Cell Division In Embryos Research Articles

TPRA40/GPR175 regulates early mouse embryogenesis through functional membrane transport by Sjögren's syndrome‐associated protein NA14

TPRA40/GPR175 is an orphan receptor whose physiological functions have not been found to date. In an attempt to generate transgenic mice that express an shRNA of TPRA40, we observed that the cell division of early mouse embryos that injected the short hairpin RNA expression vector was significantly accelerated compared with the control vector. The regulation of cell division by TPRA40 was also observed in HeLa cells. Since the C-terminal region of TPRA40 has been shown to be essential for the regulation of cell division, we performed yeast two-hybrid screening using the C-terminal region as bait. Nuclear antigen of 14 kDa (NA14), an autoantigen of Sjögren's syndrome, was identified as a binding protein to the C-terminal region of TPRA40. The binding of TPRA40 and NA14 was confirmed by GST pull-down assay and co-immunoprecipitation assay. FLAG-TPRA40 is transported from the cytosol to the plasma membrane in time-dependent manner and the translocation was inhibited by GFP-NA14DeltaN, an N-terminal deletion mutant that cannot bind to microtubules but binds to TPRA40. TPRA40DeltaC, which cannot bind to NA 14, shows impaired transport to the plasma membrane. Finally, we found that the effect of TPRA40 on mouse embryogenesis is strengthened by GFP-NA14, but not by GFP or GFP-NA14DeltaN. These observations indicate that the functional plasma membrane transport of TPRA40 that regulates cell division of mouse embryos is mediated by NA14.

FoxM1-driven cell division is required for neuronal differentiation in earlyXenopusembryos

In vertebrate embryogenesis, neural induction is the earliest step through which the fate of embryonic ectoderm to neuroectoderm becomes determined. Cells in the neuroectoderm or neural precursors actively proliferate before they exit from the cell cycle and differentiate into neural cells. However, little is known about the relationship between cell division and neural differentiation, although, in Xenopus, cell division after the onset of gastrulation has been suggested to be nonessential for neural differentiation. Here, we show that the Forkhead transcription factor FoxM1 is required for both proliferation and differentiation of neuronal precursors in early Xenopus embryos. FoxM1 is expressed in the neuroectoderm and is required for cell proliferation in this region. Specifically, inhibition of BMP signaling, an important step for neural induction, induces the expression of FoxM1 and its target G2-M cell-cycle regulators, such as Cdc25B and cyclin B3, thereby promoting cell division in the neuroectoderm. Furthermore, G2-M cell-cycle progression or cell division mediated by FoxM1 or its target G2-M regulators is essential for neuronal differentiation but not for specification of the neuroectoderm. These results suggest that FoxM1 functions to link cell division and neuronal differentiation in early Xenopus embryos.

Activation of T Cells by Cross‐Linking Qa‐2, the Ped Gene Product, Requires Fyn

Qa-2, the product of the Ped (preimplantation development) gene, regulates the rate of cell division of preimplantation mouse embryos by an unknown mechanism. Due to the limited availability of preimplantation embryos, T cells were used as a model system to assess the possible roles of Fyn and Lck, and two downstream effectors, PI-3 kinase and Akt, in Qa-2 induced cell proliferation. Resting T cells were stimulated to proliferate by treating with mouse anti-Qa-2 antibody, cross-linking with anti-mouse immunoglobulin, and adding PMA. The effects of kinase inhibitors on this proliferation were studied. Co-immunoprecipitates of T-cell lysates were analyzed for possible associations between Qa-2 and Fyn or Lck. Fyn knockout mice (Fyn-/-) were used to determine whether Fyn is required for T-cell activation induced by cross-linking Qa-2. An inhibitor of Src family kinases and inhibitors of PI-3 kinase and Akt suppressed proliferation of resting T cells induced by cross-linking Qa-2. Fyn, but not Lck, co-immunoprecipitated with Qa-2. Fyn-/- T cells failed to proliferate in response to Qa-2 cross-linking. Fyn, PI-3 kinase, and Akt are required for the activation of T cells by cross-linking Qa-2.

