CORTICAL AND SUBCORTICAL CHANGES PRECEDING FURROW FORMATION IN THE CLEAVAGE OF NEWT EGGS

In the first cleavage of the egg of the newt, Cynops(Triturus) pyrrhogaster, some sort of preparation for the furrow formation in the cortical and subcortical cytoplasm precedes the advancing tip of the cleavage furrow. This is shown by the following facts: (1) Incisions made close to the tip of the cleavage furrow do not stop the progress of furrowing, allowing the furrow to cross the incisions and appear on the farther sides, while incisions made far enough from the furrow tip always prevent the further travelling of the furrow, (2) Displacement of the subcortical cytoplasm ahead of the furrow by rubbing with a hair loop makes the furrow bend corresponding to the width of the rubbed area, and (3) Transplantation of the subcortical material of the furrow tip to lateral parts in the same egg causes a depression in the overlying cortex at the transplanted position. The linear extension of the prepared area for the furrow formation is the longest in the animal hemisphere and it decreases in gradient towards the vegetal pole.     

ON PROPAGATION OF CORTICLA FACTOR AND CYTOPLASMIC FACTOR PARTICIPATING IN CLEAVAGE FURROW FORMATION OF THE NEWT'S EGG*

From Cynops pyrrhogaster eggs just after the start of the first cleavage, a fragment of cortical layer with a small entire cleavage furrow was cut out. In the fragment, the cortex had already acquired susceptibility to and the subcortical cytoplasm had already accquired inducibility for furrow formation. The fragment was transplanted to the animal hemisphere of uncleaved fertilized eggs or eggs immediately after the onset of the first cleavage, from which a portion of the host cortex was removed. Observation was made on division of the graft, and on propagation of the cortical susceptibility and the cytoplasmic inducibility of the graft onto the host egg. The transplant divided succesively on the host egg in many cases, but the furrow of the graft never advanced to the surface of the host egg. Neither the cortical factor nor the cytoplasmic factor was transmitted across the graft to the recipient egg.     

INDUCTION OF A FURROW-LIKE DENT ON THE SURFACE OF THE NEWT EGG BEFORE THE ONSET OF CLEAVAGE*

Sawai (2) found in the amphibian egg that furrow-inducing cytoplasmic component (FIC) was localized along the cleavage furrow, which could induce a furrow on the polar surface of cleaving egg under which FIC was injected. But this procedure failed on the surface of uncleaved fertilized egg. In the present experiments, an attempt was made to induce a cleavage furrow on the surface of uncleaved egg of the newt, Cynops pyrrhogaster. A piece of the cortex was cut from the uncleaved egg, which was transplanted to the egg just before or just after the onset of the cleavage, using a fine glass needle. After the transplantation FIC was injected beneath the graft with a capillary. The graft reacted to FIC and a furrow-like dent was induced at the position. Besides, stiffness of the graft increased during the cleavage of the host egg. In contrast to the cortical grafting, a large amount of the cytoplasm excluding FIC was injected under the cortex of an uncleaved egg. After several minutes FIC was deposited at the site. A furrow-like dent was formed there in many cases.     

SURFACE CHARACTERS OF DIVIDING CELLS III. UNEQUAL DIVISION CAUSED BY STEEP TEMPERATURE GRADIENT IN GRASSHOPPER SPERMATOCYTE

Division of the spermatocytes of the grasshopper, Acrida lata, was studied under a steep temperature gradient. When a temperature gradient is applied along the spindle, the development of the aster on the warmer side is accelerated which, in turn, pushes the spindle toward the cooler side of the cell and the division becomes unequal. A condition to obtain such an unequal division is to shift the spindle by the temperature gradient and hold it eccentric during anaphase. Future cleavage plane is foreshadowed by the tongue of the mitochondria at anaphase before the cell departs from sphericity. If a temperature gradient is applied across the spindle, the spindle slides towards the cooler side sideways and the furrow is formed earlier, on the farther side of the cells from the spindle on the warmer side, although the size of the daughter cell is equal. The result indicates that the advance of the furrow is endothermically accelerated.     

