The dermomyotome of the mouse embryo is known to give rise to skeletal muscles progenitors, back dermis, to give rise to smooth muscle cells, to brown adipogenic cells and to endothelial cells. Signals from the neural tube, the ectoderm and the lateral plate mesoderm, like Shh, Wnt and BMP induce somite differentiation, by inducing several master genes of the sclerotomal compartment, or of the dermomyotomal compartment, thus restricting the pluripotence of somitic cells. Main transcription factors, which are responsible for the commitment of these pluripotent somitic cells in the myogenic lineage have been characterized. The Muscle Regulatory Factors (MRF) myf5, Myod, Mrf4 commit the cells into the myogenic fate, while myogenin is responsible for their differentiation. At the limb level, dermomyotomal cells first need to migrate in the limb bud, and the homeo-paired Pax3 gene is responsible for the delamination of these progenitors from the epithelial dermomyotome and their further migration. Pax3 and Pax7, a related paired homeoprotein, are also required for the multiplication of uncommitted myogenic progenitors at the interlimb level, and for the genesis of the pool of satellite cells that are recruited in the adult for muscle repair. Four main muscle fiber-types have been characterized in the adult skeletal muscles that are characterized by several specific physiological properties, such as their contraction speed, their oxidative or glycolytic metabolism and their resistance to exercise. How is this muscle diversity generated during development is still controversial, with some experiments demonstrating the existence of several committed myogenic populations, with clonally transmissible properties, other experiments favoring the influence on the environment to induce the fiber-type differentiation of a single homogenous myogenic population. No slow or fast-type myogenic determinant has yet been characterized in mammals. My group is interested in the mechanisms of mouse muscle development from the early stages when myogenic progenitors arise in the embryo to the later stages when fibers diversify. We have shown that Six homeoproteins initiate a genetic cascade that controls several important steps of muscle development by activating the major activators of each of these individual steps. Thus, we have shown that Six proteins are required for the genesis of hypaxial myogenic progenitors, acting upstream of the paired homeogene Pax3. Then, with Pax3, Six proteins activate Myf5, the first muscle regulatory factor known to commit the myogenic progenitor into the myogenic fate. Then, with Myf5, Six proteins activate myogenin, the muscle regulatory factor responsible for the differentiation of myogenic cells. Then, with Myf5 or with myogenin, Six proteins are responsible for the activation of a network of fast-type muscle genes like troponin, myosin, or parvalbumin. To mediate this multiplicity of effects, Six homeoproteins bind a MEF3 site in the regulatory sequences of downstream target genes. We showed in particular, by chromatin immunoprecipitation that Six1 in the embryo was bound on the enhancers of the Pax3 and Myf5 genes. We characterized several cofactors of Six and showed that these cofactors can selectively activate a number of developmental steps in synergy with the Six proteins. Some of these cofactors are expressed mainly in uncommitted progenitors, like the cofactors Eya which are required for Pax3 expression in hypaxial myogenic progenitors. Other cofactors are expressed in differentiated myocytes, like SOBP. Three bona fide Six cofactors which play a major role in the STC to drive diverse steps of muscle development are LRRFIP2, SOBP and EYA. LRRFIP2 is known to interact with Dishevelled and links Six proteins with the Wnt signaling pathway. SOBP, which is expressed in differentiating myocytes, and was previously characterized in Drosophila as a sine oculis cofactor. Eya proteins are expressed both in myogenic progenitors and in adult myofibers. These Six cofactors that can be located in the nucleus and bind directly to Six1 co-activate transcription through MEF3 binding sites. In the next four coming years we will address three questions related to the STC properties in skeletal muscle development and physiology. These projects have two main objectives: - the mechanisms that underly the determination of the stem character of dermomyotomal cells of the mouse embryo, the transcription factors that preside to the genesis of myogenic progenitors, their self-renewal and their engagement in the myogenic fate during vertebrate embryogenesis. - the mechanisms that control the genesis and the maintenance of muscle fiber-type diversity with an emphasis on the crosstalk between the slow and the fast genetic determinants that preside these antagonist fates in adult myofibers.