The AP2 transcription factors DORNRÖSCHEN and DORNRÖSCHEN-LIKE redundantly controlArabidopsisembryo patterning via interaction with PHAVOLUTA

DORNROSCHEN (DRN) (also known as ENHANCER OF SHOOT REGENERATION1; ESR1) and DRN-LIKE (DRNL; also known as ESR2) are two linked paralogues encoding AP2 domain-containing proteins. drn mutants show embryo cell patterning defects and, similarly to drnl mutants, disrupt cotyledon development at incomplete penetrance. drn drnl double mutants with weak or strong drnl alleles show more highly penetrant and extreme phenotypes, including a pin-like embryo without cotyledons, confirming a high degree of functional redundancy for the two genes in embryo patterning. Altered expression of PIN1::PIN1-GFP and DR5::GFP in drn mutant embryos places DRN upstream of auxin transport and response. A yeast two-hybrid screen with DRN followed by co-immunoprecipitation and bimolecular fluorescence complementation revealed PHAVOLUTA (PHV) to be a protein interaction partner in planta. drn phv double mutants show an increased penetrance of embryo cell division defects. DRNL can also interact with PHV and both DRN and DRNL can heterodimerise with additional members of the class III HD-ZIP family, PHABULOSA, REVOLUTA, CORONA and ATHB8. Interactions involve the PAS-like C-terminal regions of these proteins and the DRN/DRNL AP2 domain.

ElrA binding to the 3'UTR of cyclin E1 mRNA requires polyadenylation elements.

The early cell divisions of Xenopus laevis and other metazoan embryos occur in the presence of constitutively high levels of the cell cycle regulator cyclin E1. Upon completion of the 12th cell division, a time at which many maternal proteins are downregulated by deadenylation and destabilization of their encoding mRNAs, maternal cyclin E1 protein is downregulated while its mRNA is polyadenylated and stable. We report here that stable polyadenylation of cyclin E1 mRNA requires three cis-acting elements in the 3′ untranslated region; the nuclear polyadenylation sequence, a contiguous cytoplasmic polyadenylation element and an upstream AU-rich element. ElrA, the Xenopus homolog of HuR and a member of the ELAV gene family binds the cyclin E1 3′UTR with high affinity. Deletion of these elements dramatically reduces the affinity of ElrA for the cyclin E1 3′UTR, abolishes polyadenylation and destabilizes the mRNA. Together, these findings provide compelling evidence that ElrA functions in polyadenylation and stabilization of cyclin E1 mRNA via binding these elements.

Activator of G‐protein signaling in asymmetric cell divisions of the sea urchin embryo

An asymmetric fourth cell division in the sea urchin embryo results in formation of daughter cells, macromeres and micromeres, with distinct sizes and fates. Several lines of functional evidence presented here, including pharmacological interference and dominant negative protein expression, indicate that heterotrimeric G protein Gi and its interaction partner, activator of G-protein signaling (AGS), are necessary for this asymmetric cell division. Inhibition of Gi signaling by pertussis toxin interferes with micromere formation and leads to defects in embryogenesis. AGS was isolated in a yeast two-hybrid screen with G alpha i as bait and was expressed in embryos localized to the cell cortex at the time of asymmetric divisions. Introduction of exogenous dominant-negative AGS protein, containing only G-protein regulatory (GPR) domains, selectively prevented the asymmetric division in normal micromere formation. These results support the growing evidence that AGS is a universal regulator of asymmetric cell divisions in embryos.

Delayed embryo development in the ARABIDOPSIS TREHALOSE‐6‐PHOSPHATE SYNTHASE 1 mutant is associated with altered cell wall structure, decreased cell division and starch accumulation

