Neural oscillations, the byproduct of periodic fluctuations in synchronized brain activity, are a defining feature of neural circuits. While oscillatory activity is closely correlated with both an animal’s behavioral and cognitive state, it is unclear whether oscillations are causally involved in behavior and cognition. In this research proposal, we will focus on thalamo-cortical spindle oscillations, examining their possible causal role in memory consolidation. Our research proposal consists of two main research aims: 1) quantifying the causal relationship between thalamo-cortical spindles and memory-related oscillatory dynamics known as hippocampal sharp-wave ripples and 2) behaviorally measuring the causal relationship between spindle generation and memory consolidation. This will be accomplished by a synergistic merger of molecular-biology (Halassa lab) and a computational toolbox (Bendor lab), while performing large-scale neural recordings (Bendor and Halassa lab). Our combined, state of the art toolbox provides a more temporally and spatially precise method of recording, stimulating and blocking spindle activity, while directly addressing causal interactions using sophisticated Bayesian-based computational methods. Using these methods we will directly test two hypotheses, that thalamo-cortical spindles and hippocampal ripples have causal, bi-directionally interactions (aim 1), and that the amount of thalamo-cortical spindle activity is related to the strength of the encoded memory, with this effect being both modality-specific and sensitive to the timing of hippocampal ripples (aim 2).

Inorganic polyphosphate (polyP) is a biopolymer that serves multiple critical functions in biology. PolyP has been mainly studied in bacteria and shown to be involved in such diverse processes as the bacterial stress response, biofilm formation, plasmid uptake and antibiotic resistance. In comparison, its functions in mammals are significantly less well understood. Even so, polyP has been linked to multiple important physiological phenomena, such as blood clotting, chaperone function, posttranslational protein modification and neuronal signaling.
While in bacteria the enzymes that generate polyP are known (polyphosphate kinases Ppks), the mammalian enzymes remain elusive to date. In fact, we know that polyP is present in different cell types and that it is enriched in different organelles, but we do not know how and where it is made and how it is shuttled. Moving polyP research forward will critically require identification of the elusive enzymes that make and traffic polyP. Currently, a dearth of chemical tools has precluded such deeper insight into mammalian polyP physiology.
Our proposed research will culminate in new technologies required to make, modify, analyze, transport and deliver polyP both ex and in vivo. We will develop and combine multiple novel synthetic approaches to generate a range of modified polyP analogs that are not yet available. Furthermore, we will develop technologies to deliver and release those synthetic analogs in a controlled way outside and inside of cells. Together, these technologies will provide valuable novel insight into how cells make, regulate and distribute polyP. We will use these tools to identify the mammalian enzymes involved in polyP turnover, investigate how polyP levels are affected by other signaling molecules such as inositol pyrophosphates, and try to understand how polyP affects different biological processes. For example, an in-depth evaluation of the role of polyP in blood clotting will be achieved using our novel approaches. These studies will lay the foundation for further research into polyP physiology. By virtue of the combination of synthetic organic chemistry, drug delivery technology, and cell and animal biology, we will be able to develop and apply powerful novel tools dedicated to the elucidation of polyP physiology in mammals.

Genome architecture is emerging as a key epigenetic property providing either positional or topological cues that regulate gene expression. By following certain folding principles and controlling gene positioning within the nucleus or chromosomal interactions, the three-dimensional organization of chromatin seems to exert a pivotal role in coordinating the emergence of unique cellular phenotypes. During early mouse embryo development, the nuclear volume decreases ten times, while encompassing the same amount of genetic material. At the same time, transcriptional networks undergo drastic changes, switching from maternally-controlled to zygote-specific programs. While these expression patterns must unfold in a robust manner from one stage of development to the next, they also need to incorporate some degree of flexibility and cell-to-cell variability, eventually making every cell unique throughout differentiation. Deciphering how the interplay between genome organization and expression operates during this highly dynamic period in a multicellular organism’s lifespan is a formidable challenge. Here, I propose to establish a pioneering framework for assessing various aspects of genome organization while monitoring gene expression profiles in exactly the same single blastomeres, tracing their astounding dynamics while the embryo is developing into a blastocyst. By combining multi-assay measurements in the same single blastomeres with high-dimensional correlative data analyses, I aim at identifying general design principles governing DNA folding, as well as the crosstalk between genome architecture and gene expression.

