1 Department of Cell Biology, University of Alabama at Birmingham, Birmingham,
AL 35294, USA
2 Department of Medicine, Division of Cardiovascular Disease, University of
Alabama at Birmingham, Birmingham, AL 35294, USA
3 High Resolution Imaging Facility, University of Alabama at Birmingham,
Birmingham, AL 35294, USA
4 Department of Medicine, Division of Nephrology, University of Alabama at
Birmingham, Birmingham, AL 35294, USA
5 Nephrology Research and Training Center, University of Alabama at Birmingham,
Birmingham, AL 35294, USA
6 Department of Physiology and Biophysics, University of Alabama at Birmingham,
Birmingham, AL 35294, USA
* Author for correspondence (e-mail: byoder{at}uab.edu)
Accepted 7 October 2005
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SUMMARY |
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Key words: Cilia, Hydrocephalus, Tg737, Intraflagellar transport, Choroid plexus, Ependyma
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Introduction |
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The CSF is produced largely by the choroid plexus (CP), a highly
vascularized secretory neuroepithelium found in the lateral, third and fourth
ventricles of the brain. These CP cells contain numerous microvilli associated
with their highly secretory nature, and small tufts of cilia of unknown
function (Doolin and Birge,
1966). The CSF is produced through the net directional transport
of bicarbonate, chloride and sodium, with subsequent water movement through
apical aquaporin 1 channels (Brown et al.,
2004
). The rates of CSF production and reabsorption must be in
equilibrium; disturbances in the equilibrium lead to increased intracranial
pressure and hydrocephalus.
The CSF circulates within the brain ventricles, from the lateral ventricle
to the third ventricle, through the aqueduct of Sylvius into the fourth
ventricle, and finally along the spinal channel and subarachnoid space where
CSF is reabsorbed into the blood or lymphatic system
(Weller et al., 1992).
Although the mechanism(s) of CSF circulation remains poorly understood, one
factor thought to have an important role is the orchestrated beating of cilia
on the ependymal cells that line the ventricles and interventricular
connections (Ibanez-Tallon et al.,
2004
).
Data from several studies suggest that impaired CSF flow generated by
motile cilia on the ependyma results in aqueduct stenosis and a
non-communicative form of hydrocephalus; however, it remains controversial
whether the blockage of the duct is a primary cause or a consequence of
compression exerted by the expanding ventricles
(Ibanez-Tallon et al., 2004).
In addition to the obstructive hydrocephalus, there are communicating forms
where the duct remains patent. In this form of hydrocephalus, the defect is
thought to reside in excess CSF production by the CP or abnormal reabsorption
by arachnoid villi (Britz et al.,
1996
).
Much of our understanding of hydrocephalus has come from the analysis of
animal models. H-Tx rats develop congenital hydrocephalus. The mechanism
leading to the pathology remains controversial: some studies indicate the
primary defect is caused by duct obstruction, while others emphasize that the
hydrocephalus develops prior to impaired CSF flow
(Jones and Bucknall, 1988;
Kiefer et al., 1998
). Mice
lacking the E2f5 transcription factor exhibit communicating
congenital hydrocephalus that has been attributed to the increased secretory
activity of the CP; however, the role of E2f5 in CSF production
remains unknown (Lindeman et al.,
1998
). The hydrocephalus that develops in L1 neural adhesion
molecule deficient mice, a molecule which is mutated in human forms of
X-linked hydrocephalus, is initially associated with a patent aqueduct;
however, duct stenosis occurs when the pathology becomes more severe
(Rolf et al., 2001
). There are
also several mouse models of hydrocephalus that have been attributed to cilial
dysfunction. Disruption of the outer dynein arm protein Mdnah5 (Dnahc5
Mouse Genome Informatics) results in impaired cilia motility on ependymal
cells. The subsequent loss of CSF flow is thought to contribute to aqueduct
closure during early postnatal development, leading to hydrocephalus
(Ibanez-Tallon et al., 2004
).
An analogous mechanism may be involved in the WIC-Hyd rats that also have
impaired cilia motility (Torikata et al.,
1991
). In addition, mice with mutations in the cilia proteins
Spag6 or hydin, or the transcription factor Hfh4 (Foxj1 Mouse Genome
Informatics) that lack ependymal cell cilia, all exhibit hydrocephalus
(Chen et al., 1998
;
Davy and Robinson, 2003
;
Sapiro et al., 2002
). Finally,
cilia function in the CSF ventricular system is also important in humans, as
evidenced by the incidence of hydrocephalus in human patients with primary
ciliary dyskinesia (Bush,
2000
). However, it should be noted that the effect of the cilia on
the CP epithelium has not been evaluated in any of these models.
