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First published online April 24, 2009
doi: 10.1242/10.1242/dev.020735
,
1 Cardiovascular Research Center, Massachusetts General Hospital, 149 13th
Street, Charlestown, MA 02129, USA.
2 Princeton University, Department of Molecular Biology, Princeton, NJ 08544,
USA.
Authors for correspondence (e-mail:
fabrizio.serluca{at}novartis.com;
rburdine{at}princeton.edu)
Accepted 16 March 2009
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SUMMARY |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Zebrafish, seahorse, Lrrc6l, Cilia motility, Asymmetry, Pronephros, Cysts, Kupffer's vesicle
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INTRODUCTION |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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).
Defects in ciliary structure and function have also been implicated in
mammalian cystic kidney disease (Yoder,
2007). In the mammalian kidney, cells produce a single primary
cilium that extends into the renal lumen. This cilium is thought to function
in cell signaling events and/or to sense and respond to luminal flow
(Praetorius and Spring,
2005).
Similarly, studies in zebrafish have shown that cilia motility and
structure are essential for proper kidney development, and that early defects
in cilia correlate with pronephric cyst formation
(Kramer-Zucker et al., 2005;
Omori and Malicki, 2006;
Sullivan-Brown et al., 2007;
Zhao and Malicki, 2007). In
contrast to the immotile cilia observed in the mammalian kidney, the cilia in
the zebrafish pronephros are motile and thought to regulate fluid exit from
the body (Kramer-Zucker et al.,
2005). When fluid flow is disrupted in zebrafish, cyst formation
can occur. However, recent research suggests that cilia function may have even
earlier roles in regulating kidney morphogenesis
(Sullivan-Brown et al.,
2007).
Cilia motility has also been implicated in the specification of the
left-right axis in both zebrafish and mammals (reviewed by
Bisgrove and Yost, 2006). In
vertebrates, a transient ciliated structure known as the node in mammals and
Kupffer's vesicle (KV) in zebrafish is essential for proper left-right axis
development. Severe defects in cilia motility within the node/KV result in
incorrect expression of the normally left-sided gene nodal, which
subsequently results in abnormal placement of organs about the left-right
axis. Without cilia motility, directional flow of fluid inside the node/KV
fails to occur (Essner et al.,
2005; Kramer-Zucker et al.,
2005; Okada et al.,
1999), but the specific mechanism of how directional fluid flow
within the node/KV restricts nodal expression to the left side of the
organism remains unclear.
Because cilia defects result in a large and diverse group of disorders, it is important to understand the molecular and cellular basis of how cilia motility, structure and function influence physiology and development. We have taken the approach of cloning and characterizing genes that function in pathways influenced by cilia. These genes will give us a starting point for determining the downstream signaling pathways regulated by cilia and will provide insights into the structure and function of cilia components. Here, we report the cloning and characterization of two alleles of zebrafish seahorse (sea), seatg238a and seafa20r, that have been isolated from two independent screens. sea encodes a leucine-rich repeat-containing protein, Lrrc6l. We show that the above mutations in lrrc6l do not affect cilia structure, apical position of the basal body or the ability to interact with Disheveled, but result in severe cilia motility defects in the pronephros and neural tube that range from slow and disorganized cilia motility to immotile cilia. Although these two sea alleles result in a fully penetrant pronephric cyst phenotype, they surprisingly show a low incidence of left-right patterning defects. sea mutants have variable effects on fluid flow in KV, ranging from loss of flow, to flow that is indistinguishable from wild type. Thus, here we provide the first experimental evidence that lrrc6l is required for cilia motility in vivo. Although these mutations affect cilia motility, they have different effects on downstream cilia-related phenotypes, thereby showing that the function of Sea in cilia motility and cilia-related phenotypes is genetically separable.
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MATERIALS AND METHODS |
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DISCUSSION
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). Initial
linkage to chromosome 2 was found using marker Z1406 and the region narrowed
using additional markers. The closest marker, Z8448, was used to initiate a
BAC walk towards the mutation as described
(Liao and Zon, 1999). To
determine the molecular lesions in each allele, mutant cDNAs or genomic DNAs
were amplified and subjected to sequencing (details of primer sequences are
available upon request).
