGE and Immunoblotting
Proteins were separated by SDS-PAGE (Laemmli, 1970 In experiments using the sFv 1E12 mAb, bacterial periplasm was diluted 1:2 for use as the primary antibody, after the membrane had been
blocked as above. The periplasmic solution was incubated with the membrane for 1 h, followed by multiple PBS washes, and then incubated with
the 9E10.2 mAb (American Type Culture Collection), which recognizes
the c-myc epitope tag on the sFv. After multiple PBS washes, rabbit anti-
mouse polyclonal antibodies (1:3,000) were incubated with the membrane,
as an amplification step. Again the membrane was washed and incubated
with an HRP-conjugated goat anti-rabbit antibody (1:2,000). After final
washing in multiple changes of PBS, the membrane was treated with chemiluminescence reagents and exposed to x-ray film. Homogeneously pure
chicken gizzard The 1E12 Epitope Colocalizes with Actin Filaments in
Explanted Aortic Cells Grown on Planar Substrates
To establish the intracellular distribution of the 1E12 antigen
with respect to a known component of the cytoskeleton,
we compared 1E12 with smooth muscle The images in Fig. 1 show double immunofluorescence
images of cultured embryonic aortic wall cells. Fig. 1, a and
b, shows low magnification views depicting a field of cells
that had recently migrated from the explanted aortic tissue. All cells were reactive with the SM
The cells in Fig. 1, a and b, were motile, spindle-shaped
cells, except for the "deepest" cell layer, which was in direct contact with the planar glass surface. The latter cells,
depicted in Fig. 1 c, are a population of SM A high magnification view shows that the 1E12 antigen
codistributes with actin microfilament bundles when the
embryonic vessel wall cells are in contact with a planar substrate and assume an extended flattened morphology (Fig.
1, e and f). The two fluorescent images (actin and 1E12 antigen) are essentially superimposable (compare e and f).
Thus, under nonphysiological conditions where embryonic
aortic wall cells assume a conspicuously flattened morphology, the 1E12 antigen codistributes with large actin
microfilament bundles (also called "stress fibers").
The 1E12 Distribution Pattern in Cultures of
Contraction-competent Smooth Muscle Cells Is Not
Coincident with That of SM Embryonic amniotic SMCs maintain the ability to contract
when cultured using defined serum-free conditions (Bowers and Dahm, 1993 When individual cells from contraction-competent cultures were examined at high magnification using laser scanning confocal microscopy (LSCM), the two fluorescent
antigen patterns were not superimposable. This was determined by projecting a series of 10 1-µm optical sections
into a single virtual image plane. Instead of microfilament
bundles, the SM
Of greater interest is the fact that the 1E12 antigen presents a markedly different immunostaining pattern compared with SM In Situ Immunostaining of 3-d-old Quail Embryos
Demonstrates Embryonic Vessel Wall Structure, As
Well As a Unique Expression Pattern for 1E12
To examine the expression of the 1E12 antigen in primordial vascular smooth muscle cells within an intact embryonic vessel wall (i.e., cells that had not been subjected to a
tissue-culture environment or physically sectioned), we examined thick (200 µm), sagittal sections of acrylamideembedded, whole-mounted embryos by LSCM. The image
in Fig. 3 a is a low magnification projection of 10 1-µm optical sections into one image plane. The tissue, viewed in
the sagittal plane, contains undisturbed 1E12-positive cells
embedded in the extracellular matrix (ECM) of the developing aortic wall (stage 20 quail embryo). For the purposes of orientation, we have provided a schematic representation of this section in Fig. 3 b.
The parasagittal section shown in Fig. 3 a passed through
the layer of vessel wall cells most adjacent to the endothelium. It is clear from this low magnification image that primordial VSMCs in the embryonic vessel walls are not
organized in a regular pattern. The image in Fig. 3 c, corresponding to the boxed area in Fig. 3 a, is a fortuitous optical section that passed parallel to and through the main
axis of several adjoining embryonic VSMCs. Other randomly oriented, 1E12-positive cells are also visible in this
microscopic field. This image is a series of 10 1-µm optical sections projected into one image plane. One cell within
this image plane shows intracellular 1E12 labeling concentrated at the distal tips of cellular projections, possibly sites
of cell attachment. Higher magnification of a single 1-µm
optical section through this same cell is shown in Fig. 3 d;
this image clearly demonstrates discrete intracellular
staining in which the 1E12 antigen presents as fine, wisplike structures. The cell within the box in Fig. 3 c is magnified further in Fig. 3 e. This primordial VSMC exhibits several, small, organized foci of 1E12 staining arranged as
circular clusters of fluorescence that lie in a single 1-µm
plane (Fig. 3 e). These clusters bear a striking resemblance to adhesive structures termed "rosettes" or "podosomes"
(David-Pfeuty and Singer, 1980 Partial Purification of the 1E12 Epitope
The 1E12 IgM mAb proved to be unsuitable for affinity
chromatography and immunoprecipitation. However, 1E12
can be used for immunoblotting if, before incubation with
electroblotted antigen, this IgM antibody is pretreated with
a buffered solution containing dilute SDS (Hungerford et al.,
1996
Encouraged by finding the strongly immunoreactive
polypeptide band at 100 kD, preparative amounts of chicken
gizzards were processed and the resulting extracts were
subjected to perfusion-flow ion exchange chromatography. A typical chromatograph showed multiple adsorbance
peaks at 280 nm across the entire sodium chloride gradient (data not shown; see Hungerford, 1995 The peak fractions were pooled, concentrated, fractionated using SDS-PAGE under reducing conditions, and
transferred to nitrocellulose membranes. The resulting immunoblots showed a strongly reactive polypeptide band at
100 kD similar to the band detected in crude extracts displayed in Fig. 4 (data not shown). Companion immunoblots treated in an identical fashion were prepared, and
the nitrocellulose containing the 100-kD band was excised
for protein microsequence analysis. Five tryptic fragments
from the 100-kD band were successfully sequenced by tandem mass spectrometry analysis, and found to match the
sequences reported for the smooth muscle and fibroblastic isotypes of Table I.
