Henri de Lacaze-Duthiers and the ascidian hypothesis.

In 1830, Cuvier and Geoffroy Saint-Hilaire confronted each other in a famous debate on the unity of the animal kingdom, which permeated the zoology of the 19th century. From that time, a growing number of naturalists attempted to understand the large-scale relationships among animals. And among all the questions, that of the origin of vertebrates was one of the most controversial. Analytical methods based on comparative anatomy, embryology and paleontology were developed to identify convincing homologies that would reveal a logical sequence of events for the evolution of an invertebrate into the first vertebrate. Within this context, several theories have clashed on the question of the identity of the ancestor of vertebrates. Among the proposals, a group of rather discrete organisms, the ascidians, played a central role. Because he had discovered an ascidian with a particularly atypical larval development, the Molgula, Henri de Lacaze-Duthiers, a rigorous and meticulous naturalist, became involved in the ascidian hypothesis. While the visionary mind of Lacaze-Duthiers led him to establish a particularly innovative methodology and the first marine biology station in Europe, at Roscoff, the tailless tadpole of the Molgula prevented him from recognizing the ancestor of vertebrates. This old 19th century story echoes the ever-present questions driving the field of Eco-Evo-Devo.

Control of Cdc6 accumulation by Cdk1 and MAPK is essential for completion of oocyte meiotic divisions in Xenopus.

Vertebrate oocytes proceed through the first and the second meiotic division without an intervening S-phase to become haploid. Although DNA replication does not take place, unfertilized oocytes acquire the competence to replicate DNA one hour after the first meiotic division by accumulating an essential factor of the replicative machinery, Cdc6. Here, we show that the turnover of Cdc6 is precisely regulated in oocytes to avoid inhibition of Cdk1. At meiosis resumption, Cdc6 is expressed but cannot accumulate owing to a degradation mechanism that is activated through Cdk1. During transition from the first to the second meiotic division, Cdc6 is under the antagonistic regulation of B-type cyclins (which interact with and stabilize Cdc6) and the Mos-MAPK pathway (which negatively controls Cdc6 accumulation). Because overexpressing Cdc6 inhibits Cdk1 reactivation and drives oocytes into a replicative interphasic state, the fine-tuning of Cdc6 accumulation is essential to ensure two meiotic waves of Cdk1 activation and to avoid unscheduled DNA replication during meiotic maturation.

The phosphorylation of ARPP19 by Greatwall renders the auto-amplification of MPF independently of PKA in Xenopus oocytes.

Entry into mitosis or meiosis relies on the coordinated action of kinases and phosphatases that ultimately leads to the activation of Cyclin-B-Cdk1, also known as MPF for M-phase promoting factor. Vertebrate oocytes are blocked in prophase of the first meiotic division, an arrest that is tightly controlled by high PKA activity. Re-entry into meiosis depends on activation of Cdk1, which obeys a two-step mechanism: a catalytic amount of Cdk1 is generated in a PKA and protein-synthesis-dependent manner; then a regulatory network known as the MPF auto-amplification loop is initiated. This second step is independent of PKA and protein synthesis. However, none of the molecular components of the auto-amplification loop identified so far act independently of PKA. Therefore, the protein rendering this process independent of PKA in oocytes remains unknown. Using a physiologically intact cell system, the Xenopus oocyte, we show that the phosphorylation of ARPP19 at S67 by the Greatwall kinase promotes its binding to the PP2A-B55δ phosphatase, thus inhibiting its activity. This process is controlled by Cdk1 and has an essential role within the Cdk1 auto-amplification loop for entry into the first meiotic division. Moreover, once phosphorylated by Greatwall, ARPP19 escapes the negative regulation exerted by PKA. It also promotes activation of MPF independently of protein synthesis, provided that a small amount of Mos is present. Taken together, these findings reveal that PP2A-B55δ, Greatwall and ARPP19 are not only required for entry into meiotic divisions, but are also pivotal effectors within the Cdk1 auto-regulatory loop responsible for its independence with respect to the PKA-negative control.

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.

Henri de Lacaze-Duthiers (1821-1901), the father of experimental zoology and founder of the marine stations of Roscoff and Banyuls.

On the occasion of the 200th anniversary of the birth of Henri de Lacaze-Duthiers, one of the most curious and active scientific minds among 19th century naturalists, this article retraces his scientific career and recalls the long-term changes he made in the practice of science: promotion of experimental zoology, foundation of a modern scientific journal and establishment of the marine stations of Roscoff and Banyuls.

