Understanding how cells self-assemble into 3D, spatially-organized structures and how structure impacts the function of the final multicellular assembly is important in many fields, from fundamental biology to regenerative medicine. Intercellular cooperation is one of the most important functional behaviors of multicellular assemblies and also one of the least-understood, from an evolutionary perspective, since cooperative cell assemblies are vulnerable to invasion from “cheaters,” cells that reap the benefits of cooperation without paying the costs. Yet cooperation is ubiquitous in cell assemblies. Theory and in silico studies suggest that spatial structure can play a crucial role in stabilizing cooperative behavior. The key questions addressed in this proposal are: How does spatial structure arise in multicellular assemblies? - and how does this structure impact cooperation within the assembly?
We will use a powerful combination of laboratory experiments and computer simulations. Our model organism is the bacterium Pseudomonas aeruginosa, an opportunistic human pathogen that readily forms biofilms, interactive communities of bacteria that are physically bound to each other and, usually, to a surface. P. aeruginosa is well known as a model for cooperative behavior, which is regulated by intercellular quorum sensing signals. Our simulations will use a spatially resolved, 3D, individual-based model for biofilm growth.
We will first determine how 3D spatial structure develops in P. aeruginosa biofilms, with particular emphasis on how the initial configuration of cells on the surface affects the final structure of the biofilm. We will systematically manipulate the initial cell configuration both by using optical tweezers, and by using a controllable mixture of single cells and preformed cell aggregates. We hypothesize that cells in regions with initially high cell density (such as in aggregates), will initiate cooperation through quorum sensing earlier, enhancing their growth and leading to domination of those cells’ progeny in the final biofilm. We will next determine how spatial structure influences cooperation, by tracking the extent of quorum sensing in biofilms of different structure, and with evolution experiments. We hypothesize that spatially structured cell aggregates are more cooperative and more resistant to invasion by cheats. Finally, we will use the design rules that we have uncovered to create cell assemblies that are optimized for particular functions, such as resistance to or susceptibility to cheats – as a first step towards the design of targeted 3D cell assembles tailored for particular biotechnological or clinical applications.
This project is inherently collaborative, and will lead to new insights into the biology and biophysics of multicellular 3D assemblies and the interplay between spatial structure and cooperation, with relevance well beyond our bacterial biofilm model system.

As an organism develops from a fertilized egg, cells undergo a sequence of differentiation events, acquiring ever more specific identities. A fundamental problem in biology is to understand how self-renewal and cellular differentiation is carried out by intracellular genetic circuits and cell-cell signaling. Embryonic stem cells, with their remarkable ability to self-renew indefinitely, while retaining the potential to differentiate into a diverse range of specialized cell types upon stimulation (pluripotency) provide a powerful in vitro model system for studying the molecular mechanisms underlying self-renewal and cell fate specification. Addressing this problem, however, has been challenging, because most stem cell populations are inherently heterogeneous, and tools are lacking that can monitor cellular states quantitatively and dynamically at the single-cell level. Here, we employ recent advances in quantitative time-lapse microscopy and stem cell engineering to systematically examine how cellular components are regulated dynamically during pluripotency and differentiation in individual stem cells, and to use this information to understand cell state transitions. To reach this goal, we assembled a team with expertise in stem cells, cell line engineering, quantitative time-lapse microscopy, single-cell dynamics, systems biology, and mathematical modeling. We will use mouse embryonic stem cells as a model system. First, we will generate a library of fluorescently labeled mouse embryonic stem cell lines with endogenous reporters for key signaling pathways, regulatory genes, and other cellular components. Second, using this library, we will use quantitative time-lapse microscopy to analyze the dynamics of proteins in the pluripotent state, studying the temporal and spatial variation in proteins between genetically identical cells. Finally, we will use the same library to study specific cellular differentiation processes, mapping dynamic events that occur during differentiation, and analyzing when and how cell fates become established. A major focus in this work will be the role of heterogeneity, or “noise”, in cellular circuit components. Together, these efforts will enable us to discover new dynamic phenomena concealed by conventional population studies, and enable us to elucidate the effective regulatory architecture of the stem cell.

