EFFECTS OF THE SURFACTANTS ON THE CLEAVAGE AND FURTHER DEVELOPMENT OF THE SEA URCHIN EMBRYOS II. DISTURBANCE IN THE ARRANGEMENT OF CORTICAL VESICLES AND CHANGE IN CORTICAL APPEARANCE

After fertilization, two types of cortical vesicles ware examined under the electron microscope (the cortical vesicle I and II) and the light microscope (pigment granules and another kind of vesicles). The cortical vesicle I corresponds to the pigment granule and the cortical vesicle II does to the other vesicle.
The unequal division of the sea urchin embryo which occurs at the fourth cleavage was modified to an equal cleavage pattern by the treatment with sodium lauryl sulfate (SLS) or cetyl trimethyl ammonium bromide (CTAB). But other surfactants such as sodium deoxycholate, Tween 80, Lubrol PX did not have such an effect. The cell surface of the embryo which had been treated either SLS or CTAB became rough or smooth. Cortical vesicles and pigment granules disappeared and/or were dislocated from the cortex. However, cell organelles were as normal as the control. On the other hand, the cortical appearance of other surfactant-treated embryos showed no disturbance and cell organelles were also more or less normal. Therefore, the equalization of unequal cleavage is caused by the disturbance in the cortex and thus the cortex plays a major role on the micromere formation at the 16-cell stage and on the further sea urchin development.     

SPICULE FORMATION IN VITRO BY THE DESCENDANTS OF PRECOCIOUS MICROMERE FORMED AT THE 8-CELL STAGE OF SEA URCHIN EMBRYO*

The micromeres at the 16-cell stage of sea urchin embryo have already been endowed with a faculty to self-differentiate into spicule-forming cells (11). The present experiment was designed to test whether the factor(s) necessary for such self-differentiation had already been localized at the 8-cell stage in an area corresponding to the presumptive micromere region in Hemicentrotus pulcherrimus. Since the blastomeres at the 8-cell stage are all equal in size in normal embryo, unequal 3rd cleavage, by which small blastomeres are pinched off toward the vegetal pole (precocious micromeres), was experimentally induced either by treatment with 4NQO (4-nitroquinoline-1-oxide) at the 2-cell stage or by continuous culture in Ca-free sea water. The precocious micromeres were cultured in vitro in natural sea water containing horse serum. Descendants of the precocious micromeres formed spicules. In comparison their spicule formation with that by the descendants of the micromere of normal embryo, no differences were found regarding 1) time of initiation of spicule formation, 2) rate of growth of spicule, 3) size and shape of resultant spicule and 4) percentage of clones which formed spicule. The fact indicates that factor(s) indispensable for self-differentiation into spicule-forming cells have already been localized near the vegetal pole as early as the 8-cell stage.     

Unequal cleavage and the differentiation of echinoid primary mesenchyme

The role of unequal cleavage in echinoid micromere determination was investigated by equalizing the fourth and fifth cleavages with brief surfactant treatment. The surfactant sodium dodecyl sulfate was found to be effective in equalizing fourth cleavage when generally applied to 4-cell stage embryos of all species tested. Embryos of the sand dollar Dendraster excentricus developed normally when equalized at the fourth and fifth cleavages by surfactant treatment, as did untreated equally cleaving embryos of the sea urchin Strongylocentrotus droebachiensis. Embryos of the sea urchins Lytechinus pictus and S. purpuratus were animalized by the treatment but were capable of forming spicules after treatments which equalized the fourth cleavage. In addition, orientation of the fourth division spindles was found to have no effect on differentiation of the primary mesenchyme in D. excentricus. The results confirm that micromere determination in echinoids does not depend upon a strict cleavage pattern at the 16-cell stage.     

Effect on Inhibition of Micromere Segregation on the Mitotic Pattern in the Sea Urchin Embryo

Detergent treatment of sea urchin eggs at the mid 4-cell stage results in prevention of micromere segregation at the fourth cleavage. In these embryos not only the formation of the primary mesenchyme is suppressed, but synchrony of cell division, which is the rule during the first four cleavage cycles, continues for several cycles after the 16-cell stage while the typical mitotic phase wave that sets in after micromere segregation is abolished.
These results support the hypothesis that micromeres act as coordinators of the mitotic activity of the embryo.     

