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Fig. 6. Inhibition of XGrhl1 activity or expression blocks XK81A1 keratin expression in vivo. (A) Expression of {Delta}227XGrhl1 blocks endogenous XK81A1 expression specifically. Embryos were injected in one animal blastomere at the four-cell stage with XGrhl1 (2 ng) and/or {Delta}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 {Delta}227XGrhl1 expression. (B) {Delta}227XGrhl1-encoding transcripts (1 ng) do not block expression of other BMP4 signaling pathway components. The product of a factor chimera [{Delta}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 {Delta}227XGrhl1-expressing mutants in Fig. 5.





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