Su Guo, Ph.D. - US grants

Dopaminergic (DA) neurons represent a rather small but important population of cells in our brain. Their death leads to Parkinson's disease (POD), a neurodegenerative disorder characterized by rigidity, difficulty in initiating movements, and a resting tremor. Molecular genetic studies of families with autosomal dominant inheritance of PD have identified mutations in alpha-synuclein, a phospho-protein normally found in all presynaptic neurons, to be responsible for the disease phenotype. Alpha- nuclein is also the main component of Lewy bodies, the pathological hallmark of PK. Whereas the fruit flies that over-express alpha-synuclein represent a good invertebrate model for PD, efforts to create such models in mammals only had limited success. In this exploratory grant, we propose to develop a vertebrate model of PD by over-expressing alpha- synuclein in the zebrafish, Danio rerio. The feasibility of generating transgenic fish expressing exogenous proteins in neurons and the small size and transparent nature of juvenile zebrafish (the fry) provides us with an excellent opportunity to determine the cellular mechanisms underlying (the fry) provides us with an excellent opportunity to determine the cellular mechanisms underlying alpha-synuclein aggregate formation and DA neuron degeneration. Furthermore, the facile genetic analysis that can be carried out with zebrafish will allow us to subsequently identify genes and pathways that are involved. The ability of zebrafish to produce a large number of offspring also permits large- scale in vivo screens for pharmaceutical compounds that can halt DA neuron degeneration. In this proposal, we would like to create double transgenic fish that express alpha-synuclein-GFP (green fluorescent protein) fusion protein in all the neurons and Tau-RFP (red fluorescent protein) in DA and noradrenergic (NA) neurons and use this model to achieve the following specific aims: 1. Determine the location of alpha-synuclein aggregates and the time course of their formation in living transgenic fish. We would like to test whether the formation of alpha-synuclein aggregates is genetically controlled and therefore occurs in a consistent pattern of whether their formation occurs in a stochastic fashion, which would suggest epigenetic control. 2. Compare the kinetics of DA neuronal degeneration to that of alpha- synuclein aggregate formation and determine if modulation of the level of neuronal oxidative stress will affect the time course of alpha- synuclein aggregation and DA neuron degeneration. 3. Determine the behavioral consequence of over-expressing alpha- synuclein in zebrafish.

DESCRIPTION (provided by applicant): Dopaminergic (DA) neurons synthesize and release neurotransmitters dopamine. The importance of DA neurons is underscored by their involvement in multiple human neurological disorders, for instance, Parkinson's disease. Despite their functional significance, the mechanisms determining the development of these neurons are not well understood. Elucidation of these mechanisms is essential to defining and interpreting the causes of disorders affecting DA neurons and developing regenerative therapy for treating Parkinson's disease. Meanwhile, understanding the development of DA neurons will also shed light on fundamental mechanisms governing cell identity and diversity and neural circuit formation in the vertebrate nervous system. The long-term goal of this project is to understand the molecular mechanisms that control the identity and connectivity of subtypes of DA neurons in vertebrates. We are taking a genetic approach in zebrafish, a vertebrate model organism that offers a unique combination of excellent genetics and embryology. We have localized major DA neuronal subtypes in developing zebrafish. By carrying out a genetic screen based on immunohistochemistry, we have identified mutations in three genes that are required for proper development of subtypes of DA neurons. Molecular cloning of the foggy gene revealed the importance of regulated transcription elongation in DA neuron development. Thus, we shall explore how this previously under-appreciated mode of gene regulation is involved in DA neuron development. Phenotypic analysis suggested that the motionless and twin-of-motionless mutations disrupt a signal important for DA neuron induction. Therefore, their molecular identity will be determined. By analysis of cloned genes and existing mutations, we will identify essential machinery involved in controlling DA neuron development. These molecules will not only provide important insights into vertebrate neural development, but may also help develop regenerative therapy for treating neurological disorders such as Parkinson's disease.

