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First published online 14 May 2008
doi: 10.1242/dev.017558

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Ca²⁺ signals coordinate zygotic polarization and cell cycle progression in the brown alga Fucus serratus

¹ Marine Biological Association of the UK, The Laboratory, Citadel Hill, Plymouth PL1 2PB,UK.
² University of Newcastle-upon-Tyne, Institute of Cell and Molecular Biosciences, Medical School, Framlington Place, Newcastle NE2 4HH, UK.
³ Department of Biological Sciences, Lancaster Environment Centre, Lancaster University, Lancaster LA1 4YQ, UK.

View larger version (55K): [in this window] [in a new window]   Fig. 1. GFP-PCNA is a single-cell marker of S phase in Fucus serratus zygotes. (A) Eggs were biolistically co-loaded with Texas Red dextran (red images) and GFP-PCNA (green images), and visualized using 2-photon microscopy. Fertilization was confirmed using Calcofluor white (CFW), shown as the white edge in the red images. The same representative zygote is shown 0.5 hours (left) after fertilization (AF) and 3.5 hours after fertilization (right). (B) Nuclear GFP-PCNA/Texas Red dextran ratios (R) were plotted relative to their starting values (R0) for zygotes (white circles) and unfertilized eggs (black circles). Zygotes displayed significant nuclear GFP-PCNA increases between 3 and 5 hours after fertilization. Data are mean±s.e.m.; *P<0.03. (C) The nuclear GFP-PCNA increase seen 4 hours after fertilization (controls are white bars; `Fert', zygotes; `Un', unfertilized eggs) is completely abolished following treatment with the DNA polymerase inhibitor aphidicolin (20 µM; black bars). Data are mean±s.e.m.; *P<0.05.

View larger version (57K): [in this window] [in a new window]   Fig. 2. Fertilization-associated events in F. serratus. (A) The first indicator of fertilization was the secretion of cell wall components, imaged using 2-photon microscopy of the cellulose stain CFW. CFW staining was evident within 15 minutes of sperm and egg mixing, and propagated across the egg within 5 minutes. (B) Sperm pronuclear ('sp') motion towards the egg pronucleus ('ep'), visualized using 2-photon microscopy of Hoechst 33342-labelled sperm. This representative series of images shows the sperm pronucleus as a light blue dot against a green autofluorescent background. The final image was just before pronuclear fusion. (C) Time courses during the first 3 hours after fertilization for cell wall secretion (upper plot, grey circles, n=8), sperm pronuclear migration (upper plot, white circles, n=10), cytosolic [Ca2+] (lower plot, white circles, n=8) and nuclear [Ca2+] (lower plot, black circles, n=8). Cell wall secretion is given in degrees of coverage of zygote circumference. Migration of the sperm pronucleus is given as distance into the zygote. [Ca2+] was measured using Texas Red/fura dextran and divided into cytosolic and nuclear components using the regions of interest drawn in D, below. An initial sustained elevation of both cytosolic and nuclear [Ca2+] was followed by a nuclear [Ca2+] elevation at the time of pronuclear fusion. Data are mean±s.e.m. (D) Representative pseudocolour-ratiometric Texas Red/fura dextran images of post-fertilization [Ca2+] changes. Regions `c' and `n' on the left image define, respectively, the mutually exclusive cytosolic and nuclear regions of interest. The `gp' region is an artefact where the gold pellets (biolistic microcarriers) have gathered and can be ignored.

View larger version (55K): [in this window] [in a new window]   Fig. 3. Ca2+ buffers inhibit actin nucleation and DNA replication, but not pronuclear motion, in F. serratus zygotes. (A) The speeds at which sperm pronuclei progress towards egg pronuclei did not differ significantly between FITC-loaded and BAPTA dextran-loaded zygotes. Data are mean±s.e.m. (B) Pronuclear fusion in zygotes loaded with the Ca2+ buffer BAPTA dextran (0.4 mM). In these representative images, the sperm pronucleus ('sp') was labelled with 100 µM Hoechst 33342 and visualized using 2-photon microscopy. The left-hand image, taken 120 minutes after fertilization, shows the sperm pronucleus as a bright blue dot sitting next to the egg pronucleus ('ep') against a green FITC background (co-loaded with BAPTA dextran). In the right-hand image, taken 20 minutes later, the sperm pronucleus is less bright and has started to decondense, indicating that pronuclear fusion has started. (C) Untreated polarized 24-hour-old zygotes stained with Texas Red phalloidin to show actin nucleation in a cone at the germinating rhizoid apex. (D) Zygotes loaded biolistically with the Ca2+ buffer BAPTA dextran (0.4 mM) 30 minutes after fertilization and stained with Texas Red phalloidin 24 hours after fertilization. Neither zygotic polarization nor actin nucleation was observed (n=8). (E) Zygotes treated continuously with the CDK inhibitor olomoucine (100 µM) did not display zygotic polarization, but did show non-polarized cortical actin nucleation when stained with Texas Red phalloidin 24 hours after fertilization. (F) The Ca2+ buffer BAPTA dextran (0.4-0.8 mM) inhibited S-phase progression when loaded into zygotes 30 minutes after fertilization. Zygotes were biolistically loaded with GFP-PCNA, Texas Red dextran and BAPTA dextran and nuclear GFP-PCNA/Texas Red ratios (R) were plotted relative to their starting values (R0) for control (white circles) and BAPTA dextran-treated zygotes (black circles). Control zygotes displayed significant nuclear GFP-PCNA increases between 3 and 5 hours after fertilization (see also Fig. 1B), but these increases were completely abolished following treatment with BAPTA dextran. Data are mean±s.e.m.; *P<0.05.

