SUPPORTING ONLINE MATERIAL

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1 SUPPORTING ONLINE MATERIAL MATERIAL AND METHODS Fly stocks All Drosophila stocks were grown at 25 C on yeast-containing corn meal/molasses medium. The nanos (nos) alleles nos 18 and nos 53 have been previously described (S1). nos 18 is a null allele resulting from a splicing mutation in the first exon/intron junction, whereas nos 53 has a point mutation in one of the two zinc-finger domains critical for nos function. Their allelic combination has a strong ovarian phenotype and thus was used as the nos mutant throughout this paper. The pumilio alleles pum ET1 and pum ovt1 were described in (S2) and (S3), respectively. The nos-myc transgenic flies were kindly provided by Dr. Robin Wharton (S4). y 1 w; P{ry t7.2 =neofrt}82b Sb 1 /TM6, w; P{ry t7.2 =neofrt}82b P{w mc =ovod 1-18 }3R/st 1 betatub85d D ss 1 es/tm3, Sb 1 and w; Sb 1 /TM3, P{w mc =ActGFP}JMR2, Ser 1 stocks were obtained from the Bloomington Stock center (B#2051, B#2149, and B#4534). DNA cloning and generation of transgenic flies The nanos full-length cdna was a gift from Dr. Robin Wharton. To construct a fulllength rescue construct, a DraI-XbaI ( ) fragment was inserted downstream of the hsp70 promotor of pcasperhs P-element vector (S5). The resulting hs-nos construct was subsequently injected into w 1118 embryos and the mini-w gene was used as a marker to screen for transformants. S1

2 Heat-shock nos-rescue schemes To rescue nos mutant phenotype, several independent lines of hs-nos; nos 18 /nos 53 transgenic flies were either heat-shocked in a programmable incubator (Echo Therm chilling incubator IN35, Torrey Pines Scientific Inc.) or in a regular water bath, with a heat shock regime of alternating 37 C (1 hour) and 25 C (11 hours), starting from the embryonic stage until 4 days after eclosion (Fig. 1E, scheme b). The ovaries were then dissected, stained, analyzed by immuno-fluorescence microscopy (see below). Some of their siblings were tested for fertility as described below. A single copy of hs-nos largely rescued the mutant ovarian phenotype and the egg-laying ability of nos 18 /nos 53 flies (Fig.1E; fig.s1b-f, and below). In these flies, GSCs and oogenesis were fully restored in 66.4 (4.7)% of ovarioles, with each ovariole consisting of a morphologically normal germarium and a continuous array of egg chambers (Class I ovarioles; Fig. 2A). In 24.7 (4.1)% of ovarioles, GSCs appeared to be only partially restored, since these ovarioles contained a germarium with fewer cysts and only a few egg chambers (Class II ovarioles; Fig. 2B). GSCs were not restored in only 8.8 (2.2)% of ovarioles, since these ovarioles contained a germlineless germarium associated with either zero or only a few egg chambers (Class III ovarioles; Fig. 2C). To examine whether nos activity is continuously required for adult GSC maintenance, the same incubator and program was used to heat shock hs-nos; nos 18 /nos 53 flies from embryo to eclosion (Fig. 1E, scheme a). The flies were then transferred to a 25 C incubator and maintained there for the desired lengths of time (see Results) before dissection. To study the requirement during pre-adult germline development, the same heat shock program was used to heat shock hs-nos; nos 18 /nos 53 from the embryonic, 1 st -, 2 nd -, 3 rd -instar S2

3 larval, and newly eclosed adult stage to 4 days after eclosion (Fig. 1E, scheme b). Ovaries were then dissected and analyzed. To further investigate the effect of removing Nos from PGCs on their development, the same heat shock program was used to heat shock hs-nos; nos 18 /nos 53 from embryonic to 1 st instar larval stage, and ovaries dissected at the 3 rd instar larval stage to examine PGC development (Fig. 1E, scheme c). Fertility test Each female was crossed to two w 1118 males. Eggs laid within 24 hours were counted under dissection scope. Ten hs-nos; nos 18 /nos 53 females were tested and compared to 10 hs-nos; nos 18 / controls. Germline clonal analysis To study the phenotype of nos mutant clones, FLP-mediated recombination was done as described preciously (S6). Specifically, the nos 18 mutation was recombined onto the FRT82B chromosome. The presence of nos 18 mutation was selected according to its homozygous lethality and verified by sterility over the nos 53 mutation, while the FRT sequence was detected by PCR. To generate germline clones, w; FRT82B Ovo D1 /TM3 males were crossed to w hsflp 12 ; FRT82B nos 18 /TM3 virgin females, and their progenies were heat shocked at 37 C three times with 11 hour intervals starting at newly eclosed adult stage. Eight days after the last heat shock, ovaries of w hsflp 12 ; FRT82B nos 18 /FRT82B Ovo D1 were dissected, stained with DAPI, and analyzed. S3

