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First published online 18 April 2007
doi: 10.1242/dev.02851
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2 and Ncx4a regulate zebrafish left-right patterning
1 Department of Molecular, Cell and Developmental Biology, University of
California, Los Angeles, CA 90095, USA.
2 Molecular Biology Institute, University of California, Los Angeles, CA 90095,
USA.
3 Jonsson Cancer Center, University of California, Los Angeles, CA 90095,
USA.
4 Cardiovascular Research Laboratory, University of California, Los Angeles, CA
90095, USA.
Author for correspondence (e-mail:
chenjn{at}mcdb.ucla.edu)
Accepted 14 March 2007
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SUMMARY |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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2 and Ncx4a function upstream of Nodal signaling to regulate LR
patterning in zebrafish. Knocking down Na,K-ATPase 2 and Ncx4a activity
in dorsal forerunner cells (DFCs), which are precursors of Kupffer's vesicle
(KV), is sufficient to disrupt asymmetric gene expression in the lateral plate
mesoderm and randomize the placement of internal organs, indicating that the
activity of Na,K-ATPase 2 and Ncx4a in DFCs/KV is crucial for LR
patterning. High-speed videomicroscopy and bead implantation experiments show
that KV cilia are immobile and the directional fluid flow in KV is abolished
in Na,K-ATPase 2 and Ncx4a morphants, suggesting their essential role
in KV ciliary function. Furthermore, we found that intracellular
Ca2+ levels are elevated in Na,K-ATPase 2 and Ncx4a
morphants and that the defects in ciliary motility, KV fluid flow and
placement of internal organs induced by their knockdown could be suppressed by
inhibiting the activity of Ca2+/calmodulin-dependent protein kinase
II. Together, our data demonstrate that Na,K-ATPase 2 and Ncx4a
regulate LR patterning by modulating intracellular calcium levels in KV and by
influencing cilia function, revealing a previously unrecognized role for
calcium signaling in LR patterning.
Key words: Left-right patterning, Calcium homeostasis, Cilia, Zebrafish
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INTRODUCTION |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Capdevila et al., 2000;
Bisgrove and Yost, 2001).
Although the role of signals expressed asymmetrically in the LPM is
relatively clear, the mechanisms that initially break LR symmetry are only
beginning to be revealed. In Xenopus, the break in LR symmetry is
evident as early as the first few cell divisions when 14-3-3 and
H+/K+-ATPase proteins and serotonin become
asymmetrically localized (Levin et al.,
2002; Bunney et al.,
2003; Fukumoto et al.,
2005). However, similar asymmetric protein localization at this
early developmental stage has not been noted in other vertebrate species,
suggesting that mechanisms for the break of LR symmetry might have diverged
among vertebrates (Levin et al.,
2002; Kawakami et al.,
2005; Adams et al.,
2006).
In the chick, mouse and zebrafish models, the node has an essential role in
LR patterning (Essner et al.,
2002). In chick, a transient accumulation of extracellular
Ca2+ on the left side of Henson's node induced by a gradient of
H+/K+-ATPase-dependent membrane potential is required
for the left-specific activation of Notch
(Raya et al., 2004). In
mammals, motile nodal cilia generate a leftward fluid flow
(Nonaka et al., 1998;
Okada et al., 2005).
Disrupting the biogenesis or function of cilia in the node blocks nodal flow
and induces LR patterning defects in the mouse
(Nonaka et al., 1998;
Marszalek et al., 1999;
Okada et al., 1999;
Takeda et al., 1999;
McGrath et al., 2003;
Okada et al., 2005). An
asymmetric Ca2+ flux in tissues on the left edge of the node has
been noted. The two-cilia model proposes that this asymmetric Ca2+
flux is a result of the activation of polycystin-2 (also known as polycystic
kidney disease 2) cation channels by the leftward nodal flow
(McGrath and Brueckner, 2003;
McGrath et al., 2003;
Tabin and Vogan, 2003), but
the recent observation of leftward transportation of morphogens across the
node offers an alternative view of how asymmetric Ca2+ flux might
be initiated around the node (Tanaka et
al., 2005; Raya and Belmonte,
2006; Shiratori and Hamada,
2006). In zebrafish, Kupffer's vesicle (KV), the equivalent of the
node, also has a crucial role in LR patterning. Loss-of-function of
intraflagellar transport proteins such as Ift88 (Polaris) and Ift57 (Hippi)
inhibits the directional fluid flow in KV and induces randomization of organ
placement in zebrafish (Amack and Yost,
2004; Bisgrove et al.,
2005; Essner et al.,
2005; Kramer-Zucker et al.,
2005). As with the mouse model, an asymmetric Ca2+ flux
is observed in cells surrounding the KV in zebrafish. Disrupting this
asymmetric calcium signaling by treatment with thapsigargin, an inhibitor of
sarco/endoplasmic reticulum ATPase (SERCA; also known as Atp2a1 - ZFIN), or by
knocking down the activity of ipk1 (ippk - ZFIN), causes
abnormal LR patterning (Sarmah et al.,
2005).
Ca2+ is an important signaling molecule for many biological
processes. The levels of free Ca2+ in the cytoplasm are generally
low in resting cells, but can be increased drastically upon stimulation
(Berridge et al., 2000).
