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First published online 14 March 2007
doi: 10.1242/dev.02827
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Department of Molecular Biology, Princeton University, Princeton, NJ 08550, USA.
* Author for correspondence (e-mail: rburdine{at}princeton.edu)
Accepted 1 February 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Zebrafish, pkd2, Asymmetry, Left-right axis, Kidney, Pronephros, Laterality, Cilia
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Ramsdell, 2005). Heterotaxia,
where one or more organs are positioned incorrectly, can have catastrophic
effects on the development of the organism. Approximately 1 in every 8,000
births will result in heterotaxia, which also often manifests as CHD
(Mercola and Levin, 2001).
The information necessary for the proper positioning of the viscera is
provided by earlier asymmetric events including the left-side-specific
expression of Nodal-related signaling factors. In the mouse embryo, asymmetric
activation of Nodal transcription occurs at the ventral node during
gastrulation. The node is lined by an epithelium that has one primary cilium
protruding from the surface of each cell. Although cilia located at the node
have 9+0 axonemes and are generally thought to be immotile, the presence of
Left-right dynein (Ird; Dnahc11 - Mouse Genome
Informatics) is thought to enable the central population of cilia to rotate in
a clockwise direction. This ciliary rotation drives a leftward fluid flow over
the node that is necessary for the proper establishment of sidedness, as
mutations that compromise ciliogenesis or inhibit the mobility of these cilia
result in randomization of the viscera
(McGrath and Brueckner, 2003).
In teleosts, Kupffer's vesicle is thought to be analogous to the mouse node in
the establishment of left-right patterning
(Amack and Yost, 2004). A
conserved role for ciliary flow in the establishment of left-right asymmetry
is supported by motile cilia and flow found at Kupffer's vesicle in zebrafish
and Medaka embryos (Essner et
al., 2005; Kramer-Zucker et
al., 2005; Okada et al.,
2005). Ciliary flow in the mouse has also been shown to be
upstream of an asymmetric calcium flux in the epithelial cells adjacent to the
left side of the node (McGrath and
Brueckner, 2003), an event that appears to be conserved in chick
and zebrafish (Raya et al.,
2004; Sarmah et al.,
2005). This left-specific intracellular calcium release is thought
to be elicited by the flow-induced stretch activation of the calcium channel,
polycystic kidney disease 2 (Pkd2), which localizes to the cilia of the mouse
node (McGrath et al.,
2003).
Human PKD2 encodes a six-pass transmembrane,
Ca2+-activated, non-specific cation channel, termed polycystin 2
(PC2) (Gonzalez-Perrett et al.,
2001; Hanaoka et al.,
2000; Vassilev et al.,
2001). Heterozygous PKD2 mutations are found in
approximately 15% of human autosomal dominant polycystic kidney disease
patients (Wu et al., 1997).
Mouse embryos homozygous for an inactivated allele of Pkd2 exhibit
severe cardiac and kidney malformations and are embryonic lethal between E13.5
and parturition (Wu et al.,
2000). Pkd2-/- mice also display laterality
disturbances in Nodal-related signaling genes, embryonic turning,
visceral organs and heart morphogenesis. Most critically, without PC2
activity, no left-specific calcium expression is detected at the node and
asymmetric activation of Nodal transcription does not occur,
resulting in right isomerism (McGrath et
al., 2003; Pennekamp et al.,
2002). Therefore, current models for the initiation of a
left-right symmetry breaking event include a role for PC2 upstream of
asymmetric Nodal gene transcription. The link connecting this calcium
transient with Nodal activation in the mouse is still unclear,
although evidence exists for the involvement of the Notch pathway
(Raya et al., 2004). In this
paper, we present evidence for an alternative role of pkd2 in the
development of the left-right axis.
Here we describe our analysis of curly up (cup), which encodes the zebrafish ortholog of polycystic kidney disease 2. We characterized two alleles of cup, and even though one allele of cup appears to be a null, cup mutants do not display defects in kidney patterning nor do they develop kidney cysts. cup alleles display left-right defects in organ positioning that resemble human heterotaxia, as well as abnormalities in asymmetric gene expression in the lateral plate mesoderm (LPM) and dorsal diencephalon of the brain. Surprisingly, nodal expression patterns in the LPM are different in the mouse and zebrafish pkd2-/- mutants. In the majority of cup embryos, spaw is activated bilaterally in LPM. Although defects in the development of the notochord can result in bilateral transcription of nodal, we find that the midline structures in cup embryos are not compromised. Thus, in zebrafish, PC2 is not responsible for the activation of spaw transcription but is required for a mechanism to bias spaw expression towards the left half of the embryo.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Haffter et al., 1996). Both
cup mutations were generated in the TU strain and maintained by
outcrossing to AB, WIK and PWT. PWT is a wild-type strain generated in our
laboratory that has a very low background of left-right defects. The
cup phenotypes and differences in severity between
cuptc321 and cupty30b have been
observed in multiple generations.
