Sexual behavior is fundamental for evolution and an important component of human well-being. Ejaculation is a critical mechanism in male sexual function, which is hypothesized to be controlled by a neural circuit known as the 'spinal ejaculation generator' (SEG), located in the lumbar spinal cord. The SEG has also been hypothesized to control the post-ejaculatory refractory period, a phase of sexual satiety, through ascending projections to the brain. However, the mechanisms by which the SEG controls ejaculation and the refractory remain elusive. We will tackle this problem first by using anatomical tracing to find the spinal neurons that provide input to the ejaculatory muscles and immunochemistry to characterize their molecular identity. We will use this information to provide specific genetic access to SEG neurons. By expressing fluorescent probes in these neurons, we will be able to obtain targeted electrophysiological recordings and use them to characterize the intrinsic and synaptic properties of the SEG circuitry in vivo. By expressing genetically encoded calcium indicators, which provide fluorescence probes of activity, we will monitor the activity of the SEG during sexual behaviour. Finally, by expressing and optically stimulating excitatory and inhibitory opsins in SEG neurons or their terminals, we will test their causal role in triggering both ejaculation and the post-ejaculatory refractory period. This project will provide the first mechanistic description of the key neural circuitry controlling male ejaculation and the refractory period, with potential implications for the treatment of sexual dysfunction.

Microbial communities affect us in many important ways, from health and disease to food production, industrial fouling, agriculture, and the environment. There is a great need, therefore, to both understand and control microbial communities. The challenge is that these communities often contain many dynamically-interacting species, making them complex systems that are difficult to predict. To understand microbial communities, we need breakthroughs in advanced theoretical methods. The current state-of-the-art treats microbes as static networks but microbial communities are dynamic systems where species interactions can rapidly change and shift a community to an undesirable state. To deal with this, I will develop a new dynamic network theory to both understand and control microbial communities. Firstly, I will analyse how the key properties of microbial communities are influenced by different sources of network dynamics. I will study both microbe-driven changes in network structures, e.g. ecological succession, and changes driven by external factors, including the abiotic environment and host factors for symbiotic communities. I will then apply control theory to these dynamic networks in order to understand how to stabilize dynamic networks and shift networks from an undesirable (e.g. disease) to a desirable state. As a final step, I will work with my host laboratory to design experiments and test the ability of my theory to both predict and engineer gut communities for specific outcomes. In this way, I aim to make major contributions to both microbiology and complex system theory.

Complex human diseases are the result of a combination of genetic, environmental and lifestyle factors, and the development of new treatments for such diseases has been challenging. Large-scale screens in pharmaceutical industry have essentially not changed over the last 50 years and many drug candidates fail in clinic, as animal testing is mostly performed on animals with a single genetic background, compromising translational utility. While multiplexing experiments in vertebrate animal models with different genetic backgrounds seems unrealistic, small model organisms, such as C. elegans, are emerging as valid alternatives to cells and inbred mouse models in drug development, significantly lowering time and costs of research. My host laboratory has developed an automated platform for roundworm culture and high-throughput phenomic data collection and analysis. I here propose to use this platform to map mitochondrial stress pathways, such as the UPRmt, in a large C. elegans genetic reference population and in depth characterize selected candidate hits from this genetic screen through the study of loss-of-function worm models in vivo. This will allow me at the same time to develop and validate protocols for high-content and high-throughput phenotypic analysis of C. elegans. Furthermore, my project will spur replacement, reduction, and refinement (3Rs) of vertebrate animal testing and will promote the roundworm (and eventually other small organisms) as new model organism for high-throughput biology as well as for drug discovery.

Mammalian pregnancy is a unique physiological context that undergoes substantial hormonal, metabolic, microbiota and immunological changes. Epidemiology studies have shown that pregnant women have altered susceptibility to various infections. These environmental exposures could have profound and long-term effects on the offspring with impaired growth and disorders. Despite ample evidence linking the maternal immune perturbation to offspring outcomes, the mechanistic pathways for this in utero influence remain unclear. Further, little is known about the impact of these microbial exposures on the offspring immune system. Moreover, how distinct microbiota could imprint the offspring immune system for the long term remains unknown. The objective of this grant is to set up murine maternal immune activation models via infectious diseases that commonly occur during human pregnancy, including influenza virus and Listeria monocytogenes infections, to understand the impact on immune ontogeny, microbiota composition, infectious and inflammatory diseases susceptibility of offspring in neonatal and adult stages. We will dissect the mechanistic links of maternal-fetal crosstalk through vertical transmission of maternal cytokines, microbiota and/or epigenetic regulation with an ultimate goal to rescue the potential detrimental consequence in offspring. Further integration of our findings from murine models to human samples will provide significant impact in the clinic. Insight into maternal-fetal immune crosstalk is a fundamental line of research with profound implications in understanding and prevention of complex immune disorders that are on the rise in high-income countries.

