A pharmacological screen identified the H+ and K+ ATPase transporter as obligatory for normal orientation of the left-right body axis in Xenopus. Maternal H+/K+-ATPase mRNA is symmetrically expressed in the 1-cell Xenopus embryo but becomes localized during the first two cell divisions, demonstrating that asymmetry is generated within two hours postfertilization. Although H+/K+-ATPase subunit mRNAs are symmetrically localized in chick embryos, an endogenous H+/K+-ATPase-dependent difference in membrane voltage potential exists between the left and right sides of the primitive streak. In both species, pharmacologic or genetic perturbation of endogenous H+/K+-ATPase randomized the sided pattern of asymmetrically expressed genes and induced organ heterotaxia. Thus, LR asymmetry determination depends on a very early differential ion flux created by H+/K+-ATPase activity.

Biophysical signaling, an integral regulator of long-term cell behavior in both excitable and non-excitable cell types, offers enormous potential for modulation of important cell functions. Of particular interest to current regenerative medicine efforts, we review several examples that support the functional role of transmembrane potential (V(mem)) in the regulation of proliferation and differentiation. Interestingly, distinct V(mem) controls are found in many cancer cell and precursor cell systems, which are known for their proliferative and differentiation capacities, respectively. Collectively, the data demonstrate that bioelectric properties can serve as markers for cell characterization and can control cell mitotic activity, cell cycle progression, and differentiation. The ability to control cell functions by modulating bioelectric properties such as V(mem) would be an invaluable tool for directing stem cell behavior toward therapeutic goals. Biophysical properties of stem cells have only recently begun to be studied and are thus in need of further characterization. Understanding the molecular and mechanistic basis of biophysical regulation will point the way toward novel ways to rationally direct cell functions, allowing us to capitalize upon the potential of biophysical signaling for regenerative medicine and tissue engineering.

All cells possess long-term, steady-state voltage gradients across the plasma membrane. These transmembrane potentials arise from the combined activity of numerous ion channels, pumps and gap junction complexes. Increasing data from molecular physiology now reveal that the role of changes in membrane voltage controls, and is in turn controlled by, progression through the cell cycle. We review recent functional data on the regulation of mitosis by bioelectric signals, and the function of membrane voltage and specific potassium, sodium and chloride ion channels in the proliferation of embryonic, somatic and neoplastic cells. Its unique properties place this powerful, well-conserved, but still poorly-understood signaling system at the center of the coordinated cellular interactions required for complex pattern formation. Moreover, disregulation of ion channel expression and function is increasingly observed to be not only a useful marker but likely a functional element in oncogenesis. New advances in genomics and the development of in vivo biophysical techniques suggest exciting opportunities for molecular medicine, bioengineering and regenerative approaches to human health.

In addition to biochemical gradients and transcriptional networks, cell behavior is regulated by endogenous bioelectrical cues originating in the activity of ion channels and pumps, operating in a wide variety of cell types. Instructive signals mediated by changes in resting potential control proliferation, differentiation, cell shape, and apoptosis of stem, progenitor, and somatic cells. Of importance, however, cells are regulated not only by their own Vmem but also by the Vmem of their neighbors, forming networks via electrical synapses known as gap junctions. Spatiotemporal changes in Vmem distribution among nonneural somatic tissues regulate pattern formation and serve as signals that trigger limb regeneration, induce eye formation, set polarity of whole-body anatomical axes, and orchestrate craniofacial patterning. New tools for tracking and functionally altering Vmem gradients in vivo have identified novel roles for bioelectrical signaling and revealed the molecular pathways by which Vmem changes are transduced into cascades of downstream gene expression. Because channels and gap junctions are gated posttranslationally, bioelectrical networks have their own characteristic dynamics that do not reduce to molecular profiling of channel expression (although they couple functionally to transcriptional networks). The recent data provide an exciting opportunity to crack the bioelectric code, and learn to program cellular activity at the level of organs, not only cell types. The understanding of how patterning information is encoded in bioelectrical networks, which may require concepts from computational neuroscience, will have transformative implications for embryogenesis, regeneration, cancer, and synthetic bioengineering.

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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.

Understanding how organisms establish their form during embryogenesis and regeneration represents a major knowledge gap in biological pattern formation. It has been recently suggested that morphogenesis could be understood in terms of cellular information processing and the ability of cell groups to model shape. Here, we offer a proof of principle that self-assembly is an emergent property of cells that share a common (genetic and epigenetic) model of organismal form. This behaviour is formulated in terms of variational free-energy minimization—of the sort that has been used to explain action and perception in neuroscience. In brief, casting the minimization of thermodynamic free energy in terms of variational free energy allows one to interpret (the dynamics of) a system as inferring the causes of its inputs—and acting to resolve uncertainty about those causes. This novel perspective on the coordination of migration and differentiation of cells suggests an interpretation of genetic codes as parametrizing a generative model—predicting the signals sensed by cells in the target morphology—and epigenetic processes as the subsequent inversion of that model. This theoretical formulation may complement bottom-up strategies—that currently focus on molecular pathways—with (constructivist) top-down approaches that have proved themselves in neuroscience and cybernetics.

BackgroundControl of stem cell behavior is a crucial aspect of developmental biology and regenerative medicine. While the functional role of electrophysiology in stem cell biology is poorly understood, it has become clear that endogenous ion flows represent a powerful set of signals by means of which cell proliferation, differentiation, and migration can be controlled in regeneration and embryonic morphogenesis.Methodology/Principal FindingsWe examined the membrane potential (Vmem) changes exhibited by human mesenchymal stem cells (hMSCs) undergoing adipogenic (AD) and osteogenic (OS) differentiation, and uncovered a characteristic hyperpolarization of differentiated cells versus undifferentiated cells. Reversal of the progressive polarization via pharmacological modulation of transmembrane potential revealed that depolarization of hMSCs prevents differentiation. In contrast, treatment with hyperpolarizing reagents upregulated osteogenic markers.Conclusions/SignificanceTaken together, these data suggest that the endogenous hyperpolarization is a functional determinant of hMSC differentiation and is a tractable control point for modulating stem cell function.