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First published online 22 March 2006
doi: 10.1242/dev.02341
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1 The Forsyth Center for Regenerative and Developmental Biology, and Department
of Developmental Biology, Harvard School of Dental Medicine, 140 The Fenway,
Boston, MA 02115, USA.
2 Department of Biological Sciences, Purdue University, West Lafayette, IN
47906, USA.
3 Department of Cytokine Biology, The Forsyth Institute, 140 The Fenway, Boston,
MA 02115, USA.
4 Cardiovascular Research Center, Massachusetts General Hospital, Harvard
Medical School, Charlestown, MA 02129, USA.
Author for correspondence (e-mail:
mlevin{at}forsyth.org)
Accepted 27 February 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Left-right asymmetry, H+-V-ATPase, V-ATPase, Xenopus, Chick, Zebrafish, Axial patterning, Cytoplasmic pH, Membrane voltage
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Kosaki and Casey, 1998;
Levin, 1999;
Levin et al., 1996). Although
the genetic cascades of secreted signals that occur on only one side of the
embryo are being elucidated (Burdine and
Schier, 2000; Whitman and
Mercola, 2001), upstream mechanisms remain poorly understood
(Levin, 2005;
Yost, 2001). Recent findings
shed light on the events which function upstream of asymmetric transcription
(Bunney et al., 2003;
Fukumoto et al., 2005b;
Kramer et al., 2002;
Levin and Mercola, 1998;
Levin and Mercola, 1999;
Tanaka et al., 2005), and it
is now known that ion flux is involved in determining the laterality of
several vertebrates and invertebrates
(Duboc et al., 2005;
Hibino et al., 2006;
Levin et al., 2002;
McGrath et al., 2003;
Raya et al., 2004;
Shimeld and Levin, 2006). To
gain molecular insight into endogenous ion flows that control asymmetry, we
performed a loss-of-function drug screen
(Adams and Levin, 2006),
implicating the H+/K+-ATPase
(Levin et al., 2002). Here, we
characterize another crucial component of early embryonic physiology: the
H+-V-ATPase.
The H+-V-ATPase complex (see Fig. S1A in the supplementary
material) or V-ATPase (Nishi,
2002), is found in the membranes of vacuoles where it acidifies
the intravesicular environment (Inoue et
al., 2003; Kawasaki-Nishi et
al., 2003; Nishi,
2002). In many cell types, the H+-V-ATPase is also
present in the plasma membrane (Klein et
al., 1997; Narbaitz et al.,
1995; Nishi,
2002), where, by pumping protons out of the cell, it affects the
pH of the cytoplasm and immediate extracellular environment
(Brown and Breton, 2000;
Kawasaki-Nishi et al., 2003;
Scarborough, 2000). The
H+-V-ATPase is also strongly electrogenic
(Harvey, 1992;
Wieczorek, 1999).
Interestingly, besides the `housekeeping' functions of the
H+-V-ATPase and other electrogenic proteins, their activity has
been implicated in the control of cell proliferation, migration and
differentiation (Levin, 2003a;
Nuccitelli, 2003). One
important example of morphogenesis relying upon precisely orchestrated cell
behavior is the generation of consistent left-right asymmetry
(Cooke, 2004;
Levin, 2005), and a number of
biophysical mechanisms based on bioelectricity and fluid movement have been
implicated (Hamada et al.,
2002; Levin,
2003c; Tabin and Vogan,
2003). Moreover, the conservation of early LR mechanisms is highly
controversial (Burdine and Schier,
2000; Essner et al.,
2002; Levin,
2003c). Although ion flux is crucial in early frog and chick
embryos (Levin, 2005), ciliary
motion has been implicated in rodents
(McGrath and Brueckner, 2003;
McGrath et al., 2003) and
zebrafish (Amack and Yost,
2004; Essner et al.,
2005; Kramer-Zucker et al.,
2005). To understand how ion fluxes participate in asymmetry, it
is necessary to characterize the endogenous behavior of the relevant pumps in
embryos and to place their function in the context of known LR patterning
mechanisms. Here, we explore the properties of H+-V-ATPase function
in several vertebrate embryos. Through endogenous localization of the
H+-V-ATPase and gain- and loss-of-function experiments in chick,
frog and zebrafish, we identify the H+-V-ATPase as a novel,
conserved, obligate component of LR patterning upstream of asymmetric gene
expression.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) in
0.1x Modified Marc's Ringers (MMR) pH 7.8 + 0.1% Gentamicin.
Xenopus embryos were staged according to Nieuwkoop and Faber
(Nieuwkoop and Faber, 1967).
Chick embryos from Charles River Laboratories, maintained at 38°C, were
staged according to Hamburger and Hamilton
(Hamburger and Hamilton,
1992). Zebrafish embryos
(Westerfield, 1995) were
maintained at 28.5°C in water containing 1 drop per gallon Methyl
Blue.
Assaying organ situs
Xenopus embryos at stage 45 were analyzed for position
(situs) of three organs: the heart, stomach and gallbladder
(Levin and Mercola, 1998).
Heterotaxia was defined as reversal in one or more organs
(Fig. 1K,L). Only embryos with
normal dorsoanterior development (DAI=5) and clear left- or right-sided organs
were scored; percent heterotaxia was calculated as number heterotaxic divided
by the number of scorable embryos, i.e. embryos normal in all other ways, with
DAI=5. The proportion of unscorable embryos (number unscorable over total
number of embryos), is reported as a measure of treatment toxicity, but has no
influence on the heterotaxia score. The autofluorescence of gallbladder and
pancreas was used to score situs in zebrafish embryos. A Pearson
2 (increased stringency) was used to compare absolute counts
of heterotaxic embryos.
