Immunological memory is a cardinal feature of adaptive immunity and forms the molecular basis of vaccines. Memory T cells provide long-term antigen-specific protection against intracellular pathogens. Circulating memory T cells provide de-centralized immune surveillance, whereas tissue-resident memory T cells (Trm) provide highly potent immune responses on-site that are tailored to the tissues in which they reside. Trm cells are highly abundant in barrier sites and enable rapid immune responses upon antigen re-encounter and are thus an integral part of the first line of defense against pathogens. In the lung, Trm cells are important to fend off recurring respiratory pathogens, but they are hampered by poor longevity. Here, I propose to combine disease-relevant infection model systems with cutting-edge single cell technologies to chart a map of transcriptional and epigenetic networks that govern Trm cell ontogeny and function in the lung. Genetic perturbation and molecular dissection of putative regulators of Trm cell identity in vivo will help to unravel novel therapeutic targets to boost Trm cell formation and/or functionality. Employing superinfection models with heterologous pathogens will help to expand those findings beyond antigen-specific immune responses. Together, the insights gained in this interdisciplinary project will broaden our understanding of adaptive tissue immunity towards respiratory pathogens and advance our portfolio of therapeutic prevention or intervention strategies.

Mass-extinction events in Earth history can provide us with crucial baselines by which ongoing biodiversity loss can be compared and its selectivity calibrated. All mass-extinction events are connected to extreme perturbations of the carbon cycle, including changes in greenhouse gas concentrations and associated global warming/cooling. These major changes in the carbon cycle are thought to be driven by interconnected episodes of widespread volcanism, rapid increase or burning of biomass, burial, erosion or oxidation of carbon, destabilization of methane from the seafloor and permafrost, and marine anoxia. A recently discovered phenomenon associated with mass-extinction events is an increase of teratological microfossils (pollen, spores, organic-walled phyto- and zooplankton) with aberrations in morphology and texture. These malformed fossils allow us to make direct inferences about the proximate mechanisms behind these biotic crises. Unique in the fossil record, these tell-tale signatures co-occur in, and can be recovered from both the marine and terrestrial realms, allowing for the integration of these records and a search for commonality. With this interdisciplinary project we aim to test a set of interrelated hypotheses that link malformation to either metal toxicity (Hg, Cd, Ni, Pb), or increased UV-B radiation due to ozone loss, or to environmental stress related to climate change. Here, we propose to use these microfossils and their modern analogues in experimental and field settings to explore the true potential of teratology as a proxy to test, integrate and refine the many existing models for biotic crises across time and space. The deep time perspective is provided by work packages led by Ghent University and Utrecht University working on Paleozoic and Mesozoic events characterized by the presence of abundant malformed microfossils reflecting both marine and terrestrial ecosystems. Planned work will tease out the relative influence of metal toxicity related to marine anoxia and volcanic activity from geochemical and micropaleontological analyses of core and outcrop material complemented by studies of modern analogs. Work at UC Berkeley and University of Nottingham, focusing on the role of UV-B radiation, metals and temperature, will ground-truth observations from deep time via growth experiments using nearest living relatives.

Bacteria can self-organize into a myriad of morphologies and thrive in diverse ecosystems by developing survival strategies that protect the community from external threats. It has been recently proposed that understanding the collective mechanisms of prokaryotic cells could pave the way to fathoming the strategies developed by eukaryotic communities, yet this connection remains unclear. Both systems grow in heterogeneous habitats, namely organs or tissues. In this project, through experiments and mathematical modeling I aim to unravel the collective behavior and cooperativeness that bacteria and eukaryotic cells have in common in complex environments. To this end, several bacterial species, as Escherichia coli and Pseudomonas aeruginosa, and MDCK eukaryotic cells, will be cultivated in different 2D and 3D media mimicking realistic conditions, exposing them also to different external stimuli and heterogeneities, namely chemical and thermal gradients, shear flows, or mechanical forcing. From the theory side, I will employ and derive continuum nematic models, and active vertex models in the case of eukaryotic systems. The interplay between experiments, theory and simulations will be essential to understand the underlying cooperative mechanisms and the collective response of active matter in competitive and heterogeneous habitats, a research direction that remains almost virgin to date. Natural outcomes stemming from this proposal are a better understanding of cancer pathogenesis and antibiotic resistance, or the production of living and synthetic meta-materials at our whim by controlling the morphogenic response of active systems.

