Introduction. Calcium signals and developmental patterning

Calcium ions generate ubiquitous cellular signals. Calcium signals play an important role in development. The most obvious example is fertilization, where calcium signals and calcium waves are triggered by the sperm and are responsible for activating the egg from dormancy and cell cycle arrest. Calcium signals also appear to contribute to cell cycle progression during the rapid cell cycles of early embryos. There is increasing evidence that calcium signals are an essential component of the signalling systems that specify developmental patterning and cell fate. This issue arises from a Discussion Meeting that brought together developmental biologists studying calcium signals with those looking at other patterning signals and events. This short introduction provides some background to the papers in this issue, setting out the emerging view that calcium signals are central to dorsoventral axis formation, gastrulation movements, neural specification and neuronal cell fate.     

ERK1 activation is required for S-phase onset and cell cycle progression after fertilization in sea urchin embryos

Fertilization of sea urchin eggs results in a large, transient increase in intracellular free Ca2+ concentration that is responsible for re-initiation of the cell division cycle. We show that activation of ERK1, a Ca2+-dependent MAP kinase response, is required for both DNA synthesis and cell cycle progression after fertilization. We combine experiments on populations of cells with analysis at the single cell level, and develop a proxy assay for DNA synthesis in single embryos, using GFP-PCNA. We compare the effects of low molecular weight inhibitors with a recombinant approach targeting the same signalling pathway. We find that inhibition of the ERK pathway at fertilization using either recombinant ERK phosphatase or U0126, a MEK inhibitor, prevents accumulation of GFP-PCNA in the zygote nucleus and that U0126 prevents incorporation of [3H]-thymidine into DNA. Abrogation of the ERK1 signalling pathway also prevents chromatin decondensation of the sperm chromatin after pronuclear fusion, nuclear envelope breakdown and formation of a bipolar spindle.     

Cell cycle in the fucus zygote parallels a somatic cell cycle but displays a unique translational regulation of cyclin-dependent kinases

In eukaryotic cells, the basic machinery of cell cycle control is highly conserved. In particular, many cellular events during cell cycle progression are controlled by cyclin-dependent kinases (CDKs). The cell cycle in animal early embryos, however, differs substantially from that of somatic cells or yeasts. For example, cell cycle checkpoints that ensure that the sequence of cell cycle events is correct have been described in somatic cells and yeasts but are largely absent in embryonic cells. Furthermore, the regulation of CDKs is substantially different in the embryonic and somatic cells. In this study, we address the nature of the first cell cycle in the brown alga Fucus, which is evolutionarily distant from the model systems classically used for cell cycle studies in embryos. This cycle consists of well-defined G1, S, G2, and M phases. The purine derivative olomoucine inhibited CDKs activity in vivo and in vitro and induced different cell cycle arrests, including at the G1/S transition, suggesting that, as in somatic cells, CDKs tightly control cell cycle progression. The cell cycle of Fucus zygotes presented the other main features of a somatic cell cycle, such as a functional spindle assembly checkpoint that targets CDKs and the regulation of the early synthesis of two PSTAIRE CDKs, p32 and p34, and the associated histone H1 kinase activity as well as the regulation of CDKs by tyrosine phosphorylation. Surprisingly, the synthesis after fertilization of p32 and p34 was translationally regulated, a regulation not described previously for CDKs. Finally, our results suggest that the activation of mitotic CDKs relies on an autocatalytic amplification mechanism.     

Regulated Ran-binding protein 1 activity is required for organization and function of the mitotic spindle in mammalian cells in vivo.

Ran-binding protein (RanBP) 1 is a major regulator of the Ran GTPase and is encoded by a regulatory target gene of E2F factors. The Ran GTPase network controls several cellular processes, including nucleocytoplasmic transport and cell cycle progression, and has recently also been shown to regulate microtubule nucleation and spindle assembly in Xenopus oocyte extracts. Here we report that RanBP1 protein levels are cell cycle regulated in mammalian cells, increase from S phase to M phase, peak in metaphase, and abruptly decline in late telophase. Overexpression of RanBP1 throughout the cell cycle yields abnormal mitoses characterized by severe defects in spindle polarization. In addition, microinjection of anti-RanBP1 antibody in mitotic cells induces mitotic delay and abnormal nuclear division, reflecting an abnormal stabilization of the mitotic spindle. Thus, regulated RanBP1 activity is required for proper execution of mitosis in somatic cells.     

