Cell fate determination is crucial for development, tissue maintenance, and disease progression. It's the process by which cells commit to specific roles while excluding other potential fates. This ability of cells to choose different paths, despite containing identical genetic material, has long fascinated scientists. Recent discoveries reveal that the 3D organization of the genome plays a pivotal role in this process, adding complexity to our understanding of cellular decision-making. 3D genome organization refers to how DNA is folded within the cell nucleus. This arrangement is highly structured, with specific genome regions in close contact. These interactions influence gene activity, affecting cell fate decisions. It's like complex origami of genetic material, where folding patterns expose or hide genetic instructions. My research explores cell fate decisions, focusing on DNA organization within the nucleus. Imagine noodles too big for a small bowl - analogous to our DNA, which carries genetic information but is too large to fit inside a cell's nucleus without folding. For decades, scientists overlooked this folding, assuming it didn't affect cell function. However, advanced technologies have revealed the importance of DNA's 3D organization inside cells. Research now explores how 3D arrangement affects cellular processes, particularly gene activity. This adds complexity to our understanding of cell function and decision-making. Traditional research examined DNA folding and gene activity separately or in large cell groups, missing crucial individual cell details. My research utilizes cancer cells for studying cell fate choice, which fascinates due to their ability to adapt and resist treatment despite having the same genetic material as normal cells. In the context of 3D genome organization, it's as if they are rewriting their own recipe. To study this, I have developed \"FateWatch,\" an innovative tool combining DNA barcoding with advanced sequencing techniques. This approach allows me to track thousands of cells simultaneously, observing their lineages, gene activity, and 3D genome organization in a single cell at unprecedented detail. FateWatch uses DNA barcoding, introducing unique DNA sequences into each cell as genetic tags. These barcodes are passed to daughter cells during division, enabling us to trace cellular lineages and track the fate of specific cells and their descendants throughout the experiment. By combining this barcoding technique with single-cell RNA sequencing (for gene expression) and Hi-C (for mapping 3D genome organization), we create a powerful system for observing cellular behavior. My approach captures the interplay between genome structure, gene expression, and cell fate. I will investigate how treating cancer cells with drugs affects their DNA arrangement and gene activity. By comparing \"twin\" cell populations, we can determine if the same cells consistently become resistant or if it's a more random process, helping us understand if certain cells are predisposed to resistance - like how some noodles might absorb sauce based on their position. Furthermore, I aim to identify key proteins that help cells maintain their identity during division and examine whether cells are more likely to become resistant at specific stages of their cell cycle, particularly during the G1 phase when cells are more sensitive to their environment. This could reveal critical windows of vulnerability or resilience in cell cycle phases for cell fate determination. The FateWatch framework allows us to gather rich, multi-dimensional data on cellular behavior, providing insights into the complex processes of cell fate determination impossible with conventional bulk analysis methods. Ultimately, the insights from this research could revolutionize our understanding of cell fate determination, with far-reaching implications for cancer biology, developmental biology, and stem cell research.

In humans, a paper cut takes days to seal and a limb amputation takes >8 weeks to heal and months to rehabilitate. In contrast, the axolotl salamander can seamlessly close wounds within hours of injury and regenerate a functional arms or legs within a month after amputation. This prodigious regenerative ability captivated scientist for more than 150 years. In a recent breakthrough discovery we demonstrated this regenerative ability depends at least partly on the axolotl’s ability to quickly make new proteins at the site of injury. This rapid accumulation of proteins - the building blocks of the cell – seems to allow skin cells at the edges of the wound to migrate and seal the injury. This in turn allows the animal to regrow a functional limb in the weeks that follow. While we know that protein production is important, we don’t know what proteins are being made and where or why they are important. We hypothesize that axolotls produce special proteins that act as \"band aids\" to help seal wounds more quickly. In this proposal we use the unique and complementary skills of four world-class labs to visualize how skin cells in live axolotls, tune protein production to respond to injury by using high-resolution imaging and novel protein probes to visualize when and where axolotls make their “band aids”. We will compare the dynamics of this process to that of 'dorsal closure' in the fly - a well-studied example of cells closing a naturally occurring ‘hole’ at the back of the embryo. This classic example has been used to understand cell migration however the role of dynamic protein accumulation has not been studied. Next, we will apply a newly developed “single cell Ribo-Seq” approach to understand what building blocks are being produced in skin cells that are actively engaged in protein production. Finally, using a cutting-edge protein identification approach, we will determine the location and identity of axolotl band aid proteins and study their function in wound healing and regeneration using genetically engineered axolotls. Our multidisciplinary approach will (1) elucidate mechanisms of wound healing; (2) provide insight into how different species use proteins production to drive cell rearrangements and (3) create a pipeline for studying how changes in protein production contribute to cell movement in diverse biological contexts like cancer metastasis.

