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Fig. 5. otx2 regulation of target gene expression. (A) Experimental scheme. Wild-type embryos were injected with otx2/3-GR RNA at the two-cell stage. Blastula (stage 9) animal caps were isolated from injected embryos and incubated alone or in dexamethasone (dex) and/or cycloheximide (CHX) or retinoic acid (RA) for 5 hours or for 20 hours beginning at early/mid-gastrula (stage 11). Arrows indicate the stage treatment was initiated, triangles indicate the stage of harvest. (B) XCG, XAG and otx2 expression in animal caps. Embryos were injected in both blastomeres with 50 pg otx2/3-GR RNA. Animal caps were dissected at stage 9, and pools of ten explants were left untreated or were incubated in dex without or with CHX or RA for 5 hours (lanes 1-6) or 20 hours (lanes 7-10) beginning when control embryos reached stage 11. XCG, XAG and otx2 expression were analyzed by RT-PCR using EF-1a as a control. Since cement gland markers are expressed at very high levels in the mature cement gland compared to their initial expression levels, samples were assayed at high number of PCR cycles (lanes 1-6) or low number of PCR cycles (lanes 7-10) in order to remain in the linear range of amplification at the stage assayed. Early expression is not detectable at low cycle numbers. Comparable results were obtained with cycloheximide in eight independent experiments and by in situ hybridization (three independent experiments), and with RA in four independent experiments. Lane 1, untreated; lane 2, +dex; lane 3, +CHX; lane 4, +CHX+dex; lane 5, +RA; lane 6, +RA+dex; lane 7, untreated; lane 8, +dex; lane 9, +RA; lane 10, +RA+dex. (C) Western blot analysis of cycloheximide-treated explants. Embryos were injected in both blastomeres with 50 pg MT-otx2-GR RNA and animal caps dissected at stage 9. When control embryos reached late gastrula (stage 12.5), caps were treated without or with dex and/or CHX for 3 hours, a period of time encompassed by treatments described in (B), and were lysed in groups of ten for western blot analysis with an anti-myc antibody. Two cap equivalents of protein were analyzed per lane. Ponceau S staining indicated that all lanes were loaded equally (data not shown). Lane 1, untreated; lane 2, +CHX; lane 3, +dex; lane 4, +CHX+dex. 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. Larger Image Printer Friendly View

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