Fig. S4

Effect of ephb4b knockdown on DFCs clustering and KV are unrelated to apoptosis or defective cell proliferation. (A-D) Effect of ephb4b knockdown on apoptosis of DFCs and KV cells. 10 ng cMO or ephb4b-MO2 was injected into the yolk of Tg (sox17: GFP) transgenic embryos at the 512-cell stage and harvested at the bud stage (A,B) and the 5-somite stage (C,D) for immunostaining using anti-GFP and anti-(active) Caspase 3 antibodies. The signal of active Caspase 3 was hardly detected in KV of either wild-type embryos (A,C) or ephb4b morphants (B,D). (E) Effect of ephb4b knockdown on heart jogging in wild-type or tp53-/- mutant embryos. 10 ng ephb4b-MO2 was injected into wild-type (WT) or p53-/- mutant embryos at the one-cell stage. The embryos were collected at 28 hpf for probing cmlc2 expression in the heart. The ratio of embryos for each category of heart jogging was calculated. (F-J) Effect of ephb4b knockdown on DFCs and KV cells proliferation. 10 ng cMO or ephb4b-MO2 was injected into the yolk of Tg (sox17: GFP) transgenic embryos at the 512-cell stage and harvested at the bud stage (F,G) and the 5-somite stage (H,I) for immunostaining using anti-GFP and anti-pH3 antibodies. The immunostained embryos were observed by confocal microscopy. The anterior (A)-posterior (P) axis was indicated. The proportion of pH3-positive KV cells was calculated (J). n, the number of observed embryos. ns, statistically non-significant at P > 0.05%. Scale bar: 30 µm.