Regulators specifying cell fate activate cell cycle regulator genes to determine cell numbers in ascidian larval tissues.

In animal development, most cell types stop dividing before terminal differentiation; thus, cell cycle control is tightly linked to cell differentiation programmes. In ascidian embryos, cell lineages do not vary among individuals, and rounds of the cell cycle are determined according to cell lineages. Notochord and muscle cells stop dividing after eight or nine rounds of cell division depending on their lineages. In the present study, we showed that a Cdk inhibitor, Cdkn1.b, is responsible for stopping cell cycle progression in these lineages. Cdkn1.b is also necessary for epidermal cells to stop dividing. In contrast, mesenchymal and endodermal cells continue to divide even after hatching, and Myc is responsible for maintaining cell cycle progression in these tissues. Expression of Cdkn1.b in notochord and muscle is controlled by transcription factors that specify the developmental fate of notochord and muscle. Likewise, expression of Myc in mesenchyme and endoderm is under control of transcription factors that specify the developmental fate of mesenchyme and endoderm. Thus, cell fate specification and cell cycle control are linked by these transcription factors.

The Starfish Asterina pectinifera: Collection and Maintenance of Adults and Rearing and Metamorphosis of Larvae.

Here we describe methods for (a) collecting starfish during their breeding period; (b) maintaining adults with fully grown gonads in laboratory aquaria; (c) rearing fertilized eggs to brachiolaria larvae, and (d) inducing larvae to metamorphose into juveniles under laboratory conditions. Such protocols should facilitate various analyses of starfish development throughout the entire life cycle of these model organisms.

SGK phosphorylates Cdc25 and Myt1 to trigger cyclin B-Cdk1 activation at the meiotic G2/M transition.

The kinase cyclin B-Cdk1 complex is a master regulator of M-phase in both mitosis and meiosis. At the G2/M transition, cyclin B-Cdk1 activation is initiated by a trigger that reverses the balance of activities between Cdc25 and Wee1/Myt1 and is further accelerated by autoregulatory loops. In somatic cell mitosis, this trigger was recently proposed to be the cyclin A-Cdk1/Plk1 axis. However, in the oocyte meiotic G2/M transition, in which hormonal stimuli induce cyclin B-Cdk1 activation, cyclin A-Cdk1 is nonessential and hence the trigger remains elusive. Here, we show that SGK directly phosphorylates Cdc25 and Myt1 to trigger cyclin B-Cdk1 activation in starfish oocytes. Upon hormonal stimulation of the meiotic G2/M transition, SGK is activated by cooperation between the Gßγ-PI3K pathway and an unidentified pathway downstream of Gßγ, called the atypical Gßγ pathway. These findings identify the trigger in oocyte meiosis and provide insights into the role and activation of SGK.

SGK regulates pH increase and cyclin B-Cdk1 activation to resume meiosis in starfish ovarian oocytes.

Tight regulation of intracellular pH (pHi) is essential for biological processes. Fully grown oocytes, having a large nucleus called the germinal vesicle, arrest at meiotic prophase I. Upon hormonal stimulus, oocytes resume meiosis to become fertilizable. At this time, the pHi increases via Na+/H+ exchanger activity, although the regulation and function of this change remain obscure. Here, we show that in starfish oocytes, serum- and glucocorticoid-regulated kinase (SGK) is activated via PI3K/TORC2/PDK1 signaling after hormonal stimulus and that SGK is required for this pHi increase and cyclin B-Cdk1 activation. When we clamped the pHi at 6.7, corresponding to the pHi of unstimulated ovarian oocytes, hormonal stimulation induced cyclin B-Cdk1 activation; thereafter, oocytes failed in actin-dependent chromosome transport and spindle assembly after germinal vesicle breakdown. Thus, this SGK-dependent pHi increase is likely a prerequisite for these events in ovarian oocytes. We propose a model that SGK drives meiotic resumption via concomitant regulation of the pHi and cell cycle machinery.

MPF-based meiotic cell cycle control: Half a century of lessons from starfish oocytes.

In metazoans that undergo sexual reproduction, genomic inheritance is ensured by two distinct types of cell cycle, mitosis and meiosis. Mitosis maintains the genomic ploidy in somatic cells reproducing within a generation, whereas meiosis reduces by half the ploidy in germ cells to prepare for successive generations. The meiotic cell cycle is believed to be a derived form of the mitotic cell cycle; however, the molecular mechanisms underlying both of these processes remain elusive. My laboratory has long studied the meiotic cell cycle in starfish oocytes, particularly the control of meiotic M-phase by maturation- or M phase-promoting factor (MPF) and the kinase cyclin B-associated Cdk1 (cyclin B-Cdk1). Using this system, we have unraveled the molecular principles conserved in metazoans that modify M-phase progression from the mitotic type to the meiotic type needed to produce a haploid genome. Furthermore, we have solved a long-standing enigma concerning the molecular identity of MPF, a universal inducer of M-phase both in mitosis and meiosis of eukaryotic cells.

