Results: 4

1.
Fig 3

Fig 3. P granule components are segregated equally to germline and somatic blastomeres in pptr-1 mutants. From: Cytoplasmic partitioning of P granule components is not required to specify the germline in C. elegans.

A) Fixed wild-type and pptr-1 embryos stained with DAPI (blue) and OIC1D4 (red). Images are maximum projections of confocal Z-stacks (spanning entire embryo), except for P4 and Z2/Z3 images, which show single planes.
B) Wild-type and pptr-1 embryos stained for nos-2 and cey-2 RNAs (black) by in situ hybridization. In pptr-1 embryos, nos-2 and cey-2 RNAs are present at equal levels in P3 and C, indicative of symmetric segregation during the P2 division. In pptr-1 embryos, as in wild-type, nos-2 and cey-2 RNAs levels are lower in all other blastomeres, indicative of rapid degradation of these RNAs in somatic lineages.

Christopher M. Gallo, et al. Science. ;330(6011):1685-1689.
2.
Fig 1

Fig 1. Segregation of the P granule component PGL-1 in C. elegans embryos. From: Cytoplasmic partitioning of P granule components is not required to specify the germline in C. elegans.

A) Abbreviated embryonic lineage showing the divisions that give rise to somatic (AB, EMS, C and D) and germline (P1-P4) blastomeres. All cells are shown in interphase except for the zygote, which is shown in mitosis. Circles represent PGL-1 molecules: open circles represent PGL-1 diffuse in cytoplasm and closed circles represent PGL-1 assembled into granules visible by microscopy. Pink is MEX-5, which promotes granule disassembly; localized granule assembly ensures that the majority of PGL-1 segregates with the germline (Fig. 2). B) In pptr-1 mutants, PGL-1 granules disassemble at each mitosis and equal numbers of dispersed PGL-1 molecules are segregated to all cells (Fig. 2). During interphase, PGL-1 granules reform in all cells, except in MEX-5-positive somatic blastomeres (pink). Note that somatic PGL-1 granules are not equivalent to true P granules, as they do not contain P granule-associated mRNAs, which are degraded in somatic lineages (Fig. 3).

Christopher M. Gallo, et al. Science. ;330(6011):1685-1689.
3.
Fig 4

Fig 4. pptr-1 mutants specify a germline, but a minority are sterile at high temperatures. From: Cytoplasmic partitioning of P granule components is not required to specify the germline in C. elegans.

A) Embryos expressing GFP::PIE-1 or stained with NOS-1 and PGL-1 antibodies. Patterns are identical in wild-type and pptr-1, except that pptr-1 embryos lack GFP::PIE-1 foci seen in wild-type (arrows), and have lower PGL-1 levels.
B) Adult wild-type and pptr-1 hermaphrodites expressing GFP::PGL-1. Gonads are outlined. Most pptr-1 hermaphrodites develop a full gonad with gametes (top panel), but at high temperatures a minority (20%) are sterile, with no gametes (lower panel). Fertile pptr-1 gonads are smaller than wild-type and yield a reduced number of progeny; unlike the P granule defect, however, the brood size defect is partially rescued by zygotic pptr-1 (Sup. Fig. 7).
C) Percentage of sterile hermaphrodites and total numbers scored. Wild-type have 2 maternal and 2 zygotic pptr-1 copies (M2Z2). Mutant pptr-1 hermaphrodites (M0Z0) were crossed with wild-type males to generate M0Z1, which were allowed to self-fertilize to generate M1Z0, M1Z1 and M1Z2. Only M0Z0 and M0Z1 hermaphrodites show significant sterility, demonstrating maternal requirement. pptr-1 is also required maternally for P granule partitioning (Sup. Fig. 3).

Christopher M. Gallo, et al. Science. ;330(6011):1685-1689.
4.
Fig. 2

Fig. 2. P granule dynamics require par-1, mex-5/6, and pptr-1. From: Cytoplasmic partitioning of P granule components is not required to specify the germline in C. elegans.

A-E) Time-lapse images of zygotes expressing GFP::PGL-1. Image are maximum projections of confocal Z-stacks spanning 8┬Ám (~half of embryo depth). P granule numbers are shown in Sup. Fig. 1. MEX-5 and MEX-6 are uniformly distributed in par-1 embryos and PAR-1 localizes to a reduced-size posterior domain in mex-5;mex-6 zygotes (9, 10).
F) GFP::PGL-1 levels over time in wild-type and pptr-1(tm3103) zygotes. Error bars are standard deviation of mean values from 3 zygotes. In wild-type embryos, GFP::PGL-1 fluorescence does not decrease in the anterior during interphase, even though the number of visible granules decreases (Fig. 2A), consistent with granule disassembly. During mitosis, GFP::PGL-1 fluorescence and P granule size increase in the posterior (Sup. Fig. 1), consistent with granule assembly.
G) Fold-enrichment of GFP::PGL-1 and GFP::GLH-1 in each P blastomere over its somatic sister. Enrichment becomes most pronounced with each division in wild-type. No enrichment is observed at any division in pptr-1 mutants. Error bars are standard deviation from values obtained from 3 focal planes in 3 embryos.

Christopher M. Gallo, et al. Science. ;330(6011):1685-1689.

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