1E12 Antigen Amino Acid Sequence Data
; Thyberg et al., 1990
; Owens, 1995
). Therefore, within
the developing vessel wall, the VSMC plays both a synthetic and contractile role. The ability of VSMCs to modulate their phenotype is an integral part of the VSMC differentiation program.
,b; Ontell et al., 1995
). Marker proteins for the various events of skeletal muscle maturation
have been defined, and several families of regulatory
genes that control the skeletal muscle differentiation program have been identified. In contrast, specific marker
proteins for the initial stages of smooth muscle cell (SMC)
development have not been identified, despite extensive effort. The smooth muscle
-actin (SM
A) gene that encodes for the major contractile protein isoform found in
mature VSMCs (Garrels and Gibson, 1976
; Fatigati and
Murphy, 1984
; Owens and Thompson, 1986
) is transiently
expressed as an integral part of the differentiation program for both cardiac and skeletal muscle (Ruzicka and
Schwartz, 1988
; Sawtell and Lessard, 1989
; Sugi and
Lough, 1992
). Expression of the smooth muscle myosin
heavy chain gene appears to be tissue specific based on in
situ hybridization studies; however, its presence is not detected until after the initial events that establish a multilayered vessel wall (Miano et al., 1994
). Similarly, other presumptive "differentiation marker genes," such as h1calponin and SM22
, are expressed at detectable levels
only after a multilayered aortic vessel wall has been established (Duband et al., 1993
). Moreover, most of these
genes are also transiently expressed as part of the cardiac
(calponin and SM22
) and skeletal (SM22
) muscle differentiation programs during early development (Li et al.,
1996
; Miano and Olson, 1996
; Samaha et al., 1996
).
A , smooth muscle myosin heavy chain, and SM22
genes, all of which appear to be regulated by distinct transcriptional mechanisms when compared with non-smooth
muscle cells (Shimizu et al., 1995
; White and Low, 1996
;
Kallmeier et al., 1996; Li et al., 1996
). Several transcription
factors have been implicated in the process of SMC differentiation, although there is no indication of which smooth
muscle genes are specifically activated by their presence.
For example, members of the myocyte enhancer binding factor-2 family of transcription factors have been shown to
play a role in the differentiation of skeletal, cardiac, and
visceral muscle in Drosophila (Lilly et al., 1995
), and thus
may be involved in the regulation of a differentiation program common to all muscle lineages (Olson et al., 1995
).
Two other diverse families of transcription factors, the homeobox genes and GATA genes, may also have members that are important in the regulatory mechanisms of SMC
phenotype (Miano et al., 1996
; Morrisey et al., 1996
). Given
these findings, there is to date no clear explanation for
how these potential mechanisms are integrated to coordinate the temporal and spacial regulation of smooth muscle-specific genes during SMC differentiation. Clearly, there
is a need to define both specific markers and regulators of
SMC development and differentiation.
). Our goal was to identify
novel proteins that are expressed as mesodermal cells become committed to the SMC lineage. One mAb, 1E12,
specifically labels SMCs in the descending aorta during early avian development (Hungerford et al., 1996
). Unlike
SM
A, the 1E12 antigen is not expressed as a component
of the differentiation program for cardiac and skeletal
muscle cells; moreover, expression of the 1E12 antigen has
been observed in all smooth muscle tissues within the developing avian embryo (Hungerford, 1995
; Hungerford et al.,
1996
). The 1E12 antigen is first observed 8-12 h after the
onset of SM
A expression; furthermore, the 1E12 mAb
labels only a subset of the SM
A positive cells, specifically
those mesodermal cells most adjacent to the aortic endothelium (stage 17-18). The 1E12-positive cells are presumably more mature than the more peripherally located cells
that stain with only the SM
A mAb. In this report we address the hypothesis that mab1E12 defines cells committed to the smooth muscle differentiation program, and also
discuss the identity of its cognate tissue-specific antigen.
Materials and Methods
). After 5-7 d in
culture, the cells were fixed by either the cold methanol/acetone procedure described above or in Omnifix (Zymed Labs, San Franscico, CA),
followed by a short (5-s) incubation in ice-cold acetone. Fixed, permeabilized cells were then immunolabeled and analyzed by laser scanning confocal microscopy.
), and fixed in methacarn
(60% methanol, 30% chloroform, 10% glacial acetic acid). To facilitate
diffusion of the antibodies and wash solutions, the fixed tissue was further
dissected. Embryos were truncated at the head and tail. Limb buds were
removed and a lengthwise cut was made on the ventral surface of the embryonic body wall. Immunolabeling of the embryos was performed as described by Drake et al. (1992)
, using supernatant from the 1E12 hybridoma and fluorochrome-conjugated (Cy5) secondary antibodies.
.
A mAbs were obtained from Sigma Chemical Co. (clone 1A4) and
used diluted (1:200 or 1:400) with PBS. The 1E12 mAb was produced and
isotyped as previously described (Hungerford et al., 1996
) Undiluted hybridoma culture supernatant was used for immunofluorescent labeling.
1E12 ascites fluid was partially purified on a protamine agarose column
(Hudson and Hay, 1980
) and concentrated in Aquacide III (CalbiochemNovabiochem Corp., La Jolla, CA) for use in immunoblotting experiments.
All secondary antibodies were purchased from Jackson Immunoresearch
Laboratories (West Grove, PA) and used at 15 µg/ml. Appropriate controls were used for all of the immunolabeling studies presented in this
work. Preimmune (when available) or nonimmune sera were used as a
negative control for the primary antibody. Secondary antibody controls
entailed labeling of embryos, or cultured cells, with this antibody only.