Segmentation of embryonic tissues into serially repeated structures is widespread during development of most bilaterian organisms. Despite this near ubiquity, the evolutionary conservation among segmentation mechanisms from various bilaterian phyla remains controversial, as does the evolutionary origin of segmentation. Classical evolutionary scenarios derive segmentation of coelomic pouches in bilaterians from the cnidarian body plan. In particular, the gastric endoderm of scyphozoan and anthozoan cnidarians is characterized by segmentation into a series of morphologically similar mesenteries which are proposed to be the antecedents of coelomic segments. The anthozoan Nematostella vectensis has recently emerged as a model system for studying molecular mechanisms of development in Cnidaria. Recent work has shown that several well-studied developmental regulatory genes, including signaling pathway components and transcription factors, are expressed in the developing endoderm of Nematostella vectensis. However, the cellular and molecular mechanisms that drive Nematostella endoderm segmentation remain unexplored.
The work proposed has two aims: 1. Utilizing modern developmental genetics techniques to test candidate patterning signals and determinants that are expressed during endoderm segmentation. 2. Establishing gene-trapping transgenesis as an unbiased approach to discover genes involved in endoderm segmentation. Discovering the molecular mechanisms underlying endoderm segmentation in Nematostella and comparing with mechanisms from well-studied bilaterians will allow rigorous experimental evaluation of the evolutionary origin of segmentation during animal development.

Human embryonic stem cell (hESC) cultures are heterogeneous, and consist of subpopulations that represent distinct cellular substates of pluripotency. These subpopulations reside within specific spatial domains in stem cell colonies. Much evidence now suggests that intercellular communication between cells in these compartments helps to drive cell fate decisions, just as communication between cells governs fate choice in the peri-implantation embryo. ES cell cultures therefore provide a model for studying how cell interactions govern transitions through state space within a regulatory network. Currently we do not understand how extrinsic signaling governs transitions between cell states, how self renewal and pluripotency are regulated, or how intercellular signaling between different compartments effects spatial patterning. Unraveling these complexities will require a systems biology approach. By combining time-lapse video microscopy and single-cell analytical technologies with mathematical modeling, we will study the spatial and temporal dynamics of signaling pathways that regulate cell fate decisions in hESC culture. Quantitative models that describe the dynamic interplay between signaling pathways will allow us to understand how human embryonic stem cells regulate themselves and how they respond to environmental signals. This knowledge will allow us to predict the optimal combinations, dosage, and time course of exogenous signals to achieve precise and reproducible control of the cell state.

The physiology and behavior of mammals are subject to daily oscillations that are driven by an endogenous circadian clock. The mammalian circadian timing system is composed of a central pacemaker in the brain that synchronizes subsidiary oscillators in virtually all cells of the body. While the brain’s master clock is entrained by daily light-dark cycles, feeding time appears to be the dominant timing cue for peripheral clocks. Compelling evidence points toward a functional interplay between circadian rhythms and cellular metabolism. Circadian clocks play a major role in orchestrating daily metabolism and their disruption can evoke metabolic diseases such as diabetes and obesity. Concomitantly, clocks are tightly coupled to cellular metabolism and respond to feeding cycles. The molecular mechanisms through which metabolism regulates clocks’ function are just starting to emerge. Recent work of ours and others revealed that NAD+/NADH are implicated in this process, yet the molecular mechanisms involved are largely unknown. This research proposal is aimed to thoroughly address the involvement of NAD+/NADH in the function of circadian clocks. We will establish an NAD+/NADH reporter in cultured cells and living animals. In addition, we will identify novel nuclear NAD+/NADH binding proteins and test their potential roles in the core clock circuitry. Finally, we will dissect the involvement of enzymes and metabolites in the NAD-biosynthesis pathways in the function of circadian clocks. These projects address fundamental aspects of circadian clocks in connection with metabolism and certainly will be valuable in studying many metabolic processes in various fields of biology.

