Bing Ye - US grants

DESCRIPTION (provided by applicant): The overall goal of this project is to investigate the translational regulatory machinery in dendrite development. I hypothesize that the translational control machinery first discovered in oogenesis and embryogenesis of Drosophila are conserved in neurons. Candidate genes that are known to be components of such machinery will be examined for possible involvement in dendrite morphogenesis. The following specific aims are proposed: 1) Examine the expression of Nanos, Pumilio, and Hunchback in Drosophila PNS. 2) Overexpression study of nanos, pumilio, and hunchback in dendrite development. 3) Loss-of-function analysis of nanos, pumilio, and hunchback in dendrite development. 4) Translational regulation of nanos in da neuron dendrite development. The proposed experiments will provide a start-point for further investigation of translational control machinery that regulates dendrite morphogenesis.

My career goal is to understand the mechanisms of neuronal compartmentalization and how this process contributes to nervous system function and to the pathogenesis of neurological disorders. I will pursue this goal by working in an academic institution as an independent investigator. During my postdoctoral training in the laboratory of Dr. Yuh Nung Jan at UCSF, I have been using Drosophila PNS neurons as a model system to study the mechanisms that differentiate the development of dendrite from axon, two major compartments of a neuron. This training complements my doctoral training in vertebrate neurobiology. I plan to combine the strength of Drosophila (in vivo and superb genetics) and cultured rat hippocampal neurons (wellcharacterized cell biology) to study neuronal compartmentalization. The objective of this research is to examine the roles of the secretory pathway in differentiating dendrite and axon development. From a genetic screen in Drosophila, we isolated several mutants (dar mutants) with reduced dendritic arbors but normal axons. Dar2, 3, and 6 regulate the secretory pathway, suggesting that this pathway differentiates dendritic and axonal growth. I propose two aims. First, I will determine cell biological mechanisms through which the secretory pathway differentially controls dendritic and axonal growth. New techniques will be developed to complement existing ones to identify such mechanisms. Membrane traffic through the secretory pathway will be monitored in live wild-type and mutant Drosophila embryos/larvae and cultured hippocampal neurons. Second, I will identify and characterize genes that control the differential development of dendrites and axons by regulating key players of the secretory pathway. Dar7 (genetically interacts with dar2 and 3), darl (genetic interaction untested), and Trailer Hitch (regulates the secretory pathway) will be studied. Their mammalian homologs will be examined in cultured neurons to determine if the mechanisms are conserved in mammals. This research will provide much-needed information for understanding the causes of neurological disorders characterized by preferential damage to dendrites (e.g., Rett's syndrome) or by defective Golgi function (e.g., amyotrophic lateral sclerosis). Such information will also allow the design of therapeutic approaches.

DESCRIPTION (provided by applicant): Information processing in the nervous system relies on the separation of dendrites and axons. However, little is known about how dendrites and axons develop into distinct compartments. The long-term goal of this application is to define how neuronal compartmentalization is achieved during the development of neural circuits and how defects in that process lead to neurological and psychiatric diseases. The objective of this application is to delineate the signaling pathways that separate dendrite and axon development. Recent genetic studies on Drosophila have demonstrated that the fibroblast growth factor (FGF) receptors differentially control dendrite and axon development. The central hypothesis of this application is that the FGF receptors activate distinct signaling pathways to differentially control dendrite and axon development. We will test this hypothesis by pursuing three specific aims: 1) ) Identify the signaling pathway through which FGF receptors control dendrite-specific development; 2) Determine whether FGF receptors regulate axon development through pathways different from dendrite development; 3) Determine whether the roles of FGF receptors in the differential development of dendrites and axons are conserved in mammalian neurons. The approach is innovative because it takes advantage of genetic analysis to investigate the developmental differences between dendrites and axons in vivo and combines both Drosophila and mammalian systems to study evolutionarily conserved mechanisms. The proposed research is significant because it is expected to advance knowledge of the signaling mechanisms underlying the differential development of dendrites and axons. That knowledge is needed to develop strategies that will allow preferential or specific manipulations of dendrite or axon development in disease conditions and in animal models to interrogate the functions of the nervous system.