The tps1 mutant, which is disrupted in the TREHALOSE-6-PHOSPHATE SYNTHASE 1 gene, has been previously characterized as a recessive embryo lethal. tps1 embryos do not develop past late torpedo or early cotyledon stage. We report here that at the ultrastructural, biochemical, and transcriptional levels tps1 exhibits many features typically associated with the maturation phase. The appearance of storage reserve transcripts and organelles follows the same temporal pattern in tps1 and wild-type (WT) embryos in the same silique as does accumulation of storage lipid and protein. The mutant plastids accumulate large starch granules that persist until the end of seed development, in contrast with WT plastids where starch accumulation is transient. The transcriptome of tps1 embryos shows a coordinate downregulation of genes involved in starch and sucrose degradation. Interestingly, genes involved in lipid mobilization and gluconeogenesis are induced in tps1 embryos. The cell walls of tps1 embryos show a remarkable degree of thickening at the ultrastructural level and immunodetection of cell wall components shows that altered deposition of pectins accounts for this altered morphology. Consistent with this at the transcriptome level, genes involved in sugar nucleotide and pectin metabolism are altered in the mutant. The frequency of cell division in tps1 embryos is half that of the wild type at the heart and torpedo stages. These results suggest that TPS1 may play a major role in coordinating cell wall biosynthesis and cell division with cellular metabolism during embryo development.

Calcium at Fertilization and in Early Development

Fertilization calcium waves are introduced, and the evidence from which we can infer general mechanisms of these waves is presented. The two main classes of hypotheses put forward to explain the generation of the fertilization calcium wave are set out, and it is concluded that initiation of the fertilization calcium wave can be most generally explained in invertebrates by a mechanism in which an activating substance enters the egg from the sperm on sperm-egg fusion, activating the egg by stimulating phospholipase C activation through a src family kinase pathway and in mammals by the diffusion of a sperm-specific phospholipase C from sperm to egg on sperm-egg fusion. The fertilization calcium wave is then set into the context of cell cycle control, and the mechanism of repetitive calcium spiking in mammalian eggs is investigated. Evidence that calcium signals control cell division in early embryos is reviewed, and it is concluded that calcium signals are essential at all three stages of cell division in early embryos. Evidence that phosphoinositide signaling pathways control the resumption of meiosis during oocyte maturation is considered. It is concluded on balance that the evidence points to a need for phosphoinositide/calcium signaling during resumption of meiosis. Changes to the calcium signaling machinery occur during meiosis to enable the production of a calcium wave in the mature oocyte when it is fertilized; evidence that the shape and structure of the endoplasmic reticulum alters dynamically during maturation and after fertilization is reviewed, and the link between ER dynamics and the cytoskeleton is discussed. There is evidence that calcium signaling plays a key part in the development of patterning in early embryos. Morphogenesis in ascidian, frog, and zebrafish embryos is briefly described to provide the developmental context in which calcium signals act. Intracellular calcium waves that may play a role in axis formation in ascidian are discussed. Evidence that the Wingless/calcium signaling pathway is a strong ventralizing signal in Xenopus, mediated by phosphoinositide signaling, is adumbrated. The central role that calcium channels play in morphogenetic movements during gastrulation and in ectodermal and mesodermal gene expression during late gastrulation is demonstrated. Experiments in zebrafish provide a strong indication that calcium signals are essential for pattern formation and organogenesis.

