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Fig. S3
Efficiency of mRNA and morpholino injection at midblastulation stages. (A,B) Injection of 250 pg ephb4b 5′utr-mCherry fusion mRNA, which contained 5′ untranslated sequence including the ephb4b-MO2 binding site, into one-cell Tg(sox17:GFP) transgenic embryos led to global expression of mCherry including DFCs. When Tg(sox17:GFP) embryos were first injected with 250 pg ephb4b 5′utr-mCherry mRNA at the one-cell stage and then injected with 10 ng ephb4b-MO2 at the 512-cell stage, mCherry in GFP-positive DFCs was drastically reduced while it was retained in surrounding cells, suggesting that ephb4b-MO2 was restricted to DFCs. (C) Injection of 400 pg ephb4b-mCherry mRNA, which contains full-length ephb4b coding sequence, into 256-cell to 512-cell Tg(sox17:GFP) embryos led to mCherry expression specifically in DFCs. All of injected embryos were observed by confocal microscopy focusing on the DFC region at the 75% epiboly stage. The ratio of embryos with representative pattern was indicated. Scale bar: 30 µm.