Three-dimensional Analysis of Laser Scanning Confocal Microscope Sections Reveals an Array of Microtubules in the Cleavage Furrow of Sea Urchin Eggs.

Our recent observation that microtubules (MTs) are required for completion of division (abscission) led us to analyze MT organization during cytokinesis. Although many studies of MTs in sea urchin eggs have been done, computer-aided analysis of optical sections described herein reveals a new MT assemblage, which we call furrow MTs. This assemblage comprises bundles of MTs that lie in the cleavage furrow. Furrow MTs become apparent when the furrow has progressed approximately one-third of the way through the egg and persist to abscission. Furrow MTs are 8-24-&mgr;m long and arc across the base of the cleavage furrow. Acetylated tubulin is localized primarily in the furrow suggesting a distinct MT population. Three-dimensional analysis of optical sections suggests that furrow MTs are spatially distinct from midbody and astral MTs.

Microtubules are required for completion of cytokinesis in sea urchin eggs.

Completion of cytokinesis, abscission, has been studied little despite the intensive studies of the onset and contractile mechanism of the earlier phases of division. It has been well documented that microtubule (MT) disruption before furrow stimulation prevents furrowing, while MT disruption after furrow stimulation allows division to proceed. We have confirmed those findings using the MT inhibitors, nocodazole and demecolcine. In addition, we have found that MT disruption after furrow stimulation but before completion of division prevents abscission as evidenced by the observation that prospective daughter cells in MT-disrupted eggs maintain electrical continuity. Continued observation of eggs revealed that the furrow in MT-disrupted eggs did not result in abscission, but rather held steady until the time when controls underwent second cleavage, at which point the furrows regressed. These findings extend the recent reports that MTs are required for completion of division in mammalian tissue culture cells and frog eggs, to invertebrates, suggesting a common mechanism of abscission for animal cells.

Ventral cell rearrangements contribute to anterior-posterior axis lengthening between neurula and tailbud stages in Xenopus laevis.

Studies of morphogenesis in early Xenopus embryos have focused primarily on gastrulation and neurulation. Immediately following these stages is another period of intense morphogenetic activity, the neurula-to-tailbud transition. During this period the embryo is transformed from the spherical shape of the early stages into the long, thin shape of the tailbud stages. While gastrulation and neurulation depend largely on active cell rearrangement and cell shape changes in dorsal tissues, we find that the neurula-to-tailbud transition depends in part on activities of ventral cells. Ventral explants of neurula lengthen autonomously as much as the ventral sides of intact embryos, while dorsal explants lengthen less than the dorsal sides of intact embryos. Analyses of cell division, cell shapes, and cell rearrangement by transplantation of labeled cells and by time lapse recordings in live intact embryos concur that cell rearrangements in ventral mesoderm and ectoderm contribute to the autonomous anterior-posterior axis lengthening of ventral explants between neurula and tailbud stages.

Requirement for microtubules in new membrane formation during cytokinesis of Xenopus embryos.

In cleaving Xenopus eggs, exposure to nocodazole or cold shock prevents the addition of new plasma membrane to the cleavage plane and causes furrows to recede, suggesting a specific role for microtubules in cytokinesis. Whole-mount confocal immunocytochemistry reveals a ring of radially arranged, acetylated microtubule bundles at the base of all advancing cleavage furrows, from the first cleavage through the midblastula stage. We hypothesize that this novel microtubular structure is involved in transporting maternal stores of membrane in the subcortex to a site of membrane addition near the leading edge of the furrow.

Inhibition of morphogenetic movement during Xenopus gastrulation by injected sulfatase: implications for anteroposterior and dorsoventral axis formation.

In order to explore the role of morphogenetic movement in the establishment of anteroposterior and dorsoventral axes, we sought to identify novel in vivo inhibitors of gastrulation movements in Xenopus laevis. Injection of hydrolytic sulfatase into the blastocoels of gastrula stage embryos resulted in severe anteroposterior truncation, without a corresponding truncation of the dorsoventral axis. Confocal microscopy of whole embryos revealed that gastrulation movements are severely disrupted by sulfatase; in addition, sulfatase dramatically inhibited chordomesodermal cell elongation and convergent extension movements in planar dorsal marginal zone explants. The phenotype of anteroposterior reduction elicited by sulfatase is distinctly different from commonly generated dorsoanterior phenotypes (e.g., ultraviolet irradiation of the vegetal cortex prior to cortical rotation or suramin injection), and the two varieties of phenotype appear to result from inhibition of distinct, separable components of the axis-generating machinery.

