J. Embryo!. exp. Morph. Vol. 26, 1, pp. 37-49, 1971
Printed in Great Britain
37
The relationship between cleavage and blastocoel
formation in Xenopus laevis
I. Light microscopic observations
By MARVIN R. KALT 1
From the Department of Anatomy, Case Western Reserve University
SUMMARY
Blastocoel formation in Xenopus laevis was investigated by light microscopy using serial
sections of epoxy-embedded, staged embryos. The earliest manifestation of the blastocoel
in the embryo appeared during the first cleavage as a modification in the animal pole furrow
tip. This modification consisted of an expansion of a localized area of the furrow. As the
blastocoel became a distinct entity, it remained stationary, while the furrow tip continued to
advance inwardly. In contrast, no such furrow cavity was observed in the vegetal pole furrow
during its formation. During subsequent cleavages, up to the late morula stage, furrows on
opposite sides of any given blastomere had different morphologies. As further divisions occurred, the mode of furrow formation became identical regardless of location in the embryo. It is
suggested that the cytokinetic pattern in early amphibian embryos is modified to allow for the
formation of the blastocoel. After the blastocoel has formed, the cytokinetic pattern changes
to one which is concerned solely with cell division.
INTRODUCTION
Classically, the amphibian zygote was thought to divide by a process involving vesiculation of the cytoplasm (Selman & Waddington, 1955; Motomura,
I960, 1966; Zotin, 1964). Recently however, two electron microscopic studies
(Selman and Perry, 1970; Bluemink, 1970) have demonstrated the presence of a
filamentous 'contractile ring'-like layer, which is presumably responsible for
division, around the first cleavage furrow. These authors suggested that the
plane of vacuoles reported in previous light microscopic descriptions of division
furrow formation was in fact a series of moniliform dilatations between otherwise
closely apposed membranes of an already formed furrow. Regardless of the
actual structures involved in the division process, all studies to date on amphibian
cleavage still recognize that subsurface components of the cytoplasm are capable
of influencing the formation of the furrow (Selman & Waddington, 1955;
Motomura, 1960; Zotin, 1964; Kubota, 1966, 1969; Bluemink, 1970; Sawai,
Kubota & Kojima, 1969).
1
Author's Address: Department of Anatomy, Case Western Reserve University, School of
Medicine, Cleveland, Ohio 44106, U.S.A.
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M. R. KALT
No serious attempts have been made to correlate cleavage with an event that
occurs during the same period, blastocoel formation. To determine what, if any,
relationships exist between the two phenomena, a study of the basic morphology
of blastocoel formation and cleavage was undertaken. Special attention was
given to a comparison of both animal pole and vegetal pole furrows, since
previous descriptions of amphibian cleavage appear sometimes to have been
based on the unfounded assumption that both sides of the cleavage plane form
in a similar manner. The results of both this and an electron microscopic study
(Kalt, 1971) have indicated that an intimate relationship exists between blastocoel formation and the cleavage process. Blastocoel formation is an active
process which begins during the first cleavage, and modifies the pattern of cytokinesis seen during this period when compared to cytokinesis observed in later
stages of development. Furthermore, the mode of blastocoel formation is such
that it must be taken into account when proposing any mechanism of cleavage.
MATERIALS AND METHODS
Eggs of Xenopus laevis were obtained by induced ovulation, fertilized, and
fixed at 10 min intervals up to late blastula stages. Timing of stages was carried
out by observing the time of external furrow formation in a group of synchronously dividing zygotes. The time between visible furrow formation in each
division cycle was considered to represent one cleavage period, and events were
sequenced in terms of the fraction of elapsed time between one period and the
next. Room temperature was maintained at 21-23 °C.
In order to reduce artifacts resulting from paraffin embedding, fixation and
embedding methods used in electron microscopy were employed. Specimens
were fixed in 3 % glutaraldehyde in 0-1 M-cacodylate buffer at pH 7-2 for 4 h,
followed by post-fixation in 2 % osmium tetroxide in phosphate buffer at pH 7-2
for 3 h. Embryos were then dehydrated in ethanol and embedded in Maraglas,
The direction of the animal pole in relation to the micrograph is indicated by an
arrow above the figure number. All sections are cut along a vertical axis unless otherwise indicated.
