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First published online 12 November 2008
doi: 10.1242/dev.029835
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1 Department of Biology, Eberly College of Science, The Pennsylvania State
University, 201 Life Science Building, University Park, PA 16802, USA.
2 Howard Hughes Medical Institute and Department of Pediatrics, University of
Colorado Health Sciences Center, 12800 E. 19th Avenue, Aurora, CO 80045,
USA.
3 The Programs of Cell and Developmental Biology, Genetics and Neuroscience,
Huck Institutes of the Life Sciences, The Pennsylvania State University, 201
Life Science Building, University Park, PA 16802, USA.
* Author for correspondence (e-mail: AXL25{at}psu.edu)
Accepted 22 October 2008
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SUMMARY |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Mouse, Cilia, Hedgehog signaling, Gli3, C2 domain, C2cd3, Embryonic patterning, Basal body
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INTRODUCTION |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). Many potential ciliary proteins have been identified in
various species in recent years using biochemical, comparative genomic and
proteomic methods. Nevertheless, the spectrum of factors required for the
formation and/or function of cilia, as well as the molecular mechanisms
underlying the regulation of cilia biogenesis, have yet to be fully
revealed.
Two multiprotein complexes, the intraflagellar transport (IFT, complex A
and B) complexes, are present in the green alga Chlamydomonas
reinhardtii (Rosenbaum and Witman,
2002). The IFT complexes move within the flagella, suggesting that
they are likely to be involved in the transportation of molecules inside the
flagella. Mutations in protein components of the IFT complexes (the IFT
proteins), as well as in the microtubule motor proteins kinesin II and
cytoplasmic dynein, result in the degeneration of flagella, indicating that
IFT is required for flagella formation
(Pan et al., 2005).
Cilia have been implicated in the pathogenesis of many human genetic
diseases, such as polycystic kidney disease (PKD), Bardet-Biedl syndrome
(BBS), Meckel-Gruber syndrome (MKS) and Joubert's syndrome (JBTS)
(Fliegauf et al., 2007). Most
of the proteins known to be connected with these diseases are localized to the
cilia or to the basal bodies, centrosome-like structures from which cilia
originate. The identities of additional genes, such as those mutated in MKS2
(Roume et al., 1998) and JBTS2
(Valente et al., 2005)
patients, are yet to be discovered.
The discovery that cilia play essential roles in signal transduction in
multiple pathways, especially the Hedgehog (Hh) pathway, greatly advanced our
understanding of both the function of cilia and the mechanism of intracellular
signaling (Bisgrove and Yost,
2006). The Hh proteins, a family of secreted proteins, regulate
the development of multiple organ systems in both vertebrates and
invertebrates (Hooper and Scott,
2005). Loss of Hh signaling in mammals results in disruption of
left-right asymmetry, loss of ventral cell fate in the central nervous system
(CNS), loss of digits and many other defects
(Chiang et al., 1996).
In Drosophila, Hh regulates the activities of the transcription
factor Cubitus interruptus (Ci) (Methot
and Basler, 2001). Ci is a dual-function protein that acts as both
a transcriptional activator and repressor. In the absence of Hh, Ci is
proteolytically processed into a transcriptional repressor that maintains
repression of Hh target genes. When Hh is present, proteolytic processing of
Ci is inhibited and Ci acts as a transcriptional activator that turns on the
transcription of Hh target genes. The signal from Hh is transmitted to Ci
through a signaling cascade that starts with the binding of Hh ligand to its
cell surface receptor, Patched (Ptc). As a result, the G-protein-coupled
receptor-like protein Smoothened (Smo) is activated, leading to the inhibition
of Ci processing and activation of Ci activator function.
Many components of the mammalian Hh pathway serve similar functions to
their Drosophila counterparts
(Hooper and Scott, 2005).
