The oocyte is a cell of unique importance for all sexual reproducing animals, providing a haploid ge-nome, all the cellular constituents and the ground plan for embryonic development. Tight regulation of cytoplasmic, cytoskeletal and nuclear events during successive steps of oogenesis is thus essen-tial to avoid both infertility and defects in embryo development. Much of this regulation remains un-derstudied, with a significant limiting factor being experimental access to oocytes developing within the female gonad. An exciting route to overcome such limitations is currently being opened by alter-native model organisms, notably ones selected from marine environments, offering a huge range of natural diversity. Marine organisms are typically well suited for imaging approaches, however particu-lar constraints relating to their cool, salt-water environment currently restrict the application of ad-vanced imaging technologies. Our interdisciplinary project aims to overcome these constraints by developing an innovative, tailored light sheet imaging system. We will use this to exploit a jellyfish model, Clytia hemisphaerica, that has high but as yet untapped potential for experimental manipulation and live imaging of oogenesis with-in the completely transparent animal or in autonomously functioning isolated ovaries. Our research plan includes two technical innovation work-packages and two that address oocyte animal-vegetal polarity establishment, a key process that presages the larval body plan: In WP1 we will develop adaptable custom light sheet microscopes with integrated image acquisition and analysis workflows tailored for marine organisms. In WP2 we will build a suite of molecular tools for expression of tagged proteins and mRNAs during Clytia oogenesis, including transgenic lines. In WP3 we will use these tools in live Clytia ovaries to visualise and dissect functionally how the microtubule cytoskeleton establishes animal-vegetal polarity during the growth phase of oogenesis, focussing on the reposi-tioning of the nucleus and localisation of Fz1 mRNA, one of three key Wnt-pathway axis determi-nants. In WP4 we will address the mechanisms driving segregation of the two other determinant mRNAs, Fz3 and Wnt3, to opposite cortical domains during oocyte meiotic maturation. For WPs 3 and 4 we will image the cytoskeleton, mRNAs and other key cellular components live in 3D with high spa-tial and temporal resolution through oocyte growth and maturation. We will combine these with mo-lecular perturbations using inhibitors, morpholinos and genetic tools, as well as physical micromanipu-lations. Our findings will reveal key conserved mechanisms and also illuminate their evolutionary history. More widely, this project will make a significant contribution to oogenesis research by establishing Clytia as a powerful model system, and provide tailored light sheet microscopes to exploit the potential of ma-rine model organisms for mechanistic studies in biological research.

Cell division is a crucial aspect of cellular physiology and plays a pivotal role in biological processes, such as reproduction, development, tissue repair and growth. The cellular choice to commit to division is a complex decision that is driven by various molecular signaling pathways. One of the emerging layers involved in this decision making is de novo protein translation, which provides new proteins required for cell division. Despite its importance, the regulation of de novo protein translation during cell division remains poorly understood. The study of this process has been limited by the intricate interplay between transcription and translation regulations, making it challenging to isolate the effects of specific changes in the translation program on cell division. To overcome this drawback, the OffOnCYTE project aims to use meiotic division as a cell division paradigm and Xenopus laevis oocytes as an experimental system. In response to an external physiological stimulus, vertebrate oocytes undergo a translation reprogramming that triggers entry into M-phase, the phase of cell division. This translation reprogramming occurs in the absence of transcription, making it an ideal system for studying the regulation of de novo protein translation during cell division. Xenopus laevis oocytes are giant cells, containing high amounts of proteins and RNAs and easy to be microinjected, therefore highly suitable for studying protein translation. Thanks to these features, this model system provides the unique opportunity to combine single-cell approaches with large-scale biochemical and genomic techniques. By coupling unbiased genome-wide approaches and functional studies, the OffOnCYTE project aims to unravel how the cell decision to enter M-phase is coded into a translation switch. Specifically, the project will identify novel mRNAs whose translation controls the entry into cell division and will highlight new molecular mechanisms regulating mRNA translation. To achieve these goals, the OffOnCYTE project will employ a range of cutting-edge techniques, including ribosome profiling, mass spectrometry and RNA sequencing. Coupled with well-established functional approaches in the Xenopus oocyte, these techniques will allow to comprehensively analyze changes in the translation program during cell division and to identify the key molecular players that regulate this process. Overall, the OffOnCYTE project will provide important insights into the regulation of de novo protein translation that triggers cell division. The understanding of the molecular mechanisms that control this process will pave the way for the development of new strategies for controlling cell division in various biological contexts, such as oncology and biology of reproduction.

Mitochondria are essential for cells and organisms’ viability through the mitochondrial functions for energy supply and several signaling pathways (apoptosis, calcium signaling). Despite a small number of genes on the mitochondrial genome (mtDNA), when mutated, it’s associated with a large spectrum of severe mitochondrial diseases with complex transmission modes as a consequence of mtDNA heredity and the impact of the fraction of mutated genomes. Despite the conservation in evolution of the transmission of mtDNA as uniparental and maternal, the mechanisms preserving homoplasmy against challenging threats, like the entry of sperm mtDNA at fertilization or the appearance of mutations, are still largely uncharacterized. The impact of disturbing the uniparental and maternal inheritance are still unclear mostly due to the lack of available model(s) for bi-parental inheritance. We will address these two long-standing fundamental questions by the engineering and the characterization of powerful and unique experimental model(s) for biparental mitochondria inheritance in C. elegans. This new animal model(s) will arise from an unbiased genetic screen. Thanks to a positive selection strategy will identify mutant males able to transmit their sperm mtDNA to the descendance. We will then have access and test, for the first time, the functional consequences of biparental mitochondria inheritance over generations. Beyond the fundamental knowledge provided by this work, our experimental model(s) of biparental mitochondria heredity will give us the critical knowledge to predict the potential consequences of advanced methods in animal cloning, medically assisted reproduction and therapies to cure mitochondrial diseases. Our results will therefore very likely receive a lot of attention from the scientific and public audiences.

