The coarse targeting specified by protein gradients can then be refined by additional mechanisms, such as class-specific labeling molecules (Hong

et al., 2009, Kaneko-Goto et al., 2008 and Serizawa et al., 2006), which further segregate the olfactory circuit into discrete glomeruli. A P-element insertion sema-2aP2 was used for all sema-2a loss-of-function analysis ( Ayoob et al., 2006). Two alleles of sema-2b were used—a pBac insertion f02042 ( Thibault et al., 2004) and FRT-mediated deletion #078 (Sema-2bC4) ( Wu et al., 2011). A P-element insertion KG00878 into PlexB was used for loss-of-function analysis ( Ayoob et al., 2006). UAS-sema-2a RNAi transformant ID #15811 was used for all RNAi experiments (VDRC). To GSK1210151A chemical structure label Mz19+ PN dendrites, Mz19-GAL4 or Mz19-mCD8GFP were used as previously described ( Berdnik et al., 2006). To label DL3 dendrites, the enhancer trap line HB5-43-GAL4 (kindly provided by U. Heberlein and screened by E.C. Marin) was used. PD-1/PD-L1 inhibitor 2 To label VM2 dendrites, NP5103-GAL4 was used as previously characterized ( Komiyama et al., 2007). Other transgenes were from the following sources: pebbled-GAL4 ( Sweeney et al., 2007); UAS-HA-PlexA ( Terman et al., 2002); UAS-PlexB ( Ayoob et al., 2006); UAS-Sema-2a-TM ( Wu et al., 2011);

UAS-Sema-2a ( Winberg et al., 1998a). MARCM analysis to label DL1 single cell clones was performed as previously described (Komiyama et al., 2003 and Lee and Luo, 1999). In summary, flies were heat shocked for

1 hr at 37°C between 2 and 26 hr after larval hatching. QMARCM labeled DL1 single cell clones (Potter et al., 2010) were similarly generated but were heat shocked for an additional hour to increase clone frequency. VM2 anterodorsal neuroblast clones were generated between 0, 24, 48, and 72 hr after larval hatching and clone frequency was increased by performing two 1-hr heat shocks with 30 min at room temperature in between heat shocks. Additionally, flies were raised at 18°C after heat shocking for 24 hr. Or83b-GAL4 ( Larsson et al., 2004), which is expressed in all larval ORNs and ∼70%–80% adult ORNs, was used to drive the expression until of diphtheria toxin (DTI) ( Han et al., 2000). To specifically ablate larval ORNs and leave adult ORNs intact, tub-GAL80ts ( McGuire et al., 2003) was used to suppress GAL4 expression after puparium formation. Animals were raised at 25°C for 2 hr to allow egg laying and then shifted to 29°C to inactive GAL80, thus allowing GAL4 and DTI expression to ablate all larval ORNs. Animals were shifted back to 18°C at 0 hr APF to activate GAL80 and to allow the survival of adult ORNs. Immunostaining was performed according to previously described methods (Sweeney et al., 2007). Mouse anti-Sema-2a (Developmental Studies Hybridoma Bank) was used at a concentration of 1:50.