Reducing inositol lipid hydrolysis, Ins(1,4,5)P3 receptor availability, or Ca2+ gradients lengthens the duration of the cell cycle in Xenopus laevis blastomeres.

We have microinjected a mAb specifically directed to phosphatidylinositol 4,5-bisphosphate (PIP2) into one blastomere of two-cell stage Xenopus laevis embryos. This antibody binds to endogenous PIP2 and reduces its rate of hydrolysis by phospholipase C. Antibody-injected blastomeres undergo partial or complete arrest of the cell cycle whereas the uninjected sister blastomeres divided normally. Since PIP2 hydrolysis normally produces diacylglycerol (DG) and inositol 1,4,5-triphosphate (Ins[1,4,5]P3), we attempted to measure changes in the levels of DG following stimulation of PIP2 hydrolysis in antibody-injected oocytes. The total amount of DG in antibody-injected oocytes was significantly reduced compared to that of water-injected ones following stimulation by either acetylcholine or progesterone indicating that the antibody does indeed suppress PIP2 hydrolysis. We also found that the PIP2 antibodies greatly reduced the amount of intracellular Ca2+ released in the egg cortex during egg activation. As an indirect test for Ins(1,4,5)P3 involvement in the cell cycle we injected heparin which competes with Ins(1,4,5)P3 for binding to its receptor, and thus inhibits Ins(1,4,5)P3-induced Ca2+ release. Microinjection of heparin into one blastomere of the two-cell stage embryo caused partial or complete arrest of the cell cycle depending upon the concentration of heparin injected. We further investigated the effect of reducing any [Ca2+]i gradients by microinjecting dibromo-BAPTA into the blastomere. Dibromo-BAPTA injection completely blocked mitotic cell division when a final concentration of 1.5 mM was used. These results suggest that PIP2 turnover as well as second messenger activity influence cell cycle duration during embryonic cell division in frogs.

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Probing and manipulating embryogenesis via nanoscale thermometry and temperature control

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1922730117 ◽

2020 ◽

Vol 117 (26) ◽

pp. 14636-14641 ◽

Cited By ~ 6

Author(s):

Joonhee Choi ◽

Hengyun Zhou ◽

Renate Landig ◽

Hai-Yin Wu ◽

Xiaofei Yu ◽

...

Keyword(s):

Cell Cycle ◽

Cell Division ◽

Cell Communication ◽

Cycle Duration ◽

Cell Stage ◽

C Elegans ◽

Local Perturbations ◽

Cell Division Timing ◽

Nanoscale Thermometry

Understanding the coordination of cell-division timing is one of the outstanding questions in the field of developmental biology. One active control parameter of the cell-cycle duration is temperature, as it can accelerate or decelerate the rate of biochemical reactions. However, controlled experiments at the cellular scale are challenging, due to the limited availability of biocompatible temperature sensors, as well as the lack of practical methods to systematically control local temperatures and cellular dynamics. Here, we demonstrate a method to probe and control the cell-division timing inCaenorhabditis elegansembryos using a combination of local laser heating and nanoscale thermometry. Local infrared laser illumination produces a temperature gradient across the embryo, which is precisely measured by in vivo nanoscale thermometry using quantum defects in nanodiamonds. These techniques enable selective, controlled acceleration of the cell divisions, even enabling an inversion of division order at the two-cell stage. Our data suggest that the cell-cycle timing asynchrony of the early embryonic development inC. elegansis determined independently by individual cells rather than via cell-to-cell communication. Our method can be used to control the development of multicellular organisms and to provide insights into the regulation of cell-division timings as a consequence of local perturbations.

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PAR-4/LKB1 regulates DNA replication during asynchronous division of the early C. elegans embryo

The Journal of Cell Biology ◽

10.1083/jcb.201312029 ◽

2014 ◽

Vol 205 (4) ◽

pp. 447-455 ◽

Cited By ~ 18

Author(s):

Laura Benkemoun ◽

Catherine Descoteaux ◽

Nicolas T. Chartier ◽

Lionel Pintard ◽

Jean-Claude Labbé

Keyword(s):

Cell Cycle ◽

Dna Replication ◽

Cell Cycle Progression ◽

Molecular Mechanisms ◽

Control Cell ◽

Cycle Duration ◽

Cell Stage ◽

C Elegans ◽

Stage Embryo ◽

Regulation Of Cell Cycle

Regulation of cell cycle duration is critical during development, yet the underlying molecular mechanisms are still poorly understood. The two-cell stage Caenorhabditis elegans embryo divides asynchronously and thus provides a powerful context in which to study regulation of cell cycle timing during development. Using genetic analysis and high-resolution imaging, we found that deoxyribonucleic acid (DNA) replication is asymmetrically regulated in the two-cell stage embryo and that the PAR-4 and PAR-1 polarity proteins dampen DNA replication dynamics specifically in the posterior blastomere, independently of regulators previously implicated in the control of cell cycle timing. Our results demonstrate that accurate control of DNA replication is crucial during C. elegans early embryonic development and further provide a novel mechanism by which PAR proteins control cell cycle progression during asynchronous cell division.

