Living organisms constantly make important decisions in their dynamic environments in order to survive. Animals make effective decisions in response to sensory stimuli by recruiting both innate and learned information in their nervous systems, thereby guiding them to approach or avoid cues that are appetitive or aversive respectively. Importantly, in order to adapt to a changing environment, animals also need to update their memories over time. Understanding how the nervous system invokes previously stored information to dictate future decisions has been a central and historical goal in neuroscience. Recently, significant progress has been made in identifying the connectivity and functionality of specific circuits involved in distinct aspects of learning and memory. However, a challenging task has been to obtain a comprehensive and systems-level understanding of neural circuit dynamics by which memories are retained, extinguished or expanded and integrated with innate information to eventually drive various behavioural actions. In this proposal I aim to address this problem by using a novel setup to record the activities of multiple individual neurons that function at different levels downstream of the learning and memory centre in the insect brain in freely behaving animals subject to specific tasks over time. Importantly, by combining this data with synaptic-level circuit maps generated by electron microscopy, mathematical modelling to produce reliable circuit motifs, and precise optogenetic manipulations to test the models in vivo, we will obtain both a global and high-resolution understanding of memory-based and context-dependent action-selection.

Epigenetic modifications regulate development. In particular, genome-wide DNA methylation patterns undergo dynamic changes at cornerstones of mammalian development. While DNA methylation is essential for normal embryogenesis, how it regulates and maintains cell fate and function in vivo remains unclear. A key methylation-regulated developmental process is parental imprinting and perturbations to methylation imprints result in embryonic lethality. We discovered that paternal deletion of an essential imprinting control region in mammals results in a temporal-specific compensatory methylation switch onto the opposite maternal allele, minimising detrimental developmental defects. This suggests a novel paradigm of epigenetic plasticity with wider implications for the epigenetic control of genome function. Here, we will utilize cutting-edge DNA and RNA single-cell sequencing methods and in vivo knockout models to study the functional roles of parental-origin specific epigenetic plasticity during development. We will determine whether loss of imprinting drives compensating changes on the other allele and decipher the associated mechanism of regulation, maintenance and hierarchical interactions. We will investigate the genome-wide plasticity of imprinting by relating imprinted gene dosage dynamics with developmental time points and tissue expression, with specific focus on particular developmental niches. Our approach will elucidate a new avenue of epigenetic control during development and disease, contribute to our understanding of the evolution and flexibility of imprinting and provide novel insights into regenerative medicine, reproduction and epigenetic inheritance.

Sleep is widely acknowledged to be essential to brain function, including learning and memory. Although learning relies on synaptic plasticity, there is surprisingly no consensus on the nature and function of the synaptic changes sleep would mediate. The controversy in the field partly derives from the complexity of sleep structure, as sleep is composed of REM and non-REM stages suggested to hold different functions that are poorly investigated at the synaptic level. To address this issue, I propose to use a recently-developed 3D two-photon microscope in order to continuous monitor synaptic changes during learning and specific sleep stages. The mechanisms underlying sleep stage-dependent synaptic plasticity will be further investigated by combining Ca2+ imaging, light-induced CaMKII inhibitor and optogenetics. The data obtained will provide new insights into the role of sleep in synaptic plasticity, and might further reveal fundamental mechanism underlying learning and memory.

The regeneration of the adult skeletal muscle relies on the activity of resident muscle stem cells (satellite cells). These PAX7-positive (PAX7+) cells are predominantly quiescent in uninjured muscles, while being able to rapidly proliferate upon injury. Mouse satellite cells are known to be heterogeneous, with subpopulations exhibiting graded quiescent states and variable regenerative potential. However, the molecular identities and developmental trajectories of satellite cell subsets are not well established. Moreover, due to the limited access to human tissues, little is known about the biology of human PAX7+ cells. The host laboratory has recently developed robust differentiation protocols that enable efficient generation of myofibers and PAX7+ cells from pluripotent stem cells in vitro. In this project, I plan to use single-cell transcriptomics to chart the dynamic cellular landscape of human in vitro myogenic differentiation, as well as mouse muscle development in vivo, with the aim of revealing the identities of emerging cells during myogenesis, developmental roadmap for cellular heterogeneity and molecular signatures of quiescence. In addition, preliminary results implicate the retinoic acid (RA) signaling in controlling satellite cell quiescence. Assisted by single-cell profiling, the role of RA will be characterized in detail. To further uncover in an unbiased fashion the gene networks linked to quiescence, I will also conduct functional genomic screens based on the in vitro platform. Overall, this study will use diverse techniques and model systems to provide new insights into muscle stem cell biology and potentially benefit the treatment for muscle diseases.

