Rounds Of Nuclear Division Research Articles

Dynamics of the cell fate specifications during female gametophyte development in Arabidopsis.

The female gametophytes of angiosperms contain cells with distinct functions, such as those that enable reproduction via pollen tube attraction and fertilization. Although the female gametophyte undergoes unique developmental processes, such as several rounds of nuclear division without cell plate formation and final cellularization, it remains unknown when and how the cell fate is determined during development. Here, we visualized the living dynamics of female gametophyte development and performed transcriptome analysis of individual cell types to assess the cell fate specifications in Arabidopsis thaliana. We recorded time lapses of the nuclear dynamics and cell plate formation from the 1-nucleate stage to the 7-cell stage after cellularization using an in vitro ovule culture system. The movies showed that the nuclear division occurred along the micropylar-chalazal (distal-proximal) axis. During cellularization, the polar nuclei migrated while associating with the forming edge of the cell plate, and then, migrated toward each other to fuse linearly. We also tracked the gene expression dynamics and identified that the expression of MYB98pro::GFP-MYB98, a synergid-specific marker, was initiated just after cellularization in the synergid, egg, and central cells and was then restricted to the synergid cells. This indicated that cell fates are determined immediately after cellularization. Transcriptome analysis of the female gametophyte cells of the wild-type and myb98 mutant revealed that the myb98 synergid cells had egg cell-like gene expression profiles. Although in myb98, egg cell-specific gene expression was properly initiated in the egg cells only after cellularization, but subsequently expressed ectopically in one of the 2 synergid cells. These results, together with the various initiation timings of the egg cell-specific genes, suggest complex regulation of the individual gametophyte cells, such as cellularization-triggered fate initiation, MYB98-dependent fate maintenance, cell morphogenesis, and organelle positioning. Our system of live-cell imaging and cell type-specific gene expression analysis provides insights into the dynamics and mechanisms of cell fate specifications in the development of female gametophytes in plants.

Insights Into the Role of Ubiquitination in Meiosis: Fertility, Adaptation and Plant Breeding.

Ubiquitination is a post-translational modification process that plays a central role in protein degradation in eukaryotic cell cell division, including meiosis. This modification affects different cellular processes on a global scale by its pleiotropic ability to modify numerous proteins. Meiosis is essential for sexual reproduction and involves two rounds of nuclear division following a single round of DNA replication to produce haploid gametes. Unlike mitosis, meiosis has a unique prophase I, which involves homologous chromosome interaction including pairing, synapsis, recombination and segregation. Over the last several decades, molecular genetic studies have identified many proteins that participate in meiotic progression. In this review, we focus on the recent advances regarding the role of ubiquitination during plant meiotic cell cycle progression and recombination, especially the role played by the Anaphase-Promoting Complex and E3 ligases in modulating crossover formation and its impact on evolution and plant breeding.

Shaping meiotic chromosomes with SUMO: a feedback loop controls the assembly of the synaptonemal complex in budding yeast.

The synaptonemal complex (SC) is a meiosis-specific chromosomal structure in which homologous chromosomes are intimately linked through arrays of specialized proteins called transverse filaments (TF). Widely conserved in eukaryote meiosis, the SC forms during prophase I and is essential for accurate segregation of homologous chromosomes at meiosis I. However, the basic mechanism overlooking formation and regulation of the SC has been poorly understood. By using the budding yeast Saccharomyces cerevisiae, we recently showed that SC formation is controlled through the attachment of multiple molecules of small ubiquitin-like modifier (SUMO) to a regulator of TF assembly. Intriguingly, this SUMOylation is activated by TF, implicating the involvement of a positive feedback loop in the control of SC assembly. We discuss the implication of this finding and possible involvement of a similar mechanism in regulating other processes.

Functional significance of nuclear export and mRNA binding of meiotic regulator Spo5 in fission yeast.

