Unequal cleavage in the early Tubifex embryo

Unequal cleavage that produces two blastomeres of different size is a cleavage pattern that many animals in a variety of phyla, particularly in Spiralia, adopt during early development. This cleavage pattern is apparently instrumental for asymmetric segregation of developmental potential, but it is also indispensable for normal embryogenesis in many animals. Mechanically, unequal cleavage is achieved by either simple unequal cytokinesis or by forming a polar lobe at the egg's vegetal pole. In the present paper, the mechanisms for unequal cytokinesis involved in the first three cleavages in the oligochaete annelid Tubifex are reviewed. The three unequal cleavages are all brought about by an asymmetrically organized mitotic apparatus (MA). The MA of the first cleavage is monastral in that an aster is present at one pole of a bipolar spindle but not at the other. This monastral form, which arises as a result of the involvement of a single centrosome in the MA assembly, is both necessary and sufficient for unequal first cleavage. The egg cortex during the first mitosis is devoid of the ability to remodel spindle poles. In contrast to the non-cortical mechanisms for the first cleavage, asymmetry in the MA organization at the second and third cleavages depends solely on specialized properties of the cell cortex, to which one spindle pole is physically connected. A cortical attachment site for the second cleavage spindle is generated de novo at the cleavage membrane resulting from the first cleavage; it is an actin-based, cell contact-dependent structure. The cortical microtubule attachment site for the third cleavage, which functions independently of contact with other cells, is not generated at the cleavage membrane resulting from the second cleavage, but is located at the animal pole; it may originate from the second polar body formation and become functional at the 4-cell stage.     

Developmental control of cell division in leech embryos

During embryogenesis, cell division must be spatially and temporally regulated with respect to other developmental processes. Leech embryos undergo a series of unequal and asynchronous cleavages to produce individually recognizable cells whose lineages, developmental fates and cell cycle properties have been characterized. Thus, leech embryos provide an opportunity to examine the regulation of cell division at the level of individual well-characterized cells within a community of different types of cells. Isolation of leech homologues of some of the highly conserved regulators of the cell division cycle, and characterization of their patterns of maternal and zygotic expression, indicate that the cell divisions of early leech embryos are regulated by cell type-specific mechanisms. These studies with leech embryos contribute to the emerging appreciation of the diverse mechanisms by which animals regulate cell division during early development.     

Regional specification in the early embryo of the brittle star Ophiopholis aculeata

Early embryogenesis has been examined experimentally in several echinoderm and hemichordate classes. Although these studies suggest that the mechanisms which underlie regional specification have been highly conserved within the echinoderm + hemichordate clade, nothing is known about these mechanisms in several other echinoderm classes, including the Ophiuroidea. In this study, early embryogenesis was examined in a very little studied animal, the ophiuroid Ophiopholis aculeata. In O. aculeata, the first two cleavage planes do not coincide with the animal-vegetal axis but rather form approximately 45 degrees off this axis. A fate map of the early embryo was constructed using microinjected lineage tracers. Most significantly, this fate map indicates that there is a major segregation of ectodermal from endomesodermal fates at first cleavage. The distribution of developmental potential in the early embryo was also examined by isolating different regions of the early embryo and following these isolates though larval development. These analyses indicate that endomesodermal developmental potential segregates unequally at first, second, and third cleavage in O. aculeata. These results provide insight into the mechanisms of regional specification in O. aculeata and yield new material for the study of the evolution of echinoderm development.     

Cell lineage and timing of fate restriction, determination and gene expression in ascidian embryos

Tadpole larvae of ascidians show the basic body plan of chordates. An ascidian larva consists of only few types of cells and has a relatively small number of cells. Cell lineages are simple and invariant among individuals and have been described in detail. The clonal restriction of developmental fate takes place considerably early in development. I review here the temporal relationship between fate restriction, determination and initiation of lineage-specific gene expression during ascidian embryogenesis. In several cases, determination and initiation of gene expression precede fate restriction and occur during the last cell cycle before fate restriction. Such a phenomenon contradicts the traditional view of fate specification and has several important implications for the understanding of the way in which cells execute the developmental pathway.     

Patterning the C. elegans embryo: moving beyond the cell lineage.

The Caenorhabditis elegans embryo undergoes a series of stereotyped cell cleavages that generates the organs and tissues necessary for an animal to survive. Here we review two models of embryonic patterning, one that is lineage-based, and one that focuses on domains of organ and tissue precursors. Our evolving view of C. elegans embryogenesis suggests that this animal develops by mechanisms that are qualitatively similar to those used by other animals.     

