Supplementary Methods

Transcription

1 Caussinus and Gonzalez Supplementary Methods Mitotic clones Clones of NBs homozygous for numb 03235, mira ZZ176, pros 17 or apkc k06403 were generated by FLP/FRT mediated mitotic recombination (1). As a first step, each of these mutants was recombined to the corresponding proximal FRT site (2). Then, the following crosses were set: Clones that do not express GFP. numb P{neoFRT}40A/ T(2;3)TSTL, CyO: TM6B, Tb 1 males x P{hsFLP}1;P{Ubi- GFP(S65T)nls}2L P{neoFRT}40A females. P{neoFRT}82B mira ZZ176 /T(2:3)TSTL, CyO: TM6B, Tb 1 males x P{hsFLP}1; P{neoFRT} 82B P{Ubi-GFP(S65T)nls}3R females. P{neoFRT}82B pros 17 /T(2:3)TSTL, CyO: TM6B, Tb 1 males x P{hsFLP}1;P{neoFRT}82B P{Ubi-GFP(S65T)nls}3R females. P{neoFRT}42D apkc k06403 /T(2:3)TSTL, CyO: TM6B, Tb 1 males x P{hsFLP}1; P{neoFRT}42D P{Ubi-GFP(S65T)nls}2R females. Clones that express GFP-alpha-Tubulin and EYFP-Histone2 (3). The following crosses were set: UbiGFP-tub84B UbiHis2avD-EYFP;numb P{neoFRT}40A/T(2:3)TSTL, CyO: TM6B, 1

2 Tb 1 males x P{hsFLP}1;P{Ubi-GFP(S65T)nls}2L P{neoFRT}40A females. UbiGFP-tub84B UbiHis2avD-EYFP;P{neoFRT}82B miranda ZZ176 /T(2:3)TSTL, CyO: TM6B, Tb 1 males x P{hsFLP}1;P{neoFRT}82B P{Ubi-GFP(S65T)nls}3R females. UbiGFP-tub84B UbiHis2avD-EYFP;P{neoFRT}82B pros 17 /T(2:3)TSTL, CyO: TM6B, Tb 1 males x P{hsFLP}1;P{neoFRT}82B P{Ubi-GFP(S65T)nls}3R females. From these crosses larvae heterozygous for the mutant of interest, carrying both the hsflp and the chosen combination of fluorescent proteins transgenes were selected. Expression of FLP and mitotic recombination was induced by a 1 hour heat-shock at 37 C, 24 hours after egg laying, Transplantation into adult hosts Tissue dissection and injection into adult hosts was carried out following the protocol developed by Hadorn and colleagues (4) with minor modifications (J. Szabad, personal communication). A glass needle was pulled to a diameter of around 90µm, cut, and sharpened by breaking the glass with small tweezers. Then, a constriction was made with a microforge, about 1 mm away from the tip of the needle. A simple pressureinjection system was made inserting the needle through a holder into a piece of silicon tubing that has a mouthpiece at the other end. Third instar larvae, kept at 25 C, were collected 5 days after egg laying, except for brat and l(3)mbt ts1 larvae, which were collected 9-12 days after egg laying and, in the case of l(3)mbt ts1, grown at 29 C. The larvae were washed 3 times in distilled water and their brains dissected in PBS and transferred into small drops of PBS on a siliconized microscope slide where the brain 2

3 lobes were separated. Prior to transplantation, young female adult hosts were etherized and stuck on a microscope slide, ventral side up, with double-sided sticky tape. One brain lobe per adult host was collected with the tip of the needle and injected tangentially in the mid-ventral abdomen. After recovering from anesthesia, the hosts were kept under standard Drosophila culture conditions at 25 C. When tumors developed, they were dissected, cut in small pieces and re-transplanted into new hosts following the same protocol. Adult-fly paraffin sectioning Embedding, sectioning and mounting of adult fly body parts was done as previously described (5), except that 4 flies were processed at the same time, 8 µm sections were cut, and the preparations were mounted in Vectashield (Vector Laboratories) containing DAPI. Karyotyping The karyotype of NBs from third instar larva brains was determined as described in (6). To karyotype the cells growing in the implanted tumors, the hosts abdomens were cut open and incubated in a drop of tri-sodium citrate 0.5% (w/v) on a siliconized microscope slide, for 5 min. at room temperature before fixation of the tumor mass. Tumor immunostaining Immunostaining of tumor cells was carried out as described for larval NBs (6) with minor modifications. Hosts abdomens were cut open and incubated in methanol-free, 3.7% 3

4 formaldehyde (Polysciences) in PBS for 30 minutes. The tissues within the abdomen were then dissected out and fixed for a further 30 minutes in the same solution. Incubation in a solution containing Alexa Fluor 660 phalloidin (Molecular Probes) (1:40), 0.3µg/ml DAPI, 1% BSA, 0.1% Tween in PBS was used to stain Actin and DNA. 4

5 Supplementary Methods REFERENCES (1) Golic, K.G. & Lindquist, S. The FLP recombinase of yeast catalyzes site-specific recombination in the Drosophila genome. Cell, 59, (1989). (2) Xu, T. & Rubin, G.M. Analysis of genetic mosaics in developing and adult Drosophila tissues. Development, 117, (1993). (3) Rebollo, E. Llamazares, S. Reina, J. & Gonzalez, C. Contribution of noncentrosomal microtubules to spindle assembly in Drosophila spermatocytes. PLoS Biol., 2, (2004) (4) Ashburner, M. Drosophila: a Laboratory Handbook (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1989), pp (5) Ashburner, M. Drosophila: a Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1989), pp (6) Gonzalez, C. Glover, D. in Cell Cycle: a Practical Approach, P. Fantes, R. Brooks, Eds. (Oxford Univ. Press, New York, 1993), pp