The adult mammalian stomach epithelium is constantly renewed by proliferative isthmus stem/progenitor cells (ISPCs) and maintains its homeostasis under hostile luminal conditions. In the gastric corpus epithelium, differentiated Troy+ chief cells were identified as a novel stem cell population with high plasticity. Interestingly, Troy+ stem cells (TroySCs) can switch between a dormant and active state. TroySCs proliferate very slowly in homeostasis, whereas they rapidly divide upon ISPC loss facilitated by 5-FU treatment. This suggests its role as a reserve stem cell for the ISPC. However, TroySCs are also activated upon parietal cell loss despite the presence of actively cycling ISPCs. Thus, the role of TroySCs still remains vague in homeostasis and regeneration from various injuries. Here, I propose to investigate a potential role of TroySCs, beyond that of the reserve stem cells, by combining state-of-the-art mouse genetics, single cell transcriptome analysis, and mathematical modeling. First, I will characterize the population dynamics of the corpus epithelial cells under homeostasis and regeneration by lineage tracing and single cell analysis in mice. Second, based on the cell population data, I will construct a mathematical model for the corpus epithelium and theoretically investigate the function of TroySCs by perturbing their dynamics. Third, I will experimentally examine the role of TroySCs using in vivo and in vitro functional genetics. This work will reveal an essential role of quiescent stem cells in homeostasis and pathogenesis and shed light on a fundamental question about why quiescent and active stem cell populations coexist in various adult tissues.

Engineering of recombinant pathways in microbial hosts is a powerful tool to provide access to sustainable building blocks for the chemical industry. Such a bottom-up synthesis route has recently been established for monomeric phenylpropanoids. However, the yield and diversity of products obtained are still lagging behind their natural plant biosynthetic pathways. The aim of the herein described project is to explore and develop tools for the reconstitution of plant metabolic pathways in bacterial hosts: namely, tools for the availability of bacterial strains as framework, for protein engineering and for identifying novel pathway enzymes for crucial product tailoring steps.

Therefore, I propose to investigate the applicability of Corynebacterium glutamicum as a bacterial chassis for plant metabolic pathways in general and the phenylpropanoid pathway in particular. I am going to develop genetic tools based on the CRISPS/Cas9 system to allow the fast incorporation and screening of various pathway enzymes and promoter combinations. I furthermore propose to investigate the effect of tethering Cytochrome P450 (P450) enzymes – a typical bottleneck in reconstituted pathways - to their redox partners, on in vivo P450 activity. Lastly, I propose to investigate the correlation of sequence, structure and function of crucial product tailoring O-methyltransferases from various organisms to enable the classification of such enzymes based on sequence homology.

The proposed study will not only increase the efficiency of the synthetic phenylpropanoid pathway but provide tools to further our abilities in manipulating microbial hosts for fermentative production of valuable chemicals.

The biggest challenge in glioblastoma research is to understand the inherent cellular heterogeneity. While much research has been dedicated to glioblastoma stem-like cells (GSCs), a small population of tumor cells that resides at the apex of a differentiation hierarchy and underlies therapy resistance, differentiated glioblastoma cells have not received the same attention.

I hypothesize that glioblastoma is characterized by a previously underestimated extent of cellular differentiation. Dedicated differentiated cell types provide the tumor with a selective advantage, e.g. by promoting angiogenesis and evading the immune system, and by supporting GSCs with essential maintenance and cell division cues. Therefore I propose to accurately define the cellular composition of primary human glioblastoma using microfluidics-enabled transcriptome profiling of thousands of individual cells. Using sophisticated computational methods, this dataset will reveal previously unrecognized cell types, and inform on their roles in tumor formation. Additional epigenetic characterization of tumor cell subpopulations will define cell type specific gene regulatory networks and inform on the molecular consequences of common genetic alterations.

I further hypothesize that tumors of different patients are composed of varying stem-like and differentiated cell types and that cell type frequencies differ between patients. Incorporating the new information on tumor heterogeneity will allow me to redefine glioblastoma subclassification. This will lead to more accurate patient prognoses, and ultimately to the development of subtype-specific therapies and the design of more targeted clinical trials.

