Fig. 5. REPO and PNTP1 cooperate to activate the transcription of loco. (A-C) Whole-mount in situ hybridization of stage 16 embryos using the loco-c1 probe. Ventral views. Wild-type embryos (A) express loco-c1 mRNA in two rows of longitudinal glial cells (arrowheads). loco-c1 mRNA expression in longitudinal glia was undetectable in repo mutant embryos (B) or in pointed mutant embryos (C). Anterior is leftwards. (D) Structure of the loco-c1 promoter-luciferase reporter genes used for transfection assays. AEE-luc carries a 0.7 kb loco promoter fragment, which contain two CAATTA motifs (square). Single base changes (asterisk) were introduced in both motifs in AEE*-luc reporter. AES-luc has a 1.4 kb loco fragment, which includes an Ets-binding site (triangle) and a GCM-binding site (oval), both identified by Granderath et al. (Granderath et al., 2000). The glial enhancer fragment (Rrk) used by Granderath et al. (Granderath et al., 2000) is shown above the map. The exact position of the transcriptional start site is not known. (E) REPO activates transcription through the CAATTA motif in the loco-c1 promoter. S2 cells were transfected with AEE-luc (black) or AEE*-luc (gray) reporter and REPO-expressing plasmid (REPO) or the empty vector (ACT). Luciferase activity obtained after the transfection of the effector constructs was normalized to the activity of the AEE-luc reporter co-transfected with the empty vector. (F) REPO and PNTP1 cooperates on the expression of the loco promoter. S2 cells were co-transfected with the AES-luc reporter gene and effector constructs that expressed GCM (column 2), REPO (column 3), PNTP1 (column 4) or REPO and PNTP1 (column 5). Luciferase activity was normalized to the value obtained with the empty vector (column 1). (G-L) REPO and PNTP1 has synergistic effects on loco expression. (G-K) lacZ expression of rC56, an enhancer trap insertion into the loco locus. The following transgenes were misexpressed in the entire neuroectoderm using the scabrous-GAL4 strain: (G) none; (H) GCM; (I) REPO; (J) PNTP1; (K) REPO and PNTP1. (L) Number of rC56-positive cells upon misexpression using the engrailed-GAL4 driver. The number of rC56-positive cells in the engrailed-positive region in each segment were scored (n=15).

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