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Files in this Data Supplement:
Fig. S1. The acy-4 locus. (A) Intron-exon structure of acy-4 showing the regions encoding the two catalytic domains and the two transmembrane clusters. The regions disrupted by ok1806 and tm2510 are shown. The acy-4 coding sequence was defined using RT-PCR (primer sequences used are available on request). Reaction products were cloned and sequenced. The WRM061bE10 fosmid rescues both acy-4(lf) alleles. (B) Predicted membrane topology of ACY-4. (C) ClustalW alignment of ACY-4 and human ADCY5 (accession number NP_899200) and ADCY6 (accession number NP_066193), which encode type V and type VI adenylate cyclases, respectively. The underlined regi
Fig. S2. Nonspecific phosphodiesterase inhibitors weakly suppress the acy-4(lf) meiotic maturation defect. A DIC photograph of an acy-4(ok1806) young adult hermaphrodite grown on NGM medium containing a final concentration of 30 mM caffeine. This treatment results in a brood size of ∼4-8. Arrows indicate embryos in the uterus. By contrast, acy-4(ok1806) hermaphrodites are sterile and meiotic maturation is not observed in young adult hermaphrodites (see Fig. 1F and Table S4 in the supplementary material). Similarly, fertility of acy-4(ok1806) adults was also observed on NGM medium containing 10 mM isobutylmethylxanthine. Likewise, treatment of sterile gsa-1(RNAi) adults on NGM medium containing 30 mM caffeine suppresses the sterility and produces brood sizes of ∼50.
Fig. S3. MSP receptor clustering in sheath cells in unmated females. MSP-FITC (green) binding to dissected gonads from unmated fog-2(oz40) females at 22°C (A) or at 4°C (B), which is known to block receptor-mediated endocytosis. Actin is shown in red. At 22°C, maximal MSP-FITC binding is seen in optical sections taken at the level of the sheath-oocyte interface. MSP-FITC binding is observed in ring-like structures at 4°C also, and thus does not depend on endocytosis. MSP-FITC binding is observed in ring-like structures in vab-1(dx31); acy-4(lf) hermaphrodites (C), suggesting that the VAB-1 MSP/EPH receptor is not required for their formation. The fact that sheath cells can directly bind MSP in the absence of oocytes was further confirmed by analyzing MSP binding to fem-3(q20gf) gonads that produce sperm only, and not oocytes (J.A.G. and D.G., unpublished). (D) MSP-FITC binding to gsa-1(RNAi) treated hermaphrodites. (E) MSP-FITC binding to unmated fog-2(oz40) female gonads (blue line) and acy-4(ok1806) hermaphrodite gonads (red line) is saturable and the saturation binding curves can be superimposed. Binding experiments were conducted at 22°C at various MSP-FITC concentrations and binding was quantified in arbitrary fluorescence units. At least five gonads were analyzed for each concentration.
Fig. S4. inx-14 is required for germline proliferation and gametogenesis. Germ cells expressing PGL-1::GFP (arrows) are reduced in number in inx-14(tm2864) homozygotes and after inx-14(RNAi). DIC (left) and GFP fluorescence (right) images of L4-stage larvae. inx-14(tm2864) homozygous progeny of a heterozygous parent (m+ z−) have ∼20 germ cells per gonad arm. inx-14 mutant germ cells apparently do not enter meiosis, and no sperm are observed. Microinjection of inx-14 dsRNA was also used and F1 animals were examined. inx-14(RNAi) animals (presumably m− z−) have approximately 0 to 3 germ cells. Therefore, maternal inx-14 may be required for germ cell generation or survival. inx-14(tm2864) males are also sterile. Scale bar: 10 µm.
Fig. S5. Genetic mosaic analysis of inx-14. A complex array rescuing inx-14(tm2864) was generated using an 8 kb PstI genomic fragment containing inx-14(+) genomic sequences, including 5.6 kb of presumptive promoter sequences. The coinjected marker was sur-5::gfp (pTG96). The array rescued the inx-14 sterility defect, however, brood sizes were low. Fertile genetic mosaics were sought using a fluorescence-dissecting microscope. The four mosaics shown resulted from multiple consecutive losses in the lineage. The points at which the losses occurred are indicated by a triangle (mosaic 1), a square (mosaic 2), a hexagon (mosaic 3) or a star (mosaic 4). DIC microscopy of the mosaics confirmed that neither gonad arm displayed an inx-14 mutant phenotype. Mosaic 4 was isolated after mutagenesis with ethylmethanesulphonate. The germline requirement for inx-14 is consistent with the INX-14 expression pattern as detected using antibodies (see Fig. 4C) and a rescuing GFP construct (T.A.S. and D.G., unpublished).
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