Grant Mastick - US grants

DESCRIPTION (From the Applicant's Abstract): The long-term objective of this application is to increase our knowledge of how the growth and guidance of axons is regulated during embryonic development. The functioning nervous system requires precise interconnections between neurons, and this application aims to dissect the guidance mechanisms of a set of axons that pioneers the first longitudinal tract in the mouse forebrain, the tract of the postoptic commissure (tpoc). This tract is foundational, as it lays down the pathway followed by a multitude of later ascending and descending axons. Therefore the tpoc is crucial to understanding the formation of neural circuitry in the basal forebrain. Recently, we discovered that the tpoc axons make severe guidance errors in embryos mutant for the transcription factor Pax6. This mutant provides an opportunity to discover how early patterning genes, such as Pax6, might provide positional information in the form of guidance cues. The main approach will be to challenge the axons by creating altered patterns of Pax6 in the developing brain. Analysis of axon responses will reveal where and how Pax6 acts to influence axon outgrowth. To alter Pax6 expression patterns, several different strategies will be used. A new electroporation method will be used to target gene expression in intact embryos, including restoration of Pax6 to local regions of Pax6 mutant embryos, expansion of Pax6 expression in wild type embryos, and interference with endogenous Pax6 by targeted expression of dominant negative alleles of Pax6. The effect of altered Pax6 expression will also be examined in chimeric embryos with a mixture of wild type and Pax6 mutant cells, and in mutant mice that selectively lack Pax6 in only one cell type. In an initial analysis of downstream targets of Pax6, the axon guidance role of the Pax6-dependent cell adhesion molecule R-cadherin will also be examined by in vitro approaches and embryo electroporation experiments. This work will contribute to our knowledge of both the normal and mutant developing nervous system. Developmental defects underlie many devastating neurological disorders, and understanding the mechanisms of axon pathfinding and neuronal patterning may eventually lead to strategies for prevention or treatment of birth defects. In addition, the mechanisms of axon growth during development may give insight into how regeneration of the nervous system may be stimulated following trauma or disease.

DESCRIPTION (provided by applicant): The overall goal of this proposal is to define the molecular mechanisms that establish the first longitudinal axon pathways in the embryonic vertebrate brain. Longitudinal axons form the main communication highways in the CNS, transmitting all signals between brain regions and spinal cord. During early brain development, pioneer axons establish an initial simple array of longitudinal tracts by choosing specific pathways and accurately growing long distances. Our preliminary results have identified Slit/Robo signaling as a major determinant of longitudinal guidance, specifically the Slit1 and Slit2 secreted signaling proteins and their Robo1 and Robo2 receptors. We show that all pioneer longitudinal tracts are severely disrupted in mouse embryos carrying Slit or Robo mutations, due to extensive dorsal-ventral wandering and other errors. The overall goal of the proposal is to define how Slit/Robo signaling controls longitudinal guidance. The experimental system is the simple organized array of pioneer axons in early mouse and chick embryos, using a range of functional assays in intact embryos and with cultured axons. Aim 1. Determine the function of secreted Slit cues in guiding longitudinal axons. Our initial analysis of Slit mutant embryos identifies Slit1 and 2 as providing critical cues. We hypothesize that Slits function either as direct long-range instructive signals to orient and position axons, or as obligatory permissive signals for axons to respond to other guidance cues. To distinguish between these mechanisms, we will study axon projections in Slit mutant mice, challenge axons with Slits in explant cultures, and mis-express Slits in vivo. Aim 2. Test the function of the Robo family of Slit receptors in longitudinal guidance. Our evidence shows that axons mutant for Robo1 and 2 are unable to navigate along precise pathways. These results indicate that Robo1 and 2 mediate Slit signaling. We propose that Robos set the position of different longitudinal tracts via modulation of either specific combinations of isoforms or by different expression levels. To test these Robo mechanisms, we will study axons in Robo mutant mice, and mis-express Robos in specific axon populations. The main goal of this proposal is to define how neural wiring can form during embryonic brain development. New insights into neuron growth mechanisms during development will provide significant insights into birth defects in the brain, as well as how functional regeneration of the nervous system could be stimulated and guided following trauma or disease.