The endosomal system is regulated by the interplay of distinct endosomal classes and its interactions with other cytoplasmic organelles within the cell. Identifying the molecular players and mechanisms that regulate the endomembrane system is essential because defects in the system are implicated in human disorders. The previous genome-wide RNAi screen performed in the host lab found numerous metabolic and signaling genes known to be localized to other cellular organelles that affected epidermal growth factor (EGF) and transferrin trafficking, underscoring the importance of inter-organelle communication. However, little is known about the underlying molecular mechanisms. In this proposal, I will perform a focused RNAi screen using a quantitative multiparametric analysis in order to identify factors governing intra-cellular positioning and functional interactions between distinct endosomal classes, such as early and recycling endosomes. I will also search for organelle-specific genes belonging to endoplasmic reticulum, mitochondria, lipid droplets, and peroxisomes that are functioning at the membrane interface with endosomes for inter-organelle tethering and signaling. Finally, I will investigate the physiological function of candidate genes in a primary cell culture system. The project outcome will lead to a better understanding of the endosomal system and the molecular mechanism of inter-organelle communication.

Neuroscience research using rodents as an animal model relies on the assumption that results should generalize across species to primates and ultimately to humans. However, many brain areas, including the neocortex, have species specific functional organizations. The mouse lemur (Microcebus murinus), the World’s smallest primate, has the potential to become an ideal animal model bridging the gap between rodents and primates. It has most the advantages of the rodent model (small brain size, quick reproduction, relatively short life cycle), but additionally offers the evolutionary closeness of primates. Therefore, the mouse lemur promises to revolutionize the transferability of experimental results from small sized animal models to human applications. In this project, three labs will combine their expertise in primate behavior, in-vivo optical imaging, and cutting edge histology and molecular biology to explore the functional organization of the mouse lemur cortex, as well its behavioral and cognitive capacity. This collaborative project will lay the groundwork to establish the mouse lemur as a novel neuroscience model system.

In order to ensure genome stability, DNA repair is highly regulated by the cell division cycle. A major, but largely unexplored question pertains to mechanisms that promote genome stability when DNA damage occurs on segregating chromosomes. My host laboratory discovered that cells suppress the repair of DNA double-strand breaks (DSBs) in mitosis to prevent chromosome segregation errors. These occur as a consequence of repair-mediated telomere fusions. The overarching objective of my project is to address the biological significance of mitotic DSB repair inhibition in vivo by generating a transgenic mouse in which I can conditionally de-suppress DSB repair in mitosis. This system will enable me to identify the tissues and developmental processes sensitive to the genome instability caused by mitotic DSB repair and to test whether inhibition of mitotic DNA repair is a tumour suppression mechanism. In parallel I will elucidate the molecular mechanism that causes the observed telomere fusions. Based on previous results, I hypothesize that the kinase Aurora-B mediates mitotic telomere uncapping through targeting of the telomeric Shelterin complex. I will test whether active Aurora-B localizes to telomeres using a FRET-based activity sensor and identify its target among the Shelterin subunits. Since telomere fusions are enhanced upon exposure to ionizing irradiation, I will also clarify the crosstalk between Aurora-B and the DNA damage response. This project comprises phenotypic animal studies and mechanistic cell based studies and thus represents a major departure from my previous work on the genomics of yeast DNA replication.

Muscle resident stem cells, Satellite Cells (SCs), maintain tissue homeostasis and regenerate skeletal muscles. In the event of an injury or stress, SCs break from quiescence, proliferate and differentiate or self-renew. Metabolic activity controls stem cell proliferation and differentiation in young healthy mice. However, whether metabolic activity is altered during aging in SCs and/or niches is poorly studied. Furthermore, whether cell autonomous or non-autonomous metabolic effects drive age-dependent SC functional decline is completely unknown. Our objective of this proposal is to use metabolic profiling in SCs and their niche during aging to identify a novel stem cell-to-niche communication mechanism. We speculate that upon aging, SCs accumulate lactate that causes a loss of stem cell function. In addition, we speculate that shuttling of lactate, the end product of glycolysis, from muscle fiber and taken up by SCs is a major contributor to the SC metabolic state and functional decline during aging. In this proposal, we will generate 1) metabolic profiles of adult and aged SCs and their niches, 2) in vivo inducible cell-specific genetic mouse models to delete lactate dehydrogenase A (LDHA) from SCs and niche, and 3) in vitro approaches to disrupt lactate signaling between the niche and SCs using lentiviral knockdown constructs targeting lactate transporters (MCT1-4) and receptors (GPR81). We will use muscle regeneration assays to evaluate the role of lactate signaling on stem cell function and tissue repair during aging in vivo. These data will provide novel insights toward stem cell-niche interactions as well as further our understanding of aging biology.

