Christian Sardet

Free calcium pulses following fertilization in the ascidian egg

Using the calcium-specific, chemiluminescent photoprotein aequorin, we have measured changes in the concentration of free cytosolic calcium at fertilization in single eggs of the ascidians Phallusia mammillata and Ciona intestinalis. Shortly after insemination, the free calcium concentration rises within a minute from a resting level of about 90 nM in the unfertilized egg to a peak level of about 7 microM in Phallusia and about 10 microM in Ciona. The total duration time of this fertilization transient is 2-3 min. It is immediately followed by a series of 12 to 25 briefer calcium transients with peak levels of about 1-4 microM. These postfertilization pulses occur at regular intervals of 1-3 min during the completion of meiosis, and they stop as soon as the second polar body is formed at about 25 min. An interesting exception to this pattern was observed in eggs from Ciona that had been raised at lower temperatures during the winter months. Insemination in the absence of external calcium in Phallusia results in a pulse pattern very similar to the normal pattern. From this result we infer that the bulk (if not all) of the calcium required for both the fertilization pulse and the meiotic oscillations is released from internal sources.

Function and characteristics of repetitive calcium waves associated with meiosis

BACKGROUNDnInternal calcium waves and oscillations are now recognized as universal features of cellular activation, but their exact role remains uncertain. In mammalian and ascidian eggs, a large, sperm-triggered calcium activation wave crosses the egg at fertilization, followed by a series of periodic increases in intracellular calcium concentration ([Ca2+]i). We have previously shown that, in eggs of the ascidian Phallusia mammillata, these periodic, post-activation [Ca2+]i increases are in the form of waves, the origin of which relocalizes to a pacemaker region, and that they stop seconds before the completion of meiosis.nnnRESULTSnWe show here that the origin of the first one to four post-activation calcium waves in P. mammillata eggs transfers progressively from the site of sperm entry, usually in the animal hemisphere, towards an endoplasmic reticulum (ER)-rich contraction pole in the vegetal hemisphere, a process that takes about five minutes. Once the origin of these repetitive post-activation calcium waves has reached the contraction pole, all subsequent calcium waves originate from the domain of ER concentrated there, which acts as a pacemaker. The first few post-activation calcium waves are faster than the activation wave and, like the activation wave, they propagate homogeneously throughout the cytoplasm. Approximately five to ten minutes after fertilization, the post-activation calcium waves begin to propagate preferentially in the egg cortex. By manipulating intracellular calcium levels with caged inositol 1,4,5 trisphosphate (InsP3) and a competitive inhibitor of InsP3-induced calcium release, we show that the activation wave induced by the sperm is sufficient to induce extrusion of the first polar body, but that additional [Ca2+]i increases are necessary for completion of the second meiotic division. However, periodic calcium waves per se do not seem to be strictly necessary for the completion of meiosis, as a persistent and homogeneous increase in calcium, induced by the calcium ionophore ionomycin, is sufficient to cause second polar body formation and allow completion of meiosis on time.nnnCONCLUSIONnThese results clearly show that, in the ascidian egg, post-activation calcium waves are required to complete meiosis. They also show that following a period of progressive relocalization of the wave origin, which lasts approximately five minutes, an ER-rich domain at the contraction pole finally becomes a pacemaker from which the calcium waves originate. Once their origin becomes stably localized, the calcium waves begin to propagate preferentially around the cortex of the egg rather than throughout the egg cytoplasm.

Tracing of cell lineage in embryonic development of Phallusia mammillata (ascidia) by vital staining of mitochondria

Vital staining of mitochondria with a fluorescent dye 3,3-diethyloxacarbocyanine was used to follow cell lineage in embryos of Phallusia mammillata. The results agree in general with the plan established by Conklin in 1905. Strong fluorescence migrated after fertilization similarly to the pigment of the yellow crescent in Styela. Later, fluorescence segregated into muscle cell primordia, but not into mesenchyme cells. An animal hemisphere cell, b 8.17 also exhibited strong fluorescence and joined a group of muscle primordia, very likely becoming a muscle cell itself. In the tadpole, all the tail muscle cells were fluorescent. Fluorescence was also noticed in nerve cell primordia of the vegetal hemisphere, particularly in the cell A 8.16 whose descendants appeared to become part of the sensory vesicle which was strongly fluorescent in the tadpole. The usefulness of this type of vital staining in following cell lineage of colorless embryos is stressed.

