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Fig. 1. XBF-1 induces neural tissue autonomously, at the expense of epidermis and in the absence of mesodermal signals. XBF-1 induces ectopic XSox3 even when cell division is blocked. Embryos were injected with a high dose of XBF-1 plus lacZ RNA or lacZ RNA alone (A or B respectively; light blue) and hybridised with a probe for epidermal keratin (dark magenta). Other embryos (C,D) were injected with XBF-1 plus lacZ RNA and were analysed at the tadpole stage for lacZ (C,D; light blue), N-tubulin (C) and muscle actin (D) expression (magenta). Note that ectopic N-tubulin expression is not accompanied by a corresponding expansion or ectopic expression of muscle actin. XBF-1 injected animal caps do not express collagen type II (E), which in the embryo is expressed in the somites and notochord (F). In contrast, XBF-1 injected (G), but not control uninjected (H), animal caps express XSox3. In this experiment, XBF-1 injected animal caps were combined with control RLDx labelled caps (E,G,H,J). Fluorescent (J) and bright-field (K) images of a sectioned animal cap demonstrate that XSox3 expression is localised in the XBF-1 injected part of the XBF-1/RLDx animal cap conjugate. Embryos (L,M) were injected with a high dose of XBF-1 and lacZ RNA and some of these were treated with HUA at the early gastrula stage (M). All embryos shown (L-N) are hybridised with XSox3. Embryos injected with lacZ and treated with HUA have normal XSox3 expression while both XBF-1 injected (L) and XBF-1 injected plus HUA (M) treated embryos show an expansion of the XSox3 expression domain. The expansion of the XSox-3 expressing domain in HUA- treated XBF-1 injected embryos can be clearly seen in section (O). HUA treated embryos (N, embryo on the right) have larger nuclei than sibling controls (N, embryo on the left) illustrating that cell division has been effectively blocked by the HUA treatment.
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Fig. 2. XBF-1 induces tissue outgrowths in the ectoderm. In all panels, a red arrowhead indicates convoluted and/or thickened ectoderm on the side that has been injected with XBF-1 RNA (all panels except E and F) or DNA (F). Whole embryo (A) and transverse sections (B,C) of neurula embryos hybridised with N- tubulin (B) and XSox3 (C; C is also lightly stained for BrdU incorporation). Note the dramatic thickening of the ectopic neural tissue (red arrowhead) compared with the normal neural tissue on the control side (black arrowhead). The dotted line demarcates the dorsal midline (A) or the notochord (B,C), separating the injected (left) from the uninjected side. Tadpole stage embryos were hybridised with N-tubulin (D, the injected side; E, the control side; F, high magnification of a section through D) and muscle actin (G) (magenta staining in all). In G, note that there is no ectopic muscle actin expression in the lateral protrusion. In embryos shown in D-G, XBF-1 RNA or DNA was coinjected with lacZ RNA (light blue).
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Fig. 3. XSox3 and N-tubulin expression marks proliferating and post- mitotic neural cells, respectively. Embryos were processed either for BrdU incorporation alone (left panels) or simultaneously for BrdU incorporation, XSox3 (light blue or green) and N-tubulin (magenta) expression (right panels), as described in Materials and Methods. Top panels show the whole mounted embryos and the bottom panels the respective transverse sections. XSox3 expression overlaps with BrdU incorporating cells while N-tubulin is expressed in cells that do not incorporate BrdU. Note that the deep layer of the epidermal ectoderm is also heavily dividing at this stage. A dotted line demarcates the underlying somitic and lateral plate mesoderm. d.e., deep layer of the ectoderm; l.p., lateral plate mesoderm; s.e., superficial layer of the ectoderm; som., somitic mesoderm.
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Fig. 4. XBF-1 affects BrdU incorporation in the ectoderm and co-operates with XSox3. (A) Embryos were injected with either a high or a low dose of XBF-1 RNA, as indicated, in one blastomere of the two-cell stage embryo and were processed for double whole-mount in situ hybridisation for XSox3 (blue/green) and N-tubulin (magenta), followed by BrdU incorporation (brown). Whole mounts and transverse section are shown. In the sections a black line indicates the dorsal midline, separating the injected (right) from the uninjected side (left). Black and blue arrowheads delimit the XSox3 and N-tubulin expression domains, respectively. In the high-dose sections, arrows show an area of increased BrdU incorporation on the injected side. In the low-dose sections, an asterisk on the injected side (right) shows an area of the ectoderm where dividing cells should have been located, when compared with the equivalent region of the control side (asterisk on control side, left). Also note that in the control side N-tubulin positive cells are located within a wider area of BrdU negative cells while in the experimental side N- tubulin positive and BrdU positive cells are intermingled. (B) Embryos were injected with either low-dose XSox3, low- dose XBF-1, or both, and processed for N-tubulin (purple) and BrdU incorporation (brown). A low dose of XSox3 did not affect the expression of N-tubulin, whereas low-dose XBF-1 induced additional ectopic N-tubulin positive cells. Coinjection of both low-dose XSox3 and low-dose XBF-1 suppressed endogenous and induced ectopic N-tubulin, in the same way that a high-dose XBF-1 affects N-tubulin expression (see also Table 1).
