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First published online 14 January 2009
doi: 10.1242/dev.028464
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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.
Author for correspondence (e-mail:
cat24{at}bath.ac.uk)
Accepted 11 December 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Primary cilia, Centrosome, Hedgehog signalling, Ciliopathies, Talpid3, Chicken, Neural tube, Embryo
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Davey et al., 2006;
Ede and Kelly, 1964a;
Ede and Kelly, 1964b).
Development of all the regions affected in the talpid3
mutant embryo requires Hedgehog (Hh) signalling and analysis of the developing
mutant limb bud and neural tube has shown that it is the response to Hh
signalling that is defective (Lewis et
al., 1999). Expression of some downstream Shh target genes in
talpid3 mutant limb buds, head, neural tube and somites is
lost, whereas other genes are expressed more widely, suggesting that there is
failure both to activate gene expression in response to Shh and to repress
gene expression in its absence (Buxton et
al., 2004; Davey et al.,
2006; Lewis et al.,
1999). These opposing effects can be understood in terms of the
bifunctionality of the Gli proteins, the transcriptional effectors of
vertebrate Hedgehog (Hh) signalling, which function as activators (mainly
Gli1, Gli2) or are processed to short forms (mainly Gli3) that function as
repressors (Aza-Blanc et al.,
2000; Bai et al.,
2002; Marigo et al.,
1996; Ruiz i Altaba,
1999). Indeed, direct analysis of Gli3 proteins in
talpid3 mutant tissues showed that Gli3 processing is
abnormal, although translocation to the nucleus still occurs
(Davey et al., 2006). We have
identified KIAA0586 (Talpid3) as the gene affected in
talpid3 mutant embryos
(Davey et al., 2006).
KIAA0586 is ubiquitously expressed and encodes a novel protein with
no previously known function (Davey et
al., 2006).
The phenotype of talpid3 mutant chicken embryos,
including the inability to process Gli3, is strikingly similar to that of
mouse embryos with mutations in genes encoding centrosomal or intraflagellar
transport (IFT) proteins, such as Arl13b, OFD1, Polaris, IFT172, Kif3a,
Dnchc2 and Ftm (Caspary et
al., 2007; Ferrante et al.,
2006; Haycraft et al.,
2005; Huangfu et al.,
2003; May et al.,
2005; Vierkotten et al.,
2007). These mouse mutants lack normal primary cilia, the site
where cells receive Shh signals and other cell-cell signals
(Corbit et al., 2005;
Eggenschwiler and Anderson,
2007; Rohatgi et al.,
2007). An increasing number of human syndromes, collectively known
as ciliopathies, which affect cilia formation and function, also have
phenotypic features similar to talpid3 mutant chickens,
such as polydactyly (Badano et al.,
2006; Bisgrove and Yost,
2006; Eley et al.,
2005; Fliegauf et al.,
2007; Pazour and Rosenbaum,
2002; Tobin and Beales,
2007). Interestingly, KIAA0586 was identified in a human
centrosome proteome (Andersen et al.,
2003). We have therefore investigated whether the mechanism
underlying abnormal Hh signalling in the talpid3 mutant
involves a failure of primary cilia formation and whether the Talpid3 protein
is enriched in the ciliary apparatus.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). Talpid3 carriers were maintained as
described previously (Davey et al.,
2006).
Section immunohistochemistry
Embryos were fixed in 4% PFA for 2 hours at room temperature (RT) and
genotyped as described previously (Davey
et al., 2006). Selected embryos embedded in 10% sucrose, 7.5%
gelatine were sectioned at 10 µm or 40 µm. Sections were stained as
described previously (Das et al.,
2006). Primary antibodies were as follows: for visualisation of
primary cilia, rabbit anti-
-tubulin 1:1000 (Sigma) and mouse
anti-acetylated tubulin 1:1000 (Sigma); for microtubules, mouse anti
-tubulin 1:1000 (Sigma); mouse anti-Islet1 1:10 (Developmental Studies
Hybridoma Bank; DSHB), mouse anti-NKX2.2 1:5 (DSHB), mouse anti-PAX6 1:2
(DSHB), mouse anti-PAX7 1:10 (DSHB); and for filamentous actin, Alexa Fluor
546 phalloidin 1:100 (Molecular Probes). Secondary antibodies were:
Alexa-Fluor-488 conjugated anti-mouse 1:500 (Molecular Probes),
Alexa-Fluor-546 conjugated anti-rabbit 1:500 (Molecular Probes) and
Alexa-Fluor-610 conjugated anti-rabbit 1:1000 (Molecular Probes). Sections
counterstained with DAPI, viewed using Leica DMR compound microscope or Zeiss
LSM510 or Nikon eC1 laser scanning confocal microscope.
