Diversin is essential for heart formation and gastrulation movements in zebrafish embryogenesis. (A) Diversin domain structure and deletion mutants used for mRNA injections. Numbers indicate amino acid positions of mouse Diversin. (B) Zebrafish embryo (at 48 h after fertilization) that was injected with 75 pg of Div-ΔANK mRNA at the one- to two-cell stage shows two separate hearts (marked by arrows and dashed lines). Live beating hearts in this embryo are shown in Movie 1. (C–E) In situ hybridization of Nkx2.5 of zebrafish embryos at 20 h after fertilization indicates single or double heart primordia in the injected embryos (arrows, dorsal view). (C) Heart primordia of uninjected wild-type embryos are paired at the dorsal midline. (D and E) Embryos injected with 75 pg of Div-ΔANK or Dvl- ΔDEP mRNA show two heart primordia located laterally of the dorsal midline. (F) Diversin controls heart formation via the RhoA signaling pathway. Shown is quantification of cardia bifida phenotypes of zebrafish embryos that were injected with the indicated mRNAs. Amounts of injected mRNAs: Div-ΔANK and Dvl-ΔDEP, 75 pg in single injections and 37.5 pg in double injections; RhoA(N19), 30 pg; RhoA(V14), 2 pg. (G) The ankyrin repeat domain of Diversin is crucial for regulation of gastrulation movements in zebrafish embryos. In situ hybridizations of krox20 and myoD of flat-mounted zebrafish embryos are shown (12- to 15-somite stage, dorsal view). One- to two-cell-stage embryos were injected with antisense MO against zebrafish Diversin (Div MO, 1.5 ng) or against zebrafish Wnt11 and Wnt5a (Wnt11/5a MO, 1 + 2 ng), or were coinjected with MO and 50 pg of the indicated mouse Diversin mRNAs. (H and I) Quantification of experiments in G. Embryos were classified as with CE phenotype (not rescued) when anterior/posterior body axis length, shape of the notochord, and compression of somites were similar to MO-injected embryos (in G compare embryos 2 and 5 with embryos 1, 3, 4, and 6). (J) Diversin lacking the ankyrin repeat domain, Diversin-ΔANK, synergistically enhances the induction of CE defects induced by Wnt11 and Wnt5a MO. Zebrafish embryos were injected with Wnt11 and Wnt5a MO (0.5 + 1 ng) or Diversin-ΔANK mRNA (50 pg) or coinjected with both (experiments were quantified as described for G and H). n, number of embryos.
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