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Fig. 4.
Overexpression of five out of six selected vegetally enriched mRNAs reduces PGC number. One-cell embryos were injected in the vegetal region with GFP (control) or the indicated transcripts (0.5 ng). Tailbud embryos (stage 32-35) were analyzed for xpat expression by WISH, and representative images are shown (A-G). The number of PGCs per embryo was quantified (H). GFP, n=16; spire1, n=39; e2f1, n=64; otx1, n=27; parn, n=35; rras2, n=48; wwtr1, n=35. *P<0.05, compared with GFP control. Analysis based on at least two independent experiments and shown as a box and whisker plot. |
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Fig. 5.
MO-mediated knockdown of a subset of vegetally enriched mRNAs increases PGC number. One-cell embryos were injected in the vegetal region with MOs (15 ng) targeting otx1 or wwtr1. Tailbud embryos (stage 32-35) were analyzed for xpat. Representative images are shown (A-C). The number of PGCs per embryo was quantified (D). Uninjected control (ctrl), n=32; otx1-MO, n=31; wwtr1-MO, n=25. *P<0.05 compared with control. Analysis based on at least two independent experiments. |
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Fig. 6.
Altering sox7 expression reduces PGC number. One-cell embryos were injected in the vegetal region with sox7dCEnR RNA (200 pg) and Xtsox7 RNA (200 pg), alone and in combination. Tailbud embryos were analyzed for xpat expression by WISH. Representative images are shown (A-D). The number of PGCs per embryo was quantified (E). Uninjected control (ctrl), n=28; sox7dCEnR, n=24; Xtsox7, n=27; sox7dCEnR and Xtsox7, n=28. *P<0.05 compared with uninjected control. #P<0.05 compared with sox7dCEnR. Analysis based on at least two independent experiments. |
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Fig. 7.
efnb1 is required for normal PGC number and migration. One-cell embryos were injected in the vegetal region with (A) efnb1-FL alone (200 pg), or efnb1-FL (200 pg) and efnb1-MO (16 ng); (B) scrambled-MO (16 ng), efnb1-MO alone (16 ng), or efnb1-MO (16 ng) and efnb1-FL-rescue (200 pg). Tailbud embryos were analyzed for xpat expression by WISH. Representative images are shown on the left. (A) The number of PGCs per embryo was quantified. (B) Percentage of embryos with mislocalized PGCs was calculated. Black lines indicate the boundaries of normal PGC location, between somites 5 and 11. Uninjected control (Ctrl), n=28; efnb1-FL, n=28; efnb1-FL and efnb1-MO, n=28; scrambled-MO, n=25; efnb1-MO, n=27; efnb1-MO and efnb1-FL-rescue, n=28. *P<0.05 compared with uninjected control. #P<0.05 compared with efnb1-FL (A) or efnb1-MO (B). Analysis based on at least two independent experiments. |
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Fig. 8.
Inhibition of p300 reduces PGC number. (A) Schematic of GeneGo-predicted p300 regulatory interconnections from our data set. (B-G) One-cell embryos were incubated with ≤0.04% DMSO (ctrl, n=31) or increasing concentrations of the p300 inhibitor C646: 0.2 μM, n=38; 0.5 μM, n=34; 1 μM, n=34; 2 μM, n=34. Tailbud embryos were analyzed for xpat expression by WISH. The average number of PGCs per embryo was quantified (B). *P<0.05 compared with uninjected control. |
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Figure S3. Morpholino-mediated sox7 inhibition reduces PGC number in tailbud embryos.
A) Schematic of the sox7 morpholino target region (top). Wheat germ extract was incubated with
sox7-FL (500ng) in the presence of increasing concentrations of sox7-MO and subject to anti-flag
western blot analysis. Quantification of respective flag expression is shown (bottom). B-D) Onecell
embryos were injected in the vegetal region with sox7-MO (16ng). Tailbud embryos were
analyzed for xpat expression by WISH. The number of PGCs per embryo was quantified (B).
Representative images are shown (C-D). Uninjected control (ctrl) n=36, sox7-MO n=43. *
statistically significant compared to ctrl (p<0.05). Analysis based on at least two independent
experiments. |
