Oliver Hobert - US grants

DESCRIPTION: (from applicant's abstract) Our overall goal is to define and characterize genes that are required for defined neurons to attain characteristics that allow them to function in dedicated neural circuits. Work from several laboratories including our own laboratory has identified LIM homeobox (Lhx) genes as transcriptional regulatory factors required for individual neurons to function correctly in defined neural circuits. Our aim is to deepen our understanding of the function of Lhx genes in C.elegans, which given the significant degree of structural conservation across phylogeny, can be expected to reveal basic mechanisms of Lhx gene function and brain patterning in different organisms. Our research application focuses on the study of a single Lhx gene, lim-6, which is required for a defined motor neuron, DVB, to function appropriately. There are two specific aims: First, we will attempt to describe the anatomical defects associated with loss of lim-6 function, using a variety of markers that will allow us to visualize certain aspects of DVB motor neuron structure. Second, we describe genetic approaches to identify molecular components that may act together with lim-6 to affect DVB motor neuron development; this approach may lead to the identification of long-sought target genes of Lhx proteins. The data obtained so far suggest that lim-6 affects a specific, but as yet unknown aspect of terminal differentiation of motor neuron development. Potential scenarios that could be envisioned are that lim-6 regulates the steps of neuron-target recognition or of neuron-target communication. These studies may give important insights into the function of the vertebrate homologs of lim-6 as well and may reveal common themes of Lhx gene action.

Bilateral symmetry is a common feature of nervous system anatomy across phylogeny. Morphological symmetry is contrasted by the lateralization of many different nervous system functions, likely caused by individual cell types adopting different fates and functions along the left/right (L/R) axis. The molecular mechanisms of creating cellular diversity along the L/R axis in the nervous system are, however, poorly understood. The nervous system of the nematode Caenorhabditis elegans displays several examples of lateralization, including the L/R asymmetry displayed by the two ASE taste receptor neurons, ASE left (ASEL) and ASE right (ASER). While bilaterally symmetric in regard to all known morphological criteria, these two neurons display distinct chemosensory capacities which correlate with the L/R asymmetric expression of three putative chemoreceptors. We aim to understand how L/R asymmetric cell fate is created in the ASEL and ASER neurons. We hypothesize that L/R asymmetry is induced by a signal of unknown molecular nature. We propose to use laser ablation to identify the cellular source of the signal (Aim #1) and we propose to understand the molecular nature of the signal by characterizing mutants which we isolated from a genetic screen for L/R asymmetry defects (Aim #2). Our previous genetic analysis has already revealed a cascade of gene regulatory factors (including homeobox genes and miRNAs) that act in ASEL and ASER to diversify their respective fates. Future genetic studies will determine how the asymmetric activity of these gene regulatory factors is triggered. Our studies may provide novel insights into the creation of cellular diversity in the nervous system.

DESCRIPTION (provided by applicant): Nervous system laterality represents one of the most fundamental, yet also least studied phenomena in developmental neurobiology. Nervous systems are generally bilaterally symmetric on an architectural level, yet display significant degrees of functional left/right (L/R) asymmetry. How the left and right side of a brain are made to become different from one another is mechanistically not understood. We propose here to study one of the examples of neuronal laterality in the nematode C.elegans, a system particularly well amenable to study this problem since neuronal laterality can be observed on a single cell level and can be genetically dissected using mutant screening approaches. We will genetically analyze the L/R differential execution of two neuronal specification programs that derive from two bilaterally symmetric blast cells, ABp1papp and ABprpapp. Each blast cell divides several times to produce a distinct set of unpaired, unilateral neurons. We will isolate mutants in which these L/R asymmetric differentiation programs are disrupted. These studies may reveal fundamental principles of neuronal development and may shed light onto the elusive mechanisms of generating L/R asymmetry in the nervous system.

