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Mari Iwabuchi - One of the best experts on this subject based on the ideXlab platform.

  • lamin b receptor mediated chromatin tethering to the nuclear envelope is detrimental to the xenopus Blastula
    Journal of Biochemistry, 2020
    Co-Authors: Haruka Oda, Satsuki Kato, Keita Ohsumi, Mari Iwabuchi
    Abstract:

    In the nucleus of eukaryotic cells, chromatin is tethered to the nuclear envelope (NE), wherein inner nuclear membrane proteins (INMPs) play major roles. However, in Xenopus Blastula, chromatin tethering to the NE depends on nuclear filamentous actin that develops in a Blastula-specific manner. To investigate whether chromatin tethering operates in the Blastula through INMPs, we experimentally introduced INMPs into Xenopus egg extracts that recapitulate nuclear formation in fertilized eggs. When expressed in extracts in which polymerization of actin is inhibited, only lamin B receptor (LBR), among the five INMPs tested, tethered chromatin to the NE, depending on its N2 and N3 domains responsible for chromatin-protein binding. N2-3-deleted LBR did not tether chromatin, although it was localized in the nuclei. We subsequently found that the LBR level was very low in the Xenopus Blastula but was elevated after the Blastula stage. When the LBR level was precociously elevated in the Blastula by injecting LBR mRNA, it induced alterations in nuclear laminar architecture and nuclear morphology, and caused DNA damage and abnormal mitotic spindles, depending on the N2-3 domains. These results suggest that LBR-mediated chromatin tethering is circumvented in the Xenopus Blastula, as it is detrimental to embryonic development.

  • chromatin tethering to the nuclear envelope by nuclear actin filaments a novel role of the actin cytoskeleton in the xenopus Blastula
    Genes to Cells, 2017
    Co-Authors: Haruka Oda, Keita Ohsumi, Natsuki Shirai, Naoko Ura, Mari Iwabuchi
    Abstract:

    The Xenopus oocyte is known to accumulate filamentous or F-actin in the nucleus, but it is currently unknown whether F-actin also accumulates in embryo nuclei. Using fluorescence-labeled actin reporters, we examined the actin distribution in Xenopus embryonic cells and found that F-actin accumulates in nuclei during the Blastula stage but not during the gastrula stage. To further investigate nuclear F-actin, we devised a Xenopus egg extract that reproduces the formation of nuclei in which F-actin accumulates. Using this extract, we found that F-actin accumulates primarily at the subnuclear membranous region and is essential to maintain chromatin binding to the nuclear envelope in well-developed nuclei. We also provide evidence that nuclear F-actin increases the structural stability of nuclei and contributes to chromosome alignment on the mitotic spindle at the following M phase. These results suggest the physiological importance of nuclear F-actin accumulation in rapidly dividing large Xenopus Blastula cells.

Haruka Oda - One of the best experts on this subject based on the ideXlab platform.

  • lamin b receptor mediated chromatin tethering to the nuclear envelope is detrimental to the xenopus Blastula
    Journal of Biochemistry, 2020
    Co-Authors: Haruka Oda, Satsuki Kato, Keita Ohsumi, Mari Iwabuchi
    Abstract:

    In the nucleus of eukaryotic cells, chromatin is tethered to the nuclear envelope (NE), wherein inner nuclear membrane proteins (INMPs) play major roles. However, in Xenopus Blastula, chromatin tethering to the NE depends on nuclear filamentous actin that develops in a Blastula-specific manner. To investigate whether chromatin tethering operates in the Blastula through INMPs, we experimentally introduced INMPs into Xenopus egg extracts that recapitulate nuclear formation in fertilized eggs. When expressed in extracts in which polymerization of actin is inhibited, only lamin B receptor (LBR), among the five INMPs tested, tethered chromatin to the NE, depending on its N2 and N3 domains responsible for chromatin-protein binding. N2-3-deleted LBR did not tether chromatin, although it was localized in the nuclei. We subsequently found that the LBR level was very low in the Xenopus Blastula but was elevated after the Blastula stage. When the LBR level was precociously elevated in the Blastula by injecting LBR mRNA, it induced alterations in nuclear laminar architecture and nuclear morphology, and caused DNA damage and abnormal mitotic spindles, depending on the N2-3 domains. These results suggest that LBR-mediated chromatin tethering is circumvented in the Xenopus Blastula, as it is detrimental to embryonic development.

