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Cervera J, Manzanares JA, Levin M, Mafe S. Oscillatory phenomena in electrophysiological networks: The coupling between cell bioelectricity and transcription. Comput Biol Med 2024; 180:108964. [PMID: 39106669 DOI: 10.1016/j.compbiomed.2024.108964] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/07/2024] [Revised: 07/04/2024] [Accepted: 07/27/2024] [Indexed: 08/09/2024]
Abstract
Morphogenetic regulation during embryogenesis and regeneration rely on information transfer and coordination between different regions. Here, we explore theoretically the coupling between bioelectrical and transcriptional oscillations at the individual cell and multicellular levels. The simulations, based on a set of ion channels and intercellular gap junctions, show that bioelectrical and transcriptional waves can electrophysiologically couple distant regions of a model network in phase and antiphase oscillatory states that include synchronization phenomena. In this way, different multicellular regionalizations can be encoded by cell potentials that oscillate between depolarized and polarized states, thus allowing a spatio-temporal coding. Because the electric potential patterns characteristic of development and regeneration are correlated with the spatial distributions of signaling ions and molecules, bioelectricity can act as a template for slow biochemical signals following a hierarchy of experimental times. In particular, bioelectrical gradients that couple cell potentials to transcription rates give to each single cell a rough idea of its location in the multicellular ensemble, thus controlling local differentiation processes that switch on and off crucial parts of the genome.
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Affiliation(s)
- Javier Cervera
- Dept. Termodinàmica, Facultat de Física, Universitat de València, 46100, Burjassot, Spain.
| | - José A Manzanares
- Dept. Termodinàmica, Facultat de Física, Universitat de València, 46100, Burjassot, Spain
| | - Michael Levin
- Dept. of Biology, Tufts University, Medford, MA, 02155, USA; Allen Discovery Center at Tufts University, Medford, MA, 02155, USA; Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, 02215, USA
| | - Salvador Mafe
- Dept. Termodinàmica, Facultat de Física, Universitat de València, 46100, Burjassot, Spain; Allen Discovery Center at Tufts University, Medford, MA, 02155, USA
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2
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Li J, Xie Y, Liu G, Bahatibieke A, Zhao J, Kang J, Sha J, Zhao F, Zheng Y. Bioelectret Materials and Their Bioelectric Effects for Tissue Repair: A Review. ACS APPLIED MATERIALS & INTERFACES 2024; 16:38852-38879. [PMID: 39041365 DOI: 10.1021/acsami.4c07808] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 07/24/2024]
Abstract
Biophysical and clinical medical studies have confirmed that biological tissue lesions and trauma are related to the damage of an intrinsic electret (i.e., endogenous electric field), such as wound healing, embryonic development, the occurrence of various diseases, immune regulation, tissue regeneration, and cancer metastasis. As exogenous electrical signals, such as conductivity, piezoelectricity, ferroelectricity, and pyroelectricity, bioelectroactives can regulate the endogenous electric field, thus controlling the function of cells and promoting the repair and regeneration of tissues. Materials, once polarized, can harness their inherent polarized static electric fields to generate an electric field through direct stimulation or indirect interactions facilitated by physical signals, such as friction, ultrasound, or mechanical stimulation. The interaction with the biological microenvironment allows for the regulation and compensation of polarized electric signals in damaged tissue microenvironments, leading to tissue regeneration and repair. The technique shows great promise for applications in the field of tissue regeneration. In this paper, the generation and change of the endogenous electric field and the regulation of exogenous electroactive substances are expounded, and the latest research progress of the electret and its biological effects in the field of tissue repair include bone repair, nerve repair, drug penetration promotion, wound healing, etc. Finally, the opportunities and challenges of electret materials in tissue repair were summarized. Exploring the research and development of new polarized materials and the mechanism of regulating endogenous electric field changes may provide new insights and innovative methods for tissue repair and disease treatment in biological applications.