Phenotypic evolution concurs with spatial and temporal gene expression changes. Most work have been focused on spatial gene expression variations, yet little has been known about whether the temporal alteration of gene expression, termed heterochrony, also contributes to morphological variations. In this proposed work, I aim to address this question using forelimb (FL) and hindlimb (HL) development in the mouse embryo as a model, due to the ease of experimental manipulation of this system. Although gene regulatory network underlying limb development is highly conserved, the morphology of FL and HL differs. An important distinction in their development is the relative duration of gene expression time – nearly all limb regulators express for a shorter period in HL, correlates with its curtailed differentiation time-scale comparing to FL. Whether and to what extent do such temporal gene expression differences relate to morphological divergence remains unclear. To this end, I will systematically and quantitatively characterize the kinetics of key limb regulators’ expression during the FL and HL development in the mouse embryo; then combining in vitro culture assays and sequencing methods to investigate the mechanisms causing temporal gene expression differences between the two appendages; finally, I will functionally assess the role of such heterochrony to limb morphology by altering the gene expression timing, first experimentally, in mouse and chick embryos; and then theoretically, by using mathematical modelling. Together, this proposed work can advance our understanding of the connection from genotype to phenotype and will also aid biomedical research on limb defects.

Keen observers of nature have often wondered why diverse species seem to behave similarly. For example, different species of Hawaiian spiders spin similar web architectures, diverse anoles lizard species bob their heads with the same styles and speeds, and distinct species of damselflies avoid predators using the same techniques. These and many other striking examples are thought to represent the convergent evolution of behaviors. Does this convergence reflect changes in the same genes or does evolution act through many genetic routes to create the same behaviors? Genetic differences clearly play a role in behavioral variation, but it remains challenging to identify the genes that underlie evolution of behaviors. However, convergence in the genetic basis of developmental or physiological traits has been discovered with many specific examples of genes and mechanisms, so it is possible to use studies of convergence to discover how behaviors evolve. Therefore, we will use a powerful comparative system to discover the genes and molecular mechanisms that underlie convergent evolution of behaviors for the first time across divergent animals.
The Caenorhabditis nematodes offer a unique experimental platform to connect behavioral differences to genetic differences. Starting with the keystone model organism, C. elegans, and existing data, we will characterize and classify genetic differences across wild isolates from three species of Caenorhabditis - C. briggsae, C. elegans, and C. tropicalis. Whole-genome genotype data combined with high-throughput, high-content imaging of behaviors from these same wild isolates will be input into unsupervised machine learning algorithms to create a high-resolution genotype-phenotype map for a range of natural behaviors and examples of convergence. This map will be queried for signatures of shared genetic changes at orthologous genes to identify which variants are most important evolutionarily. The result will provide the first systematic glimpse into the genomic “knobs” that control behaviors at single-variant resolution across species and insights into the repeatability of the evolution of behaviors.

Sea urchins are marine animals genetically close to the vertebrate lineage. Being eye-less and lacking a central nervous system (NS), these animals instead feature dermal photoreceptors dispersed over their spherical body surface and feeding into a decentralized NS. However, sea urchins can visually resolve objects and move towards them, and they can detect looming visual stimuli from any direction and accurately point their spines towards them. Such performance is normally associated with proper eyes feeding information into a brain. Sea urchins thus offer access to a unique visual system of a type that to date has not been studied in terms of its information processing. This alternative solution to vision may also have potential biomimetic applications for robotic miniaturization, smart probes, and intelligent materials where dispersed light detectors control the properties of the material.
The core of the proposed project is to investigate and model the neural mechanisms of information processing, which enables sea urchins to perform spherical vision by deploying an obviously very different mechanism from today's technology, and also very different from visually guided behavior in most other animals. Our study includes molecular and morphological identification of cell types, measurements of behavioral responses and electrophysiological photoreceptor responses, mapping the connectome of sea urchin photoreceptors and NS, and theoretical modelling of the information processing underlying visually guided behavior. We will map the connectomics of the NS and record the activity from key positions in the processing of visual information and generation of locomotory responses. The data will be used for computational modelling of the entire process from visual input to motor control. Special focus will be given to behavioral decisions where small changes in stimuli cause behavioral switches. We will also use genetic approaches to test the agreement between theoretical models and actual behavior.

