|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
doi: 10.1242/10.1242/dev.00407
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
Author for correspondence (e-mail:
hamada{at}fbs.osaka-u.ac.jp)
Accepted 13 January 2003
 |
SUMMARY |
|---|
 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
C)
including the ankyrin repeats also localized to the node cilia and rescued the
left-right defects of Inv/Inv mutants. Although no obvious
abnormalities were detected in the node monocilia of Inv/Inv embryos,
the laterality defects of such embryos were corrected by an artificial
leftward flow of fluid in the node, suggesting that nodal flow is impaired by
the Inv mutation. These results suggest that the Inv protein
contributes to left-right determination as a component of monocilia in the
node and is essential for the generation of normal nodal flow.
Key words: Cilia, Inv, Left-right asymmetry, Node, Mouse
|
INTRODUCTION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
). The process
by which LR asymmetry is established can be divided into three phases: (1) the
initial determination of LR polarity; (2) LR asymmetric expression of
signaling molecules; and (3) LR asymmetric morphogenesis induced by these
signaling molecules (Beddington and
Robertson, 1999; Capdevila et
al., 2000; Wright,
2001; Yost, 2001;
Hamada et al., 2002).
In spite of recent progress, our knowledge of LR determination remains
limited. In particular, the mechanism by which symmetry is initially broken
remains unknown, although several models have been proposed
(Brown and Wolpert, 1990;
Brown et al., 1991). Cilia and
fluid flow have been implicated in LR determination
(Nonaka et al., 1998;
Okada et al., 1999). Monocilia
on the node pit cells rotate in a clockwise direction and thereby generate a
leftward flow of extra-embryonic fluid (referred to as nodal flow). We
recently directly demonstrated a role for nodal flow in LR determination by
examining the effects of artificial flow
(Nonaka et al., 2002). An
artificial rightward flow that was fast enough to reverse the endogenous
leftward flow was thus able to reverse LR patterning in mouse embryos.
Although it remains unclear how nodal flow contributes to LR determination, it
is possible that the flow transports an unknown morphogen toward the left side
of the embryo. Alternatively, mechanical stress induced by the flow might be
sensed by cells in or near the node.
Various mouse mutants with defects in the initial determination of LR
polarity have been identified. The absence of nodal flow, however, may account
for the situs defects of most of these mutants. Mutant mice with impaired
nodal flow can be classified into two groups: those lacking the node cilia and
those with immotile node cilia. The first group includes mice deficient in the
kinesin superfamily proteins KIF3A
(Marszalek et al., 1999;
Takeda et al., 1999) or KIF3B
(Nonaka et al., 1998). Other
mutants, such as those deficient in polaris
(Murcia et al., 2000),
probably also belong to this group. The second group includes the
Iv/Iv mutant (Supp et al.,
1997; Supp et al.,
1999) and, possibly, mice that lack DNA polymerase 
(Kobayashi et al., 2002) or
HFH4 (Chen et al., 1998;
Brody et al., 2000).
The Inv (Invs — Mouse Genome Informatics) mutation
is unique in that homozygous mutant mice exhibit LR reversal instead of LR
randomization (Yokoyama et al.,
1993). Unexpectedly, nodal flow in the Inv mutant embryo
was shown to be leftward, although it was slow and turbulent
(Okada et al., 1999). It
remains unclear how such a slow, turbulent flow might result in LR reversal,
although plausible models have been proposed
(Okada et al., 1999). The
Inv/Inv mouse may thus be the only mutant whose defects in LR
patterning cannot be simply explained by nodal flow. The Inv gene is
expressed ubiquitously in developing mouse embryos and encodes a protein that
contains ankyrin repeats and calmodulin-binding motifs
(Mochizuki et al., 1998;
Morgan et al., 1998).
Overexpression of the mouse Inv protein in frog embryos perturbed LR decision
making (Yasuhiko et al.,
2001), but the precise functions of this protein remain
unclear.
We have now generated transgenic mice that express a fusion construct of Inv and green fluorescent protein (Inv::GFP). Our observations of the subcellular localization of this fusion protein suggest that Inv contributes to LR determination as a component of monocilia in the node.
|
MATERIALS AND METHODS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
)
(kindly provided by S. Nagata) under the control of the elongation factor
1
(EF1) gene promoter, to yield BOS-Inv::GFP and to
ensure that the fusion gene was expressed ubiquitously and robustly. The
InvC::GFP construct was generated by deleting the 3'
part of the coding region of the Inv cDNA at the NheI site.
Transgenic mice were generated by pronuclear injection of DNA into fertilized
eggs obtained from the mating of inv/+ FVB mice
(Saijoh et al., 1999).
Crossing of Inv/+, Inv::GFP mice with Inv/+ mice
yielded Inv/Inv, Inv::GFP animals. The presence of the transgene was
verified by Southern blot analysis with the GFP coding sequence as a probe.