. The only modification of this process was that the
wells were blocked for 1 h at 37°C in 1% BSA/PBS.
. The digestion solution
was withdrawn and saved, and the band was rinsed twice in 100 µl of fresh
digestion buffer. Pooled digestion solutions were acidified (1% acetic acid)
and concentrated to 40 µl in a Speed Vac (Savant Institute, Hicksville, NY).
Aliquots of the digest were analyzed as previously described (Hunt et al.,
1992
) on a Finnigan-MAT TSQ-70 triple-quadrupole mass spectrometer, equipped with an APCI source (San Jose, CA). This device was interfaced to a polyimide-coated fused-silica microcapillary HPLC column (i.d. 75 µm; o.d. 200 µm; Polymicro Technologies, Phoenix, AZ), packed with Poros R2H material (Perspective Biosystems). The samples were eluted into
the mass spectrometer with an Applied Biosystems 140B Solvent Delivery
System (Foster City, CA). In addition to the putative 1E12-reactive band,
a blank piece of nitrocellulose and a band containing 2 µg of BSA were
also cut from the PVDF membrane and used for internal controls.
end of the
message encoding the VH is incorporated in the cDNA. The linkered variable region PCR products were generated using the appropriate primers
that were fused to a sequence, which, when overlapped with the homologous sequences from the other chain variable region product, encoded the
[(Gly)4Ser]3 linker sequence between the two variable domains. The linkered variable domain PCR products were gel purified, annealed with their
corresponding partner, and extended in a recombinant PCR reaction for
seven cycles in the absence of synthetic primers. A second round of PCR
using the 5
- and 3
-most primers results in the production of the intact sFvs.
- and 3
-most primers incorporated SfiI and NotI sites, respectively, for cloning into a pHEN-1 plasmid (previously described by Hoogenboom et al., 1991
). The recombinant sFv, which was cloned between
the SfiI and NotI sites, was produced as a fusion with the c-myc epitope
tag, which is recognized by the 9E10.2 mAb (submitted to the American
Type Culture Collection [Rockville, MD] by J.M. Bishop [G.W. Hooper
Research Foundation, University of California, San Francisco] and G.I.
Evan [Cell Nucleus Laboratory, Imperial Cancer Research Fund, London,
UK]; Evan et al., 1985
). It was further fused to the gene III sequences of the filamentous phage in a supE strain of Escherichia coli with constructs
that allow secretion of sFvs into the culture media of normal E. coli strains
(see Hoogenboom et al. [1991] for details).
-d-thiogalactopyranoside to a final concentration of 1 mM, as described by Hoogenboom et al. (1991)
. Cultures were grown
overnight at 30°C, and the periplasm containing the soluble 1E12 sFvs was
collected and used for immunoblotting experiments.
), and then transferred to nitrocellulose membrane (MSI, Inc., Westboro, MA) by the
method of Towbin et al. (1979)
. Membranes were blocked, incubated in
primary antibody, washed, and then incubated with HRP-conjugated secondary antibodies (Jackson Immunoresearch Laboratories). After washing, the membrane was incubated with chemiluminescent reagents (ECL
Western blotting kit; Amersham Life Science, Arlington Heights, IL) for
1 min and immediately exposed on x-ray film. 1E12 ascites were preincubated in 0.5% SDS at 40°C for 3 min before incubation with the membrane. The final concentration of SDS for incubation was 0.0005%.
-actinin was a gift (Dr. Carol Otey, University of Virginia, Charlottesville; purified according to Fermamisco and Burridge, 1980).
Results
-actin distribution in cultured embryonic smooth muscle cells using double immunofluorescence microscopy. Previous low magnification views of early embryonic tissue sections showed
that the distribution of the 1E12 antigen is initially restricted to those presumptive VSMCs most adjacent to the
aortic endothelium. In addition, these images suggested that the antigen was concentrated in the peripherial cytoplasm of such cells (see Fig. 7 in Hungerford et al., 1996
).
A mab, whereas
far fewer cells immunostain with both the SM
A antibody
and 1E12. The images in Fig. 1, a, c, and e, show an optical
field in which wide-band excitation wavelengths suitable
for both fluorescein and rhodamine were used. The redorange fluorescence depicts cells immunoreactive for SM
A,
while the yellow staining designates cells immunopositive
for both SM
A and 1E12. The green fluorescence in Fig.
1, b, d, and e, designates only those cells immunoreactive
with 1E12, when the same optical fields are illuminated
with a narrow band-pass fluorescein filter set.
Fig. 1.
The 1E12 antigen is located intracellularly and codistributes with SMA in aortic cells cultured from explants on a planar glass
substrate. The cells are double labeled with mAbs to SM
A and 1E12. (a, c, and e) Images of cells examined with simultaneous excitation in both fluorescein and rhodamine wavelengths. (b, d, and f) The same fields of cells under fluorescein only excitation, which denotes those cells that are 1E12 positive. (a and b) Low magnification views of aortic cells migrating from explanted tissue. The majority
of cells in these fields are labeled with SM
A (arrowhead, red cells). A subset of cells are double labeled with SM
A and 1E12 (arrow,
yellow cells). In this field the majority of cells are motile, spindle-shaped cells and, as such, do not show extensive stress fiber arrays, as
shown in c and d. (c and d) The explanted aortic cells have migrated onto the planar glass and have formed extensive microfilament
(stress fiber) arrays. As above, all the cells in this field were positive for SM
A, while a subset were positive for both SM
A and 1E12.
(e and f) A high magnification view of the embryonic aortic cells shows the 1E12 antigen codistributed with actin microfilament bundles.
