Many sexually reproducing microorganisms are self-fertile, but in-breeding and out-breeding strategies have different outcomes on genetic diversity. Recent work from the Martin lab showed that self-fertile fission yeast S. pombe cells exhibit an exploratory polarity patch at their periphery, which allows them to preferentially pair with non-sister cells. However, the mechanism of this preference and its prevalence in nature are unknown. Here, I will study whether out-breeding is a general feature of fission yeast cell mating and dissect the molecular mechanism and genetic basis of this phenomenon. I will first develop a zygote FACS sorting method, which I will use together with high-resolution live-cell microscopy to study how pheromone concentration, cell size and transcriptional timing influence exploratory polarization and mate choice. I will then investigate whether out-breeding is a general feature amongst the recently sequenced collection of natural variants. If I detect pre-zygotic barriers amongst these strains, I will use pedigree analysis to probe for possible causative mutations. Finally, I will combine FACS sorting with experimental evolution to isolate strains favoring in- or out-breeding and uncover the genetic basis of the mating preference via comparative genomics/transcriptomics. Along the evolutionary experiment, I will also follow the exploratory polarization patterns by live-cell microscopy to relate the molecular mechanism of mating preference alteration with genetic changes. Finally, I intend to integrate the identified mutations into wild type lab strain to reconstruct the altered Cdc42 dynamic patterns and breeding preference.

Adult mammalian tissues are composed of heterogeneous cell types generated during development. To maintain homeostatic function, the identity of each of those cell types remains stable over time, and tissue renewal is normally sustained by dedicated stem cells. After injury, however, differentiated cells in many tissues can exit their quiescent state, acquire stem cell features and cross developmental identity boundaries to participate in the repair process. In the adult mammalian brain, injury-induced cellular plasticity (i.e. the acquisition of a new identity) remains poorly understood.
Here I plan to identify which brain cells exit their normally quiescent state upon stroke and study the molecular regulation of this process in situ. To this end I propose a novel strategy combining unbiased genetic fate mapping of stroke-generated cells with single-cell and spatially-resolved gene expression analyses. Specifically, stroke-generated cells will be marked with a genetic tag, identified using single-cell RNA-seq, and their contribution to repair traced over time. Next, we will use spatial transcriptomics to identify molecular triggers of cell plasticity, as well as their cellular source within their native environment. Finally, the identified molecules will be functionally tested for their capacity to enhance endogenous recovery after stroke by increasing cellular plasticity. This study will shed light on the cellular and molecular mechanisms governing the self-repair capacity of the brain.

Some animals have great regenerative capability to regrow whole bodies even from small fragments. This ability relies on their pluripotent adult stem cells, which are capable of self-renewal and differentiating into any missing body part. During regeneration, positional signals such as Wnt signaling are crucial for stem cell regulation and specification. In planarians, inhibition of Wnt signaling promotes formation of head-specific cell types. However, the molecular control of stem cell fate by Wnt signals is unclear. In part, this is due to the limitation of lineage tracing in the well-established planarian system. Here I propose to use the acoel, Hofstenia miamia, as an alternative system to dissect this process. Hofstenia is an early diverging bilaterian and evolutionarily distant to planarians, and yet possesses whole-body regenerative ability and similar patterning pathways. In particular, Hofstenia produces large amounts of accessible embryos that are injectable, making lineage tracing of stem cells possible. I will first identify conserved Wnt signaling components by generating spatial transcriptomes along the anterior-posterior axis. Next, I will test the role of Wnt signaling in stem cell fate specification by using RNAi of Wnt pathway genes together with single-cell RNA sequencing. These data will reveal distinct transcriptional landscapes and identify Wnt-responsive cells within stem cell populations. Finally, I will develop lineage tracing methods for live imaging of stem cell differentiation and behavior. Together, this study will provide a basic understanding of how stem cell fate is regulated by positional signals during regeneration.