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Affiliation(s)
- Junfei Li
- School of Materials Science and Engineering, University of Science and Technology Beijing, Beijing 100083, China
| | - Yajie Xie
- School of Materials Science and Engineering, University of Science and Technology Beijing, Beijing 100083, China
| | - Guodong Liu
- School of Materials Science and Engineering, University of Science and Technology Beijing, Beijing 100083, China
| | - Abudureheman Bahatibieke
- School of Materials Science and Engineering, University of Science and Technology Beijing, Beijing 100083, China
| | - Jianming Zhao
- School of Materials Science and Engineering, University of Science and Technology Beijing, Beijing 100083, China
| | - Jia Kang
- School of Materials Science and Engineering, University of Science and Technology Beijing, Beijing 100083, China
| | - Jian Sha
- School of Materials Science and Engineering, University of Science and Technology Beijing, Beijing 100083, China
| | - Feilong Zhao
- School of Materials Science and Engineering, University of Science and Technology Beijing, Beijing 100083, China
| | - Yudong Zheng
- School of Materials Science and Engineering, University of Science and Technology Beijing, Beijing 100083, China
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McMillen P, Levin M. Collective intelligence: A unifying concept for integrating biology across scales and substrates. Commun Biol 2024; 7:378. [PMID: 38548821 PMCID: PMC10978875 DOI: 10.1038/s42003-024-06037-4] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/30/2023] [Accepted: 03/11/2024] [Indexed: 04/01/2024] Open
Abstract
A defining feature of biology is the use of a multiscale architecture, ranging from molecular networks to cells, tissues, organs, whole bodies, and swarms. Crucially however, biology is not only nested structurally, but also functionally: each level is able to solve problems in distinct problem spaces, such as physiological, morphological, and behavioral state space. Percolating adaptive functionality from one level of competent subunits to a higher functional level of organization requires collective dynamics: multiple components must work together to achieve specific outcomes. Here we overview a number of biological examples at different scales which highlight the ability of cellular material to make decisions that implement cooperation toward specific homeodynamic endpoints, and implement collective intelligence by solving problems at the cell, tissue, and whole-organism levels. We explore the hypothesis that collective intelligence is not only the province of groups of animals, and that an important symmetry exists between the behavioral science of swarms and the competencies of cells and other biological systems at different scales. We then briefly outline the implications of this approach, and the possible impact of tools from the field of diverse intelligence for regenerative medicine and synthetic bioengineering.
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Affiliation(s)
- Patrick McMillen
- Department of Biology, Tufts University, Medford, MA, 02155, USA
- Allen Discovery Center at Tufts University, Medford, MA, 02155, USA
| | - Michael Levin
- Department of Biology, Tufts University, Medford, MA, 02155, USA.
- Allen Discovery Center at Tufts University, Medford, MA, 02155, USA.
- Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, 02115, USA.
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Rouleau N, Levin M. The Multiple Realizability of Sentience in Living Systems and Beyond. eNeuro 2023; 10:ENEURO.0375-23.2023. [PMID: 37963652 PMCID: PMC10646883 DOI: 10.1523/eneuro.0375-23.2023] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/24/2023] [Accepted: 10/23/2023] [Indexed: 11/16/2023] Open
Affiliation(s)
- Nicolas Rouleau
- Department of Health Sciences, Wilfrid Laurier University, Waterloo, Ontario N2L 3C5, Canada
- Department of Biomedical Engineering, Tufts University, Medford, MA 02155
- Allen Discovery Center at, Tufts University, Medford, MA 02155
| | - Michael Levin
- Allen Discovery Center at, Tufts University, Medford, MA 02155
- Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02215
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Cervera J, Levin M, Mafe S. Correcting instructive electric potential patterns in multicellular systems: External actions and endogenous processes. Biochim Biophys Acta Gen Subj 2023; 1867:130440. [PMID: 37527731 DOI: 10.1016/j.bbagen.2023.130440] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/15/2023] [Revised: 06/19/2023] [Accepted: 07/28/2023] [Indexed: 08/03/2023]
Abstract
BACKGROUND Transmembrane electrical potential differences in cells modulate the spatio-temporal distribution of signaling ions and molecules that are instructive for downstream signaling pathways in multicellular systems. The local coupling between bioelectricity and protein transcription patterns allows dynamic subsystems (modules) of cells that share the same bioelectrical state to show similar biochemical downstream processes. METHODS We simulate theoretically how the integration-segregation pattern formed by the different multicellular modules that define a biosystem can be controlled by multicellular potentials. To this end, we couple together the model equations of the bioelectrical network to those of the genetic network. RESULTS The coupling provided by the intercellular junctions and the external microenvironment allows the restoration of the target bioelectrical pattern by changing the transcription rate of specific ion channels, the post-translational blocking of these channels, and changes in the environmental ionic concentrations. CONCLUSIONS The simulations show that the single-cell feedback between bioelectrical and transcriptional processes, together with the coupling provided by the intercellular junctions and the environment, can correct large-scale patterns by means of suitable external actions. GENERAL SIGNIFICANCE This study provides a theoretical advancement in the understanding of how the multicellular bioelectric coupling may guide repolarizing interventions for regenerating a tissue, with potential implications in biomedicine.