Planar cell polarity (PCP) is an organized differentiation process in which cells within a developing tissue all polarize along one planar direction. In mammals, PCP is crucial for proper development of the inner ear, kidneys, lungs, neocortex, and more. While some of the core mammalian PCP proteins such as Vang2 / Fz3/6 / Dvl2/3 and their regulators have recently been identified, there is still no clear picture of how PCP is generated. Furthermore, this coordinated polarity often involves drastic morphological changes such as convergent-extension (CE), cellular differentiation, and asymmetric cell divisions. It is unclear how these processes interact with PCP patterning. We hypothesize that PCP patterning involves feedback between intercellular signaling and morphology, namely, that intercellular signaling both affects, and is affected by, cellular morphology. Here, we propose a systems-level approach that integrates gene expression, cellular morphology, and asymmetric cell divisions into a comprehensive model of PCP patterning. We will study PCP and its interaction with tissue morphology in two complementary model systems: the inner ear, where PCP is involved in the uniformly oriented differentiation pattern of hair cells and supporting cells; and the neocortex, where PCP is involved in asymmetric division of neuronal progenitors. Our approach combines several unique experimental and theoretical studies. First, we will use state-of-the-art comparative transcriptome analyses on subpopulations of cells (hair and supporting cells for the inner ear; progenitors and differentiated neurons for the neocortex) from wild type and mutant mice for identifying essential regulatory nodes in the PCP network. Second, we will take advantage of the ability to grow live cochlear cultures and neocortex slices in culture dishes to perform real time tracking of PCP development using quantitative time-lapse microscopy. These techniques will be based on the development and characterization of wild type and mutant transgenic mice expressing a combination of fluorescently tagged proteins. Finally, we will employ a novel mathematical modeling strategy that will integrate reaction diffusion models of PCP and cellular differentiation circuits with mechanical vertex models for describing tissue morphology. Overall, these investigations are aimed at obtaining an integrated morphodynamic picture of PCP patterning.

Cellular organization is essential for carrying out the chemistry of life. At its most basic level, organization is compartmentalization by lipid membranes, around which cells install protein networks that establish further complexity. To manage these proteins networks, cells have evolved sophisticated machinery to insert membrane proteins into bilayers and move unfolded secreted proteins across them. The Sec translocon, an integral membrane protein complex, is the main machine used for both processes in all kingdoms. Despite its importance, a detailed description of its mechanism has remained elusive. This research seeks to understand protein translocation and membrane insertion by the Sec translocon.
Membrane proteins are challenging to characterize structurally, and, as the translocon likely adopts a variety of conformational states, its study is complicated by the presence of heterogeneity. These hurdles will be overcome by the use of recent advances in solid-state nuclear magnetic resonance spectroscopy (ssNMR) and dynamic nuclear polarization (DNP). These methods make it possible to probe both the structure and dynamics of the translocon in native Escherichia coli membranes. Changes in conformation upon addition of substrates and interaction partners will reveal the residues and motions essential for functioning of this biological machine in its native environment. This research will elucidate the mechanism of how the translocon distinguishes between secreted and transmembrane proteins. On a more fundamental level, it will provide a deeper understanding of the principles underlying the structure, folding, and function of membrane proteins in general.

