Adam C. Miller - US grants

DESCRIPTION (provided by candidate): All of brain function, from sensory perception to behavior, is derived from the pattern and properties of the synaptic connections among billions (in humans) of individual neurons. The long-term goal of this project is to understand molecular pathways that regulate synapse formation in vivo using a vertebrate model with a focus on the underappreciated electrical synapse. Electrical synapses are sites of direct communication between neurons that allow the passage of ions and small molecules. They are formed in a regulated manner between only a subset of potentially available partners and are composed of neuronal gap junction channels. Electrical synapses contribute extensively to neural circuits during development as well as to adult circuits from sensory perception to processing to motor output. However, the molecular mechanisms underlying the formation of the gap junction channels that form the electrical synapse are unknown. This proposal utilizes the Zebrafish Mauthner (M) circuit to investigate the genetics of electrical synapse formation. The M neurons are individually identifiable and their pre and postsynaptic partners, synapses, and function are exquisitely visualized in a living, vertebrate embryo. A forward genetic screen for mutations causing defects in the stereotyped M electrical synapses was performed that identified two distinct classes of mutations: 1) the Disconnect (Dis) class, which disrupts synapse formation, and 2) the Amped (Amp) class, which causes ectopic synapses to form along the M axon. Using an RNA-seq-based approach all three Dis mutations were positionally mapped, and one of the Dis mutants was found to be due to the loss of the autism- associated gene neurobeachin (nbea). This proposal will investigate Nbea's role in electrical synapse formation (Aim1), will clone the other Dis and Amp mutations identified in the pilot screen (Aim2), will examine the effect of the mutations on synapse function and behavior (Aim3), and will expand the pilot screen to elucidate further genes and pathways required for synaptogenesis (Aim4). During the two year mentored phase I will develop the model system by characterizing how the genes regulate electrical synapse formation in several ways: What are the temporal and spatial properties of synaptic cargo localization during in vivo synaptogenesis? How do the mutants affect the function of the synapse? How do the mutants affect neural network function and behavior? In Cecilia Moens' lab at the Fred Hutchinson Cancer Research Center (main mentor), I will learn to perform live cell imaging of fluorescently-tagged, synaptic proteins using spinning disc confocal microscopy. This technique will be applied to all mutants and will be the first live investigation of electrical synapse formation in vivo. To investigate M synapse and circuit function I will visit Joe Fetcho's lab at Cornell University to learn to perform electrophysiology n the M neural circuit and I will visit Michael Granato's lab at the University of Pennsylvania Perelman School of Medicine to learn behavioral analysis of the M-mediated escape behavior. The skills acquired will be brought back to Seattle where I will perform experiments on the mutants. For electrophysiology I will work with Rachel Wong at the University of Washington (main co-mentor) where I will receive ongoing training in electrophysiology and will have access to equipment for experiments. For behavior I will work in the Moens lab where we have the high- speed camera necessary to capture the M-mediated escape response. The electrophysiological and behavioral analysis will be applied to all mutants and will be essential for linking the cell-biological defects to functional deficits in the circuit. The training in the Fetcho and Granato las will be short and intensive, but both mentors will be available to me on an ongoing basis for technical expertise and guidance. The mentoring in the Moens and Wong labs will be ongoing, with extensive interaction and support. With this training I will have the necessary experience and a powerful set of tools and techniques to establish my own independent research group. During the independent phase of the project I will utilize the acquired skills to illuminate the molecular mechanisms that build gap junctions at the electrical synapse. The proposed studies will provide a detailed molecular, cellular, and functional view of how neural circuits form in a vertebrate in vivo. Disorders that cause neural circuit miswiring or synaptic imbalance are the basis of many neurological diseases including autism and epilepsy. In the case of autism, several molecular pathways (including Nbea examined here in Aim1) have been associated with the disorder. However a unifying theory explaining how these genes fit together to explain the syndrome remains elusive. Investigating the genetic pathways required for neural circuit wiring and synapse formation will lend insight into disease states that will ultimately allow for the identification of targets for therapy.