STUDIES ON UNEQUAL CLEAVAGE IN SEA URCHINS I. MIGRATION OF THE NUCLEI TO THE VEGETAL POLE

It has been known for nearly a century that at the 16-cell stage of sea urchin embryos, the animal 4 cells divide equally and horizontally, whereas the vegetal 4 cells cleave unequally and practically vertically into macromeres and micromeres. Recently, more careful observations were made on the process of micromere formation and it has been revealed that a primary cause for the inequality lies in the migration of the 4 vegetal nuclei to the vegetal pole of the embryo which brings about excentricity of the mitotic apparatus. Records of this phenomenon are given in the present paper.     

ANALYSIS OF THE VARIABILITY IN CLEAVAGE TIMES AND DEMONSTRATION OF A MITOTIC GRADIENT DURING THE CLEAVAGE STAGES OF NOTHOBRANCHIUS GUENTHERI

Living embryos of the annual cyprinodont fish Nothobranchius guentheri were observed under the microscope. Detailed records were made of the time of cell division, disappearance of the nucleus and of the position of each cell within the blastoderm up to and including the sixth cleavage. Combination of these data revealed the presence of a mitotic gradient, a cell division gradient and a gradient of cell cycle duration in the 8-cell, 16-cell and 32-cell stage. Comparison of the variabilities in the duration of the interphase and mitosis reveals that differences between sister cell intercleavage times in the 8-, 16- and 32-cell stage are, for the most part, due to the variability in the duration of the mitotic process. It is concluded that the DNA-division cycle is composed of at least two parallel series of events. We found the random transition model of cell cycle control, originally based on the analysis of intermitotic times of mammalian cells in tissue culture, helpful also in analysing intercleavage time variability in embryonic cells.     

Cleavage and differentiation in the sea urchin embryo transplantation studies of micromeres

Summary Micromeres isolated from the 16-cell stage were implanted on mesomeres or macromeres from the same larva. The process of coalescence and the cleavage pattern of the transplanted micromeres were studied by means of light and electron microscopy.The transplanted micromere shows the same cleavage pattern as the micromerein situ. A close contact is established between the micromere and the host cell and cytoplasmic bridges are found between the cells.The micromere is dependent on its adjoining blastomere(s) and the rate of cleavage is slowed down when the micromere is isolated. Macromeres and mesomeres are not subjected to a similar change in rate of cleavage when isolated from the rest of the embryo.The ratio mitochondria/yolk in micromeres is different from that observed in macro- or mesomeres and the possible consequences of this fact are discussed.     

Studies on Unequal Cleavage in Sea Urchins III. Micromere Formation under Compression

When sea urchin embryos at 2-cell stage are flattered between agar plates, the direction of cleavage is rotated by 90° in each division in reference to the preceding cleavage and no micromere is formed. But under this condition, micromeres are formed in 2 cases; 1) When the egg axis is parallel to the plane of flattening, 2 micromeres are formed on one side of a square 16-cell stage. 2) when the egg axis is perpendicular to the plane, 4 micromeres are formed at the center of the square.
When put into a groove, a string of 4 cells is formed showing that the spindle direction is further deflected by the groove. In the following 16-cell stage in the groove, which consists of 2 layers of 8 cells, cases with 2 micromeres on one side and 4 micromeres at the center are still found. If the 2-cell stage is introduced into a groove after the formation of mitotic apparatus, the spindle direction can no longer be changed and the 4-cell stage becomes like 4 pancakes stuck in 2 layers, indicating that 2 asters are holding the ends of a spindle in fixed positions.     

