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The morphogenesis of tissues and embryos results from a tight interplay between gene expression, biochemical signaling and mechanics. Although sequencing methods allow the generation of cell-resolved spatio-temporal maps of gene expression in developing tissues, creating similar maps of cell mechanics in 3D has remained a real challenge. Exploiting the foam-like geometry of cells in embryos, we propose a robust end-to-end computational method to infer spatiotemporal atlases of cellular forces from fluorescence microscopy images of cell membranes. Our method generates precise 3D meshes of cell geometry and successively predicts relative cell surface tensions and pressures in the tissue. We validate it with 3D active foam simulations, study its noise sensitivity, and prove its biological relevance in mouse, ascidian andC. elegansembryos. 3D inference allows us to recover mechanical features identified previously, but also predicts new ones, unveiling potential new insights on the spatiotemporal regulation of cell mechanics in early embryos. Our code is freely available and paves the way for unraveling the unknown mechanochemical feedbacks that control embryo and tissue morphogenesis.

Although it has been studied for more than a century, the question of how one cell divides into two equal parts is still not fully resolved. Zygotes have provided much of the mechanistic insight into how the mitotic apparatus finds the center of the cell since the centrally-located mitotic apparatus is created from a large sperm aster that forms at the cortex and thus far from the zygote center. Here we show that in ascidians, the sperm aster extends throughout the cytoplasm during interphase yet remains located near the cortex and does not migrate towards the zygote center. It is only at mitotic entry, when the sperm aster has duplicated and the mitotic apparatus is being assembled, that most of the migration and centration occurs. This temporal pattern of centration behavior is mirrored by primate zygotes (including human). The current mechanisms of aster centration include cytoplasmic pulling that scale with microtubule (MT) length, MT pushing against the proximal cortex or MT-based cortical pulling. However, it is not yet known whether and how these 3 mechanisms are coordinated to prevent aster migration during interphase and trigger migration at mitotic entry. By monitoring quantitatively all three mechanisms (cytoplasmic pulling, pushing and cortical pulling) we have discovered that cortical pulling is switched off as the zygote enters mitosis while both cytoplasmic pulling and proximal cortical pushing remain active. Physical simulations could recapitulate both the static and migratory aspects of sperm aster and mitotic apparatus behavior. We therefore surmise that the reduction in cortical pulling at mitotic entry represents a switch that allows proximal cortical pushing forces and cytoplasmic pulling forces to center the nascent mitotic apparatus.Graphical abstractHighlightsSperm aster/mitotic apparatus centration occurs at entry into first mitosisMT-based cortical pulling is active during interphase and switched off at mitotic entryLoss of cortical pulling at mitosis entry facilitates centration of the asterMT-based cytoplasmic pulling is active during both interphase and mitosisAgent-based simulations advocate the need for cytoplasmic pulling, a switch in cortical pulling and a minor role of pushing for aster centration at mitotic entry.

Predicting the potential for species adaption to climate change is challenged by the need to identify the physiological mechanisms that underpin species vulnerability. Here, we investigated the sensitivity to ocean acidification in marine mussels during early development, and specifically the trochophore stage. Using RNA and DNA sequencing and

One challenge in global change biology is to identify the mechanisms underpinning physiological sensitivities to environmental change and to predict their potential to adapt to future conditions. Using ocean acidification as the representative stressor, molecular pathways associated with abnormal larval development of a globally distributed marine mussel are identified. The targeted developmental stage was the trochophore stage, which is, for a few hours, pH sensitive and is the main driver of developmental success. RNA sequencing and in situ RNA hybridization were used to identify processes associated with abnormal development, and DNA sequencing was used to identify which processes evolve when larvae are exposed to low pH for the full duration of their larval stage. Trochophores exposed to low pH exhibited 43 differentially expressed genes. Thirteen genes, none of which have previously been identified in mussel trochophores, including three unknown genes, were expressed in the shell field. Gene annotation and in situ hybridization point to two core processes associated with the response to low pH: development of the trochophore shell field and the cellular stress response. Encompassing both of these processes, five genes demonstrated changes in allele frequency that are indicative of rapid adaptation. Thus, genes underpinning the most pH-sensitive developmental processes also exhibit scope to adapt via genetic variation currently maintained in the mussel population. These results provide evidence that protecting species’ existing genetic diversity is a critical management action to maximize the potential for rapid adaptation under a changing environment.

International audience; Functional approaches for studying embryonic development have greatly advanced thanks to the CRISPR-Cas9 gene editing technique. Previously practiced in just a few organisms, these knockout techniques are now widely applied. Here we describe simple techniques for applying the CRISPR-Cas9 system to study the development of the nerve cord in the ascidian Phallusia mammillata.

Cell division orientation is thought to result from a competition between cell geometry and polarity domains controlling the position of the mitotic spindle during mitosis. Depending on the level of cell shape anisotropy or the strength of the polarity domain, one dominates the other and determines the orientation of the spindle. Whether and how such competition is also at work to determine unequal cell division (UCD), producing daughter cells of different size, remains unclear. Here, we show that cell geometry and polarity domains cooperate, rather than compete, in positioning the cleavage plane during UCDs in early ascidian embryos. We found that the UCDs and their orientation at the ascidian third cleavage rely on the spindle tilting in an anisotropic cell shape, and cortical polarity domains exerting different effects on spindle astral microtubules. By systematically varying mitotic cell shape, we could modulate the effect of attractive and repulsive polarity domains and consequently generate predicted daughter cell size asymmetries and position. We therefore propose that the spindle position during UCD is set by the combined activities of cell geometry and polarity domains, where cell geometry modulates the effect of cortical polarity domain(s).Graphical abstractHighlightSpindle tilting in anisotropic cell shape induces unequal cell divisionCortical polarity domain can exert attractive or repulsive effect on spindleCell geometry and polarity domain cooperate to position the spindleCell geometry modulates the effect of polarity domain

International audience; In recent years, pollution of surface waters with xenobiotic compounds became an issue of concern in society and has been the object of numerous studies. Most of these xenobiotic compounds are man-made molecules and some of them are qualified as endocrine disrupting chemicals (EDCs) when they interfere with hormones actions. Several studies have investigated the teratogenic impacts of EDCs in vertebrates (including marine vertebrates). However, the impact of such EDCs on marine invertebrates is much debated and still largely obscure. In addition, DNA-altering genotoxicants can induce embryonic malformations. The goal of this study is to develop a reliable and effective test for assessing toxicity of chemicals using embryos of the ascidian (Phallusia mammillata) in order to find phenotypic signatures associated with xenobiotics. We evaluated embryonic malformations with high-content analysis of larval phenotypes by scoring several quantitative and qualitative morphometric endpoints on a single image of Phallusia tadpole larvae with semi-automated image analysis. Using this approach we screened different classes of toxicants including genotoxicants, known or suspected EDCs and nuclear receptors (NRs) ligands. Manuscript File Click here to view linked References 2 The screen presented here reveals a specific phenotypic signature for ligands of retinoic acid receptor/retinoid X receptor. Analysis of larval morphology combined with DNA staining revealed that embryos with DNA aberrations displayed severe malformations affecting multiple aspects of embryonic development. In contrast EDCs exposure induced no or little DNA aberrations and affected mainly neural development. Therefore the ascidian embryo/larval assay presented here can allow to distinguish the type of teratogenicity induced by different classes of toxicants.

