1 Department of Molecular, Cell and Developmental Biology, University of
California, Los Angeles, Los Angeles, CA 90095, USA
2 Molecular Biology Institute, University of California, Los Angeles, Los
Angeles, CA 90095, USA
3 Jonsson Cancer Center, University of California, Los Angeles, Los Angeles, CA
90095, USA
4 Cardiovascular Research Laboratory, University of California, Los Angeles, Los
Angeles, CA 90095, USA
5 Department of Pediatrics, University of California, Los Angeles, Los Angeles,
CA 90095, USA
6 Developmental Biology Laboratory, Massachusetts General Hospital, Charlestown,
MA 02129, USA
7 Institute of Molecular and Cell Biology, College of Life Science, National
Taiwan University, Taipei, Taiwan
* Author for correspondence (e-mail: chenjn{at}mcdb.ucla.edu)
Accepted 29 August 2003
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Heart development, Zebrafish, Na, K-ATPase
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Na,K-ATPase is an integral membrane protein that transports Na+
and K+ across the plasma membrane to establish proper chemical and
electrical gradients (for reviews, see
Blanco and Mercer, 1998;
Therien and Blostein, 2000
).
Its activity is essential for maintenance of the physiological function of
many cell types. In the heart, it is believed that Na,K-ATPase regulates
cardiac function through interaction with the Na+/Ca2+
exchanger. Blocking Na,K-ATPase activity increases intracellular
Na+ concentration, which inhibits the activity of
Na+/Ca2+ exchanger, increases intracellular
Ca2+ concentration and, thereby, enhances cardiac contractility
(for a review, see Schwinger et al.,
2003
). In fact, Na,K-ATPase inhibitors, such as cardiac
glycosides, are often used to enhance cardiac contraction in heart failure
patients, and abnormal expression levels of Na,K-ATPase in the heart have been
detected in heart failure and arrhythmia patients
(Mohler et al., 2003
;
Schwinger et al., 1999
).
Both the cation- and ATP-binding sites essential for catalytic and
transport activity of Na,K-ATPase are both located in the subunit but
the enzyme activity requires dimerization of the
- and ß-subunits
(Therien and Blostein, 2000
).
Four isoforms of the Na,K-ATPase
subunit have been identified in
mammalian cells (Blanco and Mercer,
1998
). These isoforms exhibit overlapping but distinct expression
patterns, and have dramatically different affinities to cardiac glycosides,
such as ouabain, suggesting specific functional roles for these isofoms in
regulating cardiac contraction, despite the high degree of similarity in their
sequences and enzymatic properties. In fact, mice heterozygous for the
1 and
2 isoforms have opposite physiological responses in the
heart and the skeletal muscle (He et al.,
2001
; James et al.,
1999
). The hearts of
2 heterozygous mice are
hypercontractile, whereas the hearts of
1 heterozygotes are
hypocontractile. The enhanced contraction activity noted in the cardiomyocytes
of
2 heterozygotes correlates with the increased intracellular
Ca2+ level. No such fluctuation in cardiac cells was noted in
1 heterozygotes. These findings lead to the hypothesis that Na,KATPase
1 and
2 isoforms conduct unique functions, and that the
2, but not
1, isoform modulates Ca2+ signaling during
cardiac contraction.
Although the requirement for Na,K-ATPase in adult hearts has been
intensively studied, very little is known about its role in embryonic hearts.
Genetic studies in the mouse suggest that Na,K-ATPase activity is important
during embryogenesis, as 1 homozygous mice are embryonic lethal and
2 homozygotes die during the first day after birth
(James et al., 1999
). However,
whether the lethality is caused by defects in heart development requires
further investigation. Gene expression analyses in the chick and zebrafish
suggest that Na,K-ATPase may have an important role in embryonic heart
formation. In the zebrafish, three Na,K-ATPase isoforms,
1B1 (also
known as
1a.1),
2 and ß1a, are expressed in the developing
heart (Rajarao et al., 2001
;
Serluca et al., 2001
). In the
chick, Na,K-ATPase is also expressed in cardiac precursors. More
interestingly, the localization of Na,K-ATPase protein switches from an
initial even distribution, to a polarized lateral position on the plasma
membrane of cardiac precursors at the time of heart tube formation
(Linask, 1992
), which suggests
that Na,K-ATPase may be involved in guiding the growth of the primitive heart
tube.