Tuberculosis is an ancient disease caused by Mycobacterium tuberculosis that still exerts a massive burden on public health in many parts of the world. The success of M. tuberculosis is primarily due to its ability to survive within macrophages for months and even years in an asymptomatic latent state. It is still largely unknown what makes this pathogenic invader successful, or what determines if the immune cells eliminate the assault or succumb to it. Thus far studies of host-pathogen encounters were performed at the population level, averaging molecular signatures, while masking any cell-to-cell variation. The heterogeneous, stochastic, and dynamic nature of both host and pathogen suggest that descriptions of average behavior may fail to accurately characterize these interactions. Here, I propose to take a systematic quantitative approach to study the process of infection by M. tuberculosis, establishing a fluorescent method to specifically differentiate the outcomes of infection, and combining it with single-cell RNA-seq. Using this method I will examine whether variation in transcriptional profile of individual M. tuberculosis-macrophage encounters translate to distinct infection outcomes. I will apply this method to determine the degree to which genetic diversity among clinical strains is reflected in cell-to-cell variability of the transcriptional profile and translates to distinct of infection outcomes among these strains. Collectively my studies will expose mechanisms by which transcriptional heterogeneity affects the outcome of infection, and provide a holistic view on the process of M. tuberculosis infection.

Birds are paedomorphic dinosaurs: they exhibit in their adult skeleton some traits that were present in the juveniles of their dinosaur ancestors. The mechanisms underlying this paedomorphosis are largely unknown. We hypothesize that it is caused by differences in the rates of skeletal growth and differentiation, which are known to be higher in birds than in dinosaurs. The aim of this research proposal is to investigate the mechanisms that control how fast skeletal cells differentiate, and how their rates of differentiation influence the morphology of the skeleton. Most of the vertebrate skeleton develops by replacement of cartilage extracellular matrix by bone, a process called endochondral ossification. We propose to undertake this investigation by comparing endochondral ossification in embryos of two species of birds with different rates of development – quails and zebra finches – to embryos of alligators, which will be used as out-group. We will use two novel experimental approaches: (1) the adaptation of recently developed whole-mount techniques and cutting edge imaging technologies (micro-computed tomography and confocal light microscopy) to the study of skeletal development, and (2) the compared analysis of in vitro culture of cartilage cells obtained from the three species. Those experiments will allow us to characterize, in each species, the temporal and spatial expression of molecular regulators of endochondral ossification, its relation to changes in the shape and growth of the skeleton, the relative duration of cell cycle progression, and the rates of chondrocyte differentiation.

Upon antigen recognition, T cells switch from a fast migratory to a stationary state. T-cell receptor (TCR) recognises specific peptide bound to major histocompatibility complex (pMHC) molecules on the surface of antigen-presenting cells (APCs). TCR interaction with pMHC of high affinity leads to a migration stop and robust TCR signalling. However, how a molecular recognition event can coordinate global cellular changes remains unclear. Here, I propose that membrane tension is the cellular coordinator that translates antigen recognition into a stop signal and ultimately enables T cell activation. I hypothesize that TCR engagement in motile cells creates mechanical stress that increases membrane tension, leading to extracellular calcium entry through mechanosensitive ion channels, which results in arrest of locomotion, cytoskeletal rearrangement and TCR signalling. Using a FRET-based force sensor and micro-fabricated migration channels, I will quantify the mechanical forces that act on the TCR when motile T cells encounter immobilised pMHC molecules. I will then measure membrane tension with optical tweezers and membrane topography with 3D super-resolution microscopy as a function of pMHC affinity. Next, I will investigate the role of mechanosensitive ion channels and image cytoskeletal remodelling and trafficking of signalling vesicles in T cell during the transition phase. Taken together, the proposed project will provide a better understanding of how mechanical forces derived from cell motility mediate antigen-induced T cell arrest and activation.

