Leonid L. Moroz - US grants

DESCRIPTION: (Applicant's Abstract) A central challenge to modern neuroscience is to understand mechanisms of interneuronal communications, and the regulation and synthesis of endogenous sigal molecules in the brain, in both normal and pathological conditions. Unlike classical neurotransmitters and neuropeptides, gaseous nitric oxide (NO) is synthesized and released without the intermediary of special storage, subsequently freely crossing membrane barriers and affecting targets relatively large distances away, by direct covalent bonding. Although NO is crucial for most of the major neuronal functions (including learning, memory, differentiation and apoptosis), the resulting NO action depends on its local concentrations and the local microenvironment. NO can act either as a versatile signal molecule, and neuroprotective agent, or as a prominent neurotoxic intermediate. The development of postschemic brain injury, stroke, and neurodegenerative diseases are directly associated with a prominent overproduction of NO. NO synthase (NOS) is accepted as the only source of NO synthesis in the nervous system, and, although NOS inhibitors show promise as pharmacological instruments to prevent overproduction of NO, their effectiveness is controversial. However, since all these pathologies are generally associated with tissue acidification, we propose an alternative NOS-independent mechanism of NO formation in the nervous system, the non-enzymatic NO synthesis from nitrites in acidified and reducing micro-environments. This synthetic pathway may account for the excess O in these pathologies. Nitrites themselves are the main product of NO oxidation and can be accumulated in specific cells and tissues. Furthermore, due to the relatively high endogenous nitrite concentrations and the substantial pH transients associated with neuronal activity, this pathway is likely an additional mechanism for tonic NO production under normal conditions. The long-term objectives of this proposal are to analyze the distribution and functional significance of this complimentary NOS-independent pathway of NO formation in the nervous tissues, and, specifically, to characterize nitregic (NO producing) neuron and their postsynaptic targets. To separate enzymatic and non-enzymatic No synthesis we will use selective NOS inhibitors and microchemical analysis of major metabolites involved in these two pathways. Microelectrode electrical recording and pH1 measurements will provide further functional chracterization of individual nitregic neurons. Thus, significant gains can be made in our understanding of the synthesis of this gaseous messenger in the brain. This work will also contribute to our understanding of the neural functions in normal and pathological conditions.

This Nanoscale Interdisciplinary Research Team (NIRT) award supports a group of five faculty, including four at the University of Florida and one at Cornell University, to develop tools capable of measuring the distribution and concentration of specific messenger RNA molecules (mRNAs) in defined subcellular regions of single nerve cells. Initial effort will use neurons from the model organism, Aplysia. Use of the tools will then be extended to neurons from higher organisms with the goal of understanding how neurons establish new connections or synapses. Using electron beam technology, the team will fabricate one dimensional (1-D) DNA nanoarrays for the capture and direct assay of the mRNAs. Detection will employ molecular beacons to generate a fluorescent signal in the presence of specific target mRNAs; the beacons are fluorescent nanoparticles consisting of self-assembling branched DNA nanostructures designed using an artificially expanded genetic alphabet (AEGIS). Nanofluidics and dip-pen nanolithography will be tasked with delivery of the nanoparticles to specific sites in the DNA array. Fluorescence will be detected by optical imaging. Software specialized for analyzing biological molecules will archive and interpret recovered mRNA sequences using interpretive proteomics tools developed from evolutionary models. The project will benefit from a collaborative setting where students at all levels engage multiple disciplines. If successful, this will provide an educational paradigm for the training of the scientists of the future, as well as demonstrating the utility of nanoscience and engineering in the study of classical problems in biology.

This research will illuminate one of the most challenging aspects of the evolution of neuronal circuits: genomic mechanisms underlying cell-specific adaptive modifications and the origin of novel behaviors. The evolutionary approach is less developed in modern neuroscience. However, it is crucial to understand how complex networks and brains are formed or to answer "why" questions (e.g. why different subsets of signal molecules were selected in distinct neuronal circuits). The evolution of centralized complex brains occurs in parallel, where distinct neural patterning might emerge independently in different lineages but use similar molecular building blocks or toolkits.

