In order to gain something, sometimes you have to lose something. This is true in our lives, and it is also true for cells to gain special functions. The individual component of the phloem sieve tube, the sieve element (SE), loses the cytoplasm, organelles, and even part of the cell wall to be able to transport photosynthetic products and signaling molecules over long distance. However, the molecular mechanisms behind these dramatic changes have been shrouded in mystery for more than 60 years since the discovery. The main reason for this is due to the small size of SEs and their deep location in the tissue, which makes them extremely difficult to observe with confocal live imaging. In this proposal, I aim to reveal the autolytic mechanisms of SEs by establishing a novel SE induction system that overcomes such technical hurdles. This research provides new insights into the coupling between autolysis and cell specialization. It also has the potential to expand the field of plant science, as plant development have rarely been studied in terms of degradation control.

Protein quality control systems have been perfected through evolution to guard the proteome. Their capacity, however, decreases during ageing, which in turn threatens proteostasis and exposes humans to plethora of age-related disorders. Currently, a vast majority of these diseases - including neurodegeneration - remain incurable. The next scientific frontier is to convert the broad protein quality control knowledge into tangible benefit to humans by harnessing this intrinsically protective cellular system to foster cellular and organismal resilience to pathogenesis. An unbiased search for compounds that promote cell survival in stress pointed at the phosphorylation of the eIF2a translation initiation factor as a potent modulator of cellular fitness. Thus, eIF2a phosphorylation emerges as a key player in cellular resilience with its manipulation being potentially beneficial in many conditions. This cross-disciplinary proposal aims at using thorough and unbiased set of orthogonal approaches to delineate the role of phosphorylated-eIF2a in human pathogenesis. While exploiting human and mouse genetics, cell biology and pharmacology, I will: i) study the mechanistic bases of diseases genetically linked to eIF2a phosphorylation and the cellular consequences of its alterations; and ii) genetically modulate levels of phosphorylated-eIF2a in neurodegeneration to unambiguously define its role in disease pathogenesis. Together, this project will provide a fundamental, mechanistic framework to define how to manipulate the cellular cost-benefit ratio of eIF2a phosphorylation in pathogenesis and point at conditions that may profit from its modulation.

Over the past decade, programmable gene editing has been transformed by the discovery and development of prokaryotic adaptive immune systems known as CRISPR-Cas. These systems, which evolved to eliminate foreign genetic elements such as plasmid and phage via RNA-guided nucleic acid degradation, have been powerfully redeployed for many gene editing applications. Recently, these systems have been expanded with the discovery of nuclease-deficient CRISPR-Cas systems that direct RNA-guided transposition. Transposon-encoded CRISPR–Cas systems can insert large genetic payloads into bacterial genomes without relying on DNA double-strand breaks or host repair factors, eliminating the major limitations of current integration approaches that rely on CRISPR-Cas9. Nonetheless, the molecular mechanisms of key steps in this newly discovered RNA-guided transposition pathway remain uncharacterized, restricting the applications of this potentially powerful technology. Specifically, the mechanisms through which the transposase machinery is recruited following DNA targeting, the integration orientation of donor DNA is selected, and the megadalton protein-DNA transpososome complex is disassembled, are yet to be elucidated. To determine these mechanisms, I propose to employ a powerful combination of genetic, biochemical, and single-molecule biophysical approaches that is made uniquely possible by integrating the expertise in the Sternberg and Gonzalez laboratories. The results of my studies will transform RNA-guided transposition into a highly tractable, precise, and efficient RNA-guided DNA integration tool for next-generation genome engineering.

Although diseases are clearly harmful, many organisms carry genetic variation that causes disease. Why is this the case? My research proposes to combine techniques from field ecology and population genomics to investigate the forces maintaining genetic variants that cause cancer in swordtail fishes. In Mexico, it was recently discovered that some populations of swordtail fish have high frequencies of genes causing melanoma. Yet, these populations show no signs of losing these genes. I propose to conduct a large field experiment where I will manipulate the two major forms of selection - natural and sexual selection - to identify mechanisms that lead to the preservation of melanoma-causing genes in these populations. I hypothesize that reduced survival due to melanoma is countered by with increased growth rate and attractiveness to mates. My experiment will involve the construction of artificial streams where we can manipulate factors such as exposure to predators, mate choice, and parasitism. Using cutting edge techniques in population genetics, I will quantify directly how our manipulations change selection on genes. I anticipate that the results of this interdisciplinary experiment will yield novel and surprising insights into the forces that maintain disease-causing genetic variation in natural vertebrate populations.

