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Fig. 3 Laminin and semaphorins are important guidance cues for Ti1 neurons. The schematic summarizes the expression pattern and defects resulting from blocking distinct semaphorin family members and the laminin 7 chain. For details, see text.

Fig. 3 Laminin and semaphorins are important guidance cues for Ti1 neurons. The schematic summarizes the expression pattern and defects resulting from blocking distinct semaphorin family members and the laminin 7 chain. For details, see text.

Sema-2a is restricted to the sema domain, as antibodies recognizing regions at the C terminus of Sema-2a had little effect on axon pathfinding (Isbister et al., 1999). While these experiments demonstrated the repulsive activity of Sema-2a, they did not address relevance of the graded distribution of Sema-2a. Comparisons of the slope of the Sema-2a gradient and Ti1 pathfinding error indicate that the slope of the gradient rather than the absolute concentration is important for Ti1 steering (Isbister et al., 2003).

While the role of Sema-2a is to discourage Ti1 axons from making incorrect guidance decisions through repulsion, an alternative role was found for another semaphorin family member in the developing limb bud. Semaphorin-1a (Sema-1a) is distinct from Sema-2a in that it is membrane bound through a transmembrane domain (Fig. 2; Kolodkin et al., 1992). The intracellular domain is relatively short and contains no identifiable signaling motifs, with the possible exception of a putative PDZ-binding site (Fig. 2; Wong et al., 1999). During Ti1 outgrowth, Sema-1a expression is restricted to a circumferential band of epithelium in the trochanter segment where the Ti1 pioneer neurons make the ventral turn (Fig. 2; Kolodkin et al., 1992). Later in development, the Sema-1a expression pattern expands to include complete circumferential epithelial bands in the tibia [just proximal to subgenual organ (SGO) neurons] and proximal tarsus (Singer et al., 1995; Wong et al, 1997; Isbister et al, 1999).

To block Sema-1a function during establishment of the Ti1 pathway, a monoclonal antibody was applied to grasshopper embryos. This treatment resulted in marked defasciculation and axon branching of Ti1 axons in the region of the trochanter epithelium (Fig. 3; Kolodkin et al., 1992). Notably, although the axons were defasciculated, they were still capable of turning ventrally and completing the pathway to the CNS. Interpretation of these results could lead to either an attractive or a repulsive function for Sema-1a. As discussed earlier, compared to other regions in the developing limb bud, Sema-1a expressing epithelium appears to be more adhesive for Ti1 growth cones, suggesting the possibility that Sema-1a may, in part, be mediating this adhesiveness (Condic and Bentley, 1989b; Isbister and O'Connor, 1999). Alternatively, mass defasciculation at the trochanter could also be interpreted as a loss of function of an inhibitory molecule. In this scenario, the repulsiveness of Sema-1a may normally act to promote Ti1 axon fasciculation (Kolodkin et al., 1992). However, it is unlikely that Sema-1a functions as a repulsive cue that collapses growth cones or retards their growth, as observations suggest that Sema 1a may be an attractive cue. For example, a later arising neuronal group, the SGO neurons, initiate axons that travel within and extend across Sema-1a expressing epithelium until contact with the Ti1 pathway is established (Wong et al., 1997). In the absence of Ti1 neurons (removed by either heat shock or mechanical ablation), SGO neurons grow to the edge of the Sema-1a expressing epithelium but do not continue further. When Sema-1a blocking antibodies are applied, SGO outgrowth is entirely inhibited. These results strongly suggest that Sema-1a is not only adhesive (SGO neurons cannot leave the Sema-1a expressing epithelium) but also may be required for axon initiation (Wong et al., 1997). In addition, when ectopic Sema-1a is expressed by S2 or COS cells in the limb bud, Ti1 axons repeatedly choose to reorient growth toward Sema-1a expressing cells (Wong et al., 1999). Thus, Sema-1a may be functionally complex, acting as a preferred or high-affinity guidance cue, while maintaining sufficient repulsive activity to encourage axon fasciculation.

In the developing limb bud, semaphorin family members have distinct roles in establishment of the Ti1 pathway. When both Sema-1a and Sema-2a are blocked simultaneously, the results are additive as opposed to synergistic. For example, some neurons exhibit a Sema-2a-specific defect, whereas others exhibit Sema-1a-specific defects (Isbister et al., 1999). This suggests a stepwise process of Ti1 axon guidance to the CNS. First, after axonogenesis, a distal-to-proximal gradient of Sema-2a repels Ti1 axons proximally toward the CNS. Before the turn in the trochanter, a dorsal-to-ventral Sema-2a gradient is partially responsible for directing the ventral turn of the axons. Upon turning in the trochanter, Sema-1a may encourage the ventral outgrowth of Til axons while ensuring that they do not wander proximally or distally in the limb before contacting the Cxl cells.