We report the identification of a zebrafish mutation, heart and
mind (had), which is defective in the Na,K-ATPase 1B1
isoform. The had mutation causes severe abnormalities in primitive
heart tube extension, cardiomyocyte differentiation and embryonic cardiac
function, indicating crucial roles for the Na,K-ATPase
1B1 isoform in
zebrafish heart development. In addition, we found that the
1B1 and
2 isoforms conduct different functions in developing zebrafish hearts.
Despite the high degree of homology in
1B1 and
2 coding regions,
had phenotypes can only be rescued by wild-type
1B1
mRNA. Blocking translation of the
2 isoform does not cause significant
defects in early cardiac patterning or embryonic heart function, but disturbs
the establishment of cardiac laterality, further support that the
1 and
2 isoforms of Na,KATPase are not functionally equivalent.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Linkage and sequence analyses
We established the had map cross by mating a male had
heterozygote to a female fish from the EK strain. We analyzed linkages between
had and simple sequence-length polymorphism (SSLP) markers
(Shimoda et al., 1999).
Genomic DNA samples were extracted from pools of 50 homozygous mutant embryos
and their wild-type siblings. mRNA was extracted from pools of 50 homozygous
mutant embryos and their wild-type siblings (RNAwiz, Ambion) for cDNA
synthesis (ACCESS RT-PCR system, Promega). Five sets of primers, forward (F)
and reverse (R), were used to amplify the coding region of Na,KATPase
1B1:
PCR and RT-PCR products were subcloned using TOPO TA Cloning Kit (Invitrogen) for subsequent sequencing analysis.
In situ hybridization and antibody staining
Embryos for in situ hybridization and immunohistochemistry were raised in
embryo medium supplemented with 0.2 mM 1-phenyl-2-thiourea to maintain optical
transparency (Westerfield,
1995). Whole-mount immunohistochemistry using monoclonal antibody
S46 (from F. Stockdale, Stanford University) was carried out as described
(Chen and Fishman, 1996
).
Whole-mount in situ hybridization was performed as described
(Chen and Fishman, 1996
). The
antisense RNA probes used in this study were Na,K-ATPase
1B1, wt1, pax2 (from F. Serluca), cmlc2, vmhc,
versican (from D. Y. Stainier), nkx2.5 and irx1.
Histology
Fixed embryos were dehydrated, embedded in plastic (JB-4, polysciences),
sectioned at 8 µm and stained with Hematoxylin.
Ouabain treatment
Wild-type zebrafish embryos were raised in embryo media for the first 5
hours of development. At 5 hpf, ouabain (Sigma) was added to the embryo media
to a final concentration of 1 mM. These embryos were grown in the presence of
ouabain until 24 hpf or 50 hpf, and then were fixed in 4% paraformaldehyde for
whole-mount in situ hybridization and immunohistochemistry.
Morpholino injections
Morpholino antisense oligonucleotides (Gene-Tools), complementary to the
translation start site and its flanking sequence of the Na,KATPase 1B1
(
1B1MO, 5'-CTGCCAGCTCATATTGTTCTCGGCC-3') and
2
(
2MO, 5'-TTTCATGTCCGTACCCTTTCCCCAT-3') isoforms, were
synthesized to block the translation of the
1B1 and
2 isoforms.
Morpholino oligonucleotides with a 5-base pair mismatch to
1B1MO
(5'-CTGgCAcCTCATAaTGTTgTCGcCC-3') and to
2MO
(5'-TTTgATcTCCGTAgCCTTTgCCgAT-3') were synthesized as controls
(lowercase letters indicate mismatched bases). Wild-type embryos were each
injected with 2 ng of the morpholino oligoculeotide at one- to two-cell stage.
Cardiac phenotypes were examined by whole-mount in situ hybridization at 24
and 50 hpf.