ON THE SYSTEM CONTROLLING THE TIME OF MICROMERE FORMATION IN SEA URCHIN EMBRYOS

The previously reported observation that micromere formation after cleavage suppression is not linked with the number of blastomeres present but rather with the time schedule of the fourth cleavage of the normal embryos has been confirmed. A hypothesis is advanced that a rhythmical fluctuation of the sulfhydryl contents of the egg proteins is the clock system, and micromere formation is connected with the fourth SH cycle after fertilization. The hypothesis was tested under 3 conditions:

• (i) Conditions which stop the nuclear activities but preserve the SH cycle, followed by a release from these conditions.
• (ii) Conditions which “freeze” both nuclear and cytoplasmic rhythms, and later removal of the conditions.
• (iii) Conditions which leave nuclear activities intact but prevent the cytoplasmic rhythms, followed by normal culturing.

The results came out as anticipated by the hypothesis.     

Cell lineage-specific inhibition of cytokinesis by concanavalin A in a molluscan embryo (Nassarius reticulatus,Gastropoda)

Summary The effects of the lectin concanavalin A (Con A) on cleavage were studied in early embryos of the gastropodNassarius reticulatus. Progression of the first cleavage furrow is inhibited by incubating eggs before the first cleavage with 0.3–20 μg/ml Con A. Treatment with 1.0–20 μg/ml Con A during first cleavage causes regression of the cleavage furrow. Treatment with low concentrations (0.3–1.0 μg/ml) during the same period does not affect first cleavage. However, when further development of such eggs is followed, one finds that second cleavage is inhibited typically in only one of the two blastomeres of the 2-cell stage, i.e. the CD-blastomere. As a result, a 3-cell embryo is formed. At third cleavage of such embryos, the CD-blastomere forms either one double-sized micromere (1cd-micromere) or two normal-sized micromeres (1c and 1d) simultaneously. Sometimes micromere formation in the CD-blastomere is inhibited. Con A binding does not affect karyokinesis, nor does it affect the division asynchronies typical for normal development. On the basis of these and other results it is argued that binding of Con A to sites located at the vegetal pole of the egg is responsible for the cell lineage-specific inhibition of cleavage by Con A. This effect is most probably mediated by changes in the organization of the egg cortex.     

CLEAVAGE PATTERNS AND MESENTOBLAST FORMATION IN THE GASTROPODA: AN EVOLUTIONARY PERSPECTIVE

The larger gastropod taxa are characterized by distinctive cleavage patterns. The cell stage at which the mesentoblast is formed appears to be crucial. In none of the taxa is it formed earlier than the 24- and not later than the 63-cell stage. A heterochronic shift from late to early mesentoblast formation appears to coincide with successive steps in gastropod evolution. Comparison of the early cleavage patterns appears to be a powerful method for investigating the evolutionary relations between major gastropod taxa.     

Cleavage and gastrulation of the euphausiacean<Emphasis Type="Italic"> Meganyctiphanes norvegica</Emphasis> (Crustacea,Malacostraca)

According to the Articulata hypothesis the cleavage of arthropods must be derived from spiral cleavage. However, arthropods show a great variety of cleavage modes with a widespread occurrence of superficial cleavage. In the Malacostraca, holoblastic cleavage occurs in some taxa such as Amphipoda, Euphausiacea and Dendrobranchiata. In particular, the cleavage of euphausiaceans has been proposed to be a modified spiral cleavage. The cell lineage of early stages up to blastoderm formation of the euphausiacean Meganyctiphanes norvegica is reconstructed using recent methods of fluorescent staining. Only the oblique angle of the mitotic spindles during the transition from the 2- to the 4-cell stage resembles the spiral cleavage mode. At the 8-cell stage, four cells each form a pattern of two interlocking bands which is preserved until the 122-cell stage. One blastomere is delayed in division and shows an oblique division from the fourth cleavage on. It is the precursor cell of two enlarged and cleavage-arrested cells at the 32-cell stage. At the 62-cell stage, these two cells are surrounded by eight cells following a specific cell division pattern during the subsequent division cycles. The cleavage pattern of M. norvegica occurs in two mirror images. A comparative approach reveals distinct similarities between the early cleavage patterns of Euphausiacea and Dendrobranchiata which are suggested to be homologous. Furthermore, the relationships to non-malacostracan cleavage patterns are discussed. It is shown that the early cleavage pattern of M. norvegica does not offer an example of a spiral cleavage within arthropods.     