DESCRIPTION (provided by applicant): The goal of this project is to carry out a systematic genetic analysis in the zebrafish Danio rerio, to understand the biological effects of alcohol on cell signaling. Understanding the molecular mechanisms that mediate cellular responses to acute or chronic ethanol treatment is not only important for understanding the complex signal transduction pathways and their cross regulation inside cells, but is also critical for understanding alcoholism, alcohol abuse, and the medical complications of excessive drinking. Simple and robust assays have been developed in zebrafish to assess the effects of acute as well as chronic alcohol exposure on cell signaling. By using these simple and powerful assays, we have carried out a pilot mutagenesis screen and identified mutations that display abnormal responses to either acute or chronic treatment of ethanol. These molecular genetic analyses will provide fundamental insights into signal transduction mechanisms and its modulation by acute and chronic alcohol exposure. Future behavioral and pathophysiological analyses can be done using the isolated zebrafish mutants. Further, molecules identified in zebrafish will provide candidate genes for association study of human alcoholism and alcoholic medical disorders.

[unreadable] DESCRIPTION (provided by applicant): With the complete human genome sequence information, we are faced with the enormous challenge of deciphering its function in relation to understanding the biology of human development health and disease. Protein coding sequences account for less than 2% of the human genome. Cross species sequence comparisons have revealed many conserved non-coding elements (CNEs) throughout the vertebrate genome, most of which are likely to have important functions that are currently un-defined. The goal of this project is to systematically identify and functionally analyze CNEs in the zebrafish Danio rerio, a vertebrate model organism that is not only good for forward genetics but also suitable for large-scale functional analysis. The proposed study will not only provide insights into transcriptional regulation of vertebrate brain development, the methodology developed in this study can also be applied to functionally define CNEs for other developmental processes of interest. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The suitability for large-scale screening and the transparent nature during embryonic and larval stages make zebrafish an attractive system for small molecule screening in an intact organism, with aims both to identify therapeutic compounds and to understand biological mechanisms. Dopaminergic (DA) neurons play important roles in regulating movement, motivational behaviors, and hormonal homeostasis. Deregulation of the development and/or function of DA neurons have been implicated in neuropsychiatric disorders such as addiction, schizophrenia, attention deficit hyperactivity disorder (ADHD), as well as the neurodegenerative disorder Parkinson's disease. In this proposal, we seek to identify small molecule compounds that can regulate the in vivo commitment and differentiation of naive progenitor cells toward DA neurons, and to understand the mechanisms of action of these compounds. This study shall not only provide a better understanding of the in vivo mechanisms underlying stem cell commitment/differentiation toward the DA neuronal lineage, but may also discover small molecule tools that can be used for developing useful therapeutics to combat dopamine-system related human disorders. PUBLIC HEALTH RELEVANCE: Our research aims to identify small molecule "drug"-like compounds that can regulate the commitment and differentiation of pluripotent stem cells into dopaminergic (DA) neurons. The importance of DA neurons is underscored by their involvement in multiple human neurological disorders including Parkinson's disease, schizophrenia, addiction, and autism. Our proposed studies are likely to have a long-term impact on understanding in vivo stem cell behavior and moreover to identify therapeutic compounds that can combat DA system-related disorders.

DESCRIPTION (provided by applicant): The suitability for large-scale screening and the transparent nature during embryonic and larval stages make zebrafish an attractive system for small molecule screening in an intact organism, with aims both to identify therapeutic compounds and to understand biological mechanisms. DA neurons degenerate in human Parkinson's disease (PD), a devastating neurodegenerative disorder for which there is currently no cure. Zebrafish, as a prominent vertebrate model organism with the ability to produce a large number of transparent embryos and larvae, is ideally suited for discovering small molecules that can regulate the development, maintenance, or regeneration of DA neurons. However, key limitations with whole organism-based chemical screening is low throughput and low resolution. In this proposal, we will advance whole organism screening by developing and integrating fast speed high-resolution whole-organism imaging with an improved microfluidics- based technology, using the DA neuron assay that we have already established. If successful, this platform will revolutionize the whole-organism screening capability and lead to novel small molecule compounds that can modify CNS development and function and provide leads for potential treatment of PD.

DESCRIPTION (provided by applicant): Disturbance in neuronal circuits critical for mediating reward and aversion underlies a number of neuropsychiatric disorders. However, genes and pathways that control the assembly and function of the reward and aversion neuronal circuits remain poorly defined. In this exploratory R21 application, we propose to employ larval zebrafish to study neuronal circuits underlying aversion, because the system offers great opportunity for high throughput genetic and drug screening in the context of a developing vertebrate brain. We have discovered that larval zebrafish find the dark environment aversive thereby preferring to stay in the light side. Our central hypothesis is that this dark avoidance behavior represents an excellent paradigm for understanding the development of neuronal circuitry underlying aversion at molecular and cellular levels.

