Fig. 4

Nlz1 is downstream of Foxj1a and is regulated by canonical Wnt signaling. (A)–(D), (A′)–(D′) nlz1 expression in foxj1a morphants (B, B′, n=48/50), wnt8a morphants (C, C′, n=52/55), and LiCl treated foxj1morphants embryos (D, D′, n=25/28) compared to control MO (A, A′, n=30/30), as seen in whole mount in situ hybridization of embryos at 1S stage. (E)–(F) Cilia are labeled with anti-acetylated α tubulin antibody (green) in the KV at the 5S stage. Complete lack of KV cilia was observed in foxj1a morphants (E, n=20/20); injection of nlz1 mRNA in foxj1a morphants rescues KV cilia (F, n=12/30). Quantification of expression patterns of cmlc2 (G), and lft2 (H) in Foxj1a MO (cmlc2, n=50; lft2, n=50) and Foxj1a MO+nlz1mRNA (cmlc2, n=50; lft2, n=50) injected embryos. (A)–(F) dorsal view, (A)–(D) anterior dorsal, (A′)–(D′) posterior dorsal, (E), and (F) posterior to the top. (H), (J) Embryos were treated with LiCl as specified. (I)–(L) Whole mount in situ hybridization at 95% epiboly (I), (J) and 21 h (K), (L) resulted in expansion of nlz1 expression (J, n=45/50; L, n=15/19) compared to control (I n=40,K n=40, without LiCl). (I), (J) dorsal view, (K), (L) lateral view, anterior to left. (M) Nlz1 repressed Wnt/β-catenin signaling activated by either LiCl or β-catenin in a TOPflash luciferase assay. RLU, relative luciferase units. Ab: absent; Bi: bilateral, M: midline; R: right; L: left. Scale bar (black) in (A)–(D′), 100 µm; (I)–(J), 60 µm; (K)–(L), 75 µm. Scale bar (white) in (E)–(F), 50 µm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)