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Affiliation(s)
- Javier Cervera
- Dept. Termodinàmica, Facultat de Física, Universitat de València, E-46100 Burjassot, Spain.
| | - Michael Levin
- Dept. of Biology and Allen Discovery Center at Tufts University, Medford, USA; Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, USA
| | - Salvador Mafe
- Dept. Termodinàmica, Facultat de Física, Universitat de València, E-46100 Burjassot, Spain
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Pio-Lopez L, Bischof J, LaPalme JV, Levin M. The scaling of goals from cellular to anatomical homeostasis: an evolutionary simulation, experiment and analysis. Interface Focus 2023; 13:20220072. [PMID: 37065270 PMCID: PMC10102734 DOI: 10.1098/rsfs.2022.0072] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/17/2022] [Accepted: 03/02/2023] [Indexed: 04/18/2023] Open
Abstract
Complex living agents consist of cells, which are themselves competent sub-agents navigating physiological and metabolic spaces. Behaviour science, evolutionary developmental biology and the field of machine intelligence all seek to understand the scaling of biological cognition: what enables individual cells to integrate their activities to result in the emergence of a novel, higher-level intelligence with large-scale goals and competencies that belong to it and not to its parts? Here, we report the results of simulations based on the TAME framework, which proposes that evolution pivoted the collective intelligence of cells during morphogenesis of the body into traditional behavioural intelligence by scaling up homeostatic competencies of cells in metabolic space. In this article, we created a minimal in silico system (two-dimensional neural cellular automata) and tested the hypothesis that evolutionary dynamics are sufficient for low-level setpoints of metabolic homeostasis in individual cells to scale up to tissue-level emergent behaviour. Our system showed the evolution of the much more complex setpoints of cell collectives (tissues) that solve a problem in morphospace: the organization of a body-wide positional information axis (the classic French flag problem in developmental biology). We found that these emergent morphogenetic agents exhibit a number of predicted features, including the use of stress propagation dynamics to achieve the target morphology as well as the ability to recover from perturbation (robustness) and long-term stability (even though neither of these was directly selected for). Moreover, we observed an unexpected behaviour of sudden remodelling long after the system stabilizes. We tested this prediction in a biological system-regenerating planaria-and observed a very similar phenomenon. We propose that this system is a first step towards a quantitative understanding of how evolution scales minimal goal-directed behaviour (homeostatic loops) into higher-level problem-solving agents in morphogenetic and other spaces.
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Affiliation(s)
- Léo Pio-Lopez
- Allen Discovery Center, Tufts University, Medford, MA, USA
| | | | | | - Michael Levin
- Allen Discovery Center, Tufts University, Medford, MA, USA
- Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115, USA
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Mathews J, Chang A(J, Devlin L, Levin M. Cellular signaling pathways as plastic, proto-cognitive systems: Implications for biomedicine. PATTERNS (NEW YORK, N.Y.) 2023; 4:100737. [PMID: 37223267 PMCID: PMC10201306 DOI: 10.1016/j.patter.2023.100737] [Citation(s) in RCA: 11] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 05/25/2023]
Abstract
Many aspects of health and disease are modeled using the abstraction of a "pathway"-a set of protein or other subcellular activities with specified functional linkages between them. This metaphor is a paradigmatic case of a deterministic, mechanistic framework that focuses biomedical intervention strategies on altering the members of this network or the up-/down-regulation links between them-rewiring the molecular hardware. However, protein pathways and transcriptional networks exhibit interesting and unexpected capabilities such as trainability (memory) and information processing in a context-sensitive manner. Specifically, they may be amenable to manipulation via their history of stimuli (equivalent to experiences in behavioral science). If true, this would enable a new class of biomedical interventions that target aspects of the dynamic physiological "software" implemented by pathways and gene-regulatory networks. Here, we briefly review clinical and laboratory data that show how high-level cognitive inputs and mechanistic pathway modulation interact to determine outcomes in vivo. Further, we propose an expanded view of pathways from the perspective of basal cognition and argue that a broader understanding of pathways and how they process contextual information across scales will catalyze progress in many areas of physiology and neurobiology. We argue that this fuller understanding of the functionality and tractability of pathways must go beyond a focus on the mechanistic details of protein and drug structure to encompass their physiological history as well as their embedding within higher levels of organization in the organism, with numerous implications for data science addressing health and disease. Exploiting tools and concepts from behavioral and cognitive sciences to explore a proto-cognitive metaphor for the pathways underlying health and disease is more than a philosophical stance on biochemical processes; at stake is a new roadmap for overcoming the limitations of today's pharmacological strategies and for inferring future therapeutic interventions for a wide range of disease states.
