Intracellular signaling pathways are controlled by chemical signals, but physical signals have recently emerged as important regulators of signal transduction. Currently, our understanding of the molecular events that connect changes in membrane electrical potential (Vm) to signaling pathways in non-excitable cells is limited. Our preliminary results demonstrate that the Wnt/ß-catenin signaling pathway is activated during membrane depolarization. However, how this change in Vm contributes to the activation of the Wnt/ß-catenin signaling remains unknown. In this project, we aim to address this question at the molecular, cellular, and multicellular levels by investigating the following issues. (1) What is the molecular mechanism that establishes the link between membrane depolarization and the activation of the Wnt/ß-catenin signaling? (2) What are the molecular effectors responsible for the depolarization-induced activation of the Wnt pathway? (3) How does a change in membrane potential (Vm) influence the regulation of Wnt pathway activity? To answer these questions, we will combine various approaches, including biochemistry, cell biology, optogenetics, and electrophysiology. To understand the effects of Wnt/ß-catenin signaling activation associated with membrane depolarization, we will use colon-derived organoids. These results will shed light on a novel regulatory mechanism of Wnt signaling in non-excitable cells, thereby enhancing our understanding of the involvement of this signaling pathway in epithelial physiology and associated pathologies.

The increasing prevalence of overweight and obesity has reached “epidemic” proportions as documented by overweight (BMI > 25 kg/m2) in more than 1.9 billion people among which at least 600 million are clinically obese (BMI > 30 kg/m2). This causes major costs for our health care systems. Increase in body weight is accompanied by an increase in the mass of white adipose tissue (WAT) resulting from an imbalance in energy intake and energy expenditure. Pharmacological remedies or lifestyle interventions normalizing this imbalance with significant long-term success are not available. During the last two decades, the major scientific and financial contributions have focused on the identification of genetic modifiers of the response to”obesogenic'' environments. The results obtained so far appear rather disappointing with respect to public health issues. Regarding energy expenditure, brown adipose tissue (BAT) in rodents for a long time has been implicated in the dissipation of caloric excess through diet-induced thermogenesis. It is now established that healthy adult humans also possess active BAT, localized in small depots at various anatomical sites. Importantly, BAT activity is inversely correlated with BMI. Furthermore, in rodents and humans, islands of brown-like adipocytes emerge within WAT depots after cold or ß3-adrenergic receptor stimulation. These adipocytes, termed “brite” (brown-in-white) or “beige” adipocytes, differ by embryonic origin from genuine brown adipocytes but are functional, i.e., thermogenically active. Therefore, the identification of factors increasing mass/activity of human BAT would be of great interest for the treatment of overweight/obesity and associated diseases such as type 2 diabetes. In the absence of effective and safe pharmaceutical treatments of obesity, the development of nutritional interventions to modulate metabolic functions of adipose tissues are a promising alternative approach. Differences in fatty acid composition of dietary fat contribute to adipose tissue development, in particular with respect to the relative intake of omega6 to omega3 poly-unsaturated fatty acids (PUFAs). Quality and quantity of dietary PUFAs determine the nature and diversity of fatty acid metabolites synthesized in the organism. These metabolites, named oxylipins or eicosanoids in mammals, are involved in various physiological and inflammatory processes, particularly in adipose tissue development and function. Here, we will employ nutritional interventions targeted to modulate oxylipin metabolism. We aim to identify distinct oxylipins in murine and human adipose tissues, which are associated with brite adipogenesis and thus able to increase energy expenditure. Metabolite analyses will be backed up by transcriptome analyses of adipose tissues and gut microbiota sequencing in order to decipher the pathways modulated by nutritional interventions within adipose tissues, and related changes in the gut microbiome. Oxylipins of interest will be validated in vitro for their capacity to induce brite adipogenesis and/or activity of human and murine cells. Additionally, pathways of oxylipin synthesis with confirmed bioactivity will be analyzed in vitro using pharmacological inhibition and siRNA or shRNA mediated knockdown of key enzymes. In vivo validation of selected oxylipins will be performed using implantable pellets in situ inWAT allowing a constant release of the molecule. Finally, oxylipins with the most potent bioactivities will be validated in dietary intervention studies. Our research program will shed light on the potential role of dietary lipid composition in the prevention of excess body weight gain and obesity. Our research program is related to fundamental and medical research and aims to gain a better understanding of the mechanisms underlying the role of oxylipins in brite adipocyte formation, paving a way for the development of new therapies and commercial applications.