  • chromatin tethering to the nuclear envelope by nuclear actin filaments a novel role of the actin cytoskeleton in the xenopus Blastula
    Genes to Cells, 2017
    Co-Authors: Haruka Oda, Keita Ohsumi, Natsuki Shirai, Naoko Ura, Mari Iwabuchi
    Abstract:

    The Xenopus oocyte is known to accumulate filamentous or F-actin in the nucleus, but it is currently unknown whether F-actin also accumulates in embryo nuclei. Using fluorescence-labeled actin reporters, we examined the actin distribution in Xenopus embryonic cells and found that F-actin accumulates in nuclei during the Blastula stage but not during the gastrula stage. To further investigate nuclear F-actin, we devised a Xenopus egg extract that reproduces the formation of nuclei in which F-actin accumulates. Using this extract, we found that F-actin accumulates primarily at the subnuclear membranous region and is essential to maintain chromatin binding to the nuclear envelope in well-developed nuclei. We also provide evidence that nuclear F-actin increases the structural stability of nuclei and contributes to chromosome alignment on the mitotic spindle at the following M phase. These results suggest the physiological importance of nuclear F-actin accumulation in rapidly dividing large Xenopus Blastula cells.

Christian Gache - One of the best experts on this subject based on the ideXlab platform.

  • structure of the gene encoding the sea urchin Blastula protease 10 bp10 a member of the astacin family of zn2 metalloproteases
    FEBS Journal, 1996
    Co-Authors: Guy Lhomond, Thierry Lepage, Christian Ghiglione, Christian Gache
    Abstract:

    Blastula protease 10 (BP10), a metalloprotease of the astacin family, is secreted at the Blastula stage by the sea urchin embryo. The BP10 gene shows a precise temporal and spatial regulation during embryo-genesis. It has been cloned from a sea urchin λ genomic library and the transcription unit has been entirely sequenced. It spans 6 kb and contains seven exons (2.8 kb) and six introns (3.2 kb). Sequence comparison and phylogeny analysis show that BP10 belongs to a sub-family of modular proteins which all play a role during development. In the two cases where the exon/intron organization of the gene is known (BP10 and tolloid), the modular structure of the protein is not reflected at the gene level, which indicates that this sub-family probably did not evolve by exon shuffling.

  • Spatial and temporal expression pattern during sea urchin embryogenesis of a gene coding for a protease homologous to the human protein BMP-1 and to the product of the Drosophila dorsal-ventral patterning gene tolloid.
    Development, 1992
    Co-Authors: Thierry Lepage, Christian Ghiglione, Christian Gache
    Abstract:

    A cDNA clone coding for a sea urchin embryonic protein was isolated from a prehatching Blastula lambda gt11 library. The predicted translation product is a secreted 64 × 10(3) Mr enzyme designated as BP10. The protein contains several domains: a signal peptide, a putative propeptide, a catalytic domain with an active center typical of a Zn(2+)-metalloprotease, an EGF-like domain and two internal repeats similar to repeated domains found in the C1s and C1r serine proteases of the complement cascade. The BP10 protease is constructed with the same domains as the human bone morphogenetic protein BMP-1, a protease described as a factor involved in bone formation, and as the recently characterized product of the tolloid gene which is required for correct dorsal-ventral patterning of the Drosophila embryo. The transcription of the BP10 gene is transiently activated around the 16- to 32-cell stage and the accumulation of BP10 transcripts is limited to a short period at the Blastula stage. By in situ hybridization with digoxygenin-labelled RNA probes, the BP10 transcripts were only detected in a limited area of the Blastula, showing that the transcription of the BP10 gene is also spatially controlled. Antibodies directed against a fusion protein were used to detect the BP10 protein in embryonic extracts. The protein is first detected in early Blastula stages, its level peaks in late cleavage, declines abruptly before ingression of primary mesenchyme cells and remains constant in late development. The distribution of the BP10 protein during its synthesis and secretion was analysed by immunostaining Blastula-stage embryos. The intracellular localization of the BP10 staining varies with time. The protein is first detected in a perinuclear region, then in an apical and submembranous position just before its secretion into the perivitelline space. The protein is synthesized in a sharply delimited continuous territory spanning about 70% of the Blastula. Comparison of the size and orientation of the labelled territory in the late Blastula with the fate map of the Blastula stage embryo shows that the domain in which the BP10 gene is expressed corresponds to the presumptive ectoderm. Developing embryos treated with purified antibodies against the BP10 protein and with synthetic peptides derived from the EGF-like domain displayed perturbations in morphogenesis and were radialized to various degrees. These results are consistent with a role for BP10 in the differentiation of ectodermal lineages and subsequent patterning of the embryo. On the basis of these results, we speculate that the role of BP10 in the sea urchin embryo might be similar to that of tolloid in Drosophila. We discuss the idea that the processes of spatial regulation of gene expression along the animal-vegetal in sea urchin and dorsal-ventral axes in Drosophila might have some similarities and might use common elements.

