Our consortium combines the expertise of 3 partners in developmental and cellular biology, mouse and human genetics. We aim to decipher the role of the ADAR1 enzyme and Adenosine to Inosine (A-to-I) RNA editing in the development of Schwann cells and in the maintenance of myelin of the peripheral nervous system (PNS), both in normal and pathological conditions. Catalyzed by enzymes of the ADAR family, A-to-I deamination is one of the most prevalent form of RNA base modification in mammals. Because Inosines are subsequently recognized as Guanosines, this modification is a well-known contributor to transcriptomic and, to a lesser extent, proteomic diversity. To date, ADAR1 dysfunction was shown to induce recognition of unedited RNAs by cytosolic sensors, Mda5 in particular, subsequent upregulation of hundreds of interferon (IFN)-stimulated genes (ISG) expression, and cell death. On line with this model, Partner 3 discovered ADAR1 mutations as a cause of Aicardi-Goutières syndrome (a genetically determined inflammatory encephalopathy, ascribed to the group of type 1 interferonopathies). Heterozygous mutations in this gene also lead to dyschromatosis symmetrica hereditaria (characterized by hyper- and hypo-pigmented macules). These pigmentation defects suggested an important role for ADAR1 during the development of melanin-producing neural crest (NC)-derived melanocytes, but the role of this enzyme in other NC derivatives, including myelinating-Schwann cells, remained to be defined. To explore this possibility, partner 1 (P1) generated a mouse model that undergo NC-restricted deletion of Adar1. Consistent with the phenotype observed in humans, these mutants exhibit overall depigmentation due to apoptosis of melanocytes. Mutants also present with total absence of myelin along peripheral nerves, resulting from altered differentiation of Schwann cells. As in melanocytes, this defect occurs shortly after ISG upregulation. Supported by a recent clinical description indicating that a demyelinating sensorimotor neuropathy constitutes part of the clinical spectrum related to ADAR1 mutations in humans, these results demonstrate an essential role of ADAR1 and A-to-I RNA editing during the myelination of the PNS in humans and mice. To identify the deregulated pathways that contribute to these alterations, P1 performed RNA-seq on the sciatic nerves from Adar1cKO versus control animals, as well as a first serie of in vitro rescue experiments. Published and unpublished data collected by the consortium lead to hypothesize that the myelination defects observed in Adar1cK0 animals might result from i) deregulation of Mda5/Mavs signaling pathway; ii) the maintenance/up-regulation of a small number of transcription factors (known as repressors during myelination); and iii) formation of stress granules (cytosolic aggregates of proteins and RNA). Over the four years of the project, we propose to combine the expertise of the consortium to: 1) Determine the implication of the Mda5/Mavs signaling pathway, and the contribution of 3 differentially-expressed transcription factors, in the myelination defects observed in Adar1cKO animals (rescue experiments in vitro and in vivo, Task performed by partners 1 and 3), 2) Decipher the role of stress granules, along with their RNA and protein content, in phenotype genesis (their mechanism of formation in vitro and in vivo and OMICS, Partners 2 and 1), 3) Define the requirement for ADAR1 and RNA editing in the maintenance of myelin in adult mice (conditional knockout in adulthood, Task performed by partner 1, proposed on the basis of “post-natal onset” of clinical manifestations observed in some AGS patients), 4) Interrogate the mechanisms identified in mice in the human context (use of induced pluripotent stem cells from patients with ADAR1 mutations, Partners 1 and 3). Hence, our proposal will address questions of both fundamental research and of translational importance in clinics.