Activation of plasma membrane calcium channels allows a small Ca2+
influx, which in turn leads to the activation of InsP3 receptors or
ryanodine receptors and the release of a large quantity of Ca2+
from the endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR). Once its
signaling function has ended, Ca2+ is rapidly removed from the
cytoplasm by SERCA, which returns Ca2+ to the ER/SR, and by the
plasma membrane Ca2+-ATPase (PMCA) and sodium-calcium exchanger
(NCX), which extrude Ca2+ from the cell. Furthermore, as the
Ca2+-transporting activity of NCX is modulated by the levels of
intracellular Na+, Na,K-ATPase is also considered to be a
calcium-regulating molecule. In fact, inhibition of Na,K-ATPase activity
reduces the activity of NCX and results in an increase in the concentration of
intracellular Ca2+ (for a review, see
Blanco and Mercer, 1998),
indicating that NCX and Na,K-ATPase cooperate to maintain a low intracellular
Ca2+ level in resting cells.
We showed previously that morpholino (MO) knockdown of Na,K-ATPase
2 (atp1a2a - ZFIN) induced cardiac laterality defects
in zebrafish (Shu et al.,
2003) and recent studies on the heart and mind
(Na,K-ATPase 1B1; atp1a1 - ZFIN) mutant also
revealed an incompletely penetrant laterality phenotype in the visceral organs
(Ellertsdottir et al., 2006),
suggesting a role for the sodium pump in early LR patterning in zebrafish.
Here, we investigate the mechanism by which the sodium pump regulates LR
patterning in zebrafish. Our data show that Na,K-ATPase 2 and its
functional partner Ncx4a, are involved in a very early step of LR patterning
in zebrafish. Downregulation of Na,K-ATPase 2 and Ncx4a in dorsal
forerunner cells (DFCs) and KV is sufficient to immobilize KV cilia, perturb
directional fluid flow in KV, disrupt the asymmetric expression patterns of
zebrafish laterality genes in the brain and LPM, and randomize the placement
of all internal organs. We provide evidence that Na,K-ATPase 2 and
Ncx4a are capable of modulating the intracellular calcium levels in zebrafish,
consistent with the roles of these molecules defined previously by numerous
biochemical and physiological studies in cultured cells and mammalian model
organisms. We show that the phenotypes of those embryos with elevated
intracellular Ca2+ levels induced by pharmacological means are
identical to those observed in Na,K-ATPase 2 and Ncx4a morphants.
Additionally, inhibiting calcium/calmodulin-dependent protein kinase II
(CaMKII) activity was able to suppress the LR defects caused by A23187,
thapsigargin, Na,K-ATPase 2-MO and Ncx4a-MO treatment, revealing a
previously unacknowledged role for Na,K-ATPase 2/Ncx4a/CaMKII-mediated
Ca2+ signaling in zebrafish LR patterning.
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MATERIALS AND METHODS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). Embryos
for in situ hybridization were raised in the presence of 0.2 mM
1-phenyl-2-thiourea to maintain optical transparency
(Westerfield, 1995).
Molecular cloning of Ncx4a
Total RNA was isolated from 80% epiboly embryos using the RNAqueous-4PCR
Kit (Ambion, TX). The ncx4a fragment (equivalent to aa 443-609) was
amplified by RT-PCR (95°C for 30 seconds, 60°C for 60 seconds,
72°C for 60 seconds, for 32 cycles) with the following primers:
5'-ACTATCGCACAGAGGATGGCA-3' and
5'-GTCTCATCATTGAGGAACTCC-3'.
The 3' sequence of ncx4a (equivalent to aa 84-938) was amplified by RT-PCR (95°C for 30 seconds, 58°C for 60 seconds, 72°C for 120 seconds, for 35 cycles) with primers: 5'-CTCATCTACATGTTTCTGGGC-3' and 5'-CTGTCTGCGTAAGAAGCGACATGT-3'. The 5' sequence of ncx4a (equivalent to aa 1-473) was derived from 5'-RACE using the SMART RACE cDNA Amplification Kit (BD Biosciences, CA) with the primer 5'-TGAGCTCCTTAACCGTCTCTCCTG-3'. All PCR products were cloned into the pCRII-TOPO vector (Invitrogen, CA) and confirmed by sequencing. pCRII-TOPO-Ncx4a/443-609 was linearized with BamHI and T7 RNA polymerase was used to make the antisense probe for ncx4a.
Morpholinos
Morpholino antisense oligonucleotides (MOs) complementary to the
translation start site and its flanking sequence of mRNAs encoding Na,K-ATPase
2 (2-MO, 5'-TTTCATGTCCGTACCCTTTCCCCAT-3') and Ncx4a
(Ncx4a-MO, 5'-AAAGGCGCAGATGAAACATGGTGGC-3') or to the
splice-acceptor sites of Na,K-ATPase 2 (2-SP-MO,
5'-CAACCTATGAAAGACAGACAAGTGG-3') and Ncx4a (Ncx4a-SP-MO,
5'-CAACCTGCATACAGGAGCAGTGTTT-3') were synthesized by Gene-Tools.