Metabolic diseases have reached an epidemic dimension. This increase is alarming since obesity and diabetes in addition to be severe pathologies represent risk factors for development of cardiovascular diseases and cancer. Increasing evidence suggests that the circadian system which synchronises our physiology with the light/dark cycle is intimately linked to metabolic homeostasis. Epidemiological data show for example that alterations of circadian system caused by shift work (17% of working population in France) are correlated with metabolic disorders. Moreover mice mutants for clock genes display metabolic alterations. Our main line of research is to identify the molecular, cellular and physiologic mechanisms by which circadian clock controls metabolism. We recently showed that the transcriptional repressor KLF10 displays a strong rhythmic expression in mouse liver and is a direct target of molecular clock. In addition we reported that male mutant mice having a systemic deletion of Klf10 display hyperglycemia due to increased hepatic gluconeogenesis. This phenotype is explained by the direct regulation of the Pepck gene (rate limiting enzyme of gluconeogenesis) by KLF10. We also reported that Klf10 mutant female mice display hypertriglyceridemia and a shifted circadian expression of lipogenesis genes. Our data strongly suggest that KLF10 participates in the timing of hepatic metabolism, thus linking circadian clock and metabolism. To better understand the role of KLF10 in circadian regulation of liver metabolism, and its impact in physiopathology, we will pursue two main complementary objectives: (1) to determine the role of KLF10 in the rhythmic control of glucose and lipid metabolism in liver, which is a major organ for these process; 2) to determine the physiopathological consequences of the alteration of the KLF10 dependent clock-metabolism interaction. To address these questions, we will first identify at the genomic scale the direct targets of KLF10 in liver by ChIP -Seq. This approach will be completed by the analysis of the liver transcriptome from mice bearing an inducible -liver specific deletion of Klf10 that we will generate. In parallel, we will develop another mouse model with antiphasic expression of Klf10 in liver in order to determine the importance of the timing of Klf10 expression. The analysis of metabolic and circadian physiology of these two animal models coupled to the identification of the KLF10 directed gene network in liver will provide a mechanistic understanding of the KLF10 dependent regulation of metabolic pathways. Further, we will challenge these animals with either high-fat diet or temporal restricted feeding protocols to understand to which extent the alteration of circadian coordination mediated by KLF10 result in metabolic alterations or decreased synergy between circadian and food synchronisation. This project will provide the molecular and physiologic bases to understand a key interaction in physiology and will be useful to develop preventive or therapeutic approaches taking into account the temporal organization of metabolism.

In vivo, mRNAs are packaged together with regulatory proteins into ribonucleoprotein particles (RNP) that control their fate and undergo extensive remodeling in response to developmental cues or environmental stresses. Cytoplasmic RNPs of different sizes, composition and regulatory properties have been described, including large macromolecular complexes such as P-bodies, stress granules, or germ cell granules. In neurons, so-called neuronal granules have been implicated in the long-distance transport of mRNAs to axons or dendrites, and in their local translation in response to external cues. To date, surprisingly little is known about the factors controlling the assembly and regulation of RNP granules, specifically neuronal granules. This has so far prevented a detailed understanding of the molecular mechanisms underlying the formation and turnover of these granules, and of how they are physiologically regulated. Here, we propose to study the molecular bases underlying the assembly and regulation of RNA granules, using the highly conserved IMP-containing granules as a paradigm. Cytoplasmic RNP granules characterized by the presence of IMP family members (called IMP, IGF2BP, ZBP1, Vg1RBP or VICKZ) have been described in a wide-range of organisms and cell types, where they are implicated in subcellular mRNA targeting, and/or in the spatio-temporal regulation of mRNA translation. Surprisingly, although IMP neuronal granules are considered as a major class of neuronal granules, in vivo study of their function and regulation has lagged behind, and the physiological importance of IMP granules during brain maturation has for a long time remained unclear. Recently, IMP granules dynamically transported to axons have been observed in vivo in zebrafish embryonic neurons (F. Giudicelli, unpublished). Furthermore, it was discovered that IMP