Cells continually encounter a variety of suboptimal conditions which restrict growth and proliferation. In response to such stressors, proper adaptive mechanisms are typically activated. Adaptive mechanisms can be categorized into two groups, specific and general. The specific responses, such as DNA repair or unfolded protein response, directly deal with the primary cause. By contrast, a general response is assumed to inhibit growth and render cells highly tolerant to the stress as a dormant state of an organism. While most studies have focused on the specific effectors, little is understood about how cells initiate and maintain the central stress response pathway. By analyzing high-throughput sequencing data available in our lab on various types of stress conditions, I have found several genes that are commonly regulated in mammalian cells. I hypothesize the common genes may modulate a general stress response, which protects cells from stressors. To understand the role of those candidates, I will 1) reveal molecular mechanisms underlying the common stress program and 2) evaluate their biological significance in vivo. With biochemical and computational approaches combined with loss of function studies, I will determine their targets to unveil regulatory networks established in the common stress program. Finally, I will perform in vivo experiments with various stresses to confirm whether the candidates function in the physiological context. By understanding the core stress response, this research will address an important but often overlooked aspect of stress response and provide a fundamental understanding of cell survival and maintenance.

Post embryonic growth and regeneration require new cells to integrate into the network of existing cells. In the case of the brain it is extremely important to maintain and restore circuit function, such that behaviors can be executed correctly. To study such processes a system that fulfills both post-embryonic growth as well as regeneration is needed. Furthermore, since neural circuit functions are highly dynamic the need for in vivo approaches is evident. In mammalian systems where neurogenesis occurs throughout life in restricted brain regions such approaches are technically challenging. Here, I propose to use the axolotl (Ambystoma mexicanum) brain to understand the dynamic remodeling of neural circuits through the addition of newborn neurons during post-embryonic growth and regeneration. I will establish a high resolution intra-vital in vivo imaging approach to address the dynamics of neurogenesis and remodeling of neural circuits in the telencephalon and the optic tectum. Using this setup in combination with genetically encoded calcium indicators I will generate activity maps of these regions in the presence or absence of defined stimuli and address their functional remodeling during growth and their restoration after injury. Furthermore, I will develop and adapt methods to visualize the functional connections of neurons in axolotl. Together, this work will provide fundamental insights into the functional maintenance and regeneration of neural circuits and the dynamics of how new neurons integrate in vivo which has not yet been achieved in any vertebrate system.

Basic biological processes such as cell migration, tissue morphogenesis, and hearing rely on mechanotransduction, i.e., the conversion of mechanical forces into electrical and biochemical signals that can be used by individual cells or multicellular organisms to survive. Adhesion complexes bearing mechanical force are often needed in mechanotransduction, with several protein families that include integrins and cadherins directly transmitting force signals to sites of transduction. Classical cadherins, key for tissue morphogenesis and integrity, provide calcium-dependent bonds involved in cell-cell contact formation, in signaling to the actomyosin cytoskeleton, and in mechanical stabilization of cell-cell adhesion. Non-classical members of the family are also involved in mechanotransduction, with cadherin-23 and protocadherin-15 forming an adhesive “tip link” essential for the vertebrate senses of hearing and balance. Multiple structural and biophysical studies have elucidated the unbinding, unbending, and unfolding strength of single molecules and adhesive complexes, including those formed by cadherins. However, to the best of our knowledge, nothing is known about how the mechanical properties of adhesion complexes have evolved. Here we aim to study the mechanical evolution of adhesion complexes using inner-ear cadherin tip links as a model system. Tip links initiate sensory perception by directly conveying tension to inner-ear mechanotransduction channels on a microsecond time scale. Fast and effective transmission of mechanical force must be one of the selective factors that dictate the molecular evolution of tip links. We will sequence, synthesize, and study tip-link proteins from diverse extant species and will carry out evolutionary analyses both to reconstruct the sequences of ancestral, “resurrected” tip links, and to identify potentially stronger tip links from species in which positive selection is reported. In parallel, we will use biophysical techniques to determine equilibrium binding affinities and structural properties of modern tip links from various species and ancestral tip links at different evolutionary stages. High-speed force spectroscopy and molecular dynamics simulations will be combined to probe tip link mechanics and strength across species and during evolution. Our interdisciplinary work will provide a unique evolutionary view of adhesion mechanics.

