Development ofDentalium following removal of D-quadrant blastomeres at successive cleavage stages

Summary Each primary micromere and macromere of the D-quadrant ofDentalium was deleted, through the mesentoblast stage, to investigate the way in which the polar lobe cytoplasm exerts its influence on development.-D and -1D embryos form an apical tuft but no posttrochal structures.-2D embryos form an apical tuft and a reduced posttrochal region without a shell. -3D and -4D are externally similar to control embryos. -1d embryos and -1c embryos have an apical tuft with a reduced number of cilia. Embryos in which both 1c and 1d are deleted lack the apical tuft.-2d embryos lack shell and most other posstrochal structures. -3d and-4d embryos appear externally equivalent to controls.The polar lobe cytoplasm exerts its influence sequentially, and as inIlyanassa the maximal effect is at the third quartet 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.     

Dorsoventral polarity and mesentoblast determination as concomitant results of cellular interactions in the mollusk Patella vulgata.

In mollusks with an equal four-cell stage, dorsoventral polarity becomes noticeable in the interval between the formation of the third and fourth quartet of micromeres, i.e., between the fifth and sixth cleavage. One of the two macromeres at the vegetal cross-furrow then partly withdraws from the surface and becomes located more toward the center of the embryonic cell mass than the other three macromeres. Only this specific macromere (3D) contacts the micromeres of the animal pole, divides with a delay, and develops into the stem cell of the mesentoblast (4d). After suppression of the normal contacts between micromeres and macromeres either by dissociation of the embryos or by deletion of first quartet cells, the normal differentiation of the macromeres fails to appear. By deleting a decreasing number of first quartet cells, an increasing percentage of embryos shows the normal differentiation pattern. Deletion of one of the cross-furrow macromeres does not preclude formation of the mesentoblast, which then originates by differentiation of an other macromere. It is concluded that initially the embryo is radially symmetrical and that the four quadrants have identical developmental capacities; mesentoblast differentiation from one macromere is induced through the contacts of the first quartet cells and that single macromere.     

Phorbol ester disrupts the cleavage pattern in sea urchin embryos

Phorbol-12-myristate-13-acetate (PMA) treatment (9-170 nM) of S. mirabilis embryos from the fertilization to the IV cleavage division resulted in morphogenesis disturbances, such as decrease of the intercellular contacts' extent of blastomeres during the first cleavage divisions, inhibition of micromere formation, and formation of multiple blastulae. After washing from PMA embryos remained viable up to the pluteus stage. Morphogenetic disturbances were not accompanied by changes of intracellular pH or mechanical properties of blastomeres' surface during cleavage. Protein kinase C is suggested to control adhesion of the cell surface and intracellular nucleus movements.     

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.     

The cleavage program in the 2d cell lineage of Tubifex embryos

Early development in clitellate annelids is characterized by a highly stereotyped sequence of unequal, spiral cleavages. Cell 2d (i.e., the second micromere of the D quadrant) in the oligochaete Tubifex tubifex also undergoes an evolutionarily conserved sequence of cell division to produce four bilateral pairs of ectodermal teloblasts that act as embryonic stem cells. This study was conducted to characterize each of the 15 rounds of cell division that occur in the 2d cell lineage in this clitellate. After its occurrence, cell 2d undergoes three rounds of highly unequal divisions, giving off the first smaller daughter cell toward the posterior right of the larger daughter cell, the second cell toward the posterior left, and the third cell toward the anterior side of the cell; the larger daughter cell that results from the third division (i.e., the great-granddaughter cell of 2d) then divides equally into a bilateral pair of NOPQ proteloblasts. Cell NOPQ on either side of the embryo undergoes 11 rounds of cell division, during which ectoteloblasts N, Q, and O/P are produced in this order. After its appearance, NOPQ undergoes highly unequal divisions twice cutting off the smaller cells toward the anterior end of the embryo and then divides almost equally into ectoteloblast N and proteloblast OPQ. After its appearance, OPQ undergoes highly unequal divisions twice giving off the first smaller cell toward the anterior and the second smaller cell toward the posterior of the embryo and then divides almost equally into ectoteloblast Q and proteloblast OP. Finally, OP undergoes highly unequal division four times after its birth budding off the smaller cells toward the anterior and then cleaves equally into ectoteloblasts O and P. In the unequally dividing cells of the 2d cell lineage, the mitotic apparatus (MA), which forms at the cell's center, moves eccentrically toward the cortical site where the smaller cell will be given off. The moving MA is oriented perpendicular to the surface it approaches, and its peripheral pole becomes closely associated with the cell cortex. In contrast, the MA involved in the equal divisions remains in the cell center throughout mitosis. The key features of the cleavage program in the 2d cell lineage are discussed in light of the present observations. The mechanical aspects of unequal cleavage in the 2d cell lineage and the modes of specification of MA orientation are discussed. A comparison of the cleavage mode in the 2d cell lineage is also performed among six selected clitellate annelid species.     