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Spindlin, a major maternal transcript expressed in the mouse during the transition from oocyte to embryo

Development ◽

10.1242/dev.124.2.493 ◽

1997 ◽

Vol 124 (2) ◽

pp. 493-503 ◽

Cited By ~ 5

Author(s):

B. Oh ◽

S.Y. Hwang ◽

D. Solter ◽

B.B. Knowles

Keyword(s):

Cell Cycle ◽

Mitotic Cell ◽

Meiotic Spindle ◽

Post Translational Modification ◽

Gene Encoding ◽

Cell Stage ◽

Initial Development ◽

Maternal Transcript ◽

Stage Embryo ◽

Cycle Dependent

Timely translation of maternal transcripts and post-translational modification of their gene products control the initial development of preimplantation-stage embryos. We have isolated and characterized a gene encoding a stage-specific embryonic protein. This novel gene, spindlin (Spin), is an abundant maternal transcript present in the unfertilized egg and 2-cell, but not 8-cell, stage embryo. Spin exhibits high homology to a multicopy gene, Y-linked spermiogenesis-specific transcript (Ssty), and together they form a new gene family expressed during gametogenesis. We find that spindlin associates with the meiotic spindle and is modified by phosphorylation in a cell-cycle-dependent fashion. Furthermore, it comigrates with the previously described 30x10(3) Mr metaphase complex which is posttranslationally modified during the first mitotic cell cycle. Our data suggest that spindlin plays a role in cell-cycle regulation during the transition from gamete to embryo.

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Imaging patterns of calcium transients during neural induction in Xenopus laevis embryos

Journal of Cell Science ◽

10.1242/jcs.113.19.3519 ◽

2000 ◽

Vol 113 (19) ◽

pp. 3519-3529 ◽

Cited By ~ 6

Author(s):

C. Leclerc ◽

S.E. Webb ◽

C. Daguzan ◽

M. Moreau ◽

A.L. Miller

Keyword(s):

Xenopus Laevis ◽

Calcium Signalling ◽

Neural Induction ◽

Calcium Transients ◽

Free Calcium ◽

Treated Animal ◽

Cell Stage ◽

Subsequent Formation ◽

Xenopus Laevis Embryos

Through the injection of f-aequorin (a calcium-sensitive bioluminescent reporter) into the dorsal micromeres of 8-cell stage Xenopus laevis embryos, and the use of a Photon Imaging Microscope, distinct patterns of calcium signalling were visualised during the gastrulation period. We present results to show that localised domains of elevated calcium were observed exclusively in the anterior dorsal part of the ectoderm, and that these transients increased in number and amplitude between stages 9 to 11, just prior to the onset of neural induction. During this time, however, no increase in cytosolic free calcium was observed in the ventral ectoderm, mesoderm or endoderm. The origin and role of these dorsal calcium-signalling patterns were also investigated. Calcium transients require the presence of functional L-type voltage-sensitive calcium channels. Inhibition of channel activation from stages 8 to 14 with the specific antagonist R(+)BayK 8644 led to a complete inhibition of the calcium transients during gastrulation and resulted in severe defects in the subsequent formation of the anterior nervous system. BayK treatment also led to a reduction in the expression of Zic3 and geminin in whole embryos, and of NCAM in noggin-treated animal caps. The possible role of calcium transients in regulating developmental gene expression is discussed.

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Protein kinase C acts downstream of calcium at entry into the first mitotic interphase of Xenopus laevis.

Cell Regulation ◽

10.1091/mbc.1.3.315 ◽

1990 ◽

Vol 1 (3) ◽

pp. 315-326 ◽

Cited By ~ 51

Author(s):

W M Bement ◽

D G Capco

Keyword(s):

