David Van Vactor - US grants

DESCRIPTION: Structural analogies strongly suggest that DLAR acts as a receptor for an unknown navigational cue. This proposal aims to elucidate the structural requirements for DLAR to function correctly in guidance decisions, to characterize interactions between DLAR and components of better characterized signaling pathways, and to characterize a new mutation that phenocopies mutations in DLAR. In the first specific aim a number of engineered alterations in DLAR will be tested for their ability to rescue DLAR function in a DLAR mutant background. Constructs will be engineered into multiple lines by P-element insertion and then crossed into a DLAR mutant line that expresses GAL4 in postmitotic neurons. GAL4 activates a driver that causes the expression of the construct so that it can be tested for rescuing activity. Rescue has already been achieved with intact DLAR. Constructs to be tested include: extracellular domain deleted, cytoplasmic domain deleted, deletion of each of the two phosphatase domains individually, and inactivating point mutations within each of the phosphatase domains. So long as the engineered constructs are appropriately expressed, these experiments will likely determine whether DLAR acts as a receptor and whether its phosphatase activity is required for its guidance function. At the suggestion of a previous reviewer, DLAR will be tested directly for phosphatase activity by an appropriate assay. The only difficulty here is that it is hard to know what the appropriate substrate is for DLAR to act upon. There are two separate parts to specific aim #2. The first depends upon the observation that the DLAR phenotype is synergized by a weak mutation in Rac and that a human relative of DLAR affects Rho and Rac activity through a guanine nucleotide exchange factor named Trio. It is therefore proposed to further study the effects of dominant negative and constitutively active (or overexpressed) Rho, Rac, and CD42 on motor neuron advance, guidance, and the DLAR phenotype. A GAL4-UAS system would be used to drive expression of the constructs appropriately. The second set of proposed experiments are motivated by the finding that the DLAR phenotype is suppressed by a mutation in the abl tyrosine kinase. It is possible that DLAR's phosphatase activity is somehow in competition with kinase activity in the able signaling pathway. This possible interaction will be further examined by the construction of double mutants with DLAR and other known members of the abl pathway. Kinase-active and kinase-inactive forms of abl will also be compared for their ability to suppress the DLAR phenotype. These experiments offer the potential of better characterizing the signaling pathway involved in DLAR function. Specific aim #3 involves the further analysis of a new mutation, circumfirential, that phenocopies DLAR. Several important issues will be addressed: the mutation will be mapped, the specificity of the navigation errors it evokes will be better characterized, new alleles will be collected, and a mosaic analysis will be performed to determine if circumfirential acts cell autonomously. This last experiment is very important. If circumfirential is required in the motor neuron, then it could be a member of the DLAR signaling pathway. If it is required outside the motor neuron, it could be the still elusive ligand for DLAR. All of these studies will use techniques that are standard in the field and are feasible for this investigator.

Thorough analyses of molecular mechanisms in cellular and developmental neuroscience requires an array of imaging tools and expertise. The purpose of this program project core is to provide the instrumentation and technical support to enable research staff in the three program laboratories to perform a wide range of imaging applications and image processing to extract quantitative mophometric data. The applications range from three types of confocal microscopy to wide-field fluorescence time-lapse microscopy to standard format imaging of fixed tissues.

The long-term goal of this research project is to elucidate the constituents and functional organization of signaling pathways that control directed cell motility. In the context of neural development, directed cell movement is necessary for the appropriate positioning of cell soma, and for the precise map of axonal connections between different populations of neurons. Studies in different systems suggests that such pathways form a link between signals at the cell surface and the dynamic cytoskeleton that drives forward movement. Among many proteins implicated in the cellular motility machinery, protein tyrosine kinases (PTKs) are key regulators of axon guidance. Previous work on the Drosophila PTK abl, orthologue of the vertebrate Abelson proto- oncogene, identified Disabled (dab), an abl-interacting phosphoprotein required for axonal development. Recent studies have shown that the mammalian counterpart of dab (mdab) plays a crucial role in the neuronal cell migration that underlies corticogenesis. These data suggest that a thorough understanding of the Abl pathway may provide tools and treatments relevant not only oncogenesis, but also for disease and injury of the nervous system. Unfortunately, the molecular events that mediate the cell motility functions of abl and dab are not understood in any system. We have designed and implemented a genetic screen for novel components in the Abl pathway and have identified several new loci that interact with abl and dab. One of these genes (filamin) is known to bind both cell surface receptors and actin cytoskeleton, and is required for cortical neuron migration in humans. Having established a functional assay for the role of abl in axon outgrowth, we will first dissection the structural features of abl necessary for this role. An equivalent analysis of dab will be pursued in parallel. Having discovered a genetic interaction between dab and filamin, and axonal phenotypes in filamin mutants,. Our second goal will be to define the relationship between filamin and other abl pathway components, and to dissect the contribution to define the relationship between filamin and other abl pathway components, and to dissect the contribution of different structural domains to the function of filamin during axogenesis. Finally, we have identified tetanic as a gene required for axonal development that interacts with both abl and dab. Thus, our third goal will be to determine the sequence of the tetanic gene and to explore the genetic and biochemical interactions between tetanic and other pathway components in order to define its mechanism of action within the developing nervous system.

