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Zygote
2015 Jun 01;233:426-46. doi: 10.1017/S0967199414000033.
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Ca²⁺ influx-linked protein kinase C activity regulates the β-catenin localization, micromere induction signalling and the oral-aboral axis formation in early sea urchin embryos.
Yazaki I
,
Tsurugaya T
,
Santella L
,
Chun JT
,
Amore G
,
Kusunoki S
,
Asada A
,
Togo T
,
Akasaka K
.
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Sea urchin embryos initiate cell specifications at the 16-cell stage by forming the mesomeres, macromeres and micromeres according to the relative position of the cells in the animal-vegetal axis. The most vegetal cells, micromeres, autonomously differentiate into skeletons and induce the neighbouring macromere cells to become mesoendoderm in the β-catenin-dependent Wnt8 signalling pathway. Although the underlying molecular mechanism for this progression is largely unknown, we have previously reported that the initial events might be triggered by the Ca2+ influxes through the egg-originated L-type Ca2+ channels distributed asymmetrically along the animal-vegetal axis and through the stretch-dependent Ca2+channels expressed specifically in the micromere at the 4th cleavage. In this communication, we have examined whether one of the earliest Ca2+ targets, protein kinase C (PKC), plays a role in cell specification upstream of β-catenin. To this end, we surveyed the expression pattern of β-catenin in early embryos in the presence or absence of the specific peptide inhibitor of Hemicentrotus pulcherrimus PKC (HpPKC-I). Unlike previous knowledge, we have found that the initial nuclear entrance of β-catenin does not take place in the micromeres, but in the macromeres at the 16-cell stage. Using the HpPKC-I, we have demonstrated further that PKC not only determines cell-specific nucleation of β-catenin, but also regulates a variety of cell specification events in the early sea urchin embryos by modulating the cell adhesion structures, actin dynamics, intracellular Ca2+ signalling, and the expression of key transcription factors.
Figure 1. Inhibition of PKC interferes with the normal distribution of actin cytoskeleton in blastomeres. (A) Preparation of the HpPKC inhibitor (HpPKC-I). The amino acid sequence of Hemicentrotus pulcherrimus PKC was aligned with Lytechinus pictus (LpPKC) and human PKC (HuPKC) (1–68) to define the pseudosubstrate region (underlined nine residues). Asterisks denote identical amino acid residues. The synthetic peptides of the pseudosubstrate were myristoylated to use as HpPKC-I. (B) Effects of PKC on actin distribution. F-actin was visualized with Alexa Fluor 488-conjugated phalloidin; 8 nM PMA was added to the embryos (P. lividus) for 20 min starting from 7 min before the 4th division; 5 μM of HpPKC-I was added 20 min before the 4th division. Optical (upper) and fluorescent images (lower) were taken from the same confocal plane. In either case, cell specifications were modified, but cell division progressed normally.
Figure 3. Distribution of Hpβ-catenin during mitosis. Embryonic cells at various mitotic stages (H. pulcherrimus) were stained with Hpβ-catenin antibody (green) and PI (red). Cells marked with asterisks in each embryo of (A), (B), (C) and (D) respectively were enlarged in the right-side panel (a, b, c, d). (A) At the 56-cell stage, most macromere and all mesomere derivatives shown were in prophase. (B) Cells at metaphase and anaphase in the 28-cell stage embryo. (C) Cells at telophase in the 28-cell stage embryo from the ventral view. Macromeres have just divided to be in telophase, showing an irregular, chambered structure in the process of reconstruction of the daughter nuclei. (D) A ventral view of 28-cell stage embryo. Macromeres were at interphase.
Figure 4. Localization of the nuclear β-catenin in the 16-cell to 56-cell stage embryos. (A1–E1) double staining of propidium iodide (PI) (red) and β-catenin antibody (green). (A2–E2) images of β-catenin staining only. (A3–E3) Drawing of (A1–E1) images to illustrate the nuclei and the contour lines of blastomeres. Abbreviations: mic; micromeres, Mac; macromeres, meso; mesomeres. (A) H. pulcherrimus embryo at the 16-cell stage: all cells were at interphase. Nuclear β-catenin was preferentially present in Mac. (B) Late 16-cell stage: mic remained at the interphase, but Mac progressed to anaphase. No β-catenin was found in mic nuclei. (C) Early 28-cell stage: Mac divided to the telophase, and mic formed chromosomal plate of metaphase. (D) At the 28-cell stage: mic at metaphase or anaphase. Mac and meso derivatives were at interphase. Nuclear β-catenin was detected only in Mac derivatives. (E) At the 56-cell stage: s-mic, small micromeres; l-mic, large micromeres. Veg1 and Veg2 are the macromere derivatives locating at the animal side (an) or next to l-mic, respectively. All cells shown were at interphase, and nuclear β-catenin was detected in every cell except for ‘an (animal side)’ cells. Abbreviations: An, animal side; Vg, vegetal side.