In silico analysis
GCG SeqWeb version 1.1 was used to identify the presence of leucine-rich
repeats (MEME) and coiled-coil domain (CoilScan). Protein alignments were
performed by NCBI BLAST (Blosum 62 substitution matrix). PSORT II
(http://psort.ims.u-tokyo.ac.jp)
was used to identify nuclear localization signals and CBS NetNES algorithm
(http://www.cbs.dtu.dk/services/NetNES)
was used to identify nuclear export signals.
sea cDNA cloning
The published cDNA for sea (NCBI AY618925) contains a 101 bp
5' UTR, a 1323 bp coding sequence and a 334 bp 3' UTR. A search of
The Institute for Genomic Research (TIGR) expressed sequence tag database
revealed a possible 168 bp extension (cluster fj66c12.x1 AW077770) beyond the
published 3' UTR terminus. Primers based on the extended sequence
amplified the sea cDNA using Expand Hi-fidelity enzyme (Roche,
Indianapolis, IN, USA) and a zebrafish 24 hpf cDNA library constructed using
the Marathon cDNA Amplification Kit (Clontech, Mountain View, CA USA). The 1.9
kb product was amplified, confirming the authenticity of the 3'UTR
extension, and subcloned into pBluescript SKII(+) (Stratagene, La Jolla, CA
USA) creating plasmid BL289.
In vitro transcription and in situ hybridization
mRNA transcripts were synthesized using mMessage mMachine kits (Ambion,
Austin TX, USA) and quantified by UV spectrophotometry. Plasmid BL289 was
linearized with NotI and transcribed using T3 RNA polymerase, and 500
pg of mRNA per embryo was injected at the one-cell stage. Plasmid BL355,
containing a partial sea cDNA, was linearized with NotI and
transcribed with T7 RNA polymerase to generate antisense in situ hybridization
probe. In situ hybridization was performed as previously described
(Thisse et al., 1993). Probes
used include: southpaw (Long et
al., 2003), cardiac myosin light chain (cmlc2/myl7)
(Yelon et al., 1999),
forkhead 2 (fkd2/foxa3)
(Odenthal and Nusslein-Volhard,
1998), preproinsulin
(Milewski et al., 1998),
lefty1 and lefty2
(Bisgrove et al., 1999),
lov (Gamse et al.,
2003), wt1 (Bollig et
al., 2006; Serluca and
Fishman, 2001), pax2.1
(Krauss et al., 1991), and
-tropomyosin (Ohara et
al., 1989).
Morpholinos and RT-PCR
Four different morpholino antisense oligonucleotides (MO1-MO4) directed
against sea were purchased from GENE Tools (Philomath, OR USA;
sequences available upon request). Only MO1 (seae5i5)
5'-TTAGACACTCACTGGTTTATTTCAG-3' gave specific phenotypes upon
injection, and thus was used in this report. MO1 (2 nl of 1 mM) was injected
into one-cell stage embryos for phenocopy experiments. For low-level MO
injections, 400 pl of 1 mM MO1 was injected. To determine the extent of splice
blocking, RT-PCR was performed and the resulting products were sequenced. cDNA
libraries used in RT-PCR to determine stage of sea expression or
extent of MO splice-blocking were created from total RNA with the SuperScript
First-Strand Synthesis System (Invitrogen, Carlsbad, CA, USA).
Genotyping
PCR was performed on DNA extracted from embryos or fin clips using primers
for seatg238a (5'-TTTGCTTGAAAAGTGTGATGTGA-3'
and 5'-AAGGTTGTGCTTCAGCG-3') or for seafa20r
(5'-CAGAGAACTTGTACCTGTGTTTTGGATGAA-3' and
5'-TCTCCCAGAATTCCCTCTCCTCG-3'). The fa20r mutation
creates an AluI restriction site, which cleaves the 114 bp PCR
product into 81 bp and 33 bp fragments
(Fig. 1C,C',D). The
tg238a mutation abolishes the FspBI site preventing cleavage
of a 176 bp fragment into 114 and 62 bp fragments
(Fig.
1C',C''',D').
Histology and immunohistochemistry
Immunostaining for the Na+/K+ ATPase and acetylated
tubulin were performed as described
(Sullivan-Brown et al., 2007).
Nuclei were stained with DRAQ5 (Axxora, San Diego, CA USA). Histological
analysis and sectioning of immunostained embryos were performed as described
previously (Sullivan-Brown et al.,
2007).