DESCRIPTION (provided by applicant): The intracellular mechanisms that control growth cone navigation to appropriate target cells are required to build networks of functional neural connections during nervous system development and regeneration. Our long-standing interest in the cytoskeletal signaling machinery downstream of multiple guidance receptors has led us to a series of conserved microtubule-associated proteins that appear to mediate different types of guidance behavior. Analysis of the effector protein CLASP has defined a pathway from the repellent factor Slit and its Roundabout receptors, to the Abelson tyrosine kinase, to CLASP, as a means of impeding microtubule and leading edge advance. How Abl acts to coordinate microfilament and microtubule dynamics, and control the activity of CLASP or its associated partners is unknown. Preliminary data indicates that several proteins directly or indirectly linked to CLASP are required for accurate midline repulsion, whereas other microtubule effector proteins appear to play distinct roles in early axon guidance decisions. We will use a combination of genetics, cell biology and biochemistry to determine each protein's function in axon guidance and to dissect the signaling mechanisms that regulate the key effector activities.

DESCRIPTION (provided by applicant): This is the first competing renewal application for a Program Project to support and expand interactions that focus on the signaling mechanisms that control the directed outgrowth of neuronal processes and the directed movement of neuronal cells. Together these two properties are essential to neuronal function, since the precise location of neurons and proper interconnection of their axons and dendrites underpins all neuronal signaling and plasticity. Abnormal patterns of neuronal migration have been implicated in human neurological disorders associated with mental retardation, epilepsy, and dyslexia, while congenital disorders of mental retardation are increasingly recognized as having major disorders of axonal and dendritic connections. Project 1, headed by Christopher Walsh, focuses on the analysis of genes implicated in neuronal migration and process outgrowth based on human genetic studies. Project 3, headed by John Flanagan, focuses on mechanisms of action of neural guidance factors in animal models. Project 2, headed by Davie Van Vactor, focuses on analysis of genes that control axon guidance in Drosphila, some of which correspond to human disease genes. There are two cores, one of which is dedicated to supporting state-of-the-art imaging.

High quality, quantitative microscopy and image analysis is essenfial for each of the projects in this program. The research goals in each lab are also expanding into new and more challenging imaging applicafions such as live fime-lapse microscopy and quantitative morphometry. Each of the program labs has accumulated different tools and some expertise over time, however, it will be essential to create a formal process to give access to these tools, exchange this expertise and provide computational tools for image analysis. In this way, and maintain a continuous flow of information. As part of a recent reconflguration of the microscopy resources for the Artavanis-Tsakonas and Van Vactor groups, the major microscopes owned by these two labs can now be located in a common space under supervision of Dr. David Van Vactor. Dr. Van Vactor also serves as the Chair of the Microscopy Committee of the Cell Biology Department, providing faculty oversight for many other shared instruments housed in the Nikon Imaging Center at Harvard Medical School (NIC(gHMS), a state-of-the-art facility that will be made accessible to all participants in this program project. With the recent shift to digital microscopy required to support dynamic and quantitative imaging projects and the need for a range of confocal imaging techniques. Dr. Van Vactor and the Cell Biology Department created a core facility for state-of-the-art imaging. The result was an unprecedented partnership between Harvard Medical School (HMS) and Nikon Instruments, Inc. This facility (NIC(gHMS) now houses five instruments specialized for different types of confocal applications: two laser-scanning confocals, two spinningdisc confocals (one with environmental control chamber), and a total internal reflection fiuorescence (TIRF) microscope (using evanescent wave illumination from a laser source). Dr, Van Vactor designed the facility, negofiated with the supporting vendors (eight in all), and provides confinued faculty oversight. The NIC@HMS is located in the LHRRB building near his lab allowing him to provide frequent supervision and interacfion with the facility Director, Dr. Jennifer Waters. The NIC@HMS resource is very valuable, particularly for confocal applicafions, however, there is a major drawback: the user group includes all HMS Departments. This limits the use of the facility to a weekly maximum for each group (8 hours or less). While such limits are not and issue for limited confocal projects, they are very problematic for the routine image acquisition and processing required by the program project labs for analysis of screen data. Therefore, it is vital for the program project to maintain its own, complementary resources for microscopy and image processing. Since both our own core in Dr. Van Vactor's lab and the NIC(gHMS are in the same building, it is easy for our research staff to move quickly from one to the other. As is evident from the description of each project all four research teams have a need for high quality, quantitative microcopy and image analysis. We are thus expecfing that all four groups will be using this core.