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Fig. 5. p27XIC1 is normally expressed in non-dividing cells. Expression of XBF-1 in the anterior neural plate coincides with the expression of XSox3, but not that of p27XIC1. Neural plate-stage embryos were analysed for the expression of p27XIC1 (A-C). The sectioned embryo (B) was also labelled for BrdU incorporation (brown nuclear staining). p27XIC1 is expressed in non-BrdU incorporating cells both in the mesoderm and in the neural ectoderm (B, arrow). In the anterior neural plate (C), p27XIC1 was expressed in a band in a similar region to that of XBF-1 (D) and XSox3 (D,E) (marked by asterisks; D, XBF-1 in purple and XSox3 in light blue and E, XSox3 only in purple/brown). (Lower panels) Sections of double in situ hybridisations in light blue and purple, showing that XSox3 and XBF-1 are largely coexpressed and that p27XIC1 is expressed more rostrally than XBF-1 in a more comparable region to that of neurogenin, X-ngnr-1. p27XIC1 was expressed in BrdU negative cells in the anterior neural plate (bottom right panel).
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Fig. 6. p27XIC1 blocks cell division when misexpressed. p27XIC1 misexpression abolishes BrdU incorporation in the ectoderm. Embryos were injected with p27XIC1 RNA at the two-cell stage and processed for p27XIC1 expression by in situ hybridisation (blue/purple) and BrdU incorporation (brown nuclei) in transverse sections. In situ hybridisation with a p27XIC1 probe revealed both the expression of the endogenous gene and the distribution of the injected p27XIC1 RNA, both of which coincide with a complete absence of BrdU staining. The numbers of BrdU positive cells on the p27XIC1 injected side in comparison with the uninjected side are also represented as a column chart. The BrdU count is represented as BrdU positive cells per section, over the neural fold area and was averaged from 15 sections over three embryos in two independent experiments. Sections scored were from the anterior spinal cord level. Error bars were too small to illustrate.
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Fig. 7. XBF-1 regulates p27XIC1 in a dose-dependent manner. Embryos were injected with either a high or a low dose of XBF-1 RNA and analysed for expression of p27XIC1 or N-tubulin. A high dose of XBF-1 suppressed p27XIC1 and repositioned residual p27XIC1 expression laterally (top right panel, dorsal view) while a low dose induced ectopic p27XIC1 in the lateral ectoderm (bottom middle panel, side view), in the same way that a low dose of XBF-1 induces ectopic N-tubulin (shown for comparison in bottom right panel). XBF-1 RNA was coinjected with lacZ RNA as a lineage marker; embryos injected with lacZ alone were normal (left panels).
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Fig. 8. p27XIC1 misexpression does not alter the pattern of expression of N-tubulin or XSox3. p27XIC1 misexpression has no effect in inducing ectopic XSox3 (A,C) or N-tubulin (B,D) expression. Embryos were injected with p27XIC1 RNA and were analysed for p27XIC1 (magenta), XSox3 (light blue, A,C) and N-tubulin expression (light blue, B,D) and for BrdU incorporation (brown nuclei). Magenta colour shows primarily the ectopic p27XIC1 expression although some endogenous expression can be seen in B. Cells expressing p27XIC1 did not incorporate BrdU; however cells in the neural plate that were expressing injected p27XIC1 still expressed XSox3, excluding the possibility of non-specific toxic effects of p27XIC1. Arrow in D, shows a cell within the neural plate that has received p27XIC1 RNA but does not express N-tubulin.
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Fig. 9. p27XIC1 is a direct target of XBF-1. Dexamethasone-inducible XBF-1 RNA suppresses p27XIC1 in the presence (bottom panels) and absence (top right) of the protein synthesis inhibitor, cycloheximide. In the absence of both dexamethasone and cycloheximide, XBF-1- GR did not suppress p27XIC1 expression (top left panel).
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