DESCRIPTION (provided by applicant): The hypothalamus acting through the pituitary is important for organismal adaptation to homeostatic challenges. Dysfunction in these systems impairs health, increases addictive behaviors and is a common cause of relapse. Elucidating the molecular cellular mechanisms is therefore critical for understanding, preventing, or treating a variety of associated disorders including substance abuse. The hypothalamic corticotrophin-releasing factor (CRF) and pituitary pro-opiomelanocortin (POMC) neurons are evolutionarily conserved across vertebrates. They control organismal responses to aversive stimuli through regulating circulating neuropeptides and glucocorticoids. However, it remains poorly understood how CRF and POMC neurons are regulated by both external environment and internal neural states. Neuromodulatory systems are involved, but because of the pleiotropic action of most neuromodulatory systems, it has been difficult to understand their role in CRF-POMC regulation in cell type- and gene-specific manners. Dopamine (DA) is a classical neurotransmitter that is best known for its role in signaling reward. Dopamine also plays a critical but poorly understood role in hypothalamic-pituitary regulation in a variety of species including humans, dogs, rats, mice, and fish. Recently, we have uncovered that dopamine (DA) regulates CRF-POMC function in larval zebrafish through both D1 and D2 receptors. This study will employ the transparent and highly accessible larval zebrafish system and advanced molecular genetic technologies to understand the mechanisms by which dopamine regulates CRF-POMC function. This study will unveil new molecular and cellular mechanisms on how DA neurons interact with CRF and POMC neurons to regulate their function. The findings will provide new insights into the development function and evolution of these important neural systems in the context of organismal survival and wellbeing. Equally importantly, this proposal will establish broadly applicable tools for genetically dissecting neuromodulatory systems in complex behaviors.

? DESCRIPTION (provided by applicant): Asymmetric division of progenitor/stem cells plays an important role in cell fate specification and tissue morphogenesis during development. This process is also critical for tissue homeostasis and repair in adulthood. Since dys-regulation of asymmetric division can lead to a variety of developmental and intellectual disabilities, it is critical to understand the underlying cellular and molecular mechanisms. Studies in invertebrate systems have identified important cortical polarity regulators, which ensure proper segregation of fate determinants into two daughter cells. Compared to these advances, much less is understood about the regulation of asymmetric division and subsequent daughter fate choice in vertebrates. Radial glia (RG) progenitors, the principal neural stem cells (NSCs) in the vertebrate brain, divide asymmetrically to balance self-renewal and differentiation. Although the Par-3 complex is asymmetrically localized to the apical side of dividing RGs, the RG cleavage plane is largely perpendicular to the crescent, resulting in the inheritance of Par-3 complex by both daughters. Therefore, how do the Par complexes generate distinct daughter cell fate under such conditions? In this application, we propose to use the zebrafish developing brain as a model to address this question. We will test the central hypothesis that cortical polarity establishes centrosome asymmetry to regulate asymmetric daughter cell fate in vertebrate RG progenitors. Innovative methods and techniques, including SIM and STORM microscopy, in vivo time-lapse imaging, and biochemical approaches (e.g. BioID) will be employed to test this hypothesis. The expected outcome of the proposed work is significant new insights into asymmetric division and stem cell fate during vertebrate development. These findings should have a positive impact on revealing fundamental principles, and laying groundwork for elucidating disease etiology and stimulating new therapeutic development.