However, significant differences do exist. One difference is the duplication
of most Hh pathway genes and their subsequent functional divergence in
vertebrates. For example, there are three mammalian homologs of Ci, which
constitute the Gli family (Gli1, Gli2 and Gli3). Gli1 does not appear to be
subject to proteolytic processing. Therefore, Gli1 functions as a
transcriptional activator only. Both Gli2 and Gli3 undergo proteolytic
processing in vivo, but Gli3 is much more efficiently processed than Gli2,
making it the major repressor (Pan et al.,
2006; Wang et al.,
2000). Hh pathway regulation between Drosophila and
vertebrates is also divergent in that some vertebrate-specific Hh pathway
components, such as Hip (Hhip - Mouse Genome Informatics) and Rab23, have been
identified (Chuang and McMahon,
1999; Eggenschwiler et al.,
2001).
In recent years, we and others have found that mouse and zebrafish mutants
with cilia defects exhibit compromised Hh signaling (reviewed by
Bisgrove and Yost, 2006;
Tobin et al., 2008). Our
detailed analysis indicates that IFT-related proteins are crucial for both Gli
activator and repressor functions (Liu et
al., 2005). Recent protein localization studies suggest that
multiple components of the mouse Hh signaling pathway are localized in the
primary cilia (Corbit et al.,
2005; Haycraft et al.,
2005; Rohatgi et al.,
2007). By contrast, Drosophila cilia mutants do not
exhibit defects in Hh signaling (Han et
al., 2003; Sarpal et al.,
2003). Therefore, roles for cilia in Hh signal transduction are
likely to be restricted to vertebrates.
Calcium signaling was first associated with cilia function with the
discovery that the intracellular calcium level rises upon the bending of cilia
on canine MDCK cells (Praetorius and
Spring, 2001). It was later shown that polycystin 2 (PC2; Pkd2), a
calcium-channel protein localized to the cilia, and its binding partner
polycystin 1 (PC1; Pkd1), are essential for initiating the calcium influx that
triggers the calcium level change in renal epithelia
(Nauli et al., 2003). A
PC2-dependent intracellular calcium surge was also observed on the left side
of the embryonic node at E8, after node cilia-mediated nodal flow is initiated
(McGrath et al., 2003).
However, PC1 is not expressed in the node and is not required for establishing
left-right asymmetry in the mouse, suggesting that the mechanism involved in
opening the PC2 channel in the embryo is different from that in the kidney
(Karcher et al., 2005).
Shh might play a role in PC2 activation because overexpression of
Shh leads to an increase in the intracellular calcium level on the
left side of the node (Tanaka et al.,
2005).
Additional studies suggest further roles for calcium during gastrulation
and left-right axis determination. In chicken, a high level of extracellular
calcium is observed on the left side of Hensen's node, and this asymmetric
distribution of calcium is translated into asymmetry of the embryo through
Notch signaling (Raya et al.,
2004). Calcium waves are also observed in the zebrafish and frog
organizers during gastrulation (reviewed by
Webb and Miller, 2006). It has
been suggested that calcium waves might be important for convergent extension
movements (Wallingford et al.,
2001). However, the roles for calcium in cilia formation have not
been investigated, despite the fact that several calcium-binding proteins
(e.g. calmodulin, calcineurin, centrin) are localized to the cilia or basal
body.
In the current study, we have identified C2cd3, a novel vertebrate-specific C2 domain-containing protein, as an essential regulator of ciliogenesis in the mouse. Through the study of mouse mutants carrying two different loss-of-function alleles of this gene, we show that C2cd3 is essential for mouse embryonic development through regulating the intracellular transduction of Hh signals and proteolytic processing of Gli3. Cilia biogenesis is severely disrupted in the absence of C2cd3. We speculate that C2cd3, a putative calcium-dependent lipid-binding protein that is localized at the basal body of cilia, mediates calcium-dependent vesicular transport and/or recruitment of proteins, including Hh pathway components, during cilia biogenesis. Therefore, the discovery of C2cd3 might lead to a better understanding of the connection between calcium signaling, cilia formation and cilia-dependent signal transduction.
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MATERIALS AND METHODS |
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SUMMARY
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MATERIALS AND METHODS
RESULTS
DISCUSSION
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) or using the same SSLP marker primers described above to
distinguish the mutant chromosome derived from the ES cells (129/Ola origin)
and the wild-type chromosome (C3H/HeN origin).
