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. These findings are interpreted as indicating that ectoderm cells contract rather than expand leading to disturbed epibolic movement.

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 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).

Eggs of homozygous ac/ac females were obtained at the Laboratoire de Biologie générale, Université Paul Sabatier, Toulouse. After spawning eggs were kept under aquarium conditions at a temperature of 18°C. Late blastulae 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-collidine-buffered fixatives. For scanning electron microscopy (SEM) fixed eggs were critical-point dried and handled as described by Kelley, Dekker & Bluemink (1973).

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.

Fig. 1

Camera-lucida drawings (LM) of sections, 1 μm thick, (a) Meridional cross-section of a young gastrula. (scale bar 0·5 mm), (b) 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 0·1 mm).

Fig. 1

Camera-lucida drawings (LM) of sections, 1 μm thick, (a) Meridional cross-section of a young gastrula. (scale bar 0·5 mm), (b) 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 0·1 mm).

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+ 46), neighbouring cells are relatively smooth (scale bar 10 μ m). (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 μ m), (c) Higher magnification of b, the bottom cells have a narrow apical surface with many microvilli (scale bar 10 μ m), (d) Early gastrula stage: the alignment of ectoderm cells near the pit looks less tight (scale bar 40 μ m), (e) Advanced gastrula stage: pits have developed into grooves (scale bar 100 μ m). (J) As in e, lower magnification: view of the distorted animal half lying on top of the oversized yolk plug (arrow) (scale bar 200 μ m).

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+ 46), neighbouring cells are relatively smooth (scale bar 10 μ m). (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 μ m), (c) Higher magnification of b, the bottom cells have a narrow apical surface with many microvilli (scale bar 10 μ m), (d) Early gastrula stage: the alignment of ectoderm cells near the pit looks less tight (scale bar 40 μ m), (e) Advanced gastrula stage: pits have developed into grooves (scale bar 100 μ m). (J) As in e, lower magnification: view of the distorted animal half lying on top of the oversized yolk plug (arrow) (scale bar 200 μ m).

At late gastrula stages the whole animal half often acquires a brain-like appearance (Fig. 2f) 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 f, 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 cells in the grooves become bottle-shaped and have a very narrow apex (Fig. 3b). Cells forming the inner wall of the furrow often are of intermediate shape but have a smooth apical surface with a few microvilli.

Fig. 3

Camera-lucida drawings (LM) of 0·1 μm-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 0·1 mm).

Fig. 3

Camera-lucida drawings (LM) of 0·1 μm-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 0·1 mm).

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.

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. 2a) (scale bar 1 μ m).

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. 2a) (scale bar 1 μ m).

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 μ m.)

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 μ m.)

Ectodermal cellular morphology and cellular rearrangements during amphibian gastrulation have been described by Keller (1978), Keller & Schoenwolf (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 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 al. 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 and Mr Pirn van Maurik for assistance in electron microscopy.

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