[unreadable] DESCRIPTION (provided by applicant): Nervous systems are characterized by an astounding degree of cellular diversity, yet our insights into the mechanisms of creating this diversity are limited. The molecular correlate to neuronal diversity are neuron-type specific gene expression programs. Sets of co-expressed genes that characterize an individual cell type are commonly referred to as gene batteries which are defined by their linkage to conserved, common c/s-regulatory elements. The nature of gene batteries for individual neuronal subtypes as well as their linked cis-regulatory elements are poorly described on a genome-wide level. We propose here to systematically decode cis-regulatory information on a genome-wide level and to determine the identity of a single neuron-specific gene expression program. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): MicroRNAs are gene regulatory factors whose abundance (~1% of genes in animal genomes) has only recently become apparent. In spite of their abundance, little is known about their expression patterns and the physiological contexts in which they function. The few animal miRNAs that have been functionally studied to date suggest that miRNAs are embedded in gene regulatory cascades that determine cell fate specification. Moreover, it is conceivable that miRNAs may follow the precedent of transcription factors and act in a combinatorial manner, such that specific combinations of miRNAs expressed in a given cell type may define and actively determine the identity of the cell type. To substantiate this hypothesis, the investigator will determine miRNA expression profiles both on a genome-wide level as well as with a single cell resolution using the nematode C. elegans as a model system. His studies will (1) build a catalogue of miRNA expression profiles, (2) provide a guideline for future phenotypic analysis of miRNA knockouts, and (3) provide supportive evidence for the concept of "miRNA codes", and therefore supply further testable hypothesis about miRNA function in the generation of cellular diversity in a developing organism. Moreover, it will (4) validate the "sensor strategy" to determine miRNA expression profiles in C. elegans, and (5) build a toolkit of potentially useful gfp cell fate markers. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): MicroRNAs are gene regulatory factors whose abundance (~1% of genes in animal genomes) has only recently become apparent. In spite of their abundance, little is known about their expression patterns and the physiological contexts in which they function. The few animal miRNAs that have been functionally studied to date suggest that miRNAs are embedded in gene regulatory cascades that determine cell fate specification. Moreover, it is conceivable that miRNAs may follow the precedent of transcription factors and act in a combinatorial manner, such that specific combinations of miRNAs expressed in a given cell type may define and actively determine the identity of the cell type. To substantiate this hypothesis, the investigator will determine miRNA expression profiles both on a genome-wide level as well as with a single cell resolution using the nematode C. elegans as a model system. His studies will (1) build a catalogue of miRNA expression profiles, (2) provide a guideline for future phenotypic analysis of miRNA knockouts, and (3) provide supportive evidence for the concept of "miRNA codes", and therefore supply further testable hypothesis about miRNA function in the generation of cellular diversity in a developing organism. Moreover, it will (4) validate the "sensor strategy" to determine miRNA expression profiles in C. elegans, and (5) build a toolkit of potentially useful gfp cell fate markers. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Stem cells play key roles throughout the life cycle of most multicellular organisms. This application requests funds to support five predoctoral and two postdoctoral trainees per year, in a university-wide Training Program on the biology of Stem Cells and Cell Lineage Specification. Training focuses on the analysis of cellular, molecular and genetic aspects of stem cells and the specification of individual cell lineages to which stem cells give rise. The establishment of a specific Training Program in this research area falls in line with initiatives Columbia University aimed strengthening and expanding its research program in stem cell biology. The training faculty consists of a small and distinguished group of 15 principal investigators that are affiliated with eight departments and programs. Reflecting a good mix of junior and senior faculty, four faculty members are HHMI Investigators and four are junior faculty. Six faculty members study the vertebrate nervous system, five faculty members use a variety of cell fate decisions in simple, genetically tractable model organisms and four faculty members study stem cells and cell lineages outside the nervous system, namely the immune system, epidermis and cancer cells. The training faculty, therefore, does not only reflect a strong cross-section of the outstanding neuroscience community here at Columba University, but also reflects a multifaceted approach to the subject in a variety of different cell types, using a wide spectrum of different methodologies and model systems. Predoctoral trainees will be selected from a pool of candidates that have been recruited via acceptance through a number of strong and selective graduate programs at Columbia University and postdoctoral candidates will be selected via application to the training committee. Specifically tailored course work, a seminar series, a program retreat and a New York City Stem Cell Discussion Group will ensure high quality training and broad exposure of the trainees to recent advances in the field. Faculty and trainees will interact intensively through a variety of mechanisms. With its focus on stem cell biology, our Program will not only prepare trainees for a productive career in the sciences but will also give them the opportunity to be prepared for a future engagement in an area of the biomedical sciences that is of obvious clinical relevance. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): "A genome-wide RNAi screen for neuronal cell fate mutants" The regulatory mechanisms that generate individual neuronal cell types in the nervous system are incompletely understood. Genetic mutant screens in model systems as well as reverse genetic approaches in vertebrates have yielded valuable insights into the mechanisms of neuronal diversification as these approaches have uncovered genes involved in these processes. We propose here to use a genome-wide RNA interference (RNAi)-based reverse genetics approach in the amenable model organism C. elegans to uncover genes involved in controlling the specification of a pair of sensory neurons, called the ASE neurons. The questions that we will address are: (1) what are the genes required for the ASE neurons to be generated and correctly specified; (2) as the two ASE neurons display the intriguing feature of showing left/right asymmetric features in the form of left/right asymmetrically expressed chemoreceptor genes, we wish to identify genes involved in this left/right asymmetric developmental program. So far, we have finished RNAi analysis of the chromosome I (2446 clones) and have identified 14 RNAi clones that lead to a reproducible defect in the development of the ASE neurons. We propose here to scale up this approach to all six chromosomes, leading to an estimated recovery of 6 x 14 = 84 genes potentially involved in ASE neuron development. The framework of an exploratory two-year R03 grant provides us with the opportunity to build such a collection of genes with a role in ASE development. This collection will provide an invaluable resource and starting point for a in-depth and hypothesis-driven gene-by-gene analysis in the future, eventually leading to a detailed understanding of the developmental program that leads to the specification of a single neuron class. PUBLIC HEALTH RELEVANCE: "A genome-wide RNAi screen for neuronal cell fate mutants" This grant proposal sets out to identify genes involved in neuronal development using the nematode C. elegans as a model system. We will use an RNA interference-based screen to identify genes that are required for an individual sensory neuron class to develop and to adopt its left/right asymmetric features in an appropriate manner. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Dopaminergic (DA) neurons control a variety of distinct brain functions and their malfunction or loss results in specific disease states. DA neurons are defined by the expression of a battery of specific terminal differentiation markers, including dopamine synthesizing enzymes and transporters. Little is known about how the expression of these terminal differentiation markers, and hence dopaminergic fate, is regulated. Understanding the regulatory mechanisms of DA neuron differentiation has wide-spread implications not only for basic but also clinical research. We propose here to employ the genetic amenability of the model system C. elegans, combined with state-of-the art technological advances, to genetically dissect the regulatory logic of DA neuron specification on a single neuron level in live animals through a combination of transgenic and genetic loss-of-function approaches. Our preliminary data has revealed the requirement of transcription factor (an ETS domain factor) and its cognate cis-regulatory target sequence, present in terminal markers of DA fate, for appropriate DA neuron differentiation. However, these regulatory components are not sufficient to explain the adoption of dopaminergic fate. We have obtained preliminary evidence for the involvement of other transcriptional regulators of the homeobox gene family and their cognate cis-regulatory motifs in controlling DA neuron differentiation in conjunction with the ETS domain factor and we test the hypothetical involvement of these homeobox genes by standard mutant analysis (Aim #1). Furthermore we use unbiased genetic mutant screens, which have already revealed several, as yet uncloned regulators of DA fate ("dopy" genes), to identify DA fate regulators in an unbiased manner (Aim #2). Factors found through our genetic approaches to be required for the regulation of DA fate will also be tested for whether they are sufficient to reprogram cells into a DA neuron-like state (Aim #3). PUBLIC HEALTH RELEVANCE: The project proposes to study the molecular mechanisms that control the development of dopaminergic neurons, a clinically important class of neurons. We use genetic approaches in the simple model organism C.elegans to address how the expression of genes that are normally expressed in dopaminergic neurons is regulated.

