Greg J. Bashaw - US grants

DESCRIPTION (provided by applicant): How axons in the developing nervous system successfully navigate to their correct targets is a fundamental problem in neurobiology. Axons are guided by both attractive and repulsive cues, which are members of evolutionarily conserved protein families. We are interested in the signaling mechanisms that function during attractive and repulsive axon guidance. The midline of the Drosophila embryonic CNS provides an ideal system to address these questions. Like its structural analog, the vertebrate floor plate, the fly midline is an intermediate target for many classes of navigating axons, which must decide whether or not to cross the midline. In the Drosophila CNS, the conserved guidance cue, Slit, functions to prevent axons from abnormally crossing the midline. Slit repulsion is mediated by the conserved family of Roundabout (Robo) receptors. The major aims of this application are: 1) to delimit and characterize the regions of the Robo receptor's cytoplasmic domain that are necessary and sufficient for Robo-mediated axon repulsion in response to the Slit ligand, 2) to assess the potential role of the SH3-SH2 adaptor protein Dreadlocks (Dock) and associated proteins in contributing to Robo repulsion, 3) to identify additional components involved in Slit and Robo signaling using a Drosophila genetic screen. A well established Drosophila transgenic approach will be used to determine which regions of Robo's cytoplasmic domain are required for repulsion. Classical genetic and biochemical techniques, including genetic interaction tests, mutant analysis, yeast two hybrid and co-immunoprecipitation will be used to investigate the potential role of Dock and associated proteins in Robo repulsion. In addition, established cell culture techniques will be used to address whether Slit stimulation of the Robo receptor regulates Dock/Robo interactions. To identify additional molecules involved in Robo function, a sensitized genetic screen will be performed. The proposed genetic screening strategy has already been successfully used on a small scale to identify genes that may play important roles during Robo repulsion. Deciphering the mechanisms that mediate Robo repulsive axon guidance will give important insight into how the nervous system is correctly wired during development and may have implications for nerve regeneration.

DESCRIPTION (provided by applicant): How axons in the developing nervous system successfully navigate to their correct targets is a fundamental problem in neurobiology. Axons are guided by both attractive and repulsive cues, which are members of evolutionary conserved protein families. We propose to study the signaling mechanisms that function during repulsive axon guidance. The midline of the Drosophila embryonic central nervous system (CNS) provides an ideal system to address these questions. Like its structural analog, the vertebrate floor plate, the fly midline is an intermediate target for many classes of navigating axons, which must decide whether or not to cross. In the Drosophila CNS, the conserved guidance cue Slit and its neuronal receptors the Roundabouts (Robo), play multiple roles in patterning axonal connections at the midline, acting primarily as axonal repellants. The primary aims of this proposal are to 1) test the hypothesis that proteolytic processing directly contributes to Robo repulsion and to signal termination, 2) to investigate how the identified Robo signaling components Abelson, Son of Sevenless and CrossGAP function together to coordinate signaling downstream of Robo and 3) to dissect the mechanisms underlying the distinct repulsive functions of the three Drosophila Robo family members. We have established genetic and direct biochemical links between a specific metalloprotease and regulation of Robo repulsion and have the necessary genetic, biochemical and cell biological assays to investigate the mechanism by which proteolytic processing influences repulsion. Similar kinds of approaches well established in our laboratory will allow for our continued investigation of how signaling molecules that function downstream of Robo coordinately regulate axon repulsion. A previously successful chimeric receptor approach where different portions of the Robo1, Robo2 and Robo3 receptors are exchanged with each other and then assayed for function in transgenic flies will be used to understand how different Robo receptors lead to distinct repulsive events. Together these studies promise to enrich our understanding of Slit-Robo signaling during normal development and may provide new therapeutic targets for diverse human health problems, ranging from developmental disorders of the nervous system to spinal cord injury and stroke.