Bioinformatics
Information on the cDNA, exon-intron structure and open reading frames was
obtained from the Ensembl database
(http://mouse.ensembl.org).
C2cd3 protein structure was predicted using the SMART program
(http://smart.embl-heidelberg.de).
Searches for C2cd3 orthologs were performed by BLAST search against NCBI
(http://www.ncbi.nlm.nih.gov/BLAST),
the Chlamy Center
(http://www.chlamy.org),
the Cilia proteome
(http://www.ciliaproteome.org)
(Gherman et al., 2006) and the
ciliome
(http://www.sfu.ca/~leroux/ciliome_home.htm)
(Inglis et al., 2006), using
predicted mouse C2cd3 protein sequence. The C2cd3 gene-trap ES cell
line (AG0177) was identified through a BLAST search against the database of
the International Gene-Trap Consortium
(http://www.genetrap.org)
using the C2cd3 cDNA sequence.
Analysis of Hty mutant embryos
For immunohistochemical studies, mouse embryos were fixed in 4%
paraformaldehyde in PBS for 1 hour at room temperature, washed in PBS and
processed for cryosections at 10 µm. The sections were incubated with
primary and secondary (Cy3-conjugated) antibodies, each followed by a series
of washes in PBS containing 0.1% Triton X-100 and 1% goat serum. The slides
were mounted with DABCO (Sigma) and visualized using a Nikon E600 fluorescent
microscope. The X-Gal staining on whole-mount embryos has been described
previously (Liu et al.,
1998).
Scanning electron microscopy (SEM)
E8.0 mouse embryos were fixed overnight in 2.5% glutaraldehyde, washed in
PBS and dehydrated through an ethanol series. A small portion of the embryo
was removed for genotyping before dehydration. The dehydrated samples were
critical-point dried, mounted to metal mounds with the embryonic node facing
up, sputter-coated with silver and visualized with a JEOL JSM 5400 SEM at the
Penn State EM facility.
Mouse embryonic fibroblast (MEF) culture, transfection and visualization of cilia
E10.5 embryos were dissected in sterile PBS, the cells dissociated by
passage through gauge-20 needles and then plated in DMEM supplemented with 10%
fetal bovine serum (FBS), non-essential amino acids, sodium pyruvate, Glutamax
(Invitrogen) and antibiotics, at 37°C and 5% CO2. To visualize
the cilia, MEFs were passed onto gelatin-coated glass coverslips one day
before being transferred to medium containing 0.5% FBS for 48 hours. The cells
were then labeled with antibodies against acetylated tubulin (Sigma, T7451) or
-tubulin (Sigma, T5326), and visualized under a Nikon E600 microscope.
C2cd3 was tagged with GFP by cloning into the pEGFPN1 and pEGFPC3 mammalian
expression vectors (Clontech). Transient transfection of the C2cd3-GFP
expression construct was carried out using Lipofectamine 2000
(Invitrogen).
Western blot
Protein lysate was prepared from individual E10.5 mouse embryos and 20
µg from each sample loaded onto 7% SDS-PAGE gels for western blotting as
described (Wang et al., 2000).
Anti-β-tubulin antibody (Sigma, T4026) was used as loading control. The
result of the western blot was quantitated using ImageJ (NIH). Additional
antibodies were obtained from the Developmental Studies Hybridoma Bank:
anti-Shh, anti-Foxa2, anti-Nkx2.2, anti-Isl1, anti-Lhx3, anti-En1, anti-Pax6
and anti-Pax7.
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DISCUSSION
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), a recessive mouse mutation that disrupts embryonic
development and causes embryonic lethality between E11 and E13
(Table 1). Some Hty
homozygous mutants exhibit exencephaly in the midbrain and posterior
forebrain, a twisted body axis and pericardial edema (hence the name
Hty) (Fig. 1A,B;
Table 2). The Hty
mutants that undergo neural tube closure present with a characteristic tight
mesencephalic flexure (Fig.