DESCRIPTION (provided by applicant): PROJECT SUMMARY "Genetic and functional analysis of left/right asymmetric neuron size" Nervous systems are generally bilaterally symmetric on a structural level but are strongly lateralized (left/right asymmetric) on a functional level. Molecular and morphological correlates of functional laterality are sparse and, therefore, functional lateralization is poorly understood. However, it has been noted that symmetrically positioned, bilateral groups of neurons in functionally lateralized brain regions differ in the size of their soma. The genetic mechanisms that program these left/right asymmetric soma size differences are unknown. In fact, it is generally not well understood how the soma size of neurons is controlled throughout the nervous system (which contains neurons that differ in size by several orders of magnitude). We propose here to utilize the nematode C.elegans to study how the size difference of two, otherwise bilaterally symmetric chemosensory neuron is genetically controlled. To identify genes, we visualize the size difference in the neuron pair in a large panel of different genetic mutant backgrounds, which affect previously known pathways that control cell size in other systems. We will examine where these presumptive size regulators are expressed and how their expression is regulated. We will test whether altering the size of the two neurons impacts on specific response parameters to sensory cues. In sum, we combine two exciting and actively pursued areas of research that have not been combined before - the study of left/right asymmetry in the nervous system and the study of size control, using a model system that is particularly amenable to study these problems. PUBLIC HEALTH RELEVANCE: PROJECT NARRATIVE "Genetic and functional analysis of left/right asymmetric neuron size" This grant proposal sets out to begin an analysis of the genetic regulation and functional relevance of cell size differences in the nervous system, using the nematode C.elegans as a model system. Lateral differences in neuron cell size are conserved across phylogeny, can be observed in humans and are disrupted in psychiatric disorders, illustrating the overall importance of this study.