DESCRIPTION (provided by applicant): Dysfunction in the molecular pathways that regulate synapse form and function leads to a number of neurological disorders, including epilepsy, autism and mental retardation. Our goal is to explore the molecular machinery that mediates synapse development and morphogenesis. This fundamental knowledge will be important for our understanding of neurological disease and for the conception of future therapeutic tools. Using the Drosophila neuromuscular junction (NMJ) as a genetic model system, we have discovered that miR- 8, a member of the highly conserved miR-200 family of microRNAs (miRNAs), is essential for the normal growth and complexity of the synapse. Animals lacking miR-8 display NMJ defects at different stages of development. During larval stages, when NMJs dramatically expand under control of multiple stimuli and regulatory pathways, miR-8 is required in muscle cells to promote the growth of presynaptic terminals. Our analysis suggests that miR-8 is required for the normal architecture of the cytomatrix which defines subsynaptic reticulum (SSR) of the NMJ, a structure analogous to the postsynaptic density marked by PSD-95 in mammals. Multiple screens to define downstream effectors suggest that miR-8 regulates the expression of several target genes implicated in synaptogenesis and cytoskeletal biology. To better define the mechanisms downstream of miR-8, we have shown that postsynaptic repression of the actin-associated protein Enabled (Ena) plays an important role in controlling NMJ growth in late larval stages, consistent with the localization of Ena to the SSR. Ena is predicted to be a direct target of miR-8, and controls aspects of cytoskeletal structure and dynamics during cell movement and cell junction formation, but its role(s) at the synapse are not understood. Preliminary data also indicate that although miR-8 is expressed in the central nervous system (CNS), its activity is somehow suppressed in neurons relative to other tissues. Moreover, genetic epistasis reveals that miR-8 is required for NMJ expansion induced by activation of a key presynaptic pathway that limits synapse morphogenesis (the Fragile-X Mental Retardation gene, FMR1), suggesting that some type of trans- synaptic communication is involved upstream of postsynaptic miR-8. Together, these findings reveal a fascinating mechanism that regulates synapse development, and a wonderful opportunity to exploit a powerful and well-defined model system to understand the logic of miRNA control over synapse form and function. However, many additional studies will be required to define the developmental, cellular and molecular mechanisms required for miR-8 to exert its effects at the NMJ.

Through an intimate set of collaborative interactions with three other laboratories working in invertebrate and mammalian systems, we have developed a genome-wide approach to define the conserved functional interactome for Survival of Motor Neurons (SMN), the highly conserved gene family causal to over 95% of Spinal Muscular Atrophy (SMA), one of the most common degenerative motor neuron diseases in humans. Through recent studies, Drosophila has emerged as a promising genetic model for Smn with the key hallmarks of human SMA, from failure of neuromuscular junctions to the degeneration of neurons and muscles. Despite an emphasis in the SMA field to focus on mouse models, the innovative use of invertebrate species to understand SMN biology and define conserved and potentially druggable effector pathways was recently supported by a patient advocacy group (the SMA Foundation). Our analysis of Smn modifier mutations and compounds/factors derived from chemical and genetic screens in Drosophila, C. elegans and human cells has identified several strong and conserved candidate pathways, including the Bone-Morphogenic Protein (BMP)-family signaling pathway known to control neuromuscular junction (NMJ) development in flies. Modulation of this BMP retrograde synaptic signaling pathway alone can potently attenuate key NMJ phenotypes of Smn loss in Drosophila. Therefore, in addition to completion of our secondary screens to define the Smn functional network, we here propose parallel levels of analysis to determine precisely how loss of Smn disrupts BMP signaling, and to what extent manipulation of the highly conserved elements in the BMP pathway can reverse the defects in neuromuscular structure and function resulting from reduced Smn activity. We will apply a combination of genetic, biochemical and developmental analyses to answer these questions, as we continue to identify the other conserved effectors downstream of SMN.

