Caudal expansion of fore- and midbrain markers in ace mutants and SU5402 inhibitor-treated embryos. All views are rostral to the left; (A-F,K-L) lateral views; (G-J,M-R) dorsal views. (A-J) Analysis and comparison of otx2 expression by whole-mount in situ hybridization in wild-type, ace mutant and SU5402-treated embryos, at early somitogenesis (A-D), and at the pharyngula period (E-J). (A,B) In the mutant embryos, expression of otx2 is caudally shifted well before the period when the anatomical isthmic constriction should form. (C-F) In SU5402-treated embryos, expression of otx2 (blue) is markedly shifted, similar to ace mutants, whereas expression of pax2a (red) is reduced. The expanded otx2 territory is also characteristic for later developmental stages, both in ace mutants (E-H) and SU5402 inhibitor-treated embryos (I-J), as illustrated at 28 hpf and 26 hpf, respectively. The black arrowhead (A,C,E,G,I) points to the caudal expression limit of otx2 at the MHB of wild-type embryos. The two arrowheads (B,D,F,H,J) mark the caudally expanded territory expressing otx2 in ace mutant and SU5402-treated embryos. (K-N) As with otx2, expression of dmbx1 is markedly expanded at early somitogenesis in the mutant embryos, as illustrated at the 7- and 9-somite stage. The single arrow (K,M) labels the caudal limit of dmbx1 expression in the developing wild-type mesencephalic tectum. The two arrows (L,N) indicate the expanded expression of dmbx1 in ace mutant siblings. (O-R) Analysis of wnt4 expression reveals an early upregulation, and expansion of the fore-midbrain territory toward caudal coordinates of the mesencephalic alar plate in ace mutants. (O-P) The initially broad, expanded expression domain of wnt4 narrows to the dorsomedial parts of the alar plate, and is extended to the caudally enlarged tectal compartment at later somitogenesis stages, as illustrated at the 20-somite stage. The black arrows (O,Q) indicate the caudal limit of the enriched expression of wnt4 at the fore-midbrain region in wild-type embryos. The two arrows (P,R) show the markedly upregulated and expanded expression of wnt4 in ace mutants.
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