A driving goal of regenerative medicine is to use stem cells to replace worn-out cells, tissues and organs. Stem cells could in principle be instructed to generate all cell types required. However, two crucial questions must first be addressed: 1) How can we precisely target specific cell types with these signals to control differentiation? 2) What molecular signals instruct cells to differentiate, sequentially, into cell types of interest? These fundamental questions have been difficult to address in a generalizable way. However, two new advances open up powerful new ways to address them. First, a new design principle for signaling systems shows how combinations of seemingly redundant ligands and receptors could be used to target signals to specific cell types. Second, a new technique called MEMOIR enables cells to actively record their own signaling histories within their genomes, providing information on which signals are perceived within each cell lineage. Here, I propose to combine my own experience in stem cell research with Michael Elowitz’s expertise in synthetic biology to develop technology for mapping and manipulating the processes by which cells acquire their identities. More specifically, I will analyze promiscuous receptor-ligand interactions in the FGF or ErbB receptor families to better understand how the use of ligand combinations could enable more cell type specific activation of these pathways. In addition, I will implement MEMOIR in developing neurons to extract the signaling histories that accompany their differentiation. Knowledge of the way differentiating cells process signals may enable more precise control over cell identity.

The role of midbrain dopamine neurons in learning has been extensively studied over the last two decades, mainly focused on the commonly observed reward prediction error(RPE) activity, i.e. the discrepancy between expected and obtained reward. Still, the field is going through a critical turning point as increasing evidence shows heterogeneity of dopamine neurons’ responses, opening the possibility that dopamine could modulate learning in multiple ways besides through a RPE. This proposal aims to understand if distinct dopamine projections to the striatum, a structure involved in motivation and action selection, play differential roles in learning. For example, dopamine projections to the ventral striatum signaling a RPE could drive the encoding of value observed in this area, while those to the dorsal striatum could influence the encoding of performance vigor, and those projecting to the tail of the striatum could involve this area in attention. I will also test the hypothesis that corticostriatal plasticity follows common rules across the striatum, but striatal activity will vary across regions due to heterogeneous information conveyed by dopamine innervations. I will use molecular techniques to image simultaneously the activity of dopamine axons and of principal neurons in various striatal regions with two-photon microscopy while mice perform a sensorimotor go/no-go task to achieve rewards and avoid punishment by modulating their gait. This proposal will elucidate fundamental mechanisms of dopaminergic control of associative learning and motor activity, providing relevant results for the understanding of neurodegenerative diseases such as Parkinson and Huntington’s.

Trypanosomatids are parasitic protozoa causing a range of devastating diseases. Many mitochondrial genes of these pathogens are transcribed as precursor mRNAs that require insertion and/or deletion of uridylate residues to become functional. The RNA editing reactions are catalysed by the editosome, a large molecular machinery present in the trypanosomatids mitochondria. Editosomes are parasite-specific complexes representing ideal therapeutic targets, however their structural organisation and mode of action are not fully understood. The aim of the proposed research is to obtain a comprehensive structural and functional characterisation of the trypanosomatids RNA editing machinery. To achieve this, I will first produce genetically modified Trypanosoma brucei cell lines expressing tagged editosomal proteins that will allow for rapid and efficient purification of editosome variants under native conditions. I will then use cryo-electron microscopy and single particle analysis to determine high-resolution structural models describing the organisation and the interaction network of the purified editosomes. By determining the structure of the complexes actively engaged in the editing reactions, I also aim at elucidating the conformational states associated with the individual steps of the enzymatic cascade. The proposed studies will provide a broad characterisation of the molecular architecture of the editosomes that will be crucial both for the fundamental understanding of RNA editing and the development of therapeutic strategies against trypanosomatids pathogens.

