Figure 4 Cell speciﬁcity of trans- vection. (A) Maximum confocal z projection of a wandering third in- star larval ventral nerve cord from a w; LexAop-rCD2::GFP/pJFRC21- 10XUAS-IVS-mCD8::RFP(attP40); R24B02-LexA(attP2)/R58G08-Gal4 (attP2) animal. White boxes indi- cate the regions from which the partial stacks in Figure 3, B and C were taken. (B) Transvection oc- curs between R24B02-LexA and R58G08-GAL4 in hemilineages 12A and 3A in T2. (C) Transvection occurs between R24B02-LexA and R58G08-GAL4 in hemilineage 3A in segment T3, but only weakly in hemilineage 12A in segment A1 (a few cells of the 12A hemi- lineage are present in T3 in some animals, and these also show lim- ited transvection). (D) No transvec- tion is seen in cells in the optic lobe that express R24B02-LexA. UAS-mCD8::GFP (attP2) with R58G08-GAL4 (attP40) resulted in expression of LexAop-mCD8::RFP in much of the R58G08 pattern (Figure 3D). This is particularly notable in that there is no sequence homology between the core elements of the R24B02-LexA and UAS-mCD8::GFP trans- genes (although there is ﬂanking homology from the inte- gration construct and integration site; see Figure 1A). That the GAL4 protein produced by R58G08-GAL4(attP40) was sufﬁcient to activate transcription in trans from its target UAS sites suggests that transvection can occur wherever a transcriptional activator is targeted to sequences present in trans to a functional core promoter. This result also sug- gests that there is no additional requirement for a transvection- mediating protein to bind a CRM for it to function in trans. The inability of some CRMs to drive transcription in trans must then be due to either (1) active repression by proteins recruited by the trans CRMs or (2) the inability of the trans promoter to initiate transcription (see Discussion). Trans-repression can occur between transgenes integrated at attP sites We observed trans-repression in at least 1 of our 60 trans- vection test crosses, in the case where 23G07-LexA was paired with R11G08-GAL4 in attP2. R11G08-GAL4 normally drives robust expression in hemilineages 12A and 11A (Fig- ure 5B). R23G07 drives expression in lineage 11 and in other neuron types, but is what we call a “stochastic” CRM (Figure 5A). The expression patterns of stochastic CRMs vary between individuals in that a subset of lineages may or may not be labeled in a given individual. The on/off de- cision appears to be independent for each lineage, because we typically see bilateral asymmetry. The on/off expression decision also appears to occur early in the life of the neuro- blast and then is epigenetically inherited by its daughters, so 1136 D. J. Mellert and J. W. Truman that either all of the cells in a lineage or hemilineage express the transgene or they all do not. Although R11G08-GAL4 consistently drives expression in all 11A hemilineages when present alone, after pairing with R23G07-LexA, R11G08-GAL4 expression was very weak or absent in some 11A hemilineages. Interestingly, in six of the seven samples of the R23G07-LexA/R11G08-GAL4 pairing, hemilineage 11A expression of R11G08-GAL4 was suppressed in the same lineages that were not expressed in the stochastic R23G07-LexA pattern (Figure 5, C and D). In contrast, all ﬁve of the siblings in which R11G08-GAL4 was present alone (R11G08-GAL4/TM6B) showed typical, nonstochastic expres- sion of R11G08-GAL4 in all 11A hemilineages. We can draw two conclusions from these data. First, the stochasticity seen with the R23G07 CRM is probably due to stochastic repres- sion, rather than activation. Second, the mechanism for repressing R23G07-LexA in some lineages can act in trans to similarly repress R11G08-GAL4. We do not know whether this trans interaction occurs early in the life of the neuroblast, coincident with the R23G07-LexA silencing event or whether the repression persists through development. Finally, we ob- served that pairing R11G08-GAL4 with R23G07-LexA also re- duced expression of R11G08-GAL4 in hemilineage 12A, although this repression was not as severe as in hemilineage 11A. R23G07 is never expressed in 12A, thus transgenes may affect the expression of a paired transgene in cell types that fall outside of their own typical expression pattern. Discussion Insights into transvection from using the phiC31 integrase system One issue that complicates the study of transvection is the difﬁculty in assessing the effect at the single cell level. Most