The hippocampal formation is determinant in the establishment of episodic memories and in spatial navigation. The strong correlates of principal cells in this area with the position of the animal in its environment would be the neurophysiological substrate of such behavioral capabilities: in the hippocampus cells fire in one particular position whereas in the entorhinal cortex, the main output and input structure of the hippocampus, cells fire at the nodes of a grid covering the environment. However, both lesions and neurophysiological studies have failed so far to determine the hierarchy of integration of spatial information between the two structures. In this project, we propose to use recently developed optogenetic tools to selectively inhibit neurons from one structure that directly project to the other while network activity will be monitored with dense extracellular recordings. Moreover, the relationship between the two structures could depend on the remoteness of the spatial memory of the particular environment the animal is exploring. To test the effect of spatial learning on this interaction, we propose to compare the effect of selective inhibition in familiar environments and in new places previously unreachable. Thus, this project will shed new light on the neurophysiological substrates of spatial navigation and of the formation of long-term episodic memories.

The cerebral cortex is the latest structure to appear in the evolution of the nervous system, and its circuitry constitutes one of the most complex known arrangements. Studies of model circuits in various CNS regions have identified a handful of molecules that participate in basic guidance decisions that direct axons to their targets. To address the principles of axon pathfinding in the elaborate circuitry of the cerebral cortex, I propose to examine how callosal axons are guided to homotopic regions of the contralateral cortex after crossing the midline.

While the paths of homotopic projections display great heterogeneity, they follow a simple common rationale, backtracing their initial trajectory, from the soma towards the midline, to form a bilaterally symmetric projection. This simple architecture may allow the molecular dissection of the cell-surface mechanisms linking positional cues and cellular signaling.

Combining retrograde tracing with FACS purification, neurons projecting to distinct cortical areas can be isolated. Using surgical methods, neurons projecting to one area will be ectopically transplanted to assay commitment of projection route. In a complementary approach combining post-sort cell culture, membrane proteins differentially present in neurons projecting to different cortical areas will be identified using quantitative mass spectrometry. Candidate molecules arising from the proteomic screen will be validated using in vivo knockdown and tracing to examine the fidelity of homotopic axon projections. The aim of this study is to identify molecular mechanisms that allow axons to navigate the cortical plane and form circuitry within the cerebral cortex.

Phenotypic evolution results often from changes in gene expression patterns, which are caused, usually, by alterations in cis-regulatory regions. Cis-regulatory regions integrate inputs from multiple gene-regulatory networks by encoding transcription factor binding sites. The Drosophila shavenbaby (svb) gene provides a superb model for studying cis-regulatory structure, function, and evolution. The svb gene encodes a transcription factor that directs cuticular trichome development. Six enhancer modules located in the cis-regulatory region of svb together recapitulate the entire svb embryonic expression pattern. Multiple nucleotide changes in these enhancers have led to the evolutionary loss of many trichomes in D. sechellia, a sibling species to D. melanogaster. Surprisingly, none of these nucleotide changes map to known transcription factor binding motifs.
The central aim of this study is to identify the transcription factors that bind and regulate the D. melanogaster svb enhancers in vivo. I plan to extend existing high-throughput methods of chromatin immunoprecipitation and mass spectrometry to screen the svb enhancers in vivo for their associated proteins. I will complement these experiments with a genetic screen using lines carrying RNAi constructs targeting Drosophila transcription factors, to identify factors regulating svb enhancer activity.
My project will lead to a deeper understanding of the relationship between cis-regulatory structure and function. In addition, my work will identify the individual transcription factors that bind differentially to nucleotide sites in D. melanogaster and D. sechellia and that have contributed to morphological evolution.

When reaching for a cup of coffee, people are strikingly variable in their performance but almost never spill. This contrast leads to a fundamental question: why are inconsistent movements so consistently successful? Recent theories of sensorimotor control suggest that this inconsistency reflects an extremely sophisticated control process that preferentially corrects those errors that are relevant to succeeding at the task. The proposed research will test the merits of this concept, termed the minimum intervention principle, in the context of human object manipulation. We will determine whether the nervous system is more sensitive to some errors than others and explore whether the resulting corrections are consistent with the minimum intervention principle by comparing our results to biologically-realistic mathematical models. Critically, we will induce errors with a novel experimental approach whereby disturbances are injected directly into the sensorimotor apparatus via electrical muscle stimulation rather than the traditional approach of applying an external load to the hand. This approach provides several important advantages. First, we expect that internal perturbations will mimic sensorimotor noise and thus provide insight into how the nervous system compensates for its own disturbances. Second, since the subject will not be constrained by the experimental apparatus, we will be able to study many naturalistic object manipulation tasks. And last, since our approach is compatible with a range of physiological techniques including functional magnetic resonance imaging we will be able to elucidate the underlying neural mechanisms of the minimum intervention principle.

