QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 14 January 2009
doi: 10.1242/dev.028464

This Article Summary Full Text Full Text (PDF) Supplementary Material Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Yin, Y. Articles by Tickle, C. Search for Related Content PubMed PubMed Citation Articles by Yin, Y. Articles by Tickle, C. Social Bookmarking                         What's this?

The Talpid3 gene (KIAA0586) encodes a centrosomal protein that is essential for primary cilia formation

Yili Yin^1,*, Fiona Bangs^2,*, I. Robert Paton³, Alan Prescott⁴, John James⁴, Megan G. Davey³, Paul Whitley², Grigory Genikhovich⁵, Ulrich Technau⁵, David W. Burt³ and Cheryll Tickle^2,

¹ Division of Cell and Developmental Biology, Wellcome Trust Biocentre, The University of Dundee, Dundee DD1 5EH, UK.
² Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY, UK.
³ Department of Genetics and Genomics, The Roslin Institute and Royal (Dick) School of Veterinary Studies, The University of Edinburgh, Midlothian EH25 9PS, UK.
⁴ Centre for High Resolution Imaging and Processing, The University of Dundee, Dundee DD1 5EH, UK.
⁵ Developmental Biology Section, Faculty for Life Sciences, University of Vienna, Althanstrasse 14, 1090 Wien, Austria.

View larger version (124K): [in this window] [in a new window]   Fig. 1. Primary cilia defect in talpid3 mutant embryos. Immunostaining of sections of wild type (A,C,E,G) and talpid3 mutant (B,D,F,H) chicken embryos; anti- tubulin (red) for centrosome; anti-acetylated tubulin (green) for ciliary axoneme. (A) Wild-type neural tube, primary cilia (arrows) protruding into lumen (*) from centrosomes. (B,D) talpid3 mutant neural tube (centrosomes are indicated with arrowheads), ciliary axonemes absent, compare with A. (C) Wild-type mesonephric duct; primary cilia (arrows) protruding from centrosomes into lumen (*). (E) Wild-type limb bud; primary cilia on mesenchyme cells (arrow). (F) talpid3 mutant limb bud; centrosomes are indicated with an arrowhead on mesenchyme cells, cilia axonemes are absent (compare with E). (G) Wild-type notochord; primary cilia project from centrosomes (arrows). (H) talpid3 mutant notochord; centrosomes are indicated with arrowheads, ciliary axonemes are absent, compare with G. (I-L) SEM of dorsal surface of wing bud and luminal surface of neural tube from HH24 embryos. (I) Wild-type wing bud; black circles indicate primary cilia. (I') A higher magnification of primary cilium. (J) talpid3 mutant wing bud; no primary cilia visible. (K) Wild-type neural tube; black arrows indicate primary cilia emerging from pits on apical surface of cells lining lumen. (L) talpid3 mutant neural tube; no primary cilia visible, although there are protrusions from apical surface of cells. (M,N) Sections of the mesonephric kidney (Mn) at 7 days of development stained with Haematoxylin and Eosin. (M) Wild-type embryo. (N) talpid3 mutant embryo, note cysts (*). M, mullerian duct; G, gonad. Scales bars: 5 µm in A-F; 10 µm in G-L; 500 nm in I'; 500 µm in M,N.

View larger version (72K): [in this window] [in a new window]   Fig. 2. Rescue of primary cilia. (A,B) Rescue of primary cilia in talpid3 mutant neural tube after electroporation with construct encoding full-length chicken Talpid3. (A) Section of electroporated side of neural tube expressing RFP, showing rescue of primary cilia, indicated with arrows, projecting into lumen (*) and stained with acetylated tubulin (green). (A') Higher magnification of outlined area in A; rescued primary cilia are indicated with arrows. (B) Section of non-electroporated side of neural tube, no primary cilia present. (C-E) Rescue of primary cilium formation in talpid3 mutant CEFs. (C) Wild-type CEF with primary cilium axoneme stained with acetylated tubulin, (green; arrow) protruding from centrosome stained with -tubulin (red). (D) talpid3 mutant CEF no primary cilia; acetylated tubulin staining (green) almost entirely overlaps with centrosome staining (red, arrow). (E) talpid3 mutant CEF transfected with a construct encoding full-length chicken Talpid3 shows rescue of primary cilium, arrow indicates axoneme protruding from centrosome. Scale bars: 8 µm in A,B; 4 µm in A'; 3 µm in C-E.

