Knockdown of ttc26 disrupts ciliogenesis of the photoreceptor cells and the pronephros in zebrafish

Abstract

In our effort to understand genetic disorders of the photoreceptor cells of the retina, we have focused on intraflagellar transport in photoreceptor sensory cilia. From previous mouse proteomic data we identified a cilia protein Ttc26, orthologue of dyf-13 in Caenorhabditis elegans, as a target. We localized Ttc26 to the transition zone of photoreceptor and to the transition zone of cilia in cultured murine inner medullary collecting duct 3 (mIMCD3) renal cells. Knockdown of Ttc26 in mIMCD3 cells produced shortened and defective primary cilia, as revealed by immunofluorescence and scanning electron microscopy. To study Ttc26 function in sensory cilia in vivo, we utilized a zebrafish vertebrate model system. Morpholino knockdown of ttc26 in zebrafish embryos caused ciliary defects in the pronephric kidney at 27 h postfertilization and distension/dilation of pronephros at 5 d postfertilization (dpf). In the eyes, the outer segments of photoreceptor cells appeared shortened or absent, whereas cellular lamination appeared normal in retinas at 5 dpf. This suggests that loss of ttc26 function prevents normal ciliogenesis and differentiation in the photoreceptor cells, and that ttc26 is required for normal development and differentiation in retina and pronephros. Our studies support the importance of Ttc26 function in ciliogenesis and suggest that screening for TTC26 mutations in human ciliopathies is justified.

FIGURE 1:

Expression of Ttc26 in cultured mIMCD3 cells, rodent tissues, and zebrafish embryos. (A) Northern blot of Ttc26 in adult mouse tissues. Ttc26 expression is high in the testis, with a 2.5-kb transcript exclusively seen in testis and a 4.2-kb transcript detected in testis, brain, heart, lung, kidney, and retina. The 11.4-kb band may represent unprocessed RNA. (B) Developmental expression of ttc26 in zebrafish embryos. RT-PCR data revealed ttc26 transcription was active at the one-cell stage and maintained at a relatively stable level through the developmental process (note that samples of one-cell and four-cell embryos were not loaded on the same gel). (C) Top, V5-Ttc26 fusion protein (red) colocalized with cilia (green) in SSTR3-EGFP-mIMCD3 cells. Ttc26 protein is most concentrated in the ciliary base. Middle, Cep164 (red), a distal appendage protein in basal body, is expressed in cilia (green) of SSTR3-EGFP-mIMCD3 cells. Bottom, V5-Ttc26 protein is partially overlapped with Cep164 in the basal bodies of mIMCD3 cells. Right, merged images. Note that the two proteins locate closely but not overlap. Insets, enlarged images of the cilia. (D) Neonatal rat photoreceptor cells transfected by in vivo electroporation with the plasmid pCAG-V5-Ttc26-IRES-EGFP are revealed by IRES-EGFP expression (green). Ttc26 recombinant protein, detected by immunostaining of the V5 tag, is localized to the transition zone of the transfected photoreceptor cells (red, arrows) on the top of the inner segments filled with water-soluble EGFP. Right, merged images.

FIGURE 2:

Knockdown of Ttc26 by shRNA caused shortened cilia with altered morphology in mIMCD3 cells. (A) An immunoblot assay using anti-V5 antibody verified knockdown of expressed V5-Ttc26 cotransfected with shRNA constructs in CHO cells. All three Ttc26 shRNA constructs showed efficient knockdown of the V5-Ttc26 transcript as compared with two control shRNA constructs. (B) Immunofluorescence image showing cilia (red) detected immunostaining for acetylated α-tubulin in mIMCD3 cells transfected with Ttc26 shRNA (green) and in nontransfected cells. (C) Quantification of Ttc26 knockdown effects on ciliary length. Error bars represent SDs between three independent experiments (n = 829 for Ttc26 shRNA-transfected or nontransfected cells; n = 275 for control shRNA-transfected and nontransfected cells; *, p < 0.01 by t test). (D) Cells transfected with nontarget control shRNA exhibit green fluorescence but do not show the effects of Ttc26 knockdown (arrows point to cilia in B and D). (E) Scanning electron micrographs of mIMCD3 cells treated with Ttc26 shRNA or nontarget control shRNA. Defective cilia were identified as being shortened with enlarged ends compared with the long and slender cilia in cells treated with control shRNA.