Specification of secondary mesenchyme-derived cells in relation to the dorso-ventral axis in sea urchin blastulae

To learn how the dorso-ventral (DV) axis of sea urchin embryos affects the specification processes of secondary mesenchyme cells (SMC), a fluorescent dye was injected into one of the macromeres of 16-cell stage embryos, and the number of each type of labeled SMC was examined at the prism stage. A large number of labeled pigment cells was observed in embryos in which the progeny of the labeled macromere were distributed in the dorsal part of the embryo. In contrast, labeled pigment cells were scarcely noticed when the descendants of the labeled macromere occupied the ventral part. In such embryos, free mesenchyme cells (probably blastocoelar cells) were predominantly labeled. CH3COONa treatment, which is known to increase the number of pigment cells, canceled such patterned specification of pigment cells and blastocoelar cells along the DV axis. Pigment cells were also derived from the ventral blastomere in the treated embryo. In contrast, a similar number of coelomic pouch cells was derived from the labeled macromere, irrespective of the position of its descendants along the DV axis. After examination of the arrangement of blastomeres in late cleavage stage embryos, it was determined that 17-20 veg2-derived cells encircled the cluster of micromere descendants after the 9th cleavage. From this number and the numbers of SMC-derived cells in later stage embryos, it was suggested that the most vegetally positioned veg2 descendants at approximately the 9th cleavage were preferentially specified to pigment and blastocoelar cell lineages. The obtained results also suggested the existence of undescribed types of SMC scattered in the blastocoele.     

C. elegans HAM-1 positions the cleavage plane and regulates apoptosis in asymmetric neuroblast divisions

Asymmetric cell division occurs when a mother cell divides to generate two distinct daughter cells, a process that promotes the generation of cellular diversity in metazoans. During Caenorhabditis elegans development, the asymmetric divisions of neural progenitors generate neurons, neural support cells and apoptotic cells. C. elegans HAM-1 is an asymmetrically distributed cortical protein that regulates several of these asymmetric neuroblast divisions. Here, we show that HAM-1 is a novel protein and define residues important for HAM-1 function and distribution to the cell cortex. Our phenotypic analysis of ham-1 mutant embryos suggests that HAM-1 controls only neuroblast divisions that produce apoptotic cells. Moreover, ham-1 mutant embryos contain many unusually large cell-death corpses. An investigation of this corpse phenotype revealed that it results from a reversal of neuroblast polarity. A misplacement of the neuroblast cleavage plane generates daughter cells of abnormal size, with the apoptotic daughters larger than normal. Thus, HAM-1 regulates the position of the cleavage plane, apoptosis and mitotic potential in C. elegans asymmetric cell divisions.     

Quantitative and ultrastructural analysis of the chondriome in ovogenesis and embryogenesis of the sea urchin Paracentrotus lividus. 2. Growth and proliferation of mitochondria in embryogenesis.

The dynamics of structural changes of the chondriome in the early development of the sea urchin Paracentrotus lividus was studied. Mature eggs and embryos at various stages of cleavage were used for quantitative and ultrastructural analysis based on computerized 3D reconstruction from serial ultrathin sections. The following structural transformations of the chondriome were shown to occur in the course of embryogenesis: (i) 15 min after fertilization, mitochondrial clusters disintegrate, and mitochondrial division is induced. At the stage of two blastomeres the population of mitochondria increases twofold; (ii) the mitochondria divide by means of the contraction of both outer and inner membranes. The forming furrow divides the "parental" mitochondrion into two equal "daughter" parts; (iii) at the four-cell stage the division ceases, and mitochondria start to grow, so that the mitochondrial length increases; (iv) cell differentiation further stimulates elongation of rod-shaped mitochondria, and the ratio of rod-shaped to spherical mitochondria changes; (v) in an unfertilised egg, the mitochondria are in a condensed form; after fertilisation all the mitochondria acquire a conventional form. Modern concepts of chondriome proliferation in eukaryotic cells are discussed.     