GFP fluorescence in newborn mice was observed with a stereomicroscope (Leica
MZ FLIII). For Inv::GFP, ten lines of transgenic mice were initially
obtained. Eight of them expressed Inv::GFP and their transgenes were able to
rescue the situs defects and polycystic kidney of the
Inv/Invmouse. In the remaining two lines, the transgene failed to
rescue these Inv/Inv defects. For InvC::GFP, four
transgenic mice were established. In three of them, the transgene could rescue
situs defects but not polycystic kidney of the Inv/Inv mouse. Among
these transgenic lines produced, those expressing the transgenes at a
relatively high level, B27 (Inv::GFP) and N105
(InvC::GFP), were mainly used in this study. Founder mice for
the B27 and N105 lines were Inv/+ and +/+, respectively.
Preparation of antibodies to Inv
A 0.6 kb PvuII fragment of the mouse Inv cDNA
corresponding to the region of Inv located downstream of the ankyrin repeats
was subcloned into the pGEX-4T vector (Pharmacia). The encoded glutathione
S-transferase (GST)-Inv fusion protein was expressed in Escherichia
coli strain AD202 (Akiyama and Ito,
1990), purified by chromatography on glutathione-Sepharose 4B
(Pharmacia), and injected into rabbits. Polyclonal antibodies specific for Inv
were isolated by preabsorption of rabbit serum with GST followed by affinity
purification.
Generation of primary fibroblasts and cell culture
Primary fibroblasts expressing the Inv::GFP fusion protein were established
from the skin of newborn transgenic animals. The cells were cultured in
plastic dishes with Dulbecco's modified Eagle's medium supplemented with 10%
fetal bovine serum and antibiotics. For immunofluorescence staining, the cells
were cultured on cover slips for several days, fixed with 2% paraformaldehyde
and stained with specific antibodies as described below.
Western blot analysis
Primary fibroblast cells were lysed in cell lysis buffer containing 20 mM
Tris Hcl, 1% TritonX-100, 0.25% Deoxicorate, 250 mM NaCl, 5 mM EDTA, 1 mM
PMSF. The lysates containing 20 µg protein were loaded on 10%
SDS-polyacrylamide gels. An affinity-purified polyclonal anti-Inv antibody and
a horseradish peroxidase-conjugated goat anti-rabbit antibody (Jackson) were
used as the primary and secondary antibodies, respectively. Immune complexes
were detected with ECL Western blot Detection Systems (Amersham).
Immunofluorescence analysis
Freshly isolated tissues were frozen in OCT compound (Tissue-Tec) and then
cryosectioned (at 8 µm). After fixation with 2% paraformaldehyde, the
sections were incubated with rabbit polyclonal antibodies to Inv, to GFP (MBL)
or to calmodulin (Zymed), or with mouse monoclonal antibodies to acetylated
tubulin (Sigma) or to 
-tubulin (Sigma). Antibodies were diluted in
phosphate-buffered saline containing 5% skim milk and 0.05% Tween 20. All
antibodies were diluted 1/1000 for staining sections, or 1/5000 for
whole-mount staining. Immune complexes were detected with Alexa 488-conjugated
goat antibodies to rabbit immunoglobulin G (Molecular Probes) or Alexa
568-conjugated goat antibodies to mouse immunoglobulin G (Molecular Probes).
For whole-mount immunofluorescence analysis, freshly isolated embryos were
fixed with ice-cold acetone and then incubated consecutively with primary and
secondary antibodies at 4°C for 48 and 24 hours, respectively, in
phosphate-buffered saline containing 5% skim milk and 1% Triton X-100.
Confocal images were obtained with a Carl Zeiss confocal microscope (LSM 510)
and three-dimensional images were reconstructed with the LSM 510 software
(version 2.5).
Histological analysis
Kidneys were fixed in Bouin's solution, dehydrated and embedded in paraffin
wax. Serial sections (5 µm) were prepared and stained with Hematoxylin and
Eosin according to standard procedures.
|
RESULTS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
),
which confers ubiquitous and high-level expression. The BOS-Inv::GFP
transgene was microinjected into mouse fertilized eggs (+/+ or
+/Inv), resulting in the generation of several transgenic lines
expressing Inv::GFP. Unless indicated otherwise, the B27 line, which expresses
Inv::GFP at a relatively high level, was used in the present study. Mice
harboring the Inv::GFP transgene (+/+ or +/Inv, Inv::GFP)
were normal and fertile. Transgenic embryos expressed the fusion protein in a
ubiquitous manner (Fig. 1B;
data not shown), and newborn transgenic mice were readily recognized by the
presence of fluorescence in their limbs
(Fig. 1C).
|
). The laterality defects of Inv/Inv newborn
mice are evident from the position of the stomach, which is located on the
right side (Fig. 1D). The lung
lobation pattern is also reversed, with one lobe on the right and four lobes
on the left (Fig. 1E). The
defects caused by the Inv mutation were rescued in Inv/Inv
mice by expression of the Inv::GFP transgene. Thus, in Inv/Inv,
Inv::GFP mice, the stomach was located on the left
(Fig. 1D) and lung lobation was
normal (one lobe on the left, four lobes on the right)
(Fig. 1E). Moreover,
Inv/Inv, Inv::GFP mice grew normally and were fertile, and they did
not develop the polycystic kidney disease and jaundice characteristic of
Inv/Inv animals (Fig.
