Judith Eisen - US grants

The candidate's long term goals are to understand how neuronal fate becomes specified and how neurons find and recognize their appropriate synaptic partners during development. The goal of the current application is two-fold: 1) to examine how environmental cues guide the growth cones of identified neurons to cell-specific targets during development and 2) to enable the candidate to learn and implement molecular and genetic approaches in her lab. These new approaches will complement the cellular approach currently used in the candidate's lab by allowing her to address both the phenomenology and the mechanisms of neuronal developmental. The candidate is fortunate to work in the University of Oregon Institute of Neuroscience, where she has daily interactions with other labs that also investigate neuronal development. The Institute is very supportive of her desire to receive additional training so that she can broaden her research, and she has received a commitment for a reduction in teaching and other academic responsibilities if she receives this award. The proposed studies will be carried out in a simple vertebrate, the embryonic zebrafish, where individual neurons can be identified and their development observed directly in living embryos. The candidate proposes to examine whether target muscles produce permissive signals that promote elongation of the growth cones of all motoneurons or instructive signals that guide the growth cones of particular motoneurons as they pioneer cell-specific pathways to cell-specific targets. The relationship between identified motoneurons and their target muscles will be altered by transplanting muscle precursors and motoneurons, and the subsequent pathway choices made by the growth cones of identified motoneurons will be observed in living embryos. The candidate proposes to compare the pathfinding abilities of two populations of motoneurons that develop at different times, to learn whether their growth cones use different cues to reach the same target muscle. Pioneering motoneurons will be ablated using laser-irradiation to learn whether their presence is required for proper pathfinding by follower motoneurons. The candidate proposes to use genetic and molecular approaches to isolate putative guidance molecules and study the mechanisms by which they direct pathfinding, and to study the mechanisms underlying the specification of neuronal fate.

DESCRIPTION (provided by applicant): For the nervous system to function properly, it is crucial that embryonic neurons acquire appropriate identities so they can develop the properties necessary for their later functions. The longterm goal of our laboratory is to understand the mechanisms underlying these processes. These mechanisms will be investigated by studying development of individually identified primary motoneurons in embryonic zebrafish. Our hypothesis is that the identities of primary motoneurons are specified by a series of signals that regulate expression of transcription factors that control neuronal identity and later features of development, such as axonal pathfinding. A series of experiments to test aspects of this hypothesis are proposed. Zebrafish primary motoneurons initially express a specific transcription factor, islet1, which is then downregulated; later islet1 or a related gene, islet2 is expressed in each specific primary motoneuron. The roles of the distinct phases of islet gene expression in motoneuron identity and axonal pathfinding will be tested using mutants and morpholino antisense oligonucleotides to knock down gene function. Retinoic acid, a well-known teratogen that can have devastating effects on human embryos, is synthesized in zebrafish during the time that primary motoneurons are being specified. Exposure to exogenous retinoic acid alters motoneuron numbers and patterning. The role of endogenous retinoic acid in motoneuron specification, and its interactions with other signals including the Hedgehog and Delta/Notch pathways, will be tested by a series of genetic gain- and loss-of-function experiments. The effects of retinoic acid on regulation of somite-derived signals that affect motoneuron identity will also be examined; potential signals will be identified using differential molecular screens. Interactions between specific primary motoneurons and the muscle fibers they innervate regulate their identities and survival. The role of the Delta/Notch signaling pathway in this process will be tested using a genetic approach. Although many genes regulating motoneuron development are known, many remain undiscovered. A mutagenesis screen will be undertaken to identify new genes involved in specifying motoneuron identities.