Cellular signalling pathways usually involve cascade processes. For some of these processes, ensemble average analyses cannot answer biological questions originating from stochastic biomolecular interactions. Though single-molecule analyses are sometimes helpful but still the oligomeric forms of many membrane protein complexes remain controversial. Protein or polymer scaffold lipid discs have emerged as platforms for monodispersed proteins. It is difficult to manipulate them to study protein-protein stoichiometric interactions. To overcome this, and motivated from DNA origami approaches, I propose to use DNA scaffolds to form lipid nanodiscs (DSN). Using DSN I will develop a novel programmable platform to study membrane protein-protein interactions in an isolated environment. First, I will create DNA nanorings, functionalised along their inner circumference with hydrophobic groups to hold a lipid membrane. Computational approaches will be used to optimise the designs along with experimental characterisation. The functional nature of DSNs will be established with an embedded P450 oxidoreductase enzyme. To develop a programmable platform, embedded proteins will be tethered at the edges of DSN with DNA linkers. Using DNA strand-displacement methodology proteins will be released to diffuse freely and interact in the membrane. As a proof of concept, an experiment will be set up using two formyl peptide receptors. Interactions will be analysed using AFM, single-molecule spectroscopy etc. Such platform, if achieved, could prove revolutionary for single-molecule analysis of protein oligomerization for studying, e.g., signal pathways, interaction energetics, metabolism etc.

Sleep has roles in memory consolidation. Memory consolidation is thought to include systems consolidation which reflects communication among different brain regions, as well as synaptic consolidation at the local circuitry level. I found systems consolidation during sleep among separated regions in cerebral cortex (Miyamoto et al., Science, 2016), however, synaptic consolidation pattern is still unknown. Net cortical synaptic strength changes homeostatically through wake/sleep cycle: average synaptic strength increases during wakefulness and decreases during sleep (Tononi & Cirelli, Neuron, 2014). While skill learning is known to potentiate subsets of synapses in motor cortex for encoding skill memory (Hayashi-Takagi et al., Nature, 2015), plasticity of those synapses through sleep is unknown. If sleep depresses memory-encoding potentiated synapses, it may result in impairing memory consolidation. If sleep depresses other non-potentiated synapses relatively, it may enhance signal/noise ratio and memory consolidation. Here I will use complex-wheel task for studying synaptic mechanisms of sleep-dependent skill memory consolidation in mice. Through skill learning and sleep, I will perform in vivo two-photon imaging in motor cortex to visualize fluorescence tagged AMPA receptors, since the expression of these excitatory glutamate receptors is an established index of synaptic strength. To visualize plasticity in synaptic population, I will analyze time-course of fluorescence intensity change at single-synapse resolution. This study will help clarifying how sleep favors both memory consolidation and synaptic homeostasis.

The formation of higher order chromatin structure is crucial for DNA replication, transcription, recombination, repair and chromosome segregation. The cohesin-complex is thought to contribute to formation of chromatin loops and of topologically associating domains (TADs) by entrapping two different regions of the same chromatids. Its binding along genomic DNA is thought to be controlled by two of cohesin’s cofactors, CTCF and Wapl. Cohesin has been proposed to regulate the long-range chromosomal cis-interaction by a “loop extrusion” mechanism, in which cohesin can regulate chromatin loop formation by translocating along genomic DNA. However, how cohesin controls chromatin structure remains unclear. To address this question, I will first establish assays based on FISH, Cas9-FISH in living cells, oligopaint strategy and super resolution microscopy (SRM) to visualize chromatin loops and TADs. Second, I will employ these assays and Hi-C to analyze chromatin structure in cells in which the genomic distribution of cohesin is experimentally altered. For this, I will use knowledge from my host lab which recently discovered that the distribution of cohesin in mammalian genomes depends on the DNA binding protein CTCF, the cohesin release factor Wapl and transcription. Third, I will monitor the transition of cohesin re-location and of conformational change of chromatin induced by rapid degradation of Wapl and CTCF in real time. I will then test prediction of the loop extrusion model by comparing my experimental data with the simulations recently described in the literature.

