All existing life on Earth is the result of natural selection acting upon earlier forms of life, shaping the lineages over time. In order for selective advantages to accumulate over successive generations, the offspring must retain some attributes of the parent. Thus at some point early in life’s origins, a sustainably propagating cell must have emerged, one that could divide and produce offspring with reasonable fidelity. While genetic information propagation is largely understood--nucleic acids or their precursors preserve information by sequence complementarity--it is less obvious how the other properties of primitive cells, such as their membrane composition, could have been preserved across many generations. This remains a key open question in the emergence of life on Earth. We propose to answer this question by taking a bottom-up approach, in which we attempt to build a synthetic cell that can accomplish the essential tasks of stable propagation--membrane growth, feeding, and division--using the simplest components possible. In order to succeed, we will address a number of questions fundamental to biology. For example, to create a synthetic cell that can grow its own membrane, we must discover the mechanisms governing phospholipid synthesis, which could be determined by the rates of molecular motion or chemical reactions, the availability of nutrients, or incompatibilities between the molecules involved. Similarly, to feed our synthetic cell to sustain its growth, we must elucidate the physical rules governing membrane permeability. Finally, to induce division, we must understand the fundamental physics governing membrane bending, curvature, and fusion. We will combine cutting-edge techniques and perspectives from across all three fields of science to succeed in this ambitious endeavor. Kuruma, a biologist, will synthesize membrane material from simple building blocks in vesicles. Wang, a chemist, will analyze the properties of membranes to understand lipid packing, linking together membrane permeability with membrane fusion. And, Rogers, a physicist, will explore the physics of membrane bending and devise new strategies to induce cell fission. By working together, we will paint a comprehensive picture of how stable cell propagation could have emerged, as well as solve one part of the puzzle of how life as we know it could have come to exist.

How organs control their growth to achieve the correct final size is an enduring mystery of biology. In wildtype Drosophila, for example, left and right wing areas rarely differ by more than 1%. We previously found that the hormone Dilp8 controls this developmental precision, as in absence of the hormone, the fluctuating asymmetry (FA) of bilateral organs is strongly increased. In this proposal, we hypothesize that the hormone Dilp8 and cell death cooperate to limit wing size variability. To test this idea and explore its consequences, we will integrate methods from developmental biology with quantitative data analysis and modeling from physics. In particular, we will show how changes in cell death in dilp8 mutants are quantitatively related to changes in final wing size. Our results will provide new insights into how organ size is controlled and refined in biological systems.

Brain-wide representations of ongoing behavior were recently observed in neuronal activity patterns measured form various organisms such as worms, flies, and mice. Surprisingly, also primary sensory areas, like the V1 visual cortex in mice, previously thought to exclusively represent sensory information are also strongly modulated by behavioral variables like running speed. While this phenomenon seems to be prevalent, its functions and underlying neuronal mechanisms are elusive. I hypothesize that motor commands are integral components of sensorimotor transformations. Like others have suggested for the V1 area, I propose that this sensorimotor integration allows behavior-dependent sensory gain control, and more speculatively also involves predictive coding, i.e. that perception is tuned to unexpected outcomes of the animal's explorative movements. To address these hypotheses, I will use the tractable model organism C. elegans which is amendable to the cutting-edge whole-brain calcium imaging technology. I will develop new whole-brain imaging assays in freely-moving worms. I will then use these assays to search for gain-control circuits where the sensitivity of sensory neurons is modulated by different behavioral states. I will also look for predictive-coding circuits involving error-coding neurons, sensitive to unexpected stimuli. To study the behavioral relevance of these mechanisms, e.g. during navigation, I will interfere with the neuronal activity of the relevant circuits via optogenetics and other transgenic inhibition tools. Therefore, by combining these different approaches I aim to uncover generalizable functions and mechanisms of brain-wide motor representations.

Naturalistic behaviors are refined and stabilized throughout an animal’s life. Their formation is dependent upon multiple internal and external cues, giving rise to both stereotypical intraspecific responses and individually-unique behavioral patterns. The striatum has been widely implicated in controlling voluntary action and encoding behavioral modules and sequences in adulthood. However, how striatal dynamics might support and structure the evolution of behavior throughout development remains surprisingly unexplored. Here, I propose to incorporate longitudinal, unsupervised behavioral phenotyping with high-throughput in-vivo neuronal recordings in freely-behaving mice in order to characterize the evolution of naturalistic behavioral modules and their neural correlates from weaning to adulthood. To explore the interplay between external cues, circuit dynamics and behavioral plasticity, I will confound environmental features during development and study how behavioral adaptation is reflected in the underlying single-cell and population striatal codes. Finally, I will utilize this large-scale dataset to investigate the emergence of behavioral variation and to identify the neural correlates of individuality.