This project proposes to identify and characterize cellular homologs within defined neural circuitries across opisthobranch species (e.g. Pleurobranchaea and Tritonia) to understand how changes in the genomic organization of homologous neurons lead to adaptive modifications of networks underlying escape and other behaviors. As a result, it will lead to conceptually new approaches when nervous system evolution can be portrayed on an entire genomic scale with single-cell resolution. The hypothesis about whether divergent evolution of neural circuits resulted in the appearance of novel signaling systems and other neuron-specific markers will be tested. Alternatively, novel network properties and connections might emerge as modular rearrangements of preexisting molecular components.

Training opportunities for interdisciplinary students will arise during the development of a nation-wide comparative genomic database that will be searchable for neuronal markers and signal transduction pathways. The extensive collection of transcripts will also allow testing evolutionary relationships across neurobiological models with different levels of centralization of their nervous systems. The proposed approaches and methodologies can be generalized to any system and thus will dramatically increase both the information and education opportunities that can be gained from studying classical electrophysiological preparations. The research will provide a long desired marriage of neuroscience and comparative genomics to understand of how specific neuronal networks are organized and evolved.

Proposal #: CNS 08-21622
PI(s): Fortes, Jose A.
McIntyre, Lauren M.; Moroz, Leonid L.; Principe, Jose C.; Sanchez, Justin C.
Institution: University of Florida
Gainesville, FL 32611-2002
Title: MRI/Acq.: Instrumentation for Coupled Experimental-Computational Neuroscience and Biology Research

Project Proposed:
This project, acquiring a virtualized multicomputer instrument with shared-memory subsystems and storage capacity, intends to use this instrument as a shared instrument whose resources can be virtualized, reserved, and configured on demand for different research activities related to neuroscience, computational biology, and cyberinfrastructure. Its configurations can also be dedicated and tightly coupled to in vivo experiments using network connections to in situ instrumentation used for experiments. It can simultaneously support multiple research activities because of its unique capability to support real-time computer-in-the-loop experiments, its ability to run many concurrent computation threads, its shared memory and storage subsystems, and its use of virtualization technology to manage the coexistence of multiple computing environments. To develop and validate autonomic computing nodes and techniques for multiuser virtualized computational systems, the instrument provides traces of performance and other needed monitored data. Research activities in brain-machine interfaces, neurogenesis, genomics, bioinformatics, signal processing, cyberinfrastructure, autonomic computing and other areas include:
- Brain-machine interfaces where cortex models for motor control are dynamically learned and applied in real-time,
- Experimental drug discovery through real-time analysis of large amounts of genetic data and many thousands of compounds,
- Analysis of Terabytes of genetic data captured in real-time as a neuron grows, learns, and remembers, and
- Online learning algorithms using dynamic filter topologies with online computation requirements that increase over time.
The activities have transformative goals, including the introduction of real-time, and address the high performance computation into closed-loop experiments and/or systems whose behavior is driven by complex processing of sensed data. Another goal involves providing off-line computing capabilities to match the unprecedented rate and volume of genetic data produced by massively parallel DNA sequencing technology.

DESCRIPTION (provided by applicant): Homeostatic processes are involved in the maintenance of unique neuronal phenotypes and circuit function in the face of plastic changes or injury. Neuronal homeostasis is a result of orchestrated activity of multiple gene products and, evidently, some of these can be neuron-specific. Here, we propose to use one of the best described "simple" neural circuits, the pyloric central pattern generator (CPG) in the stomatogastric ganglion of the crab (Cancer borealis), to address how gene expression patterns differ across different neuron types and how changes in gene expression maintain circuit function in response to changes in activity and modulatory state. We will start with two synaptically coupled, unambiguously identifiable neuron types that are known to be crucial for the production of rhythmic motor patterns controlling foregut movements. We propose 2 conceptually overlapping aims that will lead to the unbiased genome-wide view of neuron identity and function: Aim 1) Using sequencing-by-ligation &pyrosequencing platforms adapted to the single cell level, we will tag and quantify the majority of gene products expressed in both cholinergic (PD) and glutamatergic (LP) motoneurons, and identify which genes are differentially expressed between them, and which genes are relevant to neuronal excitability and rhythmic properties of the CPG circuit. Aim 2) We will determine which genes are involved in homeostatic regulation and functional recovery of the stereotypic rhythmic properties of the circuit. The decentralization of the stomatogastric ganglion by deprivation of descending modulatory inputs results in silencing of pyloric motor activity. However, the isolated circuit is able to restore its excitability and rhythmic properties within 2-3 days. This recovery requires changes in gene expression that can be both cell-specific and "universal". We will profile the gene expression patterns at different time points during circuit silencing and recovery of functional activity. As a result, we will identify candidate genes crucial for such functional rescue of the endogenous motor rhythms. We also hypothesize that there are evolutionarily conserved subsets of genes involved in these recovery/homeostatic mechanisms that can be shared between arthropods and mammals. PUBLIC HEALTH RELEVANCE: Here, we will characterize molecular mechanisms of how individual neurons maintain their specific properties and connections to meet the functional demands in a neural circuit controlling rhythmic foregut movements. Specifically we will describe homeostatic processes underlying functional recovery in a neural circuit following silencing and deprivation of modulatory inputs. Although we mainly develop these approaches in a model Cancer preparation where identifiable and experimentally accessible neurons allow such a proof of principle, the methods and related biological questions are of broad, general importance and their applicability to mammals will be tested as the project develops.