Aedes aegypti mosquitoes are endemic to many regions throughout the world and are vectors of arboviral pathogens that cause Dengue fever, yellow fever, Chikungunya, and Zika. Ae. aegypti are supremely adaptable and can be found breeding in a wide variety of urban and rural aquatic sites associated with human habitation, likely requiring physiological and behavioral adaptability to thrive in these diverse environments. Ae. aegypti decide where to lay eggs based on sensory cues in liquid water to maximize survival of their offspring. The aims of my proposal are to 1) Establish colonies of Ae. aegypti derived from wild strains that exhibit differential preference for egg-laying water mixtures. 2) Perform neuronal imaging to quantify differences in sensory neuron tuning to different egg-laying water mixtures. 3) Use RNA-seq to quantify any changes in expression or sequence of chemoreceptor gene function across tissues involved in egg-laying in strains with divergent preference. This work will build on previous work by my host supervisor and my PhD training in neuroscience and chemical ecology to understand egg-laying preference in Ae. aegypti at multiple biological levels and also to determine why Ae. aegypti (and not other species of mosquitoes) have become so intertwined with human settlement. I will also build genetic tools that will allow researchers to probe many aspects of mosquito biology to support critically important mosquito control efforts around the world.

Saccharomyces cerevisiae (Sce) has seen widespread use by humans throughout history for winemaking, brewing, and bakery. However, a process fundamental for this yeast’s evolution still remains only partially understood: interstrain mating (outbreeding), which potentially results in strains bearing new genomic settings and fitness. While outbreeding is easily achievable in laboratory settings, it is extremely rare in nature. In fact, we have only recently discovered the first environment where it can occur: within wasp guts. Comprehending what makes the insect gut an environment suitable for Sce mating would provide us with a better understanding of Sce evolution and expand our knowledge beyond the unnatural lab settings. Outbreeding is achieved through a multi-step process encompassing sporulation, germination, ascus break, and mates encounter. These steps may be promoted within wasp guts thanks to the sequence of drastically different chemical and mechanical stresses peculiar to this environment. We have set up a team of experts in all the fields necessary to tackle this hypothesis: microbiology, genetics, chemistry, physics, and computational biology. We will carry out in vivo experiments to assess the wasp gut environment by using dedicated sensors and Sce genes fundamental for germination and sporulation in this environment. These data will be instrumental to develop a genome-scale mathematical model exploring yeast genetics, metabolic and environmental features favoring germination and sporulation. In vitro high-throughput assays assessing both the yeast response and metabolites measured by dedicated intra- and extra-cellular sensors will provide further data to calibrate the model. Physical forces required for Sce ascus break or mate encounter will be measured through up-to-date biophysical techniques: cylindrical Couette chambers, micropipette force sensors, and microfluidic droplets. We will bring together the information that we gather about each stage to develop microfluidic devices emulating the structure and physiology of wasp guts and investigate there the entire process leading to outbreeding. This project will unveil the key factors of Sce evolution by providing fundamental insights on the biological mechanisms leading to outbreeding in natural settings, thus potentially revolutionizing our current understanding of the process.