Safe, effective therapies generally target specific disease-related molecules that appear only in disease-related cell types. The problem: Gaps in our knowledge include a comprehensive definition of cell types in any vertebrate species over developmental time and knowledge of which genes each cell type expresses at what levels. Genes currently known only by sequence might provide unique targets for cell therapies if we knew which cell types express them. A way forward: It has recently become possible to identify the transcriptional profile of individual, single cells with unprecedented molecular precision using single-cell RNA sequencing (scRNA-seq) coupled with powerful highly dimensional-reducing software that groups cells into bioinformatically identified clusters containing cell types with closely related gene expression profiles. The goals of this project are first, the comprehensive identification of transcriptionally unique cell types over developmental time in zebrafish, a major medical model, and second, the release of these data as a resource to the research community in a convenient searchable format through the Zebrafish Information Network (ZFIN). Approach: Aim 1 is to define single cell transcriptome phenotypes for various stages of wild-type zebrafish embryos, larvae, and juveniles and to locate these annotated cell types by in situ hybridization experiments displaying the expression of cell type-specific marker genes on whole mounts and histological sections. Aim 2 is to define the single cell transcriptome phenotype for all major organs in wild-type zebrafish adult males and females and to identify prominent cell types in vivo by in situ hybridization for cell type-specific marker genes on histological sections. Aim 3 is to develop an automated bioinformatic pipeline to identify cell types in scRNA-seq clusters by comparing gene expression profiles to existing resources, including ZFIN, other model organism databases (AGR, Alliance of Genome Resources), and human gene expression data. Aim 4 is to develop an interface in ZFIN to enable the research community to easily query zebrafish scRNA- seq data. Innovation: No animal species currently has a comprehensive compendium of cell types organized by gene expression patterns on a genome-wide scale during development. Significance: This R24 application will develop resources and related materials that will 1) enhance, further characterize, and improve a critical animal model for the investigation of human disease mechanisms; 2) facilitate access to data generated from the use of animal models of human disease; and 3) address the research interests of many categorical NIH Institutes and Centers that focus on various organ systems and disease types. This resource will identify previously unknown cell types, thus facilitating the precision targeting of cell types for potential therapies; will associate previously unknown genes with specific cell types, thus increasing potential molecular targets for drug therapies; and will suggest hypotheses for gene expression networks, thus improving our knowledge of cellular mechanisms in health and deepening our understanding of gene interaction webs in disease etiology.

The nervous system uses two forms of fast synaptic transmission, chemical and electrical, that both contribute to the dynamic computations that create thought, feelings, and actions. Chemical synapses are well studied, and the biochemical mechanisms by which neurotransmitter is released and received are well understood. By contrast, we know relatively little about the macromolecular complex of the electrical synapse. Electrical synapses are made from tens to thousands of gap junction channels that create direct, low-resistance routes of cytoplasmic communication between neurons. They contributed to sophisticated function in neural circuit computation, they display plasticity through a number of short- and long-term mechanisms, and their assembly is regulated during development. Together, this all suggest a complex macromolecular structure that controls their formation and function, yet a critical barrier to progress in the field remains in the identification of the proteins of the electrical synapse. The overarching goal of the proposal is to establish zebrafish electrical synapses as a model to understand their proteomic diversity. Aim1 will demonstrate that electrical synapse proteins can be identified using genome engineered zebrafish that express electrical synapse proteins tagged with TurboID. TurboID is an evolved E.coli protein that allows for in vivo, proximity-depending labeling of proteins with biotin. Such biotinylated proteins can then be efficiently isolated from the animal and analyzed using mass spectrometry. Aim2 will then assess the biochemical interactions and cellular localization of the identified proteins using expression systems for protein-protein interactions and in vivo immunohistochemistry in zebrafish. If successful, this grant will fundamentally shift the understanding of electrical synapses, revealing proteins involved in trafficking pathways, synaptic structure, and functional regulation. The proposed studies will provide novel insight into the molecular complexes of the electrical synapse in a model vertebrate, providing a foundation for the identification of targets for therapy of neurodevelopmental disorders.