1C,D). The heart loops in a leftward direction in wild-type
embryos, but turns rightward in some Hty mutants
(Fig. 1C,D, insets). At E12.5,
all Hty mutants exhibit severe polydactyly in all four limbs (7-9
digits) (Fig. 1E,F).
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;
Meno et al., 1996). In
Hty mutant embryos, both genes were expressed in both left and right
lateral plate mesoderm, suggesting a loss of left-right asymmetry
(Fig. 1H,J).
Hty encodes a C2 domain-containing protein
Through examination of 3692 meioses, we genetically mapped the Hty
mutation to a 
700 kb region between 107.27 Mb and 107.97 Mb on mouse
chromosome 7 (Fig. 2A). By
sequence analysis of all 12 previously uncharacterized genes in this region,
we found that C2cd3 (C2 calcium-dependent domain-containing 3)
contains a mutation in the first nucleotide of the fourth intron, changing a G
to an A in Hty mutants (Fig.
2B). As the first nucleotide of an intron is essential for correct
splicing of the preceding exon, we predicted that this point mutation would
disrupt splicing of the C2cd3 mRNA. Indeed, reverse transcriptase PCR
(RT-PCR) using primers in the third and fifth exons of C2cd3 revealed
multiple abnormal transcripts in Hty mutants
(Fig. 2C). Sequence analysis of
these abnormal RT-PCR products indicated that the three longest transcripts
(Fig. 2C, a-c) include two stop
codons that were originally part of the fourth intron, and hence would encode
a severely truncated C2cd3 protein (comprising the N-terminal 235 residues)
(Fig. 2D). Two shorter
transcripts (Fig. 2C, d,e) lack
part of exon 4 and would encode C2cd3 proteins with small deletions (five
residues in d, and 25 residues in e) in the N-terminal region
(Fig. 2D; data not shown). We
did not find any normal C2cd3 transcript in the Hty mutants,
even after two rounds of PCR amplification (data not shown).
The C2cd3 locus spans 98 kb and includes 34 exons. The predicted
full-length transcript is 7809 nt, encoding a 2322-residue cytoplasmic protein
with five C2 domains (Fig. 2D).
The C2 domain was named after the second functional domain of protein kinase
C, which is required for its calcium-dependent lipid-binding capability
(Nalefski and Falke, 1996). C2
domains were later identified in many proteins and found to mediate
interactions not only with membrane lipids, but also with other proteins.
We found C2cd3 orthologs in all representative vertebrate species including human, rat, chicken, Xenopus and zebrafish, yet none has been previously characterized. We also discovered that no other mouse protein shares overall sequence similarity to C2cd3, suggesting that C2cd3 does not belong to a protein family. Interestingly, we did not find a C2cd3 ortholog in Drosophila, C. elegans or in the flagellated green alga Chlamydomonas, suggesting that C2cd3 is vertebrate specific.
By whole-mount RNA in situ hybridization using various regions of the C2cd3 transcript as probes, we found that C2cd3 is ubiquitously expressed in mouse embryos between E8.5 and E10.5 (Fig. 2E; data not shown), consistent with our observation that Hty mutants exhibit defects in the patterning of multiple tissues at E10.5 (see Fig. 1 and below).
A second Hty mutant allele exhibits similar developmental defects
In order to confirm that C2cd3 is indeed the Hty gene, we
generated another C2cd3 mutant allele. We identified a gene-trap ES
cell line (AG0177) that harbors a bacterial β-galactosidase
(lacZ) insertion in the third intron of C2cd3
(Fig. 2D). This lacZ
insertion is predicted to ablate C2cd3 protein function by disrupting
C2cd3 transcription and splicing, resulting in a fusion protein
between the N-terminal 161 residues of C2cd3 and β-galactosidase. We
confirmed the lacZ insertion in the C2cd3 gene by RT-PCR and
sequence analysis (data not shown) and named this gene-trap allele
C2cd3GT.
