Wesley Grueber, Phd - US grants

DESCRIPTION (provided by applicant): The goal of this project is to understand how neurons establish their proper dendritic fields. For this purpose, we study the phenomenon of dendritic tiling in the peripheral nervous system (PNS) of Drosophila melanogaster. In tiled systems, such as the mammalian retina and Drosophila PNS, the dendritic fields of functionally similar neurons show strict avoidance, and thus partition a large receptive area (the retina or body wall, respectively) into several mini-domains. The multidendritic neurons in the Drosophila PNS comprise several independent tiled systems. We are testing the hypothesis that each of these systems provides and responds to distinct repulsive signals that allow like dendrites to recognize and avoid each other without recognizing non-like dendrites. We have developed a series of cell-type specific markers that uniquely label the distinct subsets of tiling neurons with Green Fluorescent Protein (GFP). These markers will be used to: 1) visualize neurons for ablation studies in live embryos and examine changes to the dendritic field areas of neighboring neurons, 2) examine candidate genes for their role in tiling and, 3) conduct a mutagenesis screen to identify and characterize novel genes involved in tiling and dendritic morphogenesis. These studies will identify how dendritic arbors decide when to stop growing and when to continue or resume their growth, processes that are essential for the proper wiring of nervous systems during development and regeneration.

DESCRIPTION (provided by applicant): The territories of dendritic and axonal arbors determine how neurons receive and transmit information in the nervous system, and disruptions of dendritic and axonal morphology are associated with numerous neurological disorders. Many types of dendrites and axons are organized according to the principles of tiling and self-avoidance, in which arbors of neurons expand until they abut neighboring arbors. Both self-avoidance and tiling ensure that sensory or synaptic inputs in the nervous system are sampled completely and non-redundantly, a critical requirement for functional neuronal circuits, however we still know little about how such territories are built. We propose to examine the mechanism of dendritic tiling in the Drosophila sensory system, a powerful model for studies of the molecular control of neuronal morphogenesis. We propose to examine the molecular basis for dendrite-dendrite interactions and dendrite-substrate interactions that control tiling behavior of dendrites To accomplish our aims we will perform laser cell ablations to eliminate neurons and their dendrites to determine whether dendrite-dendrite interactions are important for field formation. We will use gene knock out approaches to eliminate the function of genes and assess necessity for tiling, and, conversely, misexpress genes to determine whether they are sufficient to promote tiling. We will study the re-use of signals that are important for dendritic tiling during axon tilng to explore whether dendrites and axons are patterned by similar cellular and molecular mechanisms. We expect that these results will be highly relevant for understanding the mechanisms by which proper dendritic and axonal patterning is achieved in the nervous system, and how alteration or disruption of molecular programs might underlie developmental defects.

DESCRIPTION (provided by applicant): Modern neuroscience seeks to identify, and manipulate with precision, specific cell types in the brain to identify mechanisms of circuit assembly, function, and neural disease. These technologies are a prerequisite to a large number of fields of study, from systems neuroscience to molecular developmental analysis, and are key to providing insights not only into neural function, but also disease processes, including aging, neurodegeneration, and neural repair developing tools to label and manipulate specific neuronal types is generally costly and time-consuming. The objective of this application is to develop and release to the scientific community an extensive and versatile toolkit for manipulation of the nervous system of Drosophila, a powerful genetic model that has generated many fundamental insights into nervous system function and development. This application has three central aims. First, we propose to generate 4,000 new transgenic lines based on the recently developed InSITE system. These strains will allow cell-specific manipulations of gene expression in the vast majority of neurons in the fly. Each of these insertion sites will be molecularly mapped, and the expression pattern captured by each line will be described in both larval and adult fly brains. Second, using this collection, we will derive cell-type specific intersectional tools, as well as independent transcriptional regulatory systems, allowing gene expression to be manipulated in multiple individual cell types, independently. Third, we will create a database describing these data that will enable the community to make full use of this resource to address a wide range of scientific questions. This collection will provide a platform for introducing a versatile collection of markers and drivers of gene expression into a large number of the neuronal types present in the larval and adult nervous systems for release to the Drosophila community. Once this resource is established, the methods for using and extending the collection involve only basic genetic crosses that can be adapted in any lab. We expect that this collection will seed an expanding, highly versatile, and cost-effective collection of tools fo manipulation of the nervous system and gene discovery.