Abstract While current efforts in the analysis of neural circuits focus on interneuronal connectivity mediated by chemical synapses, less is known about the contribution of electrical synapses. Electrical transmission is mediated by neuronal gap junctions, which are widely distributed throughout the vertebrate brain. However, the extent and subcellular distribution of electrical synapses within neural circuits has been difficult to assess because: 1) antibodies targeting connexins (gap junction forming proteins) vary in their specificity, resulting in false positive or negative staining, and therefore potentially generating wrong or incomplete maps of connectivity, and 2) current electron microscopy protocols used to generate connectomes are unfavorable for detecting gap junctions, thus biasing the description of neuronal interconnection to chemical synapses. To overcome this problem, we propose to develop transgenic-based methods that will allow investigating the presence and contribution of electrical synapses in zebrafish, a model organism that has been identified as particularly advantageous for the analysis of neural circuits by the Brain Initiative. More specifically, we propose to create a Library of Transgenic Zebrafish to study Electrical Synaptic Transmission which will make it possible to generate, for the first time, a complete map of the distribution of electrical synapses in a vertebrate nervous system. The proposal involves generating three types of fish at which connexins and/or its promoters are tagged with fluorescent proteins or functional sensors that, combined, will allow comprehensive examination of the functional contributions of electrical synapses to circuits underlying various behaviors with cell specificity. Aim 1 is to generate transgenic zebrafish at which the promoters of neuronal connexins are linked to reporter fluorescent proteins. The availability of these animals will allow for the establishment of the presence of a particular gap junction protein in a cell or circuit of interest, a notoriously challenging problem, as cells expressing a particular connexin will be fluorescently labeled. Aim 2 is to generate transgenic zebrafish at which zebrafish neuronal connexins are tagged with fluorescent proteins. We will engineer the endogenous neuronal connexin proteins with fluorescent proteins or affinity tags to assess the number and subcellular location of electrical synapses of a cell with its connected neighbors. Because of the design of the constructs, tagged connexins can be imaged by diverse methods including single or 2-photon imaging of living animals or tissues, or chemical enhancements suitable for electron microscopic analysis. Finally, Aim 3 is to generate transgenic zebrafish to study functional contributions of electrical synapses to neuronal circuits. We propose to generate transgenic fish in which neuronal connexins are linked to Ca++ sensors that will make possible detecting active electrical synapses as well those undergoing plastic changes. The proposed approach represents a significant improvement over current methods of analysis and, if successful for analysis of zebrafish neural circuits, could be potentially applied to analysis of electrical transmission in mammalian species.

PROJECT SUMMARY/ABSTRACT Cell fate diversification is a critical step in producing the many neuronal subtypes required for functional circuitry in the vertebrate brain. We know a considerable amount about how neural progenitors diversify fates to form distinct daughter neurons. However, much less is known about later processes that diversity the fates of postmitotic neurons. One reason for this knowledge gap is that in most vertebrate models it is impossible to recognize and study precisely the same neurons in separate individuals, limiting predictive and statistical power. We can overcome these limitations in zebrafish by studying two adjacent, individually identifiable, spinal motoneurons. We discovered that these neurons are initially equivalent, and that interactions between them, in addition to interactions with an identified set of muscle fibers, breaks the equivalence between the two neurons and causes them to adopt distinct fates. Moreover, one of these neurons then typically dies, an important fate for sculpting brain architecture and circuitry. This is a unique situation in vertebrates, in which we can observe fate diversification as it is occurring in living embryos and predict that a neuron will die well before the process occurs, yet we still do not know the underlying molecular events. We will overcome this barrier using a variety of tools that enable us to manipulate the fates of these two neurons and single cell RNA sequencing at defined stages during the developmental process. This combination with enable us to discover genes involved in neuronal cell fate diversification as well as genes that predict neuronal cell death and survival. We plan to validate these genes using quantitative RNA in situ hybridization techniques. We will then test validated candidates using an innovative F0 CRISPR screen. Our proposed studies will reveal genes previously unknown to function during neuronal cell fate diversification, survival, and death. If successful, they will uncover genetic mechanisms of neuronal cell fate diversification with unprecedented precision, and thus will open new avenues of inquiry and deepen our understanding of neurodevelopmental mechanisms.