Another mouse model that develops hydrocephalus is the
Tg737orpk mutant. These mice exhibit hydrocephalus, cystic
kidney disease, sterility, biliary and bile duct hyperplasia in the liver,
acinar cell atrophy in the pancreas, retinal degeneration, and skeletal
patterning abnormalities (Cano et al.,
2004; Moyer et al.,
1994
; Taulman et al.,
2001
; Zhang et al.,
2003
; Zhang et al.,
2005
). The gene Tg737 encodes a conserved protein called
polaris that localizes to both motile and immotile cilia
(Taulman et al., 2001
).
Analysis of polaris in mouse, as well as of its homologs in multiple
organisms, indicates that its function is required for normal cilia formation.
Polaris is a component of a large complex known as the Intraflagellar
Transport (IFT) particle, which mediates the bidirectional movement of
proteins from the base of cilia to the cilia tip
(Haycraft et al., 2001
;
Pazour et al., 2000
;
Scholey, 2003
). Here, we
examine the connection between cilia defects and hydrocephalus in
Tg737orpk mutants, and evaluate the effects of cilial
dysfunction on both ependymal and CP epithelium. As seen in Mdnah5
mutants, the cilia defects on the ependymal cells in
Tg737orpk mice result in asynchronous beating and impaired
fluid flow across the ependymal cell surface. However, our analysis indicates
that abnormal cilial beating in Tg737orpk mutants is
unlikely to be the initiating factor, as hydrocephalus develops prior to the
formation of motile cilia on the wild-type ventricular ependyma. In addition,
the pathology is present in the absence of duct stenosis, indicating that
blockage of CSF flow is also not the causative factor. Rather, our data
support a model where cilia dysfunction leads to alterations in ion transport
activity of the CP epithelia and, subsequently, to a marked increase in CSF
production. Thus, we propose that the hydrocephalus in
Tg737orpk cilia mutants is not only a result of disrupted
ependymal cilia-generated CSF flow, but is, primarily, the result of
abnormalities involving cilia-regulated ion transport and CSF production by
the CP.
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Materials and methods |
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Morphological and histological analysis
Brains were fixed within the skull by removing the skull in the parietal
region to allow formalin penetration into the tissue. Twenty-four-hour
postfixation brains were removed and photographed. Fixed brains were then
embedded into paraffin blocks and sectioned in coronal plane. Sections were
stained with Hematoxylin and Eosin and photographed.
Magnetic resonance imaging (MRI)
Tg737orpk mutant and wild-type littermates at postnatal
day 1 and 6 were anesthetized using 1% isofluran. MRI was performed on a
Bruker-Biospin 8.5T vertical wide-bore DRX-360 (UAB 8.5T Small Animal NMR
Facility) with an AVANCE console, a Paravision 3.0.1 software platform, and a
Mini0.5 imaging system equipped with a 56 mm inner diameter gradient set
(Billirica, MA). Mice were positioned in a 20-mm birdcage resonator. Images
were coronal T2 weighted RARE (8 echoes, rare factor 8) with the following
parameters: TR 4.5 sec, effective TE 60ms, FOV 2.5 cm, 256x256 matrix,
slice thickness 0.45 mm, in plane resolution 98 µm, four averages. The body
temperature was maintained at 37°C. T2 RARE imaging allows detection of
the fluid compartments without requiring the use of contrasting agents.
Relative ventricular volume was calculated based on the intensity difference
using ImageJ software (NIH).
Immunofluorescence microscopy
Mouse brains were isolated from wild-type and Tg737orpk
animals and processed for immunofluorescence microscopy as described
previously (Taulman et al.,
2001). Primary antibody dilutions were as follows: mouse
anti-acetylated
-tubulin, 1:1500; rabbit anti-
-catenin, 1:500
(Sigma, St Louis, MI, USA); rabbit anti-polaris antibody, 1:500 (BY1700,
Sigma-Genosys against amino acids LEIDEDDKYISPSDDPHTN); rabbit
anti-polycystin-1 antibody, 1:300
(Ibraghimov-Beskrovnaya et al.,
1997
); and rat anti-zonula occludens, 1:40 (from Dr Daniel
Balkovetz, UAB). Secondary antibodies conjugated to FITC and rhodamine Red-X
were used at 1:500 (Jackson ImmunoResearch, West Grove, PA). Sections were
analyzed by immunofluorescence using an inverted Nikon TE200 microscope and
images were captured on a CoolSnap HQ/FX (Roper Scientific) CCD camera.