Localization and segregation of lineage-specific cleavage potential in embryos of Caenorhabditis elegans

Summary Early embryogenesis of the nematode Caenorhabditis elegans is characterized by the continuous visibility of a germline and the stepwise separation of all somatic cells from it. Germline and somatic cells exhibit different cleavage patterns. Typical for the germline is a series of stemcell-like, unequal cleavages generating blastomeres, which differ in size, cell cycle periods, and fate. Typical for members of somatic cell lineages during early development are their equal and synchronous cleavages generating cells of similar appearance. Using a laser microbeam various experiments have been carried out to investigate the conditions that lead to the two different types of cleavage. Development of partial embryos demonstrates that the potential for germline-like cleavage is localized in the posterior region of the fertilized egg prior to both the formation of pronuclei and the posterior aggregation of germline-specific granules. Experimental alteration of the cleavage plane can result in a switch from unequal to equal cleavage, with an apparent correlation between the orientation of the mitotic spindle and the type of cleavage. Nuclear transfer experiments indicate that nuclei and centrioles are not involved in the decision as to which type of cleavage will be executed. Cytoplasmic transfer from soma-like to germline-like cleaving cells and vice versa does not alter the cleavage type in the recipient cell. But if separation of germline from soma is delayed after the removal of a centrosome, germline-like cleavage may be completely suppressed, all cells thereafter dividing soma-like.Dedicated to Professor A. Egelhaaf on the occasion of his retirement     

Systems‐level quantification of division timing reveals a common genetic architecture controlling asynchrony and fate asymmetry

Coordination of cell division timing is crucial for proper cell fate specification and tissue growth. However, the differential regulation of cell division timing across or within cell types during metazoan development remains poorly understood. To elucidate the systems‐level genetic architecture coordinating division timing, we performed a high‐content screening for genes whose depletion produced a significant reduction in the a synchrony of d ivision between s ister cells (ADS) compared to that of wild‐type during Caenorhabditis elegans embryogenesis. We quantified division timing using 3D time‐lapse imaging followed by computer‐aided lineage analysis. A total of 822 genes were selected for perturbation based on their conservation and known roles in development. Surprisingly, we find that cell fate determinants are not only essential for establishing fate asymmetry, but also are imperative for setting the ADS regardless of cellular context, indicating a common genetic architecture used by both cellular processes. The fate determinants demonstrate either coupled or separate regulation between the two processes. The temporal coordination appears to facilitate cell migration during fate specification or tissue growth. Our quantitative dataset with cellular resolution provides a resource for future analyses of the genetic control of spatial and temporal coordination during metazoan development.     

Changes in voltage-dependent ion currents during meiosis and first mitosis in eggs of an ascidian.

Different patterns of voltage-dependent ion currents are present in mature eggs and in early embryos of the ascidian Boltenia villosa, as if each ion current is regulated in a different manner between fertilization and the early cleavages of embryogenesis. The ion currents appear and/or disappear with precise timing suggesting that they play important roles at specific times during early development. We investigated changes in three voltage-dependent ion currents (an inwardly rectifying chloride current, a calcium current, and a sodium current) and membrane surface area over time between the resumption of meiosis (with fertilization or activation) and the first mitotic cleavage. Using time-lapse video recordings made during whole-cell patch-clamp experiments, we were able to correlate electrophysiological changes with morphological changes and cell cycle related events. Between fertilization and first cleavage, INa was lost exponentially, the density of ICa remained relatively constant, and the amplitudes of both ICl and membrane surface area fluctuated in time with the cell cycle. ICl and surface area increased whenever the cell began dividing--with the polar body extrusions and the formation of the first cleavage furrow. This suggested that the values of ICl and surface area were largest during interphase and smallest during M-phase of each cell cycle. This hypothesis was supported by an experiment in which entry into M-phase was blocked in fertilized eggs by inhibiting protein synthesis. This prevented the decreases of ICl and surface area but allowed the increases to occur normally. Patterns of change in ion currents are current specific and, as is the case with ICl, are tightly correlated with developmental events.     

Mitochondria, redox signaling and axis specification in metazoan embryos

Mitochondria are not only the major energy generators of the eukaryotic cell but they are also sources of signals that control gene expression and cell fate. While mitochondria are often asymmetrically distributed in early embryos, little is known about how they contribute to axial patterning. Here we review studies of mitochondrial distribution in metazoan eggs and embryos and the mechanisms of redox signaling, and speculate on the role that mitochondrial anisotropies might play in the developmental specification of cell fate during embryogenesis of sea urchins and other animals.     

