Sigg MA et al. (2017), Evolutionary Proteomics Uncovers Ancient Associ...

XB-ART-54467

Dev Cell 2017 Dec 18;436:744-762.e11. doi: 10.1016/j.devcel.2017.11.014.

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Evolutionary Proteomics Uncovers Ancient Associations of Cilia with Signaling Pathways.

Sigg MA , Menchen T , Johnson J , Jungnickel MK , Choksi SP , Garcia G , Busengdal H , Dougherty GW , Pennekamp P , Werner C , Rentzsch F , Florman HM , Krogan N , Wallingford JB , Omran H , Reiter JF .

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Cilia are organelles specialized for movement and signaling. To infer when during evolution signaling pathways became associated with cilia, we characterized the proteomes of cilia from sea urchins, sea anemones, and choanoflagellates. We identified 437 high-confidence ciliary candidate proteins conserved in mammals and discovered that Hedgehog and G-protein-coupled receptor pathways were linked to cilia before the origin of bilateria and transient receptor potential (TRP) channels before the origin of animals. We demonstrated that candidates not previously implicated in ciliary biology localized to cilia and further investigated ENKUR, a TRP channel-interacting protein identified in the cilia of all three organisms. ENKUR localizes to motile cilia and is required for patterning the left-right axis in vertebrates. Moreover, mutation of ENKUR causes situs inversus in humans. Thus, proteomic profiling of cilia from diverse eukaryotes defines a conserved ciliary proteome, reveals ancient connections to signaling, and uncovers a ciliary protein that underlies development and human disease.

