J. Embryo!. exp. Morph. Vol. 26, 1, pp. 37-49, 1971 37

Printed in Great Britain

The relationship between cleavage and blastocoelformation in Xenopus laevis

I. Light microscopic observations

By MARVIN R. KALT1

From the Department of Anatomy, Case Western Reserve University

SUMMARYBlastocoel formation in Xenopus laevis was investigated by light microscopy using serial

sections of epoxy-embedded, staged embryos. The earliest manifestation of the blastocoelin the embryo appeared during the first cleavage as a modification in the animal pole furrowtip. This modification consisted of an expansion of a localized area of the furrow. As theblastocoel became a distinct entity, it remained stationary, while the furrow tip continued toadvance inwardly. In contrast, no such furrow cavity was observed in the vegetal pole furrowduring its formation. During subsequent cleavages, up to the late morula stage, furrows onopposite sides of any given blastomere had different morphologies. As further divisions occur-red, the mode of furrow formation became identical regardless of location in the embryo. It issuggested that the cytokinetic pattern in early amphibian embryos is modified to allow for theformation of the blastocoel. After the blastocoel has formed, the cytokinetic pattern changesto one which is concerned solely with cell division.

INTRODUCTION

Classically, the amphibian zygote was thought to divide by a process involv-ing 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 afilamentous 'contractile ring'-like layer, which is presumably responsible fordivision, around the first cleavage furrow. These authors suggested that theplane of vacuoles reported in previous light microscopic descriptions of divisionfurrow formation was in fact a series of moniliform dilatations between otherwiseclosely apposed membranes of an already formed furrow. Regardless of theactual structures involved in the division process, all studies to date on amphibiancleavage still recognize that subsurface components of the cytoplasm are capableof 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 ofMedicine, Cleveland, Ohio 44106, U.S.A.

38 M. R. KALT

No serious attempts have been made to correlate cleavage with an event thatoccurs during the same period, blastocoel formation. To determine what, if any,relationships exist between the two phenomena, a study of the basic morphologyof blastocoel formation and cleavage was undertaken. Special attention wasgiven to a comparison of both animal pole and vegetal pole furrows, sinceprevious descriptions of amphibian cleavage appear sometimes to have beenbased on the unfounded assumption that both sides of the cleavage plane formin a similar manner. The results of both this and an electron microscopic study(Kalt, 1971) have indicated that an intimate relationship exists between blasto-coel formation and the cleavage process. Blastocoel formation is an activeprocess which begins during the first cleavage, and modifies the pattern of cyto-kinesis seen during this period when compared to cytokinesis observed in laterstages of development. Furthermore, the mode of blastocoel formation is suchthat 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, andfixed at 10 min intervals up to late blastula stages. Timing of stages was carriedout by observing the time of external furrow formation in a group of syn-chronously dividing zygotes. The time between visible furrow formation in eachdivision cycle was considered to represent one cleavage period, and events weresequenced in terms of the fraction of elapsed time between one period and thenext. Room temperature was maintained at 21-23 °C.

In order to reduce artifacts resulting from paraffin embedding, fixation andembedding methods used in electron microscopy were employed. Specimenswere 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 7-2for 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 anarrow above the figure number. All sections are cut along a vertical axis unless other-wise indicated.Fig. 1. Brightfield phase micrograph of the ectoplasmic region in the vicinity of theanimal pole in a newly fertilized egg illustrating the cortex (C) and the subcorticalregion (SC). x 540.Fig. 2. Brightfield phase micrograph of the boundary region between the subcorticalectoplasm (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 eggshowing the cortex (C), an ill-defined subcortical region (SC), the underlyingendoplasm (EN), and the external jelly coat (JC). x 250.Fig. 4. Brightfield phase micrograph of the boundary region between the subcorticalectoplasm (SC) and endoplasm (EN) in the midregion of a zygote 30 min afterfertilization, x 540.

Light microscope study of blastocoel formation 39

40 M. R. KALT

DER 732 according to the method of Erlandson (1964). Serial sections 2 /*m inthickness 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 corticaland subcortical cytoplasm containing pigment granules, vacuoles, and somesmall, scattered yolk platelets (Figs. 1-3). The contents of the ectoplasm aredistinct from that of the underlying endoplasm, in which larger yolk plateletspredominate, a compositional difference present in both animal and vegetalhemispheres (Figs. 2-4). The ectoplasmic layer varies from approximately 30 [imin 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 thecortical region. The distribution of inclusions in the ectoplasm is non random,with pigment and vacuoles increasing progressively from the vegetal to theanimal pole, while yolk platelets increase in the opposite direction. Also, inany given region the cortex usually contains more pigment and less yolk perunit area than the subcortical ectoplasm of the same region.