2. Laminin

The basal lamina is a thin extracellular array that separates tissue types, withstands tension, acts as a molecular sieve for secreted molecules, and is a scaffold for migrating cells during embryonic development. A complex network of several molecules, the basal lamina includes type IV collagen, perlecan, nido-gen, and laminin (Timpl and Brown, 1996). The structure of the basal lamina relies on highly ordered physical interactions of individual components that are required in order to serve the functions described earlier. Laminin binds to a number of proteins in the basal lamina, such as perlecan and nidogen (Timpl and Brown,

1996). Importantly, laminin binds to itself, resulting in polymerization that is calcium and concentration dependent (Yurchenco et al., 1985; Cheng et al.,

1997). Type IV collagen also self-assembles, forming a collagen network (Yurchenco and Furthmayr, 1984). The laminin and collagen networks are spatially distinct within the basal lamina; however, linker molecules, such as perlecan and nidogen, connect the two networks. Within the context of the basal lamina, laminin exists as a heterotrimer within a network that associates with many other molecules, suggesting a complex conformation that may have functional ramifications.

The laminin heterotrimer consists of three distinct subunits, a, ft, and 7 (Fig. 2). Each subunit has a similar domain structure of heptad repeat-containing domains

I and II, followed by an EGF repeat-containing domain III, a globular domain IV, another EGF repeat-containing domain V, and a final globular domain VI (Fig. 2). This general structure has a few subunit-specific modifications: the a chain contains a globular domain adjacent to domain I, as well as duplications of domain III and IV. The ft chain has an a domain between I and II. Also, several variant chains of a, ft, and 7 have been cloned that have truncations in various domains (Colognato and Yurchenco, 2000). The mature laminin molecule has a cruciform structure characterized by long and short arms (Fig. 2). Domains I and

II of all three subunits come together to form a coiled-coil, which is strengthened by disulfide bonding. All laminin isoforms examined to date contain one each of the three subunits.

a. Basal Lamina Establishment

In grasshopper, laminin ft and 7 chains are distributed in the limb bud basal lamina as early as 30% of embryonic development (Bonner and O'Connor, 2001). This stage corresponds to Ti1 differentiation and axonogenesis. Laminin is distributed uniformly within the basal lamina and appears to be tightly adherent to the epithelium throughout the duration of Ti1 pathway establishment (Fig. 3). However, the epithelium does not secrete laminin, as this tissue is negative with both in situ hybridization and laminin immunocytochemistry (Bonner et al., 2002).

Interestingly, laminin is secreted into the basal lamina by randomly migrating hemocytes. Thus the basal lamina is established by a tissue that is only transiently associated with it at any given moment. This is in stark contrast to establishment of the basal lamina in vertebrates and other invertebrates such as Drosophila. In these organisms, laminin is secreted by tissue that stably abuts the basal lamina and this tissue likely plays a role in its assembly and anchorage.

b. Role of Laminin in Ti1 Axon Guidance

To determine the role of laminin during Til outgrowth and pathfinding, the nidogen-binding site on the 7 chain of laminin was disrupted with blocking antibodies and blocking peptides (Fig. 2). Disruption of the nidogen recognition site on laminin 7 had a profound effect on Til pathfinding. Whereas Til neurons in control cultures were capable of completion of the pathway to the CNS, Til neurons cultured in the presence of blocking antibodies could not navigate the ventral turn, resulting in stalled axons (Fig. 3; Bonner and O'Connor, 2001). Axons stalled predominantly in the trochanter limb segment, at the site of the ventral turn. This result was specific for the turn as Til neurons of laminin-blocked cultures arrived at the trochanter at the same time as control cultures and stalled Til axons could not overcome the cessation of growth with a longer culture period. Important in the interpretation of these results is the uniform distribution of laminin compared to the specificity of the location of the defect. While it is likely that Til axons require laminin and other guidance cues simultaneously, it appears that when laminin is blocked, Til growth cones are still responding to Sema-2a signals from the distal epithelium, as evidenced by the fidelity of the proximal extension. As the trochanter limb epithelium is more adhesive for Til neurons compared to more distal limb bud regions (Condic and Bentley, l989c; Isbister and O'Connor, l999), it is possible that laminin is required in this region to overcome this adhesion and to turn ventrally. Thus, perhaps laminin is required for the integration of other guidance signals at the turn, such as a cue that directs the ventral turn. Although the identity of a functional laminin interactor is unknown, there are several molecules whose function remains to be determined in the guidance of Til axons.

3. Other Molecules

Although the functions of laminin and semaphorins have been partly elucidated, the roles of other molecules expressed in the limb bud remain to be determined. Annulin, a transglutaminase, is restricted to epithelial bands in the developing limb bud (Singer et al., l992; Bastiani et al., l992; Singer et al., l995). G-innexin-l, a connexin gap junction protein, is expressed at the trochanter-coxa segment boundary (Ganfornina et al., l999). REGA-l, an Ig superfamily member, is dynamically expressed by the mesoderm and is also found on the epithelium in the developing limb bud (Seaver et al., l996). A second Ig superfamily member, lachesin, and lazarillo, which encodes a novel member of the lipocalin family, are found on the Ti1, Fe1, and Tr1 neurons (Karlstrom et al., 1993; Sanchez et al., 1995). A promoter of Ti1 fasciculation, fasciclin I is expressed by Ti1 neurons and is also found in the epithelium (Bastiani et al., 1987; Diamond et al., 1993).

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