The Relationship Between Cleavage and Blastocoel Formation in Xenopus Laevis I

J. Embryo!. exp. Morph. Vol. 26, 1, pp. 37-49, 1971 37 Printed in Great Britain

The relationship between cleavage and blastocoel formation in Xenopus laevis I. Light microscopic observations

By MARVIN R. KALT1 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 occur- red, 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 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 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. 38 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 syn- chronously 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 40 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 41 42 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 accompli- shed 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 the first vegetal 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 43

11 44 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 contin- uous 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 45

15 46 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 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 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 48 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 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 '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.

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{Manuscript received 4 November 1970)

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