The neural crest (NC) lineage is a specialised multipotent embryonic tissue, which contributes notably to the development of the human peripheral and enteric nervous system, craniofacial structures, pigment cells, as well as many other tissues and organs. Neurocristopathies, diseases of NC-derived tissues are an acute societal problem.
The aim of the NEUcrest project is to train 15 creative and innovative PhD students within a novel, ambitious and interdisciplinary research program. This network is aimed at considering the overall genetic, molecular and epigenetic regulation of the NC tissue in human health. It involves 11 main interacting participants and 9 associated partners, from hospitals, academic laboratories and small and medium-sized enterprises, from 7 European or EU-associated countries: France, Ireland, Spain, The Netherlands, United Kingdom, Austria and Israel. Their cumulative expertise will provide an exceptional environment for PhD students, both for excellent research training and multiple transferable skills. This will allow the PhD students optimal career development and employability, through their acquired expertise in manipulating cells and embryos in various animal models, analysing biological images and datasets, and modelling complex genetic interactions.
With an exceptionally rich training environment and regular network-wide events, our aim is to train 15 PhD students to be part of the next generation of leading European young scientists, highly proficient in the multidisciplinary range of scientific skills and technological expertise required for a comprehensive view of development and diseases : developmental and stem cell biology, cell and molecular biology, cancer biology, imaging, bioinformatics, human and animal genetics, and drug screening.
In addition to the excellent doctoral training that each beneficiary will follow at his/her University, the network will provide an active training environment, by bringing together selected partners from the academic, industrial and communication sectors, and will stimulate fruitful interactions between the NEUcrest PhD students themselves through regular network-wide events.
There will also be a strong emphasis on communication skills and public/patient/clinician outreach. Thus, the NEUcrest ITN will enable the selected PhD students to master scientific and transferable competences indispensable for addressing complex questions in Life Sciences and Human Health, and position them for leading roles in European academia and industry.
Partner 1 and Network Coordinator : Prof. Anne-Hélène Monosoro-Burq
Partner 2: Dr. Karen Liu laboratory
Partner 3: Dr. Grant Wheeler laboratory
Partner 4: Dr Gerhard Schlosser laboratory
Partner 5: Prof. Angela Nieto laboratory
Partner 6: Dr. Irene Mathijssen
Partner 7: Prof. Igor Adameyko laboratory
Partner 8: Prof. Carmit Levy laboratory
Partner 9: Dr. Vivian Lee laboratory
Partner 10: Dr. Karima Kissa laboratory
Partner 11: Dr. Nadège Bondurand laboratory
Vidéo 1: Oscillations spontanées de deux touffes ciliaires de l’oreille interne (saccule) de la grenouille taureau. Les touffes ciliaires sont vues de dessus à travers l’objectif d’un microscope optique. Le film est en temps réel.
Vidéo 2: La rigidité et la friction apparentes d’une touffe ciliaire sont modifiées par l’activation des canaux ioniques responsables de la transduction mécanoélectrique. Une force est appliquée à la touffe ciliaire en appliquant un mouvement à une fibre de verre flexible qui lui est accrochée. Un mouvement d’aller-retour triangulaire résulte en une relation force-position hystérétique qui traduit la dissipation d’énergie par la touffe ciliaire. Il y a un intervalle de positions près de l’origine où la pente de la relation force-position est plus faible et la hauteur du cycle d’hystérèse plus importante, ce qui correspond respectivement à une plus faible rigidité et à une plus haute friction de la touffe ciliaire. En réponse à une drogue (la gentamicine) qui bloque les canaux ioniques, les deux branches les plus longues du cycle deviennent linéaire et le cycle s’affine. Ces observations indiquent en retour que l’activation de ces canaux ioniques réduit la raideur apparente et augmente la friction apparente de la touffe ciliaire. Ces effets sont interprétés comme la conséquence du mécanisme de mécanosensibilité de la touffe ciliaire, qui résulte de l’activation mécanique directe de canaux ioniques par des liens élastiques dont la tension est modulée par le stimulus.
Video 1: Spontaneaous hair-bundle oscillations. Two hair bundles from the inner ear (saccule) of the bullfrog are imaged from the top under the objective of an optical microscope. The movie is in real time.