James A Coffman - One of the best experts on this subject based on the ideXlab platform.

  • runx expression is mitogenic and mutually linked to wnt activity in Blastula stage sea urchin embryos
    PLOS ONE, 2008
    Co-Authors: Anthony J Robertson, Alison Coluccio, Peter Knowlton, Carrie Dickeysims, James A Coffman
    Abstract:

    Background The Runt homology domain (Runx) defines a metazoan family of sequence-specific transcriptional regulatory proteins that are critical for animal development and causally associated with a variety of mammalian cancers. The sea urchin Runx gene SpRunt-1 is expressed throughout the Blastula stage embryo, and is required globally during embryogenesis for cell survival and differentiation. Methodology/Principal Findings Depletion of SpRunt-1 by morpholino antisense-mediated knockdown causes a Blastula stage deficit in cell proliferation, as shown by bromodeoxyuridine (BrdU) incorporation and direct cell counts. Reverse transcription coupled polymerase chain reaction (RT-PCR) studies show that the cell proliferation deficit is presaged by a deficit in the expression of several zygotic wnt genes, including wnt8, a key regulator of endomesoderm development. In addition, SpRunt-1-depleted Blastulae underexpress cyclinD, an effector of mitogenic Wnt signaling. Blastula stage cell proliferation is also impeded by knockdown of either wnt8 or cyclinD. Chromatin immunoprecipitation (ChIP) indicates that Runx target sites within 5′ sequences flanking cyclinD, wnt6 and wnt8 are directly bound by SpRunt-1 protein at late Blastula stage. Furthermore, experiments using a green fluorescent protein (GFP) reporter transgene show that the Blastula-stage operation of a cis-regulatory module previously shown to be required for wnt8 expression (Minokawa et al., Dev. Biol. 288: 545–558, 2005) is dependent on its direct sequence-specific interaction with SpRunt-1. Finally, inhibitor studies and immunoblot analysis show that SpRunt-1 protein levels are negatively regulated by glycogen synthase kinase (GSK)-3. Conclusions/Significance These results suggest that Runx expression and Wnt signaling are mutually linked in a feedback circuit that controls cell proliferation during development.

  • sprunt 1 a new member of the runt domain family of transcription factors is a positive regulator of the aboral ectoderm specific cyiiia gene in sea urchin embryos
    Developmental Biology, 1996
    Co-Authors: James A Coffman, Carmen V Kirchhamer, Michael G. Harrington, Eric H. Davidson
    Abstract:

    In this paper we present a structural and functional characterization of a new sea urchin embryo transcription factor, SpRunt-1. This factor was isolated by means of its specific interaction with acis-regulatory target site of theCyIIIagene. Here we show that this target site, the P7I site, is required for normal embryonic activation ofCyIIIa·CATreporter gene constructs. An oligonucleotide affinity column bearing the P7I target site purifies a 21-kDa polypeptide from Blastula-stage nuclear extracts, and the amino acid sequence obtained from this polypeptide was used to generate a nucleic acid probe with which the corresponding cDNA was cloned. The cDNA encodes an approximately 60-kDa protein, SpRunt-1, which includes a “runt domain” that is closely homologous to those ofDrosophilaand mammalian runt domain transcription factors. RNA and genomic blots show that SpRunt-1 is represented by a single embryonic transcript, encoded by one of possibly two runt-domain-containing genes. By RNA probe protection we found that transcripts of SpRunt-1 increase in concentration dramatically after the Blastula stage of development, suggesting that the up-regulation ofCyIIIathat occurs after Blastula stage is a function of zygotically transcribed SpRunt-1. These results are discussed with reference to known features of the runt domain family of transcription factors.

