/ . Embryol. exp. Morph. Vol. 34, 3, pp. 669-685, 1975
Printed in Great Britain
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Studies on the gastrulation of amphibian embryos:
Light and electron microscopic observation of
a urodele Cynops pyrrhogaster
By NORIO NAKATSUJ1 1
From the Department of Zoology, University of Kyoto
SUMMARY
The course of gastrulation in embryos of a urodele, Cynops pyrrhogaster, was studied with
1 /*m Epon sections and transmission and scanning electron microscopy. During the initial
period of gastrulation, the bottle cells and a groove are formed in the dorsal part. The outer
ends of the bottle cells have many microvilli, an electron-dense layer and many small vesicles.
Microtubules are present parallel to the long axis of the bottle cells, and a yolk-platelet-free
region is observed at the inner end. Thereafter, the archenteric roof makes contact with the
inner surface of the blastocoelic wall. Cells of the archenteric roof form lobopodia, filopodia
and lamellipodia. These cells make many focal contacts, having gaps of less than 20 nm,
with the blastocoelic wall. Invaginating mesodermal cells of the lateral and ventral parts
also form pseudopodia, and are in contact with the blastocoelic wall. Some of these cells
appear to flatten against the wall. These observations suggest that, after the bottle cells and
the blastoporal groove are formed, the invaginating cells actively migrate along the inner
surface of the blastocoelic wall, and that these locomotive forces have an important role in
the morphogenetic movements during gastrulation.
INTRODUCTION
The 'sinking in' activity of the bottle cells (Holtfreter, 1944; Baker, 1965) and
the epibolic expansion of the ectodermal layer (Holtfreter, 1944) have been
proposed as the forces that drive the morphogenetic movements during gastrulation in amphibian embryos. Besides these two forces, the present author has
proposed active cell locomotion along the inner surface of the blastocoelic wall
in anuran embryos, from the observation that cells of the foregut endoderm and
mesoderm form pseudopodia coinciding with their movement toward the
animal pole along the blastocoelic wall (Nakatsuji, 1974, 1975). Because the
process of gastrulation in urodele embryos has many differences from that in
anuran embryos, it is necessary to investigate whether the cells form pseudopodia and whether active cell locomotion is suggested also in urodele gastrulation.
Observations of urodele gastrulation reported hitherto are either those with
1
Author's address: Department of Zoology, Faculty of Science, University of Kyoto,
Sakyo-ku, Kyoto 606, Japan.
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NORIO NAKATSUJI
paraffin sections (Vogt, 1929) or those with electron microscopy but restricted
to a small part such as the bottle cells (Perry & Waddington, 1966). In this report,
the course of gastrulation of a urodele, Cynops pyrrhogaster, is observed by use
of semi-thin (about 1 fim) Epon sections and with transmission and scanning
electron microscopy. It is shown that the invaginating cells form pseudopodia
and it is suggested that active cell locomotion along the inner surface of the
blastocoelic wall probably plays an important role in urodele gastrulation.
MATERIAL AND METHODS
Embryos
Fertilized eggs of Cynops pyrrhogaster (formerly named Triturus pyrrhogaster)
were obtained from natural ovipositions by females collected at Kojoro Pond of
Mt. Hira in Shiga Prefecture. The stages of the embryos were determined
according to Okada & Ichikawa (1947). Embryos of stage 9-16 (early blastula
to initial neurula) were divested of the jelly layer and chorion manually.
Semi-thin and ultrathin sections
Five or six embiyos at each stage (total of 58 embryos) were fixed for half a
day at 3-5 °C in a buffered (0-05 M phosphate buffer, pH 7-2) 2-5 % glutaraldehyde solution containing paraformaldehyde (2 %) and picric acid (0-1 %) (Ito &
Karnovsky, 1968). Fixed blastulae were cut into halves with two razor blades,
in the plane passing the animal and vegetal poles, using a binocular microscope.
