Figure 2. XMeis3 gain-of-function.

Embryos were injected into the animal hemisphere at the one-cell stage with 2 ng synthetic mRNA containing the full-length coding region of XMeis3, and analysed by whole-mount in situ hybridisation. In each panel, control embryos are shown on top, the XMeis3 injected embryos are shown on the bottom. Each letter indicates at least a pair of images: one embryo injected with XMeis3 mRNA (experimental, labeled XMeis3), one not (control, unlabelled). The label above on each image indicates the gene being assayed; the label below, if present, indicates XMeis3 injection (or no injection, if not present). For D and E, there are only images of intact embryos processed for whole mount in situ hybridization. For A, B, and C, two whole mounts are shown on the left hand side, and sections of these embryos are shown on the right hand side of each panel. Each of these letters thus represents four images. The plane of sectioning is depicted by the dotted line in the insets of A, B, and C. (A) Expression of Hoxd1, whole mounts are shown in dorsal view, with anterior to the top, at stage 10.5. Lateral expression of Hoxd1 in injected embryos is stronger and in a broader domain, the gap in expression on the dorsal mesoderm is closed and a streak of expression in dorsal mesoderm is observed. (B) Expression of Hoxb4, whole mounts are shown in lateral view, with dorsal to the left, at stage 11. Lateral expression of Hoxb4 is not affected by injection of XMeis3, the black arrow points to a patch of ectopic expression in ectoderm. This is joined to the mesodermal expression domain by a very faint streak of expression. (C) Expression of Hoxc6, whole mounts are shown in dorsal view, with anterior to the top, at stage 10.5. Injected embryos show extensive early ectopic expression of Hoxc6 in dorsal mesoderm, prior to initiation of endogenous expression of Hoxc6. Please note that this early induced expression of the Hox genes is clearly mesodermal (internal to Brachy's cleft) and not ectodermal (surface expression) (D) Expression of Xbra, embryos at stage 10.5 are shown in vegetal view with dorsal to the top. No change can be observed in the expression of the mesodermal marker Xbra as a result of injection of XMeis3. (E) Expression of Xcad3, embryos at stage 17 are shown in dorsal view with anterior to the top. The anterior expression boundary of the posterior marker Xcad3 is shifted to a more anterior position following injection of XMeis3. Spemann's organizer is indicated by the crescent stripe, bottom centre, in the upper left panels of Figs. 2A and 2C.