Phenotypic rescue
Capped mRNA for Na,K-ATPase 1B1 and
2 was synthesized by in
vitro transcription with the mMESSAGE mMACHINETM Kit (Ambion). Embryos
from had heterozygote crosses were injected with 100 pg of mRNA at
the one- to two-cell stage. Cardiac phenotypes of the injected embryos were
examined by whole-mount in situ hybridization at 24 hpf. All injected embryos
were genotyped using primers flanking the deletion site of the had
allele (6F, 5'-GGGATTGTCCTGTAATCGTCA-3'; 6R,
5'-TTCTTCGGTGTTCAACAGCAG-3'). The wild-type and mutant alleles can
be distinguished by the size of PCR products. A 258 bp fragment is amplified
from the wild-type allele, whereas a 201 bp fragment is amplified from the
had allele.
Cell count
Embryonic cells are dissociated at 24 hpf using the mechanical dissociation
method previously described (Westerfield,
1995). EGFP-positive cells were counted using a Zeiss Axioplan2
microscope.
Ventricular contractility analysis
Mutant embryos of had and their wild-type siblings were
anesthetized for 5 minutes with Tricaine (0.16 mg/ml). These embryos were then
transferred to a recording chamber perfused with modified Tyrode's solution
(136 mM NaCl, 5.4 mM KCl, 0.3 mM NaH2PO4, 1.8 mM
CaCl2, 1 mM MgCl2, 10 mM HEPES, 5 mM glucose, pH 7.3) at
48 hpf. Cardiac contractions were recorded with a high-resolution video camera
(Panasonic WV BL202) for 5 minutes. The lengths of ventricles in diastolic and
systolic conditions were measured to calculate the ventricular shortening
fraction (VSF). Values are presented as mean±s.e.m.
![]() |
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
had encodes Na,K-ATPase 1B1
We mapped had to zebrafish Linkage Group 1 (LG1)
(Shimoda et al., 1999). No
recombination between had and the genetic marker z6384 was detected
in 848 meiosis (Fig. 5A). A
zebrafish EST (fa03c03), which shows significant homology to the Na,K-ATPase
subunit, is also mapped to the same region of LG1
(http://zfrhmaps.tch.harvard.edu/ZonRHmapper/Maps.htm).
Nine isoforms of the Na,K-ATPase
subunit have previously been cloned
in the zebrafish (Blasiole et al.,
2002
; Rajarao et al.,
2001
; Serluca et al.,
2001
). Interestingly, expression of the Na,K-ATPase
1B1
isoform (also known as
1a.1) was detected in the developing zebrafish
heart (Canfield et al., 2002
;
Serluca et al., 2001
), and was
previously mapped to LG1 (Rajarao et al.,
2001
; Serluca et al.,
2001
). We therefore considered Na,K-ATPase
1B1 to be a good
candidate gene for had. To further analyze whether the had
lesion resides in
1B1, we amplified
1B1 from wild-type and
had mutant embryos by the RT-PCR approach. Whereas a single fragment
was amplified from wild-type RNA using primers 5F and 5R, two truncated
fragments were obtained from had mutant RNA
(Fig. 5D). Sequencing of
genomic DNA revealed 4 exons between primers 5F and 5R (depicted as exon A-D
in Fig. 5B) in the wild-type,
and a 2 bp insertion followed by a 59 bp deletion in had mutants.
This deletion encompasses the 3' exon-intron boundary of exon C and
presumably results in aberrant splicing.
|
Blocking Na,K-ATPase activity phenocopies had
To confirm that the mutation in 1B1 causes had phenotypes,
we blocked Na,K-ATPase activity by applying ouabain, a Na,K-ATPase inhibitor,
to wild-type zebrafish embryos, and by injecting a morpholino antisense
oligonucleotide targeting the
1B1 translation initiation site
(
1B1MO) to wild-type zebrafish embryos at the one-cell stage
(Nasevicius and Ekker, 2000
).
Both ouabain treatment and
1B1MO injection yielded embryos essentially
identical to had mutants (Fig.