View larger version (61K): [in this window] [in a new window]   Fig. 4. Cytoskeletal actin disruption does not inhibit DNA replication. (A) Untreated 24-hour-old control zygotes showing zygotic polarization and mitotic division. Zygotes were loaded with 1.0 µM DiOC6(3) for 15 minutes to visualise daughter nuclei (yellow arrows) by negative staining. (B) Representative 24-hour-old zygote (n>100) treated with 0.1 µM latrunculin B continuously from 20 minutes after fertilization and showing complete mitosis, but no zygotic polarization or cytokinesis (no dividing cell wall). The zygote was imaged with two separate confocal scans to allow for the different depths of each daughter nucleus (yellow arrows). (C) Representative 24 hour old zygote (n>100) treated continuously with 1.0 µM latrunculin B. The nucleus is displaced from the usual central location, but the chromosomes are condensed, indicating M-phase entry. (D) Enlarged section from C highlighting condensed chromosomes. (E) Cytoskeletal actin disruption does not inhibit entry into M phase. Zygotes were treated with 0.1 µM latrunculin B continuously from 30 minutes after fertilization (black circles) or not treated (white circles), and scored for entry into M phase by the presence of condensed nuclei (visualized by staining with 5 µM fluorescein diacetate and 100 µM Hoechst 33342). Data are mean±s.e.m. (F) Cytoskeletal actin disruption does inhibit polarization. Length/width ratios were measured for the zygotes in E. Control zygotes (white circles) displayed steady growth which was completely inhibited in 0.1 µM latrunculin B-treated zygotes (black circles). Data are mean± s.e.m.; *P<0.05.

View larger version (53K): [in this window] [in a new window]   Fig. 5. Ca2+ buffers inhibit cell cycle progression and reveal differential effects on zygotic polarization. (A) The effect of Ca2+ buffers on cell cycle progression. Zygotes were treated 1 hour after fertilization (white circles) or 7 hours after fertilization (black circles) with one of four treatments: biolistic FITC loading, biolistic FITC+BAPTA dextran loading, biolistic FITC+Br2BAPTA loading or superfusion with TPEN. The effect of Ca2+ buffers on cell cycle progression was quantified by counting the percentage of 24-hour-old zygotes that were binucleate and had clearly undergone mitosis. Buffer concentrations were lognormally distributed so the x-axis is logarithmic. Data are means±s.e.m. with n>5 for each point. (B) The effect of Ca2+ buffers on zygotic polarization when applied 1 hour after fertilization. The logistic regressions of A were used to calculate cell cycle rates for each zygote and predicted length/width ratios were subtracted from measured length/width ratios to give length/width residuals (circles). Linear regressions are shown as thick black lines, with 99% confidence limits as broken black lines. The FITC dextran and Br2BAPTA regressions do not differ significantly from the null model (dotted horizontal line), but those for BAPTA dextran and TPEN are significantly lower than the null model. (C) The effect of Ca2+ buffers on zygotic polarization when applied 7 hours after fertilization. Data are presented as in B. The regression lines for FITC, BAPTA dextran and Br2BAPTA do not differ significantly from the null model. However, the regression line for TPEN is significantly lower than the null model. (D) Representative images to show the maximum inhibition of polarization seen in binucleate zygotes. Cell cycle progression and polarization may be uncoupled either by BAPTA dextran treatment 1 hour, but not 7 hours, after fertilization or by TPEN treatment either 1 or 7 hours after fertilization.

View larger version (30K): [in this window] [in a new window]   Fig. 6. Interspecific comparisons of zygotic cell cycle checkpoints. (A) Summary model suggested by the data in Fig. 5. Regulatory processes are represented by magenta circles and broken magenta arrows (see text for details). (B) The present study suggests that regulation of zygotic development in F. serratus is accomplished by a system of checkpoints that lie intermediate between those of the tightly coupled budding yeast (C) and the loosely coupled Drosophila embryo (D). Zygotic polarization and cell cycle progression in F. serratus are coordinated by Ca2+ dependent processes, but there is no dependence of the cell cycle on polarization. (C) In budding yeast, cell cycle progression and polarization are made interdependent by the G1/S checkpoint and the morphogenesis checkpoint. (D) In Drosophila embryos, cell cycle progression and zygotic polarization are independent, run in parallel and may be uncoupled from each other by inhibitor treatments.

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