Fig. 1. Brightfield phase micrograph of the ectoplasmic region in the vicinity of the
animal pole in a newly fertilized egg illustrating the cortex (C) and the subcortical
region (SC). x 540.
Fig. 2. Brightfield phase micrograph of the boundary region between the subcortical
ectoplasm (SC) and the endoplasm (EN) of a zygote 15 min after fertilization, x 250.
Fig. 3. Darkfield micrograph through the vegetal region of a newly fertilized egg
showing the cortex (C), an ill-defined subcortical region (SC), the underlying
endoplasm (EN), and the external jelly coat (JC). x 250.
Fig. 4. Brightfield phase micrograph of the boundary region between the subcortical
ectoplasm (SC) and endoplasm (EN) in the midregion of a zygote 30 min after
fertilization, x 540.
Light microscope study of blastocoel formation
39
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M. R. KALT
DER 732 according to the method of Erlandson (1964). Serial sections 2 /*m in
thickness were cut on a Servall MT-1 ultramicrotome, mounted on glass slides,
and stained in 1 % Alcian blue at pH 3 or 1, or in 1 % toluidine blue at pH 4.
RESULTS
The newly fertilized egg of Xenopus possesses an ectoplasmic layer of cortical
and subcortical cytoplasm containing pigment granules, vacuoles, and some
small, scattered yolk platelets (Figs. 1-3). The contents of the ectoplasm are
distinct from that of the underlying endoplasm, in which larger yolk platelets
predominate, a compositional difference present in both animal and vegetal
hemispheres (Figs. 2-4). The ectoplasmic layer varies from approximately 30 [im
in thickness at the animal pole to 5-15/tm in thickness at the vegetal pole.
Most of the variation in thickness occurs in the subcortical, rather than in the
cortical region. The distribution of inclusions in the ectoplasm is non random,
with pigment and vacuoles increasing progressively from the vegetal to the
animal pole, while yolk platelets increase in the opposite direction. Also, in
any given region the cortex usually contains more pigment and less yolk per
unit area than the subcortical ectoplasm of the same region.
By two thirds of the time through the first cleavage period, significant changes
in the distribution and location of the ectoplasm are evident. These changes are
probably the result of movements which occur in the ectoplasmic layer in
response to the initiation of the cleavage process. The subcortical region of the
ectoplasm has begun to expand in the animal region ahead of the cortex, and
has penetrated inward into the zygote in a plane corresponding to the presumptive furrow region (Fig. 5), forming a diastema region similar to that which
occurs in Ambystoma (Bluemink, 1970). By the time the furrow is well developed
externally, it has penetrated rather deeply into the ectoplasm. As furrowing
Fig. 5. Darkfield micrograph of the animal pole region 40 min after fertilization.
The ectoplasm (EC), which appears light due to the presence of pigment granules,
has started to penetrate into the underlying endoplasm (EN), x 150.
Fig. 6. Brightfield phase micrograph of the animal pole furrow 90 min after fertilization. The presumptive blastocoel cavity (PB) has been formed by the close apposition of the sides of the furrow near the original animal pole region (A). Several
vacuoles (V), some of which contain metachromatic material, are seen beneath the
furrow tip. x 250.
Fig. 7. Brightfield phase micrograph of the lower animal pole furrow region 60 min
after fertilization. Metachromatic material is present in the blastocoel (B). The
furrow (F) extends from the floor of the blastocoel, and shows a slight enlargement
at its tip (FT). The furrow, barely discernible, is indicated by arrowheads, x 250.
Fig. 8. Darkfield micrograph similar to Fig. 7, but from a more lateral section.
At the top of the micrograph, the blastocoel (B) is visible. At the bottom, a furrow tip
(FT) with a small furrow extension (F) is present. Connecting the two structures is a
curved band of cytoplasm (CB), which shows no evidence of a furrow. V=vacuole.
x325.
