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Distribution of laminin collagen type IV collagen type I and fibronectin in chicken cardiac jellybasement membrane.

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THE ANATOMICAL RECORD 224:417-425 (1989)
Distribution of Laminin, Collagen Type IV,
Collagen Type I, and Fibronectin in Chicken
Cardiac Jelly/Basement Membrane
CHARLES D. LITTLE, DOMINIQUE M. PIQUET, LYNN A. DAVIS,
LUANNE WALTERS, AND CHRISTOPHER J. DRAKE
Department of Anatomy and Cell Biology, School of Medicine, University of Virginia,
Charlottesville, Virginia 22908
ABSTRACT
Light microscopic immunolabeling studies were designed to identify and locate structural components within the cell-free extracellular matrix
which lies between the embryonic endocardial and myocardial tubes. Affinitypurified antibodies were used to examine stage 15-22 embryonic chicken hearts.
Specimens were immunolabeled by using three different methodologies: 1) postembedding labeling of 10 pm cryostat sections, 2) preembedding labeling (en bloc) of
whole hearts, and 3) postembedding labeling of ethanol/acetic acid-fixed paraffin
sections. Our results establish the spatial distribution of collagen type I and demonstrate for the first time the presence of collagen type IV and laminin in the
myocardial-basement-membranekardiac
jelly.
The formation of a vertebrate heart involves cell-cell data strongly support the contention of Markwald and
adhesion, cell-substrate adhesion, and cellular-motile his colleagues that the ECM, commonly referred to as
activity. Our interest is centered on the cell surface- cardiac jelly, is in fact an expanded basement memextracellular matrix (ECM) adhesions that occur dur- brane (Markwald et al., 1984; Kitten et al., 1987).
ing the formation of endocardial cushions. Kitten et al.
MATERIALS AND METHODS
(1987) showed that fibronectin is abundant in the ECM
En Bloc lmmunolabeling
through which endocardial-mesenchymal cells migrate
Stage
15-21
embryos
were removed from the egg
as they begin formation of the cushion tissue. Previous
work showed that hyaluronic acid (Markwald and Ad- and placed in phosphate-buffered saline (PBS). The
ams-Smith, 19721, glycosaminoglycans (Manasek et hearts were dissected from the embryo and then transal., 19731, glycoproteins (Manasek, 19771, and collagen ferred to a fresh dish of cold PBS. Prior to incubation in
I (Johnson et al., 1974) are also present in this ECM. primary antibody the hearts were carried through each
The term myocardial-basement-membrane was re- of the steps outlined below. Following each step the
cently proposed by Kitten and his colleagues (1987) to hearts were washed three times for 5 min in PBS: 1)
replace the term cardiac jelly as a name for the ex- Fixation in fresh 3% paraformaldehydelPBS (weight/
panded cell-free ECM through which the endocardial- volume), pH 7.5, for 30 min. 2) Incubation in 0.1 M
mesenchyme cells migrate. In this communication, the lysinePBS, pH 7.5 for 30 min. 3) Incubation in 0.4%
term myocardial-basement-membranekardiac jelly Triton X-lOO/PBS (v/v) for 30 min. 4) Incubation in 1
(MBM/CJ), will be use to refer to the expanded extra- mg/ml bovine serum albumidPBS for 24 hr at 4°C.
cellular matrix which lies between the primitive myo- Next, the hearts were immersed in PBS containing primary antibody (10 pg/ml), 1 mg/ml of bovine serum
cardial and endocardial tubes.