International audience; Nuclear Receptors (NRs) are a superfamily of transcription factors specific to metazoans that have the unique ability to directly translate the message of a signaling molecule into a transcriptional response. In vertebrates, NRs are pivotal players in countless processes of both embryonic and adult physiology, with embryonic development being one of the most dynamic periods of NR activity. Accumulating evidence suggests that NR signaling is also a major regulator of development in marine invertebrates, although ligands and transactivation dynamics are not necessarily conserved with respect to vertebrates. The explosion of genome sequencing projects and the interpretation of the resulting data in a phylogenetic context allowed significant progress toward an understanding of NR superfamily evolution, both in terms of molecular activities and developmental functions. In this context, marine invertebrates have been crucial for characterizing the ancestral states of NR-ligand interactions, further strengthening the importance of these organisms in the field of evolutionary developmental biology.

Bisphenol A-BPA, a widespread plastic additive, is an emerging contaminant of high concern and a potential endocrine disruptor in mammals. BPA also represents a potential threat for aquatic species, especially for larval stages. In the marine bivalve Mytilus galloprovincialis, BPA has been previously shown to affect early larval development and gene transcription. In this work, the effects of BPA (0.05–0.5–5 μM) were further investigated at different times post fertilization (24–28–32–48 hpf). BPA induced concentration-dependent alterations in deposition of the organic matrix and calcified shell at different larval stages, as shown by double calcofluor/calcein staining, resulting in altered phenotypes at 48hpf. Transcription of Tyrosinase-TYR, that plays a key role in remodelling of the shell organic matrix, and of HOX1, a member of homeobox genes involved in larval shell formation and neurogenesis, were evaluated by In Situ Hybrydization-ISH. BPA altered the spatial pattern of expression of both genes, with distinct effects depending on the concentration and developmental stage. Moreover, BPA affected the time course of mRNA levels for TYR from 24 to 48hpf. BPA impaired development of serotonin-5-HT-immunoreactive neurons at different times pf; at 48hpf, the reduction in the number of serotoninergic neurons was associated with developmental delay and downregulation of the 5-HT receptor-5-HTR. All the effects were observed from the lowest concentration tested, corresponding to detectable BPA levels in contaminated coastal waters. These data demonstrate that BPA interferes with key processes occurring during the first developmental stages of mussels, thus representing a potential threat for natural populations.

Polar body (PB) formation is an extreme form of unequal cell division that occurs in oocytes due to the eccentric position of the small meiotic spindle near the oocyte cortex. Prior to PB formation, a chromatin-centered process causes the cortex overlying the meiotic chromosomes to become polarized. This polarized cortical subdomain marks the site where a cortical protrusion or outpocket forms at the oocyte surface creating the future PBs. Using ascidians, we observed that PB1 becomes tethered to the fertilized egg via PB2, indicating that the site of PB1 cytokinesis directed the precise site for PB2 emission. We therefore studied whether the midbody remnant left behind following PB1 emission was involved, together with the egg chromatin, in defining the precise cortical site for PB2 emission. During outpocketing of PB2 in ascidians, we discovered that a small structure around 1 &micro, m in diameter protruded from the cortical outpocket that will form the future PB2, which we define as the &ldquo, polar corps&rdquo, As emission of PB2 progressed, this small polar corps became localized between PB2 and PB1 and appeared to link PB2 to PB1. We tested the hypothesis that this small polar corps on the surface of the forming PB2 outpocket was the midbody remnant from the previous round of PB1 cytokinesis. We had previously discovered that Plk1::Ven labeled midbody remnants in ascidian embryos. We therefore used Plk1::Ven to follow the dynamics of the PB1 midbody remnant during meiosis II. Plk1::Ven strongly labeled the small polar corps that formed on the surface of the cortical outpocket that created PB2. Following emission of PB2, this polar corps was rich in Plk1::Ven and linked PB2 to PB1. By labelling actin (with TRITC-Phalloidin) we also demonstrated that actin accumulates at the midbody remnant and also forms a cortical cap around the midbody remnant in meiosis II that prefigured the precise site of cortical outpocketing during PB2 emission. Phalloidin staining of actin and immunolabelling of anti-phospho aPKC during meiosis II in fertilized eggs that had PB1 removed suggested that the midbody remnant remained within the fertilized egg following emission of PB1. Dynamic imaging of microtubules labelled with Ens::3GFP, MAP7::GFP or EB3::3GFP showed that one pole of the second meiotic spindle was located near the midbody remnant while the other pole rotated away from the cortex during outpocketing. Finally, we report that failure of the second meiotic spindle to rotate can lead to the formation of two cortical outpockets at anaphase II, one above each set of chromatids. It is not known whether the midbody remnant of PB1 is involved in directing the precise location of PB2 since our data are correlative in ascidians. However, a review of the literature indicates that PB1 is tethered to the egg surface via PB2 in several species including members of the cnidarians, lophotrochozoa and echinoids, suggesting that the midbody remnant formed during PB1 emission may be involved in directing the precise site of PB2 emission throughout the invertebrates.

Global tissue tension anisotropy has been shown to trigger stereotypical cell division orientation by elongating mitotic cells along the main tension axis. Yet, how tissue tension elongates mitotic cells despite those cells undergoing mitotic rounding (MR) by globally upregulating cortical actomyosin tension remains unclear. We addressed this question by taking advantage of ascidian embryos, consisting of a small number of interphasic and mitotic blastomeres and displaying an invariant division pattern. We found that blastomeres undergo MR by locally relaxing cortical tension at their apex, thereby allowing extrinsic pulling forces from neighboring interphasic blastomeres to polarize their shape and thus division orientation. Consistently, interfering with extrinsic forces by reducing the contractility of interphasic blastomeres or disrupting the establishment of asynchronous mitotic domains leads to aberrant mitotic cell division orientations. Thus, apical relaxation during MR constitutes a key mechanism by which tissue tension anisotropy controls stereotypical cell division orientation.

Tetrabromobisphenol A-TBBPA, a widely used brominated flame retardant detected in aquatic environments, is considered a potential endocrine disruptor-ED for its reproductive/developmental effects in vertebrates. In aquatic invertebrates, the modes of action of most EDs are largely unknown, due to partial knowledge of the mechanisms controlling neuroendocrine functions. In the marine bivalve Mytilus galloprovincialis, TBBPA has been previously shown to affect larval development in the 48 h larval toxicity assay at environmental concentrations. In this work, the effects of TBBPA were further investigated at different times post-fertilization. TBBPA, from 1 μg/L, affected shell biogenesis at 48 hours post fertilization-hpf, as shown by phenotypic and SEM analysis. The mechanisms of action of TBBPA were investigated at concentrations of the same order of magnitude as those found in highly polluted coastal areas (10 μg/L). At 28-32 hpf, TBBPA significantly affected deposition of both the organic matrix and CaCO3 in the shell. TBBPA also altered expression of shell-related genes from 24 to 48 hpf, in particular of tyrosinase, a key enzyme in shell matrix remodeling. At earlier stages (24 hpf), TBBPA affected the development of dopaminergic, serotoninergic and GABAergic systems, as shown by in situ hybridization-ISH and immunocytochemistry. These data contribute draw adverse outcome pathways-AOPs, where TBBPA affects the synthesis of neutrotransmitters involved in key events (neurodevelopment and shell biogenesis), resulting in phenotypic changes on individuals (delayed or arrested development) that might lead to detrimental consequences on populations.