granules are actively and specifically transported to the axons of remodeling neurons during Drosophila brain maturation. Both the function and the transport of these granules are required for proper axonal remodeling (F. Besse; Medioni et al., 2014). The main objectives of this proposal are i) to systematically identify the molecular factors that regulate the assembly and the turnover of IMP-containing RNP complexes in Drosophila cultured cells, ii) to test their physiological importance in vivo, in the developing fly nervous system, and iii) to investigate their functional conservation in the zebrafish embryo. Specifically, we propose to perform an unbiased genome-wide RNAi screen on Drosophila cultured cells to identify mutant conditions in which the organization and/or distribution of IMP-containing granules is altered. To quantitatively and statistically analyze mutant conditions, and to define precise and coherent classes of mutants, we will combine high throughput microscopy with the development of a computational pipeline optimized for automatic analysis and classification of images. The function of positive hits isolated in the screen will then be validated in vivo in Drosophila neurons using fly genetics and imaging techniques, and characterized at the molecular and cellular levels using biochemical assays, in vitro phase transition experiments and live-imaging. Finally, the functional conservation of identified regulators will be tested in zebrafish embryos combining gene inactivation and live-imaging techniques. This integrative study will provide the first comprehensive analysis of the functional network that regulates the properties of the conserved IMP RNA granules. Our characterization of the identified regulators in vivo in neuronal cells will be of particular significance in the light of recent evidence linking the progression of several degenerative human diseases to the accumulation of non-functional RNA/protein aggregates. This work will thus shed new insight into the mechanisms controlling RNP particle assembly and disassembly in both wild-type and pathological contexts.

Among the signaling molecules involved in animal morphogenesis are the Hedgehog (Hh) family proteins which act at a short and long range to direct cell fate decisions in invertebrate and vertebrate tissues. We propose a fundamental research project aiming at a better understanding of tissue morphogenesis controlled by Hh during animal development by focusing on the mecanisms that control Hh transport to target cells in Drosophila. One of the challenge in the morphogen field is to decipher the spatio-temporal dynamics of secreted signals involved in cell fate decisions. In particular, we showed that Hh secreted proteins are not randomly distributed in the extracellular space cells but are rather differentially secreted on apical and basolateral spaces (partner 1: Pascal Thérond, IBV, Nice). We previously showed that Hh gradient is a composite of pools secreted by different routes: an apically secreted pool with long range activity and a more basolateral secreted pool with short-range activity. Moreover, several extra-cellular Hh carriers have been identified (lipoprotein particles; membrane extension called cytonemes; exovesicles) but their individual contribution to the spreading of the different Hh pools is still unresolved. Therefore, the ability to quantitatively analyze and control the spatio-temporal and dynamical properties of the secreted Hh gradient taken as a whole is essential for understanding Hh morphogenetic gradient. The overall goal of this proposal is to understand how Hh is secreted and evaluate the contribution of the multiple carriers to Hh transportation in Drosophila melanogaster. To study these mecanisms, we will develop new methods to modify the spatio-temporal and dynamical properties of the extra-cellular Hh gradient and separate the contribution of the apical versus basal Hh pools in two different epitheliums of the drosophila larvae. This will allow us to analyze the contribution of the different carriers in the establishment of the Hh gradient (P. Thérond and partner 2: Xavier Descombes, INRIA Sophia-Antipolis). This will be completed with a genome-wide screen to identify additional genes and related cellular processes responsible for Hh release (P. Thérond). With these tools in hands, we propose to classify the multiple pools of Hh and develop accurate tracking algorithm to compare trajectories of different Hh pools transportation in live animals (X. Descombes). The particular interest of this collaboration lies in the combination of development of algorithm to analyze Hh distribution and trajectories with extremely powerfull genetics, ease of in vivo manipulation and lack of genetic redundancy of Drosophila. Our project could uncover novel avenues for the discovery of therapeutic tools relevant for human health as deregulated Hh activity impairs stem cell and tissue homeostasis.