143 IN VITRO DEVELOPMENT OF OVINE OOCYTES FROM EWES OF CONTRASTING VITAMIN B12 STATUS

Suboptimal circulating concentrations of vitamin B12 are commonly found in cattle and sheep grazing cobalt-deficient pastures. Vitamin B12 is a co-factor for enzymes involved in energy metabolism (methylmalonyl CoA mutase) and DNA synthesis/methylation (methionine synthase), and vitamin B12 status may therefore impact on cell division and gene expression in early embryos. The aim of this study was to determine the effect of vitamin B12 status on the in vitro development of ovine oocytes to the blastocyst stage. Mature Scottish Blackface ewes from cobalt-deficient farms were housed for ∼ four months and fed a cobalt-deficient diet (0.06 mg cobalt kg DM−1). At housing, 55 of the ewes were given an intra-ruminal slow-release cobalt bolus to compensate for the dietary deficit, and 52 remained untreated. The ovaries of all ewes were recovered at slaughter within the natural breeding season. Oocytes were aspirated and those with evenly granulated cytoplasm and >3 layers of cumulus cells were pooled according to ewe cobalt treatment, matured, fertilized, and cultured in vitro (∼20 oocytes per 50-μL drop under mineral oil). Oocytes were matured for 24 h in M199 + 10% fetal calf serum at 38.5°C in a humidified atmosphere of 5% CO2 in air prior to co-incubation for 18 h with frozen-thawed semen from a single ejaculate (1 × 106 live sperm mL−1). Presumptive zygotes were cultured for 7 Days in synthetic oviduct fluid + 0.4% fatty acid-free BSA (5% CO2, 5% O2, 90% N). Blastocysts formed at the end of the culture period were fixed and stained (Hoechst 33258) to count cell numbers. Data were analyzed by ANOVA and chi-square. For cobalt-supplemented and non-supplemented ewes, circulating concentrations of vitamin B12 at the time of slaughter were 1244 ± 52.5 and 372 ± 27.9 pmol L−1 (P < 0.001), respectively. Numbers of small (<5 mm) follicles per ewe were 17.6 ± 1.22 and 17.1 ± 1.31, and large (>5 mm) follicles per ewe were 1.8 ± 0.16 and 1.6 ± 0.18, respectively (NS). Cobalt-supplemented ewes yielded a lower proportion of matured oocytes that cleaved but an increased proportion of cleaved oocytes that formed blastocysts by Day 6 of the culture period (Table 1). The proportion of grade 1 and 2 blastocysts was also increased but cobalt treatment did not affect blastocyst cell numbers. In conclusion, results suggest that cleaved eggs derived from ewes of adequate, compared to suboptimal vitamin B12 status have improved developmental competence in vitro. Table 1. Effect of cobalt/vitamin B12 status on the in vitro development of ovine oocytes This work was funded by the Scottish Executive Environment and Rural Affairs Department.

Acquisition of essential somatic cell cycle regulatory protein expression and implied activity occurs at the second to third cell division in mouse preimplantation embryos

It is clear that G1–S phase control is exerted after the mouse embryo implants into the uterus 4.5 days after fertilization (E4.5); null mutants of genes that control cell cycle commitment such as max, rb (retinoblastoma), and dp1 are embryonic lethal after implantation with proliferation phenotypes. But, a number of studies of genes mediating proliferation control in the embryo after fertilization-implantation have yielded confusing results. In order to understand when embryos might first exert G1–S phase regulatory control, we assayed preimplantation mouse embryos for the acquisition of expression of mRNA, protein, and phospho-protein for max, Rb, and DP-1, and for the proliferation-promoting phospho-protein forms of mycC (thr58/ser62) and Rb (ser795). The key findings are that: (1) DP-1 protein was present in the nucleus as early as the four-cell stage onwards, (2) max protein was in the nucleus, suggesting function from the four-cell stage onwards, (3) both mycC and Rb all form protein was present at increasing quantities in the cytoplasm from the 2 cell and 4/8 cell stage, respectively, (4) the phosphorylated form of mycC phospho was present in the nucleus at high levels from the two-cell stage through blastocyst-stage, and (5) the phosphorylated form of Rb was detected at low levels in the two-cell stage embryo and was highly expressed at the 4/8-cell stage through the blastocyst stage. Taken together, these data suggest that activation of mycC phospho/max dimer pairs, (E2F)/DP-1 dimer pairs, and repression of Rb inhibition of cell cycle progression via phosphorylation at ser795 occurs at the earliest stages of embryonic development. In addition, the presence of max, mycC phospho, DP-1, and Rb phospho in the nuclei of embryonic and placental lineage cells in the blastocyst and in trophoblast stem cells suggests that a similar type of cell cycle regulation is present throughout preimplantation development and in both embryonic and extra-embryonic cell lineages.

Characterization of mid-spindle microtubules during furrow positioning in early cleavage period zebrafish embryos

We report evidence to suggest that during the first few meroblastic cell divisions in zebrafish embryos a dynamic population of central-spindle microtubules serve a crucial function in positioning the cleavage furrow at the surface of the blastoderm. Originating from the mid-zone of the mitotic spindle they develop into what we term a mid-spindle 'pre-furrowing microtubule array' that expands upward and outward from the spindle mid-zone towards the blastodisc surface. We suggest that this structure transmits positional information to the blastodisc cortex that results in the correctly positioned assembly of the cytokinetic contractile apparatus. We also propose that the pre-furrowing microtubule array then develops into a furrow-ingression microtubule array that helps direct and assemble the deepening furrow as it cuts its way through the blastodisc. Due to the location of its origin, the pre-furrowing microtubule array serves to successfully separate the daughter nuclei and thus equally divide the blastoderm. Furthermore, co-localization with elements of the cortical endoplasmic reticulum and their inositol 1,4,5-trisphosphate receptors suggests that the pre-furrowing microtubule array may also play a role in organizing localized Ca2+ transients that have been shown to be essential to the furrow positioning, propagation and deepening process during cytokinesis in zebrafish embryos.