BackgroundMeiotic cells undergo two rounds of nuclear division and generate gametes. Previous studies have indicated that a number of transcription factors modulate the transcriptome in successive waves during meiosis and spore formation in fission yeast. However, the mechanisms underlying the post-transcriptional regulation in meiosis are not fully understood. The fission yeast spo5 + gene encodes a meiosis-specific RNA-binding protein, which is required for the progression of meiosis II and spore formation. However, the target RNA molecules of Spo5 are yet to be identified. Characterization of meiosis-specific RNA-binding proteins will provide insight into how post-transcriptional regulation influence gene expression during sexual differentiation.ResultsTo assess the functional significance of RNA-recognition motifs (RRMs) of Spo5, we constructed a series of new spo5 truncated mutants and previously reported spo5 missense mutants. In addition, we isolated novel spo5 missense mutants. The phenotypic characteristics of these mutants indicated that the RRMs are essential for both the localization and function of the protein. Interestingly, Spo5 is exported from the nucleus to the cytoplasm via the Rae1-dependent mRNA export pathway, but is unlikely to be involved in global mRNA export. Furthermore, cytoplasmic localization of Spo5 is important for its function, which suggests the involvement of Spo5 in post-transcriptional regulation. We identified pcr1 + mRNA as one of the critical targets of Spo5. The pcr1 + gene encodes an activating transcription factor/cAMP response element binding (ATF/CREB) transcription factor family. Among the four family members, namely Pcr1, Atf1, Atf21, and Atf31, only the mRNA encoding Pcr1 binds to Spo5.ConclusionsSpo5 is exported from the nucleus with mRNAs via the Rae1-dependent pathway. RRMs are necessary for this process and also for the function of Spo5 after the nuclear export. Spo5 appears to influence the activity of pcr1 + mRNA, and the mechanism of how Spo5 stimulates the mRNA to promote the progression of meiosis II and spore formation remains an intriguing question for future research.

Monopolin subunit Csm1 associates with MIND complex to establish monopolar attachment of sister kinetochores at meiosis I.

Sexually reproducing organisms halve their cellular ploidy during gametogenesis by undergoing a specialized form of cell division known as meiosis. During meiosis, a single round of DNA replication is followed by two rounds of nuclear divisions (referred to as meiosis I and II). While sister kinetochores bind to microtubules emanating from opposite spindle poles during mitosis, they bind to microtubules originating from the same spindle pole during meiosis I. This phenomenon is referred to as mono-orientation and is essential for setting up the reductional mode of chromosome segregation during meiosis I. In budding yeast, mono-orientation depends on a four component protein complex referred to as monopolin which consists of two nucleolar proteins Csm1 and Lrs4, meiosis-specific protein Mam1 of unknown function and casein kinase Hrr25. Monopolin complex binds to kinetochores during meiosis I and prevents bipolar attachments. Although monopolin associates with kinetochores during meiosis I, its binding site(s) on the kinetochore is not known and its mechanism of action has not been established. By carrying out an imaging-based screen we have found that the MIND complex, a component of the central kinetochore, is required for monopolin association with kinetochores during meiosis. Furthermore, we demonstrate that interaction of monopolin subunit Csm1 with the N-terminal domain of MIND complex subunit Dsn1, is essential for both the association of monopolin with kinetochores and for monopolar attachment of sister kinetochores during meiosis I. As such this provides the first functional evidence for a monopolin-binding site at the kinetochore.