The reshuffling of nuclear architecture and chromatin landscape is a recurring theme orchestrated in most developmental transitions. Plants display extraordinary capacities enabling them to adjust such transitions to external cues, an adaptive feature at the nexus of their high cell fate plasticity and fitness in changing environments - especially with regard to light conditions. To tackle the fundamental determinants of genome regulation and their declinations in plants, our long-term research axis exploits Arabidopsis photomorphogenesis, a developmental switch that initiates upon the initial light perception by a germinating seedling. We recently unveiled that the transition involves 1) the release from a transcriptionally quiescent status in darkness, 2) nucleus expansion, 3) the formation of subnuclear heterochromatic foci (chromocenters) aggregating most silent repeats, 4) wide changes in the epigenome landscape linked to gene expression reprogramming. In recent collaborative studies with the University of Zurich, we further identified that light sensing induces non-random changes in genome 3D topology and the formation of transcription clusters near the nucleus periphery. This transition being synchronously achieved in most cells of embryonic leaves constitutes a dynamic system perfectly suited to tackle the questions addressed by the ChromatinPhotoDynamics proposal. One such objective is to decipher the molecular frameworks that integrate light signaling at chromatin to reprogram genes during the photomorphogenic transition using both candidate factors and agnostic approaches. The second task is to determine how the sequential orchestration of heterochromatin rearrangements, 3D changes in genome topology, and epigenome dynamics, interplay to modulate both the general transcriptional regime and gene-specific expression patterns. The project further pertains to evolutionary questions on how variation in genome sequence and/or the epigenome contribute to nucleus organization “acclimation”, thereby opening long-term perspectives on the chromatin-level functions subjected to environmental and genetic constraints. Addressing both plant-specific and conserved chromatin regulation pathways, this study will contribute to current intense efforts devoted to deciphering the mechanisms underpinning cellular and organismal adaptive responses.

Cells proliferate by means of the mitotic cell cycle, a process underlying growth and development in all living organisms. Entry into mitosis depends on the activation of MPF (M-phase Promoting Factor, or Cdk1-cyclin B complex), the universal mitotic inducer in eukaryotic cells, which induces the phosphorylation of numerous proteins implicated in mitosis progression. Understanding the control of the cell cycle is pivotal to diverse and important biological problems and has thus fascinated biologists for many years. Despite this long-standing preoccupation, there is a surprising lack of knowledge on key points of the cell cycle, notably the control the G2-M transition. The OOCAMP project aims to improve our understanding of the G2-M transition by a physiological approach using oocytes of an amphibian (Xenopus) and a cnidarian (the jellyfish Clytia), simple and powerful experimental systems characterised by natural cell cycle arrest points released by defined stimuli to induce cell cycle progression. During oogenesis, oocytes enter meiosis and stop in prophase of the 1st meiotic division (equivalent to G2). External stimuli release this arrest to promote oocyte maturation by initiating signal transduction pathways, ultimately activating MPF. cAMP is a key immediate regulator, but intriguingly it acts in opposite ways in different species, creating the cAMP paradox. In vertebrates, cytoplasmic cAMP concentrations, and consequently activity of the kinase PKA, is maintained high in the arrested oocyte and must be down regulated to promote meiotic resumption (a situation reminiscent of the cAMP control exerted on the G2 phase of the mitotic cell cycle). In contrast, in many invertebrates including Clytia, elevation of cAMP levels and PKA activity are required to promote release from the G2 block. We plan to gain insight into cAMP-based regulation by comparing and cross-testing molecular regulation of cAMP modulators and targets between powerful experimental model species, Xenopus and Clytia, representing the two opposite modes of cAMP action. We will specifically address two key unsolved questions: the nature of the receptor systems that respond to oocyte maturation stimuli upstream of cAMP, and the identity of the PKA substrate that provokes MPF activation downstream of cAMP. Our approach will involve classical biochemical and molecular biology manipulations of candidate participant proteins combined with microinjection into oocytes of both species and monitoring of the physiological and molecular responses by microscopy and biochemical assays. We will focus on a small protein called ARPP19 (or ENSA), indicated by our preliminary results in Xenopus to be an excellent candidate for the key element maintaining prophase arrest as a PKA substrate. A distinct phosphorylation of ARPP19 triggered by the Greatwall kinase converts ARPP19 into a powerful MPF activator, essential for M-phase entry. We have identified a Clytia ortholog of ARPP19 with conserved Gwl and PKA phosphorylation sites, which will be functionally compared with its Xenopus counterparts at the cellular and molecular levels. In a parallel study we will attempt to identify the receptor of the peptide that stimulates oocyte maturation in Clytia by a novel transcriptomics-approach. The two OOCAMP partners, both with confirmed experience in the study of meiotic maturation, show excellent complementarity both in their favored animal models and their technical approaches and expertise (biochemical/molecular vs cell biology/imaging), which will drive the accomplishment of the project. To conclude, the OOCAMP program aims at understanding the biochemical systems that connect the extracellular maturation-inducing stimuli and the activation of MPF. The study of this key physiological process should continue to provide important contributions to knowledge of the hormonal regulation of reproduction, cell cycle regulation, signal transduction and mechanisms of oncogenesis.