Positional cloning of curly up
Mapping of the cup locus was performed with SSLP markers as
described (Liao and Zon,
1999). The cup genomic interval was narrowed between
markers z10888 and z25625 on chromosome 1. Sequence surrounding these SSLP
markers was identified through the Ensembl Sanger Sequence Database.
Additional genomic sequence from the region was identified using BAC ends and
BAC mate pair analysis to identify overlapping clones and assemble a genetic
contig surrounding the cup locus. New SSLP markers used to narrow the
cup genetic interval were created based on the identified genomic
sequence: z9697.9 and z11984.6 (z9697.9F,
5'-GGTCCGCTTTTGGTGCAGAC-3'; z9697.9R,
5'-CTCTCAGCTCCACAAGTCGC-3'; z11984.6F,
5'-CGCTCTCCAGAGAAAACCAC-3'; z11984.6R,
5'-GTCTGATCGCAGCGGCGG-3'). The following SNP primers were used to
confirm linkage with pkd2: pkd2LSNPf,
5'-GATGTGCTTGGACGGATCCTG-3'; pkd2LSNPr,
5'-CACACCTAAAGACACTGTCC-3'.
Sequencing of cuptc321 and cupty30b and cloning and RT-PCR of pkd2
The zebrafish pkd2 gene was amplified from a 24-hour cDNA library
created with the Marathon cDNA Amplification Kit (BD Biosciences) using
cup5Bgl2f (5'-CAGATCAGATCTATGAGCTCCAGTCGCGTTCG-3') and cup3PmeIr
(5'-TCTGACGTTTAAACTCACAAGTGGGCGGGGC-3'). The PCR product was cut
with BglII and PmeI, and inserted into the BglII
and EcoRV sites of vector T7TS
(Cleaver et al., 1996). For
production of an in situ hybridization probe, a BglII-ApaI
fragment from the T7TS-pkd2 plasmid was cloned into pBluescript II SK (+)
using the BamHI and ApaI sites. Full-length sequence was
obtained by 5' and 3' RACE. Mutations in
cuptc321 and cupty30b were identified
by sequencing genomic DNA.
RT-PCR of the pkd2 transcript was performed with total RNA isolated from 1-cell, 256-cell, 1K-cell, sphere, and 18-somite stage embryos. cDNA libraries were made with the SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen) and primers located in exon 3 and exon 7 (pkd2 1F, 5'-GTGGAGAGCCAACCAACTTC-3'; pkd2 2R, 5'-CTGAAGCCGAGCCAGCGGCT-3') were used to amplify a 700 bp fragment of pkd2.
RNA probes and whole-mount in situ hybridization
DIG-labelled RNA probes were transcribed from linearized DNA templates and
used in RNA in situ hybridization by standard methods. Antisense probes
included cardiac myosin light chain (cmlc2; myl7 -
ZFIN) (Yelon et al., 1999),
forkhead 2 (fkd2; foxa3 - ZFIN)
(Odenthal and Nusslein-Volhard,
1998), preproinsulin (ins)
(Milewski et al., 1998),
southpaw (spaw) (Ahmad et
al., 2004), paired-like homeodomain transcription factor
(pitx2) (Yan et al.,
1999), polycstic kidney disease 2 (pkd2),
sonic hedgehog (shh)
(Krauss et al., 1993;
Thisse and Thisse, 1999),
lefty1 (Bisgrove et al.,
1999), lefty2
(Bisgrove et al., 1999),
SRY-box containing gene 17 (sox17)
(Alexander and Stainier, 1999),
alpha-tropomyosin (Ohara et al.,
1989), orthodenticle homolog 5 (otx5)
(Gamse et al., 2002),
leftover (lov; kctd12.1 - ZFIN)
(Gamse et al., 2003), and
charon (char) (Hashimoto
et al., 2004).