As shown in f, 1E12 labels the entire length of the actin stress fibers in these cells. The faint staining in the other cells is due to optical
bleed-through from the rhodamine channel. Bars: (a and b) 50 µm; (c and d) 50 µm; (e and f) 10 µm.
[View Larger Version of this Image (96K GIF file)]
A- and 1E12labeled cells that have attached to the planar glass surface,
flattened, and formed actin microfilament bundles. Every
cell is SM
A positive; however, only a subset of the
SM
A-positive cells in Fig. 1 c also immunostain with the
1E12 antibody (d). Careful examination of multiple explants suggested that the cells expressing the 1E12 antigen
were primarily located at the upper layer of migratory
cells that had most recently emigrated from the aortic segment although this impression was not subjected to quantitative analysis.
A
). As demonstrated by these studies,
contractile amniotic smooth muscle cells exhibit a more
rounded and plump appearance than cells grown in the
presence of serum. To examine the 1E12 antigen under
more physiologically relevant conditions, the embryonic amniotic SMCs were simultaneously immunolabeled with
SM
A and 1E12 mAbs. When examined with epifluorescence microscopy, all cells expressed both antigens. This is
in distinct contrast with the serum-containing aortic cultures described above (Fig. 1 and data not shown).
A expression pattern resembled fine filamentous whorls surrounding a nonlabeled nuclear compartment (Fig. 2 a). Also, fine retraction fibers extending
from the cell periphery, at the level of the glass coverslip,
immunostained with the SM
A mAb. These structures are
clearly shown by the digitally generated, surface relief image of the cell shown in Fig. 2 c. It is readily apparent from
this and similar images that these contraction-competent
cells do not form the typical stress fiber morphology of
cells grown on planar substrates in the presence of serum.
Fig. 2.
This figure shows an amniotic SMC double labeled with SMA (a) and 1E12 (b). These images were obtained by LSCM. Each
image is a series of 10 1-µm optical sections through the long axis of the cell projected into a single image plane. (a) In these contractioncompetent cells, SM
A expression is observed in fine, filamentous "whorls" surrounding the nucleus (arrow). Retraction fibers, which
are present at the surface of the coverslip, also are labeled with SM
A (arrowhead). (b) 1E12 expression is distributed in the same area
as SM
A (arrow) with the cell. However, 1E12 expression appears in a punctate and granular pattern. Additionally, the 1E12 antigen is
distributed to discrete patches of the cell (arrowhead), reminiscent of cell-substrate contact sites. As a means of visualizing the morphology of the amniotic SMC shown in a and b, digital image processing software was used to render the actin immunofluorescence pattern
as a surface relief object. From this topographical representation, it is clear that the actin microfilaments extend from the cell body as
long processes (arrowheads), which are reminiscent of retraction fibers. 1E12 immunolabeling was not present in these structures. Bar,
10 µm.
[View Larger Version of this Image (29K GIF file)]
A. While some fluorescence is present in
the perinuclear cytoplasm, the 1E12 immunostaining appears as a punctate and granular pattern, and it is not detected coincidently with the SM
A-positive retraction fibers
(compare Fig. 2, a and b). Moreover, and in distinct contrast with SM
A, the 1E12 antigen is distributed in discrete focal patches at the (presumptive) leading and trailing edges of the cell; these structures are reminiscent of
cell-substratum contact sites (e.g., see Chen and Singer,
1982
).
Fig. 3.
The distribution pattern of 1E12 was examined in primordial VSMCs within an intact embryonic vessel wall by whole-mount
immunolabeling. The stage 20 quail embryo shown in this figure was embedded in acrylamide, sectioned, and viewed by LSCM. (a) Low
magnification view of a projection of a series of 10 1-µm sagittal sections through the vessel wall. Primordial VSMCs are labeled with
1E12. At this early developmental stage, the VSMCs are loosely arrayed, with open spaces between the cells. The boxed area is magnified in c. (b) For purposes of orientation, a schematic view of the preparation is shown. The drawing depicts a side view of the aorta with
the anterior (A) to posterior (P) axis, and dorsal (D) to ventral (V) axis indicated. The area shaded in green is representative of the fluorescent image shown in a. (c) High magnification view of the boxed area in a and a series of 10 1-µm optical sections projected into one
image plane. A fortuitous optical section through the main axis of a cell (arrowhead) within the plane of the section shows that 1E12 labeling is concentrated at the distal tips of cellular projections, possible points of attachment. The cell marked with the box is magnified
further in e. (d) 1-µm optical section through the cell denoted by the arrowhead in c. 1E12 labeling is seen intracellularly in fine, short,
threadlike structures. (e) A high magnification view of the cell denoted by the box in c. Small 1E12-labeled clusters (arrowheads) are
similar to adhesive structures termed rosettes or podosomes. Bars: (a) 50 µm; (c and d) 10 µm; (e) 7.5 µm.
[View Larger Version of this Image (72K GIF file)]
; Tarone et al., 1985
;
Marchisio et al., 1987
; Burridge et al., 1988
). Close inspection of multiple optical planes through the embryonic vessel wall revealed that 1E12 rosettes were detected in other
smooth muscle cells (data not shown).
). Using this immunoblotting method, we probed extracts of adult chicken gizzard (an abundant source of
smooth muscle antigens) for the 1E12 antigen. When homogenized gizzard tissue was extracted with TBS, a 100-kD
band was detected (Fig. 4, lane c). If the resulting pellet
was reextracted with the same buffer containing 6 M urea,
additional 1E12 antigen was detected (Fig. 4, lane d). This
suggested that at least some of the 1E12 antigen remained
insoluble at neutral pH and normal ionic strength. Lanes a
and b show the results of probing two similar samples with
antibodies to SM
A. In addition to the 100-kD polypeptide band recognized by 1E12, a doublet at ~40 kD was
detected in some preparations but not in others. This doublet migrates well ahead of SM
A (compare lanes a and b
with c and d). Also, based on the urea extraction data, the
1E12 antigen appears to be less soluble than SM
A.