Xwnt-8 and lithium can act upon either dorsal mesodermal or neurectodermal cells to cause a loss of forebrain in Xenopus embryos.

When Xenopus gastrulae are made to misexpress Xwnt-8, or are exposed to lithium ions, they develop with a loss of anterior structures. In the current study, we have characterized the neural defects produced by either Xwnt-8 or lithium and have examined potential cellular mechanisms underlying this anterior truncation. We find that the primary defect in embryos exposed to lithium at successively earlier stages during gastrulation is a progressive rostral to caudal deletion of the forebrain, while hindbrain and spinal regions of the CNS remain intact. Misexpression of Xwnt-8 during gastrulation produces an identical loss of forebrain. Our results demonstrate that lithium and Wnts can act upon either prospective neural ectodermal cells, or upon dorsal mesodermal cells, to cause a loss of anterior pattern. Specifically, ectodermal cells isolated from lithium- or Wnt-exposed embryos are unable to form anterior neural tissue in response to inductive signals from normal dorsal mesoderm. In addition, although dorsal mesodermal cells from lithium- or Wnt-exposed embryos are specified properly, and produce normal levels of the anterior neural inducing molecules noggin and chordin, they show a greatly reduced capacity to induce anterior neural tissue in conjugated ectoderm. Taken together, our results are consistent with a model in which Wnt- or lithium-mediated signals can induce either mesodermal or ectodermal cells to produce a dominant posteriorizing morphogen which respecifies anterior neural tissue as posterior.

Relocation of mitochondria to the prospective dorsal marginal zone during Xenopus embryogenesis.

Dorsal-ventral axis formation in Xenopus laevis begins with a cytoplasmic rotation during the first cell cycle and culminates in a series of cell interactions and movements during gastrulation and neurulation that lead to the formation of dorsal-anterior structures. Evidence reported here indicates that mitochondria are differentially redistributed along the prospective dorsal-ventral axis as a consequence of the cortical-cytoplasmic rotation during the first cell cycle. This finding reinvigorates a possibility that has been considered for many years: asymmetries in cytoplasmic components and metabolic activities contribute to the development of morphological asymmetries.

Provisional bilateral symmetry in Xenopus eggs is established during maturation.

Dorsal-ventral patterning in the Xenopus egg becomes established midway through the first cell cycle during a 30 degree rotation of the subcortical yolk mass relative to the egg cortex. This 'rotation of symmetrisation' is microtubule dependent, and its direction is thought to be cued by the usually eccentric sperm centrosome. The fact that parthenogenetically activated eggs also undergo a directed rotation, despite the absence of a sperm centrosome, suggests that an endogenous asymmetry in the unfertilised egg supports the directed polymerisation of microtubules in the vegetal cortex, in the way that an eccentric sperm centrosome would in fertilised eggs. Consistent with this idea, we noticed that the maturation spot is usually located an average of more than 15 degrees from the geometric centre of the pigmented animal hemisphere. In parthenogenetically activated eggs, this eccentric maturation spot can be used to predict the direction of rotation. Although in most fertilised eggs the yolk mass rotates toward the sperm entry point (SEP) meridian, occasionally this relationship is perturbed significantly; in such eggs, the maturation spot is never on the same side of the egg as the SEP. In oocytes tilted 90 degrees from upright during maturation in vitro, the maturation spot developed 15 degrees or more from the centre of the pigmented hemisphere, always displaced towards the point on the equator that was up during maturation. This experimentally demonstrated lability is consistent with an off-axis oocyte orientation during oogenesis determining its eccentric maturation spot position, and, in turn, its endogenous rotational bias.

Deep cytoplasmic rearrangements in ventralized Xenopus embryos.