Central nervous systems constantly detect environmental stimuli and integrate them into long-lasting behavioral changes. Adaptations to experience, for example associating a context with a positive or negative outcome represents an important form of learning. Such contextual learning is achieved in part by the modification of transcriptional programs in individual neurons. A broad range of evidence supports that immediate early gene transcription factor (IEG-TF)-dependent induction of gene expression controls various aspects of synaptic plasticity, and learning and memory. However, functions of the downstream activity-dependent gene products themselves are still poorly understood. Behaviorally-induced neuronal activity of hippocampal excitatory neurons was recently discovered to result in a bidirectional adaption of neuronal inhibition by parvalbumin- and cholecystokinin-positive interneurons. This requires a neuropeptide precursor SecretograninII (Scg2), downstream of the IEG-TF Fos, highlighting an unexpected role for a little-studied neuropeptide. In this research proposal I will use a combinatorial approach of molecular, electrophysiological, behavioral and imaging methods to identify how Scg2 mediates behaviorally-induced control of synaptic plasticity and connectivity in the mouse hippocampal microcircuit. In parallel, I will investigate the involvement of Scg2 in establishing neuronal assemblies for encoding memories and behavioral plasticity. Together, these studies will enhance our understanding how gene expression programs and, in particular, neuropeptide signaling regulate synaptic specificity and memory traces in neuronal circuits.

Neurons can be studied by monitoring intracellular calcium ion levels using fluorogenic calcium ion sensors. However, current sensors produce transient signals, potentially missing integral neuron activity and making it challenging to measure accumulated neuron activity over time. Alternatively, genetically encoded calcium ion signal integrators accumulate and integrate neuron activity over a defined time. However, these signal integrators are dependent on a light-assisted photoconversion process and only integrate neuron activity over a short time period. Furthermore, imaging cannot take place during irradiation and even irradiation must be ensured. We propose a calcium ion signal integrator that functions independently of photoconversion for monitoring neuron activity that has accumulated over extended time periods to address these issues. This novel tool consists of a genetically encoded protein sensor (GEPS) that undergoes a conformational change from an opened state to a closed state upon binding to calcium ions. After binding to calcium ions and adopting its closed conformation, the GEPS brings together two cysteines, which both react with a complementary and fluorogenic di-acrylamide probe. Labelling the closed GEPS with this fluorogenic probe slowly produces an irreversible and accumulating fluorescent signal independent of photoconversion but depends on calcium ion concentration. The integrated signal can be later imaged by fluorescence microscopy to identify activated neurons and their relative activity. This novel calcium ion signal integrator will greatly contribute to the future understanding of neuron development and diseases such as stroke.

As the causative agents of toxoplasmosis and malaria, the apicomplexan parasites Toxoplasma gondii and Plasmodium spp. are responsible for a massive burden of disease worldwide. However, our understanding of how these parasites function at a molecular level is incomplete, limiting development of therapeutic interventions. The apicomplexan ubiquitination system represents one such major gap in our knowledge. Ubiquitination is an essential eukaryotic post-translational regulatory system that mediates protein homeostasis and activity. In apicomplexans, ubiquitination is linked to many essential parasite processes, and represents a potential source of drug candidates. However, apicomplexans are far removed from humans and yeast, the species in which ubiquitination is best studied, precluding bioinformatic assignment of critical functions. Recent advances in T. gondii genome engineering provide the opportunity to systematically investigate the apicomplexan ubiquitination system. My goal is to systematically map the entire ubiquitination system of T. gondii and assess the contribution of each component to infection. I will further determine the interactome of components deemed essential, or conditionally essential during stress, and attempt to determine their specific role in regulating parasite growth and infectivity. This project will lay the foundation for research into potential treatments and improve our understanding of apicomplexan biology.

All species need to navigate, forage, and interact with other animals in a constantly changing threedimensional world. For such behaviors, vision is one of the most important senses as it allows animals to map their immediate surroundings from a distance which in turn helps coordinating ongoing and future motor actions. To construct such a map, the visual system uses a wide variety of depth cues such as object size, binocular disparity, texture, accomodation, shading, perspective, motion parallax, and occlusions. However, despite extensive research into the function of visual systems, currently, it still remains unclear what the exact perceptual algorithms and circuit mechanisms underlying depth perception are. Therefore, I propose to investigate how larval zebrafish process depth information and use such cues during hunting behavior. Given its small size, genetic tractability, optically accessible brain, and rapid development of visually driven behaviors, the larval zebrafish is an ideal vertebrate model system to tackle these questions. Starting at 5 days post fertilization, larvae visually hunt small protists. At the start of a hunting sequence, they almost invariably select the closest prey, which suggests that they posses a way of visually estimating distance. In order to investigate how larvae perform this feat, I plan to design and build a novel virtual reality system than can provide both monocular and binocular depth cues to a freely swimming or restrained zebrafish larva. Using this VR system, I then aim to determine which depth cues are utilized and investigate their neuronal representation using whole-brain two-photon imaging.