MITOSIS IN THE OCTAFLAGELLATE PRASINOPHYTE,PYRAMIMONAS AMYLIFERA (CHLOROPHYTA)1

Cell division is described in the octaflagellate prasinophyte Pyramimonas amylifera Conrad and is compared in related genera. Basal bodies replicate at preprophase and move toward the poles. Cells remain motile throughout division. The nuclear envelope disperses and chromosomes begin to condense at prophase. Pairs of multilayered kinetochores are evident on the chromosomes of the metaphase plate. Spindle microtubules extending from the region of the basal bodies and rhizoplasts attach to the kinetochores or extend from pole to pole. Numerous vesicles and ribosomes have entered the nuclear region and the incipient cleavage furrow invaginates. The chromosomes move toward the poles at anaphase leaving a broad interzonal spindle between the two chromosomal plates. The nuclear envelope reforms first around the chromatin on the side adjacent to the spindle poles and later on the interzonal side. The cleavage furrow progresses into the interzonal spindle at telophase. By late telophase the nucleoli have reformed and the chromosomes have decondensed. The interzonal spindle has not been observed late in telophase. As the cleavage furrow nears completion the cells begin to twist and contort, ultimately separating the two cells.     

Effect of Microtubular Poisons on Cleavage Furrow Formation and Induction of Furrow-like Dent in Amphibian Eggs

The effects of the microtubular poisons colchicine, vinblastine and nocodazole, on cleavage furrow formation and induction of furrow-like dents in eggs of the newt, Cynops pyrrhogaster , were examined.
Solutions of the poisons were injected beneath the cortex around the small initial furrow, or around the advancing tip of the furrow of eggs during the first cleavage. This resulted in prompt block of the progress of the furrow at the injection site, and subsequent total regression of the furrow or incomplete cleavage.
The ability of the cortex of a cleavage-arrested blastomere to form a furrow-like dent was tested by inhibiting furrow formation of one blastomere of two-cell embryos by injection of the microtubular poisons, and then transplantation of the blastomere under the cortex of the animal half with furrow-inducing cytoplasm (FIC) taken from normally cleaving eggs. No dent was formed. Moreover, FIC from eggs treated with a poison had no ability to induce a dent on the surface of normally cleaving eggs.
These results show that microtubule structures are directly involved in formation of a cleavage furrow.     

The cytoskeletal involvement in cellularization of theDrosophila melanogaster embryo

Summary The distribution and arrangement of cytoskeletal components in the early embryo ofDrosophila melanogaster were examined by thin-section electron microscopy to elucidate their involvement in the formation of the cellular blastoderm, a process called cellularization. During the final nuclear division in the cortex of the syncytial blastoderm bundles of astral microtubules were closely associated with the surface plasma membrane along the midline where a new gutter was initiated. Thus the new gutter together with the pre-formed ones compartmentalized the embryo surface to reflect underlying individual daughter nuclei. Subsequently such gutters became deeper by further invagination of the plasma membrane between adjacent nuclei to form so-called cleavage furrows. Nuclei simultaneously elongated in the direction perpendicular to the embryo surface and numerous microtubules from the centrosomes ran longitudinally between the nucleus and the cleavage furrow. Microtubules often appeared to be in close association with the nuclear envelope and the cleavage furrow membrane. The plasma membrane at the advancing tip of the furrow was always undercoated with an electron-dense layer, which could be shown to be mainly composed of 5–6 nm microfilaments. These microfilaments were decorated with H-meromyosin to be identified as actin filaments. As cleavage proceeded, each nucleus with its perikaryon became demarcated by the furrow membrane, which then extended laterally to constrict the cytoplasmic connection between each newly forming cell and the central yolk region. The cytoplasmic strand thus formed possessed a prominent circular bundle of microfilaments which were also decorated with H-meromyosin and bidirectionally arranged, similar in structure to the contractile ring in cytokinesis. These observations strongly suggest that both microtubules and actin filaments play a crucial role in cellularization ofDrosophila embryos.     

Hydrogen ion-induced curvature in cucumber hypocotyls

A strip of filter paper saturated with a pH 3.0 buffer solutionand placed on the upper side of a light-grown cucumber hypocotylseedling can eliminate the negative geotropic curvature of horizontallyplaced seedlings, by promoting elongation of the upper side.These filter paper strips also caused strong curvature of verticallyplaced seedlings and induced cell wall loosening of the epidermis. (Received February 16, 1973; )     

The dynamic surface of dividing cyanelles and ultrastructure of the region directly below the surface in <Emphasis Type="Italic">Cyanophora paradoxa</Emphasis>