ANISEED (https://www.aniseed.cnrs.fr) is the main model organism database for the worldwide community of scientists working on tunicates, the vertebrate sister-group. Information provided for each species includes functionally-annotated gene and transcript models with orthology relationships within tunicates, and with echinoderms, cephalochordates and vertebrates. Beyond genes the system describes other genetic elements, including repeated elements and cis-regulatory modules. Gene expression profiles for several thousand genes are formalized in both wild-type and experimentally-manipulated conditions, using formal anatomical ontologies. These data can be explored through three complementary types of browsers, each offering a different view-point. A developmental browser summarizes the information in a gene- or territory-centric manner. Advanced genomic browsers integrate the genetic features surrounding genes or gene sets within a species. A Genomicus synteny browser explores the conservation of local gene order across deuterostome. This new release covers an extended taxonomic range of 14 species, including for the first time a non-ascidian species, the appendicularian Oikopleura dioica. Functional annotations, provided for each species, were enhanced through a combination of manual curation of gene models and the development of an improved orthology detection pipeline. Finally, gene expression profiles and anatomical territories can be explored in 4D online through the newly developed Morphonet morphogenetic browser.

The endocrine disruptor Bisphenol A (BPA), a widely employed molecule in plastics, has been shown to affect several biological processes in vertebrates, mostly via binding to nuclear receptors. Neurodevelopmental effects of BPA have been documented in vertebrates and linked to neurodevelopmental disorders, probably because some nuclear receptors are present in the vertebrate brain. Similarly, endocrine disruptors have been shown to affect neurodevelopment in marine invertebrates such as ascidians, mollusks or echinoderms, but whether invertebrate nuclear receptors are involved in the mode-of-action is largely unknown. In this study, we assessed the effect of BPA on larval brain development of the ascidian Phallusia mammillata. We found that BPA is toxic to P. mammillata embryos in a dose-dependent manner (EC50: 11.8μM; LC50: 21μM). Furthermore, micromolar doses of BPA impaired differentiation of the ascidian pigmented cells, by inhibiting otolith movement within the sensory vesicle. We further show that this phenotype is specific to other two bisphenols (BPE and BPF) over a bisphenyl (2,2 DPP). Because in vertebrates the estrogen-related receptor gamma (ERRγ) can bind bisphenols with high affinity but not bisphenyls, we tested whether the ascidian ERR participates in the neurodevelopmental phenotype induced by BPA. Interestingly, P. mammillata ERR is expressed in the larval brain, adjacent to the differentiating otolith. Furthermore, antagonists of vertebrate ERRs also inhibited the otolith movement but not pigmentation. Together our observations suggest that BPA may affect ascidian otolith differentiation by altering Pm-ERR activity whereas otolith pigmentation defects might be due to the known inhibitory effect of bisphenols on tyrosinase enzymatic activity.

Polar body (PB) formation is an extreme form of unequal cell division that occurs in oocytes due to the eccentric position of the small meiotic spindle near the oocyte cortex. Prior to PB formation, a chromatin-centered process causes the cortex overlying the meiotic chromosomes to become polarized. This polarized cortical subdomain marks the site where a cortical protrusion or outpocket forms at the oocyte surface creating the future PBs. We observed that PB1 becomes tethered to the egg via PB2, indicating that the site of PB1 cytokinesis directed the precise site for PB2 emission. We therefore studied whether the midbody remnant left behind following PB1 emission was involved, together with the egg chromatin, in defining the precise cortical site for PB2 emission. During outpocketing of PB2 in ascidians, we discovered that a small corps around 1μm in diameter protruded from the center of the cortical outpocket that will form the future PB2, which we call the “polar corps”. During emission of PB2, this small polar corps became localized between PB2 and PB1 and appeared to link PB2 to PB1. We tested the hypothesis that this small polar corps on the surface of the forming PB2 was the midbody remnant from the previous round of PB1 cytokinesis. We had previously discovered that Plk1::Ven labeled midbody remnants in ascidian embryos. We therefore used Plk1::Ven to follow the dynamics of the PB1 midbody remnant during meiosis II. Plk1::Ven strongly labeled the small polar corps that formed on the surface of the cortical outpocket that created PB2. Following emission of PB2, this polar corps was rich in Plk1::Ven and linked PB2 to PB1. By labelling actin (with LifeAct::mCherry/GFP or TRITC-Phalloidin) we also demonstrated that actin accumulates at the midbody remnant and also forms a cortical cap around the midbody remnant in meiosis II that prefigured the precise site of cortical outpocketing during PB2 emission. Phalloidin staining of actin and immunolabelling of anti-phospho aPKC during meiosis II in eggs that had PB1 removed showed that the midbody remnant remained within the egg following emission of PB1. Dynamic imaging of microtubules labelled with Ens::3GFP, MAP7::GFP or EB3::3GFP showed that one pole of the second meiotic spindle was located near the midbody remnant while the other pole rotated away from the cortex during outpocketing. Finally, we report that failure of the second meiotic spindle to rotate can lead to the formation of two cortical outpockets at anaphase II, one above each set of chromatids. It is not known whether the midbody remnant of PB1 is involved in directing the precise location of PB2 in other species as in ascidians. However, a review of the literature indicates that PB1 is tethered to the egg surface via PB2 in a number of species including members of the cnidarians, lophotrochozoa and echinoids, suggesting that the midbody remnant formed during PB1 emission may be involved in directing the precise site of PB2 emission throughout the invertebrates.

Bivalve biomineralization is a highly complex and organized process, involving several molecular components identified in adults and larval stages. However, information is still scarce on the ontogeny of the organic matrix before calcification occurs. In this work, first shell formation was investigated in the mussel Mytilus galloprovincialis . The time course of organic matrix and CaCO 3 deposition were followed at close times post fertilization (24, 26, 29, 32, 48 h) by calcofluor and calcein staining, respectively. Both components showed an exponential trend in growth, with a delay between organic matrix and CaCO 3 deposition. mRNA levels of genes involved in matrix deposition (chitin synthase; tyrosinase- TYR) and calcification (carbonic anhydrase; extrapallial protein) were quantified by qPCR at 24 and 48 hours post fertilization (hpf) with respect to eggs. All transcripts were upregulated across early development, with TYR showing highest mRNA levels from 24 hpf. TYR transcripts were closely associated with matrix deposition as shown by in situ hybridization. The involvement of tyrosinase activity was supported by data obtained with the enzyme inhibitor N-phenylthiourea. Our results underline the pivotal role of shell matrix in driving first CaCO 3 deposition and the importance of tyrosinase in the formation of the first shell in M. galloprovincialis .

