Steeve Cruchet

The Notch pathway controls fibrotic and regenerative repair in the adult heart

Aims In the adult heart, Notch signalling regulates the response to injury. Notch inhibition leads to increased cardiomyocyte apoptosis, and exacerbates the development of cardiac hypertrophy and fibrosis. The role of Notch in the mesenchymal stromal cell fraction, which contains cardiac fibroblasts and cardiac precursor cells, is, however, largely unknown. In the present study, we evaluate, therefore, whether forced activation of the Notch pathway in mesenchymal stromal cells regulates pathological cardiac remodelling. Methods and results We generated transgenic mice overexpressing the Notch ligand Jagged1 on the surface of cardiomyocytes to activate Notch signalling in adjacent myocyte and non-myocyte cells. In neonatal transgenic mice, activated Notch sustained cardiac precursor and myocyte proliferation after birth, and led to increased numbers of cardiac myocytes in adult mice. In the adult heart under pressure overload, Notch inhibited the development of cardiomyocyte hypertrophy and transforming growth factor-β/connective tissue growth factor-mediated cardiac fibrosis. Most importantly, Notch activation in the stressed adult heart reduced the proliferation of myofibroblasts and stimulated the expansion of stem cell antigen-1-positive cells, and in particular of Nkx2.5-positive cardiac precursor cells. Conclusions We conclude that Notch is pivotal in the healing process of the injured heart. Specifically, Notch regulates key cellular mechanisms in the mesenchymal stromal cell population, and thereby controls the balance between fibrotic and regenerative repair in the adult heart. Altogether, these findings indicate that Notch represents a unique therapeutic target for inducing regeneration in the adult heart via mobilization of cardiac precursor cells.

Mechanosensory interactions drive collective behaviour in Drosophila

Collective behaviour enhances environmental sensing and decision-making in groups of animals. Experimental and theoretical investigations of schooling fish, flocking birds and human crowds have demonstrated that simple interactions between individuals can explain emergent group dynamics. These findings indicate the existence of neural circuits that support distributed behaviours, but the molecular and cellular identities of relevant sensory pathways are unknown. Here we show that Drosophila melanogaster exhibits collective responses to an aversive odour: individual flies weakly avoid the stimulus, but groups show enhanced escape reactions. Using high-resolution behavioural tracking, computational simulations, genetic perturbations, neural silencing and optogenetic activation we demonstrate that this collective odour avoidance arises from cascades of appendage touch interactions between pairs of flies. Inter-fly touch sensing and collective behaviour require the activity of distal leg mechanosensory sensilla neurons and the mechanosensory channel NOMPC. Remarkably, through these inter-fly encounters, wild-type flies can elicit avoidance behaviour in mutant animals that cannot sense the odour—a basic form of communication. Our data highlight the unexpected importance of social context in the sensory responses of a solitary species and open the door to a neural-circuit-level understanding of collective behaviour in animal groups.