DESCRIPTION (provided by applicant): The overall objective of this project is to identify the mechanisms that set the position of motor neuron cell bodies, and to control motor axon exit out of the CNS. Motor neurons form early in embryonic development, and their cell bodies cluster in precise positions near the ventral midline, the floor plate. The positioning of motor neuron cell bodies is of fundamental importance for motor input and output. In mouse embryos with mutations in the Robo axon guidance receptors, we discovered that motor neuron cell bodies shift into the floor plate. This shift reveals a surprising ability of motor neuron cell bodies fro all levels of the neuraxis to migrate extensively. The hypothesis to be tested is that motor neuron migration is regulated by two opposing sets of guidance signals from the floor plate: repellent Slit/Robo signals and attractive Netrin/DCC signals. Our model is that these guidance signals have closely balanced effects, trapping motor neurons in specific positions to form motor nuclei, and also guiding their axons out of the CNS. This exploratory application will use a range of approaches including mutant mice and in vitro migration assays for motor neurons to define the basic mechanisms that control their migration. The aims of the project are to test the function of floor plate guidance signals in controlling motor neuron migration, specifically the opposing Slit repellent and Netrin attractant signals, and to identify the cellular mechanisms that control migration responses to the cues. A related aim will explore the mechanisms of guidance cues in controlling where the motor axons exit the central nervous system. Our experiments will also define a novel function of Slit/Robo signals in a set of cells, the boundary cap, that prevent motor neuron cell bodies from ectopically migrating out along the motor nerve. The outcome of the project will be to develop a new viewpoint of motor neuron differentiation, by defining the mechanisms in which motile motor neuron cell bodies are constrained in static positions by a balance between opposing sets of guidance cues. PUBLIC HEALTH RELEVANCE: Motor neurons form early in brain development as clusters in precise positions in the brain stem and spinal cord. This project will define how two sets of molecular signals are used to set the position of motor neurons, and to determine how these signals also regulate motor axon exit out of the central nervous system.

? DESCRIPTION (provided by applicant): The overall goal of this project is to identify molecular mechanisms that control the guidance of the oculomotor nerve. The oculomotor nerve innervates four of the six eye muscles. How the nerve wires up to each muscle is fundamental to the precise timing and coordination of motor signals, and drive the sophisticated set of eye movements required for vision. The oculomotor system is highly susceptible to developmental errors, causing eye movements or alignment in 2-5% of human infants. However, the basic mechanisms of how the oculomotor nerve navigates to the eye and targets specific muscles remain largely unknown. Our preliminary studies have identified the Slit/Robo repulsive guidance cues system as key regulators of several steps in oculomotor development, including axon projections to the eye, axon interactions with muscle precursors, and a unique motor neuron cell body migration across the midline of the midbrain. In the proposed project, we will use a range of complementary approaches, including studies of oculomotor nerve development in mutant mice, axon responses in culture, and transcriptomic analysis to identify new markers and candidate regulators of guidance. The aims of the project will be: 1) Determine the role of Slit/Robo signals in guiding the oculomotor nerve to the eye muscles; 2) Determine how another guidance system, the Semaphorin/Neuropilin signals, guide the oculomotor nerve, and how these signals interact with Slit/Robo signals. 3) Determine how Slit/Robo repulsion controls the midline migration of a specific subset of oculomotor neurons. Overall, this study will define a new experimental model for understanding the molecular basis for neuromuscular targeting, and identify novel mechanisms for the development of the oculomotor system

Axon guidance is the study of how developing nerves navigate in response to external signals. A remarkably small number of navigational signals have been identified, raising the question of how the complexity of the brain is generated. One solution is that a single ligand can signal in different ways either through the presence of different receptors or through processing that alters receptor binding. Slit is a large secreted protein that typically repels growing axons using Robo receptors. Slit is cleaved into two fragments, Slit-N and Slit-C, which display new biological activities in the nervous system and that in many other tissues. However, the role of the Slit fragments is controversial. The Slit-N fragment contains the Robo binding site and has been shown to be repel axons in in vitro culture systems and so is thought to be the active signaling molecule. Multiple lines of in vivo preliminary evidence in Drosophila suggest that only the full-length (Slit-FL) protein repels axons. This proposal takes an in vivo molecular genetic approach in flies and mice to separate the signaling of Slit-FL from that of the Slit fragments.These two systems will allow us to address the effects of Slit on axon growth, axon branching and regulated adhesion (fasciculation). This work will allow us to determine how a single signal can achieve different biological outcomes. The proposed work has implications for a diverse range of fields, including infectious disease, cancer and nerve regeneration.