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???displayArticle.abstract??? Anterior-posterior patterning of the embryo requires the activity of multiple homeobox genes among them Hox, caudal (Cdx, Xcad) and Otx2. During early gastrulation, Otx2 and Xcad2 establish a cross-regulatory network, which is an early event in the anterior-posterior patterning of the embryo. As gastrulation proceeds and the embryo elongates, a new domain forms, which expresses neither, Otx2 nor Xcad2 genes. Early transcription of the Xenopus Gbx2 homologue, Xgbx2a, is spatially restricted between Otx2 and Xcad2. When overexpressed, Otx2 and Xcad2 repress Xgbx2a transcription, suggesting their role in setting the early Xgbx2a expression domain. Homeobox genes have been shown to play crucial roles in the specification of the vertebrate brain. The border between the transcription domains of Otx2 and Gbx2 is the earliest known marker of the region where the midbrain/hindbrain boundary (MHB) organizer will develop. Xgbx2a is a negative regulator of Otx2 and a weak positive regulator of Xcad2. Using obligatory activator and repressor versions of Xgbx2a, we demonstrate that, during early embryogenesis, Xgbx2a acts as a transcriptional repressor. In addition, taking advantage of hormone-inducible versions of Xgbx2a and its antimorph, we show that the ability of Xgbx2a to induce head malformations is restricted to gastrula stages and correlates with its ability to repress Otx2 during the same developmental stages. We therefore suggest that the earliest known step of the MHB formation, the establishment of Otx2/Gbx2 boundary, takes place via mutual inhibitory interactions between these two genes and this process begins as early as at midgastrulation.
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Fig. 1. Xgbx2a expression domain is located between Otx2 and Xcad2 expression domains. Double and triple in situ hybridization of normal embryos were performed in order to determine the relative position of the expression domains of Xgbx2a, Otx2 and Xcad2. (A) Embryos hybridized with Otx2 (turquoise) and Xgbx2a (magenta) specific probes. Gastrula stages, vegetal view. (B) Hybridization of embryos with Xgbx2a (magenta) and Xcad2 (turquoise) probes. Vegetal view, gastrula stages. (C,D) Triple in situ hybridization of gastrula stage embryos with probes specific for Otx2 (turquoise), Xgbx2a (magenta) and Xcad2 (turquoise). (C) Vegetal view. (D) Lateral view. All embryos shown are at stages 11.5–12. Xgbx2a is expressed in the region between Otx2 and Xcad2.
Fig. 5. Manipulation of the level of Xgbx2a activity. (A–D) Phenotypes caused by injection of the two chimeric proteins including the homeodomain region of Xgbx2a attached to viral activation domain (VP16/Xgbx2a) or engrailed repression domain (Xgbx2a/en). (A) Control embryo, stage 36. (B) Phenotype caused by over-expression of Xgbx2a. The embryo lacks the anteriorhead region. (C) Embryo injected by Xgbx2a/en mRNA. The anteriorhead is deleted, and the embryo is shorter. (D) Embryo overexpressing the VP16/Xgbx2a protein. The embryo is macrocephalic and exhibits enlargement of the cement gland (arrow). (E–H) Analysis of the effect on Otx2 expression. (E) Stage 11 embryos injected with LacZ mRNA as a control. Two phases of Otx2 expression can be seen: the early gastrula expression at the dorsal lip (right embryo) and the later expression at the anterior part of the embryo (left embryo). (F) Embryos injected with 400 pg of antisense Xgbx2a RNA. The expression of Otx2 at the dorsal lip widens. (G) Embryos injected with 300 pg of Xgbx2a/en RNA. The embryos exhibit down-regulation of Otx2 expression. (H) Embryos injected with 40 pg of VP16/Xgbx2a/GR mRNA. The expression of Otx2 is increased compared to (H) both at the dorsal lip and in the anterior domain of Otx2 expression. (I–L) The effect of Xgbx2a and its protein variants on the expression of head molecular markers. The expression of XCG1, Pax6, Wnt1 and Krox20 was examined at stage 25. (I) Control embryo. The transcripts of XCG1 (magenta, black arrow) marks the cement gland. Pax6 (blue) is expressed in the forebrain and in the eyes. Wnt1 (magenta, arrowhead) is expressed as a transverse stripe, in the MHB. Krox20 (magenta, red arrows) marks the third and the fifth rhombomeres. (J) Embryo injected with 120 pg of Xgbx2a mRNA. XCG1 and Wnt1expression is absent, Pax6 and expression is significantly down-regulated. (K) Embryo injected with 40 pg of Xgbx2a/en RNA. The regions of XCG1 and Pax6 are slightly enlarged. (L) Embryo injected with 40 pg of VP16/Xgbx2a/GR RNA. The XCG1-expressing region is significantly enlarged. The expression level of Pax6 is decreased. The expression of Wnt1 and Krox20 is absent.
Fig. 7. Xgbx2a and its antimorph affect Otx2 expression mainly during gastrulation. Embryos were unilaterally injected with either 60 pg of Xgbx2a/GR RNA (B,E,H) or 20 pg of VP16/Xgbx2a/GR mRNA (C,F,I). (A,D,G) Control uninjected embryos. LacZ RNA was co-injected as a lineage tracer (turquoise). Dex was added at stage 11.5 (A–C), stage 13 (D–F) or stage 15 (G–I). Each group was incubated with dex for 2 h and processed for in situ hybridization with Otx2 probe (magenta). (A–C) Treatment of the embryos with dex at stage 11.5 results in strong effects on Otx2 expression in the injected embryos. (D–F) Activation of the inducible proteins at stage 13 results in mild effects on Otx2 expression. (G–I) Addition of dex at stage 15 had no effect on the Otx2 pattern of expression.
