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Development 133 (9): 1657-1671
Early, H+-V-ATPase-dependent proton flux is necessary for consistent left-right patterning of non-mammalian vertebrates
Dany S. Adams1,
Kenneth R. Robinson2,
Takahiro Fukumoto3,*,
Shipeng Yuan4,
R. Craig Albertson3,
Pamela Yelick3,
Lindsay Kuo3,
Megan McSweeney3, and
Michael Levin1,
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.
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Fig. 1. Heterotaxia induced by H+-V-ATPase inhibitors.
Xenopus embryos were soaked in inhibitors of various ion
transporters. The percent of embryos considered heterotaxic (defined as a
reversal of at least one from the heart, gut or gall bladder) is calculated
relative to the total number of embryos, most of which showed normal
laterality. Inhibitors of H+-V-ATPase (A) caused significant
levels of heterotaxia, while inhibitors of other proton pumps
(B,C) or of other transporters (D-J) had no effect on
laterality. Inhibitor names and sample sizes are listed above the bars;
targets and doses of drugs in B-J are listed in
Table 1. Complete randomization
of three organs would lead to a maximum heterotaxia rate of 87.5%, as, by
chance, organ situs will appear to be wild type in 12.5% of embryos.
(K) A wild-type embryo, ventral view, showing the normal arrangement of
the gut (yellow arrowhead), heart apex (pink arrowhead) and gall bladder
(green arrowhead). (K') Higher magnification of normal heart. (L) A
heterotaxic embryo (ventral view) showing reversal of all three organs, i.e.
situs inversus. (L') Close-up of reversed heart. Image contrast has been
enhanced for clarity, and the loop of the heart has been outlined with black
dots in K and L. Drugs used for this screen were titered to determine a dose
that will cause heterotaxia without causing other morphological defects;
(M) an example titration curve for concanamycin. The asterisk in M
corresponds to the datum used in A (115 nM). There is a degree of variability
among sensitivity of embryos obtained from different females; toxicity
(defined as the percent of embryos dying post-gastrulation and/or developing
with significant morphological defects) increases at larger concentrations,
and there is only a narrow range of useful doses. The dose that is toxic to
50% of embryos (TD50; corrected for control background lethality of
9%) was 234 nM.
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Fig. 2. In situ hybridization for XNr1 in untreated and
concanamycin-treated Xenopus embryos. Treatment of
Xenopus embryos with the H+-V-ATPase inhibitor
concanamycin causes significant levels of heterotaxia that can be seen in the
situs of organs at stage 45 (Fig.
1L), but disruptions of laterality can be detected much earlier by
the expression patterns of normally left-sided markers. (A-D) Sectioned
embryos processed for in situ hybridization with an XNr1 antisense
probe. In wild-type embryos, XNr1 is restricted to the left
(A); however, in inhibitor-treated embryos, its expression is
randomized (B-D). Green arrows indicate normal position of staining;
red arrows indicate ectopic expression domain; white arrows indicate lack of
expression in normal region. Dorsal is upwards in all panels; left and right
sides correspond to those of the reader.
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Fig. 3. Immunohistochemistry of H+-V-ATPase subunit A.
Immunohistochemistry with an alkaline phosphatase conjugated secondary
antibody for subunit A of the H+-V-ATPase. Embryos were oriented
based on pigmentation and cleavage patterns - a technique that consistently
reveals biased asymmetry among the L and R blastomeres with respect to a
number of properties (Fukumoto et al.,
2005b ). Positive signals are blue to purple; LR orientation of
embryos is only possible in four- and eight-cell embryos. (A) The
molecular motor (Gross et al.,
2002) myosin V (AV section i.e. parallel to AV axis) and
(B) the ciliary protein (Bonnafe et
al., 2004) RFX3 (flat section, i.e. perpendicular to the AV axis)
are examples of symmetrically distributed proteins. (C) Western blot
showing that the antibody detects a single band of approximately the right
size (predicted: 69 kDa) for subunit A. Green and white arrows indicate
positive and a lack of signal, respectively. (D-H) Immunostaining for
subunit A. (D) Two-cell embryo, AV section, showing one common staining
pattern: `fingers' reaching animal-ward from the pool in the vegetal
cytoplasm. Although this pattern is not exclusive to H+-V-ATPase
subunits (Qiu et al., 2005),
it is not found for many proteins (e.g. compare with A). (E) Flat section of a
two-cell embryo showing another common staining pattern in which one
blastomere is more heavily stained than its contralateral counterpart. (F) The
asymmetry in staining seen in the flat sections is still visible at the
four-cell stage when it is right-sided. (G) A flat section through a
latrunculin-treated (actin depolymerized) embryo fixed at the four-cell stage,
showing disruption of the normal pattern and loss of asymmetry in subunit A
staining (compare with F). (H) A flat section through a nocodazole-treated
(microtubules depolymerized) embryo showing that localization of subunit A can
appear relatively unchanged by depolymerization of microtubules (compare with
F).
