Intrinsic apoptosis is evolutionarily divergent among metazoans.

Apoptosis is regulated cell death that depends on caspases. A specific initiator caspase is involved upstream of each apoptotic signaling pathway. Characterized in nematode, fly, and mammals, intrinsic apoptosis is considered to be ancestral, conserved among animals, and depends on shared initiators: caspase-9, Apaf-1 and Bcl-2. However, the biochemical role of mitochondria, the pivotal function of cytochrome c and the modality of caspase activation remain highly heterogeneous and hide profound molecular divergence among apoptotic pathways in animals. Uncovering the phylogenetic history of apoptotic actors, especially caspases, is crucial to shed light on the evolutionary history of intrinsic apoptosis. Here, we demonstrate with phylogenetic analyses that caspase-9, the fundamental key of intrinsic apoptosis, is deuterostome-specific, while caspase-2 is ancestral to bilaterians. Our analysis of Bcl-2 and Apaf-1 confirms heterogeneity in functional organization of apoptotic pathways in animals. Our results support emergence of distinct intrinsic apoptotic pathways during metazoan evolution.

Extrinsic apoptosis participates to tail regression during the metamorphosis of the chordate Ciona.

Apoptosis is a regulated cell death ubiquitous in animals defined by morphological features depending on caspases. Two regulation pathways are described, currently named the intrinsic and the extrinsic apoptosis. While intrinsic apoptosis is well studied and considered ancestral among metazoans, extrinsic apoptosis is poorly studied outside mammals. Here, we address extrinsic apoptosis in the urochordates Ciona, belonging to the sister group of vertebrates. During metamorphosis, Ciona larvae undergo a tail regression depending on tissue contraction, migration and apoptosis. Apoptosis begin at the tail tip and propagates towards the trunk as a polarized wave. We identified Ci-caspase 8/10 by phylogenetic analysis as homolog to vertebrate caspases 8 and 10 that are the specific initiator of extrinsic apoptosis. We detected Ci-caspase 8/10 expression in Ciona larvae, especially at the tail tip. We showed that chemical inhibition of Ci-caspase 8/10 leads to a delay of tail regression, and Ci-caspase 8/10 loss of function induced an incomplete tail regression. The specificity between apoptotic pathways and initiator caspase suggests that extrinsic apoptosis regulates cell death during the tail regression. Our study presents rare in vivo work on extrinsic apoptosis outside mammals, and contribute to the discussion on its evolutionary history in animals.

Comparative transcriptomic analysis reveals gene regulation mediated by caspase activity in a chordate organism.

BACKGROUND: Apoptosis is a caspase regulated cell death present in all metazoans defined by a conserved set of morphological features. A well-described function of apoptosis is the removal of excessive cells during development and homeostasis. Recent studies have shown an unexpected signalling property of apoptotic cells, affecting cell fate and/or behaviour of neighbouring cells. In contrast to the apoptotic function of cell elimination, this new role of apoptosis is not well understood but seems caspase-dependent. To deepen our understanding of apoptotic functions, it is necessary to work on a biological model with a predictable apoptosis pattern affecting cell fate and/or behaviour. The tunicate Ciona intestinalis has a bi-phasic life cycle with swimming larvae which undergo metamorphosis after settlement. Previously, we have shown that the tail regression step during metamorphosis, characterized by a predictable polarized apoptotic wave, ensures elimination of most tail cells and controls primordial germ cells survival and migration. RESULTS: We performed differential transcriptomic analysis between control metamorphosing larvae and larvae treated with the pan-caspase inhibitor Z-VAD-fmk in order to explore the transcriptional control of apoptotic cells on neighbouring cells that survive and migrate. When caspase activity was impaired, genes known to be involved in metamorphosis were downregulated along with other implicated in cell migration and survival molecular pathways. CONCLUSION: We propose these results as a confirmation that apoptotic cells can control surrounding cells fate and as a reference database to explore novel apoptotic functions in animals, including those related to migration and differentiation.

Apoptosis and cell proliferation during metamorphosis of the planula larva of Clytia hemisphaerica (Hydrozoa, Cnidaria).

