Micromere Formation Research Articles

The evolutionary modifications of a GoLoco motif in the AGS protein facilitate micromere formation in the sea urchin embryo.

The evolutionary introduction of asymmetric cell division (ACD) into the developmental program facilitates the formation of a new cell type, contributing to developmental diversity and, eventually, to species diversification. The micromere of the sea urchin embryo may serve as one of those examples: An ACD at the 16-cell stage forms micromeres unique to echinoids among echinoderms. We previously reported that a polarity factor, Activator of G-protein Signaling (AGS), plays a crucial role in micromere formation. However, AGS and its associated ACD factors are present in all echinoderms and across most metazoans, leaving a question of what evolutionary modification of AGS protein or its surrounding molecular environment contributed to the evolutionary acquisition of micromeres only in echinoids. In this study, we learned that the GoLoco motifs at the AGS C-terminus play critical roles in regulating micromere formation in sea urchin embryos. Further, other echinoderms AGS or chimeric AGS that contain the C-terminus of AGS orthologs from various organisms showed varied localization and function in micromere formation. In contrast, the sea star or the pencil urchin orthologs of other ACD factors were consistently localized at the vegetal cortex in the sea urchin embryo, suggesting that AGS may be a unique variable factor that facilitates ACD diversity among echinoderms. Consistently, sea urchin AGS appears to facilitate micromere-like cell formation and accelerate the enrichment timing of the germline factor Vasa during early embryogenesis of the pencil urchin, an ancestral type of sea urchin. Based on these observations, we propose that the molecular evolution of a single polarity factor facilitates ACD diversity while preserving the core ACD machinery among echinoderms and beyond during evolution.

Micromere formation and its evolutionary implications in the sea urchin.

The micromeres of the sea urchin embryo are distinct from other blastomeres. After they arise through an asymmetric cell division at the 8- to 16-cell stage, micromeres immediately function as organizers. They also commit themselves to specific cell fates such as larval skeletogenic cells and primordial germ cells, while other blastomeres remain plastic and uncommitted at the 16-cell stage. In the phylum Echinodermata, only the sea urchin (class Echinoidea) embryo forms micromeres that serve as apparent organizers during early embryogenesis. Therefore, it is considered that micromeres are the derived features and that modification(s) of the developmental system allowed evolutionary introduction of this unique cell lineage. In this chapter, we summarize the both historic and recent observations that demonstrate unique properties of micromeres and discuss how this lineage of micromeres may have arisen during echinoderm evolution.

Evolutionary modification of AGS protein contributes to formation of micromeres in sea urchins

Evolution is proposed to result, in part, from acquisition of new developmental programs. One such example is the appearance of the micromeres in a sea urchin that form by an asymmetric cell division at the 4th embryonic cleavage and function as a major signaling center in the embryo. Micromeres are not present in other echinoderms and thus are considered as a derived feature, yet its acquisition mechanism is unknown. Here, we report that the polarity factor AGS and its associated proteins are responsible for micromere formation. Evolutionary modifications of AGS protein seem to have provided the cortical recruitment and binding of AGS to the vegetal cortex, contributing to formation of micromeres in the sea urchins. Indeed, introduction of sea urchin AGS into the sea star embryo induces asymmetric cell divisions, suggesting that the molecular evolution of AGS protein is key in the transition of echinoderms to micromere formation and the current developmental style of sea urchins not seen in other echinoderms.

A comprehensive fate map by intracellular injection of identified blastomeres in the marine polychaete Capitella teleta

BackgroundThe polychaete annelid Capitella teleta (formerly Capitella sp. I) develops by spiral cleavage and has been the focus of several recent developmental studies aided by a fully sequenced genome. Fate mapping in polychaetes has lagged behind other spiralian taxa, because of technical limitations.ResultsTo generate a modern fate map for C. teleta, we injected 1,1'-dioctadecyl-3,3,3'3'-tetramethylindocarbocyanine perchlorate (DiI) into individual identified blastomeres through fourth-quartet micromere formation. Confocal laser scanning microscopy at single-cell resolution was used to characterize blastomere fates during larval stages. Our results corroborate previous observations from classic studies, and show a number of similarities with other spiralian fate maps, including unique and stereotypic fates for individual blastomeres, presence of four discrete body domains arising from the A, B, C and D cell quadrants, generation of anterior ectoderm from first quartet micromeres, and contributions to trunk ectoderm and ventral nerve cord by the 2d somatoblast. Of particular interest are several instances in which the C. teleta fate map deviates from other spiralian fate maps. For example, we identified four to seven distinct origins of mesoderm, all ectomesodermal. In addition, the left and right mesodermal bands arise from 3d and 3c, respectively, whereas 4d generates a small number of trunk muscle cells, the primordial germ cells and the anus. We identified a complex set of blastomere contributions to the posterior gut in C. teleta, which establishes the most complete map of posterior gut territories to date.ConclusionsOur detailed cellular descriptions reveal previously underappreciated complexity in the ontogenetic contributions to several spiralian larval tissues, including the mesoderm, nervous system and gut. The formation of the mesodermal bands by 3c and 3d is in stark contrast to other spiralians, in which 4d generates the mesodermal bands. The results of this study provide a framework for future phylogenetic comparisons and functional analyses of cell-fate specification.

