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In this paper we study Xotx2, a Xenopus homeobox gene related to orthodenticle, a gene expressed in the developing head of Drosophila. The murine cognate, Otx2, is first expressed in the entire epiblast of prestreak embryos and later in very anterior regions of late-gastrulae, including the neuroectoderm of presumptive fore- and mid-brain. In Xenopus, RNase protection experiments reveal that Xotx2 is expressed at low levels throughout early development from unfertilized egg to late blastula, when its expression level significantly increases. Whole-mount in situ hybridization shows a localized expression in the dorsal region of the marginal zone at stage 9.5. At stage 10.25 Xotx2 is expressed in dorsal bottle cells and in cells of the dorsal deep zone fated to give rise to prechordal mesendoderm, suggesting a role in the specification of very anterior structures. In stage 10.5 gastrulae, Xotx2 transcripts start to be detectable also in presumptive anteriorneuroectoderm, where they persist in subsequent stages. Various treatments of early embryos cause a general reorganization of Xotx2 expression. In particular, retinoic acid treatment essentially abolishes Xotx2 expression in neuroectoderm. Microinjection of Xotx2 mRNA in 1-, 2- and 4-cell stage embryos causes the appearance of secondary cement glands and partial secondary axes in embryos with reduced trunk and tail structures. The presence of the Xotx2 homeodomain is required to produce these effects. In particular, this homeodomain contains a specific lysine residue at position 9 of the recognition helix. Microinjected transcripts of Xotx2 constructs containing a homeodomain where this lysine is substituted by a glutamine or a glutamic acid residue fail to cause these effects.
Fig. 3. Localized Xotx2 expression. (A) Stage 9.5. Whole-mount in situ hybridization, animal pole view. (Aâ²) Sagittal section.
(Aâ²â²) Detailed view of the sagittal section. (B) Stage 10.25. Whole- mount in situ hybridization, vegetal view. (Bâ²) Sagittal section.
(Bâ²â²) Detailed view of the sagittal section. (C) Stage 10.5. Whole- mount in situ hybridization, vegetal view. (Câ²) Sagittal section.
(Câ²â²) Detailed view of the sagittal section. Hybridization signal is now detectable in presumptive anterior neuroectoderm. Open arrows delimit Xotx2 expression in neuroectoderm; arrowheads point to Brachetâs cleft. (D-F) Stage 12. Whole-mount in situ hybridization, posterior-dorsal (D), lateral (E) and dorsal (F) view. Embryos in C and F were not cleared. yp, yolk plug; ar, archenteron; bc, bottle cells.
Fig. 4 A. Xotx2 expression in stage 14 embryos. (A) Sagittal section. The three germinal layers (ect, ectoderm; mes, mesoderm; end, endoderm) in the dorsoanterior portion of the embryo are indicated. cga, cement gland anlage; sha, stomodeal-hypophyseal anlage; pp, prechordal plate
Fig. 4.B Xotx2 expression in stage 14 embryos. (B) Detailed view of the sagittal section. The three germinal layers (ect, ectoderm; mes, mesoderm; end, endoderm) in the dorsoanterior portion of the embryo are indicated. cga, cement gland anlage; sha, stomodeal-hypophyseal anlage; pp, prechordal plate
Fig. 5. Analysis of experimentally treated embryos at gastrula stage. (A) Whole-mount in situ hybridization, vegetal view. U, untreated stage 10.5 gastrula. LiCl, LiCl-treated embryo. Xotx2 expression has become radially symmetric. RA, RA-treated embryo (1 μM, continuous treatment starting at the 2-cell stage). Xotx2 expression is reduced but still detectable and localized in mesendoderm. Open arrows delimit the neuroectodermal region where Xotx2 transcripts are present in untreated but not in RA-treated gastrulae. UV, Xotx2 expression is essentially abolished. (B,C) Lack of Xotx2 expression in RA-treated embryos at stage 21. Whole-mount in situ hybridization in lateral view (C), compared to a normal embryo (B)
Fig. 6. Example of exogastrulae hybridized with Xotx2. An hybridization signal in a position corresponding to the prechordal plate is indicated by an open arrow. No hybridization is detectable in the ectoderm (solid arrow)
Fig. 7. Phenotypes of embryos injected with Xotx2. The embryos shown were injected with 5 ng of either âXotx2 (A) or Xotx2 (B-E) RNA. Injected embryos developed until sibling controls reached stage 34. Frequencies of occurrence of the various phenotypes are shown in Table 1. The embryo in A shows no phenotypic abnormalities. (B) Embryo with bent axis. (C) Embryo with posterior defects. (D) Embryos with additional cement glands (arrowheads). The embryo on the left side presents a secondary cement gland fused laterally to the original gland. (E) Embryo with axis duplication. The partial secondary axis is delimited by two arrows.
(F) Albino embryos microinjected with
Xotx2 and hybridized with XCG-1, a
probe specific for the developing
cement gland. An uninjected control
embryo is shown at the bottom.
Figure 7 G) Whole-mount immunostained embryos with neural specific monoclonal antibody 2G9 visualized with HRP-conjugated secondary antibody. At the top is shown a control embryo at stage 40; at the bottom an embryo of the type shown in C, showing ectopic neural tissue (arrow).
Fig. 8. Histological analysis of embryos microinjected with Xotx2. (A,B) Sagittal sections of embryos of the type in Fig. 7C and D, respectively at a stage corresponding to 34 in control embryos. Arrowheads point to cement glands, whereas an arrow points to ectopic structures located in the vicinity of the secondary cement gland. (C,D) Frontal sections at different levels of embryos of the type in Fig. 7D at a stage corresponding to 32 in control embryos. Fixed embryos were first whole-mount stained with monoclonal antibody 2G9, revealed with an HRP-conjugated secondary antibody and later processed for standard histology. Strong 2G9 staining is detectable among ectopic derivatives (arrows) where, in addition to a few mucus-secreting cement gland cells (arrowhead), muscles (m) and notochord (n) are visible. (cns), central nervous system; (e), eye; (end), endoderm; (p), pharynx; (som), somites.
Fig. 9. RNase protection assay on normal and Xotx2-microinjected animal caps and whole embryos. Lane 1, uninjected animal caps; lane 2, injected animal caps; lane 3, injected embryos; lane 4, normal embryos. Animal caps were cut from embryos at stage 8 and were collected and analyzed for goosecoid (gsc) at stage 12.5 (left panel) and for muscle actin (ms actin) at stage 24 (right panel); rpS8 and cytoskeletal actin (c actin) are the internal standard controls.
Fig. 10. Diagrammatic comparison of Xotx2 and goosecoid expression, indicated by stippling, in early Xenopus embryos: top, dorsal-posterior view; bottom, sagittal view. Areas 1 to 6 represent: 1, prechordal plate; 2, bottle cells; 3, chordamesoderm; 4, presumptive spinal cord; 5, presumptive rostral brain; 6, presumptive epidermis.
Fig. 2. Expression of Xotx2. (A) RNase protection analysis of Xotx2 expression during development from unfertilized egg (ue) to stage 25. The X. laevis gene for ribosomal protein S8 (rp S8) (Mariottini et al., 1988) is analyzed as an internal standard. P, probes; M, marker. (B) RNase protection analysis of Xotx2 expression in stage 8 total embryos and in isolated animal caps (AC) of the same stage.
