24 hours to allow completion of embryonic development and cuticle deposition. Cuticles were prepared as described previously [59]. Briefly, embryos were washed, devitellinized in1:1 heptane:- methanol, and placed on a slide. 3:1 lactic acid:water was added to the embryos and the slide incubated at 60uC for 24 hours. Cuticles were imaged on a Zeiss Axioscope microscope in darkfield with a 106 objective. Preparation of adult wings Ser-Gal4 virgin females were crossed with transgenic males containing UAS constructs encoding Gro deletion variants or with control w1118 males and incubated at 25uC. Shortly after eclosion, adult wings from the F1 generation were dissected from the flies, washed in methanol, and mounted in 70% glycerol. Adult wings were imaged on a Zeiss Axioscope microscope in brightfield with a 46 objective. Immunofluorescence For subcellular localization of Gro variants, S2 cells stably transformed with the pMK33 vectors encoding the FLAG tagged Gro variants and induced with 0.5 mM CuSO4 were stained with 1:250 diluted mouse anti-FLAG antibodies (Sigma), and wing discs expressing Myc-tagged Gro variants were stained with 1:400 diluted mouse anti-Myc antibodies (Santa Cruz Biotechnology). Secondary antibodies were goat anti-mouse conjugated with Alexa Fluor 568 (Molecular Probes). DNA was stained with 1 ug/ml DAPI. Confocal images of S2 cells and imaginal discs were obtained on a TCS SPE confocal laser-scanning microscope (Leica Microsystems, Heidelberg) using the 1006 and 206 objectives, respectively. Fluorescence in situ Hybridization Mat-Gal4 virgin females were crossed with transgenic males containing UAS constructs encoding Gro deletion variants or with control w1118 males and incubated at 25uC. Embryos containing maternally overexpressed Gro deletion variants were subjected to multiplex fluorescence in situ hybridization (FISH) as described previously [60] using a mixture of digoxigenin-11-UTP (DIG) labeled hkb antisense RNA probe, Biotin-16-UTP (BIO) labeled tll antisense RNA probe, and Fluorescein-12-UTP (FITC) labeled sna antisense RNA probes (Roche). Primary antibodies were 1:300 diluted sheep anti-DIG and 1:500 diluted mouse anti-FITC. Secondary antibodies were 1:400 diluted donkey anti-sheep conjugated to Alexa Fluor 555, 1:400 diluted rabbit anti-mouse conjugated to Alexa Fluor 633, and 1:400 diluted anti-BIO References 1. Hasson P, Muller B, Basler K, Paroush Z (2001) Brinker requires two corepressors for maximal and versatile repression in Dpp signalling. The EMBO journal 20: 5725–5736. 2. Paroush Z, Finley RL, Jr., Kidd T, Wainwright SM, Ingham PW, et al. (1994) Groucho is required for Drosophila neurogenesis, segmentation, and sex determination and interacts directly with hairy-related bHLH proteins. Cell 79: 805–815. 3. Dubnicoff T, Valentine SA, Chen G, Shi T, Lengyel JA, et al. (1997) Conversion of dorsal from an activator to a repressor by the global corepressor Groucho. Genes & development 11: 2952–2957. 4. Jimenez G, Paroush Z, Ish-Horowicz D (1997) Groucho acts as a corepressor for a subset of negative regulators, including Hairy and Engrailed. Genes & development 11: 3072–3082. 5. Zhang H, Levine M, Ashe HL (2001) Brinker is a sequence-specific transcriptional repressor in the Drosophila embryo. Genes & development 15: 261–266. 6. Chen G, Courey AJ (2000) Groucho/TLE family proteins and transcriptional repression. Gene 249: 1–16. 7. Hasson P, Paroush Z (2006) Crosstalk between the EGFR and other signalling pathways at the level of the global transcriptional corepressor Groucho/TLE. British journal of cancer 94: 771–775. Groucho Central Domains Instruct Development conjugated to Alexa Fluor 488 (Molecular Probes). Embryos were mounted with ProLong Gold antifade reagent and DAPI (Invitrogen) and images were obtained on a TCS SPE confocal laser-scanning microscope (Leica Microsystems, Heidelberg) using a 206 objective. Quantitative Reverse-Transcriptase Polymerase Chain Reaction (qRT-PCR) RNA was isolated from 0–3 hour embryos containing mater- nally overexpressed Gro deletion variants using Trizol reagent (Invitrogen), subjected to RQ1 DNase treatment (Promega), and repurified by repeating the Trizol and phenol-chloroform extractions followed by isopropanol precipitation. 3 ug of RNA was used to make cDNA with an oligo(dT)12–18 primer (Invitro- gen), M-MLV Reverse Transcriptase (Invitrogen), and RNase- OUT (Invitrogen). cDNA was then diluted 10-fold and added to a 3.6 uM mixture of primer pairs and 26 FastStart SYBR Green mix (Roche). cDNA levels were quantified by qPCR using an Opticon Monitor 3 system (Bio-Rad) and normalized to RpL32 [25]. Fold repression was then determined by dividing these values into the RpL32 normalized levels from control RNA made from embryos lacking maternally overexpressed Gro variants. Statistical significance of each value relative to the value in embryos lacking overexpressed Gro was determined from the two-tailed unpaired Student’s T-test. Primers used for gro, zen, twi, dpp, ftz, hkb, tll, sna, kni, Rpt3, and cin are given in Table S2. Supporting Information Primers used to generate Gro constructs. Primers used for qRT-PCR. Table S1 (DOC) Table S2 (DOC) Acknowledgments We thank Alberto Ponce and Pak Kwong for their technical assistance, Matthew Smith for his critical reading of the manuscript, Michael Chambers, Minghua Nie and Girish Ratnaparkhi for their advice, and Koen Venken for his input on recombineering. Author Contributions Conceived and designed the experiments: WTJ. Performed the experi- ments: WTJ. Analyzed the data: WTJ. Contributed reagents/materials/ analysis tools: WTJ. Wrote the paper: WTJ AJC. 8. Gasperowicz M, Otto F (2005) Mammalian Groucho homologs: redundancy or specificity? Journal of cellular biochemistry 95: 670–687. 9. Buscarlet M, Stifani S (2007) The ‘Marx’ of Groucho on development and disease. Trends in cell biology 17: 353–361. 10. Dresch JM, Liu X, Arnosti DN, Ay A (2010) Thermodynamic modeling of transcription: sensitivity analysis differentiates biological mechanism from mathematical model-induced effects. BMC systems biology 4: 142. 11. Hong JW, Hendrix DA, Papatsenko D, Levine MS (2008) How the Dorsal gradient works: insights from postgenome technologies. Proceedings of the National Academy of Sciences of the United States of America 105: 20072–20076. 12. Kaplan T, Li XY, Sabo PJ, Thomas S, Stamatoyannopoulos JA, et al. (2011) Quantitative models of the mechanisms that control genome-wide patterns of transcription factor binding during early Drosophila development. PLoS genetics 7: e1001290. 13. Mann RS, Lelli KM, Joshi R (2009) Hox specificity unique roles for cofactors and collaborators. Current topics in developmental biology 88: 63– 101. 14. Stifani S, Blaumueller CM, Redhead NJ, Hill RE, Artavanis-Tsakonas S (1992) Human homologs of a Drosophila Enhancer of split gene product define a novel family of nuclear proteins. Nature genetics 2: 119–127. PLoS ONE | www.plosone.org 14 February 2012 | Volume 7 | Issue 2 | e30610