Equal Inheritance: Genome Management for Proliferating Parasites

Of the 1,400 or so known human pathogen species, those belonging to the Apicomplexa phylum represent a special challenge to researchers. These singlecelled protists cause some of the world’s most prevalent parasitic diseases, including malaria (Plasmodium falciparum) and toxoplasmosis (Toxoplasma gondii). They are challenging to treat for two reasons. First, the parasites are eukaryotic and thus more similar to human cells than bacterial pathogens, making it difficult to find treatments that kill the parasite without harming human host cells. Second, the parasite cells reside within human host cells for much of their life cycle, evading detection by the host’s immune system. Apicomplexan parasites are named for a unique structure, the apical complex, which is present on the apical end of the parasite cell and enables the parasite to penetrate a host cell within seconds of contact. Once within a host cell, the parasite replicates and divides. T. gondii exhibit the simplest pattern of cell division, generally assembling two daughter cells within a mother cell, which then splits to produce the two daughter cells. Other species, like P. falciparum, may undergo multiple consecutive rounds of nuclear division, resulting in many copies of the parasite’s DNA within one engorged cell, before splintering into as many as tens or thousands of daughter cells. The number of daughter cells can depend on the stage of the parasite and on which type of cell it has invaded. A P. falciparum parasite produces 10 to 20 daughter cells within a red blood cell and thousands of daughter cells within a liver cell. This peculiar form of replication leads to interesting questions. With potentially tens or thousands of nuclei waiting in the replicating cell, how does the parasite know how many daughter cells it will need to accommodate the nuclei? And how does it distribute the nuclei to the daughter cells? In this issue of PLOS Biology, Maria Francia, Boris Striepen, and colleagues describe a fiber-like structure in T. gondii cells that appears to match each newly formed nucleus to a daughter cell, ensuring that each new parasite receives exactly one nucleus. The team found that two proteins called TgSFA2 and TgSFA3 together formed two short fibers in the dividing parasite cell. Further microscopic work suggested these fibers may be important for cell division, because the SFA fibers appeared to form near the centrosome. In animal cells, centrosomes serve as organizing centers for the spindle, a set of microtubule protein fibers that reach into the mass of replicated chromosomes and pull the chromosomes apart into two equal sets during cell division. In fact, the team observed that the SFA fibers formed only after the centrosome split in two, a key event occurring at the start of cell division. The fibers eventually grew longer, extending away from the centrosomes and reaching into the developing daughter cells, ultimately terminating on their apical end. Further experiments showed that the SFA fiber did not merely link the centrosome and developing daughter cells, but was required for the proper formation of the daughter cells. The researchers con-

How to halve ploidy: lessons from budding yeast meiosis.

Maintenance of ploidy in sexually reproducing organisms requires a specialized form of cell division called meiosis that generates genetically diverse haploid gametes from diploid germ cells. Meiotic cells halve their ploidy by undergoing two rounds of nuclear division (meiosis I and II) after a single round of DNA replication. Research in Saccharomyces cerevisiae (budding yeast) has shown that four major deviations from the mitotic cell cycle during meiosis are essential for halving ploidy. The deviations are (1) formation of a link between homologous chromosomes by crossover, (2) monopolar attachment of sister kinetochores during meiosis I, (3) protection of centromeric cohesion during meiosis I, and (4) suppression of DNA replication following exit from meiosis I. In this review we present the current understanding of the above four processes in budding yeast and examine the possible conservation of molecular mechanisms from yeast to humans.

Meiotic nuclear divisions in budding yeast require PP2ACdc55-mediated antagonism of Net1 phosphorylation by Cdk

During meiosis, one round of deoxyribonucleic acid replication is followed by two rounds of nuclear division. In Saccharomyces cerevisiae, activation of the Cdc14 early anaphase release (FEAR) network is required for exit from meiosis I but does not lead to the activation of origins of replication. The precise mechanism of how FEAR regulates meiosis is not understood. In this paper, we report that premature activation of FEAR during meiosis caused by loss of protein phosphatase PP2A(Cdc55) activity blocks bipolar spindle assembly and nuclear divisions. In cdc55 meiotic null (cdc55-mn) cells, the cyclin-dependent kinase (Cdk)-counteracting phosphatase Cdc14 was released prematurely from the nucleolus concomitant with hyperphosphorylation of its nucleolar anchor protein Net1. Crucially, a mutant form of Net1 that lacks six Cdk phosphorylation sites rescued the meiotic defect of cdc55-mn cells. Expression of a dominant mutant allele of CDC14 mimicked the cdc55-mn phenotype. We propose that phosphoregulation of Net1 by PP2A(Cdc55) is essential for preventing precocious exit from meiosis I.

