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Ultrastructural immunocytochemical localization
of fibronectin in the early chick embryo
ByE. J. SANDERS1
From the Department of Physiology, University of Alberta
SUMMARY
Fibronectin was localized using peroxidase and biotin-avidin-ferritin techniques in
developmental stages of the chick embryo from laying to primitive streak formation. The
primary location of fibronectin at all stages examined was the basal lamina of the epiblast
and its associated extracellular material. The remaining tissues showed little or no immunochemical deposit. At the primitive streak stage of development there were some regional
differences in the density of the deposit on the basal lamina. Closely adjacent to the primitive
streak, where the basal lamina is fragmentary, deposit was sparse or absent. In this, and other
regions, mesoderm cell surfaces did not stain except where they closely approached the
stained basal lamina or interstitial bodies. Staining was variable in the basal lamina anterior
to the primitive streak, but in a number of cases particularly heavy deposit was noted overlying the crescent of late hypoblast. This area seemed to correspond with the anterior fibronectin-rich band reported by others using immunofluorescence localization.
INTRODUCTION
The morphogenetic movements of gastrulation involve the coordinated and
directed movements of several early embryonic cell populations (Vakaet, 1970;
Nicolet, 1971). It is currently widely held that extracellular materials, present in
the immediate vicinity of these migrating cells, exert some controlling influence
over the extent and direction of these movements. One of the most strongly
implicated substances is fibronectin which, in a number of different developmental situations, is ideally positioned both spatially and temporally to modulate morphogenetic migration (Wartiovaara, Stenman & Vaheri, 1976; Wartiovaara et al., 1978; Wartiovaara, Leivo & Vaheri, 1979; Spiegel, Burger &
Spiegel, 1980; Armstrong & Armstrong, 1981; Lesot, Osman & Ruch, 1981;
Silver, Foidart & Pratt, 1981; Thesleff et al., 1981).
In the chick embryo, fibronectin has been identified in a number of significant
locations at developmental stages subsequent to gastrulation (Linder, Vaheri,
Ruoslahti & Wartiovaara, 1975; Newgreen & Thiery, 1980; Waterman &
Balian, 1980; Mayer, Hay & Hynes, 1981). Immunofluorescent technique on
whole mounts also indicates the presence of fibronectin in chick embryos at
1
Author's address: Department of Physiology, University of Alberta, Edmonton, Alberta,
Canada T6G 2H7.
156
E. J. SANDERS
Fig. 1. Diagrammatic representation of the stage-5 chick embryo showing the
regressing primitive streak and the anterior crescent of 'late hypoblast'. The
figures refer to the levels at which these embryos were routinely sectioned.
gastrulation stages, principally associated with the basal lamina of the epiblast
and most particularly in a band of fibres in the anterior of the area pellucida
(Critchley, England, Wakely & Hynes, 1979; Wakely & England, 1979), where
it may influence the spreading and migration of cells. Furthermore, in vitro
studies have clearly shown that the presence of fibronectin on the substratum
markedly increases the rate of spreading of mesoderm taken from primitivestreak-stage embryos (Sanders, 1980). This enhancement was observed both on
substrata pretreated with plasma fibronectin and on substratum-attached
material derived from early embryonic epithelia (endoblast and hypoblast).
In order to further study the possible role of fibronectin in gastrulation, and
the migration of mesoderm cells in particular, we have sought to localize this
substance in the early embryo more precisely. Ultrastructural techniques were
used on developmental stages from laying to gastrulation. The techniques used
were the peroxidase-antiperoxidase (PAP) method (Sternberger, 1979) and
Fig. 2. Transmission electron micrograph (TEM) showing the dorsal surface of the
epiblast of a stage-5 embryo incubated under PAP control conditions. There is
some membrane intensification, x 37100.
Fig. 3. TEM showing the basal lamina on the ventral surface of the epiblast of a
stage-XIII embryo incubated under PAP control conditions, x 37100.
Fig. 4. Light micrograph (LM) of a section through a PAP-incubated stage XI
embryo. The basal lamina on the ventral surface of the epiblast is stained, but
the dorsal surface of the epiblast is not. x 480.