9410460 Westerfield This three-year award supports U.S.-U.K. cooperative research in neurobiology between two research groups at the University of Oregon and University of London. The U.S. investigators are Monte Westerfield and Judith Eisen and the British investigators are Nigel Holder and Stephen Wilson. The cooperative effort extends recent research by both groups in anterior central nervous system of zebra fish embryos. Newly discovered genes, have been identified either by cloning or mutation, as candidates for regulating developmental processes in the central nervous system of zebra fish embryos. The objective of this new effort is to study the potential roles of these genes in the anterior central nervous system and brain segments of zebra fish. Zebra fish are ideal vertebrates for studying embryonic and genetic analyses since a large number of their regulatory genes have already been identified. The results from this project, when compared with complementary studies in mammals, will advance our knowledge about the evolution of the anterior regions of the vertebrate central nervous system in general, the genetic mechanisms that regulate patterning, and gene expression that demarcate brain subdivisions. The project takes advantage of complementary expertise of the U.S. and British investigators and provides access to mutant zebra fish and genetic sequencing techniques developed by both groups.

zebrafish; animal care; building /facility design /renovation; biomedical facility;

vertebrate embryology; developmental neurobiology; neural plate /tube; early embryonic stage; developmental genetics; cell differentiation; phenotype; alternatives to animals in research; regulatory gene; dorsal root; motor neurons; cell cell interaction; cell migration; neural crest; gene expression; green fluorescent proteins; genetically modified animals; confocal scanning microscopy; embryo /fetus culture; embryo /fetus transplantation; cell transplantation; zebrafish;

Understanding embryonic development depends critically on learning how cells acquire their fates. Cancers can cause cells to take on aspects of two different fates, thus, learning the mechanisms underlying cell fate specification may lead to therapies for this devastating disease. Screens in model genetic organisms for mutations that suppress or enhance the phenotype of a known mutation have significantly augmented our understanding of how interesting gene networks affect cell fate; such screens have not yet been undertaken in vertebrates. This application proposes to use an existing model genetic organism, the nematode worm, Caenorhabditis elegans to design enhancer/suppressor screens that can then be applied to a new vertebrate genetic organism, the zebrafish, Danio rerio. The zebrafish floating head mutation causes notochord cells to take on aspects of two different cell fates. The pie-1 mutation has a similar effect on a specific C elegans blastomer, P2. In both cases, the affected cells retain a normal early signaling ability but then differentiate into a completely different cell type. The C elegans mom-2 mutation is partially penetrant and the mom-2 gene may encode the P2 signal. Worms carrying the mom-2 mutation will be mutagenized and screened for new mutations that enhance or suppress the mom-2 phenotype. The proposed experiments will provide new insights into cell fate specification in C. Elegans as well as providing experience designing and assessing such screens, as a prelude to sesigning similar screens for genes interacting during zebrafish development.

[unreadable] DESCRIPTION (provided by applicant): The aim of this proposal is to improve animal husbandry in the University of Oregon's zebrafish and stickleback facilities. The University is a world-leader in establishing zebrafish and stickleback as model organisms for research in vertebrate development, genetics and evolution. More recently the University has begun to focus on using zebrafish as a model for specific human diseases, including genetic diseases and diseases that may result from interactions with gastrointestinal bacteria. [unreadable] [unreadable] The success of the zebrafish and stickleback research programs at the University of Oregon has resulted in a shortage of aquaria, the need for an improved procedure room and the need for additional monitoring equipment to ensure environmental stability. This application proposes the following aims to rectify these situations: [unreadable] [unreadable] To meet the increasing demand for zebrafish aquaria, we propose to replace many of our old, dilapidated racks with high-capacity racks. This will significantly increase the number of zebrafish we can house and provide more corrosion resistant and ergonomically-designed housing. [unreadable] [unreadable] To improve our ability to study the role of gastrointestinal bacteria in gut development and function, we propose to renovate a zebrafish procedure room. This will significantly upgrade our ability to produce and study germ free zebrafish reared in the absence of gastrointestinal bacteria. [unreadable] [unreadable] To ensure environmental stability in our stickleback facility, we propose to add monitoring equipment that will contact us if environmental conditions within the facility change. This will significantly increase our ability to ensure the health of our many genetically-distinct, and therefore irreplaceable lines of stickleback. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): For all children to have the opportunity to achieve their full potential for healthy lives, free from disease, it is essential to understand mechanisms underlying developmental patterning and how this patterning can go awry in human disease. The goal of this program project is to elucidate one such mechanism - the function of reciprocal intercellular signaling that specifies embryonic cells to traverse particular developmental pathways and express restricted fates. This goal will be achieved for three sets of cell fates in three component projects, using the zebrafish, a widely-utilized animal model organism pioneered by this group at the University of Oregon. The projects take advantage of the attributes of the zebrafish for developmental genetic analyses, including gene expression studies, genetic mosaic investigations, and loss- and gain-of-function experiments that will establish the nature of the interactions. The projects include screens for new mutations and genes important in these signaling pathways, facilitated by the unique Zebrafish Facility, one of four core services. Project I, "Reciprocal signaling in skeletogenesis", tests hypotheses about the functioning of signaling molecules in patterning the shape of the palatal skeleton and the pathway of chondral bone development. Results will improve understanding of signaling pathways between cranial epithelia and mesenchyme, and between cartilage and bone progenitors. They will thereby inform our understanding of cleft palate, one of the most common human birth defects, and osteoarthritis that will affect nearly one in five Americans during the coming decade. Project II, "Reciprocal signaling in synaptogenesis", tests a novel hypothesis that Usher genes encode proteins that interact in a complex mediating reciprocal signaling between sensory cells and neurons with which the sensory cells form synaptic connections. The analyses will identify the critical components of the Usher gene network and provide an integrated understanding of Usher syndrome, the most frequent cause of deafness and blindness. Project III, "Reciprocal signaling in gastrointestinal tract development", explores the hypothesis that gut microbiota influence cell fate decisions in gut epithelium and enteric nervous system by modulating a highly conserved molecular signal, Notch. The work will elucidate reciprocal signaling and how it goes awry in disorders such as inflammatory bowel disease and related disorders that together affect more than 10% of the U.S. population.