Following fertilization in Xenopus, dramatic rearrangements of the egg cytoplasm relocalize maternally synthesized egg components. During the first cell cycle the vegetal yolk mass rotates relative to the egg surface, toward the sperm entry point (SEP) (J. P. Vincent, G. F. Oster, and J. C. Gerhart, 1986, Dev. Biol. 113, 484-500), while concomitant deep cytoplasmic rearrangements occur in the animal hemisphere (M. V. Danilchik and J. M. Denegre, 1991, Development 111, 845-856). In this paper we examine the role of vegetal yolk mass rotation in producing the animal cytoplasmic rearrangements. We inhibited rotation by uv-irradiating embryos during the first cell cycle, a treatment that yields an extremely ventralized phenotype. Both uv-irradiated embryos and unirradiated control embryos show cytoplasmic rearrangements in the animal hemisphere during the first cell cycle. Cytoplasmic rearrangements on the SEP side of the embryo associated with the path of the sperm pronucleus, plus a swirl on the anti-SEP (dorsal) side, are seen, whether or not yolk mass rotation has occurred. This result suggests a role for the expanding sperm aster in directing animal hemisphere cytoplasmic movements. In unirradiated control embryos the anti-SEP (dorsal) swirl is larger than that in uv-irradiated embryos and often extends into the vegetal hemisphere, consistent with the animal cytoplasm having been pulled dorsally and vegetally by the sliding vegetal yolk mass. Thus the yolk mass rotation may normally enhance the dorsalward cytoplasmic movement, begun by the sperm aster, enough to induce normal axis formation. We extended our observations of unirradiated control and uv-irradiated embryos through early cleavages. The vegetal extent of the anti-SEP (dorsal) swirl pattern seen in control embryos persists through the early cleavage period, such that labeled animal cytoplasm extends deep into dorsal third-tier blastomeres at the 32-cell stage. Significantly, in uv-irradiated embryos, which have not undergone vegetal rotation, most of this labeled material remains more equatorial.

Deep cytoplasmic rearrangements in axis-respecified Xenopus embryos.

In fertilized eggs of the frog Xenopus, the vegetal yolk mass rotates away from the future dorsal side (J. P. Vincent and J. Gerhart, 1987, Dev. Biol. 123, 526-539), and a major rearrangement of the deep animal hemisphere cytoplasm produces a characteristic swirl in the prospective dorsal side (M. V. Danilchik and J. M. Denegre, 1991, Development 111, 845-856). The relationship between this swirl and determination of the dorsal-ventral axis was further investigated by attempting to experimentally separate the positions of the swirl and the dorsal-ventral axis. Eggs were obliquely oriented in the gravity field to respecify the direction of yolk mass rotation and the position of the dorsal-ventral axis. When yolk mass rotation occurred in the absence of a sperm, as in activated eggs, a swirl pattern formed on the side away from which the yolk mass had rotated. In fertilized eggs tipped with the sperm entry point (SEP) down or to the side, swirl patterns were always found to form on the side away from which the yolk mass was displaced. However, in eggs tipped SEP up, in which the yolk mass was forced to rotate away from the SEP, more complicated rearrangements were observed in addition to the rotation-oriented swirl. Because the direction of yolk mass rotation was found to be influenced by both gravity and the actual position of the SEP in obliquely oriented eggs (SEP to the side), such complicated rearrangement patterns may result from opposing forces generated by both yolk mass rotation and the expanding sperm aster. Thus, except in cases in which the influences of SEP position and unit gravity opposed each other, it was not possible to experimentally separate the position of the deep cytoplasmic swirl from the direction of yolk mass rotation, and therefore the position of the prospective dorsal side.

Dynamics of germ plasm localization and its inhibition by ultraviolet irradiation in early cleavage Xenopus embryos.

In the spawned Xenopus egg, germ plasm is found as cytoplasmic islands spread over a wide cortical region of the vegetal pole. However, by the blastula stage, the same material is found concentrated into a few large blastomeres at the floor of the blastocoel. Components of the germ plasm can be specifically labeled with a fluorescent dye, DiOC6(3), permitting the dynamic movements of germ plasm localization to be followed in live embryos via time-lapse confocal scanning optical microscopy. During the first cell cycle, the small islands initially appear to be fixed to the vegetal yolk mass and to move with it during the cortical rotation. After rotation, the islands appear to be released from the vegetal yolk mass and to begin fusing with one another. During early cleavages, the germ plasm aggregates into large islands at the vegetal pole in a movement dependent on microtubules. Two distinct actions can be discerned: (1) a continuous process of local fusing and (2) periodic surface contraction waves (SCWs) that gather the islands toward the vegetal pole. These SCWs are inhibited by ultraviolet irradiation of the vegetal pole. Near the vegetal pole, germ plasm patches ingress into the embryo's interior along the cleavage furrow in periodic movements contemporaneous with the SCWs.

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.

The first cleavage plane and the embryonic axis are determined by separate mechanisms in Xenopus laevis. I. Independence in undisturbed embryos.