Spatial expression of the hatching enzyme gene in the sea urchin embryo

The sea urchin embryo at the blastula stage hatches from its protective fertilization envelope which is degraded by a secreted protease, the hatching enzyme. We have previously purified the hatching enzyme from Paracentrotus lividus (Lepage and Gache (1989). J. Biol. Chem. 264, 4787-4793), cloned its cDNA, and analyzed the temporal expression of its gene (Lepage and Gache (1990). EMBO J. 9, 3003-3012). We study here the temporal and spatial expression of the hatching enzyme gene in whole embryos by immunolabeling with an affinity-purified polyclonal antibody and by in situ hybridization using nonradioactive RNA probes. The timing of expression is consistent with our data on the activation of the gene, the mRNA accumulation in the blastula, and the role of the enzyme. Immunolabeling was observed only in blastula stage embryos; neither before the 128-cell stage nor after hatching. The distribution of the enzyme varies with time from a diffuse labeling around the nucleus to a punctate localization between the nucleus and the apical face of the blastomeres, and finally at the time of hatching, to a submembranous apical location. Not all the cells of an embryo are labeled. The presence of the hatching enzyme is restricted to a sharply delimited continuous territory spanning about two-thirds of the blastula. The orientation of this territory has been determined with respect to the animal-vegetal axis of the embryo using as a landmark the subequatorial pigmented band of the P. lividus species. The synthesis of the hatching enzyme only takes place in the animal-most two-thirds of the blastula. By in situ hybridization, the mRNA coding for the hatching enzyme is only detected in early blastulas, in a limited area having the same size and shape as the territory in which the protein is found. Thus the hatching enzyme gene is likely to be spatially controlled at the transcriptional level: its expression is restricted to a region of the blastula that corresponds roughly to the presumptive ectoderm territory. To date, the hatching enzyme gene products constitute the earliest molecular markers of the sea urchin embryo spatial organization along the primordial egg axis.

Kinesin II mediates Vg1 mRNA transport in Xenopus oocytes

The subcellular localization of specific mRNAs is a widespread mechanism for regulating gene expression. In Xenopus oocytes microtubules are required for localization of Vg1 mRNA to the vegetal cortex during the late RNA localization pathway. The factors that mediate microtubule-based RNA transport during the late pathway have been elusive. Here we show that heterotrimeric kinesin II becomes enriched at the vegetal cortex of stage III/IV Xenopus oocytes concomitant with the localization of endogenous Vg1 mRNA. In addition, expression of a dominant negative mutant peptide fragment or injection of a function-blocking antibody, both of which impair the function of heterotrimeric kinesin II, block localization of Vg1 mRNA. We also show that exogenous Vg1 RNA or Xcat-2, another RNA that can use the late pathway, recruits endogenous kinesin II to the vegetal pole and colocalizes with it at the cortex. These data support a model in which kinesin II mediates the transport of specific RNA complexes destined for the vegetal cortex.

Mitochondrial respiration and Ca2+ waves are linked during fertilization and meiosis completion

Fertilization increases both cytosolic Ca2+ concentration and oxygen consumption in the egg but the relationship between these two phenomena remains largely obscure. We have measured mitochondrial oxygen consumption and the mitochondrial NADH concentration on single ascidian eggs and found that they increase in phase with each series of meiotic Ca2+ waves emitted by two pacemakers (PM1 and PM2). Oxygen consumption also increases in response to Ins(1,4,5)P3-induced Ca2+ transients. Using mitochondrial inhibitors we show that active mitochondria sequester cytosolic Ca2+ during sperm-triggered Ca2+ waves and that they are strictly necessary for triggering and sustaining the activity of the meiotic Ca2+ wave pacemaker PM2. Strikingly, the activity of the Ca2+ wave pacemaker PM2 can be restored or stimulated by flash photolysis of caged ATP. Taken together our observations provide the first evidence that, in addition to buffering cytosolic Ca2+, the eggs mitochondria are stimulated by Ins(1,4,5)P3-mediated Ca2+ signals. In turn, mitochondrial ATP production is required to sustain the activity of the meiotic Ca2+ wave pacemaker PM2.