DESCRIPTION (provided by applicant): This project will create a Summer Research Program at the University of Oregon (UO), supporting 15 undergraduate students per year for 5 years for summer research projects. The program is designed to provide opportunities for undergraduates to participate in research projects that focus on child health and human development, with the long range goal of enhancing career opportunities for talented students in biomedical research. The principal aims of the program include contributing to national efforts to diversify the pool of highly trained biomedical researchers, enhancing access to research careers for students with limited access to research facilities and experience; and training graduate students and postdoctoral fellows to be effective mentors. The PIs objectives for individual interns include: a) acquiring experience-based education in science research; b) developing appreciation of experimental approaches, strategic design, and creative reasoning; c) developing methodological skills; d) enhancing deductive and inductive reasoning skills; e) enhancing scientific communication skills; f) boosting personal confidence by professional and social interactions with faculty, postdoctoral fellows, and graduate students, and g) gaining inspiration to pursue a career in biomedical research. The NICHD R25 Summer Research Program interns will be closely mentored by host faculty, and they will work side by side with graduate and postdoctoral researchers at the bench. Interns will have abundant opportunities to interact with faculty, postdocs, graduate students, UO undergraduates, and fellow interns in the NICHD R25 program and other campus summer research programs. This program would complement the PIs' current REU Program in Molecular Biosciences funded by the National Science Foundation. Program activities include, in addition to the closely mentored research project, a Mentoring Workshop, Welcoming Orientation for interns, mentors, and faculty, a Professional Development Workshop Series, a Faculty Seminar and Discussion Series, Intern Progress Reports, Feedback and Evaluation Meetings, a weekly Social hour, Field trips, cultural, social and recreational activities, an Undergraduate Research Symposium, and Program Evaluation.

Gnotobiology Core: The Gnotobiology Core will rear germ-free zebrafish and stickleback in designated space within the UO Zebrafish Facility. This space will house sterile isolators in proximity to a fume hood for ventilation of Clidox gas used for sterilization. There is also a biosafety cabinet for aseptic manipulation of zebrafish. The Gnotobiology Core staff will have access to a new autoclave featuring custom programs optimized for sterilization of liquid food.