? DESCRIPTION: Recently, legalization of marijuana use has been a topic of significant public attention, yet our understanding of the fundamental underlying mechanisms remains rudimentary. ?9-tetrahydrocannabinol (THC), the main psychoactive ingredient in marijuana, can interact with the endogenous cannabinoid system (eCBs), which includes cannabinoid receptors, endogenous ligands, and enzymes involved in the synthesis and metabolism of these ligands. The finding that developmental cannabis exposure in humans is associated with psychiatric vulnerability and addiction later in life suggests that eCBs may influence brain development and plasticity in significant ways. However, how eCBs exert such influences remains poorly understood. This is in large part due to the broad expression of eCBs both spatially and temporally, making constitutive knockout phenotypes difficult to interpret, due to developmental compensation and lack of specificity. Thus, it requires new tools and technologies to elucidate cell type-specific and time-dependent functions of eCBs. The zebrafish Danio rerio is a vertebrate model organism well suited to connect brain development with function. The transparent and easily accessible embryos and larvae offer the opportunity to observe brain development at single-cell levels. As early as 5 days post fertilization (dpf), larva zebrafish need to hunt for food and escape from predators, thus a simple yet functional reward system is in place. Embryonic and larval zebrafish can be conveniently exposed to chemicals (e.g. drugs of abuse) for studying their biological effects. The ability to control gene activity ith spatiotemporal precision shall greatly facilitate new discoveries in biology and medicine. The recent development of a novel RNA-guided genome-editing technology named CRISPR offers unprecedented ease for studying gene function in vivo. In this exploratory R21 proposal, we will adapt the in vitro CRISPR gene regulation technologies for spatiotemporally controllable gene silencing and activation in neurons in vivo using light, which we term Opto-CRISPR. The specific hypothesis that we will test with this new technology is: the cannabinoid signaling regulates the development of dopaminergic (DA) neurons related to reward circuitry. Impact: If successful, this project will exert a major impact on the following research fields: 1) It will advance technologies for spatiotemporally targetable gene silencing and activation in neurons in vivo. We will make this technology widely available to the research community. 2) It will reveal whether eCBs regulate the development of DA neurons related to reward circuitry. Such new knowledge will help inform policy decisions and foster innovations for treating drug abuse and addiction. Suitability for CEBRA: (1) This project will test a highly novel and significant hypothesis, aimed at evaluating the role of eCBs in the development of DA neurons related to reward circuitry. At the moment, there are scant precedents or preliminary data regarding this topic, however, if confirmed, it would have a substantial impact on current thinking of eCBs signaling in the reward circuit development. (2) This project will develop innovative techniques for gene silencing and activation with high spatiotemporal precision. This will have promising applicability to drug abuse research and beyond.

PROJECT SUMMARY Addiction is a chronic relapsing brain disorder that is characterized by compulsive self-administration of abused substances despite their negative consequences. Significant progress has been made in elucidating the molecules and behavioral effects underlying each major drug of abuse. Given that addiction is a disease of interconnected brain networks, examination of network interactions is critical for understanding addiction- related dysfunctions. Resting-state functional connectivity (rsFC), which measures correlations among distinct neurophysiological events in human fMRI studies, has provided insights, but the lack of cellular resolution and inability to carry out experimental perturbation in humans precludes further cause-effect relationship studies. At present, network level analyses are scarce in animal models, resulting in a significant knowledge gap between molecular actions of drugs of abuse and their effects on behavior. In this exploratory application, we aim to uncover how drugs of abuse alter rsFC at systems levels with cellular resolution, through employing brain-wide calcium imaging and computational analyses in larval zebrafish. As a vertebrate genetic model organism, zebrafish shares considerable neuroanatomical and genomic similarity with humans. Larval zebrafish, with a transparent brain of ~100,000 neurons (as compared to ~75 million in the mouse, and ~1 billion in the human brain), is particularly suitable for single-cell resolution analysis of neural circuit activity in vivo. We have established brain-wide calcium imaging and computational data analysis platforms to characterize rsFC at single-cell resolution in larval zebrafish brain. We wish to to test whether single or repeated exposure to morphine will significantly alter rsFC. Impact: If successful, this project will establish a new paradigm to uncover, at systems level and with cellular resolution, how addictive substances alter brain states. Future efforts can expand to all major substances of abuse, which will potentially reveal distinct functional connectivity signatures in acutely or chronically drugged brains. It is also possible to use the platform to examine genetically modified brains and perform small molecule drug screens. We will make technologies including image acquisition, processing, and computational algorithms available to the broad research community. Suitability for CEBRA: (1) This project will develop a new platform to examine the in vivo effects of addictive substances at systems level and with cellular resolution. Such advances are critical to bridge the gap between molecules and behavioral effects of drugs of abuse. At the moment, there are scant precedents or preliminary data regarding this topic, however, if confirmed, it would have a substantial impact on current approaches to understand drugs of abuse. (2) This project will develop innovative technological platforms for neural activity imaging, including image acquisition, processing, and computational algorithms for data analyses. This will have promising applicability to drug abuse research and beyond.