Identification of jellyfish neuropeptides that act directly as oocyte maturation-inducing hormones.

Oocyte meiotic maturation is crucial for sexually reproducing animals, and its core cytoplasmic regulators are highly conserved between species. By contrast, the few known maturation-inducing hormones (MIHs) that act on oocytes to initiate this process are highly variable in their molecular nature. Using the hydrozoan jellyfish species Clytia and Cladonema, which undergo oocyte maturation in response to dark-light and light-dark transitions, respectively, we deduced amidated tetrapeptide sequences from gonad transcriptome data and found that synthetic peptides could induce maturation of isolated oocytes at nanomolar concentrations. Antibody preabsorption experiments conclusively demonstrated that these W/RPRPamide-related neuropeptides account for endogenous MIH activity produced by isolated gonads. We show that the MIH peptides are synthesised by neural-type cells in the gonad, are released following dark-light/light-dark transitions, and probably act on the oocyte surface. They are produced by male as well as female jellyfish and can trigger both sperm and egg release, suggesting a role in spawning coordination. We propose an evolutionary link between hydrozoan MIHs and the neuropeptide hormones that regulate reproduction upstream of MIHs in bilaterian species.

MPF, starfish oocyte and cell-free extract in the background - an interview with Takeo Kishimoto.

Professor Takeo Kishimoto's research has an enormous impact on the cell cycle field. Although his favorite model has always been a starfish oocyte, he has used many other model organisms in his research. Cell-free extracts have been wildly used in his laboratory as a very useful tool to answer cell cycle research questions. Recently, professor Kishimoto discovered the identity of the M-phase promoting factor (MPF) that was thought for years to be cyclin-dependent kinase 1 (CDK1). However, Takeo Kishimoto found that MPF consists in fact of two kinases: CDK1 and Greatwall kinase. While CDK1 phosphorylates mitotic substrates, Greatwall kinase allows these substrates to persist in their phosphorylated state because it regulates phosphatase PP2A, which dephosphorylates the majority of CDK1 substrates. When I started to interview Prof. Kishimoto, I was mostly interested in his experiences with cell-free extracts. However, as you will see below we almost immediately turned to the problem of the identity of MPF. This is fully understandable because the identity of MPF seems to be a major interest in Takeo's scientific career. I hope readers will enjoy this interview and will be able to learn about many aspects of scientific research, which do not usually appear in regular research papers.

Two new competing pathways establish the threshold for cyclin-B-Cdk1 activation at the meiotic G2/M transition.

Extracellular ligands control biological phenomena. Cells distinguish physiological stimuli from weak noise stimuli by establishing a ligand-concentration threshold. Hormonal control of the meiotic G2/M transition in oocytes is essential for reproduction. However, the mechanism for threshold establishment is unclear. In starfish oocytes, maturation-inducing hormones activate the PI3K-Akt pathway through the Gßγ complex of heterotrimeric G-proteins. Akt directly phosphorylates both Cdc25 phosphatase and Myt1 kinase, resulting in activation of cyclin-B-Cdk1, which then induces meiotic G2/M transition. Here, we show that cyclin-B-Cdk1 is partially activated after subthreshold hormonal stimuli, but this triggers negative feedback, resulting in dephosphorylation of Akt sites on Cdc25 and Myt1, thereby canceling the signal. We also identified phosphatase activity towards Akt substrates that exists independent of stimuli. In contrast to these negative regulatory activities, an atypical Gßγ-dependent pathway enhances PI3K-Akt-dependent phosphorylation. Based on these findings, we propose a model for threshold establishment in which hormonal dose-dependent competition between these new pathways establishes a threshold; the atypical Gßγ-pathway becomes predominant over Cdk-dependent negative feedback when the stimulus exceeds this threshold. Our findings provide a regulatory connection between cell cycle and signal transduction machineries.

Entry into mitosis: a solution to the decades-long enigma of MPF.

