Fig. 2

a Effects of smad1/5/9 triple knockdown in Tg(piwil1:mCherry-CAAX-nanos3 3′UTR) embryos. Injected doses were 0.5 ng smad1 MO, 0.5 ng smad5 MO, and 3 ng smad9 MO. Left top, schematic of the knockdown strategy; left bottom, representative images showing embryonic morphologies and PGC numbers at 2 dpf, with red and green arrows indicating yolk stalk regression and ventral tail fin reduction, respectively; right, box plot showing PGC numbers per embryo. The median is indicated by the center line. The bottom, top edges, and whiskers represent the min and max values and 1.5 times the IQR, respectively. b Effect of smad1 knockdown alone. Left, embryonic morphology and PGC location of representative 1 dpf embryos injected with 0.5 ng control or smad1 MO; right, bar graph showing the percentage of mislocated PGCs. c Impact of smad1 knockdown on ddx4 expression in 2 dpf embryos, examined by ISH. Each embryo was injected with 0.5 ng MO at the 1-cell stage. d Impact of smad1 knockdown on PGC numbers in Tg(piwil1:mCherry-CAAX-nanos3 3′UTR) embryos at 2 dpf. Left, representative images with PGCs in red; right, box plot showing PGC counts per embryo. The median is indicated by the center line. The bottom, top edges, and whiskers represent the min and max values and 1.5 times the IQR, respectively. Each embryo was injected with 0.5 ng MO. e Effect of smad1 knockdown on pSmad1/5/9 (magenta) immunostaining. Tg(kop:gfp-CAAX-nanos3 3′UTR) embryos were injected with 0.5 ng MO at the 1-cell stage, and collected at 75% epiboly stage for measurement. Left, representative immunostaining images with PGCs labeled by GFP (green); right, quantification of nuclear pSmad1/5/9 intensities normalized to cytoplasmic signals, with each dot representing one PGC. Error bars represented SD with the mean indicated by the center line. f Effect of smad9 knockdown on PGC numbers in 2 dpf Tg(piwil1:mCherry-CAAX-nanos3 3′UTR) embryos injected with 16 ng MO at the 1-cell stage. Left, representative images with PGCs (mCherry, red); right, box plot showing PGC counts per embryo. The median is indicated by the center line. The bottom, top edges, and whiskers represent the min and max values and 1.5 times the IQR, respectively. Statistical significance was determined using two-sided Student’s t-test unless otherwise indicated (a, d–f). Two-sided Fisher’s exact test was used in (b). Nc, number of observed PGCs. Ne, number of observed embryos. Source data are provided as a Source Data file.