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In order to study the regional specification of neural tissue we isolated Xotx2, a Xenopus homolog of the Drosophila orthodenticle gene. Xotx2 is initially expressed in Spemann's organizer and its expression is absent in the ectoderm of early gastrulae. As gastrulation proceeds, Xotx2 expression is induced in the overlying ectoderm and this domain of expression moves anteriorly in register with underlying anterior mesoderm throughout the remainder of gastrulation. The expression pattern of Xotx2 suggests that a wave of Xotx2 expression (marking anterior neurectoderm) travels through the ectoderm of the gastrula with the movement of underlying anterior (prechordal plate) mesoderm. This expression of Xotx2 is reminiscent of the Eyal-Giladi model for neural induction. According to this model, anterior neural-inducing signals emanating from underlying anterior mesoderm transiently induce anterior neural tissues after vertical contact with the overlying ectoderm. Further patterning is achieved when the ectoderm receives caudalizing signals as it comes in contact with more posterior mesoderm during subsequent gastrulation movements. Functional characterization of the Xotx2 protein has revealed its involvement in differentiation of the anterior-most tissue, the cement gland. Ectopic expression of Xotx2 in embryos induces extra cement glands in the skin as well as inducing a cement gland marker (XAG1) in isolated animal cap ectoderm. Microinjection of RNA encoding the organizer-specific homeo-domain protein goosecoid into the ventral marginal zone results in induction of the Xotx2 gene. This result, taken in combination with the indistinguishable expression patterns of Xotx2 and goosecoid in the anterior mesoderm suggests that Xotx2 is a target of goosecoid regulation.
Fig. 3. Whole-mount in situ hybridization of Xotx2 during Xenopus gastrulation. (A) Vegetal view of a stage 10 embryo. (B) Dorsovegetal view of a stage 12 embryo. (C-E) Dorsal portion of midsagittal sections of whole-mount in situ hybridized stage 10+, 10.25, and 12 embryos, respectively. Anterior is to the right in all panels except F. Xotx2 staining in C is confined to deep mesoderm, while in D and E the expression in ectoderm is apparent (arrowheads in D indicate a region of nuclear ectodermal Xotx2 staining). In E Xotx2 expression in both ectoderm and mesoderm are approximately in register. Note the ectodermal thickening marking the presumptive anterior neurectoderm (arrow). The anterior border of ectodermal staining extends further anteriorly into presumptive cement gland (see also Jamrich and Sato, 1989). (F) Xotx2 is induced in ventrolateral mesoderm expressing goosecoid. Single C-tier blastomeres were coinjected with goosecoid RNA and the lineage tracer LRD at the 32-cell stage. At the early gastrula stage, embryos containing LRD at a position greater than 120° away from the dorsal midline were subjected to whole-mount in situ hybridization for Xotx2. A vegetal pole view is shown and the primary and secondary sites of Xotx2 expression are indicated.
Fig. 5. Ectopic expression of Xotx2 induces ectopic cement glands in the skin of developing embryos. (A) Xotx2 injected (left) and uninjected (right) embryos (stage 20). Note the induction of ectopic cement gland (arrow). (B) Xotx2-injected embryos (stage 22) are left and middle, and an uninjected embryo is right. Arrows indicate ectopic cement glands. (C) Xotx2-injected embryo grown until early tadpole stage. An ectopic cement gland is present in the ventral/lateralectoderm. (D) Transverse section through head of normal tailbud stageembryo. Note that the normal cement gland shows characteristic columnar cells with dark pigmentation (arrow). (E) Transverse section through the trunk of an Xotx2- injected embryo. Box indicates the area of an ectopic cement gland. (F) High magnification view of the boxed area in E. Note the presence of characteristic columnar cells associated with pigment granules (arrow). (G) Incomplete blastopore closure resulting from injection of Xotx2 into a dorsal blastomere. Such embryos will later develop spina bifida. (H) Uninjected control embryo (top) and short-tailembryo (bottom) resulting from incomplete blastopore closure.
Fig. 6. Xotx2 induces cement gland marker expression in isolated animal caps. (A) RT-PCR analysis of RNA derived from stage 21 embryos (lane 1) and animal caps isolated from uninjected (lane 2) and Xotx2-injected embryos (lane 3) at stage 8 blastula, and developed until sibling embryos reached stage 21. RNA was subjected to RT-PCR analysis using oligonucleotides specific for the cement gland marker XAG1 and histone H4. (B) RT-PCR analysis of RNA derived from stage 21 embryos (lane 1) and animal caps isolated from uninjected (lane 2), noggin-injected (lane 3) and Xotx2- injected embryos (lane 4) at stage 8 blastula, and developed until sibling embryos reached stage 21. RNA was subjected to RT-PCR analysis using oligonucleotides specific for Xotx2, N-CAM, and histone H4. Lanes marked E, A, N, and X contain PCR products from normal embryos, animal (uninjected control) caps, noggin- injected caps, and Xotx2-injected caps, respectively.
Fig. 7. Ectodermal expression of Xotx2 can be induced by vertical signals. (A) Schematic representation of construction of mesoderm-ectoderm conjugates. Albino embryos were injected with lysinated-rhodamine dextran (LRD) into the marginal zone of all blastomeres of 2-4 cell stage embryos. At early gastrula stage (10.25), involuted mesoderm was dissected out and wrapped in stage 10.25 gastrula ectoderm from uninjected albino embryos. Conjugates were developed until siblings reached stage 15, fixed, and subjected to whole-mount in situ hybridization using Xotx2 probe. (B) Light (left) and fluorescence (right) micrographs of sections through the whole-mount in situ hybridized stage 15 equivalent conjugate shown in C (left). Arrows mark the boundary between the labelled mesoderm and unlabelled animal caps. Note the ectodermal expression of Xotx2 surrounding the mesoderm. Incubation of ectoderm cap alone did not induce ectopic Xotx2 expression (C, right).
Fig. 8. Planar signals are sufficient to induce Xotx2 expression in ectoderm. Keller sandwiches were prepared from stage 10−, 10.0, 10+, 10.25 and 10.5 embryos. Sandwich explants were developed to stages 17-19, fixed, and subjected to whole-mount in situ hybridization using Xotx2 and Krox20 probes. (A) Schematic diagram illustrating the arrangement of the anteriormesoderm (stippled) of a stage 10- embryo before involution and a representative Keller sandwich derived from stage 10- embryos. (B) Schematic diagram illustrating the arrangement of the anteriormesoderm (stippled) of a stage 10.5gastrula and a representative Keller sandwich derived from a stage 10.5embryo. The dotted lines indicate the positions of incisions made to prepare the Keller explants. In both right panels, ectoderm is progressively more anterior toward the left and the mesoderm is progressively more anterior toward the right. Notochord is designated N. The boundary of mesoderm and ectoderm is indicated by the arrowhead. Xotx2 staining (open arrow) of the ectoderm is anterior to the characteristic striped pattern of Krox20 (filled arrow). In the stage 10− sandwich (A), there is clear Xotx2 staining in the anterior notochord.