The function of most genes is achieved through the production of a protein that binds other molecules and/or catalyzes biochemical reactions inside the cell. A large fraction of the cellular machinery is dedicated to producing these active proteins in a timely manner and in appropriate amounts. Depending on the gene, protein abundance in cells is set to anywhere from 1 copy to around 1 million copies per cell. Despite this large variance, relatively small changes in the expression of any given gene can cause disease and/or drive evolutionary adaptation. Thus, measuring protein abundance across genes, cells, and different environmental conditions has been the subject of extensive research for the past 50 years. However we still lack a clear picture of how much or how little of a protein is necessary to support cell growth and function. Consequently, we know which ingredients are vital for making functional cells and but not how much is needed. Evolution can change the ingredients and their quantity, but we do not know which mechanism is favored and why. We join our expertise in evolutionary biology (Landry), cell biology (Moriya) and molecular biophysics (Reynolds) to understand how molecular-scale variation in protein activity and abundance contributes to the organism-level fitness (growth) of cells. Our goals are to: (1) determine how changes in protein activity and abundance influence growth rate within a biophysical framework (2) examine how natural systems navigate this space through mutations and selection and (3) better understand why sometimes evolution is limited in what it can change because cells cannot overcome the toxicity of some proteins and their mutations. We will combine quantitative, high-throughput experiments (in both the bacterium E. coli and yeast) with computational modeling. Together, our work will provide a quantitative framework for considering the “design” of protein systems. It will also offer a quantification of the conditions within which cellular systems evolve and a mechanistic understanding as to why protein abundance can range several order of magnitudes while a given protein is allowed to exist in a very tiny abundance window by evolution.

All large organisms on Earth belong to a single domain of life, the eukaryotes. Many eukaryotes form large multicellular structures that allow resources to be harvested from different locations and combined. For example, the structure of trees allows the combination of water and minerals from below the soil surface with light and gases from above. The ability to form such structures to exploit resources from multiple environments is generally assumed to be lacking in the other domains of life, the bacteria and archaea. However, we have identified multiple, nitrogen fixing photosynthetic cyanobacteria which form centimetre scale plant-like structures. These species, all sheathed bacteria, incorporate filamentous, interconnected cells within a rigid polysaccharide sheath, which allows them to attach to surfaces via a biofilm and form aerial shoot and subsurface root like structures. Sheathed bacteria are widely found in nature, predominantly in freshwater and coastal ecosystems, but also in saltmarshes, rocks and lava deposits, where they play an important role in preventing erosion, act as a food source and adapt the environment to render it suitable for plant growth. Utilising a novel model system we have shown that cells can migrate within sheaths at a speed equivalent to a 25 metre carriage train travelling 7 km/hour and that sheaths can bind together to form the shoot-like structures. What drives this plant-like growth and rapid cell migration is unknown but like plants is probably linked to exploitation of resources from different environments. Our multidisciplinary team will apply its expertise in microbiology, analytical polymer science and mechanical engineering to understanding the role of the aerial, biofilm and subsurface cells, how nutrients, energy and cells travel up and down sheaths and how the properties of sheaths allow the formation of large structures and transport of cells and nutrients. Specifically, we will analyse gene and protein expression in environmental samples and our model system to understand the role of aerial, biofilm and subsurface cells and whether a novel form of nitrogen fixation is occurring in biofilm cells, microscopy to analyse the architectural properties necessary for plant-like growth and movement of filaments and nutrients, and structural and chemical analysis of sheaths to elucidate their role in these processes.

Surface proteins, such as checkpoint receptors and co-stimulatory receptors, within the tumor microenvironment (TME) play important roles in regulating tumor growth and immunosuppression. Profiling the dynamic of the surface proteome of cancer cells and tumor-infiltrating T cells, including chimera antigen receptor T (CAR-T) cells, within the TME in animal cancer models can provide a comprehensive understanding of the molecular mechanisms by which tumor cells grow, metastasize, and evade immune detection. Proximity labeling (PL) has emerged as one of the most promising approaches for such studies due to its rapid labeling kinetic (10 minutes) and high spatial resolution (10 nm). PL employs engineered promiscuous enzymes fused to specific protein baits to generate reactive species, resulting in the covalent tagging of proximal proteins for proteomic analysis. However, existing PL enzymes have significant limitations in profiling cell surface proteomes in vivo. For instance, peroxidases require cytotoxic H2O2 as a co-oxidant, while biotin ligases strictly need ATP, which is present in low amounts at the cell surface. To overcome these technological barriers and gain a deeper understanding of surface proteomes related to tumor-immune interaction, I propose to develop a novel PL enzyme with surface-selective, non-toxic activity. My host lab has recently engineered a multicopper oxidase, LaccID, which leverages non-toxic oxygen as a co-oxidant to generate phenoxyl radicals for PL on the cell surface. While potentially compatible with in vivo applications, this enzyme requires 1-2 hours to achieve sufficient labeling and is susceptible to endogenous substances such as thiols and halides. Therefore, additional engineering is needed to make it a truly useful tool for in vivo surface proteome mapping. By implementing protein engineering methods such as directed evolution, I will enhance LaccID for improved activity and resistance to endogenous inhibitors. By expressing the next-generation LaccID on the surface of cancer cells, T cells, or CAR-T cells in mouse cancer models, I will use proximity labeling and mass spectrometry to map surface proteome changes during tumor progression/evasion and immune cell activation/exhaustion. This approach aims to uncover novel protein-mediated signaling axes between cancer and immune cells in mouse models, offering avenues for therapeutic intervention. Ultimately, this project will provide (i) a transformative technology for profiling the surface proteomes of specific cell types in complex, in vivo environments, with high temporal resolution (minutes) and minimal toxicity, and (ii) proteome insight into the mechanisms by which tumor-immune interactions reshape the cell surface landscape, influencing functions related to tumor killing or immune evasion.