Fig. 5. TEM of the basal lamina of a stage-XI embryo. PAP technique, x 37100.
Fig. 6. LM of a section through a PAP-incubated stage-XIII embryo. The basal
lamina stains but the other surfaces of the epiblast and hypoblast do not. x 480.
Fig. 7. TEM of the basal lamina of a stage-XIII embryo. PAP technique, x 37100.
Fibronectin in the early chick embryo
157
EMB 71
158
10
12
E. J. SANDERS
Fibronedin in the early chick embryo
159
biotin-avidin-ferritin (BA) method (Heitzmann & Richards, 1974; Skutelsky,
Danon, Wilchek & Bayer, 1977). The results show that fibronectin is present in
the embryo from the time of laying, mainly in the epiblast basal lamina and the
associated interstitial bodies. Some regions of the area pellucida showed more
intense immunolabelling than others and the basal lamina appeared devoid of
fibronectin adjacent to the primitive streak.
MATERIAL AND METHODS
Embryos were used at three different stages of development: (i) unincubated,
at stage X or XI of Eyal-Giladi & Kochav (1976); (ii) 6 h incubation, at approximately stage XIII; and (iii) 24 h incubation at stage 5 of Hamburger & Hamilton
(1951). The embryos were dissected free of the vitelline membrane and yolk in
Pannett & Compton's saline and fixed with 0-5% glutaraldehyde in 0-1 M
cacodylate buffer, pH 7-4, for 10 min at room temperature. Trials with fixative
concentrations from 0-1 % to 2-5% showed that 0-5% was most satisfactory in
terms of retaining acceptable morphology and producing the least non-specific
labelling. After this brief fixation, the embryos were washed overnight in vials
on a rotating mixer with three changes of phosphate-buffered saline (PBS)
containing 1% normal goat serum (g.s.) and 0-1 M lysine. The goat serum,
present at several stages of the preparation, was used to help prevent subsequent
non-specific immunolabelling (Sternberger, 1979), and the lysine was used to
quench the glutaraldehyde and available aldehyde groups. In some experiments,
the goat serum was replaced by 1 % bovine serum albumin. This substitution
made no difference to the quality of the control samples.
For the PAP method, embryos were incubated in rabbit anti-human fibronectin antiserum (Collaborative Research Inc.), diluted 1:100 with PBS + g.s.,
for 2 h. All incubations and washes were carried out at room temperature on a
Fig. 8. LM through the primitive streak of a stage-5 embryo. PAP technique. The
basal lamina is the most densely stained structure, and this terminates in the region
of mesoderm invagination x 125.
Fig. 9. LM of a conventionally stained section through the anterior region of the
area pellucida of a stage-5 embryo. The section is taken at approximately level 1
indicated in Fig. 1. The arrowheads mark the boundary of the centrally located
endoblast and the peripherally located late hypoblast. The latter is thrown into
characteristic folds, x 40.
Fig. 10. TEM of the basal lamina of a stage-5 embryo. Note the large interstitial body
which stains heavily at its periphery. PAP technique, x 37100.
Fig. 11. TEM of the dorsal surface of the epiblast of a stage-5 embryo. PAP technique x 37100.
Fig. 12. TEM of the stage-5 basal lamina overlying the anterior crescent of late hypoblast. PAP technique, x 37100.
6-2
160
E. J. SANDERS
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Fibronectin in the early chick embryo
161
rotating mixer. This was followed by an overnight wash with five changes of
PBS + g.s. The specimens were then incubated for 1 h in goat anti-rabbit IgG
antiserum (Miles Laboratories Inc.), diluted 1:10 in PBS + g.s. After rinsing for
1 h with two changes of PBS + g.s., the embryos were incubated in PAP reagent
(Miles), diluted 1:50 with PBS + g.s. for 30 mins and then rinsed twice with
PBS alone for 10 min. Specimens were then immersed in diaminobenzidine
(DAB) reagent for 20 min and then washed with PBS for 30 min with three
changes. The DAB reagent was freshly made in clean glassware by adding
0-01 % hydrogen peroxide to 0-05% DAB in PBS. The material was postfixed
with 1 % osmium tetroxide in 0-1 M phosphate buffer, pH 7-4, for 1 h, dehydrated
in ethanol and propylene oxide and embedded in Araldite. Three different
controls were run: (i) fibronectin antiserum replaced by normal rabbit serum;
(ii) fibronectin antiserum replaced by PBS; and (iii) all steps up to the DAB
reagent replaced with PBS + g.s., with a wash in PBS alone immediately before
the DAB treatment. Penetration of the PAP reagents into the tissue spaces did
not appear to present any problem, and reaction product was frequently seen on
tissue surfaces that were closely apposed.