How genetic programs regulate mammalian morphogenesis is one of the central problems in developmental biology. Digit patterning, a representative model to study mechanisms of morphogenesis, is regulated by multiple factors including cytokines and Homeobox-containing genes, or Hox genes, particularly Hoxa13 and Hoxd13. Recently, it has been illustrated that a reaction-diffusion model can explain this patterning in which the interactions of Sox9, a transcription factor, and WNT and BMP, cytokines, generate the periodic digit pattern. Interestingly, this periodic wave is modulated by FGF signaling and Hox genes. However, little is known about the molecular basis how Hox genes contribute to the proper digit patterning by regulating these interactions. In this project, to elucidate the role of Hox genes in mammalian digit patterning and autopod tissue identity, I will conduct integrative analyses including genetic, biochemical, and computational approaches. Firstly, single-cell RNA sequencing will be performed on both of wild type and Hoxa13 and/or Hoxd13 knockout mice to discover a gene network consisting the periodic wave pattern and analyze dosage effects of Hox genes on it. Secondly, HOXD11/D12/D13 will be epitope-tagged with genome editing tools. Thirdly, they will be immunoprecipitated to comprehensively analyze their binding specificity to the genome and other proteins. Lastly, these data are integrated with the reaction-diffusion model and investigate mechanisms of the modulation of BMP and WNT signaling by Hox genes to generate the periodic digit pattern, and consequently, establish the identity in autopod regions.

A myocardial infarction in mammals typically displays limited regeneration and instead produces significant cardiomyocyte death. This loss of contractile cells is instead replaced with a permanent non-contractile fibrotic scar. In contrast, the pro-regenerative zebrafish displays increased cardiomyocyte proliferation and fibrotic scar resolution following injury. In order to find candidate genes, other labs have RNA sequenced whole mammalian hearts prior to and after injury. The spatial resolution however is lost, and other unrelated genes may mask candidate genes responsible for regeneration. This project will use a cardiac injury zebrafish model and at critical stages during regeneration, apply a novel genomic technique “RNA Tomography (Tomo-seq)” to spatially resolve gene expression at a genome-wide level. In addition, Tomo-seq concepts will be applied to develop a new genomic technique to examine the regulatory regions surrounding genes. Bioinformatic analysis will identify candidate genes and their regulatory regions associated with different phases of regeneration, and spatially correlate them with different areas in the injury site. Finally potential targets are functionally validated in zebrafish. By identifying pro-regenerative candidate genes at a spatial-temporal resolution in zebrafish, we can further understand and possibly modulate signals to increase the limited regenerative capacities in mammals.

The gut is under constant surveillance by immune cells, which ensures the homeostasis of commensal gut microbiota whilst conferring protection from invading enteric pathogens. B lymphocytes are central to maintaining balanced responses through production of huge quantities of the Immunoglobulins IgA and IgG. Ig production is the outcome of specialized germinal center (GC) responses, which form spontaneously in gut-associated lymphoid tissues in response to chronic exposure to microbiota. Despite being the precursor to Ig secretion, gut GC responses are entirely uncharacterized. Recently, when mesenteric lymph nodes (mLNs) were visualized using multicolor fate mapping (MCFM) large proportions of spontaneous GCs became monoclonal, an unexpectedly specific outcome in the face of exposure to such a diverse range of antigens. We intend to combine MCFM with in vivo models of commensal colonization to track the kinetics and clonal diversity of GC and Ig responses in mLNs through multiphoton microscopy. After GC isolation, we will use sequencing approaches to determine which bacterial species predominantly induce clonal GCs and map subsequent Ig specificity. Following this, we will investigate GC responses during enteric infection. We are especially interested in whether newly activated pathogen-specific B cells utilize pre-existing spontaneous GCs for their own affinity maturation, and how this is altered by composition of the current or past bacterial community. These data could have wide-reaching implications for both the fundamental biology of gut immunology as well as universal principles of GC dynamics in the presence of multiple antigens.

Previous studies have suggested that embryogenesis is robust to environmental, genetic, or stochastic perturbations. However, little is understood about the mechanisms that enable multicellular organisms to develop robustly, and about the extent to which – and the way in which – they changes their developmental patterns in response to harmful perturbations. Especially, how organisms adapt to environmental stresses during embryogenesis is not well defined, partly because in most cases experimental organisms have been cultured under favorable conditions. Considering that organisms usually live in harsh conditions in their wild habitat, it is highly likely that they have evolved the undiscovered ability to deal with environmental stresses.
In this project, I will employ a high-performance cell tracing system and sophisticated computational techniques to accurately track the positions of ALL cell nuclei in developing embryos of the nematode Caenorhabditis elegans. By this approach, I will comprehensively examine the effects of nutrition deficiency on cell division and differentiation during embryogenesis. Additionally, I will elucidate the developmental basis of transgenerational starvation response. After the detection of the developmental differences caused by nutritional stress, I will clarify how the differences are induced (or suppressed) at the molecular level using powerful molecular genetics and mathematical analyses.
The success of this project will provide fundamental insights into embryogenesis and might lead to understanding of the mechanisms underlying teratogenesis, spontaneous abortion, and neonatal death in nutritionally challenged humans.