Maturation or M phase-promoting factor (MPF) is the universal inducer of M phase common to eukaryotic cells. MPF was originally defined as a transferable activity that can induce the G2/M phase transition in recipient cells. Today, however, MPF is assumed to describe an activity that exhibits its effect in donor cells, and furthermore, MPF is consistently equated with the kinase cyclin B-Cdk1. In some conditions, however, MPF, as originally defined, is undetectable even though cyclin B-Cdk1 is fully active. For over three decades, this inconsistency has remained a long-standing puzzle. The enigma is now resolved through the elucidation that MPF, defined as an activity that exhibits its effect in recipient cells, consists of at least two separate kinases, cyclin B-Cdk1 and Greatwall (Gwl). Involvement of Gwl in MPF can be explained by its contribution to the autoregulatory activation of cyclin B-Cdk1 and by its stabilization of phosphorylations on cyclin B-Cdk1 substrates, both of which are essential when MPF induces the G2/M phase transition in recipient cells. To accomplish these tasks, Gwl helps cyclin B-Cdk1 by suppressing protein phosphatase 2A (PP2A)-B55 that counteracts cyclin B-Cdk1. MPF, as originally defined, is thus not synonymous with cyclin B-Cdk1, but is instead a system consisting of both cyclin B-Cdk1 that directs mitotic entry and Gwl that suppresses the anti-cyclin B-Cdk1 phosphatase. The current view that MPF is a synonym for cyclin B-Cdk1 in donor cells is thus imprecise; instead, MPF is best regarded as the entire pathway involved in the autoregulatory activation of cyclin B-Cdk1, with specifics depending on the experimental system.

Cyclin B-Cdk1 inhibits protein phosphatase PP2A-B55 via a Greatwall kinase-independent mechanism.

Entry into M phase is governed by cyclin B-Cdk1, which undergoes both an initial activation and subsequent autoregulatory activation. A key part of the autoregulatory activation is the cyclin B-Cdk1-dependent inhibition of the protein phosphatase 2A (PP2A)-B55, which antagonizes cyclin B-Cdk1. Greatwall kinase (Gwl) is believed to be essential for the autoregulatory activation because Gwl is activated downstream of cyclin B-Cdk1 to phosphorylate and activate α-endosulfine (Ensa)/Arpp19, an inhibitor of PP2A-B55. However, cyclin B-Cdk1 becomes fully activated in some conditions lacking Gwl, yet how this is accomplished remains unclear. We show here that cyclin B-Cdk1 can directly phosphorylate Arpp19 on a different conserved site, resulting in inhibition of PP2A-B55. Importantly, this novel bypass is sufficient for cyclin B-Cdk1 autoregulatory activation. Gwl-dependent phosphorylation of Arpp19 is nonetheless necessary for downstream mitotic progression because chromosomes fail to segregate properly in the absence of Gwl. Such a biphasic regulation of Arpp19 results in different levels of PP2A-B55 inhibition and hence might govern its different cellular roles.

Antibody inhibition of protein activity in starfish oocytes.

Antibodies are widely utilized in cell and molecule biology for immunoblots, immunostaining, immunoprecipitation, immunoaffinity purification, and immunoassay. Some antibodies can be used for in vivo inhibition experiments. These antibodies bind to their target molecules and neutralize their functions, providing functional information in the study of their biological role. Here, we describe our methods for obtaining inhibitory antibodies against desired proteins. We then describe in the starfish oocyte system how to inhibit a target protein, even in the nucleus, by injection of antibody into the cytoplasm, and how to evaluate antibody inhibition of cell cycle regulators in small numbers of oocytes.

The Mos-MAPK pathway regulates Diaphanous-related formin activity to drive cleavage furrow closure during polar body extrusion in starfish oocytes.

Maintenance of spindle attachment to the cortex and formation of the cleavage furrow around the protruded spindle are essential for polar body extrusion (PBE) during meiotic maturation of oocytes. Although spindle movement to the cortex has been well-studied, how the spindle is maintained at the cortex during PBE is unknown. Here, we show that activation of Diaphanous-related formin mediated by mitogen-activated protein kinase (MAPK) is required for tight spindle attachment to the cortex and cleavage furrow closure during PBE in starfish (Asterina pectinifera) oocytes. A. pectinifera Diaphanous-related formin (ApDia) had a distinct localization in immature oocytes and was localized to the cleavage furrow during PBE. Inhibition of the Mos-MAPK pathway or the actin nucleating activity of formin homology 2 domain prevented cleavage furrow closure and resulted in PBE failure. In MEK/MAPK-inhibited oocytes, activation of ApDia by relief of its intramolecular inhibition restored PBE. In summary, this study elucidates a link between the Mos-MAPK pathway and Diaphanous-related formins, that is responsible for maintaining tight spindle attachment to the cortex and cleavage furrow closure during PBE.

Greatwall kinase and cyclin B-Cdk1 are both critical constituents of M-phase-promoting factor.

