J. Embryol. exp. Morph. Vol. 70, pp. 189-196, 1982
Printed in Great Britain © Company of Biologists Limited 1982
\ 89
The effects of partial hypoblast removal
on the cell morphology of the epiblast in the
chick blastoderm
By CH. VANROELEN 1 , P. VERPLANKEN AND
L. C. A. VAKAET 1
From the Department of Anatomy and Embryology,
State University Centre, Antwerp, Belgium
SUMMARY
The hypoblast of early-primitive-streak-stage chick blastoderms was partially removed.
This experiment provokes a reaction in the epiblast which curls up and becomes even at its
ventral surface. The basal lamina underlying the epiblast is also dependent upon the presence
of hypoblast. During culture after partial hypoblast removal, active hypoblast wound
healing is observed. Where the hypoblast underlies the epiblast again, the effects of the
removal disappear and normal development proceeds. The results suggest that the normal
epiblast morphology is dependent upon the presence of hypoblast. This influence of hypoblast on epiblast is thought to be concerned with the morphology of the epiblast and not
directly with its morphogenesis.
INTRODUCTION
Several authors have studied the inductive action of the early hypoblast on
the epiblast in the chick blastoderm (Waddington, 1932, 1933; Spratt & Haas,
1960a, b; Eyal-Giladi & Wolk, 1970; Azar & Eyal-Giladi, 1979, 1981). These
experiments were concerned with the study of the role of the hypoblast in the
morphogenesis of the epiblast.
In the present investigation, we will not deal with these morphogenetic
influences, but with the immediate dependency of the normal epiblast morphology on the presence of hypoblast. To this end, the hypoblast was partially
removed and the effects of the intervention on the epiblast were studied by
light and electron microscopy. We compared an area of epiblast deprived of
hypoblast (1) with a symmetric area in the same embryo, and (2) with the
homologous areas of blastoderms of the same stage. These precautions were
thought to be necessary since cell and tissue architecture are extremely stageand area-dependent. The mechanism of wound healing of the hypoblast will
not be considered here (see Mareel & Vakaet, 1977). The mechanism by which
the cell morphology of the epiblast is influenced by the presence of hypoblast, is
currently under investigation.
1
Authors address: Rijksuniversitair Centrum Antwerpen, Groenenborgerlaan 171, B-2020
Antwerpen, Belgium.
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EMB 70
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CH. VANROELEN AND OTHERS
Control area
Experimental area
Epiblast
Mesoblast
Hypoblast
Fig. 1. Outline of a blastoderm after the hypoblast was partially removed.
MATERIALS AND METHODS
Fertilized eggs from a commercial stock were incubated for 12 h at 38 °C
to yield blastoderms of stage 3 (Hamburger & Hamilton, 1951, or Vakaet, 1970).
All the blastoderms were explanted according to the technique of New (1955).
The hypoblast was partially removed as shown in Fig. 1. The blastoderms were
either (1) immediately fixed, or (2) further cultured on egg white at 38 °C for
20 min, 100 min, 3 to 5 h, and then fixed. As a control, some blastoderms were
fixed without experimental manipulation. A total of 95 blastoderms was
studied.
Most blastoderms were fixed 'on ring' (New, 1955). Some others were fixed
after detachment from the vitelline membrane. Fixation and tissue preparation
were carried out as described earlier (Vanroelen & Vakaet, 1981). As a fixative,
a solution containing 1% (w/v) tannic acid (mol.wt 1701) and 1% (w/v)
glutaraldehyde in 0 1 M cacodylate buffer at pH 7-4 was used. Postfixation
was carried out in a 1 % (w/v) osmium tetroxide solution in the same buffer.
After embedding in Epon 812, 2/*m sections were stained according to the
technique of Kiihn (1970). Ultrathin sections were stained in a 2 % (w/v) aqueous
solution of uranyl acetate for 30 min, and in lead citrate for 30 s (Reynolds,
1963). The ultrathin sections were examined in a JEOL 100B electron microscope.
RESULTS
In the following description, the area deprived of hypoblast will be termed
experimental area, while control area refers to the area symmetric to the experimental area.
Partial removal of hypoblast and subsequent culture initiates active wound
healing (Figs. 2 A and B). Although dependent upon its dimensions, closure
Hypoblast removal in the chick blastoderm
A
*
4A
191
A
T
4B
Fig. 2. In toto photograph of (A) a blastoderm immediately after the hypoblast was
partially removed, and of (B) the same blastoderm after 100 min of culture. Note
the closure of the wound after culture (arrows). Scale bar is 1 mm.
Fig. 3. Besides the small undulations in control area (arrowheads), curling up of the
epiblast in the experimental area can always be observed (arrows). Scale bar is
500 /im.
Fig. 4. Transverse section of the epiblast of a blastoderm fixed immediately after
partial hypoblast removal (A) in the experimental area, and (B) in the control area
of the same germ. Note the even ventral surface in the experimental area (arrows)
and the uneven ventral surface in the control area (arrowheads). Scale bar is 50 fim.
7-2
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CH. VANROELEN AND OTHERS
Fig. 5. Transverse section of a blastoderm after partial hypoblast removal and
100 min of culture. The dark line on the ventral surface of the epiblast is absent in
the experimental area (arrows) and this line is present in the healed area (arrowheads
Scale bar is 50 (im.
of the wound is, in our experimental conditions, complete approximately
3-4 h after the intervention. Further normal development of the blastoderm
then occurs.
Comparison of the cell morphology of the epiblast between the control area
and the homologous areas (left and right) of blastoderms of the same stage and
without any prior intervention does not reveal differences in morphology.
