/ . Embryo!, exp. Morph. Vol. 63, pp. 67-74, 1981
Printed in Great Britain © Company of Biologists Limited 1981
An ultrastructural study of the maternal-effect
embryos of the ac/ac mutant of Pleurodeles
waltl showing a gastrulation defect
By JOHN G. BLUEMINK AND JEAN-CLAUDE BEETSCHEN
From the Hubrecht Laboratory, International Embryological Institute, Utrecht,
and the Laboratoire de Biologie generate, Universite Paul Sabatier, Toulouse
SUMMARY
Embryos of the ac/ac maternal-effect mutant in Pleurodeles waltl show disturbed epibolic
movement during gastrulation. At the early gastrula stage, ectoderm cells begin to sink in at
random sites in the animal half of the embryo. At the advanced gastrula stage the ectodermal
pits develop into grooves. Electron microscopical analysis shows that many cells in the bottom
of the pits and grooves have narrowed apices and bear many microvilli, while the cortical
cytoplasm is dense, filamentous and underlain by a stratum of vesicles. Thesefindingsare
interpreted as indicating that ectoderm cells contract rather than expand leading to disturbed
epibolic movement.
INTRODUCTION
Abnormal gastrulation in the ac/ac ('ascite caudale') maternal-effect mutant
of Pleurodeles waltl has been described by Beetschen(1970, 1976), Beetschen &
Fernandez (1979), and Fernandez (1979). After normal cleavage, all the progeny
shows the same syndrome at the beginning of gastrulation. The ectoderm of the
animal half of the embryo becomes pitted, the depth and frequency of the pits
varying within any one batch. As gastrulation proceeds the ectodermal pits
develop into grooves which finally become continuous, giving the animal hemisphere a brain-like appearance. The epibolic movement of the ectoderm is disturbed whereas blastoporal invagination begins normally but remains incomplete. The blastopore finally develops into a deep circumferential indentation
leaving an oversized yolk plug outside.
Many embryos exogastrulate, but a more or less complete axis system still
may develop depending on the extent of gastrulation. The intense ectodermal
furrowing does not correlate with an increase in cell proliferation; on the contrary, the ac maternal effect induces a decrease in cell number in the ectoderm
(Beetschen & Fernandez, 1979). In this study deficient gastrulation in the ac
1
Author's address: Hubrecht Laboratory, International Embryological Institute,
Uppsalalaan
8, 3584 CT Utrecht, The Netherlands.
8
Author's address: Laboratoire de Biologie generate, Universite Paul Sabatier, 31077
Toulouse, France.
68
J. G. BLUEMINK AND J.-C. BEETSCHEN
7\
(b)
(a)
(c)
Fig. 1. Camera-lucida drawings (LM) of sections, 1 fim thick, (a) Meridional crosssection of a young gastrula. (scale bar 0-5 mm). (6) Arrangement of cells in the
ectoderm during pit formation: bulging cells, cells with narrowed apices (arrows)
(scale bar 01 mm), (c) Arrangement of cells forming a pit: bottle-shaped bottom
cells (asterisks) (scale bar 01 mm).
mutant was investigated to find out whether or not active contraction is involved
in the aberrant shape changes of the ectoderm. For a description of normal
development of Pleurodeles waltl see Gallien & Durocher (1957).
MATERIAL AND METHODS
Eggs of homozygous ac/ac females were obtained at the Laboratoire de
Biologie generate, Universite Paul Sabatier, Toulouse. After spawning eggs
were kept under aquarium conditions at a temperature of 18 °C. Late blastulae
Fig. 2. Scanning electron micrographs of the animal half, (a) Mid-blastula stage:
ectoderm cells. The cell in the centre has a narrow apical surface with many microvilli (compare with Fig. 4a + 4b), neighbouring cells are relatively smooth (scale
bar 10 fim). (b) Early gastrula stage: pit in the ectoderm showing smooth cells along
the wall, villated cells at the bottom (compare with Fig. 4 c) (scale bar 10 fim). (c)
Higher magnification of b, the bottom cells have a narrow apical surface with
many microvilli (scale bar 10 fim). (d) Early gastrula stage: the alignment of ectoderm
cells near the pit looks less tight (scale bar 40 fim). (e) Advanced gastrula stage:
pits have developed into grooves (scale bar 100 fim). (J) As in e, lower magnification: view of the distorted animal half lying on top of the oversized yolk plug
(arrow) (scale bar 200 fim).
Gastrulation defects in maternal-effect mutant in Pleurodeles
69
70
(a)
J. G. BLUEMINK AND J.-C. BEETSCHEN
(b)
Fig. 3. Camera-lucida drawings (LM) of 01 /xm-thick sections, (a) Meridional
cross-section of a late gastrula, showing extensively folded ectoderm, collapsed
blastocoel (arrow), oversized yolk plug (Y) (scale bar 0-5 mm), (b) Arrangement
of cells in the folded ectoderm: bottle-shaped cells (asterisks) (scale bar 01 mm).
to late gastrulae of three different batches were used. Eggs were dejellied with
fine forceps and prepared for light microscopy (LM) and transmission electron
microscopy (TEM) according to Bluemink's (1972) method B, using s-collidinebuffered fixatives. For scanning electron microscopy (SEM) fixed eggs were
critical-point dried and handled as described by Kelley, Dekker & Bluemink
(1973).