Cell cycle transition in early embryonic development of Xenopus laevis

This article reviews cell cycle changes that occur during midblastula transition (MBT) in Xenopus laevis based on research carried out in the authors' laboratory. Blastomeres dissociated from the animal cap of blastulae, as well as those in an intact embryo, divide synchronously with a constant cell cycle duration in vitro, up to the 12th cell cycle regardless of their cell sizes. During this synchronous cleavage, cell sizes of blastomeres become variable because of repeated unequal cleavage. After the 12th cell cycle blastomeres require contact with an appropriate protein substrate to continue cell division. When nucleocytoplasmic (N/C) ratios of blastomeres reach a critical value during the 13th cycle, their cell cycle durations lengthen in proportion to the reciprocal of cell surface areas, and cell divisions become asynchronous due to variations in cell sizes. The same changes occur in haploid blastomeres with a delay of one cell cycle. Thus, post-MBT cell cycle control becomes dependent not only on the N/C relation but also on cell surface activities of blastomeres. Unlike cell cycle durations of pre-MBT blastomeres, which show monomodal frequency distributions with a peak at about 30 min, those of post-MBT blastomeres show polymodal frequency distributions with peaks at multiples of about 30 min, suggesting 'quantisement' of the cell cycle. Thus, we hypothesised that MPF is produced periodically during its unit cycle with 30 min period, but it titrates, and is neutralized by, an inhibitor contained in the nucleus in a quantity proportional to the genome size; however, when all of the inhibitor has been titrated, excess MPF during the last cycle triggers mitosis. At MBT, cell cycle checkpoint mechanisms begin to operate. While the operation of S phase checkpoint to monitor DNA replication is initiated by N/C relation, the initiation of M phase checkpoint operation to monitor chromosome segregation at mitosis is regulated by an age-dependent mechanism.     

Activating the DNA damage checkpoint in a developmental context

BACKGROUND: Studies in unicellular systems have established that DNA damage by irradiation invokes a checkpoint that acts to stall cell division. During metazoan development, the modulation of cell division by checkpoints must occur in the context of gastrulation, differential gene expression and changes in cell cycle regulation. To understand the effects of checkpoint activation in a developmental context, we examined the effect of X-rays on post-blastoderm embryos of Drosophila melanogaster. RESULTS: In Drosophila, DNA damage was previously found to delay anaphase chromosome separation during cleavage cycles that lack a G2 phase. In post-blastoderm cycles that included a G2 phase, we found that irradiation delayed the entry into mitosis. Gastrulation and the developmental program of string (Cdc25) gene expression, which normally regulates the timing of mitosis, occurred normally after irradiation. The radiation-induced delay of mitosis accompanied the exclusion of mitotic cyclins from the nucleus. Furthermore, a mutant form of the mitotic kinase Cdk1 that cannot be inhibited by phosphorylation drove a mitotic cyclin into the nucleus and overcame the delay of mitosis induced by irradiation. CONCLUSIONS: Developmental changes in the cell cycle, for example, the introduction of a G2 phase, dictate the response to checkpoint activation, for example, delaying mitosis instead of or in addition to delaying anaphase. This unprecedented finding suggests that different mechanisms are used at different points during metazoan development to stall cell division in response to checkpoint activation. The delay of mitosis in post-blastoderm embryos is due primarily to inhibitory phosphorylation of Cdk1, whereas nuclear exclusion of a cyclin-Cdk1 complex might play a secondary role. Delaying cell division has little effect on gastrulation and developmentally regulated string gene expression, supporting the view that development generally dictates cell proliferation and not vice versa.     

Evolutionary implications of the mode of D quadrant specification in coelomates with spiral cleavage

In annelids, molluscs, echiurans and sipunculids the establishment of the dorsal-ventral axis of the embryo is associated with D quadrant specification during embryogenesis. This specification occurs in two ways in these phyla. One mechanism specifies the D quadrant via the shunting of a set of cytoplasmic determinants located at the vegetal pole of the egg to one blastomere of the four cell stage embryo. In this case, at the first two cleavages of embryogenesis there is an unequal distribution of cytoplasm, generating one macromere which is larger than the others at the four cell stage. The D quadrant can also be specified by a contact mediated inductive interaction between one of the macromeres at the vegetal pole with micromeres at the animal pole of the embryo. This mechanism operates at a later stage of development than the cytoplasmic localization mechanism and is associated with a pattern of cleavage in which the first two cleavages are equal. An analysis of the phylogenetic relationships within these phyla indicates that the taxa which determine the D quadrant at an early cleavage stage by cytoplasmic localization tend to be derived and lack a larval stage or have larvae with adult characters. Those taxa where the D quadrant is specified by induction include the ancestral groups although some derived groups also use this mechanism. The pulmonate mollusc Lymnaea uses an inductive mechanism for specifying the D quadrant. In these embryos each of the four vegetal macromeres has the potential of becoming the D macromere; however under normal circumstances one of the two vegetal crossfurrow macromeres almost invariably becomes the D quadrant. Experiments are described here in which the size of one of the blastomeres of the four cell stage Lymnaea embryo is increased; this macromere invariably becomes the D quadrant. These experiments suggest that developmental change in relative blastomere size during the first two cleavages in spiralian embryos that normally cleave equally may have provided a route that has led to the establishment of the cytoplasmic localization mechanism of D quadrant formation.     

