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The Xenopus embryo undergoes 12 rapid synchronous cleavages followed by a period of slower asynchronous divisions more typical of somatic cells. This change in cell cleavage has been termed the midblastula transition (MBT). We show that at the MBT the blastomeres become motile and transcriptionally active for the first time. We have investigated the timing of the MBT and found that it does not depend on cell division, on time since fertilization or on a counting mechanism involving the sequential modification of DNA. Rather, the timing of the MBT depends on reaching a critical ratio of nucleus to cytoplasm. We view the MBT as a consequence of the titration of some substance, originally present in the egg, by the exponentially increasing nuclear material. When this substance is exhausted a new cell program is engaged, leading to the acquisition of several new cell properties.
Figure 1. Dissociation of Cleaving Blastomeres
Dividing blastomeres grown in dissociation medium after removal of
their jelly coats and vitelline membranes. Eggs after cleavage 5 (A) and after cleavage 8 (B).
Figure 2. Characterization of the Cell Cycle, Rate of DNA Synthesis, Rate of RNA Synthesis and Ceil Motility at the MBT
(A) Length and synchrony of the cell cycle during early embryogenesis was measured from time-lapse videotapes of dividing dissociated blastomeres made during the first 10 hr after fertilization. The cell cycles of individual cells from different parts of a dissociated embryo were measured over a 4-5 hr period and found to be 35 + 2 min long until cleavage 11. At the MBT (cleavage 12) the cell cycle times of different cells appeared to differ substantially. However, daughter cells originating from a common ancestor at the MBT appeared to remain synchronized for at least 2-3 hr after MET. Lines: average time of these individual cell cycles. Brackets: variation in length of time among individual cell cycles. The rate of DNA synthesis was measured by continuous labeling of embryos during the first 10 hr of development in 0.5 mCi 3H-thymidine in 0.5 ml MMR at 23°C. Eggs (ten) were collected at various stages, and DNA was isolated as described in the Experimental Procedures. As expected for a synchronized, exponentially growing population of cells, the logarithm of the amount of TCA-precipitable cpm increases linearly until the MBT. From the slope of this line, one obtains average values for the cell cycle of 35 min for the period leading up to the MBT, in good agreement with the data collected via time-lapse video recording of dissociated embryos. (open circle) Average cleavage period as a function of cleavage number; (solid circle) DNA synthesis as a function of time.
(B) Onset of cell motility and RNA synthesis at the MBT. Dissociated embryos were observed via time-lapse video recording, and cells were counted as motile if they exhibited either cytoplasmic blebbing or pseudopod formation. To quantitate this parameter accurately at stages past the MBT it was necessary to dissociate large clumps of cells by either gently bumping the dish in which they lay or carefully moving the embryo once or twice with a wide bore siliconized Pasteur pipette. During this procedure most of the largest vegetal cells (about 5% of the total cells) were broken, The onset of motility and percentages of cells motile during these stages did not differ when the embryos had been injected with 50 pg/ ml cv-amanitin (final concentration in the egg) at the single-cell stage. The rate of RNA synthesis as a function of cleavage stage was quantitated by counting the number of 3H-dependent grains per nucleus. After autoradiography, 50-l 00 cells from embryos at different stages were counted. The number of grains per nucleus of different nuclei from the same embryo varied as much as threefold. The cleavage stage and elapsed time are given in the abscissa. (open circle) RNA synthesis: (solid circle) cell motility.
Figure 3. Demonstration of Motility Coincident with the MBT.
Embryos that had been growing in dissociation media were further disrupted by gentle shaking after cleavage 12. The cells were trans- ferred onto a microscope slide and photographed every 60 set at 160X. The small cytoplasmic bleb on the outer periphery of the cell will normally progress in a single direction around the cell for several revolutions before disappearing and then reappearing. (A) 0 min; (B) 1 min; (C) 2 min; (D) 3 min. At least 70%-80% of the cells defined as motile in Figure 2B exhibit this type of motility pattern. The remaining 20%-30% of motile cells undergo a much slower pseudopodal ex- tension and retraction motility. Final magnification 370X.
Figure 5. Onset of the MBT in the presence of the cleavage Inhibitor Cytochalasin B.
Eggs (50) were transferred 40 min after fertilization into 0.5 ml MMR
containing 5 fig/ml cytochalasin B and 0.5 mCi of either ‘H-thymidine
or 3H-uridine. Control eggs were incubated in the absence of cyto- chalasin B. Samples were removed at the indicated times and pre- pared for either autoradiography (RNA synthesis) or TCA precipitation (DNA synthesis). (open circle) DNA synthesis in controls; (solid circle) DNA synthesis in blastomeres treated with cytochalasin B. RNA synthesis was quanti- tated as described in Figure 28. (open square) RNA synthesis in controls: (solid square) RNA synthesis in blastomeres treated with cytochalasin B.
Figure 6. Schematic Representation of the partial constriction experiment
(A-C) A fertilized egg is constricted such that only half of the embryo inherits a nucleus. The nucleated side divides until a nucleus moves into the uncleaved half of the embryo (D), inducing cleavage to begin in this half(E). By (F), the right side has reached the MBT and begun asynchronous division, whereas the retarded half of the embryo (left) continues to divide synchronously for two more divisions.
Figure 7. Partial Constriction Experiment
(A) Constricted egg showing two cleavages on the right side of the
egg and none on the left. (B) Constricted egg at cleavage 6 on the right side of the egg and at cleavage 4 on the left.
Figure 8. Premature Onset of Transcription ir. Polyspermic Eggs Polyspermic fertilization was performed by the method of Grey et al. (1982). The number of sperm nuclei per egg was determined by counting the number of pigmented sperm entry points. Eggs were blocked in cleavage by centrifugation and were injected with 1 &I w3’P-rUTP 60 min after fertilization. At various times eggs were collected and analyzed for RNA transcripts as described in Figure 4. (Lanes a-e) Polyspermic eggs; (lanes f-j) normal eggs. Times after fertilization were as follows: 5 hr (lanes a and f); 6 hr (lanes b and g); 7 hr (lanes c and h); 8 hr (lanes d and i); 9 hr (lanes e and j).