Endocrine Disrupting Chemicals (EDCs) are molecules able to interfere with the vertebrate hormonal system in different ways, a major one being the modification of the activity of nuclear receptors (NRs). Several NRs are expressed in the vertebrate brain during embryonic development and these NRs are suspected to be responsible for the neurodevelopmental defects induced by exposure to EDCs in fishes or amphibians and to participate in several neurodevelopmental disorders observed in humans. Known EDCs exert toxicity not only on vertebrate forms of marine life but also on marine invertebrates. However, because hormonal systems of invertebrates are poorly understood, it is not clear whether the teratogenic effects of known EDCs are because of endocrine disruption. The most conserved actors of endocrine systems are the NRs which are present in all metazoan genomes but their functions in invertebrate organisms are still insufficiently characterized. EDCs like bisphenol A have recently been shown to affect neurodevelopment in marine invertebrate chordates called ascidians. Because such phenotypes can be mediated by NRs expressed in the ascidian embryo, we review all the information available about NRs expression during ascidian embryogenesis and discuss their possible involvement in the neurodevelopmental phenotypes induced by EDCs.

10 pages, 4 figures, supplementary material https://dx.doi.org/10.6084/m9.figshare.c.4325828.-- Data are archived on BCO-DMO, https://www.bco-dmo.org/project/720349, Coastal marine ecosystems experience dynamic fluctuations in seawater carbonate chemistry. The importance of this variation in the context of ocean acidification requires knowing what aspect of variability biological processes respond to. We conducted four experiments (ranging from 3 to 22 days) with different variability regimes (pHT 7.4–8.1) assessing the impact of diel fluctuations in carbonate chemistry on the early development of the mussel Mytilus galloprovincialis. Larval shell growth was consistently correlated to mean exposures, regardless of variability regimes, indicating that calcification responds instantaneously to seawater chemistry. Larval development was impacted by timing of exposure, revealing sensitivity of two developmental processes: development of the shell field, and transition from the first to the second larval shell. Fluorescent staining revealed developmental delay of the shell field at low pH, and abnormal development thereof was correlated with hinge defects in D-veligers. This study shows, for the first time, that ocean acidification affects larval soft-tissue development, independent from calcification. Multiple developmental processes additively underpin the teratogenic effect of ocean acidification on bivalve larvae. These results explain why trochophores are the most sensitive life-history stage in marine bivalves and suggest that short-term variability in carbonate chemistry can impact early, This research was funded by the US National Science Foundation (NSF; OCE-1521597 to L.K.). L.K. was also supported by the European Commission Horizon 2020 Marie Skłodowska-Curie Action (No. 747637). M.C.B. was supported by US Department of Education (Grant No. 200A150101) and NSF Graduate Research Fellowship (No. 1000198423). A.M. was supported by an Erasmus+ traineeship scholarship (University of Genova) and by the Observatoire Océanologique de Villefranche

Ascidians (tunicates; sea squirts) are marine animals which provide a source of diverse, bioactive natural products, and a model for toxicity screenings. Compounds isolated from ascidians comprise an approved anti-tumor drug and many others are potent drug leads. Furthermore, the use of invertebrate embryos for toxicological screening tests or analysis offers the possibility to image a large number of samples for high throughput screens. Ascidians are members of a sister clade to the vertebrates and make a vertebrate-like tadpole larva composed of less than 3000 cells in 18 hours. The neural complex of the ascidian larva is made of only 350 cells (of which 100 are neurons) and functional genomic studies have now uncovered numerous GRNs underpinning neural specification and differentiation. Numerous studies showed that brain formation in ascidians is sensitive to toxic insults especially from endocrine disruptors making them a suitable model to study neurodevelopmental defects. Modern techniques available for ascidians, including transgenic embryos where 3D time lapse imaging of GFPexpressing reporter constructs can be analyzed, now permit numerous end-points to be evaluated in order to test the specific mode of action of many compounds. This review summarizes the key evidence suggesting that ascidian embryos are a favorable embryological model to study neurodevelopmental toxicity of different compounds with molecular and cellular end-points. We predict that ascidians may become a significant source of marine blue biotechnologies in the 21st century.

The ascidian embryo is an ideal system to investigate how cell position is determined during embryogenesis. Using 3D timelapse imaging and computational methods we analyzed the planar cell divisions in ascidian early embryos and found that spindles in every cell tend to align at metaphase in the long length of the apical surface except in cells undergoing unequal cleavage. Furthermore, the invariant and conserved cleavage pattern of ascidian embryos was found to consist in alternate planar cell divisions between ectoderm and endomesoderm. In order to test the importance of alternate cell divisions we manipulated zygotic transcription induced by β-catenin or downregulated wee1 activity, both of which abolish this cell cycle asynchrony. Crucially, abolishing cell cycle asynchrony consistently disrupted the spindle orienting mechanism underpinning the invariant cleavage pattern. Our results demonstrate how an evolutionary conserved cell cycle asynchrony maintains the invariant cleavage pattern driving morphogenesis of the ascidian blastula. DOI: http://dx.doi.org/10.7554/eLife.19290.001, eLife digest The position of cells within an embryo early in development determines what type of cells they will become in the fully formed embryo. The embryos of ascidians, commonly known as sea squirts, are ideal for studying what influences cell positioning. These embryos consist of a small number of cells that divide according to an “invariant cleavage pattern”, which means that the positioning and timing of the cell divisions is identical between different individuals of the same species. The pattern of cell division is also largely the same across different ascidian species, which raises questions about how it is controlled. When a cell divides, a structure called the spindle forms inside it to distribute copies of the cell’s genetic material between the new cells. The orientation of the spindle determines the direction in which the cell will divide. Now, by combining 3D imaging of living ascidian embryos with computational modeling, Dumollard et al. show that the spindles in every equally dividing cell tend to all align in the long length of the cell’s “apical” surface. Such alignment allows the cells to be on the outside of the embryo and implements the ascidian invariant cleavage pattern. The cells in the embryo do not all divide at the same time. Indeed, the shape of the cells (and especially their apical surface) depends on two layers of cells in the embryo not dividing at the same time; instead, periods of cell division alternate between the layers. A network of genes in the embryo regulates the timing of these cell divisions and makes it possible for the cells to divide according to an invariant cleavage pattern. However, this network of genes is not the only control mechanism that shapes the early embryo. A structure found in egg cells (and hence produced by the embryo’s mother) causes cells at the rear of the embryo to divide unequally, and this influences the shape of all the cells in the embryo. Thus it appears that maternal mechanisms work alongside the embryo’s gene network to shape the early embryo. The next step will be to determine how physical forces – for example, from the cells pressing against each other – influence the position of the embryo’s cells. How do gene networks relay the biomechanical properties of the embryo to help it take shape? DOI: http://dx.doi.org/10.7554/eLife.19290.002