We generated C2cd3GT carrier mice following blastocyst injection of the C2cd3GT ES cells. In a complementation assay, we bred a C2cd3GT carrier mouse with carriers of the original Hty mutant allele. The resulting transheterozygous (C2cd3GT/Hty) embryos exhibited similar defects to Hty homozygous mutants (twisted body axis, pericardial edema, etc.), indicating that C2cd3 is indeed the Hty gene (Fig. 2F). Subsequent analysis of the C2cd3GT homozygous mutants indicated that this allele exhibits similar, but more severe defects in embryonic development (Table 1 and below).
Dorsal-ventral patterning of the CNS is disrupted in Hty mutant embryos
In all Hty and C2cd3GT mutants with
exencephaly, the basal plates of the midbrain are flat
(Fig. 1B, inset), suggesting
potential defects in ventral CNS patterning. Therefore, we examined
dorsal-ventral (DV) patterning in the spinal cord at E10.5. Shh
expression in the floor plate, the ventral-most region of the spinal cord, was
absent in both Hty mutant alleles
(Fig. 3A-C). Another floor
plate marker gene, Foxa2, was also absent in the Hty mutant
spinal cord, confirming the absence of the floor plate (data not shown).
Despite the loss of Shh in the floor plate, Shh was
expressed in the underlying notochord in both Hty mutant alleles,
indicating that the loss of the floor plate is not due to the absence of
inductive signals from the notochord (Fig.
3B,C). Immediately dorsal to the floor plate are V3 interneurons
and their precursors that express Nkx2.2
(Fig. 3D). In Hty
mutants, the number of Nkx2.2-positive V3 interneurons appeared to be
reduced and these cells were mislocalized to the ventral midline
(Fig. 3E).
Isl1-expressing motoneurons, which are normally located dorsal to the
V3 interneurons (Fig. 3G), were
expanded ventrally in Hty mutants, such that some
Isl1-positive cells were found in the ventral midline of the spinal
cord (Fig. 3H). Interestingly,
the Nkx2.2-expressing V3 interneurons and most
Isl1-expressing motoneurons failed to form in the
C2cd3GT spinal cords
(Fig. 3F,I). The loss of
ventral cell types in the spinal cord of both Hty mutant alleles was
accompanied by a ventral expansion of more lateral cell types, such as the
Lhx3-expressing V2 interneurons (see
Fig. 6; data not shown).
Pax6, a gene normally expressed in the precursors of motoneurons and
more dorsal cell types, was expressed throughout the DV aspect of the spinal
cord of both Hty mutant alleles
(Fig. 3J-L). These results
indicate that C2cd3 is required for normal patterning of the ventral
CNS. The more severe loss of ventral cell types in the
C2cd3GT spinal cord also suggests that Hty is
likely to be a hypomorphic allele, possibly owing to residual activity of the
mutant C2cd3 proteins with small in-frame deletions.
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), all
of which are disrupted in Hty mutant embryos. The fact that ventral
spinal cord patterning is disrupted in Hty mutants despite the
presence of Shh-expressing notochord suggests that signal
transduction downstream of Hh might be defective. In order to determine
whether the Hty phenotype is associated with a disruption in Hh
signal transduction, we examined the expression of Ptch1, one of the
direct transcriptional targets of Hh signaling
(Goodrich et al., 1997). Using
a Ptch1-lacZ reporter mouse, we found that Ptch1 expression
is downregulated in multiple tissues, including the CNS and gut
(Fig. 4A,B) and in the
posterior region of the limb buds (Fig.
4E,F). The reduction of Ptch1-lacZ expression in the
spinal cord was confirmed in sections (Fig.
4C,D). We next examined the expression of Gli1, another
target gene of Hh signaling (Bai et al.,
2004). Gli1 expression was downregulated in all tissues
of E10.5 Hty mutant embryos, including brain, otocysts
(Fig. 4G,H), spinal cord
(Fig. 4I,J) and limb buds
(Fig. 4K,L). The reduction of
Ptch1 and Gli1 expression in multiple tissues of
Hty mutant embryos indicates that C2cd3 is an important
regulator of Hh signal transduction in the mouse.