DESCRIPTION (provided by applicant): In every cell the genome operates as a three-dimensional integrative unit where different chromosomes occupy distinct territories or compartments within a nucleus but where the precise architecture and functional consequences of such organization remain elusive. We hypothesize that physical interactions between distant chromatin regions do occur in a neuron-specific manner and contribute to establishment of unique neuronal phenotypes and plasticity within a circuit. As a result, the preexisting three-dimensional (3-D) position coding can be a factor in genome-wide integration of the activity of thousands of genes, including establishing crucial epigenetic marks, and can be one of the mechanisms coordinating the complex transcriptional output of a cell. The major aims of this proposal are (1) to map long-range interactions of the cellular genome in synaptically coupled identified neurons and, (2) to characterize the dynamics of the 3-D reorganization of the nuclear genome following standard learning tests and synaptic stimulation. Here, using large accessible sensory, modulatory and motor neurons of the simpler defensive circuit in Aplysia, we will implement a novel Hi-C (chromosome conformation capture) approach to probe the 3-D architecture of the whole genome at the level of single neurons. The method is based on the combination of proximity-based ligation and selective capture of distinct anatomical regions of a nucleus with massive parallel sequencing. Thus, we will map interactive regions of the neuronal genome both in control conditions and following well established long-term plasticity tests (such as 5-HT applications). First, such spatial mapping of the genome conformation will allow us to unbiasedly characterize the location within a single nucleus of distinct mutually interacting chromatin compartments. Second, we will correlate their positions (e.g. central vs. peripheral) to the expression level of genes and their regulatory regions located within these compartments. Finally, we will correlate the expression level of selected genes with 5-cytosine methylation patterns (methylome) within gene regulatory regions, focusing upon components of 5-HT mediated signal transduction. This approach can be extended to other epigenetic marks (e.g. using chromatin immunoprecipitation for selective histone posttranslational modification events as activation and repression marks respectively) to probe mechanisms of integrative activity of neurons following synaptic inputs or drug administration. This paradigm can serve as a powerful proof-of-concept platform to characterize mechanisms of this most elusive cellular and genomic process leading to integrative activity of neurons, with broad implications to fundamental and clinical studies from drug abuse mechanisms to memory research. PUBLIC HEALTH RELEVANCE: Knowing the spatial organization of DNA methylation and transcriptional units within functionally characterized neurons is crucial for understanding the mechanisms of integrative activity of neurons. Indeed, in every cell the genome operates as a three-dimensional integrative entity where different chromosomes occupy distinct territories or compartments within a nucleus. Yet physical interactions between distant chromatin regions do occur to regulate gene activity, form epigenetic marks and coordinate complex transcriptional output of a cell. Here, we will characterize the 3-D architecture of long-range interactions of the cellular genome and its dynamics in uniquely identified neurons as they learn and remember. Information about positional coding within the nuclear genome is central to developing targeted therapies for the broad spectrum of pathological processes associated with drug abuse and memory loss.