The Personalized, Regenerative Medicine of the future will rely on being able to make replacement cells and tissues of choice at will and in a robust, predictive manner. However, key challenges have to be overcome before the promise of personalized stem cell therapeutics becomes a reality. This is because stem cell renewal/differentiation are stochastic processes, precluding the differentiation of a stem cell population into a homogeneously differentiated desired cell type, but also leading to spurious differentiation during renewal. This is thought to partly arise from heterogeneous single-cell signaling states among different cells of a population, which are not measurable using classical ‘population-average’ biochemical methods. A mechanistic understanding of how dynamic signaling processes control differentiation/renewal fates at the single-cell level might therefore significantly improve our capacity to robustly and precisely manipulate cell fates for tissue engineering purposes. We propose to use an integrated interdisciplinary strategy to map the dynamic, single-cell signaling programs that control differentiation/renewal using human Pluripotent Stem Cell (hPSC) differentiation into neural stem cells as a differentiation paradigm. Using multiplexed, genetically-encoded biosensors, we will quantitate hPSC single-cell dynamic signaling states by large-scale, multi-color, multi-day timelapse microscopy across millions of cells, to reveal with unparalleled precision how heterogeneous signaling states correlate with renewal/differentiation fates. Using computer vision approaches, we will automatically segment, track and lineage at scale each of the cells that were induced to self-renew or differentiate, and we will extract a panel of signaling, cell-cycle, pluripotency state, and cell morphodynamics features that quantify these dynamic processes. We will then mine these high-dimensional feature sets to build computational models that identify dynamic single-cell signaling patterns associated with robust fate transitions and predict actionable interventions that might cause those transitions. Lastly, using drug perturbations, and/or microfluidic/optogenetic actuators, we will quantitatively test those predictions by evoking synthetic dynamic signaling states that induce robust fate transitions. Our approach will help to significantly clarify the mechanistic basis of signaling-mediated human stem cell fate decisions, providing new avenues to robustly control stem cell fate. This might help establish a larger framework, broadly applicable to other hPSC lines and differentiation routes.

Embryos develop precisely at the multicellular level. Yet, stochasticity at the single cell level generates local variability in behaviors (e.g. in cell division, cell positioning, and gene expression). How is this apparent contradiction resolved? Do embryos compensate or possibly exploit local variability to adjust or correct patterns?
In mammalian embryos, the first developmental axis forms in the blastocyst when the outer trophoblasts (the future placenta) form a globe with an axis of proliferation/differentiation originating from the cluster of inner embryonic cells (the future embryo).
Here, we will investigate the principles underlying axis formation through a unique combination of stem cell-based embryology, quantitative imaging of the phenome of trophoblasts, and computational and statistical modeling. Using a novel blastocyst model formed solely with stem cells (Nicolas Rivron, The Netherlands), we will tune the embryonic signals and richly quantify the impact on trophoblast phenotypes, and their variability and precision in space (Shantanu Singh, USA), to model cells’ coordination during axis formation (Kyogo Kawaguchi, Japan).
This unique synergy will reveal how individual stem cells resolve the contrasting forces of single cell variability and multicellular guidance (e.g. embryonic inductions, neighbor coupling), to adjust and achieve a level of precision during the generation of an axis.