The cyanelles of glaucocystophytes are probably the most primitive of known extant plastids and the closest to cyanobacteria. Their kidney shape and FtsZ arc during the early stage of division define cyanelle division. In order to deepen and expand earlier results (Planta 227:177–187, 2007), cells of Cyanophora paradoxa were fixed with two different chemical and two different freeze-fixation methods. In addition, cyanelles from C. paradoxa were isolated to observe the surface structure of dividing cyanelles using field emission scanning electron microscopy (FE-SEM). A shallow furrow started on one side of the division plane. The furrow subsequently extended, covering the entire division circle, and then invaginated deeply, becoming clearly visible. The typical FtsZ arc was 2.3–3.4 μm long. This length matches that of the cleavage furrow observed using FE-SEM. The cyanelle cleavage furrows are from one-fourth to one-half of the circumference of the division plane. The shallow furrow that appears on the cyanelle outer surface effectively changes the division plane. Using freeze-fixation methods, the electron-dense stroma and peptidoglycan could be distinguished. In addition, an electron-dense belt structure (the cyanelle ring) was observed inside the leading edge at the cyanelle division plane. The FtsZ arc is located at the division plane ahead of the cyanelle ring. Immunogold-TEM localization shows that FtsZ is located interiorly of the cyanelle ring. The lack of an outer PD ring, together with the arch-shaped furrow, suggests that the mechanical force of the initial (arch shaped) septum furrow constriction comes from inside the cyanelle.     

CELL DIVISION AND MORPHOGENESIS OF THE CENTRIC DIATOM CHAETOCEROS DECIPIENS (BACILLARIOPHYCEAE) II. ELECTRON MICROSCOPY AND A NEW PARADIGM FOR TIP GROWTH

Valve morphogenesis starts when the silica deposition vesicle (SDV) expands across a cleavage furrow covered by an unidentified layer, which may aid in its shaping. A labiate process (LP) is present only in the outer valve of terminal cells in the filament. Before these particular cells form setae, a layered "labiate process apparatus" (LPA) appears on the SDV in the exact center of the forming valve, near the microtubule center arising after cleavage. The LPA thereafter surmounts the lips of the LP as it forms. After the girdle bands separate slightly, two lateral protrusions develop in the corners of the cell. These nascent setae are lined internally by a cylindrical, fibrous band (sleeve), which assembles immediately ahead of the expanding edge of the SDV, very close to the plasmalemma. Then these protrusions, lined by the fibrous band, the SDV, and the forming silica wall, grow through two gaps in the girdle bands. The cytoplasm at the tip of the growing seta is naked. Immediately behind the tip, this fibrous band is adpressed to the plasmalemma and thereby apparently defines the diameter of the seta; it extends to internally ensheath the tipmost edge of the SDV for a short distance, like a tight-fitting inner sleeve. This structure is considered the major organelle involved in seta morphogenesis. Microtubules (MTs), while present, are variable in extent and disposition within the seta. Turgor pressure is considered irrelevant in driving seta growth. Instead, a new paradigm proposed for tip-growing cells generally, may apply to seta morphogenesis, as follows. If, as is suspected, the fibrous band contains actin, cycling of this actin (as in animal cells undergoing ruffling or filopodial extension) could drive seta extension via attachment of the band to the just-formed silica wall. The band is visualized as a molecular treadmill whose support base, the new wall, is being continually extended; extension is controlled and generated strictly at the tip.     

FINE STRUCTURE OF MITOSIS AND CLEAVAGE IN FRIEDMANNIA ISRAELENSIS (CHLOROPHYCEAE: CHLOROSARCINACEAE)1

Observations on the ultrastructure of Friedmannia israelensis Chantanachat & Bold revealed the presence of a phycoplast and zoospores with cruciate rootlets. During mitosis, the nuclear envelope partially disintegrates and the basal bodies remain at the cell surface on either side of the developing cleavage furrow. The events during mitosis and cleavage in Friedmannia resemble those reported in the other green algae, Platymonas and Pleurastrum.     

