2% or 30.8% of hemisegments, respectively; Figure 4B). Knockdown of pbl in all muscles using 24B-GAL4 resulted in no significant ISNb pathfinding defects ( Figure 4B). To address whether pbl axon guidance and cytokinesis functions are separable, we knocked down pbl gene function using the postmitotic driver Elav-GAL4. Embryos overexpressing pbl RNAi[v35350] under the control

of Elav-GAL4 exhibited ISNb defects in 38% of hemisegments ( Figure 4B). A similar phenotype was observed with the t28343 RNAi line LBH589 under the control of two copies of Elav-GAL4. Since the GAL4/UAS system is temperature-sensitive, we allowed these embryos to develop at 29°C to increase GAL4-mediated expression of pbl RNAi and observed

an increase in the penetrance of motor axon pathfinding defects as compared to 25°C (55.8% versus 41.2%; Figures 4B, S3C, and S3D). These data strongly suggest that neuronal Pbl is required postmitotically for normal motor axon pathfinding. Since we observed that p190, like Pbl, also exhibits a strong physical association with Sema-1a and that two potential p190 enhancer GAL4 lines drive reporter expression in the CNS ( Figures S3H–S3J), we examined the role played by p190 in motor axon pathfinding using transgenic RNAi lines ( Billuart et al., 2001). Overexpression of the p190 RNAi transgene using Elav-GAL4 resulted in premature defasciculation of ISNb axons prior to reaching muscle

13, and sometimes muscle 6: reflecting either increased defasciculation or a defect in muscle target recognition (∼20% selleck kinase inhibitor of hemisegments; Figures 3J, 3K, 4C, and S6). This premature branching phenotype was rescued to wild-type levels when one copy of a UAS-mycp190 transgene ( Billuart et al., 2001) was introduced along with p190 RNAi Ribonucleotide reductase (5.9% of hemisegments; Figure 4C). Furthermore, when premature branching is observed in wild-type embryos it is qualitatively distinct from what we observe following p190 LOF, often occurring between the ventral and dorsal surfaces of muscle 13 rather than prior to ISNb arrival at muscle 13 (compare arrowhead in Figure 3A to arrows in Figures 3J and 3K). In addition, premature ISNb branching phenotypes qualitatively and quantitatively similar to those we observe in p190 RNAi lines were noted in p1902 maternally and zygotically-derived null alleles, and total ISNb defects were significantly rescued by reintroduction of the neuronal mycp190 transgene ( Figure 4D). These results show that neuronal p190 is required postmitotically for motor axon pathfinding. To test whether pbl plays a role in Sema-1a-mediated motor axon guidance, we investigated genetic interactions between pbl and Sema-1a, PlexA, and PlexB. When either a PlexA or PlexB null allele was introduced into pbl2 heterozygotes, total ISNb and premature branching defects were not significantly affected.