A holistic approach to marine eco-systems biology.

The structure, robustness, and dynamics of ocean plankton ecosystems remain poorly understood due to sampling, analysis, and computational limitations. The Tara Oceans consortium organizes expeditions to help fill this gap at the global level.

Dual mechanism controls asymmetric spindle position in ascidian germ cell precursors.

Mitotic spindle orientation with respect to cortical polarity cues generates molecularly distinct daughter cells during asymmetric cell division (ACD). However, during ACD it remains unknown how the orientation of the mitotic spindle is regulated by cortical polarity cues until furrowing begins. In ascidians, the cortical centrosome-attracting body (CAB) generates three successive unequal cleavages and the asymmetric segregation of 40 localized postplasmic/PEM RNAs in germ cell precursors from the 8-64 cell stage. By combining fast 4D confocal fluorescence imaging with gene-silencing and classical blastomere isolation experiments, we show that spindle repositioning mechanisms are active from prometaphase until anaphase, when furrowing is initiated in B5.2 cells. We show that the vegetal-most spindle pole/centrosome is attracted towards the CAB during prometaphase, causing the spindle to position asymmetrically near the cortex. Next, during anaphase, the opposite spindle pole/centrosome is attracted towards the border with neighbouring B5.1 blastomeres, causing the spindle to rotate (10 degrees /minute) and migrate (3 microm/minute). Dynamic 4D fluorescence imaging of filamentous actin and plasma membrane shows that precise orientation of the cleavage furrow is determined by this second phase of rotational spindle displacement. Furthermore, in pairs of isolated B5.2 blastomeres, the second phase of rotational spindle displacement was lost. Finally, knockdown of PEM1, a protein localized in the CAB and required for unequal cleavage in B5.2 cells, completely randomizes spindle orientation. Together these data show that two separate mechanisms active during mitosis are responsible for spindle positioning, leading to precise orientation of the cleavage furrow during ACD in the cells that give rise to the germ lineage in ascidians.

Bi-polarized translation of ascidian maternal mRNA determinant pem-1 associated with regulators of the translation machinery on cortical Endoplasmic Reticulum (cER).

Polarized cortical mRNA determinants such as maternal macho-1 and pem-1 in ascidians, like budding yeast mating factor ASH1 reside on the cER-mRNA domain a subdomain of cortical Endoplasmic Reticulum(ER) and are translated in its vicinity. Using high resolution imaging and isolated cortical fragments prepared from eggs and embryos we now find that macho-1 and pem-1 RNAs co-localize with phospho-protein regulators of translation initiation (MnK/4EBP/S6K). Translation of cortical pem-1 RNA follows its bi-polarized relocalization. About 10 min after fertilization or artificial activation with a calcium ionophore, PEM1 protein is detected in the vegetal cortex in the vicinity of pem-1 RNA. About 40 min after fertilization-when pem-1 RNA and P-MnK move to the posterior pole-PEM1 protein remains in place forming a network of cortical patches anchored at the level of the zygote plasma membrane before disappearing. Cortical PEM1 protein is detected again at the 4 cell stage in the posterior centrosome attracting body (CAB) region where the cER-mRNA domain harboring pem-1/P-MnK/P-4EBP/P-S6K is concentrated. Bi-polarized PEM1 protein signals are not detected when pem-1 morpholinos are injected into eggs or zygotes or when MnK is inhibited. We propose that localized translation of the pem-1 RNA determinant is triggered by the fertilization/calcium wave and that the process is controlled by phospho-protein regulators of translation initiation co-localized with the RNA determinant on a sub-domain of the cortical Endoplasmic Reticulum.

Cortical anchorages and cell type segregations of maternal postplasmic/PEM RNAs in ascidians.

