We propose to apply cell-free synthetic biology to study fundamental aspects of transcriptional regulation, gene regulatory network evolution, and network robustness. Cell-free synthetic biology emerged recently as an in vitro alternative for network engineering. Cell-free synthetic biology implements biological systems in a coupled transcription – translation reaction and therefore is a well-defined environment that is easy to control and interrogate. Since genetic networks can be implemented in vitro as linear dsDNA templates, as opposed to plasmids, it also circumvents time consuming cloning and transformation steps, enabling rapid prototyping of genetic systems. We have recently implemented cell-free systems on a microfluidic microchemostat array, which performs transcription-translation reactions at steady state for 30+hours, allowing us to implement complex genetic networks in vitro. Here we propose to apply this novel concept to study fundamental principles of genetic networks. We will interrogate two different types of oscillators (phase and relaxation) and determine how cis- and trans-changes to the network impact network function and robustness. Specifically, we plan to map the precise cis-binding energy landscape of these networks by characterizing a large number of network variants with modified transcription factor binding sites. To implement trans-element changes we will employ synthetic Zn-finger transcriptional regulators with defined specificity landscapes to determine how changes to the trans-element influence network performance and robustness. Finally, we will also attempt to rationally evolve network function through a promiscuous transcription factor intermediate. To determine how robust these networks are to external perturbations, we will change reaction temperatures, and independently modulate transcription and translation rates. Finally, we will characterize a subset of our networks on the single cell level in vivo to evaluate network robustness in a regime were stochasticity dominates. Gene regulatory networks are complex systems that are difficult to quantitatively characterize in vivo. The in vitro cell-free synthetic biology approach proposed here has the potential to contribute significantly to the mechanistic understanding of complex biological systems.

There is a common perception that larger brains mediate higher ‘intelligence’ or cognitive capacity. Many insects, however, demonstrate that sophisticated social structures and cognition are possible with very small brains. Foraging honeybees, for example, memorize flower signals and display higher-order learning such as categorization, concept learning and ‘counting’. How do bees perform these feats with small nervous systems? Despite the extensive knowledge on the cognitive abilities of bees, to date, no study has attempted to elucidate the neural mechanisms underpinning their higher-order learning. Here we will pool our expertise in cognitive, computational and neuroscience methods to unravel the neural circuits and architecture underpinning such cognitive performances in the honeybee. We will combine behavioral recordings of bees walking stationarily on a locomotion compensator in a virtual environment with access to its brain via multielectrode recordings. Data will be fed into computational models providing testable hypotheses about minimal neural architectures for cognition, and ultimately working towards whole-brain modeling (connectomics).
This project will expand the currently available information on the neurobiology of insect learning, and will provide the first complete computational model for the neuronal mechanisms that underlie cognition in a miniature nervous system. Our expected results have far-reaching implications for psychophysics, neuroscience and behavioral biology as well as the potential to revolutionize the field of visual processing in animals and robotics.

The aim of this project is to understand how molecular forces ‘scale-up’ to generate biological outcomes. We focus on understanding the contractile actomyosin cell cortex, a sub-micron thick network that lies beneath the plasma membrane that supports cellular and tissue morphogenesis. Ultimately, functional outcomes and active mechanical behaviour at the larger scale must arise from the intrinsic biophysical properties of the force-generating molecules of the cortex. However, how these properties emerge is unclear. Our consortium will implement novel experimental approaches solutions to tackle this question. First, we will develop myosin mutants that allow us to modulate specific parameters of motor function (e.g. force, step-size, directionality, torque; Bryant) under control by light; we call this ‘Optomechanics’. Second, we will develop hydrodynamic active-gel theory for describing essential physical behaviors arising from molecular-scale force and torque generation in the cortex (Grill). We will apply these approaches to understand the contribution of the cortex to cellular morphogenesis during cytokinesis (Grill) and junctional tissue homeostasis (Yap). For this purpose, we bring expertise in myosin biophysics and molecular bioengineering (Bryant), cortical mechanics and theory (Grill), and cell junctions and morphogenesis (Yap). The bioengineering team will generate validated reagents that will be implemented in cells by the biological teams; results from the latter will reveal how modifications in myosin force generation at the single molecule level relate to in-vivo function. We will combine optical imaging, laser nanodissection with hydrodynamic theory to develop quantitative descriptions of cortical mechanics during cytokinesis in the C. elegans zygote. In parallel, we will define the impact of cadherin on cortical mechanics at mammalian epithelial cell-cell junctions, and how this influences mechanical coherence of populations. This combination of molecular manipulation with theory will allow us to describe how actomyosin mechanical activity at cell and tissue scales emerges from myosin motor function at the molecular scale, and how myosin molecular force drives cell- and tissue-level mechanics.

