Loss of wnt signaling centers at the rhombomere boundaries in Zic1 and Zic4 morphants affects ventricle morphogenesis. (A–F) wnt1 expression in controls (A, C, E) and Zic1 + 4 morphants (B, D, F) at 16 hpf (A, B), 19 hpf (C, D) and 24 hpf (E, F), lateral view (A–D) and dorsal view (E, F). krox20 (red in panels A, B), provides a marker for r3 and r5. In hindbrain, wnt1 is expressed in roof plate (RP) (black arrow in panel A) at elevated levels in rhombomere boundaries (white arrow in panel C) and in dorsal hindbrain regions (black arrowhead in panel E). We detected no change in wnt1 expression in 16 hpf Zic1 + 4 morphants (B) compared to controls (A). By 19 hpf elevated expression of wnt1 in rhombomere boundaries (white arrow in panel C) is lost in Zic1 + 4 morphants (white arrow in panel D indicates uniform expression in dorsal hindbrain). Reduced wnt1 expression in rhombomere boundaries persists at 24 hpf in Zic1 + 4 morphants (F, black arrowhead) compared to controls (E). Note that altered wnt1 expression (F) is accompanied by a dorsal fused ventricle phenotype. (G–L) rfng expression in controls (G, I, K) and Zic morphants (H, J, L), lateral view (G–J) and dorsal view (K, L). Expression is not changed in 16 hpf Zic1 + 4 morphants (H) compared to controls (G). At 19 hpf and 24 hpf, down-regulation of wnt1 expression in rhombomere boundaries in Zic1 + 4 morphants is accompanied by expansion of rfng, expression into the rhombomere territory (J, L) compared to controls (I, K). No expansion is observed into r4 (J, L). (M–R) zic1 expression in 18 hpf and 24 hpf controls (M, P), Wnt1 morphants (N, Q) and Rfng morphants (O, R), dorsal view, and (V, Y) zic4 expression in 24 hpf controls (V), Wnt1 morphants (W), and Rfng morphants (Y), dorsal view. The expression of zic1 and zic4 is not altered in the hindbrains of Wnt1 morphants (N, Q, W) and Rfng morphants (O, R, Y) compared to controls (M, P, V). Wnt1 knockdown (Q, W) and Rfng knockdown (R, Y) cause dorsal fused phenotypes similar to Zic and Lmx morphants.
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