Ascidian postplasmic/PEM RNAs constitute a large class of cortical maternal RNAs which include developmental determinants (macho-1 and pem-1). We have analyzed the localization, cortical anchorage and cell type segregation of postplasmic/PEM RNAs in Ciona intestinalis and Phallusia mammillata using very high-resolution fluorescent in situ hybridization. We also compared RNAs extracted from whole oocytes and from isolated cortices using microarrays and localized RNAs possessing clusters of xCACx motifs in their 3'UTRs. Based on these combined approaches we conclude that: (1) the vast majority of the 39 postplasmic/PEM RNAs (including vasa) are localized in the egg cortex. (2) Many postplasmic/PEM RNAs 3'UTR are enriched in xCACx motifs, allowing us to identify 2 novel postplasmic/PEM RNAs (PSD and MnK). (3) Postplasmic/PEM RNAs anchored to cortical Endoplasmic Reticulum (cER) and those associated with granules have different cell destinations. We propose that there are 2 distinct categories of postplasmic/PEM RNAs on the basis of their cortical anchorages and cell destinations: (1) macho-1-like postplasmic/PEM RNAs anchored to cER which segregate into somatic B8.11 cells. (2) vasa-like postplasmic/PEM RNAs associated with granules which in addition to B8.11 cells segregate into B8.12 germ cells.

Cortical and cytoplasmic flows driven by actin microfilaments polarize the cortical ER-mRNA domain along the a-v axis in ascidian oocytes.

Cellular mechanisms generating the polarized redistribution of maternal Type I postplasmic/PEM mRNAs in ascidian oocytes remain unknown. We have previously shown that PEM-1 mRNA is associated with a network of rough cortical Endoplasmic Reticulum (cER) polarized along the animal-vegetal (a-v) axis forming a cER-mRNA domain in mature oocytes. We now investigate the a-v polarization of this cER-mRNA domain during meiotic maturation using H. roretzi and C. intestinalis. We show that the cER and Hr-PEM-1 aggregate as interconnected cortical patches at the cell periphery before maturation, which uniformly spread out during maturation and form a reticulated organization enriched in the vegetal hemisphere at the end of maturation. Time-lapse video recordings coupled with micromanipulations reveal that stereotyped surface, cortical and cytoplasmic flows accompany the vegetal shift of the cER-mRNA domain and mitochondria-rich myoplasm. Treatments with cytochalasin B and nocodazole indicate that both polarization of the cER-mRNA domain and mitochondria-rich myoplasm and cortical and cytoplasmic flows depend on actin cytoskeleton, but not microtubules. Using cortical fragments prepared from maturing oocytes coupled with high resolution immuno/in situ localization, we have further analyzed the effects of these inhibitors on the reorganizations the cER network and Hr-PEM-1 mRNA. We show that before maturation starts, Hr-PEM-1 mRNAs are already associated with the cER, and actin cytoskeleton inhibitors disturb their association. Finally, we hypothesize that Germinal Vesicle Break Down (GVBD) triggers an actomyosin-dependent cortical flow which directs the a-v polarization of ascidian oocytes.

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.

Comprehensive model of annual plankton succession based on the whole-plankton time series approach.

Ecological succession provides a widely accepted description of seasonal changes in phytoplankton and mesozooplankton assemblages in the natural environment, but concurrent changes in smaller (i.e. microbes) and larger (i.e. macroplankton) organisms are not included in the model because plankton ranging from bacteria to jellies are seldom sampled and analyzed simultaneously. Here we studied, for the first time in the aquatic literature, the succession of marine plankton in the whole-plankton assemblage that spanned 5 orders of magnitude in size from microbes to macroplankton predators (not including fish or fish larvae, for which no consistent data were available). Samples were collected in the northwestern Mediterranean Sea (Bay of Villefranche) weekly during 10 months. Simultaneously collected samples were analyzed by flow cytometry, inverse microscopy, FlowCam, and ZooScan. The whole-plankton assemblage underwent sharp reorganizations that corresponded to bottom-up events of vertical mixing in the water-column, and its development was top-down controlled by large gelatinous filter feeders and predators. Based on the results provided by our novel whole-plankton assemblage approach, we propose a new comprehensive conceptual model of the annual plankton succession (i.e. whole plankton model) characterized by both stepwise stacking of four broad trophic communities from early spring through summer, which is a new concept, and progressive replacement of ecological plankton categories within the different trophic communities, as recognised traditionally.

[Polarization of eggs and embryos: some common principles]. / Polarisation des oeufs et des embryons: principes communs.

