These data therefore provide cell biological support for previous studies of Dscam1 in the control of contact-mediated recognition and repulsion, and reveal an important role for substrate interactions in promoting self-avoidance. da neurons, and class IV neurons in particular, have become a model for studies of dendritic self-avoidance
and tiling mechanisms. Separating two causes of crossing in these cells should enable the identification of key molecules that regulate repulsive signaling between dendrites, as well as mechanisms that establish relationships between dendrites and other surrounding cell types that impact dendrite development and, perhaps also, function. Alleles used were mys1 (Bloomington Stock Center), and mysXG43 (linked to markers y, w, f), mewM6 ifk27e (linked to markers y, f), and ifk27e Selleck PARP inhibitor (linked to marker f) on FRT19A, and rheatendrils13-8 on FRT2A (all provided by Dr. M. Krasnow, Stanford University) ( Levi selleck chemicals et al., 2006), and Dscam123 on FRT42D ( Matthews et al., 2007). GFP protein trap lines were provided by Drs. L. Cooley (Princeton University) and B. Ohlstein (Columbia University). RNAi lines were obtained from the Vienna RNAi Collection ( Dietzl et al., 2007). UAS-αPS2 (if), UAS-βPS (mys) flies were provided
by Dr. K. Broadie (Vanderbilt University). 221-Gal4, ppk-Gal4, and clh201-Gal4 lines have been described previously ( Grueber et al., 2003, Grueber et al., 2007 and Hughes and Thomas, 2007). MARCM experiments were performed as described (Grueber et al., 2002 and Lee and Luo, 1999) by crossing FRT lines to either
hsFLP, C155-Gal4, UAS-mCD8::GFP; enough FRT2A tubPGal80 or hsFLP, tubPGal80, FRT19A; 109(2)80-Gal4, UAS-mCD8::GFP. For time-lapse analysis of MARCM clones, we examined mid-stage second instar larvae for the presence of dorsal cluster clones. Selected animals were imaged live under halocarbon oil (Sigma, St. Louis, MO) and a coverslip, recovered to yeasted grape plates, raised to late third instar at 25°C, then dissected and labeled with anti-HRP, anti-GFP, and anti-Coracle. Larvae were processed for immunohistochemistry largely as described (Grueber et al., 2002). Antibodies and dilutions used were CF.6G11 (anti-βPS, 1:10; developed by D. Brower), DK.1A4 (anti-αPS1, 1:10; developed by D. Brower), CF.2C7 (anti-αPS2, 1:10; developed by D. Brower), c556.9 and c615.16 (anti-Coracle, 1:20; developed by R. Fehon), 4F3 (anti-discs large, 1:10; developed by C. Goodman). These antibodies were obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa, Department of Biology. Other primary antibodies were chicken anti-GFP (Abcam; 1:1,000) and goat anti-HRP (Sigma; 1:200). Species-specific fluorophore-conjugated secondary antibodies (Jackson Immunoresearch) were used at 1:200 in PBS with 0.3% Triton X-100 (PBS-TX).