Pharmacological treatments
Xenopus embryos were incubated from 45 minutes post-fertilization
to stage 6-7 in drugs (Fig. 1)
at doses reported in Table 1.
All of the reagents used were selected on the basis of high specificity for
known electrogenic targets (Drose et al.,
1993; Shen et al.,
2003; Wheatly and Gao,
2004), and were titered to ensure that the DAI of the treated
embryos was normal, thus avoiding confounding randomization caused by midline
defects (Danos and Yost,
1996). Embryos were transferred to 0.1x MMR at stage 6-7 and
scored at stage 45. For all data shown, normal midline development and DAI
were observed. For cytoskeleton disruption experiments
(Fig. 3), embryos were treated
with 50 nM nocodazole or with 0.7 µM latrunculin, in 0.1x MMR
(Qiu et al., 2005).
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) was placed into the egg. Eggs were wrapped with Scotch tape,
incubated at 37.5°C, then fixed in 4% paraformaldehyde. Danio rerio embryos were incubated at 28.5°C in 1 µM lobatomide A16 or 250 nM concanamycin during stages indicated.
Self referencing ion selective probe measurements
For self referencing ion selective probe (SERIS) measurements of proton
flux (Smith et al., 1999),
healthy Xenopus embryos were cultured in 0.1x MMR at pH 7.0.
H+ flux measurements were made to the left and right, equidistant
from the ventral midline, near the AV midline, slightly on the animal side,
and approximately in the middle of the left-ventral quadrant and the
right-ventral quadrant of the embryo. The displacement of the electrode was
along a line that lay 45° from the normal to the surface of the embryo.
This was true for both measuring positions. We calculate a correction factor
of 105 for the presence of 0.5 mM HEPES buffer and have applied that factor to
generate the absolute flux values reported in the Results section
(Arif et al., 1995). The ratio
of the fluxes across the ventral midline is unaffected by this correction.
(Contact the authors for further details.)
Membrane voltage sensitive dye DiBAC4(3)
Bis-(1,3-dibarbituric-acid)-trimethine-oxanol [DiBAC4(3),
Molecular Probes] accumulates in proportion to membrane voltage; the more
depolarized a cell, the greater the accumulation and fluorescence intensity of
DiBAC4(3). Stock DiBAC4(3) (1 mg/ml in DMSO) was diluted
1:10 in distilled water, then 1:100 in 0.1x MMR (1.9 µM final).
Xenopus embryos were soaked for 30 minutes, then imaged submerged in
dye, using a Leica TCS SP2 Spectral Confocal Imaging System. The dye was
excited at 488 nm and a 20 nm band of emission wavelengths centered at 515 was
collected. On any given day, photomultiplier tube gain was kept constant to
allow comparisons among images.
Expression analysis
Whole mount in situ hybridization (WISH) on Xenopus was performed
using a standard protocol (Harland,
1991) using XNr1
(Lohr et al., 1998;
Lowe et al., 1996a), chick
Shh (Levin et al.,
1995) and chick Nodal
(Levin et al., 1995) probes.
WISH analysis was performed on zebrafish embryos as described
(Albertson and Yelick, 2005).
After staining, embryos were refixed in 4% paraformaldehyde, and dehydrated in
100% methanol overnight to remove background staining. Images were manipulated
with Adobe Photoshop to increase clarity; data were neither added nor
removed.
Immunocytochemistry was performed as described
(Levin, 2004). Briefly,
Xenopus embryos were fixed overnight at 4°C in MEMFA; chick
embryos were fixed overnight in 4% PFA. Some embryos were embedded for 40
µm sectioning on a vibratome. Samples were blocked with 20% goat
serum+0.2%BSA and incubated overnight at 4°C with primary antibody:
anti-subunit A, 1:500 (Kawa et al.,
2000); anti-myosin V, 1: 500 (Chemicon #AB5887), anti-RFX3, 1:500
(Bonnafe et al., 2004);
anti-subunit F, 1:500 (Peng et al.,
1996); anti-subunit c, 1:500 (against peptide DAGVRGTAQ,
Invitrogen). After washing, samples were incubated overnight at 4°C with
alkaline phosphatase-conjugated secondary antibodies. The chromogenic reaction
was timed to optimize signal to noise ratio. Standard no-primary, no-secondary
and peptide-adsorbed primary controls were used and resulted in no staining.
Patterns reported for localization of V-ATPase subunits represent a consensus
of data obtained from at least 15 embryos. Images were manipulated with Adobe
Photoshop to increase clarity; data were neither added nor removed.
Microinjection of mRNAs
Xenopus embryos were injected with capped, synthetic mRNAs
(Sive et al., 2000) into the
animal hemisphere of one-cell embryos 45-90 minutes post-fertilization. NHE3
constructs were injected into the vegetal hemisphere within 60 minutes of
fertilization. Zebrafish embryo microinjection was performed as described
previously (Payne-Ferreira and Yelick,
2003). Results of injections are reported as: % of otherwise
normal embryos that were heterotaxic; sample size (n); % of injected
embryos that died or were abnormal after gastrulation and not scored; and
2 and P values comparing treated with controls.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Fukumoto et al., 2005a;
Fukumoto et al., 2005b;
Levin et al., 2002;
Mitchison, 1994). A panel of
inhibitors targeting a wide variety of transporters
(Fig. 1B-J), like many others
reported previously (Levin et al.,
2002), induced a very low incidence of laterality phenotypes,
ruling out the corresponding targets from important roles in asymmetry and
demonstrating the general stability of LR patterning. By contrast, inhibitors
of the H+-V-ATPase all induced strong heterotaxia (compare
Fig. 1K with 1L) in the absence
of changes in dorsoanterior character or general toxicity
(Danos and Yost, 1995;
Danos and Yost, 1996). For
example, the potent and highly-specific V-ATPase blockers concanamycin
(Huss et al., 2002) and
bafilomycin (Bowman et al.,
1988) induced heterotaxia in 35% (n=100) and 36%
(n=100) of the embryos respectively. Importantly, inhibition of the
other two major classes of H+ transporters, carbonic anhydrase
(Fig. 1B) and the
sodium-hydrogen exchanger (NHE, Fig.