Embryonic development depends on the establishment of polarities which define the axial characteristics of the body. In a small number of cases such as the embryo of the fly drosophila, developmental axes are established well before fertilization while in other organisms such as the nematode worm C. elegans these axes are set up only after fertilization. In most organisms the egg posesses a primary (A-V, Animal-Vegetal) axis acquired during oogenesis which participates in the establishment of the embryonic axes. Such is the case for the eggs of ascidians or the frog Xenopus whose AV axes are remodelled by sperm entry to yield the embryonic axes. Embryos of different species thus acquire an anterior end and a posterior end (Antero-Posterior, A-P axis), dorsal and ventral sides (D-V axis) and then a left and a right side.

[Establishment and expression of embryonic axes: comparisons between different model organisms]. / Etablissement et expression des axes embryonnaires : comparaisons entre différents organismes modèles.

In an accompanying article (C. Sardet et al. m/s 2004; 20 : 414-423) we reviewed determinants of polarity in early development and the mechanisms which regulate their localization and expression. Such determinants have for the moment been identified in only a few species: the insect Drosophila melanogaster, the worm Caenorhabditis elegans, the frog Xenopus laevis and the ascidians Ciona intestinalis and Holocynthia roretzi. Although oogenesis, fertilization, and cell divisions in these embryos differ considerably, with respect to early polarities certain common themes emerge, such as the importance of cortical mRNAs, the PAR polarity proteins, and reorganizations mediated by the cytoskeleton. Here we highlight similarities and differences in axis establishment between these species, describing them in a chronological order from oocyte to gastrula, and add two more classical model organisms, sea urchin and mouse, to complete the comparisons depicted in the form of a Poster which can be downloaded from the site http://biodev.obs-vlfr.fr/biomarcell.

Purification of mitochondrial proteins HSP60 and ATP synthase from ascidian eggs: implications for antibody specificity.

Use of antibodies is a cornerstone of biological studies and it is important to identify the recognized protein with certainty. Generally an antibody is considered specific if it labels a single band of the expected size in the tissue of interest, or has a strong affinity for the antigen produced in a heterologous system. The identity of the antibody target protein is rarely confirmed by purification and sequencing, however in many cases this may be necessary. In this study we sought to characterize the myoplasm, a mitochondria-rich domain present in eggs and segregated into tadpole muscle cells of ascidians (urochordates). The targeted proteins of two antibodies that label the myoplasm were purified using both classic immunoaffinity methods and a novel protein purification scheme based on sequential ion exchange chromatography followed by two-dimensional gel electrophoresis. Surprisingly, mass spectrometry sequencing revealed that in both cases the proteins recognized are unrelated to the original antigens. NN18, a monoclonal antibody which was raised against porcine spinal cord and recognizes the NF-M neurofilament subunit in vertebrates, in fact labels mitochondrial ATP synthase in the ascidian embryo. PMF-C13, an antibody we raised to and purified against PmMRF, which is the MyoD homolog of the ascidian Phallusia mammillata, in fact recognizes mitochondrial HSP60. High resolution immunolabeling on whole embryos and isolated cortices demonstrates localization to the inner mitochondrial membrane for both ATP synthase and HSP60. We discuss the general implications of our results for antibody specificity and the verification methods which can be used to determine unequivocally an antibody's target.

Localization and anchorage of maternal mRNAs to cortical structures of ascidian eggs and embryos using high resolution in situ hybridization.