Video 2: Hair-bundle compliance and friction from gating of mechanoelectrical transduction channels in the hair bundle. Force is applied to a hair bundle by moving the base of an attached flexible fiber. A back-and-forth triangular motion of the fiber results in a force-position relation that displays hysteresis as the result of energy dissipation. There is a range of positions where the slope of the relation is lower and the height of the cycle is larger, corresponding to lower stiffness and higher friction of the hair bundle, respectively. Upon application of a channel blocker (the aminoglycoside gentamicin), the two long branches of the cycle become linear and the hysteretic cycle shrinks. These observations suggest in return that gating of the channels reduces the stiffness and increases the friction of the hair bundle. This effect is interpreted as an inherent consequence of hair-bundle mechanosensitivity from direct mechanical activation of ion channels by force. See Bormuth et al (2014) PNAS 111:7185.
Confinement and substrate topology strongly affect the behavior of cell populations and, in particular, their collective migration. In vitro experiments dealing with these aspects require strategies of surface patterning that remain effective over long times (typically several days) and ways to control the surface topology in three dimensions. Here, we describe protocols addressing these two aspects. High-resolution patterning of a robust cell-repellent coating is achieved by etching the coating through a photoresist mask patterned directly on the coated surface. Out-of-plane curvature can be controlled using glass wires or corrugated “wavy” surfaces.
We study the competition for space between two cell lines that differ only in the expression of the Ras oncogene. The two cell populations are initially separated and set to migrate antagonistically towards an in-between stripe of free substrate. After contact, their interface moves towards the population of normal cells. We interpret the velocity and traction force data taken before and after contact thanks to a hydrodynamic description of collectively migrating cohesive cell sheets. The kinematics of cells, before and after contact, allows us to estimate the relative material parameters for both cell lines. As predicted by the model, the transformed cell population with larger collective stresses pushes the wild type cell population.
Ingénieur de recherche : Etude des propriétés du noyau par des outils d’imagerie et d’analyse d’images avancées.
Postdoc : Développement et installation d’un microscope interférométrique pour l’étude des vésicules
Postdoc : Detection de default surfacique par thermographie infrarouge
Postdoc : Etude de surface par Imagerie microscopique
Postdoc : Etude de surface par Imagerie microscopique, Microscopie à reflectance, Ellipsométrie, Electrochimie.
Thèse de doctorat : Etude et conception d’une cavité Fabry Pérot à 4 miroirs de haute finesse
Cytoskeletal filaments assemble into dense parallel, antiparallel, or disordered networks, providing a complex environment for active cargo transport and positioning by molecular motors. The interplay between the network architecture and intrinsic motor properties clearly affects transport properties but remains poorly understood. Here, by using surface micropatterns of actin polymerization, we investigate stochastic transport properties of colloidal beads in antiparallel networks of overlapping actin filaments. We found that 200-nm beads coated with myosin Va motors displayed directed movements toward positions where the net polarity of the actin network vanished, accumulating there. The bead distribution was dictated by the spatial profiles of local bead velocity and diffusion coefficient, indicating that a diffusion-drift process was at work. Remarkably, beads coated with heavy-mero-myosin II motors showed a similar behavior. However, although velocity gradients were steeper with myosin II, the much larger bead diffusion observed with this motor resulted in less precise positioning. Our observations are well described by a 3-state model, in which active beads locally sense the net polarity of the network by frequently detaching from and reattaching to the filaments. A stochastic sequence of processive runs and diffusive searches results in a biased random walk. The precision of bead positioning is set by the gradient of net actin polarity in the network and by the run length of the cargo in an attached state. Our results unveiled physical rules for cargo transport and positioning in networks of mixed polarity.
Sound analysis by the cochlea relies on frequency tuning of mechanosensory hair cells along a tonotopic axis. To clarify the underlying biophysical mechanism, we have investigated the micromechanical properties of the hair cell’s mechanoreceptive hair bundle within the apical half of the rat cochlea. We studied both inner and outer hair cells, which send nervous signals to the brain and amplify cochlear vibrations, respectively. We find that tonotopy is associated with gradients of stiffness and resting mechanical tension, with steeper gradients for outer hair cells, emphasizing the division of labor between the two hair-cell types. We demonstrate that tension in the tip links that convey force to the mechano-electrical transduction channels increases at reduced Ca. Finally, we reveal gradients in stiffness and tension at the level of a single tip link. We conclude that mechanical gradients of the tip-link complex may help specify the characteristic frequency of the hair cell.
