Anterior migration of PCT and morphogenesis of the neck segment are impaired in taz morphants. (A) In situ hybridization of embryos with the PCT-specific probe, slc20a1a. Note the absence of anterior migration and convolution of PCT in taz morphants. (B) Neck region morphogenesis is arrested in taz morphants. In situ hybridization using pax2a probe (red), followed by anti-BrdU staining (green) to visualize the neck region morphologies and detect proliferating cells in the neck regions in control embryos (a, c, e, g, i) and taz morphants (b, d, f, h, j). Note in f, the neck regions remained aligned with the A–P axis in the taz morphant, rather than repositioned to align with the mediolateral axis as in the normal embryo (e). (g, h) Individual optical sections of stacks. (i, j) Projected images of stacks. Anterior is to the top. Bars, 20µm.

Length and position of the ET11-9 segments relative to their corresponding somite blocks in 24 hpf embryos. Anti-GFP staining of the ET11-9 segments (red) was followed by MF20 staining of somites (green). (A, B) Control embryo; (C, D) 24 hpf taz morphant. White arrows indicate position of the cloacae.

The ET11-9 segment is shortened in 24 and 48 hpf taz morphants. (A) The relative lengths of the ET11-9 segment in the normal pronephric tubule. The ET11-9 segment was stained first with anti-GFP (red), followed by α6F antibody (green). The α6F antibody stains the pronephric tubule and duct, but the red fluorescence staining of the ET11-9 segments blocked subsequent α6F green fluorescence staining, so the α6F appeared to stain the two tubular domains flanking the ET11-9 segment. The ET11-9 segments were shortened and PCT segments lengthened in 24 hpf taz morphants. The PCT segment also became dilated and the ventrally bent DL segments were slightly shortened in the 48 hpf taz morphant. (B) Reduced cell proliferation in the ET11-9 segments of the taz morphant at 24 hpf. (a, b) Individual optical sections; (c, d) projected images of stacks. Embryos were stained with anti-GFP (red), followed by anti-BrdU (green) staining of proliferating cells. Bars, 20µm.

Multiciliated cells in pronephric tubules of control embryos and taz morphants. Anti-GFP staining of the multiciliated cells (red) followed by α6F staining of the pronephric tubules (green). Note that in the 48 hpf taz morphant, the multiciliated cells were clustered in the shortened regions.

Effects of RA and DEAB on patterning of the pronephric tubule in control embryos and taz morphants. Visualization of pronephric tubule segments in 24 and 48 hpf zebrafish embryos by anti-GFP (red) staining of the ET11-9 segment, followed by α6F staining of the pronephric tubule (green). Panels show untreated (DMSO) control zebrafish embryos and taz morphants, as well as embryos treated with RA or DEAB in DMSO. RA treatment shortened the DL segments in both control (D) and 24 hpf taz morphant (H), but extended the ET11-9 segments in control embryo (D) but not in the 24 hpf taz morphant (H). DEAB extended the DL segments in both control (I, J) and 24 hpf taz morphants (M, N) as well as in control (Q, R) and taz morphants at 48 hpf (U, V), and reversed PCT dilation in 48 hpf taz morphants (V compared with T).

taz mRNA expression in zebrafish embryos. In situ hybridization with antisense taz RNA. (A, B) Expression of taz in the pronephric progenitor field (white arrows in A and B) at the bud stage. (A) Lateral view, anterior is to the left, dorsal is up. (B) Posterodorsal view, anterior is to the left. (C) Expression of taz in somites but not in pronephric progenitor field at the 11-somite stage.

Representative bright field images of hTAZ-rescued- (A) vs. un-rescued (ΔWW)(B) zebrafish embryos.