DESCRIPTION (provided by applicant): GABAergic neurons are a prominent class of neurons whose dysfunction can results in various disease states. The developmental mechanisms that result in terminal differentiation of GABAergic neurons and the functional properties of many GABAergic neurons are incompletely understood. We propose here to use the experimental advantages of the nematode C.elegans to decipher the gene regulatory events - both on the level of cis-regulatory elements and trans-acting factors - that induce the expression of terminal identity markers of a single GABAergic interneuron (Aim #1) and we propose to study a potential role of this GABAergic interneuron in controlling metabolic states and life span of C. elegans (Aim #2). PUBLIC HEALTH RELEVANCE: We propose to study development and function of a single GABAergic interneuron in the nematode C. elegans using a combination of genetic, molecular and physiological approaches.

DESCRIPTION (provided by applicant): The ability of transcription factors to reprogram the identity of cells is usually very limited and hampers our ability to generate cells types for various different applications. In this grant proposal, entitled "Generating neurons through cellular reprogramming", we propose to conduct genetic screens in the nematode C. elegans that will identify factors that normally act to prevent the ability of transcription factors to reprogram cellular identities. In preliminary work, two such "reprogramming brakes" were identified. Further genetic screens may reveal more of these reprogramming inhibitors and help us better understand the plasticity of cellular, and specifically neuronal identity. This grant proposal makes specific use of the C. elegans model system as a gene discovery tool. PUBLIC HEALTH RELEVANCE: This research proposal studies the molecular mechanisms that underlie the reprogramming of the identity of individual cells. Conceptually, the reprogramming of cellular identity has enormous potential for cell replacement therapies of various human diseases, including neurological diseases, in which individual cells are lost and in which one wishes to replace those cells with intact, reprogrammed cells.

? DESCRIPTION (provided by applicant): Driver lines that direct Cre protein to specific neuron types have proven to be invaluable tools to not only visualize specific neuron types but also to manipulate their activity through the Cre- mediated activation of optogenetic probes or to assess gene function by Cre-mediated gene knockout. Most Cre driver lines, such as BAC-based Cre drivers or knock-ins of Cre into specific loci, monitor the complete expression pattern of entire genetic loci. However, very few genes are exclusively expressed in very small populations of specific neuron types and this lack of cellular specificity limits the use of these driver lines. W propose here to develop transgenic mouse driver lines that direct Cre expression to very restricted numbers of neuronal cell types in different regions of the mouse brain, thereby providing tools to precisely map their function and molecular composition. To achieve this aim, we aim to test the hypothesis - built from our past work in the nematode C.elegans - that the cis-regulatory control elements of the mouse loci that encode the vesicular transporters for the four main neurotransmitter systems in the vertebrate central nervous system, glutamate and -aminobutyric acid (GABA) and acetylcholine (ACh) are composed of a modular assembly of individual, highly cell type-specific cis-regulatory elements. We will experimentally test the hypothesis that the expression of individual, isolated cis-regulatory elements may subdivide cholinergic, glutamatergic and GABAergic domains into restricted and perhaps novel domains of the mouse central nervous system and thereby constitute reproducible and highly specific drivers for directing the expression of genes that allow the genetic manipulation of neurons and neuronal circuits. This cis-regulatory dissection approach may solve the specificity problem of most currently available driver lines that are unable to exclusively target restricted numbers of cells.

