Atsunori Shinagawa

Marked alteration at midblastula transition in the effect of lithium on formation of the larval body pattern of Xenopus laevis

Embryos of Xenopus laevis were exposed to 0.4 M LiCl for 5 min at various stages of development. The effect of lithium on the larval body pattern could be detected from the 2‐cell to the late gastrula stage, but changed from reduction of posterior structures (“anteriorization”) to reduction of anterior structures (“posteriorization”) just after the 12th cleavage, the time of midblastula transition (MBT). Temporal coincidence of MBT with alteration of the effect of lithium was observed even with embryos derived from half‐egg fragment, in which MBT occurs just after the 11th cleavage. These results suggest the existence of a mechanism for formation of the basic plan of the larval body whose function changes at MBT. Combinations of the pre‐ and post‐MBT exposures to lithium induced marked posteriorization in most larvae, indicating that the basic plan is not irreversibly determined until MBT, but is fixed during post‐MBT stages.

Relative Timing of Stiffening with Various Combinations of Nucleate and Enucleated Egg Fragments of Xenopus laevis

Previously, I found that in Xenopus eggs, the surface contraction wave (SCW) can arise at a point close to a male, a female or a zygote nucleus or a top portion of the egg (9). This finding suggested that all types of nucleus and cytoplasm of a Xenopus egg have the potential to determine the point of initiation of the SCW. Since stiffening is closely associated with the SCW, to determine the factors governing this phenomenon, I compared the times of egg stiffening of pairs of fragments containing different types of nuclei or cytoplasm. I found that the stiffening occurs earlier in fragments containing any type of nucleus than in those containing no nucleus, and earlier in fragments containing a male nucleus than in those containing a female nucleus. These results are consistent with the notion that either a nucleus or cytoplasm that induces the SCW earliest determines the point of initiation of the SCW close to itself. I also found that DNA replication is essential for the earlier occurrence of stiffening in a fragment containing a nucleus.

Nuclear Involvement in Localization of the Initiation Site of Surface Contraction Waves in Xenopus Eggs

The initiation site of surface contraction waves (SCWs) was examined in fertilized, parthenogenetically activated and enucleated Xenopus eggs after either rotation through 90° off the vertical axis or injection of colchicine. In enucleated eggs, SCWs always started from a top site of the egg under all conditions examined. In fertilized or activated eggs, SCWs started, depending on the experimental conditions, from either the sperm entry point, the animal pole region located sideward or the top site of the egg. Histological examinations of fertilized and activated eggs revealed that the nucleus was in most cases positioned close to the initiation site of SCWs under various experimental conditions. It is suggested from these results that the egg cytoplasm has an intrinsic capability of causing the surface to generate SCWs, and that the nucleus is generally involved in localizing the initiation site of SCWs in fertilized or activated Xenopus eggs. A possible mechanism for localizing the initiation site of SCWs in Xenopus eggs is proposed.

Periodic changes in the rigidity of activated anuran eggs depend on germinal vesicle materials

The eggs of Xenopus laevis and Bufo bufo japonicus matured in vitro by progesterone were denuded and activated by electrical shock or pricking, and their diameters were determined periodically until the postactivation time equivalent to the 8- to 16-cell stage. Normally matured eggs showed a quick rounding-up and flattening immediately after activation (activation response), followed by a periodic flattening and rounding-up (postactivation response) corresponding to the intervals of early cleavage cycles. When denuded eggs that were induced to mature after removal of the germinal vesicle (GV) were activated, they showed a normal activation response, but retained the rounded-up state without exhibiting the periodic postactivation changes. Injection of GV materials back into enucleated Bufo oocytes and determination of the postactivation flattening 120 min after activation revealed that the extent of flattening correlated well with the amount of GV materials injected, provided injection was made before the first polar body was emitted. Thus, postactivation flattening, but not activation flattening, is an active property of eggs established under the influence of the GV and is linked to the periodical changes of rigidity during early cleavage cycles.