An MO with a 5 bp mismatch to 2-MO (CTL-MO,
5'-TTTgATcTCCGTAgCCTTTgCCgAT-3') was used as a control (lowercase
letters indicate mismatched bases). Wild-type embryos were injected with 2 ng
2-MO, 4.5 ng 2-SP-MO, 4 ng Ncx4a-MO or 5 ng Ncx4a-SP-MO at the
1-cell stage. The injected embryos were fixed at the 15- to 16-somite stage
(for spaw), 21- to 22-somite stage (for lft1/2), 23- to
24-somite stage (for pitx2), or after 2 days of development (for
cmlc2 and fkd2) for whole-mount in situ hybridization to
analyze gene expression patterns and internal organ laterality.
To knockdown the activities of Na,K-ATPase 2 and Ncx4a specifically
in DFCs, 2-MO or Ncx4a-MO tagged with fluorescein was injected into the
yolk of 128-cell stage embryos. After 2 days of development, the laterality of
heart and visceral organs was examined by whole-mount in situ hybridization
using cmlc2 and fkd2 probes in those injected embryos having
fluorescent signals in DFCs/KV.
Primer pairs used in RT-PCR to investigate the knockdown efficiency of
2-SP-MO and Ncx4a-SP-MO are: 5'-CTTCAGATAAATGCAGAGGAGG-3'
and 5'-ACCCAAAGTCTCCACAGCTTC-3';
5'-TAGGAAGGTGGTGAGCTGCC-3' and
5'-CCAGTTTGGTGTGCTCTCCC-3'; these pairs yield 875 bp (normal
splicing) or 360 bp (skip exon 2), and 573 bp (normal splicing) or 455 bp
(skip exon 7), respectively.
Constitutive activation of CaMKII
The coding region for CaMKII
CT287D (kindly provided by J. Brown,
University of California, San Diego, La Jolla, CA) was subcloned into the
EcoRI site of the pcGlobin2 vector
(Ro et al., 2004) to create
pcGlobin2-CaMKIICT287D, which was then linearized with ApaI.
mRNA of CaMKIICT287D was transcribed using mMESSAGE mMACHINE (Ambion)
and injected into zebrafish embryos at the 1-cell stage.
In situ hybridization
Whole-mount in situ hybridization was performed as described
(Chen and Fishman, 1996). The
laterality of the heart and visceral organs was determined by double in situ
hybridization with cmlc2 and fkd2. The other antisense RNA
probes used in this study were spaw (from M. Rebagliati, University
of Iowa, Iowa City, IA), lrdr, lft1, lft2, pitx2 (from H. J. Yost,
University of Utah, Salt Lake City, UT), ntl, Na,K-ATPase
2 and ncx4a.
Immunostaining
KV cilia were visualized by immunostaining using anti-acetylated tubulin
(Sigma, MO) as previously described
(Essner et al., 2002). Images
were acquired with an LSM510 confocal microscope (Zeiss, Germany).
Chemical treatment
Chemicals used in this study are thapsigargin (0.5 µM, Sigma), A23187
(13 nM, Sigma) and KN62 (20 µM, Calbiochem, CA). Unless otherwise
indicated, chemicals were added to the embryo media (5 mM NaCl, 0.17 mM KCl,
0.33 mM CaCl2, 0.33 mM MgSO4) at 3 hours
post-fertilization (hpf) and washed away at 6 hpf by extensive rinsing with
embryo medium. Embryos were then fixed at day 2 and the laterality of the
heart and visceral organs examined as described above.
Fluorescent bead injections
Fluorescent beads of 0.2 µm diameter (Fluorspheres 580/605, Molecular
Probes) were diluted 1:100 in 1xPBS. About 0.3 nl of beads were injected
into KV at the 7- to 10-somite stage. Embryos were imaged on a Zeiss Axioplan
2 microscope equipped with a 40x water immersion lens.
Calcium imaging
Embryos used for calcium imaging were injected with 1 nl of a mixture of 50
mM Calcium Green dextran (as a calcium indicator) and 0.05% (w/v)
tetramethylrhodamine dextran (as an internal control for injection) (Molecular
Probes). 2 ng of 2-MO or 4 ng of Ncx4a-MO were co-injected with the
mixture of Calcium Green and rhodamine dextran to create Na,K-ATPase 2
and Ncx4a morphants. Images of Calcium Green and rhodamine in blastomeres of
control embryos and Na,K-ATPase 2 and Ncx4a morphants were acquired
with an LSM510 confocal microscope (Zeiss, Germany) at the blastula stage.
Fluorescence intensities of Calcium Green and rhodamine in each embryo were
analyzed with MetaMorph (Molecular Device Corporation). To normalize for
variations between injections, the calcium intensity among embryos was
compared based on the ratio of the intensity of the Calcium Green and
rhodamine signals.