1C), did not affect laterality. Specific and dose-dependent
(Fig. 1M) randomization of LR
asymmetry, induced by loss-of-function reagents targeting the
H+-V-ATPase, but not other H+ pumps or a wide range of
other ion transporters, implicate endogenous H+-V-ATPase in the LR
pathway in Xenopus.
|
). Misexpression of this dominant-negative construct
specifically induced 20% heterotaxia [(n=191), controls=4%
(n=422), 2=40.6, P<<0.001]. By contrast,
injection of mRNAs encoding the Xenopus H,K-ATPase 
subunit
(Mathews et al., 1995), or the
dominant-negative Kir2.2 subunit (Zobel
et al., 2003) did not cause heterotaxia (1% heterotaxia each,
n=85,93 respectively). These data are consistent with the screen
results, demonstrate that embryonic asymmetry is not generally labile with
respect to microinjection per se (even when performed during cytoplasmic
rotation), and strongly support the hypothesis that H+-V-ATPase
function is necessary for LR patterning.
H+-V-ATPase inhibitors disrupt localization of normally left-sided transcripts
To determine the relationship between H+-V-ATPase and the
asymmetric gene cascade, we analyzed the expression of the earliest known
asymmetric gene in Xenopus: Nodal
(Hyatt et al., 1996). In
embryos treated with the potent and specific heterotaxia-causing
H+-V-ATPase inhibitor concanamycin
(Table 1), the situs of the
left-sided marker XNr1 (Fig.
2A) was effectively randomized
(Fig. 2B-D), exhibiting
aberrant sidedness of expression in 54% of the embryos
(Table 2). We conclude that
H+-V-ATPase functions upstream of the asymmetric transcription of
Nodal and feeds into the known asymmetric gene cascade.
|
|
;
Levin et al., 2002), we
focused our immunohistochemical analysis on the localization of
H+-V-ATPase subunits during the first few hours of development in
Xenopus. In early embryos, most proteins are symmetrically localized
with respect to the LR axis (Fig.
3A,B). Immunohistochemistry revealed that at the two-cell stage,
H+-V-ATPase subunits (Fig.
3D) exhibited `fingers' extending up from a pool in the vegetal
cytoplasm into the animal half. Such patterns are specific to a number of
LR-relevant proteins but are not a general feature of all protein localization
in the early frog embryo (Qiu et al.,
2005) (Fig. 3A,B).
In sections perpendicular to the AV axis at the two-cell stage, one cell was
often more heavily stained (>80% of the embryos examined,
Fig. 3E) (see Fig. S1I-K in the
supplementary material), exhibiting both a cytoplasmic pool and localization
at the cell cortex. At the four-cell stage, staining was still asymmetrical,
and was on the right side in embryos that could be oriented by the cleavage
furrows and pigmentation (Fig.
3F, see Fig. S1 in the supplementary material). Consistent with
the proposed role of the H+-V-ATPase in LR asymmetry, these
electrogenic protein subunits are novel markers of LR asymmetry as early as
the first two cleavages in Xenopus.
To probe upstream mechanisms (Qiu et
al., 2005; Yost,
1991), we asked whether the asymmetric localization of
H+-V-ATPase required organized microtubules/microfilaments.
Treatment of one-cell embryos with the actin depolymerizer latrunculin
(Ayscough, 1998) caused
apparently random localization of subunit A
(Fig. 3G). By contrast,
treatment with the microtubule disrupting agent nocodazole
(De Brabander et al., 1986)
had no obvious effect on the LR distribution of subunit A
(Fig. 3H). Embryos treated with
low levels of these specific cytoskeletal disruptors develop normally to later
stages, but, consistent with these data, displayed randomized asymmetry
(Qiu et al., 2005).
Early Xenopus embryos drive H+-V-ATPase-dependent, asymmetric H+ flux
In light of the loss-of-function and localization data, we asked whether
the activity of H+-V-ATPase (H+ flux) might provide a
consistently asymmetric physiological signal to the L and R sides of the body.
Using a SERIS probe (Hotary et al.,
1992; Smith et al.,
1999; Smith and Trimarchi,
2001), we detected a large net efflux of protons from the cleavage
furrow of the two-cell stage. This efflux averaged 12.7±22 pmole
cm-2s-1 (n=5) at about the midpoint of
cleavage. Importantly, we also found evidence for asymmetry of
H+-flux. As early as the four-cell stage, a distinct difference in
proton efflux was detected across the ventral midline, with larger efflux
occurring on the right side, consistent with the immunological localization of
H+-V-ATPase subunits on one side
(Fig. 3F, see Fig. S1F,G,K in
the supplementary material). In measurements on 15 embryos made between the
four-cell stage and stage 6, the average proton efflux from the middle of the
right ventral quadrant was 4.1±0.48 pmole cm-2s-1
and from the left ventral quadrant was 1.9±0.29 pmole
cm-2s-1. The average ratio of the efflux from the right
side to efflux from the left side was 2.3±0.3.