In order t o pursue our studies on the cell-ECM ad- albumin, and 0.02%Triton X-100, and then they were
hesions which occur during the migration of mesenchy- incubated for 48 hr at 4°C with gentle rocking. The
ma1 cells into the MBM/CJ, it was necessary to deter- specimens were then washed in a large excess of PBS
mine the identity and spatial distribution of ECM (4°C) for 24 hr. The last steps were then repeated with
components within this tissue. Accordingly, immuno- an appropriate fluorochrome-conjugated secondary anfluorescence localization studies were undertaken us- tibody (Cooper Biomedical, Malvern, PA). In the case of
ing affinity-purified antibodies to chicken collagen I, laminin immunolabeling a few specimens (Fig. 3f)
chicken collagen IV, chicken fibronectin, and mouse were immunolabeled with a tertiary antibody. A fluolaminin. By using three different immunolabeling pro- rescein-conjugated donkey antigoat IgG (Jackson Imtocols we were able to visualize the distribution of each munoresearch Laboratories, Inc., West Grove, PA) was
ECM component listed above in stage 15-22 embryonic applied after the secondary antibody (fluorescein-conchicken MBM/CJ. These results show that in addition jugated goat antirabbit IgG). The specimens were then
to previously reported macromolecules, collagen IV
and laminin are present in this ECM. Consequently,
Received August 3, 1988; accepted January 11, 1989.
collagen IV and laminin can be added to the list of
Address remint reauests to Charles D. Little. DeDt. of Anatomy and
putative substrate adhesion molecules for the migra- Cell Biology,-Box 436, Medical Center, University'of Virginia, Chartory endocardial-mesenchymal cells. Moreover, these lottesville, VA 22908.
0 1989 ALAN R. LISS, INC.
C.D. LITTLE ET AL.
418
washed as described above. After immunolabeling, the
hearts were infused with 5% sucrose/PBS (w/v) overnight at 4°C and then incubated with 15% sucrose/PBS
(w/v) for 1 h r a t 4°C. The specimens were mounted in
Tissue Tek OCT mounting medium (Miles Sci., Inc.,
Naperville, IL), brought to -2o"C, and sectioned in the
frozen state on a DamodIEC Harris-Cryostat (Inter.
Equip. Co., Needham Heights, MA). Sections (10 pm)
were placed on gelatin-coated microscope slides and
then allowed to thaw and dry at 37°C for 30 min. The
sections were washed free of OCT mounting medium
and sucrose and then mounted in 90% glycerol/lO%
PBS (v/v) under a #1 glass coverslip. All solutions contained 0.01% sodium azide (w/v) to prevent microbial
growth.
Postembedding lmmunolabeling of Frozen Sections
This method is essentially the same as described
above except that the hearts were not treated with Triton X-100 or antibodies (en bloc) after fixation. Instead,
the fixed hearts were sectioned in the frozen state
(above), air dried, and then incubated with 10 pg/ml of
primary antibody for 1 h r at room temperature. The
sections were washed extensively with PBS, incubated
with fluorochrome-conjugated secondary antibodies,
washed as before, and mounted (above) for light microscopy.
Acid/Alcohol-Fixed Paraffin Sections
Embryonic hearts were prepared essentially according to Saint-Marie (1962). Briefly, specimens were
fixed overnight in 99 parts of 95% ethanol and 1 part
glacial acetic acid at 4°C. The hearts were washed 2
times with 4°C absolute ethanol for 30 min and then 2
times with 4°C xylene for 30 min. Next, they were incubated in a 5 0 5 0 mixture of xylene:paraffin, followed
by a 25:75 mixture and finally 100% paraffin overnight. Sections were cut at 10 pm thickness and placed
on gelatin-coated microscope slides. The sections were
cleared of paraffin by 3 x 5 min washes in ice-cold
xylene and then rehydrated in a graded series of cold
ethanollwater washes, followed by a 2 h r incubation in
PBS at 4°C. After this step the sections were immunolabeled as described above.
Immunological Reagents
against immobilized native chicken collagen I and then
affinity purified on a n agarose-chicken collagen IV column. Western immunoblot analyses showed that the
cross-adsorbed rabbit antichicken type IV collagen antibodies recognized the immunogen but not chicken
type I collagen (data not shown; the complete characterization of this antibody is the subject of a manuscript in preparation).