DESCRIPTION (provided by applicant): Damage to neuronal dendrites is a key component of ethanol-induced neural injury. However, the mechanisms underlying ethanol-induced dendrite defects are poorly understood. Knowledge of these mechanisms is essential if we are to understand the effects of alcohol on neuronal development and design strategies for preventing the damaging effects of ethanol on developing neurons. Our long-term goals are to define the mechanisms underlying dendrite and axon development and to determine how defects in dendrites and axons lead to human diseases. The objective of the proposed research is to delineate the mechanisms underlying ethanol-induced dendrite growth defects. Previous studies have demonstrated the importance of the secretory pathway in dendrite development. Although ethanol is known to cause ER stress in various cell types, its effects on ER and Golgi, which are pivotal for the trafficking and glycosylation of membrane and secreted proteins and for cellular signaling, is much less understood. The applicant has established a unique system that is genetically tractable for studying the neuronal secretory pathway in Drosophila. The central hypothesis is that ethanol-induced ER stress leads to ER reorganization and Golgi fragmentation and consequently reduces dendritic growth. This hypothesis is based on preliminary findings from the applicant's laboratory. This hypothesis will be tested by pursuing two specific aims: 1) Identify the mechanism underlying ethanol-induced ER and Golgi defects in neurons; 2) Identify the mechanism underlying ethanol-induced dendrite growth defects. Under the first aim, genetic techniques and cell biological assays, which have been established as feasible in the applicant's lab, will be applied to delineate the roles of ER stress and related responses in ethanol-induced defects in ER and Golgi. Under the second aim, the applicant will take advantage of his expertise in analyzing dendrite development to delineate the roles of ER stress and Golgi fragmentation in ethanol-induced dendrite growth. The results of the proposed research are expected to define a causal relationship among ethanol, the secretory pathway, and dendrite development. The approach is innovative because it introduces a genetically tractable in-vivo system proven to be powerful for molecular and genetic analysis of cell biological problems into ethanol research on cellular organelles. The proposed research is significant because it will fill the gap in our understanding of ethanol effects on the secretory pathway and lead to a mechanistic understanding of ethanol-induced dendrite development. It will also establish an in-vivo system for identifying compounds that block ethanol-induced damage on the secretory pathway and neural development. Thus, it will lay the ground not only for extensive investigation of the role of ethanol on cellular organelles, but also for developing therapeutic strategies to cure ethanol-induced developmental defects.

? DESCRIPTION: During development, stem/progenitor cells replicate and differentiate into many lineages, which give rise to precise number and subtypes of cells. Defects in lineage development can cause severe developmental diseases. Currently, the state-of-the-art lineage analysis uses mosaic labeling techniques to study one or a few lineages at a time to avoid ambiguity. While the small number of highlighted cells can be investigated extensively, complications in the unlabeled adjacent lineages are hidden from analysis. The ability of unambiguously labeling large number of lineages in situ is highly desired, since it is extremely exhausting, if not impossible to use the available tools to study the precise spatial-temporal relationship of all related lineages in one animal. We propose to develop a two-photon compatible multispectral and subcellular-coding system (MACS), which permits unambiguous labeling of large number of cell lineages in the same animal. We will validate MACS by mapping all of the ~100 neural lineages in single Drosophila central brain and depict the developmental processes of all Drosophila embryo neural lineages precisely in space and time. If success, MACS can be easily adapt to other transgenic animal models, including fish, mouse and rat. MACS will create new opportunities in lineage studies, such as investigating lineage variations among individuals, and between hypomorphic alleles or different sex; as well as cell non-autonomous effects of gene mutations in stem/progenitor cells.