Hypoxia plays a central role in the pathogenesis of myocardial infarctions by causing immediate cellular damage within the short time scale of minutes to hours. Pathways that govern the acute response of heart tissue to hypoxia are poorly characterized. Currently, the procedure for stopping imminent tissue damage due to heart attack is percutaneous coronary intervention. Thus, systematically elucidating pathways involved in acute hypoxic response will lead to novel pharmacologic interventions aimed at protecting against the adverse cellular effects of hypoxia. One major challenge for the systematic study is that hypoxia response involves diverse processes, affecting cellular changes to metabolic, epigenetic, and transcriptional states, and hypoxia response is dynamic and can affect spatial organization within the cell. An appealing approach to overcome this challenge is phenotypic profiling, which offers a scalable, microscopy-based approach to convert complex cellular response to perturbations into high-dimensional vectors, referred to as phenotypic “profiles”. Here, I propose to apply phenotypic profiling to conduct a systematic study of the sensing and regulatory network governing acute cellular response to severe hypoxia in cardiomyocyte cells. My specific aims are to: (1) Establish a scalable platform for phenotypically profiling acute cellular response to severe hypoxia; and (2) Identify novel hypoxia sensors and regulatory pathways that govern acute cellular response to hypoxia using this platform.

It is a longstanding goal to decipher cell fate decisions during human development. Stem cell-based culture systems, combined with single-cell sequencing and CRISPR-Cas technologies, offer exciting possibilities to track human cell identities at unprecedented resolution and to engineer specific cell types with high precision. In this proposal, I will develop novel methods to record cell histories, trace lineages, and program human cell fates, with a focus on the human retina. In the first aim, I will develop a fate recording system in induced pluripotent stem cells (iPSCs) that unites evolvable barcodes with molecular memory recorders (Cas1/2) that capture expressed RNA into DNA. This will be used to simultaneously record real transcriptome and lineage histories along the path of cell fate acquisition. In the second aim, I will adopt the CRISPR-Cas12a(Cpf1) system that enables multiplexed gene activation to engineer cell fates based on predictions learned from single-cell transcriptome dissection of organ development. I will focus my project on human retina development where we have a well-characterized three-dimensional organoid culture system, and extensive data analyzing cell fates that emerge in these organoids. This focus will provide insights into the molecular mechanisms controlling human retinogenesis, and at the same time identify methods to engineer specific retinal cell types for therapeutic screening. The strategies and technologies developed in this project are applicable beyond the retina and will contribute to our understanding of general cell fate decisions in humans and guide cell type engineering with clinical potential.

Fertilization, the fusion of two highly specialized cells, the egg and sperm, marks the beginning of life of all sexually reproducing organisms. Despite its fundamental role, we still understand very little about how sperm-egg interaction is controlled. Research so far has mostly focused on identifying proteins that mediate gamete attachment and fusion while largely neglecting the fact that fertilization is an inherently dynamic process, during which the two gametes get together, interact and fuse. Although the sperm is in general viewed as the ‘active’, motile partner during gamete encounter and interaction, a key open question remains whether the egg does also play an ‘active’ role in the fertilization process, e.g. by generating forces that might actively engulf the sperm. To tackle this fundamental gap in our knowledge, we propose a two-pronged, cross-disciplinary approach to 1) characterize the mechanosensitive properties of the egg before, during and after fertilization by state-of-the-art biophysical techniques; and 2) pioneer the development of a bottom-up fertilization assay that we term ‘synthetic fertilization synapse’. As such, we envision to reconstitute sperm-egg interaction dynamics in a simplified in vitro system, in which we will replace the sperm by specific proteins in micro-structured lipid bilayers to study the impact of ligand mobility, number, and spatio-temporal organization on egg activity. In combination, we will use a controlled micro-confinement-based force actuator approach to perform global and local mechanical stimulation of the egg. Mechano-chemical responses of the egg will be measured by state-of-the-art high-resolution imaging and quantitative force measurement techniques. Zebrafish gametes will serve as main model systems, which will be complemented by experiments in mouse gametes. Tackling this highly challenging problem of the biophysical control of fertilization becomes only feasible by combining our different core expertise in an interdisciplinary approach. Our research will provide fundamental insights into the dynamics of sperm-egg interaction and its mechanosensitive regulation. These insights promise to transform our currently rudimentary knowledge of the mechanism of fertilization.