DESCRIPTION (provided by applicant): Spinal Muscular Atrophy (SIVIA) is a devastating inherited neurodegenerative disease causing progressive loss of motor functions due to malfunction of neuromuscular junctions (NMJs) and eventual loss of motor neurons. SMA is caused by loss of Survival of Motor Neuron (SMN1), a component of the nuclear gemin complex which is thought to mediate assembly and transport of snRNP complexes and thus control the synthesis and delivery of key synaptic proteins. However, the identity and function of relevant SMN target genes and the precise molecular role of SMN at the NMJ remain largely a mystery. The proposed project focus will be to use simple genetic model systems to dissect the mechanism(s) by which SMN controls synaptic form and function, and thus identify likely targets for interventions to attenuate SMA in mammalian models or human patients. We will be using genetic approaches in Drosophila to identify functional modifiers of SMN mutations and will study them in both Drosophila as well as C.elegans (Artavanis-Tsakonas, van Vactor and Hart Laboratories). Mammalian cell assays (Rubin laboratory) will extend and corroborate the studies in invertebrates while possible functional relationships and pharmacological interventions identified in mammalian cells will be tested using the sophisticated genetic tools that C elegans and Drosophila offer. Each system has unique experimental advantages and the integration of the proposed analysis across vertebrates and invertebrates offers exceptional promise for an in depth understanding of SMN biology and pathology while, importantly, it carries the promise of identifying novel therapeutic avenues.

Core B Project Leader: Van Vactor, David L. Project Summary / Abstract This program will rely on an innovative and growing resource of transgenic tools for in vivo analysis of microRNA (miR) function and activity. We have produced a partial collection of transgenic miR-SPonge (miR- SP) strains that are designed expressly to address the challenge of defining the cellular and developmental specificity of miR function within complex neural circuits and tissues with many constituent cell types. Preliminary data show that these tools are effective, however, accumulated data also show that many new sponge strains are needed to make this resource more comprehensive. Recent availability of many miR deletion alleles and UAS-miR mimic strains provides an important complement to our miR-SP resource, however, many new and selective null mutations are needed for a thorough and specific analysis of miR functions identified in our unpublished screens. In addition, informatics and expression analysis identifies promising target gene candidates that may define underlying downstream mechanisms for miRs that we find required in multiple neural circuits, however, new sensor reagents will be required to validate these and study their regulation in vivo. Core B will make and quality control these three types of reagents before they are distributed to our collaborators and the community by Core A.