The alpha, beta, gamma, delta-unsaturated aldehyde 2-trans-4-trans-decadienal disturbs DNA replication and mitotic events in early sea urchin embryos.

The polyunsaturated aldehydes are highly reactive products of fatty acid peroxidation and combustion of organic materials, and they have been documented to have diverse cyctotoxic and genotoxic effects. The alpha,beta,gamma,delta-unsaturated aldehyde 2-trans-4-trans-decadienal is produced by marine microalgae, and it is known to inhibit cell proliferation and induce apoptosis in several different cell types. However, the molecular basis for the cell cycle arrest is not fully understood. We used sea urchin embryos to examine how some of the key events of the mitotic cell division were influenced by this polyunsaturated aldehyde. We found that cell divisions in embryos of Sphaerechinus granularis were inhibited by 2-trans-4-trans-decadienal in a dose dependent manner with an EC50 of 1.3 microM. Mitotic events in the nondividing eggs were characterized using immunofluorescent staining. DNA labelling revealed that pronuclear migration was inhibited, and a total absence of incorporation of the DNA-base analogue 5-bromo-2-deoxyuridine indicated that no DNA replication had occurred. Staining of alpha-tubulin subunits showed that tubulin-polymerization was disrupted and aberrations were induced in mitotic spindles. Furthermore, we monitored the activity of the G2-M promoting complex cyclin B-Cdk1 in newly fertilized sea urchin eggs, and found that this complex was not activated in embryos treated with 2-trans-4-trans-decadienal despite the accumulation of cyclin B.

Glyphosate-based pesticides affect cell cycle regulation

Cell-cycle dysregulation is a hallmark of tumor cells and human cancers. Failure in the cell-cycle checkpoints leads to genomic instability and subsequent development of cancers from the initial affected cell. A worldwide used product Roundup 3plus, based on glyphosate as the active herbicide, was suggested to be of human health concern since it induced cell cycle dysfunction as judged from analysis of the first cell division of sea urchin embryos, a recognized model for cell cycle studies. Several glyphosate-based pesticides from different manufacturers were assayed in comparison with Roundup 3plus for their ability to interfere with the cell cycle regulation. All the tested products, Amega, Cargly, Cosmic, and Roundup Biovert induced cell cycle dysfunction. The threshold concentration for induction of cell cycle dysfunction was evaluated for each product and suggests high risk by inhalation for people in the vicinity of the pesticide handling sprayed at 500 to 4000 times higher dose than the cell-cycle adverse concentration.

Copepod egg production and hatching success is reduced by maternal diets of a non-neurotoxic strain of the dinoflagellate Alexandrium tamarense