To better understand the role of C2cd3 in Hh signal transduction,
we performed double-mutant analyses between Hty and known regulators
of Hh signal transduction. Rab23, which encodes a small GTPase
mutated in the mouse mutant Open brain (Opb and
Opb2), negatively regulates Hh signaling in the mouse
(Eggenschwiler et al., 2001).
In the Opb2 mutant spinal cord, Hh signaling is broadly activated,
resulting in severe ventralization of the CNS. As a result, although the
Foxa2-expressing floor plate remained ventrally restricted
(Fig. 5A,B), other ventral cell
types, such as the Nkx2.2-expressing V3 interneurons and their
precursors, were dorsally expanded to occupy most of the spinal cord
(Fig. 5E,F). By contrast,
Pax6, which is expressed in the dorsal two-thirds of the wild-type
spinal cord, was only expressed in the dorsal-most region of the spinal cord
in Opb2 mutants (Fig.
5I,J). In mouse embryos doubly mutant for Hty and
Rab23 (Hty/Opb2), as in Hty single mutants, the
floor plate was missing (Fig.
5C,D) and a reduced number of Nkx2.2-expressing V3
interneurons was present in the ventral midline
(Fig. 5G,H). Pax6 was
expressed throughout the spinal cord in both Hty single and
Hty/Opb2 double mutants (Fig.
5K,L). The indistinguishable spinal cord phenotype of Hty
and Hty/Opb2 double mutants suggests that C2cd3 acts
genetically downstream of Rab23. Rab23 acts cell-autonomously to
regulate the response of spinal cord cells to Hh and acts genetically
downstream of Ptch1 and Smo
(Eggenschwiler and Anderson,
2000; Eggenschwiler et al.,
2006). Therefore, C2cd3 is likely to be an intracellular
regulator of Hh signal transduction inside Hh target cells. Consistent with
this proposal, a double-mutant analysis between Hty and
Ptch1 indicated that C2cd3 is also genetically downstream of
Ptch1 in the Hh signal transduction pathway (see Fig. S1 in the
supplementary material).
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C2cd3 is required for ciliogenesis in the mouse
The multiple defects in mouse embryonic development and the disruption of
Hh signaling in the Hty mutants closely resemble those seen in
mutants for Ift88 and Ift52, which exhibit severe loss of
cilia [compare figs 3,
4,
5 in Liu et al.
(Liu et al., 2005) with Figs
3,
4,
5,
6 of this report]. Therefore,
we sought to determine whether C2cd3 also plays a role in the
formation of cilia. Primary cilia (node cilia) are present on all cells of the
ventral embryonic node in wild-type embryos
(Fig. 7A). In Hty
mutants, cilia failed to form in most cells in the node
(Fig. 7B). In
C2cd3GT mutant embryos, cilia were absent in nearly all
node cells (Fig. 7C),
suggesting that it is a more severe loss-of-function allele than the original
Hty allele.
To further address whether C2cd3 directly regulates the intrinsic capability of the cells to form primary cilia, we examined cilia formation in wild-type and Hty mutant mouse embryonic fibroblasts (MEFs) in culture. Wild-type cells kept in G0 for 48 hours developed primary cilia efficiently (Fig. 7D,F) (78% ciliated, n=373). By contrast, cilia formation in Hty mutant cells under identical conditions was greatly compromised (Fig. 7E,F) (21% ciliated, n=365). Therefore, C2cd3 is required intrinsically for the formation of cilia.
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). We have previously shown that mutations in mouse IFT genes
lead to inefficient processing of Gli3
(Liu et al., 2005). In order
to better understand the mechanisms underlying the roles of C2cd3 in
regulating Hh signaling and Gli activities, we examined Gli3 processing in
Hty mutants by western blot, using an antibody against the N-terminus
of Gli3 that interacts with both the full-length (Gli3-190) and processed
short (Gli3-83) forms of Gli3 (Wang et
al., 2000). As previously reported, in wild-type E10.5 embryos
Gli3 is efficiently processed into its short repressor form (Gli3-83)
(Fig. 8A, lanes 1 and 3). By
contrast, in both Hty and C2cd3GT homozygous
mutant embryos at the same stage, more Gli3 protein was present as
unprocessed, full-length form (Gli3-190)
(Fig. 8A, lanes 2 and 4). As a
result, the ratio of Gli3-190/Gli3-83 was dramatically increased in the two
Hty mutant alleles (from 0.36 in wild type to 1.34 in Hty
and 1.47 in C2cd3GT)
(Fig. 8B). This suggests that
C2cd3 regulates Hh signaling (at least) partly through the regulation
of Gli3 proteolytic processing.