Are roots in the dark? Darwin proposed that the “brain” of a plant resides in its roots. Subsequent research has shown that roots integrate environmental information about resource availability, competitors, etc., to determine critical life history traits of the shoot, such as the timing of resource allocations to reproduction, growth, defense and senescence. During domestication, this environmentally sensitive “brain” is commonly disabled to make crops less environmentally sensitive, easier to transplant, and better synchronized to facilitate harvesting. We propose to explore the importance of a new sensory modality of roots, that of light-perception, in enhancing the ecological performance of a native plant in its native habitat.
Plants use light as both an energy source and a source of signals to alter its physiology to adapt to current or anticipated environmental changes. Forward and reverse genetics have identified photoreceptors and their associated signaling cascades. Surprisingly, some photoreceptors are highly and specifically expressed in roots, which are generally thought to develop in the total darkness of the soil. Light penetration in most soils is limited to a few millimeters close to the soil surface but roots that express a plethora of photoreceptors grow deep in the soil. Since natural selection rapidly winnows non-functional genes from genomes, these root- expressed photoreceptors are likely being maintained in the genomes of higher plants because they play an important role; the goal of this interdisciplinary proposal is to uncover the function of these root-expressed photoreceptors.
While the idea of light-piping through vascular bundle was suggested three decades ago, theoretical and empirical examinations of ‘light-piping’ remains largely unexplored and the question of its relevance for plant function has simply been out of the grasp of single experts working alone or in loose collaborations with other experts. By combining state-of-the-art optical physics and molecular biology, we propose to characterize this transmitted light, uncover how plants respond to this light, and determine if the responses are relevant for the optimization of ecological fitness during the plant’s entire life in the field. Building on the impressive knowledge of light signaling in the model plant, Arabidopsis thaliana community, we will use a wild tobacco, Nicotiana attenuata to uncover the ecological relevance of having “sighted roots” in the plant’s natural habitat. We will employ the versatile molecular and biochemical tools available in Arabidopsis and incorporate the relevant components into an ecological model plant, N. attenuata. We have developed analytical, molecular and ecological platform in N. attenuata to study its rich ecological interaction in the Great Basin Desert of Utah. We propose that the intense light of the desert will illuminate the ecological function of light signaling in roots.

We propose an interdisciplinary project in the areas of neuroscience and developmental robotics. The objective is to devise a novel computational and biological framework for understanding intrinsically motivated curiosity, active learning and attention. This approach will fill large gaps that currently exist in the study of these topics in these different domains. In neuroscience, countless studies have examined learning and attention in highly simplified experimental contexts but few have examined the active control of these processes, i.e. the ways in which agents independently select what they are interested in and what they wish to learn. In developmental robotics, new algorithms have been developed for controlling active exploration in complex environments, but have not yet been validated against biological or psychological observations. We will fill these gaps by testing and extending the computational models that I developed in my doctoral work, based on behavioral and neural observations from rhesus monkeys. Specifically, I will record activity in parietal and frontal attention-related areas and in midbrain dopaminergic neurons, to understand their role in the active control of attention and interest. The project will greatly complement my training in robotics by allowing me to learn an entirely new set of techniques for behavioral and neural recordings in rhesus monkeys. In addition, the results are likely to have a transformative impact on neuroscience and developmental robotics by establishing a first computationally tractable framework for understanding intrinsically motivated curiosity and linking it with systems of attention and intrinsic motivation.

Embryonic stem (ES) cells are immortal, yet karyotypically normal cell lines derived from the mammalian blastocyst that can be differentiated into all the lineages of the future embryo. In this proposal, we will address the mechanisms governing cell fate choice in ES cells using a combination of emerging technologies for single cell analysis and mathematical modeling. While ES cells appear morphologically uniform, the advent of sensitive fluorescent reporter lines show that ES cell cultures exhibit dynamic and heterogeneous gene expression that reflects the cell types of the blastocyst from which they are derived. Specifically, undifferentiated ES cell cultures appear to contain at least two populations of cells that are both capable of self-renewal, but poised to differentiate into distinct fates:. One population is primed to become the embryonic lineages or epiblast while the other is primed to become the extra-embryonic primitive endoderm. In the embryo, similar dynamic states exist, but only transiently. As ES cells perpetuate these dynamic states indefinitely, they provide unique experimental access the process of lineage specification. By fully exploiting this model, we can elucidate the molecular mechanism that controls the progress of a cell between primed states and yet prevents them from entering further into differentiation. In both ES cells and embryos, the activation of Erk signaling downstream of Fgf modulates the transition between epiblast and endoderm and can also drive ES cells into differentiation. Is this process of priming and commitment deterministic or stochastic in nature? How does Fgf signaling impact on the fate of individual cells? To answer these questions we take a quantitative approach with single cell resolution. We have assembled a multi-disciplinary team to determine how Fgf signaling acts on these transitions in individual ES cells, how Fgf signaling interacts with the pathways that normally prevent ES cells from differentiating, and explore how key ES cell specific transcription factors affect these Fgf dependent transitions. To achieve those aims, we will exploit new fluorescent reporters that can detect low-level lineage specific gene expression as a tool to identify Fgf-driven transitioning cells. We will develop a novel microfluidics platform to measure variations in fluorescent readouts from these reporters in single cells, under defined Fgf dosage, and in response to perturbations in the Fgf pathway. Microfluidic devices will enable culturing, laser transfection, and time-lapse imaging of single cells in individually addressable growth chambers, allowing for the delivery of different agents combinatorially. The measured dynamic distribution of single cell responses will be correlated with a whole genome expression data. Mathematical modeling will be used to develop a predictive model for the relationship between Erk activation, reversible priming towards endoderm, and endodermal differentiation.