Light microscope observations
Immediately after hypoblast removal, i.e. within the time necessary for
fixation, the epiblast in the experimental area tends to curl up (Fig. 3). This
curling up, though not very pronounced if compared to the control area, is very
constant. If the margin of overgrowth partially loses contact with the vitelline
membrane during the intervention, the curling up is more pronounced. Because
random folds in the epiblast can then be observed, and because the hypoblast
Fig. 6-8. Electron micrographs of transverse sections of the ventral surface of the
epiblast of blastoderms fixed immediately after partial hypoblast removal. The
basal lamina remains present during the intervention. Scale bar is 1 /*m.
Fig. 6. The experimental area. A meshwork of microfilaments is present at the inner
ventral surface of the epiblast cells (arrows).
Fig. 7. The experimental area. The ventral plasma membrane of some epiblast cells
is irregular.
Fig. 8. The control area.
Hypoblast removal in the chick blastoderm
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CH. VANROELEN AND OTHERS
wounds in these conditions will not heal normally, only results obtained from
blastoderms firmly attached to the vitelline membrane and fixed 'on ring' will
further be shown. Whereas in the experimental area the ventral surface is
even (Fig. 4A), in the control area the ventral surface is uneven (Fig. 4B).
After staining according to the technique of Kiihn (1970), a purple line is
observed in the control area on the ventral surface of the epiblast, where
previous studies have shown the presence of a basal lamina (Low, 1967;
Sanders, 1979; Vakaet, Vanroelen & Andries, 1980).
This positivity is still observed in the experimental area of blastoderms fixed
immediately after the intervention (Fig. 4 A), but is less obvious after 100 min
of culture (Fig. 5). During culture, the hypoblast wound closes, and epiblast
that was deprived of hypoblast, gradually becomes underlain with hypoblast
again. In these areas where hypoblast underlies the epiblast again, the morphology of the epiblast cannot be distinguished from the morphology of the
epiblast in the control area (Fig. 5). Indeed, curling up of the epiblast and
evenness of its ventral surface can no longer be observed. The purple line at the
site of the basal lamina in the healed area is again present.
Electron microscope observations
After partial hypoblast removal and within the time necessary for fixation,
a meshwork of microfilaments becomes visible close to the ventral inner surface
of most epiblast cells in the experimental area (Fig. 6). Moreover, the ventral
surface of some cells is irregular to a degree never observed in the control area
(Fig. 7). In the latter area, the meshwork of microfilaments cannot be seen
(Fig. 8). Sometimes sparse microfilament bundles can be observed at the basis
of blebs or other cell extensions.
After culture, some disorganization at the level of the basal lamina (as
suggested from the light microscope study) can be observed in the experimental
area, but these changes are less obvious than in the light micrographs.
DISCUSSION
When the effects of the removal of hypoblast on the cell shape of the epiblast
are studied, comparison with a reliable control area is necessary. It is a minimal
prerequisite that the experimental and the control area belong to the same germ,
because changes in cell shape occur during development. Comparison of the
effects of the intervention between the experimental and the symmetric control
area is, however, only valid if we assume (1) that during this period of development, the blastoderm is completely symmetric, and (2) that the removal of
hypoblast on one side of the blastoderm does not alter the control area.
Comparison of our control area with the two (left and right) homologous
Hypoblast removal in the chick blastoderm
195
areas of blastoderms fixed without experimental manipulation does not show
differences.
Our experiments revealed an influence of hypoblast removal on the overlying
epiblast both at the light and electron microscope level. Curling up of the epiblast
was also observed by Kohonen & Paranko (1980) after excision of the ectoderm
of the gastrula of the newt. These observations may reflect the presence of
differences in tension in the epiblast when the experimental area and the control
area are compared. This idea is corroborated by the observation that the curling
up is more obvious in blastoderms fixed after removal from the vitelline membrane, than in blastoderms fixed 'on ring' (New, 1955). Indeed, in blastoderms
fixed after detachment from the vitelline membrane, the radial tension exerted
by the margin of overgrowth is lost. In this respect, the visualization of a
mesh work of microfilaments (see also Spooner & Wessels, 1970) may be
correlated with the curling up and with the evenness of the ventral surface, if a
contractile activity of microfilaments may be assumed. The reversibility of
the effects at the healed edge of the wound and the normal further development
after closure suggests that the normal cellular morphology depends upon the
presence of hypoblast.
The effects observed in the basal lamina remain unclear. It is without doubt
that the basal lamina also undergoes changes after hypoblast removal. These
changes may reflect that a role is played by the basal lamina as an intermediate,
or that the basal lamina changes are an accompanying event beside the described
cell morphological changes. The observation that the basal lamina changes
were more obvious in the light microscope after staining according to Kiihn
(1970), than in the electron microscope after routine staining, suggests that
different products were visualized. In this respect, the hypoblast removal
showed the disorganization of these basal lamina products stained with the
technique of Kiihn.
Concluding, besides the morphogenetic influences of the hypoblast on the
young epiblast (see Introduction), which are assumed to be irreversible in their
effect, the present study has demonstrated the existence of hypoblast influences
on epiblast cell morphology. These were shown to be reversible and are suggested
to differ from the morphogenetic influences.
The authors would like to thank Mrs G. Haest-Van Neuten and Mr Ch. De Schepper for
technical assistance, Mr J. Van Ermengem for photographic processing of the micrographs,
Mr F. De Bruyn for artwork and Mrs N. Van den hende-Bol for typing the manuscript.
This work was supported by grant no. 3.9001.81 of the Belgian Fonds voor Geneeskundig
Wetenschappelijk Onderzoek.
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{Received 16 November 1981, Revised 24 February 1982)