BACKGROUND: Metamorphosis in marine species is characterized by profound changes at the ecophysiological, morphological, and cellular levels. The cnidarian Clytia hemisphaerica exhibits a triphasic life cycle that includes a planula larva, a colonial polyp, and a sexually reproductive medusa. Most studies so far have focused on the embryogenesis of this species, whereas its metamorphosis has been only partially studied. RESULTS: We investigated the main morphological changes of the planula larva of Clytia during the metamorphosis, and the associated cell proliferation and apoptosis. Based on our observations of planulae at successive times following artificial metamorphosis induction using GLWamide, we subdivided the Clytia's metamorphosis into a series of eight morphological stages occurring during a pre-settlement phase (from metamorphosis induction to planula ready for settlement) and the post-settlement phase (from planula settlement to primary polyp). Drastic morphological changes prior to definitive adhesion to the substrate were accompanied by specific patterns of stem-cell proliferation as well as apoptosis in both ectoderm and endoderm. Further waves of apoptosis occurring once the larva has settled were associated with morphogenesis of the primary polyp. CONCLUSION: Clytia larval metamorphosis is characterized by distinct patterns of apoptosis and cell proliferation during the pre-settlement phase and the settled planula-to-polyp transformation.

Comparative study of Hippo pathway genes in cellular conveyor belts of a ctenophore and a cnidarian.

BACKGROUND: The Hippo pathway regulates growth rate and organ size in fly and mouse, notably through control of cell proliferation. Molecular interactions at the heart of this pathway are known to have originated in the unicellular ancestry of metazoans. They notably involve a cascade of phosphorylations triggered by the kinase Hippo, with subsequent nuclear to cytoplasmic shift of Yorkie localisation, preventing its binding to the transcription factor Scalloped, thereby silencing proliferation genes. There are few comparative expression data of Hippo pathway genes in non-model animal species and notably none in non-bilaterian phyla. RESULTS: All core Hippo pathway genes could be retrieved from the ctenophore Pleurobrachia pileus and the hydrozoan cnidarian Clytia hemisphaerica, with the important exception of Yorkie in ctenophore. Expression study of the Hippo, Salvador and Scalloped genes in tentacle "cellular conveyor belts" of these two organisms revealed striking differences. In P. pileus, their transcripts were detected in areas where undifferentiated progenitors intensely proliferate and where expression of cyclins B and D was also seen. In C. hemisphaerica, these three genes and Yorkie are expressed not only in the proliferating but also in the differentiation zone of the tentacle bulb and in mature tentacle cells. However, using an antibody designed against the C. hemiphaerica Yorkie protein, we show in two distinct cell lineages of the medusa that Yorkie localisation is predominantly nuclear in areas of active cell proliferation and mainly cytoplasmic elsewhere. CONCLUSIONS: This is the first evidence of nucleocytoplasmic Yorkie shift in association with the arrest of cell proliferation in a cnidarian, strongly evoking the cell division-promoting role of this protein and its inhibition by the activated Hippo pathway in bilaterian models. Our results furthermore highlight important differences in terms of deployment and regulation of Hippo pathway genes between cnidarians and ctenophores.

Metamorphosis in solitary ascidians.

Embryonic and postembryonic development in ascidians have been studied for over a century, but it is only in the last 10 years that the complex molecular network involved in coordinating postlarval development and metamorphosis has started to emerge. In most ascidians, the transition from the larval to the sessile juvenile/adult stage, or metamorphosis, requires a combination of environmental and endogenous signals and is characterized by coordinated global morphogenetic changes that are initiated by the adhesion of the larvae. Cloney was the first to describe cellular events of ascidians' metamorphosis in 1978 and only recently elements of the molecular regulation of this crucial developmental step have been revealed. This review aims to present a thorough view of this crucial developmental step by combining recent molecular data to the already established cellular events.

Release from meiotic arrest in ascidian eggs requires the activity of two phosphatases but not CaMKII.

The fertilising sperm triggers a transient Ca(2+) 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 Ca(2+) transient and its degradation is triggered by the Ca(2+)-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 Ca(2+) 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 Ca(2+)-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 Ca(2+) 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 PP2A inhibitor I2PP2A is essential for sister chromatid segregation in oocyte meiosis II.