MEPS Marine Ecology Progress Series Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsTheme Sections MEPS 280:199-210 (2004) - doi:10.3354/meps280199 Copepod egg production and hatching success is reduced by maternal diets of a non-neurotoxic strain of the dinoflagellate Alexandrium tamarense Adrianna Ianora1,*, Jefferson T. Turner2, Francesco Esposito1, Ylenia Carotenuto1, Giuliana d¹Ippolito1,3, Giovanna Romano1, Angelo Fontana3, Cástor Guisande4, Antonio Miralto1 1Ecophysiology Laboratory, Stazione Zoologica A. Dohrn, Villa Comunale 80121 Naples, Italy 2School for Marine Science and Technology, University of Massachusetts Dartmouth, 706 South Rodney French Boulevard, New Bedford, Massachusetts 02744, USA 3Istituto di Chimica Biomolecolare (ICB) del CNR, Via Campi Flegrei 34, 80078 Pozzuoli (Naples), Italy 4Facultad de Ciencias del Mar, Universidad de Vigo, Lagoas-Marcosende, 36200 Vigo, Spain *Email: ianora@szn.it ABSTRACT: A unialgal diet of a non-neurotoxic strain of the dinoflagellate Alexandrium tamarense strongly modified egg production and hatching success in the copepod Temora stylifera, even though grazing rates were relatively constant over time. Both exponential and stationary cultures of A. tamarense reduced egg production and hatching success, but the effect of stationary cultures on hatching success was dramatic, with egg viability dropping to 0% after 24 h of feeding. HPLC analyses revealed that the A. tamarense clone was non-neurotoxic, with a mean toxin content of about 0.005 fmol per cell, indicating that adverse effects were not due to saxitoxins or neosaxitoxins. 1H NMR analyses also revealed that diatom-derived PUSCAs (polyunsaturated short-chain aldehydes) were not responsible for reduced hatching rates, since these compounds were absent in A. tamarense as compared to the diatom Skeletonema costatum. Extracts of A. tamarense and the diatom-derived PUSCA 2-trans-4-trans-decadienal were also assessed in terms of biological effects on sea urchin embryo cell divisions. A. tamarense did not show anti-mitotic properties, as extracts did not block first-cell cleavage compared to decadienal. However, A. tamarense extracts did block fertilization success when sea urchin oocytes were first incubated for 30 min in extracts and then fertilized, as opposed to S. costatum extracts, which did not affect normal elevation of the fertilization membrane. This is the first report that dinoflagellates produce antiproliferative compounds, other than PUSCAs, that can potentially reduce copepod egg production and hatching success. KEY WORDS: Dinoflagellate · Copepods · Egg production · Hatching success · Antiproliferate compounds Full text in pdf format PreviousNextExport citation RSS - Facebook - Tweet - linkedIn Cited by Published in MEPS Vol. 280. Online publication date: October 14, 2004 Print ISSN: 0171-8630; Online ISSN: 1616-1599 Copyright © 2004 Inter-Research.

Two new analogues of the cytotoxic substance BE-52211 from Streptomyces sp.

Two new beta-hydroxy acetamides, BE-52211 B and BE-52211 C, which were structural analogues of BE-52211, were obtained as an inseparable mixture from an actinomycete, Streptomyces sp. Their structures were elucidated on the basis of spectroscopic data. They inhibited cell division of starfish embryos at a concentration of 2.5 microg/mL or greater.

G-proteins for spindle geometry

![Graphic][1] Loss of both apical pathways (bottom) allows astral microtubules (arrows) to form apically and basally in neuroblasts.On [page 623][2], Yu et al. demonstrate that unequal cell division in fly embryos results from two apical complexes that somehow affect microtubule

Anti-mitotic activity towards sea urchin embryos in extracts from the marine haptophycean Phaeocystis pouchetii (Hariot) Lagerheim collected along the coast of northern Norway

The marine bloom-forming alga Phaeocystis pouchetii is suspected to produce some toxic compound responsible for reduced growth, fecundity and survival of other marine organisms. Sea urchin early development was used as a model to investigate the degree and nature of toxicity. Colonial cells of P. pouchetii were collected during its spring-bloom along the coast of northern Norway and maintained in culture for a short period of time in order to evaluate the concentration of toxic compounds present inside the cells or excreted to the surrounding seawater medium. Cells were harvested by filtration and toxins were extracted separately from the collected cells and the filtrate using organic solvents. We found that extracts from the filtered seawater at a concentration corresponding to 9.0×10 5 cells ml –1 completely blocked cell divisions in embryos of the sea urchin Sphaerechinus granularis, whereas extracts from intact algal cells were only mildly cytotoxic. When the extracts from seawater culture medium were purified by RP-HPLC, cytotoxic activity towards S. granularis embryos was recovered in three consecutive fractions. Moreover, unfertilised eggs incubated in the active HPLC fractions became unproductive, whereas incubation of sperm gave a reduced fertilisation rate. This anti-proliferative effect was further characterized by immunofluorescence staining of sea urchin embryos. DNA labelling revealed that incubating sea urchin embryos in the purified algal extracts inhibited both pronuclei migration and fusion. Incorporation and detection of the DNA-base analogue 5-bromo-2-deoxyuridine showed that DNA-replication was blocked. Furthermore, staining of α-tubulin subunits demonstrated that embryonic tubulin organisation was altered. We conclude that P. pouchetii produce some anti-mitotic compound, and that senescent colonial cells to a great extent excrete this compound to their surroundings.

