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Fig. 6. HoxD1 is an XMeis3 direct-target gene that interacts with XMeis3 to activate hindbrain marker expression. (A) One-cell stage embryos were injected in the animal hemisphere with 1.6 ng of inducible XMeis3-GR encoding RNAs. Fifty-four and seventy-two animal cap explants were respectively removed from uninjected and injected groups of blastula embryos (stage 8 – 9). Eighteen explants from each group were grown in cyclohexamide and/or dexamethasone (see Materials and methods) to stage 12.5 and total RNA was isolated. RT-PCR analysis was performed with the markers: Krox20 and HoxD1. EF1a served as a control for quantitating RNA levels in the different samples. For controls, RT-PCR and -RT-PCR was performed on total RNA isolated from normal embryos. (B) One-cell stage embryos were injected in the animal hemisphere with either 1.6 ng of XMeis3 or HoxD1 encoding RNAs. Eighteen animal cap explants were removed from uninjected and injected groups of blastula embryos (stages 8 – 9). Explants from each group were grown to stage 18 and total RNA was isolated. RT-PCR analysis was performed with the markers: Krox20 and HoxB3. EF1a served as a control for quantitating RNA levels in the different samples. For controls, RT-PCR and -RT-PCR was performed on total RNA isolated from normal embryos. (C) Two-cell albino embryos were injected unilaterally into the animal hemisphere of one blastomere with RNAs encoding XMeis3 or HoxD1 proteins. In situ hybridization to Krox20 was performed in neurula stage embryos. All embryos are injected on the left side, viewed dorsally, and are oriented anterior (top), posterior (bottom). Upper left panel: control embryo. Upper right panel: XMeis3 injected (0.8 ng), 70% of the embryos had expanded Krox20 expression (62/89). HoxD1 injected (0.8 ng), none of the embryos had expanded Krox20 expression (0/27). XMeis3 and HoxD1 co-injected (0.8 ng), 68% of the embryos had ectopic Krox20 expression (28/41). (D) Embryos were co-injected with the rpt3-luc or rpt3-CAT reporter constructs (see Materials and methods) along with RNAs encoding HoxD1, Xpbx1, or XMeis3 proteins. One representative experiment is shown. At early-mid gastrula stages, 10 embryos were lysed per injection group and luciferase activity was assayed. The bar graph describes relative luciferase activity in each sample, with the control embryos expressing only the rpt3-luc reporter taken as 1. A mutant version of the rpt3-luc reporter did not activate luc transcription (not shown) in injected embryos. |