Fixed gastrulae and neurulae were cut in the mid-sagittal or equatorial plane.
These halves were fixed in the same fresh fixative solution additionally for a day,
rinsed in the same buffer that contained sucrose (4%), and were post-fixed for
2 h at 2 °C in a buffered (pH 7-2-7-4) 1 % osmium tetroxide solution that
contained sucrose (2-25 %). The samples were dehydrated in a graded series of
FIGURE 1
Micrographs of the initial period of gastrulation.
(A) A section of a blastula.
(B) A light micrograph of the marginal zone of a blastula. A lobopodium is formed.
(C) An electron micrograph of the lobopodium shown in (B).
(D) A mid-sagittal section of an embryo at stage 12a.
(E) A light micrograph of the dorsal lip region at stage 12a. Yolk-platelet-free
regions are present at the inner ends of the bottle cells (arrows).
(F) An electron micrograph of a yolk-free region shown in (E).
(G) An electron micrograph of the outer end of the bottle cells.
(H) rAn electron micrograph of a portion of two bottle cells.
Microscopic observation of urodele gastrulation
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ethanol and transferred through propylene-oxide, propylene-oxide Epon mixtures and into Epon. Finally the samples were embedded into Epon moulds.
One /*m sections were mounted on glass slides that had been previously
coated with 0-5% collodion (Aoki & Gutierrez, 1967) and were stained with
1 % toluidine blue (in 0-1 M phosphate buffer, pH 7-4) for 30 min at 90 °C or
with 0-5 % methylene blue and 0-5 % azur n (in 0-5 % sodium borate) (Richardson, Jarett & Finke, 1960) for 10 min at 90 °C. They were examined with a
bright field microscope. After the 1 /*m sections were cut and examined, blocks
were trimmed for the desired region, and ultrathin sections were cut. The
ultrathin sections were mounted on 100-mesh Formvar-film-coated copper
grids, double stained with 1% uranyl acetate (in 10% methanol) and lead
citrate (Reynolds, 1963), and examined with a Hitachi electron microscope
(Model H U - I I D - 1 ) operated at 75 KV.
Scanning electron microscopy
Embryos for scanning electron microscopy were fixed for a day in the same
pre-fixation solution described above. They were rinsed in the phosphate buffer
(0-05 M, pH 7-2) that contained sucrose (4%), and were dehydrated in a graded
series of ethanol. Samples were transferred to iso-amyl acetate and allowed to
dry in dust-free air. The dried embryos were fractured carefully with fine forceps
using a binocular microscope. Pieces consisting of the blastocoelic wall and the
invaginating cells attached to it were picked up and placed so that the invaginating cells were uppermost. Some of the invaginating cells were removed with
a hair loop until the optimal number remained on the inner surface of the
blastocoelic wall. In some embryos, fracturing was done before the dehydration.
In this case, pieces of the embryos were dehydrated and dried as described above.
Dried pieces for scanning electron microscopy were mounted on specimen
holders using an electrically conductive paint, Dotite paint D-550 (Fujikura
Kasei Co. Ltd, Tokyo), and coated with carbon and gold (or gold:palladium
= 60:40) in a vacuum evaporator. They were photographed with a JEOL
JSM SI scanning electron microscope.
FIGURE 2
Light micrographs of two embryos at stage 12b.
(A) A mid-sagittal section.
(B) An enlarged micrograph of the area shown by the small rectangle in (A).
(C) An equatorial section.
(D) An enlarged micrograph of the area shown by the rectangle (left) in (C). An
invaginating cell sends a cell process (arrow) toward the blastocoelic wall.
(E) An enlarged micrograph of the rectangle (right) in (C). ar, archenteron.
(F) An enlarged micrograph of the rectangle in (E).
Microscopic observation of urodele gastrulation
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RESULTS
Stage 9-10 (early to late blastuld)
Some blastomeres are observed to form blebs or lobopodia (Fig. 1B). Electron
micrographs show that these lobopodia are poor in yolk platelets and oil droplets (Fig. 1C).
