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.

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 (V mem ) regulation. Varying either pH or V mem , 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 V mem -dependent model of the early physiology of LR patterning. Development 133, 1657Development 133, -1671Development 133, (2006 DEVELOPMENT 1658 necessary to characterize the endogenous behavior of the relevant pumps in embryos and to place their function in the context of known LR patterning mechanisms. Here, we explore the properties of H + -V-ATPase function in several vertebrate embryos. Through endogenous localization of the H + -V-ATPase and gain-and loss-offunction experiments in chick, frog and zebrafish, we identify the H + -V-ATPase as a novel, conserved, obligate component of LR patterning upstream of asymmetric gene expression. KEY WORDS: Left-right asymmetry, H + -V-ATPase, V-ATPase, Xenopus, Chick, Zebrafish, Axial patterning, Cytoplasmic pH, Membrane voltage MATERIALS AND METHODS Animal husbandryXenopus embryos were collected according to standard protocols (Sive et al., 2000) in 0.1ϫ Modified Marc's Ringers (MMR) pH 7.8 + 0.1% Gentamicin. Xenopus embryos were staged according to Nieuwkoop and Faber (Nieuwkoop and Faber, 1967). Chick embryos from Charles River Laboratories, maintained at 38°C, were staged according to Hamburger and Hamilton (Hamburger and Hamilton, 1992). Zebrafish embryos (Westerfield, 1995) were maintained at 28.5°C in water containing 1 drop per gallon Methyl Blue. Assaying organ situsXenopus e...

SUMMARYUnderstanding the mechanisms that coordinate stem cell behavior within the host is a high priority for developmental biology, regenerative medicine and oncology. Endogenous ion currents and voltage gradients function alongside biochemical cues during pattern formation and tumor suppression, but it is not known whether bioelectrical signals are involved in the control of stem cell progeny in vivo. We studied Xenopus laevis neural crest, an embryonic stem cell population that gives rise to many cell types, including melanocytes, and contributes to the morphogenesis of the face, heart and other complex structures. To investigate how depolarization of transmembrane potential of cells in the neural crest’s environment influences its function in vivo, we manipulated the activity of the native glycine receptor chloride channel (GlyCl). Molecular-genetic depolarization of a sparse, widely distributed set of GlyCl-expressing cells non-cell-autonomously induces a neoplastic-like phenotype in melanocytes: they overproliferate, acquire an arborized cell shape and migrate inappropriately, colonizing numerous tissues in a metalloprotease-dependent fashion. A similar effect was observed in human melanocytes in culture. Depolarization of GlyCl-expressing cells induces these drastic changes in melanocyte behavior via a serotonin-transporter-dependent increase of extracellular serotonin (5-HT). These data reveal GlyCl as a molecular marker of a sparse and heretofore unknown cell population with the ability to specifically instruct neural crest derivatives, suggest transmembrane potential as a tractable signaling modality by which somatic cells can control stem cell behavior at considerable distance, identify a new biophysical aspect of the environment that confers a neoplastic-like phenotype upon stem cell progeny, reveal a pre-neural role for serotonin and its transporter, and suggest a novel strategy for manipulating stem cell behavior.

SUMMARY Biophysical signaling is required for both embryonic polarity and regenerative outgrowth. Exploiting endogenous ion transport for regenerative therapies will require direct regulation of membrane voltage. Here, we develop a pharmacological method to target ion transporters, uncovering a novel role for membrane voltage as a key regulator of anterior polarity in regenerating planaria. Utilizing the highly specific inhibitor, SCH-28080, our data reveal that H+,K+-ATPase-mediated membrane depolarization is essential for anterior gene expression and brain induction. H+,K+-ATPase-independent manipulation of membrane potential with ivermectin confirms that depolarization drives head formation, even at posterior-facing wounds. Using this chemical genetics approach, we demonstrate that membrane voltage controls head-vs.-tail identity during planarian regeneration. Our data suggest well-characterized drugs (already approved for human use) might be exploited to control adult stem cell-driven pattern formation during the regeneration of complex structures.

The Xenopus tadpole is able to regenerate its tail, including skin, muscle, notochord, spinal cord and neurons and blood vessels. This process requires rapid tissue growth and morphogenesis. Here we show that a focus of apoptotic cells appears in the regeneration bud within 12 h of amputation. Surprisingly, when caspase-3 activity is specifically inhibited, regeneration is abolished. This is true of tails both before and after the refractory period. Programmed cell death is only required during the first 24 h after amputation, as later inhibition has no effect on regeneration. Inhibition of caspase-dependent apoptosis results in a failure to induce proliferation in the growth zone, a mispatterning of axons in the regenerate, and the appearance of ectopic otoliths in the neural tube, in the context of otherwise normal continued development of the larva. Larvae amputated during the refractory stage exhibit a much broader domain of caspase-3-positive cells, suggesting a window for the amount of apoptosis that is compatible with normal regeneration. These data reveal novel roles for apoptosis in development and indicate that a degree of apoptosis is an early and obligate component of normal tail regeneration, suggesting the possibility of the existence of endogenous inhibitory cells that must be destroyed by programmed cell death for regeneration to occur.