Fig. 3

nlz1is expressed in Kupffer’s vesicle and is required for cilia formation in zebrafish. Whole mount in situ hybridization at the one somite (1S) stage with one or two RNA probes as indicated in wild type (A), and (B), control MO injected (C), and Nlz1 MO injected (D) embryos. (B) Cells in the KV co-express nlz1 and sox32, marking dorsal forerunner cells. In nlz1 morphants cha (D, n=28/35) was absent in the KV region, whereas sox32 (red) remained unaltered as compared to control MO injected embryos (C). Cilia were marked with acetylated-α tubulin (green) and nuclei were counterstained with DAPI (blue) in different regions of the embryos as indicated. Complete absence of KV cilia was observed in nlz1 morphants (F, n=15/18) compared to control (E, n=15/15). Similarly, at later stages, the number and length of the cilia in the neural FP (I, n=18/23) and in the PN (K, n=27/30) were reduced in nlz1 morphants compared to control MO injected embryos (H), and (J). Arrows indicate motile cilia. KV cilia in nlz1 morphants were rescued by nlz1 mRNA injection (G, n=45/52). (L) Quantification of cilia number in KV embryos injected with MO±mRNA. (A), and (B) dorsal view, anterior to left; (C) and (D) dorsal view, posterior to top. (D), (F) and (G) injected with 2.5 ng Nlz1 MO, (I), and (K) injected with 2 ng Nlz1 MO. Inset (A), (C), and (D) shows blow up of KV region. KV; Kupffer’s vesicle; s, somite; FP, floor-plate; PN, pronephros. Scale bar in (A)–(D), 100 µm; (E)–(G), 12.5 µm; (H)–(K), 50 µm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)