View larger version (58K): [in this window] [in a new window]   Fig. 3. Ciliogenesis and actin organisation in wild type and talpid3 mutant cells. (A-E) TEM sections through neuroepithelium of stage HH24 chicken embryos. (A,B) Wild-type embryo; primary axonemes project from pits in apical cell surfaces into lumen (*); basal bodies (black arrow) with satellites (arrowhead); s, sister centrioles. (C-E') talpid3 mutant neuroepithelium. No primary cilia project from cell surface into lumen. In C, a few short microtubules are seen at distal end of basal body (bracket). In E, vesicle (v) present near basal body; (E') Higher magnification of basal body in E showing vesicle near but not fused with basal body (bracket indicates gap). In C,D, the basal body is not orientated towards the apical surface. (F-H) Section showing one side of neural tube from stage HH24 embryos stained with phalloidin (red) and -tubulin (green). (F) Wild-type embryo; an even continuous band of phalloidin staining is present at apex of cells abutting lumen (*). (G) talpid3 mutant embryo; uneven phalloidin staining is present. (H) talpid3 mutant embryo electroporated with a construct encoding full-length chicken Talpid3; rescue of phalloidin staining can be seen, compare with F. (I-N) Actin cytoskeleton of wild-type and talpid3 mutant cells from limb buds in primary culture. Compare I with J (actin staining), and K with L (phalloidin staining). Arrows in J,L indicate actin-containing filopodia. Fewer focal adhesions expressing Vinculin (arrows in M) in talpid3 mutant cells (N) compared with wild-type cells (M) 24 hours after seeding. Scale bars: 589 nm in A-D; 721.5 nm in E; 294 nm in E'; 11 µm in F-H; 5 µm in I-N.

View larger version (44K): [in this window] [in a new window]   Fig. 4. Localisation of Talpid3 protein to centrosome. (A-I) Co-immunofluorescence staining in serum-starved CEFs; anti -tubulin (green) marks the centrosome; anti-Talpid3 is in red; merged images are shown in the right-hand column. (A-C) Localisation of Talpid3 to centrosome. (D-F) Higher power image of centrosome shown in A-C, Talpid3 is localised to both centrioles. (G-I) Talpid3 is not detected at centrosome in talpid3 mutant cells. (J-U) Co-immunofluorescence staining in HEK293T cells. Pericentrin (red) marks the centrosomes (first column) (J,M,P,S); Myc (K,N) or Flag (Q,T) tagged Talpid3 protein (green) is shown in the second column; merged images are shown in third column (L,O,R,U). DNA is stained with DAPI (blue). Scale bars: 5 µm in A-C,G-I; 0.13 µm in D-F; 3 µm in J-U. Arrows indicate centrosomes.

View larger version (114K): [in this window] [in a new window]   Fig. 5. Microtubule dynamics in wild-type and talpid3 mutant cells in culture. Microtubule organisation visualised with anti -tubulin (green), nuclei stained with DAPI (blue). CEFs from either wild-type (E,F,I,J,M,N) or talpid3 mutant cells (G,H,K,L,O,P) were treated with nocodazol and microtubule regrowth assessed. (A-D) Untreated cells. (E-H) Microtubule organisation is lost when wild-type and talpid3 cells were treated with nocodazol. (I-L) Ten minutes after nocodazol removal, microtubule nucleation has begun in wild-type (I,J) and talpid3 mutant cells (K,L). (M-P) Sixty minutes after nocodazol removal, microtubules fully reformed in wild-type cells (M,N), but regrowth delayed in talpid3 mutant cells (O,P). Scale bars: 20 µm in A,C,E,G,I,K,M,O; 5 µm in B,D,F,H,J,L,N,P.

