Thomas E. Schroeder

Cytoplasmic filaments and morphogenetic movement in the amphibian neural tube

Abstract Cells of the medullary plate and groove were studied with the electron microscope during neurulation in Hyla regilla and Xenopus laevis in an attempt to locate structures that might be involved in morphogenetic movement. The apices of the neural plate cells contain a thin band of filaments aligned parallel to the outer surface of the plate. As neurulation proceeds, the groove narrows and deepens; correspondingly, the cell apices narrow, the apical band becomes broader and crowded with oriented filaments, and the cell body assumes a long columnar or bottle shape. In Xenopus filaments are arranged circularly around the cell neck. The cell apices are tightly joined to one another. It is proposed that filament contraction causes the neural plate to invaginate. This theory is discussed in relation to other theories of neurulation and morphogenetic movements.

Surface area change at fertilization: Resorption of the mosaic membrane☆

Abstract Eggs of Strongylocentrotus purpuratus (sea urchin) have a surface area of 41,000 μm 2 before fertilization as determined by quantitative transmission and scanning electron microscopy. Within a minute after fertilization 18,000 cortical vesicles contribute an additional 57,000 μm 2 to form a mosaic membrane with the original plasma membrane. However, by 16 min after fertilization the total area of the egg is only 45,000 μm 2 , indicating a rapid resorption of surface. Calculations of surface area depend in large part upon the numbers and dimensions of microvilli, after careful compensations are made for specimen shrinkage. The 134,000 microvilli per egg are 0.35 μm long before fertilization. They elongate to 1.0 μm in the first few minutes and then soon shorten to 0.5 μm. Even at their longest, microvilli do not accommodate all of the surface area of cortical vesicle membrane. The merger of cortical vesicle membranes and the plasma membrane was demonstrated many years ago and is not in doubt; however, this study indicates that the resulting mosaic membrane is not a long-lived, simple arithmetic combination of its components. Rather, the mosaic membrane undergoes a rapid and dynamic shrinkage by a mechanism which is not apparent on the basis of egg topography alone. The absolute values of egg surface area and dynamic changes in the surface are discussed in relation to physiological events accompanying fertilization.

Ionophore A23187, calcium and contractility in frog eggs

Abstract Ionophore A23187 elicits rapid cortical contractions in frog eggs at 10 −6 M conc. in the presence or complete absence of extracellular calcium. Intracellular EDTA or EGTA inhibits contractility, suggesting the importance of intracellular calcium for the response. Similarities and differences between these contractions, cell cleavage, and wound-closure are discussed.

Microvilli on sea urchin eggs: A second burst of elongation

Abstract A scanning EM study reveals about 300,000 microvilli on each egg of the sea urchin Strongylocentrotus droebachiensis . The microvilli are about 0.2 μm long before fertilization, elongate to about 0.5 μm soon after fertilization (the “first burst” of microvillus elongation), and subsequently elongate again about midway between fertilization and first cell division (the “second burst” of elongation). The second burst occurs during a discrete 30-min period and results in some microvilli being as long as 10 μm, although the average length is about 1.8 μm. The surface area of the egg following the second burst is about 2.7 times the area of the unfertilized egg.

Morphological changes during maturation of starfish oocytes: Surface ultrastructure and cortical actin☆

The cell surface and extracellular investments of oocytes of the starfish Pisaster ochraceus are analyzed by Nomarski differential interference contrast microscopy and by scanning electron microscopy. The investing coats include a thin sheet of follicle cells, a jelly coat, and a vitelline layer; their morphologies are described. Methods are outlined for systematically removing them without altering the behavior of the oocyte so that the cell surface can be examined directly. The topography of denuded oocytes changes dramatically when they are treated with the maturation-inducing hormone, 1-methyladenine. The major topographical change is the early and transient formation of prominent surface spikes. These structures arise due to the rapid, reversible polymerization of actin into stout bundles. Polymerization and subsequent depolymerization of cortical actin is monitored by epifluorescence microscopy of oocytes stained with NBD-phallacidin, a stain which is specific for polymerized actin. Based on scanning electron microscopy, spikes apparently utilize preexisting plasma membrane of microvilli, and plasma membrane is apparently lost when spikes collapse. Long after microvilli are eliminated due to spike formation, the number of microvilli is somewhat restored, especially around the animal pole where the polar body forms. A chronology of events observed during oocyte maturation is discussed with reference to the possible mechanisms and implications of polymerization and depolymerization of cortical actin.

Fourth cleavage of sea urchin blastomeres: Microtubule patterns and myosin localization in equal and unequal cell divisions

This study traces the morphological appearance, organization, and disappearance of the cytoskeletal machinery for cell division during the fourth cell cycle of isolated sea urchin blastomeres by immunolocalization of tubulin and myosin. Mesomere-mesomeres (which divide equally) and macromere-micromeres (which divide unequally) are compared in terms of their asters (both mitotic and so-called interphase asters), spindle apparatus, and contractile ring. The results suggest that the distinctive nuclear positioning of these blastomeres is established in late interphase, that centrosomal alignment occurs in prophase, that all of the dominant astral configurations in the cell cycle belong to a single cycle of assembly-disassembly, that a second interphase-specific cycle of assembly-disassembly is confined to a diffuse cytoplasmic reticulum, and that contractile ring myosin concentrates and disperses in precise coincidence with the beginning and end of cleavage furrowing.