Like most metazoans, eggs of echinoderms and tunicates (marine deuterostomes, there is no data for the cephalochordates) arrest awaiting fertilization due to the activity of the Mos/MEK/MAPK cascade and are released from this cell cycle arrest by sperm-triggered Ca2+ signals. Invertebrate deuterostome eggs display mainly three distinct types of cell cycle arrest before fertilization mediated by potentially different cytostatic factors (CSF): one CSF causes arrest during meiotic metaphase I (MI-CSF in tunicates and some starfishes), another CSF likely causes arrest during meiotic metaphase II (amphioxus), and yet another form of CSF causes arrest to occur after meiotic exit during G1 of the first mitotic cycle (G1-CSF). In tunicates and echinoderms these different CSF activities have been shown to rely on the Mos//MAPK pathway for establishment and on Ca2+ signals for their inactivation. Despite these molecular similarities, release of MI-CSF arrest is caused by APC/C activation (to destroy cyclin B) whereas release from G1-CSF is caused by stimulating S phase and the synthesis of cyclins. Further research is needed to understand how both the Mos//MAPK cascade and Ca2+ achieve these tasks in different marine invertebrate deuterostomes. Another conserved feature of eggs is that protein synthesis of specific mRNAs is necessary to proceed through oocyte maturation and to maintain CSF-induced cell cycle arrest. Then activation of development at fertilization is accompanied by an increase in the rate of protein synthesis but the mechanisms involved are still largely unknown in most of the marine deuterostomes. How the sperm-triggered Ca2+ signals cause an increase in protein synthesis has been studied mainly in sea urchin eggs. Here we review these conserved features of eggs (arrest, activation and protein synthesis) focusing on the non-vertebrate deuterostomes.

The EMBO workshop ‘Oocyte maturation and fertilization: lessons from canonical and emerging models’ was held at the Oceanologic Observatory of Banyuls in France in June 2013 and was organized by Anne-Marie Geneviere, Olivier Haccard, Peter Lenart and Alex McDougall. A total of 78 participants shared their research on germline formation, oocyte development, sperm, fertilization and early development. Here, we report the highlights of this meeting.

Ecological interactions in the marine environment are now recognized to be partly held by chemical cues produced by marine organisms. In particular, sponges are sessile animals thought to rely on the bioactive substances they synthesize to ensure their development and defense. However, the mechanisms leading the sponges to use their specialized metabolites as chemical cues remain unknown. Here we report the constant release of bioactive polycyclic guanidinic alkaloids by the Mediterranean sponge Crambe crambe into the dissolved and the particulate phases using a targeted metabolomics study. These compounds were proven to be stored into already described specialized (spherulous) sponge cells and dispersed into the water column after release through the sponge exhaling channels (oscula), leading to a chemical shield surrounding the sponge. Low concentrations of these compounds were demonstrated to have teratogenic effects on embryos of a common sea squirt (ascidian). This mechanism of action called spherulization may therefore contribute to the ecological success of encrusting sponges that need to extend their substrate cover to expand. Part of this work was funded by the region PACA (Plateau MALLABAR), the ECOS Nord project C15U01 by the EMR, a partnership between the UPMC, the Laboratoires Pierre Fabre and CNRS. peer-reviewed

Heat Shock Factor 1 (HSF1) is a transcription factor whose loss of function results in the inability of Hsf1(-/-) females to produce viable embryos, as a consequence of early developmental arrest. We previously demonstrated that maternal HSF1 is required in oocytes to regulate expression of chaperones, in particular Hsp90alpha, and is essential for the progression of meiotic maturation. In the present work, we used comparative morphological and biochemical analytic approaches to better understand how Hsf1(-/-) oocytes undergo irreversible cell death. We found that the metaphase II arrest in mature oocytes, cortical granule exocytosis and formation of pronuclei in zygotes were all impaired in Hsf1(-/-) mutants. Although oogenesis generated fully grown oocytes in follicles, intra-ovarian Hsf1(-/-) oocytes displayed ultrastructural abnormalities and contained dysfunctional mitochondria as well as elevated oxidant load. Finally, the apoptotic effector, caspase-3, was activated in most mutant oocytes and embryos, reflecting their commitment to apoptosis. In conclusion, our study shows that early post-ovulation events are particularly sensitive to oxidant insult, which abrogates the developmental competence of HSF1-depleted oocytes. They also reveal that Hsf1 knock-out mice constitute a genetic model that can be used to evaluate the importance of redox homeostasis in oocytes.

Energy homeostasis of the oocyte is a crucial determinant of fertility. Following ovulation, the oocyte is exposed to the unique environment of the Fallopian tube, and this is reflected in a highly specialised biochemistry. The minute amounts of tissue available have made the physiological analysis of oocyte intermediary metabolism almost impossible. We have therefore used confocal imaging of mitochondrial and cytosolic redox state under a range of conditions to explore the oxidative metabolism of intermediary substrates. It has been known for some time that the early mouse embryo metabolises external pyruvate and lactate but not glucose to produce ATP. We now show at the level of single oocytes, that supplied glucose has no effect on the redox potential of the oocyte. Pyruvate is a cytosolic oxidant but a mitochondrial reductant,while lactate is a strong cytosolic reductant via the activity of lactate dehydrogenase. Unexpectedly, lactate-derived pyruvate appears to be diverted from mitochondrial oxidation. Our approach also reveals that the level of reduced glutathione (GSH) in the oocyte is maintained by glutathione reductase, which oxidises intracellular NADPH to reduce oxidised glutathione. Surprisingly, NADPH does not seem to be supplied by the pentose phosphate pathway in the unfertilised oocyte but rather by cytosolic NADP-dependent isocitrate dehydrogenase. Remarkably, we also found that the oxidant action of pyruvate impairs development, demonstrating the fundamental importance of redox state on early development.

During embryonic development and maternal meiotic maturation, positioning of the mitotic/meiotic spindle is subject to control mechanisms that meet the needs of the particular cell type. Here we review the methods, molecular tools, and the ascidian model we use to study three different ways in which centrosomes or spindles are positioned in three different cellular contexts. First, we review unequal cleavage in the ascidian germ lineage. In the germ cell precursors, a large macromolecular structure termed the centrosome-attracting body causes three successive rounds of unequal cleavage from the 8- to the 64-cell stage. Next, we discuss spindle positioning underlying the invariant cleavage pattern. Ascidian embryos display an invariant cleavage pattern whereby the mitotic spindle aligns in a predetermined orientation in every blastomere up to the gastrula stage (composed of 112 cells). Finally, we review methods and approaches to study meiotic spindle positioning in eggs.

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.

At fertilisation, repetitive increases in the intracellular Ca2+concentration, [Ca2+]i, drive the completion of meiosis and initiate the development of the quiescent egg into an embryo. Although the requirement for an ATP supply is evident, the relative roles of potential ATP sources remains unclear in the mammalian egg, and the specific role of mitochondria in [Ca2+]i regulation as well as in the sperm-triggered [Ca2+] oscillations is unknown.We have used fluorescence and luminescence imaging to investigate mitochondrial activity in single mouse eggs. Simultaneous imaging of mitochondrial redox state (NADH and flavoprotein autofluorescence) and[Ca2+]i revealed that sperm-triggered [Ca2+]oscillations are transmitted to the mitochondria where they directly stimulate mitochondrial activity. Inhibition of mitochondrial oxidative phosphorylation caused release of Ca2+ from the endoplasmic reticulum because of local ATP depletion. Mitochondrial ATP production is an absolute requirement for maintaining a low resting [Ca2+]i and for sustaining sperm-triggered [Ca2+] oscillations. Luminescence measurements of intracellular [ATP] from single eggs confirmed that mitochondrial oxidative phosphorylation is the major source of ATP synthesis in the dormant unfertilised egg. These observations show that a high local ATP consumption is balanced by mitochondrial ATP production, and that balance is critically poised. Mitochondrial ATP supply and demand are thus closely coupled in mouse eggs. As mitochondrial ATP generation is essential to sustain the[Ca2+] signals that are crucial to initiate development,mitochondrial integrity is clearly fundamental in sustaining fertility in mammalian eggs.