Osteoderms are hard calcified tissues that form within the skin of some animals. They resemble bone, hence the name, but are fundamentally different in several respects. Crocodile and armadillo skin plates, and turtle shells are among the most familiar examples, reportedly forming a protective armour against external predators and aiding locomotion. However, although less visible, osteoderms are also present in many lizards.
In terms of their shape, spatial distribution, and interaction, lizard osteoderms show the highest diversity in the animal kingdom, yet we know little about what drives this extraordinary diversity, how it is controlled, or how it originated. It could be a biproduct of other genetic differences or, more likely, a natural optimization to enhance osteoderm function, protective or otherwise, under conditions specific to each lizard type.
This project brings together a multidisciplinary team of expert engineers, developmental and evolutionary biologists from the UK, Canada and France to investigate the mechanisms underlying the development, patterning, and evolution of osteoderms in lizards. The team will use a range of advanced techniques (e.g. genetic analysis, material testing, imaging, and computer simulations) to investigate lizard osteoderms from the first molecular signalling events and cellular interactions, through to organismal level. Osteoderm mechanical properties will be characterised both as single units and as sheets so as to understand their function during feeding and locomotion.
This is a basic science project focused on a novel biological tissue and its evolutionary implications, but with a systems approach that may shed light on pathological calcifications, as well as aiding the development of biomimetic materials and structures. Most importantly it will train the next generation of scientists, in a multidisciplinary and international setting, providing them with a fundamental knowledge of biological tissues and a diverse skillset with which to address the global challenges of 21st century.

The gastrointestinal (GI) tract is efficiently organized to protect the host from potential dangerous stimuli and to tolerate commensal microbiota and food antigens. It hosts as many neurons as the spinal cord and more immune cells than all other compartments together. The innervation of the digestive tract is involved in determining the patterns of its movements, control of gastric acid secretion, release of gut hormones, modifying nutrient handling and interacting with the gut immune system. Infections of the GI tract can result in neuronal damage and dysregulation of these functions. The host laboratory has recently demonstrated that upon infection, in response to specific neurotransmitters, macrophages – a subset of innate immune cells – can acquire tissue-protective phenotype and minimize neuronal loss. Both immune and nervous systems are equipped with mechanisms allowing to store and recall information on previously encountered events. On this basis, I aim to examine whether GI infections trigger a neuro-immunological memory supporting tissue repair or response to subsequent infections. This is of fundamental and clinical interest, as interactions between immune and neuronal cells are proposed to be part of several disease processes, ranging from multiple sclerosis to irritable bowel syndrome (IBS).

The cell membrane is a double layer of lipid molecules. It plays a critical role in protecting the cell from its environment and in separating the different processes that take place within its interior. Membranes must change their shape in order for the cell to function, especially during cell division, and this depends on membrane curvature. At present, cells are only known to induce curvature by accumulating lipids in one of the layers or using specialised proteins. Our goal is to investigate a new mechanism of inducing membrane curvature by accumulation of hydrocarbons in the middle of the lipid layers that has not been observed before in nature.
These hydrocarbons are like the components of diesel fuel, and are found in photosynthetic cyanobacteria and algae – some of the most abundant and widespread organisms on Earth. Production of hydrocarbons in cyanobacteria or other microbes could substitute for liquid fuels derived from petroleum. As well, cyanobacteria and algae release hydrocarbons into the environment, where they are degraded by other bacteria that clean up oil spills. However despite their environmental and biotechnological importance, the exact cellular role of hydrocarbons has not been determined.
We recently discovered that hydrocarbons are essential for maintaining optimal cell size, growth and division, processes that require cell membranes to curve and bend, and found that cells lacking hydrocarbons have lipid membranes that are less curved or flexible. We showed that hydrocarbons integrate into the cell membranes, and used computer simulations to predict that this induces membrane curvature. To investigate this further, we have assembled a team of scientists from the UK, Sweden and New Zealand. By combining our different skills, we will analyse how hydrocarbons affect the physical properties of algal and cyanobacterial membranes by 1) running computer simulations; 2) studying membranes purified from algae and cyanobacteria; and 3) carrying out experiments on live cells. Together, these simulations and experiments will allow us to explore and quantify how hydrocarbons affect curvature and other membrane properties, and so conclusively establish the role of hydrocarbons in cells. As well as improving our understanding of biology, this information will assist the use of microbes for biofuel production and oil spill cleanup.