For the biotin-avidin-ferritin method, embryos were fixed and washed as
before. With stage-5 embryos the endoblast was dissected off with tungsten
needles either before or after fixation, in order to facilitate penetration of the
avidin-ferritin to the basal lamina. Incubation with antifibronectin antiserum
and the subsequent wash was performed as in the PAP method. Embryos were
then incubated in biotinylated antirabbit IgG (Vector Laboratories Inc.),
diluted 1:5 with PBS + g.s., for 1 h. After washing with PBS + g.s. for 1 h with
two changes, the specimens were immersed in ferritin-congugated avidin D
(Vector), diluted 1:10 with PBS 4-g.s., for 30 min. Embryos were then washed
with PBS alone, postfixed with osmium tetroxide, dehydrated and embedded as
described above. Three different controls were run: (i) fibronectin antiserum
replaced by normal rabbit serum; (ii) fibronectin antiserum replaced by PBS;
and (iii) fibronectin antiserum and biotinylated IgG replaced by PBS. This last
mentioned control was used to check for endogenous avidin-binding activity
(Wood &Warnke, 1981).
Specimens were thin sectioned for electron microscopy without staining, and
examined on copper grids with a Philips 300 microscope. Specimens at stages XI
Fig. 13. LM of a stage-5 embryo through the late hypoblast crescent. PAP technique.
The basal lamina is most heavily stained over the folded late hypoblast, (H).
Ep. = epiblast. x 300.
Fig. 14. TEM of the basal lamina in the primitive streak region of a stage-5 embryo.
Note that the basal lamina is poorly organized and fails to stain. PAP technique,
x37100.
Fig. 15. TEM through the anterior region of a stage-5 embryo showing the epiblast
(Ep) and endoblast (En). PAP technique, x 4460.
162
E. J. SANDERS
f
20
Fibronectin in the early chick embryo
163
and XIII were sectioned in the centre of the area pellucida. Stage-5 embryos
were examined in detail by sectioning in at least five different regions (see Results).
For light microscopy, 1 /*m sections were examined either unstained (PAP
method) or stained with methylene blue and azure B.
RESULTS
The unincubated embryo is characterized by a single epithelial layer, the
epiblast, underlaid by an organized but incomplete basal lamina (Low, 1967;
Sanders, 1973, 1979) and scattered hypoblast cells (Eyal-Giladi & Kochav,
1976). By 6 h incubation, both the basal lamina and hypoblast have been
completed. The stage-5 embryo (Fig. 1) is characterized by a regressing primitive
streak (Nicolet, 1971) and a mesoderm layer underlaid by endoblast which is
displacing the hypoblast (Sanders, Bellairs & Portch, 1978). The four tissues
which have been examined here are therefore: epiblast, hypoblast, endoblast
and mesoderm.
PAP Technique
The control samples run with every experiment were largely negative for
electron-dense deposit (Figs. 2 and 3). In some control samples the dorsal
surface of the epiblast and the endoblast surfaces showed an intensification of
membrane staining (Fig. 2) but was of a low level and was taken into consideration in interpreting results. The controls in which DAB was used without prior
antibody incubation showed no deposit and therefore no random adsorption
of DAB.
(i) Stage XL At this stage the ventral surface of the epiblast stained for
fibronectin while the dorsal surface did not (Fig. 4). Details of the basal lamina
on the ventral surface were obscured by the dense and uneven layer of deposit
(Fig. 5).
(ii) Stage XIII. Despite the presence of a hypoblast layer in this stage, the
only surface to react positively was the basal lamina of the epiblast (Fig. 6).
As in stage XI details of the lamina were obliterated by the dense deposit (Fig. 7).
