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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
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.
Key words: Left-right asymmetry, H+-V-ATPase, V-ATPase, Xenopus, Chick, Zebrafish, Axial patterning, Cytoplasmic pH, Membrane voltage
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