À l'occasion du 200ème anniversaire de la naissance de Henri de Lacaze-Duthiers, l'un des esprits scientifiques les plus curieux et les plus actifs parmi les naturalistes du XIXe siècle, cet article retrace sa carrière scientifique et rappelle les tournants qu'il a durablement imprimés à la pratique de la science : promotion de la zoologie expérimentale, fondation dune revue scientifique moderne et édification des stations marines de Roscoff et de Banyuls.

The M-phase regulatory phosphatase PP2A-B55δ opposes protein kinase A on Arpp19 to initiate meiotic division.

Oocytes are held in meiotic prophase for prolonged periods until hormonal signals trigger meiotic divisions. Key players of M-phase entry are the opposing Cdk1 kinase and PP2A-B55δ phosphatase. In Xenopus, the protein Arpp19, phosphorylated at serine 67 by Greatwall, plays an essential role in inhibiting PP2A-B55δ, promoting Cdk1 activation. Furthermore, Arpp19 has an earlier role in maintaining the prophase arrest through a second serine (S109) phosphorylated by PKA. Prophase release, induced by progesterone, relies on Arpp19 dephosphorylation at S109, owing to an unknown phosphatase. Here, we identified this phosphatase as PP2A-B55δ. In prophase, PKA and PP2A-B55δ are simultaneously active, suggesting the presence of other important targets for both enzymes. The drop in PKA activity induced by progesterone enables PP2A-B55δ to dephosphorylate S109, unlocking the prophase block. Hence, PP2A-B55δ acts critically on Arpp19 on two distinct sites, opposing PKA and Greatwall to orchestrate the prophase release and M-phase entry.

Identification of structural and functional O-linked N-acetylglucosamine-bearing proteins in Xenopus laevis oocyte.

O-Linked N-acetylglucosaminylation (O-GlcNAcylation) (or O-linked N-acetylglucosamine (O-GlcNAc)) is an abundant and reversible glycosylation type found within the cytosolic and the nuclear compartments. We have described previously the sudden O-GlcNAcylation increase occurring during the Xenopus laevis oocyte G(2)/M transition, and we have demonstrated that the inhibition of O-GlcNAc-transferase (OGT) blocked this process, showing that the O-GlcNAcylation dynamism interferes with the cell cycle progression. In this work, we identified proteins that are O-GlcNAc-modified during the G(2)/M transition. Because of a low expression of O-GlcNAcylation in Xenopus oocyte, classical enrichment of O-GlcNAc-bearing proteins using O-GlcNAc-directed antibodies or wheat germ agglutinin lectin affinity were hard to apply, albeit these techniques allowed the identification of actin and erk2. Therefore, another strategy based on an in vitro enzymatic labeling of O-GlcNAc residues with azido-GalNAc followed by a chemical addition of a biotin alkyne probe and by enrichment of the tagged proteins on avidin beads was used. Bound proteins were analyzed by nano-LC-nano-ESI-MS/MS allowing for the identification of an average of 20 X. laevis oocyte O-GlcNAcylated proteins. In addition to actin and beta-tubulin, we identified metabolic/functional proteins such as PP2A, proliferating cell nuclear antigen, transitional endoplasmic reticulum ATPase, aldolase, lactate dehydrogenase, and ribosomal proteins. This labeling allowed for the mapping of a major O-GlcNAcylation site within the 318-324 region of beta-actin. Furthermore immunofluorescence microscopy enabled the direct visualization of O-GlcNAcylation and OGT on the meiotic spindle as well as the observation that chromosomally bound proteins were enriched in O-GlcNAc and OGT. The biological relevance of this post-translational modification both on microtubules and on chromosomes remains to be determined. However, the mapping of the O-GlcNAcylation sites will help to underline the function of this post-translational modification on each identified protein and will provide a better understanding of O-GlcNAcylation in the control of the cell cycle.

Managing the Oocyte Meiotic Arrest-Lessons from Frogs and Jellyfish.