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Species referenced: Xenopus laevis
Genes referenced: actb cep164 enkur pitx2 rpe shh tuba4b
GO keywords: G-protein coupled receptor signaling pathway [+]
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	Figure 5. ENKUR Is a Conserved Ciliary Protein Expressed by Cells with Motile Cilia (A) Whole-mount in situ hybridization for Enkur in N. vectensis embryos at various developmental stages. Enkur is expressed throughout the embryo and is enriched at the aboral pole (arrowhead) in 48- and 74-hr embryos. Scale bar, 50 mm. (B) In situ hybridization for Enkur in S. purpuratus embryos at mesenchyme blastula (MB), early gastrula (EG), late gastrula (LG), and prism (PM) stages. Scale bar, 50 mm. Enkur is expressed in all cells and enriched at the apical pole (arrowhead) in EG embryos. (C) In situ hybridization of stage-23 Xenopus laevis embryo shows expression of Enkur in epidermal cells. (D) qRT-PCR measurement of Enkur detected expression in isolated adult mouse lungs, trachea, and testis. Error bars represent SDs from 6 technical replicates. Expression was validated using 2 distinct primer pairs. (E) Immunofluorescent staining of S. rosetta ENKUR fused to GFP (green), cilia (TUBac, red), and the basal body (CEP164, blue) expressed in IMCD3 cells. Scale bar, 2.5 mm. (F) Immunofluorescent staining of cilia (ARL13B, red) and a fusion of S. purpuratus ENKUR with GFP (green) expressed in RPE-1 cells. ENKUR-GFP localizes to cilia. Nuclei are stained with Hoechst (blue). Scale bar, 2.5 mm. (G) A multiciliated epidermal cell of a stage-23 X. laevis embryo expressing X. laevis ENKUR fused to GFP (green), membrane-red fluorescent protein (red), marking the plasma and ciliary membranes, and Centrin 4 (CENT4)-blue fluorescent protein (BFP, blue) to mark the basal bodies. ENKUR localizes along the length of cilia. Scale bar, 10 mm. (H) Immunofluorescent staining of primary cultured mouse tracheal epithelial cells for ENKUR (green), cilia (TUBac, red), and basal bodies (CEP164, white), and imaged using structured illumination microscopy. ENKUR localizes to mouse tracheal epithelial cilia. Scale bar for whole cells (top and center panels), 5 mm. Scale bar for cilia (right panel), 2.5 mm. (I) Immunoblotting for ENKUR protein in testis and trachea lysates from littermate control and Enkur -/- mice. ENKUR is expressed in testis and trachea, whereas Enkur -/- mice do not produce detectable ENKUR. See also Figures S4 and S5.
	Figure 6. ENKUR Is Required for Left-Right Axis Patterning in Mice (A) Whole-mount in situ hybridization of a stage-17 X. laevis embryo for Enkur. Ventral view of dorsal resection of embryo. Only the posterior region of the embryo is shown. Enkur is expressed in the gastrocoel roof plate (GRP). (B) Fluorescence imaging of X. laevis GRP cilia expressing GFP-ENKUR (green) and CENT4-BFP (blue). CENT4-BFP marks basal bodies. Scale bar, 5 mm. (C) In situ hybridization for Pitx2c of a stage-28 X. laevis control embryo and an Enkur knockdown (KD) embryo. Pitx2c is expressed in the left lateral plate (arrowheads and high-magnification images) of control embryos but is absent in the Enkur KD embryo. (D) Quantification of Pitx2c expression patterns in control and Enkur KD embryos. (E) In situ hybridization of somite stage 2â€“4 littermate control and Enkur -/- mouse embryos for Enkur. At this stage, Enkur is expressed exclusively in the node. Enkur -/- embryos do not express detectable Enkur. Scale bars, 50 mm (left panels) and 25 mm (right panels). (F) Immunofluorescence staining of somite stage 2â€“4 mouse embryos for ENKUR (green) and cilia (TUBac, red). ENKUR localizes to the cilia of the node and is not detectable in the cilia of Enkur -/- nodes. Nuclei are stained with Hoechst (blue). Scale bars, 10 mm (left 2 panels) and 2.5 mm (right 6 panels). (G) Photographs of thoracic and abdominal organ positions. The right (R) and left (L) side of the body are indicated. Some Enkur / mice display situs ambiguus, illustrated here by abnormal heart (H) apex orientation, midline liver (L), and right-sided spleen (Sp). Some Enkur -/- mice display situs inversus, a complete reversal of the left-right axis, including a right sided stomach (St) and inversion of the left lateral (LLL), middle (ML) and right lateral (RLL) liver lobes. Scale bar, 1 cm. (H) Quantification of situs in littermate control and Enkur / mice.
	Figure S5. ENKUR supplemental data, Related to Figure 5 and 6. (A) Whole mount in situ hybridization of N. vectensis embryos at the indicated developmental stages for Enkur. Surface view indicates the heterogeneous expression of Enkur. Arrowhead marks aboral pole. Scale bar, 50Î¼m. (B) In situ hybridization of sea urchin embryos at mesenchyme blastula (MB), early gastrula (EG), late gastrula (LG) and prism (PM) stage using the sense probe for Enkur. Scale bar, 50 Î¼m. (C) Immunofluorescence imaging of primary cultured mouse tracheal epithelial cells for cilia (TUBac, red) and ENKUR (green). Nuclei are labeled with Hoechst (blue). Cells transfected with 4 different shRNAs against Enkur display reduced ENKUR immunofluorescence at cilia. Scale bar, 5Î¼m. (D) Immunofluorescent staining of cilia (ARL13B, red) and ENKD1 fused to GFP (green) expressed in IMCD3 cells. ENKD1- GFP co-localizes with the basal body component CEP164 (blue). Scale bar for whole cell image, 5 Î¼m. Scale bar for cilia only image, 2.5 Î¼m. (E) A schematic diagram of the wild type mouse Enkur allele (+) and the targeted allele (-). The targeting vector was designed to replace part of exon 2 with a neomycin-resistance and thymidine kinase fusion. (F) Southern blot confirming homologous recombination at the mouse Enkur locus (wild type, +/+; heterozygote, +/-; homozygous mutant, -/-). (G) RT-PCR of X. laevis control embryos and those injected with 10 or 20 ng Enkur morpholino. Enkur expression is reduced in morphants. (H) Quantification of Pitx2c expression patterns in control embryos, embryos injected with Enkur MO and embryos injected with Enkur MO plus plasmid encoding sea urchin Enkur-GFP. (I) Immunofluorescence image of the Xenopus GRP depicting ACTIN (red) and cilia (TUBac, green). Ciliation of the GRP in embryos with KD of Enkur expression is similar to control embryos. Scale bar, 100Î¼m. (J) Representative images of Pitx2 in situ hybridization in which Pitx2c expressed is on the right side, left side, both sides or is absent in Xenopus embryos (ventral view). (K) In situ hybridization of 1-4 somite stage mouse embryos using the sense probe for Enkur. Asterisk marks the node. Scale bar, 100Î¼m. (L) A chest X-ray of affected individual OP 1605-II2 demonstrating dextracardia. The heart is indicated with an â€œHâ€, right (R) and left (L) side of the body are indicated.

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