Ciliopathy-associated IQCB1/NPHP5 protein is required for mouse photoreceptor outer segment formation

Abstract

Null mutations in the human IQCB1/NPHP5 (nephrocystin-5) gene that encodes NPHP5 are the most frequent cause of Senior-Løken syndrome, a ciliopathy that is characterized by Leber congenital amaurosis and nephronophthisis. We generated germline Nphp5-knockout mice by placing a β-Geo gene trap in intron 4, thereby truncating NPHP5 at Leu87 and removing all known functional domains. At eye opening, Nphp5^-/- mice exhibited absence of scotopic and photopic electroretinogram responses, a phenotype that resembles Leber congenital amaurosis. Outer segment transmembrane protein accumulation in Nphp5^-/- endoplasmic reticulum was evident as early as postnatal day (P)6. EGFP-CETN2, a centrosome and transition zone marker, identified basal bodies in Nphp5^-/- photoreceptors, but without fully developed transition zones. Ultrastructure of P6 and 10 Nphp5^-/- photoreceptors revealed aberrant transition zones of reduced diameter. Nphp5^-/- photoreceptor degeneration was complete at 1 mo of age but was delayed significantly in Nphp5^-/-;Nrl^-/- (cone only) retina. Nphp5^-/- mouse embryonic fibroblast developed normal cilia, and Nphp5^-/- kidney histology at 1 yr of age showed no significant pathology. Results establish that nephrocystin-5 is essential for photoreceptor outer segment formation but is dispensable for kidney and mouse embryonic fibroblast ciliary formation.-Ronquillo, C. C., Hanke-Gogokhia, C., Revelo, M. P., Frederick, J. M., Jiang, L., Baehr, W. Ciliopathy-associated IQCB1/NPHP5 protein is required for mouse photoreceptor outer segment formation.

Keywords: Leber congenital amaurosis; Senior-Løken syndrome; nephrocystins; nephronophthisis.

Figure 1.

Generation of Nphp5^−/− mice. A) Diagram of the 15 exon Nphp5 gene. Translation of NPHP5 initiates in exon 3 and the β-Geo gene trap is located in intron 4, truncating NPHP5 at Leu87. A loxP site was placed in intron 5. B) IQCB1/NPHP5 functional domains. NPHP5 has BBSome interaction sites (BBS; yellow), 3 CC domains (green), 2 IQCB motifs in exons 10 and 12 (IQ; blue), respectively, and a CEP290/NPHP6-binding site (Cep). Position of a frameshift mutation in exon 12 of the Crd2 dog associated with SLS is noted (14). Mutations associated with SLS (red) and nonsyndromic LCA (black) are indicated. C) X-gal staining of embryonic d 13.5 heterozygous and homozygous embryos shows ubiquitous expression of NPHP5. D) Presence of β-Geo trap was determined by PCR with LAR3/R (294 bp), whereas WT Nphp5 gene was identified with F/R (452 bp). E) Immunohistochemistry of Nphp5^+/− and Nphp5^−/− retina cryosections at postnatal day (P)6, P10, and P15. Sections were probed with anti–NPHP5-KK antibody (red) and contrasted with DAPI (blue) to reveal ONL nuclei; absence of NPHP5 immunoreactivity in Nphp5^−/− retina confirms germline knockout. IS, inner segment.

Figure 2.

Phenotype of Nphp5^−/− retinas. A, B) Scotopic (left) and photopic (right) electroretinography of Nphp5^+/− and Nphp5^−/− mice at P14 (A) and P18 (B). Lack of response (red) indicates that Nphp5^−/− mice are blind early. C) Fundoscopy of WT, Nphp5^+/−, and Nphp5^−/− retinas at P28. Knockout mouse fundus shows peripheral and central mottling of the retina.

Figure 3.

Immunolocalization of rod OS proteins in Nphp5^+/− and Nphp5^−/− retina. A–D) Expression of rhodopsin (red) at P6 (A), P10 (B), P15 (C), and P30 (D) in Nphp5^+/− (left) and Nphp5^−/− retina (right). Note that rhodopsin mistraffics in the Nphp5^−/− ONL as early as P6. E–J) Localizations of rod Tα (E, F), cGMP phosphodiesterase (PDE6) (G, H) and rhodopsin kinase (GRK1) (I, J) at P10 (E, G, I) and P15 (F, H, J) in Nphp5^+/− (left) and Nphp5^−/− retina (right). K) Statistical evaluation of Nphp5^+/− and Nphp5^−/− ONL thickness at P6, P10, P15, and P30. IS, inner segment; ns, not significant; OPL, outer plexiform layer. ***P < 0.001.

Figure 4.

Localization of cone OS proteins in Nphp5^+/− and Nphp5^−/− retina. A–C) Localization of S-opsin in heterozygous controls (left) and knockouts (right) at P6 (A), P10 (B), and P15 (C). D–F) Immunolocalization of ML-opsin in heterozygous controls (left) and knockouts (right) at P10 (D), P15 (E), and P30 (F). G–L) Immunolocalization of cone PDE6 (G, H), cone Tγ (I, J), and cone arrestin (K, L) in heterozygous (left) and homozygous knockouts (right) at P10 (G, I, K) and P15 (H, J, L). IS, inner segment; OPL, outer plexiform layer.

