J. Embryol exp. Morph. Vol. 67, pp. 59-70, 1982
Printed in Great Britain © Company of Biologists Limited 1982
59
Localization of microfilament bundles
in the upper layer of the primitive-streakstage chick blastoderm
By L. VAKAET1-2 AND CH. VANROELEN 1
From the Laboratory of Anatomy and Embryology, R.U.C.A., Antwerp
SUMMARY
Transmission electron microscopy of medium-streak-stage chick blastoderms revealed the
presence of 4-6 nm microfilament bundles just under the apical surface of upper layer cells,
in close association with the junctional complex. The presence of these microfilament bundles
was not restricted to the centre of the primitive streak, but they were also observed in the
presumptive neural plate and lateral to the anterior half of the primitive streak. The microfilament bundles are thought to be involved in every morphogenetic movement in the upper
layer and not to be restricted to groove formation.
INTRODUCTION
In several investigations microfilament bundles have been observed just under
the apical surface of the neural plate cells of amphibian embryos (Baker &
Schroeder, 1967; Burnside, 1971; Lofberg, 1974). Their presence was correlated
with the apical constriction of the cells, and with neural tube formation. Similar
observations have been made in chick neurulae (Karfunkel, 1972; Bancroft &
Bellairs, 1975; Nagele & Lee, 1980). During these studies it was suggested that
microfilament bundles might be characteristic of all cells lining grooves in early
morphogenesis.
In the present investigation, this hypothesis was tested in the primitive-streakstage chick blastoderm. During avian gastrulation, cells from the upper layer
ingress along an anteroposterior axis, resulting in the formation of a groove.
Examination of the primitive-streak region in the chick blastoderm revealed
that as the upper layer cells approach the primitive streak, many of them
elongate and have an irregular shape (Balinsky & Walther, 1961; Ruggeri, 1966
and 1967). Variations in apical cell morphology between cells destined to
ingress in the groove and cells situated more laterally were observed by scanning
electron microscopy (SEM) (Bancroft & Bellairs, 1974; Jacob, Christ, Jacob &
Bijvank, 1974). SEM studies of the primitive streak revealed that the cells
1
Authors' address: Laboratory of Anatomy and Embryology, Rijksuniversitair Centrum,
Groenenborgerlaan 171, 2020-Antwerpen, Belgium.
2
Author to whom reprint requests should be sent.
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L. VAKAET AND CH. VANROELEN
Entophyllic crescent
Deep layer
Upper layer
Primitive streak
Middle layer
Fig. 1. Drawing of a stage-5 (Vakaet, 1970) chick blastoderm (inspired by De Vos
& Van Gansen, 1980). Regions selected for ultrastructural observation: transverse
sections of the primitive streak (detail in Fig. 2), lateral to the primitive streak
(detail in Fig. 3) and the neural plate (detail in Fig. 4); a tangential section of the
primitive streak (Fig. 7).
become progressively elongated and flask-shaped (Solursh & Revel, 1978). This
technique did not allow presumptive mesoblast to be distinguished from the
presumptive endoblast cells during ingression (Wakely & England, 1977). A
basal lamina is present in close contact with the ventral surface of the upper
layer (Low, 1967; Mestres & Hinrichsen, 1974; Sanders, 1979; Wakely &
England, 1979). It is continuous in the lateral region, gradually becomes
interrupted towards the primitive streak, and only sparse fragments of basal
lamina can be recognized in the primitive streak (Vakaet, Vanfoelen & Andries,
1980). Examination of the junctional complexes between the upper layer cells
revealed the presence of gap and tight junctions in the region close to the dorsal
surface (Bellairs, Breathnach & Gross, 1975).
The aim of the present investigation was to study the architecture of the
microfilament bundles within the upper layer cells, and the distribution of the
upper layer cells showing microfilament bundles. These results will allow us to
test the hypothesis that microfilament bundles are restricted to cells lining
grooves.
Microfilament bundles in upper layer of the chick blastoderm
61
Fig. 2. Drawing made from mounted electron micrographs of the primitive streak
region. Arrows indicate the regions of which electron micrographs are shown.
Scale bar is 50 fivn.