Cytokinin receptors in sporophytes are essential for male and female functions in Arabidopsis thaliana

Arabidopsis has three cytokinin receptors genes: CRE1, AHK2, and AHK3. Availability of plants that are homozygous mutant for these three genes indicates that cytokinin receptors in the haploid cells are dispensable for the development of male and female gametophytes. The triple mutants form a few flowers but never set seed, indicating that reproductive growth is impaired. We investigated which reproductive processes are affected in the triple mutants. Anthers of mutant plants contained fewer pollen grains and did not dehisce. Pollen in the anthers completed the formation of the one vegetative nucleus and the two sperm nuclei, as seen in wild type. The majority of the ovules were abnormal: 78% lacked the embryo sac, 10% carried a female gametophyte that terminated its development before completing three rounds of nuclear division, and about 12% completed three rounds of nuclear division but the gametophytes were smaller than those of the wild type. Reciprocal crosses between the wild type and the triple mutants indicated that pollen from mutant plants did not germinate on wild-type stigmas, and wild-type pollen did not germinate on mutant stigmas. These results suggest that cytokinin receptors in the sporophyte are indispensable for anther dehiscence, pollen maturation, induction of pollen germination by the stigma, and female gametophyte formation and maturation.

Trying to Avoid Your Sister

Connections between chromosomes are essential for theiraccurate segregation during cell division. In somatic cells dividingby mitosis, newly replicated sister chromatids are connected bycohesin proteins. When the sister chromatids become attached tomicrotubules emanating from opposite poles of the spindle,cohesins resist the pulling forces and the ensuing tension stabilizesthe chromatid–microtubule attachments. In this way, each pair ofsister chromatids achieves a stable bipolar attachment to thespindle. Consequently, when cohesion is destroyed at the onset ofanaphase, sister chromatids are pulled to opposite poles and eachnew cell receives a full complement of maternal and paternalchromosomes.In the germline, meiosis employs two successive rounds ofnuclear division to produce gametes containing half the number ofchromosomes as the original precursor cell. During the firstdivision, sister chromatids remain connected while the paternaland maternal homologs are segregated (one homolog comprises apair of sister chromatids). Homolog segregation during meiosis isgoverned by the same mechanical principles as sister segregationduring mitosis and, as such, homologs must be connected. Theseconnections are called chiasmata, and they are established via aprocess called homologous recombination, a DNA repair processthat involves interaction between a broken chromosome and ahomologous template chromosome.To ensure that each pair of homologs is connected by at leastone chiasma, homologous recombination during meiosis isregulated at several levels. A key aspect of this regulation is thechoice of recombination template. The sister chromatid is thepreferred template for recombinational repair in cells dividing bymitosis. However, during meiosis this bias must be overcome sothat homologs recombine and become connected by chiasmata.How template choice is regulated remains unclear, but studies ofmeiotic recombination in budding yeast have suggested a numberof possible mechanisms.

Promoter-driven splicing regulation in fission yeast

The meiotic cell cycle is modified from the mitotic cell cycle by having a pre-meiotic S phase that leads to high levels of recombination, two rounds of nuclear division with no intervening DNA synthesis and a reductional pattern of chromosome segregation. Rem1 is a cyclin that is only expressed during meiosis in the fission yeast Schizosaccharomyces pombe. Cells in which rem1 has been deleted show decreased intragenic meiotic recombination and a delay at the onset of meiosis I (ref. 1). When ectopically expressed in mitotically growing cells, Rem1 induces a G1 arrest followed by severe mitotic catastrophes. Here we show that rem1 expression is regulated at the level of both transcription and splicing, encoding two proteins with different functions depending on the intron retention. We have determined that the regulation of rem1 splicing is not dependent on any transcribed region of the gene. Furthermore, when the rem1 promoter is fused to other intron-containing genes, the chimaeras show a meiotic-specific regulation of splicing, exactly the same as endogenous rem1. This regulation is dependent on two transcription factors of the forkhead family, Mei4 (ref. 2) and Fkh2 (ref. 3). Whereas Mei4 induces both transcription and splicing of rem1, Fkh2 is responsible for the intron retention of the transcript during vegetative growth and the pre-meiotic S phase.