DESCRIPTION (provided by applicant): Experience-dependent synaptic plasticity and underlying gene regulation are crucial for normal brain development and learning, and are disrupted in a broad array of disorders of development and learning such as schizophrenia, Alzheimer's disease, drug addiction and age related memory loss. To date, most studies of the cellular and molecular mechanisms of synaptic plasticity and gene regulation have taken a highly reductionist approach in very simplified preparations in vitro. However, practically nothing is known about genomic mechanisms of memory that occur during actual behavioral learning in the intact brain. In particular, the reductionist approach makes it difficult or impossible to study the integration of different inputs and pathways that is critical for many aspects of learning, and is a hallmark of neurodevelopment and associative learning. For these reasons, our long-term objectives are to characterize genome-wide mechanisms of long-term plasticity at the level of single identified neurons during behavioral learning. For this challenging task we will use the well-defined model system of the Aplysia withdrawal reflex, with a nearly complete mapping of a simple memory-forming circuit in a simplified behavioral preparation. We will record the activity of key individually identified neurons in that circuit and the synaptic connections between them during both a nonassociative form of learning (sensitization) and an associative form of learning (classical conditioning). And, for the first time, we will monitor the operation of the entire genome within specific individual neurons as they learn and remember. As a result, we will link neural activity to gene expression, plasticity, and behavior. We will also identify neuron type specific cellular signaling mechanisms and gene regulatory pathways during sensitization and conditioning, and test their roles in long-term plasticity. Based on our previous results, we hypothesize that three signaling pathways (5-HT, NO, and activity) act synergistically to produce more specific and longer-lasting memory traces during conditioning than during sensitization. Furthermore, 5-HT and NO can each change expression at least a thousand genes (some of which overlap) and induce large-scale chromatin remodeling. These findings have raised a fundamental question: how are these different inputs integrated at the level of genome-wide gene regulation in individual neurons in the circuit for conditioning? We will identify critical molecular targets (including promoters, enhancers and relevant transcription factors) leading to such integration, and examine their roles as decision points in the formation of long-lasting memories. These studies will significantly advance our understanding of synaptic and genomic mechanisms that contribute to circuit formation, learning, and memory, and their possible dysfunction in diseases that affect neurodevelopment and memory. PUBLIC HEALTH RELEVANCE: While substantial progress has been made in reductionistic analyses of nonassociative forms of memory in vitro, the molecular and genomic mechanisms of associative forms of memory in vivo are more elusive. We will investigate those mechanisms at all levels (from cell-to-circuit-to-behavior) by directly analyzing molecular and genomic events in individual neurons of the underlying neuronal circuit during behavioral learning. These studies will substantially advance our understanding of normal memory processes and their possible dysfunction in memory related diseases.

DESCRIPTION (provided by applicant): The objective of the proposed research is to conduct a thorough single-cell and cell-compartment gene expression study through the application of high throughput genomic technologies to identify the genomic bases of neuronal identity, polarity and plasticity. Utilizing the well-studied gill withdrawal reflex memory circuit from the model organism Aplysia californica, our goal is to define systematically the molecular repertoire (genomic blueprint) of the neurites and individual synapses of the key neurons that make up this cellular ensemble. We will define the compartmental transcriptomes (the sets of mRNAs, miRNAs and other ncRNAs) within the components of the functional circuit (cells and synapses), which are reconstituted in vitro by co- culture of 2-4 of its best characterized cells (L7 motor neuron, sensory neuron, stimulatory and inhibitory interneurons). This fully operational neural circuit reconstructed in cell culture bears many important properties of the intact circuit, and has been used with great success to ascertain the molecular underpinnings of memory formation in Aplysia, numerous aspects of which are conserved within the animal kingdom, including in the human brain. The systems biology approach will be applied to reveal gene regulatory networks and their potential role in the establishment and maintenance of long-term memory using learned fear as an experimental paradigm, focusing on synaptic mechanisms of long-term facilitation (LTF) and depression (LTD). We will use this genomic and systems biology approach to explore the following three fundamental brain mechanisms: (1) the molecular basis of neuronal identity, by revealing those transcripts that are unique to or shared among these neurons or specialized synapses; (2) the molecular signals controlling cellular polarity and the formation of the precise pattern of interconnections which underlie behavior, in part directed by the distribution of mRNAs in the central and peripheral compartments of these cells; and (3) the molecular basis of synapse-specific neuronal plasticity and neuronal growth, with special attention paid to the mRNA repertoire within the individual synapses at the junctions between pairs of pre- and post-synaptic neurons. The combined approach will take advantage of an already established team of experts in genomics, bioengineering, neuroscience, and bioinformatics. Though these paradigms will be established in the large well-characterized neurons of Aplysia, the mechanisms revealed and the technologies developed will have a broad impact in the biology of any polarized cell type with asymmetric distribution of RNAs and proteins. PUBLIC HEALTH RELEVANCE: Using Aplysia, a model organism that has proved exemplary in the study of learned behaviors and memory formation, we will be able for the first time to study how genes are expressed in individual cell sub- compartments. The multi-component system analyses to be undertaken will be used to understand how neurons and synapses operate in the context of learning and memory and how the activities of thousands of genes are distributed and integrated within a single cell. The molecular and systems-level discoveries made will have a broad impact in biology and biomedical research, including but not limited to memory loss and neurodegenerative diseases.