1F,G). These observations thus indicated that the Inv::GFP protein
is fully functional.
Localization of Inv::GFP to the primary cilia of fibroblasts
We examined the subcellular localization of Inv::GFP in primary fibroblasts
established from newborn Inv/Inv, Inv::GFP mice. In nonfixed cells,
GFP fluorescence was detected in rod-like structures protruding from the cell
body (Fig. 2A-C). To determine
whether these structures were primary cilia, we subjected fixed fibroblasts to
immunofluorescence staining for acetylated tubulin, a marker of primary cilia
and centrioles. The rod-like structures that were positive for GFP were indeed
detected by antibodies to acetylated tubulin
(Fig. 2D-I). These results thus
indicated that, in primary fibroblasts, Inv::GFP is preferentially localized
to primary cilia.
|
|
-tubulin revealed several distinct patterns of centriolar staining
(Fig. 4A).
|
50
minutes).
Localization of Inv::GFP to monocilia of the node
Given that the monocilia on the node pit cells are implicated in LR
determination (Nonaka et al.,
1998), we next examined the localization of Inv::GFP in the node.
Observation of the node of Inv/Inv, Inv::GFP embryos from the ventral
side without fixation (Fig. 5A)
revealed a dot-like pattern of GFP fluorescence signals within the node
(Fig. 5B). Lateral observation
of the node revealed rod-shaped fluorescence signals protruding from the
ventral node cells into the node cavity
(Fig. 5C). Examination of the
cells on the ventral side of the node at high magnification showed that
Inv::GFP was present within the monocilia of these cells
(Fig. 5D). The fusion protein
was detected uniformly along the entire length of all the monocilia.
Immunohistological analysis confirmed the preferential localization of
Inv::GFP to monocilia of the ventral node cells. Monocilia that were detected
with antibodies to acetylated tubulin (Fig.
5E) were thus also stained with antibodies to GFP
(Fig. 5F). Together, these
results demonstrated the preferential localization of Inv protein in node
monocilia.
|
|
-tubulin but not in the cilia
(Fig. 6M-R). These observations
thus revealed that, in cells with 9+2 cilia, Inv::GFP localized either to the
cytoplasm or to the basal body, but did not accumulate preferentially in the
ciliary body.
Inv is expressed ubiquitously in developing mouse embryos
(Mochizuki et al., 1998).
Inv::GFP was also expressed widely in transgenic mice because the transgene
was placed under the control of the Ef1a gene promoter. In those
cells without cilia, Inv::GFP was detected predominantly in the cytoplasm
(Fig. 6I), but was occasionally
detected in nuclei of some cell types such as skeletal muscle cells (data not
shown). At adult stage, the transgene expression was detected in many organs,
including the brain, muscle and testis (data not shown).
The mouse Inv protein contains two potential calmodulin binding (IQ) motifs
(Mochizuki et al., 1998;
Morgan et al., 1998) and
interacts with calmodulin in vitro
(Yasuhiko et al., 2001;
Morgan et al., 2002).
Expression of the wild-type Inv protein in frog embryos perturbed LR
determination, whereas expression of mutant Inv proteins that lack either of
the two calmodulin binding motifs did not
(Yasuhiko et al., 2001). These
observations suggested that Inv may function through direct interaction with
calmodulin. To test this hypothesis, we examined whether calmodulin
colocalizes with Inv in cilia. Immunostaining of wild-type mouse embryo
tissues with antibodies to calmodulin revealed the presence of this protein in
9+2 cilia, including those in the trachea
(Fig. 7A-C), oviduct and
ependyma (data not shown). By contrast, calmodulin was not detected in any of
the 9+0 cilia examined, including those in the kidney
(Fig. 7D-F), node
(Fig. 7G-I), and pituitary
gland (data not shown). Thus, whereas Inv is localized to 9+0 cilia,
calmodulin is localized to 9+2 cilia.
|
C::GFP, a chimeric protein
in which the N-terminal region of Inv is fused to GFP
(Fig. 8A). One of these lines
(N105) that expressed InvC::GFP at a relatively high level was used for
the studies described below. InvC::GFP protein appears to be unstable
since the level of InvC::GFP protein detected in fibroblasts from
Inv/Inv; InvC::GFP mice was always much lower than that of
Inv::GFP in Inv/Inv; Inv::GFP fibloblasts
(Fig. 3A).
|
C::GFP transgene derived from N105 mice was
transferred to the Inv/Inv background. Although Inv/Inv,
InvC::GFP mice exhibited normal LR patterning of visceral organs,
including the stomach and the lung, they developed polycystic kidney disease a
few weeks after birth (Fig.
8B). The InvC::GFP protein was detected in the primary
cilia of cultured fibroblasts derived from Inv/Inv, InvC::GFP
mice as well as in the monocilia of the node
(Fig. 8C). It was not apparent,
however, in the 9+0 cilia of the kidney
(Fig. 8C), consistent with the
failure of the transgene to rescue the kidney defect of Inv/Inv mice.