MATERIALS AND METHODS
Eggs from commercial stock were incubated for 16 h at 38 °C. Blastoderms
of stage 5 of Vakaet (1970) (corresponding to stage 3+ of Hamburger &
Hamilton, 1951) were selected. The blastoderms were removed from the eggs
and fixed as described by Sanders (1979). Summarizing, a freshly prepared
solution of 1 % (w/v) glutaraldehyde and 1 % (w/v) tannic acid (mol. wt. 1701)
in 0-1 M cacodylate buffer at pH 7-4 was used as a fixative, since previous
observations suggest that this hypotonic fixative results in an improved visualization of microfilament bundles (Vanroelen & Vakaet, 1981). The embryos
were fixed at room temperature for 2 h. After a thorough rinse, the embryos
were post-fixed in a 1 % (w/v) osmium tetroxide solution in the same buffer for
1 h. After dehydration in a graded series of ethanol, the embryos were embedded
in Epon 812. Ultrathin sections were stained in a 2 % (w/v) aqueous solution of
3
EMB 67
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L. VAKAET AND CH. VANROELEN
Microfilament bundles in upper layer of the chick blastoderm 63
uranyl acetate for 30min and in lead citrate for 30 s (Reynolds, 1963). The
sections were examined with a JEOL 100 B electron microscope. Low-magnification electron micrographs (x 1700) of each ultrathin section were mounted.
A drawing was made from these mounted micrographs in order to obtain the
exact localization of the details. The thickness of the microfilaments was
estimated on electron micrographs at a final magnification factor of x 100000
and x 200000, using a Peak scale 10 x magnifier. Sections of 14 blastoderms
were examined.
RESULTS
A drawing of a stage-5 (Vakaet, 1970) chick blastoderm is represented in
Fig. 1. The embryos were studied in three regions of the area pellucida: (1) the
primitive streak, (2) lateral to the primitive streak, and (3) the neural plate. An
account on the fine structure of the upper layer cells in the different regions has
been reported by others (see Introduction), and will therefore not be repeated
in detail here.
Our technique is not adequate for the localization of individual microfilaments. Therefore, only the localization of microfilament bundles will be described.
The primitive-streak region
Figure 2 is a drawing made from mounted low-magnification electron micrographs of a transverse section of the primitive streak. It demonstrates that the
upper layer cells in the primitive streak do not have a regular shape. The cells
are situated above one another, some of them are extremely elongated. In the
centre of the streak, some of the upper layer cells appear narrower near the
dorsal surface.
Micrographs at higher magnification show clearly delineated plasma membranes. Just under the apical surface, the lateral plasma membranes show a
junctional complex, together with plasma-membrane-associated dense accumulations. In all the cells, microfilament bundles are present in close association
with these dense accumulations. High-magnification electron micrographs reveal
that the diameter of the microfilaments varies from 4 to 6 nm. In some cells,
the microfilament bundles appear to be restricted to a 0-5 jam array from the
junctional complex (Fig. 5B and 6 A). We have not seen transverse sections of
microfilament bundles in the apical cytoplasm at some distance from the
junctional complex. In other cells, the microfilament bundles extend from one
junctional complex to the other, within the cell apex. This is especially apparent
in the cells of the centre of the primitive streak. These cells are extremely
elongated and often very narrow at their apical surface (Fig. 5 A).
Figs. 3 and 4. Drawings made from mounted electron micrographs of the lateral
area (Fig. 3) and anterior to Hensen's node (Fig. 4) are represented. Scale bar is
50 /tm.
3"2
L. VAKAET AND CH. VANROELEN
Microfilament bundles in upper layer of the chick blastoderm 65
Fig. 6. Electron micrographs of the plasma-membrane-associated dense accumulations in transverse sections of the area pellucida. (A) The primitive streak region.
(B) Lateral to the primitive streak. (C) The neural plate. Note the presence of
microfilament bundles near the junctional complex in the three regions. Scale bar
is 2 fim.
Microfilament bundles extending from one junctional complex to the other
are also observed in a few cases in presumptive columnar cells. In these cases
the possibility exists that the sections are tangential to the lateral plasma membrane of the upper layer cell (Fig. 5C). Small microfilament bundles were also
observed at the bases of apical surface projections and in microvilli.