Although many of the molecules that determine animal body plans have been uncovered in recent years, we still cannot predict the final design of an animal from a diagram of how these molecules interact. This limitation stems in great part from the fact that current technologies to visualize development rely on dead embryos or employ reporter systems that cannot capture the dynamics that underlie developmental processes. Measurements of these dynamics constitute just one step toward predictability; in order to sharpen our understanding, theoretical models that generate quantitative predictions must accompany such measurements. Our interdisciplinary team, which consists of researchers with extensive expertise in developmental biology (Andrew Oates, Francis Crick Institute), the theory and measurement of gene expression (Hernan Garcia, UC Berkeley), and protein engineering (Roberto Chica, University of Ottawa), is uniquely positioned to establish a predictive understanding of the gene regulatory networks that govern vertebrate body plans. We will focus on the segmentation clock in zebrafish, in which the length of body segments is thought to be determined by a network of oscillating genes. We will develop new technology to simultaneously monitor mRNA and protein concentrations in real time during segmentation of the zebrafish embryo. Furthermore, we will develop theoretical models that will leverage this new information to predict how molecular interactions lead to oscillations with a prescribed period and amplitude. These predictions will be tested via the creation of synthetic oscillators with engineered dynamical properties that will be used to generate embryos with altered body plans. We anticipate that these iterations of model and experiment will set the stage for a new paradigm in synthetic biology that enables the rational design of multicellular organisms.

Adipose tissue is now appreciated to be an endocrine organ, owing to the discovery of hormones such as leptin, which functions as an negative feedback neuroendocrine signal that keep weight in a narrow window of variation. The sympathetic nervous system (SNS) innervates all known organs, but the neuroanatomical origin of adipose innervation and its functional role in regulating adipose tissue phenotype and systemic metabolism remain largely unstudied.
Brain mapping owes its existence to ease of brain dissection and serial slicing. In contrast to this methodological ease, the anatomical location of the SNS along the anterior side of the verterbrae poses a difficult challenge for conventional histology, preventing a systematic study of these neurons. We here propose to perform whole body imaging using optoacoustic tomography, coupled with transgenic viral tracing for mapping subsets of SNS neurons projecting to brown (BAT), beige and white adipose tissue (WAT). These neurons will be functionally probed using chemogenic techniques.
We assembled a multidisciplinary research team that will work together closely on each phase of the project: Paul Cohen is a molecular biologist with expertise in white, beige and brown adipose biology. Ana Domingos is a neurobiologist with expertise in optogenetics who recently demonstrated the existence of neuro-adipose junctions. Daniel Razansky is a bio-engineer who has developed novel non-invasive methods for high performance molecular imaging, particularly opto-acoustic technologies.

Biological systems have evolved protein self-assemblies to generate efficient high-order nanoscale architectures and molecular machines. Translating biological organizational principles directly into synthetic protein cage-like nanostructures capable of sensing and acting on the environment in a programmable way, would constitutes a unprecedented scientific achievement and a formidable opportunity to interface and interrogate biological systems. However, it remains extremely challenging to unveil fundamental processes guiding three dimensional self-assembly of protein building blocks. Despite intense scrutiny, bottom-up methodologies enabling the de novo design and construction of tailored protein nanocages with programmable structure, dynamics and functionalities, are inexistent. The intention of this project is to take advantage of the recent progress of computational protein design to examine how complex and precise functional protein nanocage architectures can be rationally built. Specifically, I hypothesize that novel computational frameworks can be developed to engineer self-assembling nanorobots with important design space, capable of executing programmed tasks, such as triggered bioactuation, packaging and delivery of information to target structures. To this aim, we will expand protein-protein docking and interface modeling in order to establish libraries of modular and robust multifunctional components with specified architectures size and metastability, which will be experimentally validated in vitro. Establishing protein nanorobot programming as a universal framework would bring tremendous possibilities for basic research as well as biomedical applications.

Ethanol, a toxic substance consumed in many cultures, affects microbiota composition and chronic excessive consumption can lead to severe end-organ damage. Curiously, the role of microbiota in ethanol metabolism remains under-studied. Moreover, the selective pressure of ethanol on the microbiota, as well as its impact on the gut immune defences is under-investigated. I hypothesise that specific microbial clades are critical players in ethanol metabolism and that ethanol-driven microbial alterations are caused by ethanol resistance and by changes in the oxygen levels in intestinal lumen. Furthermore, these shifts and specifically microbially-generated ethanol metabolites impact gut and hepatic immune defences. Using isotopically labelled ethanol, I will determine the role of gut microbes in ethanol metabolism by ethanol metabolic flux analyses and identify the microbes that use ethanol as a carbon source. Next, I will investigate if anoxic-to-oxic shifts in the gut lumen and ethanol resistance drive the observed microbial shifts by employing cultivation devices with oxygen or ethanol gradients. I will also assess if chronic ethanol consumption results in intestinal angiogenesis. Finally, I will evaluate the impact of chronic ethanol consumption on gut immune responses using flow cytometry and confocal microscopy with a focus on innate immune cell population and function. Use of gnotobiotic mice with defined microbial communities informed by my microbiome-profiling experiments will further enlighten my immunologic analyses. This multidisciplinary approach will provide mechanistic insight for future studies aimed at reducing ethanol toxicity.