Maturation/M-phase-promoting factor is the universal inducer of M-phase in eukaryotic cells. It is currently accepted that M-phase-promoting factor is identical to the kinase cyclin B-Cdk1. Here we show that cyclin B-Cdk1 and M-phase-promoting factor are not in fact synonymous. Instead, M-phase-promoting factor contains at least two essential components: cyclin B-Cdk1 and another kinase, Greatwall kinase. In the absence of Greatwall kinase, the M-phase-promoting factor is undetectable in oocyte cytoplasm even though cyclin B-Cdk1 is fully active, whereas M-phase-promoting factor activity is restored when Greatwall kinase is added back. Although the excess amount of cyclin B-Cdk1 alone, but not Greatwall kinase alone, can induce nuclear envelope breakdown, spindle assembly is abortive. Addition of Greatwall kinase greatly reduces the amount of cyclin B-Cdk1 required for nuclear envelope breakdown, resulting in formation of the spindle with aligned chromosomes. M-phase-promoting factor is thus a system consisting of one kinase (cyclin B-Cdk1) that directs mitotic entry and a second kinase (Greatwall kinase) that suppresses the protein phosphatase 2A-B55 which opposes cyclin B-Cdk1.

Antisense morpholino targeting just upstream from a poly(A) tail junction of maternal mRNA removes the tail and inhibits translation.

Gene downregulation by antisense morpholino oligonucleotides (MOs) is achieved by either hybridization around the translation initiation codon or by targeting the splice donor site. In the present study, an antisense MO method is introduced that uses a 25-mer MO against a region at least 40-nt upstream from a poly(A) tail junction in the 3'-untranslated region (UTR) of maternal mRNA. The MO removed the poly(A) tail and blocked zebrafish cdk9 (zcdk9) mRNA translation, showing functional mimicry between miRNA and MO. A PCR-based assay revealed MO-mediated specific poly(A) tail removal of zebrafish mRNAs, including those for cyclin B1, cyclin B2 and tbp. The MO activity targeting cyclins A and B mRNAs was validated in unfertilized starfish oocytes and eggs. The MO removed the elongated poly(A) tail from maternal matured mRNA. This antisense method introduces a new application for the targeted downregulation of maternal mRNAs in animal oocytes, eggs and early embryos.

A primer on meiotic resumption in starfish oocytes: the proposed signaling pathway triggered by maturation-inducing hormone.

This short review updates the maturation-inducing hormonal signaling in starfish oocytes. In this system, the activation of cyclin B-Cdc2 kinase (Cdk1) that leads to meiotic resumption does not require new protein synthesis. The key intracellular mediator after hormonal stimulation by 1-methyladenine is the protein kinase Akt/PKB, which in turn directly downregulates Myt1 and upregulates Cdc25 toward the activation of cyclin B-Cdc2. Mitotic kinases including Aurora, Plk1 and Greatwall are activated downstream of cyclin B-Cdc2. The starfish oocyte thus provides a simple model system for the study of meiotic resumption.

Phosphorylation of Claspin is triggered by the nucleocytoplasmic ratio at the Xenopus laevis midblastula transition.

At the Xenopus midblastula transition (MBT), cell cycles lengthen, and checkpoints that respond to damaged or unreplicated DNA are established. The MBT is triggered by a critical nucleocytoplasmic (N/C) ratio; however, the molecular basis for its initiation remains unknown. In egg extracts, activation of Chk1 checkpoint kinase requires the adaptor protein Claspin, which recruits Chk1 for phosphorylation by ATR. At the MBT in embryos, Chk1 is transiently activated to lengthen the cell cycle. We show that Xenopus Claspin is phosphorylated at the MBT at both DNA replication checkpoint-dependent and -independent sites. Further, in egg extracts, Claspin phosphorylation depends on a threshold N/C ratio, but occurs even when ATR is inhibited. Not all phosphorylation that occurs at the MBT is reproduced in egg extracts. Our results identify Claspin as the most upstream molecule in the signaling pathway that responds to the N/C ratio and indicate that Claspin may also respond to an independent timer to trigger the MBT and activation of cell cycle checkpoints.

A single starfish Aurora kinase performs the combined functions of Aurora-A and Aurora-B in human cells.

