Cortical Rotation Research Articles

4 Microtubules and Cytoplasmic Reorganization in the Frog Egg

Publisher Summary This chapter discusses the establishment of organization in the frog egg cytoplasm in the unfertilized egg and its subsequent reorganization during the first cell cycle, in the hope that examination of these processes may reveal the nature of developmentally important differences and the mechanisms, by which they are established. The chapter describes the role of microtubules (MT) in these events. Descriptions of MT redistribution that correlate with cytoplasmic reorganization, and the disruption of cytoplasmic reorganization, by agents that affect microtubule stability, have implicated MTs in both steps of organization. Microtubules in the frog egg play essential roles in both cell cycle events (mitosis, cleavage) and key processes of the cytoplasmic organization, including: the establishment of animal-vegetal polarity and the cortical rotation that specifies the dorsal-ventral axis.

Evidence for the involvement of microtubules, ER, and kinesin in the cortical rotation of fertilized frog eggs.

During the first cell cycle, the vegetal cortex of the fertilized frog egg is translocated over the cytoplasm. This process of cortical rotation creates regional cytoplasmic differences important in later development, and appears to involve an array of aligned microtubules that forms transiently beneath the vegetal cortex. We have investigated how these microtubules might be involved in generating movement by analyzing isolated cortices and sections of Xenopus laevis and Rana pipiens eggs. First, the polarity of the cortical microtubules was determined using the "hook" assay. Almost all microtubules had their plus ends pointing in the direction of cortical rotation. Secondly, the association of microtubules with other cytoplasmic elements was examined. Immunofluorescence revealed that cytokeratin filaments coalign with the microtubules. The timing of their appearance and their position on the cytoplasmic side of the microtubules suggested that they are not involved directly in generating movement. ER was visualized with the dye DiIC16(3) and by immunofluorescence with anti-BiP (Bole, D. G., L. M. Hendershot, and J. F. Kearney, 1986. J. Cell Biol. 102:1558-1566). One layer of ER was found closely underlying the plasma membrane at all times. An additional, deeper layer formed in association with the microtubules of the array. Antibodies to sea urchin kinesin (Ingold, A. L., S. A. Cohn, and J. M. Scholey. 1988. J. Cell Biol. 107:2657-2667) detected antigens associated with both the ER and microtubules. On immunoblots they recognized microtubule associated polypeptide(s) of approximately 115 kD from Xenopus eggs. These observations are consistent with a role for kinesin in creating movement between the microtubules and ER, which leads in turn to the cortical rotation.

Patterns of microtubule polymerization relating to cortical rotation in Xenopus laevis eggs

Following fertilization, the Xenopus egg cortex rotates relative to the cytoplasm by 30 degrees about a horizontal axis. The direction of rotation, and as a result the orientation of the embryonic body axes, is normally specified by the position of sperm entry. The mechanism of rotation appears to involve an array of aligned microtubules in the vegetal cortex (Elinson and Rowning, 1988, Devl Biol. 128, 185-197). We performed anti-tubulin immunofluorescence on sections to follow the formation of this array. Microtubules disappear rapidly from the egg following fertilization, and reappear first in the sperm aster. Surprisingly, astral microtubules then extend radially through both the animal and vegetal cytoplasm. The cortical array arises as they reach the vegetal cell surface. The eccentric position of the sperm aster gives asymmetry to the formation of the array and may explain its alignment since microtubules reaching the cortex tend to bend away from the sperm entry side. The radial polymerization of cytoplasmic microtubules is not dependent on the sperm aster or on the female pronucleus: similar but more symmetric patterns arise in artificially activated and enucleate eggs, slightly later than in fertilized eggs. These observations suggest that the cortical microtubule array forms as a result of asymmetric microtubule growth outward from cytoplasm to cortex and, since cortical and cytoplasmic microtubules remain connected throughout the period of the rotation, that the microtubules of the array rotate with the cytoplasm.