Tangential sections of the apical surface of the primitive-streak cells
illustrate the close association of the microfilament bundles with the junctional
complexes (Fig. 7). As the apical surface is very irregular and shows a lot of
Fig. 5. Electron micrographs of transverse sections of the primitive streak region.
Scale bar is 5 /*m. (A) The centre of the primitive streak. The dorsal surface membrane is folded. The lateral membranes show interdigitations with plasma-membrane-associated dense accumulations and microfilament bundles extending from
one junctional complex to the other (arrowheads) within the cell. (B) Outside the
centre of the primitive streak. Presumptive columnar cells with apical plasmamembrane-associated dense accumulations and microfilament bundles restricted to
a 0-5 fim region from the junction (arrowheads). (C) Outside the centre of the
primitive streak. Presumptive columnar cell with microfilament bundles extending
from one junctional complex to the other (arrowheads). Note that the lateral plasma
membrane is sectioned tangentially (arrows).
66
L. VAKAET AND CH. VANROELEN
Fig. 7. Electron micrograph of a section tangential to the dorsal surface of the
primitive-streak cells. Note the microfilament bundles close to cell borders (arrowheads). Scale bar is 5 fim.
projections, perfectly tangential sections are hard to obtain. This perhaps
explains why we were not able to demonstrate circumferential rings of microfilament bundles in the cell apex, although their presence is inferred from the
observations obtained from our transverse sections. Mitochondria, small
vacuoles (0-1 fim), and microtubules are observed in the apical cytoplasm.
Lateral to the primitive streak
The region studied is represented in Fig. 3. In this region, an epithelium with
cells situated above one another is observed beside an epithelium with columnar
cells covering the entophyllic crescent. The presence of cells situated above one
another in the section does not exclude the possibility that these cells, as well,
are extending from the dorsal to the ventral surface of the upper layer. Indeed,
it may be the result of an even small angle between the columnar cell axis and
the knife during sectioning.
All the cells have apical microfilament bundles in close contact with the
junctional complexes (Fig. 6B). When the upper layer cells are narrower at their
apical surface, the microfilament bundles extend from one junctional complex
to the other. Transverse sections of microfilament bundles could not be detected.
However, some differences to the results from the primitive streak appear:
(a) microfilament bundles are less obvious, (b) the junctional complex, if compared to the lateral plasma membranes, is not so highly contrasted, and (c) the
surface projections seem to be different from these observed in the groove. The
latter is confirmed by previous SEM observations (Bancroft & Bellairs, 1974;
Vakaet, unpublished).
Microfilament bundles in upper layer of the chick blastoderm
67
The neural plate
The region selected for ultrastructural observation is represented in Fig. 4.
The cells in this layer are elongated, extending fiom the dorsal to the ventral
surface, or they appear above one another. Rounded cells in mitosis can be
recognized near the apical surface.
In this region, microfilament bundles are also ultrastructurally observed in
close contact with the junctional complex (Fig. 6C). Cells, apparently narrower
near the apical surface, show microfilament bundles extending from one
junctional complex to the other.
DISCUSSION
Do all epithelial cells lining grooves during morphogenesis possess microfilament bundles at their apical ends? Microfilament bundles have been observed during neurulation in the chick (Karfunkel, 1972; Nagele & Lee, 1980)
and in Amphibia (Baker & Schroeder, 1967; Bumside, 1971; Lofberg, 1974),
just under the apical surface of the folding neural plate cells. Bancroft &
Bellairs (1977) showed the presence of microfilament bundles in cells which will
form the otic, nasal and lens placodes, but could not detect them in cells destined
to form the head epidermis. Balsinky & Walther (1961) suspected the presence
of microfilament bundles in the centre of the primitive-streak-stage chick
blastoderm, and described them as 'strips of denser cytoplasm arranged transversely to the long axis of the cell'. Our study demonstrates that microfilament
bundles are present just under the apical surface of the cells in the centre of the
primitive streak, where they can be observed as dense bands of microfilaments
in TEM sections. Cells in the centre of the primitive streak are very narrow at
their dorsal surface. They are wedge- or flask-shaped, as was shown by SEM
studies of this array (Wakely & England, 1977; Solursh & Revel, 1978; Vakaet,
unpublished). Moreover, the nucleus of these cells frequently was observed in
the same plane of the section as the narrow apical end. The microfilament
bundles seem to form a continuous meshwork beneath the apical surface of
these cells.