Distinct Nuclear and Cytoplasmic Functions of the S. pombe Cdc14-like Phosphatase Clp1p/Flp1p and a Role for Nuclear Shuttling in Its Regulation

Cdc14-like phosphatases regulate a variety of cell cycle events by dephosphorylating CDK sites. Their cell cycle-dependent changes in localization may be important to carry out distinct functions. Work in budding and fission yeast suggested that Cdc14-like phosphatases are inhibited by nucleolar sequestration. In S. cerevisiae, Cdc14p is released from the nucleolus by the FEAR network and Cdk1, whereas the S. pombe CDC14-like phosphatase Clp1p (also known as Flp1p) is released at mitotic entry by an unknown mechanism. The mitotic exit network (MEN) in S. cerevisiae and its homologous network, the septation initiation network (SIN), in S. pombe act through an unknown mechanism to keep the phosphatase out of the nucleolus in late mitosis. SIN-dependent cytoplasmic maintenance of Clp1p is thought to be essential for the cytokinesis checkpoint, which blocks further rounds of nuclear division until cytokinesis is completed. By targeting Clp1p to the nucleus or the cytoplasm, we demonstrate distinct functions for these pools of Clp1p in chromosome segregation and cytokinesis, respectively. Our results further suggest that the SIN does not keep Clp1p out of the nucleolus by regulating nucleolar affinity, as proposed for S. cerevisiae Cdc14p, but instead, Clp1p may be regulated by nuclear import/export.

The Clp1p/Flp1p phosphatase ensures completion of cytokinesis in response to minor perturbation of the cell division machinery in Schizosaccharomyces pombe.

Fission yeast mutants defective in actomyosin ring formation and function exhibit a prolonged G2 delay following cytokinesis failure. This G2 delay depends on the SIN, a signaling network essential for cytokinesis, and the non-essential Cdc14p family phosphatase, Clp1p/Flp1p and has been proposed to signify a cytokinesis checkpoint mechanism. However, the physiological relevance of this proposed Clp1p/Flp1p-dependent checkpoint is unclear because all previous studies were carried out using mutations in essential actomyosin ring components under fully restrictive conditions and thus these cells would have died regardless of the presence of the checkpoint. Here we show that delays in cytokinesis caused by minor perturbations to different components of the cytokinetic machinery, which normally cause only mild defects, become lethal when Clp1p/Flp1p is inactivated. In addition, we show that Clp1p/Flp1p does not function simply to inhibit further rounds of nuclear division, but also allows damaged actomyosin rings to be maintained to facilitate completion of cell division. Ectopic activation of the SIN significantly bypasses the requirement of Clp1p/Flp1p for G2 delay as well as for completion of cytokinesis. We conclude that the Clp1p/Flp1p-dependent cytokinesis checkpoint provides a previously unrecognized cell survival advantage when the cell division apparatus is mildly perturbed.

The NOMEGA gene required for female gametophyte development encodes the putative APC6/CDC16 component of the Anaphase Promoting Complex in Arabidopsis.

Development of the female gametophyte involves several rounds of nuclear divisions during which nuclei are rearranged and finally cellularized to form a mature seven-celled embryo sac. During these nuclear divisions, key proteins involved in the cell cycle need to be degraded quickly in order to facilitate both the metaphase-anaphase transition stage and late anaphase. Here, we report the characterization of an Arabidopsis mutant nomega, which results in arrest of the embryo sac development at the two-nucleate stage. The NOMEGA gene product shows high homology to the APC6/cell division cycle (CDC)16 subunit of the Anaphase Promoting Complex/Cyclosome (APC/C). The phenotype of the nomega mutant is quite different from that of the hobbit mutant, which had suggested a role for the plant APC/C in auxin signalling. We show that nomega mutant embryo sacs are unable to degrade Cyclin B, an important APC/C substrate, providing further evidence of a role for the NOMEGA gene product and the plant APC/C in cell cycle progression during gametophyte development.

SSP2, a sporulation-specific gene necessary for outer spore wall assembly in the yeast Saccharomyces cerevisiae.

Sporulation in yeast consists of two highly coordinated processes. First, a diploid cell that is heterozygous at the mating-type locus undergoes meiosis, in which one round of DNA replication is followed by two rounds of nuclear division. Second, the meiotic products are packaged into spore cells that remain within the mother cell. A large number of genes are induced specifically during sporulation, and their products carry out different sporulation-specific events. Expression of these sporulation-specific genes is controlled by several regulators which function at different stages of the sporulation program, resulting in a cascade of gene expression following induction of meiosis. Here we describe one sporulation-specific gene, SSP2, which is induced midway through meiosis. Ssp2 shows significant homology to the predicted product of a hypothetical ORF in Candida albicans. Homozygous mutant ssp2 diploid cells fail to sporulate. In the mutant background, meiotic recombination and nuclear divisions remain normal; however, viability declines rapidly. Following meiosis, ssp2 cells form the prospore membrane, but fail to form the outer layer of the spore wall. The Ssp2 protein localizes to the spore wall after meiosis II. In addition, the ssp2 defect is also associated with delayed and reduced expression of late sporulation-specific genes. Our results suggest that SSP2 function is required after meiosis II and during spore wall formation.

