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
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SUMMARY |
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Key words: Cilia, Inv, Left-right asymmetry, Node, Mouse
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INTRODUCTION |
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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.
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MATERIALS AND METHODS |
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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.
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RESULTS |
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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.
|
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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.
|
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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.
|
|
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.
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DISCUSSION |
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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 Inv
C::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
Inv
C::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.
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ACKNOWLEDGMENTS |
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Footnotes |
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