aida Induces Ventralization and Inhibits axin-Induced Dorsalization in Zebrafish Embryos (A) Effect of aida overexpression or knockdown on expression of the dorsal and ventral markers. Embryos were injected with 300 pg of aida mRNA or 10 ng of aida-MO and examined for marker expression at the shield stage by in situ hybridization. Ectopic expression of aida repressed expression of gsc and chd, but expanded the expression of bmp2, eve1, and gata2. Knockdown of aida by MO injection caused reversed effects, which could be attenuated by coinjection with 100 pg of aida mRNA. (B) Statistical data for (A). (C) aida-induced ventralization of various degrees at 24 hpf. (Ca) Uninjected embryos. (Cb–Cd) Embryos were injected with 300 pg of aida mRNA. Of the injected embryos, 32% showed an increased blood island (Cb, indicated by an arrow), short tail, and closer eyes (Cb, indicated by arrowhead); 26% showed an increased blood island (Cc, indicated by arrowhead), reduced head, and eye fusion; and 4% showed complete loss of head and notochord (Cd). (D) aida-MO causes dorsalization in embryos at 24 hpf. Embryos were injected with 10 ng of aida-MO. Arrowhead marks a reduced blood island. (E) Depletion of aida protein by antibody injection leads to formation of dorsalized embryos. One-cell embryos were similarly injected with affinity-purified anti-Aida polyclonal antibody or with the antibody preincubated with recombinant aida protein, and in situ analysis of the expression of chd and gsc in 30% epiboly embryos, and eve1 in shield-stage embryos, was performed. (F) One-cell embryos were injected as in (D). Photos were taken at the six-somite stage. (G) Statistical data for aida inhibition of axin-induced dorsalization. Injection with 300 pg of aida mRNA, but not with 300 pg of aidaΔAxin mRNA, decreased the percentage of 24 hpf embryos that were dorsalized by injection of wild-type axin. (H) Ratios of embryos with normal, expanded, or reduced expression of various markers indicated, after injection of axin mRNA, coinjection of axin with aida, or aidaΔaxin mRNA. (I) aida-MO increases the ratio of dorsalized embryos induced by axin. axin mRNA (300 pg) and 10 ng of aida-MO were injected either alone or together. The ratios of different embryos were calculated based on morphological observations of embryos at 24 hpf. (J) Ratios of different embryos determined by in situ analysis of marker genes at the shield stage after injection of axin mRNA (300 pg) or coinjection of axin with aida-MO (10 ng). (K) Statistical data for the effect of aida mRNA or aida-MO on axinΔaida-induced dorsalization. Injection with 300 pg of aida mRNA or 10 ng of aida-MO had no significant effect on the percentage of 24 hpf embryos that were dorsalized by injection of axinΔaida. (L) Ratios of embryos with normal, expanded, or reduced expression of various markers indicated, after injection of axinΔaida mRNA, coinjection of axinΔaida with aida mRNA (300 pg), or coinjection of axinΔaida with aida-MO (10 ng).