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5��LMF��2}.TPi, FNi, FPi, and TNi correspond to true-positive, false-negative, false-positive, and true-negative values at given LMF cutoff value i, respectively. The Hh miRNA-target network was constructed using at chosen cutoff value (LMF �� 0.62). The network is visualized with Cytoscape software (http://www.cytoscape.org/). Immunostainings of larval wing imaginal discs were performed as previously described (Belenkaya et?al., 2004). Primary antibodies were mouse anti-Ptc (1:40; DSHB, Apa-1), mouse anti-Smo (1:50; DSHB, 20C6), rabbit anti-Hh (1:50; kindly provided by Dr. Xinhua Lin), and rabbit anti-��-Gal (1:1000; Cappel). Primary antibodies were detected by anti-mouse this website or anti-rabbit secondary antibodies conjugated to Alexa-Fluor 594 and 647 (1:1,000; Invitrogen). Fluorescent images were acquired with a Leica TCS SP2 AOBS. Images were processed using Adobe Photoshop. Third instar larvae were heat killed (70��C for 10�C15 s), mounted in 50% glycerol and examined under a Zeiss Axioskop 2 compound fluorescence microscope. Average values and their corresponding SDs were calculated, and t test analysis was performed with Microsoft Excel. We thank Drs. M. Krasnow, X. Lin, and M. Scott for reagents. We thank the Transgenic Caspase activity assay RNAi Resource Project and the Bloomington Drosophila Stock Center for flies, the Developmental Studies Hybridoma Bank for monoclonal antibodies, and the Drosophila RNAi Screening Center (Harvard Medical School) for plate-reader equipment. We also thank C. Pitsouli, Y. Kwon, D. Yan, and R. Binari for critical comments Ceftiofur on the manuscript and reagents. This work was supported by the National Institute of Health (5P01CA120964 and 5R01DK088718). N.P. is a Howard Hughes Medical Institute investigator. ""In recent years, methods allowing manipulation of genes of interest within living organisms have enabled a detailed understanding of numerous genes�� function across diverse phyla. The ability to alter the mouse genome has been especially critical for the investigation of gene function in?vivo and has contributed immensely to our understanding of many fundamental questions in developmental biology and biomedical sciences. In particular, the development of site-specific recombinase systems in mice, including the Cre/loxP and FLP/FRT systems, has allowed the inactivation of genes of interest with both spatial and temporal control. As a result, conditional gene disruption has become a critical tool for understanding gene function during development, homeostasis, as well as in?disease states ( Rajewsky, 2007?and?Schmidt-Supprian and Rajewsky, 2007). Fluorescent reporter alleles have proved to be another key resource in the dissection of gene function by allowing direct visualization and isolation of molecularly defined subsets of cells ( Hadjantonakis et?al., 2003).