These results thus suggest that InvC is able to localize to 9+0 cilia
and to function as an LR determinant. The absence of InvC::GFP in the
kidney is most probably due to the unstable nature of this protein.
Rescue of the laterality defects of Inv/Inv embryos by
artificial nodal flow
The localization of the Inv::GFP protein in 9+0 cilia prompted us to
examine the node monocilia of Inv/Inv embryos. Scanning electron
microscopy revealed that the cilia of the Inv/Inv embryos were
indistinguishable from those of wild-type embryos in their length, thickness
and shape (data not shown). Video microscopy also revealed that the node cilia
of Inv/Inv embryos rotate at a speed of 600 rpm in a clockwise
direction, just like those of wild-type embryos. Thus, we were not able to
detect any abnormalities of the node monocilia in Inv/Inv mice.
We recently developed an experimental system for the culture of mouse
embryos under conditions of artificial flow of the culture medium
(Nonaka et al., 2002). With
this system, we showed that artificial nodal flow is able to determine LR
patterning of wild-type embryos in a manner dependent on the direction and
speed of flow and on the developmental stage of the embryo
(Nonaka et al., 2002). We
therefore tested whether artificial nodal flow was able to rescue the
laterality defects of Inv/Inv mice. Exposure of Inv/Inv
embryos to a fast leftward flow resulted in normal heart looping and normal
embryonic turning (Fig. 9).
This rescue of the LR patterning defects of Inv/Inv mice by
artificial leftward flow suggested that nodal flow is impaired in
Inv/Inv embryos. This conclusion is consistent with previous
observations that nodal flow is slow and turbulent in Inv/Inv embryos
(Okada et al., 1999), and it
supports the idea that Inv plays a role in LR decision making as a component
of the node monocilia.
|
|
DISCUSSION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
)
reported the presence of Inv immunoreactivity in nuclei and at the cell
membrane, but not in the primary cilia, of cultured Madin-Darby canine kidney
(MDCK) cells, which resemble kidney epithelial cells. However, transfection of
MDCK cells with pBOS-Inv::GFP revealed that the Inv::GFP protein was localized
to the monocilia (data not shown). The reason for this apparent discrepancy is
not clear. We also observed the nuclear localization of Inv::GFP in certain
cell types, such as skeletal muscle cells, of transgenic mice, suggesting that
the subcellular distribution of Inv may be dependent on cell type.
In mammals, primary cilia are present in a variety of organs at embryonic
and adult stages. With a few exceptions, such as the role of node monocilia in
LR patterning, the precise functions of these cilia remain unknown. Although
node monocilia are motile (Sulik et al.,
1994; Nonaka et al.,
1998), most primary cilia are thought to be immotile. For example,
monocilia present in the proximal tubule of the kidney are relatively long
compared with the diameter of the collecting tube and coexist with numerous
microvilli, making it unlikely that they are able to move actively. Such
immotile primary cilia have been proposed to function as signal sensors, in
which case Inv may be required not only for the movement of primary cilia (as
in the node cilia) but also for the reception of external signals by these
structures.
Role of Inv in LR determination
Substantial evidence implicates the node monocilia and nodal flow in LR
determination. The localization of Inv to 9+0 monocilia further supports a
role for node cilia in LR decision making. However, the mechanism by which
nodal flow directs LR patterning is not known. As previously suggested
(Nonaka et al., 1998), nodal
flow may transport a molecule that acts as a LR determinant toward the left
side. Alternatively, the direction of or mechanical stress generated by the
flow may be sensed differentially by cells on the two sides of the embryo. The
Inv protein may thus be required for correct movement of the node cilia or for
receiving signals generated by the flow. Our observations favor the former
possibility but do not exclude the latter.
The mouse Inv protein possesses two calmodulin binding (IQ) motifs, both of
which indeed bind calmodulin in vitro
(Yasuhiko et al., 2001;
Morgan et al., 2002). Whereas
mouse Inv perturbed LR determination when ectopically expressed in
Xenopus embryos, mutant Inv proteins lacking either of the two
calmodulin binding motifs exhibited no such activity
(Yasuhiko et al., 2001). These
observations were thus suggestive of a functional interaction between Inv and
the Ca2+-binding protein calmodulin. However, our data do not
support a role for such an interaction in LR patterning. First, we showed that
these two proteins are not colocalized in the mouse embryo; rather, they show
reciprocal expression patterns in that calmodulin is present in 9+2 cilia,
whereas Inv is localized to 9+0 cilia. Second, in contrast to the observations
made with frog embryos (Yasuhiko et al.,
2001), the InvC::GFP protein, which lacks the C-terminal
region (including one of the calmodulin binding motifs) of Inv, was able to
rescue the LR defects of Inv/Inv mice. Nonetheless, it is formally
possible that Inv interacts with low levels of calmodulin because the
InvC::GFP protein retains the N-terminal calmodulin binding motif. Our
data do not exclude a role for Ca2+ in Inv function, in which
regard mice lacking polycystin 2, a putative Ca2+ channel, have
been shown to exhibit LR defects
(Pennekamp et al., 2002).