Throughout evolution, the human genome has been attacked by retrotransposons, parasitic DNA elements that spread through our genome by a copy-paste activity. I previously showed that SVA elements, the youngest class of retrotransposons in our genome, harbour a strong gene-regulatory potential, which is normally repressed by KRAB zinc finger protein ZNF91 (Jacobs et al., 2014, Nature). However, for reasons unknown, repression of retrotransposons is much less efficient in neurons, resulting in the activation of the hidden enhancer potential of SVA elements spread throughout the human genome. The importance of these SVA insertions for the evolution of human neural gene-regulatory networks, and how many genes have come to depend on SVA's regulatory influence, remains elusive. This research program uses 'cortical organoids’; 3-dimensional brain tissues derived from human and primate stem cells, to investigate how recent SVA insertions have impacted human neuronal gene expression. Furthermore, I will investigate how changes of the epigenetic landscape in neurons affect the activity of retrotransposons and the influence they have on nearby neural genes. Finally, I will explore the possibility that loss of epigenetic silencing of retrotransposons is responsible for dysregulation of genes associated with neurological diseases. Preliminary findings suggest a potential role for retrotransposons in susceptibility loci for Alzheimer's and Parkinson's disease. Finding further support for this notion in the current research program, will form the basis of a novel concept that can explain how changes in the epigenetic landscape can uncover a dormant genetic predisposition to disease.

The drive to seek rewards controls almost every aspect of our behavior, from stereotypic reflexive responses to complex voluntary actions. Thus it comes as no surprise that the symptomatology of neurological disorders that interfere with reward include deficits in the capacity to make even simple movements. To drive behavior the brain uses two major subcortical networks, the basal ganglia and the cerebellum. Experimental data and computational models suggest that the basal ganglia and cerebellum play different roles in the control of behavior. In the basal ganglia, reward signals guide action selection, but how reward signals are translated into behavior remains unclear. The cerebellum uses sensory error signals to optimize motor commands; however, whether the signaling in the cerebellum depends on rewards and how reward might be incorporated into the motor commands have yet to be deciphered.

We will explore whether and how reward signals interact with motor commands in the basal ganglia and cerebellum. We will take advantage of primate smooth pursuit eye movements as a powerful system to study the relationship between reward processing and motor behavior. We will record and manipulate activity in both the basal ganglia and cerebellum while animals are involved in novel behavioral tasks. The tasks extend the current state of the art concerning what is known about sensory or motor processing to study how reward is translated into behavior. The overarching goal is to unify research on the basal ganglia and cerebellum to understand the transformations and computations that translate reward drive into action and their implementations in the basal ganglia and cerebellum.

To understand how brains integrate sensory information and generate behavioural responses is a central goal of systems neuroscience. Larval Zebrafish have emerged as a very promising vertebrate model system to address this challenge because their small size and transparency enable optical access to the majority of neurons within the brain at cellular resolution. Important insights on circuit function have already been gained by two-photon imaging of neuronal populations in restrained larvae behaving in virtual environments – however, critical limitations of virtual environments as a replacement for real environments are widely acknowledged and remain a barrier to progress. An ideal solution to this problem would be to image larvae during natural, unrestrained behaviour, and this is the goal we aim to achieve in this project. It has so far not been possible to track naturally moving zebrafish, nor any other vertebrates, while imaging at cellular resolution. Here we propose to overcome this limitation by forming a novel collaboration across disciplines and three different laboratories. We will pool our collective expertise in optical systems design, wavefront-shaping, electrical engineering, and zebrafish neuroscience to perform an experiment that has until now been impossible: imaging comprehensive neural activity in a freely moving, untethered vertebrate. We will collectively design and validate a real-time optical tracking and imaging system to measure brain activity with single neuron resolution in a freely moving vertebrate. This will allow us to monitor, with unprecedented detail, the population activity of the reticulospinal system in larval zebrafish and understand how this population of ~ 300 supraspinal neurons combinatorially codes for the full range of locomotor and postural behaviors.