Distinct roles for pbs21 and pbs25 in the in vitro ookinete to oocyst transformation of Plasmodium berghei

We have developed an in vitro culture system for early sporogonic stages of Plasmodium berghei, which can be used to study developmental events normally taking place in the midgut of an infected mosquito. These include penetration of insect cells by the mature ookinete, transformation into oocysts and the early development of the latter, sustained through several rounds of nuclear division. The system, based upon co-culture of enriched ookinetes with several established insect cell lines, was used to study the development of mutant ookinetes lacking both the Pbs21 and Pbs25 surface proteins. Motility and entry of double knockout and Pbs21 single knockout ookinetes into the insect cells are normal, but the number of ookinetes successfully transforming into oocysts expressing the CSP protein are substantially reduced. Finally, using the yeast two-hybrid system we also show that Pbs25 has the capacity to homodimerise as well as to form heterodimers with Pbs21.

SSP1, a gene necessary for proper completion of meiotic divisions and spore formation in Saccharomyces cerevisiae.

During meiosis, a diploid cell undergoes two rounds of nuclear division following one round of DNA replication to produce four haploid gametes. In yeast, haploid meiotic products are packaged into spores. To gain new insights into meiotic development and spore formation, we followed differential expression of genes in meiotic versus vegetatively growing cells in the yeast Saccharomyces cerevisiae. Our results indicate that there are at least five different classes of transcripts representing genes expressed at different stages of the sporulation program. Here we describe one of these differentially expressed genes, SSP1, which plays an essential role in meiosis and spore formation. SSP1 is expressed midway through meiosis, and homozygous ssp1 diploid cells fail to sporulate. In the ssp1 mutant, meiotic recombination is normal but viability declines rapidly. Both meiotic divisions occur at the normal time; however, the fraction of cells completing meiosis is significantly reduced, and nuclei become fragmented soon after meiosis II. The ssp1 defect does not appear to be related to a microtubule-cytoskeletal-dependent event and is independent of two rounds of chromosome segregation. The data suggest that Ssp1 is likely to function in a pathway that controls meiotic nuclear divisions and coordinates meiosis and spore formation.

SepB: an Aspergillus nidulans gene involved in chromosome segregation and the initiation of cytokinesis.

In Aspergillus nidulans conidia, cytokinesis (septation) is delayed until three rounds of nuclear division have been completed. This has permitted the identification of essential genes that are involved in the coordination of cytokinesis with nuclear division. Conditional mutations in the sepB gene block septation but allow germinating spores to complete the first three rounds of nuclear division at restrictive temperature. sepB3 mutants demonstrate transient delays in M-phase, accumulate aneuploid nuclei and show defects in chromosome segregation. Molecular analysis of the sepB gene reveals that it is essential and possesses limited similarity to the CTF4 gene of Saccharomyces cerevisiae. Using temperature-shift analysis we show that sepB is required after the first nuclear division but before the onset of cytokinesis. A failure to execute the sepB function results in a block to nuclear division and leads to cell death at a time when wild-type cells would be undergoing cytokinesis. Finally, we demonstrate that sepB is also required for the uninucleate cell divisions of developing conidiophores. Our results suggest that sepB3 mutants accumulate specific nuclear defects that do not arrest mitosis, but block the initiation of septum formation. Thus, proper chromosome segregation and a functional sepB gene are required to initiate cytokinesis.

