Figure 2 Transvection between transgenes inserted into attP2. (A–E) Partial z projections through the left T2 hemisegment in the ventral nerve cord of the indicated genotypes. Only the second and third chromosome genotypes are shown, all X chromosome geno- types are w1118. Neurons belonging to hemilineage 12A are sur- rounded by a yellow box, whereas neurons belonging to hemilineage 3A are surrounded by a blue box. Asterisks indicate ectopic expres- sion. (A) When no transgenes are paired, transvection does not occur. Shown is a partial stack through the cell body layer of the same CNS as shown in Figure 1. Hemilineages 12A and 3A are independently labeled with rCD2:: GFP and mCD8::RFP, respectively, without crosstalk. In addition to being labeled by different ﬂuores- cent proteins, the cell bodies can be assigned to their respective hemilineage based on their posi- tion and all of the neurites of a hemilineage form a fasciculated bundle (arrowheads). (B) An ex- ample of transvection between R24B02-LexA and R58G08-GAL4 when both transgenes are inserted in attP2. In contrast to the example in A, neurons from both hemilineages are labeled by both ﬂuorescent proteins. (C) An example of a sibling from the ge- notype in B that carries CyO instead of UAS-mCD8::RFP. Neurons from both hemilineages express LexAop- rCD2::GFP. (D) Swapping CRMs between R24B02-LexA and R58G08-GAL4 to make R58G08- LexA and R24B02-GAL4 does not affect transvection when both are inserted in attP2. (E) Moving both R58G08-LexA and R24B02-GAL4 to attP40 does not eliminate transvection. (F) Schematic summarizing transvection that occurs in B and C. Green-shaded enhancers are active in the indicated cell population. Green arrows indicate activation. Solid arrows from promoter indicate transcription. When R24B02-LexA and R58G08-GAL4 are inserted into the same integration site and paired, each CRM can drive transcription off of both promoters in some cell types. In T2 hemilineage 12A (yellow box in image panels; left schematic), the R24B02 CRM activates both promoters, leading to the transcription of both GAL4 and LexA. In T2 hemilineage 3A (blue box in image panels; right schematic), the R58G08 CRM activates both promoters. suggesting the interaction occurs between the CRMs/pro- moters of the two transgenes. We reasoned that transvection might be the cause of the interaction between R58G08-GAL4 and R24B02-LexA at attP2. If so, the effect should be pairing dependent, and inserting either transgene into a distant site should elimi- nate the interaction. To address this, we examined larvae in which no two transgenes were inserted into the same attP site. To generate these animals, we ﬁrst integrated R24B02- LexA into attP40. We also used UAS/LexAop reporters that were inserted into the genome by P-element–mediated transformation. We were then able to examine ﬂies that carried four transgenes at four different locations: R24B02- LexA (attP40), a third chromosome insertion of P{LexAop- CD2::GFP} (Lai and Lee 2006), R58G08-GAL4(attP2), and a second chromosome insertion of P{UAS-mCD8::mRFP.LG}. In this genotype, we observed RFP and GFP patterns in the larval VNC that were distinct from each other and labeled only the speciﬁc neuronal hemilineages expected for inde- pendent expression of each CRM (Figure 2A). Thus, the in- teraction we had previously observed between the CRM transgenes was completely disrupted when they were moved to distant genomic locations, as would be expected if trans- vection were the basis for the effect. 1132 D. J. Mellert and J. W. Truman