Microscopy of biological specimens is often performed at low levels of illumination to avoid damage. Under these conditions images are dominated by shot noise, the statistical fluctuations in the number of detected probe particles (e.g., photons or electrons). Better images can be obtained when correlated probe particles are used, which allows extracting more information per interaction with the sample. A way to do so is to pass the illumination multiple times through the specimen [1, 2]. We propose to design and construct a microscope where the specimen is placed at the center of a self-imaging cavity, in which the probe particles interact with the sample multiple times. An optical setup will be used to demonstrate the main benefits of a multipass microscope: contrast enhancement, sub-shot noise imaging, and imaging at low damage. Fast electro-optical switching techniques will be employed to, for the first time, show sub-shot noise imaging without postselection of the probe particles. Various imaging modalities will be explored, such as phase, absorption and polarization microscopy in both bright- and darkfield configurations. Finding the right imaging modality for specific specimens (e.g., living cells) will be at the focus of the project. In parallel, the feasibility of a multipass electron microscope will be explored using charged particle tracing simulations. We will construct and test a prototype as a first step towards the imaging of organic molecules and proteins with atomic resolution. [1] Giovannetti V, Lloyd S and Maccone L, Nat Photon 5, 222-229 (2011). [2] Higgins B L, Berry D W, Bartlett S D, Wiseman H M and Pryde G J, Nature 450, 393-396 (2007).

Both wild and crop plants must contain pathogen infections to survive. Prevailing theories on the evolution and spread of plant pathogens are based primarily on observations of nearly clonal hypervirulent pathogen lineages in agricultural systems, which are characterized by dense stands of genetically uniform individuals and a paucity of other species. Surprisingly little is known about how pathogens evolve in natural settings, where emergence of hypervirulence appears to be much less prevalent.

The central goals of this proposal are to reveal (i) the dynamics of bacterial pathogen colonization in natural plant populations, and (ii) the influence of several factors—pathogen diversity, host genetics and host microbiomes—on pathogen infection dynamics. My specific plan is to investigate the evolution and spread of the endemic plant pathogen Pseudomonas syringae in wild populations of Arabidopsis thaliana, using innovative high-throughput methods for culturing and genome comparisons of bacterial lineages. These observations will reveal associations of pathogen spread with geographic isolation of hosts, host microbiome composition, host genetics, and pathogen genome diversity. I will then test causal relationships for a subset of these associations through controlled laboratory infections. A unique aspect of my proposal is the hierarchical approach—identifying infection dynamics at the leaf, individual plant, and population level—and the integration of experiments in the laboratory with high-throughput investigation of natural infections.

Innate behaviors such as aggression and reproduction are essential for the survival of animals. Neuropeptides are implicated in the regulation of various innate behaviors by changing their basal arousal state and motivation level via modulating neural activity. Oxytocin (OXT) and vasopressin (AVP) are involved in various social behaviors such as aggression, mating, pair-bonding and social recognition. However, how these peptides exert their functions at the neural circuit level is not well understood.
I plan to study the role of OXT/AVP on the neural circuit of aggressive behaviors by addressing the following questions: (1) Whether if OXT/AVP is actually released onto the ventrolateral subdivision of the ventromedial hypothalamus (VMHvl), which was previously shown to control both the appetitive and consummatory phases of fighting and mating? (2) What is the role of the neural activity patterns responding to OXT/AVP release during aggression? (3) How is the activity of OXT / AVP neurons controlled during aggression and from where do the OXT/AVP neurons receive their input? (4) Whether if OXT/AVP control other social behaviors in VMHvl in addition to aggressive behaviors?
In contrast to my previous research experience in neuroendocrinology, the proposed research will focus on how neuropeptides modulate innate behaviors at the circuit level. Therefore, this will be a good opportunity to extend my training from endocrinological to behavioral neuroscience.