Metazoan development relies on tightly regulated gene expression patterns. Hence, the regulatory landscapes of developmental genes are complex and often contain several simultaneously active enhancers. Recent studies reported that the combined enhancer activities in such loci can either be additive, super-additive or sub-additive compared to the sum of the individual activities. Yet, the principles that might govern enhancer cooperativity remain elusive. To fill this gap, I propose measuring the activity of large collections of systematically paired developmental enhancers using STARR-Seq (500x500 enhancers), which was developed in my host lab to map individual enhancers genome-wide. I expect to identify an unprecedented number of additive, super- and sub-additive enhancer pairs and to reveal the sequence features associated with these distinct behaviors. To test all the genomic elements that could potentiate and/or interfere with developmental enhancer activity, I will use another STARR-Seq variant in which selected developmental enhancers will be individually paired with a comprehensive library of genomic DNA fragments (1xall elements). This will address how housekeeping enhancers, which form a functionally distinct class of enhancers, and other types of genomic elements impact developmental enhancer activities. Finally, I will validate my results in situ using CRISPR/Cas9 genome editing and test whether the genes’ endogenous regulatory landscapes influence enhancer cooperativity. Overall, this original approach will address a key aspect of developmental gene regulation which, despite its relevance for the study of development and pathogenesis, remains elusive.

The neonatal period is witness to fast physiological changes. The, up until birth, largely sterile environment becomes colonized by microbes, crucial for imprinting durable and specific immune responses, a period named “window of opportunity”. Postnatal immune imprinting involves extensive inter-organ trafficking of immune cells, and hence their interaction with endothelial cells of blood and lymphatic vessels. Still, to date virtually nothing is known about immune-vascular cross talk during postnatal period and the role of endothelial cells in immune imprinting. By using a mouse model that expresses a photoconvertible fluorescent protein, I will first characterize the immune subsets trafficking from gut, an essential organ for postnatal immune education, to other organs. Second, I will study the molecular changes in endothelial cells in this neonatal period to understand how this affects inter-organ communication and the pre-programming of long-lasting immune responses. This will be done by flow cytometry, high resolution 3D imaging and single cell RNA sequencing. Moreover, while there is a significant knowledge on the impact of environmental factors such as nutrition, exposure to antibiotic and farm animals on future health, much less is known about postnatal education of blood and lymphatic vasculature. Accordingly, I will investigate the impact on diet and microbial colonization in the neonatal period and their effects on both immune and vascular components. Altogether, I wish to unveil how early life events impact the immune-vasculature crosstalk and future health imprinting, thus helping to unlock intricate disease mechanism and their prevention in the future.

T cells are key effectors of adaptive immunity that are constantly moving, can enter most tissues, and operate in large crowds. Millions of densely packed T cells continuously roam through lymphatic tissue in search of foreign antigen; activated T cells divide vigorously, flock to tissues, mount local immune responses to pathogens or cancer cells, and remain there to guard against further intrusions. Crowding is normally expected to deter motion and lead to jamming, an effect confirmed in very diverse systems ranging from pedestrians and vehicular traffic to cellular monolayers and granular material. Yet T cells defy this general principle and are capable of fluid motion even in extremely dense environments such as lymphatic tissue and the epidermis, where there is no apparent room to maneuver. How they achieve this is unclear: although novel microscopy modalities can visualize migrating T cells in living tissue, studies of T cell movement have primarily focused on individual or few cells and have not addressed potential crowding effects. We hypothesize that (1) T cells have evolved to cooperate effectively in large groups and avoid crowding issues, such that (2) impaired T cell crowd operation is largely confined to anomalous tissue environments such as tumors. Here, we aim to unravel the mechanisms that facilitate fluid, “jam-free” motion of densely packed T cells, and determine in which situations T cell traffic might nevertheless be disturbed by emerging detrimental crowding effects. We will study T cell crowds in a wide range of conditions in silico, in vitro and in vivo: we will (1) conduct simulations to predict how T cell crowds operate in challenging conditions and which emerging crowd behavior is expected; (2) design microfluidic devices to expose T cell crowds to hallmark crowding scenarios, identify molecules involved in cellular crosstalk within crowds, and identify transcriptional signatures associated with efficient crowd operation; (3) examine T cell crowds in human and mouse tissues to test whether dynamic or static patterns indicating jamming can be confirmed in physiological or pathological conditions in vivo. Thus, by combining our unique joint expertise in crowd dynamics, immunology, and computational biology, we will shed light on how T cell crowds, a system quite unlike any other many-particle system studied so far, maintain motion with such remarkable efficiency in many conditions and what it would take to disrupt their smooth flow.