During oocyte development, meiosis arrests in prophase of the first division for a remarkably prolonged period firstly during oocyte growth, and then when awaiting the appropriate hormonal signals for egg release. This prophase arrest is finally unlocked when locally produced maturation initiation hormones (MIHs) trigger entry into M-phase. Here, we assess the current knowledge of the successive cellular and molecular mechanisms responsible for keeping meiotic progression on hold. We focus on two model organisms, the amphibian Xenopus laevis, and the hydrozoan jellyfish Clytia hemisphaerica. Conserved mechanisms govern the initial meiotic programme of the oocyte prior to oocyte growth and also, much later, the onset of mitotic divisions, via activation of two key kinase systems: Cdk1-Cyclin B/Gwl (MPF) for M-phase activation and Mos-MAPkinase to orchestrate polar body formation and cytostatic (CSF) arrest. In contrast, maintenance of the prophase state of the fully-grown oocyte is assured by highly specific mechanisms, reflecting enormous variation between species in MIHs, MIH receptors and their immediate downstream signalling response. Convergence of multiple signalling pathway components to promote MPF activation in some oocytes, including Xenopus, is likely a heritage of the complex evolutionary history of spawning regulation, but also helps ensure a robust and reliable mechanism for gamete production.

The G2-to-M transition from a phosphatase perspective: a new vision of the meiotic division.

Cell division is orchestrated by the phosphorylation and dephosphorylation of thousands of proteins. These post-translational modifications underlie the molecular cascades converging to the activation of the universal mitotic kinase, Cdk1, and entry into cell division. They also govern the structural events that sustain the mechanics of cell division. While the role of protein kinases in mitosis has been well documented by decades of investigations, little was known regarding the control of protein phosphatases until the recent years. However, the regulation of phosphatase activities is as essential as kinases in controlling the activation of Cdk1 to enter M-phase. The regulation and the function of phosphatases result from post-translational modifications but also from the combinatorial association between conserved catalytic subunits and regulatory subunits that drive their substrate specificity, their cellular localization and their activity. It now appears that sequential dephosphorylations orchestrated by a network of phosphatase activities trigger Cdk1 activation and then order the structural events necessary for the timely execution of cell division. This review discusses a series of recent works describing the important roles played by protein phosphatases for the proper regulation of meiotic division. Many breakthroughs in the field of cell cycle research came from studies on oocyte meiotic divisions. Indeed, the meiotic division shares most of the molecular regulators with mitosis. The natural arrests of oocytes in G2 and in M-phase, the giant size of these cells, the variety of model species allowing either biochemical or imaging as well as genetics approaches explain why the process of meiosis has served as an historical model to decipher signalling pathways involved in the G2-to-M transition. The review especially highlights how the phosphatase PP2A-B55δ critically orchestrates the timing of meiosis resumption in amphibian oocytes. By opposing the kinase PKA, PP2A-B55δ controls the release of the G2 arrest through the dephosphorylation of their substrate, Arpp19. Few hours later, the inhibition of PP2A-B55δ by Arpp19 releases its opposing kinase, Cdk1, and triggers M-phase. In coordination with a variety of phosphatases and kinases, the PP2A-B55δ/Arpp19 duo therefore emerges as the key effector of the G2-to-M transition.

Translational Control of Xenopus Oocyte Meiosis: Toward the Genomic Era.

The study of oocytes has made enormous contributions to the understanding of the G2/M transition. The complementarity of investigations carried out on various model organisms has led to the identification of the M-phase promoting factor (MPF) and to unravel the basis of cell cycle regulation. Thanks to the power of biochemical approaches offered by frog oocytes, this model has allowed to identify the core signaling components involved in the regulation of M-phase. A central emerging layer of regulation of cell division regards protein translation. Oocytes are a unique model to tackle this question as they accumulate large quantities of dormant mRNAs to be used during meiosis resumption and progression, as well as the cell divisions during early embryogenesis. Since these events occur in the absence of transcription, they require cascades of successive unmasking, translation, and discarding of these mRNAs, implying a fine regulation of the timing of specific translation. In the last years, the Xenopus genome has been sequenced and annotated, enabling the development of omics techniques in this model and starting its transition into the genomic era. This review has critically described how the different phases of meiosis are orchestrated by changes in gene expression. The physiological states of the oocyte have been described together with the molecular mechanisms that control the critical transitions during meiosis progression, highlighting the connection between translation control and meiosis dynamics.

The greatwall kinase is dominant over PKA in controlling the antagonistic function of ARPP19 in Xenopus oocytes.