6B,C,D). We further analyzed the effects of these treatments on
heart tube extension and cardiac chamber differentiation by in situ
hybridization using the vmhc probe. After one day of development, 63%
of ouabain-treated embryos (n=104) and 94% of
1B1MO-injected
embryos (n=106) show severe defects in heart tube extension similar
to those observed in had mutants
(Fig. 6G,H,I). After two days
of development, cardiac expression of vmhc is no longer restricted to
the ventricle in 74% of ouabain-treated embryos (n=82) and 82% of
1B1MO injected embryos (n=99)
(Fig. 6M,N), as was observed in
had mutants (Fig. 6L). No such phenotypes were observed in untreated control embryos
(n=105), nor in embryos injected with the 5 bp-mismatch
1B1
control morpholino (n=72). Therefore, inhibition of Na,K-ATPase
1B1 activity produces had mutant phenotypes, strongly
supporting the hypothesis that mutation in
1B1 is responsible for the
had phenotype. Furthermore, we noted that in order to completely
phenocopy had phenotypes, ouabain treatment needed to be conducted
prior to the onset of gastrulation, suggesting that Na,K-ATPase
1B1
activity is required during early embryogenesis.
|
|
To investigate the role of the Na,K-ATPase 2 isoform in zebrafish
heart development we created a morpholino antisense oligonucleotide targeting
the
2 translation initiation site (
2MO). We tested the
inhibition ability of
2MO in vivo by injecting
2MO together with
2-RFP mRNA.
2-RFP is a chimera of Na,K-ATPase
2 with the coding region of Red Fluorescent Protein (RFP) fused in
frame at the C terminus. We detected a strong RFP signal in embryos injected
with 50 pg
2-RFP mRNA, but this signal is completely
suppressed when co-injected with 2 ng of
2MO (see Fig. S1 at
http://dev.biologists.org/supplemental/).
This data demonstrates the effectiveness of
2MO in inhibiting
translation of the Na,K-ATPase
2 isoform in vivo.
We injected one-cell stage wild-type zebrafish embryos with the 2MO
to inhibit the translation of Na,K-ATPase
2 isoform, and observed
neither primitive heart tube extension defects nor a reduction in the
contractility of the embryonic hearts (n=120). The gross morphology
of the
2MO-injected embryos appeared normal after two days of
development (Fig. 6E). However,
upon more careful analysis, we discovered a novel role for the
2
isoform in regulating cardiac laterality. In the zebrafish, the primitive
heart tube is placed on the left side of the embryo by 24 hpf (cardiac
jogging). This left-jogged heart gradually swings back to the midline before
the ventricle bends to the right of the atrium (cardiac looping)
(Chen et al., 1997
). We found
that 51% of
2MO-injected embryos analyzed failed to jog to the left
(18% right, 33% midline, n=88)
(Fig. 6J). 49% of
2MO-injected embryos analyzed (n=113) exhibited abnormal
looping (18% had a reversed looping and 31% failed to loop and remained as a
straight heart tube) (Fig. 6O). Such laterality defects were not observed in the control groups. Only 3% of
uninjected control embryos (n=71) and 10% of embryos injected with
the
2 5-bp-mismatch control morpholino oligonucleotide (n=96)
exhibit cardiac looping abnormality. These results indicate that
1B1
and
2 isoforms are not functionally equivalent and that
2
activity is required for establishing cardiac laterality.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Na,K-ATPase activity is required for embryonic cardiac function
A large number of physiological studies have firmly established Na,K-ATPase
as a crucial component in regulating postnatal cardiac function (for a review,
see Schwinger et al., 2003).
Recent studies on cardiac and skeletal muscle contraction of Na,K-ATPase
1 and
2 heterozygous mice suggest that these isoforms have
different roles in regulating Ca2+ signaling, which lead to the
opposite physiological effects observed in
1 and
2 heterozygotes
(He et al., 2001
;
James et al., 1999
). The
bradycardiac and hypocontractile phenotypes observed in had mutants
are similar to the reduced contractility phenotype observed in adult
1
heterozygous mice (James et al.,
1999
), suggesting that the zebrafish Na,K-ATPase
1B1
isoform may regulate embryonic cardiac function in a Ca2+
independent manner, as mouse
1 does in adult hearts.