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). For SEM,
samples washed in 100% acetone for 10 minutes before critical point drying,
flushed five times to ensure excess acetone was removed, dried, then placed on
carbon mounts, coated with Au/Pd and imaged using an ESEM at 15 kV, 10 mm WD
in high vacuum mode. For TEM sections cut between 70-100 nm and viewed with
FEI Tecnai 12 transmission electron microscope.
Histology
Seven-day-old embryos fixed 4% PFA overnight, washed in PBS, dehydrated
into 100% xylene, then wax embedded overnight. Sections were cut at 5 µm,
dewaxed, stained with Hematoxylin and Eosin, and photographed using Leica DMR
compound microscope.
Neural tube electroporation
The lumen of the neural tube of stage HH10-14 talpid3
mutant flock embryos was injected with 1 µg/µl of construct encoding
either full-length chicken Talpid3 (ggKIAA0586) or fragments of chicken
Talpid3 in pCAGGs, plus 0.1 µg/µl pCAGGS-RFP and 0.02% Fast Green, and
electroporated with square wave current of 25 mV applied five times for 15
mseconds (CUY21 electroporator, Protech International, TX, USA). Embryos were
incubated for a further 48 hours, which is when mutant embryos can be
identified, then fixed and sectioned.
Cell culture for examination of cilia formation, cilia rescue and cytoskeletal organisation
Chick embryonic fibroblasts (CEFs) were generated from HH24 embryo bodies
by cutting the tissue into small pieces before trypsinising for 1-2 minutes.
Trypsin was inhibited with serum, tissue disaggregated, cells spun down and
then resuspended in fresh DMEM/Ham's F12 (Gibco) plus 10% foetal calf serum
(FCS). Cells were passaged twice before use. Primary cell cultures were
generated from HH24 limb buds, by trypsinisation for 1-2 minutes, tissue
disaggregated, then cells spun down and resuspended in 20 µl of DMEM and
seeded onto fibronectin (SIGMA) coated 13 mm coverslips. Cells were left to
adhere for 1 hour before flooding with DMEM/Ham's F12+10% FCS. HEK293T cells
were maintained in DMEM+10% FCS under normal cell culture conditions.
In test for rescue of primary cilia, CEFs were seeded onto coated 13 mm coverslips at 1x106/ml and transfected with a construct encoding full-length chicken Talpid3 in pCAGGS (ggKIAA0586) using Fugene6 (Roche) for 48 hours. When near confluence, cells were synchronised in serum-free medium for 2 days, then fixed and stained.
For localisation of tagged Talpid3, HEK293T cells were seeded onto 13 mm coverslips at 1x106/ml, then grown for 18-24 hours before transfection with 1 µg ggKIAA0586::Myc or Flag::ggKIAA0586. For localisation of GFP fusion construct, primary chicken cell cultures or HEK293T cells were transfected with 0.5 µg/µl ggKIAA0586ex11/12::GFP or hsKIAA0586ex11/12::GFP, respectively, in pCAGGS using Fugene6, then observed after 5-7 hours for GFP expression, fixed in 4% PFA 10 minutes, washed three times in PBS and stored for 24 hours at 4°C, then blocked in 5%FCS/PBS+0.5% Triton for 30 minutes.
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An antibody was raised against C-terminal peptide (DSDSSGADTF) of chicken Talpid3 in rabbit. Serum from fifth bleed was used to detect overexpressed HA::ggKIAA0586 by western blot analysis, producing band of 200 kDa. Antibody was then affinity purified by coupling peptide to HiTrap NHS-activated HP column (Amersham) and tested by immunofluorescence in HEK293T cells transfected with HA::ggKIAA0586. All transfected cells were recognised by both anti-Talpid3 and anti-HA antibodies. Purified Talpid3 antibody was diluted 1:2 for cell immunofluorescence staining.