During the past 25 years, the characterization of sperm-triggered calcium signals in eggs has progressed from the discovery of a single calcium increase at fertilization in the medaka fish to the observation of repetitive calcium waves initiated by multiple meiotic calcium wave pacemakers in the ascidian. In eggs of all animal species, sperm-triggered inositol (1,4,5)-trisphosphate[Ins(1,4,5)P3] production regulates the vast array of calcium wave patterns observed in the different species. The spatial organization of calcium waves is driven either by the intracellular distribution of the calcium release machinery or by the localized and dynamic production of calcium-releasing second messengers. In the highly polarized egg cell, cortical endoplasmic reticulum (ER)-rich clusters act as pacemaker sites dedicated to the initiation of global calcium waves. The extensive ER network made of interconnected ER-rich domains supports calcium wave propagation throughout the egg. Fertilization triggers two types of calcium wave pacemakers depending on the species: in mice, the pacemaker site in the vegetal cortex of the egg is probably a site that has enhanced sensitivity to Ins(1,4,5)P3; in ascidians, the calcium wave pacemaker may rely on a local source of Ins(1,4,5)P3 production apposed to a cluster of ER in the vegetal cortex.

Time-lapse 4D imaging of fluorescently tagged proteins to follow the dynamics of cellular structures (chromosomes, microtubules, actin, centrosomes, cortical structures like the CAB in ascidians, etc.) combined with targeted gene knockdown during embryonic development is a powerful technique to understand the mechanisms of embryonic development. The eggs and embryos of the primitive marine chordate Phallusia mammillata are an excellent model system for combining live cell imaging with gene knockdown experiments. Here we describe simple methods for microinjecting Phallusia eggs with mRNA encoding fluorescent fusion proteins combined with 4D time-lapse imaging techniques we use to follow all of embryonic development from the egg to late tailbud stage.

During the transition from maternal to zygotic control of development, cell cycle length varies in different lineages, and this is important for their fates and functions. The maternal to zygotic transition (MZT) in metazoan embryos involves a profound remodeling of the cell cycle: S phase length increases then G2 is introduced. Although β-catenin is the master regulator of endomesoderm patterning at MZT in all metazoans, the influence of maternal β-catenin on the cell cycle at MZT remains poorly understood. By studying urochordate embryogenesis we found that cell cycle remodeling during MZT begins with the formation of 3 mitotic domains at the 16-cell stage arising from differential S phase lengthening, when endomesoderm is specified. Then, at the 64-cell stage, a G2 phase is introduced in the endoderm lineage during its specification. Strikingly, these two phases of cell cycle remodeling are patterned by β-catenin-dependent transcription. Functional analysis revealed that, at the 16-cell stage, β-catenin speeds up S phase in the endomesoderm. In contrast, two cell cycles later at gastrulation, nuclear β-catenin induces endoderm fate and delays cell division. Such interphase lengthening in invaginating cells is known to be a requisite for gastrulation movements. Therefore, in basal chordates β-catenin has a dual role to specify germ layers and remodel the cell cycle.

International audience; The fertilising sperm triggers a transient Ca2+ increase that releases eggs from cell cycle arrest in the vast majority of animal eggs. In vertebrate eggs, Erp1, an APC/C-cdc20 inhibitor, links release from metaphase II arrest with the Ca2+ transient and its degradation is triggered by the Ca2+-induced activation of CaMKII. By contrast, many invertebrate groups have mature eggs that arrest at metaphase I, and these species do not possess the CaMKII target Erp1 in their genomes. As a consequence, it is unknown exactly how cell cycle arrest at metaphase I is achieved and how the fertilisation Ca2+ transient overcomes the arrest in the vast majority of animal species. Using live-cell imaging with a novel cyclin reporter to study cell cycle arrest and its release in urochordate ascidians, the closest living invertebrate group to the vertebrates, we have identified a new signalling pathway for cell cycle resumption in which CaMKII plays no part. Instead, we find that the Ca2+-activated phosphatase calcineurin (CN) is required for egg activation. Moreover, we demonstrate that parthenogenetic activation of metaphase I-arrested eggs by MEK inhibition, independent of a Ca2+ increase, requires the activity of a second egg phosphatase: PP2A. Furthermore, PP2A activity, together with CN, is required for normal egg activation during fertilisation. As ascidians are a sister group of the vertebrates, we discuss these findings in relation to cell cycle arrest and egg activation in chordates.

The completely transparent eggs and embryos of the ascidian Phallusia mammillata are well suited for imaging-based studies of how cell cycle control mechanisms have been integrated into the processes of meiosis, fertilization, and embryonic development. Several cell cycle-related issues that pertain to reproduction and development have been addressed using the ascidian model. For example, how are sperm-triggered calcium oscillations controlled by cell cycle kinases? How is chromosome segregation during meiosis regulated? What processes does the Mos/MAPK signaling cascade control in eggs in addition to CSF-mediated cell cycle arrest? Following fertilization ascidians blastomeres display cell cycle asynchrony, oriented cell division, and unequal cleavage resulting in the formation of a distinctive gastrula composed of precisely 112 cells. Here, we shall summarize these topics and where possible show how the mechanisms identified in ascidians compare to those identified in other organisms.

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.

In ascidians the cell cycle machinery has been studied mainly in oocytes while ascidian embryos have been used to dissect the mechanism that controls asymmetric cell division (ACD). Here we overview the most specific and often exceptional points and events in cell cycle control in ascidian oocytes and early embryos. Mature stage IV eggs are arrested at metaphase I due to cytostatic factor (CSF). In vertebrates, unfertilized eggs are arrested at metaphase II by CSF. Meta II-CSF is mediated by the Mos/MEK/MAPK/Erp1 pathway, which inhibits the ubiquitin ligase APC/Ccdc20 preventing cyclin B destruction thus stabilizing MPF activity. CSF is inactivated by the fertilization Ca2+ transient that stimulates the destruction of Erp1 thus releasing APC/Ccdc20 from inhibition. Although many of the components of CSF are conserved between the ascidian and the vertebrates, the lack of Erp1 in the ascidians (and indeed other invertebrates) is notable since the Mos/MAPK pathway nonetheless mediates Meta I-CSF. Moreover, since the fertilization Ca2+ transient targets Erp1, it is not clear how the sperm-triggered Ca2+ transient in ascidians (and again other invertebrates) stimulates cyclin B destruction in the absence of Erp1. Nonetheless, like mammalian eggs, sperm trigger a series of Ca2+ oscillations that increases the rate of cyclin B destruction and the subsequent loss of MAPK activity leading to meiotic exit in ascidians. Positive feedback from MPF maintains the Ca2+ oscillations in fertilized ascidian eggs ensuring the eventual loss of MPF stimulating the egg-to-embryo transition. Embryonic cell cycles in the ascidian are highly stereotyped where both the rate of cell division and the orientation of cell division planes are precisely controlled. Three successive rounds of ACD generate two small posterior germ cell precursors at the 64 cell stage. The centrosome-attracting body (CAB) is a macroscopic cortical structure visible by light microscopy that causes these three rounds of ACD. Entry into mitosis activates the CAB causing the whole mitotic spindle to rotate and migrate toward the cortical CAB leading to a highly ACD whereby one small cell is formed that inherits the CAB and approximately 40 maternal postplasmic/PEM RNAs including the germ cell marker vasa.