Diabetes is a condition that affects over 500 million patients worldwide, significantly impacting their quality of life and posing a substantial burden on healthcare systems. This disease is caused by the gradual destruction of insulin-producing ß-cells, which reside within the islets of the pancreas. Without these cells, the body struggles to regulate blood sugar levels, leading to serious health problems and diabetes symptoms. This research project explores the possibility of restoring, or regenerating, these insulin-producing cells by studying the concept of cell plasticity in the human pancreas. Cell plasticity refers to a cell’s ability to change its function or identity, potentially taking over the role of other cells. In animals like mice and zebrafish, certain pancreatic cells can switch to producing insulin when ß-cells are lost, helping to maintain normal blood sugar levels and preventing diabetes symptoms. However, it is still unclear whether human pancreatic cells have a similar ability, and if this can be enhanced to recover lost ß-cells in diabetes patients. To investigate this, the project uses lab-grown human pancreatic islet organoids, which are 3D clusters of cells that mimic the structure of the human pancreatic islet and contain all the different types of islet cells. These organoids resemble the pancreas during fetal development, a stage of life that is known for its greater plasticity and ability to regenerate. The organoids thereby provide an ideal model to study whether human pancreatic cells can indeed undergo plasticity and thus regenerate insulin-producing cells, as well as to identify any genetic or epigenetic factors that might block this process. First, the research will focus on zebrafish, which is known for its impressive regenerative abilities. By using advanced single-cell sequencing technologies, the study will identify which genes and molecular mechanisms are activated when zebrafish ß-cells are lost and how other pancreatic cells can switch states to take over insulin production. Specifically, this project will investigate if epigenetic factors are involved in this process, as these might block cell plasticity in the human pancreatic islet. Once this knowledge is gathered, the project will investigate whether similar mechanisms exist in the human islet organoids, by artificially causing ß-cell loss in this model and analyzing the response with single-cell sequencing technologies. This will reveal if the islet organoids, which mimic the fetal stage, indeed have a higher regenerative potential. Alternatively, if the organoids don’t show the same plasticity as the zebrafish, this research will pinpoint to the barriers that prevent this, such as the absence of specific genes, epigenetic factors, or other biological issues. Building on this, the project will use innovative gene-editing technologies like CRISPR to modify specific genes in the human organoids, aiming to enhance their cell plasticity. By testing different factors discovered through the zebrafish and organoid studies, this research intends to further boost the ability of these organoids to regenerate insulin-producing cells. This could be a key step toward developing a treatment that helps diabetes patients to naturally recover their insulin production. If successful, this research could transform diabetes care by offering a regenerative therapy that allows patients to regain insulin production, potentially reducing or eliminating the need for insulin injections. Moreover, it would create a new model for studying tissue regeneration in humans, with insights that could extend beyond diabetes to other diseases and organs.

How did the early Earth environment influence the chemistries that led to life as we know it? If minerals were indeed the predecessors to enzymes, how did they support complex reaction sequences? Did they collaborate with emerging enzymes? In this proposal, we tackle these questions from three disciplinary angles: catalysis of key metabolic reactions at mineral surfaces, chemistry within porous environments, and resurrection of ancient enzyme structures. The focus of these three approaches centers on organic cofactors, crucial helper molecules that can support catalysis within the enzyme active site and at the mineral surface. These versatile molecules, which are absolutely conserved across the tree of life, are relics of metabolism before enzymes. By binding to early peptides, we hypothesize that cofactors and minerals may have helped spark the emergence of the earliest enzyme structures, allowing them to fold and function. We also posit that patterning of pore networks may have established the earliest reaction “pathways”. To study these processes, we are building a reaction system in which the interacting roles of spatial structure, mineral composition, cofactors, and primitive enzymes can be disentangled. In doing so, we are interrogating the history of reaction control and bias, the properties of the earliest proteins, and the nature of self-sustaining reaction networks. Ultimately, the goal of this work is to construct a pre-cellular, proto-enzymatic, assembly line in which a common cofactor participates in its own synthesis.

Among marine invertebrates, many animals have formed a mutual symbiosis with photosynthetic partners, like algae. In this relationship, the animal provides protection and nutrients, and in exchange the algae provide energy through photosynthesis. This partnership is highly prevalent in corals and sea anemones, underpinning the productivity and biodiversity of coral reef ecosystems, which are home to nearly 25% of all marine life. Like plants, photosynthesis represents an important energy source in photosymbiotic animals. Since the mid-19th century, researchers have observed that marine animals living in symbiosis with photosynthetic algae, such as sea anemones, flatworms, and sea slugs, move in response to light, a phenomenon known as phototaxis. Such adaptations may have allowed these organisms to orient themselves toward a specific light environment conducive to optimal photosynthetic activity, allowing them to thrive in shallow marine habitats. While the survival of both organisms relies on a mutually beneficial partnership, nothing is known about how these organisms communicate nor what underlying signal is used to induce these fascinating light-driven behaviours in the animal. Our proposed research will explore the mechanisms that connect algal photosynthesis with animal behaviour. We will do this by investigating the host nervous system and the physiology and metabolism of the photosynthetic algae. We will conduct experiments to explain how photosynthetic partners influence the behaviour of their hosts in order to gain a new understanding of this fascinating process, providing new insights into the fundamentals of the photosymbiotic relationship in animals. Photosymbiosis has been fundamental to the success of coral reef ecosystems, sustaining their growth into one of the most diverse biomes on Earth. However, with climate change threatening their extinction by the end of this century, there is an urgent need to develop innovative techniques and approaches to ensure the survival of corals in the future. The importance of this work therefore extends beyond understanding how one partner manipulates the other; it addresses fundamental aspects of the evolution of photosymbiosis and how this important partnership thrives.