Cell motility is an integrated process implicated in many cellular functions such as embryogenesis, immunological response, wound healing, and growth cone path finding. For a long time, the study of cell migration has mainly focused on the observation of molecular components responsible for the formation of cell protrusion: actin networks and actin regulatory proteins. Recent studies propose that membrane biophysics, in particular membrane tension, could control actin network formation and cell migrations. Thus, in this project I propose to study the interplay between membrane tension and actin networks during cell migration. To do so, I will use a tension-sensitive fluorescent probe (FliptR probe) to measure membrane tension, together with a cell permeable fluorogenic F-actin label (SiR-actin) suitable for live labelling of the actin cytoskeleton. Using these probes, I will measure the local membrane tension variations during cell migration and monitor how these affect and/or are affected by actin cytoskeleton. By combining these approaches with the cutting-edge technique of adhesion protein micro-patterning, I will create a controlled and reproducible environment to finely tune cell migration. This will allow us to understand how the cellular environment affects the interplay between membrane tension and actin cytoskeleton. All together, I propose an innovative approach that will enable us for the first time to study the impact of local membrane tension on cell migration. The findings of this project will shed light into the cell migration, actin and lipid membrane fields.

The intestinal epithelium is a mucosal tissue reliant on its regenerative capacity as it is perpetually exposed to cell death whether it be during homeostasis or insult. Defective regeneration of the epithelium is associated with multiple diseases such as Intestinal Bowel Disease. Macrophages can act to sense damage and enhance tissue repair in the gut. Whether they can integrate the recognition of dying cells with intestinal epithelial regeneration remains unknown. We previously reported that the recognition of apoptotic cells by macrophages enhances their capacity to promote tissue repair. However, depending on the nature of intestinal injury, intestinal cells can undergo divergent cell death modalities including apoptosis, necroptosis and pyroptosis. Thus, we propose a model wherein macrophages directly participate in the integration of cell death with tissue repair. We aim to characterize cell death modalities, including that of stem cells, in models of intestinal injury. We additionally plan to identify the relevant cell death receptors for macrophage detection of apoptotic, necroptotic or pyroptotic cell death and correlate these findings with regeneration/repair or its failure during disease. Current treatment strategies focus on resolving inflammation during intestinal disease instead of enhancing regenerative responses of the epithelium. Thus, uncovering the nature and consequence of cell death in the gut may provide valuable insight into the treatment of gastrointestinal diseases.

Sex determination through the X and Y chromosomes is among the novelties that characterise mammals. The genetic dimorphism generated by the different content of sex chromosomes between males (XY) and females (XX) had led X-dosage compensatory mechanisms to arise. One of the two X chromosomes in females needs to be silenced in order to avoid a double dose of X-related genes in females as compared to males. The way to achieve X-chromosome inactivation (XCI) has evolved differently between two mammalian infraclasses: eutherians (placentalia) and metatherians (marsupialia). A series of epigenetic modifications and the coordinated activity of lncRNAs led by the master regulator Xist, are key for XCI in eutherians. In marsupials, Xist is not conserved and little is known about how this process occurs. James Turner’s group found that another lncRNA, namely RSX, is specifically expressed from the inactive X in the opossum Monodelphis domestica and may drive XCI in metatherians. In this project, I will investigate the epigenetic mechanisms responsible for achieving XCI in the opossum during embryonic development. Specifically, I will focus on the role that repressive histone modifications play in the germline and in the early embryo to regulate imprinted XCI. I will also interrogate if the link between pluripotency and biallelic expression of X-linked genes is maintained in the opossum and how the regulatory landscape of Xist and RSX has evolved between eutherians and metatherians. This project will be important to gain insight into how genomic imprinting and the epigenetic landscape is established in metatherians and how it has evolved since the divergence from eutherian mammals.