The control of phenotypic variance, known as phenotypic robustness, is a fundamental characteristic of living organisms. This assures a relative stability of phenotypes even when organisms are exposed to stressful internal or external environments. However, it has been extensively documented that stressful conditions decrease organismal robustness resulting in an increase of phenotypic variance at the population level. Although ubiquitous in biology, the underlying genetic factors driving such shift in variance are poorly understood. This project specifically addresses this question by studying the genetic basis of gene expression variability in Drosophila melanogaster exposed to dietary stress. For this, genomic loci associated with transcriptional variability will be identified (variance-eQTL or v-eQTL); these are loci whose allelic state predicts the amount of variability around the expected mean. Recently developed outbred Drosophila populations will be used; this will allow the exploration of more genetic diversity than ever before. Thousands of flies will be exposed to two dietary conditions, standard food and high-sugar diet. v-eQTL maps will be generated for both conditions and will be compared with two objectives: 1) to reveal cryptic genetic variants only relevant under stress; and 2) to explore how stress disturbs the co-expression networks present in normal conditions. The analysis of this data will reveal the genomic loci associated with variation in transcriptional robustness between individuals, and will ultimately offer a deeper evolutionary and medical understanding of phenotypic robustness.

Despite its significance, the transgenerational inheritance adaptive behaviors through germline modifications are poorly established and understood in terms of the underlying biological mechanisms. Here, by employing a novel social learning paradigm in Drosophila I aim to understand how predator-threat information is successfully inherited by subsequent generations through an epigenetic reprogramming of parental germline cells. In fruit flies, exposure to an endoparasitoid wasp elicits preferences for alcohol containing food and oviposition depression. Visual input mediated predator presence induces germline apoptosis resulting in both acute and learned oviposition depression in wasp-exposed females. Strikingly, daughters of these exposed females are preprogrammed to prefer egg-laying in alcohol enriched media even in the absence of predator. In addition, the altered food preference behavior in response to an anticipated predator-threat persists for up to five generations. These interesting observations set us to understand the mechanisms for predator-threat induced social learning in terms of (i) cellular and molecular signals, which trigger apoptosis in developing oocytes of wasp-exposed females, and (ii) epigenetic reprogramming in exposed flies that alters germline information conferring transmission of behaviors to subsequent generations. While the current proposal questions the classical conception of learning, the outcomes of this study in terms of the neurophysiological basis for inheritance of social behaviors will radically transform the way we think about learning, memory and behavior.

Learning to associate fear with stimuli predicting aversive outcomes is key for adaptation and survival. However, experiencing fear in maladaptive contexts can be crippling and disabling. Around 4.5% of the global population suffers from anxiety disorders such as post-traumatic stress disorder, generalized anxiety disorder and specific phobia. One of the most influential ways to overcome feared associations is extinction, which consists of exposure to previously feared stimuli in neutral circumstances, thus reducing the fear response. Extinction, frequently manipulated in the laboratory context, putatively stands at the base of effective psychological exposure therapy. In this proposal, I plan to adapt a new experimental paradigm, targeted memory reactivation (TMR), to strengthen fear extinction in humans. TMR, which has effectively facilitated several different types of memory, involves the subliminal presentation of previously associated cues during slow-wave sleep. Employing the lab's expertise in TMR procedures, I will try to strengthen extinction in a controlled setting and reveal the underlying neural mechanism by which TMR affects learnt memories using neuroimaging techniques (electroencephalogram, functional MRI) and electrophysiological recordings (electrocorticography). With this project, I aspire to bridge between the cognitive literature on the features and limitations of human memory and the more mechanistic animal-based literature on consolidation and memory formation. This can only be achieved by combining a well-controlled and manipulated behavioral paradigm together with state-of-the-art high-resolution brain-imaging techniques.