Fig. 4.
Chicken gizzard homogenates were separated by 10%
SDS-PAGE, transferred to nitrocellulose, and probed with antibodies to either SMA (lanes a and b) or the 1E12 antigen (lanes
c and d). Gizzard homogenates were prepared by extraction in
TBS alone (lanes a and c) or reextraction of the TBS-insoluble
pellet in 6 M urea/TBS (lanes b and d). Immunoreactivity was determined by enhanced chemiluminescence techniques. 1E12 ascites were incubated with 0.5% SDS before incubation with the
membrane and used at a final dilution of 1:1,000. 1E12 does not
immunoreact with actin, but is strongly immunoreactive with a
band migrating at Mr = 100,000 (arrow). In addition, a doublet
migrating ahead of actin on the gel is often observed to be immunoreactive (lane d).
[View Larger Version of this Image (52K GIF file)]
). A sample from
each absorbance peak was tested for 1E12 immunoreactivity by ELISA. A series of strongly immunoreactive peaks
eluted between 0.5 and 1.3 M NaCl (data not shown).
-actinin (Baron et al., 1987
; Arimura et al.,
1988
, respectively). Table I lists the sequences of the five
tryptic fragments (left column). The middle column indicates where the tryptic fragment starts within the sequence
of the smooth muscle cell (SMC) and nonmuscle cell
(NMC) isoforms of
-actinin. The right column lists the
functional domain of
-actinin, in which each fragment is
located.
To address the complication that some immunoblots
showed the variable doublet at 40 kD, and to obtain an immunoreagent that was easier to use than the parent IgM
molecule, we took advantage of recombinant protein technology to produce a single chain antibody (sFv) with 1E12
specificity (see Materials and Methods). Using the sFv immunoreagent, crude chicken gizzard extracts were analyzed by immunoblotting (Fig. 5). Polypeptides in lanes a-d
were separated by 10% SDS-PAGE under reducing conditions, transferred to nitrocellulose, and probed with the
sFv 1E12. Adult chicken gizzard was extracted in TBS (Fig.
5, lane a) or TBS containing 6 M urea (lane b). A single,
strongly immunoreactive band, Mr = 100,000, was observed in the crude gizzard homogenates extracted under
both mild and stringent conditions. No bands at the 40-kD
migration position were detected with the sFv immunoreagent. As shown in lane c, the sFv 1E12 was also immunoreactive with homogeneously pure chicken gizzard -actinin.
In contrast, a control sample containing chicken serum proteins showed no reactive bands (lane d). Also, neither the
secondary (anti-myc mAb) nor the tertiary antibodies
(goat anti-mouse IgG), used for this immunoblot, exhibited cross-reactivity with the chicken gizzard extracts or
the purified
-actinin (data not shown).
The goal of this study was to characterize further a novel
SMC-specific protein marker, the 1E12 antigen (Hungerford et al., 1996). The 1E12 antigen is expressed very early
in the SMC differentiation program; we hypothesized therefore that the protein plays an important role in establishing the cytoskeletal machinery characteristic of the SMC
lineage. In this study we have examined the intracellular localization of the 1E12 antigen in three distinctly different embryonic SMC preparations. The use of cell biological, immunological, and biochemical approaches identified
the 1E12 antigen as a member of the
-actinin family of
cytoskeletal proteins and as an unequivocal marker for
specification to a smooth muscle fate.
1E12 Expression in Cultured SMCs
The phenotypic modulation of SMCs in culture has been
well documented (Chamley-Cambell et al., 1981; Thyberg
et al., 1990; Thyberg, 1996
). Indeed, a myriad of studies
has shown that the expression and subsequent distribution
of smooth muscle proteins in cultured cells is dependent
upon many factors, including the contents of the culture
media and the type of substratum (for review see Owens,
1995
). We decided therefore to examine expression of the
1E12 antigen using two different methods of culturing embryonic SMCs. One method yields a well-spread noncontractile phenotype, and the other method results in more
rounded cells that are capable of contractility.
Actin microfilament bundles, also called stress fibers, are
formed when animal cells are cultured on highly adhesive
planar substrates (see Trinkaus, 1984, for discussion). Consistent with this fact, cells that emigrate from explanted
embryonic aortae onto glass, in the presence of serum-derived
substrate attachment factors, assume a characteristic stress
fiber morphology (Fig. 1 e). The plasma membrane appears
to be stretched across the culture substratum in these strongly
adherent cells (not shown). In contrast, embryonic amniotic SMCs isolated and maintained by a different method
(Dahm and Bowers, 1993) retain the ability to contract in
culture, have a more rounded morphology, and do not exhibit exaggerated actin microfilament bundles (Fig. 2 a).
Our immunofluorescence analysis of both types of cultured primordial SMCs indicates that the 1E12 epitope is
located intracellularly. As expected, the distribution pattern of 1E12, relative to that of SM
A, was different depending on the phenotype/morphological characteristics
of the cultured cell. In the highly flattened cells from aortic
explant cultures, the 1E12 staining was distributed along
the length of the actin-containing microfilament bundles.
In contrast, the distributions of 1E12 vs SM
A in rounded,
functionally contractile, amniotic SMCs were distinct. Actin was distributed in a whorled pattern surrounding the
nucleus and in fine filaments extending from the cell periphery (Fig. 2 a, arrowhead), whereas 1E12 staining appeared as a granular perinuclear stain and as small circular
patches at the proximal aspect of lamellapodia-like protrusions (Fig. 2 b). Digital image processing software was used
to render the actin immunofluorescence pattern as a surface relief object (Fig. 2 c). This topographical representation demonstrates that actin microfilaments extend within
cellular protrusions a considerable distance from the cell
body (Fig. 2, arrowheads). The latter structures are reminiscent of retraction fibers created by motile fibroblasts (see Trinkaus, 1984
). In addition, broad lamellapodia-like
protrusions appear to extend from opposite sides of the
cell body (Fig. 2 c). It is noteworthy that neither actin nor
1E12 staining was present in stress fiber-like arrangements in the contraction-competent SMC cultures.
