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Fig. 2. The effects of mesoderm-inducing factors on early Hox expression. (A) Levels of Hoxd-1 in explanted animal cap sandwiches from stage 11.5 noninjected embryos, embryos injected with Activin and embryos injected with Activin and the dominant interfering construct Xbra-EnR. Lightcycler PCR was used to quantitatively measure the levels of Hoxd-1, which were normalised to ODC levels and are shown as a percentage of the endogenous levels in whole embryos (WE). (B, C) The growth factor Activin was injected into the blastocoel of stage 8 embryos. In situ hybridisations (lateral views, organiser to the right) are shown for Hoxd-1 at stage 11 in noninduced control (B) and Activin-injected (C) embryos. In induced embryos, the Hoxd-1 expression is expanded in the animal direction. Arrowheads point to the animal border of Hoxd-1 expression. (D–K) Xbra was ectopically expressed in the animal region. Hox expression was analysed by in situ hybridisation. Lateral views (organiser is to the right) of noninjected control embryos (ni) (D, F, H, J) and Xbra-injected embryos (E, G, I, K) stained for Hoxd-1 at stage 11 (D, E), Hoxb-4 at stage 11.5 (F, G), Hoxc-6 at stage 12 (H, I) and Hoxb-9 at stage 12.5 (J, K). Compared to corresponding controls, the expression of all analysed Hox genes in the Xbra-injected embryos is expanded in the animal direction. (L) The ability of Xbra to induce Hox genes in stage 11.5 explanted animal cap sandwiches (AC) was analysed by RT-PCR. All the Hox genes examined (Hoxd-1, Hoxb-4, Hoxc-6 and Hoxb-9) were induced by Xbra. The endogenous expression in whole embryos (WE) is also shown. |