Phosphorylation of Mcl‐1 by CDK1–cyclin B1 initiates its Cdc20‐dependent destruction during mitotic arrest

The balance between cell cycle progression and apoptosis is important for both surveillance against genomic defects and responses to drugs that arrest the cell cycle. In this report, we show that the level of the human anti‐apoptotic protein Mcl‐1 is regulated during the cell cycle and peaks at mitosis. Mcl‐1 is phosphorylated at two sites in mitosis, Ser64 and Thr92. Phosphorylation of Thr92 by cyclin‐dependent kinase 1 (CDK1)–cyclin B1 initiates degradation of Mcl‐1 in cells arrested in mitosis by microtubule poisons. Mcl‐1 destruction during mitotic arrest requires proteasome activity and is dependent on Cdc20/Fizzy, which mediates recognition of mitotic substrates by the anaphase‐promoting complex/cyclosome (APC/C) E3 ubiquitin ligase. Stabilisation of Mcl‐1 during mitotic arrest by mutation of either Thr92 or a D‐box destruction motif inhibits the induction of apoptosis by microtubule poisons. Thus, phosphorylation of Mcl‐1 by CDK1–cyclin B1 and its APC/C^Cdc20‐mediated destruction initiates apoptosis if a cell fails to resolve mitosis. Regulation of apoptosis, therefore, is linked intrinsically to progression through mitosis and is governed by a temporal mechanism that distinguishes between normal mitosis and prolonged mitotic arrest.     

Widespread RNA segregation in a spiralian embryo

Asymmetric cell divisions are a crucial mode of cell fate specification in multicellular organisms, but their relative contribution to early embryonic patterning varies among taxa. In the embryo of the mollusc Ilyanassa, most of the early cell divisions are overtly asymmetric. During Ilyanassa early cleavage, mRNAs for several conserved developmental patterning genes localize to interphase centrosomes, and then during division they move to a portion of the cortex that will be inherited by one daughter cell. Here we report an unbiased survey of RNA localization in the Ilyanassa embryo, and examine the overall patterns of centrosomal localization during early development. We find that 3-4% of RNAs are specifically localized to centrosomes during early development, and the remainder are either ubiquitously distributed throughout the cytoplasm or weakly enriched on centrosomes compared with levels in the cytoplasm. We observe centrosomal localization of RNAs in all cells from zygote through the fifth cleavage cycle, and asymmetric RNA segregation in all divisions after the four-cell stage. Remarkably, each specifically localized message is found on centrosomes in a unique subset of cells during early cleavages, and most are found in unique sets of cells at the 24-cell stage. Several specifically localized RNAs are homologous to developmental regulatory proteins in other embryos. These results demonstrate that the mechanisms of localization and segregation are extraordinarily intricate in this system, and suggest that these events are involved in cell fate specification across all lineages in the early Ilyanassa embryo. We propose that greater reliance on segregation of determinants in early cleavage increases constraint on cleavage patterns in molluscs and other spiralian groups.     

Notch and Prospero Repress Proliferation following Cyclin E Overexpression in the Drosophila Bristle Lineage

Understanding the mechanisms that coordinate cell proliferation, cell cycle arrest, and cell differentiation is essential to address the problem of how “normal” versus pathological developmental processes take place. In the bristle lineage of the adult fly, we have tested the capacity of post-mitotic cells to re-enter the cell cycle in response to the overexpression of cyclin E. We show that only terminal cells in which the identity is independent of Notch pathway undergo extra divisions after CycE overexpression. Our analysis shows that the responsiveness of cells to forced proliferation depends on both Prospero, a fate determinant, and on the level of Notch pathway activity. Our results demonstrate that the terminal quiescent state and differentiation are regulated by two parallel mechanisms acting simultaneously on fate acquisition and cell cycle progression.     

C.elegans野生型和par突变体早期胚胎卵裂的观察

不对称分裂在动植物的发育中起到了非常重要的作用。Caenorhabditis elegans（C．elegans）胚胎最早的两次卵裂为研究控制不对称分裂的机制提供了很好的机会。用普通光学显微镜观察了野生型胚胎早期卵裂和par-1、par-2、par-3、par-4突变体胚胎的早期卵裂。野生型胚胎最早的分裂是不等的,产生了两个不同大小的子细胞。两个子细胞又以不同的方向进行第二次分裂。在C．elegans中任意一个par基因的缺失会使胚胎的第一次卵裂丧失不对称性。这会导致一些发育调控因子不能在特定的胚胎细胞中准确地定位,造成细胞分裂纺锤体方向的异常。par类基因参与不对称性的建立,这种不对称性决定了C．elegans身体的前后轴。