Immunoprecipitation of 35 S - methionine - labeled
stage 22 chicken embryonic heart explants was accomplished a s follows: Sixty hearts were dissected free, cut
into several small portions, and placed in a 100 mm
tissue culture dish with 2 ml of Dulbecco's modified
Eagle's Medium plus 10% chicken serum. The explants
were allowed to attach overnight; then the medium
was replaced with 2 ml of methionine-free DMEM, to
which was added 100 pCi/ml of 35S-methionine. The
explants were returned to the tissue culture incubator
for 24 hr. At the end of the labeling period, the explants
were scraped from the dish into 500 pl of radioimmunoprecipitation assay buffer (RIPA) composed of 1%
Triton-X 100, 1% sodium deoxycholate, 0.5% sodium
dodecylsulfate, 0.1 M Tris/HCl, pH 8.0, and 0.1 M
NaC1. The mixture was homogenized with a loose pestle and centrifuged at 10,OOOg for 5 min. A 50 p1 aliquot of cleared lysate was mixed with 10 pg of nonimmune rabbit IgG for 15 min at room temperature,
followed by tumbling with Sepharose protein A for 15
min. The beads were pelleted by centrifugation and
discarded; the supernatant was transferred to a new
tube. Affinity-purified rabbit antimouse laminin (10
pg) or 10 p1 of mouse hybridoma 31-2 ascites fluid was
added to the sample and tumbled gently 1h r at 4°C. In
the case of the mouse 31-2 antibody a second antibody
(rabbit antimouse IgG) was added to the sample and
tumbled for 1hr. This was followed by incubation with
Sepharose protein A beads for 1 hr. The beads were
pelleted and washed repeatedly in RIPA buffer, resuspended in SDS sample buffer, heated to 100°C for 5
min, then pelleted. The resulting supernatant was subjected to SDS-polyacrylamide gel (5%)electrophoresis
according to Laemmli (1970). Immunoreactive polypeptides were analyzed by fluorography. To serve as a
positive immunological control, a n embryonic mouse
epithelial cell culture was also labeled with 35-methionine, lysed in RIPA, and treated a s above.
Antibodies to chicken collagen I and fibronectin were
RESULTS
characterized previously (Little and Chen, 1982).
Antibody Specificity
Laminin was purified from the Englebreth-HolmSwarm (EHS) mouse tumor (Kleinman et al., 1982,
Laminin purified from the mouse EHS-tumor ap1983) and injected into a rabbit, and the resulting an- pears as two diffuse Coomassie blue-stained bands a t
tibodies were affinity purified on a laminin-Sepharose approximately 440 kDa (A chain) and 220 kDa (B
column as described (Little and Chen, 1982). A mouse chains) when analyzed on 5% SDS-polyacrylamide
antichicken laminin monoclonal antibody (31-2) was slab gels (Fig. l a ; lane 1). Rabbit antibodies raised
generously provided by Dr. E. Godfrey, Medical College against a similar preparation of laminin were affinity
of Wisconsin.
purified on immobilized antigen columns and then
Collagen IV was purified from pepsinized adult used to probe 35S-methionine-labeled cultures of an
chicken breast muscle by using the method of Mayne embryonic mouse epithelial cell line. Electrophoresis of
and Zettergren (1980). A method developed for prepa- immunoprecipitates shows two major polypeptide
ration of antibodies to mouse type IV collagen (Chen bands. The radiolabeled polypeptides migrated to
and Little, 1985, 1987) was adapted for isolation of an- slightly lower molecular weight positions relative to
tibodies to chicken antigen. Briefly, the purified colla- the EHS-tumor laminin chains (Fig. la: lane 2). A congen IV fragments were injected subcutaneously in a trol reaction (Fig. l a : lane 3) using nonimmune rabbit
rabbit. The elicited antibodies were cross-adsorbed IgG shows that the bands present in lane 2 are the
BASEMENT MEMBRANE ANTIGENS IN EARLY HEART
1
2
3
1
2
3
4
-B
Fig. 1.
Results from two 5% SDS-polyacrylamide gels (a and b)
are shown. Some lanes were stained with Coomassie blue and some
show fluorographically detected 35S-methionine-labeledpolypeptides.