Swarming is among nature's most complex and poorly understood collective behaviors and is found in diverse animals, from birds and fish to insects. Although prior work on bird flocking and fish schooling has suggested that the collective behavior is due to relatively simple algorithms, research in mosquito swarms has not yet identified the principles of their swarming behavior. Mosquitoes are the world’s deadliest creatures and impact a billion people per year with the pathogens of disease. For mosquitoes, swarms are critical for their reproduction and, hence, the spread of disease. At dusk, thousands of mosquitoes are thought to be attracted to visual markers to form a swarm. Once in the swarm, mosquitoes mate in-flight in the complex 3D swarms where individuals must approach mates while avoiding their competitors. However, little is known about the sensory mechanisms of swarm formation and the processes that enable these intricate within-swarm behaviors. Malaria-mosquitoes Anopheles gambiae are suitable organisms for tackling these questions and determining the sensory mechanisms at different scales: long-distance attraction to swarm markers (>10 m) and close-range attraction to mates (0.1 m). Previous work has shown that male mosquitoes are attracted to, and form a swarm that fewer females enter above a visually contrasting object on the ground just before dusk for mating, but it remains unclear what features of the visual marker (color, size, contrast) attract the mosquitoes to cause the swarm formation. Once in the swarm, males distinguish males and females based on sex-specific flight tones, and recent work has shown that males avoid collision with other males based on visual and acoustic cues. However, the exact visual features that mediate mating interactions and male-male avoidance have not been tested. Despite significant improvements in our understanding of the ethology of swarming in mosquitoes, the sensory and neural bases of swarming behaviors and visual-acoustic processing are poorly understood. Recent developments in computer vision, quantitative behavior, and neuroscientific and genetic approaches now make it possible to establish a systematic methodology for conducting behavioral and neural experiments on flying mosquitoes. I propose to study the behavioral and neural mechanisms of visual-acoustic-based mating behaviors in the swarm by utilizing an innovative experimental pipeline across organizational scales. This pipeline and associated computational analyses will enable testing the visual markers for swarm formation and analysis of mosquito trajectories using a state-of-the-art 3D tracking system to investigate stereotypic behaviors during swarm formation and within-swarm interactions. A cutting-edge virtual reality setup with tethered mosquitoes will enable me to recapitulate the visual-acoustic features of the swarm, enabling time-series observations of behavioral and physiological changes during localization behavior. Last, I will study the neural basis of visual-acoustic integration by recording male mosquitoes' neural activity when exposed to these features. This study will provide a new understanding of the functional neuro-mechanics of mating swarms, and provide crucial knowledge about the mechanisms that underlie the fecundity of malaria vectors that cause >400,000 human deaths each year. Therefore, my project will directly support the development of malaria-vector control strategies/methods, such as acoustic and visual lures. Finally, swarming is an important topic in aerial robotics research; my study may provide bio-inspiration for novel algorithms in swarming robots.

Patients with dementia continue to increase as the global population ages. Alzheimer's disease is the most common form of dementia and is characterized by neuroinflammation induced by brain immune cells called microglia. They are essential for maintaining brain homeostasis by removing pathogens and dead cells, but once activated, they induce neuroinflammation and exacerbate neurodegeneration. Whereas the importance of neuroinflammation has been pointed out, there has been no treatment for Alzheimer's disease targeting neuroinflammation to date. One reason for this is that the microglia of rodent models widely used differ significantly in nature and function from human microglia, as they do not have orthologs of the risk genes for Alzheimer's disease. Therefore, a model of human microglia is urgently needed for neurodegenerative disease research. In this study, \"neuroimmune organoids\" will be applied for the first time to investigate microglia in Alzheimer's disease. Organoid technology is a technique for culturing three-dimensional tissue from stem cells on a dish. The neuroimmune organoids are the latest technology ideal for studying neurodegeneration because, in this technique, human brain tissue with microglia is xenotransplanted into mouse brains, allowing human microglia to be cultured for long periods. They can recapitulate the physiological conditions in the human brain and overcome the limitations of earlier rodent models. Meanwhile, the Y chromosome has been considered a \"genetic wasteland\" because it has lower gene density than the X and autosomal chromosomes. Blood cells lose their Y chromosome with aging, and recent studies suggested that loss of the Y chromosome exacerbates heart failure and cancer and is prevalent in the microglia in patients with Alzheimer's disease. However, it is unclear whether microglia which has lost the Y chromosome worsen Alzheimer's disease. This study primarily aims to elucidate the effect of the loss of the Y chromosome in microglia on Alzheimer's disease. Why do microglia lose their Y chromosomes as they age? The fundamental mechanism of neurodegeneration may be related to the answer to this question. It is believed that there are distinct upstream mechanisms in cancer and neurodegenerative diseases. Cancer characteristic is uncontrolled growth induced by genomic instability, which causes DNA damage and mutations. Although abnormal protein aggregation is essential for neurodegeneration, recent studies have also detected genomic instability in neurodegenerative disease lesions. Furthermore, the loss of the Y chromosome in blood cells has been linked to abnormalities in cancer-driver genes. This project will investigate how genomic instability induces Y-chromosome loss in microglia and identify common upstream mechanisms in cancer and neurodegeneration. In summary, this study will use cutting-edge brain organoids to define a new role for the Y chromosome in the human brain. We also propose genomic instability as a common cause of cancer and neurodegeneration. This study has the potential to accelerate Alzheimer's disease research by applying the extensive knowledge and tools developed in cancer research to date, leading to benefiting millions of patients.