During development tissue remodeling leads to shape changes of organs and parts of the organism: for example tissues can invaginate, elongate, or stretch. Distinct signaling pathways regulate these behaviors through the control of the actin cytoskeleton and of myosin-based tension required for force generation and cell shape change, as well as the control of cell adhesion. Although some of the basic mechanisms responsible for shape changes are known, it is not understood how they are coordinated within groups of cells or tissues. What is the spatiotemporal pattern
of forces that orient tissue morphogenesis? What is the nature of the generated forces and how do they originate? I propose to use the ventral furrow formation of the Drosophila embryo as a model system to investigate how different tissues coordinate and interact to drive morphogenesis.

Number of biological processes (such as replication, splicing, transcription, translation, transport, degradation…) involve the generation of mechanical forces and torques. These functions are performed by molecular motors –enzymes that couple chemical energy sources such as nucleoside triphosphates or electrochemical gradients into mechanical work. Many bacteriophages and certain animal viruses actively package their genomes into small proteinaceous capsids, compressing the nucleic acid to near-crystalline densities and macroscopic pressures. At the heart of this process is a multimeric molecular complex known as the packaging motor. This machine catalyzes the hydrolysis of adenosine triphosphate (ATP) and converts the chemical energy released into the mechanical work necessary to surmount the large energetic barriers associated with genome compaction. Phi 29 presents the ideal system for the study of DNA packaging for several reasons: it is one of the simplest of the double-stranded DNA bacteriophages, it has been extensively characterized via bulk techniques and exhibits a slow and homogeneous translocation. In the frame of this HFSP fellowship, I will utilize the tools of single-molecule manipulation (such as optical tweezer) to study the fundamental mechanical processes that are at the core of enzymatic activity. A detailed understanding of structural and functional dynamics in the packaging motor of Phi 29 promises to provide insight into the general mechanism by which these crucial cellular ATPases convert the chemical energy released in ATP hydrolysis into the mechanical work necessary for their varied biological tasks.

The dynamics of hippocampal place cells changes dramatically according to the behavior of the animal. During a run through a cell’s place field, spikes are emitted at progressively earlier phases of the underlying theta rhythm. During awake immobility and slow-wave sleep, rapid orderly activation of place cells expresses a possible or experienced trajectory of the animal (replay). When the animal transiently stops at turning points in a T-maze, sweeps of place cells activity probes all possible routes from that point. Moreover, non-spatial context experienced during running contributes to the encoding and long-term storage of newly acquired memories following sleep. Common mechanisms underlying these dynamical properties are poorly understood and cannot be accommodated by existing network models. We propose that such a diverse range of behaviors can be explained by endowing a network, storing a map of the environment in its recurrent collaterals, with short-term synaptic depression. We shall combine theoretical analysis, model-based experiments and data analysis to (i) test the hypothesis that short-term synaptic plasticity plays a fundamental role in the diverse hippocampal regimes of activity (ii) investigate how non-spatial cues (e.g. auditory or olfactory) triggers and affects the replay events in coordination with cortical inputs to the hippocampus. The interaction between this novel theory of hippocampal dynamics and experiments will provide a deeper understanding of spatial coding and memory mechanisms in the hippocampus.