The origin of neurons is one of the most fundamental events in the development of complex animal organization. In the broader sense, it is also essential for our understanding of the origin of biological complexity and mind. This project is designed to identify and characterize signal molecules that are responsible for the development and formation of simple neural circuits and behavior. The hypothesis to be tested is that neurons arose independently in different early animals, and therefore the nervous systems of today's animals might include cells of diverse ancestry. By using the tools of modern genomics and physiology, these processes can be reconstructed in the descendants of these early animals, such as ctenophores (comb jellies). These processes can then used to repair or even design novel neural circuits. The sea gooseberry, Pleurobrachia bachei will be used as the major ctenophore model. As the broad impact, this research program will integrate education in Neuroscience with Genome Biology to decipher the molecular toolkits that controlled formation of the earliest behaviors.

The approaches and methodology that we will develop can be generalized to any system. As such, the project will conceptually change the interpretation of data gained from studying classical animal models, and will probe unique mechanisms of how to "make" a neuron, a neural circuit, and an elementary brain. It is essential for charting new directions both in synthetic biology and regeerative medicine. In addition to their value as models in neuroscience, comb jellies are significant parts of marine ecology and biological fishery control. Thus, identification of chemical signaling components in these animals will advance our understanding of their biology and contribute to monitoring and controlling the health of marine habitats.

The University of Florida (UF) is awarded a grant to purchase a state-of-the art solid-state semiconductor DNA sequencer for the Whitney Lab for Marine Bioscience and a new high bandwidth internet connection to the main UF campus. Situated some 90 miles from the main campus in Gainesville, the Whitney lab is a leader in utilizing marine biodiversity to address fundamental problems in biomedical science. The new equipment will allow the nine resident research labs as well as visiting scientists from around the world to utilize their existing strengths in cellular and molecular approaches to understanding neural systems and development. Researchers at Whitney utilize a variety of different animals found in local waters to exploit their favorable properties (such as the large size, optical properties, and fecundity) to address important issues in sensory biology, the neural control of rhythmic activity, circadian behavior, and embryonic development at cellular resolution. A number of pioneering molecular techniques of individual cells and total genome sequencing have been initiated by Whitney researchers.

The capacity to perform "third" generation sequencing at the interface between marine and terrestrial environments at the Whitney lab allows the ability to study organisms at the molecular level in their natural environment. As a research facility of UF, Whitney trains undergraduate, graduate, and postdoctoral students throughout the year, as well as REU students in the summer (for the last 26 years!). In addition, Whitney has a number of STEM programs for K-12 students in the St. Johns and Flagler county areas and a highly successful ?Evenings at Whitney? adult seminar series in our 260 seat Center for Marine Studies building that brings exciting benefits of scientific research to our local community. The high bandwith "Lambda Rail" internet connection will allow us not only to share our research data with colleagues around the world, but leverages the high performance computing capabilities located at the UF main campus in Gainesville for data analysis. More information about the Whitney Lab can be found on its website (http://www.whitney.ufl.edu/).

This INSPIRE project is jointly funded by the Organization Program in the Neural Systems Cluster in the Division of Integrative Organismal Systems, the Systematics and Biodiversity Science Cluster in the Division of Environmental Biology, both in the Biological Sciences Directorate, and by the Biologial Oceanography Program in the Division of Ocean Sciences in the Geosciences Directorate, the Office of International Science and Engineering, and the Office of Integrative Activities.