Expansion of short nucleotide repeats is the cause of over 30 inherited neurological diseases. These expansions could result in neurotoxicity through a loss of protein function or accumulation of misfolded repeat-containing proteins. Repeating-containing RNA, which accumulates in aberrant nuclear foci, can also be a driver of pathogenesis by sequestering RNA processing proteins and producing toxic peptides through non-canonical translation. Recently, RNA containing these disease-associated repeats (DARs) has been demonstrated to intrinsically undergo phase transition to form an aggregated state through multivalent base-pairing, providing a model for the mechanism of RNA foci formation in repeat expansion diseases. The genome of the social amoeba Dictyostelium discoideum contains an exceptionally high number of short nucleotide repeats, many of which occur in coding regions and resemble human DARs. My work will develop Dictyostelium as a model for studying evolutionarily derived mechanisms which keep DAR-containing proteins and RNA from becoming pathogenic. Specifically, this proposal presents experimental strategies for identifying factors which prevent the aggregation of DAR-containing proteins and RNA, and non-canonical translation of DAR-containing RNA. The efficacy of these factors in suppressing DAR toxicity will be investigated in mammalian cells and animal disease models. Besides their relevance to disease pathogenesis and therapeutic potential, insights derived from the proposed study will also shed light on basic principles regulating RNA and protein homeostasis.

Chemotherapy-induced senescence (TIS) is an alternative form of senescence, which is triggered by inhibiting the cell-cycle (senescence after growth arrest or SAGA) with CDK4/6 inhibitors, such as palbociclib. MacroH2As (a non-canonical histone variant of H2A) are enriched in during senescence and is believed to maintain heterochromatin and repression of cell cycle genes. The nucleosome-nucleosome interactions of macroH2A, which influence compaction and transcription, remains largely unknown. This is mainly due to the limitation of current techniques and the challenges which heterochromatin pose. However, the structural mechanisms of macroH2A and its subtypes (1.1, 1.2, and 2) play to maintain stable gene repression during senescence is largely unknown. Using a TIS model liposarcoma cell line and by developing chromatin targeted probes to induce spatially correlated breaks to infer structural information on the nanoscale (tri-nucleosome) level, I will investigate the structural-function relationship in heterochromatin and macroH2A in SAGA induced liposarcoma cells. The findings of this work will provide a better insight into this novel mode of senescence (TIS) by understanding how chromatin reorganization and cell growth genes are repressed and maintained by macroH2A.

Shigella is an important human pathogen responsible for 164,000 deaths per year, and cases of antimicrobial resistance are rising. In the lab, Shigella has emerged as a paradigm of cellular microbiology whose investigation has enabled landmark discoveries in infection and cell biology. Septins, a poorly understood component of the cytoskeleton recognised for their roles in cell division, can entrap cytosolic Shigella in cage-like structures for host defence. For cage entrapment, new work has shown that septins recognise micron-scale curvature presented by Shigella. Despite recent insights, the physical and molecular determinants of septin recruitment and cage assembly required for Shigella entrapment are mostly unknown. My proposal will investigate biophysical cues that underlie Shigella-septin interactions, and illuminate their role in cell-autonomous immunity. Using state-of-the-art fluorescence microscopy techniques, I will first investigate the spatial dynamics of Shigella virulence proteins and cell biophysics in broth culture and during septin cage entrapment in vitro using purified proteins. Next, I will resolve the impact of biophysical cues presented by Shigella which trigger cell-autonomous immunity during infection of human epithelial cells. Together, these integrative experiments will provide valuable insights about biophysical cues sensed by cell-autonomous immunity to defend against intracellular bacterial pathogens, bridging our views of biophysics and host defence. The outcomes of my research plan are expected to discover novel concepts in infection biology and cellular homeostasis, and may inspire septin-based approaches to counteract bacterial infection.

Cellular identity is defined by the expression of specific gene sets. Once established, the information to maintain gene expression programs is stored in alternative chromatin states, which are thought to be propagated through cell divisions to maintain cellular identity. However, although cellular memory is widely accepted as an important aspect underlying development, empirically it is yet to be demonstrated to exist at the single-cell level. Indeed, one possibility is that functional chromatin states are epigenetically propagated through mitosis, and a cellular memory operates at the single-cell level. Alternatively, cell fates could be inherently probabilistic, depending on the distribution of regulatory factors between nascent cells. In this project I propose to investigate this topic by combining advanced imaging, single-cell multi-omics and prospective lineage tracing. To study molecular states in cell pedigrees I will take separate approaches, using mouse embryonic stem cells as model. First, to test how much of the transcriptional identity of a parent is maintained in progenies, I will analyse transcriptional states of individual cells in traced lineages. Second, to understand if parental chromatin structures that underlie locus activity can be reproduced following mitosis, I will study similarities in locus conformation in related cells. Finally, I propose to investigate how transmission of molecular states changes upon lineage commitment, and in the absence of the epigenetic regulator, Polycomb. Altogether, this comprehensive study will uncover how developmentally important functional chromatin states are maintained through mitosis at the single-cell level.