To confirm that the asymmetric efflux was due to asymmetric H+-V-ATPase function, we compared H+ efflux in control embryos with embryos treated with ion flux inhibitors. Neither the absolute net proton fluxes nor their right/left ratios were affected by the application of omeprazole (0.27 µM), an inhibitor of H+/K+-ATPase. However, the H+-V-ATPase inhibitor concanamycin (0.28 µM) reduced the proton efflux on the right side to about half of its original value and eliminated the LR asymmetry in the proton fluxes [ratio went from 2.1±0.2 to 1.1±0.05 (n=3), t=4.85, P<0.05]. Taken together, these data reveal the existence of consistent H+ flux asymmetry in the four-cell embryo, and confirm that, as suggested by the localization data, the asymmetry in the flow of H+ ions out of the early blastomeres is due specifically to differential H+-V-ATPase activity.
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), we also used
the reporting dye DiBAC4(3) to determine whether there is a
consistent asymmetry to Vmem
(Epps et al., 1994). Embryos
marked with Alexa 647 dextran in the right ventral cell (RV) at the four-cell
stage were monitored using DiBAC4(3) at 4-32 cells
(Fig. 4). Although no pattern
was obvious in 4-, 8- or 32-cell embryos, at 16 cells, we observed
hyperpolarization of the RV quadrant, relative to the LV quadrant
(Fig. 4A, yellow shading). An
example of an embryo in which this difference is easily observed is shown in
Fig. 4B-D. This pattern and its
embryological timing are consistent with the SERIS data, and with the
hypothesis that higher net H+ efflux on the right side at the
four-cell stage results in a difference in membrane potential across the
ventral midline by the 16-cell stage.
That this Vmem gradient is detectable in 16-cell embryos implies
that H+-flux-affecting reagents must be active by this stage in
order to affect LR patterning. To test this prediction, we injected YCHE78
mRNA, the dominant-negative H+-V-ATPase subunit E, into the LV or
RV cell at the four-cell stage, which results in no detectable expression
until the 
64-cell stage. This had no effect on organ situs [controls=0%
(n=38, unscorable=4%), LV=0% (n=62, unscorable=10%), RV=1%
(n=67, unscorable=12%)], 2=0.0, P=0.9),
although co-injected GFP was expressed normally (DNS). Similarly, we injected
YCHE78 DNA at the one-cell stage, which leads to protein expression only after
zygotic transcription begins at mid-blastula transition. Co-injected
ß-gal DNA was expressed (and caused higher toxicity than mRNA
injections), but injections had no effect on organ situs [ß-gal DNA
alone=0% (n=38, unscorable=65%), YCHE78 DNA=3% (n=29,
unscorable=70%), 2=2.0, P=0.155]. These data are
consistent with a dependence of LR asymmetry on very early, but not later,
activity of H+-V-ATPase.
Exogenous plasma-membrane H+-pump activity causes heterotaxia
We next examined the effects of an excess, symmetrical H+-flux
across the plasma membrane on both sides of the midline. We induced ectopic
H+ flux by expressing a well-characterized single-subunit
plasma-membrane H+-pump, PMA1.2
(Masuda and Montero-Lomeli,
2000). Use of this construct also allowed us to address whether it
is the balance of H+ flux at the cell membrane that is important
for LR asymmetry, as the H+-V-ATPase is also known to occur in
vacuoles, while PMA1.2 functions only in the cell membrane. Ubiquitous early
misexpression of this H+ pump at cell surfaces caused significant
heterotaxia [PMA1.2=21% heterotaxic (n=135, unscorable=22%),
untreated=2% (n=187, unscorable=3%), 2=28.3,
P<<0.001]. These data are consistent with the SERIS probe
measurements and the importance of differential H+ flux, and
suggest that it is the flow of hydrogen ions across the cell, not vacuole,
membrane that is important for LR asymmetry.
Manipulation of pH causes heterotaxia
To gain insight into the mechanisms by which the H+-V-ATPase
controls downstream events, we separately tested its two physiological roles -
regulation of pH and membrane voltage - by experimentally altering these two
parameters independently of direct manipulations of the
H+-V-ATPase. We first examined the effect of changing the pH of the
external environment of the embryo by raising or lowering the pH of the medium
during cleavage stages (Table
3). Although pH 7-11 had no effect on asymmetry, pH 5-6 caused a
low level of LR patterning defects (6%, P=0.003), and pH 4 caused a
significant level of heterotaxia (19%, P<<0.001). This is
consistent with inhibition of the activity of H+-extruding pumps by
high external proton concentrations. To alter embryonic pH without changing
Vmem, we used the electroneutral OH-/Cl-
exchanger tributyltin chloride (TBT, Table
1) to raise internal pH
(Matsuya et al., 2000;
Simchowitz et al., 1991).
Treatment with 10 µM TBT from stages 1 to 13 caused heterotaxia in 17% of
embryos [(n=151); untreated 1% (n=361);
2=44.7, P<<0.001].
|
;
Sabirov et al., 1999).
Injection of this antiporter mRNA within 1 hour of fertilization caused
heterotaxia [NHE3=16% (n=77, unscorable=32%), untreated=3%
(n=114, unscorable=5%), 2=8.9, P=0.003].
This confirms the importance of pH for LR asymmetry, and, similar to the
PMA1.2 data, indicates that the relevant H+ flux occurs at the cell
membrane, not in vesicles. Interestingly, NHE3 was active in causing
heterotaxia only when injected in the vegetal hemisphere and when injected
within 60 minutes of fertilization; later injections had no effect. Assuming
that NHE3 mRNA is translated 1.5 hours after injection (based on
detection of ß-gal/GFP translation), the temporal window during which
NHE3 can cause heterotaxia ends by 2.0 to 2.5 hours post-fertilization at
18°C. Consistent with expression data, the asymmetric membrane voltages at
16-cell stage, and the lack of effect on asymmetry of late YCHE78 injection or
DNA injection, we conclude that the LR-relevant gradients in cell membrane
H+ flux occur during the first cleavages, and that normal
asymmetric pH at the cell membrane is crucial for LR patterning.