Primary antibodies used were as follows: for visualisation of primary
cilia, rabbit anti--tubulin 1:1000 (Sigma) and mouse anti-acetylated
tubulin 1:1000 (Sigma); for actin and focal adhesions, anti-actin 1:1000
(Sigma), Alexa-Fluor-488 phalloidin 1:1000 (Molecular probes), anti-vinculin
1:1000 (Sigma); for tagged Talpid3 constructs, monoclonal mouse anti-Myc
1:1000 (Sigma) and mouse anti-Flag antibody 1:1000 (Sigma); for centrosomes,
rabbit anti-pericentrin 1:5000 (abcam); for transfected cells, mouse anti-HA
1:2000 (Sigma); and for microtubules, mouse anti--tubulin 1:1000
(Sigma). All antibodies were applied for 1 hour at room temperature, then
removed with three 5-minute washes in PBS/0.2% Tween20. Secondary antibodies
were: Alexa-Fluor-488 conjugated anti-mouse 1:500 (Molecular Probes),
Alexa-Fluor-546 conjugated anti-rabbit 1:500 (Molecular Probes) and
Alexa-Fluor-610 conjugated anti-rabbit 1:1000 (Molecular Probes), and were
incubated for 1 hour at room temperature. Samples were DAPI stained and
mounted, and viewed on Zeiss LSM510 confocal microscope.
Microtubule re-growth assay
CEFs were seeded onto cover slips in DMEM/Ham's F12 at
0.75x106/ml. 24 hours later treated with 25 µM nocodazol
at 37°C for 1 hour. After nocodazol removal, cells were incubated for 0,
10 or 60 minutes, then fixed in 50% methanol/50% acetone for 10 minutes, and
stained with mouse anti--tubulin as above.
Cloning of the Nematostella vectensis Talpid3 homologue
Nvtalpid3, the cnidarian homologue of Talpid3, was cloned
from Nematostella vectensis by extending an EST sequence using ORF
predictions available for Nematostella genome and by RACE PCR. Primer
sequences are available upon request. Putative full-length clone 6058 bp
(Accession Number FJ428244) encoding conceptually translated 1708 amino acid
protein, was validated by RT-PCR and sequencing.
Bioinformatics analyses of polypeptide sequences
EMBOSS sequence analysis system (Rice
et al., 2000) was used to extract amino acid sequences (SEQRET),
to derive peptide statistics (PEPSTATS), to create plots of sequence
conservation based on multiple sequence alignments (PLOTCON) and to produce
helical wheel diagrams (PEPWHEEL) to visualise distribution of polar and
non-polar residues in alpha helical regions. Multiple alignments of amino acid
sequences were made using MUSCLE (Edgar,
2004) and viewed using JALVIEW
(Clamp et al., 2004). PFAMSCAN
(Finn et al., 2006) and PSCAN
(Gattiker et al., 2002) were
used to scan amino acid sequences for sequence motifs. PCOILS was used to
predict coiled-coil regions (Gruber et
al., 2006). Secondary structure of primary protein sequences were
predicted using SABLE (Porollo et al.,
2004; Wagner et al.,
2005); results were displayed using POLYVIEW
(Porollo et al., 2004). DOMPRO
(Yoo et al., 2008),
SCOOBY-DOMAIN (Pang et al.,
2008) and DOMAINATION (George
and Heringa, 2002) were used to predict domain boundaries,
globular domains and protein domains from local gapped alignments generated
using PSI-BLAST, respectively. GLOBPLOT
(Linding et al., 2003) was
used as described previously (Davey et
al., 2006).