? DESCRIPTION (provided by applicant): Synaptic connectivity constitutes an integral part of neuronal identity. The recent reconstruction of the connectome of the C.elegans male and its comparison to the long known connectome of the hermaphrodite (a derived female) reveal a sexually dimorphic dimension of neuronal identity: Some defined neuron types that are present in both hermaphrodites and males show sexually dimorphic synaptic connectivity patterns. We propose to dissect the regulatory programs that specify sexual dimorphic identity, as manifested by dimorphic synaptic connectivity features. Specifically, we propose here to (1) reliably and easily visualize sexually dimorphic synaptic connectivity patterns in transgenic animals using GFP-based reporter systems; (2) study aspects of the establishment, maintenance and autonomy of these dimorphic synapses and (3) identify molecules through a candidate gene approach and unbiased profiling approach that genetically program these dimorphic patterns of connectivity and identity. We expect that our studies will provide novel insights into the currentl little explored sexual dimension of neuronal identity.

? DESCRIPTION (provided by applicant): In this resource-building proposal, we follow the map-building tradition of the Caenorhadbitis elegans field, aiming to generate an extensive expression atlas of sensory receptors of the G-protein- coupled receptor family. We predict that such an expression pattern will reveal: (a) patterns of left/right asymmetric gene expression, pointing to functional lateralization of neuron pairs; (b) patterns of sexually dimorphic gene expression, pointing to neurons involved in sexually dimorphic behaviors; (c) temporally controlled patterns of gene expression, pointing to neurons that alter their function over time. The genetic programs that instruct left-right asymmetric, sexually dimorphic and temporally controlled features of the nervous system are poorly understood and we anticipate that our identification of gene expression patterns that display such features will provide powerful entry points into studying how these features are programmed.

PROJECT SUMMARY There is a current lack of understanding of differential gene expression within the nervous system. Ideally one would like to know, across all neuron types, exactly how the genome is transcribed and processed into functional RNAs. This information is fundamentally important because differential gene expression defines the form and function of individual neurons, determines how individual neurons contribute to circuit physiology and behavior, and influences how individual neurons are affected by injury and disease. Further, detailed and complete knowledge of differential gene expression within the nervous system would help elucidate the logic and cellular mechanisms that generate neuronal diversity, including regulation of gene expression, alternative splicing, and miRNA function. Yet progress in this area has been limited: For most nervous systems, the exact number of distinct types of neurons is unknown and therefore a global map of neuron-specific gene expression is not achievable. Here we propose to address this problem in a project to discover and analyze the C. elegans Neuronal Gene Expression Map & Network (CeNGEN). The C. elegans nervous system contains precisely 302 total neurons comprising 118 classes of distinct neuronal types. We propose to exploit this unique attribute to analyze gene expression with high accuracy in every individual neuronal type. CeNGEN proceeds in four specific aims. Aim 1) Establish 118 transgenic strains, each one expressing fluorescent markers that uniquely label a single type of neuron. Aim 2) Use innovative cell dissociation and FACS methods to isolate each type of neuron from age-matched adults, and use RNA-seq approaches to assess global coding transcript and miRNA expression, as well as splicing diversity. Aim 3) Utilize single cell sequencing technology to precisely map gene expression over multiple parameter spaces. Aim 4) Build cell-centered and gene-centered expression maps, and seek connections with other uniquely known features of the C. elegans nervous system including the wiring diagram, the cell lineage, neurotransmitter identity, and function. CeNGEN represents a paradigmatic advance in neurogenetics, and provides a unique opportunity to elucidate the global control of neuron-specific gene expression and to relate gene expression to neuronal wiring and function. Expected significant outcomes include: Identification of conserved regulatory mechanisms that generate neuronal specificity and diversity; Detailed understanding of alternative splicing and miRNA function across the nervous system; Relationship of differential gene expression to neuronal lineage, anatomy, function and connectivity. CeNGEN will also serve as a resource for future studies in C. elegans neuroscience, and will provide a framework for addressing global differential gene expression in more complex nervous systems that are currently not amenable to this comprehensive approach.