In several species, axis formation and tissue differentiation are the result of developmental cascades which begin with the localization and translation of key maternal mRNAs in eggs. Localization and anchoring of mRNAs to cortical structures can be observed with high sensitivity and resolution by fluorescent in situ hybridization coupled with labeling of membranes and macromolecular complexes. Oocytes and embryos of ascidians--marine chordates closely related to vertebrates--are ideal models to understand how maternal mRNAs pattern the simple ascidian tadpole. More than three dozen cortically localized maternal mRNAs have been identified in ascidian eggs. They include germ cell markers such as vasa or pem-3 and determinants of axis (pem-1), unequal cleavage (pem-1), and muscle cells (macho-1). High resolution localization of mRNAs, proteins, and lipids in whole eggs and embryos and their cortical fragments shows that maternal mRNA determinants pem-1 and macho-1 are anchored to cortical endoplasmic reticulum and segregate with it into small posterior somatic cells. In contrast, mRNAs such as vasa are associated with granular structures which are inherited by the same somatic cells plus adjacent germ cell precursors. In this chapter, we provide detailed protocols for simultaneous localization of mRNAs and proteins to determine their association with cellular structures in eggs and embryos. Using preparations of isolated cortical fragments with intact membranous structures allows unprecedented high resolution analysis and identification of cellular anchoring sites for key mRNAs. This information is necessary for understanding the mechanisms for localizing mRNAs and partitioning them into daughter cells after cleavage.

Atypical protein kinase C controls sea urchin ciliogenesis.

The atypical protein kinase C (aPKC) is part of the conserved aPKC/PAR6/PAR3 protein complex, which regulates many cell polarity events, including the formation of a primary cilium at the apical surface of epithelial cells. Cilia are highly organized, conserved, microtubule-based structures involved in motility, sensory processes, signaling, and cell polarity. We examined the distribution and function of aPKC in the sea urchin embryo, which forms a swimming blastula covered with motile cilia. We found that in the early embryo aPKC is uniformly cortical and becomes excluded from the vegetal pole during unequal cleavages at the 8- to 64-cell stages. During the blastula and gastrula stages the kinase localizes at the base of cilia, forming a ring at the transition zone between the basal body and the elongating axoneme. A dose-dependent and reversible inhibition of aPKC results in mislocalization of the kinase, defective ciliogenesis, and lack of swimming. Thus, as in the primary cilium of differentiated mammalian cells, aPKC controls the growth of motile cilia in invertebrate embryos. We suggest that aPKC might function to phosphorylate kinesin and so activate the transport of intraflagellar vesicles.

Embryological methods in ascidians: the Villefranche-sur-Mer protocols.

Ascidians (marine invertebrates: urochordates) are thought to be the closest sister groups of vertebrates. They are particularly attractive models because of their non-duplicated genome and the fast and synchronous development of large populations of eggs into simple tadpoles made of about 3,000 cells. As a result of stereotyped asymmetric cleavage patterns all blastomeres become fate restricted between the 16- and 110 cell stage through inheritance of maternal determinants and/or cellular interactions. These advantageous features have allowed advances in our understanding of the nature and role of maternal determinants, inductive interactions, and gene networks that are involved in cell lineage specification and differentiation of embryonic tissues. Ascidians have also contributed to our understanding of fertilization, cell cycle control, self-recognition, metamorphosis, and regeneration. In this chapter we provide basic protocols routinely used at the marine station in Villefranche-sur-Mer using the cosmopolitan species of reference Ciona intestinalis and the European species Phallusia mammillata. These two models present complementary advantages with regard to molecular, functional, and imaging approaches. We describe techniques for basic culture of embryos, micro-injection, in vivo labelling, micro-manipulations, fixation, and immuno-labelling. These methods allow analysis of calcium signals, reorganizations of cytoplasmic and cortical domains, meiotic and mitotic cell cycle and cleavages as well as the roles of specific genes and cellular interactions. Ascidians eggs and embryos are also an ideal material to isolate cortical fragments and to isolate and re-associate individual blastomeres. We detail the experimental manipulations which we have used to understand the structure and role of the egg cortex and of specific blastomeres during development.

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/.

Establishment of animal-vegetal polarity during maturation in ascidian oocytes.