The crucial importance of secreted extracellular scaffolding proteins (ESPs), such as precerebellins, neuronal pentraxins, and C1q-like molecules, for the formation and function of neuronal synapses in vivo has only recently been discovered. These molecules share a modular structure, with domains that specifically interact with pre- and postsynaptic cell surface receptors to bridge neurons across the synaptic cleft. Guided by structural information of known ESPs, we aim to develop synthetic connector molecules that will expand the range and affinity of trans-synaptic interactions. These synthetic ESPs will likely become new and powerful tools to manipulate the number and properties of excitatory and inhibitory synapses, and to regulate synaptic communication and cognitive abilities in mouse models in vivo.
To achieve our goals, we will develop and implement a highly multi-disciplinary approach based on the diverse and complementary expertise and state-of-the-art techniques established in the applicants’ laboratories. Armed with structural and biophysical knowledge about physiological ESPs and their receptor interactions, we will design hybrid molecules that contain novel combinations of binding domains and thus promote a novel spectrum of molecular interactions across neuronal synapses. The synthetic ESPs will be further refined by mutagenesis or by replacing native domains with nanobodies of desired specificity. These novel neuronal connectors will be recombinantly produced, purified and delivered into subregions of the hippocampus and the cerebellum by acute or chronic injections using osmotic pumps. We will also express genes encoding the designer ESPs using viral vectors with neuron type-specific promoters. Electrophysiology, confocal microscopy, nanoscopy and behavioral analyses will be then employed to verify the targeting and functional efficacy of synthetic ESPs at the synaptic, neural network and organismic levels. Their effects on motor and spatial learning, reversal learning, pattern separation and completion will be studied in combination with recordings of neural activity oscillations and single-cell firing.
Considering the huge potential variety and versatility of these novel tools, we anticipate an immediate impact onto basic neuroscience and, in longer term, potential new avenues for the treatment of neuropsychiatric disorders.

The tiny nematode worm C.elegans has been instrumental for identifying genes that increase lifespan. Although most of the genes increasing lifespan in C.elegans are also present in humans, only very few have been associated with human longevity. This is mainly due to the fact that humans are more complex than worms. Instead of studying single genes we will investigate a multi-gene approach where we assume that lifespan in the worm can be predicted by the behaviour of all these genes together. We do this by viewing the worm genetic system as an ecological system. Similar to the fact that ecological systems display characteristic behaviors before they can collapse, we will test if the gene-regulatory network of worms displays characteristic behavior when approaching a collapse leading to death. This will provide insight in the mechanisms of aging and might help to translate the results to humans.

1. Astrocytes, classically considered as simply supportive cells for neurons, are emerging as relevant elements in brain information processing through their ability to regulate synaptic activity. Indeed, the tripartite synapse formed by pre- and post-synaptic neurons and surrounding astrocyte structures has been proposed as a functional unit of brain processes. These novel and important functions of astrocytes are under control of G protein signaling-dependent processes, which trigger astrocyte Ca2+ signals eventually leading to the release gliotransmitters and other mediators regulating synaptic functions.
2. Recent evidence indicates that the roles of mitochondria in the brain may go beyond the mere needs of energy supply for cell survival and maintenance, being possibly involved in the regulation of synaptic functions. It is conceivable that mitochondrial G protein signaling participates in these processes. Various subtypes of G protein-coupled receptors (GPCRs) and the associated signaling molecular elements are present within mitochondria, suggesting the existence of mitochondrial G (mtG) protein signaling pathways.

Thus, astroglial mtG signaling potentially plays an important role in the regulation of tripartite synapse and hence brain functions. However, no studies have addressed this issue so far.
The present project proposes to investigate the consequences of the activation of astroglial mtG signaling pathways in brain physiology, identifying the underlying mechanisms at cellular, network, behavioral and theoretical modeling levels. We propose to generate and develop novel pharmacogenetic tools (DREADDs specifically expressed by astroglial mitochondria, mtDREADDs) that will allow the specific control of astroglial mtG protein activity. We will experimentally and theoretically analyze the consequences of activation of different mtG proteins (via mtDREADDs) on neuronal, synaptic, and network activity as well as brain functions in living animals.
The expected results will reveal novel processes of cellular signaling in the CNS, and will identify new regulatory mechanisms mediated by astroglial cells in brain function.

