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Three different calcium wave pacemakers in ascidian eggs

Bio Mar Cell, Unité de Biologie du Développement UMR 7009 CNRS/Paris VI, Observatoire, Station Zoologique, Villefranche sur Mer, 06230 France

View larger version (118K): [in a new window]   Fig. 1. Distribution and reorganization of organelles, cytoplasm and cortex in Phallusia eggs and zygotes. Unfertilized egg (A); zygote, 3 minutes after fertilization (B); and zygote, 10 minutes after fertilization (C). The distribution of yolk platelets (blue) and mitochondria (green) in confocal sections 3 µm below the surface (A) or near the equator (B,C) oriented along the animal-vegetal (a-v) axis. The sperm aster region (sa, arrowhead) is a mitochondria-free, yolk platelet-free zone. In B, a contraction pole (cp) has formed. Bar, 23 µm. (D-F) The overall organization of the unfertilized egg (D); zygote, 3 minutes after fertilization (E); and zygote, 10 minutes after fertilization (F). ER network, red; yolk platelets, blue; mitochondria-rich domain, green; microtubules, green lines. Chromosomes (blue) are shown in the sperm aster, in the meiotic spindle and in the polar body (pb1). (G,H) Unfertilized egg: distribution of ER in confocal grazing tangential sections (1 µm (G) or 3 µm (H) below the surface) of an egg oriented along the animal-vegetal (a-v) axis. er, ER-rich domain; er arrow, corridor of ER-rich domain traversing the subcortical ER-poor and mitochondria-rich domain (m in green). (I) Unfertilized egg: distribution of cytosol visualized with injected Ca Green dextran (same egg and confocal section as that seen above in H). Yolk platelets are seen negatively as black round vesicles excluding the dextran dye. Bar, 15 µm.

View larger version (58K): [in a new window]   Fig. 2. Series I and series II physiological calcium wave pacemakers (PM1 and PM2) are associated with two distinct ER-rich domains. (A) Variations of [Ca2+]c displayed as normalized ratio CG/TR fluorescence (DFCG/TR/FCG/TR(0)) (1 image every 14 seconds). The [Ca2+]c scale on the right corresponds to earlier quantitative measurements of [Ca2+]c using aequorin (see Speksnijder et al., 1990a; Speksnijder et al., 1990b; Roegiers et al., 1999). Fertilization elicits series I calcium oscillations (PM1 boxed in blue) and the extrusion of the first polar body (pb1). Series II calcium oscillations (PM2 boxed in pink) can be observed after the extrusion of pb1, in the last 15-20 minutes, and end just before the extrusion of the second polar body (pb2). (B) Only the red lines, which represent the ER network, and the blue chromosomes are depicted. The red arrows show the origin and direction of calcium waves initiated by the different pacemakers. (Left) PM1-confocal ratio imaging of cytosolic calcium (CG/TR) (1 image every 9 seconds). The ratios are shown as pseudocoloured images: higher [Ca2+]c concentrations are in red and yellow; lower [Ca2+]c in blue. The CG/TR ratio image sequence shows the initiation and propagation of the second calcium wave triggered by the moving series I pacemaker (PM1, white arrowhead at 4 minutes 0 seconds (4'00'')). All calcium waves in the series start in a peripheral ER-rich domain corresponding to the sperm aster (sa; arrowhead in TR image), which moves with the cortical contraction. The TR image shown (6'08'') corresponds to the third calcium wave initiated by pacemaker PM1. (Right) PM2-ratio CG/TR sequence of a series II calcium oscillation (1 image every 14 seconds). All waves are initiated from the same vegetal cortical region of the egg (contraction pole area; white arrowhead at t=12'15''). The sperm aster (sa, arrowhead in TR image) has enlarged and has moved into the interior of the egg away from the calcium wave pacemaker (PM2) located in the contraction pole which, in this case, is below the imaged confocal plane.

