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Fig. 5. FoxB1 is required for the formation of posterior neural tissue and the suppression of anterior tissue. (A–N) Together with β-gal mRNA, either Control MO (34 ng, left columns) or FoxB1 MO (34 ng, right columns) were injected unilaterally into the 2-cell stage embryos. The expression of Otx2 (fore- and mid-brain; A and B), Rx2A (retina; C and D), En2 (mid-hindbrain boundary; E and F), Krox20 (hindbrain; G and H), HoxB9 (spinal cord; I-J′), HoxC6 (spinal cord; K and L), and MyoD (paraxial mesoderm; M and N) genes analyzed by in situ hybridization is shown in purple and β-gal is stained in red. In embryos injected with FoxB1 MO, the expression of Otx2, Rx2A, En2 and Krox20 (corresponding to rhombomere 3) on the injected side was expanded (arrowheads; B, D, F and H) and the expression of HoxB9 (J and J′) and HoxC6 (L) was decreased and shifted posteriorly (the horizontal lines mark approximate anterior limits of the expression). The expression pattern of MyoD was not significantly affected by FoxB1 MO (N). The injected side of the embryo is indicated by brackets. A–H: anterior (front) view with dorsal to the top; I–N: dorsal view with posterior to the top. (O and P) Expression of anterior–posterior marker genes in dorsal ectodermal explants. Embryos were injected with Control or FoxB1 MO (34 ng/blastomere) at 2-cell stage and cultured until late gastrula stage (st. 12.5). Dorsal ectodermal explants were isolated from a presumptive posterior neural plate region indicted by a black square (O). The isolated explants were cultured until the neurula stage (st. 21) and analyzed by QPCR (P). The relative level of expression (arbitrary units) normalized to ODC expression is shown for each gene. Each experiment was repeated more than twice and gave similar results. (Q-X′) FoxB1 acts together with Wnt and FGF signaling to promote posterior neural development. Together with β-gal mRNA, the 2-cell stage embryos were injected unilaterally with a mixture of MO and mRNA or DNA as indicated: FoxB1 MO alone (34 ng; Q and Q′); FoxB1 MO with Nmut-FoxB1 mRNA (1000 pg; R–S′), XWnt-8 DNA (500 pg; U and U′), eFGF mRNA (1 pg; V and V′) or both XWnt-8 DNA (500 pg) and eFGF mRNA (1 pg; W and W′); Control MO (34 ng) with eFGF mRNA (1 pg; X and X′). Expression of the HoxB9 gene analyzed by in situ hybridization is shown in purple and β-gal is stained in red. Nmut-FoxB1 partially but significantly rescued the reduction of HoxB9 expression by FoxB1 MO (horizontal lines, compare Q′ with R′ and S′). Moreover, in embryos injected with FoxB1 MO and XWnt-8 DNA, the expression of HoxB9 was rescued partially (horizontal lines, U′) and coexpression of XWnt-8 and eFGF showed slightly better rescuing activities (W and W′). eFGF alone (1 pg; V and V′) was not able to rescue the expression of HoxB9 in the anterior–posterior (AP) direction. In some embryos, HoxB9 expression was even expanded in the dorsal-ventral (DV) direction (arrowheads, S–X). The injected side of the embryo is indicated by brackets. All panels show dorsal views with posterior to the top. (Y) Summary of phenotypes obtained from several experiments shown in (I)–(J′) and (Q)–(X′). The degree of recovery in the HoxB9 expression domains was scored according to the direction of change along the AP or DV axis as shown in (T), and categorized into three types: reduced along the AP axis (AP reduced), reduced along the AP axis with expansion along the DV axis (AP reduced/DV expanded), and expansion along the DV axis (DV expanded). Numbers of embryos per experimental group are indicated above the bars. |