Na,K-ATPase 1B1 regulates embryonic cardiac patterning
Our studies on had mutants revealed previously undiscovered roles
of Na,K-ATPase in cardiac morphogenesis. We found that the Na,K-ATPase
2 isoform is important for establishing cardiac laterality, and that
the
1B1 isoform is required for primitive heart tube extension and
cardiomyocyte differentiation. The discovery of the involvement of Na,KATPase
1B1 in primitive heart tube extension is an exciting one, because it
provides a handle for future molecular and cellular studies on mechanisms
governing heart tube extension. There are two equally plausible, and not
mutually exclusive, cellular mechanisms by which Na,K-ATPase regulates
primitive heart tube extension. One possibility is that Na,K-ATPase
1B1
regulates primitive heart tube extension by rearranging the cytoskeleton, as
the Na,K-ATPase
subunit is known to be associated with multiple
cytoskeletal proteins (for a review, see
Therien and Blostein, 2000
).
The other possibility is that the polarity of cardiomyocytes is involved in
primitive heart tube formation. Na,K-ATPase assumes a polarized position in
epithelial cells, as well as in cardiac precursors during primitive heart tube
formation in the chick (Linask,
1992
). A recent study has shown that the basolateral distribution
of Na,K-ATPase requires functional atypical PKC
(Suzuki et al., 2001
).
Therefore, we are intrigued to note that mutation in the zebrafish atypical
PKC
results in phenotypes similar to those caused by the heart
and mind mutation. Both mutations manifest in a primitive heart tube
extension defect, as well as brain defects and an upwardly curved body
(Horne-Badovinac et al., 2001
;
Peterson et al., 2001
;
Stainier et al., 1996
;
Yelon et al., 1999
).
Similarities in the spectrum of phenotypes shared between these two mutants
suggest that Na,K-ATPase
1B1 and aPKC
may direct primitive
heart tube extension by regulating the polarity of cardiac cells.
Divergent functions of Na,K-ATPase isoforms in heart development
Multiple isoforms of Na,K-ATPase are expressed in mammalian hearts, and
these isoforms conduct different functions in regulating cardiac function
(James et al., 1999). We found
a similar situation in the developing zebrafish hearts. Both the
1B1
and
2 isoforms are expressed in the developing zebrafish heart and they
have distinct roles in heart development, as demonstrated by the morpholino
knockdown and mRNA rescue experiments. The
1B1 isoform regulates the
early patterning and contractility of developing zebrafish hearts, whereas the
2 isoform is required for proper cardiac laterality. It is not clear
how molecules sharing such a high degree of similarity in their coding
sequences and enzymatic activities assume such different functions.
Identifying interacting proteins and signaling networks of each Na,KATPase
isoform will provide further understanding of cardiomyocyte function and
differentiation. Moreover, recent studies suggest that Na,K-ATPase isoforms
have significant differences in their affinities for cardiac glycosides
(Muller-Ehmsen et al., 2001
),
and that cellular physiological responsiveness to ouabain is dosage dependent
(Aizman et al., 2001
). As
Na,K-ATPase inhibitors are often used to enhance cardiac contractility,
studying the diverse functions of the Na,K-ATPase isoforms and their signaling
network may lead to better treatment for heart diseases, and to the design of
drugs that have more precisely targeted actions.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Aizman, O., Uhlen, P., Lal, M., Brismar, H. and Aperia, A.
(2001). Ouabain, a steroid hormone that signals with slow calcium
oscillations. Proc. Natl. Acad. Sci. USA
98,13420
-13424.