The issues related to the basis of the neural code are of fundamental interest in understanding how the brain processes and decodes sensory or cognitive information. In the central nervous system, neurons transduce information by using brief pulses of electrical activity, called “action potentials”. Trains of action potential constitute the physiological substrate for inter-neuronal communication. However, the relevant features that are used for information coding remain unclear. Experimental evidences are lacking to explain how the network processes dynamical information.
Here, I propose to reconstitute in vitro neuronal networks in a controlled environment to examine how the network’s topology affects the dynamical properties of the circuit. Therefore, I will develop a micro-fabricated platform for culturing neurons in interconnected compartments that will mimic neuronal modules of cortical networks. Using this innovative technology, the precise control of cell types, connection pattern, and connectivity will shed light on general principles that govern information processing in neuronal networks. Microelectrode array will enable the simultaneously observation of hundreds of neurons. The dynamical state of the system will be characterized at rest or in response to an external stimulus to reveal why neuronal networks are flexible devices that can process multimodal information. Because, this experimental model is finely defined in terms of network’s architecture, we will use computer simulations to confront experimental and theoretical results to unravel mechanisms that define the basis of the neural code.

Infectious diseases have always been a major health problem throughout the world. Despite the recent development of vaccines and antibiotics, there are still nearly 15 million deaths every year attributable to the effects of infectious diseases. Although a significant proportion of inter-individual variation in susceptibility to particular microbes can be attributed to environmental factors such as malnutrition or poor hygienic conditions, a substantial portion is due to host genetic factors. The importance of host genetic factors on susceptibility to infectious diseases has been shown by comparative studies involving twins and adopted persons. In addition, multiple rare single-gene mutations leading to severe immunodeficiencies have also been linked with susceptibility to different infectious agents. Although genetic studies of infectious diseases have identified important pathways involved in protective immunity, very little is known about the underlying genetic factors contributing to differences in susceptibility to infectious diseases at the population level. Here, I propose to use a unique combination of genomic, immunological and statistical approaches, including gene expression profiling and quantitative trait loci mapping in infected primary cell cultures, to decipher the genetic basis and mechanisms underlying inter-individual variation in immune responses to infection. Understanding the genetic basis of variation in immune responses to infection is a key step toward improving diagnostic accuracy and implementing novel preventive strategies against infectious diseases and other immune-related disorders.