Ir40a neurons are not DEET detectors

N,N-Diethyl-meta-toluamide (DEET) is the most widely used insect repellent, but it requires repeated application at high, potentially harmful, concentrations, which is prohibitively impractical and costly in the countries suffering most from insect vector-borne diseases1; understanding DEET’s mode of action might help identify improved alternatives. Kain et al.2 characterized ionotropic receptor 40a (Ir40a)-expressing olfactory sensory neurons (OSNs)—located in the sacculus of the Drosophila antenna3—as comprising a key pathway mediating aversion to both DEET and chemoinformatically predicted ‘DEET-like’ compounds, using calcium imaging, a transcription-based neural activity reporter, Ir40a RNA interference and behavioural experiments. We are unable to reproduce evidence for physiological activation of Ir40a OSNs by these repellents, and find that Ir40a mutant flies avoid DEET. These results call into question the importance of this sensory pathway in DEET detection. There is a Retraction accompanying this Brief Communication Arising by Kain, P. et al. Nature 534, http://dx.doi.org/10.1038/ nature18613 (2016). Ir40a-expressing OSN dendrites innervate sacculus chambers I and II (Fig. 1a), and their axons project to the ‘arm’ and ‘column’ in the antennal lobe (Fig. 1b)4. Using a calcium imaging-based screen for natural stimuli capable of activating these neurons (Extended Data Fig. 1), we identified ammonia as an agonist that evokes dose-dependent responses in the axon termini of all Ir40a neurons (Fig. 1c, d). This chemical specificity is consistent with the phylogenetic grouping of Ir40a with other members of the IR family of variant ionotropic glutamate receptors that detect amines4. Surprisingly, we were unable to reproduce the reported activation of Ir40a neurons by DEET or DEETlike compounds (butyl anthranilate, ethyl anthranilate and methyl N,N-dimethylanthranilate)2 (Fig. 1e). To rule out insufficient stimulus concentration as a cause for the lack of responses, we presented these chemicals directly to the antenna by manual puffing from a syringe, as described2. Using this method, we detected strong responses to ammonia, but not to DEET or DEET-like chemicals in Ir40a neurons (Fig. 1f). We also visualized calcium levels in Ir40a OSN soma, as described2, but this again revealed responses to ammonia but not DEET or DEET-like compounds (Fig. 1g and Supplementary Videos 1–8). Ir40a neurons were initially implicated as DEET sensors through their activation of a transcriptional reporter of neural activity (calciumdependent nuclear import of LexA, CaLexA; ref. 5)—expressed in all antennal neurons with the driver elav-Gal4—upon long-term DEET exposure2. The reliability of this reporter is, however, unclear, as less than half (2 to 3 out of 6) presumed Ir40a OSNs in chamber I and no chamber II OSNs were described to exhibit DEET-evoked CaLexA activity2. This seems not to be due to insufficient DEET, as exposure to 10% or 100% DEET led to similar CaLexA reporter results2. We therefore repeated these experiments and found, unexpectedly, that among the entire population of antennal neurons, chamber I neurons are particularly susceptible to background CaLexA signals, which were equivalent in flies exposed to no odour, DEET or ammonia (Fig. 1h). Such signals might be due to high basal calcium levels and/or basal nuclear translocation of abundantly expressed CaLexA reporter in these cells; regardless, our observations indicate that, under these experimental conditions, CaLexA cannot be used to report on stimulusevoked activity of sacculus chamber I neurons. To assess the function of Ir40a, we generated Ir40a antibodies and, by CRISPR/Cas9 genome editing, an Ir40a mutant (Extended Data Fig. 2a, b). In wild-type flies, Ir40a co-localizes with the co-receptor Ir25a (ref. 6) in OSN soma and dendrites, suggesting that these function together in sensory detection; in Ir40a mutants, anti-Ir40a immunoreactivity is absent (Extended Data Fig. 2b), confirming this allele to be a protein null. Consistent with these observations, physiological responses of Ir40a OSNs to ammonia are abolished in both Ir40a and Ir25a mutant animals (Extended Data Fig. 2c). We tested behavioural responses of Ir40a and Ir25a mutants in a DEET olfactory aversion assay (Fig. 1i–j). Both mutants avoid DEET as robustly as wild-type controls; in contrast, mutants for the odorant receptor co-receptor Orco no longer avoid DEET (Fig. 1j), confirming previous data in both Drosophila7 and mosquitoes8,9 that olfactory detection of DEET is OR-dependent. In summary, our data fail to reproduce evidence for physiological activation of Ir40a neurons by DEET, or to demonstrate a genetic requirement for either Ir40a or the co-receptor Ir25a in behavioural avoidance of this repellent. We propose that this sensory pathway is not relevant for DEET detection; future work will determine its role in behavioural responses to ammonia, an olfactory cue of ecological importance for many insects10,11.