Localised MPF regulation in eggs

In this review we discuss the evidence that activation and inactivation of M-phase promoting factor (MPF), the universal mitotic activator, are regulated locally within the cell, and consider the mechanisms that might be responsible. Localised initiation of MPF activation has been demonstrated in Xenopus eggs and egg fragments by examination of the timing of surface contraction waves (SCWs), indicators of MPF activity, and confirmed by direct measurement of MPF in such fragments. Both the timing and the site of SCW initiation relate to the presence of nuclei and of associated centriole-nucleated microtubules. Localised MPF activation is likely to occur in the perinuclear cytoplasm as well as within the nucleus. Studies in a number of cell types show that the perinuclear/centrosomal region is the site of accumulation of MPF itself (the cyclin B-Cdc2 kinase complex) and of many of its molecular regulators. It also harbours calcium-regulating machinery, and in sea urchin eggs is the site of transient calcium release at the onset of mitosis. During mitosis MPF, regulatory molecules and calcium signalling components associate with spindle structures. Inactivation of MPF to end mitosis has been shown to be initiated locally at the mitoic spindle in Drosophila embryos. In sea urchin and frog eggs, calcium transients are required for both mitotic entry and exit and in mouse eggs, MPF inactivation requires both a calcium signal and an intact spindle. It thus appears that calcium signals coinciding with localised accumulation of MPF regulators are required first to set off and/or amplify the MPF activation process around the nucleus, and later to promote MPF inactivation via cyclin B destruction. Calcium release from sequestering machinery organised around nuclear and astral structures may act co-operatively with localised MPF regulatory molecules to trigger both mitotic entry and exit.     

Microdomains bounded by endoplasmic reticulum segregate cell cycle calcium transients in syncytial Drosophila embryos

Cell cycle calcium signals are generated by the inositol trisphosphate (InsP3)-mediated release of calcium from internal stores (Ciapa, B., D. Pesando, M. Wilding, and M. Whitaker. 1994. Nature. 368:875-878; Groigno, L., and M. Whitaker. 1998. Cell. 92:193-204). The major internal calcium store is the endoplasmic reticulum (ER); thus, the spatial organization of the ER during mitosis may be important in shaping and defining calcium signals. In early Drosophila melanogaster embryos, ER surrounds the nucleus and mitotic spindle during mitosis, offering an opportunity to determine whether perinuclear localization of ER conditions calcium signaling during mitosis. We establish that the nuclear divisions in syncytial Drosophila embryos are accompanied by both cortical and nuclear localized calcium transients. Constructs that chelate InsP3 also prevent nuclear division. An analysis of nuclear calcium concentrations demonstrates that they are differentially regulated. These observations demonstrate that mitotic calcium signals in Drosophila embryos are confined to mitotic microdomains and offer an explanation for the apparent absence of detectable global calcium signals during mitosis in some cell types.     

Non-equivalence of embryonic and somatic cell nuclei affecting spindle composition in clones

Cloning by nuclear transfer remains inefficient but is more efficient when nuclei from embryonic cells or embryonic stem cells (ECNT) are employed as compared with somatic cells (SCNT). The factors determining efficiency have not been elucidated. We find that somatic and embryonic nuclei differ in their ability to organize meiotic and mitotic spindles of normal molecular composition. Calmodulin, a component of meiotic and mitotic spindle chromosome complexes (SCCs), displays sharply reduced association with the SCC forming after SCNT but not ECNT. This defect persists in mitotic spindles at least through the second mitosis, despite abundant calmodulin expression in the cell, and correlates with slow chromosome congression. We propose that somatic cell nuclei lack factors needed to direct normal SCC formation in oocytes and early embryos. These results reveal a striking control of SCC formation by the transplanted nucleus and provide the first identified molecular correlate of donor stage-dependent restriction in nuclear potency.     

