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

• DOI: 10.1242/dev.02341
• Publication Date: 2006-03-23T01:33:34Z
• Authors: Dany S. Adams; Kenneth R. Robinson; Takahiro Fukumoto; Shipeng Yuan; R. Craig Albertson; Pamela Yelick; Lindsay Kuo; Megan McSweeney; Michael Levin
• Keywords: 0301 basic medicine; Embryo, Nonmammalian; Time Factors; Xenopus; Cell Membrane; Gene Expression Regulation, Developmental; Proton Pump Inhibitors; Chick Embryo; Hydrogen-Ion Concentration; Immunohistochemistry; Models, Biological; Actins; H(+)-K(+)-Exchanging ATPase; 03 medical and health sciences; Vertebrates; Animals; Enzyme Inhibitors; Protons; Zebrafish; Body Patterning

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 (Vmem) regulation. Varying either pH or Vmem, 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 Vmem-dependent model of the early physiology of LR patterning.