We examined the spatial relationships between the meridian of sperm entry the plane of first cleavage, and the embryonic axis (defined by the neural groove) in eggs of Xenopus laevis. Direct measurement of the angular separations between these embryonic structures in gelatin-embedded eggs confirmed the classical conclusion that the sperm entry point and neural groove tend to form on opposite sides of the egg, and also revealed that the first cleavage plane has a nearly random orientation with respect to the neural groove. We next examined the distortion of the first cleavage plane that results from the normal processes of convergence and extension during gastrulation and neurulation. We permanently marked the first cleavage plane by injecting one blastomere of the two-cell embryo with a fluorescent lineage marker. At the start of gastrulation, the interface between the labeled and unlabeled regions was almost randomly oriented relative to the dorsal blastopore lip, confirming our first set of observations. In embryos with the interface less than 60 degrees to the plane passing through the midline of the dorsal lip, convergent movements of cells produced a confrontation of labeled and unlabeled cells along much of the dorsal midline. Thus, although the first cleavage plane and the bilateral plane were frequently not congruent, the morphogenetic movements of gastrulation and neurulation brought about an apparent congruence in many half-labeled embryos.

Differentiation of the animal-vegetal axis in Xenopus laevis oocytes. I. Polarized intracellular translocation of platelets establishes the yolk gradient.

The animal-vegetal axis of the oocyte of Xenopus laevis is recognizable not only by the pattern of surface pigmentation, but also by the distribution of yolk platelets, with the largest platelets (congruent to 14 microns in diameter) and 70% of the total yolk protein localized in the vegetal hemisphere. We have used fluorescent and radioactive vitellogenins (yolk protein precursors) to study the spatial and temporal patterns of yolk deposition along this axis. We find that the rate of uptake of vitellogenin is nearly uniform over the surface of vitellogenic oocytes of all sizes. Once formed, yolk platelets in the animal hemisphere move inward, around the germinal vesicle, and into the central region of the vegetal hemisphere. Yolk platelets of the vegetal hemisphere do not actively move but are slowly displaced from the surface by successive layers of younger platelets arising and enlarging near the surface. The oldest yolk platelets, which arise circumcortically at the beginning of vitellogenesis in stage II and III oocytes, eventually come to reside in the vegetal hemisphere of stage VI oocytes, in the upper portion of the cup-shaped region of largest platelets. The vegetal hemisphere thus gains the majority of yolk protein by directed intracellular transport from the animal hemisphere adding to the amount directly sequestered by the vegetal hemisphere.

Separate ribosomal pools in sea urchin embryos: ammonia activates a movement between pools.

Monoribosomes from unfertilized eggs of Strongylocentrotus purpuratus were shown to translate mRNA less efficiently than ribosomes derived from polyribosomes of embryos, as measured by globin synthesis in a ribosome-dependent rabbit reticulocyte lysate [Danilchik, M. V., & Hille, M. B. (1981) Dev. Biol. 84, 291-298]. Data presented in this paper show that monoribosomes from 16-cell and blastula embryos resemble monoribosomes from unfertilized eggs in translational capacity and are less active than the ribosomes associated with polyribosomes. Thus, we find two distinct populations of ribosomes in embryos. We define the less active monoribosome population as "naive" ribosomes and the more active, functioning polysome-derived ribosomes as "experienced" ribosomes. Naive and experienced ribosomes have the same elongation rates. The relationship between ionic triggers and the conversion of monoribosomes to experienced ribosomes was studied with the Ca2+ ionophore A23187, which releases intracellular Ca2+ stores, and NH4Cl, which alkalinizes the cytoplasm. We found that ribosomes in the monoribosome populations from A23187-activated eggs or from NH4Cl-activated eggs resembled naive monoribosomes from unfertilized eggs in their translational activity. In contrast, ribosomes derived from the polysomes of NH4Cl-treated eggs were as active as the experienced polysome-derived ribosomes from normal embryos. Eggs activated with A23187 did not produce polyribosomes. The presence of significant amounts of experienced ribosomes in NH4Cl-treated eggs implicates alkalinization of the cytoplasm as a stimulus for ribosome activation, which occurs slowly during initial development.

Sea urchin egg and embryo ribosomes: differences in translational activity in a cell-free system.

To determine whether ribosomes have a role in the postfertilization activation of protein synthesis in sea urchin eggs, we measured the translational activity of ribosomes isolated from unfertilized eggs and embryos of Strongylocentrotus purpuratus. Numerous previous studies have indicated few if any differences in the activity of such ribosomes. However, by using improved physiological isolation and in vitro conditions, we have found important differences in the activities of egg and embryo ribosomes. Ribosomes obtained from blastula polyribosomes were active in translating reticulocyte mRNA in a ribosome-dependent cell-free translation system, whereas ribosomes obtained from unfertilized eggs became fully active only after a characteristic, reproducible delay of up to 15 min at 26 degrees C. The extent of this delay varied with incubation pH, but not with concentrations of K+, Mg2+, initiation factors, or mRNA. However, at incubation pH between 6.90 and 7.65, the egg ribosomes were always less active than blastula ribosomes.