Figure 5.

Absence of fully developed transition zones in Nphp5^−/− photoreceptors. A, B) WT (left) and Nphp5^−/− (right) retina sections expressing EGFP-CETN2 at P10 (A) and P15 (B). CETN2-GFP serves as a centriole and transition zone marker. Insets (A, B) showing enlargements, reveal that WT basal bodies generate transition zones, but transition zones are stunted or absent in Nphp5^−/− retina. C) Immunolocalization of NPHP5 in the presence of EGFP-CETN2. P15 EGFP-Cetn2⁺;Nphp5^+/− (left) and EGFP-Cetn2⁺;Nphp5^−/− (right) retina cryosections were probed with anti–NPHP5-KK antibody (red). D) Enlargement of panel C as indicated. In EGFP-Cetn2⁺;Nphp5^+/− photoreceptors, NPHP5 is located in the proximal OS, but is undetectable in the EGFP-Cetn2⁺;Nphp5^−/− retina (right). Arrows 1–3 denote daughter centriole (1), mother centriole (2), and transition zone (3). Note transition zones are absent or stunted in Nphp5^−/− photoreceptors (C, right, insets). E) Prominin 1 (PROM1) in heterozygous (left) and homozygous (right) knockouts. PROM1 is primarily present at the proximal Nphp5^+/− OS adjacent to the CC (resembling a sickle; inset). In Nphp5^−/− photoreceptors, PROM1 mislocalizes in the inner segment (IS).

Figure 6.

CEP290/NPHP6 in Nphp5^−/− retina. A–C) Localization of CEP290/NPHP6 (red) in Nphp5^+/− control (left) and Nphp5^−/− (right) retinas at P6 (A), P10 (B), and P15 (C). D) EGFP-Cetn2⁺;Nphp5^+/− (left) and EGFP-Cetn2⁺;Nphp5^−/− (right) probed with antibody directed against CEP290. Two sets of centrioles plus transition zone, 1 set colabeled with EGFP-CETN2 and CEP290 (yellow), the other only with CETN2 (left inset). Transition zones are stunted or absent (right inset). IS, inner segment.

Figure 7.

Transition zone ultrastructure. A–C) At P6, normal basal body docking and connecting cilium structure is observed in the WT (A), heterozygote (B), and knockout (C) mice. D–F) At P10, OSs are formed in the WT (D) and heterozygote (E) animals but not in the knockout mice (F). Insets show transition zone cross-sections with the 9 + 0 microtubule arrangement in all genotypes.

Figure 8.

Cone long-term survival in Nphp5^−/− ;Nrl^−/− retina. A–E) Nphp5^−/+;Nrl^−/− (left) and Nphp5^−/−;Nrl^−/− retina sections (right) probed with anti–ML-opsin antibody at P10 (A), P15 (B), 1 mo (C), 2 mo (D), and 3 mo (E) of age. F–I) Nphp5^−/+ (left) and Nphp5^−/− (right) retina sections probed with anti–ML-opsin antibody at P10 (F), P15 (G), 1 mo (H), and 2 mo (I). Note that cone degeneration in Nrl^−/−;Nphp5^−/− retina is slowed and mutant cones still express ML-opsin at 3 mo. Panels A–C were from mice expressing EGFP-CETN2. J) Statistical evaluation ONL thickness at P10, P15, 1 mo, 2 mo, and 3 mo. K) Representative ERG traces of Nphp5^−/−;Nrl^−/− (red), Nphp5^+/− (black), and Nrl^−/− mice (green) at P15 and 1 mo. Nrl^−/− photopic responses are elevated and double-knockout cones are nonfunctional. IS, inner segment; ns, not significant; OPL, outer plexiform layer.

Figure 9.

Cilia formation in kidneys and MEFs. A) Light microscopy of Nphp5^+/− (left) and Nphp5^−/− (right) hematoxylin and eosin–stained kidney sections at 12 mo of age. No morphologic abnormalities were visible in the kidney parenchyma of any knockout mice at 12 mo of age (n = 3) or earlier (not shown). Tubules of heterozygous and homozygous knockouts are well formed. Cyst formation, interstitial fibrosis, and tubular atrophy are not detected. B, C) Confocal microscopy of kidney epithelial cells demonstrate cilia labeled with antiacetylated tubulin antibody (red). D) EGFP-Cetn2⁺;Nphp5^+/− (left) and EGFP-Cetn2⁺;Nphp5^−/− (right) MEFs form cilia of identical length, identified by Ac-α-tubulin (AC-tub) antibody (red). Basal body (yellow) indicates colabeling with CETN2 and Ac-α-tubulin. Daughter centrioles label with GFP-CENT2 only; insets show enlargements of primary cilia.