Fig. 16. TEM of the dorsal surface of the endoblast. Stage-5 embryo. PAP technique,
x37100.
Fig. 17. TEM of the ventral surface of the endoblast. Stage-5 embryo. PAP technique, x 37100.
Fig. 18. TEM of the surface of a late hypoblast cell of a stage-5 embryo. PAP
technique. Note the association of the deposit with membrane invaginations. One
invagination (arrowhead) appears to be a coated pit. x 55 600.
Fig. 19. TEM of the surface of a mesoderm cell remote from the basal lamina.
Stage-5 embryo. PAP technique, x 37100.
Fig. 20. TEM of a stage-5 embryo, showing the epiblast (Ep) and its basal lamina
with closely apposed mesoderm cell (M). PAP technique, x 11700.
164
E. J. SANDERS
22
23
Fig. 21. TEM of the ventral surface of the epiblast of a stage-XI embryo. The
interstitial bodies associated with the basal lamina show ferritin deposits. Biotinavidin technique, x 55 600.
Fig. 22. As Fig. 21, stage-XIII embryo, x 55600.
Fig. 23. TEM of the basal lamina of a stage-5 embryo. The ferritin deposit is more
uniformly distributed on the basal lamina than in earlier stages. Biotin-avidin
technique, x 46100.
Fibronectin in the early chick embryo
165
(iii) Stage 5. In view of the relative complexity of the stage-5 embryo, sections
through at least five different regions were examined on each embryo, as shown
in Fig. 1. Sections were made through the primitive streak, at various levels,
including areas lateral to this structure (Fig. 8). In addition, sections were taken
in the area pellucida anterior to Hensen's node, passing through the crescent of
loosely attached ventral cells characterized by their foamy appearance, and
sometimes known as the 'germinal crescent' (Nicolet, 1971). These cells are
termed 'late hypoblast' here, Fig. 1, and are clearly distinguishable from the
endoblast by both light and electron microscopy, by their increased yolk content
and larger size (Fig. 9).
(a) Epiblast and basal lamina. As illustrated in Fig. 8, the basal lamina was
by far the most fibronectin-rich tissue surface in the embryo in all regions
examined. Although there was some decrease in intensity from the level of
Hensen's node to more posterior regions, all areas showed deposit both on the
basal lamina proper and on the associated interstitial bodies (Fig. 10), and
occasional extracellular filaments. This was in sharp contrast to the dorsal
surface of the epiblast, which showed little or no deposit in comparison with
controls (Fig. 11).
Anterior to Hensen's node the situation was variable. However, in a number
of cases the basal lamina overlying the late hypoblast in this region presented a
much denser deposit than that overlying the endoblast (Figs 12 and 13). It is
unlikely that the variability of this region was due to impaired accessibility of
the reagents, since equally inaccessible regions showed ample deposit.
As the basal lamina enters the region of the primitive streak or Hensen's node
it first begins to fragment, and then disappears entirely in the zone of actively
invaginating cells (Low, 1967). The PAP staining was of particular interest in
this area. As expected, light microscopy showed a tapering off of the stained
basal lamina as it approached the primitive streak, Fig. 8. Ultrastructural
examination showed that nearest the streak or Hensen's node, in the fragmenting
region, the lamina failed to stain (Fig. 14), and a transition could be observed
between the stained and unstained areas of the basal lamina.
(b) Lower layer (endoblast and late hypoblast). In comparison with the basal
lamina, all other tissue surfaces showed minimal staining. The endoblast
presented a somewhat patchy deposit (Fig. 15) but both the dorsal and ventral
surfaces of this tissue largely possessed a thin, uniform layer of reaction product
(Figs 16 and 17). The late hypoblast in the anterior of the area pellucida (see
Fig. 1) showed a patchy staining with the deposit noticeably associated with
apparent pinocytotic invaginations of the cell surface (Fig. 18). In some places
the invaginations appeared to be coated pits (Fig. 18). This characteristic
distribution was not seen in stage-XIII hypoblast or in any other tissue.
(c) Mesoderm. These cells were consistent in their reaction, and showed little
or no deposit within the bulk of the tissue (Fig. 19). Where mesoderm cells were
closely apposed to the basal lamina (Fig. 20) or the dorsal surface of the endo-
166
E. J. SANDERS
blast they were frequently associated with dense patches of material, possibly
interstitial bodies.