Haploid gametes are generated through two consecutive meiotic divisions, with the segregation of chromosome pairs in meiosis I and sister chromatids in meiosis II. Separase-mediated stepwise removal of cohesion, first from chromosome arms and later from the centromere region, is a prerequisite for maintaining sister chromatids together until their separation in meiosis II [1]. In all model organisms, centromeric cohesin is protected from separase-dependent removal in meiosis I through the activity of PP2A-B56 phosphatase, which is recruited to centromeres by shugoshin/MEI-S332 (Sgo) [2-5]. How this protection of centromeric cohesin is removed in meiosis II is not entirely clear; we find that all the PP2A subunits remain colocalized with the cohesin subunit Rec8 at the centromere of metaphase II chromosomes. Here, we show that sister chromatid separation in oocytes depends on a PP2A inhibitor, namely I2PP2A. I2PP2A colocalizes with the PP2A enzyme at centromeres at metaphase II, independently of bipolar attachment. When I2PP2A is depleted, sister chromatids fail to segregate during meiosis II. Our findings demonstrate that in oocytes I2PP2A is essential for faithful sister chromatid segregation by mediating deprotection of centromeric cohesin in meiosis II.

Chromosome spreads with centromere staining in mouse oocytes.

This chapter describes a technique for performing chromosome spreads from mouse oocytes. It is based on a previously described protocol (Hodges and Hunt, Chromosoma 111: 165-169, 2002), which we have modified. Chromosomes are stained with either Propidium Iodide or Hoechst. This spreading technique allows for simultaneous immunostaining of proteins associated with chromosomes. It is very useful to stain spreads with CREST serum which labels kinetochores, to be able to distinguish bivalents (chromosome pairs), dyads or univalents (paired sister chromatids), and single sister chromatids without ambiguity.

Cyclin A2 is required for sister chromatid segregation, but not separase control, in mouse oocyte meiosis.

In meiosis, two specialized cell divisions allow the separation of paired chromosomes first, then of sister chromatids. Separase removes the cohesin complex holding sister chromatids together in a stepwise manner from chromosome arms in meiosis I, then from the centromere region in meiosis II. Using mouse oocytes, our study reveals that cyclin A2 promotes entry into meiosis, as well as an additional unexpected role; namely, its requirement for separase-dependent sister chromatid separation in meiosis II. Untimely cyclin A2-associated kinase activity in meiosis I leads to precocious sister separation, whereas inhibition of cyclin A2 in meiosis II prevents it. Accordingly, endogenous cyclin A is localized to kinetochores throughout meiosis II, but not in anaphase I. Additionally, we found that cyclin B1, but not cyclin A2, inhibits separase in meiosis I. These findings indicate that separase-dependent cohesin removal is differentially regulated by cyclin B1 and A2 in mammalian meiosis.

A critical balance between Cyclin B synthesis and Myt1 activity controls meiosis entry in Xenopus oocytes.

In fully grown oocytes, meiosis is arrested at first prophase until species-specific initiation signals trigger maturation. Meiotic resumption universally involves early activation of M phase-promoting factor (Cdc2 kinase-Cyclin B complex, MPF) by dephosphorylation of the inhibitory Thr14/Tyr15 sites of Cdc2. However, underlying mechanisms vary. In Xenopus oocytes, deciphering the intervening chain of events has been hampered by a sensitive amplification loop involving Cdc2-Cyclin B, the inhibitory kinase Myt1 and the activating phosphatase Cdc25. In this study we provide evidence that the critical event in meiotic resumption is a change in the balance between inhibitory Myt1 activity and Cyclin B neosynthesis. First, we show that in fully grown oocytes Myt1 is essential for maintaining prophase I arrest. Second, we demonstrate that, upon upregulation of Cyclin B synthesis in response to progesterone, rapid inactivating phosphorylation of Myt1 occurs, mediated by Cdc2 and without any significant contribution of Mos/MAPK or Plx1. We propose a model in which the appearance of active MPF complexes following increased Cyclin B synthesis causes Myt1 inhibition, upstream of the MPF/Cdc25 amplification loop.

Mos limits the number of meiotic divisions in urochordate eggs.

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 Ca(2+) 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.

ERK- and JNK-signalling regulate gene networks that stimulate metamorphosis and apoptosis in tail tissues of ascidian tadpoles.