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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-tubulin). Cilia were also seen on limb bud mesenchyme cells
(Fig. 1E); notochord cells
(Fig. 1G); endothelial cells of
both dorsal aorta and cardinal vein; epithelial and mesenchymal cells of
somites; gut epithelia; neuroectoderm of the developing eye; endocardium; and
extra-embryonic mesoderm (data not shown). By contrast, in sections of
talpid3 mutant embryos, no ciliary axonemes could be seen
projecting into the lumen of the neural tube, although centrosomes were
clearly visible (Fig. 1B,D);
nor could primary cilia be seen on cells in the limb bud
(Fig. 1F), notochord
(Fig. 1H) or any of the other
tissues listed above (data not shown). Scanning electron microscopy of
wild-type embryos also showed that primary cilia can readily be distinguished
projecting from the centre of many cells of the outer periderm layer of the
limb bud ectoderm (Fig. 1I,
black circles; Fig.1I')
(41/111 cells examined) and in the neural tube, projecting from pits in the
apical surface of cells lining the lumen
(Fig. 1K, arrows). In
talpid3 mutant embryos, no primary cilia could be seen
projecting from periderm cells (Fig.
1J) (132 cells examined) nor neural tube cells
(Fig. 1L).
In human ciliopathies and mouse mutants that lack primary cilia, a range of
defects can occur in addition to those associated with abnormal Hh signalling,
including polycystic kidneys (Bisgrove and
Yost, 2006; Lehman et al.,
2008). Mutant embryos from our current talpid3
flock occasionally survive for up to 10 days, thus allowing examination of
organs later in development. Histology of embryonic day 7
talpid3 mutant mesonephros (the functional embryonic
kidney in chickens) revealed multiple large cysts
(Fig. 1N, asterisks; compare
with wild-type kidney, Fig.
1M). This is comparable with the pathology of the developing
metanephric kidney seen in mice with abnormal ciliogenesis
(Lehman et al., 2008).
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). This
construct also rescued primary cilia (Fig.
2A,A',B). Furthermore, when mutant chicken embryonic
fibroblasts in culture were transfected with the same expression construct,
primary cilia were rescued (Fig.
2E) (n=5/5 transfected cells observed, compare with
Fig. 2D) and axoneme formation
was comparable with that in wild-type chicken fibroblasts
(Fig. 2C).
Ultrastructural analysis of ciliogenesis and actin organisation in talpid3 mutant cells
Stages in formation of a primary cilium have been deduced from detailed
analysis of fibroblasts and smooth muscle cells in chicken and mammalian
tissues using transmission electronmicroscopy
(Sorokin, 1962;
Sorokin, 1968). We therefore
examined the ultrastructure of talpid3 mutant cells to
gain insights into why ciliogenesis fails.
In wild-type chicken neural tube, most cells had primary cilia projecting
into the lumen (Fig. 3A,B).
Each cilium emerged from a pit in the apical cell surface, and the axoneme was
enclosed in a sheath of ciliary membrane and contained microtubules extending
along its length (Fig. 3A,B).
The basal body can be recognised by its appendages (structures including
satellites and rootlets associated with the mature basal body)
(Fig. 3A,B)
(Sorokin, 1968). In some
sections, the sister centriole located below the basal body could also be seen
(Fig. 3B). By contrast, in
cells of talpid3 mutant neural tube, no primary cilia
projected from apical cell surfaces into the lumen
(Fig. 3C-E) (3/3 mutant embryos
examined). Basal bodies (Fig.
3C-E), however, were readily identified by the presence of
associated appendages (Fig.
3C-E), and these, together with their sister centrioles(s), were
seen in the apical region of the cells. In some cases, a few short
microtubules were present distally on the basal body
(Fig. 3C) but, in most cases,
no trace of axoneme development was observed. Neither were any ciliary
vesicles associated with the basal bodies
(Fig. 3C,D) (55/58 basal bodies
observed, two embryos examined) although in three cases, a vesicle was
observed nearby (Fig.
3E,E'). Therefore in talpid3 mutant
embryos, migration of the centrioles to the apical region of neural tube cells
and maturation of the mother centriole into a basal body appeared unaffected,
but docking, which involves fusion between the ciliary vesicle associated with
the basal body and the apical cell membrane
(Dawe et al., 2007;
Sorokin, 1968), and subsequent
axoneme formation did not occur. The TEM images of talpid3
mutant cells also showed that basal bodies were frequently misorientated and
did not lie perpendicular to the apical cell surface as in normal cells
(Fig. 3C,D; see Fig. S1 in the
supplementary material) [9/18 (50%) basal bodies misorientated in one
talpid3 mutant embryo; 26/40 (65%) basal bodies
misorientated in another].