Presence of a nucleus or nucleus-deriving factors is indispensable for the formation of the spindle, the diastema and the cleavage furrow in the blastomere of the Xenopus embryo.

The present study examines the indispensability of a nucleus or nucleus‐deriving factors in the induction of cleavage in Xenopus eggs by testing cleavage in Xenopus eggs fertilized with ultraviolet (UV)‐damaged sperm and deprived of the female nucleus. These eggs, which contain only one UV‐damaged nucleus with one set of centrioles, undergo unique cleavages. Cleavage takes place in only one of the two blastomeres formed by the immediately preceding cleavage. Histologically, only one nucleus, which does not appear to be organized into typical chromosomes, is found in one of the two blastomeres formed by the immediately preceding cleavage. The typical bipolar spindle and the diastema, or a slit of astral rays, are formed in the blastomere that contains the nucleus. By contrast, only asters lacking the spindle and the diastema are formed in the remaining blastomeres, which do not contain a nucleus. The same results are obtained in eggs that contain two UV‐damaged nuclei with one set of centrioles. In these eggs, cleavage appears to occur in one or two blastomeres that contain either or both of the nuclei and one bipolar spindle. In eggs that contain one intact and one UV‐damaged nuclei, cleavage takes place quite normally with each blastomere containing one nucleus or one set of chromosomes as well as one bipolar spindle. Thus, there is a very close correlation between the presence of a nucleus and the formation of the mitotic spindle, the diastema and the cleavage furrow in the blastomeres of Xenopus embryos. We conclude that the presence of a nucleus or nucleus‐deriving factors is indispensable for the formation of the bipolar spindle, the diastema and the cleavage furrow in the blastomeres of the Xenopus embryos..

Mechanism of dorso-ventral axis specification in nuclear transplanted eggs of Xenopus laevis

The rotation of subcortical cytoplasm relative to egg surface (“subcortical rotation”, 28) is known to specify the dorso‐ventral axis of fertilized Xenopus eggs. However, except for brief descriptions (14, 15), no information has been provided on the process of the dorso‐ventral axis specification in nuclear transplanted Xenopus eggs. This study shows that (i), among the events that occur in association with dorso‐ventral axis specification in fertilized Xenopus eggs, only the subcortical rotation occurs in a normal manner in both nuclear transplanted and non‐nuclear transplanted (enucleated) Xenopus eggs, (ii), in these eggs, there is no factor that appears to affect the direction of subcortical rotation as the sperm entry point does in fertilized eggs: neither the site of pricking for activation nor that of injection for nuclear transplantation affects the direction of the subcortical rotation in those eggs and (iii) the subcortical rotation in nuclear transplanted eggs predicts very precisely the direction of their dorso‐ventral axis as it does in fertilized eggs. These results suggest that matured Xenopus oocytes have a cytoplasmic potential to induce subcortical rotation along a particular meridian in the absence of a sperm aster and that the subcortical rotation induced by this cytoplasmic potential specifies the dorso‐ventral axis in nuclear transplanted Xenopus eggs as in fertilized eggs.

Close Correlation of the Periodicities of the Cleavage Cycle and Cytoplasmic Cycle in Early Newt Embryos

The correlation between autonomous cyclic activity of the cytoplasm (cytoplasmic cycle) and the cleavage cycle was studied by using animal and vegetal half fragments of newt eggs formed by bisecting the uncleaved eggs after those eggs had been rotated through 90° off the vertical axis so as to alter the allocations of the cytoplasmic content in the two halves. When the bisection was made shortly after the rotation (Early Bisection), the resulting vegetal fragment showed 1.5 times longer intervals in the cytoplasmic cycle than its animal partner when cleavage was prevented by injection of colchicine, while when the bisection was made 30 min after the rotation (Late Bisection), the resulting pair of halves displayed equal intervals in the cytoplasmic cycle. The intervals of cell division of vegetal halves formed by the two kinds of bisection in the absence of colchicine were then examined. In these conditions, the vegetal half formed by Early Bisection still showed 1.5 times longer intervals in the cleavage cycle than its counterpart, and the half formed by Late Bisection displayed the same intervals in the cleavage cycle as its animal partner.