View larger version (70K): [in a new window]   Fig. 3. An artificial calcium wave pacemaker (PM3) is generated by uncaging of cIns(1,4,5)P3 or cgPtdIns(4,5)P2. (A) Variations of [Ca2+]c induced by local and global UV photorelease of cIns(1,4,5)P3 in activated eggs (1 image every 5 seconds; the images displayed in the CG/TR sequence correspond to the orange dots on the graph). The contraction pole (cp), first polar body (pb1, also indicated by a white arrow in a) and the size of the UV-flashed area (blue circle) are indicated. (a-c) Localized UV-flashes of increasing duration (1 second for a, 2 seconds for b, and 2.5 seconds for c) give rise to calcium waves initiated in the flashed area. The waves propagate further as increasing amounts of Ins(1,4,5)P3 are photoreleased. (d) Brief (0.5 seconds) global UV-flashes over the whole egg increase the intracellular Ins(1,4,5)P3 levels and give rise to calcium waves initiated in the egg cortex (1 image every 5 seconds). Bar, 23 µm. The bar graph shows the number of waves that emanate from the animal hemisphere cortex (PM3) or the vegetal hemisphere cortex (PM2) after flash photolysis of cIns(1,4,5)P3. (B) Global UV photorelease of gPtdIns(4,5)P2 in unfertilized eggs. A single UV-flash of long duration (red arrowhead, t=0) gives rise to calcium oscillations that resemble the physiological series I calcium oscillations and leads to the extrusion of pb1 (1 image every 10 seconds). CG/TR sequence: most gPtdIns(4,5)P2-induced calcium oscillations are waves initiated in the cortex of the animal pole region (see bar graph). The arrow in the TR image shows the meiotic spindle-associated ER-rich domain that marks the animal pole. Bar, 23 µm.

View larger version (53K): [in a new window]   Fig. 4. The artificial pacemaker (PM3) predominates over the physiological calcium wave pacemaker(PM2). (A) Variations of [Ca2+]c in a fertilized egg undergoing meiosis II (1 image every 10 seconds), in response to a global increase in gPtdIns(4,5)P2 levels (red arrows indicate the four successive 1.5-second UV flashes applied). The flashes produce a sustained calcium increase, then two oscillations that are waves initiated in the animal pole (PM3, green graph). The three subsequent waves emanate from pacemaker PM2 in the contraction pole (pink graph) and precede the extrusion of the second polar body (pb2). (B) Another experiment showing the effect of cgPtdIns(4,5)P2 uncaging during the series II calcium waves (1 image every 4 seconds). The images displayed in the CG/TR sequence correspond to the yellow dots on the graph. The physiological series II calcium wave pacemaker PM2 is located in the contraction pole (cp) after PM1 activity has ceased (calcium waves shown in i and ii; Bar, 23 µm). This particular experiment shows that global UV-flashes over the whole zygote (uv1, uv2, uv3) can elicit three different types of waves. The first global photorelease of gPtdIns(4,5)P2 (uv1, flash duration 3 seconds; red arrowhead at 7'19'') triggers an artificial calcium wave whose initiation site (PM3) is in the animal pole (a) cortex. The second UV-flash (uv2, flash duration 1 second; red arrowhead at 11'17'') triggers an artificial calcium wave initiated in the contraction pole in the vegetal hemisphere. The third UV-flash (uv3, flash duration 2 seconds, arrowhead at 13'49'') elicits a calcium wave that is initiated in both the animal and vegetal pole regions. Probabilities for the occurrence of each type of wave are shown in the bar graph.

View larger version (17K): [in a new window]   Fig. 5. Differential sensitivity of the three pacemakers to cytoskeletal inhibitors. Patterns of [Ca2+]c oscillations triggered by fertilization (A,C-E) or after photorelease of cgPtdIns(4,5)P2 (B,C,F) in ascidians eggs perfused with nocodazole (A,B) or cytochalasin B (C-F) or both (B). The green arrowheads indicate the time of perfusion of the cytoskeletal inhibitors nocodazole (noc) or cytochalasinB (cytoB). Interruptions in the graphs are due to the fact that no measurements could be made during the time of perfusion. The black arrowheads mark the time of fertilization (f), while the red arrowheads indicate the time at which a UV flash is applied to uncage cgPtdIns(4,5)P2.