Mature ascidian oocytes are arrested in metaphase of meiosis I (Met I) and display a pronounced animal-vegetal polarity: a small meiotic spindle lies beneath the animal pole, and two adjacent cortical and subcortical domains respectively rich in cortical endoplasmic reticulum and postplasmic/PEM RNAs (cER/mRNA domain) and mitochondria (myoplasm domain) line the equatorial and vegetal regions. Symmetry-breaking events triggered by the fertilizing sperm remodel this primary animal-vegetal (a-v) axis to establish the embryonic (D-V, A-P) axes. To understand how this radial a-v polarity of eggs is established, we have analyzed the distribution of mitochondria, mRNAs, microtubules and chromosomes in pre-vitellogenic, vitellogenic and post-vitellogenic Germinal Vesicle (GV) stage oocytes and in spontaneously maturing oocytes of the ascidian Ciona intestinalis. We show that myoplasm and postplasmic/PEM RNAs move into the oocyte periphery at the end of oogenesis and that polarization along the a-v axis occurs after maturation in several steps which take 3-4 h to be completed. First, the Germinal Vesicle breaks down, and a meiotic spindle forms in the center of the oocyte. Second, the meiotic spindle moves in an apparently random direction towards the cortex. Third, when the microtubular spindle and chromosomes arrive and rotate in the cortex (defining the animal pole), the subcortical myoplasm domain and cortical postplasmic/PEM RNAs are excluded from the animal pole region, thus concentrating in the vegetal hemisphere. The actin cytoskeleton is required for migration of the spindle and subsequent polarization, whereas these events occur normally in the absence of microtubules. Our observations set the stage for understanding the mechanisms governing primary axis establishment and meiotic maturation in ascidians.

Calcium signals and mitochondria at fertilisation.

At fertilisation, Ca(2+) signals activate embryonic development by stimulating metabolism, exocytosis and endocytosis, cytoskeletal remodelling, meiotic resumption and recruitment of maternal RNAs. Mitochondria present in large number in eggs have long been thought to act as a relay in Ca(2+) signalling at fertilisation. However, only recently have studies on ascidians and mouse proven that sperm-triggered Ca(2+) waves are transduced into mitochondrial Ca(2+) signals that stimulate mitochondrial respiration. Mitochondrial Ca(2+) uptake can substantially buffer cytosolic Ca(2+) concentration and the concerted action of heterogeneously distributed mitochondria in the mature egg may modulate the spatiotemporal pattern of sperm-triggered Ca(2+) waves. Regulation of fertilisation Ca(2+) signals could also be achieved through mitochondrial ATP production and mitochondrial oxidant activity but these hypotheses remain to be explored. A critically poised dynamic interplay between Ca(2+) signals and mitochondrial metabolism is stimulated at fertilisation and may well determine whether the embryo can proceed further into development. The monitoring of Ca(2+) signals and mitochondrial activity during fertilisation in living zygotes of diverse species should confirm the universality of the role for sperm-triggered Ca(2+) waves in the activation of mitochondrial activity at fertilisation.

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.

Maternal determinants and mRNAs in the cortex of ascidian oocytes, zygotes and embryos.

The peripheral region of ascidian oocytes and zygotes contains five determinants for morphogenesis and differentiation of the embryo. The determinant for the 24 primary muscle cells of the tadpole, macho1, is one of several cortical mRNAs localized in a gradient along the animal-vegetal axis in the oocyte. After fertilization these mRNAs, together with cortical endoplasmic reticulum (cER) and a subcortical mitochondria-rich domain (myoplasm), relocate in two major reorganization phases forming the posterior plasm (postplasm) of the zygote. At the 8-cell stage cortical mRNAs concentrate in a macroscopic cortical structure called the centrosome-attracting body (CAB), forming a characteristic posterior end mark (PEM) in the two posterior vegetal blastomeres. We propose to call the numerous mRNAs showing this particular cortical localization in the posterior region of the embryo postplasmic/PEM RNAs and suggest a nomemclature. We do not know how postplasmic/PEM RNAs reach their polarized distribution in the oocyte cortex but at least PEM1 and macho1 (and probably others) bind to the network of cER retained in isolated cortical fragments. We propose that after fertilization, these postplasmic/PEM mRNAs move in the zygote cortex together with the cER network (cER/mRNA domain) via microfilament- and microtubule-driven translocations. The cER/mRNA domain is localized posteriorly at the time of first cleavage and distributed equally between the first two blastomeres. After the third cleavage, the cER/mRNA domain and dense particles compact to form the CAB in posterior vegetal blastomeres of the 8-cell stage. We discuss the identity of postplasmic/PEM RNAs, how they localize, anchor, relocate and may be translated. We also examine their roles in unequal cleavage and as a source of posterior morphogenetic and differentiation factors.

