||Figure 7. FoxA4 inhibition affected anterior ectodermal/neural specification.
Embryos at early neural plate stage injected with 20 ng of CtrlMO or of FoxA4MO before the first cleavage (A, B, F, G, K–N, P–S) or unilaterally injected into 1 cell at the 2-cell stage (C, D, H, I, O, T). The injected side was determined by DOG fluorescence (insets). Embryos were hybridised with the following probes: Xanf1, en2, and hoxb7 (A–C, F–H); hoxc6 (D, I); otx2 and krox20 (K, L, P, Q); Xag1 and N-tubulin (M, N, R, S); en2 and hoxb1 (O, T). Green arrows in (A, F) point to the anterior limit of hoxb7. At neural plate stage, Xag1 marks the cement gland anlage (indicated between red arrowheads in N), and the hatching gland primordia (red arrows in N)  . Black arrowhead in (H), caudal shift of Xanf1 and fusion with the en2 stripe. Black arrow in (H), fusion of the SHA and the ANR. Red arrow in (I), down-regulation of hoxc6 on the injected side. Yellow arrowhead in (Q), diffuse CGA and anterior border of the ANR. Notice the down-regulation and the caudal shift of en2 (red arrows) and hoxb1 (green arrows) by comparing the injected side (right) with the non-injected side (left) in the FoxA4 morphant in (T) and with the CtrlMO-injected embryo in (O). (E) Ratio between the distance from the posterior limit of Xanf1 to the blastopore and the total length of the embryo in the groups shown in A, B, F, G (r-Xanf1). The ratio was significantly lower in FoxA4 morphants; P<0.0001, two-tailed t-test. (J) The ratio between the length of the hoxb7 domain and the embryo’s length was significantly lower in FoxA4 morphants, as measured in the groups shown in A, B, F, G; P<0.0001, two-tailed t-test. ANR, anterior neural ridge; CGA, cement gland anlage; SHA, stomodeal-adenohypophyseal anlage; m/h, midbrain/hindbrain boundary; m, d, presumptive mesencephalic and caudal diencephalic regions expressing otx2; r3, third rhombomere; r4, fourth rhombomere. (A, D, F, I, K, M, O, P, Q, R, T) are dorsal views; (B, C, G, H, L, N, Q, S) are anterior views.