Aurora, an essential mitotic kinase, is highly conserved during evolution. Most vertebrates have at least two Aurora kinases, Aurora-A and Aurora-B, which have distinct functions in the centrosome-spindle and inner centromere-midbody, respectively. However, some non-vertebrate deuterostomes have only a single Aurora. It remains to be verified whether the single Aurora performs the same functions as vertebrate Auroras A and B combined. We have isolated a cDNA of a single Aurora (ApAurora) from the echinoderm starfish, Asterina pectinifera, and show that ApAurora displays most features of both Aurora-A and Aurora-B in starfish oocytes and early embryos. Furthermore, ApAurora that is stably expressed in HeLa cells can substitute for both human Aurora-A and Aurora-B when either is reduced by RNAi. A single ApAurora thus has properties of both Aurora-A and Aurora-B in starfish eggs and HeLa cells. Together with phylogenetic analysis indicating that ApAurora forms a clade with all types of vertebrate Auroras and single Auroras of non-vertebrate deuterostomes, our observations support the idea that the single Aurora found in non-vertebrate deuterostomes represents the ancestor that gave rise to various types of vertebrate Auroras. This study thus provides functional evidence for phylogenetic considerations.

Proline-rich domain in dynamin-2 has a low microtubule-binding activity: how is this activity controlled during mitosis in HeLa cells?

The large GTPase dynamin is strongly accumulated in the constricted area including midzonal microtubules of dividing cells. The proline-rich domain (PRD) of dynamin has been considered as a microtubule-binding domain. However, it remains unclear how PRD controls dynamin-microtubule interaction in mitotic cells. Here, we found that the microtubule-binding activity of PRD is low in dynamin-2. One of the mitosis-specific kinase activities to PRD in HeLa cells was identified as cyclin B-Cdc2 kinase. The kinase phosphorylated PRD at Ser(764) and/or Thr(766) and reduced the microtubule-binding activity of PRD. These results suggest that phosphorylation of PRD by cyclin B-Cdc2 kinase plays an important role to control dynamin-2-microtubule interaction in mitotic HeLa cells.

Initiation of DNA replication after fertilization is regulated by p90Rsk at pre-RC/pre-IC transition in starfish eggs.

Initiation of DNA replication in eukaryotic cells is controlled through an ordered assembly of protein complexes at replication origins. The molecules involved in this process are well conserved but diversely regulated. Typically, initiation of DNA replication is regulated in response to developmental events in multicellular organisms. Here, we elucidate the regulation of the first S phase of the embryonic cell cycle after fertilization. Unless fertilization occurs, the Mos-MAPK-p90Rsk pathway causes the G1-phase arrest after completion of meiosis in starfish eggs. Fertilization shuts down this pathway, leading to the first S phase with no requirement of new protein synthesis. However, how and in which stage the initiation complex for DNA replication is arrested by p90Rsk remains unclear. We find that in G1-arrested eggs, chromatin is loaded with the Mcm complex to form the prereplicative complex (pre-RC). Inactivation of p90Rsk is necessary and sufficient for further loading of Cdc45 onto chromatin to form the preinitiation complex (pre-IC) and the subsequent initiation of DNA replication. However, cyclin A-, B-, and E-Cdk's activity and Cdc7 accumulation are dispensable for these processes. These observations define the stage of G1 arrest in unfertilized eggs at transition point from pre-RC to pre-IC, and reveal a unique role of p90Rsk for a negative regulator of this transition. Thus, initiation of DNA replication in the meiosis-to-mitosis transition is regulated at the pre-RC stage as like in the G1 checkpoint, but in a manner different from the checkpoint.

Start of the embryonic cell cycle is dually locked in unfertilized starfish eggs.

A key event in the oocyte-to-embryo transition is the start of the embryonic mitotic cell cycle. Prior to this start, the cell cycle in oocytes is generally arrested at a particular stage during meiosis, and the meiotic arrest is released by fertilization. However, it remains unclear how release from the meiotic arrest is implicated in the start of the embryonic cell cycle. To elucidate this link, we have used starfish eggs, in which G1 phase arrest occurs after completion of meiosis if the mature oocytes are not fertilized, and fertilization simply directs the start of the embryonic cell cycle. The starfish G1 arrest is known to rely on the Mos-MAPK-Rsk (p90 ribosomal S6 kinase) pathway, and inactivation of Rsk induces S phase in the absence of fertilization. However, here we show that this S phase is not followed by M phase when MAPK remains active, owing to poly(A)-independent repression of cyclin A and B synthesis. By contrast, inactivation of MAPK alone induces M phase, even when S phase is inhibited by constitutively active Rsk. Thus, there is a divergence of separate pathways downstream of MAPK that together block the start of the embryonic mitotic cycle. One is the previously known Rsk-dependent pathway that prevents S phase, and the other is a novel pathway that is not mediated by Rsk and that leads to prevention of the first mitotic M phase through suppression of protein synthesis of M phase cyclins. Release from such a 'dual-lock' by fertilization results in the start of the embryonic cell cycle.