Zebrafish Ciliopathy Screen Plus Human Mutational Analysis Identifies C21orf59 and CCDC65 Defects as Causing Primary Ciliary Dyskinesia

Abstract

Primary ciliary dyskinesia (PCD) is caused when defects of motile cilia lead to chronic airway infections, male infertility, and situs abnormalities. Multiple causative PCD mutations account for only 65% of cases, suggesting that many genes essential for cilia function remain to be discovered. By using zebrafish morpholino knockdown of PCD candidate genes as an in vivo screening platform, we identified c21orf59, ccdc65, and c15orf26 as critical for cilia motility. c21orf59 and c15orf26 knockdown in zebrafish and planaria blocked outer dynein arm assembly, and ccdc65 knockdown altered cilia beat pattern. Biochemical analysis in Chlamydomonas revealed that the C21orf59 ortholog FBB18 is a flagellar matrix protein that accumulates specifically when cilia motility is impaired. The Chlamydomonas ida6 mutant identifies CCDC65/FAP250 as an essential component of the nexin-dynein regulatory complex. Analysis of 295 individuals with PCD identified recessive truncating mutations of C21orf59 in four families and CCDC65 in two families. Similar to findings in zebrafish and planaria, mutations in C21orf59 caused loss of both outer and inner dynein arm components. Our results characterize two genes associated with PCD-causing mutations and elucidate two distinct mechanisms critical for motile cilia function: dynein arm assembly for C21orf59 and assembly of the nexin-dynein regulatory complex for CCDC65.

Figure 1

Zebrafish Knockdown Screen Identifies c21orf59, ccdc65, and c15orf26 as Essential for Cilia Motility (A–D) Morpholino knockdown of c21orf59 (A), ccdc65 (B), and c15orf26 (C) produces ciliopathy phenotypes in zebrafish, including axis curvature, hydrocephalus (arrowheads), and supernumerary otoliths (insets in A and D). Phenotypes are not observed in control injected embryos (D). (E and F) Expression of ccdc65 mRNA in ciliated tissues, visible in control (E), is eliminated by foxj1a morpholino knockdown (F). (G) Ultrastructure of wild-type cilia shows the 9+2 arrangement of microtubules with inner and outer dynein arms projecting from the A subfibers of each doublet in cross sections and averaged doublets (inset; n = 75). (H) Kymogram of wild-type olfactory cilia beat from high-speed microvideo (Movie S1) shows rhythmic cilia wave form at 31 Hz. (I) c21orf59 morphant cilia ultrastructure showing loss of dynein outer arm motor domains in computer-averaged microtubule doublets (inset; n = 30 doublets). (J) Kymogram of c21orf59 morphant olfactory cilia beat (Movie S3) shows near-complete cilia paralysis. (K) ccdc65 morphant cilia show normal dynein arm ultrastructure in averaged doublets (inset; n = 27). (L and M) Kymograms of ccdc65 morphant olfactory cilia beat shows either complete cilia paralysis (L; Movie S4) or dyskinetic, faster beat rate (48 Hz; M; Movie S5). (N) Similar to c21orf59, outer dynein arms are lost in c15orf26 morphants (inset; n = 8). (O) Kymogram of control pronephric cilia (Movie S2) shows a beat rate of 66 Hz and a wave amplitude of 8.6 μm (white bar in O). (P) Kymogram of c15orf26 morphant pronephric cilia (Movie S6) shows severely reduced beat amplitude (white bars = 1.8 μm) or paralysis. (Q–S) RNAi knockdown of the c21orf59 ortholog in planaria, FBB18. (Q) Smed-FBB18 (RNAi) significantly reduces cilia-driven gliding locomotion of planaria (Movie S7). (R and S) Ultrastructure of control (R) and Smed-FBB18 (RNAi) (S) cilia. Insets show defects in Smed-FBB18 (RNAi) ODA assembly (arrows). The scale bar represents 100 nm.