Proliferation analysis
Proliferation in CP cells from 3-day-old animals was determined by
immunostaining with an anti-phospho-histone H3 antibody (diluted 1:300;
Upstate, Lake Placid, NY). The proliferation index was assessed by counting
the number of H3-positive cell nuclei per 1000 nuclei.
Scanning electron microscopy
Freshly isolated brains from wild-type and Tg737orpk
mutant animals were processed for scanning electron microscopy as described
previously (Yoder et al.,
2002). Samples were then analyzed on either ISI SX-40 or Hitachi
2460 Variable Pressure scanning electron microscopes.
Videorecording of ependymal cilia
The function of ependymal cilia was assessed as described previously
(Ibanez-Tallon et al., 2004).
Briefly, to capture beating of the ependymal cilia, fresh brain slices from
either the lateral or fourth ventricle of 12-day-old animals were placed on a
glass coverslip. Prewarmed Phenol Red-free DMEM/F12 medium was mixed with a
suspension of red fluorescent beads (50 µm, Sigma) and added to fresh brain
slices. Cilia or particle movement was monitored by differential interference
contrast (DIC) and fluorescence microscopy on a Nikon TE200 equipped with a
CoolSnap HQ/FX CCD camera. Images were captured at 28 frames/second using
MetaMorph software. The same program was used to track particle movement and
to calculate mean speed of the tracked red fluorescent beads.
Brain ventricular injection of fluorescent DiI
Two- and 6-day-old animals were anesthetized using 100 mg/kg ketamine and 5
mg/kg xylazine, intraperitoneally. The right lateral ventricle was injected
with 1.0 µl of 0.2% DiI using the following coordinates: depth 1.8 mm,
lateral 0.9 mm crossing the line which bundle the posterior angles of orbitae
bilaterally in 2-day-old mice, 0.8 mm posterior to this point in 6-day-old
mice. Mice were then sacrificed and the brains were snap frozen and
cryosectioned. Horizontal sections of injected brains were then fixed with 4%
paraformaldehyde and nuclei stained with Hoescht. Sections were imaged using
fluorescence microscopy. The time required for the dye to pass from the
lateral ventricle into the fourth ventricle was determined by analyzing brain
sections generated from mice 5, 10, 20 and 30 minutes post-injection.
Isolation of CSF
To isolate the CSF, 18- to 23-day-old wild-type or mutant animals were
anesthetized as described above. CSF was harvested using a micromanipulator
and a Hamilton syringe, with a 26-gauge needle with the following coordinates
in mutant: Bregma 0.6 mm, lateral 1 mm, depth 1.8 mm. CSF was harvested
from wild-type mice as described previously
(DeMattos et al., 2002).
Chloride ion concentration was determined with ion selective microelectrodes
following the manufacturer's instructions (Lazarlabs, CA, USA).
Determination of [cAMP]i from isolated choroid plexus
Choroid plexi isolated from mutant and wild-type brains were immediately
frozen in liquid nitrogen. Tissue processing and intracellular cyclic AMP
content was determined using a competitive EIA assay system (Zymed
Laboratories, CA, USA), following the manufacturer's instructions. Protein
content was determined using DC Protein Assay Kit (BioRad Laboratories).
Statistical analyses
Values are means±s.e. Statistical significance was determined using
an unpaired Student's t-test.
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Results |
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Cilia are malformed on Tg737orpk mutant ependymal and choroid plexus epithelia
Previous data indicated that polaris and its homologs in
Chlamydomonas (IFT88) and C. elegans (OSM-5) function as an
IFT particle protein required for cilia formation
(Haycraft et al., 2001;
Pazour et al., 2000
). Inside
the ventricular system, cilia are found on ependymal cells that line the
ventricles, as well as on CP epithelia. Although the importance of the cilia
on the CP has not been explored, beating of the numerous motile cilia on
ependymal cells is thought to facilitate CSF movement, and data indicate that
loss of these cilia is associated with severe hydrocephalus. Thus, to further
explore a connection between the pathogenesis of hydrocephalus and cilia
defects in Tg737orpk mutants, we compared the cilia on the
ependyma and CP epithelia in mutant and wild-type mice by immunofluorescence
and by scanning electron microscopy.