Immunofluorescence
Embryos were fixed in Dent's fixative at 4°C overnight, gradually
rehydrated into PBDT (1xPBS, 0.1% Tween 20, 1% DMSO), and blocked for 2
hours in PBDT containing 10% normal goat serum (NGS). To visualize cilia,
embryos were incubated overnight at 4°C in a 1:400 dilution of monoclonal
acetylated tubulin primary antibody (Sigma #T6793) in PBDT. Six 30-minute
washes in PBDT containing 1% NGS and 0.1 M NaCl were administered before the
last 30-minute wash in PBDT containing 1% NGS. The embryos were incubated
overnight at 4°C with FITC-IgG2b at 1:500 (Southern Biotech #1090-02). The
following day, the embryos were soaked in Hoechst (Molecular Probes #H3570)
for 15 minutes at 1:1250 in PBDT before six additional 30-minute washes with
PBDT containing 1% NGS and 0.1 M NaCl. After washing, the embryos were rinsed
and stored in Slow Fade buffers (Molecular Probes #S-7461).
Microscopy
Images of live embryos were taken using the ProgressC14 digital camera
(Jenoptik) mounted on a Leica MZFL III microscope. Embryos processed for
immunofluorescence were mounted in Aqua-PolyMount (Polysciences #18606) and
visualized on a Zeiss LSM 510. Embryos processed for in situ hybridization
analysis were mounted in modified GMM
(Struhl, 1981) [100 ml Canada
Balsam (Sigma #C-1795), 10 ml methylsalicylate (Sigma #M0387-100G)],
visualized using a Leica DMRA microscope at 10x magnification, and
photographed with the ProgressC14 digital camera.
Morpholino injection
Morpholino antisense oligonucleotides (MO) (Gene Tools, LLC) were
maintained in 50 µg/µl stock solutions in water at -80°C. The
cup augMO, 5'-AGCTCATCGTGTATTTCTACAGTAA-3', spans a
portion of the 5'UTR directly upstream of the AUG start site and the
start site itself. The pkd2 augMO [previously reported as
hi4166 MO by Sun et al. (Sun et
al., 2004)], 5'-AGGACGAACGCGACTGGAGCTCATC-3', begins
at the start AUG and extends into the first exon. Although these two MOs
overlap by 8 nucleotides, only the pkd2 augMO effectively phenocopied
cup (1-4 ng). The 9697-S1 splice-site MO,
5'-GAAACGGGCCTTCTGTGAACTACAG-3', is complimentary to the intron
3-exon 4 splice junction. The 9697-S2 splice-site MO,
5'-TTAACATACGCAGTGCCATTCTTGG-3', overlaps the exon 4-intron 4
splice junction of the pkd2 transcript. Only 9697-S1, used at 9 ng
per embryo, was successful in phenocopying cup. Although this MO is
predicted to block splicing, no alterations in the pkd2 message were
observed by RT-PCR. Thus, we believe the 9697-S1 MO is more likely to be
blocking translation. In support of this, other splice-site MOs have been
shown to block translation of the message (M. Mullins, personal
communication), but formal proof will require the generation of antibodies
that recognize the Cup protein.
MOs were mixed with 5 mg/ml Phenol Red and injected into 1- to 4-cell stage
embryos as described (Gritsman et al.,
1999).
Histological analyses
Embryos were fixed in 4% paraformaldehyde (Sigma P6148) in PBS overnight at
4°C, and stored in 4% sucrose in PBS. After a gradual dehydration into
ethanol, embedding was performed according to the Electron Microscopy Sciences
protocol for JB-4 histology (EMS #14270-00). The embryos were sectioned on a
Leica RM2255 Rotary Microtome at 4 µm. Hematoxylin and Eosin staining (all
components were in non-alcohol-based solutions) was performed according to
laboratory protocols (J.S.-B., unpublished; available upon request).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). We
utilized positional cloning techniques to identify the gene affected in the
cup mutant. The cup mutation was placed on chromosome 1
using bulk segregant analysis (Talbot and
Schier, 1999). Subsequent analysis was performed on 213 meiotic
events to narrow the chromosomal location of cup to a 6.6 cM interval
between simple sequence-length polymorphism (SSLP) markers z10888 and z25625
(Liao and Zon, 1999). We
assembled a set of genomic clones that span the cup locus using
available genomic sequence from the Sanger sequencing project. New SSLP
markers were then created from these contig sequences (z9697.9 and z11984.6)
and used to further refine the location of the cup mutation to a 2.6
cM region. One likely candidate for cup in this region was the gene
pkd2, based on left-right defects observed in Pkd2 mutant
mice (Pennekamp et al., 2002).
