<i>FoxA4</i> inhibition affected anterior ectodermal/neural specification.

<p>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: <i>Xanf1</i>, <i>en2</i>, and <i>hoxb7</i> (A–C, F–H); <i>hoxc6</i> (D, I); <i>otx2</i> and <i>krox20</i> (K, L, P, Q); <i>Xag1</i> and <i>N-tubulin</i> (M, N, R, S); <i>en2</i> and <i>hoxb1</i> (O, T). Green arrows in (A, F) point to the anterior limit of <i>hoxb7</i>. At neural plate stage, <i>Xag1</i> marks the cement gland anlage (indicated between red arrowheads in N), and the hatching gland primordia (red arrows in N) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110559#pone.0110559-Wardle1" target="_blank">[98]</a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110559#pone.0110559-Theveneau1" target="_blank">[90]</a>. Black arrowhead in (H), caudal shift of <i>Xanf1</i> and fusion with the <i>en2</i> stripe. Black arrow in (H), fusion of the SHA and the ANR. Red arrow in (I), down-regulation of <i>hoxc6</i> 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 <i>en2</i> (red arrows) and <i>hoxb1</i> (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 <i>Xanf1</i> 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; <i>P</i><0.0001, two-tailed t-test. (J) The ratio between the length of the <i>hoxb7</i> domain and the embryo’s length was significantly lower in FoxA4 morphants, as measured in the groups shown in A, B, F, G; <i>P</i><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 <i>otx2</i>; 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.