Light microscope study of blastocoel formation
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M. R. KALT
continues, the tip of the advancing furrow in the animal hemisphere becomes
expanded and swollen, while at the neck of the furrow the walls become closely
apposed. At this time the furrow is surrounded by a wide layer of ectoplasm,
which at a point below the furrow tip usually contains several large vacuoles
(Fig. 6). The swollen tip represents the first manifestation of the blastocoel in
the zygote. From this time on, the nascent blastocoel remains stationary, no
longer moving as the advancing furrow tip. Instead, it continues to enlarge and
becomes filled with acid mucosubstance (Stableford and Kalt, unpublished)
(Fig. 7).
Further advance of the furrow below the stationary blastocoel is accomplished by formation of a small furrow extension in the floor of the blastocoel
cavity. This extension progresses through the cytoplasm, but shows no large
expansion at its tip, in contrast to the earlier furrow. There is instead a smaller
vacuole (Figs. 7-10), measuring 5-20 /*m in diameter, at the furrow tip. The
membranes formed by extension of the furrow tip from the blastocoel floor are
so closely apposed in some places as to be unresolvable by light microscopy
(Fig. 10).
While the external furrow continues circumferentially around the zygote,
furrowing is intiated in the vegetal region and progresses upward toward the
blastocoel. Furrow formation in the vegetal region seems to occur from the
cortex inward with no marked blastocoel-like swelling at the tip, and no granular
material can be observed in the furrow (Fig. 11). The ectoplasm bounding the
advancing furrow in the vegetal region differs from that seen in the animal
hemisphere, in that the subcortical region is inconspicuous and the cortex
shows only sparse pigmentation. The tip of the advancing furrow is seen only as
a narrow imagination of the cell surface surrounded by yolk (Fig. 11). The
first cleavage is completed as the furrow tips meet. The second cleavage furrow
may already be visible by the time the opposing blastomeres from the first
cleavage completely separate.
The second cleavage shows the same general progression of ectoplasmic
movements as the first, occurring at right angles to the first cleavage plane and
Fig. 9. Brightfield phase micrograph showing an enlargement of the lower region of
the blastocoel seen in Fig. 8. A large PAS-positive vacuole (V) is at the base of the
blastocoel (B). No clear furrow is demonstrable below this vacuole. x 850.
Fig. 10. Brightfield phase micrograph of the furrow tip (FT) shown in Fig. 8.
Microvilli (MV) are present at the cell surfaces. A highly convoluted furrow (F)
extends above the furrow tip. x 1600.
Fig. 11. Brightfield phase micrograph of thefirstvegetal pole furrow (F) 75 min after
fertilization. Small microvilli line the upper furrow region, x 1100.
Fig. 12. Darkfield micrograph of a section perpendicular both to the 2nd and to the
presumptive 3rd cleavage planes. The ectoplasm (EC), located along the region of the
second furrow (F), has started to migrate into the endoplasm (EN), away from the
central position of the embryo, x 180.
Light microscope study of blastocoel formation
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M. R. KALT
normal to the equator. An important difference, however, is that furrowing
does not start at the level of the original animal pole, but instead begins in both
blastomeres at a point ten to fifteen degrees below the animal pole. This corresponds to the area where most of the subcortical ectoplasm is now located. The
second animal pole furrow possesses an initial moderate expansion of its tip,
the expansion becoming incorporated into the blastocoel when the two structures meet. After this point, the furrow tip continues as only a small expansion,
and the furrow walls are closely apposed. Like the initial animal pole furrow,
this furrow is also surrounded by cytoplasmic vacuoles. The second vegetal
pole furrow resembles its counterpart of the first cleavage.
The slight shift in the main body of subcortical cytoplasm away from the
animal pole of the blastomeres observed during the second division becomes
more pronounced during the third cleavage, which occurs in an equatorial
plane. Due to the rotation of the mitotic apparatus, the blastocoel is now in the
same position relative to it as was the animal pole during the first two divisions.