Is the presence of microfilament bundles in epithelia during morphogenesis
restricted to cells lining grooves? From the literature (see above), it appeared
that the presence of dense bands of microfilaments is correlated with the
presence of grooves. Outside these grooves microfilament bundles were not
detected. In our study, microfilament bundles can be detected in cells outside the
centre of the groove, in the lateral region and the neural plate region. Outside
the centre of the groove, presumptive columnar cells are present beside presumptive wedge- or flask-shaped cells. In the latter cells, microfilament bundles
can be seen as dense bands (probably a continuous meshwork). The organization of microfilament bundles in a circumferential ring was widely accepted, and
recently doubted by Nagele & Lee (1980) who observed discrete microfilament
68
L. VAKAET AND CH. VANROELEN
bundles. Our experiments, however, do not allow us to reject or accept the
hypothesis of circumferential organization of the microfilament bundles. Indeed,
on the one hand, the presence of a circumferential ring of microfilament bundles
in the cell apex is probable because they were always observed near the junctional complex. Moreover, transverse sections of microfilament bundles at some
distance from the junctional complex could never be detected. Finally, the
tangential section of the dorsal surface showed half a ring. On the other hand,
microfilament bundles extending from one junctional complex to the other
within the cell, could suggest the presence of discrete bundles in the cell apex.
However, in those cases, the possibility that the section was tangential to the
lateral plasma membrane cannot be eliminated. Although present over the
whole area pellucida, the electron density of the microfilament bundles compared to that of the cytoplasm is higher in the primitive-streak region than
outside this region.
Several observations suggest that microfilament bundles are contractile. We
observed that microfilament bundles were always more obvious in cells which
were narrower at their apical surface and therefore probably constricted at their
apical end. Baker & Schroeder (1967) remarked that the apical cell membrane
is always folded, while the microfilament bundles are not. More conclusive
evidence was provided by Karfunkel (1972), who used cytochalasin B, a microfilament-modifying agent, in the chick during neurulation. The absence of
microfilament bundles and flattening of the neural folds after treatment with
this product was observed. Recently, Nagele & Lee (1980) demonstrated that,
during neurulation in the chick, actin is a component of the microfilament
bundles.
Summarizing, the presence of microfilament bundles in epithelia, during
embryogenesis, is certainly not restricted to cells lining grooves. As it appears
from our study, at least the cells beside the groove, contain microfilament
bundles in the cell apex. We think that they form an interconnected meshwork
through the junctional complexes over the complete area pellucida. They appear
as a continuous meshwork in the cell apex of presumptive wedge- and flaskshaped cells. The architecture of microfilament bundles in the apices of columnar
cells remains uncertain. Because they are ubiquitous in the area pellucida of the
primitive-streak-stage chick blastoderm, they cannot be regarded as a marker
of differentiation in this tissue. If we agree with the view that microfilament
bundles are contractile, they may be regarded as cellular organelles with a
function during every morphogenetic event in the upper layer. These not only
include the obvious phenomena, such as the formation and shortening of the
streak, and the folding of the neural groove, but also the movements in the
plane of the upper layer. Therefore, microfilament bundles may have a role in
the further determination and differentiation of the different anlage fields of the
upper layer.
Microfilament bundles in upper layer of the chick blastoderm 69
The authors would like to thank Mr Ch. De Schepper for technical assistance, Mr F. De
Bruyn for artwork, Mr J. Van Ermengem for photographic processing and Mrs N. Van den
hende-Bol for typing our manuscript.
This work was supported by grant n° 3.9001.81 of the Belgian Fonds voor Geneeskundig
Wetenschappelijk Onderzoek.
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{Received 20 February 1981, revised 20 July 1981)