Figure 5. LiCl, PKC activator and PKC inhibitors affect the distribution of β-catenin. Localization of β-catenin (green) and nuclear staining (red) in the H. pulcherrimus embryos at the 16-cell, 28-cell and 56-cell stages. (A) Control embryos. (B) Embryos treated with 40 mM LiCl starting from the 4-cell stage. (C) Embryos exposed to PKC activator (8 nM PMA for 20 min starting from 5 min before the 4th cleavage). (D, E) Embryos exposed to 5 μM HpPKC-I starting from 20 min before the 4th division (16-cell stage). (F) Embryos exposed to 400 nM Gö6976, a calcium-dependent PKC inhibitor, for the same period as HpPKC-I. Abbreviations: An, animal side; Vg, vegetal side; mic; micromeres, Mac; macromeres, meso; mesomeres.
Figure 6. Inhibition of Ca2+infux and of PKC activity delayed gastrulation, but not PMC ingression. (A) P. lividus embryos were treated with 25 μM GdCl3 for 50 min starting from 15 min before the 2nd, 4th, 5th and 7th cleavages at room temperature. Cell cycles went on every 30–35 min. Control and GdCl3-treated embryos were both fixed at the same time, and the percentage of PMC-ingressed embryos or gastrulation-initiated embryos were calculated from circa 100 embryos from two or three independent experiments, respectively. (B, C) H. pulcherrimus embryos were treated with HpPKC-I at 6 μM (B) or 5 μM (C) during the period indicated by the horizontal bars. Gastrulation levels were estimated only from the side-viewed embryos. Embryos were cultured at 15°C (B), at 18°C (C). The cell cycles of these experiments were 45–48 min (B) and 39 min (C).
Figure 7. HpPKC inhibitor caused similar morphological changes on embryos of H. pulcherrimus and P. lividus. Embryos were treated with 5 μM HpPKC-I from 20 min before the 4th cleavage to 20 min after the 6th cleavage. This period was about 120 min for H. pulcherrimus culturing at 18°C and 100 min for P. lividus at 23°C. Control and HpPKC-I-treated embryos. (A) H. pulcherrimus embryos; early blastulae at 6 h post fertilization (p.f.), gastrulae at 22 h, and plutei at 52 h (B) P. lividus; morulae at 5 h, gastrulae at 21 h, and plutei at 47 h. (C) Names of the skeletal parts of the pluteus.
Figure 8. Morphological changes of H. pulcherrimus embryos treated with HpPKC inhibitor before the 8-cell stage. (A) Control embryos. (B) Embryos treated with 5 μM HpPKC-I starting from the 4-cell stage (20 min before the 3rd cleavage) to the end of 8-cell stage. (C) Embryos treated with 5 μM HpPKC starting from 20 min before the 4th cleavage to the end of 16-cell stage. Embryos were cultured at 18°C. The hand-drawn delineations on the right panels represent the embryos marked with asterisks in (A–C). Oral ectoderm area and aboral area were indicated by white arrowheads and black arrows, respectively.
Amore,
Spdeadringer, a sea urchin embryo gene required separately in skeletogenic and oral ectoderm gene regulatory networks.
2003, Pubmed,
Echinobase
Amore,
Spdeadringer, a sea urchin embryo gene required separately in skeletogenic and oral ectoderm gene regulatory networks.
2003,
Pubmed
,
Echinobase
Angerer,
SoxB1 downregulation in vegetal lineages of sea urchin embryos is achieved by both transcriptional repression and selective protein turnover.
2005,
Pubmed
,
Echinobase
Berridge,
Neural and developmental actions of lithium: a unifying hypothesis.
1989,
Pubmed
Berridge,
Inositol trisphosphate and calcium signalling.
1993,
Pubmed
Bienz,
beta-Catenin: a pivot between cell adhesion and Wnt signalling.
2005,
Pubmed
Chun,
The biphasic increase of PIP2 in the fertilized eggs of starfish: new roles in actin polymerization and Ca2+ signaling.
2010,
Pubmed
,
Echinobase
Ciapa,
Effect of lithium on ionic balance and polyphosphoinositide metabolism during larval vegetalization of the sea urchin Paracentrotus lividus.
1993,
Pubmed
,
Echinobase
Coffman,
Oral-aboral axis specification in the sea urchin embryo III. Role of mitochondrial redox signaling via H2O2.
2009,
Pubmed
,
Echinobase
Coffman,
Oral-aboral axis specification in the sea urchin embryo. I. Axis entrainment by respiratory asymmetry.
2001,
Pubmed
,
Echinobase
Coffman,
Oral-aboral axis specification in the sea urchin embryo II. Mitochondrial distribution and redox state contribute to establishing polarity in Strongylocentrotus purpuratus.