Stage 11-12a (straight to crescent-shape blastopore)
The bottle cells and the blastoporal groove are formed in the dorsal lip
region (Fig. 1D). At the inner ends of the bottle cells, yolk-platelet-free regions
are observed to exist (Fig. IE and F). The outer ends of these cells have many
microvilli, an electron-dense layer and many small vesicles (Fig. 1G). Microtubules are present parallel to the long axis of the bottle cells (Fig. 1H).
Stage Ylb (semi-circular-shape blastopore)
The archenteron is formed in the dorsal part, and its roof appears to be in
contact with the inner surface of the blastocoelic wall (Fig. 2 A, C). The archenteric roof is more than two cells thick at this stage (Fig. 2E). The invaginating
cells are observed to form pseudopodia of various shapes, which are seen along
the inner surface of the blastocoelic wall (Fig. 2B, D, F and Fig. 3). These
pseudopodia are poor in yolk platelets. The invaginating cells make many focal
contacts having gaps of less than 20 nm with the cells of the blastocoelic wall
(Fig. 3 A-E). Focal contacts were observed between the invaginating cells as well.
Stage 12c (horse-shoe-shape blastopore)
The archenteric roof elongates and thins to become almost one-cell thick
(Fig. 4 A, B). Cells of the archenteric roof are observed to form pseudopodia of
various shapes (Fig. 4 C), and make focal contacts having gaps of less than 20 nm
with the blastocoelic wall (Fig. 4D).
FIGURE 3
Electron micrographs of the interface between the archenteric roof and the inner
surface of the blastocoelic wall of the same embryos shown in Fig. 2.
(A) A portion of Fig. 2B. The blastocoelic wall is to the left.
(B) An enlarged micrograph of the area shown by the small rectangle in (A). The
upper cell is part of the blastocoelic wall.
(C) A portion of Fig. 2B. An invaginating cell forms a long cell process (arrow).
The blastocoelic wall is to the left.
(D) An enlarged micrograph of the area shown by the small rectangle in (C).
Granular material is present in the intercellular space. The upper cell is part of the
blastocoelic wall.
(E) A portion of Fig. 2F. A fine cell process is formed by an invaginating cell.
Granular material is present in the intercellular space. Cross and oblique sections of
the cell processes are present (arrows).
Microscopic observation of urodele gastrulation
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Stage 13-14 {large to small yolk plug)
The archenteron becomes large (Fig. 5 A, C). Its roof is one-cell thick (Fig. 5B,
D). In the ventro-lateral part, the invaginating cells form pseudopodia, and
appear to be in contact with the inner surface of the blastocoelic wall (Figs. 5 E,
F and 6A, B). These pseudopodia have the shapes of filopodia and of flattened
pseudopodia or lamellipodia. An amorphous material is present in the intercellular space between the invaginating cells and the blastocoelic wall (Fig. 6C,
D).
Stage 15-16 {final gastrula to initial neurula)
The neural plate becomes conspicuous, and presumptive notochordal cells
become closely packed one on another (Fig. 7A, B, E and F). The intercellular
space between the presumptive notochordal cells and the neural plate cells
becomes much smaller than at earlier stages (Fig. 7C, D). In the dorso-lateral
part, the presumptive somite and lateral plate cells form pseudopodia and
appear to be in contact with the inner surface of the ectodermal layer (Fig. 7 G,
H).
Scanning electron microscopy
The invaginating cells of the archenteric roof were observed with a scanning
electron microscope (Fig. 8A-F). These cells form filopodia and thin lamellipodia,
and are apparently attached to the inner surface of the blastocoelic wall.