The kinesin related motor protein, Eg5, is essential for maintenance of pre-implantation embryogenesis

Eg5 is a plus end directed kinesin related motor protein (KRP) previously shown to be involved in the assembly and maintenance of the mitotic spindle. KRPs are molecular motors capable of generating forces upon microtubules (MTs) in dividing cells and driving structural rearrangements necessary in the developing spindle. In vitro experiments demonstrate that loss of Eg5 results in cell cycle arrest and defective centrosome separation resulting in the development of monopolar spindles. Here we describe mice with a genetrap insertion in Eg5. Heterozygous mutant mice appear phenotypically normal. In contrast, embryos homozygous for the Eg5 null allele recovered at embryonic days 2.5-3.5 display signs of a proliferation defect as reduced cell numbers and failure of compaction and progression to the blastocyst stage was observed. These data, in conjunction with previous in vitro data, suggest that loss of Eg5 results in abnormal spindle structure, cell cycle arrest and thereby reduced cell proliferation of early cleavage pre-implantation embryos. These observations further support the conclusion that Eg5 is essential for cell division early in mouse development, and that maternal contribution may sustain the embryo through the maternal to zygotic transition at which point supplies of functional Eg5 are exhausted, preventing further cell cleavage.     

The first cell cycle after transfer of somatic cell nuclei in a non-human primate

Production of genetically identical non-human primates through somatic cell nuclear transfer (SCNT) can provide diseased genotypes for research and clarify embryonic stem cell potentials. Understanding the cellular and molecular changes in SCNT is crucial to its success. Thus the changes in the first cell cycle of reconstructed zygotes after nuclear transfer (NT) of somatic cells in the Long-tailed Macaque (Macaca fascicularis) were studied. Embryos were reconstructed by injecting cumulus and fibroblasts from M. fascicularis and M. silenus, into enucleated M. fascicularis oocytes. A spindle of unduplicated premature condensed chromosome (PCC spindle) from the donor somatic cell was formed at 2 hours after NT. Following activation, the chromosomes segregated and moved towards the two PCC spindle poles, then formed two nuclei. Twenty-four hours after activation, the first cell division occurred. A schematic of the first cell cycle changes following injection of a somatic cell into an enucleated oocyte is proposed. Ninety-three reconstructed embryos were transferred into 31 recipients, resulting in 7 pregnancies that were confirmed by ultrasound; unfortunately none progressed beyond 60 days.     

Okadaic acid inhibits a protein phosphatase activity involved in formation of the mitotic spindle of GH4 rat pituitary cells.

Okadaic acid, a selective inhibitor of serine/threonine protein phosphatases, was utilized to investigate the requirement for phosphatases in cell cycle progression of GH4 rat pituitary cells. Okadaic acid inhibited GH4 cell proliferation in a concentration-dependent manner with a half-maximal inhibition (IC50) of approximately 5 nM. Treatment of GH4 cells with 10 nM okadaic acid resulted in a 40-60% decrease in phosphatase activity and an increase in the proportion of phosphorylated retinoblastoma (RB) protein. Cell cycle analysis indicated that okadaic acid increased the percentage of cells in G2-M, decreased proportionally the percentage of cells in G1 phase, and had little effect on the percentage of cells in S-phase. The absence of a change in the proportion of S-phase cells indicates that G1-specific phosphatases responsible for dephosphorylation of RB protein were not inhibited by 10 mM okadaic acid. Mitotic index revealed that 10 nM okadaic acid decreased proliferation of GH4 cells specifically by slowing the progression through mitosis. Immunostaining with anti-tubulin demonstrated that 10 nM okadaic acid-treated mitotic cells contained mitotic spindles; however, the spindle apparatus in these cells frequently contained multiple poles. These results suggest that the organization of spindle microtubules during prometaphase requires a protein phosphatase that is sensitive to nanomolar concentrations of okadaic acid. Chromosomes in 10 nM okadaic acid-treated cells appear to be attached to spindle microtubules and the nuclear envelope is absent. The effects of okadaic acid on the spindle differ from those elicited by the calcium channel blocker, nimodipine, indicating that this okadaic acid sensitive phosphatase is not part of the calcium signalling events which participate in mitotic progression.     