Janet Heasman - One of the best experts on this subject based on the ideXlab platform.

  • foxi1e activates ectoderm formation and controls cell position in the xenopus Blastula
    Development, 2007
    Co-Authors: Janet Heasman, Adnan Mir, Matt Kofron, Aaron M Zorn, Matej Bajzer, Mansoor Haque, Christopher Wylie
    Abstract:

    The segregation of the vertebrate embryo into three primary germ layers is one of the earliest developmental decisions. In Xenopus , where the process is best understood, the endoderm is specified by a vegetally localized transcription factor, VegT, which releases nodal signals that specify the adjacent marginal zone of the Blastula to become mesoderm. However, little is known about how the ectoderm becomes specified. In this paper, we show that the forkhead protein FoxI1e (also known as Xema) is required at the Blastula stage for normal formation of both the central nervous system and epidermis, the two early derivatives of the ectoderm. In addition, FoxI1e is required to maintain the regional identity of the animal cells of the Blastula, the cells that are precursors of ectodermal structures. In its absence, they lose contact with the animal cap, mix with cells of other germ layers and differentiate according to their new positions. Because FoxI1e is initially expressed in the animal region of the embryo and is rapidly downregulated in the neural plate, its role in neural and epidermal gene expression must precede the division of the ectoderm into neural and epidermal. The work also shows that FoxI1e plays a role in the embryo in the poorly understood process of differential adhesion, which limits cell mixing as primary germ layers become specified.

  • patterning the xenopus Blastula
    Development, 1997
    Co-Authors: Janet Heasman
    Abstract:

    This review starts from the classical standpoint that there are at least two separable processes acting with respect to axis formation and tissue specification in the early Xenopus embryo: a UV-insensitive event establishing a postgastrula embryo consisting of three concentric germ layers, ectoderm, mesoderm and endoderm, all of a ventral character; and a UV-sensitive event producing tissue of a dorsal type, including somites, notochord and neural tissue, and concomitantly establishing the dorsoventral and anteroposterior axes. The experimental evidence suggesting the molecular basis of the dorsal and ventral pathways is reviewed.

  • a functional test for maternally inherited cadherin in xenopus shows its importance in cell adhesion at the Blastula stage
    Development, 1994
    Co-Authors: Janet Heasman, D Ginsberg, Benjamin Geiger, K Goldstone, T Pratt, Chikako Yoshidanoro, C C Wylie
    Abstract:

    We report here on the consequences of reducing the expression of EP-cadherin at the earliest stages of Xenopus development. Injection of oligodeoxynucleotides antisense to maternal EP-cadherin mRNA into full-grown oocytes reduced the mRNA level in oocytes, and the protein level in Blastulae. Adhesion between blastomeres was significantly reduced, as seen in whole embryos, and in assays of the ability of blastomeres to reaggregate in culture. This effect was especially conspicuous in the inner cells of the Blastula and included the disruption of the blastocoel. The severity of the EP-cadherin mRNA depletion and of the disaggregation phenotype was dose dependent. This phenotype was rescued by the injection into EP-cadherin mRNA-depleted oocytes of the mRNA coding for a related cadherin, E-cadherin, that is normally expressed at the gastrula stage in the embryonic ectoderm.

  • adhesion systems in the xenopus Blastula
    Comptes Rendus De L Academie Des Sciences Serie Iii-sciences De La Vie-life Sciences, 1992
    Co-Authors: Janet Heasman
    Abstract:

    Des systemes d'adhesion differencies ont longtemps ete consideres etre un mecanisme probable de la formation des tissus durant l'embryogenese. Dans le cas du Xenopys deux systemes d'adhesion ont ete identifies au stade de la Blastula. Les deux sont Ca 2+ dependants. L'un des systemes fait intervenir un membre de la famille des cadherines, l'autre un hydrate de carbone porte par une molecule de glycolipide. Nous avons identifie dans la Blastula du Xenopus d'une part la cadherine, d'autre part le systeme faisant intervenir l'hydrate de carbone