PROJECT SUMMARY Complex brain disorders such as the autism spectrum disorders (ASDs), schizophrenia, depression, and anxiety disorders stem from heterogeneous genetic predispositions (and at times, with environmental influences). A common hallmark of these disorders is a systems level brain dysfunction. Although human genetic studies have identified a repertoire of disease susceptibility genes with functions ranging from transcriptional and translational regulation to synaptic structural modulation and neurotransmission, at present, little is known as to how disruption of genes and associated molecular and cellular processes alter brain connectivity that define certain behavioral features of each disorder. This exploratory R21 application aims to develop a new platform for understanding, at the basic circuit level, how disruption of genes alters brain connectivity, thereby attempting to connect molecules cells and behavior. Functional Magnetic Resonance Imaging (fMRI) studies have explored resting-state or task-related functional connectivity, which measures correlations among distinct neurophysiological events in human brains. These studies have provided a valuable framework, but the lack of cellular resolution and difficulty to carry out experimental perturbation in humans precludes further cause-effect relationship studies. To uncover brain functional connectivity at systems levels with cellular resolution, we propose to perform brain-wide calcium imaging and computational analyses employing larval zebrafish. As a vertebrate genetic model organism, zebrafish shares considerable neuroanatomical and genomic similarity with humans. Larval zebrafish, with a transparent brain of ~100K neurons (as compared to ~75 million in the mouse, and ~1 billion in the human brain), is particularly suitable for dynamic single-cell resolution imaging in vivo. In this application, through brain-wide calcium imaging and computational data analyses, we propose to determine how genetic alterations may affect brain functional connectivity at cellular resolution in larval zebrafish. Expected outcomes and impact: If successful, this project will establish a new paradigm to uncover, at systems level and with cellular resolution, how genetic changes alter brain connectivity. These studies will lay critical foundation for future understanding of mechanisms between gene alterations and circuit activity changes, as well as expanding the studies to a greater number of genes. We will make technologies including image acquisition, processing, and computational algorithms available to the broad research community. In the long run, new basic knowledge about gene and brain connectivity relationships will aid in developing novel therapeutic ideas. The high risk and high reward nature of the proposed work makes this application well suited for the R21 mechanism.

PROJECT SUMMARY How behavior is regulated is a fundamental unsolved problem. Approaching this problem requires tractable behavioral readouts. It also requires model systems that are amenable to molecular genetic and systems level dissection. The long-term goal of this project is to elucidate the molecular and cellular basis of aversive behavior, an action that is propelled by un-wanting, ?dislike?, or fear. Understanding how aversive behavior is regulated at the basic molecular, cellular, systems, and population levels shall provide fundamental insights into our understanding of brain function, and are therefore of high significance. Larval zebrafish provide a salient vertebrate system that enables the understanding of behavior from molecules to systems. For example, they exhibit a light/dark preference behavior, with dark being perceived aversive. Light/dark preference as a choice behavior is observed across the animal kingdom. The underlying mechanisms are however not understood. In mammals, light/dark preference is considered an anxiety-like trait and used to assess the anxiolytic properties of drugs. Intriguingly, treatment of larval zebrafish with the same anti-anxiety medications also significantly relieves their dark aversion. We have demonstrated heritable variation of the dark aversion behavior in larval zebrafish. In this application, we propose to exploit the unique strengths of larval zebrafish for high throughput phenotyping/genotyping and brain-wide calcium imaging. We will team up with experts in population genetics and computational science to understand the molecular and cellular basis of this behavioral variation. The central objectives of this proposal are: 1) Determine, at the molecular genetic level, the driving forces for this behavioral variation. 2) Uncover cellular and network level mechanisms that underlie this behavioral variation. Successful completion of these aims will link genes to brain and to behavior. Impact and Outcomes: Complex behaviors are observed in a spectrum across the population, with the extreme ends of the spectrum often classified as disease states. The proposed work harvests a unique resource of behavioral variation in a tractable vertebrate model organism, and is expected to uncover new and potentially evolutionarily conserved insights into behavioral regulation. These findings should have a positive impact on informing human studies of behavioral variation ranging from normal spectrum to disease states. The proposed work also lay foundation for other researchers to use zebrafish for mapping naturally existing quantitative traits.