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Affiliation(s)
- Juanita Mathews
- Allen Discovery Center at Tufts University, Medford, MA, USA
| | | | - Liam Devlin
- Allen Discovery Center at Tufts University, Medford, MA, USA
| | - Michael Levin
- Allen Discovery Center at Tufts University, Medford, MA, USA
- Wyss Institute for Biologically Inspired Engineering at Harvard University, Boston, MA, USA
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Bongard J, Levin M. There's Plenty of Room Right Here: Biological Systems as Evolved, Overloaded, Multi-Scale Machines. Biomimetics (Basel) 2023; 8:110. [PMID: 36975340 PMCID: PMC10046700 DOI: 10.3390/biomimetics8010110] [Citation(s) in RCA: 9] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/20/2022] [Revised: 02/24/2023] [Accepted: 03/01/2023] [Indexed: 03/18/2023] Open
Abstract
The applicability of computational models to the biological world is an active topic of debate. We argue that a useful path forward results from abandoning hard boundaries between categories and adopting an observer-dependent, pragmatic view. Such a view dissolves the contingent dichotomies driven by human cognitive biases (e.g., a tendency to oversimplify) and prior technological limitations in favor of a more continuous view, necessitated by the study of evolution, developmental biology, and intelligent machines. Form and function are tightly entwined in nature, and in some cases, in robotics as well. Thus, efforts to re-shape living systems for biomedical or bioengineering purposes require prediction and control of their function at multiple scales. This is challenging for many reasons, one of which is that living systems perform multiple functions in the same place at the same time. We refer to this as "polycomputing"-the ability of the same substrate to simultaneously compute different things, and make those computational results available to different observers. This ability is an important way in which living things are a kind of computer, but not the familiar, linear, deterministic kind; rather, living things are computers in the broad sense of their computational materials, as reported in the rapidly growing physical computing literature. We argue that an observer-centered framework for the computations performed by evolved and designed systems will improve the understanding of mesoscale events, as it has already done at quantum and relativistic scales. To develop our understanding of how life performs polycomputing, and how it can be convinced to alter one or more of those functions, we can first create technologies that polycompute and learn how to alter their functions. Here, we review examples of biological and technological polycomputing, and develop the idea that the overloading of different functions on the same hardware is an important design principle that helps to understand and build both evolved and designed systems. Learning to hack existing polycomputing substrates, as well as to evolve and design new ones, will have massive impacts on regenerative medicine, robotics, and computer engineering.
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Affiliation(s)
- Joshua Bongard
- Department of Computer Science, University of Vermont, Burlington, VT 05405, USA
| | - Michael Levin
- Allen Discovery Center at Tufts University, 200 Boston Ave., Suite 4600, Medford, MA 02155, USA
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McMillen P, Walker SI, Levin M. Information Theory as an Experimental Tool for Integrating Disparate Biophysical Signaling Modules. Int J Mol Sci 2022; 23:9580. [PMID: 36076979 PMCID: PMC9455895 DOI: 10.3390/ijms23179580] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/14/2022] [Revised: 08/13/2022] [Accepted: 08/14/2022] [Indexed: 11/16/2022] Open
Abstract
There is a growing appreciation in the fields of cell biology and developmental biology that cells collectively process information in time and space. While many powerful molecular tools exist to observe biophysical dynamics, biologists must find ways to quantitatively understand these phenomena at the systems level. Here, we present a guide for the application of well-established information theory metrics to biological datasets and explain these metrics using examples from cell, developmental and regenerative biology. We introduce a novel computational tool named after its intended purpose, calcium imaging, (CAIM) for simple, rigorous application of these metrics to time series datasets. Finally, we use CAIM to study calcium and cytoskeletal actin information flow patterns between Xenopus laevis embryonic animal cap stem cells. The tools that we present here should enable biologists to apply information theory to develop a systems-level understanding of information processing across a diverse array of experimental systems.