Activator of G‐protein signaling in asymmetric cell divisions of the sea urchin embryo

An asymmetric fourth cell division in the sea urchin embryo results in formation of daughter cells, macromeres and micromeres, with distinct sizes and fates. Several lines of functional evidence presented here, including pharmacological interference and dominant negative protein expression, indicate that heterotrimeric G protein Gi and its interaction partner, activator of G-protein signaling (AGS), are necessary for this asymmetric cell division. Inhibition of Gi signaling by pertussis toxin interferes with micromere formation and leads to defects in embryogenesis. AGS was isolated in a yeast two-hybrid screen with G alpha i as bait and was expressed in embryos localized to the cell cortex at the time of asymmetric divisions. Introduction of exogenous dominant-negative AGS protein, containing only G-protein regulatory (GPR) domains, selectively prevented the asymmetric division in normal micromere formation. These results support the growing evidence that AGS is a universal regulator of asymmetric cell divisions in embryos.

Formation and function of the polar body contractile ring in Spisula

Initial studies suggested that spatial organization of the putative polar body contractile ring was determined by the peripheral aster in Spisula [Biol. Bull. 205 (2003) 192]. Here we report detailed supporting observations, including testing of aster and ring function with inhibitors. The metaphase peripheral aster was confirmed to spread cortically in an umbrella-like pattern, with microtubule-poor center. The aster disassembled during anaphase, leaving the spindle docked at the F-actin-poor center of a newly generated cortical F-actin ring that closely approximated the aster in location, measured diameter range, and pattern. Cytochalasin D and latrunculin-B permitted all events except ring and polar body formation. Nocodazole disassembly or taxol stabilization of the peripheral aster produced poorly defined rings or bulging anaphase asters within the ring center, respectively, inhibiting polar body formation. Polar body extrusion occurred at the ring center, the diameter of which diminished. Ring contractility—previously assumed—was verified using blebbistatin, a myosin-II ATPase inhibitor that permitted ring assembly but blocked polar body extrusion. The data support the hypothesis that peripheral aster spreading, perhaps dynein-driven, is causally related to polar body contractile ring formation, with anaphase entry and aster disassembly also required for polar body biogenesis. Previously reported astral spreading during embryonic micromere formation suggests that related mechanisms are involved in asymmetric somatic cytokinesis.

Ca2+ regulation on the micromere formation at the 16-cell stage of sea urchin embryos

Ca2+ regulation on the micromere formation at the 16-cell stage of sea urchin embryos

Micromere Formation at Third Cleavage Is Decisive for Trochoblast Specification in the Embryogenesis ofPatella vulgata

Trochoblasts of the molluscPatella vulgatadifferentiate early in development into ciliated cells due to a cell intrinsic developmental capacity. The third cleavage appears to be decisive for their specification. Permanent inhibition of cleavage after the third cleavage, as induced by a continuous incubation with the drug Cytochalasin B, had no effect on trochoblast-specific gene expression later in development. However, permanent inhibition of cleavage before third cleavage abolished trochoblast differentiation including trochoblast-specific gene expression. Inhibition of just the third cleavage itself was sufficient to repress trochoblast-specific gene expression. In contrast, inhibiting the second or fourth cleavage did not effect trochoblast-specific gene expression. Correct formation of micromeres at the third cleavage is required to obtain trochoblast-specific gene expression as was shown in experiments in which the formation of micromeres during third cleavage was suppressed by pressure. In addition, centrifugation before or during the third cleavage disturbs trochoblast-specific gene expression, whereas centrifugation after the third cleavage does not affect trochoblast-specific gene expression. Thus, during the third cleavage a decisive step in the determination of developmental fate of trochoblasts takes place, likely resulting in a segregation of activating and inhibitory determinants. Trochoblast-specific markers inPatellawere only expressed in embryos that were division arrested after third cleavage. This suggests that a segregation of differentiation potentials has to take place before trochoblast differentiation markers are expressed. The restriction of differentiation potentials in the cleaving stage embryo is thus required to enable trochoblast-specific gene expression.

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

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.

The isotopic effects of D2O in developing sea urchin eggs.