PROJECT SUMMARY How behavior is regulated is a fundamental unsolved problem. Approaching this problem requires tractable behavioral readouts. It also requires model systems that are amenable to molecular genetic and systems level dissection. The long-term goal of this project is to elucidate the molecular and cellular basis of aversive behavior, an action that is propelled by un-wanting, ?dislike?, or fear. Understanding how aversive behavior is regulated at the basic molecular, cellular, systems, and population levels shall provide fundamental insights into our understanding of brain function, and are therefore of high significance. Larval zebrafish provide a salient vertebrate system that enables the understanding of behavior from molecules to systems. For example, they exhibit a light/dark preference behavior, with dark being perceived aversive. Light/dark preference as a choice behavior is observed across the animal kingdom. The underlying mechanisms are however not understood. In mammals, light/dark preference is considered an anxiety-like trait and used to assess the anxiolytic properties of drugs. Intriguingly, treatment of larval zebrafish with the same anti-anxiety medications also significantly relieves their dark aversion. We have demonstrated heritable variation of the dark aversion behavior in larval zebrafish. In this application, we propose to exploit the unique strengths of larval zebrafish for high throughput phenotyping/genotyping and brain-wide calcium imaging. We will team up with experts in population genetics and computational science to understand the molecular and cellular basis of this behavioral variation. The central objectives of this proposal are: 1) Determine, at the molecular genetic level, the driving forces for this behavioral variation. 2) Uncover cellular and network level mechanisms that underlie this behavioral variation. Successful completion of these aims will link genes to brain and to behavior. Impact and Outcomes: Complex behaviors are observed in a spectrum across the population, with the extreme ends of the spectrum often classified as disease states. The proposed work harvests a unique resource of behavioral variation in a tractable vertebrate model organism, and is expected to uncover new and potentially evolutionarily conserved insights into behavioral regulation. These findings should have a positive impact on informing human studies of behavioral variation ranging from normal spectrum to disease states. The proposed work also lay foundation for other researchers to use zebrafish for mapping naturally existing quantitative traits.

PROJECT SUMMARY How behavior is regulated is a fundamental unsolved problem. Approaching this problem requires tractable behavioral readouts. It also requires model systems that are amenable to molecular genetic and systems level dissection. The long-term goal of this project is to elucidate the molecular and cellular basis of aversive behavior, an action that is propelled by un-wanting, ?dislike?, or fear. Understanding how aversive behavior is regulated at the basic molecular, cellular, systems, and population levels shall provide fundamental insights into our understanding of brain function, and are therefore of high significance. Larval zebrafish provide a salient vertebrate system that enables the understanding of behavior from molecules to systems. For example, they exhibit a light/dark preference behavior, with dark being perceived aversive. Light/dark preference as a choice behavior is observed across the animal kingdom. The underlying mechanisms are however not understood. In mammals, light/dark preference is considered an anxiety-like trait and used to assess the anxiolytic properties of drugs. Intriguingly, treatment of larval zebrafish with the same anti-anxiety medications also significantly relieves their dark aversion. We have demonstrated heritable variation of the dark aversion behavior in larval zebrafish. In this application, we propose to exploit the unique strengths of larval zebrafish for high throughput phenotyping/genotyping and brain-wide calcium imaging. We will team up with experts in population genetics and computational science to understand the molecular and cellular basis of this behavioral variation. The central objectives of this proposal are: 1) Determine, at the molecular genetic level, the driving forces for this behavioral variation. 2) Uncover cellular and network level mechanisms that underlie this behavioral variation. Successful completion of these aims will link genes to brain and to behavior. Impact and Outcomes: Complex behaviors are observed in a spectrum across the population, with the extreme ends of the spectrum often classified as disease states. The proposed work harvests a unique resource of behavioral variation in a tractable vertebrate model organism, and is expected to uncover new and potentially evolutionarily conserved insights into behavioral regulation. These findings should have a positive impact on informing human studies of behavioral variation ranging from normal spectrum to disease states. The proposed work also lay foundation for other researchers to use zebrafish for mapping naturally existing quantitative traits.