Bars indicate molecular weight markers, M, = 205,000, 116,000,
97,000. a: Purified mouse-EHS laminin A chains (M, = 400,000) and
B chains (M, = 210,000) chains are shown after Coomassie blue staining (lane 1). Embryonic mouse epithelial cell cultures were radiolabeled as described in Materials and Methods and then subjected to
immunoprecipitation analysis by using the rabbit antimouse laminin
affinity-purified IgG (lane 2).A control reaction in which nonimmune
rabbit IgG was used (lane 3).b Purified mouse-EHS laminin A and
B chains stained with Coomassie blue (lane 1).Radiolabeled stage 22
embryonic chicken heart lysates were analyzed by immunoprecipitation by using rabbit anti-mouse laminin IgG (lane 2). The same sample was probed with a monoclonal mouse antichicken laminin antibody (lane 3). A control in which nonimmune rabbit IgG was
substituted for immune IgG (lane 4). The migration positions of
chicken laminin chains are designated by the arrowheads labeled A
and B. Equal aliquots of radiolabeled antigen were loaded in each
lane and then analyzed identically.
419
180 kDa, designated “A” and “B,” displayed lower electrophoretic mobility than the EHS-laminin. The 180
kDa band immunoprecipitated by the rabbit antimouse
laminin antibodies (lane b2) comigrated with the laminin B chain recognized by the mouse antichicken laminin antibodies (lane b3). A control immunoreaction in
which 10 bg/ml of nonimmune rabbit IgG was substituted for immune rabbit IgG is shown in Figure l b ,
lane 4.The control lane shows a sharp band a t approximately 200 kDa as well as faint high molecular weight
bands. These nonspecifically precipitated chicken polypeptides are present in all the experimental lanes, including duplicate lanes not shown (compare lane 4 with
lanes 2 and 3).
Based on the results shown in Figure l b , it appears
that the rabbit antibodies preferentially recognize
chicken laminin B chains. I n contrast, the mouse antibody (31-2) precipitates laminin A chains (-280 kDa)
and B chains (-180 kDa) in addition to other unknown
polypeptides. The affinity-purified rabbit IgG was more
efficient at immunoprecipitating chicken laminin B
chains than the mouse monoclonal antibody. (All experimental conditions used in lanes b2, b3, and b4 were
identical, including photographic, fluorographic, biochemical, and immunochemical.) Indirect immunofluorescence microscopy conducted by using the rabbit antimouse laminin (polyclonal) antibody resulted in
easily detectable levels of immunostaining (see below).
However, under similar conditions, immunostaining
with the mouse antichicken laminin monoclonal antibody did not result in detectable fluorescent images
(data not shown).
Western blotting of adult chicken muscle pepsin extracts with rabbit anticollagen IV antibodies yielded a
set of immunoreactive bands with the molecular
weights reported for pepsinized chicken collagen IV
(Mayne and Zettergren, 1980). The immunoreactive
bands were removed by prior treatment of samples
with bacterial collagenase. Affinity-purified rabbit antichicken collagen IV antibodies, which had been crossadsorbed against collagen type I, did not recognize
chicken collagen I on immunoblots (data not shown,
manuscript in preparation). In addition, the immunostaining patterns of type I collagen and type IV collagen were reproducibly different (see below), strongly
suggesting immunological monospecificity.
result of a specific immunoprecipitation reaction.
These immunochemical data (Fig. la) demonstrate
that the rabbit antimouse laminin antibodies will reclmmunostaining
ognize mouse laminin A and B chains in a crude cell
A differential interference contrast (DIC) microlysate.