One of the main challenges of this century is to better understand the function of the brain in order to solve its devastating disorders. We have learned a great deal about the basic components of the brain: the different types of neurons, each with its unique arborization, and the connections, termed synapses that enable neurons to communicate with each other. We also have a basic understanding of how information is transmitted through electrical and chemical signals. However, we know little about how individual neurons integrate the multiplicity of inputs, both excitatory and inhibitory, to compute a single output. Nor do we understand much about how individual neurons work in concert within a specific circuit to perform complicated computations. A major limitation has been our inability to monitor and control the electrical and synaptic activities of neurons inside the brain of a living animal. Overcoming this limitation requires the combination of different technologies to enable neuronal activity to be monitored with high sensitivity and fidelity while minimally interfering with brain function.
This grant exploits advances in optical methods, molecular biology, genetics, protein engineering, and electrophysiology and brings together tools developed in three laboratories, each with distinct expertise. These include a fiber-optic-based "micro-optrode" that can be inserted into the brain of a living animal in order to simultaneously record the electrical activity and biochemical signals. In particular, this instrument can detect and decode optical signals transmitted by probes that respond to the microenvironment inside the neuron. Such molecular probes, which are introduced into neurons through genetic manipulations, are designed to reveal how the neuron decodes synaptic messages that it receives from its connected partners in the brain of an animal receiving sensory stimulation. To dissect how signal processing within the various compartments and arbors of a neuron contributes to its function, we are exploiting a novel strategy to locate the molecular probes in specific neuronal microdomains. The combination of innovative approaches that we propose in this grant is expected to produce new knowledge about the contribution of specific neuronal subtypes and circuits to the complex processing of the brain in normal and pathological conditions.

Transposons are genetic elements that can cut themselves out of the genome and hop to a new location. Remarkably, they comprise half the human genome, but their functions are still ambiguous. Because transposition can wreak havoc in the genome, causing severe diseases, many mechanisms have evolved across the tree of life to quell transposons. Astonishingly, recent research revealed that some transposons possess functions essential to their host. They contribute to regulation of gene expression, human development and neuronal diversification. These findings have led to a paradigm shift in biology: no longer are transposons mere parasites of the genome but can contribute to host function.
A class of single celled eukaryotes, called ciliates, exploit transposons in a particularly impressive way. They employ them to clean up their genomes by tossing out junk DNA, and this produces easier-to-maintain smaller chromosomes that express most genes in the cell. We have proposed that ciliates specifically recruited transposons for this ‘house-keeping’ job, but how they managed to tame these intrinsically harmful elements so that they are essential members of the cellular machinery is unknown. To test our hypothesis and to answer these questions, we will merge a unique set of state-of-the-art interdisciplinary expertise spanning chemistry, physics, computer science and biology. We aim to track the evolutionary pathway of transposon recruitment, to understand the chemistry and biophysics of these ‘good’ transposons and to investigate how they differ from canonical ‘bad’ transposons. We will visualize the structure of the molecular machines at a resolution that resolves individual atoms, and analyze how these beneficial transposons are programmed and directed to act only at the right time and place to defend the cell from their potential harmful effects. Our work will discover if and how such potentially harmful genomic parasites can be adapted for essential biological roles in ciliates and beyond, providing unmatched insight into a fundamental biological mechanism for the acquisition of novel genetic and physiologic functions.
Moreover, the elucidation of how these eukaryotes have managed to customize and tame transposons to function efficiently but safely will provide invaluable implications for engineering transposon-based tools for genetic research and gene therapy.