When developing sea urchin eggs were treated with sea water containing 40% D2O (D2O-SW) at the 8-cell stage, the micromere formation was delayed and micromeres were larger than those seen in the control. But eggs returned to normal artificial sea water (NASW) at the 16- to 32-cell stage did not form abnormal spicules in larvae of Pseudocentrotus depressus. Little effect on the spicule formation of Hemicentrotus pulcherrimus was also noted. If the culture period in D2O-SW was extended until the hatching stage, the number of plutei with abnormal spicules increased. Primary mesenchyme cells of Pseudocentrotus depressus larvae failed to make two aggregated spicule rudiments on the ventral side of the larva and developed a ring-like spicule. This ring-like spicule, however, only occasionally occurred in the larvae of Hemicentrotus pulcherrimus. The cell cycle was longer in the presence of D2O. However, blastomeres managed to divide throughout the development. Larvae reared in 20% D2O-SW after the hatching stage developed into quasi-normal plutei but smaller than control. We found no exogastrulation in these larvae. Exogastrulation was found only in larvae continuously cultured in 40% D2O-SW from the early development. These results are inconsistent with previous reports made by other authors.

Studies on Unequal Cleavage in Sea Urchins III. Micromere Formation under Compression: (equal division/unequal division/micromere formation/Hertwig's rule/Balfour's rule).

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.

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

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.

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.

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.

EFFECTS OF THE SURFACTANTS ON THE CLEAVAGE AND FURTHER DEVELOPMENT OF THE SEA URCHIN EMBRYOS 1. THE INHIBITION OF MICROMERE FORMATION AT THE FOURTH CLEAVAGE.

Eggs of the sea urchins, Hemicentrotus pulcherrimus, Temnopleurus toreumaticus and Pseudocentrotus depressus were used as materials. Embryos were exposed to the surfactants such as SLS, CTAB, digitonin, Tween 80, sodium deoxycholate and Lubrol P. If embryos are kept in the solutions of SLS, CTAB and digitonin, 4 vegetal cells of the 8-cell stage divide equally at the fourth cleavage and consequently 16 equal-sized blastomeres are formed at the 16-cell stage. In this case, micromere formation is inhibited by the equal cleavage. The minimum effective concentration of the surfactants for the equal cleavage gradually increases as the time performing the treatment is postponed. The continuous exposure to the surfactant is unnecessary for the inhibition of micromere formation. In the egg temporarily exposed during the earlier stage, the equal cleavage occurs at the fourth division in natural seawater. Micromere formation is strongly affected by the surfactant (SLS) at the mid 4-cell stage.

Modified cleavage pattern after suppression of one mitotic division

1. 1. When one nuclear division is suppressed at the 4-cell stage by UV, DNP or 4NQQ without affecting the cytoplasmic SH cycle, the treated embryo remains in the 4-cell condition while the control embryo proceeds to the 8-cell stage. Simultaneously with the fourth cleavage of the control, the treated embryo pinches off 4 micromeres directly from the larger cells. The an and Veg materials are segregated only at the time of the fifth normal division. Consequently, the time during which the An and Veg materials remain together within the same blastomere is longer than normal by the duration of two cleavage cycles (8-16-32 cell stages of control). 2. 2. When observed at the gastrula or pluteus stage, the treated embryos are quite normal. This may mean that as long as the right schedule is observed for micromere formation, delay within two cell cycles in the separation of other blastomeres does not disturb harmonious development. 3. 3. Precocious micromeres of treated embryos contain a higher concentration of RNA during the interphase, similar to normal micromeres.

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.

Behaviour of sulfhydryl groups of sea urchin eggs under the blockage of cell division by UV and heat shock

1. 1. In the sea urchins Hemicentrotus pulcherrimus, Anthocidaris crassispina, and Clypeaster japonicus, cleavage of the eggs can be suppressed by short treatment with higher temperature (34 °C) or by UV irradiation. 2. 2. While the division of the cell body is blocked ( a) in the heat treatment, nuclear division is not affected ( b) by UV irradiation, but it is suppressed. 3. 3. During heat treatment ( a), usual cyclic change in SH amounts of 0.6 M KCl-soluble protein stops while in UV treatment ( b), it persists. 4. 4. There are two cycles present in the cell which are independent from each other: one is the nuclear cycle and the other is the cytoplasmic SH cycle. 5. 5. Since cleavage does not take place if one of the two cycles is blocked or they are pushed out of pace, synchronization of the two cycles is a necessary condition for a cleavage to occur. 6. 6. From the fact that the time of micromere formation in UV irradiated eggs always occurs simultaneously with the fourth cleavage of the untreated controls and at the fourth peak of SH, a possibility is considered that the cyclic change of SH may be a pace-maker for differentiation.