Large cell clusters were seldom observed, suggesting that these interactions are weak or limited by unknown factors. Within the CNS, Ptp10D is expressed only on axons (Sun et al., 2000; Tian et al., 1991; Yang et al., 1991) (Figure 4A). It is also expressed on the apical surfaces of tracheal cells (Jeon and Zinn, 2009) (Figure 4C). Antibody against Sas has been widely used as a selective marker for apical cell surfaces (Schonbaum et al., 1992; Wodarz et al., 1995). Sas is probably expressed at some level on the apical surfaces of all cells of epithelial origin, including high throughput screening neurons and glia. Most or all cell bodies and axons in the VNC appear to stain with anti-Sas. Sas is expressed at higher levels on some axons than

on others, in particular on the intersegmental and segmental nerve roots and on two longitudinal bundles on each side of the CNS (Figure 4B). Figure S4 demonstrates that ensheathing cells on the dorsal surface of the CNS express Sas. These are the perineurial, subperineurial, Selleck LY2157299 interface, and channel glia (Ito et al., 1995). In the tracheal

system, the patterns of staining with anti-Ptp10D and anti-Sas are essentially identical (Figures 4C and 4D) (Jeon and Zinn, 2009). Although lethal ethyl methane sulfonate (EMS) mutations in sas were isolated many years ago, little is known about the functions of the sas gene. sas mutants do not exhibit embryonic lethality, but their postembryonic growth is stunted and they usually die as second instar larvae (hence the name). Third instar escapers have convoluted tracheae ( Schonbaum et al., 1992), but we were unable to find any tracheal defects in embryos. To evaluate whether sas mutants have CNS phenotypes,

we examined embryos homozygous for a strong EMS mutation, sas15, or transheterozygotes bearing this mutation over Df(3R)ED5221, which removes the entire sas gene. We sequenced the sas gene on the sas15 chromosome, and found that sas15 introduces a stop codon after aa 642, which is N-terminal to all conserved Sas domains. These data indicate that sas15 is likely to be a null mutation. To visualize CNS axons, we used mAbs BP102 and Suplatast tosilate 1D4. BP102 labels most or all CNS axons, producing a ladder-like pattern, with two commissural tracts in each segment and two longitudinal tracts extending the length of the embryo (Seeger et al., 1993) (Figures 5B, 5D, and 5F). 1D4 recognizes the transmembrane form of FasII (Vactor et al., 1993), and stains pioneer axons at stage 14 (Figure 5A). By late stage 16, 1D4 staining within the CNS is restricted to three distinct longitudinal axon bundles on each side of the VNC, and no staining is seen on commissural axon bundles that cross the CNS midline (Figure 5E). sas15/Df transheterozygotes appear normal at stage 14 ( Figures 5G and 5H), but display weak longitudinal axon defects at stage 16. The outer 1D4 axon bundle is interrupted and the other bundles appear slightly irregular ( Figures 5I and 5K).