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Fig. 4. DiBAC staining reveals the Vmem pattern of blastomere
membranes under normal conditions and in H+-V-ATPase inhibitor.
(A) Graph showing the difference in DiBAC intensity on the left versus
the right ventral quadrants of the embryo. Positive values (yellow) indicate
that the right side is hyperpolarized with respect to the left side; negative
values indicate the inverse. At the 16-cell stage, the right side is
hyperpolarized with respect to the left. (B-D) Example of DiBAC-stained
16-cell embryo; DiBAC4(3) intensity on the left was greater than on
the right. Fluorescence intensity in C and D is pseudocolored; LUT shown
below. (B) Alexa 647-10,000 Mr dextran (Molecular Probes)
lineage labeled the right ventral quadrant. (C) Background-corrected
maximum-projected series of confocal images of DiBAC fluorescence overlaid
with lineage label to show location of left ventral (LV) and right ventral
(RV) quadrants and the position of the ventral midline (between). (D) Regions
of interest outlined in yellow. Mean pixel intensities in these two regions
were used as measures of depolarization of cells on the two sides of the
embryo. The difference between these two mean intensities was calculated to
produce data in A. (E,F) DiBAC4(3) fluorescence from
a four-cell embryo; (E) untreated and (F) treated with concanamycin.
Consistent with the prediction that inhibiting the H+-V-ATPase will
cause cells to depolarize (as H+ builds up inside the membrane),
concanamycin causes an increase in DiBAC4(3) fluorescence intensity
visible here as the larger area of red and the area of white to lavender.
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Fig. 5. Expression of H+-V-ATPase subunits in early chick
embryos. (A-D) Whole-mount in situ hybridization for
H+-V-ATPase subunits; green arrows indicate normal location of
staining. (A) Whole-mount in situ hybridization for subunit A in stage two
chick embryos. Staining is in the primitive streak. (B) Whole-mount in situ
hybridization for subunit A at stage 3 showing staining along the length and
through to the tip of the primitive streak. (C) Section through the primitive
streak of a stage 3 embryo reveals expression in the mesoderm. (D) Stage
4- embryo probed for subunit F, which, like subunits A and B, is
found in the primitive streak, extending through Hensen's node. (E-H)
Immunostaining for H+-V-ATPase subunits. (E) Stage 2+
chick embryo reveals subunit B along the length of the primitive streak. (F)
In stage 3 chicks, staining for subunit B is in the streak and the node. (G)
Cross-section through the streak of a stage 3 embryo showing subunit a
staining in the mesoderm (M); Ec, ectoderm; En, endoderm. (H) Subunit B
staining in a stage 4 chick. The streak and node are both positive.
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Fig. 6. In situ hybridization for sonic hedgehog and Nodal, and pH
imaging, in concanamycin-treated chick embryos. Embryos exposed to vehicle
alone exhibited the normal left-sided expression of Shh (A)
and Nodal (C). When exposed to the H+-V-ATPase
inhibitor concanamycin during early streak stages, the left-sided expression
becomes destabilized (see Table
4). Examples include bilateral expression of Shh
(B) or Nodal (D). Culture of chick embryos can lead to
bending of the AP axis such as that of embryo in D. In our experiments, about
15% of both control and treated embryos show this bend. Green arrows indicate
normal position of staining; red arrows indicate ectopic expression domain.
(E-H) Embryos treated with the pH indicator cSNARF-5F. Anterior is
upwards and left is leftwards in all images. White dots indicate the
approximate boundaries of the primitive streak. (E,F) Transmitted light images
of two embryos. (G) Pseudocolored image of ratiometric data representing pH,
shown in control embryo E. In most images, pH of the primitive streak is
somewhat lower than pH of the surrounding area pellucida (AP) cells; the
degree of contrast varies and is somewhat low in the image shown. (H) In
concanamycin-treated embryos, the primitive streak cells (which stain
positively for H+-V-ATPase subunits; see
Fig. 5E-H) are at a lower pH
than control cells (purple compared to green; embryo F). The AP, by contrast,
is at a higher pH than controls. The difference between pH of the primitive
streak and pH of the AP is much more pronounced in concanamycin-treated
embryos.
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Fig. 7. H+-V-ATPase subunits and heterotaxia in zebrafish.