Monoclonal antibody against dnmt1 arrests the cell division of xenopus early-stage embryos

DNA methylation plays a crucial role in embryogenesis, and Dnmt1 is known to be a key enzyme in the maintenance of DNA methylation. Dnmt1 is highly accumulated in mature oocytes and eggs. To analyze the function of the maternally accumulated Dnmt1, we injected monoclonal antibodies that specifically recognize the amino terminus of Xenopus Dnmt1 into Xenopus laevis embryos. The monoclonal antibodies inhibited the cell division of the embryos before the midblastula transition. Monoclonal antibody neither inhibited DNA methylation activity of Dnmt1 in vitro nor affected its stability in embryos. In addition, injection of α-amanitin, an inhibitor of transcription, did not rescue the cell division arrest. The results suggest that the inhibition of cell division by monoclonal antibodies was due neither to the direct inhibition of DNA methylation activity of Dnmt1 nor to aberrant transcription before the midblastula transition. The morphology of chromatin of the arrested cells showed that the cell cycle was arrested at interphase. This was supported by the biochemical analysis in which the arrested cells demonstrated low histone H1 kinase activity, which indicated that the cells had not entered M phase. Dnmt1 may have an important function other than DNA methylation activity for early embryogenesis in Xenopus laevis.

Conserved Enzymatic Production and Biological Effect of O-Acetyl-ADP-ribose by Silent Information Regulator 2-like NAD+-dependent Deacetylases

Silent information regulator 2 (Sir2) family of enzymes has been implicated in many cellular processes that include histone deacetylation, gene silencing, chromosomal stability, and aging. Yeast Sir2 and several homologues have been shown to be NAD(+)-dependent histone/protein deacetylases. Previously, it was demonstrated that the yeast enzymes catalyze a unique reaction mechanism in which the cleavage of NAD(+) and the deacetylation of substrate are coupled with the formation of O-acetyl-ADP-ribose, a novel metabolite. We demonstrate that the production of O-acetyl-ADP-ribose is evolutionarily conserved among Sir2-like enzymes from yeast, Drosophila, and human. Also, endogenous yeast Sir2 complex from telomeres was shown to generate O-acetyl-ADP-ribose. By using a quantitative microinjection assay to examine the possible biological function(s) of this newly discovered metabolite, we demonstrate that O-acetyl-ADP-ribose causes a delay/block in oocyte maturation and results in a delay/block in embryo cell division in blastomeres. This effect was mimicked by injection of low nanomolar levels of active enzyme but not with a catalytically impaired mutant, indicating that the enzymatic activity is essential for the observed effects. In cell-free oocyte extracts, we demonstrate the existence of cellular enzymes that can efficiently utilize O-acetyl-ADP-ribose.

Role of syndecan in the elongation of postoral arms in sea urchin larvae.

Ac-SYN is the core protein of a cell surface proteoglycan of the sea urchin Anthocidaris crassispina. To examine the functions of Ac-SYN, embryos were cultured in the presence of affinity-purified antibody against Ac-SYN. At the late pluteus stage, severe inhibition of elongation of the postoral arms was seen in treated embryos compared with control embryos. Blastocoeleic microinjection of the antibody did not affect morphogenesis. The relationship between the number of cells in the postoral arms and the length of the postoral rods was investigated in normal embryos. This showed that postoral arm elongation has two phases: the first phase accompanies the increase in cell numbers while the second does not. The syndecan antibody inhibited the increase in cell numbers in the postoral arms. Furthermore, in the treated embryos, cell numbers continued to increase normally until 31 h post fertilization (hpf), while cell division stopped after 31 hpf. These results suggest that Ac-SYN participates in postoral arm formation via cell division in sea urchin embryos.