DESCRIPTION (provided by applicant): Understanding how distinct classes of motor neurons are specified and guided to appropriate muscle domains is of fundamental importance to the design of therapeutic approaches to nerve regeneration and motor neuron disease. It is well established that combinatorial codes of transcription factors dictate motor axon pathway selection in both vertebrate and invertebrate nervous systems;however, the downstream targets of these transcription factor codes that control axon guidance are poorly defined. This proposal seeks to define functional links between transcriptional regulators of motor axon guidance and specific axon guidance receptors that control path finding. The Drosophila embryonic nervous system is an attractive model in which to address these questions because of the ability to label and genetically manipulate individual and uniquely identified motor neurons. In addition, many of the key transcriptional regulators as well as the guidance cues and receptors are evolutionarily conserved;thus, findings in Drosophila are very likely to be directly relevant to higher vertebrate nervous systems. The major aims of this proposal are: 1) to determine whether the even-skipped homeobox transcription factor directly or indirectly regulates the expression of the Unc-5 axon guidance receptor to guide motor axons dorsally, 2) to determine whether the Slit receptor Robo2's influence on motor axons that project to ventral muscle targets reflects a functional link with the transcriptional regulation of ventral projection, and 3) to identify additional downstream targets of the transcriptional regulators of dorsal motor axon pathway selection using complementary genetic and molecular screening approaches. Classical genetic and biochemical techniques, including genetic interaction tests, mutant analysis, and mis-expression experiments will be used to investigate the role of Slit and Netrin receptors in contributing to the readout of the transcriptional code for motor axon guidance. To identify additional determinants of dorsal motor axon projection, we will take advantage of dose-dependent effects of mis-expressing even-skipped to 1) perform genetic screens for dominant enhancers and suppressors of this mis-expression phenotypes and 2) perform mRNA expression screens using candidate genes and cDNA microarrays for genes that are differentially regulated by even-skipped in purified populations of dorsally projecting motor neurons. PUBLIC HEALTH RELEVANCE: The proposed research has the potential to make important contributions to the understanding of developmental disorders of the nervous system and may suggest new strategies to promote regeneration after brain and spinal cord injury. In addition, a more complete understanding of how neurons develop and form specific connections will be invaluable for developing stem cell therapies for neuronal replacement to treat neurological disorders ranging from Alzheimer's disease to Amyotrophic Lateral Sclerosis.

DESCRIPTION (provided by applicant): Determining how neurons are correctly specified and assembled into functional circuits will provide critical insight into developmental disorders of th nervous system and may suggest therapeutic approaches to promote nerve regeneration. To achieve this goal it is important to understand how axon responses to conserved families of axon guidance cues are regulated. Slit and Netrin, and their Robo and Fra/DCC receptors, are highly conserved signaling molecules that regulate multiple aspects of circuit development. Here, we propose to investigate how responses to Slit and Netrin are regulated by defining functional and molecular links between conserved transcriptional regulators that impart neuronal subtype identity and the cell surface axon guidance receptors for Slit and Netrin that coordinate motor and midline axon guidance. In addition, we propose to explore a newly discovered mechanism through which the Frazzled/DCC receptor intracellular domain (ICD) itself can regulate transcription to negatively regulate responses to the midline repellant Slit. Te developing Drosophila embryonic CNS is an ideal system to explore transcriptional mechanisms that regulate axon guidance because of the availability of powerful genetic approaches and the evolutionary conservation of the transcription factors and cell surface receptors that coordinate circuit assembly. The aims of this proposal are to determine how the Slit receptor Robo2 and the Netrin receptor Frazzled (Fra) are regulated by transcription factors, including Hb9, Nkx6 and Islet, and how, in turn, this regulation instructs pathway selection in defined populations of motor and interneurons. We will also use FACs sorting of defined subsets of motor neurons, together with transcript profiling in wild type and mutant backgrounds, to systematically identify additional effectors of these transcriptional programs. Finally, using a combination of robust in vitro and in vivo genetic and biochemical strategies, we will evaluate the hypothesis that in order to promote midline crossing, the Fra receptor undergoes gamma-secretase dependent proteolysis to release an intracellular domain (ICD) fragment that translocates to the nucleus to regulate its target gene commissureless (a key negative regulator of midline repulsion). Our proposed research will inform studies of homologous proteins in mammalian systems and could provide pharmacologic and genetic strategies to manipulate the specification of neuronal subtypes and receptor signaling. Furthermore, the results of our research may suggest new therapeutic targets for diverse disorders of the nervous system.

