Diversin regulates heart formation and gastrulation movements in development

Abstract

Canonical and noncanonical Wnt signaling regulate crucial events in the development of vertebrates and invertebrates. In this work we show that vertebrate Diversin, a potential orthologue of Drosophila Diego, controls fusion of heart precursors and gastrulation movements in zebrafish embryogenesis. These events are regulated by noncanonical Wnt signaling, which is independent of beta-catenin. We found that Diversin directly interacts with Dishevelled and that this interaction is necessary and sufficient to mediate signals of the noncanonical Wnt pathway to downstream effectors like Rho family GTPases and Jun N-terminal kinase. The ankyrin repeats of Diversin are required for the interaction with Dishevelled, for the activation of noncanonical Wnt signaling, and for the biological responses. The mutation K446M in the DEP domain of vertebrate Dishevelled, which mimics a classical Drosophila loss of function mutation, prevents functional interaction with Diversin's ankyrin repeats. Diversin also affects planar cell polarity in Drosophila, which is controlled by the noncanonical Wnt signaling pathway. Our data thus demonstrate that Diversin and Dishevelled function together in a mutually dependent fashion in zebrafish gastrulation and organ formation.

Fig. 1.

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.

Fig. 2.

Diversin affects PCP signaling in Drosophila. All panels show tangential sections of adult eyes of indicated genotypes, with corresponding schemes below. Arrows indicate dorsal chiral forms in black, ventral chiral forms in red, and symmetric ommatidia in green (R3/R3 type) and blue (R4/R4 type). Compared with a wild-type eye (A), overexpression of Dgo (sev→Dgo in B and sev→Diversin in C) at the time of PCP signaling leads to typical PCP phenotypes, including rotation and chirality defects. (D) Section of a dgo³⁸⁰ mutant eye. In contrast to Dgo (E), Diversin (F) cannot rescue the dgo-null mutation. Both transgenes were expressed under the control of the ubiquitous tubulin promoter.

Fig. 3.

The ankyrin repeat domain of Diversin binds to the DEP domain of Dishevelled, and both domains are required and sufficient to activate noncanonical Wnt/JNK signaling in a mutually dependent fashion. (A) Domain structure of Diversin construct and mutants used. (B) Interaction of Diversin with Dishevelled in mammalian cells (HEK293). Shown are Western blots of anti-Dishevelled or anti-Diversin immunoprecipitates with the indicated antibodies. (C) The ankyrin repeats of Diversin are functionally important for noncanonical Wnt/JNK signaling. HEK293 cells were cotransfected with the indicated Diversin constructs (0.25 μg) and either Dishevelled (0.25 μg, black bars) or empty vector (white bars). JNK-dependent transcription was measured by luciferase reporter activity. Error bars indicate standard deviations. A MAPK kinase kinase (0.25 μg) construct that activates JNK activity served as control. (D) Overview of domain structure of mouse Dishevelled-2 and of deletion constructs and mutants of Dishevelled-2. (E and F) The DEP domain of Dishevelled binds to Diversin. Western blot analysis of immunoprecipitated Diversin and Dishevelled constructs is shown. Equal expression of transfected cDNAs (3–12 μg) in HEK293 cells was verified by immunoblotting with the indicated antibodies. (G) The DEP domain of Dishevelled is essential for Diversin-dependent activation of JNK. Experiments were performed as described in C. Indicated cDNAs (0.25 μg) were cotransfected with empty vector (white bars) or Diversin (0.25 μg, black bars).