? DESCRIPTION (provided by applicant): Precise temporal and spatial regulation of gene expression is essential to many aspects of nervous system development, function and plasticity. Among several classes of gene regulatory factors, non-coding RNAs have emerged as a rich potential source of regulatory mechanism in the central nervous system. In particular, microRNAs (miRs) provide sequence-specific control over target mRNA translation and stability that can tune the levels of downstream proteins quite precisely thus improving the stability and robustness of molecular networks. However, comprehensive analysis of miR function within the intact nervous system has been very challenging, leaving open key questions such as: How complex is the miR regulatory landscape for neural circuits that mediate essential behaviors? Are these miRs acting mainly during neural development or are they reused to manage ongoing neural circuit activity and adaptation to stimuli? To what extent are miR mechanisms utilized in many parts of the brain, or do they regulate distinct sets of target genes in different cell types and/or developmental stages? In order to address these questions, we have assembled a team of accomplished investigators prepared to work in unison using multiple robust behavioral and cellular assays as part of an integrated program. Our team includes Drs. David Van Vactor (Harvard Medical School), Leslie Griffith (Brandeis University), Ronald Davis (Scripps Institute), and Dennis Wall (Stanford University), who will each assume responsibility for key components of this joint program. We will use Drosophila as a model organism that offers many sophisticated genetic tools complementary to the innovative tools we will develop. Drosophila has proven to be particularly effective for identification and dissection of cellular and molecular mechanisms underlying well conserved behaviors. This model is also accessible to a full range of techniques for determining the detailed cellular and physiological phenotypes of mutants in specific pathways, thus offering a system ideal for mapping out miR functions on a comprehensive scale followed by mechanistic dissection that will effectively leverage a wealth of tools and knowledge. Together, we will (i) build and apply new genetic tools, (ii) apply these tools to identify miRs required in multiple neural circuits, (iii) discover the mechanisms and regulatory strategies for miR function in each context, and then (iv) compare each model to distinguish general and specific strategies and examine their conservation. This will be the first analysis of its kind in the nervous system. Our preliminary findings already identify convergence between different circuits that will prioritize our detailed studies of several miRs: miR-13, miR-92, miR-190 and let-7. Preliminary analysis of miR-92 already points to a series of highly conserved downstream genes implicated in both neural circuit development and synaptic plasticity from insects to mammals, providing a set of specific mechanistic hypotheses that we will test in the three model circuits to define the regulatory logic for each validated target.

Project 1 Project Leader: Van Vactor, David L. Abstract It has become increasingly clear that neuronal connectivity, function and plasticity all rely on the post- transcriptional regulation of gene networks. Among newly discovered classes of regulatory factor likely to be vital for regulating mRNA stability and translation in neurons, small non-coding microRNAs (miRs) have tremendous potential to shape the gene expression landscape. This large class of sequence-specific regulatory molecules is highly expressed in the nervous system. However, very few miR functions have been examined in vivo where the relationship between neural circuit architecture and behavioral outputs are maintained. To address this challenge, we have recently created tools to manipulate miR function with spatial and temporal specificity, in a model organism ideal for the study of neural circuits that control a wide variety of behaviors. Drosophila offers an increasingly sophisticated set of genetic approaches and reagents that are complementary and enabling to the new tools we have produced. As part of a carefully orchestrated set of collaborations using multiple robust and fundamental behaviors in this organism, we set out to map the miRs essential in the neural circuits controlling locomotion, sleep and associative memory. Using the larval NMJ as a model synapse for genetic validation ideal for miRs that may act in multiple neural circuits or multiple stages of postembryonic development, we have shown that null mutations recapitulate 85% of our miR-SP bouton phenotypes tested to date. Our functional screens reveal four conserved miRs likely to shape the development and plasticity of neural circuits: the miR-13 and miR-92 families, miR-190 and let-7. With an array of techniques, many of which we are developing with and/or learning from our collaborators, we will determine which of these miRs are essential for neurotransmission and activity-induced remodeling at the synapse. We will use new genetic tools to define the spatial and temporal logic for each miR function. We will then use a state-of-the-art combination of transcriptome sequencing and computational informatics, followed by use of in vivo activity sensors and functional validation, to discover the downstream mechanisms for each miR that intersects our coordinated screens. With an initial focus on the miR-92 family, we have obtained preliminary informatics and expression data identifying several highly conserved target gene candidates that have been implicated in developmental and activity-dependent synaptic signaling pathways. Once we dissect the cellular and molecular mechanisms for each gene, we will then compare each miR-target relationship across our model circuits to determine which mechanisms are context-specific, and which are widely used across the brain. This combination of comprehensive discovery science and comparative molecular anatomy will address fundamental and timely questions in a way that is not yet possible to achieve in mammalian models, but is likely to identify regulatory strategies highly relevant to many neural circuits.

Core A Project Leader: Van Vactor, David L. Project Summary / Abstract This new program will apply cutting-edge genetic tools and integrative approaches to advance our knowledge of general and specific microRNA-mediate mechanisms regulating neural circuit and synapse function in a model organism ideally suited for analysis of the cellular and molecular basis of behavior. Core A will serve to provide the administrative structure and oversight required by a multi-PI program, and to provide the coordination of frequent communication and dissemination of reagents and information necessary for this highly collaborative program to reach all of its goals. To achieve this coordination, a web-based interaction interface and a schedule of regular meetings, plus distribution of materials, will be established and monitored by the PI and two staff members.