Co-immunoprecipitation
HA epitope-tagged sea wild-type and mutant cDNAs were cloned into
pcDNA3.1 (Invitrogen, Carlsbad, CA USA) using standard methods. Flag-tagged
Disheveled has been described previously
(Habas et al., 2001). For
immunoprecipitation assays, wild-type or mutant Seahorse proteins were
expressed in the presence or absence of Disheveled in Cos-7 cells using
Lipofectamine2000 transfection (Invitrogen, Carlsbad, CA USA). Cells were
lysed in a modified RIPA buffer [50 mM Tris (pH 7.4), 150 mM NaCl, 0.5% NP-40,
0.25% deoxycholic acid, 1 mM EDTA, 1 mM NaF] supplemented with 0.4 mM PMSF and
Complete Mini Protease inhibitor cocktail (Roche, Indianapolis, IN USA). The
immunoprecipitation antibody (-Flag AP M2, Sigma-Aldrich, St Louis MO,
USA) was added to the lysates at a dilution of 1:500 and incubated at 4°C
for 2 hours. Protein A-Sepharose beads (Sigma-Aldrich, St Louis MO, USA) were
added to the lysates and gently rocked for an additional two hours at 4°C.
The bead complexes were washed with modified RIPA buffer, mixed with
SDS-sample buffer, boiled and spun. All supernatants were resolved on Nu-PAGE
10% Bis-Tris Gels (Invitrogen, Carlsbad, CA USA) and proteins detected using
primary antibodies, coupled with horseradish peroxidase, against the epitope
tags (Anti-HA-HRP clone 3F10, Roche, Indianapolis, IN USA; Anti-Flag-HRP clone
M2, Sigma-Aldrich, St Louis MO, USA). Proteins were detected using LumiLight
Plus (Roche, Indianapolis, IN, USA).
Microscopy
Embryos processed for immunofluorescence were visualized on a Zeiss LSM
510. For analysis of KV cilia, embryos were mounted in 50% glycerol in PBS,
and imaged. Cilia lengths were obtained using ImageJ to analyze re-plotted
iz-stacks. Embryos processed for RNA in situ hybridization were photographed
as described (Schottenfeld et al.,
2007). Cilia motility imaging in the pronephros was performed as
described (Sullivan-Brown et al.,
2007). Embryos (48 hpf) were processed for transmission electron
microscopy as described (Majumdar and
Drummond, 2000) and imaged on the Zeiss 921AB.
Analysis of KV fluid flow
Beads were injected in KV and movement analyzed at the six-somite stage as
described (Essner et al., 2005;
Okabe and Burdine, 2008).
After imaging, embryos were raised to 48 hpf to distinguish mutants from
siblings using the CTD phenotype and heart looping was scored. Defects in
fluid flow were annotated using a blind test involving nine people who
analyzed unlabeled movies from sea siblings or mutants compared with
wild-type embryos. Flow is annotated as described previously
(Essner et al., 2005).
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MATERIALS AND METHODS
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DISCUSSION
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).
fa20r was isolated in an independent F3 screen for pronephric cyst
mutations [as previously described in Chen et al.
(Chen et al., 2001)].
Complementation crosses between fa20r and tg238a
heterozygous parents resulted in 25% CTD F1 embryos with pronephric cysts,
confirming that the mutations were allelic (data not shown). The gene mutated
in fa20r and tg238a was independently cloned in a retroviral
insertional mutagenesis screen for genes causing pronephric cysts
(Sun et al., 2004) and named
seahorse (sea, NCBI AY618925). Thus, we will refer to our
alleles as seafa20r and seatg238a. The
CTD phenotype in seafa20r and
seatg238a embryos is first detectable at 27 hours post
fertilization (hpf) and persists through later stages (data not shown and
Fig. 2A-C). Both alleles
develop pronephric cysts that are morphologically visible in all mutant
embryos by 3.5 dpf (Table 1;
Fig. 2D-F; and data not
shown).
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). Lrrc6l is a leucine-rich repeat-containing protein of 440
amino acids with a predicted molecular weight of 50.5 kDa. MEME repeat
searching identified four leucine-rich repeat (LRR) protein-protein
interaction motifs present near the N terminus
(Fig. 1Aii, green). The 22-32
amino acid consensus LRR in Lrrc6l includes the canonical repeat shared among
all LRR proteins (Fig.