In addition to confirming an intracellular localization for
the 1E12 antigen, these in vitro studies strongly support
our previous conclusion that 1E12 protein expression is an
appropriate marker to define cells committed to the VSMC
differentiation program (Hungerford et al., 1996). In the
embryonic aortic cell cultures, SM
A and 1E12 doublelabeled cells comprise a minority of the cells present in the
culture (all cells are SM
A positive, but only a few are
also 1E12 positive). Our hypothesis is that the SM
A and
1E12 double-labeled cells represent primordial VSMCs
that were further along their differentiation program relative to cells that express only SM
A. In contrast, all of the
contraction-competent, cultured, amniotic cells immunolabeled with both 1E12 and SM
A mAbs. This finding is
consistent with other studies showing that SMCs, such as
these, grown in serum-free medium often maintain more
features of their phenotypic status in vivo than those grown in the presence of serum (Chamley-Cambell et al., 1981;
Hedin and Thyberg, 1987
; Hedin et al., 1990
; Bowers and
Dahm, 1993
). Of course, we cannot be certain that SM
Apositive/1E12-negative cells did not previously express 1E12,
but subsequently modulated their smooth muscle phenotype such that 1E12 expression is no longer detectable.
As a biological control, we immunostained chicken embryonic fibroblast cultures with both 1E12 and SMA
mAbs (data not shown). The 1E12 mAb labeled a handful
of cells per confluent 100-mm culture dish, while the SM
A
mAb labeled the majority of the fibroblasts. This result is
not surprising, as it is well documented that many cell
types cultured in vitro express SM
A (Skalli et al., 1987
, 1989; Rønnov-Jessen et al., 1990
; Sappino et al., 1990
; Jahoda et al., 1991
; Lazard et al., 1993
). Thus, 1E12 recognizes only those cells in vitro that manifest a committed
smooth muscle phenotype.
1E12 lmmunolabeling In Situ: A Unique Method for Studying Structural Proteins in the Primordial Vessel Wall
LSCM images of 1E12 expression within the aortic wall of
a stage 20 quail embryo show a cytoskeletal protein array
within a primordial VSMC. Moreover, these cells have not
been dissociated from the vessel wall nor subjected to a
tissue-culture environment. We know of no other images
that depict the cells of the developing vessel wall in this
manner. Our previous study showed that the 1E12-positive primordial VSMCs are located in the cell layer(s) most adjacent to the aortic endothelium in stage 20 quail
embryos (Hungerford et al., 1996). In the present study we
show that the primordial VSMCs are loosely arranged with
considerable open space between the cytoskeletal arrays
of 1E12 antigen. Since the 1E12 antigen is clearly present
in cell processes, this image suggests that the remaining
space is largely occupied by ECM.
The somewhat acellular architecture of this early vessel wall also suggests that embryonic VSMCs do not initially form a functionally contractile tissue when recruited to the endothelium, and our unsuccessful attempts to elicit contraction of aortic smooth muscle at early stages of development are in agreement with this hypothesis (Hungerford, J., unpublished observations). Thus, it appears that at early stages VSMCs may be primarily involved in synthetic activity and not contractility. Furthermore, recent collaborative efforts are entirely consistent with the possibility that day 2.5-6 avian VSMCs are engaged in the progressive synthesis of multiple contractile proteins, while at the same time they are in a highly proliferative state (Lee et al., 1996).
The LSCM images of the 1E12 antigen distribution in
the intact vessel wall are the first images of this kind, which
makes drawing comparisons with previously published
work difficult. Standing alone, the data suggest that the
1E12 epitope is involved with an intracellular apparatus that
mediates cell adhesion. 1E12 labeling is localized to the
tips of cellular extensions, possible sites of attachment (Fig.
3 c). Moreover, the antigen is present as small circular assemblies that resemble rosettes or podosomes, adhesive
structures observed in transformed cells, osteoclasts, and
cells of the monocytic lineage (David-Pfeuty and Singer,
1980; Tarone et al., 1985
; Marchisio et al., 1987
; Burridge et
al., 1988
; Aubin, 1992
). These adhesion structures typically
contain actin microfilaments in the center, encircled by a
rosette containing vinculin,
-actinin, and talin. In cultured,
transformed cells, or cells of the monocytic lineage, podosomes are thought to confer increased migratory capabilities
to these cell types. From the images in Fig. 3, we cannot
determine whether these sites have the same organization
as podosomes or perhaps correspond to in vitro cell-ECM
adhesion sites (Chen and Singer, 1982
). It is reasonable to
hypothesize that, within the developing embryo, the adhesions made to the ECM or other cells need to be transitory
to allow for the constant rearrangements that occur during
morphogenesis. Alternatively, these 1E12-immunolabeled
structures may represent the ontogeny of cell-cell junction
complexes or the onset of dense body/dense plaque formation. Based on our identification of the 1E12 antigen as a
putative
-actinin family member, we believe that these
are all valid possibilities (see discussion below).
1E12 mAb Recognition of Smooth Muscle -Actinin
Initial attempts to immunoblot or immunoprecipitate with 1E12 using standard techniques were unsuccessful. We reasoned that, when concentrated for immunochemical studies, the large multivalent IgM antibody might be aggregating so as to prevent antigen recognition. To address this possibility, 1E12 was incubated with 0.5% SDS before probing the nitrocellulose sheets with the primary antibody solution, the rationale being to promote a more open IgM protein conformation and thereby promote antigen recognition.