DESCRIPTION (provided by applicant): Abstract We request continued NIH support for Columbia's University-wide Doctoral Program in Neurobiology in Behavior (NB&B). Founded in 1995 by John Koester and Darcy Kelley (the current Program Director), NB&B includes 50 neuroscience training faculty from 10 departments on both the Medical School and Arts and Sciences campuses that serve as Ph.D. mentors and/or participate actively in NB&B teaching and governance. Sixty-six additional faculty members serve as potential research rotation and dissertation sponsors. For the 2011-12 academic year, the Program includes 88 pre-doctoral graduate students, of which 74 are from the US or are permanent residents. An Executive Committee from participating Departments appoints the Co-Directors: Darcy Kelley, Carol Mason and Ken Miller. The Co-directors are supported by an Internal Advisory Committee of mid-career faculty members from the two campuses and a Curriculum Committee. An External Advisory Committee reviews the NB&B program every four years. Feedback on the program includes an exit interview after the Ph.D. defense; career trajectories are actively tracked post-Ph.D. In 2012, 392 applications to the NB&B program were received of which 67% were US citizens or permanent residents. Students are admitted directly to the program by a fifteen-member Admissions Committee of training faculty. We actively recruit applicants from underrepresented groups and matriculate 1 or 2 annually (~30% of offers). The number of applicants offered admission and the percentage of matriculating students is increasing: 11 matriculants in 2010 (42 offers), 13 matriculants in 2011 (43 offers), 10 matriculants in 2012 (33 offers). US students receive individual fellowship support from the NSF, DOD and NIH (pre-doctoral NRSAs). Non-US students receive external support from Fulbright, NSERC (Canada) and the HHMI. Five students each in years 1 and 2 have been supported by this training grant. Reflecting the increasing matriculation rate and very high quality of students, we now request stipend and tuition support for 11 students total in years 1 and 2. The training program opens with a Boot Camp, a two-week introduction to current research approaches, followed by a neuroscience survey course and three advanced seminars. Research rotations, typically three, are taken in the first year. In addition, students complete three professional development courses led by the NB&B Program Co-directors: a first-semester course focused on developing a research fellowship proposal, a second-semester course on responsible conduct of science and a professional development course for advanced students. Students are directly supervised by the Co-directors in years one and two. By the beginning of the second year, students choose a thesis laboratory and take a qualifying examination to advance to candidacy by the beginning of year 3. The qualifying examination committee typically constitutes the core of the thesis advisory committee that meets yearly with each student through year four and every 3 to 6 months prior to thesis defense, typically 5.5 - 6.5 years post-matriculation. All students attend the weekly NB&B research seminar, a student journal club and lab meetings. A NB&B Program-wide retreat is held biannually and students receive support to attend scientific meetings.

Maintenance of body homeostasis is critical to survival; animals must detect and correct chemical and thermal imbalances as well as eliminate toxic compounds. Internal sensory neurons innervate various internal organs and provide information about internal physiological state to the nervous system. Internal sensory neurons are conserved across species and yet are poorly understood compared to external sensory neurons. In particular, there is a large gap in knowledge of the internal sensory neural circuitry and the underlying molecular mechanisms for detecting internal state. These gaps in our knowledge may be due to the relative complexity and genetic inaccessibility of specific subpopulations of neurons in these circuits. To overcome these challenges, we propose to combine powerful anatomic, genetic, and functional methods and provide an entry point into studying internal sensory circuits in the Drosophila nervous system. In this proposal we will build on state-of-the-art serial electron microscopy that has identified synaptic connectivity of internal sensory neuron circuits and further propose to (a) use genetically-encoded indicators of neural activity to assess activity in deep, hard-to-access internal sensory neuron populations, and (b) use powerful Drosophila genetic tools to elucidate the molecular mechanisms that mediate sensory transduction of internal sensory neurons, as well as to manipulate activity of specific subcomponents of the neural circuit to elucidate their role in maintaining physiological homeostasis.

We request support for training in Columbia's University-wide Doctoral Program in Neurobiology and Behavior (NB&B). The goals of this program, established in 1995, are two-fold: First, to prepare exceptional predoctoral students for productive careers in neuroscience research and related fields; and Second, to train scientists who seek to broaden our understanding of the fundamental mechanisms of the nervous system and the many disorders that affect it. We achieve this through a multidisciplinary training program aimed to provide students with a solid foundation in rigorous experimental design and strong quantitative and analytical skills, coupled with broad based knowledge of neuroscience and allied disciplines. The first year begins with a student run orientation ? ?Bootcamp? ? which introduces trainees to current research approaches. During the first year, students take Analysis for Neuroscientists, which lays a foundation for rigorous research during three laboratory rotations and subsequent thesis research. A year-long Survey of Neuroscience course provides an in-depth introduction to Cellular and Molecular neuroscience during the Fall semester and to Systems and Developmental Neuroscience during the Spring, ensuring core knowledge for all students independent of prior academic background. By the end of their first year in the program students select a mentor to guide their thesis research. In their second year, students begin thesis research, develop a research proposal, select two advanced elective courses, and take a qualifying exam. Research progress is monitored by the mentor, Co-Directors, the student?s thesis committee, and a Student Progress Committee. Students gain professional skills in writing, presenting, and cutting-edge research techniques through research-in-progress talks, a weekly seminar series, skills-based nanocourses, journal clubs, and a program retreat. These events also ensure an active and cohesive community of mentors and students. Students participate in nationally- recognized outreach programs including NeuWrite and Columbia University Neuroscience Outreach (CUNO). The program is administered by the Department of Neuroscience, and is strongly interdisciplinary with 63 participating faculty from departments across the Health Sciences campus, Schools of Arts and Sciences/Engineering, and the Zuckerman Institute. Mentor labs provide a broad range of research topics and training opportunities for NB&B students. The program receives feedback from External, Internal, and Student Advisory Committees. Between 2013-2019 we received an average of 423 applications per year, with 64% of these training grant eligible. In 2019 we accepted 35 students and 18 will enroll, for a 51% yield. We actively recruit applicants from underrepresented groups and between 2-8 have matriculated annually over the last 10 years. Graduates go on to make substantial contributions to biomedical research within academia and in science-allied careers outside of academia. We request stipend and tuition support for 10 students total in years 1 and 2.