Subcellular localization and role of Ran1 in Tetrahymena thermophila amitotic macronucleus

Amitosis, a direct method of cell division is common in ciliated protozoan, fungi and some animal and plant cells. During amitosis, intranuclear microtubules are reorganized into specified arrays which assist in separation of nucleus, despite lack of a bipolar spindle. However, the regulation of amitosis is not understood. Here, we focused on the localization and role of mitotic spindle assembly regulator: Ran GTPase (Ran1) in macronuclear amitosis in binucleated protozoan Tetrahymena thermophila. HA-tagged Ran1 was localized in the macronucleus throughout the cell cycle of Tetrahymena during vegetative growth, and the accessory factor binding domains of Ran1 contributed to its macronuclear localization. Incomplete somatic knockout of RAN1 resulted in aberrant intramacronuclear microtubule array formation, missegregation of macronuclear chromosomes and ultimately blocked macronuclei proliferation. When the Ran1 cycle was perturbed by overexpression of Ran1T25N (GDP-bound Ran1-mimetic) or Ran1Q70L (GTP-bound Ran1-mimetic), intramacronuclear microtubule assembly was inhibited or multi-micronucleate cells formed. These results suggest that Ran GTPase pathway is involved in assembly of a specialized intramacronuclear microtubule network and coordinates amitotic progression in Tetrahymena.     

Improved efficiency of canine nucleus transfer using roscovitine-treated canine fibroblasts

The aim of this study was to investigate whether roscovitine (the cyclin-dependent kinase 2 inhibitor) effectively induces synchronization of the donor cell cycle at G0/G1 and to examine the effect of donor cell cycle synchronization protocols on canine somatic cell nucleus transfer. Canine fibroblasts were obtained from skin biopsy cultures taken from a 7-yr-old retriever. The donor cell cycle was synchronized either by culturing cells to reach confluency or by treating cells with 15 μg/mL roscovitine for 24 h. Cell cycle stages and apoptosis were analyzed by flow cytometry. After synchronization of the donor cell cycle, cells were placed with enucleated in vivo-matured dog oocytes, fused by electric stimulation, activated, and transferred into 18 naturally estrus-synchronized surrogates. There was no significant difference in cell cycle synchronization and apoptosis rates between the confluent and roscovitine groups. After transfer of reconstructed embryos, pregnancy was detected in three of nine surrogates that received cloned embryos reconstructed with roscovitine-treated cells, whereas only one of nine surrogates was pregnant after transfer of cloned embryos reconstructed with confluent cells. One pregnant female from the confluent cell group delivered one live and one dead pup, but the live one died within 5 days after birth. Three pregnant females from the roscovitine-treated cell group delivered eight live pups and one dead pup, and one of eight live pups died within 6 days after birth. In conclusion, the current results demonstrated that reconstructing embryos with roscovitine-treated cells resulted in increased efficiency of canine somatic cell nucleus transfer.     

Mobilisation of stored calcium in the neck region of human sperm--a mechanism for regulation of flagellar activity

Calcium signalling plays a pivotal role in sperm physiology, being intimately involved in the regulation of acrosome reaction, chemotaxis and hyperactivation. Here we describe briefly the mechanisms of calcium regulation in somatic cells and the ways in which these mechanisms have been adapted to function in mature spermatozoa. We then consider recent data from this and other laboratories on the responses of sperm to three compounds: progesterone and nitric oxide (both products of the cumulus oophorus) and 4-aminopyridine. All of these compounds induce calcium signals in the posterior sperm head and neck region and, when applied at appropriate concentrations, modify flagellar activity, causing asymmetric bending of the proximal flagellum. We argue that these effects reflect a common mode of action, mobilisation of calcium stored in the sperm neck region. Finally we consider the nature of calcium signalling pathways in sperm. We suggest that this highly specialised and extremely polarised cell, though working with the same calcium signalling 'tools' as those of somatic cells, employs them to generate unusually 'hard-wired' calcium signals that do not act to integrate stimuli. 'Leakage' between these calcium signalling pathways will generate inappropriate responses, compromising functioning of the cell.     