The ependymal cells of adult mice have numerous long cilia that extend into the ventricular lumen. On wild-type CP epithelium, most cells have a small tuft of cilia on the apical surface; however, there are also numerous CP cells with a single primary cilium. The functional importance of these cilia types is unknown (Fig. 3).
In agreement with the hypomorphic nature of the
Tg737orpk mutation, polaris expression and cilia were
still detected on the ependyma and CP epithelium of mutant animals. Compared
with wild-type controls, the cilia on the mutant ependyma were fewer in
number, disorganized, stunted and anisometric, and often exhibited a bulb-like
structure at their tips in which the mutant form of the polaris protein
accumulated (Fig. 3). These
bulb-like structures were also observed on the CP epithelia and, as seen on
the ependyma, the mutant form of polaris was concentrated at the tip. These
morphological differences were also evident using scanning electron microscopy
and are in agreement with recently published data showing that primary cilia
on renal collecting duct cells of Tg737orpk mutants also
have this bulb-like structure (Liu et al.,
2005).
Malformed cilia in Tg737orpk mutants result in impaired beat and reduced fluid flow
The cilia morphology defects on the ependymal cells of
Tg737orpk mutants suggest that hydrocephalus may be
associated with an altered cilia beat and, subsequently, impaired CSF
movement. To assess these possibilities, we analyzed cilia beating on freshly
isolated ependymal cells using time-lapse DIC and fluorescence microscopy with
small fluorescent beads added to track fluid movement (see Movie 1 in the
supplementary material). On wild-type ependyma, cilia beat was rapid, well
orchestrated, and produced a laminar flow across the cells. By contrast, the
movement of cilia on mutant ependyma exhibited a low frequency beat, which was
asynchronous and failed to produce a significant amount of directional fluid
flow (Fig. 4). Thus, as seen
for other mouse mutants, the defect in cilia motility in the
Tg737orpk mutants is consistent with the impaired CSF flow
through the ventricles and with the development of hydrocephalus
(Ibanez-Tallon et al.,
2004).
|
Our analysis of cilia formation on ependymal cells using serial-section immunofluorescence indicated that, in one-day-old wild-type mice, most ependymal cells lining the ventricles had only a primary cilium. The presence of the multi-ciliated cells did not occur on the ventricular walls until around postnatal day 7. This was well after the pathology develops in the Tg737orpk mutants (postnatal day 1), suggesting that the loss of these motile cilia and the subsequent flow generated by them cannot be the cause of the hydrocephalus. One exception to this was the cells lining the aqueduct interconnecting the third and fourth ventricle. Most of these cells were multi-ciliated by postnatal day one (Fig. 5). Thus, the loss of motile cilia in the aqueduct of mutants could impair flow through the duct and lead to a pathology similar to obstructive hydrocephalus.
In contrast to the ependymal cells, the cilia on wild-type CP epithelium were well formed by day 1 and were similar to those seen in the adults. Because these cilia are present when the hydrocephalus initiates, loss of their function could contribute to the pathology. Although cilia on the multi-ciliated CP epithelium are motile (data not shown), our analyses indicate that they would have a minimal effect on generating CSF flow.
Initiation of hydrocephalus in Tg737orpk mice occurs prior to aqueduct stenosis
In contrast to the ependymal cells lining the ventricular walls, motile
cilia were present on aqueduct cells prior to the onset of hydrocephalus,
raising the possibility that an impaired function of these cilia may initiate
the phenotype. This could occur by duct stenosis, which is normally inhibited
by the beating of the cilia on these cells, or by impaired CSF flow through
these narrow structures in the absence of normal cilia beat.