1A'', underline). The full LRR repeat region in Lrrc6l most
closely resembles that of the SDS22-like subfamily of LRR proteins
(Kobe and Kajava, 2001;
Ohkura and Yanagida, 1991).
Amino acids 131-146 encode the LRR cap
(Ceulemans et al., 1999), a
motif that terminates LRR domains in many LRR-containing proteins. Immediately
after the LRR cap motif, a coiled-coil domain was predicted between 148 and
186 amino acids (Fig. 1Aii).
The protein contains a possible nuclear localization signal (NLS) at 363-369
amino acids and three weak nuclear export signals (NESs) at 73-75, 295-299 and
356-358 amino acids (Fig.
1Aiii). Lrrc6l is significantly conserved among species
(Fig. 1A'). Amino acids
1-383 of Lrrc6l are homologous (39% identity, 58% similarity) to an
LRR-containing protein from T. brucei (AAF73195)
(Morgan et al., 2005).
Full-length Lrrc6l had 55% identity to X. tropicalis LRRC6
(CAJ83438.1) and 54% identity to mouse LRRC6 (AAH46277.1)
(Xue and Goldberg, 2000). Both
Xenopus and mouse orthologs extended roughly 30 amino acids beyond
the C terminus of zebrafish Lrrc6l (Fig.
1A'). Both sea alleles were sequenced to determine the molecular lesions in Lrrc6l. The fa20r mutation encodes a Q201X truncation that occurs after the predicted coiled-coil domain (Fig. 1A,B,B'). The tg238a encodes a L93P missense mutation within the fourth LRR (Fig. 1A,B'',B'''). Injection of seatg238a or seafa20r clutches with lrrc6l mRNA was able to rescue both the CTD and pronephric cyst phenotypes at 1.5 dpf and 2.5 dpf, respectively (Table 1). Finally, a morpholino directed against the exon/intron boundary of exon 5 (e5i5) phenocopied sea mutants with variable degrees of CTD and visible pronephric cysts (Table 1; Fig. 2G-J). These effects were most obvious at 1.5 dpf and 2.5 dpf, respectively. By 3.5dpf, 52% of the embryos retained cysts whereas the majority no longer displayed a CTD phenotype and even fewer resembled sea mutants at 5 dpf (Fig. 2H,J; and data not shown). This suggests that the injected MO is not able to maintain knockdown to later stages and that the MO-injected embryos can recover from both the tail curl and pronephric cyst formation. We confirmed the effects of the morpholinos on splicing by RT-PCR on MO-injected embryos at four somites, 24 hpf and 48 hpf (Fig. 2K,L).
seahorse expression pattern suggests a role for Lrrc6l in cilia motility
To determine when and where sea may function during development,
we analyzed sea mRNA expression in wild-type embryos. By RT-PCR,
sea is expressed both maternally and zygotically
(Fig. 3A). Consistent with our
RT-PCR experiments, a low level of expression was detected by RNA in situ
hybridization at 256-cell, sphere and 30-50% epiboly stages, indicating that
sea is maternally provided (data not shown). Specific expression was
observed in the dorsal forerunner cells
(Fig. 3B) and in KV
(Fig. 3C). During mid to late
somitogenesis, sea mRNA was also detected in the floor plate
(Fig. 3D). After 24 hpf,
sea expression in the floor plate was weaker in the anterior regions
but stronger at the posterior end of the tail
(Fig. 3F,G,I), expanding into
the chordoneural hinge (Fig.
3G). sea was expressed in the pronephric tubules from
mid-somitogenesis through 48 hpf (Fig.
3D-F,H-J).
All of these locations contain motile cilia
(Kramer-Zucker et al., 2005)
(this report). As further support that sea may be involved in cilia
motility, sea is expressed in a patchy pattern within the pronephros
(Fig. 3F, inset), a pattern
similar to other genes expressed in multiciliated cells in this region
(Liu et al., 2007;
Ma and Jiang, 2007).
Furthermore, at 36 hpf, sea expression localizes to the anteriormost
tubules adjacent to the glomerular region
(Fig. 3H), which may correspond
to the ciliary neck segment found in mammals and other teleosts
(Wingert et al., 2007).
|
).
However, at 3dpf variable effects on wt1 expression were observed in
sea. In sibling embryos, wt1 expression was condensed within
the fused glomerular region (Fig.