Perceptual decision formation in human and non-human primates involves accumulation of noisy sensory evidence through a process known as evidence accumulation. While evidence in favor or against a choice accumulate, the decision systems provide a continuous flow of information to the motor system and prepare it for the probable outcome. During decision formation, neurons show evolving activity patterns that are thought to represent the progression of the decision toward a choice. The circuit mechanisms that produce these decision-related dynamics, however, remain unidentified. This proposal aims at identifying the circuit motifs behind such computations. I have previously demonstrated that during olfactory decisions flies engage in an evidence-accumulation process before committing to a choice. Reaction times in a trained odor discrimination task increase, and perceptual accuracy declines, as stimulus contrast is reduced, in quantitative agreement with a drift-diffusion model of evidence accumulation to threshold. I have also demonstrated that the mushroom bodies of Drosophila play an important role in decision formation. My proposal builds on these findings and aims to understand how neurons in the decision centers interact during odor discrimination tasks. I will use a single-fly-based assay and a virtual-reality arena to understand how behavioral outputs are generated in the fly brain during decision-making. Such insights will provide neurophysiological underpinnings for the theoretical models of decision-making.

The structure of large protein complexes is difficult to obtain, due to their large molecular weight and the strenuous task of finding crystallization conditions. Another challenge is the presence of heterogeneity in the conformation of the complex, which complicates studies by solid-state NMR and makes the particle orientation more difficult to determine in single-particle cryo-electron microscopy.
The technique of cryo-electron tomography (cryoET) benefits from additional geometrical information from the tilting of the specimen and can image one-of-a-kind 3D objects. The proposed project aims to produce structures at near-atomic resolution for different sub-states of desensitized AMPA-type glutamate receptor ion channels, as this sample exhibit a strong conformational heterogeneity.
The first project objective is to improve the resolution of cryoET for small systems which do not present conformational heterogeneity. We will use direct electron detectors to correct for sample movement, and new sample schemes to reduce radiation damage.
The second project objective is to develop data processing algorithms for the treatment of sample heterogeneity. We will use two strategies: the simultaneous determination of the particle class and particle orientation, and the treatment of rigid protein domains as independent sub-particles.
The third project objective is to investigate the relationship between the structural heterogeneity of a protein complex in non-crystalline amorphous ice and its dynamics and heterogeneity at ambient temperature in solution. We will compare cryoET reconstructions to measurements of the protein dynamics by solid-state NMR at ambient temperature.

Many microscopic building blocks of living organisms have well-defined handedness and this structural information can propagate across many length scales to establish macroscopic processes and structures that are inherently chiral. However, understanding how microscopic chirality leads to macroscopically chiral structures, such as the left-right symmetry breaking in embryo development, is inherently difficult in living organism due to their inherent complexity. We propose to study the emergence of chiral structures in a simplified reconstituted system of purified microtubule filaments with Kinesin-2 molecular motors, depletant agent and adenosine triphosphate (ATP). Our system will allow us to have an accurate control over the handedness of the molecular constituents and interactions at play. First, we will focus on the ability of this active gel to self-assemble and to produce spontaneous chiral flows under confinement, as seen in cytoplasmic streaming events. A second part will focus on the emergence of chiral flows resulting from asymmetric collective beating in reconstituted micro patterned cilia-like active microtubule bundles. Finally, we will assemble doublets and arrays of artificial active cilia to study how molecular chirality affects their beating synchronization. Taken together, the in vitro reconstitution of these cellular structures and behaviors will bring quantitatively understanding and control of the chiral molecular mechanisms at play both in reconstituted microtubule and kinesin networks and cilia-like beating of synthetic active microtubule bundles. This project will broaden our understanding of the emergence of asymmetry in developing organisms.