Manipulation of Vmem causes heterotaxia
The H+-V-ATPase is electrogenic
(Gluck, 1992;
Harvey and Wieczorek, 1997)
and can therefore significantly contribute to the steady-state
Vmem. We confirmed this in Xenopus blastomeres by imaging
Vmem in vivo in control and concanamycin-treated embryos. As
predicted, inhibition of H+-V-ATPase depolarizes Vmem
(Fig. 4E,F). We conclude that
H+-V-ATPase activity is necessary for the normal pattern of
Vmem in Xenopus.
To address the role of Vmem in the absence of pH changes, we
altered Vmem directly by incubating embryos in the
Na+/K+ inhibitor palytoxin (PTX), which converts the
Na+/K+-ATPase into a non-specific ion channel, thus
dissipating the voltage gradient and depolarizing cells
(Hilgemann, 2003;
Tosteson et al., 1997;
Tosteson et al., 2003). PTX
treatment (2 nM) of embryos from stage 1-6 caused heterotaxia in the absence
of DAI change or other defects in 20% of surviving treated embryos
[(n=60, unscorable=84%), untreated=2% (n=58, unscorable=3%),
2=8.3, P=0.004]. We conclude that normal
Vmem, or patterned differences in Vmem, are necessary
for normal asymmetry.
H+-V-ATPase is present in early chick and zebrafish embryos
To explore the evolutionary conservation of an
H+-V-ATPase-dependent LR-patterning mechanism, we characterized the
expression pattern of H+-V-ATPase subunits in chick and zebrafish.
In situ hybridization analysis of subunits A, c, and F
(Fig. 5A-D), and
immunocytochemistry for subunits F and c
(Fig. 5E-H) indicated that the
H+-V-ATPase is expressed in the primitive streak of chick embryos
at stages 2-4, and in the node at stage 4 - the time-period in which chick LR
sidedness is determined by depolarization in the streak
(Levin et al., 2002).
Zebrafish embryos (Fig. 7A-F)
showed immunohistochemical staining for subunit c in two- to eight-cell stage
embryos. By the 32-cell stage, staining appeared stronger in the marginal
cells. Early in epiboly, staining was even in all cells, and subunit c was
detected surrounding some yolk syncytial nuclei. Staining was apparent in all
cells at epiboly.
|
). Inhibition of H+-V-ATPase by concanamycin
(Woo et al., 1996) or DCCD
(Finbow et al., 1993)
specifically randomized the localization of Shh and Nodal
(Fig. 6A-D) Concanamycin
induced aberrant sidedness of Shh expression in 67%, and
Nodal in 24%, of embryos (Table
4). Thus, as in Xenopus, H+-V-ATPase is
upstream of the known asymmetric gene cascade, and is required for normal
Nodal expression and therefore normal LR patterning in chick.
|
; Morley et al.,
1996), we found that in control embryos, area pellucida cells
maintain higher intracellular pH than primitive streak cells
(Fig. 6G,H). And, as predicted
by the expression of the H+-V-ATPase in the primitive streak, the
H+-V-ATPase inhibitor concanamycin lowers the pH of streak
cells.
|
H+-V-ATPase function is upstream of Kupffer's vesicle function
To further examine evolutionary conservation of the V-ATPase, we disrupted
its function in Danio rerio embryos. As in Xenopus,
microinjection into fertilized zebrafish eggs of mRNAs encoding a
dominant-negative subunit E mRNA caused a high level of heterotaxic organ
situs [YCHE78=36% (n=42, unscorable=0%), untreated=5% (n=96,
unscorable=4%), 2=19.5, P<<0.001;
Fig. 7G-I]. Injections of mRNA
for NHE3, to alter cytoplasmic pH only, caused 34% heterotaxia,
[(n=99, unscorable=6%), untreated=5% (n=94, unscorable=1%),
2=23.4, P<<0.001]. Expression of PMA1.2, to
exogenously increase cell membrane H+ flux, caused 29% heterotaxia,
[(n=52, unscorable=9%), untreated=5% (n=96, unscorable=4%),
2=14.2, P<<0.001]. Taken together, these data
support the hypothesis that specific levels and/or distributions of
H+-V-ATPase-dependant H+ transport activity at the
plasma membrane are necessary for correct LR asymmetry in zebrafish
embryos.
|
) have yet
been discovered in zebrafish asymmetry. To determine whether the
H+-V-ATPase is involved early, as in Xenopus, or later, at
stages when cilia might be involved
(Essner et al., 2005), we used
the specific H+-V-ATPase inhibitors lobatamide A16
(Shen et al., 2002) and
concanamycin (Huss et al.,
2002). As in Xenopus and chicks, initiation of
H+-V-ATPase loss-of-function between fertilization and the two-cell
stage causes significant heterotaxia [A16=30% (n=119), untreated=2%
(n=138), 2=37.0, P<<0.001;
concanamycin=30% (n=87), 2=34.1, P<<0.001,
phenotypes same as shown in Fig.
7G-I]. By contrast, exposure to lobatamide A16 beginning at three
somites (just prior to the formation of KV) did not result in a significant
level of heterotaxia [A16=5% (n=56), 2=0.0,
P=0.85]. Thus, in zebrafish embryos, H+-V-ATPase-dependent
H+ flux is important prior to the three-somite stage, i.e. prior to
KV formation, and is the earliest known step in LR patterning in
zebrafish.