C2cd3 protein is localized to the basal body of cilia
In order to better understand the roles of C2cd3 in cilia formation, we
examined its subcellular localization by overexpressing C2cd3 tagged with
green fluorescent protein at its C-terminus (C2cd3-GFP) in primary MEFs. By
labeling ciliary axoneme with an anti-acetylated tubulin antibody, we
discovered that C2cd3-GFP is present at one end of the cilia, presumably at
the basal body (Fig. 9A). By
labeling the two centrioles of the basal body with an anti--tubulin
antibody, we confirmed that C2cd3-GFP is indeed localized at the basal body
(Fig. 9B). A similar basal body
localization was observed when the GFP tag was added to the N-terminus of
C2cd3 (data not shown).
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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C2cd3, cilia formation and cilia-related human genetic diseases
The formation of functional cilia is a complex process and requires a wide
variety of genes. The lack of common structural features among proteins
regulating cilia formation has hindered the discovery of these important
regulators. Recent molecular screens utilizing comparative genomics and
proteomics have identified dozens of potential novel regulators of cilia
(Inglis et al., 2006).
However, owing to the immensely complex nature of ciliogenic regulation, these
screens have apparently failed to saturate the `ciliome'. The fact that C2cd3,
which is specific to vertebrates, is not found in the current ciliome
emphasizes the fact that our knowledge of ciliogenic regulation is far from
complete.
The identification of novel regulators of cilia not only provides new
insight into the molecular mechanism of cilia regulation, but is also
invaluable in providing new animal models for cilia-related genetic diseases
in human. A number of human genetic diseases, including BBS, MKS and JBTS,
exhibit common symptoms, such as cystic kidney and polydactyly
(Fliegauf et al., 2007). Many
patients with these diseases carry mutations in genes encoding ciliary
proteins. For many patients, the gene mutations have not been identified,
although some have been mapped to specific chromosomal regions. Interestingly,
the C2cd3 ortholog in humans is in chromosomal region 11q13.4, close
to the reported critical regions for MKS2 and JBTS2
(Roume et al., 1998;
Valente et al., 2005). It will
be interesting to determine whether the sequence or expression of the human
C2CD3 gene is disrupted in these patients.
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250 kDa, which is much larger than the reported size of
any Chlamydomonas IFT proteins
(Rosenbaum and Witman, 2002).
Finally, we have not detected C2cd3 inside the cilia, suggesting that it might
not be an integral part of the IFT complexes.
It has recently been reported that seven BBS proteins form another
multiprotein complex called the BBSome
(Nachury et al., 2007). The
BBSome is localized to the primary cilia in ciliated cells and appears to be
associated with the cell membrane, suggesting that it might be involved in
vesicular transport. It will be interesting to determine whether C2cd3 plays a
role in the function of the BBSome.
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). C2
domains have subsequently been found in other proteins, many of which are
associated with the cell membrane and carry out a variety of functions. We
speculate that C2cd3 might be involved in vesicular transport required for
cilia biogenesis and/or in the transport of membrane-associated proteins.
Alternatively, or additionally, some or all of its five C2 domains might
mediate interactions with multiple proteins to form a multiprotein complex
involved in cilia biogenesis. We are currently addressing these possibilities
biochemically.
