5%) compared to embryos heterozygous for either Sema-1a or pbl, although premature branching was apparently unaffected ( Figure 5). Similar patterns of genetic interactions were also observed with pbl3, and pbl5 null alleles ( Figure S6). Smad pathway The amino acid Val531, which is changed to aspartate in the pbl5 mutant (V531D), is located within the Pbl DH domain and is known to be required for nucleotide exchange activity in the DH domains of other GEFs ( Liu et al., 1998; Prokopenko et al., 1999). These results suggest

that Sema-1a and Pbl act together in the same signaling pathway to promote motor axon guidance through the regulation of Pbl GEF activity and, since similar genetic interactions are not observed with PlexA, that Pbl functions as an intracellular mediator downstream of Sema-1a rather than PlexA. However, heterozygosity C59 wnt for pblKG07669 significantly enhances the Sema-1a null phenotype ( Figure S6), indicating that additional Pbl guidance functions cooperate with Sema-1a signaling.

Since p190 physically associates with Sema-1a, we next asked whether or not p190 is involved in Sema-1a-mediated motor axon guidance. First, we observed that the p190 RNAi phenotype was suppressed by loss of a single copy of Sema-1a: from 22.1% to 8.2% for premature ISNb branching, and from 36.4% to 21.2% for total ISNb defects ( Figure 5). In contrast, PlexA or PlexB null alleles did not affect the p190 RNAi phenotype. These suppressive genetic interactions between p190 and Sema-1a were also observed using a different p190 RNAi line and the p1902 null allele ( Figure S6). Therefore, p190 functions to antagonize Sema-1a signaling, but not PlexA or PlexB signaling. This is consistent with physical association between p190 and Sema-1a being stronger than with either PlexA or PlexB ( Figure 1B). Taken together, these genetic interaction data suggest that Pbl and p190 exert opposing control over Sema-1a-mediated motor axon. Next, we examined whether pbl and p190 either genetically interact. When a single pbl2 mutant allele was introduced to p190 RNAi knockdown embryos, the premature branching phenotype

was significantly reduced, from 22.1% to 9.4% ( Figure 5). In these embryos, we also observed a significant increase in motor axon defasciculation defects (excluding premature branching phenotypes) at the last ISNb choice point (Figures 3L and 5). To test whether increasing Pbl levels affects premature ISNb branching phenotypes, we overexpressed HA-pbl alone or with coexpression of p190 RNAi in neurons. Increasing Pbl leads to a significant increase in premature branching phenotypes, but does not affect the p190 RNAi phenotype ( Figure S7D). These data suggest that premature ISNb branching is largely controlled by antagonistic functions of p190 and pbl. We find that Sema-1a and Pbl collaborate to induce cell contraction in vitro (Figure 2).