To begin testing these possibilities, CSF flow was evaluated by using the fluorescent dye DiI injected into one lateral ventricle of 2- and 6-day-old wild-type and Tg737orpk mutant mice. The movement of DiI through the ventricles was analyzed by serial sectioning of the brain. To initiate this analysis, we evaluated DiI movement in wild-type (day 2 and 6) mice at 5, 10, 20, and 30 minutes after injection into the lateral ventricle to determine the time needed for it to be detected in the fourth ventricle. DiI was detected at all time points except for at 5 minutes, thus all subsequent analyses were performed after 10 minutes (Fig. 6). Our analysis of 2-day-old mutants was indistinguishable from that of the wild-type controls. This confirms that the aqueduct remains patent in the early stages of the disease and that the impaired motility of the cilia lining the aqueduct at this early age does not result in an obstructed CSF flow that could cause the pathology. In contrast to the 2-day-old mutants, in 6-day-old Tg737orpk mice, DiI was not detected in the fourth ventricle, indicating that passage through the aqueduct had been compromised. Because this occurs late in the pathogenesis of the disease in these mutants, the duct stenosis and loss of flow is likely to be a consequence, rather than a cause, of the hydrocephalus.
Cell polarity on the choroid plexus epithelia of Tg737orpk mutants
Another potential pathogenic mechanism is altered cell polarity, similar to
that seen for the kidneys of Tg737orpk mice, as well as of
several other PKD mouse models, which have revealed a mislocalization of
polarized proteins such as the EGF receptor and
Na+/K+-ATPase
(Wilson, 1997). In the kidney,
this results in excess fluid accumulation in the tubules and the development
of the cystic pathology (Avner,
1993
; Wilson,
1997
). Here, we analyzed sections of brains to determine the
localization of
-catenin and ZO-1 (Tjp1 Mouse Genome
Informatics), indicators of general polarity as well as of transport proteins
such as the Na+/K+-ATPase and the anion exchanger 2
(Fig. 7). The data indicate
that all of these proteins were localized normally in the mutants and at
similar levels to in the control samples. Thus, there were no overt defects in
the organization of the tissue because of defects of the cilia.
Another aspect of polarity that we analyzed was whether the distribution of
signaling proteins in the cilia axoneme was affected. An altered localization
of proteins in the axoneme could lead to their dysfunction and impair the
sensory or signaling activity of these cilia, as has been proposed to occur in
the kidneys of cystic mutants (Olteanu et
al., 2005; Liu et al.,
2005
). Because there are no data with regards to signaling
proteins in the cilia of the CP, on the basis of previous studies of renal
cilia, we evaluated whether polycystin-1 (Pkd1 Mouse Genome
Informatics) was present in the cilia of the CP and whether its distribution
was affected by the Tg737 mutation. Polycystin-1 is an integral cilia
membrane protein involved in a fluid flow-induced calcium signaling pathway
(Nauli et al., 2003
;
Praetorius and Spring, 2003
).
As seen in primary cilia of the kidney, polycystin-1 localized predominantly
at the basal bodies in both multi- and primary ciliated cells and at lower
levels along the cilia axoneme in wild-type CP
(Fig. 8). By contrast, in
Tg737orpk mutants, polycystin-1 was concentrated in the
bulb-like structure at the tip of cilia in CP, rather than in the basal body
(Fig. 8). Although polycystin-1
mutations are not associated with hydrocephalus, this example supports the
possibility that there may be cilia-mediated signaling defects in the CP of
the Tg737orpk mutants resulting from the mislocalization
of cilia proteins in the axoneme, which may result in the subsequent
transmission of a signal from the cilia into the cell, as has been proposed
for cystic kidney disease (Sutters and
Germino, 2003
).
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Discussion |
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In support of an impaired CSF flow mechanism, our in vitro analysis of the ciliary beat and fluid flow generated by the cilia on the ependymal cells isolated from the lateral ventricle of Tg737orpk mutants revealed that the beat is disorganized and flow is impaired. However, when we correlated the time at which the pathology becomes evident (postnatal day 1) in the Tg737orpk mutants with when the motile cilia actually form on cells in the ventricles, the data do not support a direct role for impaired cilia beat as being an initiating factor. An exception to this was the cells that line the aqueduct interconnecting the third and fourth ventricles. Unlike the ependyma lining the ventricles, this ductal epithelium possesses motile cilia that are present prior to onset of the pathology. However, our in vivo analyses of CSF movement using DiI injection indicate no differences in CSF flow between the mutant and wild-type controls at early stages of the disease. Impaired CSF movement was evident only after significant expansion of the ventricles, suggesting that loss of CSF flow is a consequence of the pathology. These data raise the possibility that a mechanism other than duct obstruction or loss of CSF flow is the initiating factor leading to the development of hydrocephalus.