Figure 2

Identification of Recessive Mutations in C21orf59 in Four Families with Primary Ciliary Dyskinesia and Loss of Ciliary Dynein Arms in Individuals with PCD (A) Chest X-ray of individual A5014_087-21, showing situs inversus. Red arrow indicates the apex of the heart on the right side. (B and C) TEM of individual A5014_087-21 (B) showing shortened/missing outer (red arrows) and inner (white arrowheads) dynein arms compared to control cilia (C). (D and E) Three different C21orf59 mutations detected in four independent families with PCD. Family number (underlined), mutation (arrowhead), and predicted translational changes are indicated. For the mutations detected, arrows indicate positions in relation to exons. Sequence trace is shown for mutation above normal controls. (F) Exon structure of human C21orf59 cDNA. Positions of start codon (ATG) and stop codon (TGA) are indicated. (G) C21orf59 protein putatively contains a coiled-coil (CC) and a domain of unknown function (DUF2870) at the C-terminal end. (H and I) Immunolocalization of DNAH5 and DNALI1 in human respiratory epithelial cells from individual A5014_087-21 carrying the C21orf59 c.735C>G (p.Tyr245^∗) mutation. (H) Immunofluorescence analysis of human respiratory epithelial cells via specific antibodies directed against the outer dynein arm heavy chain DNAH5 (green). As a control, axoneme-specific antibodies against α/β-tubulin (red) were used. Nuclei were stained with Hoechst 33342 (blue). In respiratory epithelial cells from healthy probands, DNAH5 (green) localizes along the entire length of the axonemes. DNAH5 (green) absent from cilia in respiratory epithelial cells from individual A5014_087-21. (I) Immunofluorescence analysis of human respiratory epithelial cells using specific antibodies directed against the inner dynein arm intermediate chain DNALI1 (red). As a control, axoneme-specific antibodies against acetylated α-tubulin (green) were used. Nuclei were stained with Hoechst 33342 (blue). DNALI1 is localized along the entire length of the axonemes of healthy probands. In contrast, in respiratory cells of individual A5014_087-21, DNALI1 is absent from cilia. Scale bars in (H) and (I) represent 10 μm.

Figure 3

Functional Analysis of C21orf59 Alleles in Zebrafish (A and B) Control (A) or c21orf59 (B) morpholinos were injected into the transgenic Tg(ubiquitin:arl13b-GFP) zebrafish line. Laser scanning confocal imaging reveals motile, GFP-positive Kupffer’s vesicle cilia as “zigzags” (A). Cilia in c21orf59 morphant KV’s (B) were paralyzed and appear as straight lines. Scale bar represents 5 μm. (C) Quantification of the percentage of motile cilia within KV. Coinjection of c21orf59 morpholino with a full-length human C21orf59 mRNA or three C21orf59 missense alleles rescued the motility of KV cilia, whereas injection of human C21orf59 mRNA bearing the c.792_795delTTTA (p.Tyr264^∗) mutation failed to rescue cilia motility. n = 3–6 embryos per sample, as indicated. Error bars indicate SEM. (D) Injection of WT, but not c.792_795delTTTA (p.Tyr264^∗) mutant, C21orf59 human mRNA rescued heart looping morphogenesis in zebrafish c21orf59 morphants.

Figure 4

Analysis C21orf59/FBB18 in C. reinhardtii (A) Flagella from wild-type and oda1 mutant Chlamydomonas strains were extracted by detergent or freeze-thaw methods. FBB18 is found exclusively within the flagellar matrix in both strains. Note that blot exposure time was significantly longer for the wild-type strain. (B) Probing flagella extracts from wild-type (cc125) and Chlamydomonas mutant strains with an FBB18 antibody reveals that flagella protein levels are significantly enhanced in cilia mutants with severe motility defects but not in oda11, which retains most of the outer arm and exhibits close to wild-type ciliary motility. (C) FBB18 immunoblot of wild-type (cc125) and cilia mutant cell bodies shows increased FBB18 protein abundance in mutants with impaired cilia motility. (D) Fractionation of Chlamydomonas flagella matrix extract in a Superose 6 gel filtration column. FBB18 migrates in a single peak distinct from core IFT complex proteins.