A FINE STRUCTURAL ANALYSIS OF CLEAVAGE INDUCTION AND FURROWING IN THE EGGS OF ARBACIA PUNCTULATA

A fine structural study has been carried out on the various formed elements present before, during, and after the first cleavage division, not only in normally developing Arbacia eggs, but also in eggs which have been induced to cleave prematurely by high-pressure centrifugation. The aim has been to ascertain whether or not any of the morphologically identifiable components may be involved in initiating the furrowing process. Also, attention has been given to the fine structure of the cytoplasmic cortex, particulary in the walls of the furrow, in the hope of reaching a better understanding of the mechanics of cleavage. The annulate lamellae and the membranous envelope of the nucleus are the only formed elements which disappear shortly before cleavage, not only in eggs undergoing normal division, but also in eggs which have been induced to cleave ahead of schedule by high-pressure, high-force centrifugation. Therefore, it is suggested as a tentative hypothesis that materials liberated upon disintegration of the nuclear membrane and the annulate lamellae play an essential role in initiating and effecting the furrowing reaction, especially since the stratification of these elements in experimentally induced eggs corresponds to the position of the developing furrow. Another of the membranous elements in the egg, the Golgi complex, shows considerable modification as a result of high-pressure centrifugation, but these structures do not undergo disintegration. Rather, they become curled into rounded bodies. The vacuole population is not greatly affected by inducing treatments. During cleavage, both naturally occurring and experimentally induced, a considerable number of 50 A filaments appear in the denser cytoplasmic cortex, but only in the walls of the furrow. These filaments are similar to those which have been demonstrated in a number of contractile cells. Accordingly, it is suggested that this fibrillar system may be actively involved in the development of the cleavage force.     

DEVELOPMENT OF THE FLAGELLAR APPARATUS AND FLAGELLAR ORIENTATION IN THE COLONIAL GREEN ALGA GONIUM PECTORALE (VOLVOCALES)1

Vegetative cells of Gonium pectorale have a fine structure similar to that of Chlamydomonas. In addition, three zones comprise an extracellular matrix; a fibrillar sheath and tripartite boundary surround individual cells, and a fragile capsule zone surrounds the entire colony. Cytokinesis is accomplished by a phycoplast and cleavage furrow. The flagellar apparatus of the immature vegetative cell of this colonial alga is similar to that of Chlamydomonas, but the basal bodies are slightly separated at their proximal ends. The four microtubular rootlets alternate between two and four members. During development, the basal bodies become further separated and nearly parallel. The distal fiber is stretched, but it remains attached to both basal bodies. At maturity, the basal bodies of peripheral cells of the colony have rotated in opposite directions on their longitudinal axes resulting in a displacement of the distal fiber to one side, an asymmetrical orientation of the rootlets and loss of 180° rotational symmetry. Central cells remain similar to Chlamydomonas in that basal bodies do not rotate, rootlets are cruciate, the distal fiber remains medially inserted and 180° rotational symmetry is conserved. A “pin-wheel” configuration of flagellar pairs and the orientation of parallel rootlets toward the colony perimeter probably accounts for the rotation of the colonies during forward swimming. In addition, these ultrastructural features support the traditional placement of G. pectorale as an intermediate between the unicellular Chlamydomonas and the more complex colonial volvocalean genera.     

WAVE OF STIFFNESS PROPAGATING ALONG THE SURFACE OF THE NEWT EGG DURING CLEAVAGE

In the eggs of the newt, Cynops (Triturus) pyrrhogaster, change in stiffness of the cortex was measured in various regions at the time of the cleavage. Measurements were performed by Mitchison and Swann's cell elastimeter method with a modification, in which two fine pipettes were attached to the surface of one egg at the same time, in order to compare the rigidity of two regions. The stiffness of the cortex changed very little before the start of the first cleavage. However, just before the appearance of the first cleavage furrow, the stiffness increased rapidly at the animal pole region, which later returned to the former level. As the cleavage furrow progressed, a wave of high stiffness travelled meridionally as a belt along the surface from the animal pole region toward the vegetal region. At second cleavage, the cycle of change in stiffness was repeated.     

Cell cycle arrest by a gradient of Dpp signaling during Drosophila eye development

Background  

The secreted morphogen Dpp plays important roles in spatial regulation of gene expression and cell cycle progression in the developing Drosophila eye. Dpp signaling is required for timely cell cycle arrest ahead of the morphogenetic furrow as a prelude to differentiation, and is also important for eye disc growth. The dpp gene is expressed at multiple locations in the eye imaginal disc, including the morphogenetic furrow that sweeps across the eye disc as differentiation initiates.     