Why there is such an enormous diversity of neurons in our brains is the least understood and one of the most challenging problems in modern biology. Two factors contribute to this diversity. One is that different neurons have different functions, and the other, is that different neurons have different evolutionary histories. However, neither evolutionary history nor classification of neuronal types is established. The goal of this project is to reconstruct the genealogy of neurons and develop an unbiased classification of neurons in the majority of animal groups. This will provide a way to predict the properties of neurons and their connections. A novel approach to determine the kinds of molecules that are expressed in different neurons will be developed. To gain access to critically important organisms, the Principal Investigator will organize worldwide voyages on research vessels to collect marine animals that represent all major types of neuronal organization. Thousands of distinct neuronal populations across species will be collected and analyzed with novel computational and mathematical tools. The work will create a new field of NeuroSystematics. The team will develop and test a novel approach for distant training of undergraduate and graduate students to perform real-time analyses at any world location. The project will involve research collaborations with over 150 investigators from universities from many countries and will provide opportunities for international and underrepresented minority students to be involved in biodiversity research in remote oceanic laboratories. The project will also provide priceless resources and reference databases for several disciplines.

The research strategy is based upon the development of a mobile version of a nano-volume capture technology designed for massive transcriptome analysis of entire nervous systems at single-cell resolution. The focus will be on animal lineages never investigated before, including phoronids, brachiopods, xenoturbellids, and rare and fragile larvae. The team will integrate phylogenomic tools and statistical geometry to characterize transcriptional divergence in cell type evolution and reconstruct neurogenic gene regulatory circuits. To incorporate the inherent statistical uncertainty in the genome, as well as natural selection within complex systems, stochastic approaches from information theory will be used to evaluate molecular diversities across dozens of classes of neuronal architectures. The genomic nature of species boundaries will be studied to reveal environmental and neurosensory limitations to the rates of speciation. Thus, deciphering the genealogy of neurons will be integrated with the functional and ecological constraints that underlie the formation of new behaviors.

The origin of nervous systems with complex brains represents a major evolutionary transition in the history of animals. Investigators will test alternative hypotheses for brain/memory evolution to determine whether these functions have single or multiple origins. Genes expressed in Octopus brains will be studied and compared with genes expressed in the brains of other mollusks. New ways of assessing evolutionary relationships between the genes from the different animals will be developed in order to determine whether different animal groups use the same genes during brain development or whether each type of animal has a different set of genes that organize brain centers during development. Project outcomes include making available to the wider research community inexpensive gene analysis techniques with short processing turn-around times. The project will provide opportunities for students to combine research training with field-based marine biodiversity research in Puerto Rico, American Samoa, Palau, and Hawaii. Outreach activities are planned for K-12 teachers and students.

This project will investigate whether brains from diverse phylogenetic groups originate from a single ancestral cell lineage or whether neurons and the brains they form have developed from different lineages representing multiple, perhaps 9-12, cephalization events. The tripartite brain hypothesis predicts that the bilaterian brain is composed of three distinct regions which are determined by a set of conserved transcription factors, Hox, Otx, and Pax2/5/6, whereas an alternate view is that cephalopod-specific innovations may have led to independent formation of the complex brains of species in this group. Single-neuron genomics and connectivity will be used to reconstruct the evolutionary fate of homologous cell populations in cephalopod and chordate brains.

Ctenophores, also known as comb-jellies, are marine invertebrates that show an unprecedented capacity for regenerating. Their nervous system has two networks of interconnected cells and an elementary brain, and information travels from one nerve cell to another differently than in any other animal group. This research will reveal how comb-jellies build their nervous systems as compared to those of all other animals, and discover new molecules that control nerve cell communication in comb-jellies. Learning about these enigmatic organisms will open new horizons in scientific knowledge about alternative ways to build nerve cells and neural circuits, and will also decipher ways to rebuild damaged nervous systems. Data from these experiments will be deposited in publically web-accessible servers for the broader scientific community. This work will also provide unique opportunities for field education and training of students and the general public by using mobile marine laboratories and digital resources that are accessible world-wide.