? DESCRIPTION (provided by applicant): The Undiagnosed Disease Network (UDN) has identified a mutation in the Yippee-like 3 (YPEL3) gene that potentially causes a rare human condition manifesting a number of neurological symptoms. Although the discovery has opened the opportunity to diagnose and treat this rare human disease, there is an enormous knowledge gap to be filled in order to understand the pathogenesis of this human condition and for designing potential treatments. This is largely due to the lack of knowledge on the cellular and molecular functions of YPEL3. The long-term goal is to understand the mechanism of pathogenesis caused by human mutations in the YPEL3 gene and to design therapeutic approaches to treat this rare disease. The objective of this application is to establish an in vivo Drosophila model for identifying the cellular and molecular functions of YPEL3 in the development of nervous system. Drosophila is advantageous in studying YPEL3 functions because it provides excellent tools for dissecting cellular and molecular functions and enables us to avoid possible redundancy from other YPEL genes in mammals. The central hypothesis is that YPEL3 regulates peripheral nerve development by modulating pre-mRNA splicing. This hypothesis has been deduced from the nature of the human mutation, the symptoms displayed by the patient, and the literature related to the YPEL gene family. The rationale for the proposed research is that since YPEL3 is well-conserved between human and Drosophila, understanding the cellular and molecular functions of YPEL3 in Drosophila will aid to develop effective treatment of the human diseases related to YPEL3 mutations. The hypothesis will be tested under two specific aims: 1) Identify the roles of YPEL3 in nerve development; and 2) Identify the molecular functions of YPEL3. Under the first aim, loss-of-function mutants of YPEL3 will be generated and tested for morphological alterations of glial wrapping and axon branching in peripheral nerves. Under the second aim, localization and phosphorylation of nuclear splicing regulators will be determined in glia and neurons of YPEL3 mutant. The contribution of the proposed research will be significant because it will provide not only the mechanistic insight into how YPEL3 mutations lead to pathogenesis but also a genetically amenable in vivo model system for studyin YPEL3-related diseases, facilitating the development of therapeutics. The research proposed in this application is innovative, because it integrates the fragmented information into a cohesive novel concept, and because its use of Drosophila as a model organism to avoid possible redundancy caused by other YPEL homologs as is the case in mammals.

? DESCRIPTION (provided by applicant): Down syndrome (DS) is the most common genetic form of intellectual disability, affecting one in every 700- 1000 live births, but there is currenty no effective treatment for this complex neurodevelopmental disorder. DS is caused by the trisomy of human chromosome 21, which leads to overexpression of a number of genes. In consequence, a major hurdle in DS treatment is the identification of genes that are the drivers of pathogenesis and can be targeted for effective therapies. The development of the neocortex of DS patient is defective, but the underlying molecular and cellular mechanisms are poorly understood. The long-term goal is to define the mechanisms underlying neuronal development and to determine how defects in this process lead to complex brain disorders. The objective of this application is to elucidate the molecular mechanism underlying the developmental defects in the neocortex in DS. The preliminary studies in mice suggest that overexpression of the gene Down syndrome cell adhesion molecule (DSCAM), which occurs in the brains of human DS patients, leads to defects in cortical development. However, it remains unknown how DSCAM overexpression causes defects in cortical development, or whether it is responsible for any of the cortical defects in DS. Unraveling these molecular and cellular mechanisms will provide insights into the potential of targeting DSCAM and its signaling cascades for treating the cortical defects in DS. The following two specific aims are proposed to address this issue: 1) identify the signaling mechanism by which overexpressed DSCAM affects cortical development; and 2) define the role of increased DSCAM levels in cortical development in a DS mouse model. By using a Drosophila neuronal system whose development is highly sensitive to DSCAM levels, considerable progress has been made in elucidating the mechanism by which DSCAM controls neuronal development. Experiments designed for Aim 1 will test this molecular model in mouse neocortex. In Aim 2, the contribution of DSCAM and its signaling pathway to the developmental defects in neocortex will be tested in a DS mouse mode. The contribution of the proposed research will be significant because it will elucidate the molecular and cellular mechanisms underlying the cortical developmental defects associated with DS and to provide potential targets for treating the complex brain disorder in DS. The research proposed in this application is innovative because it will investigate the roles and the underlying mechanisms of increased DSCAM levels in the cortices of normal and diseased mammalian brains. It is also innovative because it uses a Drosophila system that is highly sensitive to DSCAM levels to identify signaling mechanisms downstream of overexpressed DSCAM and combines the strength of Drosophila and mouse systems in dissecting the molecular and cellular substrates of the complex brain disorders in DS.