Figure 5

Cellular Localization of C21orf59 Protein (A) C21orf59 immunostaining in rat trachea ciliated epithelial cells reveals predominant cytoplasmic localization in perinuclear puncta. (B) C21orf59 colocalizes with SAS6 in cytoplasmic puncta in rat trachea ciliated epithelial cells. Scale bars represent QA.

Figure 6

Identification of Recessive Mutations in CCDC65 in Two Families with Primary Ciliary Dyskinesia and Identification of FAP250/CCDC65 as the Causative Gene in the ida6 Mutant (A) Chest CT scan of individual A5014_493-21 showing volume loss and bronchiectasis in the right middle lobe and bronchiectasis in both lower lobes. (B) TEM of individual A5014_493-21 cilia showing normal dynein arms and central pairs. Note that upon close examination, N-DRC links are missing (red arrowheads). Microtubule disorganization (B, inset) was observed in 5%–15% of PCD individual cilia sections. (C) Control cilia TEM showing normal dynein inner arms and nexin links (red arrowheads). (D) A homozygous truncating CCDC65 mutation detected in two independent families with PCD. Family number (underlined), mutation (arrowhead), and predicted translational changes are indicated. Sequence trace is shown for mutation above normal control. For the mutations detected, arrows indicate positions in relation to exons. (E) Exon structure of human CCDC65 cDNA. Positions of start codon (ATG) and of stop codon (TAA) are indicated. (F) Domain structure of the CCDC65 protein indicating two coiled-coil (CC) domains and position of truncating stop codon mutation. (G–J) The C. reinhardtii IDA6 gene encodes FAP250/DRC2/CCDC65. (G) FAP250 maps near the ACTIN gene on the right arm of C. reinhardtii Linkage Group XIV (chromosome 13), in the vicinity of the ida6 mutation. AC206 and ACTIN have been linked to the ac206 and ida5 mutations on the left and right arms of Linkage Group XIV, respectively (bottom line). ida6 is an inner arm motility mutation located ∼6 cM from ida5 and the FAP250 gene is located ∼6.1 Mb away from the ACTIN gene. (H) The ida6 motility mutant was transformed with an ∼9.5 kb genomic subclone encoding FAP250, and transformants were screened for improved motility by phase-contrast microscopy. The forward swimming velocities of three rescued strains are shown here relative to wild-type and ida6. (I) Longitudinal views of the 96 nm repeat of axonemes from WT, pf3, ida6, and an IDA6 rescued strain (G11) were computer averaged and then compared to identify regions of statistically significant differences. The top row shows the average of the 96 nm repeat for each strain, whereas the bottom row shows the difference plots between the wild-type and each sample. A schematic diagram of the densities observed in each repeat is labeled on the bottom left. The crescent-shaped region associated with the dynein regulatory complex (DRC) is located above radial spoke 2 (RS2), between the outer arms (OA) and inner arms (IA). Both pf3 and ida6 are associated with significant defects in the assembly of the DRC and closely associated inner arm dyneins (see difference plots). These defects are not observed in the rescued ida6::IDA6 strain. The number of axonemes and 96 nm repeats analyzed for each strain are as follows: wild-type (9 axonemes, 62 repeats), pf3 (7 axonemes, 63 repeats), ida6 (4 axonemes, 35 repeats), and ida6::IDA6 (7 axonemes, 60 repeats). (J) Axonemes from WT, ida6, and a rescued IDA6 strain were analyzed on an immunoblot probed with antibodies against several flagellar proteins. Both FAP250 (DRC2) and tektin are missing or reduced in ida6 and restored to near wild-type levels in the rescued IDA6 strain. The RSP16 subunit of the radial spokes serves as a loading control. Scale bars in (A) and (B) represent 20 μm.