IFT46 plays an essential role in cilia development

Abstract

Cilia are microtubule-based structures that project into the extracellular space. Ciliary defects are associated with several human diseases, including polycystic kidney disease, primary ciliary dyskinesia, left-right axis patterning, hydrocephalus and retinal degeneration. However, the genetic and cellular biological control of ciliogenesis remains poorly understood. The IFT46 is one of the highly conserved intraflagellar transport complex B proteins. In zebrafish, ift46 is expressed in various ciliated tissues such as Kupffer׳s vesicle, pronephric ducts, ears and spinal cord. We show that ift46 is localized to the basal body. Knockdown of ift46 gene results in multiple phenotypes associated with various ciliopathies including kidney cysts, pericardial edema and ventral axis curvature. In ift46 morphants, cilia in kidney and spinal canal are shortened and abnormal. Similar ciliary defects are observed in otic vesicles, lateral line hair cells, olfactory pits, but not in Kupffer׳s vesicle. To explore the functions of Ift46 during mouse development, we have generated Ift46 knock-out mice. The Ift46 mutants have developmental defects in brain, neural tube and heart. In particular Ift46(-/-) homozygotes displays randomization of the embryo heart looping, which is a hallmark of defective left-right (L/R) axis patterning. Taken together, our results demonstrated that IFT46 has an essential role in vertebrate ciliary development.

Keywords: Cilia; Ciliopathy; IFT; IFT46; Intraflagellar transport; KO mouse; L/R defect; Zebrafish.

Fig 1. Alignment of IFT46 protein sequences using GeneDoc

The IFT46 protein sequences from human, mouse, zebrafish and Chlamydomonas are aligned using GeneDoc program. The intraflagellar transport complex B protein 46 C terminal domain is marked with blue bar, The zebrafish ift46 has 59%, 69% similarity to human IFT46 and mouse Ift46 and 69% similarity to Chlamydomonas, respectively.

Fig 2. Expression pattern of ift46 in zebrafish embryos

(A) Whole-mount in situ hybridization shows ift46 expression at 80% epiboly in the dorsal forerunner cells (DFC, arrow). (B) ift46 is strongly expressed in Kupffer’s vesicle (KV, arrow) at bud stage, (C, D) At 20-somite stage and 24 hpf ift46 expression is detected in pronephric ducts (pd), eyes, ears, spinal cord (sc) and diffusely in the brain. (E) Cross-section showing ift46 expression in the pronephric ducts (arrow) and spinal cord (asterisk). (F) At 40 hpf, ift46 is widely expressed in brain, eyes, otic vesicles (arrow) and pectoral fins. (G, H) At 72 and 96 hpf, ift46 is expressed in the olfactory pits (op) and neuromast (nm) hair cells (arrow).

Fig 3. ift46 morphants develop phenotypes associated with ciliary dysfunction

(A) Wild-type embryos show normal morphology. (B) ift46 morphants are characterized by curved body axis, the development of pronephric cyst and pericardiac edema at 96 hpf. (C–D) Cross-sections of wild-type zebrafish at the glomerular-tubular region of pronephros at 72 hpf show normal morphology of pronephric glomeruli and ducts. (C’–D’) ift46 morphant embryos display a grossly distended cyst (asterisk) in place of glomeruli (gl) and dilated pronephric ducts (pd) (arrow). Scale bar, 50 µm. (E-F’) Histological sections of the eyes of wild-type and ift46 morphants at 5 dpf. The retina in wild-type control (E and E’) has normal laminated structures, including retinal pigment epithelium (RPE), outer segment (OS), outer plexiform layer (OPL) and inner nuclear layer (INL). (E) and (F) scale bar, 200 µm. The outer segment (OS) (bracket) is thinner in ift46 morphants (F’) compared to the control (E’). Scale bar, 100 µm. (G–J) The EGFP-tagged ift46 (green) is localized to the base of primary cilia (red) in hTERT-RPE1 cells. Merged imaged are shown in (I). Arrowhead indicates the base of cilia. Scale bar, 3 µm. (J) Higher magnification view of the boxed area in (I).

Fig 4. Ciliary defects in zebraifh ift46 morphants

Immunofluorescence with antibody against acetylated α-tubulin showing cilia in the pronephric ducts, (scale bar, 10 µm) (A-A’), otic vesicle, (scale bar, 10 µm) (B-B’), olfactory placode, (scale bar, 10 µm) (C-C’) and a lateral line hair cell, (scale bar, 5 µm) (D-D’). The ift46 morphants exhibit shortened or fewer cilia compared with the control fish in all of these organs. Arrow head: tether cilia. Arrow: short cilia. (E–F’) SEM images showing the cilia in the olfactory placode of control fish (E, E’) and ift46 morphants (F, F’) at 5 dpf. Scale bar, 40 µm (E) and (F), 4 µm (E’) and (F’) (G-H’) TEM images of cilia in zebrafish pronephric ducts at 72 hpf. Ultrastructure of normal pronephric cilia shows 9+2 microtubule architecture at 72 hpf which is intact in ift46 morphants. (G’, H’) Representative views of individual cilium at higher magnification. Scale bar, 5 µm (G) and (H), 100 µm (G’) and (H’).

Fig 5. Ift46 mutant mice were generated by homologous recombination

(A) Schematic diagram indicating the structure of the mouse Ift46 gene and the gene-targeting strategy. The Ift46 mutant mice were generated by targeting exon 4. Genotyping primers are shown in the map P1–P3 (arrows). (B) Genotyping of Ift46 mutants at E9.5. The wild-type allele, Ift46 WT, produces a 161 bp fragment and the mutated allele, Ift46 MT, produces a 620 bp PCR fragment.

Fig 6. Phenotypic characterization of Ift46 knock-out mouse embryos

(A) Ift46 homozygotes exhibit anterior defects, kinks of the neural tube (bracket) and pericardial edema (arrow) at E10.5. (B) At E9.5, normal heart-looping and neural tube closure are seen in heterozygous mice. (C) The cranial neural tube (asterisk) fails to close and the heart looping is abnormal in Ift46 homozygous (−/−) (arrow). LV, left ventricle; OFT, outflow tract; RV, right ventricle; A, atrial. (D–E) Whole-mount in situ hybridization of Ift46 heterozygous and homozygous mutant embryos for Lefty-1 detects abnormal bilateral expression of Lefty-1 in homozygotes. (F–I) Scanning electron microscopy of the embryonic nodal cilia in wild-type (F, G) and Ift46 homozygous (−/−) embryos (H, I). Ventral view of the E8.0 embryos at 1,500X (F, H) and 10,000X (G, I) magnifications. Anterior to the top. Circle in (F, H) indicates magnified area in (G, I). Scales bars: 20 µm (F, H), 1 µm (G, I). (J) Analysis of mouse heart looping in Ift46(+/+) /Ift46(+/−) (n=52) and Ift46(−/−) (n=9). (K) Analysis of Lefty-1 expression in E8.5 mouse embryos (Ift46(+/+) /Ift46(+/−), n=9) (Ift46(−/−), n=2).