Next we tested the ability of the rabbit antimouse graph of a section through the outflow tract region of a
laminin antibodies to recognize chicken laminin. For stage 15 embryonic chicken heart reveals a n outer mymolecular weight comparison, a sample of purified ocardial tube (m) and inner endocardia1 tube (el. Fine
mouse EHS-tumor laminin is also included in this SDS filamentous material, observable by DIC optics, occugel (Fig. l b : lane 1).Stage 22 chicken heart explants pies the space between the two cellular tubes, (Fig. 2a).
were labeled with 35S-methionineand lysed with RIPA The corresponding epif luorescence micrograph shows
buffer, and the resulting solution was probed with the the location of immunoreactive laminin (Fig. 2b). Imrabbit antilaminin antibodies. A diffuse band a t ap- munostaining within the MBM/CJ is distributed as
proximately 180 kDa and a sharp band at 100 kDa irregular granular spots which coincide with the mawere immunoprecipitated (Fig. lb: lane 2). A similar terial seen under DIC microscopy. Considerable imradiolabeled sample was probed with a mouse mono- muno-fluorescence is associated with the heart tubes.
clonal antibody (31-2) to chicken laminin. This resulted In order to demonstrate immunostaining within the
in the detection of diffuse bands at approximately 280 MBMiCJ it was necessary to overexpose the immunoand 180 kDa and faint bands at 150 and 100 kDa (Fig. staining associated with the cell layers.
A similarly prepared stage 15 heart section is shown
l b : lane 3). The chicken laminin chains at 280 kDa and
420
C.D. LITTLE ET AL.
Fig. 2. This set of micrographs show cryostat sections (10 pm) of
stage 15 embryonic hearts in the region of the outflow tract. The
hearts were immunolabeled prior to embedding and cryosectioning. a:
The differential interference contrast (DIC) image shows sparse ECM
material lying between the myocardium (m) and the endocardium (el.
b The corresponding immunofluorescent image shows very bright
staining for laminin in the heart walls and punctate arrays of staining which codistribute with the material lying between the heart
tubes. DIC image (c) and the epiflourescent image (d) of a heart
immunostained with collagen type I antibodies show bright strands of
immunoreactive material between the primitive heart tubes. x 180.
in Figure 2c and 2d. In this case the embryonic heart
was incubated with antibodies to collagen I. The immunofluorescence shows bright fibrils of collagen
throughout the MBM/CJ. In some cases the fibrils extend across the entire acellular space. In order to
demonstate the fine fibrils within the MBM/CJ it was
necessary to overexpose the fluorescence associated
with the heart tubes. The embryonic tissue shown in
Figure 2 was immunolabeled en bloc and then sectioned.
Figure 3 also depicts the results of immunolabeling
of embryonic hearts en bloc. The DIC image (Fig. 3a)
shows the atrioventricular region of a stage 22 heart in
which cells (arrowhead) had moved from a n earlier position in the endocardium (e) and migrated into the
MBM/CJ. The cells seem to be closely associated with
conspicuous fibers, which appear to bridge the region
between the endocardium and the myocardium (m).
The epifluorescence image (Fig. 3b) shows th at fibro-
nectin antibodies strongly immunolabel the fibers
which are visible under DIC optics. The cellular endocardial and myocardial tubes are heavily labeled
with the fibronectin antibodies.
Figure 3c shows a stage 22 heart similar to the previous figure. Migratory cells (arrowhead)are in contact
with strands of material visible under DIC optics. The
distribution of immunolabeled collagen type I coincides
with these filaments. A comparison of Figure 3b and 3d
suggests that the ECM fibers visible under DIC optics
contain both immunoreactive collagen type I and fibronectin. The myocardium (m) and endocardium (el immunolabel strongly with the collagen type I antibodies.
The distribution of laminin and collagen type IV
were examined by using en bloc staining in approximately stage 19 embryonic hearts (Fig. 3e-h). The DIC
optical image (Fig. 3e) shows material intervening between the atrioventricular myocardium (m) and endocardium (e). Laminin was distributed as a n amor-
BASEMENT MEMBRANE ANTIGENS IN EARLY HEART
phous array of very fine filaments throughout the
MBM/CJ. Codistributed within this fine material were
discrete, variable-sized, immunoreactive particles (Fig.