The primary objective of the Zebrafish Facility is to support researchers using zebrafish to study vertebrate genefics, genomics, and development by employing expert husbandry techniques, supplying efficient and fimely services, and fostering a helpful, cooperative environment. This Core accomplishes these goals by providing the following services: (1) husbandry and feeding of adult fish, (2) husbandry and rearing of embryos and larvae, (3) provision of staged embryos, (4) sperm cryopreservation, (5) database tracking of all fish stocks and frozen sperm, (6) high through-put quaranfined screening for mutafions in fish from other facilities, (7) procedure rooms and equipment for embryo microinjecfion, and (8) advice and training on fish husbandry and facility set-up. Because our Zebrafish Facility has recently been expanded to provicle more procedure space, we are now adding a new services to the Zebrafish Facility, derivafion and maintenance of germ-free embryos and larvae.

The Administrative Core does the accounting, record keeping, purchasing, preparation of continuation reports and final drafts of manuscripts, seminar, meeting, and travel arrangements, and other reporting required for this Program and assists the PD in tracking oversight and compliance. Most of this activity is supported by the Institute of Neuroscience; the Program supports 15% of a Grant Administrator, 20% of an Office Specialist and 10% of the PD's salary.

Hirschsprung disease (HSCR) is a leading cause of intestinal dysmotility resulting from a reduced enteric nervous system (ENS) and leading to lack of distal intestine innervations. HSCR can be caused by mutations in several different loci and shows considerable phenotypic variation: individuals carrying the same allele can differ in the extent of intestinal denervation and severity of enterocolitis, intestinal inflammation of unknown etiology that is a serious HSCR complication. The sources of these phenotypic variations in HSCR are not well understood. If we could identify factors that make some individuals healthier than others, we would gain insight into potential therapies for those with more serious disease. We have a panel of zebrafish ENS mutants that serve as models for understanding variation in HSCR. Zebrafish is an excellent model in which to study HSCR phenotypic variation because we can manipulate all of the relevant parameters. Our zebrafish mutants have different extents of distal intestine denervation, often with considerable phenotypic variation among mutants that share the same genotype. These mutants also exhibit variation in intestinal motility, variation in their intestinal bacterial communities, and variation in intestinal inflammation. We propose that alterations in the ENS phenotype in ENS mutants, including changes in specific neuronal subtypes, result in dysmotility that causes changes in the milieu of the intestinal lumen. This, in turn, can lead to formation of altered bacterial communities that can cause inflammation by recruiting neutrophils, cells of the innate immune system that respond to bacteria and are a hallmark of intestinal inflammation. We also propose that changes in the intestinal bacterial community composition and in inflammation can feed back to the ENS to amplify variation in intestinal motility. We will test these hypotheses by manipulating the ENS phenotype, intestinal bacterial composition, and neutrophil influx and comparing all of these processes in individual animals of known genotypes. We will follow bacterial colonization, neutrophil recruitment, and intestinal motility in living animals in real time. Our proposed studies will provide new information about how the ENS regulates the composition of the intestinal bacterial community and inflammation, and how these processes feed back onto the ENS to amplify intestinal pathology. This information will provide new insights into the mechanisms of phenotypic variation in HSCR, expand our understanding of phenotypic variation in human disease, and may reveal new targets for therapies.

DESCRIPTION (provided by applicant): Both genetic and environmental factors play significant roles in influencing and perhaps causing neurodevelopmental disorders such as autism, mental retardation, and schizophrenia. Recent advances in genetic analysis have revealed a number of genes linked to familial forms of these disorders. Very little is known, however, about how the environment may contribute to these disorders, or how the environment may interact with identified genetic causes. We propose that abnormal microbial colonization of the gut during development leads to aberrant expression levels of genes that are involved in brain development, specifically in synapse formation. We hypothesize that incorrect microbial colonization of the gut may exacerbate or drive behavioral deficits by promoting aberrant synapse formation that results in altered neuronal circuitry and function. We propose to examine interactions between early gut colonization by the microbiota and expression of synaptic cell adhesion molecules, such as Neuroligins, during development in zebrafish. This model provides an unparalleled opportunity to study the consequences of altered gut microbial colonization on synapse formation, development of neuronal circuitry, neuronal activity, and behavior, because we can manipulate both host genetics and microbial communities and follow development of defined neuronal populations in real time in living juveniles and adults. We also propose to determine whether there is a critical period during which colonization by a microbiota whose members express specific traits is required for normal synapse development, and to learn how the microbiota signals to the developing brain to promote the normal synapse formation required for normal behavior. Finally, we will test directly whether a dysbiotic, pro-inflammatory microbiota can alter synapse development, formation of neuronal circuitry, neuronal activity, and behavior, in both young and adult animals. Our proposed experiments will provide the first comprehensive view of how microbial signals affect development of neuronal anatomy and physiology and how this affects behavior at later stages of development and in adults. Revealing and characterizing an interaction between the microbiota and the genes that drive synapse formation would have a dramatic impact on current treatment approaches for individuals diagnosed with neurodevelopmental disorders.