In this project we will investigate the interplay between physics and biology. We understand that biological systems are constrained by physics, and we believe that the evolution of proteins took place in the context of physical constrains. Here we will identify the proteins responsible for generating, transducing and changing osmotic pressure, and we will use a newly developed osmotic sensor to directly measure osmolarity in living organ tissues. Our work is tailored to the specific case of pancreas development in the mouse where hollow lumens emerge at embryonic day 10 and grow to become a complex tree-like structure that carry pancreatic enzymes into the gut. We will investigate physical mechanisms deployed during embryonic development. Such mechanisms may also be used later in life and even during adult organ regeneration. Our study will cement the foundation for applied research providing a reference of the physical parameters and temporal dynamics that are required for healthy pancreas formation. In our project we are taking the challenging but innovative approach of directly measuring physical parameters. Historically, we have inferred such parameters like tension, viscosity and osmolarity in cells from indirect microscopy measurements. Yet today novel sensors enable us to directly measure such parameters to avoid assumptions and accurately model biological systems. But more importantly, to measure changes over time. In this project, we paired classic laboratory experimentation with theoretical analysis to build a model that accounts for dynamic osmotic pressure and cellular feedback mechanisms that shape the organ during development. Our aims are structured to probe the development of mouse pancreas from both the physics and the biology perspective. We believe that the forefront of human knowledge today lies at the interphase between different disciplines, and we made plans to collaborate with cutting-edge technology facilities in Dresden to successfully accomplish our aims. Our work will provide a detailed description and functional test of the role of osmotic pressure during lumen formation, and we will test specific candidate proteins for their function in translating osmotic pressure (physical signal) into a cellular response (biochemical or genetic signal) and vice versa. Our research not only will advance our fundamental understanding of embryology and tissue dynamics, but also it will serve as the seed for therapeutic interventions for people who suffer from cystic fibrosis. These patients display increased osmotic pressure in lumen mucus secretions, and there is a need to understand the pathophysiological consequences of changes to osmolarity in the lumen. Our findings will reveal specific molecular players that respond to osmotic pressure and can be used for diagnostics and therapeutics to alleviate the consequences of osmotic dysregulation. Furthermore, our project will generate clear protocols to derive patient samples and probe them with osmotic sensors in culture conditions. This will open new avenues to pursue personalized medicine where time-sensitive decisions can be made from patient in vitro culture results to best design an adequate therapy. Overall, we are pushing the frontier of current knowledge of physics and biology to hopefully make fundamental discoveries for the better of humankind.

The heart is one of the first organs to form. Genes specifying heart cells (cardioblasts) are highly conserved from insects to humans. The earliest processes of heart shaping are also qualitatively similar between species: two regions of cardioblasts migrate to the embryo midline to form a simple tube, neighbored by extraembryonic tissues. Yet, it remains a major challenge to dissect the biophysical processes (e.g., the magnitude and direction of forces) that first build the heart, including the role of extraembryonic tissues. Here, we bring together two largely separate fields, evo-devo and bioengineering, to tackle this challenge. By combining the strength of comparative approaches in evo-devo with quantitative biomechanical measures of cell and tissue properties, we will be able to make advances not possible with more traditional, single approaches. We will use evo-devo methods across four insects to tackle this challenge. Our species represent diverse sizes and geometries and span over 400 million years of evolution (hemimetabolous: milkweed bug and earwig, holometabolous: fruit fly and flour beetle). Through imaging and functional perturbations, we will identify heart closure mechanisms, distinguishing species-specific from conserved elements. As part of this, we will provide an unprecedented atlas of earwig development during organ formation, providing a new powerful tool for the community, alongside new methods development to extend model system toolkits in the fly and beetle. Indirect evidence suggests early cardioblasts mechanically adjust to changes in their local environment. By leveraging bioengineering tools to measure and perturb material properties both inside intact embryos and in bioengineered microenvironments for extracted cells and tissues (in vivo, ex ovo, and for isolated cells), we will provide the first quantification of the biophysics of early heart formation across species. Subsequently, we will model how cells interact to construct the heart, using our novel focus on mechanical inputs from neighboring tissues to elucidate general principles of early heart morphogenesis. Overall, this project offers unique perspectives to tackle a longstanding problem: how does the heart robustly form? It represents a truly interdisciplinary and international team, bringing a diverse range of skills and approaches.