PROJECT SUMMARY How behavior is regulated is a fundamental unsolved problem. Approaching this problem requires tractable behavioral readouts. It also requires model systems that are amenable to molecular genetic and systems level dissection. The long-term goal of this project is to elucidate the molecular and cellular basis of aversive behavior, an action that is propelled by un-wanting, ?dislike?, or fear. Understanding how aversive behavior is regulated at the basic molecular, cellular, systems, and population levels shall provide fundamental insights into our understanding of brain function, and are therefore of high significance. Larval zebrafish provide a salient vertebrate system that enables the understanding of behavior from molecules to systems. For example, they exhibit a light/dark preference behavior, with dark being perceived aversive. Light/dark preference as a choice behavior is observed across the animal kingdom. The underlying mechanisms are however not understood. In mammals, light/dark preference is considered an anxiety-like trait and used to assess the anxiolytic properties of drugs. Intriguingly, treatment of larval zebrafish with the same anti-anxiety medications also significantly relieves their dark aversion. We have demonstrated heritable variation of the dark aversion behavior in larval zebrafish. In this application, we propose to exploit the unique strengths of larval zebrafish for high throughput phenotyping/genotyping and brain-wide calcium imaging. We will team up with experts in population genetics and computational science to understand the molecular and cellular basis of this behavioral variation. The central objectives of this proposal are: 1) Determine, at the molecular genetic level, the driving forces for this behavioral variation. 2) Uncover cellular and network level mechanisms that underlie this behavioral variation. Successful completion of these aims will link genes to brain and to behavior. Impact and Outcomes: Complex behaviors are observed in a spectrum across the population, with the extreme ends of the spectrum often classified as disease states. The proposed work harvests a unique resource of behavioral variation in a tractable vertebrate model organism, and is expected to uncover new and potentially evolutionarily conserved insights into behavioral regulation. These findings should have a positive impact on informing human studies of behavioral variation ranging from normal spectrum to disease states. The proposed work also lay foundation for other researchers to use zebrafish for mapping naturally existing quantitative traits.

PROJECT SUMMARY Asymmetric cell division (ACD) plays a critical role in fate specification and morphogenesis during development. This process is also crucial for tissue homeostasis and repair in adulthood. Dys-regulation of ACD results in developmental/intellectual disabilities and tumorigenesis, making it critical to understand the underlying cellular and molecular mechanisms. A critical aspect of ACD is the establishment of cell polarity. Studies in invertebrate systems have identified important cortical polarity regulators, which ensure proper segregation of fate determinants into two daughter cells. These studies further shed light on how distinct protein complexes establish and maintain their reciprocal cortical polarity. Despite these advances, how cortically localized proteins polarize non-cortically distributed cell fate determinants is not well understood. Research into vertebrate systems has begun to uncover new insights into the function of these evolutionarily conserved polarity regulators. Radial glia progenitors (RGPs), the principal vertebrate neural stem cells (NSCs), represent a model cell type as they predominantly undergo ACD during active neurogenesis to balance self-renewal and differentiation. Our prior work shows that in embryonic zebrafish forebrain RGPs, the key cortical polarity regulator Par-3 is critical to establish asymmetric Notch signaling activity in daughter cells. It remains unknown how Par-3 establishes such asymmetry. Recently, we uncover, for the first time to our knowledge, that Par-3 is present in the cytosol and associates with the dynein motor complex on Notch ligand-containing endosomes. Together, Par-3 and dynein are required in the mother RGP to directionally transport Notch ligand-containing endosomes to the self- renewing daughter. Additionally, we discover that cortical Par-3 domain shifts from apical at interphase toward posterior during mitosis, to align with cell division orientation along the anteroposterior embryonic axis. In this application, we wish to build upon these new findings to address the following questions: 1) how does Par-3 work together with dynein to direct polarized dynamics of Notch ligand-containing endosomes? 2) what is the dynamic relationship between cortical and cytoplasmic Par-3? 3) What mechanisms reconstruct the axis of Par-3 polarity from apicobasal during interphase to anterior-posterior during mitosis? Our central hypothesis is that both intrinsic and extrinsic mechanisms operate to reshape Par-3 cortical polarity; this cortical polarity then sets up a polarized cytoplasmic gradient of Par-3, which in turn directly facilitates endosome dynamics by activating dynein. The proposed work is expected to yield significant new insights into asymmetric division and neural stem cell fate regulation during vertebrate brain development. These findings should have a positive impact on revealing fundamental principles and laying groundwork for elucidating disease etiology and stimulating new therapeutic development.

