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Neural explant Repellent

(3) Incubate 12-18 h, analyse axon trajectories

Open brain assay

(1) Remove eye, stage 32-34

(2) Peel back epidermis

(3) Incubate 12-18 h, analyse axon trajectories

Injection

Target RNA or DNA + GFP

Analyze axon in:, trajectories in embryo

Figure 4 Common assays for axon guidance activity. A variety of in vitro and in vivo assays have been developed to assess the capacity of a molecule to influence axon guidance. Common in vitro assays include: a, the stripe assay; b, the collapse assay; c, the bead assay; d, the turning assay; and e, the explant assay. Two in vivo assays are: f, the open-brain assay; and g, the injection of RNA or DNA into blastomere-stage embryos (see text for details).

in vivo anchorage of certain proteins like the ephrins (Figure 4c).

Two techniques are also commonly used to analyze the activity of soluble guidance cues. In the turning assay soluble proteins are ejected out of a micropipette, resulting in stable diffusion gradient (Lohof etal., 199 ). An attractive cue will guide the growth cones toward the pipette, whereas a repellent molecule will result in turning away from the pipette (Figure 4d). A second test to the guidance activity of soluble proteins is the co-culture explant assay (Tessier-Lavigne et al., 1988). Cultured cells expressing and secreting the protein of interest (for example, COS (cell line from African green monkey kidney) cells or the floor-plate of the developing spinal cord) are grown near neural explants. Axons extending from the neural explant will grow toward the diffusible source if the protein secreted is an attractive one, and will grow away if the protein is exhibiting a repellent activity. Thus, there will be more axons on the side facing the cell explant in the case of an attractant and fewer axons in the case of a repellent molecule (Figure 4e).

3. If the protein of interest is essential for axon pathfinding, then interfering with its function in vivo should result in pathfinding errors. Techniques to accomplish this include the overexpression or downregulation of proteins. For example, the recent development of knockin or knockout mice provides an elegant in vivo model for studying axon pathfinding and target-mapping defects. Other methods for studying axon pathfinding and target recognition in Xenopus are the open retina and open brain preparations (Figure 4f; McFarlane et al., 1996). Either the retina or the tectum of living animals is exposed to the guidance cue of interest and the subsequent pathfinding of the axons is observed with in vivo time-lapse microscopy. Other methods for interfering with protein function are the use of siRNA or morpholino antisense oligonucleotides. These techniques are well established in C. elegans, Xenopus, and zebra fish model systems. RNA or DNA constructs interfering with the protein of interest are introduced by electroporation, injection, or lipofection into early blastomere or later-stage embryos along with the green fluorescent protein (GFP) to visualize the injected axons and to look for axon-pathfinding errors (Figure 4g).

1.12.3.2 Guidance Cue Families

Extracellular cues have long been postulated to provide positional information to axonal growth cones.

Until recently the identity of the molecules responsible for this remained a mystery. However, in the last 10-15 years, the application of a variety of powerful genetic techniques in Drosophila melano-gaster and C. elegans and the development of molecular and biochemical tools and assays to study axon growth in vertebrates have led to the identification of several families of axon guidance cues; the most prominent of them are the netrin-1s, semaphorins, Slits, and ephrins, which are discussed below (Figure 5). However, it should be noted that many other molecules have been implicated in axon guidance, such as the morphogens Wnt4 and Sonic Hedgehog, and growth factors like the fibroblast growth factor (FGF) and the glial cell line-derived neurotrophic factor (GDNF).

Perhaps the most remarkable aspect of the identification of these four families of molecules has been the extent to which their role in axon guidance has been conserved during evolution. For example, despite an evolutionary separation exceeding 600 million years, the netrin-1s have retained their role for attracting axons ventrally toward the midline in organisms as diverse as C. elegans, Xenopus, and human. A second interesting feature is the relatively small number of molecules that are used to generate the startling array of complexity found within the CNS. Here, we will describe the four major families of axon guidance molecules before discussing their multiple roles in axon pathfinding in the next section.

1.12.3.2.1 Netrin-1s and their receptors Chemo-attractants are specifically expressed in the developing embryo to guide axons toward intermediate targets and to facilitate correct pathfinding to their ultimate destination. The netrin-1s (''one who guides,'' a derivation from Sanskrit) are the best-known chemoattractants, and have been implicated in the guidance of many developing axonal populations across a variety of species. Two studies, the first analyzing genes that regulate circumferential axon guidance in C. elegans and the second searching for secreted midline attractants involved in guiding spinal commissural axons in rodents, concurrently identified the first members of this family. Gene knockout studies in both these species have since verified the importance of this family of attractants in axon guidance. For example, one of the prominent phenotypes in mice lacking netrin-1 is a failure of the spinal commissural axons to reach the normally attractive floorplate (Serafini et al., 1996). C. elegans and Drosophila contain one or two known members respectively (uncoordinated (UNC)-6 and netrin-1-A and netrin-1-B, reviewed in Tessier-Lavigne and

Typical Response

Potential modulators

Axon guidance cues

Netrins

Slits

Semaphorins

Ephrins

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