This protocol appeared successful, in that a 100-kD band was always strongly recognized by 1E12. There was concern, however, regarding the fact that in some immunoblots bands at 40 kD were variably present when crude extracts of chicken gizzard were probed as seen in Fig. 4. To circumvent problems associated with the the large, pentameric IgM structure, we generated a recombinant, single chain version of 1E12. The recombinant 1E12 recognized a single band, Mr = 100,000, in gizzard lysates extracted under both mild and strong (6 M urea) extraction conditions. This result confirmed that the 1E12 antigen displays an apparant molecular mass of 100 kD.
After partial purification on anion exchange chromatography and subsequent fractionation using SDS-PAGE, the
bands were transferred to nitrocellulose and the 100-kD
band was excised. Tandem mass spectroscopy analysis
identified the 100-kD band as the smooth muscle, or fibroblast, isotype of -actinin. These
-actinins are 98% identical, the greatest diversity being in the second half of the
first EF-hand (Blanchard et al., 1989
). As shown in Table
I, the amino acid sequences of the five tryptic fragments obtained from mass spectrometry match regions of the
smooth muscle (Baron et al., 1987
) and fibroblastic (Arimura et al., 1988
)
-actinin sequences, where the two are
identical. The nonmuscle and smooth muscle isoforms of
-actinin are alternative splice products of a single gene
(Waites et al., 1992
). The alternatively spliced region begins adjacent to the last trypic peptide we obtained in the
first EF-hand. Based on our previous study of 1E12 distribution in embryonic tissue sections (Hungerford et al., 1996
)
and our current study, we conclude that 1E12 recognizes
those cells both in vivo and in vitro that possess the phenotype of a committed SMC. Our data suggests that 1E12
distinguishes between smooth muscle and fibroblast (nonmuscle) isoforms of
-actinin. Additionally, the recombinant
1E12 immunoreagent reacts with a preparation of homogenously purified chicken gizzard smooth muscle
-actinin.
The screening criterion that resulted in cloning 1E12
was designed to be an unbiased search for antibodies that
marked the early vessel wall (Hungerford, 1995; Hungerford et al., 1996
). While we did not anticipate the identity
of the 1E12 antigen to be
-actinin, much of the immunolabeling data presented in this study and our previous
studies are consistent with this finding. The
-actinins have
been implicated as part of the intracellular machinery involved in both cell-cell and cell-matrix adhesion (for reviews see Blanchard et al., 1989
; Luna and Hitt, 1992
; Hemmings et al., 1995
), and the proteins are found in dense
bodies and dense plaques of mature SMCs (Draeger et al.,
1990
; Chou et al., 1994
). The distribution of the 1E12 antigen in embryonic tissue and cultured SMCs is highly suggestive of a molecule, such as smooth muscle
-actinin,
that is associated with actin organization and involved in
cell attachment.
The distribution of -actinin in cultured cells is variable
depending on cell type and shape, type of substratum, and
accessibility of the marker antibody to the cell-matrix or
cell-cell junctional complex (Lazarides and Burridge, 1975
;
Pavalko et al., 1995
; Crowley and Horwitz, 1995
). Typically,
-actinin labeling is observed periodically along the
actin stress fibers and in the focal adhesions, located at the
ends of the stress fibers; however, the extent to which
these structures label can differ, based on the criterion given above. In our hands, the distribution of the 1E12 antigen varies with the preparation of cultured embryonic
smooth muscle cells. In explanted, embryonic VSMCs, 1E12
immunostaining is associated with the actin stress fibers,
although the characteristic periodicity of
-actinin is not
observed. We do not see discrete labeling of the focal adhesions in these cells. In functionally contractile, embryonic, amniotic SMCs, the 1E12 antigen is concentrated in podosome-like structures at the base of broad cellular protrusions. Distinct stress fibers are not present in these
cells, but 1E12 is localized to the general area of actin distribution within the cell body.
Although detection of -actinin in many cell types may
be limited because of antibody accessibility (Palvalko et al.,
1995), distribution of
-actinin has been studied at the
level of the LSCM in embryonic chicken corneal epithelium (Khoory et al., 1993
) and by EM in later stages of development in chicken gizzard SMCs (Chou et al., 1994
).
Localization of
-actinin within the cells of the corneal epithelium of a 6-d-old chicken embryo is similar to our confocal images of the developing VSMCs within the vessel wall. In both cases, short, threadlike staining was observed
cytoplasmically, and denser labeling was found in areas of
attachment to other cells or the matrix. However, the
1E12-positive, rosette-like structures that we observed in
individual cells of the vessel wall are unique to our study.
While these structures bear a striking resemblance to the
podosomes found on cultured cells, they have not been described in situ previously.
The in vivo equivalents of adhesion structures have not
been studied extensively, there are, however, several structures in vivo, including the myotendinous and neuromuscular junctions, intercalated disks in cardiac muscle, and
dense bodies and dense plaques of smooth muscle that reflect the diversity of actin organization and the repertoire
of proteins found in adhesion structures in vitro. In the future, it will be interesting to see if known markers of in
vitro cell-cell and/or cell-matrix adhesion are localized in
situ to structures similar to those pictured in this study. Our unpublished observations suggest that, in addition to
-actinin, vinculin is also distributed in analogous circular
structures in the developing vessel wall of similarly prepared quail embryos.
While -actinin has been localized to the dense body/
dense area of mature VSMCs (Draeger et al., 1990
), we
see no evidence of 1E12 labeling in such discrete structures within the embryonic (stage 20) vessel wall. The formation and protein content of these structures has been
examined in chicken gizzard SMCs from embryonic day 10 to posthatchling stages, much later stages than the present studies (Volberg et al., 1986
; Chou et al., 1995). There are
no equivalent studies of dense body/dense plaque formation at the early stages of vessel wall development. Thus,
we do not know to what extent, if any, these structures exist within developing VSMCs at these early stages.