Cell Cycle ◽

Protein Kinase C ◽

Protein Kinase ◽

Xenopus Laevis ◽

Mitotic Cell ◽

Cortical Granule ◽

Mitotic Cell Cycle ◽

Cleavage Furrow ◽

Kinase C ◽

Cortical Granule Exocytosis

Transit into interphase of the first mitotic cell cycle in amphibian eggs is a process referred to as activation and is accompanied by an increase in intracellular free calcium [( Ca2+]i), which may be transduced into cytoplasmic events characteristic of interphase by protein kinase C (PKC). To investigate the respective roles of [Ca2+]i and PKC in Xenopus laevis egg activation, the calcium signal was blocked by microinjection of the calcium chelator BAPTA, or the activity of PKC was blocked by PKC inhibitors sphingosine or H7. Eggs were then challenged for activation by treatment with either calcium ionophore A23187 or the PKC activator PMA. BAPTA prevented cortical contraction, cortical granule exocytosis, and cleavage furrow formation in eggs challenged with A23187 but not with PMA. In contrast, sphingosine and H7 inhibited cortical granule exocytosis, cortical contraction, and cleavage furrow formation in eggs challenged with either A23187 or PMA. Measurement of egg [Ca2+]i with calcium-sensitive electrodes demonstrated that PMA treatment does not increase egg [Ca2+]i in BAPTA-injected eggs. Further, PMA does not increase [Ca2+]i in eggs that have not been injected with BAPTA. These results show that PKC acts downstream of the [Ca2+]i increase to induce cytoplasmic events of the first Xenopus mitotic cell cycle.

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Prospective Fates of Blastomeres at the 32 Cell Stage of Xenopus laevis Embryos

Proceedings of the Japan Academy ◽

10.2183/pjab1945.47.407 ◽

1971 ◽

Vol 47 (4) ◽

pp. 407-412 ◽

Cited By ~ 56

Author(s):

Osamu NAKAMURA ◽

Keiko KISHIYAMA

Keyword(s):

Xenopus Laevis ◽

Cell Stage ◽

Xenopus Laevis Embryos

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Cyclin B mRNA depletion only transiently inhibits the Xenopus embryonic cell cycle

Development ◽

10.1242/dev.111.4.1173 ◽

1991 ◽

Vol 111 (4) ◽

pp. 1173-1178 ◽

Cited By ~ 1

Author(s):

D.L. Weeks ◽

J.A. Walder ◽

J.M. Dagle

Keyword(s):

Cell Cycle ◽

Cell Division ◽

Antisense Oligonucleotides ◽

Normal Development ◽

Embryonic Cell ◽

Cyclin B ◽

Xenopus Embryos ◽

Embryonic Cleavage ◽

Embryonic Cell Cycle

The control of the cell cycle is dependent on the ability to synthesize and degrade proteins called cyclins. When antisense oligonucleotides are used to deplete Xenopus embryos of mRNA encoding cyclin B protein, embryonic cleavage is inhibited. Surprisingly, after missing several rounds of cleavage, the cell cycle and cell division resumes. These studies indicate that the early embryonic cell cycle can proceed with undetectable levels of cyclin B encoding mRNA. In contrast, other events of normal development, including the activation of embryonic transcription and gastrulation, are inhibited.

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Differential regulation of the yeast CDC7 gene during mitosis and meiosis

Molecular and Cellular Biology ◽

10.1128/mcb.8.1.293-300.1988 ◽

1988 ◽

Vol 8 (1) ◽

pp. 293-300

Author(s):

R A Sclafani ◽

M Patterson ◽

J Rosamond ◽

W L Fangman

Keyword(s):

Cell Cycle ◽

Cell Division ◽

De Novo ◽

Transcript Level ◽

Mitotic Cell ◽

Single Copy ◽

Repair Process ◽

Level Of Activity ◽

Induced Mutagenesis ◽

Complete Cell

The product of the CDC7 gene of Saccharomyces cerevisiae is known to be required in the mitotic cell cycle for the initiation of DNA replication. We show that changes in transcript levels do not account for this stage-specific function, since the steady-state mRNA concentration remains constant at 1 copy per cell throughout the cell cycle. By measuring the cell division capacity of a cdc7::URA3 mutant after loss of a single-copy plasmid containing the CDC7 gene, we show that the CDC7 protein is present in at least 200-fold excess of the amount required for a single cell division. These results appear to exclude periodic transcription or translation as a means by which CDC7 function is regulated. In contrast, the CDC7 protein is known to be dispensable for meiotic S phase, but is required for synaptonemal complex formation and recombination. We found that the CDC7 transcript level does vary during meiosis, reaching a maximum near the time at which recombination occurs. Meiotic spores containing a cdc7 null allele germinate but fail to complete cell division. Apparently the excess CDC7 product present in mitotic cells is physically excluded from the spores (or becomes inactivated) and must be produced de novo after germination. The cdc7-1 allele had previously been shown to confer a reduction in the rate of induced mutation. We show that the cloned wild-type CDC7 gene not only complements this defect, but that when the CDC7 gene is on a multiple copy plasmid, induced mutagenesis is increased. Therefore, in contrast to the excess CDC7 activity for cell division, the level of activity for some error-prone repair process may be normally limiting.

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