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Affiliation(s)
- Patrick McMillen
- Allen Discovery Center at Tufts University, Medford, MA 02155, USA
| | - Sara I. Walker
- Beyond Center for Fundamental Concepts in Science, Arizona State University, Tempe, AZ 85281, USA
- Santa Fe Institute, Santa Fe, NM 87501, USA
| | - Michael Levin
- Allen Discovery Center at Tufts University, Medford, MA 02155, USA
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Levin M. Technological Approach to Mind Everywhere: An Experimentally-Grounded Framework for Understanding Diverse Bodies and Minds. Front Syst Neurosci 2022; 16:768201. [PMID: 35401131 PMCID: PMC8988303 DOI: 10.3389/fnsys.2022.768201] [Citation(s) in RCA: 53] [Impact Index Per Article: 17.7] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2021] [Accepted: 01/24/2022] [Indexed: 12/11/2022] Open
Abstract
Synthetic biology and bioengineering provide the opportunity to create novel embodied cognitive systems (otherwise known as minds) in a very wide variety of chimeric architectures combining evolved and designed material and software. These advances are disrupting familiar concepts in the philosophy of mind, and require new ways of thinking about and comparing truly diverse intelligences, whose composition and origin are not like any of the available natural model species. In this Perspective, I introduce TAME-Technological Approach to Mind Everywhere-a framework for understanding and manipulating cognition in unconventional substrates. TAME formalizes a non-binary (continuous), empirically-based approach to strongly embodied agency. TAME provides a natural way to think about animal sentience as an instance of collective intelligence of cell groups, arising from dynamics that manifest in similar ways in numerous other substrates. When applied to regenerating/developmental systems, TAME suggests a perspective on morphogenesis as an example of basal cognition. The deep symmetry between problem-solving in anatomical, physiological, transcriptional, and 3D (traditional behavioral) spaces drives specific hypotheses by which cognitive capacities can increase during evolution. An important medium exploited by evolution for joining active subunits into greater agents is developmental bioelectricity, implemented by pre-neural use of ion channels and gap junctions to scale up cell-level feedback loops into anatomical homeostasis. This architecture of multi-scale competency of biological systems has important implications for plasticity of bodies and minds, greatly potentiating evolvability. Considering classical and recent data from the perspectives of computational science, evolutionary biology, and basal cognition, reveals a rich research program with many implications for cognitive science, evolutionary biology, regenerative medicine, and artificial intelligence.
