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A pharmacological screen identified the H+ and K+ ATPase transporter as obligatory for normal orientation of the left-right body axis in Xenopus. Maternal H+/K+-ATPase mRNA is symmetrically expressed in the 1-cell Xenopus embryo but becomes localized during the first two cell divisions, demonstrating that asymmetry is generated within two hours postfertilization. Although H+/K+-ATPase subunit mRNAs are symmetrically localized in chick embryos, an endogenous H+/K+-ATPase-dependent difference in membrane voltage potential exists between the left and right sides of the primitive streak. In both species, pharmacologic or genetic perturbation of endogenous H+/K+-ATPase randomized the sided pattern of asymmetrically expressed genes and induced organ heterotaxia. Thus, LR asymmetry determination depends on a very early differential ion flux created by H+/K+-ATPase activity.
Pharmacological Screen Implicates K+ and H+ Ion Flux in LR Patterning
(A) K+ channel (BaCl2 and Chromanol 293B) and H+ pump (lansoprazole) inhibitors induced high incidences of heterotaxia. Batches of Xenopus embryos were exposed to ion channel and pump inhibitors between fertilization and stage 16 and scored for the laterality of the heart, stomach coiling, and gallbladder at stage 45. The incidence of heterotaxia in control embryos was about 1%.
(B) H+/K+-ATPase blockers (omeprazole and SCH28080) induced heterotaxia whereas amiloride, EIPA, and cariporide (which inhibit Na+/H+ exchangers), ouabain (which inhibits Na+/K+-ATPase), and aurovertin (which inhibits the mitochondrial H+-ATPase) did not cause significant incidences of heterotaxia at doses that do not elicit anterioposterior defects.
(C–G) Analysis of the situs of the heart, gut coiling, and gallbladder position of Xenopus embryos exposed H+/K+-ATPase inhibitors. Ventral views of stage 45 embryos are shown. Red arrowheads indicate the apex of the heart, yellow arrowheads indicate the direction of gut coiling, and green arrowheads indicate the gallbladder.
(H and I) Example of normal heart in untreated and reversed heart in chick embryos injected in the albumin with lansoprazole and cultured in ovo to stage 15. Identical results were obtained with omeprazole and SCH28080.
Inhibitors of H+/K+-ATPase Activity Perturb Sidedness of Asymmetric Gene Expression in Xenopus
Examples of ectopic right-sided and absent expression of genes normally expressed in the left lateral mesoderm as detected by in situ hybridization.
Left (A–F) and right (A′–F′) flanks of embryos probed for XNr-1 (A-C′, stages 22–24), Pitx-2 (D–F′, stages 28–30) expression.
(G and H) Transverse histological sections of stages 28–30 embryos showing left-sided lefty expression in an untreated embryo (G) and bilateral expression in a treated embryo (H). In all images, arrowheads indicate normal (red), absent (white) or ectopic (blue) expression.
Aberrant Expression of Normally Asymmetrically Expressed Genes in Chick Embryos Treated with H+/K+-ATPase Inhibitors
Identical results were obtained with SCH28080, lansoprazole, and omeprazole and typical examples are shown here.
(A–G) HH stage 5 embryos probed for Shh, Wnt8C, and fgf8 expression by in situ hybridization. Treated embryos show bilateral or absent perinodal expression (B, D, E, and G).
(H and I) Nodal expression in HH stage 8 embryos.
(J and K) Pitx-2 expression seen at HH stage 11.
(L–O) Left flank and head mesenchyme expression of Caronte at HH stage 9 (L, red arrowheads). Examples of aberrant expression of both domains in embryos exposed to H+/K+-ATPase inhibitors include bilateral head and flank (M), head but not flank (N), and absent flank with bilateral head (O) expression. In all images, arrowheads indicate normal (red), absent (white), and ectopic (blue) expression.