4I). In mutant embryos, the wt1 expression was more
diffuse ranging from slight (Fig.
4J) to extreme expansion (Fig.
4K). The expansion of wt1 expression is probably an
indirect/secondary consequence of the dilations that form in this region (see
below).
|
). Consistent
with other cystic mutants, sea mutants did not show a normal pattern
of basolateral Na+/K+-ATPase localization at 3 dpf based
on immunofluorescence (Fig.
5M-O). Overall, these results are consistent with those we
obtained for other mutants with cilia motility defects
(Sullivan-Brown et al.,
2007).
seahorse mutations strongly affect cilia motility in the pronephros and neural tube
We have shown that defects in cilia motility precede tubule dilations
(Sullivan-Brown et al., 2007).
To analyze cilia motility in sea, video recordings of cilia movement
were performed as previously described
(Sullivan-Brown et al., 2007).
In sibling embryos, cilia are motile in the pronephric tubules (30 hpf) (see
Movies 1 and 2 in the supplementary material), the cloaca (2 dpf) (Movie 3 in
the supplementary material) and in the neural tube (2 dpf) (Movie 4 in the
supplementary material). At later time points, cilia in the medial tubules
bundle and display a coordinated movement (3 dpf) (Movie 5 in the
supplementary material) (Sullivan-Brown et
al., 2007). By contrast, the motility of sea mutant cilia
at these same time points and locations was affected in all embryos observed,
and ranged from slow and disorganized to completely immotile (see Movies 6-10
in the supplementary material). This result suggests that sea plays a
crucial role in regulating cilia motility in these tissues. To determine
whether sea mutations also affect cilia formation, we performed
immunofluorescence for acetylated tubulin. No differences in cilia length or
number were observed in the pronephros of sea mutants
(Fig. 4L,M). To confirm that
sea mutations do not affect cilia structure, we performed TEM
analysis of cilia within the pronephros and found no differences in axoneme
structure between mutants and siblings
(Fig. 4N-P).
seahorse mutations have weak effects on left-right patterning
Other zebrafish mutants with CTD and pronephric cyst phenotypes also have
defects in left-right patterning. This combination of phenotypes is not
surprising as these mutations often affect cilia, and cilia have been
implicated both in cyst development and left-right patterning (reviewed by
Bisgrove and Yost, 2006). As
sea is expressed in KV and affects cilia motility, we analyzed
left-right patterning in both alleles.
We analyzed visceral organ placement
(Schottenfeld et al., 2007)
and asymmetric lov expression in the brain
(Gamse et al., 2003) (see Fig.
S1 in the supplementary material). Surprisingly, we found that visceral organ
asymmetry was only affected in 9-11% of sea mutant embryos
(Table 2; see Table S1 in the
supplementary material). sea mutants showed a similarly low level of
defects in brain asymmetry (17-29%) (Table
3). These numbers are much lower than what we observe in other
mutants we have analyzed, such as curly up (pkd2), where 65%
of mutants display visceral organ defects and 59% display defects in brain
asymmetry (Schottenfeld et al.,
2007).
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seahorse mutations have variable effects on KV fluid flow
Even though sea strongly affects cilia motility and kidney
development, sea may either play a less crucial role in left-right
patterning than other cilia mutants, or have less of an effect on cilia
formation and motility in KV. To analyze the effect of sea on KV,
numbers and lengths of cilia from siblings and mutants were determined as
described in the Materials and methods. We analyzed 882 cilia in 25 embryos
(14 siblings and 11 mutants) but we did not observe a significant difference
in cilia number (P>0.28) or length (P>0.19) between
siblings and mutants (3.45±0.53 µm in length and 32±18 cilia
per KV in siblings versus 3.20±0.33 µm in length and 40±17
cilia per KV in mutants, two-tailed student t-test applied).