During development, axons are guided to their correct target by the growth cone (GC) which responds to environmental guidance cues. The axonal response to guidance cues is mediated partly by local translation (LT) in the GC. While studies so far investigating regulation of this cue-induced LT have examined regulators of general translation, the fact that different cues stimulate translation of specific subset of proteins raise the question as to how specific mRNAs are selected for translation in the GC. MicroRNAs (miRNAs) are translational regulators known to regulate LT in dendrites and to be expressed in axons. Thus, in this project, I will investigate the implication of miRNAs in axon guidance (AG) as regulators of LT specificity, using Xenopus retinal ganglion cells (RGC) projections as model.
I will therefore 1) identify axonal miRNAs populations and candidates to be involved in regulation of cue-induced LT using both a candidate approach and axonal RNA-deep sequencing approach; 2) determine which miRNA candidates are involved in AG in vitro and in vivo using morpholinos to manipulate the expression of specific miRNA candidates in RGC; 3) determine which mRNAs are susceptible to be regulated by each of the miRNAs implicated in AG using ribosomal profiling, RNA-deep sequencing, and bioinformatics; and 4) determine whether these miRNAs regulate the LT directly in the GC using live imaging to follow in the GC the cue-induced LT of identified target-mRNAs.
This work will be critical for understanding the specificity of mRNA LT in the GC during development, and should also provide new insight for axonal regeneration as some microRNAs are re-expressed after nerve lesion.

Mechanical stimulation derived from physical activity is critical to the development and regulation of the muscle tissue, whose architecture, enzymatic apparatus and innervation is largely dictated by mechanical demand. Despite the importance of this mechano feed-back process to human and animal (patho)physiology, little is currently known about the molecular mechanisms that mediate it in the cell. Members of the titin-like family of giant proteins (0.7-4 MDa) are now emerging as key mechanotransducers in the sarcomere. These proteins form elastic filaments that are embedded within the sarcoskeletal lattice of myosin-actin motors. They contain conserved kinase domains flanked by mechano-sensitive, regulatory motifs that undergo conformational deformation during myofibril stretch and, thereby, regulate kinase activity. It is believed that mechanical stress leads to kinase activation but, to date, it is unknown how and when these elastic kinases act in the developmental, reformative or atrophic stages of muscle and how they relate to other signalling pathways in muscle. Here, we will address the mechanisms of mechanotransduction in a kinase representative of this family, twitchin kinase from C. elegans, through its atomic and catalytic profiling in vitro under strain-mimicing conditions. We will then use the powerful genetics of C. elegans to engineer transgenic worms carrying in their muscles synthetic cytoskeletal filaments with genetically-encoded fluorescence reporters that will quantitatively report on kinase activation. We will use advanced imaging and data analysis techniques to correlate kinase activation state with the physiological stages of muscle (development/aging), myofibril structure and animal locomotory behaviour. The Overall Objective of this research is to establish the operational, mechanistic principles of this kinase family and use these to assess the physiological significance of their mechanosensing in vivo for muscle development and regulation.

The 19th century German embryologist Karl Ernst von Baer first noted that the embryos of animal species bear striking similarities during periods of their embryonic development. These seminal observations were extended in the 20th century to show that the period of greatest similarity occurs in the middle of embryogenesis during the so-called phylotypic period. This pattern of conservation, referred to as the hourglass model of developmental evolution, is believed to reflect a major constraint on the evolution of animal body plans. Despite an accumulation of morphological data confirming that there is often extensive divergence in the early and late embryos of species from the same phylum, the molecular underpinning of the morphological hourglass remains largely a mystery. Recently, we have shown that gene expression divergence also follows an hourglass pattern with highest conservation occurring at the arthropod phylotypic stage, providing a key insight into the molecular basis of this phenomenon.
We aim to extend these results in the current project by incorporating measures of divergence in embryos at both the morphological and DNA sequence levels. We will measure phenotypic variance between Drosophila strains and species in the length of embryonic development – an ecologically important trait which differs substantially across species – and integrate this with measures of the timing of the first appearance of key morphological landmarks. These complementary data will provide a framework within which we can identify changes in the onset and rate of developmental events, both examples of a class of evolutionary modifications known as heterochrony, widely acknowledged as an important mechanism for generating morphological novelty. We will also locate the genetic variants that underlie differences in morphology and gene expression between strains to shed light on the relationship between embryonic variance at the level of molecules and visible phenotypes.
Our interdisciplinary approach incorporates a novel blend of powerful imaging and image analysis technologies together with quantitative genetic modeling methods in order to build a comprehensive picture of how multiple biological levels impact on the evolution of Drosophila embryogenesis. Ultimately, with the unprecedented insights into the nature of the forces that shape insect development provided by this project, we aim to uncover the mechanstic basis underlying one of the fundamental laws that governs the evolution of animal species.

