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H+ pump-dependent changes in membrane voltage are an early mechanism necessary and sufficient to induce Xenopus tail regeneration
Dany S. Adams,
Alessio Masi, and
Center for Regenerative and Developmental Biology, Forsyth Institute, and
Developmental Biology Department, Harvard School of Dental Medicine, 140 The
Fenway, Boston, MA 02115, USA.
Author for correspondence (e-mail:
Accepted for publication 17 January 2007.
In many systems, ion flows and long-term endogenous voltagegradients
regulate patterning events, but molecular detailsremain mysterious. To
establish a mechanistic link between biophysicalevents and regeneration, we
investigated the role of ion transportduring Xenopus tail
regeneration. We show that activity of theV-ATPase H+ pump is
required for regeneration but not woundhealing or tail development. The
V-ATPase is specifically upregulatedin existing wound cells by 6 hours
post-amputation. Pharmacologicalor molecular genetic loss of V-ATPase
function and the consequentstrong depolarization abrogates regeneration
without inducingapoptosis. Uncut tails are normally mostly polarized, with
discretepopulations of depolarized cells throughout. After amputation,the
normal regeneration bud is depolarized, but by 24 hourspost-amputation
becomes rapidly repolarized by the activityof the V-ATPase, and an island of
depolarized cells appearsjust anterior to the regeneration bud. Tail buds in
a non-regenerative`refractory' state instead remain highly depolarized
relativeto uncut or regenerating tails. Depolarization caused by V-ATPase
loss-of-functionresults in a drastic reduction of cell proliferation in the
bud,a profound mispatterning of neural components, and a failureto
regenerate. Crucially, induction of H+ flux is sufficientto rescue
axonal patterning and tail outgrowth in otherwisenon-regenerative conditions.
These data provide the first detailedmechanistic synthesis of bioelectrical,
molecular and cell-biologicalevents underlying the regeneration of a complex
vertebrate structurethat includes spinal cord, and suggest a model of the
biophysicaland molecular steps underlying tail regeneration. Control of
H+flows represents a very important new modality that, together
withtraditional biochemical approaches, may eventually allow augmentationof
regeneration for therapeutic applications.