Interestingly, although the cilia in the pronephros of sea mutants are predominantly immotile, cilia motility in KV of sea mutants is highly variable, based on the movement of beads injected to analyze flow. We chose to analyze embryos from two different sea backgrounds as the level of defects can vary from clutch to clutch. The pair seatg238a-C produces a higher level of left-right patterning defects (28%) (see Table S1 in the supplementary material) compared with the pair seafa20r-M21, which produces a lower level (2%) (see Table S1 in the supplementary material). We predicted that flow would be more severely affected in mutant embryos from pair C than from pair M21, and this is what we observe (Table 4). In seatg238mutants, one out of five embryos had strong counter-clockwise flow, whereas four out of five showed reduced or absent flow. In seafa20r mutants, five out of eight embryos had strong counter-clockwise flow, whereas three out of eight showed reduced or absent flow (see Movies 11-13 in the supplementary material). However, defects in flow did not always correlate with defects in heart looping. Some embryos with strong flow were found to have incorrect heart looping, whereas other embryos with defects in flow had correct heart looping. This suggests that Sea may affect left-right patterning both by affecting flow and through additional undetermined mechanisms that are independent of flow.
|
). As the role for cilia in left-right patterning probably
occurs prior to this point at 
12-14 hpf, maternal contribution of Sea
could influence this event. We have not been able to formally test this
hypothesis as we have not been able to generate a functional translational
start site morpholino to knock-down maternal message without causing
additional phenotypes (B.X. and R.D.B., unpublished)
(Kishimoto et al., 2008). Another explanation for why sea mutants have weak effects on left-right patterning is that the point mutations do not completely eliminate sea function. To test this hypothesis, we examined whether the knockdown of sea mRNA using splice-site morpholino antisense oligonucleotides (MOs) produces the same left-right patterning phenotypes observed in mutants. Splice-site morpholinos typically affect only zygotic message and thus should produce comparable phenotypes with zygotic mutants. Sea MO-injected embryos have a higher level of defects in visceral organ placement compared with sea mutants (P<3x10–5 or P<5x10–6 for seatg238 and seafa20r, respectively, Chi-Square analysis applied). A higher percentage of sea MO-injected embryos also have defects in asymmetric gene expression compared with sea mutants (Table 2). These results suggest that the sea mutant alleles are hypomorphic and that mutant proteins retain some function that is eliminated by MO knockdown.
To further test this hypothesis, we injected a suboptimal dose of MO, which does not cause significant curly tail defects on its own, and assayed for enhancement of left-right phenotypes in sea mutants. We injected clutches from sea heterozygous parents and separated embryos on the basis of CTD phenotypes at 27 hpf and then on the basis of heart looping defects at 50 hpf, followed by genotyping. Although uninjected controls had the expected 25% CTD phenotypes and a low level of heart looping defects, the MO-injected clutches had higher percentages in both categories (Table 5; total numbers for injected and uninjected clutches). When separated by genotype, it is clear that the MO enhances the left-right patterning phenotypes in mutant embryos. In addition, the injection of the MO into heterozygous embryos produces a tail curl that is indistinguishable from mutants.
|
).
Kishimoto et al. found that Sea participates in Wnt/PCP signaling and
interacts with Disheveled (Dsh). Loss of Dsh in Xenopus leads to
mispositioned basal bodies and defects in ciliogenesis
(Park et al., 2008). In
theory, Sea could participate with Dsh to control these aspects of cilia
function.
To determine whether our mutant alleles affect the ability of Sea to
interact with Dsh, we performed co-immunoprecipitation experiments with
epitope-tagged Sea and Dsh constructs. Both Sea mutant proteins retained the
ability to interact with Dsh in this assay, although Seafa20r shows
a reduced ability to bind Dsh (Fig.
6). This could indicate that the truncation in this protein
affects the Dsh-binding domain. Our mutants do not affect ciliogenesis, as is
seen in Dsh loss of function (Park et al.,
2008), suggesting that the interaction between mutant Sea and Dsh
does not eliminate Dsh function in ciliogenesis. Furthermore, TEM analysis of
Sea mutants demonstrates that cilia have correct apically positioned basal
bodies (Fig. 4Q), which are
incorrectly positioned in Xenopus embryos lacking Dsh function
(Park et al., 2008). Thus, the
ability of our sea alleles to affect cilia motility appears to be
independent of interaction with Dsh.
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DISCUSSION |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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;
Omori and Malicki, 2006;
Sullivan-Brown et al., 2007;
Zhao and Malicki, 2007;
van Rooijen et al., 2008). We
first see dilations in the area we refer to as the medial tubules (this
report) (Sullivan-Brown et al.,
2007). This area of the zebrafish pronephros is analogous to the
proximal convoluted and straight tubules
(Wingert et al., 2007), a
region where cysts occur in human disease
(Nakanishi et al., 2000).