Shoot branching in plants integrates a wide range of developmental and environmental cues. During shoot branching, axillary buds transition from dormancy to growth, regulated by signaling factors such as hormones and nutrients, which are produced, transported, and consumed in different ways across the plant. In many species, the highly conserved transcription factor BRANCHED1 (BRC1) maintains axillary buds in a dormant state. The hormone strigolactone (SL) increases BRC1 activity and promotes bud dormancy, and sucrose decreases BRC1 activity and releases bud dormancy, but their relative contributions remain unclear. To clarify mutual dependence of transport and gene regulation during shoot branching, I will determine: (1) how BRC1 activity and axillary bud dormancy depend on transport of SL, sucrose, and other signaling factors, and (2) whether transport of signaling factors into/out of axillary buds depends on plasmodesmata (PD) constituents and aquaporins regulated by BRC1. I will develop a new microfabricated device to enable dynamic buffer control, while live confocal imaging axillary bud activity and growth. I will image fluorescent reporters in existing and new Arabidopsis lines, and I will develop mathematical models integrating transport and gene regulation, to describe and predict dormancy-to-growth transitions in isolated and interacting axillary buds. Few models of plant development have integrated transport mechanics and gene regulation in this way, so this work will have more general implications for plant development.

Birds are extremely agile and robust flyers, able to cope with challenging gusty wind conditions and perform remarkable aerial manoeuvres. To do so, birds take advantage of the flexibility of their feathers, which vibrate as a function of airflow, stimulating the mechanosensory neurons in the skin, to provide information about the airflow over the wings. We aim to explore this complex sensorimotor loop, first to identify what aerodynamic information is available to birds to help control their flight and understand how birds’ wings can act both as aerodynamic surfaces and as a distributed airflow sensing array and then to understand how this aerodynamic information is encoded in the nervous system. Although airflow sensing is thought to be critical for efficient and agile flight, almost nothing is known about how changes in airflow translate to feather movement and subsequent neural signaling. Thus, this work addresses a broad biological question about sensory encoding of aerodynamic information. This proposal represents an integrated, international collaboration to explore this question across multiple levels of organization, from the movements of feathers to neural activity during perturbed flight manoeuvres. Dr Windsor (University of Bristol, UK) is an expert in using computational image analysis to study animal dynamics and his team will use techniques from aerospace engineering to look at the aerodynamic and vibrational properties of feathered wings, including measurements of the wing motions of behaving birds. Dr Perkel (University of Washington, Seattle, USA), an expert in songbird neuroscience, will investigate the physiological mechanisms underlying the encoding of this mechanosensory information by the nervous system. His lab will record electrical signals from single neurons in the wing nerve and spinal cord of zebra finches and trace their neural pathways. Dr Woolley (McGill University, Montreal, Canada) is an expert in behaviour and neurophysiology. Her lab will map the regions of the brain responding to airflow stimuli based on insights gained from Dr Windsor’s and Dr Perkel’s groups and then measure neural responses in flying birds. By combining approaches from aerospace engineering and neuroscience we aim to understand the functional properties of wing mechanosensing that allow birds to “feel” their way through the air.