Artificial nodal flow was able to rescue the LR patterning defects of
Inv/Inv mice. This result is consistent with previous observations
that nodal flow of Inv/Inv embryos is leftward but slow and turbulent
(Okada et al., 1999). Inv may
thus be required for the correct movement of node cilia. Our examination of
the node monocilia of Inv/Inv mice, however, failed to reveal any
apparent anomalies in their morphology or motility. Further investigations of
the fine structure and movement of the node monocilia in these mutant animals
are thus required to detect possible abnormalities. The development of an
imaging system that allows observation of the cilia of live mouse embryos at
high magnification would facilitate this goal.
Node monocilia may also function as signal sensors. Our data with
artificial nodal flow may not favor this notion, but they do not exclude the
possibility that Inv plays dual roles in generating correct nodal flow and in
transducing signals generated by such flow. Numerous examples of immotile
cilia that function in sensory perception, including chemoreception,
photoreception and mechanoreception, have been described in nonvertebrates. In
Caenorhabditis elegans, cilia are present in 60 out of the 302
neurons, in which they function as sensory organelles
(Bargmann and Horvitz, 1991;
Dwyer et al., 1998). In
Chlamydomonas, flagella transduce signals during gamete adhesion in
mating (Solter and Gibor,
1977; Pan and Snell,
2002). Furthermore, in the mouse, the polaris protein is required
for formation both of 9+0 cilia such as node monocilia
(Murcia et al., 2000) and of
9+2 cilia such as those in brain ependymal cells. A polaris homolog in C.
elegans, OSM-5, localizes to the basal body and cilia and functions in
intraflagellar transport; in osm-5 mutants, sensory neurons lack
cilia that function as sensors of osmotic pressure
(Haycraft et al., 2001;
Taulman et al., 2001). Whether
or not node cilia (and Inv) function in sensory perception remains to be
determined.
Role of Inv in kidney development
Homozygous Inv mutant mice develop polycystic kidney disease a few
weeks after birth. Furthermore, many of the genes required for LR
determination, including Inv, polaris and polycystin 2, are also
implicated in polycystic kidney disease in humans. In general, the
histological features of polycystic kidneys are first apparent in the proximal
collecting tubules. Our examination of eight independent Inv/Inv,
Inv::GFP transgenic lines revealed that the abundance of Inv::GFP protein
in monocilia of the proximal collecting tubules in newborns correlated with
the severity of polycystic kidney disease at later stages (data not shown).
This correlation, together with the localization of Inv::GFP in the monocilia
of the kidney epithelium, suggests that Inv functions in these cilia. However,
examination of the kidney monocilia of Inv/Inv newborn mice (before
polycystic kidney disease becomes severe) revealed no apparent anomalies with
respect to the number or morphology of the cilia (data not shown). Although
the precise mechanism responsible for the development of polycystic kidney
disease in Inv/Inv mice is unknown, the kidney monocilia may function
as organelles that sense external signals, such as ion concentration, osmotic
pressure or mechanical flow, and Inv may be required for transduction of such
signals.
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
|
REFERENCES |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Akiyama, Y. and Ito, K. (1990). SecY protein, a membrane-embedded secretion factor of E. coli, is cleaved by the OmpT protease in vitro. Biochem. Biophys. Res. Commun. 167,711 -715.[CrossRef][Medline]
Bargmann, C. I. and Horvitz, H. R. (1991). Chemosensory neurons with overlapping functions direct chemotaxis to multiple chemicals in C. elegans. Neuron 7, 729-742.[CrossRef][Medline]
Beddington, R. S. and Robertson, E. J. (1999). Axis development and early asymmetry in mammals. Cell 96,195 -209.[CrossRef][Medline]
Brody, S. L., Yan, X. H., Wuerffel, M. K., Song, S. K. and
Shapiro, S. D. (2000). Ciliogenesis and left-right axis
defects in forkhead factor HFH-4-null mice. Am. J. Respir. Cell
Mol. Biol. 23,45
-51.