By two thirds of the time through the first cleavage period, significant changesin the distribution and location of the ectoplasm are evident. These changes areprobably the result of movements which occur in the ectoplasmic layer inresponse to the initiation of the cleavage process. The subcortical region of theectoplasm has begun to expand in the animal region ahead of the cortex, andhas penetrated inward into the zygote in a plane corresponding to the presump-tive furrow region (Fig. 5), forming a diastema region similar to that whichoccurs in Ambystoma (Bluemink, 1970). By the time the furrow is well developedexternally, 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 fertiliza-tion. The presumptive blastocoel cavity (PB) has been formed by the close apposi-tion of the sides of the furrow near the original animal pole region (A). Severalvacuoles (V), some of which contain metachromatic material, are seen beneath thefurrow tip. x 250.Fig. 7. Brightfield phase micrograph of the lower animal pole furrow region 60 minafter fertilization. Metachromatic material is present in the blastocoel (B). Thefurrow (F) extends from the floor of the blastocoel, and shows a slight enlargementat 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 acurved band of cytoplasm (CB), which shows no evidence of a furrow. V=vacuole.

x325.

Light microscope study of blastocoel formation 41

42 M. R. KALT

continues, the tip of the advancing furrow in the animal hemisphere becomesexpanded and swollen, while at the neck of the furrow the walls become closelyapposed. 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 inthe zygote. From this time on, the nascent blastocoel remains stationary, nolonger moving as the advancing furrow tip. Instead, it continues to enlarge andbecomes filled with acid mucosubstance (Stableford and Kalt, unpublished)(Fig. 7).

Further advance of the furrow below the stationary blastocoel is accompli-shed by formation of a small furrow extension in the floor of the blastocoelcavity. This extension progresses through the cytoplasm, but shows no largeexpansion at its tip, in contrast to the earlier furrow. There is instead a smallervacuole (Figs. 7-10), measuring 5-20 /*m in diameter, at the furrow tip. Themembranes formed by extension of the furrow tip from the blastocoel floor areso 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 theblastocoel. Furrow formation in the vegetal region seems to occur from thecortex inward with no marked blastocoel-like swelling at the tip, and no granularmaterial can be observed in the furrow (Fig. 11). The ectoplasm bounding theadvancing furrow in the vegetal region differs from that seen in the animalhemisphere, in that the subcortical region is inconspicuous and the cortexshows only sparse pigmentation. The tip of the advancing furrow is seen only asa narrow imagination of the cell surface surrounded by yolk (Fig. 11). Thefirst cleavage is completed as the furrow tips meet. The second cleavage furrowmay already be visible by the time the opposing blastomeres from the firstcleavage completely separate.

The second cleavage shows the same general progression of ectoplasmicmovements 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 ofthe blastocoel seen in Fig. 8. A large PAS-positive vacuole (V) is at the base of theblastocoel (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 the first vegetal pole furrow (F) 75 min afterfertilization. Small microvilli line the upper furrow region, x 1100.Fig. 12. Darkfield micrograph of a section perpendicular both to the 2nd and to thepresumptive 3rd cleavage planes. The ectoplasm (EC), located along the region of thesecond furrow (F), has started to migrate into the endoplasm (EN), away from thecentral position of the embryo, x 180.

Light microscope study of blastocoel formation 43

11

44 M. R. KALT

normal to the equator. An important difference, however, is that furrowingdoes not start at the level of the original animal pole, but instead begins in bothblastomeres at a point ten to fifteen degrees below the animal pole. This corres-ponds to the area where most of the subcortical ectoplasm is now located. Thesecond animal pole furrow possesses an initial moderate expansion of its tip,the expansion becoming incorporated into the blastocoel when the two struc-tures 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 vegetalpole furrow resembles its counterpart of the first cleavage.