The small protein ARPP19 plays a dual role during oocyte meiosis resumption. In Xenopus, ARPP19 phosphorylation at S109 by PKA is necessary for maintaining oocytes arrested in prophase of the first meiotic division. Progesterone downregulates PKA, leading to the dephosphorylation of ARPP19 at S109. This initiates a transduction pathway ending with the activation of the universal inducer of M-phase, the kinase Cdk1. This last step depends on ARPP19 phosphorylation at S67 by the kinase Greatwall. Hence, phosphorylated by PKA at S109, ARPP19 restrains Cdk1 activation while when phosphorylated by Greatwall at S67, ARPP19 becomes an inducer of Cdk1 activation. Here, we investigate the functional interplay between S109 and S67-phosphorylations of ARPP19. We show that both PKA and Gwl phosphorylate ARPP19 independently of each other and that Cdk1 is not directly involved in regulating the biological activity of ARPP19. We also show that the phosphorylation of ARPP19 at S67 that activates Cdk1, is dominant over the inhibitory S109 phosphorylation. Therefore our results highlight the importance of timely synchronizing ARPP19 phosphorylations at S109 and S67 to fully activate Cdk1.

Centrosome overduplication, increased ploidy and transformation in cells expressing endoplasmic reticulum-associated cyclin A2.

Cyclin A2 is predominantly, but not exclusively, localized in the nucleus from G1/S transition onwards. It is degraded when cells enter mitosis after nuclear envelope breakdown. We previously showed that a fusion protein (S2A) between the hepatitis B virus (HBV) surface antigen protein and a non-degradable fragment of human cyclin A2 (Delta152) resides in the endoplasmic reticulum membranes, escapes degradation and transforms normal rat fibroblasts. The present study investigates whether cytoplasmic cyclin A2 may play a role in oncogenesis. We show that the sequestration of non-degradable cyclin A2-Delta152 by a cellular ER targeting domain (PRL-A2) leads to cell transformation when coexpressed with activated Ha-ras. REF52 cells constitutively expressing PRL-A2 are found to have a high incidence of multinucleate giant cells, polyploidy and abnormal centrosome numbers, giving rise to the nucleation of multipolar spindles. Injection of these cells into athymic nude mice causes tumors, even in the absence of a cooperating Ha-ras oncogene. These results demonstrate that, independently of any viral context, an intracellular redistribution of non-degradable cyclin A2 is capable of deregulating the normal cell cycle to the point where it promotes aneuploidy and cancer.

Xenopus H-RasV12 promotes entry into meiotic M phase and cdc2 activation independently of Mos and p42(MAPK).

In the Xenopus oocyte, progesterone triggers M phase Promoting Factor (MPF) activation in a protein synthesis dependent manner. Although the synthesis of the p42(MAPK) activator Mos appears to be required for MPF activation, p42(MAPK) activity has been shown to be dispensable. To clarify this paradox, we attempted to activate the p42(MAPK) pathway independently of Mos synthesis by cloning and using Xenopus H-Ras in the oocyte. We demonstrate that the injection of the constitutively active Xe H-RasV12 mutant induces p42(MAPK) and MPF activation through two independent pathways. Xe H-RasV12 induces only a partial activation of p42(MAPK) when protein synthesis and MPF activation are prevented. A full level of p42(MAPK) activation is reached when MPF is activated and Mos is present. In contrast, MPF activation induced by Xe H-RasV12 is achieved independently of Mos synthesis and p42(MAPK) activation but still depends on protein synthesis. Therefore, the amphibian oocyte represents a new model system to analyse an original H-Ras pathway ending to MPF activation and distinct from the p42(MAPK) pathway. The identification of the proteins synthesized in response to Xe H-RasV12 and required for MPF activation, represents an important clue in understanding the mechanism of progesterone action.

Phosphorylation of ARPP19 by protein kinase A prevents meiosis resumption in Xenopus oocytes.

During oogenesis, oocytes are arrested in prophase and resume meiosis by activating the kinase Cdk1 upon hormonal stimulation. In all vertebrates, release from prophase arrest relies on protein kinase A (PKA) downregulation and on the dephosphorylation of a long sought but still unidentified substrate. Here we show that ARPP19 is the PKA substrate whose phosphorylation at serine 109 is necessary and sufficient for maintaining Xenopus oocytes arrested in prophase. By downregulating PKA, progesterone, the meiotic inducer in Xenopus, promotes partial dephosphorylation of ARPP19 that is required for the formation of a threshold level of active Cdk1. Active Cdk1 then initiates the MPF autoamplification loop that occurs independently of both PKA and ARPP19 phosphorylation at serine 109 but requires the Greatwall (Gwl)-dependent phosphorylation of ARPP19 at serine 67. Therefore, ARPP19 stands at a crossroads in the meiotic M-phase control network by integrating differential effects of PKA and Gwl, two kinases essential for meiosis resumption.