As the global population ages, the negative effects on health, such as increased vulnerability to infections and diseases, are becoming increasingly apparent. One major factor in this decline is the diminished efficiency of the immune system, which is closely linked to hematopoietic stem cells (HSCs). These cells are crucial for producing all types of blood cells, including those essential for immune responses. HSCs have a remarkable ability to either differentiate into various types of blood cells or self-renew to maintain their own population. Researchers historically thought of HSCs as a uniform group of cells, but advances in single-cell technologies have revealed their high diversity. This means different HSCs may have distinct roles, with some producing a balanced mix of immune cells, i.e. lymphoid and myeloid cells, and others biased towards producing more myeloid cells, which are involved in fighting infections and responding to inflammation. Unlike lymphoid cells, that are crucial for long-term immune responses, myeloid cells do not contribute to immune memory. As we age, this balance shifts towards these myeloid-biased HSCs (my-HSCs), which might contribute to the decline in immune function seen in older individuals. Recent studies have shown that partially removing these my-HSCs in mice can rejuvenate the immune system, restoring it to a more youthful state. However, the exact role of my-HSCs in aging, their origins, and their dependence on epigenetic factors—mechanisms that regulate gene activity without changing the DNA sequence—remain unclear. This project aims to address these gaps. First, we will use a specialized mouse model to deplete selectively cells that typically give rise to lymphoid cells, allowing us to observe whether my-HSCs alone drive aging. This will help determine if these cells are central to the aging process. Next, we will investigate the development and origins of my-HSCs over time to understand why they become more prevalent with age. This knowledge could reveal new targets for interventions to slow down aging. Finally, we will test whether specific interventions can either reprogram or eliminate my-HSCs to rejuvenate the immune system. Success here could open new pathways for therapies aimed at extending healthy lifespan. In an increasingly aging world, understanding and reversing the decline in immune function is more important than ever. By uncovering the role of my-HSCs in aging, this project could lead to new therapies that enhance healthy lifespan and improve the quality of life for millions of older individuals.

RNA, a molecule that acquired an intense public attention mainly during the recent Covid pandemic, is believed to be central for the origin of life, as an ancient carrier of genetic information and a catalyst. Its formation in a hostile geological environment, that likely existed on the early Earth about 4 billion years ago, is surrounded with a lot of enigmas. One of them is related to the availability of phosphates, an integral part of all known RNA molecules today. Phosphates represent the highest oxidized form of phosphorus oxosalts, nevertheless, a question arises whether these molecules could have accumulated in a reducing environment that likely existed on our planet at the times when life emerged. Interestingly, phosphate derivatives are less reactive than their reduced form variants, called phosphites. In the frame of the current proposal we set out to explore chemical and geological conditions that enabled accumulation of phosphite salts, the formation of phosphite-containing prebiotic building blocks, their polymerization processes leading to reduced form RNA molecules, as well as the oxidation of the latter to RNA-like polymers. To conduct this research we will use a unique, highly interdisciplinary combination of the expertise coming from two distant research fields: prebiotic nucleic acids chemistry and early Earth geochemistry. Our main aim is to reconstruct a scenario that accounts for all major steps of the synthetic process leading from accumulation of phosphite ions up to the emergence of short RNA molecules on our planet. Finally, we propose to study whether phosphite linkages incorporated in RNA, as likely remnants of reduced form predecessors of RNA, could give rise to the emergence of the catalytic activity of these molecules.

Mitochondria, the energy producing powerhouses of cells, play a crucial role in maintaining brain health and function. When mitochondria malfunction in the central nervous system, it can contribute to the development of devastating neurological disorders. Mitochondria are highly mobile and can move around cells to fuel biochemical processes where they are needed the most. A fascinating process called intercellular mitochondrial transfer (IMT) has been discovered that allows mitochondria to be transferred between distinct cell types. IMT is guided by tunnelling nanotubes which are membranous channels that connect cells for the exchange of various cellular signals and cargo. The transfer of mitochondria to stressed neurons appears to have striking protective effects and holds the potential to be exploited to aid recovery from brain disease or injury. Understanding IMT is therefore essential for developing new treatments. While IMT has been documented in diverse tissues and biological contexts, the exact molecular mechanisms underlying the process remain a mystery. This HFSP LT Fellowship project aims to shed light on the mechanisms guiding IMT using state-of-the-art methods. We will investigate how mitochondria are transferred between neurons and affect brain function by developing an innovative and precise platform for detecting IMT between neurons. We will use this platform to to uncover the genes and pathways that are responsible for IMT and investigate the protein-protein interactions that mediate transfer. We will use cutting-edge electron microscopy techniques to track IMT at the single molecule level and to three-dimensionally reconstruct tunnelling nanotubes transporting mitochondria in cells at high resolution. The findings from this research could lead to the development of novel therapeutic approaches for conditions like Alzheimer's, Parkinson's, and ischemic diseases and introduce new paradigms to the field of IMT.

