These results show that Sas is necessary for Ptp10D’s functions in preventing longitudinal axons from crossing the midline. However, the sas Ptp69D and Ptp10D Ptp69D 1D4 phenotypes are somewhat different. Fewer 1D4-positive bundles cross the midline in each segment in sas Ptp69D than in Ptp10D Ptp69D, and there are more distinct bundles remaining in the longitudinal tracts (compare Figure 6C to 6D). There are also complete breaks in the 1D4-positive longitudinal tracts in sas Ptp69D (

Figure 6D), which are not observed in Ptp10D Ptp69D. We further analyzed these phenotypes by staining with other signaling pathway markers for specific neurons and axons. Apterous (Ap)-GAL4 is expressed in a small number of neurons whose axons extend within a single longitudinal bundle (Garbe et al., 2007; Lundgren et al., 1995). Surprisingly, in both Ptp10D Ptp69D and sas Ptp69D these neurons do not extend axons at all, or have short processes that project in the wrong direction ( Figure S5). Anti-Connectin stains two longitudinal bundles that are distinct from the 1D4-positive bundles. Ptp10D Ptp69D and sas Ptp69D display similar phenotypes in which the outer Connectin bundle is missing and there are occasional breaks in the inner bundle ( Figure S5).

To evaluate whether Sas is likely to act together with Ptp10D in 1D4-positive axons to prevent them from crossing the midline, we expressed the Sas cDNA (-)-p-Bromotetramisole Oxalate construct from the FasII-GAL4Mz507 driver in the sas Ptp69D double mutant background, ISRIB manufacturer in order to restore Sas selectively to the same subset of CNS neurons whose phenotype is scored through analysis of the 1D4 staining pattern. We observed that Sas expression in FasII-positive neurons almost completely rescued the sas Ptp69D CNS phenotypes

( Figures 6E, 6J, and 6K). To test whether Sas must be expressed in FasII neurons in order to signal to Ptp10D, we then overexpressed Sas in glia using the Gcm-GAL4 driver in the sas Ptp69D double mutant background. This also rescued the phenotype ( Figure S5). These data, however, do not necessarily indicate that phenotypic rescue is the result of interactions between neuronal Ptp10D and Sas on glial cell surfaces. When Sas is expressed in small clusters of cells (e.g., Apterous neurons), anti-Sas antibody staining spreads out from the cell bodies in a pattern suggesting that nonmembrane-bound Sas proteins are deposited in the extracellular matrix (ECM) ( Figure S5). ECM-bound (“soluble”) Sas may be able to interact with Ptp10D in the same manner whether it is expressed on neurons or in glia. We further characterized genetic interactions between sas and Ptp10D by examining their epistatic relationships, asking whether sas gain-of-function (GOF) phenotypes are modified by LOF mutations in Ptp10D (Ptp10D has no known GOF phenotypes).