In ascidian tadpoles, metamorphosis is triggered by a polarized wave of apoptosis, via mechanisms that are largely unknown. We demonstrate that the MAP kinases ERK and JNK are both required for the wave of apoptosis and metamorphosis. By employing a gene-profiling-based approach, we identified the network of genes controlled by either ERK or JNK activity that stimulate the onset of apoptosis. This approach identified a gene network involved in hormonal signalling, in innate immunity, in cell-cell communication and in the extracellular matrix. Through gene silencing, we show that Ci-sushi, a cell-cell communication protein controlled by JNK activity, is required for the wave of apoptosis that precedes tail regression. These observations lead us to propose a model of metamorphosis whereby JNK activity in the CNS induces apoptosis in several adjacent tissues that compose the tail by inducing the expression of genes such as Ci-sushi.

Fertilization regulates apoptosis of Ciona intestinalis extra-embryonic cells through thyroxine (T4)-dependent NF-kappaB pathway activation during early embryonic development.

In Ciona intestinalis, the elimination of extra-embryonic test cells during early stage of development is delayed by a fertilization signal. Test cells undergo a caspase-dependent apoptosis event repressed by thyroxine (T4)-activated NF-kappaB. When apoptosis was experimentally blocked, the hatching stage was delayed. The incubation of unfertilized eggs with a 1-h-fertilized egg extract or purified T4 restored apoptosis in test cells at a similar timing than found in fertilized eggs. Ciona expresses specific genes forming a functional IkappaB/NF-kappaB pathway. One, Ci-p65, was transiently induced upon fertilization via T4 and found to exert its anti-apoptotic role in test cells nuclei as well as in a reconstituted cell system. Blocking NF-kappaB activity by dexamethasone-induced overexpression of Ci-IkappaB abrogated the repression of apoptosis in test cells. Overall, the data are consistent for defining a central coupling role of both T4 and NF-kappaB during early embryo development.

Ascidians as a vertebrate-like model organism for physiological studies of Rho GTPase signaling.

GTPases of the Rho family are evolutionarily conserved proteins that control cell shape dynamics during physiological processes as diverse as cell migration and polarity, axon outgrowth and guidance, apoptosis and phagocytosis. In mammals, 18 Rho proteins are distributed in 7 subfamilies. Rho, Rac and Cdc42 are the best-characterized ones, benefiting from the use of worm and drosophila, which only express these 3 subfamilies. An additional model would therefore help understand the physiological role of other mammalian subfamilies. We identified in genome databases the complete Rho family of two ascidians, Ciona intestinalis and Ciona savignyi, and showed that these families contain single ancestors of most mammalian Rho subfamilies. In Ciona intestinalis, all Rho genes are expressed and display specific developmental variations of mRNA expression during tadpole formation. Although C. intestinalis expresses five additional Rac compared to the closely related Ciona savignyi, only two appeared fully active in functional assays. Last, we identified in Ciona intestinalis database more than 50 Rho regulators (RhoGEFs and RhoGAPs) and 20 effector targets, whose analysis further supports the notion that Rho signaling components are of comparable complexity in mammals and ascidians. Since the tadpole of ascidians combines vertebrate-like developmental features with reduced cell number, particularly adapted to evolutionary and developmental biology studies, our data advocate this model for physiological studies of Rho signaling pathways.

Tail regression in Ciona intestinalis (Prochordate) involves a Caspase-dependent apoptosis event associated with ERK activation.

Two apoptotic events take place during embryonic development of Ciona intestinalis. The first concerns extra-embryonic cells and precedes hatching. The second controls tail regression at metamorphosis, occurs through a polarized wave originating from tail extremity, and is caspase dependent. This was shown by: (1) in vivo incorporation of a fluorescent marker of caspase activation in different cell types of the tail; (2) detection of an activated form of caspase 3-like protein by western blotting; and (3) failure of 30% of larvae to undergo metamorphosis after treatment of fertilized eggs with a pan-caspase inhibitor. In addition, Ciona embryos express a single ERK protein, specifically phosphorylated at metamorphosis. ERK activation was shown to be located in cells of the tail. Addition of MEK inhibitor in the culture medium prevented ERK activation and metamorphosis. In silico analysis of Ciona genome pointed to 15 caspases with high homology with humans, and a single ERK gene with high homology to both mammalian ERK1 and ERK2. It is concluded that the sequence of events leading to metamorphosis includes ERK phosphorylation followed by caspase-dependent apoptosis and tail regression.