How a neuron?s dendrites and axons develop into distinct morphology?which is fundamental to the assembly of neural circuits?is poorly understood. Understanding the mechanisms that differentiate dendrite and axon development, therefore, is a vital goal in developmental neuroscience. Several regulatory mechanisms that are dedicated to either dendrite-specific or axon-specific growth in vivo have been identified by taking advantage of a Drosophila system. In addition, a molecular pathway that suppresses dendritic growth but promotes axonal growth within the same neuron (i.e., a bimodal mechanism) has been located upstream of these dedicated mechanisms. The bimodal regulation provides a unique mechanism for generating morphological diversity in neurons, and is relevant for the design of effective strategies to regenerate an injured or diseased nervous system. The long-term goal of this research is to define how a neuron develops into distinct subcellular parts and how defects in this process lead to human disease. The objective of the proposed studies is to uncover the molecular and cellular mechanisms of bimodal controls of dendritic and axonal growth. Recent studies have shown that the evolutionarily conserved dual leucine zipper kinase/Wallenda (DLK/Wnd) pathway is a bimodal regulator of dendritic and axonal growth, and that this pathway regulates the expression levels of a transcription factor (Knot) and a cell adhesion molecule (Dscam) to control dendritic and axonal growth, respectively. Preliminary studies suggest a novel concept: Translational regulation through RNA-binding proteins is at the core of bimodal control of dendritic and axonal growth. The following model, which integrates specific molecules and regulations with their spatial locations for bimodal control, will be tested: The DLK/Wnd pathway regulates two distinct RNA-binding proteins to control PABP-dependent initiation of Dscam translation in axon terminals for axonal growth and Knot expression in the cell body for dendritic growth, respectively. This model will be tested by identifying (a) the molecular mechanism by which the DLK/Wnd pathway regulates axon-terminal development and dendritic branch development and (b) the subcellular locations at which the DLK/Wnd pathway regulates downstream factors to instruct the differential growth of dendrites and axons. The proposed research is innovative because it proposes a novel concept in the differential development of dendrites and axons and employs several innovative techniques that are well suited for this line of research. This research is significant because it is expected to offer key insights into the coordination between dendritic and axonal development, identify a critical role translational control plays in the differential development of dendrites and axons, discover novel mechanisms by which the DLK/Wnd pathway functions in neurons, and provide insights into the pathogenesis of neurodevelopmental disorders.

Sensory experiences during development profoundly influence sensory processing in mature animals. Since most of an animal?s sensory experiences are multimodal, the activity of one sensory modality often causes long-term changes in another modality. Such cross-modal plasticity not only leads to compensation for sensory functions in the case of sensory deprivation, but also allows normal individuals to respond properly to sensory stimuli in their unique habitats or situations and contributes to individual?s differences in the perception of multisensory cues. Despite the importance of cross-modal plasticity, the underlying circuit and molecular mechanisms are poorly understood. In the proposed research, a novel form of cross-modal plasticity has been discovered in Drosophila and developed into a system for studying the underlying mechanisms at the behavioral, circuit, synaptic, and molecular levels. This system allows for comparison of cross-modal and modality-specific plasticity in the same sensory system. A genetic screen has identified novel regulators of cross-modal plasticity. The objective of the proposed research is to identify the mechanisms that underlie cross-modal plasticity in the developing somatosensory system of Drosophila larvae, and provide circuit and molecular models for guiding future studies in other species. The central hypothesis is that gentle mechanosensory inputs during development strengthen serotonergic inhibition of the synaptic transmission from nociceptors to multisensory second-order neurons (MSONs), which is achieved through specific genes in the MSONs. This hypothesis will be tested by identifying the circuit (Aim 1) and molecular (Aim 2) mechanisms that underlie cross-modal plasticity. The proposed research is innovative because it proposes the novel concept of distinct mechanisms that underlie cross-modal and modality-specific plasticity and will use a novel system that is amenable to the use of genetic screens to study cross-modal plasticity. This research is significant because it is expected to: 1) elucidate how cross- modal and modality-specific plasticity co-exist in a developing sensory system and demonstrate the role of neuromodulatory interneurons in establishing cross-modal plasticity during development; 2) identify a novel molecular mechanism that underlies cross-modal plasticity, particularly one that distinguishes it from modality-specific plasticity within the same neural circuit; 3) yield a multidisciplinary, state-of-the-art experimental system for identifying the principles that govern the experience--dependent assembly of neural circuits for multisensory integration. Moreover, because a common problem of many neurodevelopmental disorders is dysregulated multisensory integration, the proposed study will offer insights into the pathogenesis of these disorders.