Unicellular eukaryotes and cultured cells from several animal species were invaluable in discovering the mechanisms that govern incorporation, handling, and excretion of copper at the cellular level. However, understanding the systemic regulation of copper availability and distribution among the different tissues of an intact multicellular organism has proven to be more challenging. This analysis is made even more difficult if the genetic variability among organisms is taken into account. The zebrafish has long been considered a powerful animal model because of its tractable genetics and embryology, but it has more recently become a player in environmental studies, pharmaceutical screening, and physiologic analysis. In particular, the use of the larvae, small enough to fit into a microtiter well, but developed enough to have full organ functionality, represents a convenient alternative for studies that aim to establish effects of environmental agents on the intact, living organism. Studies by our group and others have characterized absorption and tissue distribution of copper and have described the acute effects of the metal on larvae in terms of survival, organ stress, and functionality of sensory organs. A large body of work has shown that there is strong conservation in mechanisms and genes between fish and mammals, opening the possibility for genetic or small molecule screens or for generating fish models of human diseases related to copper metabolism. The variability within humans in terms of tolerance to copper excess or deficiency requires a genetic approach to be taken to understand the behavior of populations, because markers and vulnerabilities need to be identified. The zebrafish could represent a unique tool to move in this direction.
Mechanosensory hair cells are essential for audition in vertebrates, and in many species, have the capacity for regeneration when damaged. Regeneration is robust in the fish lateral line system as new hair cells can reappear after damage induced by waterborne aminoglycoside antibiotics, platinum-based drugs, and heavy metals. Here, we characterize the loss and reappearance of lateral line hair cells induced in zebrafish larvae treated with copper sulfate using diverse molecular markers. Transgenic fish that express green fluorescent protein in different cell types in the lateral line system have allowed us to follow the regeneration of hair cells after different damage protocols. We show that conditions that damage only differentiated hair cells lead to reappearance of new hair cells within 24 h from nondividing precursors, whereas harsher conditions are followed by a longer recovery period that is accompanied by extensive cell division. In order to characterize the cell population that gives rise to new hair cells, we describe the expression of a neural stem cell marker in neuromasts. The zebrafish sox2 gene is strongly expressed in neuromast progenitor cells, including those of the migrating lateral line primordium, the accessory cells that underlie the hair cells in neuromasts, and in interneuromastic cells that give rise to new neuromasts. Moreover, we find that most of the cells that proliferate within the neuromast during regeneration express this marker. Thus, our results describe the dynamics of hair cell regeneration in zebrafish and suggest the existence of at least two mechanisms for recovery of these cells in neuromasts.
In teleosts, the lateral line system is composed of neuromasts containing hair cells that are analogous to those present in the inner ear of all vertebrates. In the zebrafish embryo and early larva, this system is composed of the anterior lateral line (ALL), which covers the head, and the posterior lateral line (PLL), present in the trunk and tail. The mechanosensory hair cells found in neuromasts can be labeled in vivo using fluorescent dyes such as 4-di-2-Asp (DiAsp) or FM1-43. We have studied the effects of water-borne copper exposure on the function of the lateral line system in zebrafish larvae. Our results show that transient incubation of post-hatching larvae for 2h with non-lethal concentrations of copper (1-50 microM CuSO4) induces cellular damage localized to neuromasts, apoptosis, and loss of hair cell markers. This effect is specific to copper, as other metals did not show these effects. Since hair cells in fish can regenerate, we followed the reappearance of viable hair cells in neuromasts after copper removal. In the PLL, we determined that there is a threshold concentration of copper above which regeneration does not occur, whereas, at lower concentrations, the length of time it takes for viable hair cells to reappear is dependent on the amount of copper used during the treatment. The ALL behaves differently though, as regeneration can occur even after treatments with concentrations of copper an order of magnitude higher than the one that irreversibly affects the PLL. Regeneration of hair cells is dependent on cell division within the neuromasts as damage that precludes proliferation prevents reappearance of this cell type.