Polarity of the ascidian egg cortex and relocalization of cER and mRNAs in the early embryo.

The mature ascidian oocyte is a large cell containing cytoplasmic and cortical domains polarized along a primary animal-vegetal (a-v) axis. The oocyte cortex is characterized by a gradient distribution of a submembrane monolayer of cortical rough endoplasmic reticulum (cER) and associated maternal postplasmic/PEM mRNAs (cER-mRNA domain). Between fertilization and first cleavage, this cER-mRNA domain is first concentrated vegetally and then relocated towards the posterior pole via microfilament-driven cortical contractions and spermaster-microtubule-driven translocations. The cER-mRNA domain further concentrates in a macroscopic cortical structure called the centrosome attracting body (CAB), which mediates a series of asymmetric divisions starting at the eight-cell stage. This results in the segregation of determinant mRNAs and their products in posterior cells of the embryo precursors of the muscle and germ line. Using two species of ascidians (Ciona intestinalis and Phallusia mammillata), we have pursued and amplified the work initiated in Halocynthia roretzi. We have analysed the cortical reorganizations in whole cells and in cortical fragments isolated from oocytes and from synchronously developing zygotes and embryos. After fertilization, we observe that a cortical patch rich in microfilaments encircles the cER-mRNA domain, concentrated into a cortical cap at the vegetal/contraction pole (indicating the future dorsal pole). Isolated cortices also retain microtubule asters rich in cER (indicating the future posterior pole). Before mitosis, parts of the cER-mRNA domain are detected, together with short microtubules, in isolated posterior (but not anterior) cortices. At the eight-cell stage, the posteriorly located cER-mRNA domain undergoes a cell-cycle-dependant compaction into the CAB. The CAB with embedded centrosomal microtubules can be isolated with cortical fragments from eight-cell-stage embryos. These and previous observations indicate that cytoskeleton-driven repositioning and compaction of a polarized cortical domain made of rough ER is a conserved mechanism used for polarization and segregation of cortical maternal mRNAs in embryos of evolutionarily distant species of ascidians.

Formation of a large Vasa-positive germ granule and its inheritance by germ cells in the enigmatic Chaetognaths.

Chaetognaths (arrow worms) are abundant hermaphrodite marine organisms whose phylogenetic position amongst protostomes and deuterostomes is still debated. Ancient histological observations dating from a century ago described the presence in eggs of a large granule, presumed to be a germ plasm, and its probable inheritance in four primary germ cells (PGCs). Using videomicroscopy, electron microscopy and immunocytochemistry (labelling with anti-Vasa antibodies) we have followed the cycle of aggregation and dispersion of germ plasm and nuage material in eggs, embryos, PGCs and oocytes in several species of benthic (Spadella) and planctonic (Sagitta) chaetognaths. In these animals, germ cells and gametes can be observed in vivo throughout the 1-2 month life cycle. After describing internal fertilization in live animals we show that the single large (15 microm diameter) germ granule forms by a spiralling aggregation movement of small germ islands situated in the vegetal cortex at the time of first mitosis. We also demonstrate that the granule forms autonomously in unfertilized activated eggs or fertilized egg fragments. Once formed, the germ granule first associates with the cleavage furrow and is segregated into one of the first two blastomeres. The germ granule is then translocated from the cortex to the mitotic spindle during 3(rd) cleavage and remains in the single most-vegetal blastomere until the 32-cell stage. At the 64-cell stage the germ granule is partitioned as nuage material into two founder PGCs and further partitioned into four PGCs situated at the tip of the archenteron during gastrulation. These four PGCs migrate without dividing to reach the transverse septum, then proliferate and differentiate into oocytes and spermatocytes of two ovaries and two testes. We noted that germ plasm and nuage material were associated with mitochondria, the nucleus, the spindle and the centrosome during some stages of development and differentiation of the germ line. Finally, we demonstrate that a Vasa-like protein is present in the germ granule, in PGCs and in the electron-dense material associated with the germinal vesicle of oocytes. These features stress the conservation of cellular and molecular mechanisms involved in germ cell determination.