In other words, the equatorial plate of the mitotic apparatus is now vertically
aligned with the blastocoel. The first manifestation of the third cleavage occurs
in relation to the blastocoel in the form of an extensive ectoplasmic development
of pigment and vacuoles identical to that seen in the original animal pole region
during the first division (Fig. 12). As before, this ectoplasmic area progressively
condenses in a plane, bisecting the blastomere and extending from the blastocoel
outward. Subsequently, the interior cortical surface invaginates, producing a
furrow which is mildly dilated along its length. No ectoplasmic development
occurs to any large extent in the exterior cortical region of the blastomere
opposite the blastocoel. Rather, one sees an invagination of the cortex which is
Fig. 13. Brightfield micrograph of an animal pole cell sectioned parallel to the
equator, from a 16-cell-stage embryo. A presumptive fourth division cleavage furrow
(PF) may be seen extending between the blastocoel (B) and the external cortex.
The second cleavage furrow (F) and the jelly coat (JC) are also visible, x 180.
Fig. 14. Brightfield phase micrograph of a fourth division external furrow (F) in a
vegetal blastomere. The furrow walls are closely apposed, and no expansion is
visible at the furrow tip (FT), x 610.
Fig. 15. Brightfield phase micrograph of a fifth division cleavage, showing the
point of junction (J) between the internal (IF) and external (EF) furrows. The sides
of the internal furrow are well separated, while the sides of the external furrow
appear to be joined only intermittently, x 850.
Fig. 16. Brightfield phase micrograph of a sixth division furrow in a blastomere
located just below the equator of the embryo. Both the internal furrow (IF) and the
external furrow (EF), show intermittent points of close membrane apposition, and
have only slight swellings at what appear to be the furrow tips. EM observations on
an adjacent thin section, however, revealed that the furrow is completed and continuous between the visible endings. Because the membranes in this region (indicated by
arrowheads) are closely apposed, the furrow cannot be resolved by light microscopy.
x250.
Light microscope study of blastocoel formation
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M. R. KALT
reminiscent of the furrowing seen earlier in the vegetal region, producing a
furrow with closely apposed walls. Thus, the blastocoel region at this point in
time has become the site which corresponds in its ectoplasmic activity to the
original animal pole region of the zygote.
As the next several cleavages occur, a more uniform distribution of the subcortical ectoplasmic component begins to take place throughout the embryo,
in contrast to the marked asymmetries observed earlier. This increasing uniformity occurs gradually, first in the animal cells (Fig. 13), and later in those of
the vegetal region. Thus, while some differences still remain between blastomeres
with respect to their ectoplasmic component, the distribution of this material is
becoming more uniform. Increasing uniformity is also reflected in furrow formation, where a lessening of subcortical ectoplasmic development occurs in furrows
forming from the blastocoel side of the cell. This change appears to coincide
with a reduction in the degree of vacuolization, the vacuoles tending to remain
confined to the area immediately bordering the blastocoel.
By about the sixth cleavage, a further change in the morphology of division
can be observed. Indications of this change are present by the fourth or fifth
division, and become increasingly apparent in subsequent divisions. Furrows
forming anywhere in the embryo gradually converge in their morphologies, in
contrast to the earlier condition where furrows on opposite sides of a cell had
quite different morphological characteristics. Each side of the furrow in both
animal and vegetal cells shows an intermediate type of development when compared to furrows observed in the first cleavage. The furrow tips in all cells show
only a slight separation of apposing membranes, and in some cases, show no
visible separation at all (Figs. 14-16). Only a few vacuoles are observed around
furrow areas, and intermittent points of contact are present between furrow
walls along at least part of all furrows, although these contacts are more extensive in furrows originating from external surfaces than in internal furrows
originating from the blastocoel (Figs. 15, 16). Thus, as development progresses,
furrow formation gradually becomes uniform as the blastocoel reaches its full
size.
DISCUSSION
The present study strongly suggests that the process of embryonic cleavage is,
in several respects, unique as a case of cell division. The cytokinetic pattern is
modified by blastocoel formation as well as by the more obvious cytoplasmic
asymmetries of vacuole and yolk concentration. The blastocoel itself is not
formed merely by the separation of already cleaved blastomeres, but rather is
formed during the first division as a specialization of the animal pole furrow tip.