Maternal mRNAs of PEM and macho 1, the ascidian muscle determinant, associate and move with a rough endoplasmic reticulum network in the egg cortex.

Localization of maternal mRNAs in the egg cortex is an essential feature of polarity in embryos of Drosophila, Xenopus and ascidians. In ascidians, maternal mRNAs such as macho 1, a determinant of primary muscle-cell fate, belong to a class of postplasmic RNAs that are located along the animal-vegetal gradient in the egg cortex. Between fertilization and cleavage, these postplasmic RNAs relocate in two main phases. They further concentrate and segregate in small posterior blastomeres into a cortical structure, the centrosome-attracting body (CAB), which is responsible for unequal cleavages. By using high-resolution, fluorescent, in situ hybridization in eggs, zygotes and embryos of Halocynthia roretzi, we showed that macho 1 and HrPEM are localized on a reticulated structure situated within 2 μm of the surface of the unfertilized egg, and within 8 μm of the surface the vegetal region and then posterior region of the zygote. By isolating cortices from eggs and zygotes we demonstrated that this reticulated structure is a network of cortical rough endoplasmic reticulum (cER) that is tethered to the plasma membrane. The postplasmic RNAs macho 1 and HrPEM were located on the cER network and could be detached from it. We also show that macho 1 and HrPEM accumulated in the CAB and the cER network. We propose that these postplasmic RNAs relocalized after fertilization by following the microfilament- and microtubule-driven translocations of the cER network to the poles of the zygote. We also suggest that the RNAs segregate and concentrate in posterior blastomeres through compaction of the cER to form the CAB. A multimedia BioClip `Polarity inside the egg cortex tells the story and can be downloaded at www.bioclips.com/bioclip.html

The aPKC-PAR-6-PAR-3 cell polarity complex localizes to the centrosome attracting body, a macroscopic cortical structure responsible for asymmetric divisions in the early ascidian embryo

Posterior blastomeres of 8-cell stage ascidian embryos undergo a series of asymmetric divisions that generate cells of unequal sizes and segregate muscle from germ cell fates. These divisions are orchestrated by a macroscopic cortical structure, the `centrosome attracting body (CAB) which controls spindle positioning and distribution of mRNA determinants. The CAB is composed of a mass of cortical endoplasmic reticulum containing mRNAs (the cER-mRNA domain) and an electron dense matrix, but little is known about its precise structure and functions. We have examined the ascidian homologues of PAR proteins, known to regulate polarity in many cell types. We found that aPKC, PAR-6 and PAR-3 proteins, but not their mRNAs, localize to the CAB during the series of asymmetric divisions. Surface particles rich in aPKC concentrate in the CAB at the level of cortical actin microfilaments and form a localized patch sandwiched between the plasma membrane and the cER-mRNA domain. Localization of aPKC to the CAB is dependent on actin but not microtubules. Both the aPKC layer and cER-mRNA domain adhere to cortical fragments prepared from 8-cell stage embryos. Astral microtubules emanating from the proximal centrosome contact the aPKC-rich cortical domain. Our observations indicate that asymmetric division involves the accumulation of the aPKC–PAR-6–PAR-3 complex at the cortical position beneath the pre-existing cER-mRNA domain.