Therefore, we used the 3' untranslated region of the pkd2
transcript to generate a single nucleotide polymorphism (SNP) marker. SNP
mapping of cup recombinant embryos revealed no recombination between
pkd2 and the cup locus
(Fig. 1A).
|
; Hanaoka et al.,
2000; Vassilev et al.,
2001). Zebrafish pkd2 shares 67% and 65% identity, and
78% and 79% similarity, with the human and mouse orthologs, respectively (over
900 and 899 amino acid alignments; data not shown). Similarities include
signaling elements such as an EF-Hand and a coiled-coil domain that are
located in the C-terminus of the protein
(Delmas et al., 2004).
To verify that pkd2 is mutated in cup, each
Ensembl-predicted exon was sequenced from cup genomic DNA of two
different alleles, cuptc321 and
cupty30b. Comparisons between the sequence of wild-type
sibling embryos and cuptc321 embryos identified a nonsense
mutation in exon 2 that is predicted to generate a truncated protein
consisting of only the N-terminal 135 residues
(Fig. 1B). ty30b
embryos have a missense mutation in exon 5 that changes a T to a C at
nucleotide 1052, substituting a proline for a leucine in the first
extracellular loop of the protein (Fig.
1B). We originally defined cuptc321 as a
stronger allele than cupty30b based on the severity of the
tail curl phenotype (Fig. 1C).
Since tc321 is likely to be a molecular null based on its predicted
protein product, the mutations for each allele are consistent with
tc321 being a stronger loss-of-function allele than ty30b.
The mouse anti-PC2 antibodies p57 and 58 recognize an antigenic region that
includes the conserved leucine residue mutated in ty30b embryos.
These antibodies can block PC2-dependent intracellular calcium release from ER
stores (Nauli et al., 2003),
and we therefore predict that a mutation in this region of the protein would
interfere with proper channel activity.
|
). To
determine the extent of laterality defects in cup mutants, we
analyzed the position of the heart (cmlc2), liver (fkd2),
and pancreas (ins) in cup embryos at 48 hours
post-fertilization (hpf) using RNA in situ hybridization with markers for
these organs. In a wild-type zebrafish embryo, the ventricle of the heart
loops towards the right and the atrium loops towards the left, the liver is
positioned to the left of the midline and the pancreas lies to right of the
midline (Fig. 2A,D). This
wild-type pattern was observed in 35% of tc321 and 38% of
ty30b embryos, whereas 33% of tc321 and 46% of
ty30b mutants showed a complete reversal in the placement of these
organs (Fig. 2B,E,
Table 1). A heterotaxic
phenotype was seen in 32% and 16% of tc321 and ty30b
embryos, respectively (Fig.
2C,F, Table 1).
Since our mutant population has a clear increase in defects over background,
we conclude that cup is globally affecting left-right organ
placement.
|
;
Gamse et al., 2002;
Gamse et al., 2003). In
cup mutant embryos, 40-45% display denser projections on the left
side as compared with the right, whereas 55-59% show predominant expression of
habenula marker genes on the right side. Thus, cup mutants exhibit
random placement of neural asymmetric structures across the midline, although
the parapineal and larger habenula always remain paired
(Fig. 2G,H,
Table 1).
cup affects asymmetric gene expression in LPM and brain
Expression of the zebrafish nodal gene southpaw
(spaw) and downstream targets pitx2, lefty1 and
lefty2 are restricted to the left LPM during late somitogenesis prior
to proper asymmetric localization of organs at 48 hpf
(Ahmad et al., 2004). To
address how early cup affects left-right patterning, expression of
southpaw, pitx2 and lefty1/2 was examined in clutches of 20-
to 22-somite embryos from two heterozygote parents for the cup
mutation (Table 2). Although
25% of embryos within the clutch should be homozygous mutant for cup,
correct expression of spaw at Kupffer's vesicle was observed in all
embryos analyzed (data not shown). This is consistent with the finding that
the mouse Pkd2 mutation does not affect Nodal expression in
the mouse node. By contrast, both cup alleles showed increased
frequency of bilateral, right-sided, or absent spaw expression in the
LPM, with bilateral expression being the most common
(Fig. 3A-D,
Table 2). The bilateral
spaw expression can be further classified into three categories:
bilateral expression extending into the anterior cardiac LPM
(Fig. 3B,
Table 3); bilateral expression
restricted to the posterior portion of the LPM
(Fig. 7C,
Table 3); and uneven expression
on both the left and right sides, where spaw propagates anteriorly on
one side and remains posterior on the other half
(Fig. 7B,
Table 3). In embryos that
expressed spaw only posteriorly, lefty was never expressed
in the cardiac mesoderm or dorsal diencephalon
(Fig. 3H,
Table 3,
Fig. 7C,D). When spaw
did extend anteriorly, expression of lefty correlated with the
left-right placement of spaw (Fig.