The establishment of proper neuron connections during development is essential for coordinated behavior. Axons, the wires of the nervous system, are guided to their targets by attractive and repulsive cues. This project will investigate the mechanisms that promote axon attraction to the midline of the fly central nervous system. Similar to the vertebrate floor plate, the fly midline is an intermediate target for navigating axons, which must decide whether or not to cross the midline. Netrin attracts axons to the midline through receptors of the Deleted in Colorectal Carcinoma (DCC) family (frazzled in Drosophila). However, it is unclear how Frazzled/DCC signaling mediates axon attraction and what additional mechanisms contribute to midline circuit assembly. Defining these mechanisms will enrich understanding of how guidance molecules work in the intact animal and have the potential to suggest therapeutic targets for disorders of the nervous system. The project includes outreach activities that are aimed at increasing high school scientific literacy and aptitude. Each summer, a high school teacher will spend six weeks in the laboratory, where he/she will collaborate with lab personnel to conduct original research and design a teaching module to bring back to the classroom.

To define mechanisms mediating axon attraction, this proposal seeks 1) to identify genes that act with Fra to control midline guidance, 2) to determine how the novel role of Fra in activating gene transcription is regulated by receptor proteolysis, and 3) to test if nuclear localization and transcriptional activation function of the Fra intracellular domain (ICD) are required for Fra-dependent gene expression. A sensitized genetic screen will identify new components that act with Fra to promote midline crossing, and molecular genetic approaches will be used to investigate a novel role for Semaphorin1a (identified in our screen) during midline guidance. Molecular and genetic approaches will be used to test the importance of proteolysis of the Fra receptor. Candidate genes from the screen will be tested for roles in Fra-dependent transcription. In vitro assays in insect and yeast cells will be used to delimit sequences that are necessary and sufficient for nuclear localization, nuclear export, and transactivation function of the Fra ICD. Finally, in vivo requirements for these elements will be tested by generating mutated forms of the Fra receptor, expressing them as transgenes, and assaying their ability to promote midline axon attraction and target gene expression.

? DESCRIPTION (provided by applicant): Defining how neurons are assembled into functional circuits will provide insights into diverse disorders of the nervous system and may suggest strategies to promote neuronal repair. Slit and its Robo receptors comprise an evolutionary conserved family of signaling molecules that play critical roles in regulating neuronal development; however, the understanding of how Robo receptors are regulated and how they signal to direct axon guidance is incomplete. These are important questions because perturbations of Robo signaling are implicated in diseases of nervous system development. Slits and Robos also play essential developmental roles in non- neuronal tissues and Robo mis-regulation is associated with several kinds of cancer. This proposal will define the molecular mechanisms underlying Robo regulation and signaling using the genetically tractable Drosophila embryonic nervous system as a model. Using state of the art molecular, genetic, biochemical and cell biological approaches we will continue our ongoing investigation of Robo receptor biology. Specifically, we propose to characterize the role of Robo receptor cleavage and endocytosis during midline axon repulsion. Genetic evidence indicates that endocytosis positively regulates repulsion, while receptor proteolysis appears to negatively regulate repulsion. We will explore how these processing and trafficking events are spatially and temporally coordinated to control Robo receptor signaling. Additionally, we will investigate a novel role for the Robo2 receptor in antagonizing Slit-Robo signaling to allow midline crossing. Biochemical and genetic evidence supports the model that Robo2 binds to Robo to inhibit repulsive signaling and we will define the underlying mechanism using a combination of genetic, biochemical and biophysical techniques. Finally, we will investigate how Robo2 and Robo3 regulate the medial-lateral position of axons within the CNS by 1) exploring the biochemical and cell biological mechanisms through which Robo2 and 3 direct longitudinal pathway choice and 2) identifying new factors that work with Robo2 and 3 to mediate this guidance activity. Preliminary evidence indicates that distinct biochemical properties imparted by Robo receptor immunoglobulin domains confer specific guidance outputs. We will mutate these domains and determine how these manipulations affect the in vivo functions of the receptors. Our proposed research will 1) define new concepts in the molecular biology of axon guidance, 2) inform studies of homologous proteins in mammalian systems and 3) provide pharmacologic and genetic strategies to manipulate receptor signaling.