Alexander, J., Stainier, D. and Yelon, D. (1998). Screening mosaic F1 females for mutations affecting zebrafish heart induction and patterning. Dev. Genet. 22,288 -299.[CrossRef][Medline]
Blanco, G. and Mercer, R. (1998). Isozymes of the Na-K-ATPase: heterogeneity in structure, diversity in function. Am. J. Physiol. 275,F633 -F650.[Medline]
Blasiole, B., Canfield, V., Degrave, A., Thisse, C., Thisse, B.,
Rajarao, J. and Levenson, R. (2002). Cloning, mapping, and
developmental expression of a sixth zebrafish Na,K-ATPase 1 subunit
gene (atp1a1a.5). Gene Expr. Patterns
2, 243-246.[CrossRef][Medline]
Canfield, V., Loppin, B., Thisse, B., Thisse, C., Postlethwait,
J., Mohideen, M., Rajarao, S. and Levenson, R. (2002).
Na,K-ATPase and ß subunit genes exhibit unique expression
patterns during zebrafish embryogenesis. Mech. Dev.
116, 51-59.[CrossRef][Medline]
Chen, J. and Fishman, M. (1996). Zebrafish
tinman homolog demarcates the heart field and initiates myocardial
differentiation. Development
122,3809
-3816.
Chen, J., Haffter, P., Odenthal, J., Vogelsang, E., Brand, M.,
van Eeden, F., Furutani-Seiki, M., Granato, M., Hammerschmidt, M.,
Heisenberg, C., Jiang, Y., Kane, D., Kelsh, R., Mullins, M. and
Nusslein-Volhard, C. (1996). Mutations affecting the
cardiovascular system and other internal organs in zebrafish.
Development 123,293
-302.
Chen, J., van Eeden, F., Warren, K., Chin, A., Nusslein-Volhard,
C., Haffter, P. and Fishman, M. (1997). Left-right
pattern of cardiac BMP4 may drive asymmetry of the heart in
zebrafish. Development
124,4373
-4382.
Cheng, C., Hui, C., Strahle, U. and Cheng, S. (2001). Identification and expression of zebrafish Iroquois homeobox gene Irx1. Dev. Genes. Evol. 211,442 -444.[CrossRef][Medline]
He, S., Shelly, D., Moseley, A., James, P., James, J., Paul, R.
and Lingrel, J. (2001). The 1- and
2-isoforms of Na-K-ATPase play different roles in skeletal muscle
contractility. Am. J. Physiol. Regul. Integr. Comp.
Physiol. 281,917
-R925.
Horne-Badovinac, S., Lin, D., Waldron, S., Schwarz, M., Mbamalu,
G., Pawson, T., Jan, Y., Stainier, D. and Abdelilah-Seyfried, S.
(2001). Positional cloning of heart and soul reveals multiple
roles for PKC in zebrafish organogenesis. Curr.
Biol. 11,1492
-1502.[CrossRef][Medline]
Huang, C. J., Tu, C. T., Hsiao, C. D., Hsieh, F. J. and Tsai, H. J. (2003). Germ-line transmission of a myocardium-specific GFP transgene reveals critical regulatory elements in the cardiac mosin light chanin 2 promoter of zebrafish. Dev. Dyn. (in press).
James, P., Grupp, I. G., Woo, A., Askew, G., Croyle, M., Walsh,
R. and Lingrel, J. (1999). Identification of a
specific role for the Na,K-ATPase 2 isoform as a regulator of calcium
in the heart. Mol. Cell
3, 555-563.[Medline]
Linask, K. (1992). N-cadherin localization in early heart development and polar expression of Na,K-ATPase, and integrin during pericardial coelom formation and epithelialization of the differentiating myocardium. Dev. Biol. 151,213 -224.[Medline]
Lingrel, J. and Kuntzweiler, T. (1994).
Na,K-ATPase. J. Biol. Chem.
269,19659
-19662.
Mohler, P., Schott, J., Gramolini, A., Dilly, K., Guatimosim, S., duBell, W., Song, L., Haurogne, K., Kyndt, F., Ali, M. et al. (2003). Ankyrin-B mutation causes type 4 long-QT cardiac arrhythmia and sudden cardiac death. Nature 421,587 -590.[CrossRef][Medline]
Muller-Ehmsen, J., Juvvadi, P., Thompson, C., Tumyan, L.,
Croyle, M., Lingrel, J., Schwinger, R., McDonough, A. and Farley,
R. (2001). Ouabain and substrate affinities of human
Na,K-ATPase 1ß1,
2ß1, and
3ß1 when
expressed separately in yeast cells. Am. J. Physiol. Cell.