3f). Thus the laminin immunolabeling seemed to distinguish two staining patterns, one granular and the
other fine filamentous. The specimen shown in Figure
3f was immunostained by using a three-step procedure
which employed a fluorochrome-conjugated secondary
and tertiary antibody (see Materials and Methods).
In the case of collagen IV, immunolabeling showed
fine wisplike filaments throughout the acellular expanse of ECM (Fig. 3h), which were associated with
material visible by DIC optics (Fig. 3g). The fluorescence intensity of collagen IV staining was often
greater than that of laminin, based on subjective judgement.
For comparison with the preembedding labeling
technique, Figure 4 shows a stage 19 atrioventricular
region that was embedded in paraffin and then immunolabeled after sectioning. Affinity-purified rabbit antilaminin IgG and nonimmune IgG were used a t the
same concentrations. The DIC images show conspicuous ECM material lying between the endocardium and
the myocardium (Fig. 4a). This material contains immunoreactive laminin, which appears as small dots or
granules of variable size associated with fine wisplike
staining (Fig. 4b). The staining is localized to areas
where the filaments of ECM are apparent under DIC
optics. In contrast, a control specimen (Fig. 4c) shows
no specific immunostaining within the myocardial
basement membrane when the same concentrations
(10 p.g/ml) of nonimmune IgG and fluorochrome-conjugated secondary antibody are applied to the specimen
(Fig. 4d). The initial photomicrographic exposure times
and the photographic printing conditions were identical for the sections shown in Figure 4b and 4d. Hearts
prepared by the en bloc staining method, using control
antibodies, showed less nonspecific fluorescence than
paraffin-labeled hearts.
The immunostaining patterns described above are
representative of a series of examinations of various
stage hearts (15-22) using each of the antibodies (laminin, collagen I, collagen IV,and fibronectin) in the
three different labeling protocols described in Materials and Methods. Space limitations preclude presentation of all the data; however, the immunolabeling results shown were consistent with all three labeling
procedures for each affinity-purified antibody. The en
bloc staining protocol resulted in the best preservation
of the delicate ECM filaments between the myocardium and endocardium. Indeed, labeling specimens
after cryosectioning resulted in the considerable
MBM/CJ being removed during the various washes. In
each staining experiment three controls were performed: 1) Some specimens were incubated with only
fluorochrome-conjugated secondary antibodies (no primary antibody). 2) Some hearts were incubated in nonimmune primary antibodies followed by fluorochromeconjugated secondary antibodies. 3) Some specimens
were not incubated in with antibodies but were carried
through the various solutions and inspected for intrinsic fluorescence. In all cases the control specimens
showed trace amounts of diffuse fluorescence distributed evenly throughout the microscopic field, most of
which was attributable to intrinsic fluorescence.
42 1
DISCUSSION
ECM Constituents of Cardiac Jelly
The list of specifically identified components contained within the MBM/CJ has increased as more
refined techniques and immunological reagents have
become available. The current list of constituents includes hyaluronic acid, heparin sulfate proteoglycan
(HSPG), collagen I, collagen IV, fibronectin, and laminin (Markwald et al., 1984;Johnson et al., 1974; Kitten
et al., 1987; and this report).
The immunof luorescent images of MBM/CJ fibronectin in this study differed from those of Kitten and his
co-workers (1987). This may be due to the different
methods of fixation. The freeze-substitution method
used by these workers resulted in a more homogenous
distribution of ECM components whereas the paraformaldehyde fixation used here resulted in filamentous bundles of ECM material within the MBM/CJ.
The en bloc fixation and labeling method was chosen to
maximize the immunofluorescence signal-to-noise ratio. In this regard, the en bloc labeling method results
in a virtually black background.
ECM Adhesions During Cushion Mesenchyme Migration
Our interests lie in the molecular basis of cellular
adhesion to the ECM during morphogenesis. All of the
ECM components in the list above, except HSPG, are
known to have corresponding cell surface adhesion receptors (Underhill et al., 1985; Rubin et al., 1986; Buck
and Horwitz, 1987; Dedhard et al., 1987; Ruoslahti and
Pierschbacher, 1987; Hynes, 1987; Terranova et al.,
1983; Lesot et al., 1983, Brown et a]., 1983; Ogle et al.,
1986; Graf et al., 1987; Ogle and Little, 1989). Thus,
each of these macromolecules, individually or in combination, is a candidate for the adhesive scaffold to
which endocardial-derived mesenchymal cells attach
during migration into and through the MBM/CJ.