Visualization of cytoskeletal changes through the life cycle inAcetabularia

The cytoskeleton in the siphonous, marine green algaAcetabularia is visualized by immunocytochemistry using antibodies against plant alfa tubulin and animal smooth muscle actin. In the vegetative phase of the life cycle, when the cell grows a cylindrical stalk and until the reproductive cap is completed, actin forms continuous, parallel bundles that extend through the entire length of the stalk and cap rays respectively. Microtubules (MTs) cannot be detected until the primary nucleus, located in the rhizoid of the giant cell, divides to form thousands of secondary nuclei. MTs can then be seen radiating from each secondary nucleus that is encountered in the stalk on its migration upwards into the cap rays. They are oriented mostly parallel to the long axis of the cell. At arrival in the cap rays up to the “white spot” stage, when nuclei assume equidistant positions in the cap ray cytoplasm, a radiating system of MTs forms around each nucleus and dramatically increases until impressive radial arrays have developed. This phase coincides with a disappearance of actin bundles in the cap rays, but they are retained in the stalk cytoplasm. Shortly after that additional MTs appear around the disk like partitions of cap ray cytoplasm. Concomitantly, bundles of actin reappear colinearly with the circumferrential MTs eventually forming complete rings around each disk of cap ray cytoplasm. During this process the compartments of the future cysts are gradually bulging outwards and simultaneously the rings of actin sink inwards until domes are formed with the nuclei fixed in the top centers of the domes. At this stage the peripheral areas of the radiating MT systems around the nuclei start to break down, whereas the circumferrential MT systems remain intact. Subsequently, the rings of both actin and MTs decrease in diameter, and finally contract to a spot opposite the nucleus, while the cysts continue to develop their oval shape. After the cysts have become separated, they round up and enter several rounds of nuclear divisions. MTs form short radial arrays around each nucleus with minor changes due to a reduction of MTs during division followed by a reappearance after completion of each division. Actin is rearranged in the cysts to a cortical network of randomly oriented, short bundles, that is maintained until gamete formation sets in.

Segmentation and commitment in the leech embryo

Leeches are useful for studying embryogenesis. As members of the phylum Annelida, they are organized into a discrete number of metameric units called segments, akin to those of insects and other members of the closely related phylum Arthropoda. For several reasons, developmental studies of the leech often complement those of insects. Leeches have fewer segment-specific specializations and far fewer cells than do insects. Moreover, many of the cells in the mature animal-most notably the neurons-are individually identifiable and often physiologically accessible. Finally, embryogenesis in the leech proceeds via complete cleavages, so that individual cells can be identified from the start, whereas the insect egg undergoes several rounds of nuclear division without cytokinesis, forming a syncytial blastula with several thousand, apparently uncommitted, nuclei. The general pathway of events in leech embryogenesis is presented in the figure. Cells comprising the segments arise by stereotyped lineages from ten columns (bandlets) of blasts cells, longitudinally arrayed in an embryonic structure called the germinal plate. The blast cells in each bandlet are the birth-ranked (first born at the rostra1 end) progeny of a defined embryonic stem cell called a teloblast, which lies at the caudal end of the bandlet. There are five bilateral pairs of teloblasts: one mesodermal pair (M) and four ectodermal pairs (N, O/P, O/P and Q). The teloblasts themselves arise by stereotyped, holoblastic cleavages from the egg. Genesis of Tissue Type Based on the results of pioneering lineage studies with numerous invertebrates, it was predicted that each bilateral pair of teloblasts in the leech would generate only one tissue type. This hypothesis has recently been disproven (as have analogous dogmas for nematode and ascidian embryos). All leech teloblasts, including the mesodermal precursors, give rise to some neurons (traditionally regarded as ectodermal derivatives); all four ectodermal precursors generate epidermal cells and one even contributes to the ducts of nephridia (traditionally regarded as a mesodermal derivative). However, these contributions to tissue type are not made randomly (Kramer and Weisblat, J. Neurosci. 5, 388-407, 1985; We&blat et al., Dev. Biol. 704, 65-85, 1984). Corresponding to the five teloblasts on each side of the early embryo are five distinct groups of cells in each half-segment of the late embryo, called the M, N, 0, P, and CI kinship groups. The stereotyped spatial pattern of each kinship group invariably contains certain identified cells and not others. At present, there is no obvious morphological, biochemical, or functional explanation for the assignment of cells to particular kinship groups. Minireview