We propose to use DNA-based scaffolds to build lectins of pre-specified stoichiometry and precisely defined three-dimensional geometry to test mechanochemical requirements of plasma membrane invagination mediated by their membrane-resident glycosphingolipid (GSL) binding partners.
It is striking that numerous pathogenic GSL-binding lectins including the B-subunits of Shiga and cholera toxins and the VP1 protein of SV40 (i) consist of a pentameric fold (ii) bind GSLs with similar geometries (i.e., angles and distances between carbohydrate recognitions sites (CRDs)); and (iii) finally adopt a curvature-active state that enables their clathrin-independent uptake into cells despite the fact that they do not exhibit any sequence similarity. This suggests the exciting possibility that their clustering, membrane bending and invagination capacities are the result of parallel, convergent evolution.
Probing lectin functionality experimentally is infeasible using conventional approaches such as targeted mutagenesis because it is impossible to alter lectin oligomerization state and CRD geometry in a controlled manner. Therefore, we will engineer synthetic scaffolds composed of DNA to display GSL-binding CRDs in specific natural and non-natural geometries and stoichiometries. Such designer DNA-based lectins indeed offer the ability to address structural questions with unprecedented molecular precision. Innovative computational and chemical biological tools will be developed that should be of general interest to the fields of DNA devices and synthetic biology. Novel DNA-based GSL-binding scaffolds will be characterized as to their capacities to cluster on membranes, to adopt a curvature-active state (i.e. to induce or sense curvature), and to traffic into cells via defined pathways. Based on structural input, experiments will be modeled at length scales from 0.1 nm to 10 microns to provide a molecular understanding of underlying mechanistic aspects, and guide the design of new scaffolds. We expect to achieve an unprecedented understanding of GSL-based membrane mechanical processes, including the discovery of novel physical principles. Beyond the world of pathogens, our study bears relevance to the GSL-dependent functions of signaling receptors and the design of novel delivery tools for therapeutic intervention against GSL-expressing tumors.

How does a nervous system adapt to the body plan of a growing animal from birth to adult? This question is fundamental to biology, yet is currently impossible to answer without turning to small model organisms, which provide an extraordinary level of access to the nervous system at the molecular, anatomical, and physiological levels. We will explore how a nematode C. elegans manages to drive undulatory locomotion at all life stages, with different motor circuit components, using powerful new tools in serial-section electron microscopy, optical neurophysiology in freely behaving animals, super resolution light microscopy, and biophysical modeling. This research program should provide a unique data set that illuminates developmental dynamics from neural activities to animal behaviors.

Ribosomes stand amongst the most complicated structures in biology. Their assembly is a question of fundamental interest, but is still poorly understood in spite of efforts of excellent groups. We propose to address this challenging question using a quantitative approach, inspired by recent results in cell-free translation and in single-molecule biophysics. By applying a force to specially designed DNA/RNA duplexes, we can repetitively release and re-anneal RNA with full control over velocity and orientation of the strands. We can thus mimic the gradual appearance and folding of rRNA in co-transcriptional assembly of bacterial ribosomes, including varying transcription rate and pauses. Combined force and fluorescence experiments will allow us to follow in real time the binding of ribosomal (r-)proteins to the gradually released rRNA. We will focus with our single molecule measurements on the initial phase of the assembly of the large ribosomal subunit of E. coli. This involves the 23S rRNA, the five r-proteins that have been identified as essential and sufficient for the first assembly stage (L4, L13, L20, L22 and L24), and a selection of assembly helper proteins. The single-molecule studies will be complemented by coupled transcription-assembly experiments, in which the assembly will be followed in fluorescence experiments using Förster Resonance Energy Transfer between fluorescent labels on r-proteins adjacent in the ribosome and by testing the ribosomal capacity of protein synthesis. Using this combination of approaches, we will provide a dynamic view of the choreography of ribosome assembly, identify essential assembly factors among the about 15 known assembly helpers and identify their roles. Our interdisciplinary team combines two biophysics groups, well known for single-molecule studies on nucleic acids and associated proteins, and two biochemistry groups, well known in the fields of ribosome assembly and cell-free translation. Close collaboration between these four groups will bring together the expertise required to successfully perform the proposed experiments and will bring our understanding of ribosome assembly to a new level.