FIGURE 3:

Phenotypes of ttc26 zebrafish morphant larvae. Zebrafish embryos were injected with two ttc26 MOs (MO-AUG and MO-SP) or control MO. (A) Knockdown of ttc26 results in developmental defects. Panel 1, lateral view of 120-hpf larva injected with control MO. Panel 2, dorsal view of the control MO-treated larva. Panels 3–6, lateral views of 120-hpf morphant larvae injected with ttc26 morpholino MO-AUG. The ttc26 morphants displayed curled or kinked tails, precardiac edema, and shortening of the body. Panels 7–10, dorsal or lateral view of 72-hpf morphant larvae injected with ttc26 morpholino MO-SP. The curled or kinked tails and body curvature are very similar to those of MO-AUG morphants. (B) Eye morphology is altered in ttc26 morphants at 5 dpf. Histological sections are shown for eyes, central retina (CR), and retinal marginal zone (MZ) from larvae injected with control MO and ttc26 MO-AUG. Left, retina of 5-dpf fish injected with the control MO display well-formed laminae (top), with details visible in high magnification (60×) views of the central retina (CR, middle) and marginal zone (MZ, bottom). The higher magnification (60×) views of the morphant retinas reveal shortened and disorganized photoreceptor outer segments in the retina, with otherwise normal lamination. (C) TEM of the control-MO larvae at 5 dpf shows normally developed photoreceptor outer segments with densely packed disks (left). TEM of the morphant larvae shows that outer segments are smaller, shortened, and disoriented (right), consistent with the morphology seen in the light micrographs in (B). Note that a disorganized partial outer segment can be seen on the left adjacent to a cone photoreceptor (red arrow). N, nucleus; M, mitochondria; OS, outer segment. (D) RT-PCR analyses confirm that morpholino MO-SP interferes with splicing of ttc26 mRNA in larvae. Forty-eight hours postfertilization and 120-hpf larvae injected with control-MO or MO-SP were used for RNA extraction and RT-PCR. The lower band (590 base pairs) visible in both conditions represents PCR product from correctly spliced mRNA. The larger PCR product (1553 base pairs, containing the 963–base pair nonsplicing intron 1) is detected after MO-SP treatment due to the blocked splice donor site. As shown, although not complete, splice blocking is more evident at 48 hpf, consistent with some recovery from the morpholino effect at 120 hpf. (E) The binding positions of the PCR primers (above) and the MO-SP oligo (below) are illustrated in a diagram. For simplicity, only exons 1 and 2 are shown.

FIGURE 4:

Morpholino knockdown of ttc26 disrupts motile cilia and causes tubule dilation in zebrafish pronephros. (A) Cilia in the anterior and posterior regions of the pronephric kidneys in larvae at 27 hpf. Green, cilia revealed by immunostaining of acetylated α-tubulin; blue, nuclei. Top, cilia are long and well-organized in larvae injected with the control MO oligo. Middle and bottom, knockdown of ttc26 by transfection with MO-AUG or MO-SP morpholinos leads to shortened, disorganized, and disoriented cilia. (B) Kidney cross-section in a 5-dpf larva shows normal morphology after injection with control–MO (C–E) Injection with MO-AUG or MO-SP morpholino leads to distended/dilated pronephric tubes and ducts. The zebrafish larvae used are shown below the corresponding micrograph images, with the defects marked (*). gl, glomerulus; pt, pronephric tubule; pd, pronephric duct.

FIGURE 5:

ttc26 knockdown disrupts pronephric cilia beat coordination in 2-dpf larvae. Video images on the left show the dilated pronephros ducts in ttc26 morphants (B and C) vs. control (A). Dotted lines, position of the line scan. Corresponding cilia beat amplitude line scans are shown on the right. Control cilia bundles beat coordinately, but the cilia beat rate in ttc26 morphants (B and C) is increased and out of phase, resulting in loss of coordination. (See supplemental movies.)