The dysregulation of protein synthesis in favor of oncogenic proteins is a hallmark of cancer. Recent discoveries revealed that translation of upstream open reading frames (uORFs) within 5´ untranslated regions (5´UTRs) of oncogenic messenger RNAs (mRNAs) contributes substantially to increased oncogene synthesis during cancer initiation. Although we begin to understand the molecular mechanisms underlying the specific programs of protein synthesis, we lack a general picture of the order of events that lead to the remodeled protein synthesis in cancer. Here, I propose to develop, validate and apply a split-fluorescent uORF/mainORF (mORF) reporter system that recapitulates preferential oncogene translation in a squamous cell carcinoma (SCC) model. I aim at developing and characterizing this reporter system in order to record for the first time the translational remodeling in an in vivo mouse cancer model during transition from healthy tissue to different stages of tumor formation. In addition, this approach will enable me to screen for factors involved in the induction of oncogenic drivers by 5´UTR-associated mechanisms. Finally, I will also reveal the molecular mechanisms by which the newly identified factors drive translational reprogramming in cancer. My findings will substantially contribute to our understanding of protein synthesis pathways during health and disease and provide a foundation for the development of novel cancer therapies.

Maternal immune activation (MIA) in pregnant mice has been shown to induce phenotypes resembling those of Autism spectrum disorder (ASD) in offspring, among those are deficiencies in social interaction. A recent study showed social behavior deficits can be temporarily rescued by eliciting an inflammatory response. Specifically, it was shown that administration of lipopolysaccharide (LPS) induces interleukin-17a (IL-17a) or direct administration of IL-17a into primary somatosensory cortex dysgranular zone (S1DZ), promoted sociability in these mice. These results raise the following questions: (a) what is the role of S1DZ in sociability? And (b) how does IL-17a affect the neuronal activity in this brain area that rescues social behavior deficits? In my postdoctoral studies I plan to answer these questions by recording neuronal activity in S1DZ in freely moving MIA-affected and control mice during social interaction and characterize the neuronal representations in this brain area. Moreover, by administrating IL-17a I will study how this cytokine act as a neuromodulator affecting the neuronal activity in S1DZ. Identifying the role of S1DZ in social behavior and the underlying mechanisms increasing sociability that are prompt by the presence of IL-17a, will pave the way to design potential treatments for behavioral abnormalities manifested in ASD phenotypes.

RNA structure plays a crucial role in regulating gene expressions such as translation and RNA degradation. Considering the advance of high-throughput sequencing, researchers are currently able to access both in vivo RNA structures and RNA degradation information over tens of thousands of RNAs in one single experiment. However, it is still unclear which RNA structure feature/motif is generally responsive to RNA degradation in living cells. It is challenging for traditional analysis methods to process the emerging " big data" of RNA structure in vivo, and they are not able to assess the complexity of RNA structure features for the whole RNA, which is over thousands of nucleotides long on average. In this project, I aim to develop novel artificial intelligence (AI) frameworks incorporating deep learning and omics approaches to determine in vivo functional RNA structure motifs in RNA degradation. By integrated multi-omics data, I will construct deep learning models to establish the relationship between RNA structure and RNA degradation and consequently extract the potential RNA motifs. The combination of dry-bench and wet-bench methods will allow me to develop a prediction-validation working scheme to solve this multidisciplinary problem. Based on the identified functional RNA structure motifs, I will also establish corresponding web servers for the community to predict/design the RNA structure motifs for the RNA of interest. The proposed research will make major advances in our fundamental understanding of the RNA structure functionality in RNA degradation in living cells and the AI framework will enable further applications to the complex genomics dataset.