The ENCODE project and RNA deep-sequencing have revealed that the genome is pervasively transcribed, however, a considerable fraction of the RNA that is produced does not code for protein, but instead functions as a crucial layer of regulation over the messenger RNAs that do code for proteins. A major question in biology is how the activity of these regulatory non-coding RNAs (ncRNA) are controlled. Our 4 groups have now independently reported, through different fields of study, that the seemingly unruly network of ncRNAs are regulated by programmed interactions between the ncRNA themselves.
We propose to combine our expertise in RNA biology, computation, biotechnology, and oncology, to uncover the principles governing ncRNA interactions, with particular focus on ncRNA regulation of miRNAs, and to test the hypothesis that a major function of ncRNAs is to regulate other regulatory ncRNAs, particularly miRNAs by acting as competitive antagonists through cis-encoded sequences and recruitment of RNA binding proteins, and that ncRNA regulation of miRNA activity is important for controlling cancer relevant cell functions. To test our hypotheses, we will, (1) determine which ncRNAs have ceRNA/sponge activity through large-scale loss-of-function studies, (2) determine how the target site sequence encoded by an ncRNA affects the efficiency of ncRNA-mediated sponging, and (3) determine the relevance of linear and circular ncRNAs in cell proliferation and survival using a high-throughput functional approach.
These studies will assign functions to 100s of little studied non-protein coding genes, determine the mechanisms and principles of a novel form of gene regulation, and establish a new understanding of how a major part of the transcriptome, the ncRNAs, interact with each other, and potentially identify genes with relevance to cancer cell biology.

The human microbiome is increasingly recognized as a vital yet poorly understood ‘organ’, central to many aspects of host health, including auto-immune disorders, obesity, cancer and even psychiatric conditions. While the avalanche of metabolic data provides correlative evidence for potential roles of the microbiome in health and disease, insights are limited by a lack of testable models of human microbiome development, organization and functioning, and tractable experimental systems.
We aim to build a science of microbiome developmental biology, to understand how microbial consortium structure and functioning emerge from metabolic and demographic feedbacks among expanding lineages of bacterial species. Specifically, we will use an experimentally- and computationally-tractable two species ‘minimal microbiome’ platform to ask: (A) how do metabolic interactions influence microbe-microbe spatial structure (species mixing) and functional relationships (mutualism, exploitation, competition)? (B) How do demographic feedbacks influence emergent structural and functional relationships? (C) How do emergent structural and functional properties shape community antibiotic resistance? (D) How can we effectively manage microbiome health via modified nutrients, drugs and mixing?
Our explicit focus is on a metabolically interactive two-species model system composed of the opportunistic oral pathogen Aggregatibacter actinomycetemcomitans (Aa) and the common commensal Streptococcus gordonii (Sg). We will interrogate the Aa-Sg system using an integrated mix of individual-based simulations, ecological theory, picolitre-scale porous confinement structures, confocal and scanning electrochemical microscopy. Our project will break new ground by establishing the molecular and demographic mechanisms shaping causal relationships between microbiome environment (nutrients, drugs, mass-transfer regimes) and its structural and functional properties, including contributions to host health/disease.

Non-apoptotic cell death (NACD) is now recognized as tightly regulated forms of cell death, although the molecular mechanisms driving the underlying processes remain to be unveiled. Compelling evidence suggests that NACD sub-routines require oxygenated lipids as critical signals, demanding many questions to be answered: What are the enzymatic mechanisms involved in the formation of oxygenated lipids? Which of these lipid species play an active role in NACD signaling and what are these roles? Are they triggers and/or executors? And what are the targets? Lipid chemistry is remarkably diversified, harboring unlimited opportunities for a refined and meaningful signaling, but, at the same time, this diversity represents a major technical challenge for experimental studies. Here, we propose an analogy between language and biology to address this challenge in an interdisciplinary framework combining experimental and computational studies. Assisted by computational tools developed originally for language applications we plan to decipher the unspoken oxi-lipidome language as follows: Step 1) Identifying the words: We hypothesize that oxygenated lipids formed during different processes of cell death are analogous to words. To define the list of words relating to different forms of NACD, we will generate oxi-lipidome profiles using distinct cell death triggers and well-defined genetic models. Their analysis will yield a modulatory profile used to provide a signature for different cell death modes; Step 2) Deriving a lipidome dictionary: Oxidized-(phospho)lipid (oxi(P)L) heat maps as generated in Step 1 will lay the basis to characterize prominent differences between normally functioning and death-destined cells. This will lead to the identification of potential NACD oxi(P)L signals that will undergo further scrutiny through the language-inspired bioinformatics “filters”. Rational synthesis of the thus defined types of informative phospholipids will be thoroughly analyzed in the context of their interactions with target proteins known to shape the oxi-lipidome. Step 3) Deciphering the meanings of lipid words: In an iterative process integrating predictions and experimentation, we will study the interaction of many lipids with specific proteins and interactions that a specific lipid can undergo with many proteins using computational classification techniques also used in language analysis. Using biophysics and biochemistry techniques we will validate predicted covalent versus non-covalent interaction of the oxi(P)L-protein interfaces. The libraries of oxi-lipids created in step 2 will be delivered to specific intracellular destinations and tested for their signaling role in NACD pathways. Conclusively, knowledge gained by this comprehensive and highly multidisciplinary approach will provide intriguing insights on how the oxi-lipidome could emerge as a tremendous reservoir of yet unrecognized information.