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RESULTS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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2 is required for LR patterning in zebrafish2 is required
for establishing proper cardiac laterality in zebrafish
(Shu et al., 2003). To
evaluate whether Na,K-ATPase 2 regulates the laterality of visceral
organs, we injected an MO targeting the translation initiation site of
Na,K-ATPase 2 (2-MO) into wild-type zebrafish embryos at the
1-cell stage and analyzed the positions of the internal organs by in situ
hybridization using cmlc2 (myl7 - ZFIN) to visualize the
heart and fkd2 (foxa3 - ZFIN) for the gut, liver and
pancreas. After 2 days of development, the cardiac ventricle is placed to the
right of the atrium (cardiac looping), and a leftward bend of the developing
intestine (gut looping) positions the gut and liver on the left side of the
embryo and the pancreas on the right. As we reported previously, Na,K-ATPase
2 morphants (referred to as 2 morphants) do not have apparent
morphological defects besides abnormalities in cardiac looping
(Fig. 1B)
(Shu et al., 2003).
Specifically, 21% of 2 morphants had reversed cardiac looping and the
hearts of 26% never underwent looping (n=122)
(Fig. 1E-G,K). In addition to
the cardiac looping abnormality, knocking down the activity of Na,K-ATPase
2 induced laterality defects in visceral organs. 27% of 2
morphants had reversed positioning of the gut, liver and pancreas and 11% of
the 2-MO-injected embryos did not undergo gut looping (n=122)
(Fig. 1H-K). Furthermore, the
laterality defects of the heart and visceral organs were not concordant with
each other. In 45% of 2 morphants, organ positions were normal (situs
solitus), 13% were completely reversed (situs inversus) and 42% had discordant
positions of the heart and visceral organs (heterotaxia). Similar laterality
defects were also observed in embryos injected with 2-SP-MO, an MO
targeting the splice-acceptor site of exon 7 (43% abnormal cardiac and 42%
abnormal gut looping, n=96) (Fig.
1K), but such laterality defects were not observed in embryos
injected with a control 2 MO containing 5 mismatches (CTL-MO). Only 6%
of CTL-MO-injected embryos exhibited cardiac laterality defects and 5% had
reversed visceral organs (n=103). These results demonstrate that
Na,K-ATPase 2 is required for the establishment of proper laterality of
both the visceral organs and the heart in zebrafish.
To investigate whether the activity of Na,K-ATPase 2 is required for
establishing asymmetric gene expression in the LPM, we analyzed expression of
laterality genes in 2 morphants. Whereas some embryos had normal
left-sided expression of spaw and pitx2, transcripts of
these genes were detected on the right-hand side, bilaterally, or not at all
in the LPM of other 2 morphants
(Fig. 2A-H;
Table 1). The expression of
lft1 and lft2 in the left LPM was also disrupted in 2
morphants; but surprisingly, rather than showing a randomized expression
pattern, the expression of lft1/2 was absent in most of the
2 morphants. Occasionally, we detected lft1/2
expression in either the left (normal) or right (reversed) LPM of 2
morphants, but never bilaterally (Fig.
2I-N; Table 1). In
addition to the left LPM, lft1 was also expressed in the left
habenula in the developing zebrafish brain. We found that lft1
expression in the brain was also absent in a large fraction of 2
morphants (49%, n=75) (Fig.
2I-K). These data demonstrate that downregulating the activity of
Na,K-ATPase 2 disrupts the asymmetric expression patterns of zebrafish
laterality genes and randomizes the placement of multiple internal organs,
indicating that the activity of Na,K-ATPase 2 is required for an early
step of LR patterning in zebrafish.
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). However, in
2 morphants, there was an unusual discordant expression of
spaw and pitx2 (randomization) and of
lft1/2 (absent). We investigated whether this was due to a
global repression of lft1/2 expression in 2
morphants. As shown in Fig. 3,
the expression of lft1/2 during gastrulation, as well as the
midline expression of lft1 in the segmentation period, were not
affected in 2 morphants. These findings indicate that the absence of
lft1/2 expression in the LPM and habenula is a result of LR
patterning defects, rather than a general suppression of their expression
induced by the downregulation of Na,K-ATPase 2.
The fish-specific sodium calcium exchanger Ncx4a is required for LR patterning
Calcium signaling has been implicated as an early determinant of LR
patterning (Fujinaga et al.,
1994; Toyoizumi et al.,
1997; McGrath et al.,
2003; Raya et al.,
2004). One well-characterized function of Na,K-ATPase is to
regulate the intracellular Ca2+ level by modulating the activity of
NCX (Blanco and Mercer, 1998;
Blaustein and Lederer, 1999).
Therefore, we investigated whether Na,K-ATPase 2 regulates LR
patterning by functionally coupling with NCX. We identified six zebrafish
homologs of NCX from the zebrafish Ensemble database. Phylogeny analysis
indicates that these homologs represent two Ncx1s (Ncx1h and Ncx1n; ZFIN
annotation Slc8a1a and Slc8a1b, respectively)
(Langenbacher et al., 2005),
one Ncx2 (ZFIN annotation Slc8a2), one Ncx3 (ZFIN annotation Slc8a3) and two
Ncx4s (Ncx4a and Ncx4b; ZFIN annotation Slc8a4a and Slc8a4b, respectively)
(Fig. 4A). Interestingly,
whereas homologs of Ncx1, 2 and 3 exist in other vertebrate species, the Ncx4s
appear to be fish-specific (Marshall et
al., 2005).