3A-C, Fig. 7A,B,D,
Table 3). Equivalent
spaw expression in the anterior LPM resulted in randomized
lefty expression in the heartfield. pitx2 expression was
also randomized in the LPM for both alleles
(Table 2). Thus, zebrafish
cup mutants do differ from mouse Pkd2 mutants, which are
reported to exhibit a loss of Nodal in the LPM. However, the mouse
mutant expresses Pitx2 bilaterally only in the posterior LPM, similar
to the spaw phenotype in cup mutants.
|
|
). Since cup is expressed in the floorplate
in zebrafish (see below), we investigated whether notochord or floorplate
development is affected in cup mutants by examining expression of
shh and lefty1 in 12-somite and 18-somite embryos,
respectively. In wild-type embryos, shh is expressed in the notochord
and floorplate of the midline and lefty1 is present only in the
notochord. Both gene products showed normal expression patterns in
pkd2-deficient embryos (data not shown). Expression of
alpha-tropomyosin was also unaltered in mutant embryos at 8 somites
and 24 hpf, indicating that somitogenesis was proceeding normally (data not
shown). Since midline structures appeared unaffected in cup mutants,
the bilateral spaw expression cannot be explained by a compromised
midline. Thus, in the zebrafish embryo, pkd2 appears to be
responsible for restricting spaw transcription to the left side of
the LPM. In addition, because we observe a high percentage of spaw
and pitx2 expression confined to the posterior LPM, we propose that
pkd2 plays an additional role in the propagation of nodal
signals in this tissue.
|
).
cup embryos exhibited abnormal expression in the brain with the
majority of embryos showing a loss of expression of these Nodal target genes
(Fig. 3E-H,
Table 2). Thus, pkd2
may play a role in the zebrafish diencephalon in sending or receiving an
asymmetric signal from the LPM.
Expression analysis of pkd2
To determine where PC2 activity is required for proper left-right
patterning, expression patterns of pkd2 were analyzed by RNA in situ
hybridization. Whereas mouse pkd2 is expressed ubiquitously
(Pennekamp et al., 2002), we
found distinct expression domains for pkd2 in zebrafish.
Transcription of pkd2 was first detected at the onset of epiboly in
cells marking dorsal fates at dome stage
(Fig. 4B). Transcript levels
increased as gastrulation continued, with expression expanding into the dorsal
marginal cells (Fig. 4D,F).
Since transcripts cannot be detected at dome stage and at 40% epiboly in
MZoep embryos, which lack most of the mesendoderm, pkd2 is
expressed in mesodermal and endodermal precursor cells during gastrulation
(Fig. 4C,E). pkd2 also
localized to the shield and dorsal forerunner cells (DFCs)
(Fig. 4F,J). The DFCs
maintained expression of pkd2 as they migrated towards the posterior,
leading to a concentrated area of expression at the tailbud
(Fig. 4G,K). These transcripts
began to become more diffuse as the DFCs formed Kupffer's vesicle.
pkd2 mRNA was present in a ring-like pattern outlining the vesicle
during early somite formation, but became less visible as somitogenesis
proceeded. A low level of expression could be seen in the neural floorplate
and pronephric duct primordia during these later stages
(Fig. 4H,I,L).