2004,
Pubmed
,
Echinobase
Cook,
Wingless inactivates glycogen synthase kinase-3 via an intracellular signalling pathway which involves a protein kinase C.
1996,
Pubmed
Croce,
Wnt6 activates endoderm in the sea urchin gene regulatory network.
2011,
Pubmed
,
Echinobase
Croce,
Coquillette, a sea urchin T-box gene of the Tbx2 subfamily, is expressed asymmetrically along the oral-aboral axis of the embryo and is involved in skeletogenesis.
2003,
Pubmed
,
Echinobase
Dale,
Polarized distribution of L-type calcium channels in early sea urchin embryos.
1997,
Pubmed
,
Echinobase
Davidson,
A provisional regulatory gene network for specification of endomesoderm in the sea urchin embryo.
2002,
Pubmed
,
Echinobase
Dickey-Sims,
Runx-dependent expression of PKC is critical for cell survival in the sea urchin embryo.
2005,
Pubmed
,
Echinobase
Drummond,
The interaction of lithium with thyrotropin-releasing hormone-stimulated lipid metabolism in GH3 pituitary tumour cells. Enhancement of stimulated 1,2-diacylglycerol formation.
1984,
Pubmed
Duboc,
Nodal and BMP2/4 signaling organizes the oral-aboral axis of the sea urchin embryo.
2004,
Pubmed
,
Echinobase
Eichholtz,
A myristoylated pseudosubstrate peptide, a novel protein kinase C inhibitor.
1993,
Pubmed
Eliyahu,
The involvement of protein kinase C and actin filaments in cortical granule exocytosis in the rat.
2005,
Pubmed
Emily-Fenouil,
GSK3beta/shaggy mediates patterning along the animal-vegetal axis of the sea urchin embryo.
1998,
Pubmed
,
Echinobase
Fuchikami,
T-brain homologue (HpTb) is involved in the archenteron induction signals of micromere descendant cells in the sea urchin embryo.
2002,
Pubmed
,
Echinobase
Goode,
Differential regulation of glycogen synthase kinase-3 beta by protein kinase C isotypes.
1992,
Pubmed
Gumbiner,
Cell adhesion: the molecular basis of tissue architecture and morphogenesis.
1996,
Pubmed
Kawasaki,
Lim1 related homeobox gene (HpLim1) expressed in sea urchin embryos.
1999,
Pubmed
,
Echinobase
Klein,
A molecular mechanism for the effect of lithium on development.
1996,
Pubmed
Kominami,
Subequatorial cytoplasm plays an important role in ectoderm patterning in the sea urchin embryo.
2006,
Pubmed
,
Echinobase
Kyozuka,
Actin cytoskeleton modulates calcium signaling during maturation of starfish oocytes.
2008,
Pubmed
,
Echinobase
Kühl,
The Wnt/Ca2+ pathway: a new vertebrate Wnt signaling pathway takes shape.
2000,
Pubmed
Lhomond,
Frizzled1/2/7 signaling directs β-catenin nuclearisation and initiates endoderm specification in macromeres during sea urchin embryogenesis.
2012,
Pubmed
,
Echinobase
Li,
Requirement of SpOtx in cell fate decisions in the sea urchin embryo and possible role as a mediator of beta-catenin signaling.
1999,
Pubmed
,
Echinobase
Li,
Lithium regulates glycogen synthase kinase-3beta in human peripheral blood mononuclear cells: implication in the treatment of bipolar disorder.
2007,
Pubmed
Livingston,
Phorbol esters alter cell fate during development of sea urchin embryos.
1992,
Pubmed
,
Echinobase
Livingston,
Injection of myo-inositol reverses the effects of lithium on sea urchin blastomeres.
1995,
Pubmed
,
Echinobase
Logan,
Nuclear beta-catenin is required to specify vegetal cell fates in the sea urchin embryo.
1999,
Pubmed
,
Echinobase
MacDonald,
Wnt/beta-catenin signaling: components, mechanisms, and diseases.
2009,
Pubmed
Mao,
Altering cell fates in sea urchin embryos by overexpressing SpOtx, an orthodenticle-related protein.
1996,
Pubmed
,
Echinobase
Maslanski,
Lithium-sensitive production of inositol phosphates during amphibian embryonic mesoderm induction.
1992,
Pubmed
McClay,
A micromere induction signal is activated by beta-catenin and acts through notch to initiate specification of secondary mesenchyme cells in the sea urchin embryo.
2000,
Pubmed
,
Echinobase
McCrea,
Purification of a 92-kDa cytoplasmic protein tightly associated with the cell-cell adhesion molecule E-cadherin (uvomorulin). Characterization and extractability of the protein complex from the cell cytostructure.