Wild-captured animals from the genuses Pleurobrachia and Mnemiopsis are the major experimental models for this research. Neuron-specific, single-cell approaches will be used to study gene expression, metabolomics, proteomics, connectivity and function with the overall goal of identifying novel neurotransmitters and neural specification molecules in cell populations forming the two distinct neural nets (ectodermal vs mesoglea-derived) and aboral organ (the analog of the elementary brain) of diverse ctenophore species. Specific outcomes will be: (1) deciphering a new chemical language in neural systems by genome-scale annotation of neurogenic and myogenic molecular pathways and transcription factors using single-cell RNA-seq and epigenome sequencing technologies; (2) tracing neuronal identities throughout development; (3) identifying and characterizing novel signal molecules or neurotransmitters using capillary electrophoresis and mass spectrometry complemented by physiological approaches; and (4) developing potential models to explain the neural origins and evolutionary diversification of synapses.

The genetic material, or genome, first and foremost operates at the level of specific cells, and practically any animal tissue or embryo consists of thousands of highly diverse cells. How and why the same genome leads to such enormous diversity of cell types and functions are unanswered questions of modern biology. Yet, cell-specific approaches to link cause and effect are virtually absent for a majority of animal groups. This interdisciplinary project addresses these bottlenecks experimentally by developing novel genomic approaches and chemical labeling tools for genome-wide characterization of expression, classification, and mapping of thousands of individual cells in parallel. This information is used to (i) achieve a nearly complete census of cell types within a given organism, focusing on animal models critical to understanding mechanisms of learning and memory, such as Aplysia, and regeneration, such as Pleurobrachia, and (ii) generate nanoscale probes that selectively mark specific cells for genome editing, regardless of any advance knowledge about the cells' molecular diversity. Several communities are benefiting from the proposed research, including comparative neurobiology, development, biological oceanography, and the emerging field of synthetic biology. The project also affords cross-disciplinary training opportunities for trainees from the undergraduate to postdoctoral level and educational outreach activities in marine and comparative biology aimed at a diverse K-12 student body.

The grand challenge in our understanding of the genomes-to-phenomes relationships is our general inability to manipulate genome operation at the level of specific individual cells at any given location and at any given time. These obstacles are more dramatic for most invertebrates, when researchers study development or neuronal functions with little information about the cellular composition of target organs. Here, microfluidics for massive parallel single-cell capture and sequencing are integrated with novel cell selection technologies, such as aptamer-based-Cell-SELEX, for quantitative gene expression analyses and imaging of individual cells in intact tissues. Aplysia (and, once single-cell tools are validated, Pleurobrachia and/or related ctenophore species) are used to achieve nearly complete genome-wide classification of the majority of cell types in their neural systems and effector organs. The read-out(s) to measure/control gene expression in identified neurons are: scRNA-seq data with both normalized and absolute quantification of expression levels for target genes, and q-RT-PCR. Controls are neurons in which target genes are not active or silenced. First, unique resources for a diversity of cell adhesion molecules and other surface macromolecular structures critical to design and characterize cell-specific probes are generated. Then, using tools of chemical evolution, a high-throughput system to manufacture cell-specific aptamer-/molecular beacon-based fluorescent probes at a large scale is tested. Finally, hybrid nanoscale probes (e.g. made by coupling cell-specific fluorescent markers with nucleic acid analogues) are tested for their ability to self-deliver molecular constructs into target cells without direct injection, electroporation, or the need to make transgenic animals. This project is co-funded by the Chemistry of Life Processes program in the Division of Chemistry.

Marine environments harbor by far the greatest range of biodiversity on Earth and this diversity of life provides untapped opportunities to understand how organisms "work" and how they interact with their natural environment. Recent developments in molecular technology in including next-generation DNA sequencing and functional genomics provide opportunities to build a new level of understanding of the molecular basis of cellular regulation during development, neurogenesis, cancer, adaptive resiliency, and regenerative biology. Marine animals, particularly invertebrates, are ideal experimental systems for a variety of investigations into fundamental properties of animals. This proposal uses delicate marine organisms, collected and cultured at the Whitney Lab for Marine Bioscience (http://www.whitney.ufl.edu/), to leverage these new technologies for an unprecedented molecular understanding of a variety of cellular behaviors related to the interrogation of gene expression at the resolution of identified cells. This grant provides instrumentation for the isolation of pure populations of identified single cells that can be used for downstream molecular characterization (e.g. the construction of gene regulatory networks involved in the establishment of stem cell or neural identity). This equipment will be housed on site, and can be deployed for a wide array of ongoing research projects of resident and visiting researchers. All research groups at the Whitney Lab remain actively involved in the Lab's 32 year-old NSF REU program, participate in K-12 STEM outreach programs (e.g. Scientist for a Day and Whitney Lab's Traveling Zoo), summer camps for 3rd- 4th graders, and a free public lecture series ("Evenings at Whitney"). Whitney recently opened a Sea Turtle Rehabilitation Hospital that focuses on the surgical removal and cure of debilitating fibropapilloma (FP) disease that affects all species of sea turtle worldwide. Whitney faculty are periodically consulted on social and land-use management issues, and share knowledge and expertise with the local community.