Although not visible by RNA in situ hybridization, a maternal contribution
of pkd2 mRNA was detected by RT-PCR at 1-4 cells and 256 cells, both
of which are stages prior to the mid-blastula transition (1000 cells)
(Fig. 4A). Since Kupffer's
vesicle has been implicated in left-right patterning in zebrafish, we predict
that PC2 might be acting in this structure to affect left-right patterning
(Amack and Yost, 2004;
Essner et al., 2005). While
this manuscript was in preparation, Bisgrove et al. found that injection of
morpholinos against pkd2 to specifically knockdown translation of PC2
in Kupffer's vesicle resulted in left-right patterning defects, supporting a
role for PC2 in this location (Bisgrove et
al., 2005). cup embryos do have a structurally intact
Kupffer's vesicle as seen by light microscopy during mid-somitogenesis, and
sox17 expression highlights the proper migration of DFCs towards the
tailbud region in mutants during late epiboly stages (data not shown). RNA in
situ hybridization for charon, a known Nodal inhibitor expressed at
Kupffer's vesicle, also shows normal expression patterns in cup
mutant embryos (data not shown) (Hashimoto
et al., 2004). These data indicate that pkd2 is not
responsible for the formation of Kupffer's vesicle, but rather is acting from
within the vesicle to affect asymmetric spaw expression.
Morpholino knockdown of pkd2-/- results in additional cystic phenotypes in a concentration-dependent manner
We created several morpholinos against the pkd2 transcript. Two
MOs were able to effectively phenocopy the cup phenotype, one
directed against the pkd2 start site (augMO), and one directed
against an intron-exon junction (MO 9697-S1). Alterations in organ positioning
and asymmetric spaw expression were examined in order to assess the
left-right patterning abnormalities in pkd2 morphants. Although the
morphants displayed slightly milder tail curling
(Fig. 5A-D), the laterality
defects associated with the MO knockdowns were comparable to those of
cup mutants (Table 4).
Interestingly, pkd2 morphants also exhibited hydrocephalus and
dilations in the pronephric region, neither of which was displayed in
cup mutants (Fig.
5E,L). A number of genes known to cause cystic kidney phenotypes
in other model organisms when mutated have been shown to cause pronephric
cysts in zebrafish mutants and morphants
(Kramer-Zucker et al., 2005;
Liu et al., 2002;
Otto et al., 2003;
Sun and Hopkins, 2001), and
MOs to pkd2 have been reported to cause kidney cysts
(Sun et al., 2004). Thus, the
finding that the pronephric region is dilated in our pkd2 morphants
is not unexpected. However, we note that the dilations in our morphants are
more elongated and are more restricted to the glomerulus than those seen in
other pronephric mutants and morphants in the laboratory, and may not be
representative of true pronephric cysts (our unpublished data). In fact, in
cup mutants, severe edema is observed by day 6 in all tissues,
suggestive of a general loss in fluid homeostasis not restricted to the
pronephros or neural tube. Additionally, cup morphants have defects
in body size and organ morphology, suggesting that the MOs cause more global
organ patterning defects that could be the cause of the pronephric dilations
we observe (see below).
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|
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cup mutants display no obvious structural ciliary defects
Primary cilia exist on the cells that line the ventral node of the mouse
embryo. PC2 localizes to these cilia and has been shown to be responsible for
eliciting an asymmetric calcium surge in the epithelial cells adjacent to the
left side of the node (McGrath et al.,
2003). Cilia also reside on the cells of Kupffer's vesicle in the
zebrafish embryo (Essner et al.,
2002). To determine if cilia are affected in cup morphant
and mutant embryos, an antibody against acetylated tubulin was used to
visualize cilia by immunofluorescence. As the curly tail phenotype cannot be
seen in cup mutants until 33 hpf, pkd2 augMO morphants were
analyzed at 12 somites for the presence of cilia on Kupffer's vesicle. The
cilia at Kupffer's vesicle in pkd2 morphants appeared structurally
intact (Fig. 6A,B), as did the
cilia lining the collecting ducts of the kidney in cup mutants at 33
hpf (Fig. 6C,D). Because the
formation of Kupffer's vesicle is unaffected in pkd2 mutants and
morphants, it was not surprising to see the same circular distribution of
cilia in the morphant embryos. Although no obvious structural abnormalities
are detected in the cilia of pkd2 mutant zebrafish, we cannot rule
out the possibility that the cilia might be functionally compromised.
|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
). Similar to
the Pkd2 knockout mouse, cup mutants exhibit a variety of
organ laterality defects, supporting a conserved requirement for pkd2
function in left-right patterning. In pkd2 mutant zebrafish,
spaw and pitx2 are both expressed bilaterally in the LPM,
and the majority of spaw expression is restricted to the posterior.
lefty expression is absent in the cardiac LPM in the majority of
cup embryos, although we believe the absence of asymmetric
lefty is due to the inability of spaw to propagate in the
LPM in embryos lacking PC2 (Fig.
