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Supplementary Material

DEV077743 Supplementary Material

Files in this Data Supplement:

  • Supplemental Table S1 -
  • Supplemental Figure S1 -

    Fig. S1. let-7-C EcRE-1, EcRE-2 and EcRE-3 are evolutionarily conserved. Representation of the three EcREs in the let-7-C intron 1 and nucleotide conservation between 12 Drosophila species retrieved using the University of California at Santa Cruz genome web site. The EcREs are highlighted by gray boxes.

  • Supplemental Figure S2 -

    Fig. S2. let-7-C intron 1 EcREs are required for tissue-specific induction and maintenance of pri-let-7-C. (A) Schematic representation of let-7-C transcriptional reporters. The line below represents the intron 1 and EcRE deletions used for analysis. (B) β-Galactosidase staining of central nervous system (CNS) and wing imaginal disc from the let-7-C intron 1 transgenes: let-7-Cp12.5kb::LacZ and let-7-Cp12.5kbΔEcRE::LacZ (indicated on the left of the panel). Tissues were dissected from pre-wandering (PW), −18 hours,−8 hours, 0 hours and 6 hours after puparium formation (APF). Tissue-specific expression between the let-7-Cp::LacZ and let-7-CpΔEcRE::LacZ in imaginal discs demonstrates a requirement for the let-7-C EcREs in imaginal disc expression. The expression in CNS is reduced in let-7-CpΔEcRE::LacZ transgenics. (C) let-7-C EcREs are required for maintenance of let-7-C expression in the CNS. CNS and ventral nerve cord (VNC) were dissected from 24 hours APF and 72 hours APF. lacZ expression in let-7-CpΔEcRE::LacZ is drastically reduced at later developmental time points. The transgenes are indicated on the left of the panels. Scale bar: 100 µm.

  • Supplemental Figure S3 -

    Fig. S3. A 3.3 kb intronic enhancer is sufficient for correct spatiotemporal expression of a lacZ transgene. (A-F) Spatial expression pattern of the let-7-Cp3.3kb::LacZ and let-7-CpΔEcRE::LacZ transgenic lines. The central nervous system (CNS), wing imaginal disc (WD) and leg imaginal disc (LD) tissues dissected from staged third instar larvae (pre-wandering, −18 hours and −8 hours APF), pre-pupae, 6 hour pupae were immunostained for β-galactosidase. Deletion of let-7-C EcREs in let-7-CpΔEcRE::LacZ abolished induction of lacZ in imaginal discs (compare B,C with E,F) and reduced expression in CNS (compare A with D). (G-N) let-7-C EcREs are required for maintenance of let-7-C expression in the CNS. CNS and ventral nerve cord (VNC) were dissected from 24 hours APF and 72 hours APF. lacZ expression in let-7-CpΔEcRE::LacZ is drastically reduced at later developmental time points. The transgenes are indicated on the left of the panels. Scale bar: 100 µm.

  • Supplemental Figure S4 -

    Fig. S4. Kc-167 cells express EcR A, EcR B1 and EcR B2 isoforms, which bind let-7-C EcREs.15 µg of nuclear extracts prepared from Kc-167 cells treated with 20E for 6 hours was incubated with the let-7-C EcREs for 30 minutes in the presence or absence of 1 µl of EcR common, EcR B1 or EcR A antibody. Complexes were resolved on a 5% polyacrylamide gel. EcR-Usp complex and supershifted EcR-Usp complex (ss) are indicated.

  • Supplemental Figure S5 -

    Fig. S5. Expression levels of EcR in flp-out clones of EcR RNAi and EcR-DN constructs. (A) EcR and dsRed immunostaining in flp-out clones over expressing EcR RNAi construct. (B) EcR A1 immunoreactivity and dsRed expression in EcR-AW650A overexpressing flp-out clones in 3rd instar larval leg discs and CNS. (C) EcR B1 immunoreactivity and dsRed expression in EcR-B1W650A overexpressing flp-out clones in 3rd instar larval leg discs and wing discs. The genotype is as indicated on the top of each panel. Mitotic clones were induced at 72 hours AEL. Scale bar: 50 µm.

  • Supplemental Figure S6 -

    Fig. S6. The let-7-C minimal promoter constructs are functional in vitro. Kc-167 cells transfected with either the wild-type or let-7-Cp3.3kb:: cDNA minimal promoter constructs. As a control, hsp70-let-7-C cDNA lacking any promoter was also transfected. Forty-eight hours post-transfection cells were treated with 5×10−6M 20E or with the solvent control (ethanol) for 24 hours and 48 hours. Total RNA was extracted from both 20E-treated and solvent-treated cells, and analyzed by northern blot hybridization (let-7 and U6 snRNA). Kc-167 cells show expression of endogenous let-7 upon 20E treatment (lanes 2 and 3). The expression of wild-type let-7-C minimal promoter is induced upon 20E treatment, whereas the deletion of the three EcREs in the minimal promter bring down the level of induction to endogenous levels (compare lanes 2 and 3 with lanes 8 and 9). Levels of let-7 relative to U6 snRNA have been plotted in the bar graph shown in the lower panel. The constructs are noted below the histogram plot.