Diverse type of cilia are present in zebrafish

(A) Cilia in the kupffer’s vesicle (KV) of a 10-somite stage zebrafish larvae. (B-H) Confocal images showing cilia in different type of cells as indicated. The position of these cells were indicated in the top diagram. (I-J) Confocal images showing cilia in the sperm (flagellum) or spermatocyte. All the cilia were visualized with anti-acetyleated tubulin antibody. Scale bar=10 μm.

Rescue of ovl mutants with Tg(hsp70l:ift88-GFP) transgene.

(A) Schematic diagram of hsp70l:ift88-GFP construct. (B) Procedure of heat shock experiments for ovl mutants rescue assay. (C) External pheontype of 3dpf wild type, ovl mutant or ovl mutant larvae carrying Tg(hsp70l:ift88-GFP) transgene. The numbers of larvae investigated were shown on the bottom right. (D) Confocal images showing cilia in different type of organs as indicated. Red channel indicates cilia visualized by anti-acetylated α-tubulin antibody and fluorescence of Tg(hsp70l:ift88-GFP) is showed in green. Scale bars: 200 μm in panel C and 10 μm in panel D.

Intraflagellar transport in different type of cilia.

(A-E) Left, Snapshot of Intraflagellar transport videos in different cell types as indicated. Middle (A, B), Snapshot of same cilia at different time points. Arrowheads with the same color indicate the same IFT particle. Right, kymographs illustrating the movement of IFT particles along the axoneme. Horizontal scale bar: 10 μm and vertical scale bars: 10 s. Representative particle traces are marked with white lines in panels D and E. (F) Histograms displaying the velocity of anterograde and retrograde IFT in different type of cilia as indicated. “Antero.” and “Retro.” represent anterograde and retrograde transport, respectively. (G) Summary of IFT velocities in different tissues of zebrafish. Numbers of IFT particles are shown in the brackets. (H) Left, Statistics analysis of cilia length in different tissues of zebrafish. Right, anterograde and retrograde IFT average velocity in different tissues of zebrafish. (I)Anterograde and retrograde IFT velocities plotted versus cilia length. Linear fit (black line) and coefficient of determination are indicated. (J) Frequency of anterograde and retrograde IFT entering or exiting cilia.

Alterations in motor proteins, BBS proteins, or tubulin modifications have minimal effects on IFT.

(A) Left: Snapshot of IFT videos in crista cilia of 4dpf wild type or mutant larvae as indicated. Right: Kymographs showing IFT particle movement along axoneme visualized with Ift88:GFP. Horizontal scale bar: 10μm and vertical scale bar:10s. (B) Histograms showing anterograde and retrograde IFT velocity in crista cilia of control or mutant larvae. (C) Genomic structure and sequences of wild type and ttll3 mutant allele. PAM sequence of sgRNA target are indicated in blue. (D) Protein domain of Ttll3 in wildtype and ttll3 mutants. (E) Confocal images showing crista cilia in wild-type or maternal-zygotic (MZ) ttll3 mutants visualized with anti-monoglycylated tubulin antibody (green) and anti-acetylated α-tubulin antibody (red). (F) Histograms depicting IFT velocity in crista cilia of control and ttll3 mutants. Top, anterograde IFT. Bottom, retrograde IFT. (G) Histograms illustrating IFT velocity in crista cilia of ccp5 or ttll6 morphants. Scale bars: 10 μm in panel A and 5 μm in panel E. *p<0.5; *** p<0.001.

Increased size of IFT fluorescent particles in crista cilia.

(A) Representative STED images of crista (top) and spinal cord (bottom) in 4dpf Tg(hsp70l: ift88-GFP) larva. Cilia was stained with anti-monoglycylated tubulin (magenta), and IFT88-GFP particles were counterstained with anti-GFP antibody (green). Enlarged views of the boxed region are displayed on the right. (B) Dot plots showing the number of IFT particles per arbitrary unit (a.u.) in crista cilia recorded by spinning disk and STED. (C) Statistical analysis showing IFT particles size in the cilia of ear crista and spinal cord. (D) External phenotypes of 2 dpf zebrafish larvae injected with higher and lower dose of ift88 morpholinos. (E) Statistical analysis of cilia length in control or ift88 morphants. (F) STED images showing IFT particles in crista cilia of 3dpf control or ift88 morphants. Enlarged views of the boxed region are displayed on the right. (G) Dot plots showing the number of IFT particles in control and ift88 morphants. (H) Statistical analysis showing IFT particles size of crista cilia in control or ift88 morphants. (I) Left, Snapshot of IFT videos in crista cilia of 3dpf control (top) or ift88 morphant (bottom) carrying Tg(hsp70l: ift88-GFP). Right, Kymographs showing movement of IFT particles along axoneme. Horizontal scale bar: 10μm and vertical scale bar:10s. (J) Histograms showing IFT velocity in 3dpf control or ift88 morphants. (K) Model illustrating IFT with different train sizes in long and short cilia. Scale bars: 0.2 μm in panel A and F, and 500 μm in panel I. **p<0.01; *** p<0.001.

