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Fig. 6. Inhibition of XGrhl1 activity or expression blocks XK81A1 keratin expression in vivo. (A) Expression of δ227XGrhl1 blocks endogenous XK81A1 expression specifically. Embryos were injected in one animal blastomere at the four-cell stage with XGrhl1 (2 ng) and/or δ227XGrhl1 (1 ng)-encoding transcripts. In situ hybridization for XK81A1 expression (blue stain) was performed on stage 14 embryos. β-Galactosidase, a lineage tracer, stained red. The broken white lines delineate areas of δ227XGrhl1 expression. (B) δ227XGrhl1-encoding transcripts (1 ng) do not block expression of other BMP4 signaling pathway components. The product of a factor chimera [δ227XGrhl1 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 β-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 theδ 227XGrhl1-expressing mutants in Fig. 5. |
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Fig. 2. Expression of XGrhl1 is restricted to tissues with an epidermal fate. Whole-mount in situ hybridization analysis of XGrhl1 expression in staged embryos. Maternal XGrhl1 transcripts are detected in the animal pole of early cleavage (A), blastula (B-D) and gastrula (E-H) stage embryos. Embryos are in a lateral orientation except for F (dorsal) and G (ventral). Zygotic expression is observed in presumptive epidermis through neurulation (I-L; dorsal orientation), tailbud and swimming tadpole stages (Q and R respectively; lateral orientation, anterior towards the left). Transverse embryonic section at stage 15 (M) demonstrates XGrhl1 expression (blue stain) in the presumptive epidermis, this stain being absent from the neural plate (arrowhead). By stage 21 (P), the neural plate is closed and covered by epidermis. Epidermal keratin (XK81A1) and zygotic XGrhl1 have a similar pattern of expression (N,O; anterior and dorsal orientation, respectively). |