Mitochondrial dysfunction is a hallmark of several human diseases and somatic cell decline during aging. Germ cells must provide pristine, functional mitochondria to the embryo, although human oocytes can be stored decades before they are fertilized. C. elegans adults can survive for extended periods of time, during which they consume the gonad except for a few germ cells. Remarkably, these aged germ cells can regenerate the entire gonad. Here, I will use C. elegans to investigate the hypothesis that compositional differences between germline and somatic mitochondria underlie differences between these cell types. I will expand upon preliminary work showing that adult germ cell mitochondria possess a dramatically altered ratio of respiratory chain components compared to several somatic cell types. I propose three aims to test my hypothesis. I will first determine the point in development when germline and somatic mitochondrial become distinct, which is integral to understanding how such differences could be regulated. I will further examine the molecular basis of this difference, its relationship to mitochondrial activity and the consequences of altering these differences in both germline and somatic cells. Finally, I will test if engineering germ cell-specific mitochondrial characteristics in somatic cells can alter somatic function, potentially impacting the fate of cells subject to mitochondrial dysfunction during aging. These aims will uncover fundamental aspects of mitochondrial function during development and may reveal avenues through which the rejuvenating properties of germ cells could be applied to the soma.

Proteins can self-assemble to create hollow structures that encapsulate cargo. These appear in nature with various roles, such as reaction vessels for catalysis or viral capsids. Recently, the first examples of artificially engineered protein containers have appeared, presenting an exciting opportunity to develop synthetic assemblies to mimic the structure and function of their biological counterparts. The proposed research will develop a protein cage which can load nucleic acids into its core. This presents a solution to the delivery of therapeutic nucleic acids. A protein-based carrier has distinct advantages over current approaches, due to the high level of structural control and potential for site-specific modification. The encapsulation will be achieved through electrostatic interactions, exploiting the anionic nature of nucleic acids and directing them to the positively charged cavity of the protein cage. The first part of the project will focus on the design and expression of a hollow protein structure with a positively charged lumen. Secondly, the capacity for loading will be investigated, with a focus on the stability and capacity of the capsule to protect its cargo from degradation. Finally, the ability of the device to enter cells and regulate protein expression will be thoroughly investigated, including modification of the capsid surface to control cellular uptake and trafficking. This research will provide new insights into the potential of protein cages as active devices to regulate cellular function. Additionally, the resulting protein-DNA complexes provide an ideal model system for investigating the properties of naturally occurring protein compartments.

Understanding the mechanisms of longevity can provide ways to achieve healthy ageing. Naked mole rats (NMRs) are the longest-lived rodents that remain healthy until the end of their lives and are resistant to age-related diseases including cancer. One causative agent of this remarkable trait is the acquisition of high molecular mass hyaluronic acid (HMM-HA). The catalytic domain of the HA synthase gene HAS2 is highly conserved across several phyla, exhibiting two amino acid changes unique to NMRs that result in the synthesis of HMM-HA. I propose to create transgenic mice using CRISPR/Cas targeted genome editing that will contain the unique NMR AA changes engineered into the murine HAS2 gene. An orthogenic mouse model of the NMR HAS2 gene will tell whether HMM-HA can delay aging and prevent cancer in a species other than NMR. To identify genes involved in HMM-HA pathway and novel tumor suppressor mechanisms we will generate a whole-genome NMR CRISPR KO library. Primary NMR skin fibroblasts expressing SV40-LT antigen and Ras-V12 will be transduced with the library and malignantly transformed colonies will be selected. Unpublished data from the Gorbunova lab show that NMR hematopoietic stem cells (HSCs) are biased towards erythroid lineage. I will apply the NMR KO library to NMR bone marrow (BM) cells revealing genes responsible for the prevalence of erythroid development inherent to NMR HSCs. The genome scale NMR KO library will prove an invaluable tool for researchers utilizing NMR, while the discovery of new tumor suppressors and the molecular understanding of the longevity and cancer resistance of this remarkable rodent have great potential for biomedical applications.