A “micromere model” of cell-cell interactions in sea urchin early embryos

It has been shown that isolation of sea urchin blastomeres before the post-division adhesion leads mainly to the formation of equal blastomeres at the stage of 4th cleavage division, whereas isolation after adhesion results in the formation of micromeres simultaneous with that in intact embryos. Similar results were obtained in five sea urchin species. It has been concluded that there exists a critical point in the cleavage process, when blastomeres exchange information that determines the further cleavage pattern. It has been shown with this “micromere model” that serotonin and its analogs influence the cleavage pattern of half-embryos. These data have served as a basis for the hypothesis of “protosynapse,” a bilaterally symmetric structure in which the blastomeres are not only source and target of the signal but also a passive obstacle to leakage of the signal substance from the interblastomere cleft to the milieu. Such a structure may also specify the primary asymmetry of the blastomeres. The micromere model may be useful in specific pharmacological screening.     

Distribution and redistribution of pigment granules in the development of sea urchin embryos

Summary Pigment granules (PGs) are embeded in the cortex of embryos of the Japanese sea urchins,Hemicentrotus pulcherrimus and Anthocidaris crassispina. PGs in the cortex actively retreated from the vegetal pole area at the 4-cell stage and then a notable PG-distribution gradient formed along the egg axis (the polar redistribution of PGs). The polar redistribution of PGs in the cortex occurred at the same time after fertilization even in solutions of microtubule disrupting reagents such as Colcemid, vinblastine sulfate or griseofulvin. Consequently, the polar redistribution of PGs was not associated with the microtubules. However, the polar redistribution of PGs was interrupted in seawater containing cytochalasin B (CB), dithiothreitol (DTT) or tetracaine, and the distribution pattern of PGs in the cortex was definitely disturbed. Moreover, CB, DTT and tetracaine altered the division pattern of vegetal blastomeres at the 4th cleavage which is normally unequal so that all the blastomeres divided equally. Microtubule disrupting reagents did not have such an effect on the cleavage pattern. Thus the cortical movement along the egg axis reflected by the polar redistribution of PGs seems to correlate with the micromere formation.     

Primary mesenchyme cell patterning during the early stages following ingression

Sea urchin primary mesenchyme cells (PMCs) ingress into the blastocoel during an epithelial-to-mesenchymal transition (EMT), migrate along the blastocoelar wall for a period of time, and then settle into a subequatorial ring to form the larval skeleton. Fluorescent-marked blastomeres alone, or in combination with blastomere recombination, were used to track the position of PMCs during the early phases of this movement. Micromeres expressing Golgi-tethered GFP (galtase-GFP) were transplanted onto TRITC-stained hosts (in place of the endogenous micromere) to observe the progeny of a single micromere. Galtase-GFP as a Golgi marker is not transferred between PMCs when the syncytium forms. Thus, the position of cells can be followed relative to beginning position for longer periods than previously reported. The PMC progeny of a single micromere do not disperse upon ingression, but instead remain in a closely associated cluster. Generally, progeny of a single micromere remain in the quadrant of origin. In total, greater than approximately 94% of labeled PMCs remain within the local region of ingression. By contrast, when a transplanted micromere is placed at the vegetal plate after removing all 4 host micromeres, the resultant PMCs ingress and migrate into all 4 quadrants. Similarly, if 1 blastomere is injected at the 2-cell stage, and later the 2 unlabeled micromeres are removed at the 16-cell stage, the remaining PMCs ingress into all 4 quadrants of the vegetal plate. We conclude that the normal restriction of PMCs to a quadrant is due to mechanical constraint from other micromere-PMCs. If a labeled micromere is placed ectopically at the macromere/mesomere boundary, the PMC progeny ingress ectopically and migrate longitudinally along the animal-vegetal axis only. Injection of galtase-GFP into one blastomere at the 4-cell stage shows a 2-step pattern of localization. At late mesenchyme blastula and early gastrula stages, greater than 90% of GFP-expressing PMCs remain in the injected quadrant, while at mid- to late-gastrula stage and beyond, more PMCs are found outside the injected quadrant. The migration that sets up the asymmetry of the larval skeleton first occurs around mid- to late-gastrula stages, when some PMCs from an aboral quadrant migrate to the adjacent oral quadrant. In all, these data combined with previous data suggest that freshly ingressed PMCs migrate along a longitudinal path toward the animal pole and back toward the vegetal pole. Beginning at mid- to late-gastrula stage, PMCs utilize oral-aboral cues from the ectoderm for the first time. At this time, some aboral PMCs migrate into the adjacent oral quadrant to assist in the formation of the ventrolateral cluster.     