Barrier surfaces of multicellular organisms are in constant contact with the environment and infractions to the integrity of epithelial surfaces is likely a frequent event. Interestingly, components of the immune system, that can be activated by environmental compounds such as the microbiota or nutrients, are interspersed among epithelial cells or directly underlie the epithelium. It is now appreciated that immune cells continuously receive and integrate signals from the environment. Curiously, such continuous reception of stimulation does not normally trigger an inflammatory response but mediators produced by immune cells in response to such signals seem to rather promote barrier integrity and repair. The molecular mediators involved in this process are poorly understood. In recent years, the cytokine interleukin-22, produced mainly by group 3 innate lymphoid cells (ILCs), has been studied as a paradigm for how immune cells can control various aspects of epithelial cell function because expression of its receptor is restricted to non-hematopoietic cells. We will summarize here the diverse roles of IL-22 for the malignant transformation of epithelial cells, for tumor growth, wound healing and tissue repair. Furthermore, we will discuss IL-22 as a potential therapeutic target.
Numerous physical and chemical agents can destroy mechanosensory hair cells in the inner ear of vertebrates, a process that is irreversible in mammals. Few experimental systems allow the observation of hair cell death mechanisms in vivo, in the intact animal, one of these being the lateral line system in the zebrafish. In this work we characterize the behavior of dying lateral line hair cells in fish exposed to low doses of copper in the water. The concentration of copper used in our study kills hair cells in a few hours, but removal of the metal is followed by robust regeneration of new hair cells. We use a combination of membrane and nuclear live stains, ultrastructural analysis and measurement of reactive oxygen species to characterize the events leading to the death of hair cells under these conditions. Our results show that a combination of necrotic cell death, accompanied by apoptotic features such as rapid DNA fragmentation, lead to the loss of these cells. We also show that hair cells exposed to copper undergo oxidative stress and that antioxidants can protect these cells from the effects of the metal. The study of this process in the zebrafish lateral line allows rapid morphological analysis of hair cell death and may be used as an efficient end point for molecule screens aimed at preventing these effects.
Whether the recently identified innate lymphocyte population coexpressing natural killer cell receptors (NKRs) and the nuclear receptor RORγt is part of the NK or lymphoid tissue inducer (LTi) cell lineage remains unclear. By using adoptive transfer of genetically tagged LTi-like cells, we demonstrate that NKR⁻RORγt(+) innate lymphocytes but not NK cells were direct progenitors to NKR(+)RORγt(+) cells in vivo. Genetic lineage tracing revealed that the differentiation of LTi-like cells was characterized by the stable upregulation of NKRs and a progressive loss of RORγt expression. Whereas interleukin-7 (IL-7) and intestinal microbiota stabilized RORγt expression within such NKR-LTi cells, IL-12 and IL-15 accelerated RORγt loss. RORγt(+) NKR-LTi cells produced IL-22, whereas RORγt⁻ NKR-LTi cells released IFN-γ and were potent inducers of colitis. Thus, the RORγt gradient in NKR-LTi cells serves as a tunable rheostat for their functional program. Our data also define a previously unappreciated role of RORγt⁻ NKR-LTi cells for the onset or maintenance of inflammatory bowel diseases.
Copper is an essential ion that forms part of the active sites of many proteins. At the same time, an excess of this metal produces free radicals that are toxic for cells and organisms. Fish have been used extensively to study the effects of metals, including copper, present in food or the environment. It has been shown that different metals induce different adaptive responses in adult fish. However, until now, scant information has been available about the responses that are induced by waterborne copper during early life stages of fish. Here, acute toxicity tests and LC50 curves have been generated for zebrafish larvae exposed to dissolved copper sulphate at different concentrations and for different treatment times. We determined that the larvae incorporate and accumulate copper present in the medium in a concentration-dependent manner, resulting in changes in gene expression. Using a transgenic fish line that expresses enhanced green fluorescent protein (EGFP) under the hsp70 promoter, we monitored tissue-specific stress responses to waterborne copper by following expression of the reporter. Furthermore, TUNEL assays revealed which tissues are more susceptible to cell death after exposure to copper. Our results establish a framework for the analysis of whole-organism management of excess external copper in developing aquatic animals.