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Fig. 4. Cleavage mutant forms of Xnr2 retain diminished signaling activity and therefore do not function as authentic dominant negative molecules. (A) Diagram of the various Xnr2 cleavage mutant constructs tested. The gray region of the bar indicates the Xnr2 proA and proB regions, while the white regions represent the mature Xnr2 ligand. The Activin pro domain is indicated in blue. (B) RT-PCR analysis of xnr2 cleavage mutant constructs. Embryos were injected in the animal pole at the one-cell stage with 10 pg of xnr2 or 2 ng of the cleavage mutant mRNAs. Wild-type xnr2 and both CM-xnr2 and DCM-xnr2 induce mesoderm in animal caps, as indicated by the presence of xbra transcripts. An equivalent dose of CM-der shows no mesoderm inducing activity. Both xnr2 cleavage mutant constructs fail to induce extreme dorsal fates (marked by gsc). (C-T) xnr2 cleavage mutant constructs induce ectopic mesoderm in whole embryos. Embryos were injected with the 10 pg xnr2 or 2 ng cleavage mutant mRNAs in the animal pole at the one-cell stage, allowed to develop to stage 11 and analyzed by in situ hybridization. Wild-type xnr2 and all three cleavage mutant constructs cause expansion of the mesodermal marker xbra, the dorsal mesodermal marker gsc and the ventral/lateral mesodermal marker xwnt8 (F-Q). By contrast, CM-der leads to an inhibition of xbra expression and has no obvious effect on gsc and xwnt8 (R-T), presumably because of limited diffusion of the animally injected mRNA). |