The heart is driven by an electrical activation that propagates in cells and triggers their concerted contraction, which results in efficient pumping of the blood. The electrical activation sequence can be disrupted by disease, a condition referred to as conduction disorder, or arrhythmia. For example, upper chamber, i.e., atrial, fibrillation (AF) is the most prevalent arrhythmia and is associated with a 5-fold increase in risk of stroke. Moreover, ventricular conduction disorders can lead to heart failure (HF), which is the leading cause of hospitalization in patients above 65 years old, and is associated with exceptionally high morbidity and mortality rates: 50% of patients die within five years of being diagnosed. However, there exists no clinical imaging modality that can noninvasively and directly map the electrical activation of tissues to improve our understanding of these complex diseases, perform better diagnoses, and contribute to the development of novel therapeutic approaches. The acoustoelectric effect has recently been shown to provide contrast directly from current densities by detecting the signature of ultrasound-modulated electrical impedance using high frequency electrodes. A few studies have been conducted and demonstrated the potency of the approach by mapping the electrical currents in ex vivo tissues. Yet, to this day, the acoustoelectric effect associated with biological action potentials has never been observed in vivo, due to the challenges inherent in the detection of small electrical signals at high frequency and the limited imaging frame rate of conventional ultrasound emission techniques. In this study, our objective is to demonstrate the feasibility of the real-time, noninvasive, and direct mapping of the electrical activation of heart tissues in vivo. Our method of Ultrafast Acoustoelectric Imaging (UAI) can image one hundred times faster than conventional methods and relies on the latest high-performance electronics for high signal-to-noise ratio measurements. Our specific aims are A) Optimize UAI instrumentation and image formation algorithms using a Langendorf rat heart model B) Validate UAI against electrical mapping in an open-chest sheep heart model C) Demonstrate the feasibility of transthoracic Ultrafast Acoustoelectric Imaging in the normal human heart These specific aims reflect the high risk associated with the development of an imaging modality for the electrical activation of tissue. Indeed, while most imaging modalities are typically developed and optimized using ‘phantoms’, i.e., tissue-mimicking objects with controlled physical properties, no existing phantom can reproduce, even approximately, the complex biochemistry leading to the generation of action potentials. Therefore, the optimization and validation of UAI for biological application must be performed in active tissue, in which the electrical activation can be artificially controlled, such as in the heart. Specific aim 1 will determine the imaging parameters required, i.e., emission frequency, frame rate, and pressure, to obtain the optimal UAI signals and images using isolated beating rat hearts in a Langendorf model in a controlled environment. Specific Aim 2 will validate, in a large animal heart model, the agreement between the UAI images and the true electrical activation sequence measured with implanted electrodes in the in vivo setting. Finally, Specific Aim 3 will demonstrate the clinical feasibility in normal human subjects. The outcomes of this study are expected to yield the only imaging methodology for the organ-independent mapping of the electrical activation in vivo, at no or little additional cost to that of standard ultrasound imaging. No imaging modality can currently map the electrical activation of tissue directly. The high impact of this OH Risque application lies in the fact that UAI can easily be integrated into any commercial echographic system for immediate translation to the clinic.

The formidable evolutionary success of bacteria is illustrated by the fact that they inhabit almost all environmental niches on Earth. The new ecological niche is usually colonized by very small number of bacterial cells. When adaptive variants are not present among them, bacterial population will be eliminated by the natural selection. However, when the purifying selection is not very strong, population size could increase and adaptive variants can be generated thus allowing survival of the bacterial population. But adaptation is never permanent. Environments are heterogeneous in many dimensions: temporally and spatially, due to variations in the abiotic and biotic factors. Bacteria themselves constantly change their environments. One of the solutions to cope with such uncertainty is permanent multidirectional exploration of the fitness space. This evolutionary 'strategy' increases the probability of survival and of successful evolution of the bacterial populations. Causes and consequences of bacterial diversification were previously investigated in many studies. However, all these studies concerned only bacteria in the liquid environments, agitated or non-agitated, in batch cultures or in chemostats. In their natural environments, bacteria are found in structured, highly organized multicellular super-structures, such as colonies and biofilms. One prominent feature of the structured environments relative to the liquid cultures is attenuation of the clonal interference, which renders nearly impossible selective sweeps. This results in generation of much higher genetic and phenotypic diversity in structured environments. We propose to study the molecular determinants of the adaptive radiation in aging E. coli colonies. We will study spatio/temporal changes in morphology, physiology and gene expression patterns of bacterial cells inside colony using microscopy tools and flow cytometry. We intend also to sequence genomes of the interesting mutants, which will allow us to simultaneously perform genomic, transcriptomic and phenotypic analysis. This robust integrative approach will allow us to investigate the genetic basis of adaptive radiation and reconstruct its history during aging of the bacterial colony. We will also test the nature of the interaction between different mutants isolated from the same colony. Different mutants can compete for the same resources, but also some mutants can create conditions for the survival of the others. Hence, some bacteria may scavenge toxins or provide nutrients for other bacteria. This may result in the symbiotic interactions as it was described in biofilms. For this, we will reconstruct colonies using combination of different mutants derived from the same colony. If we find many mutations per genome, the identified mutations will be sequentially introduced in ancestral genome and relevant phenotypes will be studied. This will allow us to establish the nature of the epistatic interactions between different mutations. Epistasis is an important and poorly understood aspect of mutations that strongly influences the evolutionary impact of genetic variation on adaptation and fitness. Particularly important are compensatory mutations that attenuate fitness loss caused by another mutation. This biological phenomenon has important implications because it allows mutant cell to reach adaptation peak that is not accessible to the ancestral genotype. After characterization of the genetic basis of adaptive radiation in laboratory strain, we will study presence of these mutations in E. coli commensal and pathogenic strains isolated from different ecological niches. For this, we posses a large collection of E. coli natural isolates. In addition, we will test how different genotypes constrain adaptive radiation using E. coli natural isolates, whose genomes have been sequenced. These questions are of general interest to evolutionary biology, because it lends insight into the relative importance of chance, historical contingency, and natural selection in shaping the genetic outcome of adaptation. The simplicity with which both bacteria and environments can be manipulated in the laboratory allows for explicit tests of all above-mentioned hypotheses.