Physiol. 281,C1355
-C1364.
Nasevicius, A. and Ekker, S. (2000). Effective targeted gene `knockdown' in zebrafish. Nat. Genet. 26,216 -220.[CrossRef][Medline]
Peterson, R., Mably, J., Chen, J. and Fishman, M. (2001). Convergence of distinct pathways to heart patterning revealed by the small molecule concentramide and the mutation heart-and-soul. Curr. Biol. 11,1481 -1491.[CrossRef][Medline]
Rajarao, S., Canfield, V., Mohideen, M., Yan, Y., Postlethwait,
J., Cheng, K. and Levenson, R. (2001). The repertoire
of Na,K-ATPase and ß subunit genes expressed in the zebrafish,
Danio rerio. Genome Res.
11,1211
-1220.
Schwinger, R., Wang, J., Frank, K., Muller-Ehmsen, J., Brixius,
K., McDonough, A. and Erdmann, E. (1999). Reduced
sodium pump 1,
3, and ß1-isoform protein levels and
Na,K-ATPase activity but unchanged Na-Ca exchanger protein levels in human
heart failure. Circulation
99,2105
-2112.
Schwinger, R., Bundgaard, H., Muller-Ehmsen, J. and Kjeldsen, K. (2003). The Na, K-ATPase in the failing human heart. Cardiovasc. Res. 57,913 -920.[CrossRef][Medline]
Serluca, F., Sidow, A., Mably, J. and Fishman, M.
(2001). Partitioning of tissue expression accompanies multiple
duplications of the Na,K-ATPase alpha subunit gene. Genome
Res. 11,1625
-1631.
Shimoda, N., Knapik, E., Ziniti, J., Sim, C., Yamada, E., Kaplan, S., Jackson, D., de Sauvage, F., Jacob, H. and Fishman, M. (1999). Zebrafish genetic map with 2000 microsatellite markers. Genomics 28,219 -232.
Stainier, D. and Fishman, M. (1992). Patterning the zebrafish heart tube: acquisition of anteroposterior polarity. Dev. Biol. 153,91 -101.[Medline]
Stainier, D., Lee, R. and Fishman, M. (1993).
Cardiovascular development in the zebrafish. I. Myocardial fate map and heart
tube formation. Development
119, 31-40.
Stainier, D., Fouquet, B., Chen, J., Warren, K., Weinstein, B.,
Meiler, S., Mohideen, M., Neuhauss, S., Solnica-Krezel, L., Schier, A.,
Zwartkruis, F., Stemple, D., Malicki, J., Driever, W. and Fishman, M.
(1996). Mutations affecting the formation and function of the
cardiovascular system in the zebrafish embryo.
Development 123,285
-292.
Suzuki, A., Yamanaka, T., Hirose, T., Manabe, N., Mizuno, K.,
Shimizu, M., Akimoto, K., Izumi, Y., Ohnishi, T. and Ohno, S.
(2001). Atypical protein kinase C is involved in the
evolutionarily conserved par protein complex and plays a critical role in
establishing epithelia-specific junctional structures. J. Cell
Biol. 152,1183
-1196.
Therien, A. and Blostein, R. (2000). Mechanisms
of sodium pump regulation. Am. J. Physiol. Cell
Physiol. 279,C541
-C566.
Walsh, E. and Stainier, D. (2001). UDP-glucose
dehydrogenase required for cardiac valve formation in zebrafish.
Science 293,1670
-1673.
Westerfield, M. (1995). The Zebrafish Book. Eugene, OR: University of Oregon Press.
Yelon, D. (2001). Cardiac patterning and morphogenesis in zebrafish. Dev. Dyn. 222,552 -563.[Medline]
Yelon, D., Horne, S. and Stainier, D. (1999). Restricted expression of cardiac myosin genes reveals regulated aspects of heart tube assembly in zebrafish. Dev. Biol. 214, 23-37.[CrossRef][Medline]