Brown, N. A., McCarthy, A. and Wolpert, L. (1991). Development of handed body asymmetry in mammals. Ciba Found. Symp. 162,182 -196.[Medline]
Brown, N. A. and Wolpert, L. (1990). The development of handedness in left/right asymmetry. Development 109,1 -9.[Abstract]
Capdevila, J., Vogan, K. J., Tabin, C. J. and Izpisua Belmonte, J. C. (2000). Mechanisms of left-right determination in vertebrates. Cell 101,9 -21.[CrossRef][Medline]
Chen, J., Knowles, H. J., Hebert, J. L. and Hackett, B. P. (1998). Mutation of the mouse hepatocyte nuclear factor/forkhead homologue 4 gene results in an absence of cilia and random left-right asymmetry. J. Clin. Invest. 102,1077 -1082.[Medline]
Dwyer, N. D., Troemel, E. R., Sengupta, P. and Bargmann, C. I. (1998). Odorant receptor localization to olfactory cilia is mediated by ODR-4, a novel membrane-associated protein. Cell 93,455 -466.[CrossRef][Medline]
Fujinaga, M. (1997). Development of sidedness of asymmetric body structures in vertebrates. Int. J. Dev. Biol. 41,153 -186.[Medline]
Hamada, H., Meno, C., Watanabe, D. and Saijoh, Y. (2002). Establishment of vertebrate left-right asymmetry. Nat. Rev. Genet. 3,103 -113.[CrossRef][Medline]
Haycraft, C. J., Swoboda, P., Taulman, P. D., Thomas, J. H. and Yoder, B. K. (2001). The C. elegans homolog of the murine cystic kidney disease gene Tg737 functions in a ciliogenic pathway and is disrupted in osm-5 mutant worms. Development 128,1493 -1505.[Abstract]
Kobayashi, Y., Watanabe, M., Okada, Y., Sawa, H., Takai, H.,
Nakanishi, M., Kawase, Y., Suzuki, H., Nagashima, K., Ikeda, K. and Motoyama,
N. (2002). Hydrocephalus, situs inversus, chronic sinusitis
and male infertility in DNA polymerase lambda-deficient mice: possible
implication for the pathogenesis of immotile cilia syndrome. Mol.
Cell. Biol. 22,2769
-2776.
Marszalek, J. R., Ruiz-Lozano, P., Roberts, E., Chien, K. R. and
Goldstein, L. S. (1999). Situs inversus and embryonic ciliary
morphogenesis defects in mouse mutants lacking the KIF3A subunit of
kinesin-II. Proc. Natl. Acad. Sci. USA
96,5043
-5048.
Mizushima, S. and Nagata, S. (1990). pEF-BOS, a
powerful mammalian expression vector. Nucleic Acids
Res. 18,5322
.
Mochizuki, T., Saijoh, Y., Tsuchiya, K., Shirayoshi, Y., Takai, S., Taya, C., Yonekawa, H., Yamada, K., Nihei, H., Nakatsuji, N., Overbeek, P. A., Hamada, H. and Yokoyama, T. (1998). Cloning of inv, a gene that controls left/right asymmetry and kidney development. Nature 395,177 -181.[CrossRef][Medline]
Morgan, D., Turnpenny, L., Goodship, J., Dai, W., Majumder, K., Matthews, L., Gardner, A., Schuster, G., Vien, L., Harrison, W. et al. (1998). Inversin, a novel gene in the vertebrate left-right axis pathway, is partially deleted in the inv mouse Nature Genet. 20,149 -156.[CrossRef][Medline]
Morgan, D., Goodship, J., Essner, J. J., Vogan, K. J., Turnpenny, L., Yost, J., Tabin, C. J. and Strachan, T. (2002). The left-right determinant inversin has highly conserved ankyrin repeat and IQ domains and interacts with calmodulin. Hum. Genet. 110,377 -384.[CrossRef][Medline]
Murcia, N. S., Richards, W. G., Yoder, B. K., Mucenski, M. L., Dunlap, J. R. and Woychik, R. P. (2000). The oak ridge polycystic kidney (ORPK) disease gene is required for left-right axis determination. Development 127,2347 -2355.[Abstract]
Nonaka, S., Shiratori, H., Saijoh, Y. and Hamada, H. (2002). Determination of left-right patterning of the mouse embryo by artificial nodal flow. Nature 418, 96-99.[CrossRef][Medline]
Nonaka, S., Tanaka, Y., Okada, Y., Takeda, S., Harada, A., Kanai, Y., Kido, M. and Hirokawa, N. (1998). Randomization of left-right asymmetry due to loss of nodal cilia generating leftward flow of extraembryonic fluid in mice lacking KIF3B motor protein Cell 95,829 -837.[CrossRef][Medline]
Nurnberger, J., Bacallao, R. L. and Phillips, C. L.
(2002). Inversin forms a complex with catenins and N-cadherin in
polarized epithelial cells. Mol. Biol. Cell
13,3096
-3106.
Okada, Y., Nonaka, S., Tanaka, Y., Saijoh, Y., Hamada, H. and Hirokawa, N. (1999). Abnormal nodal flow precedes situs inversus in iv and inv mice. Mol. Cell 4,459 -468.[CrossRef][Medline]
Pan, J. and Snell, W. J. (2002). Kinesin-II is
required for flagellar sensory transduction during fertilization in
Chlamydomonas. Mol. Biol. Cell
13,1417
-1426.
Pennekamp, P., Karcher, C., Fischer, A., Schweickert, A., Skryabin, B., Horst, J., Blum, M. and Dworniczak, B. (2002). The ion channel polycystin-2 is required for left-right axis determination in mice. Curr. Biol. 12,938 -943.[CrossRef][Medline]
Saijoh, Y., Adachi, H., Mochida, K., Ohishi, S., Hirao, A. and
Hamada, H. (1999). Distinct transcriptional regulatory
mechanisms underlie left-right asymmetric expression of lefty-1 and
lefty-2. Genes Dev. 13,259
-269.
