H+ pump-dependent changes in membrane voltage are an early mechanism necessary and sufficient to induceXenopustail regeneration

Abstract

In many systems, ion flows and long-term endogenous voltage gradients regulate patterning events, but molecular details remain mysterious. To establish a mechanistic link between biophysical events and regeneration, we investigated the role of ion transport during Xenopus tail regeneration. We show that activity of the V-ATPase H+ pump is required for regeneration but not wound healing or tail development. The V-ATPase is specifically upregulated in existing wound cells by 6 hours post-amputation. Pharmacological or molecular genetic loss of V-ATPase function and the consequent strong depolarization abrogates regeneration without inducing apoptosis. Uncut tails are normally mostly polarized, with discrete populations of depolarized cells throughout. After amputation, the normal regeneration bud is depolarized, but by 24 hours post-amputation becomes rapidly repolarized by the activity of the V-ATPase, and an island of depolarized cells appears just anterior to the regeneration bud. Tail buds in a non-regenerative `refractory' state instead remain highly depolarized relative to uncut or regenerating tails. Depolarization caused by V-ATPase loss-of-function results in a drastic reduction of cell proliferation in the bud, a profound mispatterning of neural components, and a failure to regenerate. Crucially, induction of H+ flux is sufficient to rescue axonal patterning and tail outgrowth in otherwise non-regenerative conditions. These data provide the first detailed mechanistic synthesis of bioelectrical,molecular and cell-biological events underlying the regeneration of a complex vertebrate structure that includes spinal cord, and suggest a model of the biophysical and molecular steps underlying tail regeneration. Control of H+ flows represents a very important new modality that, together with traditional biochemical approaches, may eventually allow augmentation of regeneration for therapeutic applications.

Latent regeneration abilities persist following recent evolutionary loss in asexual annelids

“…Our study provides evidence that the potential for re-evolution of regenerative abilities is real: regenerative abilities can remain latent after being effectively lost and can be re-elicited through entirely natural means. Regeneration has been at least partly rescued in Hydra and vertebrates through experimental manipulations, for example, by activating Wnt signaling, ectopically expressing an ion pump, or suppressing the immune response (25,(30)(31)(32). Our study complements these experimental rescues by demonstrating the possibility for a "natural rescue": by con- litoralis (H, 10 dpa) restores internal and external structures present in a normal head (G).…”

Mechanisms of urodele limb regeneration

“…The gene for Sp9 , a transcription factor that plays a key role in amniote limb development by its positive regulation of fibroblast growth factor 8 (Fgf8) expression (Kawakami et al., 2004), is also expressed in the wound epidermis of regenerating axolotl limbs and may be involved in formation of the AEC (Satoh, Cummings, Bryant, & Gardiner, 2010a). Epidermal ion channels generate early signals obligatory for blastema formation, including Na + influx/H + efflux (Adams, Masi, & Levin, 2007; Jenkins, Duerstock, & Borgens, 1996). How these early signals are linked to secretory functions of the wound epidermis and the subsequent events of histolysis and dedifferentiation is not clear, but may involve upregulation of nitric oxide signals in the epidermis and stimulation of a rise in cytosolic Ca 2+ that results in the localization of protein kinase C to the plasma membrane, where it is activated by diacylglycerol to regulate transcription (Rao et al., 2009).…”

Looking proximally and distally: 100 years of limb regeneration and beyond

“…H þ efflux in the amputated tail is driven by a plasma membrane ATPase in the epidermal cells , and is likely to be important for limb regeneration as well, given that a gene encoding a v-ATPase was the most abundant clone in a suppressive subtraction cDNA library made from dedifferentiating axolotl limb tissue (Gorsic et al, 2008). These ion movements are obligatory for regeneration, since druginduced inhibition of either Na þ or H þ movements during the first 24 hr or so after amputation results in failure of blastema formation (Jenkins et al, 1996;Adams et al, 2007).…”