Micromere lineages in the glossiphoniid leech Helobdella

In leech embryos, segmental mesoderm and ectoderm arise from teloblasts by lineages that are already relatively well characterized. Here, we present data concerning the early divisions and the definitive fate maps of the micromeres, a group of 25 small cells that arise during the modified spiral cleavage in leech (Helobdella robusta) and contribute to most of the nonsegmental tissues of the adult. Three noteworthy results of this work are as follows. (1) The c"' and dm' clones (3d and 3c in traditional nomenclature) give rise to a hitherto undescribed network of fibers that run from one end of the embryo to the other. (2) The clones of micromeres b" and b"' (2b and 3b in traditional nomenclature) die in normal development; the b" clone can be rescued to assume the normal c" fate if micromere c" or its clone are ablated in early development. (3) Two qualitative differences in micromere fates are seen between H. robusta (Sacramento) and another Helobdella sp. (Galt). First, in Helobdella sp. (Galt), the clone of micromere b" does not normally die, and contributes a subset of the cells arising exclusively from c" in H. robusta (Sacramento). Second, in Helobdella sp. (Galt), micromere c"' makes no definitive contribution, whereas micromere dm' gives rise to cells equivalent to those arising from c"' and dm' in H. robusta (Sacramento).     

Lineage-specific alteration in cell cycle structure in early Tubifex embryos

Embryos of the freshwater oligochaete Tubifex exhibit asynchrony in division timing as early as the second cleavage; this cleavage asynchrony becomes pronounced as development proceeds. The present study was undertaken to elucidate the composition and duration of the cell cycles of early Tubifex embryos, with special reference to their cell lineages. No significant variations in lengths of cleavage cycles were found among early embryos. In all blastomeres up to the eighth cleavage cycle, the M phase was followed directly by a 30 min S phase, which suggested that early embryos lack G1 phase. The durations of the M phase did not change during this period of development, but did differ between cell lines. The M phase in the A and B cell lines lasted for about 130 min, while the M phase in the C and D cell lines lasted for about 95 min. An examination of chromosome cycles showed that this difference in M phase durations resulted from a longer stay by the A/B cell lines in prometaphase. Only G2 phase lengthened during early development. After several rounds of G2 phase extension, three classes of G2 phase duration were established: the most extended G2 phase (∼6 h) in the first quartette of micromeres (cells 1 a–1 d), the shortest G2 phase (∼1.58 h) in teloblasts, and an intermediate G2 phase (∼2.4 h) in the progeny of macromeres (i.e. endodermal cells). Experiments with syncytial blastomeres showed that the timing of entry into the M phase, hence the duration of the G2 phase, was affected by cytoplasmic compositions. The shortest G2 phase correlated closely with the presence of yolk-free cytoplasm called pole plasm.     

Process of pigment cell specification in the sand dollar, Scaphechinus mirabilis

The process of pigment cell specification in the sand dollar Scaphechinus mirabilis was examined by manipulative methods. In half embryos, which were formed by dissociating embryos at the 2-cell stage, the number of pigment cells was significantly greater than half the number of pigment cells observed in control embryos. This relative increase might have been brought about by the change in the arrangement of blastomeres surrounding the micromere progeny. To examine whether such an increase could be induced at a later stage, embryos were bisected with a glass needle. When embryos were bisected before 7 h postfertilization, the sum of pigment cells observed in a pair of embryo fragments was greater than that in control embryos. This relative increase was not seen when embryos were bisected after 7 h postfertilization. From the size of blastomeres, it became clear that the 9th cleavage was completed by 7 h postfertilization. Aphidicolin treatment revealed that 10-15 pigment founder cells were formed. The results obtained suggest that the pigment founder cells were specified through direct cell contact with micromere progeny after the 9th cleavage, and that most of the founder cells had divided three times before they differentiated into pigment cells.     

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.     

Cell specification and the role of the polar lobe in the gastropod mollusc Crepidula fornicata

A small polar lobe forms at the first and second cleavage divisions in the gastropod mollusc Crepidula fornicata. These lobes normally fuse with the blastomeres that give rise to the D quadrant at the two- and four-cell stages (cells ultimately generating the 4d mesentoblast and D quadrant organizer). Significantly, removal of the small polar lobe had no noticeable effect on subsequent development of the veliger larva. The behavior of the polar lobe and characteristic early cell shape changes involving protrusion of the 3D macromere at the 24-cell suggest that the D quadrant is specified prior to the sixth cleavage division. On the other hand, blastomere deletion experiments indicate that the D quadrant is not determined until the time of formation of the 4d blastomere (mesentoblast). In fact, embryos can undergo regulation to form normal-appearing larvae if the prospective D blastomere or 3D macromere is removed. Removal of the 4d mesentoblast leads to highly disorganized, radial development. Removal of the first quartet micromeres at the 8-cell stage also leads to the development of radialized larvae. These findings indicate that the embryos of C. fornicata follow the mode of development exhibited by equal-cleaving spiralians, which involves conditional specification of the D quadrant organizer via inductive interactions, presumably from the first quartet micromeres.     

