Early, H+-V-ATPase-dependent proton flux is necessary for consistent left-right patterning of non-mammalian vertebrates

Abstract

Biased left-right asymmetry is a fascinating and medically important phenomenon. We provide molecular genetic and physiological characterization of a novel, conserved, early, biophysical event that is crucial for correct asymmetry: H + flux. A pharmacological screen implicated the H + -pump H + -V-ATPase in Xenopus asymmetry, where it acts upstream of early asymmetric markers. Immunohistochemistry revealed an actin-dependent asymmetry of H + -V-ATPase subunits during the first three cleavages. H + -flux across plasma membranes is also asymmetric at the four-and eight-cell stages, and this asymmetry requires H + -V-ATPase activity. Abolishing the asymmetry in H + flux, using a dominant-negative subunit of the H + -V-ATPase or an ectopic H + pump, randomized embryonic situs without causing any other defects. To understand the mechanism of action of H + -V-ATPase, we isolated its two physiological functions, cytoplasmic pH and membrane voltage (V mem ) regulation. Varying either pH or V mem , independently of direct manipulation of H + -V-ATPase, caused disruptions of normal asymmetry, suggesting roles for both functions. V-ATPase inhibition also abolished the normal early localization of serotonin, functionally linking these two early asymmetry pathways. The involvement of H + -V-ATPase in asymmetry is conserved to chick and zebrafish. Inhibition of the H + -V-ATPase induces heterotaxia in both species; in chick, H + -V-ATPase activity is upstream of Shh; in fish, it is upstream of Kupffer's vesicle and Spaw expression. Our data implicate H + -V-ATPase activity in patterning the LR axis of vertebrates and reveal mechanisms upstream and downstream of its activity. We propose a pH-and V mem -dependent model of the early physiology of LR patterning. Development 133, 1657Development 133, -1671Development 133, (2006 DEVELOPMENT 1658 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-offunction 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. KEY WORDS: Left-right asymmetry, H + -V-ATPase, V-ATPase, Xenopus, Chick, Zebrafish, Axial patterning, Cytoplasmic pH, Membrane voltage MATERIALS AND METHODS Animal husbandryXenopus embryos were collected according to standard protocols (Sive et al., 2000) in 0.1ϫ 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 situsXenopus e...

Laterality defects are influenced by timing of treatments and animal model

“…What does complete absence of gene expression mean? Several studies report that some fraction of untreated animals do not express an asymmetric gene, yet these controls have no reported defects in organ laterality [for example, (Schweickert et al, 2007) reports absent pitx2 expression in 6% of controls and (Adams et al, 2006) and (Bunney et al, 2003b) both report absent Xnr-1 expression in 6% of controls]. Absent gene expression was common in treated animals, especially in mice (Figure 4 and Supplemental Table 1), and had a significant role when using gene expression to predict organ situs (Figure 5, compare all left panels with middle panels).…”

“…kinesin and LRD (Qiu et al, 2005)] and cell polarity proteins (Bunney et al, 2003a). Because of their LR-biased localizations, these motor and polarity proteins distribute ion transporters in a biased manner between the early blastomeres (Levin, 2006); the asymmetrically distributed cargo includes two proton pumps (Levin et al, 2002; Adams et al, 2006) and two potassium channels (Aw et al, 2008; Morokuma et al, 2008b). Coupled with a network of open gap junctions, the pH gradients and differences in membrane voltage that result from the biased localization of these ion transporters allow charged molecules such as serotonin to be distributed to the right- and ventral-most blastomere (Fukumoto et al, 2005a; Fukumoto et al, 2005b).…”

Far from solved: A perspective on what we know about early mechanisms of left–right asymmetry

“…C: By the four-cell stage, maternal mRNAs are now largely localized to the right ventral blastomere. These mRNAs encode ion transporters including two potassium channels Morokuma et al, 2008) and two proton pumps, the H þ -K þ -ATPase (Levin et al, 2002) and the H þ -V-ATPase (Adams et al, 2006). D: Together, the asymmetric localization of these transporters (blue circles) leads to a circuit establishing physiological asymmetries such as an increased pumping of positively charged ions out of the right side cell (þ), leading to a difference in transmembrane potential between the L and R blastomeres across the ventral midline.…”

“…At the protein level, mainly investigated in Xenopus, these include the Levin et al, 2002;), 14-3-3E polarity protein (Bunney et al, 2003), and a series of ''ciliary'' proteins with intracellular roles like Kinesin 3B (Qiu et al, 2005). At the level of physiology, the right ventral blastomere in the four-cell frog embryo pumps twice as many protons out as does the left ventral blastomere (Adams et al, 2006), the L and R blastomeres of ascidian embryos exhibit asymmetric calcium signaling (Albrieux and Villaz, 2000), and left-sided depolarization is observed to the left of the early (prenode) primitive streak in the chick (Levin et al, 2002). Early biophysical asymmetries include the discovery that the Xenopus egg has consistently chiral microfilament organization (Danilchik et al, 2006), as well as a cytoskeleton that has a net right-ward orientation for the guidance of intracellular transport by means of kinesin motors .…”

“…In the frog embryo, the asymmetry of the cytoskeleton drives differential localization of maternal protein cargo along the LR axis during the first cleavages. These proteins include two potassium channels Morokuma et al, 2008) and two proton pumps (Levin et al, 2002;Adams et al, 2006). The asymmetric fluxes and transmembrane potential gradients resulting from the biased localization of these transporters, together with a network of open gap junctions, allows LR morphogens such as serotonin to be distributed to the right-and ventral-most blastomere (Fukumoto et al, 2005a,b).…”