1991,
Pubmed
Miller,
Mechanism and function of signal transduction by the Wnt/beta-catenin and Wnt/Ca2+ pathways.
1999,
Pubmed
Miller,
Characterization of the role of cadherin in regulating cell adhesion during sea urchin development.
1997,
Pubmed
,
Echinobase
Miller,
Changes in the pattern of adherens junction-associated beta-catenin accompany morphogenesis in the sea urchin embryo.
1997,
Pubmed
,
Echinobase
Mitsunaga,
Probable Contribution of Protein Phosphorylation by Protein Kinase C to Spicule Formation in Sea Urchin Embryos: (sea urchin/protein kinase C/spicule formation/H-7/HA1004).
1990,
Pubmed
,
Echinobase
Miyawaki,
Nuclear localization of beta-catenin in vegetal pole cells during early embryogenesis of the starfish Asterina pectinifera.
2003,
Pubmed
,
Echinobase
Nishizuka,
Turnover of inositol phospholipids and signal transduction.
1984,
Pubmed
Oliveri,
Activation of pmar1 controls specification of micromeres in the sea urchin embryo.
2003,
Pubmed
,
Echinobase
Oliveri,
A regulatory gene network that directs micromere specification in the sea urchin embryo.
2002,
Pubmed
,
Echinobase
Rakow,
Molecular Cloning and Characterization of Protein Kinase C from the Sea Urchin Lytechinus pictus: (protein kinase C/cDNA cloning/polyclonal antibodies/developmental expression).
1994,
Pubmed
,
Echinobase
Ransick,
A complete second gut induced by transplanted micromeres in the sea urchin embryo.
1993,
Pubmed
,
Echinobase
Ransick,
Micromeres are required for normal vegetal plate specification in sea urchin embryos.
1995,
Pubmed
,
Echinobase
Shen,
A synthetic peptide of the pseudosubstrate domain of protein kinase C blocks cytoplasmic alkalinization during activation of the sea urchin egg.
1990,
Pubmed
,
Echinobase
Spiegel,
Development of cell junctions in sea-urchin embryos.
1983,
Pubmed
,
Echinobase
Stamateris,
The expression and distribution of Wnt and Wnt receptor mRNAs during early sea urchin development.
2010,
Pubmed
,
Echinobase
Su,
A perturbation model of the gene regulatory network for oral and aboral ectoderm specification in the sea urchin embryo.
2009,
Pubmed
,
Echinobase
Takacs,
Expression of an NK2 homeodomain gene in the apical ectoderm defines a new territory in the early sea urchin embryo.
2004,
Pubmed
,
Echinobase
Takeichi,
Cadherin cell adhesion receptors as a morphogenetic regulator.
1991,
Pubmed
Weitzel,
Differential stability of beta-catenin along the animal-vegetal axis of the sea urchin embryo mediated by dishevelled.
2004,
Pubmed
,
Echinobase
Wikramanayake,
Nuclear beta-catenin-dependent Wnt8 signaling in vegetal cells of the early sea urchin embryo regulates gastrulation and differentiation of endoderm and mesodermal cell lineages.
2004,
Pubmed
,
Echinobase
Wikramanayake,
Autonomous and non-autonomous differentiation of ectoderm in different sea urchin species.
1995,
Pubmed
,
Echinobase
Wikramanayake,
Multiple signaling events specify ectoderm and pattern the oral-aboral axis in the sea urchin embryo.
1997,
Pubmed
,
Echinobase
Wikramanayake,
beta-Catenin is essential for patterning the maternally specified animal-vegetal axis in the sea urchin embryo.
1998,
Pubmed
,
Echinobase
Yaguchi,
Specification of ectoderm restricts the size of the animal plate and patterns neurogenesis in sea urchin embryos.
2006,
Pubmed
,
Echinobase
Yang,
Block of stretch-activated ion channels in Xenopus oocytes by gadolinium and calcium ions.
1989,
Pubmed
Yazaki,
Ca(2+) in specification of vegetal cell fate in early sea urchin embryos.
2001,
Pubmed
,
Echinobase
Yazaki,
Mechanisms of calcium elevation in the micromeres of sea urchin embryos.
2004,
Pubmed
,
Echinobase
Yazaki,
Functional gap junctions in the early sea urchin embryo are localized to the vegetal pole.
1999,
Pubmed
,
Echinobase
Yazaki,
Polarization of the Surface Membrane and Cortical Layer of Sea Urchin Blastomeres, and its Inhibition by Cytochalasin B: (post-cleavage surface movements/cortical microfilaments/egg-surface antibody/cytochalasin B/sea-urchin blastomeres).
1991,
Pubmed
,
Echinobase
Yuh,
Brn1/2/4, the predicted midgut regulator of the endo16 gene of the sea urchin embryo.
2005,
Pubmed
,
Echinobase