DISCUSSION
From the blastula stage, some blastomeres were observed to protrude blebs
or lobopodia toward the blastocoel or intercellular spaces. Lobopodium formation was observed from the initial gastrula stage and was rare at the blastula
stage in the case of anuran embryos (Nakatsuji, 1974,1975). In anuran blastulae,
blastomeres are closely packed and the inner surface of the blastocoelic wall is
relatively smooth. Formation of lobopodia or blebs was observed from the
blastula stage in teleost embryos (Lentz & Trinkaus, 1967; Wourms, 1972a),
in which the deep cells protrude blebs toward the segmentation cavity. In the
case of echinoderm embryos, cells of the blastocoelic wall protrude bulbous
pseudopodia at the blastula stage before the filopodia are formed by the
mesenchyme cells (Gustafson & Wolpert, 1967; Gibbins, Tilney & Porter, 1969).
Electron micrographs showed that the outer ends of the bottle cells have many
microvilli, an electron-dense layer and many small vesicles. Microtubules were
observed to be present parallel to the long axis of the bottle cells. These features
of the bottle cells had been already reported in Triturus (Perry & Waddington,
1966) and in Hyla (Baker, 1965), and probably show the contraction of the
outer end and the elongation of the cell body. Yolk-platelet-free regions were
Microscopic observation of urodele gastrulation
611
D
Fig. 4. Micrographs of an embryo at stage 12c. (A) A mid-sagittal section, ar,
archenteron; 6/,blastocoel. (B) An enlarged light micrograph of thearea shown by the
small rectangle in (A), ar, archenteron. (C) An electron micrograph of the interface between the archenteric roof (lower) and the blastocoelic wall (upper) shown
in (B). (D) An enlarged electron micrograph of the area shown by the rectangle
in (Q.
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observed at the inner ends of the bottle cells. These regions resemble the hyaline
cap of the disaggregated blastomeres of vermiform or cylindrical shape (Holtfreter, 1946, 1948), which perform the peristaltic movement and uni-directional
locomotion in vitro. Formation of the blastoporal groove was shown to occur in
the isolated vegetal hemisphere of the urodele embryos (Stableford, 1948;
Doucet-de Brume, 1973). This suggests that the formation of the bottle cells
and the blastoporal groove are brought about autonomously by the cells in that
region.
Electron micrographs of the lobopodia in the blastula and the yolk-plateletfree region of the bottle cells showed that these regions are poor in large
organelles such as yolk platelets and oil droplets. Their appearances are similar
to the lobopodia in echinoderm late blastulae (Gibbins et al. 1969) and those in
teleost blastulae (Lentz & Trinkaus, 1967; Trinkaus & Lentz 1967).
At stage 12b, the archenteron is formed. Its roof is more than two-cells thick
at this stage and becomes one-cell thick shortly after. Cells of the archenteric
roof and the invaginating mesoderm of the lateral and ventral parts were observed to form pseudopodia and to be in contact with the inner surface of the
blastocoelic wall. These pseudopodia have the shapes of lobopodia, filopodia
and lamellipodia flattened against the blastocoelic wall, and are poor in yolk
platelets and oil droplets. Their morphological appearances have similarity with
the pseudopodia formed by deep cells of teleost embryos during epiboly (Lentz
& Trinkaus, 1967; Trinkaus & Lentz, 1967; Wourms, 19726; Trinkaus, 1973).
The pseudopodia shown in Fig. 6 appear to be flattened against the inner surface of the blastocoelic wall. Their ends are raised up from the wall. Their
appearances resemble the leading lamella and lamellipodium of the fibroblast
in tissue culture (Abercrombie, Heaysman & Pegrum, 1971; Heaysman &
Pegrum, 1973). Cells of the archenteric roof make many focal contacts having
gaps of less than 20 nm with the inner surface of the blastocoelic wall and with
the other invaginating cells. These focal contacts seem to have enough adhesive
FIGURE 5
Light micrographs of two embryos at stage 13.
(A) A mid-sagittal section, ar, archenteron; bl, blastocoel.
(B) An enlarged micrograph of the area shown by the rectangle in (A).
(C) An equatorial section, ar, archenteron.