Role of the sub-cellular localization of RasGAP fragment N2 for its ability to sensitize cancer cells to genotoxin-induced apoptosis

The specific sensitization of tumor cells to the apoptotic response induced by genotoxins is a promising way of increasing the efficacy of chemotherapies. The RasGAP-derived fragment N2, while not regulating apoptosis in normal cells, potently sensitizes tumor cells to cisplatin- and other genotoxin-induced cell death. Here we show that fragment N2 in living cells is mainly located in the cytoplasm and only minimally associated with specific organelles. The cytoplasmic localization of fragment N2 was required for its cisplatin-sensitization property because targeting it to the mitochondria or the ER abrogated its ability to increase the death of tumor cells in response to cisplatin. These results indicate that fragment N2 requires a spatially constrained cellular location to exert its anti-cancer activity.

Fluctuation-Driven Neural Dynamics Reproduce Drosophila Locomotor Patterns

The neural mechanisms determining the timing of even simple actions, such as when to walk or rest, are largely mysterious. One intriguing, but untested, hypothesis posits a role for ongoing activity fluctuations in neurons of central action selection circuits that drive animal behavior from moment to moment. To examine how fluctuating activity can contribute to action timing, we paired high-resolution measurements of freely walking Drosophila melanogaster with data-driven neural network modeling and dynamical systems analysis. We generated fluctuation-driven network models whose outputs—locomotor bouts—matched those measured from sensory-deprived Drosophila. From these models, we identified those that could also reproduce a second, unrelated dataset: the complex time-course of odor-evoked walking for genetically diverse Drosophila strains. Dynamical models that best reproduced both Drosophila basal and odor-evoked locomotor patterns exhibited specific characteristics. First, ongoing fluctuations were required. In a stochastic resonance-like manner, these fluctuations allowed neural activity to escape stable equilibria and to exceed a threshold for locomotion. Second, odor-induced shifts of equilibria in these models caused a depression in locomotor frequency following olfactory stimulation. Our models predict that activity fluctuations in action selection circuits cause behavioral output to more closely match sensory drive and may therefore enhance navigation in complex sensory environments. Together these data reveal how simple neural dynamics, when coupled with activity fluctuations, can give rise to complex patterns of animal behavior.

An expression atlas of chemosensory ionotropic glutamate receptors identifies a molecular basis of carbonation detection

Taste perception is thought to involve the encoding of appetitive and aversive chemical cues in food through a limited number of sensory pathways. Through expression analysis of the complete repertoire of Drosophila Ionotropic Receptors (IRs), a sensory subfamily of ionotropic glutamate receptors, we reveal that the majority of IRs is expressed in diverse peripheral neuron populations across gustatory organs in both larvae and adults, implying numerous roles in taste-evoked behaviours. We characterise Ir56d, which labels two anatomically-distinct classes of neurons in the proboscis: one represents a subset of sugar- and fatty acid-sensing neurons, while the other responds to carbonated solutions and fatty acids. Mutational analysis shows that IR56d, together with the broadly-expressed co-receptors IR25a and IR76b, is essential for physiological activation by carbonation and fatty acids, but not sucrose. We further demonstrate that carbonation is behaviourally attractive to flies (in an IR56d-dependent manner), but in a distinct way to other appetitive stimuli. Our work provides a valuable toolkit for investigating the taste functions of IRs, defines a molecular basis of carbonation sensing, and illustrates how the gustatory system uses combinatorial expression of sensory receptors in distinct neuron types to coordinate behaviour.

Sensory neuron lineage mapping and manipulation in the Drosophila olfactory system

Nervous systems exhibit myriad cell types, but understanding how this diversity arises is hampered by the difficulty to visualize and genetically-interrogate specific lineages, especially at early developmental stages prior to expression of unique molecular markers. Here, we use a genetic immortalization method to analyze the development of sensory neuron lineages in the Drosophila olfactory system, from their origin to terminal differentiation. We apply this approach to first define a fate map of all olfactory lineages and refine the model of temporal patterns of lineage divisions. Taking advantage of a selective marker for the lineage that gives rise to Or67d pheromone-sensing neurons and a genome-wide transcription factor RNAi screen, we identify the spatial and temporal requirements for Pointed, an ETS family member, in this developmental pathway. Transcriptomic analysis of wild-type and Pointed-depleted olfactory tissue reveals a universal requirement for this factor as a switch-like determinant of fates in these sensory lineages.

