Rargb negatively regulates Bmp activity and localization in vivo. (A and B) Overexpression of Bmp in heat shock inducible hs:bmp2b embryos results in midline hearts (A) and bilateral livers (B), paralleling the phenotype of rargb morphants. (C) Treatment of rargb morphant embryos with the Bmp inhibitor dorsomorphin results in a reduction in the number of bilateral livers compared to DMSO-treated control rargb morphants. (D) Western blot for Bmp effectors phospho-Smads 1/5/8 in uninjected control (Un) and rargb MO-injected embryos (MO) at 18 hpf. Band density values normalized to Actin demonstrate that loss of rargb results in increased expression of p-Smads 1/5/8. Embryos treated with the Bmp inhibitor dorsomorphin (DM) show reduced p-Smad 1/5/8 expression compared to DMSO-treated controls. (E–H) Fluorescence microscopy of Bmp response element reporter fish BRE:eGFP. At 20 hpf, control embryos display greater Bmp activity on the left side (E), whereas rargb morphants display symmetrical Bmp activity (F). Altered Bmp localization at 20 hpf corresponds to heart laterality defects at 36 hpf in rargb morphants (H). White dotted lines denote the midline (E and F) or outline the heart tube (G and H). Scale bars: 100 µm.
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