Guidance of Til Axons

A. Mechanisms

We have described three important substrates (basal lamina, epithelium, and guidepost cells) encountered by the Ti1 growth cones that have distinct effects on their behavior. How does a single apparatus, the growth cone, discriminate from these different substrates? Although the Ti1 neurons follow a relatively simple trajectory, time-lapse imaging of the neurons reveals complex growth cone behaviors depending on the substrate encountered. When traversing the femur limb segment, Ti1 growth cones typically advance by extending lamellipodia between filopodia, which is characterized by an accumulation of actin filaments in filopo-dia and lamella that are extended in the direction of growth (O'Connor et al., 1990; O'Connor and Bentley, 1993). Often, at the trochanter, Ti1 axons extend microtubule-filled branches both dorsally (the incorrect decision) and ventrally (the correct decision). The dorsal projection is subsequently retracted and ventral projection proceeds (O'Connor et al., 1990; Sabry et al., 1991). The decision to proceed ventrally is characterized by a greater accumulation of F-actin in filopodia on the ventral side of the growth cone (or in the ventral branch) compared to dorsally extending filopodia (O'Connor and Bentley, 1993). Also, ventral extending filopodia start to show an increase in net extension just prior to growth cone turning, possibly indicating that individual filopodia can sense guidance cues (Isbister and O'Connor, 1999).

A somewhat different growth cone behavior is observed when they contact guidepost cells. Reorientation of Ti1 growth cones in response to guidepost cells can be accomplished by contact with a single filopodium (O'Connor et al., 1990). Upon contact, the proximal shaft of the filopodium expands, with high amounts of F-actin accumulation followed by microtubule invasion (O'Connor et al., 1990; Sabry et al., 1991; O'Connor and Bentley, 1993). Although the extracellular signal(s) responsible for events such as filopodial formation has not been identified, downstream effectors of filopodia formation likely converge on an increase of local Ca2+, as release of caged Ca2+ results in filopodia formation in Ti1 neurons (Lau et al., 1999).

The Ti1 growth cones encounter several guidepost cells (preaxonogenesis neurons) during their migration. Guidepost cells appear to provide a high-affinity substrate for the Ti1 growth cones as they appear to always turn toward the cells after making filopodial contact. For example, the Ti1 axons contact and momentarily stall on the Tr1 cell in the trochanter limb segment before turning ventrally. In addition, after ventral extension, the Ti1 growth cones turn proxim-ally after contacting the Cx1 cells. These contacts are also specialized in that the growth cones typically form gap junctions with the guidepost cells while they do not with other cell types (Bentley et al., 1991). However, it appears that Fe1 and Tr1 guidepost cells play a nonessential role in guiding the Ti1 growth cones. Examination of precocious developing embryos where the Ti1 growth cones extend past the Fe1 cell prior to its differentiation showed little affect on growth cone guidance (Caudy and Bentley, 1986). Similarly, we have observed numerous examples of Ti1 pathways that show no evidence of contact with the Tr1 guidepost cell, suggesting that these cells are not necessary for accurate pathfinding (unpublished observations). In contrast, ablation studies have demonstrated that Cx1 cells are necessary for the proximal turn made by Ti1 growth cones as they extend toward the CNS (Bentley and Caudy, 1983).

Removal of the basal lamina with enzymatic digestion revealed an adhesive role of the basal lamina, as shown by the retraction of Ti1 axons back to the cell bodies. (Condic and Bentley, 1989c). Surprisingly, when the enzymes were washed out, the Ti1 axons again initiated axons and navigated without error to the trochanter limb segment and commenced the ventral turn (Condic and Bentley, 1989a). These experiments suggest that Ti1 axons rely on the basal lamina at least initially for adhesion, but that the epithelium is capable of compensating for the loss of the basal lamina. Interestingly, however, the reliance of Ti1 adhesion to the basal lamina is dependent on location in the limb bud. For example, when the basal lamina is removed when the growth cones are in the trochanter limb segment, the axons do not retract, instead, the Ti1 cell bodies are displaced distally, toward the growth cones (Condic and Bentley, 1989b). Similarly, after disassembling the F-actin in filopodia, those filopodia in contact with the trochanter epithelium retract significantly slower than filopodia contacting the femur epithelium, suggesting greater filopodial:substrate adhesion in the trochanter (Isbister and O'Connor, 1999). Thus, with respect to adhesion properties, the limb bud is heterogeneous. Not surprisingly, several molecules are found to be expressed in restricted bands of epithelium and may be potential mediators of differential adhesion (see later).

B. Guidance Molecules

1. Semaphorins

Although the Ti1 pathway appears to be relatively simple, the expression profile of known guidance cues in the limb bud is rather complex. For example, sema-phorin family members are expressed in restricted epithelial domains that change as development proceeds. Semaphorins are a family of conserved glycoproteins that are distinguished by a common semaphorin domain. Widely accepted as repulsive guidance cues, semaphorins repel neurons. Members of two classes of semaphorins are expressed in the developing grasshopper limb bud and contribute to Ti1 pathfinding (reviewed by Bonner and O'Connor, 2000).

Fig. 2 Domain structures of semaphorins and laminin. Semaphorin 1a is a transmembrane glycoprotein that consists of a sema domain and a short intracellular tail. Semaphorin 2a is secreted and also has a sema domain, as well as an Ig domain. Laminin is a major constituent of the basal lamina and is made up of three subunits: a, 3, and 7. The following domains are common to all laminin chains: globular domains, helical domains, and EGF repeats. The a chain also has several G domains, and the 3 chain has an a domain.

Fig. 2 Domain structures of semaphorins and laminin. Semaphorin 1a is a transmembrane glycoprotein that consists of a sema domain and a short intracellular tail. Semaphorin 2a is secreted and also has a sema domain, as well as an Ig domain. Laminin is a major constituent of the basal lamina and is made up of three subunits: a, 3, and 7. The following domains are common to all laminin chains: globular domains, helical domains, and EGF repeats. The a chain also has several G domains, and the 3 chain has an a domain.

Grasshopper semaphorin 2a, (Sema-2a), a secreted class 2 semaphorin, is composed of a conserved sema domain and an Ig domain (Fig. 2). A candidate repulsive cue, Sema-2a is expressed in two overlapping epithelial gradients in the limb bud: a distal-to-proximal gradient and a dorsal-to-ventral gradient (Fig. 3; Isbister et al., 1999). Consistent with a repulsive role in Til axon guidance, a high expression of Sema 2a correlates with areas of the limb bud that Ti1 axons avoid. In the presence of antibodies that recognize the sema domain, Til neurons extend axons into the limb epithelium, which expresses high levels of Sema-2a, areas they usually avoid (Fig. 3; Isbister et al., 1999). Importantly, the repulsive activity of

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