View larger version (115K): [in this window] [in a new window]   Fig. 6. Alignment of Talpid3 proteins and domain predictions. (A) Scale and location of talpid3 mutation with highly conserved region (purple) and coiled-coil domain (green; see Fig. S4 in the supplementary material), exons 9-12 are indicated between arrows. (B) Multiple alignments of orthologous Talpid3 sequences (amino acids 295-735) from vertebrates and Nematostella vectensis using MUSCLE displayed with JALVIEW. Predicted coiled-coil region green box between amino acids 498-529; highly conserved region purple box, amino acids 498-585. Chicken sequence is used as a reference and does not contain gaps. (C) Distant sequence homologies between amino acids 392-611 (DOMAINATION domain 3) (see Table S1 in the supplementary material) of chicken Talpid3, and amino acids 0-229 of human CCCAP. Predicted coiled-coil region from PCOILS analysis (see Fig. S4 in the supplementary material) is shown by a solid outline; the highly conserved region is indicated by a broken outline. The locations of boundaries for domains encoded by exons 9-13 are shown by arrows.

View larger version (62K): [in this window] [in a new window]   Fig. 7. Structure/function analysis of Talpid3 protein: rescue of cilia formation and neural tube patterning in talpid3 mutant embryos and centrosomal localisation. (A-H) Schematic diagrams of chicken Talpid3 protein illustrating expression constructs used for rescue experiments. Coiled-coil domain (green) highly conserved region (purple) is shown. (A) Full-length Talpid3 protein. (B) N-terminal 1-366 amino acids, note talpid3 mutation leads to premature stop codon after amino acid 366. (C) Amino acids 366-1524 containing highly conserved region. (D) Amino acids 483-1524 containing highly conserved region. (E) Amino acids 533-1524 lacking coiled-coil domain. (F) Amino acids 483-1524 containing coiled-coil domain but lacking remainder of highly conserved region. (G) Amino acids 653-1524 lacking highly conserved region. (H) Amino acids 483-653 highly conserved region only. Columns show number of talpid3 mutant embryos with cilia/number of embryos tested and number of talpid3 mutant embryos with neural tube patterning rescued/number of talpid3 mutant embryos tested. NT, not tested. (I-P) Transverse sections of talpid3 mutant neural tube electroporated with expression constructs, dorsal is upwards; ventral is downwards. Expression domains of homeodomain transcription factors (green), RFP is in red (control for transfection efficiency); nuclei stained with DAPI (blue). (I-L) Expression patterns after electroporation of construct C. (I) Nkx2.2 and (J) Islet1 (arrows) expression on electroporated side, indicated by RFP (red). (K) Pax6 is detected in a normal expression domain (between arrows), with a gap of non-Pax6 expressing cells indicated by white bracket. Pax6 is also expressed more ventrally where cells are not electroporated (RFP absent). (L) Pax7 expression is restricted more dorsally on electroporated side; compare level of arrowhead on electroporated side (RFP, red), with that on control side. (M-P) Expression patterns after electroporation with construct E, showing lack of rescue, despite successful electroporation (RFP, red). (M,N) No Nkx2.2 and Islet1 expression is induced when compared with non-electroporated side of neural tube. (O,P) Pax6 and Pax7 expression domains remain expanded ventrally on electroporated side of neural tube. (Q,R) Rescue of primary cilia in talpid3 mutant neural tube following electroporation with construct D. Primary cilia are indicated with arrows, stained with anti-acetylated tubulin (green) and centrosomes indicated with arrowheads, stained with anti--tubulin (red). Compare electroporated side (Q) with non-electroporated side (R). (S-U) HEK293T cells transfected with hsKIAA0586ex11/12::GFP. (S) -tubulin marks centrosome (red, arrowed). (T) Localisation of hsKIAA0586ex11/12::GFP fusion protein (green; arrow). (U) Merged image shows hsKIAA0586ex11/12::GFP colocalises with -tubulin in centrosome (arrow). Scale bars: 175 µm in I-P; 8 µm in Q-R; 3 µm in S-U.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter