XB-IMG-77855
Xenbase Image ID: 77855
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Fig. 1. An otx2-glucocorticoid receptor fusion activates hormone-dependent ectopic XCG expression in embryos and explants.
(A) Experimental scheme. Albino embryos were injected with otx2-GR RNA at the two cell stage. Injected embryos or blastula (stage 8 or 9) animal caps were incubated alone or with dexamethasone (dex) until embryos reached hatching (stage 32). Arrows indicate the stage treatment was initiated, triangles indicate the stage of harvest.
(B) Ectopic cement gland induction in whole embryos. Embryos were injected in one blastomere with 100 pg otx2-GR RNA mixed with 60 pg lacZ RNA. Embryos were incubated without (a) or with (b) dex beginning at early gastrula (stage 10.5) and until stage 32, when they were stained for lacZ (light blue) and for XCG expression by in situ hybridization (black). In addition to endogenous XCG (white open arrowhead), ectopic XCG was observed in the ventrolateral ectoderm (black arrowheads) and at lower frequencies in dorsal epidermis (white arrowheads). Lateral view, anterior to the center.
(C) Ectopic cement gland induction in animal caps. Embryos were injected in both blastomeres with 50 pg otx2-GR RNA. Animal caps dissected at stage 9 were left untreated or transferred to dex when control embryos reached the stages indicated. XCG expression was analyzed by in situ hybridization when control embryos reached stage 32. (a) untreated; (b) +dex st.11.5; (c) +dex st. 14; (d) +dex st. 20.
(D) Northern blot analysis of otx2-GR RNA levels in animal caps. Embryos were injected in both blastomeres with 50 pg MT-otx2-GR RNA. Animal caps were dissected at stage 8 and pools of ten caps were analyzed by northern blot for otx2 expression when control embryos reached stage 8.5 (lane 1), stage 10 (lane 2), stage 12 (lane 3), or stage 20 (lane 4). Ethidium bromide-stained 28S rRNA is a loading control.
(E) Western blot analysis of inducible protein levels in animal caps. Explants prepared as in D were harvested in groups of ten when sibling embryos reached stage 8.5 (lane 1), stage 10 (lane 2), stage 12 (lane 3), or stage 20 (lane 4). Two cap equivalents of protein were analyzed by western blot analysis using an anti-myc antibody. Ponceau S staining indicated that all lanes were loaded equally (data not shown). Image published in: Gammill LS and Sive H (1997) Copyright © 1997. Image reproduced with permission of the publisher and the copyright holder. This is an Open Access article distributed under the terms of the Creative Commons Attribution License.
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