Early, H+-V-ATPase-dependent proton flux is necessary for consistent left-right patterning of non-mammalian vertebrates.
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.
Adams
Dany S
DS
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.
Robinson
Kenneth R
KR
Fukumoto
Takahiro
T
Yuan
Shipeng
S
Albertson
R Craig
RC
Yelick
Pamela
P
Kuo
Lindsay
L
McSweeney
Megan
M
Levin
Michael
M
D000199:Actins / Q000378:metabolism; D000818:Animals; D019521:Body Patterning; D002462:Cell Membrane / Q000378:metabolism; D002642:Chick Embryo; D004625:Embryo, Nonmammalian; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018507:Gene Expression Regulation, Developmental; D017506:H(+)-K(+)-Exchanging ATPase / Q000737:chemistry* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007150:Immunohistochemistry; D008954:Models, Biological; D054328:Proton Pump Inhibitors; D011522:Protons; D013997:Time Factors; D014714:Vertebrates / Q000196:embryology*; D014981:Xenopus / Q000196:embryology; D015027:Zebrafish / Q000196:embryology
D000199:Actins; D004791:Enzyme Inhibitors; D054328:Proton Pump Inhibitors; D011522:Protons; D017506:H(+)-K(+)-Exchanging ATPase