Recent genome-wide methods of mapping functional DNA elements have uncovered a vast landscape of genomic loci that have the characteristics of enhancers. However, the identification and mapping of this complex putative enhancer landscape does not link specific genes to their enhancers, nor does it prove any causal relation between the putative enhancers and gene activation or repression. In this project I will combine a powerful screening platform that harnesses CRISPR-cas9-based screens to test the function of hundreds of enhancers, in isolation and combination, which putatively regulate gene expression of genes associated with ricin sensitivity. In the first part of the project I will identify all potentially "active" enhancers during the cell response to ricin. In the second stage I will generate a CRISPR-cas9 library to disrupt the activity of all enhancers in isolation or in designed combinations, and measure the change in the cell response to ricin. In the third phase I will focus my attention on dozens of individual enhancers that affect the cell response to ricin, and using ATAC-seq, RNA-seq and 4C-seq methods I will determine the regulatory mechanism for these individual enhancers. In the final part of the project I will build a model trying to explain enhancer behavior in gene regulation. This work will provide the first genome-wide, high-throughput investigation of enhancer function on gene regulation, and will begin to unravel the combinatorial effects of enhancers by modulating them in their native genomic location. In short, this work will put regulatory genomics on firm functional or mechanistic footing.

I seek to understand how the patterns of animal movement seen in nature emerge from the combined actions of many individuals. For group-living species, this process occurs over two scales of organization: individual decisions emerge into coordinated group movement, and the movements of groups drive population-level patterns of space use. To understand how these processes connect, we need to collect and analyze data at both of these scales, yet doing so within a single system has remained a challenge. I will address this challenge using the meerkats of the Kalahari Desert as a model system. Meerkats (Suricata suricatta) live in stable groups that remain together as they forage over large distances. Leveraging recent technology, I will collect data on the fine-scale movements and vocalizations of all individuals within meerkat groups. Through probabilistic modeling, I aim to determine what rules govern where individuals move, and how these rules give rise to coordinated group movement. Key questions include how meerkats’ calls allow groups to maintain cohesion and decide where to go, and whether certain individuals have disproportionate influence over group movements. At the group level, I will use movement and demographic data collected over 20 years at the Kalahari field site to determine what factors drive group movement, including habitat features, memory, and interactions with other groups, to understand how aggregate space-use patterns emerge. Bringing together a wealth of data from a long-term field study with newly-available high-resolution tracking methods will enable us to link the multiple spatial and temporal scales driving animal movement patterns in the wild.

The ability to precisely edit and manipulate the genome is critical for biological research and the treatment of human disease. The power of such tools is highlighted by advances in CRISPR-Cas9 mediated gene editing, but there is continual need to develop new tools. The discovery of diverse genome manipulating enzymes throughout nature highlights the importance of basic biological findings and the wealth of natural systems that may be amenable for repurpose in human cells.

One compelling biological system is the programmed genome rearrangement of the ciliate Oxytricha which involves the fragmentation and joining of over 225,000 DNA pieces to form functional genes. Remarkably, gene coding sequences are often scrambled and must be reordered during reassembly, a process thought to be mediated by RNA templates. This phenomenon warrants further investigation and hints at the existence of remarkable genome manipulating tools. Despite our knowledge of the outcome of rearrangement, how this process happens and the proteins involved is not well understood.

The objective of my research is to identify the key proteins involved in the programmed genome rearrangements of Oxytricha and to elucidate the molecular mechanism of this process.
I aim to:
1) characterize changes in the transcriptome during programmed genome rearrangement,
2) develop functional genetic tools to screen for required factors, and
3) to recapitulate genome rearrangement in vitro using cell extracts.

This work aims to elucidate the identity and basic biological properties of the enzymes involved in this process with the long-term objective to adapt and harness identified components for use in human cells.