Effect of sodium dodecyl sulfate on polar lobe formation and function in Ilyanassa obsoleta embryos

Polar lobes, anucleate vegetal pole protrusions formed by Ilyanassa obsoleta embryos, serve as a mechanism for shunting morphogenetic determinants to one cell during the first two cleavages. Polar lobe material becomes segregated in the CD cell during first cleavage and in the D cell during second cleavage, resulting in a very unequal four-cell stage. Larval structures including external shell, foot, operculum, statocysts, and eyes develop only when polar lobe material is present. Treatment with the anionic detergent sodium dodecyl sulfate (SDS) before and during the first cleavage inhibited polar lobe formation and equalized cleavage, as the lobe material was distributed to two cells. No polar lobes formed during second clevage in SDS-equalized embryos, and the four-cell stage consisted of four equal cells with reduced cell contacts. SDS inrreversibly inhibited polar lobe formation without affecting cytokinesis. Although 27% of the larvae from SDS-equalized embryos had one or more lobe-dependent structures duplicated, morphogenesis was impaired: more than 40% of such larvae failed to form shell and/or statocysts. When cells were separated after equalized first cleavage and raised as pairs, the pairs of resulting larvae duplicated lobe-dependent structures with the same frequency as whole equalized embryos. Possible explanations for impaired morphogenesis in SDS-treated embryos are discussed.     

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.     

Centrifugation redistributes factors determining cleavage patterns in leech embryos

In the normal development of glossiphoniid leech embryos, cytoplasmic reorganization prior to the first cleavage generates visibly distinct domains of yolk-deficient cytoplasm, called teloplasm. During an ensuing series of stereotyped and unequal cell divisions, teloplasm is segregated primarily into cell CD of the two-cell stage and then into cell D of the four-cell and eight-cell stages. The subsequent fate of cell D is also unique in that it alone undergoes further cleavages which generate five bilateral pairs of embryonic stem cells, the mesodermal (M) and ectodermal (N, O/P, O/P, and Q) teloblasts. Here we report studies on the effects of centrifugation on cleavage pattern and protein composition of individual blastomeres of the leech Helobdella triserialis. Centrifugation partially stratifies the cytoplasm of each cell, generating a layer of clear cytoplasm in cell CD derived largely from teloplasm. After centrifuging embryos at the two-cell stage, clear cytoplasm present in cell CD and normally inherited by cell D is redistributed and can be inherited by both cells C and D at the second cleavage. The developmental fates of cells C and D in centrifuged embryos correlate with the amount of clear cytoplasm they receive. In particular, when clear cytoplasm has been distributed roughly equally between the two cells, both cell C and cell D undergo further cleavages resembling the pattern of divisions normally associated with cell D. Likewise, non-yolk-associated proteins, normally found in higher quantities in cell D than in cell C, appear evenly disbursed between the two cells under conditions which induce this fate change. These results are consistent with the idea that the fates of cells C and D are influenced by the distribution or cellular localization of cytoplasmic components.     

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.     

Microinjection of lectins, hyaluronidase, and hyaluronate fragments interferes with cleavage delay and mesoderm induction in embryos of Patella vulgata

In embryos of Patella vulgata at the 32-cell stage, one of the four vegetally located macromeres makes contacts with overlying animal micromeres. As a result, this macromere (designated 3D) divides significantly later than the other macromeres and forms the mesodermal stem cell 4d. Shortly before and during this interaction two types of extracellular matrix are present: a basal lamina-like layer on the tips of the micromeres and a loose fibrillar meshwork in the blastocoel. In this paper we examine the role of the matrix in cleavage delay and mesoderm determination. The microinjection of extracellular matrix-binding lectins, or of hyaluronidase, or of decasaccharide fragments of hyaluronate into the blastocoel results in embryos in which either no or two macromeres are delayed in cleavage and are presumably determined as mesodermal stem cells. We suggest that the fibrillar meshwork is needed for macromere elongation toward the micromeres and that the basal lamina-like layer is involved in the determination process itself.