Ecdysteroids affect Drosophila ovarian stem cell niche formation and early germline differentiation

Previously, it has been shown that in Drosophila steroid hormones are required for progression of oogenesis during late stages of egg maturation. Here, we show that ecdysteroids regulate progression through the early steps of germ cell lineage. Upon ecdysone signalling deficit germline stem cell progeny delay to switch on a differentiation programme. This differentiation impediment is associated with reduced TGF-β signalling in the germline and increased levels of cell adhesion complexes and cytoskeletal proteins in somatic escort cells. A co-activator of the ecdysone receptor, Taiman is the spatially restricted regulator of the ecdysone signalling pathway in soma. Additionally, when ecdysone signalling is perturbed during the process of somatic stem cell niche establishment enlarged functional niches able to host additional stem cells are formed.     

Chromosome stability differs in cloned mouse embryos and derivative ES cells

The mechanisms that have evolved to maintain genome stability during cell cycle progression are challenged when a somatic cell nucleus is placed in a meiotic environment such as the ooplasm. Chromosomal spindle aberrations ensue in the majority of reconstructed oocytes within 2 h of transplantation, but it is not known if they recover or persist with the onset of embryonic divisions. We analyzed the chromosomal spindles and the karyotype of cumulus cell-derived mouse clones through the initial and hence most critical mitoses. Cloned embryos start out with less aneuploidy than fertilized embryos but surpass them after ES cell derivation, as measured by frequencies of chromosome trisomies and structural rearrangements. Despite the limited proportion of cloned mouse embryos that reach late gestation, a phenotypic mutation lacking a karyotypic mark was found in a newborn mouse cloned in 2002 and has been inherited since by its offspring. These data concur with a prevalent epigenetic, rather than genetic, basis for cloned embryo failure, but they also warn against the temptation to think that all conditions of clones are epigenetic and recover during gametogenesis. The cloning procedure is defenseless (no matter how technically refined) towards pre-existing or induced subchromosomal mutations that are below the experimental detection limit of the cytogenetic assay.     

Glycogen Synthase Kinase-3 and phospholipase C-beta signalling: Roles and possible interactions in myelodysplastic syndromes and acute myeloid leukemia

GSK-3 and PLCbeta enzymes are responsible for the regulation of several signalling pathways related to many cellular functions. In hematopoietic cells, GSK-3 deficiency is correlated with an MDS-like phenotype and with leukemogenesis, showing a prognostic potential in AML cells. GSK-3 interacts with Wnt or MAPK signalling, but it is also linked to PI3K/Akt/mTOR pathways to regulate cell proliferation and apoptosis of hematopoietic stem cell progenitors. PLCbeta enzymes are involved in cell cycle progression of hematopoietic, MDS/AML and immune cells, through activation of PKC or calcium signalling. Of note, a PLCbeta1/PKCalpha pathway is modulated during MDS pathogenesis, with a specific involvement of the inositides localized in the nucleus. Here we focus on GSK-3 and PLCbeta signalling, describing the many evidences that underline the pivotal role of both GSK-3 and PLCbeta-dependent pathways in MDS/AML, their association with therapy and their possible interactions.     

Cytoskeletal and nuclear organization in mouse embryos derived from nuclear transfer and ICSI: a comparison of agamogony and syngamy before and during the first cell cycle