EFFECT OF DNP ON ACCELERATION OF THE CLEAVAGE FOLLOWING INSUFFICIENT PARTHENOGENETIC ACTIVATION IN THE SEA URCHIN EGG*

Unfertilized eggs of sea urchins, Hemicentrotus pulcherrimus and Pseudocentrotus depressus, were treated with 4–5% butyric acid-sea water for 40–60 sec so that they were activated partheno-genetically without visible cortical changes. When these insufficiently activated eggs were inseminated 90–120 min after butyric acid-treatment, they divided much earlier than the control eggs in the first cleavage cycle. In the present paper, it becomes clear that if eggs are put into m /2,000-m /16,000 DNP-sea water at 60 min after insufficient activation and 30 min later, returned to normal sea water and then inseminated, they still show acceleration of the first cleavage in the same degree as the eggs which are not treated with DNP, while if eggs are exposed to DNP for 30 min prior to the insufficient activation or within 60 min after the activation, they do not show any acceleration of the cleavage. From these results, it may be concluded that some preparations for cleavage acceleration which are arrested by DNP become ready in the eggs at an early period in the first cleavage cycle and these preparations cannot be cancelled by DNP-treatment once they have been completed.     

Surfactant-induced lipid peroxidation in a tropical euryhaline teleost Oreochromis mossambicus (Tilapia) adapted to fresh water

Exposure to anionic (sodium dodecyl sulfate, SDS), cationic (cetyl trimethyl ammonium bromide CTAB) and non ionic (Triton X-100) surfactants at a sub lethal concentration of 1 ppm resulted in severe oxidative stress in the hepatic, renal and cardiac tissues of fresh water adapted Oreochromis mossambicus. Hepatic catalase showed significant increase (P<0.001) in all the surfactant exposed fish, but the renal enzyme was significantly increased only in CTAB dosed fish (P<0.001) and the cardiac enzyme showed significant increase in Triton (P<0.05) and CTAB dosed fish (P<0.001). SOD levels were significantly increased (P<0.001) in hepatic, renal and cardiac tissues of all the surfactant-treated fish. Glutathione reductase also was significantly increased (P<0.001) in the hepatic and renal tissues of surfactant dosed fish except cardiac tissues of CTAB exposed animals. Glutathione levels in the tissues studied were significantly higher in the surfactant treated animals (P<0.001) whereas malondialdehyde levels were significantly elevated only in the hepatic tissues of animals exposed to Triton (P<0.001). The surfactants based on their charge, antioxidant profile and in vivo metabolism may be arranged in the order of decreasing toxicity as CTAB > Triton > SDS. Thus it may be inferred from the present study that the antioxidant defenses and the in vivo metabolism of the surfactants are key factors in deciding the surfactant toxicity.     

Cellular distribution of RNA populations in 16-cell stage embryos of the sand dollar, Dendraster excentricus

RNA was extracted from pure preparations of micromeres and meso-plus macromeres isolated from 16-cell stage embryos of Dendraster excentricus. Molecular hybridization-competition experiments disclosed that the binding of 16-cell stage labeled RNA to denatured sperm DNA was competed equally well by micromere RNA, meso-plus macromere RNA, total 16-cell RNA and unfertilized egg RNA, indicating the egg-type populations were distributed almost equally in the different blastomeres. In contrast, experiments with ³H-RNA extracted from micromeres obtained from pulse-labeled 16-cell stage embryos showed qualitative differences when unfertilized egg RNA and total 16-cell stage RNA were used as competitors. Such differences in RNA populations could not be detected in ³H-RNA isolated from the meso-plus macromere fraction.