Figure 1 Independent labeling of two hemilineages in the larval ventral nervous system. (A) Schematic of a CRM- GAL4 transgene inserted into an attP site. Red-shaded boxes indicate sequences that originate from the attB- containing CRM-GAL4 vector. Yellow-shaded boxes indi- cate sequences from the attP-containing P-element. Gray boxes indicate the surrounding genomic region. attR and attL are the hybrid sites that result from phiC31-mediated recombination between attP and attB. Arrows indicate di- rection of transcription. The area above the brace indicates the sequences that vary in this study. For simplicity, we show only these elements in subsequent ﬁgures. Abbrevi- ations: P, P-element sequence; CRM, cis-regulatory module; DSCP, Drosophila synthetic core promoter; white, mini- white used to score for integrations; pUC19, plasmid back- bone from the CRM-GAL4 vector containing bacterial origin of replication and ampicillin resistance gene. (B–D) Maximum confocal z projection of the thoracic region of a wandering third instar larval central nervous system of the same genotype as in Figure 2A [w; P{UAS-mCD8::mRFP.LG}/R24B02-LexA (attP40); R58G08-GAL4(attP2)/P{LexAop-CD2::GFP}]. R24B02-LexA expression is shown in B and in green in D. It is expressed in hemilineage 12A in segments S2 to T2, and in A1. Hemilineage 12A’s cell bodies are located relatively medially, and its neurite bundle projects dorsally. In T1 and T2, the 12A bundle splits into a medial and dorsal branch (arrowheads). In T2, the dorsal branch of 12A projects across the midline. R58G08-GAL4 is shown in C and in magenta in D. It is expressed in hemilineage 3A in segments T1–T3. The cell bodies of 3A are located more laterally than those of 12A. The 3A bundle initially projects dorsally, but then turns laterally and projects to the dorsal region of the leg neuropil (arrows). The white box indicates the area shown in Figure 2A. Background staining of trachea is indicated in C. linearized plasmid was then blunted, and the plasmid was recircularized by blunt-end ligation. Transgenic ﬂy lines were generated by Genetic Services (Cambridge, MA) via phiC31-integrase–mediated insertion into attP40 or attP2. Results Transvection can occur between two transgenes inserted at the same attP site The development of the CRM-GAL4 collection that we screened for ventral nerve cord neuronal lineage expression has been previously described (Pfeiffer et al. 2008). Brieﬂy, each CRM-GAL4 contains: (1) a fragment taken from the cis-regulatory region of a preselected set of neuronally expressed genes (the CRM), (2) a synthetic core promoter that was designed to be both highly active and promiscuous (the DSCP) and (3) the yeast transcription factor GAL4, which is used to drive expression of one or more UAS trans- genes that are inserted elsewhere in the genome. The CRM- GAL4 transgenes have been inserted into the genome at deﬁned target sites via phiC31-mediated recombination (Pfeiffer et al. 2008). The target sites (attP sites) were them- selves introduced into the genome via P-element–mediated transformation. In this study, we used attP2 (Groth et al. 2004), which is located on the left arm of the third chromo- some and attP40 (Markstein et al. 2008), which is located on the left arm of the second chromsome. When a CRM-GAL4 is inserted into an attP site, the ﬁnal result is a locus that is ﬂanked by 59 and 39 P-element sequences and contains: (1) a copy of the yellow gene (the positive marker for P-element transformants), (2) one each of attL and attR sites (the out- come of recombination between attP and attB), (3) mini- white (a positive marker for phiC31 transformants), (4) the CRM-GAL4, and (5) the pUC19-derived backbone (Invitro- gen) of the vector used to introduce the CRM-GAL4. A sche- matic of an integrated CRM-GAL4 is shown in Figure 1A. While screening through CRM-GAL4s that had been in- tegrated into attP2, we identiﬁed two transgenes, R24B02- GAL4 and R58G08-GAL4, that drove expression in neuroblast hemilineages 12A and 3A (Truman et al. 2010), respectively. When examined individually, the expression patterns of R24B02- and R58G08-driven transgenes (Figure 1, B–D) are easily distinguishable from one another due to the morpho- logical characteristics of the hemilineages that they label. To independently label both hemilineage 12A and hemilineage 3A in the same ﬂy, the CRMs R24B02 and R58G08 were used to create R24B02-LexA::GADﬂ and R58G08-LexA::GADﬂ (hereafter referred to as R24B02-LexA and R58G08-LexA; GADﬂ, full-length GAL4 activation domain). This allowed us to examine two different transgene pairs: R24B02-LexA/ R58G08-GAL4 and R24B02-GAL4/R58G08-LexA. When R58G08-GAL4 and R24B02-LexA were used to drive pJFRC21-10XUAS-IVS-mCD8::RFP (hereafter referred to UAS-mCD8::RFP) and LexAop-rCD2::GFP, we expected to see hemilineages 12A and 3A labeled with their respective ﬂuorescent protein (Figures 1, B–D and 2A). Surprisingly, when we examined larvae that had both CRM drivers inserted into attP2, neurons from both hemilineages were found labeled by both ﬂuorescent proteins (Figure 2B). In sibling larvae that lacked UAS-mCD8::RFP, neurons of the R58G08-GAL4 pattern still expressed the LexAop-rCD2::GFP reporter (Figure 2C). Because R58G08-GAL4 does not acti- vate LexAop-rCD2::GFP on its own (our unpublished obser- vations; Figure 2A), we conclude that there is an interaction between the R24B02-LexA and R58G08-GAL4 transgenes themselves when they are paired in attP2. Examining the complementary pair of transgenes in which the GAL4 and LexA were swapped (R24B02-GAL4 and R58G08-LexA) resulted in the same pattern of crosstalk (Figure 2D), Transvection at phiC31 Integration Sites 1131