This project builds on previous expertise to utilize cutting edge, single cell omics techniques to understand the molecular regulation of distinct cell types. A cell labeling facility (microinjection rig) that can fluorescently label individual cells via a variety of molecular techniques, and an efficient multiuser friendly small volume fluorescent cell sorter (BD FACSMelody) will be deployed to address a wide array of ongoing research projects of resident and visiting researchers. Fluorescently labeled cells, whether it is a dextran lineage marker, nuclear markers of DNA content, mRNAs for specific genes injected in to living cells, reporter genes driven by endogenous enhancers, or CRSPR/Cas9 reporter gene knock-ins can be dissociated into single cells and sorted using a FACS (Fluorescently Activated Cell Sorter) machine. FACS machines were once large prohibitively expensive instruments needing large numbers of cells (1 x 106-9 ) and only able to sort limited wavelength channels. Newer generation FACS machines have become much more powerful with the development of multiple solid state lasers and small volume sorters (96 or 384 well formats). The capability to isolate single cells goes hand-in-hand with the ability to sequence transcriptomes and genomes of single or small numbers of molecularly identified cells, using Next Gen sequencing technology.

Project Summary All neurons are remarkably different and existing approaches do not allow de novo visualization of specific living cells in intact brains, without laborious tasks of making transgenic animals. The project will address this grand challenge: our interdisciplinary team will develop and validate novel nanoscopic probes, to rapidly (<30 min) label specific neurons within highly heterogeneous cell populations. For these applications, in vitro neuronal selection Neuro-SELEX (systematic evolution of ligands by exponential enrichment) will be used to generate libraries of nucleic acid-based probes. These aptamer-based tools will also serve as ?pull-out? molecular constructs to identify cell-specific membrane proteins associated with unique neuronal identity and wiring. As a result, this research will provide a broad spectrum of advanced nanotools to decipher the organization of neural circuits at the level of single cells and their compartments. Our preliminary data indicate that the Neuro-SELEX can produce multipurpose toolkits to uniquely map specific neurons or axons without a priori knowledge about their molecular diversity in the intact nervous system. These results, together with our published data, provide the scientific premise for three proof-of-the-concept aims. Arguably, Aplysia is a very powerful experimental model for such technology development. First, to selectively label identified neurons and glial cells, hybrid fluorescent aptamers will be generated using chemical evolution for neuron-specific selection. We will develop a high-throughput cost-efficient system to manufacture molecular probes at a large scale, targeting each key, functionally identified, neuron within a simple-memory forming circuit. Second, we will design fluorescent probes (e.g., modified nucleic acids with fluorophores) for multiplex labeling of several neuronal cell types in vivo. This bar-coding would allow simultaneous visualization of pre- and postsynaptic partners within the same circuit in real time. Furthermore, these probes will be chemically modified to self-deliver molecular constructs into hundreds of target cells without the needs of direct injection, electroporation or making transgenic animals. Third, in proteomic experiments, we will utilize these probes as specific binding tags or ligands to capture and identify membrane proteins specific for each neuronal type of the model circuit including possible synaptic components and receptors. These versatile nanoprobes, with high selectivity and high-throughput fabrication capabilities, will be resourceful to test causality relationships between cellular genomes and complex neuronal phenotypes. Technologies and infrastructure should be applicable to virtually all animal cell types and organs. In perspective, novel fluorescent markers and molecular reporters can be used in early diagnostics and therapy for a broad spectrum of neurological and cell-specific disorders as well as in personalized medicine.