Our data showed that Na,K-ATPase 2 activity is required for proper
asymmetric expression of spaw, which is already evident by the
12-somite stage in zebrafish (Long et al.,
2003). Therefore, NCXs that act as functional partners of
Na,K-ATPase 2 in LR decisions should be expressed early during
embryogenesis. In situ hybridization analysis showed that ncx1h is a
cardiac-specific gene whose expression is first detected in the bilateral
cardiac primordia at the 12-somite stage
(Langenbacher et al., 2005),
ncx2 is not expressed during embryogenesis (not shown), and
ncx4b expression is restricted to the hypaxial muscle and to some
head mesenchymes after 2 days of development
(Fig. 4C). Therefore, these
genes are not likely to cooperate with Na,K-ATPase 2 in LR patterning.
However, ncx1n, ncx3 and ncx4a are expressed ubiquitously
during gastrulation and early somitogenesis stages, and thus are potential
functional partners of Na,K-ATPase 2 in LR patterning. After 1 day of
development, ncx4a expression remains ubiquitous
(Fig. 5), whereas
ncx1n and ncx3 transcripts are predominantly detected in the
neural tissues (Langenbacher et al.,
2005) and somites (Fig.
4B), respectively.
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2 morphants. The general morphology of Ncx4a morphants was normal
(Fig. 1C), but the expression
domains of spaw and pitx2 were randomized
(Table 1). In addition, whereas
the midline expression of lft1/2 was unaffected (data not shown),
their expression in the lateral mesoderm and habenula was absent in the
majority of Ncx4a-MO-injected embryos
(Table 1). Furthermore, the
position of the heart and visceral organs was disrupted. 46% of the embryos
injected with Ncx4a-MO had laterality defects (n=180). Specifically,
23% of Ncx4a morphants had reversed cardiac looping and 14% had a straight
heart tube at the midline. 33% of the Ncx4a-MO-injected embryos had reversed
positioning of the visceral organs and the gut failed to loop in 9% of these
embryos (Fig. 1K). As we
observed in 2 morphants, the looping of the heart and gut of Ncx4a
morphants were not concordant with each other. 26% of the Ncx4a-MO-injected
embryos had heterotaxia and 21% had situs inversus. Similar laterality defects
in asymmetric gene expression and the placement of internal organs were
observed in embryos injected with Ncx4a-SP-MO, an MO targeting the
splice-acceptor site of exon 2 of ncx4a (37% abnormal cardiac, 30%
abnormal gut looping, n=126) (Fig.
1K). These data demonstrate that as with Na,K-ATPase 2, the
activity of Ncx4a is required for proper LR patterning in zebrafish.
Cooperative effect of Ncx4a and Na,K-ATPase 2 in LR patterning
The similarity of the Na,K-ATPase 2- and Ncx4a-knockdown phenotypes
and their shared physiological role in modulating intracellular
Ca2+ levels suggest that Na,K-ATPase 2 might function
cooperatively with Ncx4a to regulate LR patterning. We co-injected 2-
and Ncx4a-MOs into zebrafish embryos and analyzed their effects on LR
patterning. Whereas injecting 0.5 ng of 2-MO or 2 ng of Ncx4a-MO into
wild-type embryos did not cause significant laterality defects, co-injection
of these amounts of 2-MO and Ncx4a-MO effectively induced randomized
placement of the internal organs (44% heterotaxia, n=140)
(Fig. 5G). In addition,
spaw and pitx2 expression patterns were randomized and
lft1/2 expression was absent in 2 and Ncx4a double
morphants (Table 1). This
further supports the notion that Na,K-ATPase 2 and Ncx4a function
cooperatively to regulate LR patterning in zebrafish.
Na,K-ATPase 2 and Ncx4a in DFCs are required for directional fluid flow in KV
In situ hybridization analysis revealed that the genes encoding Na,K-ATPase
2 and Ncx4a are expressed in a broad range of tissues during the
gastrula period and early somitogenesis, including KV
(Fig. 5B,E), which is the
equivalent of the mammalian node in teleosts
(Essner et al., 2002;
Essner et al., 2005;
Kramer-Zucker et al., 2005).
To investigate whether Na,K-ATPase 2 and Ncx4a regulate zebrafish LR
patterning by modulating the formation and/or function of KV, we injected
fluorescein-conjugated 2-MO or Ncx4a-MO into the yolk at the early
blastula stage (128-cell stage) to target the precursors of KV, the DFCs
(Amack and Yost, 2004). Embryos
injected with MO at the 128-cell stage accumulated strong fluorescent signals
in DFCs (Fig. 6B), and
developed cardiac looping defects (2-MO: 15% straight and 27% reversed,
n=135; Ncx4a-MO: 13% straight and 28% reversed, n=112)
(Fig. 6A) in association with
the absence of lft1/2 and randomized spaw and
pitx2 expression (Table
1). By contrast, 91% of CTL-MO-injected embryos were normal
(n=111) (Fig. 6A). MO
injected into the yolk at the 128-cell stage can be incorporated into DFCs as
well as localized to yolk nuclei (Cooper
and D'Amico, 1996). To investigate whether the LR defects observed
were due to the knockdown of Na,K-ATPase 2 and Ncx4a activity in DFCs,
we injected fluorescein-conjugated 2-MO or Ncx4a-MO into the yolk at
the dome stage; at this time, the DFCs are no longer connected directly to the
yolk (Cooper and D'Amico,
1996). We did not detect fluorescent signals in DFCs of these
embryos (Fig. 6C) and did not
observe a randomization of the placement their internal organs (88% of
2-MO and 91% of Ncx4a-MO-injected embryos had normal situs;
n=133 and 143, respectively), indicating that the activities of
Na,K-ATPase 2 and Ncx4a are crucial in DFCs for LR patterning in
zebrafish.