Recent work in this laboratory showed that embryonic cushion-mesenchyme cells will migrate vigorously
through 3-dimensional gels of laminin. Moreover, addition of the laminin B1 chain synthetic pentapeptide
tyrosyl-isoleucyl-glycyl-seryl-arginine
to the cultures
will abolish the migration of cushion-mesenchyme
cells. Various Arg-Gly-Asp (RGDI-containingsynthetic
peptides, however, have no effect on migration of cushion-mesenchyme cells into 3-dimensional gels of laminin, collagen, or collagen + fibronectin (Davis et al.,
1989; and unpublished data). The ability of endocardial
cushion-mesenchymal cells to migrate through laminin gels in vitro, coupled with the presence of laminin
in the MBM/CJ, suggests that laminin may be one of
the ECM components to which the mesenchymal cells
adhere during formation of the endocardial cushions.
lmmunochemical Characteristics of Avian Laminin
The rabbit antimouse laminin antibodies immunoprecipitate a polypeptide with the characteristics of
laminin B chains but not of A chains. It is possible that
the affinity-purified rabbit IgG cross-reacts only with
chicken B chains whereas the mouse antibodies preferentially recognize A chain:B chain complexes. If the
foregoing is true, then “free” B chains must be present
in the embryonic heart lysates. It then follows that the
rabbit antibodies recognize epitopes on chicken lami-
422
C.D. LITTLE ET AL.
BASEMENT MEMBRANE ANTIGENS IN EARLY HEART
423
Fig. 4. Paraffin-embedded stage 19 embryonic chicken hearts were
sectioned, deparaffinized, rehydrated, and immunolabeled. a: A DIC
image showing a section of atrioventricular myocardium (m), endocardium (e), and intervening ECM. b The strands of ECM material
lying between the two cardiac tubes immunolabel with laminin antibodies. c: A DIC micrograph of the ECM within the acellular
region of a n embryonic heart similar to the specimen described above.
d The nonspecific fluorescence that results when nonimmune IgG
and second antibody is applied to the section. Except for the primary
antibodies, all conditions, including photography, are identical to the
specimen shown in b. x 500.
Fig. 3. The atrioventricular region of embryonic hearts shown in
this figure was immunolabeled prior to embedding and frozen sectioning. In each panel the primitive endocardial tube (e) and myocardial
tube (m) are shown. a: The DIC image of a stage 22 endocardial
cushion. Cells derived from the endocardium (arrowhead) have seeded
into the region between the primitive endocardium and myocardium.
x 600. b Immunostaining with fibronectin antibodies shows brightly
labelled filaments within the MBM/CJ, some of which are closely
associated with the migratory cells. The walls of the heart tubes are
heavily labeled. c: A similar cryosection showing migratory cells (arrowhead). x 500. d The pattern for collagen type I immunostaining
shows filamentous strands between the heart endocardium (e) and
myocardium (m); some of the filaments are apparently associated
with the migratory cells (arrowhead). The myocardium and endocardium are brightly labeled. e: DIC optics shows the endocardium and
myocardium of a stage 19 embryonic heart. x 375. f: Laminin immunostaining is observed throughout the MBM/CJ, a punctate granular
pattern codistributed with fine wisplike arrays. g: A stage 19 heart
that was immunolabeled with collagen type IV antibodies. x 330. h:
The collagen type IV staining pattern shows wisplike patches of fluorescence within the MBMICJ.
nin B chains which are cryptic when complexed with A
chains. Otherwise, one would expect chicken A:B complexes to be precipitated by the rabbit antimouse laminin IgG. That this may be the case is suggested by the
fact that the same rabbit antibodies which do not precipitate chicken laminin A:B complexes will precipitate mouse laminin A:B complexes.
