A Chemical Genetics Approach Reveals H,K-ATPase-Mediated Membrane Voltage Is Required for Planarian Head Regeneration

Abstract

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.

Bioelectric signaling regulates head and organ size during planarian regeneration

“…We report here that the H + ,K + -ATPase (H,K-ATPase) ion transporter regulates tissue remodeling via apoptosis during planarian regeneration. Our previous work revealed a role for H,K-ATPase in specifying anterior polarity in new tissues (Beane et al, 2011). In the present work, we demonstrate that H,K-ATPase is also required for head size and organ proportionality during regeneration, by mediating the apoptotic sculpting of original tissues.…”

“…DiBAC 4 (3) (bis-[1,3-dibarbituric acid]-trimethine oxanol) (Invitrogen), suspended in DMSO, was used at 0.475 M to measure voltage; the ratiometric dye SNARF-5F-AM (Molecular Probes S-23923), suspended in DMSO with 20% w/v Pluronic F-127, was used at 5 M to measure intracellular pH; both as previously described (Beane et al, 2011) using 3% EtOH-immobilized worms (Stevenson and Beane, 2010). Movements were quantified with a custom-made computer vision system (Hicks et al, 2006;Blackiston et al, 2010).…”

Early bioelectric activities mediate redox-modulated regeneration

“…Bioelectric and, more recently, redox activities have been shown, independently, to modulate regeneration in widespread models: planaria (Beane et al, 2011;Pirotte et al, 2015), zebrafish (Gauron et al, 2013;Monteiro et al, 2014) and amphibians (Love et al, 2013;Reid et al, 2009). Seeking a biochemical and biophysical integration, we aimed to determine whether and how these pervasive activities interact during regeneration.…”

“…V-ATPase proton efflux activity correlates with regeneration rate and ability in zebrafish caudal fin regeneration (Monteiro et al, 2014). H + /K + -ATPase-mediated V m depolarization modulates planarian head regeneration (Beane et al, 2011). In addition, ion and V m dynamics correlate with axolotl larvae tail regeneration (Özkucur et al, 2010).…”

Local and long-range endogenous resting potential gradients antagonistically regulate apoptosis and proliferation in the embryonic CNS

“…Such patterns of resting potential within living tissues (bioelectric signals) have been studied in the context of their roles in cell migration and wound healing (Cao et al, 2013, Jaffe, 1979, McCaig et al, 2005, Richard B. Borgens, 1989, Zhao et al, 2006 and have long been proposed to direct growth and form in vivo (Burr, 1932). Bioelectric signals are implicated in vertebrate appendage regeneration , Tseng et al, 2010, cancer initiation and metastasis (Binggeli and Weinstein, 1986b, Blackiston et al, 2011, Brackenbury, 2012, Chernet and Levin, 2013b, Lobikin et al, 2012b, left-right patterning (Aw et al, 2008, Aw et al, 2010, Levin et al, 2002, planarian head induction (Beane et al, 2011, Beane et al, 2013, Marsh and Beams, 1947, and eye and brain formation (Nuckels et al, 2009, Pai et al, 2015, Pai et al, 2012a, Pai et al, 2012b. Thus bioelectric signals have been shown to be important regulators of large-scale patterning and tissue and organ identity (Levin, 2009, Levin, 2013a, Levin and Stevenson, 2012, Tseng and Levin, 2013a.…”