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doi: 10.1242/10.1242/dev.00407

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The left-right determinant Inversin is a component of node monocilia and other 9+0 cilia

Daisuke Watanabe^1,*, Yukio Saijoh^1,*, Shigenori Nonaka^1,*, Genta Sasaki^1,*, Yayoi Ikawa^1,*, Takahiko Yokoyama^2, and Hiroshi Hamada^1,*,

¹ Developmental Genetics Group, Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamada-oka, Suita, Osaka 565-0871, Japan
² Department of Anatomy and Developmental Biology, Tokyo Women's Medical University, School of Medicine, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8600, Japan
^* CREST, Japan Science and Technology Corporation (JST)
Present address: Department of Anatomy and Developmental Biology, Kyoto Prefecture University of Medicine, Kawaramachi-Hirokoji, Kamikyo-ku, Kyoto 602-0841, Japan

View larger version (94K): [in a new window]   Fig. 1. Rescue of the Inv/Inv phenotype by expression of an Inv::GFP transgene. (A) Schematic representation of the structure of the Inv::GFP fusion protein. IQI and IQII represent two putative calmodulin binding motifs present in Inv. (B) Expression of the Inv::GFP fusion protein in a mouse embryo at embryonic day (E) 12.5. GFP fluorescence was detected in the transgenic embryo shown on the right but not in the non-transgenic littermate shown on the left. (C-G) Comparison of Inv/Inv mice with or without the Inv::GFP transgene at postnatal day 2. (C) Fluorescence was detected in the forelimb of an Inv/Inv, Inv::GFP mouse (right) but not in that of an Inv/Inv mouse without the transgene (left). (D) The stomach (arrowhead) was located on the right side of an Inv/Inv mouse (left embryo) but on the left side of an Inv/Inv, Inv::GFP mouse (right embryo). (E) Lung lobation pattern of an Inv/Inv mouse (left) and an Inv/Inv, Inv::GFP mouse (right). L, left; R, right. (F,G) The kidneys of an Inv/Inv mouse (F) exhibited multiple cysts (arrowheads) that were not present in the Inv/Inv, Inv::GFP mouse (G). Kidney sections were stained with Hematoxylin and Eosin.

View larger version (111K): [in a new window]   Fig. 2. Specific localization of GFP::Inv in the primary cilia of cultured fibroblasts. The subcellular localization of Inv was examined in primary fibroblasts established from Inv/Inv, Inv::GFP transgenic mice (A-I) or from wild-type (non-transgenic) mice (J-L). (A-C) Nonfixed cells were examined for GFP fluorescence (A) and by differential interference contrast (DIC) microscopy (B); the merged image of A and B is shown at higher magnification in C. GFP fluorescence was associated with rod-like structures (arrowheads) at the periphery of the nucleus (Nu). Scale bar: 5 µm. (D-I) Cells were fixed and subjected to double immunofluorescence staining with antibodies to GFP (red; D,G) and antibodies to acetylated tubulin (green; E,H). (F) Merged image of D,E. (G,H) Higher magnification views of D,E, respectively. (I) DIC image corresponding to G,H. The Inv::GFP fusion protein was specifically localized to primary cilia that were positive for acetylated tubulin (arrowheads). (J-L) Double staining with antibodies to Inv (red, J) and antibodies to acetylated tubulin (green, K). (L) Merged image of J,K. The endogenous Inv protein was also detected in the primary cilia of wild-type fibroblasts (arrowheads).

View larger version (50K): [in a new window]   Fig. 3. The endogenous Inv protein is also localized to primary cilia. (A) Primary fibroblasts established from wild-type (+/+), Inv/+, Inv/Inv, Inv/Inv, Inv::GFP mice were subjected to western blot with an anti-Inv antibody. The endogenous Inv protein (asterisk) is detected in +/+ and Inv/+ fibroblasts but not in Inv/Inv fibroblasts. The antibody also detected the Inv::GFP protein (arrowhead) in Inv/Inv, Inv::GFP fibroblasts. (B) Primary fibroblasts derived from Inv/Inv, +/+ or Inv/Inv, Inv::GFP mice were subjected to double staining with anti-acetylated tubulin (a-c, green) and anti-Inv antibody (d-f, red). Note that primary cilia are positive for acetylated tubulin (a-c, arrowheads). The anti-Inv antibody detects primary cilia in +/+ (e) and Inv/Inv, Inv::GFP (f) fibroblasts, but not those in Inv/Inv fibroblasts (d).

View larger version (57K): [in a new window]   Fig. 4. Inv::GFP localization during cilium formation. Primary fibroblasts established from Inv/Inv, Inv::GFP transgenic mice were cultured until they were subconfluent and were then subjected to double staining with antibodies to GFP (red) and antibodies to -tubulin (green). A low-magnification view is shown in A and higher magnification views of centrosomal regions are shown in B-D. Several distinct patterns of Inv::GFP staining were detected. (B) Inv::GFP is localized to one of the centrioles, the mother centriole, even before extension of a cilium becomes obvious (arrowhead). (C) Inv::GFP is restricted to the extension of a mother centriole as it begins to form a cilium. (D) Inv::GFP is present along the entire length of a fully developed cilium. Scale bar: 2.5 µm.