Consistently, treatment with the H+-V-ATPase blocker lobatamide
A16 resulted in 55% right/bilateral expression of the asymmetric (left-sided)
marker Southpaw (Long et al.,
2003) [Fig. 7J-L
(n=38), untreated=7% (n=27), 2=13.8,
P<<0.001). Similar results were obtained using concanamycin
[treated=58% (n=26), untreated=8% (n=24),
2=11.4, P=0.001;
Table 5), demonstrating that
H+-V-ATPase activity is upstream of the earliest known asymmetric
marker in zebrafish.
|
;
Kramer-Zucker et al., 2005)
and we analyzed the interaction of H+-V-ATPase function with the
cilia pathway. Although H+-V-ATPase was not detected on the surface
of KV cilia (Fig. 8A), we found
that treatment of zebrafish embryos with the H+-V-ATPase inhibitors
lobatamide A16 or concanamycin caused the KV cilia to appear short and/or
greatly reduced in number [Fig.
8B,C; untreated=15% (n=41); A16=41% (n=39),
2=5.7, P=0.017; concanamycin=52% (n=27),
2=9.1, P=0.002]. We conclude that
H+-V-ATPase activity is upstream of the LR signaling events that
are dependent on normal KV cilia.
|
; Fukumoto et al.,
2005b). In Xenopus, serotonin is initially present in all
blastomeres but is localized to one blastomere by the 16/32 cell stages
(Fig. 8D,F). By contrast,
embryos exposed to the specific H+-V-ATPase blocker concanamycin
exhibited an almost complete absence of serotonin content
(Fig. 8E,G, n=17). We
conclude that H+-V-ATPase activity is required for the maintenance
of serotonin within early blastomeres and its localization to one specific
cell, placing the activity of this pump upstream of serotonin signaling in the
LR pathway. |
DISCUSSION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
), we were able to
use inhibitors at doses that affected LR asymmetry strongly while permitting
otherwise normal development (Fig.
1L). Use of specific blockers likewise implicated the
H+-V-ATPase in the control of laterality in the chick and zebrafish
(Figs 6,
7), revealing this versatile
pump as the first endogenous asymmetry mechanism conserved in three
vertebrates.
Crucially, to unequivocally confirm the requirement for the
H+-V-ATPase, we augmented drug experiments with specific constructs
targeting the H+-V-ATPase. Misexpression of the dominant-negative E
subunit (Lu et al., 2002) in
Xenopus and in zebrafish embryos causes randomization, confirming the
requirement for H+-V-ATPase for correct asymmetry. Though changing
Vmem and pH by overexpression can randomize, only the V-ATPase is
implicated endogenously in setting LR-relevant values of these physiological
parameters, as specific inhibitors of the Na+/K+-ATPase
(Levin et al., 2002) and other
H+ pumps (Fig. 1) do
not cause heterotaxia. Because genetic loss of H+-V-ATPase activity
in mice causes early embryonic lethality
(Sun-Wada et al., 2000), the
connection between this mechanism and rodent nodal cilia is unknown.
Intriguingly, however, inhibition of carbonic anhydrase causes a LR-relevant
phenotype in mice (Brown et al.,
1989) but not in Xenopus
(Fig. 1B). Perhaps control of
pH is the common denominator, while the mechanism of that control has diverged
among phyla.
Asymmetry-relevant H+-V-ATPase activity takes place early in embryogenesis
Loss-of-function of the H+-V-ATPase in chick randomizes the
early left-sided marker Shh (Fig.
6B). This implies that, as in Xenopus, in chicks the
H+-V-ATPase functions upstream of early asymmetric gene expression.
Future efforts to model physiological events that are integrated at the node
must take into account not only electroneutral H+/K+
exchange (Levin et al., 2002)
and Ca2+ flux (Raya and
Belmonte, 2004; Raya et al.,
2004), but also pH and/or Vmem regulation by
H+-V-ATPase. In zebrafish embryos, randomization of visceral situs
and of Spaw localization, and disruption of KV cilia, was caused by
H+-V-ATPase inhibition prior to somite formation, suggesting that
H+-V-ATPase is the earliest-known component of the LR pathway in
fish embryos, and acts prior to, and upstream of, events in KV
(Amack and Yost, 2004;
Essner et al., 2005;
Kramer-Zucker et al., 2005).
This implies that ciliary action, at least in zebrafish, is an important, but
not initial, step in LR patterning (Okada
et al., 2005). Interestingly, although inhibition of
H+-V-ATPase affects KV cilia, inhibition of the LR-crucial
H+/K+-ATPase does not
(Kawakami et al., 2005),
suggesting that the zebrafish relies on ion fluxes at more than one LR
signaling step. Further study of the interaction between early ion flux and
ciliary events in fish will be interesting, as this is the first species in
which both membrane voltage/pH and ciliary action are involved.
As with the H+/K+-ATPase
(Levin et al., 2002), the
earliest asymmetries of H+-V-ATPase were observed in
Xenopus. Maternal H+-V-ATPase proteins are present, and
asymmetries in their localization, and in directly detectable
H+-V-ATPase-dependent H+ flow, exist by the 2nd cleavage
in some percent of embryos. Alteration of H+-V-ATPase function
randomized Nodal (Fig.
2), the earliest available Xenopus asymmetric marker
(Lowe et al., 1996b), placing
it upstream of the conserved cascade in frog. Global upregulation of
H+ flux using the exogenous NHE3 pump did not affect laterality
when injection of the construct took place later than 1.5 hours
post-fertilization. Likewise, later inhibition of H+-V-ATPase,
caused by injections of YCHE78 mRNA at the four-cell stage or injections of
YCHE78 DNA, also had no effect on organ situs. DiBAC fluorescence, used to
reveal differences in membrane potential show biased asymmetry at the 16-cell
stage; it is important to note, however, that because of the magnitude of, and
the variations in cell volume, DiBAC is probably not accurate at earlier
stages. Thus, a consistent asymmetry in Vmem may be present
earlier. Localization of H+-V-ATPase subunits to the right ventral
cell of four-cell embryos, vibrating probe data showing enhanced
H+-efflux from the right ventral cells of four- and eight-cell
embryos, and DiBAC fluorescence showing the right ventral quadrant to be
hyperpolarized at the 16-cell stage, all support a role for
H+-V-ATPase activity during the first few hours of frog
development.
Endogenous expression and mechanisms upstream of H+-V-ATPase asymmetries
Consistent with the loss-of-function results, expression analysis revealed
that H+-V-ATPase subunits are present in chick from streak
initiation (Fig. 5A) and in
zebrafish (Fig. 7A) from the
first cleavage. We detected no consistent asymmetries of expression in either
species, similar to the situation for H+/K+-ATPase
(Levin et al., 2002). It is
likely that protein-level regulators remain to be discovered, the asymmetries
of which confer functional asymmetries on the activity of pumps that are
present symmetrically.
Asymmetrical localization of maternal components of the
H+-V-ATPase was observed after the first cleavage in
Xenopus (Fig. 3).
Although the plane of first cleavage can be experimentally repositioned
(Black and Vincent, 1988;
Danilchik and Black, 1988), in
normal embryos the cleavage furrow usually corresponds to the future midline
of the embryo (Klein, 1987;
Masho, 1990), and injection of
one cell at the two-cell stage is routinely used to target half the embryo,
allowing the contralateral half to serve as an internal control
(Harvey and Melton, 1988;
Vize et al., 1991;
Warner et al., 1984). Thus,
consistent with the timing data, this early asymmetry of localization is
likely to be key to the involvement of the H+-V-ATPase in LR
patterning of the whole embryo.
In light of early work from the Yost laboratory suggesting that
organization of the cytoskeleton prior to 1st cleavage is required for LR
asymmetry in Xenopus (Yost,
1991), the accepted role of the cytoskeleton and motor proteins in
the localization of subcellular components, and data implicating dyneins and
kinesins (Nonaka et al., 1998;
Supp et al., 1997), we
examined the interaction of the cytoskeleton with the H+-V-ATPase
(Fig. 3F-H). Consistent with
earlier observations linking embryo-wide asymmetry with subcellular
organization (Bunney et al.,
2003), and recent work showing that H+-V-ATPase
subunits interact directly with the actin cytoskeleton
(Chen et al., 2004;
Lee et al., 1999;
Vitavska et al., 2003), we
found that the asymmetric targeting of the H+-V-ATPase is dependent
on actin, but not microtubule, organization. We propose that some as yet
uncharacterized aspect of subcellular targeting
(Al-Awqati, 1996;
Brown, et al., 1992) [perhaps
oriented by a nucleation center which fits the role of Wolpert and Brown's
`F-molecule' (Brown and Wolpert,
1990)] is upstream of the H+-V-ATPase in the LR
pathway. The ability of very early latrunculin exposure to randomize LR
asymmetry in embryos (Qiu et al.,
2005) is consistent with the observed effects on localization,
linking cytoskeletal organization with large-scale asymmetry. We are currently
investigating cytoskeletal processes that assure asymmetric localization of
ion pumps.
The physiology of H+-V-ATPase function and H+ efflux in asymmetry
As the H+-V-ATPase normally energizes both vesicle and plasma
membranes, it is important to consider which of these roles controls
asymmetry. The SERIS measurements show that significant asymmetric flux can be
detected at the cell surface. Moreover, equalizing asymmetries of
H+ flux at the cell membrane, by global overexpression of
gain-of-function constructs (NHE3 and PMA1.2) that are known to induce
H+ flux specifically from cell surfaces, alters asymmetry in both
frog and zebrafish. Thus, efflux of H+ ions from the cell surface
is one crucial function of the H+-V-ATPase complex. However, it
cannot conclusively be ruled out that subcellular-compartment pH may be
important, and vesicular transport at the node could be such a locus
(Tanaka et al., 2005).
What aspect of H+-V-ATPase activity is required for asymmetry?
We distinguished between the two H+-V-ATPase functions, pH and
Vmem regulation. Manipulating pH directly in different ways, using
changes in medium pH, TBT and ectopic overexpression of the H+ pump
NHE3 in the blastomere plasma membrane, all disrupted asymmetry, consistent
with our conclusion that control of pH by H+-V-ATPase is important.
