DEV034603 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Representation of the adult hermaphrodite gonad. Germ cells proliferate near the DTC and enter meiosis in the transition zone (TZ). The germline develops as a syncytium; germ cell nuclei share a common cytoplasmic core separated by incomplete membranes. Oocytes grow and cellularize in the proximal arm. The most proximal (−1) oocyte undergoes meiotic maturation in response to the MSP signal from sperm. Five pairs of sheath cells (1-5) surround the germline.

• Supplemental Figure S2 -

  Fig. S2. Evidence that the glp-1 Looc phenotype is separable from the differentiation versus meiotic entry decision. (A-D) Fluorescence images of DAPI-stained dissected gonads from wild-type (A), glp-1(tn777ts) (B) and glp-1(tn777ts); let-60/ras(n1046gf) (D) hermaphrodites and glp-1(tn777ts); fog-2(oz40) unmated females (C). Animals were analyzed 16 hours post-L4 at 25°C. Note that all germ cells have a cytological appearance characteristic of pachytene-stage or early meiotic germ cells in glp-1(tn777ts) mutants irrespective of the presence of the let-60/ras(n1046gf) mutation or the absence of sperm. Because a Looc phenotype is not observed in glp-1(tn777ts); let-60/ras(n1046gf) hermaphrodites or glp-1(tn777ts); fog-2(oz40) unmated females, we conclude that the regulation of oocyte growth is a separate glp-1 function than regulation of the proliferation versus meiotic entry decision. Similarly, dissected gonads from inx-22(tm1661); glp-1(tn777ts) (n=7), glp-1(tn777ts); ced-3(n717) (n=15), and glp-1(tn777ts) ced-4(n1162) (n=15) hermaphrodites were analyzed and found to contain only meiotic germ cells. For a quantification of the cytological distribution of germ cell stages in glp-1(tn777ts); fog-2(oz40) females, see supplementary material Table S1.

• Supplemental Figure S3 -

  Fig. S3. Distal oil injection produces a glp-1 Looc phenocopy. Fluorescence (bottom) and merged (top) images of an adult wild-type hermaphrodite (12 hours post-L4), which was injected with oil in the distal proliferative zone and stained with DAPI in whole mount 24 hours later. The position of oil (arrow) is shown. Distal nuclei in the proliferative zone surrounding the oil exhibit abnormal nuclear morphologies. By contrast, transition zone and pachytene nuclei appear morphologically normal. Proximal oocytes are indicated by the white outline. The −1 oocyte exhibits a Looc phenotype.

• Supplemental Figure S4 -

  Fig. S4. Localization of NMY-2 and P-rMLC. (A) Localization of NMY-2::GFP (green) and cortical actin (red) in dissected hermaphrodite gonads. (B) P-rMLC (red) localization in dissected gonads from unmated fog-2(oz40) females, and acy-4(ok1806) and gsa-1(RNAi) hermaphrodites, and fog-2(oz40) females 15 minutes after mating. DNA is shown in blue. mlc-4(RNAi) injection depletes all the cortical staining and mlc-4(RNAi) in the wild type appears to reduce cytoplasmic streaming (data not shown). Quantification of flows after mlc-4(RNAi) is problematic because ovulation is blocked and gonad arms exhibit an endomitotic oocyte phenotype and become disorganized. (C) P-rMLC (green) and cortical actin (red) localization in dissected gonads from young unmated fog-2(oz40) females. (D) P-rMLC (red) and DNA (DAPI staining, blue) in acy-4(lf); fog-3(q443); tnEx37 females. RNAi was performed at 22°C. Scale bar: 10 µm.

• Supplemental Figure S5 -

  Fig. S5. Increased phosphorylation of rMLC after mel-11(RNAi). (A) Localization of P-rMLC (red) and DNA (blue) in dissected gonads after mel-11(RNAi) or control(RNAi) on rrf-1(pk1417) hermaphrodites. L3-stage larvae were treated with RNAi using the feeding method (at 22°C) and analyzed on day 2 of adulthood. Photomicrographs were taken within three regions: the distal third of the gonad (distal), the next third of the gonad (loop), and the proximal region. Images were acquired using 200 msecond exposures and quantified in arbitrary fluorescence units using AxioVision software (B). P-rMLC levels were compared with the control for each region: *P<0.0001; ^# P<0.025; ‡ P<0.01.

• Supplemental Tables S1 - Adobe PDF

• Supplemental Tables S2 - Adobe PDF

• Supplemental Tables S3 - Adobe PDF

• Movie 1 -

  Movie 1. Time-lapse video showing cytoplasmic streaming in a wild-type hermaphrodite.

• Movie 2 -

  Movie 2. Time-lapse video showing an increased cytoplasmic streaming rate in a glp-1(tn777ts) hermaphrodite.