Figure 3 Transvection between integrated transgenes is inﬂu- enced by promoter type, but not zeste. (A–D) Images and genotypes presented as in Figure 2. (A) Transvection be- tween R24B02-Ptrans-GAL4 and R58G08-LexA is unidirectional. R24B02 drives inappropriate ex- pression of LexA in hemilineage 12A, but R58G08 does not drive expression of GAL4 in 3A. (B) Transvection between R58G08- Ptrans-GAL4 and R24B02-LexA is also unidirectional. R58G08 drives inappropriate expression of LexA in hemilineage 3A, but R24B02 does not drive expres- sion of GAL4 in 12A. (C) Same genotype as in Figure 2D, but in a yza background. Transvection still occurs in the absense of zeste function. (D) Example of unidi- rectional transvection between UAS-mCD8::GFP and R24B02- LexA. In this case, UAS-mCD8:: GFP is activated by GAL4 in hemi- lineage 3A, and this causes LexA to misexpress in hemilineage 3A. R24B02 does not activate tran- scription in UAS-mCD8::GFP. (E) The R24B02 CRM acts in trans to drive expression of GAL4 from the R58G08-Ptrans-GAL4 trans- gene if the R24B02 transgene lacks an intact core promoter. (F) Inappropriate expression of GAL4 in the R24B02 pattern due to transvection from R24B02- ΔP-LexA to R58G08-Ptrans-GAL4 is also evident from the appear- ance of 12A projections in the dorsal neuropil of T1 and T2 (arrowheads). (G) An optical slice through the 24B02-GAL4 pattern at the same dorsoventral coordi- nates as in F. (H) Schematic sum- marizing transvection between transgenes utilizing promoters of unequal strength (data shown in B and E). Green-shaded enhancers are active in the indicated cell population. Green arrows indicate activation. Solid arrows from promoter indicate transcription, with the size of the arrow indicating the strength of the promoter. Broken arrows with an X indicate no transcription. (Left) When R24B02-LexA, which uses the strong DSCP, is paired with R58G08-Ptrans-GAL4, which uses the weaker P-transposase promoter, transvection only operates in one direction. In T2 hemilineage 12A, R24B02 acts only on its cis promoter, whereas in T2 hemilineage 3A, R58G08 acts on both promoters. (Right) When R24B02 does not have a functional core promoter, it is free to activate the trans P-transposase promoter in T2 hemilineage 12A. In T2 hemilineage 3A, R58G08 acts only on its own P-transposase promoter because there is no trans core promoter. inserted at the same attP site, we assembled a panel of 21 CRM-GAL4s whose larval CNS expression patterns we had previously characterized, including R58G08-GAL4 and R24B02-GAL4. The CRM for R23G07-GAL4, also included in the panel, was used to make 23G07-LexA::GADﬂ (Pfeiffer et al. 2010) (hereafter referred to as R23G07-LexA) and, together with R58G08-LexA and R24B02-LexA, crossed to the GAL4 panel to test for transvection between 60 different pairs of transgenes at attP2. We were conservative in scoring for transvection in that we disregarded any ambiguous cases where two patterns might have true overlap in their patterns of expression. Moreover, we only focused on the ventral nerve cord of third-instar larvae, as that was the only tissue for which we had previously characterized the expression pattern of the CRMs in our panel. Our assessment of trans- vection frequency should then be considered conservative. 1134 D. J. Mellert and J. W. Truman