The main aim of this proposal is to understand the cellular and circuit mechanisms that establish spatial coding cellular ensembles in the mammalian hippocampus. The cognitive spatial map theory of the hippocampus posits that internal representations of space are implemented by a sparse subset of 'place cells' that display location-specific firing during spatial navigation, while other neurons remain silent. Moreover, this spatial code is highly dynamic, such that place cells alter their firing properties when the spatial environment changes. The cellular and circuit mechanisms that establish sparsely distributed and dynamic spatial coding schemes in the hippocampus are poorly understood. Longstanding theories posit that the hippocampal inhibitory circuitry plays a central role in the formation and segregation of spatially informative cellular ensembles. If so, the anatomical diversity of local GABAergic interneurons may further promote dynamic organization of place cell assemblies in the population by regulating active dendritic input processing at the subcellular level, which can define the output behavior of principal cells to synaptic excitation in a given environment. Understanding multimodal dynamics in the hippocampus will, therefore, have important consequences for our understanding of how cortical circuits are organized to process and store information. To directly test these hypotheses we will perform multi-level analyses requiring a multidisciplinary approach that brings together experts in neurophysiology, nonlinear optics, molecular genetics and synthetic chemistry.
Our research plan is composed of five tightly integrated projects. 1) Functional population imaging in hippocampal CA1 of behaving mice to determine the fine-scale spatial organization of hippocampal spatial coding ensembles and their dynamic reorganization during controlled changes in the spatial environment and spatial learning. 2) Assessment of multimodal organization and dynamics in synaptic microcircuits of genetically-identified interneurons. 3) Implementation of novel cellular-resolution optical, photochemical and pharmacogenetic techniques for manipulating activity of identified inhibitory circuits in vivo. 4) Dissecting multimodal population and microcircuit dynamics of spatial coding CA1 hippocampal neuronal ensembles using cellular-resolution manipulations in vivo. 5) Measurements and manipulation of subcellular integration of excitatory and inhibitory synaptic inputs in functionally-identified hippocampal CA1 pyramidal cells during spatial navigation and learning.
This project will yield a detailed understanding of the cellular and circuit mechanisms that establish spatial coding ensembles in the mammalian hippocampus. The complexity of this problem necessitates a multidisciplinary and integrated approach.

Dynamic interactions between RNA and proteins are fundamental to gene regulation. To function properly, RNA molecules and associated binding proteins form RNA/protein (RNP) complexes that further assemble into micron-sized RNP bodies, which are involved in many developmental processes. Despite their biological importance, both the precise function of RNP bodies and the biophysical principles governing their mesoscale dynamics, remain enigmatic. An emerging concept is that RNP bodies may represent ATP-dependent liquid-like assemblies of RNA and protein, and that they could thus function as intracellular micro-reactors for efficient RNA regulation. One potential source of an ATP-dependent liquidity is the activity of RNA helicases, a physiologically important class of ATP-hydrolyzing enzymes that are ubiquitous components of RNP bodies. We propose to test the hypothesis that RNA helicases play a central role in the underlying molecular dynamics and structural fluidity of RNP bodies. We will take a multi-scale approach to (1) elucidate the molecular composition of developmentally regulated RNA helicase complexes, (2) determine the molecular mechanisms of RNA helicases in these complexes, and (3) decipher how these dynamic complexes contribute to the assembly and liquid-like biophysical properties of RNP bodies. Our vision is to develop a fundamental understanding of the role of ATPase activity in RNP body assembly, and the physiological consequences for developmental gene expression programs in metazoans.