Expressions of the prefertilization polar axis in sea urchin eggs

Abstract The distribution of pigment granules in eggs of three species of sea urchins is described with reference to developmental stage and an eggs animal-vegetal axis of organization. Polarity in unfertilized sea urchin eggs has been a debated subject; present evidence demonstrates that the animal-vegetal axis is established before fertilization. The pigment pattern in some batches of Paracentrotus eggs exhibiting the celebrated “pigment band,” originally described by Theodor Boveri, is revised and is interpreted as a comparatively precocious expression of the underlying egg polarity. “Unbanded” Paracentrotus eggs and eggs of Arbacia lixula and Arbacia punctulata can be induced to exhibit the same pigment pattern by artificial activation. The induced pigment pattern aligns with an axis defined by polar bodies and the jelly canal, which are two external markers of the animal pole which are only rarely seen. It is therefore concluded that all of these eggs possess an animal-vegetal axis before fertilization even though it usually remains unexpressed until later developmental stages. Polarized changes in pigmentation are consistent with the following general mechanism: A change is triggered in the cortex of the vegetal pole; the change is programmed for a time which corresponds to the fourth mitotic division, even though mitosis itself is not involved; activation at fertilization initiates the “clock” in most cases, although in “banded” Paracentrotus eggs the “clock” is apparently started before ovulation; only the vegetal hemispheres pigment is affected by the change. The nature of the underlying axis which defines animal and vegetal poles is discussed. Aspects of the axis have been tentatively traced back to the primary oocyte stage, but its fundamental nature remains unknown.

The origin of cleavage forces in dividing eggs. A mechanism in two steps.

Abstract The mechanical behavior of sea urchin eggs is examined from soon after fertilization up to the time that the cleavage furrow begins to form. The procedure involves compressing eggs between parallel plates so that tensile forces in the egg surface can be calculated. Results show that eggs of Strongylocentrotus purpuratus at 12–13 °C undergo a strong cortical contraction which significantly precedes and is independent of mitosis and which persists through anaphase, the time when the cleavage stimulus from the mitotic asters differentiates the cortex for cleavage. The cleavage furrow begins to form when cortical tension is high, although there is no obvious incremental increase in tension which specifically correlates with the cleavage stimulus. It is therefore concluded as a tenable hypothesis that there are two separate but coordinated steps which occur while the egg is still spherical and which then lead to cleavage. The cleavage stimulus, instead of causing contraction as often believed, elicits relaxation of the polar cortex after the entire cortex has first contracted by an independent mechanism. Accordingly, the global contraction-polar relaxation hypothesis explains that the motive force for the cleavage furrow actually originates from the prior general contraction and, in a strict sense, is independent of mitosis. The mitosis-linked step is brought about by the cleavage stimulus and upsets the prior balance of high tensile forces by relaxing the poles; this channels the cortical contractility into the equator, resulting in the formation of the cleavage furrow. These ideas are contrasted with alternate hypotheses of cleavage initiation.

The Contractile Ring and Furrowing in Dividing Cells

The contractile ring is the cytoskeletal structure believed to generate the constriction force for cells that divide by furrowing, namely, all animal cells and a few plant cells. Although its function seems superficially to be reflected in its organization, in many ways it remains poorly understood. To gain an integrated picture of the contractile ring, we need to know a great deal more about what it is morphologically and biochemically, how it works mechanically to produce a furrow, where its components come from and how they are organized, and how its formation is stimulated by antecedent events in mitosis. These are challenging goals, especially in the face of three obstacles: first, the cells that divide by furrowing vary widely in character and the diversity tends to conflict with efforts to find unifying, universal principles of mechanism; second, the techniques for detecting or quantifying the necessary details of the contractile ring are often too crude and imprecise to answer clearly the experimental questions that are asked, and third, the impatience to “solve” the mechanism of furrowing has blurred the distinction between facts and imagination. In this article I will first reiterate the contractile ring’s basic organizational features, for they establish the principle framework in which a morphologist views other aspects of furrowing. I will then briefly consider possible origins of the contractile ring as well as the events that are thought to stimulate its formation. Finally, I will discuss a global contractile phenomenon of the cell cortex that precedes furrowing and that poses a special set of questions concerning furrowing and other allied constriction. I urge any reader who might be seriously interested in furrowing to consult thoroughly the extensive original literature and yet to maintain an open mind about what has been written.

Cell cleavage: Ultrastructural evidence against equatorial stimulation by aster microtubules☆

Abstract Cell cleavage is spatially and temporally coordinated with karyokinesis. In astral division, as occurs in sea urchin eggs, coordination is accomplished by the mitotic asters. We have explored the following hypotheses: 1. 1. That microtubules of the two asters cross at the cells equator. 2. 2. That because they cross, or by some other configuration, more microtubules interact with the equatorial cortex than with the polar cortex. 3. 3. That the microtubule component of astral rays differentially stimulates the equatorial cortex for cleavage contraction. Using a fixation procedure which enhances visibility of microtubules, we have determined that aster microtubules do not cross at the equatorial cortex at any stage of mitosis relevant to cleavage stimulation, contrary to the first hypothesis. Aster microtubules extend progressively farther during anaphase, but the two arrays occupy mutually exclusive hemispheres in the egg. Using another fixation procedure which results in more conventional microtubule morphology, we have systematically counted microtubules penetrating the cortex at both the equator and the poles in sections cut parallel and perpendicular to the axis of the mitotic apparatus, respectively, at all stages of mitosis. We did not observe any microtubules in the cortex of the equator during prometaphase, metaphase, early anaphase or mid-anaphase. In comparison, small numbers of microtubules were observed in the polar cortex during this time. By late anaphase there are some microtubules in the equatorial cortex but many more are observed in the polar cortex. These findings are contrary to the second hypothesis and therefore do not establish the morphological basis for the third hypothesis. We conclude that there is no positive correlation between microtubule numbers at the egg equator and the timing of cleavage stimulation. Therefore, coordination between karyokinesis and cell cleavage is achieved by some process other than the simple numerical increase of microtubules at the equatorial cortex.