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), and that apical actin enrichment is required for
ciliogenesis (Pan et al.,
2007). Confocal microscopy of transverse sections of wild-type
chicken neural tube showed an even continuous band of F-actin, stained with
phalloidin at the apex of cells (Fig.
3F), whereas in the talpid3 mutant, even
though there was strong staining at the cell apex, it was punctate and not
continuous (Fig. 3G).
Electroporation of ggKIAA0586, which rescued primary cilium formation (see
Fig. 2A), also restored the
continuous band of phallodin staining at the apex of
talpid3 neural tube cells on the electroporated side
(Fig. 3H). Abnormalities in
microfilament organisation were also seen in cultured mutant cells.
talpid3 mutant limb cells had stronger actin staining at
the ruffled membrane and fewer stress fibres than did wild-type limb cells
(compare Fig. 3I,K with
Fig. 3J,L). In addition, there
were many fine filopodia containing actin around the circumference of the
mutant cells, which were not seen in wild-type cells (compare
Fig. 3I,K with
Fig. 3J,L), a feature
previously observed in scanning electron microscopical studies on
talpid3 mutant limb bud cells in vivo
(Ede et al., 1974). Discrete
vinculin-positive focal adhesions were also less well defined in
talpid3 mutant limb cells than in wild-type cells (compare
Fig. 3M with
Fig. 3N).
Subcellular localisation of Talpid3 protein
To verify that the Talpid3 protein is present in the centrosome [as
suggested by Andersen et al. (Andersen et
al., 2003)], we raised an antibody against the C terminus of
chicken Talpid3 and used this in double immunofluorescence staining with
-tubulin as a centrosomal marker in serum-starved wild-type and
talpid3 mutant chicken embryonic fibroblasts to determine
the subcellular localisation of Talpid3. The talpid3
mutation results in a premature stop codon and, even if a truncated protein
was produced, this antibody would not recognise it. In wild-type fibroblasts,
Talpid3 antibody staining colocalised with -tubulin
(Fig. 4A-C), and was enriched
in both centrioles (Fig. 4D-F),
whereas, in talpid3 mutant fibroblasts, Talpid3 antibody
staining could not be detected in the centrosome
(Fig. 4G-I).
Cytoskeletal organisation and dynamics
The detection of Talpid3 protein in the centrosome is consistent with a
role in primary cilia formation but the centrosome also directs microtubule
organisation, including the mitotic spindle. We therefore examined
localisation of Myc- or Flag-tagged Talpid3 during the cell cycle in HEK293T
cells. Tagged Talpid3 protein (stained with antibodies against either Myc or
Flag) co-localised with Pericentrin (a centrosomal marker) during interphase
(Fig. 4J-L) and metaphase
(Fig. 4M-O). More diffuse
staining of tagged Talpid3 protein was also seen throughout the cytoplasm
during anaphase (Fig. 4P-R) and
telophase (Fig. 4S-U). Despite
the presence of Talpid3 at the centrosome in early phases of the cell cycle,
no spindle defects were observed in talpid3 mutant cells
(data not shown). In addition, there were no obvious differences in
microtubule organisation between chicken embryonic fibroblasts from wild-type
and talpid3 mutant embryos (compare
Fig. 5A,B with
Fig. 5C,D), although there was
a delay in microtubule re-growth after nocodazol treatment (compare
Fig. 5E,F,I,J,M,N with
Fig. 5G,H,K,L,O,P).
Bioinformatics and structure/function analysis of the Talpid3 protein
In order to identify functional domains in the Talpid3 protein, we extended
our previous bioinformatics analysis
(Davey et al., 2006) using
orthologous cDNA sequences of Nematostella vectensis and a predicted
homolog in the genome sequence of Strongylocentrotus purpuratus
(Putnam et al., 2007;
Sodergren et al., 2006).
Alignment of vertebrate Nematostella vectensis and
Strongylocentrotus purpuratus Talpid3 sequences revealed a distinct
highly conserved region between amino acids 498-585
(Fig. 6A,B; see Figs S2,S3 in
the supplementary material), which lies downstream of the Talpid3 mutation,
which would truncate the protein at amino acid 366. This conserved region is
predicted to contain a single coiled-coil domain between amino acids 498-529
(Fig. 6A,B; see Figs S2-S5 in
the supplementary material) and is encoded by exons 11 and 12
(Fig. 6A).