The identification of this structural specialization as the incipient blastocoel
rests on three criteria. First, as the initial division progresses, this cavity becomes
stationary while the furrow tip continues to advance. Secondly, mucosubstance
is present both in this cavity and in the definitive blastocoel (Stableford, 1967).
Light microscope study of blastocoel formation
47
Thirdly, as demonstrated in this and the following report (Kalt, 1971), enlargement of this region forms the definitive blastocoel seen later.
The above description disagrees with Selman & Perry's (1970) report of the
first cleavage in Triturus. In this organism, httle secretory material was noted in
the furrow, and the origin of a cavity from the animal pole furrow tip was not
reported. Instead, the animal pole furrow was described essentially to pass
straight down the egg. Selman & Perry mention that some differences occur
in Xenopus, but these dissimilarities from Triturus are not fully described, nor
are subsequent patterns of division.
In any case, prior to the second division in Xenopus, there is a pronounced
accumulation of subcortical ectoplasm in the region where the second cleavage
plane will be initiated. After the cleavage plane is formed, the subcortical
ectoplasm continues to accumulate in the region surrounding the blastocoel.
The concentration of ectoplasm around the blastocoel at this point may be a
means to ensure the subsequent distribution of this material to all blastomeres.
As a consequence, at the third cleavage, which is equatorial, the vegetal pole
cells are provided with a mass of highly vacuolized and pigmented ectoplasm
that they previously did not possess to any substantial degree. As the division of
the embryo progresses, this material in turn is further distributed and gradually
decreases in amount, while at the same time the pattern of cleavage changes.
The consistent spatial relationship between the ectoplasm and the incipient
furrow suggests that some component of the former, rather than of the endoplasm, may be responsible for many of the differences observed in early furrow
tip morphology. Further support for this idea may be demonstrated by examining cells at opposite ends of a mid-blastula stage embryo. Based on their endoplasmic morphology, cells at the animal pole appear to be similar to the original
animal region of the zygote, cells at the vegetal pole of the blastula appear
to be similar to the original vegetal region of the zygote. Despite these morphological differences, their furrows all show a uniform development pattern. In
these cells, the ectoplasmic component shows an even distribution with a greatly
diminished layer of vacuoles.
An inverse relationship appears to exist between vacuole population and
blastocoel formation. As the blastocoel becomes fully formed, the area of
vacuolized cytoplasm surrounding it decreases. At least some of the vacuoles
in the cytoplasm contain a mucosubstance which appears to be discharged into
the blastocoel (Motomura, 1960; Stableford, 1967), a process which implies
fusion of vacuoles with the cell surface. This suggests that the blastocoel is
being enlarged by coalescence with vacuoles. Morphological evidence of this
event will be presented in the following paper (Kalt, 1971).
Localized differences in cytoplasmic contents may influence cleavage patterns
in organisms other than Xenopus. For example, Thomas (1968), in an electron
microscopic study of division in teleost blastulas, found a vesicular type of
cleavage in some cells and a contractile ring type ('furrow cleavage') in other
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M. R. KALT
cells in the same embryo. As development progressed, a generalized transition
from vesicular to furrow cleavage was reported to occur, although no mention
was made of initial cleavages. Monne & Harde (1951) and Motomura (1966),
working on echinoderm embryos, reported that the blastocoel arises during the 2cell stage by the secretion of a mucosubstance, while Tilney & Marsland (1969)
have shown that in these forms the first furrow possesses a filamentous 'contractile ring' type structure. The same type of filaments have been reported in the
squid (Arnold, 1969) and in coelenterates (Schroeder, 1968; Szollosi, 1970).
Anteunis, Fautrez-Firlefyn & Fautrez (1961), working with the crustacean,
Artemia, observed the development of the blastocoel during the first cleavage.
From the foregoing, it is obvious that division patterns may be modified during
early embryogenesis in a number of organisms.
The necessity for treating cleavage as a highly specialized case of cell division
requires that great care be exercised in drawing generalizations based on observations made at only one point in development. This is especially important in
reference to observations made solely during the first division, when blastocoel
formation is occurring, since the size, shape, and development of the furrow may
be influenced by dynamic events occurring in the surrounding cytoplasm.