A comprehensive landscape of mRNA translation, storage and decay is required to understand the control of protein production. In eukaryotes, many regulators of these processes condense in cytoplasmic droplets called P-bodies. In a pioneer study we showed that P-bodies are primarily involved in mRNA storage, raising new issues. What is the spatiotemporal dynamic of mRNA storage and translation? What controls the differential storage of alternative mRNA isoforms? Is storage in P-bodies crucial to cell physiology and does it play a role in the recently found cases of intellectual disability (ID) with defective P-bodies? The objective of this project is to address these questions in the context of the cell cycle, using global multiscale approaches ranging from the molecular to the cellular level. Built on the main scientific interests of three partners, it capitalizes on their recent achievements and unique expertise on RNP granules, single molecule imaging and bioinformatics technologies.

The skin is a physical barrier that provides protection from the outside world. Any breach of its integrity allows potential pathogens to enter. Animals therefore have evolved mechanisms to repair tissue and induce sophisticated immune responses. In common with all invertebrates, C. elegans has only an innate immune system, but also lacks motile immune cells to help repair barrier epithelia. On the other hand, its epidermis is surrounded by a stiff apical extracellular matrix (aECM), which has features common with the plant and yeast cell wall. The aECM applies tension on the underlying tissues and counters hydrostatic pressure. In plants, cells constantly monitor the mechanical integrity of their cell wall during growth prompting the question of whether C. elegans does likewise for its aECM. Partner2 has found that mechanical coupling between the aECM and the epidermis is essential for embryonic elongation. In the adult, Partner1 has found that a decrease in skin stiffness results in the induction of immune responses in the epidermis. Here, we intend to explore how mechanical inputs control tissue repair. First, we will characterise the actin cytoskeleton and a multifold plasma membrane structure, the meisosome, with superficial similarity to the stress sensing eisosomes in yeast, as candidates for a sensing role. Second, we will assess the biophysical properties of the skin and formulate predictive models to help identify the relevant parameters involved in mechanosensing. Third, we will use the genetic power of C. elegans to run both a saturated unbiased genetic screen, and a candidate-based screen to identify the molecular sensors and mechanotransduction pathways. By combining our expertise, we expect to reveal new paradigms of mechanical surveillance after tissue injury. In the long term, this knowledge should give insight into conserved damage detection, central to innate immunity and organismal homeostasis across species.

The density of human habitation around the Mediterranean sea is the highest in the world making it an ideal site to monitor man-made pollution of a marine environment, which could also affect humans. Here we set out the case for choosing the ascidian as the ideal animal model to monitor pollution. Amongst all the invertebrates the ascidian is the closest to humans (maximizing the transfer of findings to humans). Each ascidian can generate up to 1 million swimming tadpole larva in about 12 hours following fertilization making this animal useful for rapid large-scale screening experiments. Finally, over the past 2 decades we have developed genomic/molecular/imaging tools and solutions for the ascidian Phallusia mammillata, chosen because its eggs and embryos are completely transparent and thus ideal for microscopy. We are now in a position to fully exploit the Phallusia model to tackle the problem of marine pollution by human populations. This project brings together biologists and engineers to develop new animal-based toxicological tests using state-of-the art molecular imaging techniques to monitor marine pollution. Several expression screens made by the coordinating team have revealed new proteins enriched on the DNA or in nuclei which are potentially new indicators with improved sensitivity to monitor DNA aberrations (micronuclei and DNA bridges). We aim to improve these indicators further in order to devise a completely new 2-in-1 test to detect both carcinogenicity and endocrine disrupting (EDC) activity. The carcinogenicity and the EDC activity test will use be performed simultaneously on the same embryo. Fluorescent transgenic embryos will be imaged in 3D time-lapse during the 10 hours of development from fertilization to hatching of the swimming tadpole and challenged with the several reference chemicals as defined in OECD test guidelines procedures. Carcinogenicity will be determined by scoring micronuclei and DNA bridges (as in the regulatory test OECD guideline procedure 487) while EDCs activity will be scored by imaging activation of the nuclear receptors affected by EDCs (ERR, TR, AR and PXR) using GFP-based molecular indicators