Hypothalamic tanycytes are sensory glia-like cells lining the walls of the 3rd ventricle (3V) that were proposed to respond to a variety of metabolic and environmental stimuli. The preoptic area (POA) is a hypothalamic region involved in thermoregulation whose nuclei are distributed around the 3V. The POA receives input from peripheral temperature receptors but also contains neurons that respond to deep-brain thermal changes, although the exact mechanism of this phenomenon is not clear. Due to the privileged position of tanycytes, with their somata directly contacting the cerebrospinal fluid and processes reaching portal blood vessels, we propose that tanycytes are part of the central temperature-sensing circuitry. This hypothesis is supported by the fact that they express two receptors, TRPM5 and TRPM3, which are known to be temperature-sensitive. In this project I will use transgenic mouse lines and employ a combination of electrophysiological, chemogenetic, optogenetic and imaging techniques to explore the sensory roles of tanycytes, mostly focusing on thermodetection. I will start with characterising sensory capabilities of tanycytes including their responsiveness to temperature changes and analysing their functional diversity. Then I will test whether tanycytes communicate with neurons in the POA and if so, I will attempt to identify the phenotype of these neurons. Lastly, I will perform in vivo experiments to test the effect of chemogenetic activation and inhibition of temperature-sensitive tanycytes on thermal effector responses as well as eating behaviour. Overall, I expect my study to shed light on the role tanycytes play in temperature detection and homeostasis.

Morphological plasticity is a key adaptive process allowing organisms to cope with changes in their environment. In animals, this process has been described in ephemeral organisms (e.g. flies, worms and mice) that typically experience a specific period in which development will respond to environmental signals producing long-lasting changes in animal body. In contrast, animals with extreme longevity are constantly patterning such that they must continuously adjust their developmental and physiological behaviours with the unpredictable fluctuations of food supply. To decipher the logic of such enhanced morphological plasticity, I am using the sea anemone Nematostella vectensis, a cnidarian laboratory model that exhibits a striking ability to adjust developmental patterns to the nutritional status of the environment while having a long lifespan. Based on preliminary data from the host lab, muscle is emerging as a central tissue that mediates the nutrient-dependent development of Nematostella tentacles. I will define this novel property of muscle cells in response to feeding using transgenic reporter lines. In parallel, I will also establish novel genetic tools to manipulate different muscle types and determine their specific contribution to post-embryonic development. I will also perform single-cell sequencing experiments to characterize the potential metabolic and developmental properties of muscles. Altogether, this proposed work will provide new insight into how morphological plasticity is encoded at the cellular and molecular levels in a long-lived animal.

A shared feature of many anxiety disorders is the overgeneralization of fear, the tendency to falsely link together events that have differing emotional significance. In the case of abnormal fear learning this can produce heightened levels of arousal and fear in situations that should be perceived as safe.
Previous studies indicate several distributed brain areas interacting with neuronal networks in the amygdala to modulate the fearful perception of diverse stimuli. It therefore remains largely unknown how changes in synaptic and cellular function (synaptic scale) can influence encoding of fearful stimuli in large cell networks (network scale) and how interaction between brain areas (global scale) finally leads to overgeneralized behavioral responses. Although a few studies have begun to examine neural substrates of fear generalization at the synaptic and network level, to date, no structured and integrated picture has been generated. This major gap exists because no study so far has been able to integrate experimental results across the different scales to generate a complete model of interactions that regulate fear generalization. As a consequence, new therapies and drugs to treat anxiety disorders emerge only slowly with no major discoveries in sight.
In this proposal, we aim to utilize a unique combination of expertise in neurophysiology, optogenetics and advanced brain imaging to study the mechanisms of fear generalization across spatial scales, ranging from synapses to individual neurons, to large-scale, distributed networks across the whole brain. For the first time we will investigate how local amygdalar networks regulate fear generalization within the context of their afferent signals emerging from global brain activity. To connect synaptic, network and global scales, we will develop new innovative technologies such as an fMRI compatible version of a miniaturized microscope for functional Ca2+ imaging to simultaneously monitor whole brain function and local network activity during fear generalization tests in mice. We will complement these studies with in vivo Ca2+ imaging, optogenetics, and ex vivo electrophysiology to identify the distributed inputs and local circuits in the amygdala that generate overgeneralized fear responses.
The goal of our interdisciplinary systems neuroscience/engineering approach is to provide a comprehensive understanding of the neural substrates that trigger overgeneralized fear, thereby generating a novel framework for how anxiety-related behaviors emerge across different brain scales.