The human brain is composed of billions of interconnected neurons that form highly complex neuronal circuits that process information and encode behavior. Many questions about these interconnected networks are unanswered: How variable are they from individual to individual, how do they change throughout life, how does the environment impact on them, and what are the genetic blueprints that generate these networks. Disruptions of the genetic blueprints that build neuronal networks are the likely cause of many human neurological diseases. In order to study neuronal networks in the brain, it is of paramount interest to easily visualize the patterns of connectivity of neurons, ideally in the context of live organisms. The cellular complexity of brains prevents such types of studies in complex organisms, and this project therefore uses a simple invertebrate model system, the nematode C.elegans, to visualize all the major neuronal connections of its simple nervous system. Previous studies have amply demonstrated that mechanisms of brain patterning discovered in C.elegans are conserved in other animals as well. The investigators develop and use cutting-edge fluorescent reporter technology, combined with microscopical and computer vision technology to achieve this goal. The project's construction of animals in which most neuronal connections are fluorescently labeled provides a major resource. This resource is made available to the large field of C.elegans researchers who with that resource can study the many questions that relate to circuits in the brain, including the decoding of the nervous system's genetic blueprint. In addition, the project includes cutting-edge, interdisciplinary training opportunities for undergraduate and graduate students from diverse backgrounds, as well as postdoctoral fellows.

The project entails the development and dissemination of tools that empower the C.elegans neuroscience community to study the connectome of the nematode C.elegans. In the first phase, the technology hub develops two sets of tools: One group uses fluorescent-based reporter technology (GRASP and iBlinc as potential alternative) to generate a large number of transgenic C.elegans strains in which the main "edges" of the entire wiring diagram (i.e., pairwise combinations of neurons) are visualized. As part of this project, this resource is distributed throughout the C.elegans community to enable labs with long-standing interest in various aspects of neuronal development and function and with a focus on specific neuronal circuits and behaviors to use these synaptic labels to examine variability, development, and plasticity of these connections. In parallel, the other group develops microfluidic-based and automated image analysis technologies to precisely quantify the structure of the connectome and to enable high-throughput screening of worm population for defects in synaptic wiring. Computer vision and machine learning is used to score automatically disruptions of synaptic wiring to detect subtle changes in wiring. This NeuroTechnology Hub award is part of the BRAIN Initiative and NSF's Understanding the Brain activities.

In this resource-building, map-mapping proposal, we propose to decode the molecular composition of the electrical synapse network (?electrical connectome?) of the nematode C.elegans. Its >1,000 synaptic connection, made between 302 neurons, are likely generated by 12 distinct, electrical synapse forming proteins, called innexins, that assemble into distinct channel complexes. We propose to map the expression and localization pattern of all 12 neuronal innexin genes throughout the entire nervous system, with single neuron resolution and we propose to assign specific innexins to specific electrical synapses. Our work will lay the groundwork for understanding how the electrical connectome is established and for ensuing functional studies that probe the function of individual electrical synapses.

PROJECT SUMMARY While common, evolutionarily conserved themes of early neuronal patterning in the developing embryo have emerged over the years, there are, as of now, no common organizational themes for how neuron acquire and maintain their terminally differentiated state. We propose to test here the hypothesis that each neuron class of the nematode C.elegans is uniquely defined ? and functionally specified ? by a neuron class-specific combination of transcription factors, with at least one of the transcription factors being a homeodomain-type transcription factor. The finding that homeodomain transcription factors control the identity of all neurons in a simple nervous system would have wide- ranging implications for our understanding of evolution of the nervous system and may provide means to classify (and then functionally study) neuron classes in mammalian nervous systems.

The development of the nervous system, specifically the dynamics of neuronal development and wiring to build brain architecture and their constructive role in emergent brain activity, constitutes a central unexplained phenomenon in living systems. The study of developing brains requires a comprehensive and systematic characterization of the brain of an organism at different ages and a suitable mathematical framework, able to capture the structure of the growing nervous system and the emerging networks therein. We propose to address this fundamental challenge by developing such a mathematical framework capable of characterizing underlying network changes in living brains and their consequences for functional neural activity and resulting behavior. This mathematical framework will be applied to analyze the complete nervous system, at single-cell precision, of the model organism C. elegans. To address these important challenges, we have assembled an interdisciplinary team with expertise in topology, computational biology, statistics, theoretical physics, neuroscience and biology of the model organism. Our group will develop new mathematical, statistical, and computational tools to characterize the structure of developing brain networks. This analysis will reveal shared-organizational, emergent principles of nervous-system development and function. Based on the widespread representation of biological data as complex networks and the universality of the mathematical, statistical, and computational methods we will develop, we expect wide applicability beyond the original system.