Bacteria play a ubiquitous role in the ecological success of many plants, algae, and animals. Much of this success reflects the capacity for bacteria to provide resources to their hosts that are otherwise scarce or inaccessible in the immediate environment. Microalgae of the genus Symbiodinium are found globally in tropical and temperate coastal ecosystems, forming symbioses with many invertebrates but notably reef forming corals. Over 50 years of research has shown immense functional divergence amongst the genus Symbiodinium. This functional divergence has commonly been ascribed to fundamental differences in gene acquisition and expression of resource exchange and stress tolerance. However, I hypothesize that obligate associations with bacterial consortia regulate Symbiodinium fitness and functional diversity over space and time. As such, mutualistic bacteria may act as essential “resource surrogates” for Symbiodinium when they live transiently as free-living cells outside of their host corals. The type and health of the algae–bacterial community interactions are entirely unrecognized but in fact, determine the survival of Symbiodinium, and hence the future for reef systems worldwide as they are subjected to accelerating anthropogenic stress. To address this complete unknown, I will use a combination of state of the art techniques (e.g. NanoSIMS, chemotaxis flow imagers and bioreactors) to characterize key ecologically-relevant Symbiodinium-bacterial associations and identify key metabolic networks that promote Symbiodinium ecological resilience. This study will transform our understanding of what makes a healthy coral symbiont, and hence the fitness of reef ecosystems.

Perception is shaped both by sensory information and internal variables. Our brains uses regularities of the environment to make predictions, and combines them with sensory stimuli to form percepts. However, how and where is statistical knowledge of the environment stored and learned remains unknown. Visual perception arises from a set of hierarchically-organized cortical areas. Abundant feedback inputs project from higher to lower areas but their role in perception is unknown. In this proposal, I will test if knowledge of the world is stored in the connectional specificity of feedback projections. I hypothesize that the tuning-specific wiring pattern of feedback projections terminating in mouse primary visual cortex reflects spatiotemporal visual statistics of the environment. First, I will assess if sensory experience plays a role in visual feedback wiring. To do so I will visually-deprive mice or raise them in artificial environments with altered visual statistics. Using a novel combination of dual-color optical recordings, I will measure how connectional specificity of functionally-characterized feedback axons in primary visual cortex relates to the animal’s visual experience. I will also determine if spatiotemporal patterns of correlated neuronal activity are sufficient for instructing the observed wiring organization. These experiments will unveil if feedback inputs are a neural substrate for storing learned regularities of the world. By doing so, they will shed light on the computational role of this enigmatic connections while providing a mechanistic description of the internal factors shaping perception.

Specific regulation of genes is essential for cellular life. In mammals, transcription factors play a critical role in this process as they distinctly mark regions of the genome for expression and are the pillars of cell identity. Reprogramming of a terminally differentiated cell into a pluripotent cell can be accomplished by mere expression of a few transcription factors. These transcription factors regulate developmentally silenced regions of the genome and initiate reprogramming. Fundamentally, how a transcription factor is able to target specific DNA sequences within chromatin is still largely a mystery. Oct4 is one of the key factors in this ‘pluripotent cocktail’ and it can directly interact with nucleosomes. Most transcription factors cannot interact with nucleosomes because of the inaccessibility of DNA wrapped around histones. Pioneer transcription factors, such as Oct4, are able to access these restricted regions of DNA, yet we still do not know how. I propose to use biochemical and structural techniques to elucidate the molecular assembly of Oct4 and chromatin. The results of my proposed work aim to uncover the molecular determinants of a transcription factor for association with nucleosomes and the mechanism by which a transcription factor complex may initiate nucleosome rearrangement/eviction to recreate cell identity.