PROJECT SUMMARY Determining how neurons are correctly specified and assembled into functional circuits will provide insight into developmental disorders of the nervous system and may suggest therapeutic approaches to promote nerve regeneration. Slit and Netrin, and their Robo and Fra/DCC receptors, are evolutionary conserved families of signaling molecules that play important roles in regulating neuronal development; however, the understanding of how these receptors are regulated and how they signal to direct axon growth and guidance is incomplete. These are important questions because perturbations of these signaling pathways are implicated in diseases of nervous system development. Slits, Netrins and their receptors also play essential roles outside of the nervous system and disruptions of these pathways are associated with several kinds of cancer. Our research program focuses on three broad areas related to the roles and regulation of these molecules in neuronal development using the genetically tractable Drosophila embryonic nervous system as a model. First, we are working to define functional and molecular links between conserved transcriptional regulators that impart neuronal subtype identity and the Robo and Fra/DCC receptors that coordinate axon guidance and dendrite morphogenesis in response to Slit and Netrin. Here, we will use genetic and molecular screening approaches, including Fluorescence Activated Cell sorting (FACs) of defined subsets of motor neurons, together with transcript profiling in wild type and mutant backgrounds, to systematically identify additional effectors of these transcriptional programs. Second, we are characterizing a newly discovered mechanism through which the Frazzled/DCC receptor intracellular domain (ICD) itself can act in the nucleus as a transcriptional activator to regulate commissureless expression to ensure that commissural axons avoid premature responses to midline repellent Slit. Here, we will use genetic and molecular approaches to identify factors that cooperate with the Fra ICD to regulate transcription and transcript profiling methods to identify additional targets of the Fra ICD. In addition, we will explore whether signaling from the nucleus is a common property of axon guidance receptors and through collaboration we will test if this is a conserved property of guidance receptors. Third, we are determining the molecular mechanisms underlying Robo and Fra/DCC receptor signaling during axon guidance using molecular, genetic, biochemical and cell biological approaches. Specifically, we use in vivo genetic manipulation, together with fluorescently tagged receptors and reporters of signaling molecule activity in vitro, to define the cell biological outputs of receptor activation with sub-cellular resolution. Our research program will define new concepts in the molecular biology of axon guidance, inform studies of related proteins in mammalian systems and will likely enhance our understanding of neural developmental disorders.

PROJECT SUMMARY Determining how neurons are assembled into functional circuits will provide insight into developmental disorders of the nervous system and may suggest therapeutic approaches to promote nerve regeneration. To navigate to their correct targets, axons must modulate their responses to extracellular cues, and regulated intracellular protein trafficking plays a pivotal role in this process. For example, commissural axons cross the midline despite the presence of repellant ligands in order to establish connections that are essential for coordinated motor behavior. In Drosophila, the endosomal protein Commissureless (Comm) prevents commissural axons from prematurely responding to the repellant Slit, by inhibiting surface expression of the Slit receptor Roundabout1 (Robo1). In mammals, Robo receptors are also negatively regulated in commissural axons prior to midline crossing, but the mechanisms are unknown. Unlike Slit and Robo, comm is not conserved in vertebrates; however, our preliminary data indicate that the vertebrate Nedd-4 interacting proteins (Ndfip1 and Ndfip2) can act analogously to Comm to regulate the trafficking and stability of human Robo receptors in vitro, and that loss of Ndfip1or Ndfip2 function in vivo in mice results in increased expression of Robo receptors and defects in axon guidance. We will test the hypothesis that Ndfip proteins control axon guidance in the developing brain and spinal cord by recruiting Robo receptors to endosomes and triggering their degradation through interactions with Nedd-4 E3 ubiquitin ligases. In aim 1, we will use molecular, cell biological and biochemical approaches to: 1) determine whether Ndfip proteins exhibit differential effects on intracellular trafficking of Robo receptors or other axon guidance receptors, 2) delimit the sequences that are necessary and sufficient to mediate interactions between Ndfip proteins and Robo family receptors, 3) characterize the role of HECT E3 ligase activity on receptor trafficking and 4) identify the specific Nedd4 family ligase(s) that is required for Robo receptor regulation. Aim 2 will explore the embryonic expression patterns and in vivo requirements for Ndfip proteins during commissural axon guidance by examining the trajectory of commissural axons in Ndfip1 and Ndfip2 single and double mutants, using 1) immunofluorescence for pre and post-crossing commissural axon markers, and 2) unilateral lipophilic dye tracing experiments. In addition, we will generate conditional knockouts of Ndfip1, Nedd4-1 and Nedd4-2 using Cre-lines specific for commissural neurons to investigate requirements for Nedd4-1 and Nedd4-2 in spinal commissural axon guidance. Aim 3 will assess the in vivo links between Ndfip proteins and Robo receptors by 1) testing whether neurons cultured from Ndfip mutants exhibit altered repulsive responses to exogenously added Slit proteins and 2) examining genetic interactions between Ndfip and Robo mutants. Finally, a biochemical screen will be conducted to identify novel substrates of Ndfip proteins.