The essential role for larval ORNs in PN dendrite targeting is evident from the significant difference between the dendrite targeting

defects at the two temperatures. To test whether Sema-2a derived from larval ORNs is necessary for dendrite targeting of dorsolateral-targeting PNs, we next asked whether RNAi knockdown of Sema-2a in ORNs affected PN dendrite position. Because Sema-2a and Sema-2b function redundantly (Figure 3), see more sema-2a loss-of-function alone should not cause PN dendrite mistargeting. We thus performed Sema-2a RNAi knockdown in sema-2b−/− mutant animals using the ORN-specific pebbled-GAL4 driver. We additionally included one mutant copy of sema-2a to reduce the amount of Sema-2a and sensitize the animals to RNAi knockdown. Flies heterozygous for sema-2a and sema-2b exhibited no dendrite targeting defects ( Figures 6A and 6D, compared to Figure 3J). Flies homozygous mutant for sema-2b and heterozygous for sema-2a exhibited a small but significant ventromedial shift of Mz19+ PN dendrite targeting ( Figures 6B and 6D).

However, when Sema-2a was additionally knocked down in ORNs, we found an additional significant ventromedial shift for Mz19+ PN dendrites ( Figures 6C and 6D). From this experiment alone, we cannot distinguish whether the ventromedial shift of Mz19+ dendrites is caused by Sema-2a function in RO4929097 larval ORNs, adult ORNs, or both, as both populations express pebbled-GAL4. However, several lines of evidence suggest that larval ORNs make a major contribution. First, larval ORNs contributed significantly to the Sema-2a protein distribution pattern in the ventromedial antennal lobe prior to arrival of adult ORN axons ( Figures 4D and 4E). Second, adult PN dendrite patterning occurs before arrival of adult ORN axons. Third, ablating larval ORNs caused

a ventromedial shift in dendrite targeting, just as in sema-2a sema-2b Protein kinase N1 double mutants. Taken together, these experiments strongly suggest that Sema-2a contributed by larval ORNs repels dorsolateral-targeting PNs from the ventromedial antennal lobe. To confirm that larval ORN-derived Sema-2a restricts PN targeting to the dorsolateral antennal lobe, we tested whether Sema-2a overexpression in ORNs was sufficient to rescue the mistargeting of normally dorsolateral-targeting PNs. In sema-2a−/− sema-2b−/− mutant flies, Sema-2a overexpression with pebbled-GAL4 was sufficient to rescue the ventromedial targeting defects of Mz19+ PN dendrites ( Figures 6E–6H), supporting the notion that Sema-2a from larval ORNs plays an essential role in regulating dendrite targeting of adult PNs.