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2
(Fig. 6G) and Ncx4a morphants
(not shown). The expression of KV markers such as lrdr
(dnah9 - ZFIN) is also normal in 2 and Ncx4a morphants (not
shown). Furthermore, after analyzing 798 KV cilia in 12 embryos, we did not
detect significant differences in the number or length of KV cilia between
control embryos and 2 morphants (4.12±0.22 µm in length and
71±18 cilia per KV in controls versus 3.93±0.66 µm in length
and 62±19 cilia per KV in 2 morphants, P>0.1)
(Fig. 6H,I). Together, these
data indicate that Na,K-ATPase 2 and Ncx4a do not control laterality
through a role in the formation of KV or KV cilia. We then investigated
whether Na,K-ATPase 2 and Ncx4a are required for proper function of KV,
and found that the motility of KV cilia was severely defective in 2 and
Ncx4a morphants. Of the 35 uninjected control embryos analyzed, 33 had many
motile cilia in KV (see Movie 1 in the supplementary material). However, we
did not observe any motile KV cilia in 27 of the 31 2 morphants
inspected (see Movie 2 in the supplementary material). Furthermore, whereas
strong counterclockwise fluid flow was detected inside KV in all
CTL-MO-injected embryos that were successfully implanted with fluorescent
beads (Table 2; Movie 3 in the
supplementary material), directional fluid flow was not observed in 2
(Table 2; Movie 4 in the
supplementary material) and Ncx4a (not shown) morphants. Taken together, these
data provide evidence that the activities of Na,K-ATPase 2 and Ncx4a
are required for KV ciliary motility and confirm the correlation between
directional fluid flow and LR patterning.
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).
Knocking down the activity of Ncx4a or Na,K-ATPase 2 is thus predicted
to slightly increase the level of intracellular calcium. This notion is
supported by the finding that Ca2+ levels in the blastomeres of
2 and Ncx4a morphants are 1.24-fold and 1.14-fold higher than in
control embryos (P<0.01, n=12 and P<0.05,
n=16, respectively) (Fig.
7A). In addition, the phenotypes of Ncx4a or 2 morphants
are similar to those of embryos treated with A23187, a calcium ionophore that
allows calcium influx from extracellular sources. Incubating embryos with low
levels of A23187 (13 nM) disrupted the asymmetric expression patterns of
spaw, pitx2 and lft1/2
(Table 1) and induced cardiac
looping defects (16% straight and 29% reversed, n=112), but did not
affect general morphogenesis of zebrafish embryos
(Fig. 1D), a phenotype similar
to that of 2 and Ncx4a morphants. This effect is calcium-dependent, as
the incidence of embryos with LR defects was significantly reduced when
treatment with A23187 was performed in embryo media from which calcium
chloride had been omitted (13% abnormal, n=130)
(Fig. 7B). In addition to
A23187, thapsigargin, a SERCA-inhibitor that blocks the resequestration of
Ca2+ into the sarco/endoplasmic reticulum, is often used to
manipulate intracellular Ca2+ levels. We found that treating
embryos with a low dose of thapsigargin (0.5 µM) could also induce cardiac
looping defects (8% straight and 41% reversed, n=112) without
affecting other aspects of morphogenesis
(Fig. 7B), further supporting
the notion that the intracellular Ca2+ level plays a crucial role
in LR patterning.
Calcium/calmodulin-dependent protein kinase II (CaMKII) is an essential
downstream mediator of calcium signaling
(Berridge et al., 2000). To
investigate whether CaMKII mediates Na,K-ATPase 2 and Ncx4a-dependent
signaling in LR patterning, we tested whether increased CaMKII activity could
induce LR defects. We injected mRNA encoding CaMKIICT287D, a mutant
form of CaMKII that has constitutive Ca2+-independent activity
(Brickey et al., 1994;
Pfleiderer et al., 2004), into
zebrafish embryos at the 1-cell stage and observed dosage-dependent LR
defects. Normally, the primitive heart tube protrudes to the left of the
embryo (cardiac jogging) at 24 hpf in zebrafish
(Chen et al., 1997). We found
that whereas 35% and 48% of embryos receiving 10 pg and 20 pg of
CaMKIICT287D mRNA exhibited abnormal jogging, respectively (10 pg
injection: 13% no jog and 22% right-jog, n=78; 20 pg injection: 26%
no jog and 22% right-jog, n=94), 69% of embryos receiving 50 pg of
mRNA had abnormal jogging (51% no jog and 18% right-jog, n=49),
indicating that the activation of CaMKII can indeed induce laterality
defects.
We further investigated whether inhibiting CaMKII could suppress the LR
defects induced by thapsigargin, A23187, 2-MO or Ncx4a-MO knockdown.
