Overexpression of Arl13b increases cilia length in zebrafish embryos in a dose-dependent manner. (A) Primary cilia on somitic cells of wild-type (wt) and Tg(bactin2::Arl13b-gfp) transgenic zebrafish embryos. (B) Quantitation of primary cilia length on somitic cells of wild-type (wt) and Tg(bactin2::Arl13b-gfp) transgenic zebrafish embryos (high GFP expressors). 10 embryos were analyzed from each group, 50 cilia from each embryo. (C) Increase in dosage of Arl13b-gfp mRNA resulted in a proportionate increase in primary cilia length. (D) The protein expression level of different doses of Arl13b-gfp mRNA. (E) Quantitation of primary cilia length increase in response to Arl13b overexpression. 10 embryos were analyzed from each group, 50 cilia from each embryo. Zebrafish embryos were analyzed at 15 hpf. Ciliary axonemes were labeled with anti-acetylated tubulin antibodies (red) and Arl13b was visualized with GFP fluorescence (green). In this and other figures, nuclei were labeled with DAPI (blue), scale bars=1 µm and error bars represent SEM, unless mentioned otherwise.

Consequences of increased ciliary length on signaling and motility functions of cilia. (A) The expression pattern of ptc1 in a 24 hpf wild-type embryo and an embryo that overexpressed Arl13b. (B) The quantitative comparison of gli1 and ptc1 mRNA levels between wild-type and Arl13b overexpression using qRT-PCR. The rplpo gene was used as an internal loading control, and the levels of the transcripts in wild-type embryos were arbitrarily set at 1. 6 samples from 2 independent batches of embryos were analyzed. (C) Quantitation of heart looping positions in 24 hpf wild-type and embryos that overexpressed Arl13b. 100 embryos were analyzed from each group. Left position (L), middle position (M), right position (R). (D) Motile cilia in KV of a 12 hpf wild-type embryo and an embryo that overexpressed Arl13b. (E) Quantitation of motile cilia length increase in response to Arl13b overexpression in KV. 10 embryos were analyzed from each group, 20 cilia from each embryo. (F) TEM of motile cilia in pronephric (kidney) duct of a 24 hpf wild type embryo and an embryo overexpressing Arl13b. Yellow arrows indicate the central pair, red arrow the outer microtubule doublets and green arrow the outer dynein arms. Scale bar=0.1 nM. 3 embryos were sectioned and analyzed in each category. Ciliary axonemes in panel C were stained with anti-acetylated tubulin antibodies (red), and Arl13b was visualized with GFP fluorescence (green).

Arl13b is required for the ciliogenic transcription factors, FoxJ1 and Rfx to promote ciliary length increase. (A) Primary cilia on somitic cells were lengthened in transgenic zebrafish embryos that overexpressed FoxJ1 or Rfx2. The embryos were analyzed at 18 hpf. (B) Quantitation of ciliary length increase in embryos that overexpressed FoxJ1 or Rfx2. Eight embryos were analyzed for each group, 50 cilia from each embryo. (C) Upregulation of arl13b mRNA levels after overexpression of FoxJ1 or Rfx2, a-actin mRNA served as loading control. (D) Quantification of arl13b mRNA up-regulation in response to overexpression of FoxJ1 or Rfx2 by qPCR from two individual experiments. (E) Knockdown of Arl13b abrogated primary cilia length increase by Rfx2 overexpression. Primary cilia in somitic cells of 18 hpf embryos are shown. (F) Knockdown of Arl13b abrogated increase of motile cilia length by FoxJ1. Motile cilia in the pronephric (kidney) duct of 24 hpf embryos are shown. (G) Quantitation of ciliary length alterations depicted in E. Eight embryos were analyzed from each group, 50 cilia from each embryo. Ciliary axonemes were stained with anti-acetylated tubulin antibodies (red). The green cytoplasmic staining is fluorescence of GFP expressed from an IRES element in the heat shock rfx2 and foxJ1 transgenes.

Arl13b is sufficient to restore ciliary length in FoxJ1 deficient zebrafish embryos.(A) Overexpression of Arl13b in embryos deficient in FoxJ1 is sufficient to restore the length of motile cilia. Embryos were analyzed at 24 hpf. (B) Quantitation of ciliary length in different groups of embryos. 10 embryos were analyzed for each group, 50 cilia from each embryo.

Mouse Arl13b mRNA can rescue the zebrafish arl13b morphant phenotype. (A) Mouse Arl13b mRNA rescued the shortening of cilia in arl13b morphants. Cilia on somitic cells are shown. (B) Quantitation of somitic primary cilia length in wild-type, arl13b morphants and arl13b morphants with mouse Arl13b overexpression. (C) Curled down body of arl13b morphant zebrafish is rescued by mouse Arl13b mRNA. Ciliary axonemes were labeled with anti-acetylated tubulin antibodies (red) and mouse Arl13b was visualized with GFP fluorescence (green). In this and other figures, nuclei were labeled with DAPI (blue), scale bars=1 µm and error bars indicate SEM, unless mentioned otherwise.

Overexpression of Arl13b does not have adverse effects on ciliary differentiation. Normal localization of TCTN1, CEP164 and DZIP1 (all in red, arrowheads) in cilia overexpressing Arl13b (green GFP fluorescence).

Increase in ciliary length on overexpression of Arl13b at 14 hpf. Primary cilia on somitic cells are shown. Ciliary axonemes were labeled with anti-acetylated tubulin antibodies (red) and mouse Arl13b was visualized with GFP fluorescence (green). A cilium in a cell that did not inherit the Arl13b overexpression plasmid is indicated (arrow) and serves as control.

Rfx2 overexpression could not restore ciliary length in Arl13b deficient zebrafish. Ciliary axonemes were labeled with anti-acetylated tubulin antibodies (red) and Rfx2 was visualized with GFP fluorescence (green).

Arl13b causes ciliary membrane extension in zebrafish. Ciliary axonemes were labeled with anti-acetylated tubulin antibodies (red) and mouse Arl13b was visualized with GFP fluorescence (green).