Loss of lmx1b expression in Zic morphants underlies loss of roof plate development and is linked to ventricle morphogenesis. (A–H) Expression of lmx1b.2 at 18 hpf (A–D) and 24 hpf (E–H). Dorsal view (A, B and E, F) and lateral view (C, D and G, H) of controls (A, C and E, G) and Zic1 + 4 morphants (B, D and F, H). In Zic1 + 4 morphants, the expression of lmx1b.2 in hindbrain roof plate (RP) is missing or down-regulated in 18 hpf (B, D) and 24 hpf embryos (F, H) compared to controls at 18 hpf (A, C) and 24 hpf (E, G). The expression of lmx1b.2 in roof plate is indicated in controls (A, C and E, G) by black arrows. The expression of lmx1b.2 in ventral serotonergic neurons is unaffected in Zic1 + 4 morphants (arrowheads in panels A, B). In 24 hpf Zic1 + 4 morphants, loss of lmx1b.2 expression in the roof plate correlates with points of dorsal fusion (white arrows in panel F). (G′, I′) Cross-sections at the levels indicated in panels G and I of 24 hpf embryos labeled with lmx1b.2 probes (G′) and gdf6a probes (I′) to illustrate their expression in roof plate cells (indicated by black arrows). The expression of lmx1b.2 in ventral serotonergic neurons is indicated by black arrowheads. (I, J) gdf6a expression in dorsal view of 24 hpf controls (I) and Zic1 + 4 morphants (J). In control embryos, gdf6a is expressed in the roof plate (arrow in panel I). In Zic1 + 4 morphants (J), the expression of gdf6a expression is missing or down-regulated in roof plate compared to controls (I). (K, L) gdf6a expression in 24 hpf lateral view control embryos (arrow in panel K indicates expression in the RP) and Lmx1b.2 morphants (L), shows that gdf6a expression in roof plate is missing or down-regulated (L). (M, N) zic1 expression, dorsal view of 24 hpf controls (M) and Lmx1b.2 morphants (N) shows that Lmx1b.2 knockdown causes a dorsal ventricle fusion phenotype. MHB, midbrain–hindbrain boundary.
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