BA Technique
The controls run for this method were always reliable and ferritin was
routinely found to be absent from these embryos. In the experimental series of
embryos at all stages, the only surface found labelled was the basal lamina of the
epiblast (Figs. 21, 22, 23). Labelling was consistent between individual embryos
but of relatively low density in comparison with PAP. For this reason regional
differences in density across the area pellucida were difficult to discern. Other
than the selective labelling of the ventral surface of the epiblast, the only
generalization possible with certainty was that in the early stages (XI and XIII)
and the interstitial bodies were primarily labelled (Figs. 21 and 22), while at
stage 5 label was found both on these bodies and on the basal lamina proper
(Fig. 23).
DISCUSSION
The finding that fibronectin was present in the basal lamina was not unexpected, since this has been repeatedly demonstrated in other tissues in recent work
(Wartiovaara et al. 1976, 1978; Courtoy, Kanwar, Hynes & Farquhar, 1980;
Madri, Roll, Furthmayr & Foidart, 1980; Waterman & Balian, 1980; Lesot
et al. 1981; Repesh, Furcht & Smith, 1981; Silver et al. 1981; Thesleff et al 1981).
The present results are of particular interest in view of the early stages in the
development of the basal lamina used here. Fibronectin was found in this
structure shortly after the time of laying with little difference in labelling
intensity from that in the gastrulating embryo. Formation of the basal lamina
during this developmental period has been followed in some detail (Low, 1967;
Sanders, 1979) and the gradual morphological elaboration described. It would
now be of interest to examine the basal lamina before the time of laying to
determine the relationship between the formation of this structure and the
appearance of fibronectin. Mitrani & Farberov (1981) report that no fibronectin
is detectable by immunofluorescence at in utero stages of development.
Interstitial bodies, found closely adjacent to the basal lamina, were originally
described by Bellairs (1963) and Low (1970), and have recently been shown to
contain fibronectin at later stages of development than those used here (Mayer
et al. 1981). In the present case these bodies, like the basal lamina, are shown by
the PAP method to contain fibronectin from stage XI onwards. Results with
the less sensitive BA method would indicate less fibronectin in the basal lamina
than the interstitial bodies at the earlier stages. It is possible that the interstitial
bodies reorganize and contribute material, including fibronectin, to the developing basement membrane. From a morphogenetic point of view, therefore
fibronectin is present in the tissue space, between the upper and lower layers,
well in advance of the appearance of the migratory mesoderm cells. This situation is also the case with regard to the advance deposition of fibronectin the
Fibronectin in the early chick embryo
167
migratory pathway of neural crest cells described by Newgreen & Thiery (1980).
This led these authors to conclude that the appearance of fibronectin in itself
was not sufficient to initiate cell migration, although the role of fibronectin in
the subsequent facilitation of migration is a clear possibility.
Label was not found associated with the mesodermal cell surfaces here except
where these cells came into the vicinity of the basal lamina. At this stage of
development extracellular filaments are rarely observed (Sanders, 1979). The
mass of the mesoderm is several cell layers thick at stage 5 (Ebendal, 1976;
England & Wakely, 1977) and many cells, which apparently have no contact
with the basal lamina, are therefore minimally associated with fibronectin.
Although the possibility exists that extracellular scaffolding materials, with or
without fibronectin, have not been preserved or visualized by the current
technique, it is difficult to envisage how fibronectin could be influencing cells in
the bulk of the mesoderm. It is possible that the spreading of only those mesoderm cells actually in contact with the basal lamina and/or interstitial bodies is
actively influenced by the presence of fibronectin, and these cells in turn
influence the remaining cells deeper within the mesoderm tissue. Such a spread
of influence has been suggested to operate in the interrelationship between basal
lamina and limb bud mesenchymal cells (Lunt & Seegmiller, 1980). In this way
the cells in immediate contact with the fibronectin-rich extracellular materials
may be induced to spread and migrate away from the primitive streak and this
movement of cells might stimulate mesoderm cells more remote from the
fibronectin to behave similarly, perhaps by contact guidance.