Mos kinase is a universal mediator of oocyte meiotic maturation and is produced during oogenesis and destroyed after fertilization. The hallmark of maternal meiosis is that two successive M phases (meiosis I and II) drive two rounds of asymmetric cell division (ACD). However, how the egg limits the number of meioses to just two, thereby preventing gross aneuploidy, is poorly characterized. Here, in urochordate eggs, we show that loss of Mos/MAPK activity is necessary to prevent entry into meiosis III. Remarkably, maintaining the Mos/MAPK pathway active after fertilization at near physiological levels induces additional rounds of meiotic M phase (meiosis III, IV and V). During these additional rounds of meiosis, the spindle is positioned asymmetrically resulting in further rounds of ACD. In addition, inhibiting meiotic exit with Mos prevents pronuclear formation, cyclin A accumulation and maintains sperm-triggered Ca2+ oscillations, all of which are hallmarks of the meiotic cell cycle in ascidians. It will be interesting to determine whether Mos availability in mammals can also control the number of meioses as it does in the urochordates. Our results demonstrate the power of urochordate eggs as a model to dissect the egg-to-embryo transition.

During mammalian oocyte maturation there are marked changes in the distribution of mitochondria that supply the majority of the cellular ATP. Such redistribution of mitochondria is critical for oocyte quality, as oocytes with a poor developmental potential display aberrant mitochondrial distribution and lower ATP levels. Here we have investigated the dynamics of mitochondrial ATP production throughout spontaneous mouse oocyte maturation, using live measurements of cytosolic and mitochondrial ATP levels. We have observed three distinct increases in cytosolic ATP levels temporally associated with discrete events of oocyte maturation. These changes in cytosolic ATP levels are mirrored by changes in mitochondrial ATP levels, suggesting that mitochondrial ATP production is stimulated during oocyte maturation. Strikingly, these changes in ATP levels correlate with the distribution of mitochondria undergoing translocation to the peri-nuclear region and aggregation into clusters. Mitochondrial clustering during oocyte maturation was concomitant with the formation of long cortical microfilaments and could be disrupted by cytochalasin B treatment. Furthermore, the ATP production bursts observed during oocyte maturation were also inhibited by cytochalasin B suggesting that mitochondrial ATP production is stimulated during oocyte maturation by microfilament-driven, sub-cellular targeting of mitochondria. J. Cell. Physiol. 224: 672–680, 2010. © 2010 Wiley-Liss, Inc.

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°/minute) and migrate (3 μm/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.

Mitochondria play a central and multifaceted role in the mammalian egg and early embryo, contributing to many different aspects of early development. While the contribution of mitochondria to energy production is fundamental, other roles for mitochondria are starting to emerge. Mitochondria are central to intracellular redox metabolism as they produce reactive oxygen species (ROS, the mediators of oxidative stress) and they can generate TCA cycle intermediates and reducing equivalents that are used in antioxidant defence. A high cytosolic lactate dehydrogenase activity coupled with dynamic levels of cytosolic pyruvate is responsible for a very dynamic intracellular redox state in the oocyte and embryo. Mammalian embryos have a low glucose metabolism during the earliest stages of development, as both glycolysis and the pentose phosphate pathway are suppressed. The mitochondrial TCA cycle is therefore the major source of reducing equivalents in the cytosol so that any change in mitochondrial function in the embryo will be reflected in changes in the intracellular redox state. In the mouse, the metabolic substrates used by the oocyte and early embryo each have a different impact on the intracellular redox state. Pyruvate which oxidises the cytosolic redox state, acts as an energetic and redox substrate whereas lactate, which reduces the cytosolic redox state, acts only as a redox substrate. Mammalian early embryos are very sensitive to oxidative stress which can cause permanent developmental arrest before zygotic genome activation and apoptosis in the blastocyst. The oocyte stockpiles antioxidant defence for the early embryo to cope with exogenous and endogenous oxidant insults arising during early development. Mitochondria provide ATP for glutathione (GSH) production during oocyte maturation and also participate in the regeneration of NADPH and GSH during early development. Finally, a number of pathological conditions or environmental insults impair early development by altering mitochondrial function, illustrating the centrality of mitochondrial function in embryo development.

Mitochondria have long been known to be the powerhouses of the cell but they also contribute to redox and Ca2+ homeostasis, provide intermediary metabolites and store proapoptotic factors. Mitochondria have a unique behavior during development. They are maternally transmitted with little (if any) paternal contribution, and they originate from a restricted founder population, which is amplified during oogenesis. Then, having established the full complement of mitochondria in the fully grown oocyte, there is no further increase of the mitochondrial population during early development. The localization of mitochondria in the egg during maturation and their segregation to blastomeres in the cleaving embryo are strictly regulated. Gradients in the distribution of mitochondria present in the egg have the potential to give rise to blastomeres receiving different numbers of mitochondria. Such maternally inherited differences in mitochondrial distribution are thought to play roles in defining the long-term viability of the blastomere in some cases and embryonic axes and patterning in others. Mitochondria may also regulate development by a number of other means, including modulating Ca2+ signaling, and the production of ATP, reactive oxygen species, and intermediary metabolites. If the participation of mitochondria in the regulation of sperm-triggered Ca2+ oscillations is now well established, the role of other properties of mitochondrial function during development remain largely unexplored probably due to the difficulty of accessing the mitochondrial compartment in an embryo. Maintaining a functional complement of maternally derived mitochondria is vital for the early embryo. Mitochondrial dysfunction may not only compromise developmental processes but also trigger apoptosis in the embryo. This dual role for mitochondria (to maintain life or to commit to cell death) may well represent a quality control system in the early embryo that will determine whether the embryo proceeds further into development or is quickly eliminated.

