Fig. 6. Inhibition of XGrhl1 activity or expression blocks XK81A1 keratin expression in vivo. (A) Expression of 227XGrhl1 blocks endogenous XK81A1 expression specifically. Embryos were injected in one animal blastomere at the four-cell stage with XGrhl1 (2 ng) and/or 227XGrhl1 (1 ng)-encoding transcripts. In situ hybridization for XK81A1 expression (blue stain) was performed on stage 14 embryos. ß-Galactosidase, a lineage tracer, stained red. The broken white lines delineate areas of 227XGrhl1 expression. (B) 227XGrhl1-encoding transcripts (1 ng) do not block expression of other BMP4 signaling pathway components. The product of a factor chimera [227XGrhl1 sequences linked in frame with enhanced green fluorescent protein cDNA (EGFP)] was detected in nuclei of transfected cells (extreme right panel), consistent with appropriate nuclear localization. (C) Injection of a XGrhl1-targeted MO blocks endogenous XK81A1 keratin gene expression specifically. Embryos were injected into one animal blastomere at the four-cell stage with XGrhl1MO±M-XGr (a MO-resistant XGRhl1- expressing RNA transcript) (upper left). In situ hybridization for XK81A1 expression (blue stain) and a ß-galactosidase lineage tracer (red) was performed on stage 14 embryos. The XGrhl1-MO mediated block in XK81A1 gene expression is rescued partially by co-expression of M-XGr mRNA (lower left). Coincident blue and red staining is indicated (arrowheads). A control morpholino (CMO, upper right) or M-XGr alone (lower right) failed to affect normal development. (D) Injection of XGrhl1-targeted MO induces an epidermal defect in maturing tadpole specifically. Defects in head and trunk structures representative of those observed in embryos injected with XGrhl1-specific MO in one animal pole blastomere at the eight-cell stage are shown. Epidermal and pigment changes in head and trunk are observed in XGrhl1MO-injected embryos (when compared with CMO-injected embryos) that are identical to those seen with the 227XGrhl1-expressing mutants in Fig. 5.