In this project we will uncover the evolutionary dynamics in both humans and pathogens in response to epidemics. Recent technology advances enable detection of infectious agents in ancient DNA (aDNA) samples that tie to major historical epidemics. However, we know practically nothing about the dynamic process by which genetic adaptation occurs simultaneously in both the host and pathogen as a consequence of epidemic outbreaks.
We propose to integrate aDNA and sophisticated computational approaches to investigate the selective pressures imposed by the introduction of new pathogens during European colonization of the Americas. Our goals are to characterize the changes in both pathogen and human genetic diversity before and after European colonization to describe: 1) the genetic signatures that were putatively responsible for decimating the Native population, and 2) the selective and demographic processes that conferred adaptation to the colonization environment, especially with respect to pathogen exposure. To this end we will sequence the genomes of at least 30 individuals from before and immediately after colonization, and for the Colonial period we have access to archaeological remains of individuals who were likely victims of epidemics. We will also leverage the metagenomic data produced when sequencing aDNA and quantify the pathogen genetic diversity present in these samples before and after colonization. Lastly, we will use novel statistical methods to identify loci in post-colonization samples that depart from expected proportions of Native American, European or African ancestry to test whether admixture facilitated adaptation.
Our study will leverage temporal genomic data to address a long-standing question of how pathogens have influenced human evolution. As we lack studies quantifying jointly the changes in both pathogen and human diversity across time, this project offers a unique opportunity to directly assess, for the first time, how much evolutionary pressure is experienced within a human population when encountering new pathogens. Our design integrates novel paleogenomics approaches and cutting-edge methods development to leverage longitudinal sequence data of both ancient host and ancient pathogen sequence data to address coevolution with a temporal resolution that has not previously been reached.

Despite effort, it remains incredibly difficult to identify the cellular basis, and/or the causative genetic variants, underlying complex behavior. Understanding how behavior is encoded requires solving a dual problem involving both neurodevelopment and circuit function. Genes build nervous systems; nervous systems are activated to produce behavior. Streelman and Baier will collaborate to develop a unique model system to chart the complex path from genome to brain to behavior, in vertebrates from natural populations. In Lake Malawi, male cichlid fishes construct sand ‘bowers’ to attract females for mating. Bower building is an innate, repeatable natural behavior that we quantify in the lab. Males build two bower types: 1) pits, which are depressions in the sand, and 2) castles, which resemble miniature volcanoes. Species that build these two bower types can interbreed in the lab. Remarkably, first-generation hybrids of pit- and castle- species perform both behaviors in sequence, constructing first a pit and then a castle bower, indicating that a single brain containing two genomes can produce each behavior in succession. Moreover, brain gene expression in these hybrids is biased towards pit- alleles during pit digging, and castle- alleles during castle building. This phenomenon of allele-specific expression matched to behavior is compelling and offers the chance to identify the genome regulatory logic and neural circuitry underlying complex behavior. Streelman’s group will use single-cell RNA-sequencing to pinpoint specific cell populations that mediate context-dependent allele-specific expression in male bower builders. Baier’s team will use genome editing and optogenetic tools to manipulate the neurons that matter in the brains of behaving Malawi bower builders. Our collaborative work will thus identify the neurons responsible for biased allelic gene expression matched to behavior, and then manipulate those neurons to modify behavioral output. Achieving our goals will demonstrate how the genome is activated in particular cell types to produce context-dependent natural social behaviors.

