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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 2–4 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 2–4 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. |
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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, 50μm. (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 μm. (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, 5μm. (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 μm. Scale bar for cilia only image, 2.5 μm. (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, 100μm. (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, 100μm. (L) A chest X-ray of affected individual OP 1605-II2 demonstrating dextracardia. The heart is indicated with an “H”, right (R) and left (L) side of the body are indicated. |