The aforementioned approach will be led by experiments that aim at providing multiple views of a developing network and their functional consequences to whole-brain activity. We will analyze the brain at two levels: changes to the underlying network as a consequence of extensive neural additions and connective neural (re-)wiring. We will compare the developing network at two transition periods: early maturation from the first to the second larval stage and, later, maturation of the two different sexes. In both of these developmental periods, newborn neurons grow the existing brain network, considerably, by roughly a third in size. In order to characterize the global properties of the data collected from these two different layers (neural network and brain activity) and to study the maps between them, we will develop tools based on topological data analysis (TDA) and Bayesian inference techniques. TDA provides methodology derived from algebraic topology that can be used to extract global features in large datasets. As a relatively new field, there are several major roadblocks that obstruct the wide applicability of TDA to biological systems, including the development of statistical approaches, comparison (homomorphisms) of networks (simplicial complexes), and time-series analysis. These tools will be then applied to study biological datasets that describe the developing brain network and changes to neurobehavioral activity therein. In particular, we will characterize basal networks and those for attractive and aversive behavior, for whole brains at a single-cell level, during developmental transitions that are known to restructure this behavioral network at both the level of input and output.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

Project Summary A ?connectome? describes the complete synaptic wiring diagram of a brain. The elucidation of the connectome of any animal brain and, ultimately, the human brain will have a tremendous impact on our understanding of brain function and constitutes a central goal of 21st century neuroscience, akin to the efforts to assemble the complete sequence of genomes. Current connectomic efforts are focused on determining the anatomical synaptic connections between neurons in a brain, thereby completely ignoring aspects of neuronal communication that are likely of equal importance but are not captured by anatomical connections: Neuromodulatory communication by neuropeptides and their cognate receptors. Neuropeptidergic communication is usually non-synaptic, i.e. neuropeptides are often released from non-synaptic sites and cognate neuropeptide receptors are often located distal from the source of the cognate neuropeptide. While the importance of a number of neuropeptides and their receptors in controlling behavior are well appreciated, the extent of usage of neuropeptidergic signaling is only beginning to be fully appreciated. Every neuron in an animal nervous system is now thought to express at least one neuropeptide, but the pathways of communication of these neuropeptidergic signals have not been comprehensibly mapped and, hence, our understanding of information flow in the nervous system remains limited. We propose here to use C. elegans as a model system to establish the first comprehensive neuropeptidergic connectome. The simplicity of the C. elegans nervous system allows to undertake such a comprehensive analysis and, importantly, allows to compare a neuropeptidergic connectome to that of the completely established synaptic connectome. Based on preliminary data we expect to describe a ?multilayer connectome? with substantially distinct pathways of information flow, as well as distinct and similar topological features. We will achieve to build such a connectome through (1) comprehensively defining ligand/receptor pairs through in vitro receptor activation assays, (2) defining the expression patterns of all neuropeptide and neuropeptide receptor encoding genes, (3) synthesizing these data into a neuropeptidergic network and computationally comparing the topology of this network to the synaptic connectivity network and (4) undertaking a preliminary functional validation of specific nodes and edges of this network.

Many neurons display the remarkable ability to alter specific phenotypic properties in response to changes in the internal or external environment. Such ?neuronal plasticity? phenomena must intersect with genetically hardwired regulatory programs that define the fully differentiated state of a neuron. We propose here to investigate how such hardwired gene regulatory programs are modified to enable the nervous system to change specific phenotypic aspects of a neuron under specific conditions. We study this problem with single neuron resolution in the context of the nematode C. elegans whose nervous system remodels a number of features in response to the phylogenetically conserved insulin/IGF1-like hormonal signaling system. We propose and test in this grant proposal that the insulin/IGF-1-controlled DAF-16/FoxO transcription factors acts cell autonomously in many different neuron types to control the expression of specific target genes that are up- or downregulated in response to specific environmental conditions. We propose and test that DAF-16/FoxO cooperates with neuron-type specific terminal selector transcription to either promote or antagonize the ability of these transcription factors to control their effector genes.