7). We also find discordance in the percentage of embryos
expressing bilateral spaw and right-sided pitx2 in
cup mutants. We attribute these differences to the following
possibilities. Since spaw expression is often uneven in its anterior
propagation in cup mutants, the side that receives the spaw
signal more prevalently will be the side to express pitx2.
Alternatively, although spaw is thought to act directly upstream of
pitx2, it is possible that spaw is not the sole regulator of
pitx2 in the LPM during these stages.
|
),
cilia-generated `nodal-flow' is thought to bend non-motile cilia at the left
edge of the node and activate PC2. Flow activation of PC2 leads to an increase
in calcium at the left edge of the node, which results in activation of the
Nodal signaling pathway on the left by an unknown mechanism. Aspects
of this model are based in large part on the Pkd2 knockout mouse, as
calcium is not upregulated on the left side of the node and Nodal is
not activated in the LPM (McGrath et al.,
2003; Pennekamp et al.,
2002). However, our work convincingly demonstrates that
spaw is expressed in embryos lacking the PC2 channel and suggests
that the role of PC2 is to restrict spaw to the left LPM.
|
;
Kramer-Zucker et al., 2005). A
number of calcium asymmetries surrounding Kupffer's vesicle have been reported
(Sarmah et al., 2005;
Webb and Miller, 2000).
However, which calcium asymmetries may be relevant for the establishment of
the left-right axis and how calcium asymmetries may function in this process
are not clear.
One possible explanation for the differences between mouse and zebrafish is
that the role for pkd2 in left-right patterning is conserved, but the
actual function the channel plays in each organism is not. This would be
similar to the situation for Fgf8, which has a conserved role in left-right
patterning, but is reported to act as a left-determining factor in mouse and a
right-determining factor in chick and rabbit
(Boettger et al., 1999;
Fischer et al., 2002;
Meyers and Martin, 1999). It
should be noted, however, that two out of 12 Pkd2 mutant mice
described by Pennekamp et al. exhibited bilateral expression of Nodal
in the LPM (Pennekamp et al.,
2002). Intriguingly, a large percentage of the mouse mutants do
exhibit bilateral Pitx2 expression, a downstream target of
Nodal signaling, and the domain of expression is restricted to the
posterior portion of the embryo (Pennekamp
et al., 2002), similar to the posterior expression of
spaw in cup mutants. Thus, it is fair to conclude that
Nodal signaling can be initiated in the mouse in the absence of PC2,
and that the bias of left-sided expression is now lost. This is similar to our
findings in fish and suggests that the role of PC2 in left-right patterning in
all organisms may need to be reconsidered.
In addition, the mechanism by which PC2 acts in any organism to affect
asymmetric events is still unclear. In mouse, PC2 is thought to act at the
node to affect left-right patterning. Zebrafish pkd2 is expressed in
Kupffer's vesicle, the proposed node-equivalent in teleosts, and Bisgrove et
al. report a role for pkd2 in this structure
(Bisgrove et al., 2005). A
recent model proposes that activity in the mouse node provides a subtle
asymmetry on the left side that manifests into robust expression of
Nodal in the left LPM through a combination of activating signals on
the left and repressive signals on the right
(Nakamura et al., 2006). PC2
may be acting from within Kupffer's vesicle to initially bias the activation
of spaw gene expression to the left side of the organism.
Alternatively, PC2 might be responsible for repressing Nodal signaling on the
right half of the embryo (Fig.
7). Based on pkd2 expression data, this repression could
take place at multiple sites in the embryo, including Kupffer's vesicle and
the floorplate. As no midline defect is apparent in cup mutants, we
believe that it is unlikely that PC2 in the floorplate is responsible for
restricting spaw to the left side, but this has not been formally
proven. If pkd2 is acting at multiple levels to affect the direction
of spaw activation and spaw propagation, Nakamura's model
(Nakamura et al., 2006)
predicts that the majority of embryos would display bilateral expression of
spaw, which is what we observe. This model also explains how
alterations in Nodal initiation and propagation can result in
right-sided or no expression of Nodal, which we observe in a low
percentage of cup mutants.
Our results also suggest that maternal pkd2 might play a role in
proper mesendoderm patterning of the visceral organs. Thus, it is possible
that the effects of pkd2 on left-right patterning could occur prior
to the formation of Kupffer's vesicle. Interestingly, in Xenopus,
there is evidence that indicates an earlier non-ciliary role for PC2 in
left-right patterning prior to gastrulation
(Qiu et al., 2005).