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Affiliation(s)
- Michael Levin
- Allen Discovery Center at Tufts University, Medford, MA, United States
- Wyss Institute for Biologically Inspired Engineering at Harvard University, Cambridge, MA, United States
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11
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Minh-Thai TN, Samarasinghe S, Levin M. A Comprehensive Conceptual and Computational Dynamics Framework for Autonomous Regeneration Systems. ARTIFICIAL LIFE 2021; 27:80-104. [PMID: 34473826 DOI: 10.1162/artl_a_00343] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/13/2023]
Abstract
Many biological organisms regenerate structure and function after damage. Despite the long history of research on molecular mechanisms, many questions remain about algorithms by which cells can cooperate towards the same invariant morphogenetic outcomes. Therefore, conceptual frameworks are needed not only for motivating hypotheses for advancing the understanding of regeneration processes in living organisms, but also for regenerative medicine and synthetic biology. Inspired by planarian regeneration, this study offers a novel generic conceptual framework that hypothesizes mechanisms and algorithms by which cell collectives may internally represent an anatomical target morphology towards which they build after damage. Further, the framework contributes a novel nature-inspired computing method for self-repair in engineering and robotics. Our framework, based on past in vivo and in silico studies on planaria, hypothesizes efficient novel mechanisms and algorithms to achieve complete and accurate regeneration of a simple in silico flatwormlike organism from any damage, much like the body-wide immortality of planaria, with minimal information and algorithmic complexity. This framework that extends our previous circular tissue repair model integrates two levels of organization: tissue and organism. In Level 1, three individual in silico tissues (head, body, and tail-each with a large number of tissue cells and a single stem cell at the centre) repair themselves through efficient local communications. Here, the contribution extends our circular tissue model to other shapes and invests them with tissue-wide immortality through an information field holding the minimum body plan. In Level 2, individual tissues combine to form a simple organism. Specifically, the three stem cells form a network that coordinates organism-wide regeneration with the help of Level 1. Here we contribute novel concepts for collective decision-making by stem cells for stem cell regeneration and large-scale recovery. Both levels (tissue cells and stem cells) represent networks that perform simple neural computations and form a feedback control system. With simple and limited cellular computations, our framework minimises computation and algorithmic complexity to achieve complete recovery. We report results from computer simulations of the framework to demonstrate its robustness in recovering the organism after any injury. This comprehensive hypothetical framework that significantly extends the existing biological regeneration models offers a new way to conceptualise the information-processing aspects of regeneration, which may also help design living and non-living self-repairing agents.
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Affiliation(s)
- Tran Nguyen Minh-Thai
- Lincoln University, Complex Systems, Big Data and Informatics Initiative (CSBII)
- Can Tho University, College of Information and Communication Technology
| | - Sandhya Samarasinghe
- Lincoln University, Complex Systems, Big Data and Informatics Initiative (CSBII).
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12
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Tassinari R, Cavallini C, Olivi E, Taglioli V, Zannini C, Ventura C. Unveiling the morphogenetic code: A new path at the intersection of physical energies and chemical signaling. World J Stem Cells 2021; 13:1382-1393. [PMID: 34786150 PMCID: PMC8567452 DOI: 10.4252/wjsc.v13.i10.1382] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/15/2021] [Revised: 05/16/2021] [Accepted: 09/10/2021] [Indexed: 02/06/2023] Open
Abstract
In this editorial, we discuss the remarkable role of physical energies in the control of cell signaling networks and in the specification of the architectural plan of both somatic and stem cells. In particular, we focus on the biological relevance of bioelectricity in the pattern control that orchestrates both developmental and regenerative pathways. To this end, the narrative starts from the dawn of the first studies on animal electricity, reconsidering the pioneer work of Harold Saxton Burr in the light of the current achievements. We finally discuss the most recent evidence showing that bioelectric signaling is an essential component of the informational processes that control pattern specification during embryogenesis, regeneration, or even malignant transformation. We conclude that there is now mounting evidence for the existence of a Morphogenetic Code, and that deciphering this code may lead to unprecedented opportunities for the development of novel paradigms of cure in regenerative and precision medicine.
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Affiliation(s)
- Riccardo Tassinari
- National Laboratory of Molecular Biology and Stem Cell Engineering, National Institute of Biostructures and Biosystems - ELDOR LAB, Bologna 40129, Italy
| | - Claudia Cavallini
- National Laboratory of Molecular Biology and Stem Cell Engineering, National Institute of Biostructures and Biosystems - ELDOR LAB, Bologna 40129, Italy
| | - Elena Olivi
- National Laboratory of Molecular Biology and Stem Cell Engineering, National Institute of Biostructures and Biosystems - ELDOR LAB, Bologna 40129, Italy
| | - Valentina Taglioli
- National Laboratory of Molecular Biology and Stem Cell Engineering, National Institute of Biostructures and Biosystems - ELDOR LAB, Bologna 40129, Italy
| | - Chiara Zannini
- National Laboratory of Molecular Biology and Stem Cell Engineering, National Institute of Biostructures and Biosystems - ELDOR LAB, Bologna 40129, Italy
| | - Carlo Ventura
- National Laboratory of Molecular Biology and Stem Cell Engineering, National Institute of Biostructures and Biosystems - ELDOR LAB, Bologna 40129, Italy.