Stem cells face various challenges in their differentiation process including external perturbations, such as cell-to-cell signaling and internal perturbations, such as genetic mutations. To robustly differentiate, stem cells must integrate and respond to cues, yet be tolerant of destructive ones. Previous work shows that chromatin landscape is determinant of cellular activity, hence, it may infer the cellular sensitivity to perturbations as well. However, how the dynamics of the chromatin landscape reflects the cell’s sensitivity, is still missing. My hypothesis is that sensitivity to perturbations during differentiation is facilitated by a more accessible chromatin landscape. Here, I set to (a) define the temporal sensitivity of differentiating cells to various perturbations, and (b) define how the chromatin landscape reflects the sensitivity and robustness of the cells. First, we will grow and differentiate stem cells along several trajectories while characterizing their differentiation in-depth, utilizing sequence-based technologies such as chromatin accessibility and conformation, methylation and gene expression. Second, we will perturb the several differentiation trajectories, imposing external and internal perturbations, defining the perturbations’ effect on differentiation. Finally, we will perturb the cells at dense time points in the differentiation process, generating sequence-based time-specific profiles of perturbed vs normal differentiation. This work will characterize several differentiation trajectories in great details and provide the necessary high-throughput data to investigate the causality between chromatin landscape and sensitivity to perturbations.

Liquid-liquid phase separation is key for organising cellular content dynamically without the need for membrane confinement. Phase separation is an intriguing concept to explain as yet puzzling observations in chromatin organisation. It is known to be involved in the condensation of nucleoli, heterochromatin and long-range chromatin-chromatin contacts such as super-enhancers. The cellular mechanisms that control these phenomena are still poorly understood. Studies so far were focussing mainly on biophysical characterisation rather than function or regulation. Preliminary data in the host lab points to the dual-specificity kinase DYRK3, a known regulator of condensation, to be a regulator of novel nuclear condensates containing the chromatin remodeler Set1/COMPASS complex and other factors involved in gene expression control. On this basis, I will combine proteomics, chromatin and transcriptome profiling, cell biology and high-throughput multiscale imaging to determine the composition of transcriptional condensates and how they are controlled by DYRK3. Targeted perturbations will reveal the impact of these condensates on gene expression. The mechanisms discovered for DYRK3 will form a basis for understanding kinase-controlled transcription regulation more generally. Understanding the regulation of these condensates will not only shed light on the basic biology of cellular organisation but may enable therapeutic intervention in protein-aggregation diseases and cancer.

Isopeptide bonds - covalent amide bonds between amino acid side chains - stabilize and crosslink protein folds, notably in gram-positive bacterial pili, or fibrin clots. In contrast to disulfide bonds, far less is known about the generation of isopeptides. Here, I propose to de novo design and characterize proteins that can autocatalytically form both intra- and intermolecular isopeptides. To study a family of pili subunits in gram-positive bacteria that forms intramolecular isopeptides without enzymes, corresponding autocatalytic conformations will be constructed de novo. By controlling the orientation and hydrogen bonding of catalytic amino acids in the protein, I will dissect and optimize the mechanism of isopeptide formation. Resulting protein designs will serve as minimal models to investigate the stabilizing biological functions of isopeptides. Pathogenic bacteria use proteins containing thioester bonds to attach to their host via intermolecular isopeptides - a poorly understood process that I propose to elucidate with de novo design. A reactive Cysteine-Glutamine thioester will be confined onto a designed simplified protein scaffold. Docking de novo binders into proximity of the thioester will enable me to isolate key variables that govern autocatalytic intermolecular isopeptide formation in a controlled environment. Computational design of autocatalytic reactions in proteins enables radical new ways to covalently stabilize and crosslink them, currently lacking in the Rosetta software suite. Fundamentally, I aim to reveal what drives autocatalytic isopeptide formation and what constitutes their biological function by deconstructing their de novo design principles.

Although growing evidence suggesting that typical immune molecules contribute to shaping the synaptic plasticity and regulating the neuronal activity in the brain, the cellular and molecular mechanisms of these molecules, remains unclear. Moreover, the spatial and temporal expression of these molecules in the brain during development as well as in adults remains unresolved. Here I aim to use the most advanced approaches and techniques to investigate the role of such immune molecules for regulating the synaptic plasticity, neuronal circuits, and brain function. For that, I am interested in tackling fundamental and unsolved questions in the field: Does neuron in the brain express typical immune cytokine receptors or another immune molecule? If it so, does such receptor express within the synaptic cleft? Does it express in pre or post-synaptic or both? What is the molecular function of such receptor? Does it bind their classical cytokine and mediate intracellular signaling? What is the molecular and cellular consequence of such receptor activation? Does it affect synaptic transmission, plasticity, and strength? Does it have any impacts on the neuronal function or circuits? Could the activation of such receptors influence the animal behavior? What are the molecular mechanisms regulating such receptor and cytokine expression? Tackling these fundamental questions could help us better understand the regulation of neuronal function by the immune system in the brain which could be critical for processes such as learning, memory, and behavior during development as well as in pathophysiological conditions.