It is important to note that the pronephric cyst phenotypes we observe in
our sea alleles strongly resemble those reported for an insertional
allele of sea (Kishimoto et al.,
2008). Intriguingly, the insertional allele does not affect cilia
motility, whereas both of our alleles have a strong affect on motility.
Although work has convincingly shown that defects in fluid flow can results in
pronephric dilations in zebrafish
(Kramer-Zucker et al., 2005),
the phenotype of the sea insertional mutant strongly suggests that
kidney cyst formation in zebrafish can be separable from cilia motility
defects. Thus, our alleles of Sea may affect both cilia motility and
pronephric cyst formation through different mechanisms.
Defects in cilia motility and/or flow do not correlate with left-right axis defects
As cilia motility in KV is important for proper left-right patterning
(Essner et al., 2005;
Kramer-Zucker et al., 2005),
zebrafish mutations affecting cilia motility are predicted to have strong
effects on left-right patterning. Intriguingly, we observe that sea
mutants have a weak effect on left-right patterning. One possibility for this
finding is that left-right patterning is an early developmental event and may
be influenced by the maternal contribution of Lrrc6l. We believe this is
likely, but we have been unable to generate the appropriate tools to test
this. A second possibility is that some residual function of Sea remains in
the mutant embryos. In support of sea alleles being hypomorphic, we
can obtain a stronger effect of sea on left-right patterning using MO
antisense knockdown. Interestingly, in our experiment the addition of a
suboptimal dose of MO to sea heterozygotes produced curly tail down
phenotypes that were indistinguishable from sea mutants. Taken
together, these results suggest that embryos are sensitive to the amount of
Lrrc6l that is present and that the hypomorphic alleles may result in variable
phenotypes depending on the amount of maternal protein present coupled with
influences from the genetic background. We have noticed that the penetrance of
left-right patterning defects in sea can vary depending on the
background and age of the parents (see Table S1 in the supplementary
material). However in our alleles, we have not observed variability in
pronephric cyst formation or cilia motility defects in the pronephros and
neural tube.
A more intriguing possibility to explain why mutations that affect cilia
motility do not cause pronounced left-right patterning defects is that these
functions may be separable, similar to what we describe above for pronephric
phenotypes. We do find that our sea alleles affect KV cilia motility
and flow, but embryos with obvious defects in flow do not consistently show
defects in heart looping. We also find that embryos with defects in heart
looping often have flow that resembles wild type. An explanation for these
results is that these defects in flow are not severe enough to consistently
influence asymmetric patterning. This explanation is intriguing, as mutations
in mouse that cause subtle defects in cilia motility, such as inv,
still have profound effects on left-right patterning
(Okada et al., 1999). Further
studies on sea mutants could help determine what elements of flow are
absolutely crucial to left-right patterning. Alternatively, inv
mutations have also been implicated in Wnt signaling and it may be this
function that is more crucial to left-right patterning
(Simons et al., 2005). As Sea
interacts with Dsh and is involved in Wnt/PCP signaling
(Kishimoto et al., 2008), it
is intriguing to speculate that the function of Sea in this pathway is more
crucial to left-right patterning and pronephric cyst formation. The PCP
pathway may act in parallel to flow, suggesting that whereas flow is somewhat
affected in sea mutants, the more crucial role for sea may
lie in a different aspect of left-right axis determination.
|
). As
C. elegans do not have motile cilia, lrrc6l was placed in a
subset of genes required for cilia motility. Orthologs to sea also
exist in the flagellated protozoan Trypanomsoma brucei, where RNA
interference results in aberrant basal body replication and flagellar
biogenesis, resulting in a subsequent reduction of cell size
(Morgan et al., 2005). In
humans and mice, Lrrc6l was identified as a testis-specific protein, most
abundantly expressed in pachytene and diplotene cells in meiosis I
(Xue and Goldberg, 2000).
Furthermore, mutations in sea have been identified in different
screens in zebrafish to cause cystic kidneys and defects in the kinocilia on
hair cells (McDermott et al.,
2007; Sun et al.,
2004). Thus, the function of Lrcc6l appears to be conserved in
processes involving cilia/flagella.