In particular, as the NHE3 pump is electroneutral, its ability to randomize
suggests that the normal pattern of pH in blastomeres is required for normal
LR asymmetry. Because H+-V-ATPase is also electrogenic, we explored
the effect on LR asymmetry of changing Vmem independently of
changes to the H+-V-ATPase, using palytoxin, which directly
abolishes Vmem. Like manipulation of H+-V-ATPase
activity and of pH, loss of Vmem causes significant levels of
heterotaxia. Thus, the normal pattern of Vmem is required for
normal LR patterning. Consistent with this finding, it has already been shown
that the normal asymmetric pattern of Vmem in chicks is required
for normal LR asymmetry (Levin et al.,
2002). Both pH and membrane voltage are important for downstream
steps, and we are currently working to identify the mechanisms by which
cellular ion fluxes are transduced to stable gene expression programs.
|
; Grandin
and Charbonneau, 1989). pH acts synergistically with many other
important biochemical pathways (Busa and
Nuccitelli, 1984), including Ca2+-calmodulin binding
and regulation of cAMP levels, which are important in LR patterning
(Kawakami and Nakanishi,
2001). For example, pH gates gap junctions
(Greenfield et al., 1990;
Turin and Warner, 1980),
which are essential for normal LR axis development
(Levin and Mercola, 1999). pH
regulation also has a role in gene expression and differentiation
(Sater et al., 1994;
Uzman et al., 1998) and in
other important developmental processes such as proliferation, apoptosis and
growth factor release (Bidani et al.,
1998; Lee and Steinhardt,
1981; Putney and Barber,
2003; Shrode et al.,
1997). pH also has other physiological effects that are likely to
be important during development, although a direct connection has not been
shown. For example, the G-protein-coupled receptors OGR1 and GPR4 are both
inactive at pH 7.8. At pH 6.8, however, OGR1 leads to inositol phosphate
formation, while GPR4 causes cAMP formation
(Ludwig et al., 2003). Thus,
pH changes can be coupled to two important second messengers. It is also known
that certain ion channels are pH sensitive
(Akopian et al., 2000;
Berdiev et al., 2003), and
H+-V-ATPase could exert its effect by controlling another
electrogenic component of LR patterning. There is also the possibility that
activity of the asymmetrically localized H+-V-ATPase could cause
redistribution of determinants (which may include the H+ ion
itself) by virtue of electrophoretic forces driven by the strong voltage
gradients that the H+-V-ATPase can produce. One such plausible
determinant is serotonin (5-HT). The normal localization of 5-HT to one
blastomere at the 16-cell stage is required for asymmetry
(Fukumoto et al., 2005b), and
we observed that 5-HT cannot be detected by that stage in embryos exposed to
H+-V-ATPase inhibitors (Fig.
8D-G). One possibility, based on the requirement for vesicular
monoamine transporters (VMAT) in asymmetry
(Fukumoto et al., 2005a) and
on the well-known role of the H+-V-ATPase in powering 5-HT import
into vesicles via the proton gradient-dependent transporter VMAT
(Hayashi et al., 1999), is
that 5-HT is degraded by monoamine oxidase when its import into protective
vesicles is inhibited by downregulation of the H+-V-ATPase. Thus,
one likely link between H+-V-ATPase function and downstream events
is 5-HT signaling. However, the ability of asymmetry to be perturbed by
cell-membrane modulation of pH and voltage suggests strongly that other
linking mechanisms remain to be discovered. Although many of the downstream
details remain to be worked out, we have formulated a model that accounts for
the available data.
Hypothesis for the role of H+ flux: the pepperoni model
We propose that a small charged morphogen, here hypothesized to be a
positively charged inhibitor of left-side specific gene products (`inhibitor
of leftness' or IOL), is evenly distributed in the fertilized egg, but becomes
asymmetrically localized during early cleavage stages
(Fig. 9). For this morphogen to
be active, two requirements must be met: there must be a threshold
concentration and there must be a high pH. The proposed Vmem
gradient, established by the activity of the right-sided
H+-V-ATPase, and possibly the H+/K+-ATPase
explored by Levin et al. (Levin et al.,
2002), provides the unidirectional motive force that moves the
morphogen, allowing the threshold concentration to be reached only on one side
(the right). The asymmetric activity of the H+-V-ATPase also raises
the pH high enough to activate the morphogen, again, only on the right. Under
these conditions the morphogen is active and affects the downstream genetic
cascade only on the right side.
Because of the dual requirement for both threshold concentration and pH, manipulations that affect either of these conditions will cause heterotaxia. Heterotaxia I results when Vmem is disrupted. In that situation, although pH may be high enough to activate the morphogen, the threshold concentration has not been reached, and heterotaxia results. This may happen in embryos treated with H+-V-ATPase inhibitors, expressing the electrogenic H+-pump PMA1.2, or treated with the Vmem disrupting agent PTX. Heterotaxia II results when Vmem is normal, but pH is not high enough. Under those conditions, the threshold concentration may be reached, but pH is too low, and again, the morphogen remains inactive and heterotaxia results. This describes the situation in embryos treated with H+-V-ATPase inhibitors, low pH medium or the electroneutral ionophore TBT, and in those embryos expressing the electroneutral NHE3. Testing this model and applying it to embryos of other species represents the next exciting challenge for future efforts to understand the integration of bioelectric and genetic information in left-right signaling.
|
ACKNOWLEDGMENTS |
|---|
subunit, W. Reith for
anti-RFX3 antibody, M. Tosteson for palytoxin, and D. Stone and X. S. Xie for
anti-A and anti-F subunit antibodies. We also thank Mark Mercola for important
early discussions and the encouragement of this work at the beginning. This
project was supported by NIH Training Grant 1-T32-DE-08327 and NIH Career
Transition Award 1-K22-DE016633-01A1 to D.S.A; NSF grant IOB-0087517 to K.R.R;
and NIH Research grant 1-R01-GM-0622, American Cancer Society Research Scholar
Grant RSG-02-046-01-DDC and US Department of Transportation Grant to the FRCDB
to M.L. This investigation was conducted in a Forsyth Institute facility
renovated with support from Research Facilities Improvement Grant Number
CO6RR11244 from the National Center for Research Resources, National
Institutes of Health. |
Footnotes |
|---|
Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/133/9/1657/DC1
* Present address: Weill Medical College, Cornell University, New York, NY
10021, USA 
|
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