of nuclear receptor activity. Optimization of image acquisition, data saving and management will be performed to reach an estimate of 100 embryos imaged per microscope per day at high resolution and more than 200 embryos per day at low resolution. The team of Nadine Peyrieras (UPR 3294, team 3) will develop software solutions for image analysis to score automatically DNA aberrations and fluorescent nuclei. The performance of the 2-in-1 test will be assessed using reference chemicals but also through a case study. During the last 2 years of the project, marine samples collected in local basin will be quantified by spectrometric analysis by the team of Christophe Migon (UMR 7093, team 4) after being assess for toxicity using our tests. The most sensitive indicators will be inserted in the genome of Phallusia mammillata to generate strains of fluorescent ascidians that will be maintained in our EMBRC funded marine culture facilities. Managing this task will allow us to exploit the 2-in-1 test in every part of the world where Phallusia mammillata grows. Finally the potential impact on human health of EDC exposure will be assessed by modifying the test for NRs activation. This test will show that human NRs are activated during exposure of the ascidian embryo to EDCs to demonstrate that such marine pollution can trigger the xenobiotic response and provoke endocrine disruption in humans. Beyond the molecular tests described above, the deliverables of this project will be patented by teams 1 and 3 of the consortium. Scientific publications will be completed after patent deposition in order to protect the work achieved during the 48 months of this project.

Larval settlement is a key step in the life cycles of corals and of many other marine animals, and is also thought to have had general significance in animal nervous system evolution, but its molecular regulation is poorly understood. We will study larval settlement using two complementary cnidarian species: the ecologically relevant scleractinian coral Pocillopora acuta coral and the established hydrozoan experimental model Clytia hemisphaerica. Pocillopora acuta is a promising experimental model pioneer reef-building coral species, with larvae available through monthly emissions in aquarium settings. We will specifically address the hypothesis that one or more specialised cell types at the aboral end of cnidarian larvae (called planulae) express proteins that mediate the settlement process. These include settlement-inducing GLWamide-family neuropeptides that we have already identified in both species. Our central aims are to identify all the participating cells and proteins from the larvae aboral poles, and to characterize the molecular cues from the settlement-inducing biofilms. Exploiting the complementary expertise of the project partners, we will use transcriptomics, peptidomics and metabolomics approaches to identify molecular candidates from the larval (Aim 1) and biofilm (Aim2) sides, followed by functional testing of selected candidate molecules in larval settlement assays (Aim3). For Aim 1 we will exploit our existing Clytia hemisphaerica transcriptome and genome resources, including a single cell transcriptome of the medusa and bulk RNA-seq of the aboral and oral ends of the planula larva. We will additionally produce high-resolution single cell transcriptomes for the Clytia hemisphaerica and Pocillopora acuta larvae with tens of thousands of cells from competent pre-settlement planulae using the 10X Genomics platform, and identify larval cell types using computational pipelines. Cell distributions, morphologies and connections will be determined by in situ hybridisation combined with fluorescence and electron microscopy. We will focus on characterising cell types expressing genes that are candidates for mediating the settlement response, notably small secreted proteins, GPCRs and neuropeptides. In parallel, under Aim 2, we will use mass spectrometry-based approaches to directly identify proteins and small molecules produced during settlement, including antimicrobial type peptides that we speculate may be secondarily detected by the larva and induce settlement. This approach will also allow us to identify biomarkers for larval settlement. The findings from Aims 1 and 2 will together allow us to generate a list of candidate sensory cells, proteins and small molecules that are most likely to initiate the settlement response. Under aim 3 the function of a selection of these will be investigated via the development of larval settlement assays. Established gene knockdown approaches in Clytia will enable us to test the involvement of candidate secreted proteins and their receptors. In both species we will in parallel test the activity of custom-synthesised small molecules to induce the settlement process, and of antibodies to inhibit it. Overall we anticipate that this project will generate a thorough understanding of molecules mediating cnidarian larval settlement. This will contribute to building a picture of larval nervous system evolution, and also potentially have ecological applications.