Mitochondria have long been known to be the powerhouses of the cell but they also contribute to redox and Ca2+ homeostasis, provide intermediary metabolites and store proapoptotic factors. Mitochondria have a unique behavior during development. They are maternally transmitted with little (if any) paternal contribution, and they originate from a restricted founder population, which is amplified during oogenesis. Then, having established the full complement of mitochondria in the fully grown oocyte, there is no further increase of the mitochondrial population during early development. The localization of mitochondria in the egg during maturation and their segregation to blastomeres in the cleaving embryo are strictly regulated. Gradients in the distribution of mitochondria present in the egg have the potential to give rise to blastomeres receiving different numbers of mitochondria. Such maternally inherited differences in mitochondrial distribution are thought to play roles in defining the long-term viability of the blastomere in some cases and embryonic axes and patterning in others. Mitochondria may also regulate development by a number of other means, including modulating Ca2+ signaling, and the production of ATP, reactive oxygen species, and intermediary metabolites. If the participation of mitochondria in the regulation of sperm-triggered Ca2+ oscillations is now well established, the role of other properties of mitochondrial function during development remain largely unexplored probably due to the difficulty of accessing the mitochondrial compartment in an embryo. Maintaining a functional complement of maternally derived mitochondria is vital for the early embryo. Mitochondrial dysfunction may not only compromise developmental processes but also trigger apoptosis in the embryo. This dual role for mitochondria (to maintain life or to commit to cell death) may well represent a quality control system in the early embryo that will determine whether the embryo proceeds further into development or is quickly eliminated.

International audience; The study of sperm egg interactions and the signals they trigger, have made remarkable progress from the time of the seminal discovery of calcium waves 30 years ago. In this special issue of Seminars in Cell and Developmental Biology, 3 short pieces and 10 expert reviews survey this recent past and brings us up to date with current developments in this fertile field. Jaffe, Steinhardt and Whitaker recall the historical years while Miyazaki, Dumas, Rubinstein, Swann, Fissore, Sato, Foltz, Stricker, and their co-authors bring us up to date with current developments in sperm egg interaction, egg activation and calcium signalling. Dumollard, Duchen and Sardet and Ducibella, Schultz and Ozil deal with the pleiotropic consequences of stereotyped calcium signals from the cytoplasmic and cortical reorganizations they trigger including the stimulation of mitochondrial respiration, cell cycle regulation, the activation of protein synthesis, and subsequent developmental events. A list of interesting web sites giving access to video archives of fertilization and calcium waves is also provided.

Fertilization triggers repetitive waves of cytosolic Ca(2+) in the egg of many species. The mechanism involved in the generation of Ca(2+) waves has been studied in much detail in mature ascidian eggs, by raising artificially the level of inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)] or of its poorly metabolizable analogue, glycero-myo-phosphatidylinositol 4,5-bisphosphate [gPtdIns(4,5)P(2)]. Here, we use this strategy and the experimental results it provides to develop a realistic theoretical model for repetitive Ca(2+) wave generation and propagation in mature eggs. The model takes into account the heterogeneous spatial distribution of the endoplasmic reticulum. Our results corroborate the hypothesis that Ca(2+) wave pacemakers are associated with cortical accumulations of endoplasmic reticulum. The model is first tested and validated by the adequate match between its theoretical predictions and the observed effects of localized injections of massive amounts of Ins(1,4,5)P(3) analogues. In a second step, we use the model to make some propositions about the possible characteristics of the sperm factor. We find that to account for the spatial characteristics of the first series of Ca(2+) waves seen at fertilization in ascidian eggs, it has to be assumed that, if the sperm factor is a phospholipase C, it is Ca(2+)-sensitive and highly diffusible. Although the actual state of knowledge does not allow us to explain the observed relocalization of the Ca(2+) wave pacemaker site, the model corroborates the assumption that PtdIns(4,5)P(2), the substrate for phospholipase C is distributed over the entire egg. We also predict that the dose of sperm factor injected into the egg should modulate the temporal characteristics of the first, long-lasting fertilization wave.

International audience; The egg of ascidians (urochordate), as virtually all animal and plant species, displays Ca 2+ signals upon fertilisation. These Ca 2+ signals are repetitive Ca 2+ waves that initiate in the cortex of the egg and spread through the whole egg interior. Two series of Ca 2+ waves triggered from two distinct Ca 2+ wave pacemakers entrain the two meiotic divisions preceding entry into the first interphase. The second messenger inositol (1,4,5) trisphosphate (IP3) is the main mediator of these global Ca 2+ waves. Other Ca 2+ signalling pathways (RyR and NAADPR) are functional in the egg but they mediate localised cortical Ca 2+ signals whose physiological significance remains unclear. The meiosis I Ca 2+ wave pacemaker is mobile and relies on intracellular Ca 2+ release from the endoplasmic reticulum (ER) induced by a large production of IP3 at the sperm aster site. The meiosis II Ca 2+ wave pacemaker is stably localised in a vegetal protrusion called the contraction pole. It is probable that a local production of IP3 in the contraction pole determines the site of this second pacemaker while functional interactions between ER and mitochondria regulate its activity. Finally, a third ectopic pacemaker can be induced by a global increase in IP3, making the ascidian egg a unique system where three different Ca 2+ wave pacemakers coexist in the same cell.

Fertilization increases both cytosolic Ca 2+ 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 Ca 2+ waves emitted by two pacemakers (PM1 and PM2). Oxygen consumption also increases in response to Ins(1,4,5) P 3 -induced Ca 2+ transients. Using mitochondrial inhibitors we show that active mitochondria sequester cytosolic Ca 2+ during sperm-triggered Ca 2+ waves and that they are strictly necessary for triggering and sustaining the activity of the meiotic Ca 2+ wave pacemaker PM2. Strikingly, the activity of the Ca 2+ 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 Ca 2+ , the egg9s mitochondria are stimulated by Ins(1,4,5) P 3 -mediated Ca 2+ signals. In turn, mitochondrial ATP production is required to sustain the activity of the meiotic Ca 2+ wave pacemaker PM2.

Calcium wave pacemakers in fertilized eggs of ascidians and mouse are associated with accumulations of cortical endoplasmic reticulum in the vegetal hemisphere. In ascidians, two distinct pacemakers (PM1 and PM2) generate two series of calcium waves necessary to drive meiosis I and II. Pacemaker PM2 is stably localized in a cortical ER accumulation situated in the vegetal contraction pole. We now find that pacemaker PM1 is situated in a cortical ER-rich domain that forms around the sperm aster and moves with it during the calcium-dependant cortical contraction triggered by the fertilizing sperm.Global elevations of inositol (1,4,5)-trisphosphate (Ins(1,4,5)P3) levels produced by caged Ins(1,4,5)P3 or caged glycero-myo-PtdIns(4,5)P2 photolysis reveal that the cortex of the animal hemisphere, also rich in ER-clusters, is the cellular region most sensitive to Ins(1,4,5)P3 and acts as a third type of pacemaker (PM3). Surprisingly, the artificial pacemaker PM3 predominates over the natural pacemaker PM2, located at the opposite pole. Microtubule depolymerization does not alter the activity nor the location of the three pacemakers. By contrast, blocking the acto-myosin driven cortical contraction with cytochalasin B prevents PM1 migration and inhibits PM2 activity. PM3, however, is insensitive to cytochalasin B.Our experiments suggest that the three distinct calcium wave pacemakers are probably regulated by different spatiotemporal variations in Ins(1,4,5)P3 concentration. In particular, the activity of the natural calcium wave pacemakers PM1 and PM2 depends on the apposition of a cortical ER-rich domain to a source of Ins(1,4,5)P3 production in the cortex.Movies available on-line