MMconi92827

Drosophila stocks, genetics and ‘flp-out’ technique
Flies were raised at 25°C on standard medium supplemented with yeast. Virgin females were separated in four fly stocks carrying hs-flp, Sb balanced over a TM6, Tb, Hu chromosome and mutant alleles on X-chromosome balanced over a FM7, Bar balancer chromosome or Basc, Bar chromosome. These stocks (referred to as 900, 905, 910, 920) carry different mutant alleles. Their associated genes are players in the JAK/STAT pathway (900 with hop2, 905 with osupd4 , 910 with hop3 and 920 with hopTum-l. see Table for detail). Virgin female fly stocks were crossed individually with male flies (referred to as 313) from a fly stock carrying vgBE_LacZ (marker for vg expression) and Actin, FRT, y+, FRT, wg+. Flies were kept two days for mating and egg laying. The mutant alleles were tested for function in wg-induced transdetermination. Therefore we used the flp-out technique to ectopically express Wg in imaginal disc cells. The progeny (referd to as A (hop2), B (osupd4), C(hop3) and D (hopTum-l) were heat-shocked ~60-75 h after egg deposition (AED, mid-second instar stage) for 1 h at 38°C to gain the ubiquitous Wg overexpression and induce transdetermination (blastema regeneration) in imaginal discs. The flp-out system works by fusion of shock-inducible hs promotor and the flp recombinase gene. Heat-shock activates the flp recombinase. Flp recombinase then act at FRT sites and leads to the cut out of the y+ marker. Wg expression is then activated by the constitutive actin promotor (see figure). Using this protocol we tried to achieve wg overexpression and induction of transdetermination in ~50% of the disc cells. After heat-shock, larvae developed at 25°C. Wandering larvae were collected 3-10 days later for disc dissection. Flies heterozygotous for a mutant allele can be identified by absence of Bar marker (X-chromosome). Animals heterozygotous for hs-flp, Sb were identified by the absence of Tb marker (third chromosome). Flies with Bar marker (absence of a mutant allele) were used as the control. For complete genotype descriptions and crossbred selection schemes see Table.

Imaginal disc dissection and straining
Discs of wandering stage late third instar larvae were dissected in PBS and stored on ice. Fat body and gut were removed and discs were still attached to larval mouthhooks. For x-gal straining discs were fixed for 30 min in 3% glutaralaldehyde in PBS at room temperature. Fixated discs were rinsed in PBS and washed fife times in PBT (PBS containing Triton X-100) for up to 1 h. PBT washed discs were stained in staining solution with 4% X-gal. After incubation at room temperature for 4 hours or over night, discs were washed in PBT, transferred in 50% glycerol and afterwards in 99% glycerol. Discs were mounted in 99% glycerol. For antibody staining, discs were fixed in 4% paraformaldehyde in PBS for 30 min at room temperature. Discs were washed in PBS and afterwards at least 2 times washed in PBT for at least 20 min. For storage at -20°C, discs were first equilibrated and washed in 96% EtOH and afterwards in MeOH. Incubation with antibodies was performed using standard protocols. After washing of fixated discs in PBT, discs were blocked for 1 h in PTB with 2% normal goat serum. All antibodies were diluted in 2% normal goat serum. Primary antibodies were mouse-anti-elaV (1:50) and rabbit anti-beta-galactosidase (1:200). Incubation was performed at 4°C over night or 2-4 hours at room temperature. Secondary antibody was horseradish peroxidase-conjugated mouse-anti rabbit (1:200) and fluorescence labelled secondary antibodies were Cy2-conjugated goat-anti-rabbit (1:500) and Cy3-conjugated goat-anti-mouse (1:500). Secondary antibodies were incubated at 4°C over night or 2-4 hours at room temperature. After incubation or Diaminobenzidine(DAB)/HRP staining, discs were washed in PBT and mounted in 99% glycerol. Samples were monitored using optical- and fluorescence microscopy.

Cuticle preparation
A fly stock with C765-Gal4-mediated Su(z)2 overexpression was provided. Another fly stock without Gal4 construct served as control. Flies were raised at 29°C on standard medium. Red-eyed adult animals were collected and transferred to isopropyl alcohol. Imaginal disc-derived cuticular structures (wing, halter, leg, eyes) were dissected in isopropyl alcohol and transferred on to a microscope slide. Hoyer’s-lactat was added to the cuticle structures and they were covered with a coverslip. Slides were incubated at 65°C over night to clear the cuticles. Cuticles were examined using an optical microscope.