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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 anterior head region. (C) Embryo injected by Xgbx2a/en mRNA. The anterior head 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. |
