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.
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.
Dany S
DS
Adams
Kenneth R
KR
Robinson
Takahiro
T
Fukumoto
Shipeng
S
Yuan
R Craig
RC
Albertson
Pamela
P
Yelick
Lindsay
L
Kuo
Megan
M
McSweeney
Michael
M
Levin
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