4 Immuno-staining and microscopy Drosophila ovaries were dissected and stained according to (S7). Rabbit anti-vasa antibodies (S8), a gift from Dr. Yuh-Nung Jan, was used at 1:1000 dilution to label germline cells. Monoclonal anti-1b1 antibody (S9) from the University of Iowa Hybridoma Bank was used at 1:2 dilution to outline somatic cells and to label spectrosmes/fusomes. Mouse monoclonal anti-myc antibody from the University of Iowa Hybridoma Bank was used at 1:50 dilution to visualize the NOS-MYC protein. Fluorescently labeled secondary antibodies were obtained from Jackson Immnoresearch Laboratories (West Grove, PA, USA) and were used at 1:200 dilutions from a 1mg/ml stock. The DNA dye DAPI was purchased from SIGMA (St. Louis, MO, USA) and was used at a final concentration of 0.5 mg/ml. All samples were examined by epifluorescence microscopy using a Zeiss Axioplan microscope, images were captured by a Photometrics Star 1 CCD camera (Phoenix, AZ, USA) using IP Lab software. Confocal microscopy was done with a Zeiss LSM510 system. All images were processed using Adobe Photoshop 6.0. Egg chamber analysis To determine whether nos is required for post-gsc events of oogenesis, egg chambers from hs-nos; nos 18 /nos 53 females and generated by nos mutant clones were stained by DAPI to visualize DNA, by anti-vasa antibody to label germ cells, and by anti-1b1 antibody to outline somatic cells, spectrosomes, and fusomes. These egg chambers displayed no obvious morphological defect at every level examined (e.g., Fig. 2, B and C; Fig. 3, C and D). This includes the number of nurse cells, oocytes, and follicle cells, as well as their ploidy and patterning in the egg chamber. Furthermore, follicle cell migration, nurse-cell S4

5 dumping/apoptosis, and egg chamber polarity all appear to be normal. Eventually, mature eggs were oviposited. These observations suggest that Nos is not required for post-gsc events of oogenesis. S5

6 SUPPORTING FIGURES Fig. S1. A hs-nos transgene rescues nos mutant phenotype. (A) Schematic drawing of the hs-nos construct used for the nos rescue. (B) Restoration of oviposition of the nos 18 /nos 53 mutants by heat-shock induced hs-nos expression. (C-F) low (C,D) and high (E,F) magnification views of hs-nos;nos 18 /nos 53 ovarioles without heat-shock (C,E) or with heat-shock (D,F), showing that heat-shock restores GSCs and oogenesis. The anti- VASA antibody (green) stains germline cells, while anti-1b1 antibody (red) labels spectrosomes and fusomes and outlines somatic cells. Arrow heads in (E) and (F) indicate terminal filament. Arrows in (F) point to two restored GSCs with spectrosomes (red). Scale bars in C,D=50 µm, and bars in E,F=10 µm. Fig. S2. The pum, nos and pum-nos double mutants display similar phenotypes. Ovarioles from nos 18 /nos 53 (A,D,G). pum ET1 /pum ovt1 (B, E, H), and pum ET1 nos 18 /pum ovt1 nos 53 (C,F,I) females were stained with the anti-vasa antibody (green) to label germline cells, and with anti-1b1 antibody (red) to label spectrosomes and fusomes and to outline somatic cells. Ovarioles in (A-C) are completely devoid of germ cells and were most frequently seen in all three mutants ( % for nos, % for pum, and % for nos-pum). Ovarioles in (D-F) contain a small number of germ cells in the germarium and 2-3 egg chambers (with the largest egg chamber partially shown) and were seen less frequently in all three mutants ( % for nos, % for pum, and % for nos-pum). In these ovarioles, GSCs may divide a few times, but the resulting egg chambers development is discontinuous as adjacent egg chambers are very different in age. Ovarioles in (G-I) contain a continuous array of egg chambers and were only occasionally seen in all three mutants S7

7 (4.16.5% for nos, % for pum, and % for nos-pum). In these ovarioles, GSCs are still capable of divisions. Bars in A-C=10 µm, D-F=25 µm, and G-I=50 µm. S8

8 A C P 5 white HSP70 nos M P 3 pcasperhs-nos Construct D B Eggs laid / 24hr female Genotype WT nos nos nos nos E F Transgene - - Heat Shock - - Figure S1. Wang and Lin 2003

9 nos - pum - nos - pum - Figure S2. Wang and Lin 2003

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