The cilia proteome currently contains 14 proteins with leucine-rich repeats
(including Lrrc6l) out of over 1200 proteins identified as being found in
cilia or basal bodies (Gherman et al.,
2006). The architecture of Lrrc6l places it in the SDS22-like
subfamily. Proteins in this family are diverse in function, but include
splicing factors and nuclear export proteins
(Kobe and Kajava, 2001).
Interestingly, Lrrc6l has putative nuclear import/export sequences, suggesting
that this protein may have functions inside the nucleus.
Recently, another LRR containing protein in zebrafish, which we call
switch hitter (lrrc50), has been shown to affect cilia
motility, cause kidney cysts and strongly affect left-right patterning
(Sullivan-Brown et al., 2007;
van Rooijen et al., 2008)
(J.S.-B., K.B. and R.D.B., unpublished). The number and position of the LRRs
and coiled coil domain in Lrrc6l are similar in position to those in Lrrc50.
LRR and coiled-coil domains often act as protein-protein interactions domains,
suggesting that Lrrc6l and Lrrc50 may act as scaffolding proteins. Work on the
Lrrc50 ortholog Oda7 in Chlamydomonas suggests this protein acts to
interconnect outer and inner row dyneins and coordinate their functions
(Freshour et al., 2007).
However there is additional evidence that Oda7 may also act in the cytoplasm
to allow for the proper assembly of outer row dynein complexes bound for the
cilium (Freshour et al.,
2007). Recent work finds that Lrrc6l is expressed in the cytoplasm
and not the cilium or centriole (Kishimoto
et al., 2008). Lrrc6l and Lrrc50 have similar domains
arrangements; thus, we hypothesize that Lrrc6l may be functioning analogously
to Lrrc50 in the cytoplasm to allow for correct assembly of protein complexes
bound for the cilium. oda7/lrrc50 mutations result in the
loss of outer dynein arms in the cilium, which explains why mutations in this
gene affect cilia motility (Freshour et
al., 2007; van Rooijen et al.,
2008). As we do not see defects in cilia structure in sea
mutants, it will be interesting to determine what Lrrc6l interacts with in
order to affect cilia motility. Our current work suggests that interactions
with Dsh are not affected in our alleles, suggesting other interacting
partners may be important for cilia motility. Given that mutations in both
lrrc6l and lrrc50 affect cilia motility, it will be
interesting to determine whether the other 12 leucine-rich repeat proteins in
the cilia proteome play similar roles in cilia function, kidney cystogenesis
and left-right patterning. Additionally, it will be important to determine
whether any of these proteins are acting redundantly, perhaps compensating for
loss of Sea in KV but not in the pronephros.
|
Footnotes |
|---|
Supplementary material available online at http://dev.biologists.org/cgi/content/full/136/10/1621/DC1
We thank Robert Geisler and Silke Geiger-Rudolph for the original bulked segregant analysis that placed seatg238a on chromosome 2; John Mably for advice on positional cloning; Christine Hostetter, Heather McAllister and Jaclyn Taylor for zebrafish care; Peggy Bisher for assistance with TEM; Stephan Y. Thiberge for assistance with video microscopy; Ray Habas for disheveled constructs; and the members of the Burdine and Fishman laboratories for helpful discussions. We thank Jonathan Eggenschwiler, Jack Lee, Kim Poole and Jodi Schottenfeld for participating in the blind test of KV flow. J.S.B. is supported by predoctoral award 05-2411-CCR-E0 from the New Jersey Commission on Cancer Research. S.-Y.L. is supported by a Graduate Research Fellowship from the National Science Foundation. K.M.J. is supported by postdoctoral grant 0825952D from the American Heart Association. R.D.B. is the 44th Scholar of the Edward Mallinckrodt Jr Foundation, and funds from this award were used in support of this work. Funds from awards to R.D.B. from the New Jersey Commission on Cancer Research (04-2405-CCR-E0), from the Polycystic Kidney Disease Foundation, (#117b2r) and from the National Institutes of Child Health and Human Development (1R01HD048584) were used in support of this work. Video imaging was performed in the Princeton Imaging Facility, which is supported by grant P50GM071508 from NIH/NIGMS. Deposited in PMC for release after 12 months.
* These authors contributed equally to this work 
Present address: Novartis Institutes for Biomedical Research, Cambridge, MA
02139, USA
|
REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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T
transition in exon 5 encoding a Q201X truncation (B,B').
seatg238a is a 273T