For decades, the nuclear pore complex (NPC) has been considered the sole gateway between the nucleus and the cytoplasm, allowing transport of molecules across the nuclear envelope (NE). However, in recent years NE budding emerged as a non-canonical alternative route for nuclear export of viral nucleocapsids, as well as for large ribonucleoparticles (RNPs). Yet, very little is known about the significance of this unconventional export pathway. The project proposed here aims to elucidate the role of NE budding as a non-canonical route for exporting large-RNPs. The strategy I propose is to track and perturb NE budding transport of large-RNPs. In particular, I will study: (I) the relationship between NE budding and NPC canonical export pathway; (II) its potential involvement in nuclear quality control; and (III) I will test the hypothesis that lamin and Torsin protein families participate in NE budding of large cellular complexes. To follow RNP trafficking and image NE budding events, I will apply state-of-art RNA live-imaging techniques and use a combination of cryo-electron microscopy and tomography (cryo-EMT) and high-resolution live-cell microscopy. Moreover, I will apply biotin proximity ligation assay (BioID) to identify novel RNA and protein cargos transported through NE budding in a Torsin-dependent manner. The results stemming from the work proposed here will represent a significant step towards understanding NE dynamics, and may shift the current dogma of nuclear export.

Lipid bilayer membranes define the outer boundary of a biological cell, protecting its internal volume from elements of the extracellular environment. To gain entry through these membrane barriers, some pathogens fuse their membranes with the membrane of the host cell in a process commonly known as membrane fusion. It is largely believed that placing two membranes near one another can lead to the spontaneous formation of a neck (stalk) joining the two membranes. The stalk eventually resolves into an opening (pore) for material exchange, followed by complete fusion of the two membranes into one. Dedicated proteins (fusogens) found in viruses and cell organelles accelerate this process by facilitating transitions between several intermediate states of the membrane fusion process. While our knowledge of the proteins involved in membrane fusion has steadily accumulated, much less is known about the actual mechanics of membrane fusion, as these processes occur too fast for conventional experimental approaches to capture them with sufficient resolution. This project aims to develop a new approach for studying membrane fusion mechanics by utilizing recent developments in the field of DNA nanotechnology, where molecules of DNA are reprogrammed to fold into well-defined structures of the same size as fusogenic membrane proteins. Using such DNA nanostructures as a type of a molecular scaffold, two membranes with different chemical and physical properties will be placed a prescribed distance away from one another to determine the likelihood of spontaneous fusion. Elements of fusogenic proteins will be incorporated within the DNA nanostructures to generate on-demand membrane fusion events. Advanced microscopy techniques will be combined with computer simulations to verify that such artificial DNA nanostructures can indeed capture the functionality of naturally occurring membrane fusion proteins. Finally, the method will be applied to induce and arrest fusion of a virus with a lipid membrane, providing a highly detailed view of the first step of viral infection. The fundamental insights into the mechanics of membrane fusion as well as the DNA nanotechnology platform to study such processes will open new avenues of investigations of various biological processes, from neuroscience to drug delivery.

Gaining insight into the structure and function of biological membranes is important for understanding cell organization. Yet, despite a great deal of progress with identifying lipids and proteins on biological membranes, we lack an understanding of their organization and movements. We propose to look at how proteins change shape during binding events using two innovative techniques. One technique uses acoustic waves to extract information of the size and shape of proteins determined by their hydrodynamic properties. The other, called atomic force microscopy, uses a miniature detector to rapidly scan a surface, similar to a finger reading braille text, to deliver a microscopic 3D view of the proteins and lipids and how their shapes change during binding events. Models of these events will be created to interpret how they are regulated and controlled. Cells have a complicated network of vehicles, called endosomes, which transport proteins and lipids to wherever they are needed at a moment’s notice. We study this endosomal network in fine detail to get a better idea of how it’s regulated. We build a simplified synthetic “endosome” containing some of the key lipids and proteins found on native endosomes. We monitor protein-lipid and protein-protein binding events to see how the proteins change their shape upon binding. By changing the density and patterns of the proteins on our synthetic “endosome” we seek to understand how a native endosome controls such critical events within the cell. The endosomal network impacts many diseases for example, Alzheimer’s and cardiovascular disease, as well as playing a role the entry of certain pathogens into cells. Also, drugs can be delivered to cells via the endosomal network. Thus, a better understanding of its regulation could impact human health and welfare.