THE PICEA ABIES SHOOT APICAL MERISTEM IN CULTURE. II. DEPOSITION OF POLYSACCHARIDES AND LIGNIN-LIKE SUBSTANCES BENEATH CULTURES

Excised shoot apical meristems of Picea abies seedlings grow and develop primordial leaves when cultured on Millipore (mixed esters of cellulose) filter membranes lying on a simple, defined medium gelled with agarose. When the cultures are removed from the membranes, each leaves a spot of altered light transmission, spectral characteristics, hygroscopicity, and chemical reactivity. These spots are the manifestation of deposition in the membrane pore space of polysaccharides, lignin-like components, and probably other substances. Deposition of water-insoluble, Schiff's reagent-positive substances can be detected in the filter membranes after only 3–6 hr exposure to a meristem and continues for 10–15 days or longer. Precursors of the insoluble deposition materials can diffuse through at least nine layers of Millipore membrane before deposition at a site remote from living cells. Placement of a dialysis membrane between the meristem and the Millipore membrane prevents detectable deposition in the latter. The observations are consistent with the hypothesis that apical meristems can synthesize and export mobile precursors of cell wall components as well as any substances necessary to promote their condensation or polymerization into insoluble materials at remote sites. The system may be useful in studying synthesis of cell wall components and investigating the functional role of growth regulators in shoot apical development.     

AN ELECTRON MICROSCOPIC STUDY OF THE DEVELOPMENT OF THE CLEAVAGE FURROW IN MAMMALIAN CELLS

The process of cytoplasmic cleavage has been studied in thin sections of rat erythroblasts and the cells of mouse leukemia and Walker 256 carcinoma of the rat. The development of the cleavage furrow begins in relation to the mid-body, which, earlier, appears on the equatorial plane in association with the continuous fibers of the spindle. The earliest evidence of a cleavage furrow is the presence of a vesicle or vesicles close to the mid-body. Subsequently, many smaller vesicles are seen in the equatorial plane. The cleavage furrow probably develops by the fusion of these vesicles so that a new plasma membrane is formed between the daughter cells, and extends from the telophase intercellular bridge to the cell margin. During the stage of formation of the vesicles, cisternae, believed to be part of the endoplasmic reticulum, assume an intimate relationship with the cleavage plane, and they may perhaps be involved in the formation of the vesicles.     

Furrow Formation in the Vegetal Hemisphere of Xenopus Eggs

The mechanism of furrow formation in the vegetal hemisphere of amphibian eggs was studied using Xenopus eggs. Injection of colchicine into the eggs after the furrow tip had entered the vegetal hemisphere arrested the subsequent cleavage. The effect of impairing the continuity between the animal and vegetal hemispheres was examined by squeezing the equator of uncleaved eggs from both sides with the edges of coverslips. On gentle squeezing a shallow vegetal furrow was formed at the first cleavage, whereas on strong squeezing furrowing was arrested at the equator. The mechanism of furrow formation in the vegetal hemisphere of amphibian eggs is discussed on the basis of these findings.     

Localized calcium signals along the cleavage furrow of the Xenopus egg are not involved in cytokinesis

It has been proposed that a localized calcium (Ca) signal at the growing end of the cleavage furrow triggers cleavage furrow formation in large eggs. We have examined the possible role of a Ca signal in cleavage furrow formation in the Xenopus laevis egg during the first cleavage. We were able to detect two kinds of Ca waves along the cleavage furrow. However, the Ca waves appeared after cleavage furrow formation in late stages of the first cleavage. In addition, cleavage was not affected by injection of dibromoBAPTA or EGTA into the eggs at a concentration sufficient to suppress the Ca waves. Furthermore, even smaller classes of Ca release such as Ca puffs and Ca blips do not occur at the growing end of the cleavage furrow. These observations demonstrate that localized Ca signals in the cleavage furrow are not involved in cytokinesis. The two Ca waves have unique characteristics. The first wave propagates only in the region of newly inserted membrane along the cleavage furrow. On the other hand, the second wave propagates along the border of new and old membranes, suggesting that this wave might be involved in adhesion between two blastomeres.     

THE FINE STRUCTURE OF THE MID-BODY OF THE RAT ERYTHROBLAST

The development of the mid-body has been studied in mitotic erythroblasts of the rat bone marrow by means of thin sections examined with the electron microscope. A differentiated region on the continuous spindle fibers, consisting of a localized increase in density, is observed at the equatorial plane. The mid-body seems to develop by the aggregation of such denser lengths of spindle fiber. Its appearance precedes that of the cleavage furrow. A plate-like arrangement of fibrillary material lies transversely across the telophase intercellular bridge. Later, this material becomes amorphous and assumes the form of a dense ring closely applied to a ridge in the plasma membrane encircling the middle of the bridge. Although the mid-body forms in association with the spindle fibers, it is a structurally distinct part, and the changes which it undergoes are not shared by the rest of the bundle of continuous fibers.