Hty, cilia and Hh signaling
Our previous studies indicate that IFT is required for Hh signal
transduction in the mouse (Huangfu et al.,
2003). More specifically, both the proteolytic processing and
activation of the Gli transcription factors require IFT function
(Liu et al., 2005). This is in
contrast to Drosophila, in which the mechanism of IFT is largely
conserved but only a small number of sensory neurons exhibit primary cilia and
IFT is not required for Hh signaling
(Witman, 2003). In the mouse,
some Hh signaling components, such as Ptch1, Smo, Sufu (suppressor of fused),
Gli1, Gli2 and Gli3, are localized to primary cilia
(Corbit et al., 2005;
Haycraft et al., 2005;
Rohatgi et al., 2007).
However, it is not clear whether the presence of cilia per se is enough for
normal Hh signaling, or whether IFT participates directly in transporting Hh
signaling components inside the cilia.
Hty mutants display multiple embryonic developmental defects, including disruption of CNS and left-right patterning, polydactyly in the limbs and pericardial edema. Most of these defects can be explained by abnormal Hh signaling. Indeed, our Hh target gene expression analysis and genetic studies indicate that C2cd3 regulates Hh signaling, acting downstream of Ptch1 and Rab23. The fact that C2cd3 is specific to vertebrates is intriguing as C2cd3 might be involved in transporting vertebrate-specific cargos such as Hh pathway components.
|
|
; Huangfu and Anderson,
2005; Liu et al.,
2005). Our discovery that C2cd3 is required for efficient
processing of Gli3 is consistent with its essential role in cilia biogenesis.
However, we do not have sufficient evidence to distinguish whether the
inefficient processing of Gli3 in the Hty mutants results from the
cilia defects, or whether C2cd3 plays a more direct role in Gli3
regulation.
It is paradoxical that despite the accumulation of full-length Gli3 protein
(Gli3-190), the loss of ventral cell types in the spinal cord and
downregulation of Hh target gene expression in the Hty mutants
suggest a loss of Gli activator activity. We propose that Gli3-190 in
Hty mutants is not in its active state and is likely to be subject to
additional negative regulation. It has been shown in Drosophila that
Sufu keeps Ci in a stable but inactive state
(Ohlmeyer and Kalderon, 1998).
Therefore, it will be of interest to investigate whether Sufu is responsible
for the loss of Gli activator function in Hty and other cilia
mutants.
Cilia and calcium signaling
Cilia are closely associated with intracellular calcium signaling in many
contexts. Currently, it is generally believed that cilia play a role in
regulating calcium levels in ciliated cells. In renal epithelial cells,
bending of the primary cilia opens the calcium channel, PC2, allowing calcium
to enter the cytoplasm (Wilson,
2004). In gastrulating mouse embryos, a calcium surge is observed
on the left side of the embryonic node, which might be the consequence of
cilia deformation-induced PC2 activation
(McGrath et al., 2003).
Alternatively, Shh might play a role in PC2 activation, as overexpression of
Shh leads to an increase in intracellular calcium levels on the left side of
the node (Tanaka et al.,
2005).
The presence of multiple C2 domains in the C2cd3 protein is likely to be key to an understanding of its molecular functions. It is possible that calcium plays important roles in C2cd3 function, as well as in cilia formation. In fact, many known ciliary proteins are calcium-binding proteins (e.g. calmodulin, calcineurin, centrin). Therefore, a close examination of the roles of calcium in cilia formation is needed.
Alternatively, calcium might be involved in the process of loading and unloading cargoes. One speculation is that C2cd3, as an adaptor protein between IFT complex and vesicular cargoes at the basal body, exhibits a high affinity for its cargoes in the presence of calcium, allowing their transportation to the base of cilia. A transient drop in calcium level at the destination, or the presence of a competing adaptor protein, could lead to the dissociation of C2cd3 from its cargoes, facilitating unloading.
In conclusion, we have discovered that C2cd3 is a vertebrate-specific, novel, C2 domain-containing protein essential for Hh signaling, cilia formation and mouse embryonic development. The presence of calcium-dependent lipid-binding domains in C2cd3 suggests a potential role in vesicular transport. Finally, the proximity between the human C2cd3 ortholog and certain cilia-related genetic disease loci on chromosome 11 makes it an interesting candidate for ciliopathy in humans.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/24/4049/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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