The time dependent regulation of genetic activity in eukaryotes is one of the most complex challenges of modern science. Multiple biomolecular factors constantly interact to maintain the necessary expression levels of various genes and functional RNAs. In this project, we propose to utilize the dramatic advances in live cell fluorescent microscopy and stochastic physics to study a canonic nuclear system – X-chromosome inactivation (XCI). This crucial process is necessary for female embryo development and brings about the transcriptional silencing of a whole X chromosome. Yet despite XCI’s centrality, we still lack an understanding of its temporal regulation mechanisms. The spatial dynamics of the nuclear machinery, including the noncoding Xist RNA and its partners, their interaction with chromatin and the dynamic processes leading to the loss of the transcriptional apparatus from genes during XCI, are all unknown. To answer these questions, we will track the diffusion of Xist RNA together with essential nuclear factors, in live female mouse embryonic stem cells, using super resolution microscopy (SIM, dPALM), and stochastic techniques (FCS, FRET). This includes imaging of Xist associated proteins, silenced and activated genetic loci, and transcriptional machinery. Diffusion will be analyzed using advanced mathematics to extract dynamic characteristics of tracked factors; characteristics which are crucial to fully understand XCI. From these, a biophysical model will be developed to describe the interactions, energetic landscapes and as an outcome, the mechanisms which control this fundamental genetic process.

Collective cell migration is essential in biological processes such as angiogenesis, organogenesis, and wound healing. In various cancer types deregulated migration is implicated in the formation of metastasis. Collective cell migration is meditated by leader cells, which sense multiple parameters and execute movement into the open space, followed by the cell sheet. Cells constantly monitor their environment and the sensing of nutrients has been identified as an important determinant of proliferation. The primary objective of this proposal is to investigate the impact of nutrient supply on cell migration. Thus I aim to identify the metabolic switch, a change in nutrient availability and production, sufficient to alter the migratory properties. More specifically, building on the previous findings and tools of the host laboratory, I aim to first characterize cell speed, direction, and persistence of human endothelial cells in the context of altered nutrient supply using a high-throughput live cell imaging platform. Second, by utilizing specifically designed fluorescent probes, I will dissect the spatio-temporal regulation of nutrient-sensing signaling pathways including the activation and repression of mTOR by PI3K/AKT, PLD/PA, or LKB1/AMPK, which leads to altered cytoskeleton arrangements. Finally, the integration of these findings will aid the generation of a model describing how nutrient supply alters cell migration. This model will be validated using chemical inhibitors, inducible gene deletion, or targeted perturbation. The results of this study contribute to our understanding of how the metabolic environment of a cell dictates its properties, functions, and decisions.

Lymph nodes (LNs) are situated at junctures of the blood vascular and the lymphatic system where antigens drain from peripheral tissues via afferent lymphatics. During the development of malignant breast cancer, new LNs emerge within the glandular tissues that are normally devoid of LNs. However, we do not understand the mechanism of development of these LNs and their role in antitumor immunity. We hypothesize that de novo LNs form in the vicinity of mammary carcinomas as an accessory “base camp” for the initiation and maintenance of antitumor immunity. Here, we combine and leverage our expertise in immunology, vascular biology, and bioengineering to address the hypothesis in three specific aims: (1) To molecularly dissect the pathways of tumor-induced development of accessory LNs with a particular emphasis on lymphovasculokines. (2) To assess to which extent accessory LNs support antitumor immunity. (3) To stimulate formation of accessory LNs in mammary tissues to foster antitumor immunity. The potential impact of this project is high as a deeper understanding of the interplay between accessory LNs and tumors may lead to a novel cancer treatment approach.

Cancer cells need to overcome the onerous task of adapting to cellular stress during the multi-step process of cancer development. Several studies suggest that remodeling of gene expression at the level of translation control, and in particular the major initiation complex, eIF4F, and its cap-binding subunit eIF4E, provides cancer cells with a rapid mechanism to alter expression of key transcripts that are required for this process. In a seminal study, the Ruggero group generated the first eIF4E haploinsufficient mouse and demonstrated that these mice containing 50% less eIF4E are strikingly phenotypically normal, but resistant to cancer formation. Genome-wide translational profiling experiments identified not only downregulated but surprisingly also upregulated genes. However, the role of the proteins encoded from these upregulated genes in cellular transformation remains unknown. It is the aim of my project to characterize the molecular and functional features of these mRNAs that are surprisingly selectively upregulated at the translation level upon eIF4E haploinsufficiency during oncogenic insult. I will test different hypotheses to understand translational control mechanisms of these specific mRNAs regulated by the expression level of eIF4E. In addition to the molecular characterization, I will also identify their role in cellular transformation. In summary, this project will provide invaluable information into post-transcriptional regulation underlying oncogenic transformation.