Gene regulation is fundamental to almost all biological processes and is the critical driving force for the generation of the diversity of cell types that make up a multicellular organism. Yet, we still do not fully understand how gene expression is regulated and modulated with changing cellular contexts. Gene transcription occurs in a discontinuous manner, where bursts of RNA production are interspersed by periods of gene inactivity. As the major regulators of the initiation of transcription, enhancers have been reported to modulate transcriptional bursting. However, how this is achieved remains largely unexplored. Here, I will investigate how enhancers influence transcriptional bursting by finely tuning the nuclear concentrations of specific enhancer-binding transcription factors (TFs) in a very precise and dynamic manner. To manipulate TF levels, I will exploit an optogenetic system recently developed by the Furlong lab, which will allow me to address this question at an unprecedented quantitative and temporal resolution for the first time in a living embryo. The functional impact of manipulating TF concentration on bursting will be quantified at two levels using state-of-the-art approaches. First, globally, using a spatial single-cell and single-molecule genomics method (intronic seqFISH+) to identify general regulatory principles. Second, dynamically, using live imaging and the MS2 and PP7 systems to dissect how burst frequency, duration and intensity are mediated by enhancers. Together, these findings will advance our understanding of gene regulation by providing new insights into the kinetics, regulation and general robustness of transcriptional bursting.

Protein post-translational modification (PTM) is a sophisticated form of cellular information processing, essential for the emergence of organismal complexity, such as the evolutionary expansion of the cerebral cortex. Nonetheless, it is unclear how PTMs shape brain development and understanding the interconnections between these two highly complex mechanisms may open a ground-breaking avenue for tackling neurological disorders. The host lab demonstrated that protein AMPylation and the ER-specific AMPylator FICD are enriched in the neuronal niche and that FICD-mediated protein AMPylation is critical to neuronal differentiation. However, a set of the identified AMPylated proteins in neurons is enriched in proteins with mitochondrial localization, hinting that the mitochondrial-specific AMPylator SelO has an important role in neuronal mediated AMPylation and plays a critical role in neuronal metabolism and metabolic disorders. I plan to elucidate the role of the uncharacterized mitochondrial SelO using state of the art proteomic approaches in human cerebral organoids and decipher the SelO-Dependent AMPylome. I will dissect the role of SelO-dependent AMPylation in neuronal development, using a novel live imaging technique to track AMPylated proteins in mouse cortical development and human cerebral organoids. In addition, I will investigate the role of SelO in mitochondrial and cellular metabolism, using the candidates identified in the SelO-dependent AMPylome. Overall, I aim to elucidate the role of protein AMPylation in the metabolic regulation of neurogenesis, and its importance in Mitochondrial Disease and Autism Spectrum Disorders.

Living matter represents spatially structured soft materials with internal energies far from the thermal equilibrium. The inherent order and fluidity in such systems lead to dynamic, anisotropic properties spanning orders of scales. Spontaneous symmetry breaking in structured matter nucleates local singularities called topological defects. Despite reported evidence of topological defects in living systems, their biological functions have remained grossly unexplored, denying us, to date, a holistic biophysical picture. With a focus on microbial species, I propose to develop the first experimental framework to understand topology-induced microbial physiology, based on a combination of cutting-edge methods including advanced imaging, spectroscopy and machine learning. Specifically, by tracking defect genesis and dynamics in bacterial colonies of species with diverse phenotypes (morphology and motility traits), I will quantify biological activity in the defect vicinity over short to generational timescales. Using fluorescence confocal and electron microscopy (to track molecular transport, exudates and ROS levels), and nanoSIMS technique (to quantify nutrient uptake), I will obtain seminal data linking defects to microbial physiology, and harness this mapping relation to construct machine learning algorithms that will ultimately predict the biological role of defects in multi-species communities. By discerning the biological functions of topological defects, I will be in robust position to pioneer a quantitative approach to this uncharted problem, heralding a unifying framework to predict more complex scenarios, thereby advancing our general understanding of physics of life.