A long standing problem in evolutionary biology is determining of the mechanisms that produce developmental divergence and genetic incompatibilities [1]. Several approaches have been established to address this problem: a) identification of loci responsible for performance differences in F1 offspring [2-4], b) expression analysis in F1 hybrids to quantify regulatory additivity [5-6] or the location of regulatory differences (cis/trans) [7-8], and c) comparative analysis of conserved traits in distantly related species which often shows divergence on gene function [9-10]. Although these three approaches focus on understanding species differences at various levels, it is unclear how they relate to one another. Here a novel integrated approach is proposed, covering the complexity of phenotypic levels (transcriptome and morphology) and comparing micro and macro evolutionary time scales. We study fruit development, a trait that can be related to fitness in plants. In a microevolutionary context, the fruit transcriptome will be studied in F1 hybrids of closely related lineages and analyzed for cis/trans regulatory differences and additivity, while fruit features will be quantified using morphometrics. This will be done both within Arabidopsis accessions (conserved fruit morphology), as well as tomato cultivars (divergent morphology). This data will allow a functional macroevolutionary comparison in the context of gene expression additivity and cis/trans regulatory differences within their known developmental networks. The proposed study will provide insights into the evolution of traits and their underlying developmental networks between closely and distantly related lineages.

Multipotent stem cells employ unique histone modification mechanisms to confer their ability for rapid and divergent cell fate decisions upon environmental cues. However, it remains unclear how histone epigenetic modifications work in largely quiescent tissue stem cells to modulate fate determination. Recently, Dr. Tumbar’s laboratory found that quiescence is a state in which hair follicle stem cells (HFSCs) are assigned a self-renewal or differentiation/cell death fate. Co-incidentally, at this stage histone repressive marks such as H3K27me3 are globally erased, and histone methylases mRNAs, such as Ezh2, are down-regulated. Here I am hypothesizing that the low level of epigenetic marks of tissue stem cells in quiescence confers them plasticity for subsequent flexibility in fate acquisition. To test this, I will transiently induce Ezh2 expression and block removal of epigenetic marks at two stages: (1) in quiescence, prior to fate decisions; (2) in proliferation, when hair follicle stem cells self-renew their pool. If erasure of the H3K27me3 marks were important at the quiescent but not the proliferative stage, the first but not the second Ezh2 induction would impair stem cell differentiation. In addition, ChIP sequencing analysis will reveal gene loci regulated in this process, with predicted impairment of de-methylation in differentiations specific genes upon expression of Ezh2 in quiescence. These approaches will test a model in which the recognized quiescence of tissue stem cells is functionally coupled with tissue stem cell plasticity and fate acquisition in tissue homeostasis.

AMPA, kainate and NMDA are members of the glutamate receptor family of ligand gated ion channels, these receptors are activated by glutamate the mayor excitatory neurotransmitter in the central nervous system and the presence of this receptors in the brain is essential for normal brain function, and higher order nervous system process including learning, memory and pain perception. Basically the members of this family form tetramers and each subunit is formed by four transmembrane domains, intracellular caboxy-termini and a extracellular amino-termini which form the ligand binding domain (LBD). The LBD are divided in in two lobes, the D1 and D2, and the movement of this lobes during the gating allows the channels to transit between active and desensitized state. The rapid kinetics of glutamate receptors enable them to follow the release of glutamate at rates approaching several kilohertz, but the transition between the active and desensitized states of the channel, limits their temporal sensitivity and underlies some forms of short-term depression. This type of synaptic depression contributes to neuronal computation at some synapses. However, these receptors have complex structures and differences on the conformations of activated and desensitized states is unknown. We hypothesize that there exist structural differences between those receptors on the activated and desensitized states who determine this differences on kinetics. In this application we propose a series of chemical, electrophysiological and structural experiments to find the structural determinants of activation and desensitization on AMPA and kainate receptors.

MicroRNAs (miRNAs) act as regulators of development and physiology through their ability to regulate the expression of most cellular genes. Although individual miRNAs can bind to up to a hundred of target genes, it can only bind to one RNA target at time. Thus, miRNAs that have a larger repertoire of target genes will down-regulate each individual target gene to a lesser extent than miRNAs with fewer targets. Since a single miRNA’s effectiveness is influenced by the concentrations of its target genes, it follows that the altering the expression levels of individual targets will have repercussions on all the other targets of that miRNA. Such alterations are exemplified by those commonly observed in cancer where oncogenes and tumor suppressor genes are frequently up-regulated and down-regulated, respectively.
In this proposal, it is hypothesized that any given RNA transcript, through its ability to compete for miRNA binding, can regulate the expression of other RNAs, in trans. Moreover, these non-coding functions of endogenous RNA transcripts have evolved as physiologically and pathologically relevant factors in multicellular organisms. The goal of this research proposal is to continue the characterization of the cellular relevance and physiological importance of RNA:miRNA networks by using complementary in vitro, in vivo and rational experimental or bioinformatic approaches. The investigation of these processes will shed light on novel and untapped regulatory mechanisms within the cell. Knowledge of such processes will permit the development of new means and methods to alter gene expression for therapeutic purpose and/or scientific discovery.

