Introduction

The nervous system is composed of a complex arrangement of individual neurons that make very specific functional connections. During development, neuronal cell bodies extend neurites that navigate through the embryonic environment in response to a variety of directional cues. An integral component of this navigation, also termed "pathfinding," is the anatomical specialization at the distal tip of the outgrowing neurite, called the growth cone. The growth cone is a highly motile and dynamic, actin-filled structure that actively surveys the environment by extending and retracting filopodial and lamellipodial protrusions. Upon reaching their targets, the migrating growth cones will undergo a transformation in order to form a stable connection with their target. A fundamental question in neuroscience is how the migrating tip distinguishes between the various guidance cues to forge an accurate path to the appropriate target. Generally speaking, there are two broad classes of guidance cues, attractive and repulsive; and growth cones exhibit contrasting behaviors depending on which they are in contact with. In the complex molecular landscape of the developing embryo, growth cones often encounter these diverse cues simultaneously and must therefore be able to process seemingly contradictory information in order to arrive at a pathfinding response. While a great deal of information can be collected from studying growth cone responses to cues in isolation (i.e., in vitro), it is crucial to also analyze these behaviors in vivo.

For the neuroscientist interested in the mechanisms underlying axonal guidance, the development of the grasshopper embryo provides several notable benefits. First, the relatively large size of the embryonic nervous system allows for straightforward imaging of both fixed and live neurons in vivo, including visualization of the neuronal cytoskeleton. Second, the ability to culture grasshopper embryos up to a period of 2 days combines the advantage of maintaining an intact organism with the benefits of cell culture techniques. In addition, analysis of molecules can be carried out at precise developmental stages in order to ascertain their role in axon guidance rather than their contribution to earlier developmental events. Third, the highly accessible and relatively simple nervous system is amenable to various cell biological manipulations, which are outlined later. Finally, the simplicity of the nervous system is not undermined by a lack of functional and molecular conservation, as axon guidance mechanisms in grasshoppers are conserved in vertebrates. This chapter outlines some of the methodologies pertinent to answering questions relating to axon guidance. Specifically, the focus is on one neuronal projection found in the developing grasshopper limb bud, called the Tibial-1 (Ti1) pathway.

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