(A-F) Immunohistochemistry for H+-V-ATPase subunits in
zebrafish embryos: (A) subunit F in a two-cell embryo; (B) subunit c in a
four-cell embryo; (C) subunit F in an eight-cell embryo. Brown (HRP-conjugated
secondary) indicates a positive signal. At these stages, the cortical
cytoplasm of all cells is positive for these two subunits, as is the cortical
cytoplasm of the yolk cell. (D) Thirty-two-cell zebrafish embryo stained for
subunit c. Staining (blue, AP-conjugated secondary) is heaviest in the
marginal cells of this stage, but present in all cells. (E) Early in epiboly,
subunit c staining is dark in the cells of the spreading blastoderm. In
addition, staining is visible around a ring of yolk syncitial nuclei, probably
representing vesicle staining (red arrowhead). (F) Once epiboly is more than
50% complete, immunohistochemistry for subunit c shows an even and heavy
distribution in all the cells of the blastoderm. The antibodies used in this
figure did not work in western blots on chick and fish extracts. (G-I)
Heterotaxia in zebrafish larvae. Tricaine-anaesthetized 5- to 6-day larvae
were examined on a Zeiss StemiSV11 dissecting microscope under 488/40 nm
illumination, using a 510 nm barrier filter. An embryo was considered
heterotaxic if either pancreas (orange arrowheads), gall bladder (red
arrowheads) or both organs were on the side opposite normal. (G) Normal
position of gall bladder (right) and pancreas (left). (H) Heterotaxia
involving the pancreas. (I) Heterotaxia involving both organs. When scoring
two organs, the top heterotaxia rate is 75% as, by chance, organ situs will
appear to be wild type in 25% of fully randomized embryos. (J-L)
Whole-mount in situ hybridization for the nodal-related gene southpaw
(Spaw), which is expressed in the left lateral plate mesoderm (LPM)
and tailbud of wild-type embryos from approximately 15-somite to 22-somite
stage. (J) Untreated embryos at 20-22-somite stages showed wild-type
Spaw expression in the left LPM (green arrow). Spaw
expression in the tail bud (an internal positive control for the specificity
of the treatment on Spaw asymmetry) is indicated by a blue arrowhead.
Similarly staged embryos treated early with the H+-V-ATPase
inhibitor lobatomide A16 exhibited reversed (K, red arrow) and bilateral (L,
green and red arrows) Spaw expression
(Long et al., 2003). See
Table 5 for statistical
analysis.
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Fig. 8. Effect of early H+-V-ATPase inhibition on Kupffer's vesicle
cilia and localization of serotonin. (A) Oblique confocal section
through an 8-somite zebrafish larva doubly immunostained for
H+-V-ATPase subunit A (green) and acetylated tubulin (red).
Although subunit A is obvious in cells of the overlying epithelium (Ep), no
H+-V-ATPase subunits are associated with KV cilia.
(B,C) IHC for acetylated tubulin reveals the structure of KV
cilia (green arrowheads) in five- to seven-somite stage zebrafish embryos. (B)
Untreated embryo showing the characteristic circular field of long straight
cilia, (C) KV cilia of five- to seven-somite embryos, soaked in
H+-V-ATPase inhibitors from the one-cell to shield stage, are often
reduced in number, altered in distribution or appear foreshortened relative to
controls. (D,E) Immunohistochemistry for serotonin (5-HT, green
arrows) in four-cell Xenopus embryos using an antibody previously
shown to be specific for 5-HT (Levin,
2004); (D) untreated, (E) incubated in concanamycin from the
one-cell stage. At this stage, there is no effect of H+-V-ATPase
inhibition on 5HT localization (green arrows) or level. (F,G)
Immunohistochemistry for 5HT in 32-cell Xenopus embryos; (F) normal
pattern of 5HT staining in one cell in an untreated embryo (green arrows); (G)
5HT is absent from the H+-V-ATPase inhibited embryo.
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Fig. 9. Hypothesis for the role of H+-V-ATPase in LR patterning; the
pepperoni model. We propose the following model to account for the extant
data on early LR patterning events in Xenopus. In control embryos,
asymmetric H+-V-ATPase activity is necessary for the establishment
of both a pH and a Vmem gradient. The Vmem gradient
provides the motive force to move a small charged morphogen (herein called
`inhibitor of leftness' or IOL) unidirectionally through gap junctions.
Because there are no gap junctions connecting the two cells across the ventral
midline (Levin and Mercola,
1998), the concentration of IOL will increase in the right ventral
region. This increase in concentration is required for IOL activity, i.e.
there is a threshold concentration for IOL to work. We further propose that
IOL activity also requires a particular pH to function. Again, as a result of
asymmetric H+-V-ATPase activity, only the right ventral region
reaches the required pH. Under these conditions, IOL can be active only in the
right ventral region, where it triggers side-appropriate downstream events.
Heterotaxia I: any treatment that interferes with the Vmem gradient
will prevent IOL from reaching threshold concentration. We believe this
explains the heterotaxia that results from treatments with
H+-V-ATPase inhibitors, expression of the dominant-negative subunit
E, expression of ectopic H+-pumps that change the normal pattern of
H+-flux and treatment with palytoxin, which destroys
Vmem entirely. The concentration requirement also explains the
heterotaxia caused by blocking GJC (Levin
and Mercola, 1998), although the mechanism is distinct from that
caused by changing Vmem. Heterotaxia II: This model also predicts
that any treatment pushing the pH in the right ventral region beyond an
acceptable range will cause heterotaxia. We believe this is why heterotaxia
results when embryos are treated with H+-V-ATPase inhibitors,
dominant-negative subunit E, the low pH of the medium and the electroneutral
change in pH caused by treatment with tributyltin or by expression of
NHE3.
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