Ischemic heart disease associated with acute loss of cardiomyocytes is a major cause of heart failure. While an ideal cell-based therapy to replace the lost cardiac muscle has not yet emerged, recent reports on factors able to stimulate the proliferation of mature cardiomyocytes, which normally have little self-renewal capacity, has stirred hope for new therapeutic approaches. An inflammatory microenvironment is deleterious to cardiac repair by affecting cardiomyocyte survival. Macrophages in particular play a major role in the inflammatory response. However, macrophages constitute a heterogeneous population and their roles in cardiac repair are not fully understood. Here, I propose experiments that will define the function of distinct macrophage subtypes in cardiac regeneration at cellular and molecular levels by exploiting the zebrafish, a model organism that has a well-developed immune system and can regenerate its myocardium. First, I will characterize the heterogeneity of fish macrophages with a high-resolution expression profiling technique. Second, I will generate new transgenic lines that will allow monitoring of the recruitment and phenotypic alterations of each macrophage subtype in the injured heart. Third, I will employ cell-type specific inducible ablation and gene expression approaches to address the requirement of each macrophage subtype during the multistage process of cardiac regeneration. Finally, I will generate differential expression profiles of macrophages in healthy and injured heart to identify candidate molecules that modulate the regenerative ability of cardiomyocytes, and I will analyze their contribution using loss-of-function approaches.

Intrinsically disordered proteins play crucial roles in many biological processes. In paramyxoviruses such as Sendai and measles virus, transcription and replication require an interaction between the disordered tail of the nucleoprotein (N) which encapsidates the genomic RNA and the C-terminus of the phosphoprotein (P), a cofactor of the core polymerase L. The molecular mechanisms of protein recognition and interaction in the absence of a folded structure are not well understood, and it is not clear how interaction signals can be transmitted to distant effector domains via disordered sequences. To shed light on these phenomena, a multidisciplinary structural biology approach will be employed, applying solution-state NMR in conjunction with small-angle X-ray and neutron scattering, solid-state NMR and molecular modeling to the protein domains in isolation as well as in complex and in the context of the full-length nucleocapsid. The aim of this project is to obtain a complete picture of structure and dynamics in the intrinsically disordered regions of the N and P proteins of Sendai and measles virus. Their highly dynamic nature will be accounted for by providing a description in terms of explicit and statistical ensemble properties.

The vast majority of studies in systems neuroscience focus on the brain’s response to a stimulus or task. However, the brain is very active even in the absence of explicit input or output. This intrinsically generated activity accounts for most of the brain’s energy usage and displays a highly coherent spatio-temporal structure that is altered in a number of brain diseases, yet its role in brain function remains largely unknown. In particular, its impact on the neural processing of stimuli and its importance for behaviour are not yet understood. I intend to study this spontaneous brain activity measuring local field potentials from large multi-electrode arrays in combination with single-unit recordings in awake monkeys. The research revolves around 4 key goals. First, I will analyse the spatio-temporal structure of spontaneous activity at the scale of the entire brain, while the monkeys are at rest. Second, I will determine the precise nature of the interaction between spontaneous activity and activity evoked by stimuli, while the monkeys are performing a simple task in which stimuli are repeatedly presented. Third, by manipulating the monkey’s task expectations, I will test the extent to which the structure of spontaneous activity reflects expectations about future stimulus events. Lastly, I will complicate the monkey’s task even further to study the role of spontaneous activity in determining the monkey’s behaviour. For the data analysis of these studies I will apply newly developed computational methods, in collaboration with a computational neuroscientist. Taken together, these studies will yield important insights into how and why the brain is active spontaneously.