The slight shift in the main body of subcortical cytoplasm away from theanimal pole of the blastomeres observed during the second division becomesmore pronounced during the third cleavage, which occurs in an equatorialplane. Due to the rotation of the mitotic apparatus, the blastocoel is now in thesame 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 verticallyaligned with the blastocoel. The first manifestation of the third cleavage occursin relation to the blastocoel in the form of an extensive ectoplasmic developmentof pigment and vacuoles identical to that seen in the original animal pole regionduring the first division (Fig. 12). As before, this ectoplasmic area progressivelycondenses in a plane, bisecting the blastomere and extending from the blastocoeloutward. Subsequently, the interior cortical surface invaginates, producing afurrow which is mildly dilated along its length. No ectoplasmic developmentoccurs to any large extent in the exterior cortical region of the blastomereopposite the blastocoel. Rather, one sees an invagination of the cortex which is

Fig. 13. Brightfield micrograph of an animal pole cell sectioned parallel to theequator, 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 avegetal blastomere. The furrow walls are closely apposed, and no expansion isvisible at the furrow tip (FT), x 610.

Fig. 15. Brightfield phase micrograph of a fifth division cleavage, showing thepoint of junction (J) between the internal (IF) and external (EF) furrows. The sidesof the internal furrow are well separated, while the sides of the external furrowappear to be joined only intermittently, x 850.

Fig. 16. Brightfield phase micrograph of a sixth division furrow in a blastomerelocated just below the equator of the embryo. Both the internal furrow (IF) and theexternal furrow (EF), show intermittent points of close membrane apposition, andhave only slight swellings at what appear to be the furrow tips. EM observations onan adjacent thin section, however, revealed that the furrow is completed and contin-uous between the visible endings. Because the membranes in this region (indicated byarrowheads) are closely apposed, the furrow cannot be resolved by light microscopy.x250.

Light microscope study of blastocoel formation 45

15

46 M. R. KALT

reminiscent of the furrowing seen earlier in the vegetal region, producing afurrow with closely apposed walls. Thus, the blastocoel region at this point intime has become the site which corresponds in its ectoplasmic activity to theoriginal animal pole region of the zygote.

As the next several cleavages occur, a more uniform distribution of the sub-cortical ectoplasmic component begins to take place throughout the embryo,in contrast to the marked asymmetries observed earlier. This increasing uni-formity occurs gradually, first in the animal cells (Fig. 13), and later in those ofthe vegetal region. Thus, while some differences still remain between blastomereswith respect to their ectoplasmic component, the distribution of this material isbecoming more uniform. Increasing uniformity is also reflected in furrow forma-tion, where a lessening of subcortical ectoplasmic development occurs in furrowsforming from the blastocoel side of the cell. This change appears to coincidewith a reduction in the degree of vacuolization, the vacuoles tending to remainconfined to the area immediately bordering the blastocoel.

By about the sixth cleavage, a further change in the morphology of divisioncan be observed. Indications of this change are present by the fourth or fifthdivision, and become increasingly apparent in subsequent divisions. Furrowsforming anywhere in the embryo gradually converge in their morphologies, incontrast to the earlier condition where furrows on opposite sides of a cell hadquite different morphological characteristics. Each side of the furrow in bothanimal and vegetal cells shows an intermediate type of development when com-pared to furrows observed in the first cleavage. The furrow tips in all cells showonly a slight separation of apposing membranes, and in some cases, show novisible separation at all (Figs. 14-16). Only a few vacuoles are observed aroundfurrow areas, and intermittent points of contact are present between furrowwalls along at least part of all furrows, although these contacts are more exten-sive in furrows originating from external surfaces than in internal furrowsoriginating from the blastocoel (Figs. 15, 16). Thus, as development progresses,furrow formation gradually becomes uniform as the blastocoel reaches its fullsize.

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 ismodified by blastocoel formation as well as by the more obvious cytoplasmicasymmetries of vacuole and yolk concentration. The blastocoel itself is notformed merely by the separation of already cleaved blastomeres, but rather isformed during the first division as a specialization of the animal pole furrow tip.The identification of this structural specialization as the incipient blastocoelrests on three criteria. First, as the initial division progresses, this cavity becomesstationary while the furrow tip continues to advance. Secondly, mucosubstanceis present both in this cavity and in the definitive blastocoel (Stableford, 1967).

Light microscope study of blastocoel formation 47Thirdly, as demonstrated in this and the following report (Kalt, 1971), enlarge-ment of this region forms the definitive blastocoel seen later.

The above description disagrees with Selman & Perry's (1970) report of thefirst cleavage in Triturus. In this organism, httle secretory material was noted inthe furrow, and the origin of a cavity from the animal pole furrow tip was notreported. Instead, the animal pole furrow was described essentially to passstraight down the egg. Selman & Perry mention that some differences occurin Xenopus, but these dissimilarities from Triturus are not fully described, norare subsequent patterns of division.