Mycobacterium tuberculosis (Mtb) survival and replication within, or escape from, its phagocytic host requires molecular exploitation of host proteins and lipids. However, there are still large gaps in our understanding of bacterial survival and escape, particularly regarding quantitative analysis of interactions between host and Mtb derived proteins or lipids. Super-resolution fluorescence imaging has begun to yield unprecedented insight into biological processes including protein distribution at the nanometer scale and live dynamics of proteins, lipids and the cytoskeleton. However, these techniques have rarely been applied to Mtb research. Here, super-resolution imaging complimented with proteomics will allow the spatiotemporal analysis of membrane associated proteins, including pathogen derived proteins which interact with the intra- and extracellular milieu of primary human macrophages and neutrophils. Work will also utilise live-cell imaging sensitive to membrane composition to elucidate how the pathogen modulates host cell lipids for survival and replication. Additionally, how Mtb escapes its host for transmission is poorly understood. Mtb modulation of the host’s cytoskeleton, membrane and linker proteins using high- and super-resolution imaging, will clarify this process. Quantifying the distribution or disruption of host proteins and lipids through the host-pathogen lifecycle, and investigating whether Mtb proteins themselves present on the host plasma membrane will translate to application of therapeutics. This work will generate novel hypotheses for the Mtb field, and other pathogens, in the context of host immunity and disease equilibrium.

Insect herbivores and their food sources comprise ~50% of biodiversity, and their interactions impact community structure, species’ geographical ranges, and food security. How insects choose their food sources, and how plants can defend themselves, are therefore questions of substantial ecological, evolutionary and economical relevance. However, the astounding diversity of herbivorous insects and their food sources renders them challenging to answer. We seek to address this problem by investigating an aspect of plant feeding that all herbivores have in common: the mechanical breakdown of tough plant material. Plant fracture requires the application of a force, the magnitude of which depends on the morphology of the fracturing tool, and the plant material properties. This force is generated by muscle, in a process that consumes energy. Feeding on plants is therefore both mechanically challenging and metabolically costly, to an extent defined by muscle physiology, mouthpart morphology, and plant tissue mechanical properties. We will build on this common, physical foundation to develop a mechanical and energetic framework that can be used to derive first-principles predictions of herbivore behaviour. Developing such a framework will require advances in the fields of solid mechanics and muscle physiology. We will expand fracture theory to describe the mechanics of thin sheet cutting, taking into account tool geometry, large deformations, and multi-axial stress states that occur during plant tissue fracture; and we will develop thermal imaging as a novel method to study muscle energetics. We will apply this framework to the leaf-cutter ants, iconic, polymorphic herbivores that feed on a diversity of plants and exhibit a complex foraging behaviour, making them a rich and multi-faceted model system. We will predict food preferences and cutting strategies in individual workers and ant foraging parties, and use machine learning and computer vision to test these predictions in large-scale, ecologically relevant experiments. This international, inter-disciplinary collaboration will not only (i) provide a quantitative theory to study individual as well as group feeding behaviour, (ii) advance our understanding of insect-plant co-evolution, but also (ii) have impact in other fields, from inspiration for small-scale cutting tools to developments in motor control theory.

Over the last decade, humans have made extraordinary advances in our ability to synthetically adapt biology. We are in the midst of developing the tools that can reverse genetic disease by altering DNA, and we are creating proteins that do not exist in nature to perform new functions. Yet our understanding of the basic function of proteins and how this is influenced by design is extremely primitive. Even the basic understanding of binding-based signal transduction in proteins, allostery, is arguably far from complete: we are still a long way from being able to mimic nature’s sophistication in creating functional proteins and engineering allosteric functions into proteins – the difference between a mere description and a true understanding. Our mechanistic understanding of allostery has been hindered by a lack of experimental techniques that can probe single proteins within an ensemble and the gap between experimentally observed and computationally predicted quantities. We will tackle this problem by adopting new single molecule tools, in conjunction with protein design and multiscale theoretical modelling. To understand how protein design impacts allostery, we will use and further develop a combination of theoretical (based on Ising formalism, elastic network models (ENMs), and sequence coevolution analyses) and experimental (nanoplasmonic tweezers, extraordinary acoustic Raman (EAR) spectroscopy, and molecular characterization of folding/binding/function) methods. We choose as model system tandem-repeat proteins because of their simplicity and modularity, structural malleability and adaptability to new functions allosterically driven by alterations in interfacial interactions and assembly state. Using both natural and designed TR proteins (and their mutants), we seek to close the gaps between design, theory/computations and function. The goal is a deeper understanding of the biophysics of allostery that will inform our traverse into synthetic biology while also having a specific impact on TR proteins and their essential roles in cellular pathways. Notably, an important interdisciplinary impact of this work will be to quantify the interaction of electromagnetic waves with proteins in the >10 GHz frequency range, precisely where new 5G cellular phone standards are being developed and proteins are susceptible to disruption due to their elastic resonances.