All living organisms must organize their DNA within the confines of the cell. This is achieved by a combination of “passive” factors (e.g. cell size and shape) and “active” processes (e.g. DNA folding proteins). While much is known about how DNA is organized in higher organisms, we still have little understanding of the DNA folding processes that drive chromosome organization and compaction in bacteria. Given the abundance of these organisms, and their impact on human activity, this represents a startling gap in our knowledge. In recent years nano-scale cell imaging, large-scale genomic techniques, and mathematical/physical modeling of biopolymers have emerged as new tools to study DNA biology. In this program we bring together these approaches to facilitate precise dissection of bacterial chromosome structure. Thus, in a collaborative effort between theory and experiment, we will test and expand models to describe bacterial genome organization.

Although locomotion may seem effortless, it relies on spinal circuits producing complex patterns of muscle contractions. Spinal cord central pattern generator circuits (CPGs) produce motor output autonomously as a result of descending commands from the brain and modulations caused by internal physiological cues. The cerebrospinal fluid (CSF) constitutes a fascinating interface through which chemical cues produced in the brain can reach neurons within the entire central nervous system. Located along the central canal, CSF-contacting neurons (CSFns) project a ciliary tuft into the CSF and an axon into the spinal cord. These mysterious cells were identified in over 200 species of vertebrates by Kolmer and Agduhr who hypothesized that they could act as a proprioceptive sensory neuron. The difficulties of labeling and accessing these cells have prevented the characterization of their sensory physiology or specification. However newly developed tools and experimental approaches in zebrafish provide us with a unique opportunity to overcome these technical challenges. The transparency of zebrafish at early stages makes it a powerful system for identifying neurons by their morphology and for investigating and manipulating neuronal activity of specific cells in the intact animal. Using an optogenetic approach, Dr. Wyart and colleagues discovered that CSFns could strongly modulate the activity of spinal CPGs. In this proposed project, the Delmas, Lewis and Wyart labs will combine expression profiling, channel physiology, behavioural analysis and loss-of-function assays to elucidate how CSFns are specified and how they sense chemical and mechanical cues in the CSF. The Lewis lab has pioneered the use of FAC sorting and transcriptome analysis to identify genes expressed in distinct subtypes of spinal neurons. The Delmas lab has unraveled the functions of channels underlying chemo- and mechano-sensation in mammals. The Wyart lab has combined optical and physiological approaches to identify the functions of CSFns. Together we will establish how genetic networks specify these cells and unravel the molecular mechanisms by which CSFns are physiologically activated in vivo and contribute to behaviour.

Autophagy, the process whereby cells degrade their own components, is essential for normal cell function and mammalian development, and also has been implicated in numerous human diseases. Autophagy involves a wide array of protein-protein interactions, including a large number involving ubiquitin, ubiquitin-like proteins and various adaptor modules. The ability to target and disrupt these protein-protein interactions would enable dissection of the molecular details of autophagy, which would greatly enhance our basic understanding of the process and could lead to the identification of targets for therapeutic intervention. This project will develop and apply a toolkit of protein-based modulators to explore the roles of distinct molecular networks in autophagy. We will first identify protein interaction interfaces, and subsequently, will use computational design and combinatorial protein engineering to develop high affinity sensors or inhibitors that can function in live cells. Structural studies will be performed to determine the molecular basis of their action. These tools will then be used in vivo settings, using variety of cell and mouse models, to monitor and control physiological and pathophysiological autophagy processes.