PROJECT SUMMARY Understanding how individual neurons contribute to network functions is fundamental to neuroscience. Recent years have seen exciting progresses in the reconstructions of single-neuron morphologies and wiring diagrams at the level of individual synapses. Although these progresses offer promises of understanding neuronal networks, such understandings would not be reached if we do not understand how the structural details of single neurons contribute to the network connectivity. Neuronal network connectivity, which is an emergent property generated by the connections among single neurons, has been studied in depth using graph theory and other mathematical approaches. However, most computational models have disregarded fine morphological features involved in network connectivity. For the few that did, the methods developed are either unavailable to the broad neuroscience community or not user-friendly, preventing further investigations of the link between experimental structural data and network modelling. The objective of this proposed study is to develop and validate a user-friendly toolset for discovering the rules that link neuronal morphology to network connectivity, which will allow to extract and predict neural network properties from single-neuron morphologies. This open-source computational tool will include methods for visualization and data analysis for neuronal populations derived from whole brain imaging data. It will also provide an innovative generative model for interrogation of the organizational principles underlying brain networks? architecture exploring potentially relevant network properties. In Specific Aim 1, we will develop new models and methods for analyzing the impact of single-cell morphology on network connectivity. In Specific Aim 2, we will validate the use of the toolset to predict network connectivity and pathological deviations. The contribution of the proposed research will be significant because it will: (1) provide new computational tools that allow users to fill the gap between single-cell and network properties; (2) introduce the concept of neuronal structural variation; (3) yield a toolset that can be used to generate testable predictions and new biomarkers for developing therapeutic interventions for brain disorders. The research is innovative because it will (1) develop an open-source model to generate in- silico neuronal circuits capable of incorporating neuronal reconstructions, brain region segmentations and whole-brain fluorescence imaging datasets; (2) apply the concept of multi-objective optimality to network topology at the cellular scale; (3) analyze and model within-class morphological variations; 4) use intellectual disability models to validate our tools. Accomplishing the specific aims will yield a tool for linking descriptions of neuronal structures with network modeling, allowing the exploration of multi-objective optimality theoretical frameworks and improved methodologies for circuit classification based on network topology and the discovery of fundamental wiring laws in the brain.

PROJECT SUMMARY Sensory experiences during development profoundly influence sensory processing in mature animals. Since most of an animal?s sensory experiences are multimodal, the activity of one sensory modality often causes long-term changes in another modality. Such cross-modal plasticity not only leads to compensation for sensory functions in the case of sensory deprivation, but also allows normal individuals to respond properly to sensory stimuli in their unique habitats or situations and contributes to individual?s differences in the perception of multisensory cues. Despite the importance of cross-modal plasticity, the underlying circuit and molecular mechanisms are poorly understood. The objective of the parent grant R01NS104299 is to identify the mechanisms that underlie cross-modal plasticity in the developing somatosensory system of Drosophila larvae, and provide circuit and molecular models for guiding future studies in other species. The central hypothesis is that gentle mechanosensory inputs during development strengthen serotonergic inhibition of the synaptic transmission from nociceptors to multisensory second-order neurons (MSONs), which is achieved through specific genes in the MSONs. The requested Research Supplement to Promote Diversity in Health-Related Research will support the training of an outstanding postbaccalaureate. The research proposed will supplement the originally proposed studies to ensure the successful attainment of its aims. Two studies are proposed to supplement the original Aim 2: (1) determine whether FMRP is required for cross-model behavioral plasticity; and (2) determine whether cross-modal plasticity affects neural ensemble activities. These research activities will expand the research experiences of the supplementee. Moreover, they will provide opportunities for her to learn scientific writing, oral presentation skills, and networking. These training will position her strongly for a health-related research career.