H+/K+-ATPase mRNA in Xenopus and Chick Embryos
(A) H+/K+-ATPase α mRNA is localized preferentially to the animal hemisphere of the unfertilized Xenopus egg (shown in histological section).
(B and C) Animal views showing asymmetric localization patterns seen occasionally at the 2-cell stage (B) and consistently in the 4-cell stage.
(D–F) Histological sections through animal hemisphere of 4-cell embryos confirm that the mRNA localization is preferentially localized within right ventral blastomeres (red arrowheads) and depleted from the left ventral blastomere (white arrowheads), but is symmetric in the smaller dorsal blastomeres. Dorsal is oriented to the top in (C–F).
(G) Dorsal view of an 8-cell embryo showing LR symmetrical expression in the dorsal animal blastomeres and the narrow band of mRNA on the animal side of the dorsovegetal blastomeres (arrowhead).
(H) After MBT (stage 8), H+/K+-ATPase α mRNA is expressed symmetrically throughout the animal cap, as visualized in transverse histological section.
(I) H+/K+-ATPase α mRNA is localized in neural tissues and posterior gut of tailbud stage (stage 32) embryos.
(J and K) HH stage 3+ chick embryos showing symmetrical expression of H+/K+-ATPase α and β subunit mRNAs. The mRNAs remain symmetrically expressed in the ridges of the primitive streak at all stages examined (HH stages 2+ to 4−).
Heterotaxia Following Misexpression of H+/K+-ATPase α and β Subunits and the Kir4.1 Potassium Channel
Xenopus embryos were injected at the 1-cell stage with mRNAs encoding H+/K+-ATPase α and β subunits and Kir4.1. The incidence of heterotaxia (percentage of heterotaxic embryos) was scored as in Figure 1. α and β subunits expressed together, but not alone, induced statistically significant (*, p < 0.001) incidence of heterotaxia. Kir4.1 further increased the incidence of heterotaxic embryos when coexpressed with both H+/K+-ATPase subunits.
LR Asymmetries of Membrane Potential Patterns in the Primitive Streak Area Are Modulated by BaCl2 or Omeprazole
(A–D) A transient domain of depolarization to the left side of the primitive streak in chick embryos visualized with the potentiometric fluorescent probe DiBAC4(3). The blue to green to red pseudo-color scale represents increasing fluorescence intensities reflecting increased accumulation of the anionic dye in intracellular membranes. Increased fluorescence corresponds to a less negative membrane potential (i.e., depolarized).
(E–H) Linescans of the embryos made perpendicular to the streak (red lines in A-D) show the increased left sided fluorescence. Supplemental Table S3, available at http://www.cell.com/cgi/content/full/111/1/77/DC1, summarizes the mean LR differences from all embryos tested.
(I–K) Typical LR asymmetric pattern of depolarization is seen prior to exposure (I). The streak is outlined in (I). A relative increase in right-sided fluorescence is seen in the same embryo as in (I) after exposure to BaCl2 (J). Similar increase in right-sided fluorescence after omeprazole exposure (K). Arrowheads indicate Hensen's node.
(L–N) Linescans of the embryos (made along the red lines in I–K).
Models for Involvement of H+/K+-ATPase in Early LR Asymmetry
(A) Data presented here indicate that H+/K+-ATPase activity is localized to the right side of early Xenopus and chick embryos. An initiating mechanism to orient LR asymmetry has not been described experimentally in either chick or Xenopus but is envisaged to be responsible for the asymmetric H+/K+-ATPase activities, by localization of mRNA (Xenopus) or posttranslationally (chick), as described in the text.
(B) H+/K+-ATPase function might directly regulate the secretion of an early determinant of asymmetric gene expression.
(C) Alternatively, the H+/K+-ATPase might influence the propagation of unknown, low molecular weight LR determinants (green dots) between cells. Unidirectional propagation might rely simply on electrophoresis of charged determinants through open gap junction channels (depicted) or connexon gating (see text). The segregation of determinants triggers asymmetric expression of genes in multicellular fields.