In any case, prior to the second division in Xenopus, there is a pronouncedaccumulation of subcortical ectoplasm in the region where the second cleavageplane will be initiated. After the cleavage plane is formed, the subcorticalectoplasm continues to accumulate in the region surrounding the blastocoel.The concentration of ectoplasm around the blastocoel at this point may be ameans to ensure the subsequent distribution of this material to all blastomeres.As a consequence, at the third cleavage, which is equatorial, the vegetal polecells are provided with a mass of highly vacuolized and pigmented ectoplasmthat they previously did not possess to any substantial degree. As the division ofthe embryo progresses, this material in turn is further distributed and graduallydecreases in amount, while at the same time the pattern of cleavage changes.

The consistent spatial relationship between the ectoplasm and the incipientfurrow suggests that some component of the former, rather than of the endo-plasm, may be responsible for many of the differences observed in early furrowtip morphology. Further support for this idea may be demonstrated by examin-ing cells at opposite ends of a mid-blastula stage embryo. Based on their endo-plasmic morphology, cells at the animal pole appear to be similar to the originalanimal region of the zygote, cells at the vegetal pole of the blastula appearto be similar to the original vegetal region of the zygote. Despite these morpho-logical differences, their furrows all show a uniform development pattern. Inthese cells, the ectoplasmic component shows an even distribution with a greatlydiminished layer of vacuoles.

An inverse relationship appears to exist between vacuole population andblastocoel formation. As the blastocoel becomes fully formed, the area ofvacuolized cytoplasm surrounding it decreases. At least some of the vacuolesin the cytoplasm contain a mucosubstance which appears to be discharged intothe blastocoel (Motomura, 1960; Stableford, 1967), a process which impliesfusion of vacuoles with the cell surface. This suggests that the blastocoel isbeing enlarged by coalescence with vacuoles. Morphological evidence of thisevent will be presented in the following paper (Kalt, 1971).

Localized differences in cytoplasmic contents may influence cleavage patternsin organisms other than Xenopus. For example, Thomas (1968), in an electronmicroscopic study of division in teleost blastulas, found a vesicular type ofcleavage in some cells and a contractile ring type ('furrow cleavage') in other

48 M. R. KALT

cells in the same embryo. As development progressed, a generalized transitionfrom vesicular to furrow cleavage was reported to occur, although no mentionwas made of initial cleavages. Monne & Harde (1951) and Motomura (1966),working on echinoderm embryos, reported that the blastocoel arises during the 2-cell stage by the secretion of a mucosubstance, while Tilney & Marsland (1969)have shown that in these forms the first furrow possesses a filamentous 'con-tractile ring' type structure. The same type of filaments have been reported in thesquid (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 duringearly embryogenesis in a number of organisms.

The necessity for treating cleavage as a highly specialized case of cell divisionrequires that great care be exercised in drawing generalizations based on observa-tions made at only one point in development. This is especially important inreference to observations made solely during the first division, when blastocoelformation is occurring, since the size, shape, and development of the furrow maybe influenced by dynamic events occurring in the surrounding cytoplasm.

The author wishes to thank Drs Joseph Grasso and Bernard Tandler for their helpfulcriticism of this manuscript, and Dr Louis T. Stableford of Lafayette College, who firstdirected his interest toward this problem.

This research was supported in part by grant no. AM 11896, N.I.H. (U.S.A.) awarded toJoseph Grasso. The author is the recipient of an N.S.F. Predoctoral Fellowship.

REFERENCES

ANTEUNIS, A., FAUTREZ-FIRLEFYN, N. & FAUTREZ, J. (1961). Formation de la premiereebauche 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 microscopicstudy 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 Xenopuslaevis. 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 betweenfurrow 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 theposition of the furrow. J. Embryol. exp. Morph. 21, 119-129.

MONNE, L. & HARDE, S. (1951). On the formation of the blastocoele and similar embryoniccavities. Ark. Zool. 28, 463-469.

MOTOMURA, I. (1960). Formation of the cleavage plane by the secretion of mucosubstance onthe 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 precedingfurrow formation in the cleavage of newt eggs. Development, Growth, Diff. 11, 246-254.

Light microscope study of blastocoel formation 49SCHROEDER, 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.

{Manuscript received 4 November 1970)

E M B 26