The small intestine is the major site of nutrient absorption after food intake and malabsorption (impaired nutrient uptake) affects millions of people worldwide. However, there is an unmet need to study malabsorption with physiologically relevant in vitro models. Glucose-galactose malabsorption (GGM) is a genetic disorder that affects the SGLT1 protein, which results in diarrhea and life-threatening dehydration upon glucose and galactose consumption. In my research I aim to establish a physiologically relevant platform to assess nutrient bioavailability employing intestinal organoids (biovanoids) to model GGM. GGM biovanoids will be generated by introducing a disease-associated mutation in SGLT1 (C292Y) using CRISPR/Cas9-mediated base editing. Some aged GGM patients describe an acquired glucose tolerance, which accelerated upon probiotic consumption of certain bacteria. The influence of SGLT1 mutations on host-microbe interactions and possible metabolic adaptation through e.g. age-associated hormonal changes will be studied using GGM biovanoids. This may offer an opportunity to introduce glucose tolerance in young GGM patients. Overall, biovanoids offer a promising platform to generate essential knowledge about malabsorption. To elaborate further applications, biovanoids will be validated for their potential to predict intestinal absorption and gut wall metabolism of drugs. Poor bioavailability contributes to high attrition rates of oral drugs during clinical phases of drug development and currently applied models fail to reliably predict bioavailability in humans. Biovanoids could improve these predictions to eventually increase successful drug approval to the market.

A high level of oxygen in the atmosphere is taken for granted as it is indispensable for human and most animal life. Yet, life (bacteria and unicellular eukaryotes) evolved for over 2 billion years in a low oxygen environment. Oxygen became widely available only in the last 600 million years and was likely the main factor behind the rapid diversification of most complex multicellular life forms. For elaborate multicellular tissues to form, their overall shapes however must still satisfy oxygen demands of individual cells. Oxygen consumption will rapidly reduce the oxygen level within a few cell depth into the tissue. This becomes a problem in early embryonic development before respiratory and circulatory systems are established. Many cells still need to differentiate and migrate for long distances and place themselves at the right destination. It is largely unknown what makes the varying oxygen environment and the execution of their precise differentiation and navigation within a tissue compatible. The project aims at elucidating the common basis of oxygen dependency in morphogenesis by studying how the social amoeba Dictyostelium detects and reacts to oxygen at the molecular level and how this dictates cell migration and morphogenesis. Dictyostelium is an organism evolutionarily close to the branches of multicellular evolution, and presents enormous advantages to be easily cultured in the laboratory and reversibly switched from unicellular to multicellular states in less than a day. Amoeboid cells are able to change their state (by differentiation) and place themselves in more favorable conditions for foraging and respiration (by migration). An emphasis is placed on clarifying this most fundamental and primordial characteristic with a focus on the cross-scale quantitative relationship between oxygen, metabolic states, cytoskeletal machineries and migratory modes. New screening methods based on aerotaxis responses, oxygen sensors and 3D microscopy will be combined with molecular genetics of oxygen response pathways. The role of the new mechanism unearthed will further be tested for their roles in neural crest cell migration in zebrafish. They will be formulated into multi-scale mathematical models to clarify how the coupling between oxygen consumption, sensing and migration of individual cells gives rise to robust and collective morphogenetic movements.

The skin epithelium forms a primary barrier against inflammation-inducing environmental agents and is maintained by the epithelial stem and progenitor cells (EpSCs). Stem and progenitor-cells (SCs) adapt to various conditions via crosstalk with their local microenvironment or "niche". The EpSC niche is comprised of fibroblast, neuronal, immune, and other cells that provide context-specific signals to direct SC function. However, how the EpSC niche is affected by inflammation is poorly understood. In addition to actively participating in inflammation, EpSCs also retain a memory of inflammation long thereafter, which enables more rapid responses to subsequent assaults. However, the contribution of niche factors to maintaining EpSC memory is entirely unknown. To address this knowledge gap, I propose to systematically chart the cellular constituents of the EpSC niche before, during, and after acute inflammation. Armed with this high-resolution map of the EpSC niche, I will then perform functional studies to identify niche factors that control EpSC inflammatory function and memory. Understanding inflammation-induced niche rewiring and identifying the key inflammatory niche regulators of EpSCs may illuminate mechanisms of inflammatory epithelial pathologies such as psoriasis, chronic non-healing wounds, and cancers that are rooted in SC dysfunction.