View larger version (141K): [in a new window]   Fig. 5. Localization of Inv::GFP to the monocilia of the node. The node of Inv/Inv, Inv::GFP embryos at E8.0 was examined with a confocal microscope. The Inv::GFP fusion protein was preferentially localized to the cilia of the node (arrowheads). (A) DIC image of the ventral side of the node of a nonfixed embryo. (B) GFP fluorescence image of the node region shown in A. Many dots of fluorescence are apparent in the node. A, anterior; L, left; P, posterior; R, right. (C) Lateral view of the node reconstructed from serially z-scanned confocal images of GFP fluorescence. (D) Merged GFP fluorescence and DIC images of a single node cilium. (E,F) Double staining of an embryo with antibodies to acetylated tubulin (green; E) and antibodies to GFP (red; F). The node cilia, as revealed by their content of acetylated tubulin, were also positive for GFP. Scale bars: in A 20 µm for A,B; in D, 2.5 µm for D.

View larger version (85K): [in a new window]   Fig. 6. Localization of Inv::GFP to 9+0 cilia but not to 9+2 cilia. Sections of the kidney (A-C), pituitary (D-F), retina (G,H), cerebellum (I), oviduct (J-L) and trachea (M-R) of Inv/Inv, Inv::GFP mice were subjected to double immunostaining with antibodies to GFP (red) (A,D,G,I,J,M) and antibodies to acetylated tubulin (green) (B,E,K,N) or with antibodies to GFP (red; P) and antibodies to -tubulin (green; Q). DIC images are shown at the right of corresponding fluorescence images (C,F,H,L,O,R). Note the prominent localization of Inv::GFP in 9+0 cilia (arrowheads) of kidney (A-C), pituitary gland (D-F) and retinal (G,H) cells. The fusion protein was not detected in the 9+2 cilia (arrowheads) of oviduct (J-L) or tracheal (M-O) cells, but was detected in the cytoplasm (arrow) of the Purkinje cells (I, arrow) and in the basal body (arrows) of tracheal cells (P-R, arrows). Both 9+0 and 9+2 cilia were positive for acetylated tubulin (B,E,K,N) and the basal body was positive for -tubulin (Q). Pt, proximal tubule; Rpe, retinal pigment epithelium.

View larger version (103K): [in a new window]   Fig. 7. Localization of calmodulin to 9+2 cilia. Sections both of the trachea (A-C) and kidney (D-F) of newborns and of the node (G-I) of embryos at E8.0 were subjected to double staining with antibodies to acetylated tubulin (green) (A,D,G) and antibodies to calmodulin (red) (B,E,H). Corresponding DIC images are shown on the right of each pair of fluorescence images (C,F,I). Calmodulin was localized to the 9+2 cilia (arrowheads) of tracheal cells, but not in the 9+0 cilia (arrowheads) of the kidney and node.

View larger version (56K): [in a new window]   Fig. 8. Localization of the ankyrin repeat domain of Inv to the node cilia and its rescue of LR defects of Inv/Inv mice. (A) Schematic representation of the InvC::GFP fusion protein. (B) Phenotype of Inv/Inv mice harboring the InvC::GFP transgene at 2 weeks (a,b) or 4 weeks (c) of age. The transgene rescued the LR defects of Inv/Inv mice as revealed by the normal locations of the stomach and spleen (arrows in a,c) and the normal lobation pattern (L:R, 1:4) of the lungs (b). Although the kidneys appeared normal at 2 weeks (arrowheads in a), they were polycystic at 4 weeks (arrowheads in c). (C) Localization of the InvC::GFP protein in cells of Inv/Inv, InvC::GFP mice. Kidney sections of newborn animals, primary fibroblasts and E8.0 embryos were double immunostained with antibodies to GFP (red, a-c) and antibodies to acetylated tubulin (green, d-f), as indicated. The corresponding DIC images are shown below each pair of fluorescence images (g-i). InvC::GFP was detected in the primary cilia of cultured fibroblasts and in the monocilia of the embryonic node, but not in the 9+0 cilia of the kidney. Arrowheads indicate the positions of cilia.

View larger version (99K): [in a new window]   Fig. 9. Rescue of the LR defects of Inv/Inv embryos by artificial nodal flow. Both Inv/+ and Inv/Inv embryos were exposed to artificial leftward flow as described previously (Nonaka et al., 2002) and were then examined for the directions of heart looping and embryonic turning. (A) The numbers of embryos that exhibited normal or reversed heart looping or embryonic turning, or outlooping of the heart or an unturned tail were determined. (B,C) Examples of the rescued (B) and non-rescued (C) Inv/Inv embryos are shown. Heart looping is indicated by the broken lines. Scale bar: 1 mm.

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