Drosophila Genetic Toolkit for Developmental Biology

Drosophila stocks used included apME-NLS::dsRed⁴, Df(3L)GN34²⁶, ens^HP36480 (Bloomington), ens^f07121 (Harvard), Df-ens^Δ3277, ens^ΔN, ens^ΔC (ref. 8 (link)), khc⁸, khc⁴, khc²³ (ref. 10 (link)), klc^8ex94 (ref. 27 (link)), twi-Gal4⁴, alpha-Gal4, G7-Gal4²⁸ (from K. Broadie), and UAS-ens-IR lines 106207 and 18491 (VDRC). UAS-ensHA transgenic flies were generated by BestGene Inc. Mouse cells were transfected with siRNA using lipofectamine RNAiMAX (Invitrogen) or DNA using lipofectamine 2000 (Invitrogen). Primary antibodies used were: rabbit anti-dsRed (Clontech), rat anti-tropomyosin (Abcam), mouse anti-myosin heavy chain (from S. Abmayr), rabbit anti-Zasp (from F. Schöck), chicken anti-βgal (Abcam), mouse anti-α-Tubulin (Sigma), rat anti-Ensconsin (from P. Rørth), guinea pig anti-Krüppel (from J. Reinitz), rabbit anti-Eve (from M. Frasch), mouse anti-βPS Integrin (DSHB), rabbit anti-Vestigial (from S. Carroll), FITC conjugated anti-HRP (Jackson ImmunoResearch), mouse anti-Discs large (DSHB), rabbit anti-ATP synthase^{29 (link)} (from H. Duan), rat anti-DE-Cadherin (DSHB), mouse anti-Chaoptin 24B10 (DSHB), rat anti-Elav (DSHB), Alexa Fluor 488 conjugated wheat germ agglutinin (Invitrogen), MF20 (DSHB), rabbit anti-KHC (Santa Cruz), mouse anti-c-Myc (Roche), rabbit anti-GFP (Invitrogen). Secondary antibodies were either biotinylated (Vector Laboratories and Jackson ImmunoResearch) or conjugated to Alexa Fluor 488, 555, or 647. The fusion index^{30 (link)}, sarcomere length^{15 (link)}, bouton number^{17 (link)}, and larval velocity^{14 (link)} were quantified as described with minor modifications (see methods). The yeast 2-hybrid was performed with full length Ens by Hybrigenics S.A Services using a 0–24 hour Drosophila cDNA library. Standard protocols were used for immunoprecipitation, western blot, and qPCR experiments and are described in Methods.