Life as we know it depends on the membrane compartmentalization of the cell. Membrane proteins and their organizing scaffolding proteins maintain the flow of information and materials across the membrane. This project will advance the characterization of membrane and membrane associated proteins by developing novel electron microscopy and biochemical experiments enabled by new nanomaterials.
We focus on gephyrin, the major scaffolding protein in inhibitory synapses, and the glycine receptor (GlyR), which is one of the channels that gephyrin anchors to the synapse. Currently there is no detailed molecular mechanism to how gephyrin polymerizes and forms a scaffold, although the proteins and post-translational modifications that influence clustering in cells have been identified. A major puzzle is the variability and apparent geometric inconsistency of the scaffold. For instance, gephyrin’s hexagonal/trimeric binding geometry does not match the geometry of the binding sites within GlyR, which are arranged within a pentamer. GlyR dependent synapses have important regulatory functions in the brain stem and pain pathway and thus are potential drug targets. The emergence of a recent structure of a GlyR homopentamer has left us with questions about the structure of a physiologically relevant heteropentamer channel, the cytosolic part of the protein, and most importantly about conformational states of the channel that have not been visualized. These gaps in knowledge have hampered efforts to understand gephyrin-GlyR binding as well as to design new GlyR drugs.
This project will develop i) a nanosheet synthetic scaffold that can present gephyrin with its binding motif in a precisely defined geometry (Lau:Materials Science) and ii) nanoparticles functionalized with GlyR drug targets that provide high electron contrast labeling and bi-functional crosslinkers for the structure-function measurements (Yameen:Organic Chemistry). These nanomaterials will enable assays of gephyrin scaffold formation and GlyR complex formation, and electron microscopy functional mapping of GlyR (Mim:Structural Biology). The proposed research will provide insight into gephyrin-GlyR structure-function relationship and demonstrate new tools for biological characterization.

New genetic diversity originates in each generation through genome misreplication during germline development. Despite the fundamental nature of this process, observations of germline mutation have been indirect until recently, with the advent of coupled parent-offspring sequencing. However, because germline mutations are rare, and families are inherently limited in size, statistically robust estimates of germline mutation rates need to aggregate data from hundreds of distinct pedigrees, potentially integrating over biologically meaningful inter- and intra-individual mutational heterogeneity. Here, we propose a novel approach to study the individualistic component of germline mutation, using single-cell sequencing of gametes from a single individual. To provide proof of principle, will employ the model organism Ciona intestinalis, a hermaphroditic marine invertebrate with a small genome, which enables us to observe, with unprecedented depth, mutations occurring in both the male and female germlines in a single genetic background. By also sequencing offspring from the same individual, we will test the hypothesis that gamete sequencing can be used to measure mutations entering subsequent generations. Large samples of gametes will also enable us to study intra-individual mutational heterogeneity. Mutations shared by numerous gametes will be analyzed phylogenetically to provide a portrait of the lineage tree of germline stem cells, and of the dynamic nature of mutational processes along this tree. By extending this approach to the population scale, it should be possible to examine the patterns, and ultimately, the causes of inter-individual variation in germline mutation.

Essential ecosystem functioning and services are often the product of a stable community of interacting species. The degree of stability of these communities is to a large extent determined by the network structure of interactions between the species. Until now, research has focused on understanding how the static network structure influences the stability and robustness of the community in light of biotic and abiotic perturbations. These studies are generally limited to static snapshots of the interaction network. Yet, real ecological communities are in constant flux, and a complete understanding of long-term community dynamics must take into account the intrinsic ability of species to evolve and thereby modify their local interactions, thus changing the interaction network of the community.

This project addresses both theoretical and experimental challenges to bring together ecological and evolutionary dynamics in the study of complex ecological interaction networks. My work will offer predictions of how the network within ecological communities forms as species interacting in various ways come together in different environments. I will also validate and further expand these predictions using a recently developed microbial laboratory system. In this system I will be able to measure population dynamics, network structure as well as genetic change of high-dimensional microbial interaction networks. I expect my results will have broad implications for our understanding of how ecosystems adapt in ecological and evolutionary time to the increasing pressure of anthropogenic change.