Angiogenesis is a critical process in vascular development and diseases such as cancer and ischemic coronary artery disease. Angiogeneic growth factors stimulate proangiogenic signals of endothelia cells (ECs) via ligand-induced dimerizations of receptor-tyrosine kinases (RTKs) and modulate several key processes in ECs including proliferation, migration, and assembly. Despite remarkable advances in this field, however, it is still unclear how these biomolecular regulators coordinate highly complex phenotypic events such as vascular sprouting, migration, branching, and network formation. To understand molecular mechanisms of angiogenesis with high spatio-temporal complexity, it is required to monitor angiogenesis in real-time at the single molecule level with multivariate analytical capability. Here, I propose a research program that investigates molecular mechanisms of angiogenesis at the single molecule level via in situ surface plasmon dark field microscopy. Unlike conventional microscopy that only provides a static assessment of cell status, the proposed surface plasmon microscopy allows us to visualize single molecule events of the signaling molecules continuously over the entire angiogenesis process as well as morphogenetic development. With this new technique, specifically, I will challenge the following topics: 1. In situ monitoring RTK dimerization of individual ECs and its correlation analysis with distinct morphogenesis (migration, branching, and proliferation). 2. Focused angiogenic signal activation of a single cell and its communication with surrounding cells. 3. Establishment of an artificial magnetic tweezer system for directed neovascular development.

Coral reefs are ecologically and economically important marine ecosystems that are currently under threat as a consequence of environmental degradation, global climate change and emerging diseases. An important element in determining the health of corals is the stability and function of the coral 'holobiont', the complex and diverse community of microorganisms living in partnership with the coral host. Shifts in the balance of the holobiont community composition can lead to coral disease and bleaching. However, studying the mechanisms behind these shifts is hindered by the physical and biochemical complexities of the coral holobiont. To overcome these obstacles we will for the first time apply microfluidic technology to create a "Microfabricated Coral Holobiont" (MCH). This simulated microenvironment will replicate the key physical, chemical and biological features of the coral holobiont and will represent a powerful new experimental platform to study the ecology of the coral holobiont within a controlled setting in the laboratory. We will couple this system with videomicroscopic measurements of microbial behavior and a suite of specific cell stress response probes to examine the hypothesis that specific chemical cues produced by different members of the coral holobiont can favor or impede the colonization of pathogenic bacteria involved in coral disease. We will use a second novel microfluidic-based tool, the "In Situ Chemotaxis Assay" (ISCA), to investigate the chemical ecology of natural coral-associated microbial communities within two important coral reef ecosystems, the Great Barrier Reef (Australia) and the Red Sea (Eilat, Israel). Coupled with modern molecular tools (metagenomics), the ISCA will allow for the first in situ measurement of microbial behavioral responses to chemical cues released by different compartments of the holobiont. The interdisciplinary research team is uniquely positioned to tackle this challenge, as it brings together expertise in nanotechnology, engineering, fluid dynamics, biochemistry, molecular biology, microbial and chemical ecology and oceanography. The new approach proposed here will deliver novel insights into the cellular mechanisms behind coral disease, which will ultimately aid in the management of coral reefs and in the preservation of their ecological, economic and societal value.

Decision making spans a vast range of types and complexity, from choosing your partner to deciding whether to dive left or right to save a goal. Progress in a variety of fields has led to an understanding of the mechanisms that evaluate evidence and reach a decision. Studies of simple decisions have provided insight into the neurobiological mechanisms responsible for decision making in both monkeys and humans. These studies often require a choice between two possibilities, such as leftward or rightward motion of dots moving on a screen. Human and nonhuman primate experiments support a mechanism in which a decision is made when the accumulated evidence reaches a criterion level. Such an accumulation process explains both the accuracy of the decisions made over a range of difficulty levels as well as the time required to make each decision. However, a decision in the absence of an action (or the withholding of an action) has little value, and in recent years there has been substantial progress in understanding how a task goal determines the actions that will be made. In this new framework, the movement system sets up a feedback rule that determines how the commands we send to our muscles should depend on sensory inputs we receive, so as to optimizes certain criteria such as a tradeoff between effort and accuracy.