Solter, K. M. and Gibor, A. (1977). Evidence for a role of flagella as sensory transducers in mating of Chlamydomonas reinhardi. Nature 265,444 -445.[CrossRef][Medline]
Sulik, K., Dehart, D. B., Iangaki, T., Carson, J. L., Vrablic, T., Gesteland, K. and Schoenwolf, G. C. (1994). Morphogenesis of the murine node and notochordal plate. Dev. Dyn. 201,260 -278.[Medline]
Supp, D. M., Witte, D. P., Potter, S. S. and Brueckner, M. (1997). Mutation of an axonemal dynein affects left-right asymmetry in inversus viscerum mice. Nature 389,963 -966.[CrossRef][Medline]
Supp, D. M., Brueckner, M., Kuehn, M. R., Witte, D. P., Lowe, L. A., McGrath, J., Corrales, J. and Potter, S. S. (1999). Targeted deletion of the ATP binding domain of left-right dynein confirms its role in specifying development of left-right asymmetries. Development 126,5495 -5504.[Abstract]
Takeda, S., Yonekawa, Y., Tanaka, Y., Okada, Y., Nonaka, S. and
Hirokawa, N. (1999). Left-right asymmetry and kinesin
superfamily protein KIF3A: new insights in determination of laterality and
mesoderm induction by kif3A-/- mice analysis.
J. Cell Biol. 145,825
-836.
Taulman, P. D., Haycraft, C. J., Balkovetz, D. F. and Yoder, B.
K. (2001). Polaris, a protein involved in left-right axis
patterning, localizes to basal bodies and cilia. Mol. Biol.
Cell 12,589
-599.
Wright, C. V. (2001). Mechanisms of left-right asymmetry: What's right and what's left? Dev. Cell 1, 179-186.[CrossRef][Medline]
Yasuhiko, Y., Imai, F., Ookubo, K., Takakuwa, Y., Shiokawa, K. and Yokoyama, T. (2001). Calmodulin binds to Inv protein: implication for the regulation of Inv function. Dev. Growth Differ. 43,671 -681.[CrossRef][Medline]
Yokoyama, T., Copeland, N. G., Jenkins, N. A., Montgomery, C.
A., Elder, F. F. and Overbeek, P. A. (1993). Reversal of
left-right asymmetry: a situs inversus mutation.
Science 260,679
-682.
Yost, H. J. (2001). Establishment of left-right asymmetry. Int. Rev. Cytol. 203,357 -381.[Medline]
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
This article has been cited by other articles:
|
|
 |
|
  Y. Wang, Z. Zhou, C. T. Walsh, and A. P. McMahon Selective translocation of intracellular Smoothened to the primary cilium in response to Hedgehog pathway modulation PNAS, February 24, 2009; 106(8): 2623 - 2628. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Aw and M. Levin Is left-right asymmetry a form of planar cell polarity? Development, February 1, 2009; 136(3): 355 - 366. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Shiba, Y. Yamaoka, H. Hagiwara, T. Takamatsu, H. Hamada, and T. Yokoyama Localization of Inv in a distinctive intraciliary compartment requires the C-terminal ninein-homolog-containing region J. Cell Sci., January 1, 2009; 122(1): 44 - 54. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Duan, N. Gotoh, Q. Yan, Z. Du, A. M. Weinstein, T. Wang, and S. Weinbaum Shear-induced reorganization of renal proximal tubule cell actin cytoskeleton and apical junctional complexes PNAS, August 12, 2008; 105(32): 11418 - 11423. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. S. McClintock, C. E. Glasser, S. C. Bose, and D. A. Bergman Tissue expression patterns identify mouse cilia genes Physiol Genomics, January 17, 2008; 32(2): 198 - 206. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Beckers, L. Alten, C. Viebahn, P. Andre, and A. Gossler The mouse homeobox gene Noto regulates node morphogenesis, notochordal ciliogenesis, and left right patterning PNAS, October 2, 2007; 104(40): 15765 - 15770. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Rohatgi, L. Milenkovic, and M. P. Scott Patched1 Regulates Hedgehog Signaling at the Primary Cilium Science, July 20, 2007; 317(5836): 372 - 376. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Wang and J. Nathans Tissue/planar cell polarity in vertebrates: new insights and new questions Development, February 15, 2007; 134(4): 647 - 658. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. W. Bisgrove and H. J. Yost The roles of cilia in developmental disorders and disease Development, November 1, 2006; 133(21): 4131 - 4143. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Sugiyama and T. Yokoyama Sustained cell proliferation of renal epithelial cells in mice with inv mutation. Genes Cells, October 1, 2006; 11(10): 1213 - 1224. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. F. O'Toole, E. A. Otto, Y. Frishberg, and F. Hildebrandt Retinitis pigmentosa and renal failure in a patient with mutations in INVS Nephrol. Dial. Transplant., July 1, 2006; 21(7): 1989 - 1991. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Shiratori and H. Hamada The left-right axis in the mouse: from origin to morphology Development, June 1, 2006; 133(11): 2095 - 2104. