Fig. 3

Zebrafish microinjection experiments reveal functional conservation of NvChd, NvGrm and NvDpp. (A–E) Lateral views of live zebrafish embryos at 32 h postfertilization (hpf). (A) Uninjected wild-type embryo. (B) Embryos injected with 150 pg of NvDpp mRNA are ventralized, as apparent by loss of anterior-dorsal neural structures and notochord, and expansion of ventral tail fin tissue, blood islands and posterior somites. (C) Embryo injected with 75 pg of NvChd mRNA displays mild dorsalization (C1–C2), indicated by loss of ventral tail fin tissue (arrowheads) and truncation of the tail. (D) Injection of 7.5 pg of NvGrm mRNA leads to strong dorsalization (C4), morphologically apparent by curling up of the tail. (E) Similarly, injection of 150 pg NvGDF5-like mRNA results in severe dorsalization (C3–C4). (F–M) In situ hybridization of zebrafish embryos at midgastrulation stage (80% epiboly) with the ventral marker eve1, vegetal views, ventral leftwards. (F) Uninjected wild-type embryo. (G) Embryo injected with 150 pg of NvDpp mRNA, ventral eve1 expression is expanded (17/21 embryos). (H) Injection of 7.5 pg NvGrm mRNA abolishes eve1 expression (10/19 embryos with complete loss of eve1, 9/19 with strong reduction). (I) Coinjection of 150 pg NvDpp mRNA and 7.5 pg NvGrm mRNA restores eve1 expression to near wild type (18/20 embryos). (J–M) Embryos injected with a morpholino against zebrafish bmp2b. (J) Injection of bmp2b MO alone leads to complete loss of eve1 expression (15/15 embryos), (K) which can be restored by coinjection of 200 pg NvDpp mRNA (20/22 embryos with expanded eve1 expression compared to wild type). (L, M) Rescue of eve1 expression by NvDpp mRNA can be partially (L) or completely (M) suppressed by coinjection of 30 pg NvGrm mRNA (10/27 embryos with reduction below wild type, 12/27 with complete loss).