Project Abstract This is an application for renewal and reorganization of a longstanding program for predoctoral training at Harvard Medical School designed to integrate across disciplinary boundaries. Over the last funding cycle, combined modernization and expansion of the four-decade old parent program (Cellular and Developmental Biology) have led us to rename our current program, Molecular, Cellular and Developmental Dynamics (MCD2), to signal an increase in intellectual breadth and depth, as well as multiple programmatic innovations, that will improve the quality of the training experience offered to our students. The mission of the MCD2 program (http://cellbio.med.harvard.edu/mcdd/) is to train future leaders at the forefront of discovery that will investigate the fundamental organizing principles and dynamics of molecules, cells, and tissues. Our goal is to build versatile and independent scholars capable of advancing important scientific frontiers with rigorous and novel approaches. To accomplish this, we offer a coordinated curriculum with emphasis on transferrable skills in experimental design, quantitative analysis, and project development. In addition to four compulsory Research Skills courses, our students select a series of Quantitative Analysis and Core Content courses to complement their existing strengths and scientific vocabulary. Our program has consistently led the forefront of educational innovation within the Harvard landscape, producing novel courses and training activities, such as our new Innovation Grant Program to fuel bold yet rigorous student-initiated proposals, and our new ?Big Data? curriculum to equip trainees with the analytical skills required for discoveries that will stand the test of time. MCD2 brings together a community of deeply committed scientists and educators to deliver this innovative program, and to provide our trainees with rigorous and supportive research training mentorship. Our preceptor group spans a multi-disciplinary spectrum of expertise, ranging from molecular cell biology to organismal development and physiology. This creates an environment where students learn to think across boundaries of scale, model system, and approach. To strengthen this environment, we also organize a rich array of paracurricular activities that promote scientific communication and collaboration, and enrich the community. Each year we will select a cohort of 18 MCD2 predoctoral students from a pool of over 200 talented candidates in multiple Harvard Integrated Life Science (HILS: http://www.gsas.harvard.edu/hils/) graduate programs. MCD2 members will be supported by this grant during their second and/or third year of PhD training, after Dissertation Advisor and research focus have been selected. As our trainees progress to degree completion, we also offer an assortment of professional development activities, workshops, and resources to prepare them for success at the next stage of their careers. While the majority of our trainees pursue careers in research and medicine, we also appreciate that many other professions are required to bring biomedical discoveries into clinical and other applications.

Amyotrophic lateral sclerosis (ALS), commonly known as Lou Gehrig?s disease, is a catastrophic neurodegenerative disorder that selectively involves motor neurons in the brain and spinal cord, resulting in progressive muscle weakness and atrophy. It is estimated to affect 2/10000 people, is invariably lethal within 3-5 years, and completely lacks therapeutic treatments. The underlying biology and molecular mechanisms leading to the disease are not well understood. More than 90% of the cases are of unknown genetic origin (sporadic ALS). Genome-wide association studies have identified more than twenty different genes associated with familial ALS (10% of total cases), indicating a complex underlying genetic architecture. Given the paucity of therapeutic interventions there is a great unmet need to develop novel therapies. A key to this is a better molecular and genetic understanding of the causes underlying ALS phenotypes. Through parallel genetic disease model screens in Drosophila, we found that mutations in phospho-lipase D (PLD), and at least six components in its upstream intracellular pathway, serve to suppress and control the toxic impact of TDP-43 and FUS mutations in the nervous system, indicating that the PLD pathway is a significant potential ALS target. This signaling pathway is multifunctional and has been linked to cancer as well as other neurodegenerative diseases. We also identified the VEGF/PDGF growth factor pathway (Pvf-Pvr in Drosophila), a likely upstream signaling input to PLD, as an potent modulator of ALS phenotypes. Deeper analysis to understand the specific mechanisms underlying the impact of PLD and VEGF/PDGF on ALS phenotypes is essential before they can be pursued as future therapeutic targets. Here, we propose to further leverage the strengths of Drosophila to confirm that changes in the PLD pathway modulation can prevent progressive, adult-onset neuromuscular failure. Depending on the results we will obtain, this exploratory project (R21) may form the basis to justify future analysis under R01-based mechanism.