KN62 is a well-characterized CaMKII inhibitor. We found that treatment with 20
µM KN62 did not cause noticeable morphological defects in wild-type embryos
but could significantly reduce the incidence of the cardiac looping defects
induced by 2-MO, Ncx4a-MO, thapsigargin or A23187. Only 22% of the
embryos co-treated with thapsigargin and KN62 (6% straight and 16% reversed,
n=96) and 27% of the embryos co-treated with A23187 and KN62 (6%
straight and 21% reversed, n=108) underwent abnormal cardiac looping
(P<0.05) (Fig. 7B).
Similarly, only 26% (11% straight and 15% reversed, n=85) and 24% (6%
straight and 18% reversed, n=98) of the 2 and Ncx4a morphants
treated with KN62 developed cardiac looping defects, respectively
(P<0.01) (Fig. 7B).
We further analyzed whether the suppression of the LR defects was due to the
restoration of cilia motility and directional fluid flow in KV. Of the 13
embryos analyzed by high-speed videomicroscopy, we observed active KV ciliary
rotation in nine KN62-treated 2 morphants (see Movie 5 in the
supplementary material). Moreover, 17 out of the 24 KN62-treated 2
morphants that were successfully implanted with fluorescent beads displayed
counterclockwise fluid flow (Table
2; see Movie 6 in the supplementary material). These data clearly
demonstrate that blocking the activity of CaMKII, a downstream signaling
molecule for calcium, suppresses LR defects induced by thapsigargin, A23187,
2-MO or Ncx4a-MO. These data support the notion that Na,K-ATPase
2 and Ncx4a regulate zebrafish LR patterning by controlling levels of
intracellular Ca2+ and indicate that CaMKII mediates calcium
signaling at an early step of LR patterning.
|
DISCUSSION |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
; Blaustein and Lederer,
1999), but their roles during development have remained largely
unknown. We now show that Na,K-ATPase 2 and Ncx4a function upstream of
Nodal signaling to control zebrafish LR patterning. Knocking down the
activities of these genes disrupts the normal expression patterns of
laterality genes in the LPM and randomizes the placement of the internal
organs. Since no apparent morphological defects other than the randomization
of organ laterality were noted in 2 and Ncx4a morphants, the LR defects
observed in these embryos are likely to be direct effects of downregulation of
expression of Na,K-ATPase 2 and Ncx4a rather than secondary
consequences of gastrulation and/or midline abnormalities.
We observed an unusual discordant expression pattern of spaw and
lft1/2 in 2 and Ncx4a morphants and A23187-treated embryos. In
general, the expression of lft1/2 follows that of nodal both
temporally and spatially (for a review, see
Schier, 2003). Of the
zebrafish mutants and morphants examined thus far, randomized spaw is
associated with randomized lft1/2 expression, and loss of
spaw expression is associated with the absence of lft1/2
expression (Bisgrove et al.,
2000; Bisgrove et al.,
2005; Essner et al.,
2005; Kramer-Zucker et al.,
2005). However, this concordant expression pattern is disrupted in
2 and Ncx4a morphants, where spaw and pitx2 are
randomized but the expression of lft1/2 in LPM is usually missing. It
is likely that the absence of lft1/2 expression in the lateral plate
mesoderm and habenula of 2 and Ncx4a morphants or A23187-treated
embryos is a consequence of the laterality defects because lft1/2
expression in other tissues of these embryos is unaffected. How the concordant
expression pattern of laterality genes was lost in these embryos is currently
unknown, but this unusual phenotype indicates that multiple regulatory
mechanisms are involved in the asymmetric expression of lft1/2 in the
LPM.
We present four lines of evidence supporting the notion that Na,K-ATPase
2 and Ncx4a modulate calcium signaling in DFCs/KV and thereby regulate
LR patterning in zebrafish. First, although the sensitivity of our current
imaging technology prevented us from measuring intracellular Ca2+
levels specifically in DFCs, we were able to measure Ca2+ levels in
blastomeres. We observed a significant elevation of intracellular
Ca2+ levels in blastomeres of 2 and Ncx4a morphants. This
result is consistent with the roles of NCX and Na,K-ATPase in regulating
intracellular Ca2+ levels delineated by numerous biochemical and
physiological studies in cultured cells and in mammals
(Blanco and Mercer, 1998;
Blaustein and Lederer, 1999),
and indicates that like their mammalian counterparts, Na,K-ATPase 2 and
Ncx4a are involved in maintaining calcium homeostasis in zebrafish. Second, we
manipulated intracellular Ca2+ levels with two chemicals used
widely for this purpose. A23187 is a calcium ionophore that increases
intracellular Ca2+ levels in intact cells by allowing extracellular
calcium to cross the cell membrane. Thapsigargin is a SERCA-inhibitor that
prevents Ca2+ from being resequestered into the ER/SR. Both of
these treatments elevate intracellular Ca2+ levels. We found that
embryos treated with a low dose of A23187 or thapsigargin are phenotypically
identical to 2 and Ncx4a morphants. These embryos have no apparent
morphological defects other than randomization of the placement of the
internal organs. These embryos also share the unusual discordant spaw
and lft1/2 expression phenotype with 2 and Ncx4a morphants,
implying that the LR defects of 2 and Ncx4a morphants are the result of
imbalanced intracellular calcium. Third, we showed that constitutive
activation of CaMKII causes LR defects in zebrafish, and that inhibiting
CaMKII activity with KN62 is able to suppress LR defects (as induced by
2-MO, Ncx4a-MO, A23187 and thapsigargin) at a concentration that does
not cause any additional morphological defects in the developing zebrafish
embryos. These observations indicate that the LR defects induced by
Ca2+ perturbation are at least in part mediated by CaMKII. Finally,
targeting 2- and Ncx4a-MOs to DFCs is sufficient to disrupt asymmetric
expression of spaw, lft1/2 and pitx2 and to randomize the
placement of the internal organs, demonstrating an essential role of calcium
homeostasis in DFCs/KV in zebrafish LR patterning. An emerging theme from
recent studies suggests that the directional nodal flow and asymmetric calcium
signaling around the node are fundamental to LR decisions in both mouse and
zebrafish (McGrath et al.,
2003; Bisgrove et al.,
2005; Essner et al.,
2005; Kramer-Zucker et al.,
2005; Sarmah et al.,
2005). Our findings that elevated Ca2+ levels in DFCs
can immobilize KV cilia, abolish KV fluid flow and randomize the placement of
the internal organs indicate that in addition to asymmetric signaling around
the node, calcium influences zebrafish LR patterning by regulating the
function of KV cilia.
