Bioelectric signaling in regeneration: Mechanisms of ionic controls of growth and form

Abstract

The ability to control pattern formation is critical for the both the embryonic development of complex structures as well as for the regeneration/repair of damaged or missing tissues and organs. In addition to chemical gradients and gene regulatory networks, endogenous ion flows are key regulators of cell behavior. Not only do bioelectric cues provide information needed for the initial development of structures, they also enable the robust restoration of normal pattern after injury. In order to expand our basic understanding of morphogenetic processes responsible for the repair of complex anatomy, we need to identify the roles of endogenous voltage gradients, ion flows, and electric fields. In complement to the current focus on molecular genetics, decoding the information transduced by bioelectric cues enhances our knowledge of the dynamic control of growth and pattern formation. Recent advances in science and technology place us in an exciting time to elucidate the interplay between molecular-genetic inputs and important biophysical cues that direct the creation of tissues and organs. Moving forward, these new insights enable additional approaches to direct cell behavior and may result in profound advances in augmentation of regenerative capacity.

Keywords: Bioelectricity; Ion channel; Patterning; Resting potential; Voltage.

Figure 1. Bioelectrical signaling drives pattern formation at the level of the cell, tissue, and organism

(A) Changes in transmembrane voltage are transduced (B) by a set of membrane mechanisms (voltage-powered transporters of serotonin and butyrate, voltage-gated calcium channels, voltage-regulated phosphatases, and others) into second-messenger cascades that regulate gene expression, thus directing cell behavior (C) such as, migration, proliferation, cell death, differentiation, gene expression, and shape changes. (D) In turn, these changes in cell behavior enable the creation of complex structures. Abbreviations: 5-HT, 5-hydroxytryptamine, also known as serotonin; HDAC, histone deacetylase; MAD3, Max-interacting transcriptional repressor; Akt, serine/threonine-specific protein kinase; GJC, gap junction communication; NCX, Na⁺/Ca²⁺ exchanger; VGCC, voltage-gated calcium channel; Cx, connexin; MAP kinase, mitogen-activated protein kinase. Lightning bolts represent changes in resting membrane potential. Panels A and B modified with permission (Levin, 2007b).

Figure 2. Bioelectric cues can specify pre-pattern information needed to create complex structures and direct the reprogramming of complete organs via non– cell-autonomous patterning signals

(A) Spatial distributions of resting potential gradients reveal the existence of complex prepatterns in vivo. Imaging with a voltage-sensitive fluorescent dye in the Xenopus nascent face reveals the borders of patterning compartments and organ locations prior to the induction of face-specific patterning transcripts. Anterior/face, red arrow denotes position of future right eye field. Modified with permission from (Vandenberg and Morrie, 2011). (B) Manipulation of these endogenous patterns by misexpression of ion channels can result in organ-level reprogramming. For example, targeted V_mem change, via misexpression of ion channels in the frog embryo, induces the formation of ectopic structures such as complete eyes, even in regions normally not competent to form eyes such as the gut (red arrow). Modified with permission from (Pai et al., 2012).

Figure 3. Large-scale bioelectric patterns are instructive for shape

(A-B) Limb regeneration does not normally occur in post-metamorphic froglets. After only 24 hours of exposure, an ionophore cocktail designed to specifically alter the bioelectric state of the blastema triggers growth of an entire limb (green arrowheads indicate the appearance of distal elements such as toes and toenails). Used with permission from (Tseng and Levin, 2013). (C-D) During the refractory period in Xenopus, tail regeneration does not occur. (E-F) A one-hour exposure of the animal to an ionophore cocktail induces sodium influx into the bud, which triggers the regeneration of an entire new tail. This example illustrates how a simple signal can trigger a complex, self-limiting downstream morphogenetic cascade appropriate in orientation, scaling, and location within the host organism. Exploiting such endogenous “master-regulator” triggers may be a powerful strategy for regenerative medicine, to restore complex organs long before we have the knowledge to micromanage its creation from specific cell types. Yellow arrows indicate location of amputation. Abbreviations: hpa, hours post amputation; dpa, days post amputation. Modified with permission from (Tseng et al., 2010a).

Figure 4. Large-scale bioelectric patterns direct the morphology of regenerated structures

(A) Understanding of the bioelectric circuit that controls anterior–posterior specification in a fragment of regenerating planaria can be used to design drug cocktails that alter the regenerating anatomical structures produced by adult stem cells. Using this information, the desired target morphology can be created including inducing the posterior-facing blastema to build a secondary head in planaria. Modified with permission from (Beane et al., 2011). (B) Pattern memory encoded in bioelectric circuits can be altered by manipulating bioelectric cues. Planarian head-tail polarity is regulated in part by an endogenous voltage gradient. When cut fragments are briefly exposed to reagents to alter the topology of bioelectric cues (e.g., gap junction targeting drugs or RNAi targeting innexins), their regeneration results in the creation of two headed animals. Remarkably, weeks later, when these same animals are re-cut in plain water over multiple rounds of regeneration, the two-headed worm phenotype persists. Recent work (Durant et al., 2017) shows that these two-headed forms can be re-set back to a permanent one-head state by a manipulation of the H⁺/K⁺-ATPase component of the circuit, and reveals how epigenetic long-term pattern memory that can be stored in bioelectric circuits.