The world around us is constantly changing – new elements appear all the time. How does the brain decide what novel things to explore? Animals exist in a constant push and pull between being adventurous and conservative. My objective is to understand how the brain controls the behavioral response to novel stimuli under naturalistic settings, and how those brain functions have been modified by evolution and ecology. During my fellowship I will approach the neurobiology of novelty from an interdisciplinary perspective that merges the evolution of behavior with its neurophysiology. I chose an ideal model system to study comparative neurophysiology: the deer mouse (genus Peromyscus). Deer mice are the most abundant mammal in North America (comprised of >50 species). Different Peromyscus species have evolved distinct behavioral repertoires. I will combine evolution and ecology with novel tools such as fiber photometry and wireless electrophysiological recording to understand the computations performed in the deer mouse brain under ethological conditions in response to novel stimuli. Specifically, I will study the brain of a variety of Peromyscus species with distinct behaviors under different novelty challenges. Overall, I will focus my research program on testing three general hypotheses: (i) different species or populations of Peromyscus will present differential response to novel objects, and (ii) dopaminergic neurons projecting to the tail of the striatum, controlling novelty responses behave differently in these species (iii) dopaminergic neurons projecting to the tail of the striatum shape neophobic behaviors in the wild.

Gene regulatory networks (GRNs) represent the wiring diagrams of cells and can be envisioned as the blueprint for organism development. Transient interactions of transcription factors (TFs) and DNA as well as chromatin structure fine tune gene expression and allow cells with the same genome to give rise to a myriad of cell types and tissues. My overall-goal is to elucidate this fundamental process live and investigate, how exactly transient interactions, dynamics, and variations in absolute concentrations of TFs on the molecular level shape GRNs, impact on vertebrate embryo development, and can be used to infer cellular identity. Our current understanding of gene regulation arises mostly from static measurements employing sequencing technologies and in situ hybridisation approaches. However, extracting quantitative clues such as TF concentrations and their changes over time is very challenging yet crucial to shed light on the organising principles. I propose to use quantitative imaging, fluorescence fluctuation spectroscopy (FFS), and endogenous labelling of TFs to measure absolute, native concentrations and interaction dynamics directly during live zebrafish development. Using these readouts as a new handle on cellular heterogeneity, I will elucidate how specific TFs regulate hindbrain establishment and by employing engineered TFs as reporters for DNA accessibility, how overall chromatin organisation evolves in different developmental contexts. Together with the design of an open-source microscopy-spectroscopy platform tailored for in vivo FFS, this project will result in previously unattainable, quantitative insights into vertebrate embryogenesis.

Symmetry breaking is a process during which a homogeneous system adopts asymmetry and is fundamental during initial cell fate determination in early development and regeneration. Single-cell interactions and information exchange with the environment allows for such large-scale coordinated behaviors. Identifying mechanisms driving symmetry breaking is not trivial due to frequent nonlinear relationships between the system’s lower- and higher-order dynamics. Employing approaches from cell-, systems- and biophysical fields, I will study symmetry breaking in intestinal organoids. In a uniform growth-promoting environment, only a subset of intestinal stem cells differentiates into secretory cells. Recent evidence implicates an upstream regulatory pathway of extracellular signal-regulated kinase (ERK) in contributing to proper patterning of the stem cell niche. The proposed research aims at understanding whether and how ERK mediates symmetry breaking during intestinal organoid formation. To address this fundamental question, I will: 1) analyze the spatiotemporal-specific role of ERK signaling during symmetry breaking, 2) identify ERK-mediated tissue-level coordination focusing on biochemical and mechanical regulation, 3) determine how single-cell interactions are integrated into large-scale collective behaviors using theoretical modeling and optogenetics. This research project will determine the spatiotemporal-specific role of ERK during symmetry breaking with unprecedented multiscale resolution and provide key insights into the emergence of complexity and collective behavior fundamental to processes such as mammalian development, cancer invasion and wound healing.