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).
Analysis of the neural tube from talpid3 mutant embryos
electroporated with a series of constructs encoding different fragments of
Talpid3 showed that the entire conserved region is essential for rescue
(Fig. 7A-H). Thus, constructs
containing the entire conserved region (construct D) rescued primary cilia
formation (Fig. 7Q,R) and
neural tube patterning (construct C, Fig.
7I-L), inducing expression of Nkx2.2 and Islet1
(Fig. 7I,J; compare RFP
electroporated side with non-electroporated side) and restricting Pax6 and
Pax7 expression dorsally (Fig.
7K,L). Electroporation of a construct encoding just the conserved
region (Fig. 7H), however, was
not able to rescue neural tube patterning. Neither primary cilia formation nor
neural tube pattern was rescued by constructs encoding C-terminal fragments
lacking the coiled-coil domain (construct E,
Fig. 7E), the adjacent highly
conserved region, amino acids 529-585 (construct F,
Fig. 7F) or the entire
conserved region (constructs B and G, Fig.
7B,G). Fig. 7M-P
shows a talpid3 mutant neural tube electroporated with
construct E. Despite substantial RFP expression, indicating successful
transfection, expression patterns of Nkx2.2, Islet1, Pax6 and Pax7 were
unchanged. Thus, the conserved region is required but not sufficient to rescue
dorsoventral patterning of the neural tube. It should be noted that none of
the constructs (A-H) altered neural tube patterning in wild-type embryos and
therefore do not show any dominant-negative effects, including construct B,
which encodes the fragment of Talpid3 protein predicted to be expressed in the
mutant.
To determine the function of the conserved region in centrosomal
localisation, we transfected a construct encoding this region alone from the
human protein (KIAA0586) fused to GFP (hsKIAA0586ex11/12::GFP) into HEK293T
cells. GFP expression was seen in the centrosome
(Fig. 7T) (3/3 transfected
cells observed) co-localising with -tubulin
(Fig. 7S,U). Likewise, when a
construct encoding the chicken Talpid3 conserved region fused to GFP
(ggKIAA0586ex11/12::GFP) was transfected into chicken primary culture cells,
co-localisation was also seen with -tubulin at the centrosome (data not
shown). These data indicate that the conserved region is sufficient to target
Talpid3 protein to the centrosome.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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) and
similarities with mouse mutants that lack cilia and were originally
highlighted as being defective in Hh signalling
(Haycraft et al., 2005;
Huangfu and Anderson, 2005;
Huangfu et al., 2003).
Primary cilia are absent on cells in all talpid3
chicken mutant tissues studied, including those not known to be dependent on
Hh signalling, such as the mesonephric duct. A growing number of human
conditions known as ciliopathies, including syndromes such as primary cilia
dyskinesia, Bardet-Biedl syndrome (BBS), Joubert syndrome and Meckel syndrome
(Badano et al., 2006;
Bisgrove and Yost, 2006;
Fliegauf et al., 2007;
Tobin and Beales, 2007), have
a range of defects, including those associated with abnormal Hh signalling and
also polycystic kidneys. A role for primary cilia in polycystic kidney disease
was first suggested after it was discovered that the genes affected in mice
with polycystic kidneys, e.g. the orpk and inv mice
(Lehman et al., 2008;
Moyer et al., 1994;
Shiba et al., 2005;
Siroky and Guay-Woodford,
2006), encoded cilia associated proteins. Thus, our finding that
kidneys of 7-day-old talpid3 chicken mutant embryos are
cystic is consistent with the general inability of talpid3
mutant cells to form primary cilia. Thus, we conclude that the
talpid3 chicken mutant is a new example of a ciliopathy
and a potential model for human disease. The range of defects in different
human ciliopathies varies although the reasons for this are not clear. Some
ciliopathies, such as BBS, Joubert syndrome, Meckel syndrome and oral facial
digital syndrome (OFD) have features that one would specifically associate
with Hh signalling defects, such as polydactyly, similar to the
talpid3 mutants, whereas others, such as those caused by
mutations in polycystin 1 and polycystin 2, have kidney defects. It will be
interesting to define the precise spectrum of defects in
talpid3 chicken mutants for comparison with the human
syndromes.