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. J. Schrick, P. Vogel, A. Abuin, B. Hampton, and D. S. Rice ADP-Ribosylation Factor-Like 3 Is Involved in Kidney and Photoreceptor Development Am. J. Pathol., April 1, 2006; 168(4): 1288 - 1298. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M.-a. Nakaya, K. Biris, T. Tsukiyama, S. Jaime, J. A. Rawls, and T. P. Yamaguchi Wnt3alinks left-right determination with segmentation and anteroposterior axis elongation Development, December 15, 2005; 132(24): 5425 - 5436. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. R. Davenport and B. K. Yoder An incredible decade for the primary cilium: a look at a once-forgotten organelle Am J Physiol Renal Physiol, December 1, 2005; 289(6): F1159 - F1169. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. M. Barr Caenorhabditis elegans as a Model to Study Renal Development and Disease: Sexy Cilia J. Am. Soc. Nephrol., February 1, 2005; 16(2): 305 - 312. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. V. Masyuk, B. Q. Huang, A. I. Masyuk, E. L. Ritman, V. E. Torres, X. Wang, P. C. Harris, and N. F. LaRusso Biliary Dysgenesis in the PCK Rat, an Orthologous Model of Autosomal Recessive Polycystic Kidney Disease Am. J. Pathol., November 1, 2004; 165(5): 1719 - 1730. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. J. Pazour Intraflagellar Transport and Cilia-Dependent Renal Disease: The Ciliary Hypothesis of Polycystic Kidney Disease J. Am. Soc. Nephrol., October 1, 2004; 15(10): 2528 - 2536. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Romio, A. M. Fry, P. J.D. Winyard, S. Malcolm, A. S. Woolf, and S. A. Feather OFD1 Is a Centrosomal/Basal Body Protein Expressed during Mesenchymal-Epithelial Transition in Human Nephrogenesis J. Am. Soc. Nephrol., October 1, 2004; 15(10): 2556 - 2568. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Hecksher-Sorensen, R. P. Watson, L. A. Lettice, P. Serup, L. Eley, C. De Angelis, U. Ahlgren, and R. E. Hill The splanchnic mesodermal plate directs spleen and pancreatic laterality, and is regulated by Bapx1/Nkx3.2 Development, October 1, 2004; 131(19): 4665 - 4675. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. Sun, A. Amsterdam, G. J. Pazour, D. G. Cole, M. S. Miller, and N. Hopkins A genetic screen in zebrafish identifies cilia genes as a principal cause of cystic kidney Development, August 15, 2004; 131(16): 4085 - 4093. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Q. Zhang, P. D. Taulman, and B. K. Yoder Cystic Kidney Diseases: All Roads Lead to the Cilium Physiology, August 1, 2004; 19(4): 225 - 230. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. A. Cano, N. S. Murcia, G. J. Pazour, and M. Hebrok orpk mouse model of polycystic kidney disease reveals essential role of primary cilia in pancreatic tissue organization Development, July 15, 2004; 131(14): 3457 - 3467. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Levin THE EMBRYONIC ORIGINS OF LEFT-RIGHT ASYMMETRY Critical Reviews in Oral Biology & Medicine, July 1, 2004; 15(4): 197 - 206. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Nurnberger, A. Kribben, A. O. Saez, G. Heusch, T. Philipp, and C. L. Phillips The Invs Gene Encodes a Microtubule-Associated Protein J. Am. Soc. Nephrol., July 1, 2004; 15(7): 1700 - 1710. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. L. Phillips, K. J. Miller, A. J. Filson, J. Nurnberger, J. L. Clendenon, G. W. Cook, K. W. Dunn, P. A. Overbeek, V. H. Gattone II, and R. L. Bacallao Renal Cysts of inv/inv Mice Resemble Early Infantile Nephronophthisis J. Am. Soc. Nephrol., July 1, 2004; 15(7): 1744 - 1755. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Bonnafe, M. Touka, A. AitLounis, D. Baas, E. Barras, C. Ucla, A. Moreau, F. Flamant, R. Dubruille, P. Couble, et al. The Transcription Factor RFX3 Directs Nodal Cilium Development and Left-Right Asymmetry Specification Mol. Cell. Biol., May 15, 2004; 24(10): 4417 - 4427. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. M. Guay-Woodford Murine models of polycystic kidney disease: molecular and therapeutic insights Am J Physiol Renal Physiol, December 1, 2003; 285(6): F1034 - F1049. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. J. Ward, D. Yuan, T. V. Masyuk, X. Wang, R. Punyashthiti, S. Whelan, R. Bacallao, R. Torra, N. F. LaRusso, V. E. Torres, et al. Cellular and subcellular localization of the ARPKD protein; fibrocystin is expressed on primary cilia Hum. Mol. Genet., October 16, 2003; 12(20): 2703 - 2710. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
