Fig. 5. Mutation of a conserved arginine residue in hNODAL renders the protein more active and can induce left-right asymmetry defects. (A,B) Cyc^R314Q and hNODAL^R183Q are more active than Cyc and hNODAL. Zebrafish embryos at the one-cell stage were injected with 5 or 25 pg of RNA encoding either Cyc, Cyc^R314Q, hNODAL or hNODAL^R183Q. Expression of gsc (A) or ntl (B) was examined at 50% epiboly. The Arg-Gln mutations render Cyc^R314Q and hNODAL^R183Q more active than the wild-type proteins. Animal pole views shown. Scale bar: 100 µm. (C) Overexpression of Cyc^R314Q and hNODAL^R183Q in Xenopus embryos causes perturbations in left-sided expression of Xpitx2. Xenopus embryos at the four-cell stage were injected with 25 pg of DNA expression constructs encoding either Cyc, Cyc^R314Q, hNODAL or hNODAL^R183Q in the dorsal-left or dorsal-right blastomeres and cultured until stage 28 and processed for in situ hybridization to detect Xpitx2 expression. Embryos were scored as left, right, bilateral or no Xpitx2 expression. Ventral views of stage 28 embryos with Xpitx2 expression in the cardiac primordia are shown. Injections of hNODAL^R183Q or Cyc^R314Q can induce right-sided, bilateral or no Xpitx2 expression at a higher frequency than observed after injections of hNODAL or Cyc. (D) Gut looping is also perturbed by injection of hNODAL^R183Q. Xenopus embryos at the four-cell stage were injected with 25 pg of DNA encoding either hNODAL or hNODAL^R183Q in the dorsal-left or dorsal-right blastomeres and cultured until stage 45 and scored for the direction of intestinal coiling. Injected embryos manifested either normal, reversed or abnormal looping. In some embryos, heterotaxia of intestinal coiling and cardiac laterality was observed. Right-sided injections of hNODAL^R183Q increase the frequency of reversed intestinal coiling in comparison with hNODAL.