PROJECT SUMMARY/ABSTRACT (OVERALL) The overall research goal of this Program Project Grant is to develop the knowledge base and the experimental and theoretical framework for engineering transmissible health. Since the establishment of the germ theory of disease in the late 1800s, a major public health goal has been to limit the transmission of disease-causing microbes. Microbes normally resident in hosts, in contrast, are increasingly appreciated for their health- promoting roles, which include fostering normal development, establishing appropriate immunological tone, and preventing invasion of pathogens. The potential for resident microbes to be used as therapeutic probiotics holds great promise, but current probiotic design strategies focus exclusively on administering probiotics to individual hosts, neglecting the possibility of transmission except as a threat that needs to be prevented. However, just as for pathogens, transmission of commensal microbiota between individuals and within social groups is likely to occur and may even contribute to the health benefits associated with social connectivity. In contrast, microbial isolation is a defining feature of modernized societies, which are experiencing alarming increases in autoimmune disorders and other diseases of microbiota dysbiosis. The interactions between commensal microbes and their environments both within and outside of hosts, and the ways in which these interactions shape dispersal, transmission, and host health, remain opaque, preventing design of community- level strategies to exploit the beneficial potential of our intestinal microbiota. We propose to explore the parameters of inter-host transmission of host-associated bacteria and bacterial communities that could be harnessed for therapeutic purposes. We imagine that the properties of resident bacteria can be tuned to promote health on both an individual and a population level. In particular, we propose to design smart probiotics that would sense and treat inflammation. At a local level, in individual host intestines, these microbes would be engineered to inhibit features of the host environment that favor pro-inflammatory strains. At a population level, these microbes would be engineered to successfully spread between and colonize hosts, and would limit the transmission of pro-inflammatory microbiota members, effectively conferring herd immunity to intestinal inflammation. Our use of zebrafish and their commensal microbiota as an accessible experimental platform for monitoring and manipulating host-microbe systems will provide important new insights that are crucial if we hope to use similar smart probiotic strategies to transform other multi-species systems, such as humans.

There is mounting evidence that the intestinal microbiota play an important role in normal nervous system development, although the underlying molecular mechanisms are generally unknown. We propose to investigate the role of the intestinal microbiota in development of an identified population of superficial interneurons (SINs) in the zebrafish optic tectum that are required for visually-guided prey capture behavior. Previous work showed that prey capture is significantly impaired in larvae in which SINs are genetically ablated. We find that the microbiota are necessary for SINs to differentiate their GABAergic phenotype, and consequently larvae reared germ free (GF), in the absence of bacteria, phenocopy the prey capture deficit of larvae in which SINs have been ablated. We hypothesize that members of the zebrafish-associated microbiota normally produce molecular products that promote SIN differentiation. We will test this hypothesis using an unbiased screen to identify bacterial products that affect SIN differentiation. First we will use gnotobiotic zebrafish colonized with specific zebrafish bacterial isolates to learn which bacterial species are required for normal SIN development and prey capture behavior. We will then take advantage of a pipeline we established that utilizes bacterial genetics, bioinformatics, and biochemical approaches, to learn the molecular nature of the bacterial products. Discovering the molecular mechanisms by which host-associated bacteria promote normal nervous system development will provide important new insights into a fundamental process that is not well understood, reveal how this process can go awry during intestinal dysbiosis, an imbalance that has been linked to neurodevelopmental conditions such as autism spectrum disorder and schizophrenia, and provide new possibilities for developing targeted therapies.

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.