Involvement of a urethane-sensitive system in timing the onset of gastrulation in Xenopus laevis embryos

This paper describes success in delaying the onset of gastrulation in Xenopus laevis embryos without damage to their subsequent development by temporarily arresting cleavage with urethane. Exposure of X. laevis embryos to 150 mM urethane before gastrulation resulted in cleavage arrest and its removal led to cleavage resumption. During cleavage arrest, cyclic activities including nuclear replication and the M‐phase‐promoting factor cycle continued, although their duration was lengthened to nearly 1.8‐fold that of the controls. Because of a 30‐min time lag from removal of urethane to resumption of cleavage, as well as the retardation of cyclic activities during cleavage arrest, the development of embryos after a 60‐min exposure to urethane lagged two cell cycles behind that of control embryos. Here, the two cell cycle delay is equivalent to 50 min at 22–23°C. The start of gastrulation in exposed embryos was accordingly delayed about 50 min, although the delay in mid‐blastula transition was as little as 20–25 min. Consistent results were obtained in embryos exposed to urethane for 90 or 120 min and those exposed to procaine or NH4Cl for 60 min. Although these results imply that delay in the start of gastrulation in exposed embryos is ascribed simply to delay in their development raised by cleavage arrest, at the same time they suggest that the onset of gastrulation is timed by systems sensitive to urethane, procaine and NH4Cl in X. laevis embryos.

Localization and behavior of putative blastopore determinants in the uncleaved Xenopus egg

The present study examines putative blastopore determinants in uncleaved Xenopus eggs. Deletion of marginal and lower portions of Xenopus eggs when between 30 and 50% of the first cell cycle has been completed (0.3–0.5 normalized time (NT)) results in the complete absence of the blastopore, while deletion of the vegetal hemisphere during the same period leads to the formation of a smaller blastopore. Extrusion of only yolk and deep cytoplasm of the vegetal hemisphere during 0.3–0.5 NT does not affect the formation or size of the blastopore. Consistently, transplantation of cortical and subcortical cytoplasm from marginal, but not other, sites of eggs at 0.3–0.5 NT to an animal blastomere from 16‐cell stage embryos induces an ectopic blastopore and bottle cell‐like cells. This does not occur in the same transplantation from eggs at 0.2 NT. These results suggest that the blastopore determinants become localized to the marginal cortical and/or subcortical cytoplasm during 0.2–0.3 NT. Other results suggest the involvement of a hexyleneglycol‐sensitive system in the process of localization of the blastopore determinants to the marginal region during 0.2–0.3 NT. The properties and behavior of the putative blastopore determinants are discussed in relation to those of VegT, which previously has been shown to induce ectopic blastopores.

Possible Involvement of a Cell Cycle Control System Dependent on Nuclear Activities in Establishment of the Cell Division Interval in Early Xenopus Embryos

Abstract In early Xenopus embryos, continuous exposure of embryos to aphidicolin and inactivation of the nucleus by ultraviolet-irradiation induce prolongation of the cell division interval. The extent of prolongation of the cell division interval appears to depend on how heavily DNA replication is suppressed by the treatments. Embryos showing significantly prolonged cell division intervals tend to fail normal cell divisions, often forming abortive furrows. There appears to be a critical point for the extent of prolongation of the cell division interval at 30% that divides the success and failure of normal cell division. This percentage, 30%, coincides with that for the prolongation of the interval of oscillatory activities seen in enucleated eggs. The presence of an intact nucleus that can undergo DNA replication rescues normal cell divisions with normal intervals and a normal furrow. Histologically, the nucleus in embryos showing more than 30% prolongation of cell division intervals appears to fail DNA replication and remain unduplicated with a round morphology like the interphase nucleus. A number of unduplicated asters lacking chromosomes are found in these embryos. We conclude from these results that early Xenopus embryos, like late embryos, have a cell cycle control system that is affected by DNA replication and is involved in establishing the cell division interval.