Traditionally, decision making and movement-control were believed to operate separately and sequentially, but recent evidence from cognitive psychology and neuroscience suggests a more intimate connection. A fascinating implication of this connection is that it can explain why, in the absence of additional information, a decision-maker might change his or her mind. We have recently developed a task in which we can monitor confidence in a decision and also changes-of-mind, showing that once the brain reaches a decision and commits to a choice, it can continue to analyze evidence that was in the pipeline but unavailable to impact the initial decision. Our project extends and unifies the fields of decision making and movement control with a combined behavioral, neurophysiological and theoretical approach within a common conceptual framework. Our hypothesis is that a common framework applies to the revision of both actions and ideas, and we will develop a theory of decision making and control that extends to the acts of vacillation and self-correction.

Antigen-specific B-cells form germinal centers (GC) in the lymph node follicles 1-2 weeks after antigen challenge. GCs function to generate B-cells bearing higher affinity antigen receptors, which subsequently differentiate into memory B-cells and plasma-cells. This process, termed affinity maturation, includes random hypermutation of the BCR genes that results in altered affinity for the BCR. The precise mechanism by which high affinity B cells get selected among a pool of cells bearing a range of affinities is still unclear. In GCs B-cells engage and communicate with other cells like T-helper and follicular dendritic cells (FDCs). FDCs present antigen to B-cells in form of immune complexes, whereas bone marrow derived dentritic cells may present antigen to follicular T-cells. GC is an open structure which ensures that rare high affinity B-cells will continuously enter and participate in antibody responses. The goal of the proposed work is to elucidate the immune cellular communications that govern the formation of GCs. We will use imaging in conjunction with molecular genetic and cellular immunology technologies to investigate the cellular interactions during maturation of GCs in vivo. We will use models based on knock-in mice expressing low and high affinity BCR specific for nitrophenol haptens. This antigen can be attached to ovalbumin to elicit T-cell help from endogenous Ova-specific T-cells. Cellular interactions and antigen uptake will be analyzed and tested for their role in GC formation and maintenance.

Cell differentiation during development is driven by the action of signaling molecules. Although the molecular components necessary for transduction of the differentiating signals have been described extensively, the way these signaling cascades allow the cell to integrate multiple, sometimes contradictory signals remains largely unknown. The TGF-beta pathway is central in development and composed of two antagonistic branches (BMP and TGF-beta). It controls the differentiation of the two cell lines we will study (cultured myoblast C2C12 and human Embryonic Stem Cells RUES 1 and 2 lines). We will use a microfluidic automated cell culture chip allowing precise temporal control of extracellular environment, combined with time-lapse fluorescence microscopy to follow the real time response to stimulation by TGF-beta ligands, by recording both the nuclear-translocation of the Smad transcription factors and subsequent gene expression. We will monitor the real-time pathway response, as a function of parameters like the intensity or the time course of signaling and the timing and relative proportions of antagonistic signaling (BMP vs TGF-beta). We will also consider the importance of cell-cell communication and pathway induced inhibitors. We expect that these experiments, combined with mathematical modeling of the pathway, will teach us how cells encode differentiating signals.

The functions of immune cells are governed by the intracellular calcium (Ca[2+]) levels in conjunction with other signals. Ca[2+] entry into cells is accomplished through specialized calcium release activated Ca[2+] channels (CRAC channels) which consist of two membrane proteins, namely, stromal interaction molecule 1 (STIM1) and CRAC modulator 1 (CRACM1 or ORAI1). STIM1 is an endoplasmic reticulum (ER) resident protein with a calcium sensor gating Ca[2+] entry, and ORAI1 is a plasma membrane (PM) channel protein. Cellular studies have shown that regulation of the CRAC channel is dictated by the dynamics of STIM1 and ORAI1 movements to sites of ER-PM apposition and by direct interaction of STIM and ORAI proteins. However, several important parameters have not been accessible for quantitative study in cells. The proposed research aims to address this biological question by modeling the close apposition between PM and ER using lipid vesicles tethered to a surface or a supported lipid bilayer. The dynamics of STIM1 and ORAI1 movements and CRAC channel activation in this rigorously controlled environment will be fluorescently visualized with an approach using microfabrication technology, protein engineering, and optics. The influence of critical parameters such as the concentrations of STIM1, ORAI1, tethering proteins, Ca[2+] ions and the constituents of the lipid bilayers on the kinetics of STIM-ORAI complex assembly and channel activation will be examined in detail. The study will provide a detailed accounting of factors controlling store-operated Ca[2+] entry under physiologically relevant conditions.