The movement of eukaryotic flagella and cilia is in part regulated by
intraflagellar calcium. For example, increased intracellular Ca2+
levels change the Chlamydomonas flagellar waveform
(Hyams and Borisy, 1978;
Bessen et al., 1980), whereas
the elevation of intracellular Ca2+ levels can ultimately make sea
urchin sperm flagella immobile (Brokaw,
1979; Gibbons and Gibbons,
1980). Our study showed that intracellular Ca2+ levels
also regulate ciliary motility in KV in zebrafish, suggesting a conserved role
of calcium in cilia motility. Although the precise mechanisms by which calcium
signaling regulates ciliary function in KV requires further investigation, our
study provides evidence that signaling mediated by CaMKII has an important
role. It is possible that the activation of CaMKII represses the expression of
certain genes required for ciliary function. Alternatively, calcium might
modulate ciliary motility at a post-translational level, a notion supported by
the finding that the binding of dynein to microtubules is modulated by
calcium, which in turn affects the movement of Chlamydomonas flagella
and sea urchin sperm axonemes (Nakano et
al., 2003; Wargo et al.,
2004; Wargo et al.,
2005). It will be important to investigate whether a similar
mechanism applies to KV ciliary motility. Furthermore, it has been shown that
forced expression of inversin leads to LR defects and this effect is dependent
on a physical interaction of INVS and calmodulin via the IQ domain, which is
negatively regulated by the presence of calcium
(Yasuhiko et al., 2001;
Morgan et al., 2002), raising
the possibility that calcium signaling directly modulates protein activities.
Further investigation of the mechanisms by which calcium signaling modulates
ciliary motility will deepen our understanding of LR patterning, and should
help to clarify whether this aspect of LR patterning is conserved among
vertebrates.
We show that Na,K-ATPase 2 and Ncx4a influence LR decisions in
zebrafish by modulating intracellular Ca2+ levels in DFCs/KV. The
finding that Ncx1 is asymmetrically expressed on the right-hand side of
Hensen's node during gastrulation in the chick suggests that these
calcium-handling proteins might be involved in LR decisions in other
vertebrate species (Linask et al.,
2001). Currently, NCX1, NCX2, Na,K-ATPase 1 and 2
knockout mice are available, but no LR defects in these lines have been
documented (James et al.,
1999; Cho et al.,
2000; Wakimoto et al.,
2000; Koushik et al.,
2001; Reuter et al.,
2002). It will be important to re-evaluate the expression pattern
of laterality genes in these mice to assess their roles in LR patterning in
mammals. Alternatively, using Na,K-ATPase and NCX to modulate LR-related
Ca2+ signaling might be a fish-specific mechanism. Owing to the
partial genome duplication in teleosts, more Na,K-ATPase and NCX genes exist
in the zebrafish than in the mammalian genome (nine Na,K-ATPase
-subunits and six NCX genes in zebrafish versus four Na,K-ATPase
-subunits and three NCX genes in mammals)
(Blanco and Mercer, 1998;
Blaustein and Lederer, 1999;
Rajarao et al., 2001;
Serluca et al., 2001;
Blasiole et al., 2002;
Canfield et al., 2002), and
Ncx4a belongs to a subgroup of NCX genes that only exists in fish (our study)
(Marshall et al., 2005).
Therefore, it is conceivable that calcium homeostasis in the node might be
regulated by other calcium-handling proteins in mammals. Analyzing whether
Na,K-ATPase and NCX are important for regulating the intracellular
Ca2+ level in ciliated nodal cells and whether a CaMKII-dependent
pathway modulates nodal ciliary motility in other vertebrates will provide
insight into the conservation of these pathways.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/10/1921/DC1
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
|
REFERENCES |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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