Deep cytoplasmic rearrangements during early development in Xenopus laevis

The egg of the frog Xenopus is cylindrically symmetrical about its animal-vegetal axis before fertilization. Midway through the first cell cycle, the yolky subcortical cytoplasm rotates 30 degrees relative to the cortex and plasma membrane, usually toward the side of the sperm entry point. Dorsal embryonic structures always develop on the side away from which the cytoplasm moves. Details of the deep cytoplasmic movements associated with the cortical rotation were studied in eggs vitally stained during oogenesis with a yolk platelet-specific fluorescent dye. During the first cell cycle, eggs labelled in this way develop a complicated swirl of cytoplasm in the animal hemisphere. This pattern is most prominent on the side away from which the vegetal yolk moves, and thus correlates in position with the prospective dorsal side of the embryo. Although the pattern is initially most evident near the egg's equator or marginal zone, extensive rearrangements associated with cleavage furrowing (cytoplasmic ingression) relocate portions of the swirl to vegetal blastomeres on the prospective dorsal side.

Polarity of the surface and cortex of the amphibian egg from fertilization to first cleavage

The anuran egg is polarized along its animal-vegetal axis and becomes bilaterally symmetrical before first cleavage. Functional sperm entry is regionally restricted to the animal hemisphere of the egg, and functional sperm entry does not occur after egg activation. This regional and functional restriction in sperm entry correlates with the presence of long, slender microvilli and with the presence of the filamentous component of the glycocalyx. After sperm fusion, the egg undergoes activation, including a depolarization of the membrane potential and exocytosis of granules in the cortex. Both of these activation responses are the result of a propagated increase in intracellular calcium. The egg's ability to undergo a propagated activation response develops after germinal vesicle breakdown and depends on the development of the cortical endoplasmic reticulum. Once activated, the radial symmetric egg acquires bilateral symmetry due to a rotation of the egg cortex relative to the inner cytoplasm. A transient array of parallel microtubules forms near the vegetal cortex and may be part of the motor driving the cortical rotation.

Hyperdorsoanterior embryos from Xenopus eggs treated with D 2O

Excessively dorsalized embryos of Xenopus laevis develop from eggs treated with 30–70% D 2O for a few minutes within the first third of the cell cycle following fertilization. As the concentration of D 2O and the duration of exposure are increased, the anatomy of these embryos shifts in the direction of enlarged dorsal and anterior structures and reduced ventral and posterior ones. Twinning of dorsoanterior structures is frequent. Intermediate forms include embryos with large heads but no trunks or tails. The limit form of the series has cylindrical symmetry, with circumferential bands of eye pigment and cement gland, a core of notochord-like tissue, and a centrally located beating heart. D 2O treatment seems to increase the egg's sensitivity to the dorsalizing effects of cortical rotation and to stimulate the egg to initiate two or more directions of rotation. Such eggs probably establish thereafter a widened and/or duplicated Nieuwkoop center in the vegetal hemisphere, with the subsequent induction of a widened and/or duplicated Spemann organizer region in the marginal zone, which leads to excessive dorsal development. The existence of these anatomical forms indicates the potential of the egg to undertake dorsal development at all positions of its circumference and suggests that normal patterning depends on the limited and localized activation or disinhibition of this widespread potential.

Determinants of Early Amphibian Development

The animal-vegetal organization of the amphibian egg may originate from the axis of organelles and cytoskeletal elements established in the oocyte as it divides from the oogonium. Along this axis, cytoplasmic materials are localized during oogenesis: yolk platelets, for example, are translocated toward the vegetal pole, increasing their amount and size in that region. In the first cell cycle after fertilization, the egg cortex rotates 30° relative to the cytoplasmic core, modifying animal-vegetal organization. The direction of this rotation, biased by the point of sperm entry, defines the site of development of anatomical structures of the dorsal midline of the embryo. As its immediate effect, rotation activates the cytoplasm of a subregion of the vegetal hemisphere, causing cells cleaved from this subregion to be more effective than other vegetal parts in inducing marginal zone cells to initiate gastrulation movements. The most strongly induced part of the marginal zone begins gastrulation first (the dorsal lip of the blastopore) and proceeds through a series of cell interactions leading to its determination as the anterior dorsal mesoderm of the embryo. If these cell movements are inhibited in the gastrula stage, or if vegetal induction is inhibited in the blastula stage, or if cortical rotation is inhibited in the first cell cycle after fertilization, the embryo always fails to develop dorsal structures of the anterior end of its body axis; the more inhibition, the more posterior is the level of truncation, until a radial ventralized embryo develops, derived from the animal-vegetal organization of the oocyte.