|
Fig. 4 Roles of Xenopus hwa during embryonic body axis formation. (A) Expression of hwa in oocytes and embryos by WISH detection. Red arrowheads indicate the sperm entry site. St, stage; mpf, minutes post-fertilization. The red arrow shows the cortical rotation direction; the black arrow indicates asymmetrically located hwa transcripts. (B) Quantification of hwa transcripts in two dorsal and two ventral blastomeres at four-cell stage by qRT-PCR. Left: Illustration of cutting of embryos. Right: qRT-PCR result showing average levels from three technical repeats (±SEM) normalized to odc level. The dorsal marker wnt11b serves as an indicator. **P < 0.01, ***P < 0.001. (C) Quantification of hwa mRNA levels by qRT-PCR at the indicated stages; prog, progesterone. hwa expression level is averaged from technical repeats (±SEM) and normalized to odc level at the indicated stages. (D) Induction of secondary axis by hwa in Xenopus embryos. One ventral blastomere of four-cell stage embryos was injected with different doses of hwa-myc or 20 pg of GFP mRNA plus 200 pg of β-gal mRNA and observed at stage 32. Left: Classes of embryos. Right: Ratios of classified embryos. (E) Organizer and ventral marker expression patterns detected by WISH at stage 10 (vegetal view). One ventral blastomere of four-cell stage embryos was injected with 20 pg of hwa or 100 pg of GFP mRNA plus 200 pg of β-gal mRNA. The ratio of embryos with the representative pattern is indicated. (F) Maternal knockdown effect of hwa. Oocytes were injected with different doses of antisense oligodeoxynucleotides (AS1 or AS4) and the resulting embryos were observed and categorized at stage 24. All scale bars, 1 mm.