Plants are sessile organisms whose growth and development rely on finely-tuned signaling mediated by plant hormones (phytohormones). One of the pivotal phytohormones, gibberellic acid (GA), promotes a wide range of developmental processes in plants, such as seed germination, root and shoot elongation, fiber and cambium development, flowering time and fruit patterning. It is therefore crucial for plants to tightly regulate the distribution of GA throughout their lifespan. Our original HFSP team, in parallel to colleagues in the field, demonstrated that a class of nitrate/peptide transporters (NPFs) actively transport GA and play major roles in GA delivery and response. However, we still do not know the mechanism by which the GA transporters select and move their substrates. One of the major impediments is the lack of high-resolution structures. Also, functional redundancy severely hampers genetic studies to investigate the contribution of each transporter in plant growth. To understand the mechanisms of active GA transport in plants, our team will address the following three objectives: 1) Uncover the molecular mechanisms of key novel NPF GA transporters using a combination of structural and functional studies; 2) Investigate the physiological functions of active GA transport in root development and fiber formation using genetic, cellular, and systems biology approaches; and 3) Identify novel GA transporters, beyond the NPF family. These objectives will require innovative techniques, for instance, we will overcome functional redundancy using our unique multi-targeted forward-genetic cell-type-specific transportome-scale screening. Our comprehensive studies on GA delivery at multiple levels—from the molecular and cellular mechanisms of individual transporters to the GA-trafficking protein network in the plant body—will greatly facilitate the design of GA-transporter specific modulators and deliver crucial knowledge on the mechanisms of active GA transport in plants. As genetic manipulation of GA biosynthesis and perception drove the first Green Revolution in world agriculture, controlling GA delivery through manipulation of GA transporters has the potential to generate a second Green revolution to improve agricultural traits.

Hybrids are offspring produced from mating events between two different species. Once thought to be rare, we now know that hybridization is quite common, and evidence for it is found across the tree of life, including in our own species. There has been a longstanding debate about the importance of hybridization because it can have both beneficial and harmful effects. Hybridization can help organisms adapt, but it can also drive extinction by generating harmful combinations of genes. Past work has proposed that in stressful environments, the benefits of hybridization may outweigh the costs, but this idea has not been clearly tested. How this tug-of-war between the positive and negative impacts of hybridization resolves is unknown and is an outstanding puzzle in several fields of biology. Moreover, understanding these dynamics has become more pressing as rates of hybridization have increased due to the stresses of rapidly changing environments. We will combine research approaches from mathematics to environmental chemistry to address this fundamental question. We will ask how hybrids respond to multiple environmental stressors, and what genetic tools they use to do so. To characterize the costs of hybridization we will develop new computational tools to identify gene combinations that may be harmful or helpful to hybrids. Finally, we will integrate results to build mathematical models to predict the possible consequences of hybridization. Together, this work will reveal the consequences of hybridization and allow us to predict its impacts in our changing world.

Hematopoietic stem cells (HSCs) are responsible for the production of all blood cells throughout life. In vivo, HSCs reside within heterogeneous microenvironments or niches able to promote their function. Understanding the mechanisms responsible for the niche ability to support HSCs is required to expand our knowledge of HSC-niche interaction in vivo in healthy conditions and after niche injury induced by chemotherapy. Additionally, it will pave the way to create in vitro niches allowing for HSC reprogramming, maintenance and expansion for therapeutic purposes. Using zebrafish, a powerful model due to its similarity to human developmental processes, hematopoietic master regulators and disease inducing mutations, I will: Aim 1: Unravel the cellular and molecular mechanisms providing niche cells the ability to support HSC using live imaging and single-cell multiomics. a- Characterize the 3D structure and the cellular dynamics of the HSC niche using live imaging of a newly generated multicolor zebrafish reporter line in healthy conditions and after cell-specific ablations. b- Decipher the niche transcriptional code responsible for HSC support combining single-cell RNA-seq, single-cell ATAC-seq and spatial transcriptomics. Aim 2: Characterize the clonal dynamics of HSCs and niche cells in the marrow in perturbed conditions using genetic lineage tracing. a- Analyze the clonal origin of HSCs and niche cells in the healthy marrow using the scGESTALT barcoding technology. b- Expose animals to sublethal irradiation and characterize the clonal dynamics of niche recovery after injury.