PROJECT SUMMARY An important goal in neuroscience is to elucidate with cellular and molecular clarity how neurodegeneration (ND) might occur in vivo, given the intricate signaling and interactions among neurons and glia in the brain. This application aims to understand a fundamental G Protein Coupled Receptor (GPCR) signaling pathway in both programmed neuronal death (PND) and insult-induced ND (IND). IND will be studied in in the context of Gaucher disease (GD), a multisystemic disorder including neuropathology before the age of three and Parkinson?s disease (PD). It is well known that neuronal death occurs both in development and in diseased conditions. During development, PND is critical for constructing a functional nervous system, e.g. by providing signals for the colonization of microglia. On the other hand, IND due to injury or disease processes significantly impairs the nervous system function. Studies employing invertebrate model organisms have provided insights. How PND and IND are mechanistically regulated in vertebrates, however, is not well understood. Through an unbiased whole organism-based small molecule screen employing a chemo-genetic nitroreductase/metronidazole (NTR/MTZ) dopamine (DA) neuron degeneration model in zebrafish, we have uncovered inhibitors of the renin-angiotensin system (RAS) that significantly protect neurons from both PND and IND. RAS is a peptidergic GPCR signaling system found in vertebrates, classically known to regulate blood pressure and salt retention. RAS inhibitors are widely used drugs for treating high blood pressure. The mechanism of action of RAS in ND however remains poorly understood, despite that RAS expression is detected in both neurons and glia, and altered expression is observed during aging, in multiple ND diseases, and inhibitors of RAS are in clinical trials for treating ND. We further find that inhibiting RAS signaling reduces DA ND in GD. Microglial colonization in the healthy developing brain is also significantly decreased upon RAS inhibition. Built on these preliminary data, we hypothesize that RAS signaling regulates both PND and IND outside its conventional role in the vascular system but involves neurons and glia. This hypothesis will be tested in both PND and IND, using a combination of molecular genetic, chemical genetic, and advanced microscopic imaging methods. Expected outcomes and impact: Through a systematic screen, we have uncovered a role of RAS signaling in both PND and IND in a highly accessible vertebrate model organism. The proposed research will create new fundamental knowledge to address the underlying mechanisms. Inhibitors of RAS signaling have clinical implications for treating ND diseases. By addressing the mechanisms of action for these agents, our research is well in line with NIH?s strategic plan to benefit human health through basic science research.

PROJECT SUMMARY It has been observed across species that embryonic radial glia neural progenitors undergo asymmetric cell division (ACD) to generate daughter cells with different Notch activity. The Notchhi daughter undergoes self- renewal, whereas the Notchlo daughter embarks on differentiation. Being able to molecularly define such different states will significantly advance our understanding of how self-renewal and differentiation are regulated. Although the relative Notch activity levels between embryonic daughters are correlated with their self- renewing vs. differentiation potential, the absolute Notch activity is heterogeneous across the progenitor population. This makes it impossible to simply sort single-cell RNA-seq (scRNA-seq) data based on the expression levels of Notch effectors (e.g. hes/her transcript levels). It is therefore important to track the lineage relationships among progenitors precisely at the level of sibling cells in scRNA-seq experiments. This exploratory R21 application, motivated by an important biological problem, aims to establish high resolution sib lineage-tracing and combine it with scRNA-seq. This represents a technological breakthrough that will enable comparison of gene expression profiles between sibling cells. Expected outcomes and impact: If successful, this project will establish a new and broadly applicable method in which sib cell states can be compared at the transcriptomic level both in vivo and in vitro. By applying this method to embryonic radial glia progenitors that are undergoing asymmetric cell division during active neurogenesis, we expect to uncover evolutionarily conserved core genes and pathways distinguishing Notchhi and Notchlo sib states that are shared across sib-lineages. Since Notchhi and Notchlo sib states are associated with self-renewal and differentiation respectively in embryonic progenitors, we expect to gain a glimpse into whether and how self-renewal and differentiation as distinct cellular states can be depicted at the transcriptomic level that are uncoupled from specific lineage outcomes. We will make this novel dataset with precise clonal tracking widely available to the broad research community. This project will lay foundation for a future R01, which aims to dissect the function of signature genes and pathways that define Notchhi vs. Notchlo cell states. In the long run, new basic knowledge about the underlying genetic programs will aid in developing new therapeutic ideas. The high risk and high reward nature of the proposed work makes this application well suited for the R21 mechanism.