gbx1 overexpression affects midbrain, midbrain-hindbrain boundary (MHB) and hindbrain. (A, A′) Control embryo stained for six3 (blue) and otx2 (red);. (B, B′) gbx1-injected embryo. The near absence of otx2-positive cells posteriorly to the six3-expressing domain indicates a loss of the midbrain territory in the gbx1-injected embryos. (C, C′) Control embryo stained for emx1 (blue) and otx2 (red). (D, D′) gbx1-injected embryo. The emx1 domain corresponding to forebrain remains robust, whereas the otx2 domain corresponding to midbrain is lost. (E) Wild-type (WT) embryo after in situ hybridization (ISH) with pax2.1 and (E′) with pax2.1 and otx2 (red). (F, F′) gbx1-injected embryos; pax2.1 is expressed ectopically in the anterior of the embryo. The MHB is shifted anteriorly as indicated by green arrowheads. Also, the distance between the posterior pax2.1 domain and the anterior domain is increased (black bars in (E, F)). (G) WT embryo after ISH with fgf8 and (G′) with fgf8 and otx2 (red). (H, H′) gbx1-injected embryos; fgf8 is expressed ectopically in the anterior of the embryo. The MHB is shifted anteriorly as the distance between this expression domain and the margin in increased (black bars in (G, H)). (I) WT embryo after ISH with eng2 and (I′) with eng2 and otx2 (red). (J) gbx1-injected embryo; eng2 is not expressed ectopically. (J′) Combined ISH with otx2 (red) shows that the remaining domain is located caudally to the reduced otx2 domain. (K) WT embryo after ISH with wnt1 and (K′) with wnt1 and otx2 (red). (L) gbx1-injected embryo;wnt1 is not expressed ectopically. (L′) In all gbx1-injected embryos the MHB is shifted anteriorly as indicated by green arrowheads. (M) WT embryo after ISH with hoxa1 and otx2 (red). (N) gbx1-injected embryo; the hoxa1 expression doamin is enlarged. (O) WT embryo after ISH with cdx4 and otx2 (red). (P) gbx1-injected embryo; the cdx4 expression domain is enlarged. (Q) WT embryo after ISH with val and krox20 (red). (R) gbx1-injected embryo; val expression is not affected: the krox20 expression domain corresponding to rh3 (green arrowhead) is enlarged, whereas rh5 is less affected. (A, B, E-P) Lateral views, anterior to the left; (C, D-Q, R) dorsal views, anterior to the top; (A′-L′) flat-mounted embryos, anterior to the top.
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