All of our movements require the coordination of dozens of muscles to ensure that we reach our desired goal. Indeed, the ability to perform complex motor corrections that depend on the context, termed flexible feedback control, is a fundamental aspect of human behavior. Three brain areas essential for the preparation and execution of reaching movements are the interconnected dorsal premotor, primary motor, and primary somatosensory cortices. These areas modulate their activity to reflect the context of upcoming actions, but the roles of each area and the underlying computational principles are poorly understood. To address this question, macaque monkeys will be trained to reach targets in virtual reality using a haptic feedback device in response to various mechanical loads and sensory expectations while neural activity is recorded from over 1000 sites simultaneously. To dissect the computational principals of this complex control problem, a modular neural network model of the circuit will be trained to complete goal-directed tasks by learning to control a 50-muscle biomechanical model. Finally, neural projections between these brain areas will be selectively manipulated using optogenetic stimulation to dissect the roles of each of these areas during feedback control. The results of these three experiments will provide a rich bi-directional transfer of information between theory and experiment and significantly improve our understanding of motor control.

Cell division mechanisms are highly conserved, yet mechanistic requirements vary between species and cell types. For example, while epithelial cells round up for mitosis, hematopoietic cells grow in suspension, maintaining their round shape throughout the cell cycle. Division of cells with distinct morphologies likely requires specialized machineries, yet cell type-specific mitotic mechanisms remain largely unexplored. Understanding specialized cell division machineries might profoundly improve anti-cancer therapies. Many chemotherapeutic drugs target universal cell division pathways, resulting in toxicity due to mitotic perturbation of hematopoiesis, yet the majority of cancers are of epithelial origin. Thus, exploiting cell type-specific vulnerabilities has the potential to increase the specificity of chemotherapy. The aim of this work is to explore how mitotic mechanisms adapt to diverse challenges in different human cell types and growth geometries. I plan to uncover cell type-specific mitotic pathways by genome-wide CRISPR/Cas9 screening, complemented with a candidate-based approach. First, I will use two screening approaches to compare cellular machineries required for chromosome segregation and cytokinesis. Second, I will explore how epithelial and hematopoietic cells accommodate different demands for shape changes during division by investigating cell type-specific regulation of cortex contractility during mitotic entry and cytokinesis. Overall, this project will shed light on how cell division mechanisms accommodate specialized requirements in multicellular organisms, with the tremendous potential to uncover tissue-specific targets for cancer therapy.

The aim of this proposal is to gain insight into the mechanisms of chromatin reprogramming to totipotency in mammals. Totipotency is the potential of a cell to generate all cell types of an organism including extraembryonic tissues. Whilst induced reprogramming to pluripotency by the Yamanaka factors is relatively inefficient and takes days to weeks, natural reprogramming to totipotency occurs within hours in the one-cell embryo or zygote. How zygotic reprogramming is achieved is poorly understood. We will approach this fundamental question by studying the chromatin state of totipotent cells and their precursors. We will determine how higher-order chromatin organization changes during epigenetic reprogramming in zygotes and primordial germ cells, using both mouse embryos and human/mouse primordial germ cell-like cells. We will use our understanding of the zygotic chromatin state to identify candidate pioneer factors and to test their role in promoting totipotency and genome activation. The results of the project will provide insights into how reprogramming alters genome organization and promotes establishment of a totipotent chromatin “ground state”. This project is led by a strong multi-disciplinary team bringing together expertise in polymer physics (Mirny), ovary organoids (Saitou), biochemistry and single-molecule imaging (Peters) and zygote biology (Tachibana-Konwalski).

Skin is a remarkable tissue requiring dynamic and robust behaviors. It acts as a tight bi-directional barrier while being constantly exposed to mechanical stress. The epithelial layer of the skin, the epidermis, acts as load-bearing layer. The presence of tension in the epidermis is critical for its function, however it also constantly threatens the integrity of this tissue which quickly degenerates in the presence of impaired mechanical reinforcement of cell adhesions or the cytoskeleton. Thus it is critical to uncover the poorly understood mechanisms of mechanical stress protection and mechanoadaptation in the epidermis specifically. It is becoming evident that the rheological changes in the nucleus, nuclear envelope, and modifications in chromatin architecture are critical for cellular adaptation and recovery from external mechanical cues, but the mechanisms and precise functional implications of this regulation are unclear. This project aims to investigate the mechanisms and functional consequences of dynamic remodeling of perinuclear actin and heterochromatin in regulating nuclear rheology and thereby the mechanical stress response of epidermal stem cells. Second, based on the preliminary data from the host laboratory, I will test whether strain-induced poly-ADP ribosylation is a mechanopreotective mechanism which, by placing the DNA damage repair machinery in close proximity to the sites of DNA damage enhances the efficiency of repair upon mechanical stress. These studies will unravel molecular mechanisms that allow cells to adapt to mechanical stress through dynamic regulation of nuclear rheology.