The author wishes to thank Drs Joseph Grasso and Bernard Tandler for their helpful
criticism of this manuscript, and Dr Louis T. Stableford of Lafayette College, who first
directed his interest toward this problem.
This research was supported in part by grant no. AM 11896, N.I.H. (U.S.A.) awarded to
Joseph Grasso. The author is the recipient of an N.S.F. Predoctoral Fellowship.
REFERENCES
A., FAUTREZ-FIRLEFYN, N. & FAUTREZ, J. (1961). Formation de la premiere
ebauche du blastocoele dans l'oeuf d' Artemia salina. Expl Cell Res. 25, 463-465.
ARNOLD, J. M. (1969). Cleavage furrow formation in a telolecithal egg (Loligo pealii).
I. Filaments in early furrow formation. /. Cell Biol. 41, 894-904.
BLUEMINK, J. G. (1970). The first cleavage of the amphibian egg. An electron microscopic
study of the onset of cytokinesis in the egg of Ambystoma mexicanum. J. Ultrastruct. Res.
32, 142-166.
ERLANDSON, R. A. (1964). A new Maraglas, DER 732, embedment for electron microscopy.
/. Cell Biol. 22, 704-709.
KALT, M. R. (1971). The relationship between cleavage and blastocoel formation in Xenopus
laevis. II. Electron microscopic observations. /. Embryol. exp. Morph. 26, 51-66.
KUBOTA, T. (1966). Studies of the cleavage in the frog egg. I. On the temporal relation between
furrow determination and nuclear division. /. exp. Biol. 44, 545-552.
KUBOTA, T. (1969). Studies of the cleavage in the frog egg. II. On the determination of the
position of the furrow. J. Embryol. exp. Morph. 21, 119-129.
MONNE, L. & HARDE, S. (1951). On the formation of the blastocoele and similar embryonic
cavities. Ark. Zool. 28, 463-469.
MOTOMURA, I. (1960). Formation of the cleavage plane by the secretion of mucosubstance on
the egg of the frog. Sci. Rep. Tohoku Univ. Ser. IV, Biol. 26, 53-58.
MOTOMURA, I. (1966). Secretion of a mucosubstance in the cleaving egg of the sea urchin.
Ada Embryol. Morph. exp. 9, 56-60.
SAWAT, T., KUBOTA, T. & KOJIMA, M. K. (1969). Cortical and subcortical changes preceding
furrow formation in the cleavage of newt eggs. Development, Growth, Diff. 11, 246-254.
ANTEUNIS,
Light microscope study of blastocoel formation
49
T. E. (1968). Cytokinesis: Filaments in the cleavage furrow. Expl Cell Res. 53,
272-276.
SELMAN, G. G. & PERRY, M. M. (1970). Ultrastructural changes in the surface layers of the
newt's egg in relation to the mechanism of its cleavage. /. Cell Sci. 6, 207-227.
SELMAN, G. G. & WADDINGTON, C. H. (1955). The mechanism of cell division in the cleavage
of the newt's egg. /. exp. Biol. 32, 700-733.
STABLEFORD, L. T. (1967). A study of calcium in the early development of the amphibian
embryo. Devi Biol. 16, 303-314.
SZOLLOSI, D. (1970). Cortical cytoplasmic filaments of cleaving eggs: A structural element
corresponding to the contractile ring. /. Cell Biol. 44, .192-210.
THOMAS, R. J. (1968). Cytokinesis during early development of a teleost embryo; Brachydanio
rerio. J. Ultrastruct. Res. 24, 232-238.
TILNEY, L. & MARSLAND, D. (1969). A fine structural analysis of cleavage induction and
furrowing in the eggs of Arbacia punctulata. J. Cell Biol. 42, 170-184.
ZOTIN, A. (1964) The mechanism of cleavage in amphibian and sturgeon eggs. /. Embryol.
exp. Morphol. 12, 247-262.
SCHROEDER,
{Manuscript received 4 November 1970)
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