One important issue we are continuing to address concerns reconciling the wide range of -actinin distribution
in vertebrate tissues, with the exquisite specificity 1E12
displays with respect to smooth muscle. There have been
no prior reports of such specific
-actinin expression in the
developing embryo. Previous developmental studies have
focused on
-actinin distribution during differentiation of
specific cytoskeletal structures (Endo and Masaki, 1984
;
Goncharova et al., 1992
; Chou et al., 1994
), rather than on
the onset of expression with respect to the early events of
morphogenesis. In contrast, the spacial and temporal distribution pattern of vinculin and talin, two other abundant
components of adhesion complexes, has been described in
the developing quail embryo (Duband and Thiery, 1990
).
Interestingly, both of these proteins are expressed at high
levels in both the endothelial and smooth muscle components of the vasculature during early developmental stages
that correspond to the onset of 1E12 expression. Therefore, it is not surprising that a particular variant of an actin
organizing molecule such as
-actinin would be found in
the early stages of vessel wall development.
Although the diversity of -actinin isoforms has been
well documented, a better understanding of the mechanisms that generate these variants and the relationship between them is needed. It is clear that two genes encoding
for chicken
-actinins (Parr et al., 1992
; Waites et al., 1992
)
and three genes encoding for human
-actinins (Millake
et al., 1989
; Youssoufian et al., 1990
; Beggs et al., 1992
)
have been identified, and that alternative splicing of these
gene products accounts for the major tissue-specific isoforms: nonmuscle, smooth muscle, skeletal muscle, and
cardiac
-actinins. However, identification of novel tissuespecific isoforms such as the recently isolated 115-kD, calcium-insensitive, endothelial cell-specific
-actinin (Imamura et al., 1994
; Imamura and Masaki, 1994
) suggests
that either additional alternative spliced isoforms or posttranslational modifications of the known major isoforms also contribute to the repertoire of
-actinins. The differences in calcium sensitivity between variants may also reflect tissue-specific requirements of
-actinin. Considering
all of these issues, we may be able to account for 1E12
specificity by considering that there are uncharacterized
isoforms or variants of
-actinin that have cell type-specific roles. Interestingly, up to 11 isoelectric variants of
smooth muscle
-actinin have been isolated from chicken gizzard (Endo and Masaki, 1982
). It is likely that careful
developmental studies have been hampered in part by the
multitude of
-actinin variants present in various cell types
and the lack of specific immunoreagents to target these
antigens. Indeed, even a ubiquitously distributed protein
such as fibronectin is present as an alternatively spliced
vascular form during embryogenesis (Glukhova et al.,
1989
, 1990
; Castellani et al., 1994
).
While our data strongly suggest that the identity of the
1E12 antigen is an isoform of smooth muscle -actinin, we
cannot rule out the possibility that 1E12 recognizes an
epitope common to another protein that is not abundant
in our gizzard preparation. Each of the three structural domains of
-actinin have similarities with other cytoskeletal
proteins. In particular,
-actinin belongs to a diverse family that includes the spectrins, fodrins, and dystrophins.
There is precedent for antibodies directed aginst the central repeat domains of
-spectrin and -dystrophin to crossreact with
-actinin (Blanchard et al., 1989
). In addition,
the actin binding and EF-hand motifs of
-actinin are
shared with many other proteins.
We have commented previously on the 1E12 antigen
with respect to the early avian vessel wall development
(Hungerford, 1995; Hungerford et al., 1996
). With new
and exciting findings in the vascular development field, we
feel it is important to comment further on the potential
significance of the 1E12 antigen. The molecular basis of
vessel wall formation remains enigmatic (Thayer et al., 1995
;
Folkman and D'Amore, 1996
). In particular, the mechanism by which VSMCs gather and differentiate around the
nascent endothelium is unknown. Several intriguing reports (Davis et al., 1996
; Suri et al., 1996
; Vikkula et al.,
1996
), published during preparation of this manuscript,
may provide clues for how a multilayered vessel wall forms.
While some data exist to show that a population of VSMCs
can transdifferentiate from endothelial cells (DeRuiter et al.,
1997
), these recent reports suggest a mechanism by which VSMC precursors are recruited from the surrounding mesoderm. In the model proposed by Folkman and D'Amore
(1996)
, angiopoietin-1 activates TIE2 tyrosine kinase receptors on endothelial cells, leading to secretion of a "recruiting signal." As VSMC precursors contact the endothelium, other signaling factors that induce differentiation of the VSMCs may be secreted. However, at this time, the
coordinated regulation of VSMC differentiation is not well
understood because there are few known smooth muscle-
specific proteins expressed during the earliest stages of
vessel wall development. The work presented here indicates that smooth muscle-specific proteins are present in a
nascent cytoskeletal apparatus at the time cells appear to
become specified to a smooth muscle fate. We have provided evidence that the 1E12 epitope is located on the
smooth muscle isoform of
-actinin. Perhaps expression of
smooth muscle
-actinin, 1E12 variant, is a critical event in
both the coordinated regulation of vessel wall formation
and SMC differentiation.
Received for publication 18 December 1996 and in revised form 27 February 1997.
1. Abbreviations used in this paper: ECM, extracellular matrix; LSCM, laser scanning confocal microscopy; NMC, nonmuscle cell; PVDF, polyvinyldifluoride; SMWe thank Dr. Tom Trusk for assistance with computer imaging; Dr. Carol
Otey for providing purified -actinin and helpful discussions; and Dr.
Eleanor Spicer for advice on perfusion flow chromatography. We also
thank Dr. Kumar Srikumar for helpful suggestions concerning IgM mAbs.