J007 Cardiac remodeling following chronic activation of the Notch signaling pathway

The Notch signaling pathway is a communication system between adjacent cells, mediated by transmembrane receptors (Notch1-4) and ligands (Jagged1 and 2, Delta-like1, 3, and 4). Notch is essential for the development and homeostasis of several self-renewing organ systems. Notch is also essential in the developing heart, and mutations in Notch genes cause cardiac malformations and congenital heart disease. Recently, using loss-of-function studies, we showed that the Notch1 receptor controlled the response to injury in the adult heart by limiting myocyte hypertrophy, enhancing myocyte survival, promoting precursor proliferation, controlling cardiogenic differentiation, and reducing interstitial fibrosis. In addition, our data suggested that upregulation of Jagged1 expression constituted a primary response in the stressed myocardium, suggesting that this ligand mediated Notch signaling in the postnatal heart. Therefore, to analyze the effects of a chronic Jagged1-induced Notch activation on the cardiac response to stress, we generated transgenic mice overexpressing Jagged1 in cardiomyocytes using the α-myosin heavy chain gene promoter (TGMHCJ1 mice). These mice expressed 100-500 fold Jagged1 mRNA relative to wild-type (WT) controls. The overexpression of Jagged1 on the surface of cardiomyocytes was confirmed by Western blotting and immunofluorescence staining. Consequently, the percentage of cardiac cells with an activated Notch pathway was higher in TGMHCJ1 mice than in WT animals. The expression of the Notch target genes Hes1 and Hey2 was also activated. Echocardiographic analysis under basal conditions revealed an enlargement of the right ventricle (RV) with a diminished left ventricle (LV) mass and chamber size in adults. TGMHCJ1 mice displayed increased juvenile mortality. The severity of the phenotype was dependent on the level of Jagged1 expression. Immunofluorescence analysis revealed myocyte hypertrophy and disarray in the RV of TGMHCJ1 mice whereas, myocytes in the LV were not affected. Moreover, when subjected to transaortic constriction (TAC), TGMHCJ1 mice demonstrated an impaired LV hypertrophic response to pressure overload and reduced cardiac fibrosis. These results indicate that chronic activation of Notch affects differently left and right ventricle integrity under physiological and pathological conditions.

An expression atlas of variant ionotropic glutamate receptors identifies a molecular basis of carbonation sensing

Through analysis of the Drosophila ionotropic receptors (IRs), a family of variant ionotropic glutamate receptors, we reveal that most IRs are expressed in peripheral neuron populations in diverse gustatory organs in larvae and adults. We characterise IR56d, which defines two anatomically-distinct neuron classes in the proboscis: one responds to carbonated solutions and fatty acids while the other represents a subset of sugar- and fatty acid-sensing cells. Mutational analysis indicates that IR56d, together with the broadly-expressed co-receptors IR25a and IR76b, is essential for physiological responses to carbonation and fatty acids, but not sugars. We further demonstrate that carbonation and fatty acids both promote IR56d-dependent attraction of flies, but through different behavioural outputs. Our work provides a toolkit for investigating taste functions of IRs, defines a subset of these receptors required for carbonation sensing, and illustrates how the gustatory system uses combinatorial expression of sensory molecules in distinct neurons to coordinate behaviour.Little is known about the role of variant ionotropic glutamate receptors (IRs) in insect taste. Here the authors characterise the expression pattern of IRs in the Drosophila gustatory system and highlight the role of one receptor, IR56d, in the detection of carbonation