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 blasto- coel formation and the cleavage process. Blastocoel formation is an active process which begins during the first cleavage, and modifies the pattern of cyto- kinesis 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 other- wise 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 presump- tive 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 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). 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.