(D) An enlarged micrograph of the area shown by the upper rectangle in (C).
(E) An enlarged micrograph of the lower rectangle in (C).
(F) An enlarged micrograph of the rectangle in (E). An invaginating mesodermal
cell takes a flattened shape against the inner surface of the blastocoelic wall. A fine
cell process (arrow) is formed by another cell.
Microscopic observation of urodele gastrulation
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B
Fig. 6. Micrographs of an embryo at stage 14. (A) A light micrograph of the ventrolateral region. Invaginating mesodermal cells (left) form fine cell processes (arrows)
along the blastocoelic wall (right). (B) A near region of (A). A part of a mesodermal
cell appears to flatten against the inner surface of the blastocoelic wall. (C) An
electron micrograph of a fine process shown in (A). (D) An electron micrograph
of the same cell process shown in (C) in another section. An amorphous material is
present in the intercellular space.
Microscopic observation of urodele gastrulation
681
force for the locomotion of the cells along the wall (Curtis, 1967). In the case of
chick embryos, the migrating mesoblasts make focal close 2-5-10 nm junctions
and focal tight junctions with the epiblasts and hypoblasts (Trelstad, Hay &
Revel, 1967; Revel, Yip & Chang, 1973). The deep cells in teleost embryos make
20 nm and more close contacts with the other cells during locomotion (Trinkaus
& Lentz, 1967). Focal close contacts were observed between the mesodermal
cells in anuran gastrulae (Johnson, 1970).
By use of scanning electron microscopy, invaginating cells were observed
clearly to form filopodia and lamellipodia flattened against the inner surface of
the blastocoelic wall. Filopodia and lamellipodia were observed, with scanning
electron microscopy, to be formed by the migrating cells during corneal
endothelium formation in the chick embryo (Bard, Hay & Meller, 1975; Nelson
& Revel, 1975).
The formation of pseudopodia by the invaginating cells and their contact
with the blastocoelic wall suggest that the cells migrate actively along the
blastocoelic wall and that these locomotive forces have an important role in
the morphogenetic movements during gastrulation. This concept was stated
previously in the case of anurans (Nakatsuji, 1974, 1975) from a study of the
formation of the pseudopodia by the invaginating cells of the foregut endoderm
and mesoderm. The archenteric roof of anuran embryos is more than two-cells
thick throughout gastrulation. Accordingly the suspected locomotive forces of
the cells, which are attached to the blastocoelic wall, must be transported to the
cells which line the archenteron, in order to bring about the elongation of
archenteron. On the other hand, the archenteric roof of the Cynops embryo is
almost one-cell thick. Accordingly the locomotive forces of these cells can work
immediately as the elongation force of the archenteric roof.
The scanning electron micrographs were taken by Dr T. Hattori of the Department of
Pathology, Kyoto Prefectural University of Medicine. The author is greatly indebted to him
and Professor S. Fujita for making the scanning electron microscope in their department
available for this study.
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FIGURE 7
Micrographs of two embryos at stage 16.
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(C) An electron micrograph of the interface between the presumptive notochord
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(D) An electron micrograph of the interface at higher magnification.
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(G) An enlarged light micrograph of the area shown by the upper small rectangle
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{Received 16 May 1975)
FIGURE 8
Scanning electron micrographs of the archenteric roof cells on the inner
surface of the blastocoelic wall at stage 12b. Cells are migrating
toward the left-above direction.
(A) A micrograph showing four invaginating cells attached to the blastocoelic wall.
(B) An enlarged micrograph of the portion shown by the rectangle in (A).
(C) An enlarged micrograph of the rectangle in (B).
(D) A micrograph when the tilt angle was changed from that in (A). An invaginating
cell at the centre of this figure corresponds to the uppermost cell in (A).
(E) An enlarged micrograph of the portion shown by the smaller rectangle in (D).
(F) An enlarged micrograph of the larger rectangle in (D).
Microscopic observation of urodele gastrulation
685