The basal lamina immediately adjacent to the zone of actual invagination in
the primitive streak was found to be devoid of label. This correlates with the
observation that cells newly invaginated tend to be rounded, while more
laterally they tend to be more flattened or irregularly shaped (Solursh & Revel,
1978), although such flattening is in no way comparable with that observed
in vitro (Sanders, 1980), where flattening is extreme. Wakely & England (1977)
also report changes in mesoderm cell morphology as cells move away from the
primitive streak. Such circumstantial correlations tend to strengthen the
speculation that fibronectin has an in vivo role in control of cell shape in this
situation. The factors responsible for the difference in fibronectin content of the
basal lamina in the primitive streak region are unclear.
In agreement with the present results, in vitro studies of these tissues (Sanders,
1980) have shown that the mesoderm at this stage lacks the ability to synthesize
large quantities of fibronectin, and in addition, that these cells are very responsive to substratum-attached fibronectin. Also, in culture, stage-XIII hypoblast
and stage-5 endoblast were shown to produce abundant cell surface fibronectin.
As shown here in situ, however, these two tissues are associated with relatively
little fibronectin and much less than is the basal-lamina-bearing epiblast. This
difference may be a reflection of the response of the endoblast and hypoblast to
the culture conditions.
168
E. J. SANDERS
Observations here show that the late hypoblast bound label in apparent
endocytic invaginations, some of which appeared to be coated pits. This may be
compared with the extensive coated vesicle activity in yolk-sac endoderm
(Mobbs & McMillan, 1981), since the latter tissue is thought to derive from
hypoblast via extraembryonic endoderm (Fontaine & Le Douarin, 1977). In the
yolk sac this endocytosis appears to be involved with yolk protein sequestration,
but whether the hypoblast is active in this process is unclear. It is usually
considered that the extraembryonic endoderm is the primary site of yolk
uptake (Bellairs, 1963).
Previous work on gastrulating embryos has localized fibronectin using
whole-mount immunofluorescence technique (Critchley et al 1979; Wakely &
England, 1979). One of the main findings from these studies was that fibronectin
was particularly abundant in the basal lamina in a well-defined crescent,
corresponding with the position of the 'germinal crescent' at the anterior of the
embryo at the boundary of the area pellucida and area opaca. The present
results show a general diminution of label on the basal lamina on passing from
the anterior to the posterior of the embryo, but not a consistently appearing,
well-defined, anterior band of intense label. This may be due to the high
sensitivity of the PAP method, which results in heavy deposits across the entire
basal lamina thus obscuring an anterior band and to the relatively low sensitivity of the BA method. However, in a number of embryos (Fig. 12) particularly
heavy deposit was observed in the basal lamina overlying the anterior crescent
of late hypoblast (germinal crescent). This tissue has been shown by several
studies to form from the original hypoblast by cranial compression (Vakaet,
1962; Rosenquist, 1972; Fontaine & Le Douarin, 1977), and perhaps to give
rise to primordial germ cells (Vakaet, 1962,1970). Since there is a developmental
continuity between the stage-XI hypoblast and the stage-5 anterior crescent,
and since the former shows the ability in culture to secrete large amounts of
fibronectin, it seems possible that the hypoblast may contribute to the copious
amount of fibronectin sometimes seen in the basal lamina of the anterior
crescent. The variability observed in this area could possibly be due to imprecise
staging of the embryos, although this would require that the fibronectin-rich
crescent was itself precisely timed in its appearance. Therefore, apart from the
variability, the speculations of Critchley et al. (1979) and Wakely & England
(1979), regarding a possible role for fibronectin in germ cell migration from the
anterior crescent, are not contradicted by the present observations. The reason
why these cells would require a substratum much richer in fibronectin than does
the mesoderm is not apparent, unless it is to provide the specific direction
required by the germ cells as apposed to the general centrifugal direction of the
mesoderm.
I am indebted to Mrs Sita Prasad for careful technical assistance and to Dr S. E. Zalik for
reading and criticizing the manuscript. This work was supported by a grant from the Medical
Research Council of Canada.
Fibroneclin in the early chick embryo
169
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(Received 4 January 1982)