Our goal is to unveil evolutionary and ecological roles of horizontal gene transfer (HGT) in natural ecosystems. HGT is believed to be promiscuous in nature and one of the major driving forces for microbial evolution. Yet, little is known about the frequency of HGT and its contribution to structures, interactions, and functions of natural microbial communities. HGT is often studied under artificial laboratory conditions using simple donor-recipient pairs of microbes. Furthermore, HGT events in natural communities are typically inferred from comparative genomics without experiments. Here, we propose to exploit the characteristic gut microbiota of honey bees as a versatile model to study the impact of HGT in a natural microbial community. Honey bees harbor remarkably stable and simple bacterial gut communities consisting of only eight bacterial species. Each species is represented by a number of divergent strains that can coexist. Preliminary genomic data suggests that HGT occurs within and across species in the community. In this proposal, we hypothesize that HGT confers robustness on the community level to hedge against environmental perturbations by two mechanisms: (I) HGT allows the community to maintain beneficial functions by increasing their redundancy, (II) HGT contributes to maintaining species diversity by spreading conditionally critical functions. To test these hypotheses, we will combine genomics, experimental, and theoretical approaches and investigate dynamics, functions, and consequences of HGT in the bee gut microbiota. Using in vivo screening and population genomics, we will first establish a comprehensive gene catalogue of horizontally transferred elements. We will develop an ex vivo system that will enable us to track HGT and community structure changes at the single-cell resolution in real-time in the actively maintained bee gut. Genomics and ex vivo data will be used to develop theoretical and computational models of eco-evolutionary dynamics that account for how HGT alter quantitative ecological interactions. Ultimately, we will test HGT-deficient communities of bee gut symbionts in bee colonization experiments to show the influence of identified HGT on community structure and symbiotic functions in vivo. The project will lead to novel insights into eco-evolutionary dynamics by incorporating HGT into community dynamics.

Cell fate is determined by epigenetic controls that determine patterns of gene expression. Differentiated cells are typically locked into stable steady states of restricted expression, while in cancer cells and stem cells restrictions are diminished or lost. Gene expression is determined biochemically, by transcription and chromatin remodeling factors, and physically, by access restriction or enhancement. Here, I focus on the largely unexplored physical control of gene expression. I test the hypothesis that non- equilibrium stress fluctuations in the nucleus serve to locally enhance transport and DNA accessibility. I will probe in how far enhanced fluctuations and decreased packing density in cancer cells and stem cells are directly correlated with enhanced and possibly pathologically uncontrolled transcription activity. I will develop and use new methods to track fluctuations in the nucleus of control cells and cancer cells with near infrared fluorescent single-walled carbon nanotubes (SWNTs). These extremely photostable and precisely targetable nano-probes allow us to track dynamics with millisecond resolution for up to hours. I will apply and further develop a new super-resolution microscopy technique known as Double-Helix Point Spread Function Microscopy (DH-PSF), which uses an axially extended helical point spread function to achieve simultaneous extended z-resolution and orientation tracking of multiple SWNT probes in 3D. I will use “active matter” concepts from statistical physics to analyze non-equilibrium and equilibrium fluctuations and measure local chromatin mechanical response, as well as transport through the chromatin network.

Cell reprogramming provides a potentially unlimited cell source for in vitro tissue engineering for biomedical research and regenerative medicine. Despite significant advancements in the field, it remains elusive whether the reprogrammed cells fully resemble their native counterparts. This is mainly because we lack understanding of biological pathways in single cells. The recently developed Fluorescent In Situ Sequencing (FISSEQ) allows for in situ sequencing of RNAs of single cells in tissues without pre-selection of the target RNA population. By performing in situ sequencing of DNA barcode-linked antibodies that specifically bind to target proteins, the FISSEQ method can be adapted to perform in situ proteomic study. This project aims to expend and modify the RNA FISSEQ technique to characterize chromatin-associated proteins that are important for transcriptional regulation at single cell level. We will characterize these chromatin-bound proteins in single cells of both native and lab-generated human cerebral tissues. By identifying the key regulatory factors for cell-fate determination, we will be able improve our ability to guide engineering of physiologically-relevant tissues.

Cells in developing organisms can keep track of time. They use this to make important decisions - for example on when to turn or to stop in the case of migrating axons or cells - without being instructed to do so by signals from other cells in their surroundings. But how such internal clocks work, and importantly, how they are made to be so precise, is still largely unknown. Previous work by one of the participating teams has shown that during the development of the nematode worm C. elegans, the migration of a neuroblast is regulated through the timed expression of a signaling receptor. This system provides a powerful assay to study at single cell level how an internal clock controls gene expression. The three teams will use a unique combination of genetics, evolutionary biology and mathematical modeling to gain detailed insight into the workings of this timing mechanism. They will investigate how timing is mediated at the transcriptional level, how robust this is to environmental variations and how this mechanism has evolved in other nematode species. Importantly, these results will be used in mathematical modeling to gain insight into the underlying regulatory architecture and to make predictions that will be tested in further experiments. Such interplay between experimental and theoretical analysis is a powerful and innovative approach that will enable the three teams to gain deep understanding of how cells measure time.