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Figure 1. Expression of XMeis3, Hoxd1, Hoxb4, and Hoxc6 during gastrulation.
Embryos were analysed by whole-mount in situ hybridisation for expression of XMeis3 (A and B), Hoxd1 (C and D), Hoxb4 (E and F), and Hoxc6 (G and H). Whole mounts are shown on the left side of each panel, sections of these embryos are shown on the right side of each panel, in the inset, on the bottom right corner of every panel, the dotted line indicates the plane of sectioning. Spemann's organiser is clearly visible in Figs 1A,C,E, as the gap in the Hox or Meis expression domain, facing up in the left hand panels . Embryos shown are at stage 11, vegetal views with dorsal up (A, C, E, and G) and at stage 13, dorsal views with anterior up (B, D, F, and H). XMeis3 expression overlaps with dorsolateral expression of Hoxd1, Hoxb4, and Hoxc6 in mesoderm at stage 11 (A, C, E, and G). XMeis3 expression in ectoderm at stage 13 overlaps with expression of Hoxd1 but not with expression of Hoxb4 and Hoxc6 (B, D, F, and H). At stage 11, Hox and Meis expression is limited by a sharp boundary, running parallel to the outside of the embryo. This boundary is Brachy's cleft, the boundary between involuted mesoderm and external ectoderm Brachy's cleft runs from the blastopore to the upper limit of the involuted mesoderm (and is actually visible as a cleft in the upper part of the right panel of Fig 1C). All early Hox expression is known to be inside this cleft at this stage (mesodermal, not ectodermal) and thus marks the position of the cleft. The early XMeis3 expression shows the same pattern. It is mesodermal. At a later stage (st.13, Fig 1B), [40] XMeis 3 expression is also outside Brachy's cleft (ectodermal).
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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.
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Figure 3. Effects of XMeis3 MO loss-of-function on embryonic development and the rescue of MOXMeis3.
Embryos at the one-cell stage were injected into the animal hemisphere with MOXMeis3 in amounts of 12 ng (B), 24 ng (C), and 36 ng (D), and allowed to develop until the control embryos (A) reached tadpole stages. This treatment disturbs development of the embryonic axis. At the highest concentration, the embryo is blocked during gastrulation (fig. 3D) and then disintegrates to a mass of dissociated cells contained within the vitelline membrane (not shown). The specificity of MOXMeis3 is shown by the rescue with XMeis3 synthetic mRNA. Embryos were injected with 32 ng of MOXMeis3 and 125 pg synthetic mRNA for XMeis3 and allowed to develop until the control embryos reached the tad pole stage (E), In the majority of the embryos a large part of the axis was rescued (F), in a small number of embryos the phenotype could even be reversed, not only is the axis fully rescued but the embryo shown in (G) even possesses additional trunk structures as was revealed by the presence of somites in the axis outgrowth (not shown). The most extreme MO treatment thus produced a gastrulation block. Other treated embryos were allowed to develop to comparable stages (405) as shown by development of stage specific structures, for example the cement gland (seen best in Figs 3A, B, C, F G as the black spot at the lower front end of each embryo. Front ends are left in 3A, B, E, F, G. Various directions in 3C.
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Figure 4. XMeis3 loss-of-function.
Embryos were injected at the one-cell stage with 16 ng of the MOXMeis3, and analysed by whole mount in situ hybridisation at stage 10.5/11, shown on the left side of each panel, and at stage 12, shown at the right side of each panel. Injected embryos are shown at the bottom of each panel, untreated embryos are shown on top. Shown are vegetal views with dorsal to the top. Expression of Hoxd1 (A), Hoxb4 (B), and Hoxc6 (C) is downregulated in mesoderm of injected embryos at early gastrula stages. A reduction in neurectodermal expression of the three Hox genes studied, is also observed in injected embryos at stage 12.
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Figure 5. Synergistic effect between Hoxd1 and XMeis3 in ectopic expression.
Embryos at the one-cell stage were injected into the animal hemisphere with either 100 pg Hoxd1 mRNA, 100 pg Xmeis3 mRNA, or 50 pg of both mRNA's. A single injection of 100 pg of either factor is not sufficient to induce a phenotypic effect. The combination of half the amount of Hoxd1 and XMeis3, results in posteriorisation, shown by a clear reduction of eye formation, and an anterior shift of the eye.
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Figure 6. Synergistic effects in loss-of-function of Hoxd1 and XMeis3.
(A) Embryos were injected with 362 ng of MOHoxd1 into the lateral marginal zone on the left side of embryos, rendering the un-injected side an internal control. Embryos were allowed to develop until control stage 11 and assayed by in situ hybridisation for expression of Hoxd1. Embryos are shown in vegetal view, with dorsal up. Expression of Hoxd1 is reduced on the left side of injected embryos (shown on the bottom of the panel). (B) To investigate whether there is synergy between Hoxd1 and XMeis3, 16 ng MOXMeis3 and 16 ng MOHoxd1 were injected, together and separately, into the animal hemisphere of one-cell stage embryos. The embryos were harvested at st 11 and assayed for expression of Hoxd1 by in situ hybridisation. Embryos are shown in lateral view, with dorsal to the left. Injection of either MOHoxd1 or MOXMeis3 separately leads to a reduction in the early mesodermal expression of Hoxd1. Their co-injection leads to a further reduction in early mesodermal Hoxd1 expression as compared to injection of either MOXMeis3 or MOHoxd1 separately. This suggests that Hoxd1 and XMeis3 work synergistically in mediating establishment of Hoxd1 expression in mesoderm during early gastrula stages.
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