Neurons are characterized by their unique morphology, which requires efficient sorting and transport of cargoes over long distances. One class of molecules that affect neuronal development, function and survival are neurotrophins. Neurotrophic growth factors are target tissue-derived cues that bind to their cognate receptors on the pre-synaptic membrane and are transported retrogradely to the soma, where they induce signalling events which control transcriptional programs governing neuronal differentiation and survival. Despite their importance in neuronal homeostasis, the regulation of uptake and transport of neurotrophins and related growth factors is presently unknown. Recently, the host laboratory reported that the tetanus neurotoxin requires the synaptic basement membrane protein nidogen for its entry and axonal transport in mammalian motor neurons (Bercsenyi K, et al., 2014). Importantly, the tetanus toxin is known to enter the same intracellular trafficking pathway entered by neurotrophins (Salinas S, et al., 2010). In this context, this proposal is aimed at investigating the question: Does the synaptic basement membrane play a role in regulating the uptake, transport and biological activity of neurotrophins and related growth factors? As part of the research plan, I intend to identify growth factors that bind to nidogens and other components of the basement membrane at the mammalian neuromuscular junction, genetically modulate this binding and study its effects on neurotrophic factor endocytosis and trafficking, with a view to understanding the physiological relevance of this biological process.

Immunogen specific elicitation of HIV-1 broadly neutralizing antibodies has been proposed as the key step of an AIDS vaccine. Unfortunately, such vaccination strategy remains elusive. Priming B cell receptor germline precursors with specific engineered immunogens has been already proposed to be a potential strategy to generate bNAbs. Hence, characterizing and identifying potential germline precursors of bNAbs in the B cell repertoire of patients that has been exposed to native HIV-1 or a HIV-1 mimicking immunogen for a short period of time is a necessary step for the iterative design of successful bNAbs generating HIV-1 immunogens. In this project, using the PGT121-class germline-targeting Env SOSIP trimer, I will isolate low-affinity binding germline precursors of the bNAb PGT121 via particulate presentation from samples from (1) HIV-1 freshly infected patients and from (2) HIV-1 vaccinated patients. Using single cell RNA-seq, I will compare the antibody sequence of the isolated PGT121-class germline precursors with the sequence of their corresponding mature bNAb PGT121. I aim to obtain unprecedented information about the potential generation of germline precursors of bNAbs in patients after short exposure to HIV-1. The information obtained in this project will be crucial to design vaccine immunogens that can generate immune protection against HIV-1.

Our understanding of how central and peripheral processes interact and affect balance control is limited. This project will elucidate the interplay of brain and brawn in balance control. It will use a comprehensive multi-faceted approach of laboratory and daily-life assessments using body-worn sensors, to test the hypothesis that central processes can compensate for a slowing down of peripheral processes. The overall goal of this project is to advance current knowledge by disentangling central and peripheral contributions to balance control.
Specifically, the goal of the project will be reached by addressing two major aims:
Aim 1: Establish how the interplay of central and peripheral impairments affects balance control;
Aim 2: Identify whether prefrontal brain areas govern this interplay.
This project is novel in that it will be the first to use body-worn sensors to study underlying mechanisms for balance problems in the home environment during every day life. The outcomes are expected to inform personalised interventions to prevent and restore balance problems. The novel sensor-based techniques developed in this project provide tools to identify individuals that may benefit from such interventions and can also be used to evaluate the effectiveness of these interventions.

The presence of immune cells in decidua during early to mid-pregnancy creates a specific environment at maternal/fetal interface, which is crucial in preventing rejection of the fetus. Although unique receptors and interactions have been identified for decidual immune cells, the full spectrum of their responses is still not completely understood.
In my post-doctoral research, I will study the cell types, their transcriptional regulation, and their cellular interactions established in maternal/fetal immune cross-talk. This research will be addressed by analysing the transcriptomes of decidua-infiltrating immune cells at a single-cell level. With these results and using bioinformatics tools, I will reconstruct the specific niche required to ensure success at this first stage of pregnancy.
I will pay special attention to the unique cell-cell communication occurring in this particular context. To study cellular interactions, I will develop “CellCommDB”, a curated database of known ligands and receptor proteins involved in cell-cell communication. The innovative part of “CellCommDB” will be the integration of the multiple levels of complexity found in protein receptors, like the fact that many cell surface receptors consist of multiple different protein chains.
Overall, my results will help to decode the paradigm of maternal/fetal allogenic co-existence, and to design novel, targeted therapies to increase pregnancy success. Moreover, it will pave the path for further studies to analyze the unique cellular interactions that defines the function and regulation of tissues.