Capturing the dynamic 3D atomic-scale structure of a macromolecular machine while it performs its biological function remains an outstanding goal of biology. Conventional structural tools (e.g. X-ray crystallography, NMR & cryoEM) only provide ‘snapshots’ of stable states along a reaction pathway. Reaction intermediates, and in particular short-lived intermediates, are hard to capture and characterize with such conventional techniques. Here we propose to combine (prior) information from multiple existing static structures of stable states with dynamic datasets of inter-atomic distances obtained by high-throughput non-equilibrium single-molecule FRET (smFRET) measurements in a microfluidic mixer using novel time-resolved multi-pixel single-photon avalanche diode detector. These measurements will be performed on libraries of molecular constructs, sampling multiple inter-atomic distances as function of reaction time. These measured distance distributions will then serve as multiple intra- and inter-domain distance constraints which, together with prior information (available structures), will enable the Rosetta software to achieve large-scale energy optimization-based refinement of time-resolved ‘snap shots’ of complex structures with improved accuracy. These time-resolved Rosetta structures together with intermediary molecular dynamics simulations will allow solving the 3D atomic-level structure of the macromolecule for each sampled reaction time point, eventually producing a 3D structural dynamic movie of the macromolecule in action. To demonstrate the utility of the proposed method, we will solve the dynamic structure of RNA polymerase during transcription initiation (promoter binding, bubble opening, abortive initiation, promoter clearance) and a pair of intrinsically disordered proteins (IDPs) involved in transcription regulation (ACTR and NCBD) that adopt a fully folded structure during a coupled folding and binding reaction. In addition to elucidating outstanding questions in transcription by combining detector developments, high-throughput and time-resolved out-of-equilibrium single-molecule FRET measurements with new experimentally-constrained molecular structure computational approaches, this multidisciplinary project will result in a new generic toolkit applicable to a large array of enzymes, proteins and molecular machines.

To understand how the brain represents the world is a central theme in neuroscience. Neural circuits encode information in terms of rate, timing and synchrony of action potentials arising from the activity of a complex spatial organization of excitatory/inhibitory neurons. The identity of the active neurons at any given time has a profound effect onto the final outcome and regulates fundamental human psychophysical behavior. Attempts to decode the complex behaviors in neurons has led to development of several neuromodulation and sensing tools, by which neural activity can be controlled by microelectrodes or through genetically altering specific neural circuits, ultimately to deliver electrical impulses. Unfortunately, these techniques introduce strong perturbations to the observed system, without reaching the desired spatio-temporal resolution. Experimental approaches that allow non-invasive activation of specific phenotypic groups of neurons could introduce systematic variation of the timing of signals revealing how neural ensembles encode information. Here we propose a novel (nano)tool to alter membrane potential in specific neuronal phenotypes in a spatiotemporally controlled manner.
The nFlare project will develop a novel research paradigm in neuroscience based on a new class of injectable nanodevices delivered to neurons and anchored to their membranes. These nanotools have the ability to depolarize cell membranes or to deliver active biomolecules. Electric or chemical stimulation of single cells will be achieved using deep-penetration near infrared excitation light, to reduce invasiveness.
nFlare core aims are: i) the development of nanodevice components able to generate photoelectrochemical current upon illumination with light; ii) state-of-the-art biochemical functionalization of the nanodevice to target specific neurons and to reduce inflammatory response; iii) applicability of such nanoscale devices as high spatio-temporal neuronal activity modulators in 2D and 3D in vitro neuronal networks followed by in vivo validation.
Selective modulation of neural circuits by artificial stimulation of neuronal membrane or controlled delivery of environmental factors could help answering fundamental neurobiological questions.

How cell differentiation and morphogenesis are coordinated during embryonic development is an open question in developmental biology. The accepted theory sees morphogenesis as a consequence of the cell differentiation events initiated by biochemical signals. However, recent evidence shows that embryonic cells can adjust their differentiation program according to the size and duration of their cell-cell contacts, suggesting that the interplay between biochemical signals and mechanical stimuli determines cell fate. Yet, how these stimuli are integrated in the gene regulatory networks of cell differentiation is largely unknown. Here I will pioneer an evolutionary approach to find conserved mechanisms by which cell-cell contacts regulate gene expression, by comparing two echinoderm species, sea urchin and sea star. In both species, mesendoderm differentiation depends on Wnt/ß-catenin signalling. However, they present different patterns of Wnt/ß-catenin activity and different cell-cell adhesion properties, with sea star cells being far less adhesive than sea urchin cells. Using a combination of live imaging, scRNAseq and theoretical modelling, I will determine how cell-cell contacts regulate mesendoderm cell differentiation in the two species and if variation in cell-cell contact formation underlies the divergence of tissue patterns between sea urchin and star embryos. This study will uncover basic principles of cell differentiation and tissue patterning, broadening our understanding of embryonic development and evolution.