More than 90% of human transcripts are alternatively spliced, which greatly increases the gene product repertoire of the genome. Many alternative splicing (AS) events are regulated and act as switches in important biological processes such as differentiation, apoptosis, and neuronal patterning. Aberrant AS is an underlying cause in many human diseases. AS decisions are guided by binding of splicing factors to nascent mRNA. Recently, chromatin features such as nucleosome remodeling and distinct histone methylation marks have emerged as important AS regulators.
To gain a better understanding of the roles of chromatin in AS regulation, I propose to systematically identify known and putative chromatin and transcription factors that regulate AS by means of an RNAi screen. The screen will be carried out simultaneously in mouse embryonic stem cells and fibroblasts in order to detect factors that impact chromatin-regulated AS events that are differentially regulated between these cell types. I will adapt a novel screening strategy recently developed in the host laboratory to monitor several dozen endogenous AS events in parallel using RT-PCR amplification and multiplexed deep sequencing.
For candidate hits, I will then globally identify the networks of AS events they regulate. Together with genome-wide mapping of their binding sites and mass spectroscopic identification of associated proteins, these data will be used to pinpoint likely interaction partners. Finally, I will employ established techniques to clarify whether the identified factors act directly or indirectly. The results are expected to significantly improve our understanding of chromatin function and AS regulation.

Slime molds and fungi are ubiquitous components of terrestrial biomes and often grow as dynamic networks. Individuals can reach across multiple hectares of habitat, and for example the “humongous fungus”, Armillaria bulbosa, stretches through 15 hectares of a Michigan forest soil. These organisms function as pathogens, mutualists, and decomposers, and play key roles in biogeochemical cycles and agriculture. Traditionally considered as close relatives of plants, the fungi are now understood as more closely related to animals. Reasonable estimates place the diversity of true fungi at 5 million species; slime molds may range to 2,500 species.
Filamentous slime molds and fungi forage through various substrates for scarce and heterogeneously distributed resources; organisms must dismantle and move resources across vast distances, and then coordinate their use across the individual. How are local patches of food parsed, moved, and integrated to enable an organism to grow as a coherent whole? We hypothesize integration is a function of the internal flows of networks. Communication and growth are likely to be facilitated by the fluids which carry both metabolites and organelles across a network. But different anatomical and functional constraints may facilitate or constrain these flows to a greater or lesser degree. For example, there are no internal partitions within the networks formed by slime molds, but many fungi grow as strings of cells separated by walls, and these walls may limit the movements of cellular structures. While slime molds may actively move large organelles to coordinate the processing of a newly discovered food source, fungi may rely on the passive transport of smaller metabolites. Perhaps communication across fungal networks is less effective than communication across slime mold networks. We aim to understand what organisms do to integrate organismal growth across these different kinds of networks by working with many different species from each of three groups: the acellular slime molds, ascomycete fungi and basidiomycete fungi. Targeting multiple species will enable identification of general properties, and also unique solutions taken by subsets of species. Our approach to organism self-organization involves an international team of a mycologist, a network imaging specialist, and an applied mathematician. The interplay of these disciplines is required to: (i) identify how fluids travel through different kinds of networks, (ii) discover how organisms communicate, and network architectures change, in response to new food sources, and (iii) unravel the different paths by which transport mechanisms and network architectures interact to enable the growth of an individual.

Neurogenesis is a complex developmental process in which neurons differentiate from multipotent neural progenitor cells into highly specialized cells. This process depends on intrinsic signals like transcription factors and extrinsic factors like gradients of morphogens or growth factors which together influence neural cell fate, migration, maturation, morphology, connectivity and function. I propose to test if the developmental requirements and time-dependent factors of neurogenesis in the retina can be bypassed by using a scaffold of decellularized neural tissues in which only the extracellular matrix (ECM) remains. I will seed this scaffold with stem cells or neural progenitor cells to test if these multipotent cells differentiate into the correct retinal neurons and adopt the connectivity of functional neural modules. I hypothesize that an ECM-derived decellularized scaffold maintains the spatial architecture of the retina as well as ECM-attached growth factors. Thus, the decellularized scaffold would possess the ability to induce de novo assembly of neural tissues, decoupling the creation of neural circuits from normal development. This approach to the reconstruction of retinal circuits might allow for tissue regeneration in a variety of disease states.