The processing and encoding of external stimuli is essential for all life forms to react appropriately to environmental cues. When animals move, however, they activate their own sensory systems. Expectations about self-generated sensory input and its subtraction from the total input are essential for accurate sensory processing. Organisms have optimized a solution for distinguishing between self-generated and external stimulations by involving a descending (efferent) neural pathway that modulates ascending (afferent) activity. Mechanosensory hair cells, for example, are subject to feedback regulation by cholinergic efferent neurons that originate from several nuclei in the brainstem. The present research proposal aims at understanding how efferent neurons control mechanosensory processing.
Fishes and amphibians have a superficial and accessible mechanosensory system, the lateral line, which circumvents many of these problems. The lateral line serves to detect hydromechanical variations around the animal’s body and controls two characteristic behaviors: the stereotypic fast escape responses to sudden mechanical input, and complex and highly variable obstacle avoidance and rheotaxis. The simplicity of the lateral line in the zebrafish larva makes it a particularly powerful experimental model system.
Versatile recent technological developments suggest that efferent/afferent interactions in the zebrafish lateral line can be interrogated using a combination of pharmacological and genetic manipulations of neural circuits in vivo, intravital structural and functional imaging of neuronal ensembles, and fictive behavioral paradigms. Combining the knowledge and strength of each group, we will be able to clarify efferent/afferent interactions and lay the foundations to further ask how the nervous system adjusts its sensitivity to discriminate between external- and self-stimulation. This collaborative project is based on molecular studies of efferent neurons, in toto visualization and manipulation of the zebrafish lateral line, and zebrafish neurophysiology, functional imaging and behavior.

Continuous location estimation is a difficult computational problem. Roboticists grappling with the problem of noisy sensor measurements, changing features in the external world, and novel environments, find that probabilistic representation and inference of locations yields good performance, but storing and updating a single best-guess about location does not. Animals are capable of spatial navigation performance that equals or bests that of mobile robots. Yet our understanding of animal navigation is predicated on the framework of single-hypothesis tracking; there is little exploration of the compelling possibility that the brain’s spatial representations enable probabilistic coding and inference.
In this proposal, we will directly test the general hypothesis that mammals use probabilistic representation and inference for location estimation. We will address this possibility by combining investigation, in mice and humans, with computational models of probabilistic computation on spatial variables. Applying state-of-the art techniques in computational simulation and theory, in vivo electrophysiology and genetic manipulations in mice, and ultra-high resolution neuroimaging and behavior in humans, we will determine if (i) performance of spatial behaviors, (ii) representation of spatial information by place cells, and (iii) high resolution signatures of activity obtained with fMRI, are each consistent with implementation of probabilistic computations. We will in addition seek to obtain the first evidence for molecular mechanisms that control probabilistic computation. Similar experimental paradigms for animals and humans will ensure comparability of our results between species. Implementation of close reciprocal interactions between the three participating laboratories will ensure that the computational modelling studies generate theoretical predictions for the experimental studies, the results of which will in turn help refine the theoretical models.
Taken together, our multidimensional, interactive approach has the potential to provide the first account of the neural mechanisms that allow our brains to perform accurate spatial computations in the face of sensory uncertainty. This will provide a new conceptual framework for investigation of neural circuits underlying navigation and for integration of experimental results obtained in mice and humans.

One essential element very often missing to explain cell-fate control concerns the spatiotemporal regulation of translation. How does translation at synapses regulate the growth and connection strength of individual synapses during learning process? How does localized mRNA translation contribute to maintaining the pluripotency of stem cells? To address these fundamental biological questions, we propose a multi-disciplinary approach combining tools and concepts from synthetic biology, biophysics and neurobiology. Our strategy is to rationally design functionally controllable RNA-protein (RNP) complexes to spatiotemporally control mRNA translation in living cells. One of the main objectives will be to build synthetic RNPs that can traffic, translate or degrade a mRNA molecule. Our design will integrate magnetic nanoparticles and biological molecules to combine both the functional properties of native RNPs and the capability of being manipulated in space and time using magnetic forces. We hypothesize that local translation in cells often play critical roles in cell-fate decisions, such as the establishment of polarity and regulation of synapse formation and plasticity. However, currently available techniques are limited with their capacity in regulating local translation with spatiotemporal precision. Our new engineering approaches to develop synthetic RNPs in target cells will compensate pre-existing techniques by adding precise spatiotemporal controls. With synthetic RNP-mediated spatiotemporal translational regulatory systems, we will ask fundamental biological questions that are not accessible using current cell biology and ‘omics’ approaches, including: (1) What is the specific role of local translation at a synapse in terms of synaptic plasticity and memory formation and consolidation? (2) Can we control cellular behaviors of stem cells or differentiated cells by providing spatiotemporal gene expression dimensions? The new apporach will allow us to examine the effect of localized mRNA translation in controlling cell-fate behavior for two general cell states including pluripotency of stem cells and differentiated state of neurons. Such approaches will open new directions to better understand how local translation contributes to cell-fate conversion.