RNA modifications have been proposed to affect various aspects of RNA biology, including translation and RNA stability. However, how these effects are mediated is not fully understood. Drosophila melanogaster embryonic development is an excellent model to study the effects of RNA modifications, as RNAs with critical roles during development are highly post transcriptionally regulated. I propose to develop and apply a new method to detect the RNA modification 5-methylcytosine (5mC) transcriptome wide at single nucleotide resolution in the Drosophila embryo. This strategy relies on reverse transcription of RNA and subsequent digestion of the RNA-DNA hybrid with a modification dependent restriction enzyme. The digestion sites are ligated to an adapter containing a T7 promoter, facilitating amplification of the RNA-5mC containing regions. Next, RNA library preparation and Illumina sequencing will reveal the site of modification at the start of the sequence read. The application of this technology will identify the modified RNA species together with the exact location of the modification in Drosophila embryos. In combination with spatial transcriptomics and RNA localization data, in a second step I will elucidate the role of RNA modification status on RNA localization and translation. To reveal the functional consequences of RNA-5mC, I will use affinity purification in combination with mass spectrometry to identify RNA-5mC reader proteins. The abundant genetic tools available in Drosophila will allow me to assess the functional consequences of the removal of each of the reader proteins on the modified transcripts and on a whole organism phenotypical level.

Tumor-infiltrating T cells (TILs) are critical participants in controlling tumor growth, metastatic potential, and ultimate lethality of many cancers. These immune cells act contextually within the tumor microenvironment to either promote tumor clearance or facilitate tumor growth. This is exemplified in the recent successes of the immune checkpoint inhibitors, anti-CTLA-4 and anti-PD-1. In these treatments, subsets of patients experience remarkable tumor regression mediated by rescue of ‘partially exhausted’ CD8+ T cells (peTILs) and negation of the immunosuppressive functions of regulatory T cells (Tregs). However, the dominant molecular pathways preventing TIL-mediated tumor rejection remain obscure. Identification and detailed investigation of these pathways is of fundamental importance in order to understand immune responses to aberrant tissue growth. Recently, the gene LAYN has been identified as highly expressed in the tumor microenvironment and associated with the suppressive signature of Tregs, as well as the exhausted state of CD8+ T cells. Strikingly, high LAYN expression on Tregs correlated with poor clinical outcomes in multiple cancers. Our laboratory has independently observed high LAYN expression on TILs in melanoma patients. As there is a lack of knowledge regarding layilin biology (only 17 publications to date) we propose to comprehensively dissect the functional role of layilin on tumor-infiltrating T cells. We hypothesize that layilin signaling acts to augment Treg suppressive capacity while attenuating CD8+ TIL cytotoxic activity. To address this hypothesis we have generated novel tools to delete or over express layilin in both human and mouse TILs.

Consciousness is probably the biological construct we are most intimately familiar with, yet our understanding of its possible functions remains quite poor. Various – sometimes contradicting – accounts range from denying it from having any functional role to considering it a prerequisite for all high-level functioning. Here, I propose a new experimental approach which will hopefully enable us to make the much-needed leap towards a genuine theory of consciousness. I argue that most current methodologies have drifted away from the studied phenomenon: conscious vs. unconscious processing of information. While the latter is held to occur regularly in everyday lives, it takes on a very different form in the lab, when probed using psychophysical techniques which commonly rely on artificial operationalizations having little resemblance to real-life processes. And the stimuli are typically highly degraded, arguably evoking weak signals (and, in turn, non-robust findings). Instead, I suggest two novel ways to get closer to real-life unconscious processing of stimuli with which subjects can interact: the first – a unique method we developed to suppress, for the first time, real-life, actual 3D objects. The second – a virtual reality protocol which mimics unconscious processing of stimuli in real-life environments (e.g., driving or walking). These two experimental paths enable us to examine the processing of strong, non-degraded, real-life/real-life-like stimuli. I propose 7 experiments that – if successful – promise to fundamentally change the way consciousness and its functions are studied and open a new branch of study, with far-reaching theoretical and practical implications.