Life begins with fertilization, where the embryo inherits most of its cellular contents from the oocyte, a large maternal egg cell. During their development, oocytes grow extensively and store the essential biomolecules needed for early embryonic development. Storing these materials, even in a large cell such as an oocyte, is a major challenge. To overcome this, oocytes use proteins that can change their three-dimensional structure to neatly assemble into compartments, like tiny Lego blocks, efficiently collecting and storing the components needed for embryogenic development. Disruption of these structures can lead to failure of embryonic development, highlighting their critical role in reproduction. However, the molecular mechanisms that regulate this unique organization of cellular components are only beginning to be understood and remain largely unknown. When we examined mouse oocytes using cryo-electron tomography (cryo-ET), a technique that captures detailed three-dimensional images of molecules inside cells in their natural, frozen state, we discovered a previously unrecognized compartment. Some proteins in the oocytes were stacked in well-organized intracellular crystals. The high number of these crystals, up to 10,000 per cell, suggests that they are composed of abundant proteins and may play an important role in oocyte and embryo development. However, the identity and function of these protein crystals in mammalian oocytes are still unknown. In my postdoctoral research, I aim to identify the proteins that form these crystals and understand their role in oocyte and embryo development. To do this, I will isolate crystals from mouse ovaries and analyze their composition and structure using microcrystal electron diffraction, a technique that uses electron beams, similar to X-rays, to reveal the atomic arrangement of very small crystals. Next, I will study when these crystals form and how they change during oocyte and embryo development using advanced imaging techniques such as second harmonic generation microscopy, which highlights ordered structures such as these crystals without the need for additional labeling. I will also investigate how various changes in the intracellular environment and metabolism during development affect crystallization. To understand the role of these crystals, I will deplete the crystallizing protein in mouse oocytes and observe the effects on oocyte and embryo development. I hypothesize that protein crystallization may support development by serving as a storage compartment, regulating the activity of metabolic enzymes, or protecting the cell by packaging harmful metabolic by-products into compact crystals that isolate them from other cellular components. Finally, I will use cryo-electron tomography to investigate whether similar crystals exist in human oocytes and whether mutations in crystallizing proteins are linked with early menopause or infertility. Overall, this research will provide new insights into the composition and function of protein crystals in oocytes that are currently undiscovered. These findings will deepen our understanding of oocyte biology and could lead to advances in reproductive health and in vitro fertilization technologies. In addition, the project has broader implications for the study of protein crystals in other cell types, including those associated with diseases such as asthma, cataracts, and muscle disorders, offering opportunities for their prevention.

Adult resident stem cells mediate tissue repair after injury by differentiating into the specific cell types needed to restore tissue integrity. Recent studies demonstrated that resident stem cells can acquire a memory from previous triggers, encoded in their epigenome, that enhance their responsiveness during subsequent injuries. Thus, modulating stem cell memory could be a promising way to boost stem cell potential and tissue repair, especially in tissue with limited regenerative capacity. The central nervous system (CNS) fails to heal following injury, representing a major challenge in regenerative medicine. While the CNS contains resident stem cells, their contribution is limited, raising the need for new approaches enhancing neural stem cell function in vivo. In this project, I aim to record the formation of chromatin memories in neural stem cells and characterize their functional impact, with the goal of engineering priming memories enhancing their contribution to injury repair. I will use the mouse spinal cord as a model system given that the resident stem cell population, ependymal cells (EpCs), has been well characterized. First, I will investigate the acquisition of inflammatory memory in neural stem cells after injury. The response to injury and fate of EpCs that were never triggered by injury (naive EpCs) and EpCs that were previously exposed to injury (primed EpCs) will be compared using genetic fate mapping, conditional transcriptional factor perturbations, and in vivo injury models. Then, I will characterize how memories are encoded in the chromatin of neural stem cells. The chromatin landscape of naive and primed neural stem cells will be compared to identify chromatin regions that remain accessible after EpCs recover their original identity. Spatial genomics analyses of the injury response of naive and primed cells will identify the most promising candidates that modulate the response and plasticity of neural stem cells after injury. Finally, I will engineer chromatin memories in EpCs to enhance their regenerative potential. Using CRISPR-based epigenome editing, I will implant the memory-associated chromatin modifications identified into naive EpCs and assess the impact of this artificial memory on neural stem cell fate and potential. Overall, this project brings an innovative perspective to unlock the regenerative potential of neural stem cells using targeted molecular engineering and may inspire new strategies to promote CNS repair. Editing epigenetic cell memories could open new therapeutic perspectives for numerous conditions by manipulating the in vivo behavior of resident stem cells from many tissues.

Sleep is a mysterious state of reduced environmental awareness that usually occurs when animals are still. However, some animals have evolved ways to sleep while moving through the environment. Notably, birds can sleep with one or both brain hemispheres in flight or while actively swimming, as recently discovered in Canada geese. However, it is unknown if and how birds sense and track their position in space when sleeping on the move. Our team aims to answer this question by recording from place cells – neurons in the hippocampus that track an animal’s position in space – during sleep-swimming in geese. Specifically, we will determine if the hippocampus: 1) continues to track the bird’s position, 2) retains the last known waking position and updates it upon awakening, or 3) replays previous position sequences, as described in sleeping stationary rodents. Our team consists of Niels Rattenborg (Max Planck Institute for Biological Intelligence) and Nachum Ulanovsky (Weizmann Institute of Science). Rattenborg is an expert on avian sleep who discovered that birds can sleep while flying and swimming. Ulanovsky is a hippocampal expert who has developed methods for recording and analyzing place cells in freely flying bats and birds. We will combine our expertise to study the behavior and activity of hippocampal place cells in Canada geese that will be sleep-swimming in a large, floating aviary. We will use a training protocol established recently by Rattenborg, in which geese readily display sleep-swimming. This entails imprinting young geese on the experimenters and having them swim back and forth inside a long (70 m) water channel; after completing several laps, the geese become sleepy, close their eyes, and exhibit brain waves typical of sleep. We will use custom neural-loggers developed by Ulanovsky to record place cells in awake and asleep geese on land and as they swim through the water channel. This study will produce the first recordings of hippocampal place cells in any animal moving while asleep, and will provide novel perspectives on how the brain monitors the outside world during sleep. This research promises to provide insight into sleep walking in humans, a poorly understood and potentially dangerous disorder for which no animal model exists.