Beneficial effects of HDL in atherosclerosis remains chiefly associated with reverse transport of cholesterol, even if other anti-atherogenic properties are well documented (anti-oxidant, anti-inflammatory or anti-thrombotic effects). Proteomic approaches have shown that HDL are more than lipids associated with apolipoprotein A1. We and others have reported the presence of alpha-1 antitrypsin (AAT) associated with HDL. This serine protease inhibitor is the natural inhibitor of neutrophil elastase, reported that to play a role in atherothrombotic lesions, including aortic abdominal aneurysm (AAA) in which it can be responsible for the arterial wall fragilization. The aims of this project are: - to identify new proteins associated with HDL in pathological conditions by comparing HDL from atherothrombotic patients versus controls, using a proteomic approach (task 1) - to use HDL as a vector of anti-proteases and in particular of anti-elastase in order to target diseased tissue and thus counteract the proteases responsible for degradation of the extracellular matrix and cell death (tasks 2, 3, 4) The search of biomarkers in atherosclerosis is of major importance in order to predict the onset of clinical complications, to evaluate the efficacy of treatments and to understand its pathophysiology, in order to define new therapeutic targets. In the task #1, we aim to discover new proteins/peptides associated with HDL in normal and pathological conditions. For this purpose, we will isolate HDL by two different techniques: - ultracentrifugation - immunoabsorption HDL will be isolated from plasma of coronary patient versus stable angina or control subjects (BIOCore Study “Biomarkers of COronary Events” PIs: Pr L. Feldman- Dr O. Meilhac). HDL will also be isolated from atherothrombotic samples (human carotid endarterectomy samples with or without intraplaque hemorrhages) and compared to those isolated from fatty streaks of human aortas. In the tasks #2, 3, 4, we plan to use HDL as natural vector to deliver anti-proteases within diseased areas in different pathologies. We will use 4 animal models in which leukocyte elastase plays a pivotal role: - A rat model of AAA in which the thrombus and neutrophils that it contains are a source of elastase - A mouse model of emphysema induced by elastase and characterized by an acute influx of neutrophils into the lung, leading to the destruction of alveolae, - A rat model of cerebral ischemia by injection of a thrombus, in which we already have shown that HDL are protective in acute conditions. A model of blood brain barrier will be used to get insights into the mechanisms involved in HDL protection. - A mouse model of neoangiogenesis, allowing us to test the effect of elastase on destabilization of neovessels and the potential protective effect of HDL Both fields of HDL proteomics and the use of HDL for therapeutic application emerged very recently. The aims of the proposed project are highly innovative and ambitious. This project is totally original in the context of the Inserm Unit 698 since nobody in the laboratory works on lipoproteins and HDL in particular. Albeit very original, it will provide many opportunities of collaborations with all the teams composing the Inserm Unit 698 (hemostasis, cardiovascular remodelling, bio-engineering, immunopathology and clinical research). This nascent team should perpetuate after the 36-period of this ANR funding, coinciding with the application for de novo creation of this Inserm Unit. Finally, this project has a great potential of valorisation: we already protected the use of HDL as a vector of pharmacologic coumpounds including anti-proteases by a European patent (July 09). A collaboration has been initiated with CSL Behring (specialized in commercialization of HDL and AAT). Valorisation process will be conducted in close collaboration with Inserm Tranfert.