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;
Yoshimura et al., 2007). BBS1
is part of a complex of centrosomal proteins collectively termed the BBSome,
which also includes BBS2, 4, 5, 7, 8 and 9. Whether the Talpid3 protein is
also part of the BBSome or helps to traffic Rab8a to the cilium remains to be
investigated. Another possible reason why ciliogenesis fails in the
talpid3 chicken mutant is because Talpid3 is required for
apical actin enrichment. In oviduct ciliated cells, the apical actin network
is closely associated with basal body appendages
(Chailley et al., 1989), and
recent work on Xenopus laevis epidermal cells with motile cilia has
shown that apical enrichment of actin is required for ciliogenesis
(Park et al., 2006).
Therefore, it is possible that the abnormal actin organisation in
talpid3 mutant cells results in basal body misorientation,
leading to failure of ciliogenesis. Apical actin enrichment is mediated by
activation of RhoA (Pan et al.,
2007) and it has been shown that RhoA is localised to the basal
body in multiciliated cells (Park et al.,
2008), thus providing an explanation for how a centrosomal protein
such as Talpid3 could directly affect actin organisation. Furthermore, reduced
numbers of stress fibres and focal adhesions in talpid3
mutant cells suggests that RhoA activity is decreased
(Nobes and Hall, 1999).
Localisation of RhoA to the basal body in Xenopus laevis epidermal
cells has been shown to be mediated by Inturned, an effector of the Wnt planar
cell polarity signalling pathway (Park et
al., 2008); interestingly, Inturned, together with Fuzzy, is
required for normal cilium formation (Park
et al., 2006).
We have shown that the Talpid3 protein is present in both centrioles of the
centrosome. There are several mouse ciliopathy models with mutations in genes
encoding centrosomal proteins, including BBS1, BBS2, BBS4, OFD1 and Ftm
(Davis et al., 2007;
Ferrante et al., 2006;
Mykytyn et al., 2004;
Nishimura et al., 2004;
Vierkotten et al., 2007).
BBS1, BBS2, and BBS4 mutant mice still form primary cilia, although they are
abnormal or degenerate, whereas both OFD1 and Ftm mutant mice lack primary
cilia and have a similar phenotype to talpid3 chicken
mutants, including polydactyly and dorsalised neural tube. Furthermore, OFD1
has been shown to localise to both centrioles in human undifferentiated
embryonic cells (Romio et al.,
2004). Thus, the talpid3 chicken mutant most
closely resembles OFD1 mutant mice. There are no ultrastructural studies, to
date, of cells from either OFD1 or Ftm mutants, and therefore it is not clear
whether ciliogenesis fails at the same stage in these mutants as in
talpid3. Another centrosomal protein ODF2, has been
suggested to be necessary for basal body docking. However, in
Odf-/- cells, unlike talpid3 mutant
cells, basal bodies fail to mature and lack appendages
(Ishikawa et al., 2005),
suggesting that Talpid3 acts downstream of ODF2.
|
) found
that talpid3 cells move slower than wild-type cells, but
showed that this was not due to the talpid3 mutant cells
moving intrinsically more slowly but instead spending more time at rest. This
pausing might be explained by the slower rate of microtubule re-growth
observed in talpid3 mutant cells.
We have identified a region of the Talpid3 protein that is conserved all
the way down to Nematostella vectensis. This conserved region is
sufficient for centrosomal localisation and interestingly there is significant
distant homology between this region in Talpid3 and a region in another
centrosomal protein, CCCAP (centrosomal colon cancer autoantigen protein)
(Kenedy et al., 2003)
(Fig. 6C) (PSI-Blast E value
1e-47 and sequence similarity 47%). Further structure/function analysis of the
Talpid3 protein showed that the highly conserved region is required but not
sufficient to rescue primary cilia formation, thus suggesting that other
domains in the C terminus are also required. Rescue of neural tube patterning
in the mutant provides a powerful assay with which to identify these domains.
A deeper understanding of the Talpid3 protein will give new insights into
mechanisms involved in normal ciliogenesis and may also shed light on the
basis of human ciliopathies.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/136/4/655/DC1
|
Footnotes |
|---|
* These authors contributed equally to this work 
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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