The intestinal microbiome is a species-rich, spatially heterogeneous ecosystem and core determinant of overall host health. An explosion of metagenomic studies interrogating the functions of microbiome composition in health and disease has revolutionized our view of intestinal microbial communities, but many important gaps remain in our understanding of these ecosystems. Chief among these are the combined contributions of viruses - particularly bacteriophages - to microbiome homeostasis, and of the spatial and temporal structure of the microbiome. Spatially structured communities, or biofilms, are the norm rather than the exception among microbes in the wild, including the intestinal tract, qualitatively altering their ecological and evolutionary dynamics across species but also with their host or predators. Here, we propose to fill these gaps by performing a mechanistic characterization of the physical and ecological forces driving host-microbiome-phageome ecosystem stability and dynamics. To achieve this, we will use an interdisciplinary approach combining tools and concepts form ecology, physics and engineering. First, we will develop a novel experimental framework built around the miniGut, a tube-shaped organoid that recapitulates critical biological and physical features of the intestine, including mucus secretion. We will use miniGuts to identify and quantify the critical factors governing the steady states and stability of a model microbiome, including the presence of a lysogenic phage engineered to allow us to track bacteria-phage-intestinal spatial dynamics in live samples at single-cell resolution. Underlying all aims will be the development of a dynamical systems perspective on the two- and three-way interactions where we will record ecosystem equilibrium points and trajectories in phase diagrams. Finally, using this knowledge, we will engineer lambda phage to manipulate ecosystem steady-states within miniGuts. To do so, we will transmit new functions to host microbial species via lysogeny, for example altering the bacterium’s ability to adhere to mucus, to tolerate antibiotics, and to either cooperate or antagonize other members of the model microbiome. Altogether, our interdisciplinary study will set the stage for mechanistic explorations of the role of phage and microbiome spatial structure in host physiology.

In host-microbe interaction studies, genomic technologies have significantly advanced our understanding of host responses and structure of host-associated microbial communities (microbiota). However, spatial and physiological heterogeneity in host-microbe interactions has been more challenging to study. Plants are comprised of various cell types, each of which may interact with a distinct set of microbes and, at the same time, accomplish other tasks such as growth and nutrient acquisition/utilization. To date, interactions between individual plant cell types and the microbiota are poorly understood due to our lack of knowledge about cell type-specific responses of plants and the composition of associated microbes at the cellular level. Here, I employ high-throughput single cell trasncriptomics to analyze plant cell type-specific responses to the microbiota under nutrient (phosphate) replete and starvation conditions. I will mainly investigate immune response and phosphate starvation response and crosstalks between them in individual cell types. I will also develop a method for in situ microbiota community profiling by imaging-based sequencing of bacterial 16S rRNA, which provides a spatial map of the plant microbiota at the strain-level of resolution. By linking plant gene expression and microbiota distribution at the single cell resolution, I will investigate the roles of plant genes and microbiota members that show plant cell-type specificity. This project attempts to reveal plant cell type-specific responses to biotic and abiotic environmental signals and microbiota assembly that are significant and biologically meaningful.

The proposed project aims to implement a new technology based on functional ultrasound (fUS) imaging and Ca2+ imaging using acoustic sensors of calcium (ASC). Functional ultrasound imaging is a new neuroimaging modality providing highly sensitive functional imaging of the brain by measuring cerebral blood flow. Ultrasound has excellent penetration depth, providing whole-brain coverage. However, the reliance of existing fUS methods on imaging blood flow results in an indirect readout of neuronal activity. Recently, it has become possible to connect ultrasound more directly to cellular function using genetically encoded acoustic reporters based on air-filled proteins derived from buoyant bacteria. In this project, I will use new versions of these proteins engineered to serve as dynamic sensors of Ca2+, which I will express in neurons and perform functional calcium imaging in the mouse brain using ultrasound. In addition to the ultrasonic detection of ASCs during task-evoked and resting state acquisition, I will complement the technology with simultaneous hemodynamic measurements to understand how the two signals relate to each other. This Whole-brain Acoustic Tracking of Calcium and Hemodynamics (WATCH) will provide valuable insights on neurovascular coupling and allow the interpretation of hemodynamics signals when models cannot be modified genetically with ASCs.