RESULTS
At mid-blastula/early-gastrula stage the arrangement of the ectoderm cells
becomes less regular, cells start to bulge out and their apical surfaces are no
longer smoothly aligned (Fig. 1). Most ectoderm cells are elongated and some
have a narrowed apical surface (Fig. 1 b) with many microvilli (Fig. 2a). Such
cells are often embedded somewhat more deeply in the ectodermal layer. In the
pits the cells along the wall are smooth (Fig. 2 b) whereas the bottom cells, which
have a smaller apical surface, bear many microvilli (Fig. 2 c). The alignment of
the ectoderm cells, particularly near the pits, looks less tight (Fig. 2d). At more
advanced gastrula stages the pits develop into grooves (Fig. 2e). The cells near
the groove have a smooth surface which is often stretched towards the groove.
At late gastrula stages the whole animal half often acquires a brain-like
appearance (Fig. 2 / ) as a result of the network of deep ectodermal furrows. In
the vegetal half the blastopore has extended around the egg circumference.
Invagination remains incomplete and an oversized yolk plug remains outside
(Fig. 2 / , top). Semi-thin sections have shown that the ectoderm does not thin
out during gastrulation. The animal cap as a whole is thrown into folds and
shrinks when the blastocoel collapses at an advanced gastrula stage (Fig. 3 a). As
described above, superficial ectoderm cells are elongated during pit formation.
Those forming the ridges between the grooves remain elongated whereas bottom
Gastrulation defects in maternal-effect mutant in Pleurodeles
71
Fig. 4. Transmission electron micrograph of sectioned material. Early gastrula;
ectoderm cells. Cross section of a cell having a narrow apical surface with many
microvilli. Neighbouring cells have a smooth surface (compare with Fig. 2 a)
(scale bar 1 /zm).
cells in the grooves become bottle-shaped and have a very narrow apex (Fig.
3 b). Cells forming the inner wall of the furrow often are of intermediate shape
but have a smooth apical surface with a few microvilli.
In ultra-thin sections of early gastrulae, scattered superficial cells in the
ectoderm exhibit a narrow apical surface with many microvilli (Fig. 4). The
cortical cytoplasm is dense and filamentous and is underlain by a stratum of
vesicles. These cells show the characteristic features of contracted cells. Cells in
the bottom of the grooves show the same features (Fig. 5). The narrow apical
surface that bulges out into the lumen bears many microvilli. The layer of cortical cytoplasm is dense and numerous vesicles are present. Adjacent cells forming
the wall of the groove look different in that they have no narrow apical surface,
few microvilli, and no stratum of filaments with vesicles associated with it.
However, at sites where cells contact each other lumps of filamentous material
are frequently observed.
DISCUSSION
Ectodermal cellular morphology and cellular rearrangements during amphibian gastrulation have been described by Keller (1978), Keller & Schoenwolf
72
J. G. BLUEMINK AND J.-C. BEETSCHEN
Fig. 5. As in Fig. 4., advanced gastrula: ectoderm cells in a groove. The bottom
cells bear microvilli, the dense cytoplasm is underlain by a stratum of vesicles. The
surfaces of the cells along the wall of the groove are relatively smooth (compare
with Fig. 2 c). (Scale bar 1 /urn.)
(1977) and Nakatzuji (1975a, b). In normal gastrulation ectoderm cells stretch
and flatten during epiboly, thus compensating for the inward migration of the
meso- and endoderm, so that the ectoderm cells finally constitute the whole
outer embryonic surface. In the ac/ac mutant the ectodermal cell shape changes
are opposite to what is normally expected. The cells elongate perpendicular to
the embryonic surface instead of stretching parallel to it. In contrast to the
ectoderm, the endo- and mesoderm cells begin to migrate as normal. The inward
movement progresses as far as the collapsed blastocoel will allow and without
an adequate epibolic movement invagination stops halfway. It is widely accepted
Gastrulation defects in maternal-effect in mutant Pleurodeles 73
that during epibolic movement ectodermal cell spreading is a major driving
force, but no such spreading takes place in the mutant embryos (Beetschen,
1976; Beetschen & Fernandez 1979). The blastocoelic roof remains as thick as
in younger controls. Careful analysis of mitotic activity by Beetschen & Fernandez (1979) has provided evidence that the irregular ectodermal furrowing is
not correlated with an increase in cell number; on the contrary, cell number in
the mutant embryo was found to be lower than in normal controls of the same
age.
The morphological characteristics of amphibian cells during normal contraction are a narrow apical cell surface bearing many microvilli in association
with dense, filamentous cortical cytoplasm underlain by a stratum of vesicles
(Bluemink, 1970,1972,1978; Perry, 1975; Perry, John &Thomas, 1970; Selman
& Perry, 1970). In the ac/ac embryos superficial ectoderm cells (late blastula,
early gastrula) and bottle-shaped cells in pits and grooves (advanced gastrula)
showing these characteristics can therefore be taken to undergo contraction
(see also Wessels et ah 1971). Our conclusion is that active contraction of cells
at randomly distributed sites in the ectodermal half rather than overall expansion
possibly causes the syndrome.
Since it has been found that cortical injury to the uncleaved egg partially cures
the mutant effect (Fernandez, 1979), Beetschen has suggested that an anomaly in
the regulation of cell surface permeability might be involved (Beetschen &
Fernandez, 1979). Experimental evidence exists that a change in the ion permeability of the plasma membrane causes local surface contraction in amphibian
eggs (Gingell, 1970). Whether contraction of the ectoderm cells in the embryos
of the mutant can be explained similarly needs to be investigated. To this end
the ion permeability characteristics of the embryonic surface should be analysed
using intracellular microelectrodes or the so-called 'vibrating probe' (see
Jaffe & Nuccitelli, 1974). The final question concerning the nature and the action
of the maternal factor(s) causing the syndrome, is yet too remote to be answered
more directly.
We thank Mrs Carmen Kroon-Lobo for preparing the line drawings and figures artd Mr
Pirn van Maurik for assistance in electron microscopy.
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{Received 8 October 1980, revised 12 December 1980)