A possible role for Cup in mesendoderm patterning
We find that pkd2 is expressed maternally in zebrafish, and has
restricted expression to mesendoderm during early epiboly and gastrulation.
Our work with MOs suggests that pkd2 may play a role in the
patterning of the mesendoderm. For example, at high doses of MO against
pkd2, various mesendoderm defects are apparent, including loss of
glomerular tissue in the kidney, smaller organs including the liver and gut,
and thinner body axes. These defects are variable, but were seen with two
different MOs against pkd2 and are thus likely to be specific
defects. The Pkd2 knockout mouse also displays pancreatic, liver,
kidney and cardiac complications at early developmental stages, further
supporting a function for PC2 in mesoderm and endoderm
(Wu et al., 2000).
Although low levels of MO against pkd2 phenocopied the left-right
defects seen in zygotic cup mutants, we believe cup mutants
are a better system for studying the role of pkd2 in left-right
patterning. In fact, while this manuscript was in preparation, another study
was published on pkd2 using MOs in zebrafish that came to different
conclusions on the role of pkd2 in left-right patterning
(Bisgrove et al., 2005). We
believe that the differences in our conclusions might be due in part to the
additional effects of the pkd2 MO on the mesendoderm, which are not
seen in zygotic cup mutants.
Similarly, zygotic cup embryos do not develop pronephric cysts
during larval development as would be predicted for a mutation in a gene that
causes polycystic kidney disease in other vertebrates. Although we could
detect dilations in the pronephric region of wild-type embryos injected with a
high concentration of pkd2 augMO, histological sections revealed that
the cystic tissue had an unconventional morphology as compared with other
cystic mutants in the laboratory (our unpublished data). We believe the
MO-specific phenotype in the kidney is due to the knockdown of maternal
pkd2 mRNA that is present in zygotic cup mutants, similar to
what has been proposed previously (Sun et
al., 2004), but because mesendoderm defects are apparent in
pkd2 morphants, the cystic kidney phenotype is questionable in its
origin. Although MO knockdown of pkd2 could be affecting redundancies
in pkd2 or alternative splice forms, we have found no evidence for
the existence of these to date. We attempted to analyze protein knockdown in
mutants and morphants using several antibodies against human and mouse PC2,
but none of these antibodies were able to cross-react with the zebrafish
protein as assessed by whole-mount in situ hybridization or western blotting.
Future studies on whether maternal pkd2 mRNA is affecting early
mesendoderm patterning will be needed to fully understand the dilated kidney
phenotype we observe in the morphants.
Organ laterality defects in cup mutants suggest multiple levels of regulation in left-right patterning
Visceral organ patterning is affected in cup mutants and can be
categorized into three main sub-groups: normal organ positioning, situs
inversus, and heterotaxia. It is intriguing that each phenotypic group
constitutes about one-third of the total population of cup mutants,
and we see similar segregation in other asymmetry mutants in our laboratory
(our unpublished results). It has been proposed that in the absence of
asymmetric signaling, each organ will randomly adopt a position on the
left-right axis resulting in a high degree of heterotaxia
(Burdine and Schier, 2000;
Concha et al., 2000;
Yan et al., 1999). The fact
that such a large population of mutants with altered spaw signaling
retains either wild-type or completely inverted patterning is unexpected and
suggests some global left-right information is still retained when asymmetric
Nodal signaling is disrupted. This idea is further supported by the finding
that whereas over 30% of cup mutant embryos show complete situs
inversus, only a small percentage of them show right-sided spaw
expression. We have shown that the expression of downstream asymmetric genes
depends on the magnitude of spaw expression on each side, and thus it
is important to incorporate the expression patterns of multiple Nodal pathway
signals in a model of how asymmetric nodal genes translate into the
proper positioning of the organs.
cup mutants also show a loss of asymmetric gene expression in the
dorsal diencephalon of the brain at 24 somites and randomized asymmetric
placement of the parapineal and dorsal habenula. These data are consistent
with the phenotypes of other mutants that lack Nodal signaling in the
zebrafish brain (Concha et al.,
2000). cup embryos are unique, though, in their ability
to activate Nodal pathway gene expression in the LPM but not in the
diencephalon of most mutants. It is intriguing to speculate that the
activation of nodal genes in the brain might also be dependent on the
anterior propagation of spaw in the LPM, or upon pkd2
activity in the diencephalon.
|
ACKNOWLEDGMENTS |
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