(A) A dendrogram based on ClustalW sequence alignment of Drosophila and human REEP proteins shows two branches corresponding to human REEP1-4 (CG42678) and human REEP5-6. Broken lines represent Drosophila REEP proteins that are evolving rapidly (Supplementary file 1), reflected by longer branch lengths. Sequences used are NP_075063.1, NP_057690.2, NP_001001330.1, NP_079508.2, NP_005660.4, NP_612402.1, NP_726266.1, NP_610936.2, NP_651429.1, NP_611831.2, NP_572730.1, NP_726366.1. (B) Rtnl1 genomic and transcript map, showing the region deleted in Rtnl1− by excision of P-element NP7026 (Wakefield and Tear, 2006; Figure 1—figure supplement 1), the RHD domain (Pfam 02453) and its coordinates in protein isoform G, the Rtnl1::YFP exon trap insertion CPTI001291 (green triangle), and the fragment targeted by GD RNAi 7866. Map and coordinates are from the Drosophila Genome Browser (www.flybase.org, version R6.04), here and in subsequent panels; light regions in transcripts represent coding regions, dark shaded regions represent untranslated regions. (C) ReepA genomic and transcript map showing the region deleted in ReepA− by excision of P-element CB-0501–3, the position of the DP1 domain (Pfam 03134) and its coordinates in protein isoforms H and J. GFP insertion sites for ReepA1::GFP, ReepA2::GFP and ReepA3::GFP fusions are shown with green triangles. (D) ReepB genomic and transcript map showing the region deleted in ReepB− by excision of P-element EY05130, the position of the DP1 domain (Pfam 03134) and its coordinates in protein isoforms A and D). (E–I) Confocal sections showing localization of ReepA::GFP isoforms and ReepB::GFP. (E) Overlap of ReepA1::GFP and ReepB::GFP with anti-KDEL labeling in larval epidermal cells. To facilitate display of weaker ReepA1::GFP, the GFP channel in wild-type control (WT) and ReepA::GFP images has been brightened four times as much as for ReepB::GFP. (F) Expression of ReepA3::GFP and ReepB::GFP in third instar ventral nerve cord. The GFP channels for WT and ReepA::GFP have been brightened by twice as much as for ReepB::GFP. VNCs are outlined with yellow dashed lines. Arrowheads show ReepB::GFP extending into peripheral nerves. (G) A single confocal section of a peripheral nerve, showing ReepB::GFP localized continuously along its length. (H) Double labeling of an NMJ for ReepB::GFP and the mainly postsynaptic marker Dlg, showing ReepB::GFP in an axon emerging from nerve bundles (arrowhead) to extend to the NMJ, as well as in axons traversing the muscle surface (arrow); note the dispersed ReepB::GFP staining also in the underlying muscle. (I) Double labeling of ReepA::GFP and ReepB::GFP lines for GFP and Dlg (mainly postsynaptic) shows presynaptic expression of ReepB::GFP. (J) GFP expression in a wildtype negative control (WT), or from Rtnl1::GFP expressed in two closely apposed motor neurons by m12-GAL4. Scale bars 10 µm, except F, 20 µm.