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Fig. 2. Direct targets of Sox17β. (A) A schematic of the GR:Sox17β fusion protein consisting of the hormone-binding domain of the human glucocorticoid receptor fused to Sox17β. (B) Isolated blastula animal cap explants either injected with mRNA encoding GR:Sox17β (150 pg) or uninjected were each cultured in three conditions: 1×MBS, 1×MBS + 10–6 dexamethasone (DEX) or in 1×MBS with 10–6 dexamethasone + 10 μg/ml cycloheximide (CHX). Dexamethasone activates the GR:Sox17β fusion protein and cycloheximide blocks translation. Control animal caps were treated with 5 ng/ml human activin A either with or without 10 μg/ml cycloheximide. At gastrula (stage 11) the explants were assayed by RT-PCR. GR:Sox17β induced Hnf1β, Foxa1, Foxa2 and Sox17α transcription when translation was blocked and are therefore direct Sox17 targets. A repeat of this experiment assayed by real-time RT-PCR is presented in Figs S1-S3 at http://dev.biologists.org/supplemental. (C) Whole-mount in situ hybridization to bisected gastrula (stage 11) embryos with the indicated probes confirms that Sox17 target genes are expressed in the endoderm. Dorsal/anterior towards the left. |