In this study, somatic cell nuclear transfer (SCNT) and intracytoplasmic sperm injection (ICSI) are used as models of agamogony and syngamy, respectively. In order to elucidate the reasons of low efficiency of somatic cell cloning, cytoskeletal and nuclear organization in cloned mouse embryos was monitored before and during the first cell cycle, and compared with the pattern of ICSI zygote. A metaphase-like spindle with alignment of condensed donor chromosomes was assembled within 3 hr after NT, followed by formation of pronuclear-like structures at 3-6 hr after activation, indicating that somatic nuclear remodeling depends on microtubular network organization. The percentage of two (pseudo-) pronuclei in cloned embryos derived from delayed activation was greater than that in immediate activation group (68.5% vs. 30.8%, P<0.01), but similar to that of ICSI group (68.5% vs. 65.5%, P>0.05). The 2-cell rate in NT embryos was significantly lower than that in zygotes produced by ICSI (64.8% vs. 82.5%, P<0.01). Further studies testified that the cloned embryos reached the metaphase of the first mitosis 10 hr after activation, whereas this occurred at 18 hr in the ICSI zygotes. Comparision of the pattern of microfilament assembly in early NT embryos with that in syngamic zygotes suggested that abnormal microfilamental pattern in cloned embryos may threaten subsequent embryonic development. In conclusion, agamogony, in contrast to syngamy, displays some unique features in respect of cytoskeletal organization, the most remarkable of which is that the first cell cycle is initiated ahead distinctly, which probably leads to incomplete organization of the first mitotic spindle, and contributes to low efficiency of cloning.     

Inhibiting MAP kinase activity prevents calcium transients and mitosis entry in early sea urchin embryos

A transient calcium increase triggers nuclear envelope breakdown (mitosis entry) in sea urchin embryos. Cdk1/cyclin B kinase activation is also known to be required for mitosis entry. More recently, MAP kinase activity has also been shown to increase during mitosis. In sea urchin embryos, both kinases show a similar activation profile, peaking at the time of mitosis entry. We tested whether the activity of both kinases is required for mitosis entry and whether either kinase controls mitotic calcium signals. We found that reducing the activity of either mitotic kinase prevents nuclear envelope breakdown, despite the presence of a calcium transient, when cdk1/cyclin B kinase activity is alone inhibited. When MAP kinase activity alone was inhibited, the calcium signal was absent, suggesting that MAP kinase activity is required to generate the calcium transient that triggers nuclear envelope breakdown. However, increasing intracellular free calcium by microinjection of calcium buffers or InsP(3) while MAP kinase was inhibited did not itself induce nuclear envelope breakdown, indicating that additional MAP kinase-regulated events are necessary. After MAP kinase inhibition early in the cell cycle, the early events of the cell cycle (pronuclear migration/fusion and DNA synthesis) were unaffected, but chromosome condensation and spindle assembly are prevented. These data indicate that in sea urchin embryos, MAP kinase activity is part of a signaling complex alongside two components previously shown to be essential for entry into mitosis: the calcium transient and the increase in cdk1/cyclinB kinase activity.     

Germinal vesicle material is essential for nucleus remodeling after nuclear transfer

Successful cloning by nuclear transfer has been reported with somatic or embryonic stem (ES) cell nucleus injection into enucleated mouse metaphase II oocytes. In this study, we enucleated mouse oocytes at the germinal vesicle (GV) or pro-metaphase I (pro-MI) stage and cultured the cytoplasm to the MII stage. Nuclei from cells of the R1 ES cell line were injected into both types of cytoplasm to evaluate developmental potential of resulting embryos compared to MII cytoplasmic injection. Immunocytochemical staining revealed that a spindle started to organize 30 min after nucleus injection into all three types of cytoplasm. A well-organized bipolar spindle resembling an MII spindle was present in both pro-MI and MII cytoplasm 1 h after injection with ES cells. However, in the mature GV cytoplasm, chromosomes were distributed throughout the cytoplasm and a much bigger spindle was formed. Pseudopronucleus formation was observed in pro-MI and MII cytoplasm after activation treatment. Although no pronucleus formation was found in GV cytoplasm, chromosomes segregated into two groups in response to activation. Only 8.1% of reconstructed embryos with pro-MI cytoplasm developed to the morula stage after culture in CZB medium. In contrast, 53.5% of embryos reconstructed with MII cytoplasm developed to the morula/blastocyst stage, and 5.3% of transferred embryos developed to term. These results indicate that GV material is essential for nucleus remodeling after nuclear transfer.