0% versus 58.5%), while the mEC-5xmyc truncated protein led to an intermediate reduction (25.5%; Figure 7A). However, significant but modest rescue of CNS defects was observed

in embryos carrying either mICD or Fc/mICD mutant Sema-1a proteins, which lack forward signaling activity ( Figure 7A). These results demonstrate that multimerization of the Sema-1a extracellular domain is largely sufficient to mediate Sema-1a functions in CNS axon guidance. Furthermore, we found that neuronal expression NVP-BKM120 price of either of these signaling mutant transgenes partially rescued Sema-1a null ISNb pathway phenotypes ( Figure 7A). In particular, neuronal expression of mEC/Fc-5xmyc, which should allow forward but not reverse signaling, resulted in only modest rescue of ISNb defects but significant rescue of CNS defects, suggesting an essential role for Sema-1a-mediated reverse signaling in peripheral axon guidance. Next, we performed additional rescue experiments in order to examine whether the introduction of both forward (mEC/Fc-5xmyc) Selleck GW-572016 and reverse (mICD or Fc/mICD) signaling mutant transgenes together can further rescue Sema-1a ISNb phenotypes. However, simultaneous expression of these transgenes did not lead to additional rescue of Sema-1a null PNS phenotypes, as compared to neuronal expression of either single transgene (

Figure 7A). Given that Sema-1a functions as a ligand for PlexA in the PNS (forward signaling; Winberg et al., 1998), these complementation analyses strongly suggest that both the extracellular and intracellular Sema-1a domains, Phosphoprotein phosphatase and therefore the coordinated action of bidirectional

signaling, are necessary for Sema-1a-mediated motor axon guidance. Since overexpression of wild-type Sema-1a synergistically enhances pbl misexpression phenotypes in both the PNS and CNS ( Figures 6B–6F), we reasoned that if this synergistic enhancement occurs through the potentiation of reverse signaling, the Sema-1a intracellular domain alone should recapitulate the synergistic enhancement we see when wild-type Sema-1a is coexpressed with HA-pbl. We found in our GOF analysis that expression of mICD or Fc/mICD did indeed increase pbl GOF ISNb phenotypes to a similar level as wild-type Sema-1a, but produced only mild defects in CNS patterning ( Figures 6E and 6F). However, overexpression of the Sema-1a extracellular domain (mEC-5xmyc or mEC/Fc-5xmyc) did not affect pbl GOF phenotypes in either ISNb or CNS axon guidance. These in vivo GOF data strongly suggest that Pbl mediates Sema-1a reverse signaling. To address further Sema-1a receptor function and its regulation by Pbl, we performed additional GOF experiments utilizing apterous-GAL4 (apGAL4), which drives expression of GAL4 in only three neurons per hemisegment; these neurons extend axons longitudinally and do not cross the CNS midline ( O’Keefe et al.