From Oocyte to 16-Cell Stage: Cytoplasmic and Cortical Reorganizations That Pattern the Ascidian Embryo

The dorsoventral and anteroposterior axes of the ascidian embryo are defined before first cleavage by means of a series of reorganizations that reposition cytoplasmic and cortical domains established during oogenesis. These domains situated in the periphery of the oocyte contain developmental determinants and a population of maternal postplasmic/PEM RNAs. One of these RNAs (macho‐1) is a determinant for the muscle cells of the tadpole embryo. Oocytes acquire a primary animal–vegetal (a‐v) axis during meiotic maturation, when a subcortical mitochondria‐rich domain (myoplasm) and a domain rich in cortical endoplasmic reticulum (cER) and maternal postplasmic/PEM RNAs (cER‐mRNA domain) become polarized and asymmetrically enriched in the vegetal hemisphere. Fertilization at metaphase of meiosis I initiates a series of dramatic cytoplasmic and cortical reorganizations of the zygote, which occur in two major phases. The first major phase depends on sperm entry which triggers a calcium wave leading in turn to an actomyosin‐driven contraction wave. The contraction concentrates the cER‐mRNA domain and myoplasm in and around a vegetal/contraction pole. The precise localization of the vegetal/contraction pole depends on both the a‐v axis and the location of sperm entry and prefigures the future site of gastrulation and dorsal side of the embryo. The second major phase of reorganization occurs between meiosis completion and first cleavage. Sperm aster microtubules and then cortical microfilaments cause the cER‐mRNA domain and myoplasm to reposition toward the posterior of the zygote. The location of the posterior pole depends on the localization of the sperm centrosome/aster attained during the first major phase of reorganization. Both cER‐mRNA and myoplasm domains localized in the posterior region are partitioned equally between the first two blastomeres and then asymmetrically over the next two cleavages. At the eight‐cell stage the cER‐mRNA domain compacts and gives rise to a macroscopic cortical structure called the Centrosome Attracting Body (CAB). The CAB is responsible for a series of unequal divisions in posterior–vegetal blastomeres, and the postplasmic/PEM RNAs it contains are involved in patterning the posterior region of the embryo. In this review, we discuss these multiple events and phases of reorganizations in detail and their relationship to physiological, cell cycle, and cytoskeletal events. We also examine the role of the reorganizations in localizing determinants, postplasmic/PEM RNAs, and PAR polarity proteins in the cortex. Finally, we summarize some of the remaining questions concerning polarization of the ascidian embryo and provide comparisons to a few other species. A large collection of films illustrating the reorganizations can be consulted by clicking on “Film archive: ascidian eggs and embryos” at http://biodev.obs‐vlfr.fr/recherche/biomarcell/. Developmental Dynamics 236:1716–1731, 2007.

Fertilization and early development in Beroe ovata

Fertilization in the clear egg (1 mm in diameter) of the ctenophore Beroe ovata and, in particular, the positioning and movements of pronuclei, and their relationship to the larval oral-aboral axis have been observed. Fertilization can take place anywhere on the egg surface. The sperm pronucleus remains at its entry site and becomes surrounded by a specialized zone (30-50 micron in diameter) beneath the surface referred to as the sperm pronuclear zone or SPZ and devoid of large cortical granules. Polyspermy has been observed to be frequent; each pronucleus is surrounded by its own SPZ. Only the egg pronucleus migrates with a continuous velocity (averaging 18 micron/min) and moves beneath the surface directly toward the immobile sperm pronucleus. In polyspermic eggs, the egg pronucleus can probe several SPZ, each containing a single sperm nucleus, before it finally enters one SPZ and fuses with the chosen sperm pronucleus. These migrations of the egg pronucleus occur over several millimeters and take hours, but the mechanism underlying the motion or how the egg pronucleus decides which SPZ to enter is not yet known. Under our experimental conditions the mitotic apparatus and the first cleavage plane which defines the oral-aboral axis of the larva (see Reverberi (1971). Experimental Embryology of Marine and Fresh-Water Invertebrates. North-Holland, Amsterdam. for review) pass through the point of sperm entry. During fertilization and cleavage, movements of a cortical autofluorescent material are clearly seen. This material is segregated into micromeres as cleavage progresses.