The band a t approximately 150 kDa precipitated by
the mouse monoclonal antibody is very likely entactin,
a basement membrane glycoprotein known to interact
strongly with laminin (Carlin et al., 1981; Martin and
Timpl, 1987). The identity of the band a t 100 kDa
present in both the rabbit antimouse laminin immunoprecipitate and the mouse antichicken laminin immunoprecipitate is unknown. It is important to note that
the mouse antibody (31-2j passively precipitated several components that are probably not laminin-derived
C.D. LITTLE ET AL.
424
polypeptides. Although data describing avian laminin Carlin B., R. Jaffe, B. Bender, and A.E. Chung 1981 Entactin, a novel
basal lamina associated sulfated glycoprotein. J . Biol. Chem.,
are sparse, the bands at approximately 280 kDa and
256:5209-5214.
180 kDa are almost certainly chicken laminin A and B Chen, J.-M., and C.D. Little 1985 Cells that emerge from embryonic
exDlants Droduce fibers of type
J . Cell Biol.,
chains, respectively. Chicken laminin subunits are re_ - IV collagen.
161:1175-1 181.
ported to be of lower apparent molecular weight than
J.-M., and C.D. Little 1987 Cellular events associated with lung
the corresponding mouse polypeptide (Bayne et al., Chen,
branching morphogenesis including the deposition of collagen
1984). Avian laminin may have unique features; howtype IV. Dev. Biol., 120~311-321.
ever, the general characteristics of laminin in organ- Davis, L.A., R.C. Ogle, and C.D. Little 1989 Embryonic heart mesenchymal cell migration in laminin. Dev. Biol., May Vol. 132.
isms as diverse as Drosophila (Fessler et al., 1987;
S.E., E. Ruoslahti, and M.D. Pierschbacher 1987 A cell surMonte11 and Goodman, 1988), and human (Martin and Dedhard,
face receptor complex for collagen type I recognizes the arg-glyTimpl, 1987) appear to be similar.
asp sequence. J . Cell Biol., 104:585-593.
Embryonic “Basement Membranes” Vs.
Embryonic Mesenchymes
The data reported here combined with earlier work
(Chen and Little, 1985,1987) suggest that laminin and
type IV collagen are not restricted in their distribution
to conventional embryonic basement membranes. Indeed, our unpublished observations show that laminin
and collagen IV are widely distributed in embryonic
tissues. This more general distribution of laminin and
collagen IV has been observed by others. For example,
Solursh and Jensen (1988) detected collagen IV in limb
bud mesenchyme. Moody and her colleagues have recently shown a correlation between the presence of
laminin in mandibular arch mesenchyme and the guidance pathway of the trigeminal axon in chicken embryos (Riggott and Moody, 1987). Others have reported
laminin in cranial mesenchyme (Tuckett and MorrissKay, 1986) and in early ganglia and nerve roots
(Rogers et al., 1986).
Two structural glycoproteins, laminin and collagen
IV, may now be added to the list of components known
to be present in the ECM commonly called cardiac jelly.
These two glycoproteins are generally thought t o be
restricted to conventional basement membranes.
Therefore, their presence in this region of the embryonic heart adds weight to the suggestion of Kitten et al.
(1987) that cardiac jelly is an expanded basement
membrane.
The identification of laminin and collagen IV within
such anatomically diverse mesenchymes as heart,
lung, mandible, and limb demonstrates a widespread
distribution of these “basement membrane” components throughout the embryo. As investigators continue to describe the molecular anatomy of tissue ECM,
it may well be that the current dichotomous classification system (basement membrane ECM vs. interstitial
ECM) will have to be reevaluated with regard to embryonic tissues.
ACKNOWLEDGMENTS
This work was supported by grants from the Developmental Biology Program, NSF (#85-178161, NIHLB
(#37709), and a Basic Research Grant-March of
Dimes Birth Defects Foundation (#1-10661, to C.D.L.
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