Another possible mechanism involves defects in the CP. The CP is a
specialized secretory organ located within the brain ventricles, and its
primary functions are the production and homeostasis of the CSF
(Strazielle and Ghersi-Egea,
2000). Our analyses of the CP cells indicate that there are two
populations, one that has small tufts of motile cilia and another that has a
single primary cilium. The function(s) of either of these types of cilia on
the CP has not been explored. To our knowledge, this is the first description
of primary cilia on the CP, and we speculate that these cilia have sensory
roles similar to that shown in the embryonic node and in the renal
tubules.
Although not as common as obstructive hydrocephalus, where CSF movement is impaired, communicative forms of this disease have also been described that result either from a delayed reabsorption by arachnoid granulae or an excess CSF accumulation, such as in the case of CP tumors. In most cases where there are defects in reabsorption, MRI analysis reveals an expansion in the subarachnoid space. This is not evident in the Tg737orpk mutants, which suggests that impaired reabsorption is not the cause. As there is no overgrowth or increased proliferation of the CP in Tg737oprk mutants, any effects on CP function would likely occur at the level of a pathway regulating the secretory behavior of these cells. Thus, it is intriguing that our analysis of CSF composition indicates a significant increase in the level of chloride. Chloride is transported through the activity of an unidentified, apically localized, inwardly rectifying chloride channel that is regulated by cAMP. Thus, the increased chloride level in the CSF is supported by the elevated intracellular cAMP concentration in the CP epithelium. The elevated chloride level in the CSF suggests that the altered ion transport properties of the CP result in an increased fluid movement and an excess CSF production that would contribute to the development of hydrocephalus. The physiology causing this increased chloride transport and the connection to cilial function is currently under investigation.
Intriguingly, in the E2f5 mutants, defects in CP secretory
behavior are thought to cause a communicating form of hydrocephalus, as seen
in early Tg737orpk mutants
(Lindeman et al., 1998). This
may be analogous to the mechanism of renal cyst development in mice and humans
with cilia dysfunction (Guay-Woodford,
2003
). Several studies have shown that elevated cAMP signaling
caused by the vasopressin receptor type 2 results in excess fluid secretion
across cystic epithelium, the inhibition of which abrogates the cystic
pathology. Thus, it will be interesting to evaluate whether a similar
mechanism is involved in the hydrocephalus pathology in
Tg737orpk mice
(Sullivan et al., 1998
;
Torres, 2004
).
Overall, the brain pathology in the Tg737orpk mutants
appears to be a consequence of several cilia dysfunction-mediated events. The
first, which we believe is an initiating factor, involves altered ion
transport across the CP epithelium and an increase in the production of CSF.
How impaired cilia or polaris function in the CP epithelium affects the
localization, expression or activity of proteins involved in ion movement, and
which proteins are specifically involved, is being evaluated. One possibility
is that the loss of normal polaris function in the mutants results in an
altered distribution of a transporter/channel/exchanger in the cilia axoneme,
which, subsequently, leads to their aberrant function. The precedent for this
has been established by the case of polycystin 1. Polycystin 1 is required for
the flow-induced calcium signaling mediated by the deflection of the primary
cilium on renal epithelium, and, recently, it has been shown that this
flow-induced calcium signal is similarly abrogated in perfused tubules from
Tg737orpk mutants (Liu
et al., 2005). Thus, we expect that the loss of, or deformed,
cilia on cells of the CP may alter the function of proteins involved in ion
transport and CSF production, similar to that which occurs in the renal
epithelia of cystic kidney diseases. It is interesting to speculate that
similar defects might occur in the epithelia of other tissues (i.e. the
biliary duct and pancreatic duct) affected in the
Tg737orpk mutants. Thus, understanding how cilia organize
directional ion transport and CSF production in the CP may provide important
insights into the pathogenesis of several other diseases involving cilia
dysfunction.
The second event is likely to be the loss of cilia beat on the ependymal cells lining the ducts and ventricles. Previous studies in mice, such as in the Mdnah5 mutant, indicate that motile cilia do have important roles in CSF movement and that the loss of these motile cilia leads to hydrocephalus. Based on our analysis of when and where motile cilia form in relation to disease pathogenesis in the Tg737orpk mutants, it is likely that the progression of the disease is exacerbated by the impaired CSF movement through the ducts connecting the ventricles. This would result in increased intracranial pressure, ventricular expansion and duct stenosis, with rapid progression of the disease.
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ACKNOWLEDGMENTS |
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Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/23/5329/DC1
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