Rescue of ciliogenesis defects in ovl (ift88) mutants via Tg(hsp70l:ift88-GFP)

(A) Rescue of ciliogenesis defects in ear macula, olfactory placode and pronephric duct. MC, multicilia. Red channel shows cilia visualized by anti-acetylated α-tubulin antibody. Fluorescence of Tg(hsp70l:ift88-GFP) is showed in green. Scale bar=10μm. (B) External phenotypes of adult ovl homozygotes rescued with Tg(hsp70l:ift88-GFP) transgene.

Generation of Tg (βactin2:tdTomato-ift43) transgene for IFT imaging

(A) Schematic diagram showing βactin2:tdTomato-ift43 transgenic construct. (B-C) Left, Snapshot of Intraflagellar transport videos in cilia of ear crista and pronephric duct epithelial cells. Right, kymographs illustrating the movement of IFT particles along the axoneme. Horizontal scale bar=10 μm. Vertical scale bar=10 s. (D) Summary of the anterograde and retrograde velocities of IFT measured in embryos expressing Tg(βactin2:tdTomato-ift43). Numbers of IFT particles are shown in the brackets.

IFT in the cilia of neuromast hair cells of different zebrafish mutants.

(A)(Left) Snapshot of IFT videos in neuromast cilia of 4dpf control, kif17 or bbs4 mutants. (Right) Kymographs showing IFT particle movement along axoneme.(B) Histograms showing anterograde and retrograde IFT velocity in neuromast cilia in control and mutants. Horizontal scale bar=10μm. Vertical scale bar=10s

Complete loss of tubulin glycylation in ttll3 mutants.

(A) Immunostaining with anti-monoglycylated tubulin antibody in 4dpf ttll3 mutants showing complete absence of monoglycylation modification in cilia of neuromast, olfactory placode and pronephric duct. (B) Immunostaining with anti-polyglycylated tubulin antibody revealed complete elimination of polyglycylation modification in multicilia of olfactory placode and pronephric duct. Red channel shows cilia visualized with anti-acetylated α-tubulin antibody. Scale bar=5 μm.

Validation of the efficiency of ttll6 and ccp5 morpholinos.

(A, B, E ,F) External phenotypes of control, ttll6 and ccp5 morphant embryos at 2 dpf. (C) Agarose gel electrophoresis of RT-PCR amplicons using primer pairs as indicated in panel (D). The PCR product size in the ttll6 morphants was significantly larger than that in the control (con.). (D) Schematic diagram of ttll6 sp MO target sites (orange) and RT-PCR primer positions. Sequencing results indicated that ttll6 morphant exhibited incorrect splicing, with intron12 being wrongly included in the mature mRNA.(G) Agarose gel analysis of RT-PCR amplicons using primer pairs as indicated in panel (H). The PCR product size in the ccp5 morphant was significantly larger than that in the control (con.). (H)Schematic diagram illustrating the splice donor sites targeted by antisense morpholinos (orange). Injection of ccp5 MO resulted in the production of aberrant transcripts, wherein intron 5 was mis-spliced into the mature mRNA. Scale bar =500 μm.

Generation of ATP reporter transgenic line.

(A) Schematic diagram of βactin2: arl13b-mRuby-iATPSnFR1.0 transgenic construct. (B) Confocal images showing cilia of crista and epidermal cell in 4dpf transgenic embryos. (C) Statistical results of relative fluorescence intensity in cilia of crista and epicell cells (ratio of red fluorescence intensity vs green fluorescence intensity).

Summary of IFT average velocities in different zebrafish muants or morphants.