Fig. 3. XRnf12 suppresses axis duplication elicited by Xlim-1/Ldb1 and antagonizes head organizer activity in a RING-dependent manner. (A) XRnf12 blocks secondary axis formation elicited by Xlim-1/Ldb1, whereas the RING mutant XRnf12(HC>AA) does not. Embryos injected with the mRNAs indicated were scored for axis development at the tailbud stage and categorized as secondary axis (dark blue bars), normal (white bars), or others (hatched bars). ß-globin serves as a negative control. n, total number of injected embryos; expt, number of independent experiments. Amounts of mRNAs injected (ng/embryo): Xlim-1, 0.25; Ldb1, 0.5; XRnf12 constructs, 0.25; ß-globin (coinjected), 0.25; ß-globin (alone), 1.0. (B-D) Overexpression of XRnf12 in the dorsal marginal zone leads to head defects. While XRnf12(HC>AA) has little or no effect (B), XRnf12 overexpression in the dorsal region results in reduced head structures (C). ß-globin has no effect (D). Amounts of mRNAs injected were 2.0 or 4.0 ng/embryo.