We are living on a fast-changing planet with an unprecedented climate crisis and the growing emergence of pathogens, pollutants, and inflammatory irritants that we have never encountered before. Additionally, our ozone layer is thinning, increasing our exposure to the sun’s harmful rays. Not surprisingly, chronic inflammatory disorders and skin cancers are on the rise, because bearing the brunt of these environmental assaults is our skin epidermis: it is at the interface between our body and the outside world. To keep us as protected as possible from scratches, microbes, and the sun’s ultraviolet light, barrier tissues such as the skin epithelium exhibit a remarkable ability to regenerate and adapt to these external challenges. These tasks are only possible through the function of specialized tissue cells called epithelial stem cells (EpSCs), which are long-lived, and able to self-renew and regenerate the skin’s barrier. EpSCs work in coordination with our immune system to repair damage and prevent infections. However, when mutations in EpSCs cause this communication to go awry, the susceptibility to chronic inflammatory skin disorders and cancer emerge, posing ever-increasing global health problems. Recently, we have been learning that stem cells in our skin can remember previous experiences of scratches or infections that happened months, even years ago. Physiologically, this memory can result in a swift and enhanced response to subsequent attacks, enabling our skin to heal faster. While a fast response to tissue injury is beneficial, these memories can be maladaptive, leading to chronic inflammatory disorders and cancers. Patients with chronic inflammatory disorders, such as psoriasis, atopic dermatitis (eczema), and inflammatory bowel disease are at increased risk of cancer, but why this is the case remains largely a mystery. My research seeks to shed light on these clinically relevant observations. More than 200 cases per hour of squamous cell carcinoma (SCC) of the skin are diagnosed in the United States alone, and this has increased by more than 200 percent in the past 30 years. I plan to use excellent mouse models to explore how epithelial stem cells in our skin respond to different types of inflammation, such as those observed in psoriasis and eczema, and how these inflammatory challenges could then impact organismal fitness. Here, I plan to investigate how localized skin inflammation may impact other organs, such as the lungs. This question is important because babies suffering from eczema in the skin later in life develop inflammatory syndromes in the airway (i.e. asthma), a phenomenon called ‘Atopic March’. In addition, patients with eczema are at increased risk of cancer, but again why this happens remains unexplored. My proposed research could have enormous implications for the long-term consequences of inflammation in health and fitness and for our knowledge of how tissue stem cells change in inflammation and cancer.

Our bodies host trillions of microorganisms, collectively known as the microbiome, which play a crucial role in maintaining our health by aiding digestion and supporting our immune system. Recent studies have revealed that not only the types of microbes, but also how they are spatially arranged in our gut, can significantly impact their interactions with human cells. These interactions are linked to conditions such as colorectal cancer, obesity, and inflammatory bowel disease (IBD), a chronic gut disorder affecting nearly 1 in 100 people in the Western world, for which there is currently no cure. Gaining a better understanding of how microbes are arranged in both healthy and diseased guts could provide new insights into their role in human health and open up novel treatment options for currently incurable diseases. In this project, I will explore how the 3D arrangement of gut microorganisms influences their interactions with the cells lining the gut wall, in both healthy and IBD-affected individuals. To investigate this, I will employ advanced 3D printing technology to recreate the human gut environment in the lab, printing gut bacteria alongside the slimy mucus layer they live in, arranged in precise 3D patterns. To model the gut wall, I will grow human \"mini guts\" in a petri dish using a cutting-edge, non-animal experimental approach. By carefully positioning bacteria that play a key role in IBD into different patterns within the mucus layer and combining them with lab-grown \"mini guts,\" I will, for the first time, study how these different 3D arrangements affect gut wall function. Specifically, I will examine whether changes in microbial patterns influence the leakiness of the gut barrier and its ability to trigger immune responses, both critical factors in preventing inflammatory conditions like IBD. This project aims to establish a new, improved method for studying bacterial communities in the lab, enhancing our ability to understand and manipulate these complex systems. This method has the potential to serve as an alternative to using mice in biomedical research, which, despite being widely used, has significant limitations. In the future, this approach could replace many current uses of mice in research, providing a more accurate and humane model. This bold, high-risk, high-reward project sits at the intersection of microbiology, cell biology, bioengineering, and computational biology. With support from world-leading laboratories at Imperial College London, I will have access to the expertise and resources necessary to bring this vision to life. While developing 3D bioprinting techniques to study microbial arrangement is a significant departure from traditional methods, the potential benefits are enormous, potentially uncovering key disease mechanisms and leading to groundbreaking new treatments that could benefit people globally. Beyond understanding gut health, this research will highlight the broader importance of 3D microbial arrangement in other areas of the body, such as the skin and respiratory tract, where microbes play vital roles. The potential for breakthroughs in multiple fields, driven by this research, could ultimately help prevent and treat a wide range of disease conditions influenced by microorganisms.