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13
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Abstract
Increased control of biological growth and form is an essential gateway to transformative medical advances. Repairing of birth defects, restoring lost or damaged organs, normalizing tumors, all depend on understanding how cells cooperate to make specific, functional large-scale structures. Despite advances in molecular genetics, significant gaps remain in our understanding of the meso-scale rules of morphogenesis. An engineering approach to this problem is the creation of novel synthetic living forms, greatly extending available model systems beyond evolved plant and animal lineages. Here, we review recent advances in the emerging field of synthetic morphogenesis, the bioengineering of novel multicellular living bodies. Emphasizing emergent self-organization, tissue-level guided self-assembly, and active functionality, this work is the essential next generation of synthetic biology. Aside from useful living machines for specific functions, the rational design and analysis of new, coherent anatomies will greatly increase our understanding of foundational questions in evolutionary developmental and cell biology.
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Affiliation(s)
- Mo R. Ebrahimkhani
- Department of Pathology, School of Medicine, University of Pittsburgh, A809B Scaife Hall, 3550 Terrace Street, Pittsburgh, PA 15261, USA
- Department of Bioengineering, Swanson School of Engineering, University of Pittsburgh, Pittsburgh, PA, USA
- Pittsburgh Liver Research Center, University of Pittsburgh, Pittsburgh, PA, USA
- McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA, USA
| | - Michael Levin
- Allen Discovery Center at Tufts University, 200 Boston Avenue, Suite 4600, Medford, MA 02155, USA
- Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, USA
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14
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Levin M. Bioelectrical approaches to cancer as a problem of the scaling of the cellular self. PROGRESS IN BIOPHYSICS AND MOLECULAR BIOLOGY 2021; 165:102-113. [PMID: 33961843 DOI: 10.1016/j.pbiomolbio.2021.04.007] [Citation(s) in RCA: 42] [Impact Index Per Article: 10.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/30/2020] [Revised: 04/26/2021] [Accepted: 04/29/2021] [Indexed: 10/21/2022]
Abstract
One lens with which to understand the complex phenomenon of cancer is that of developmental biology. Cancer is the inevitable consequence of a breakdown of the communication that enables individual cells to join into computational networks that work towards large-scale, morphogenetic goals instead of more primitive, unicellular objectives. This perspective suggests that cancer may be a physiological disorder, not necessarily due to problems with the genetically-specified protein hardware. One aspect of morphogenetic coordination is bioelectric signaling, and indeed an abnormal bioelectric signature non-invasively reveals the site of incipient tumors in amphibian models. Functionally, a disruption of resting potential states triggers metastatic melanoma phenotypes in embryos with no genetic defects or carcinogen exposure. Conversely, optogenetic or molecular-biological modulation of bioelectric states can override powerful oncogenic mutations and prevent or normalize tumors. The bioelectrically-mediated information flows that harness cells toward body-level anatomical outcomes represent a very attractive and tractable endogenous control system, which is being targeted by emerging approaches to cancer.
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Affiliation(s)
- Michael Levin
- Allen Discovery Center at Tufts University, 200 Boston Ave., Suite 4600, Medford, MA, 02155, USA.
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15
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Abstract
Embryogenesis, as well as regeneration, is increasingly recognized to be orchestrated by an interplay of transcriptional and bioelectric networks. Spatiotemporal patterns of resting potentials direct the size, shape, and locations of numerous organ primordia during patterning. These bioelectrical properties are established by the function of ion channels and pumps that set voltage potentials of individual cells, and gap junctions (electrical synapses) that enable physiological states to propagate across tissue networks. Functional experiments to probe the roles of bioelectrical states can be carried out by targeting endogenous ion channels during development. Here, we describe protocols, optimized for the highly tractable Xenopus laevis embryo, for molecular genetic targeting of ion channels and connexins based on CRISPR, and monitoring of resting potential states using voltage-sensing fluorescent dye. Similar strategies can be adapted to other model species.
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Affiliation(s)
- Vasilios Nanos
- Department of Biology, and Allen Discovery Center, Tufts University, Medford, MA, USA
| | - Michael Levin
- Department of Biology, and Allen Discovery Center, Tufts University, Medford, MA, USA.
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Mumford TR, Roth L, Bugaj LJ. Reverse and Forward Engineering Multicellular Structures with Optogenetics. CURRENT OPINION IN BIOMEDICAL ENGINEERING 2020; 16:61-71. [PMID: 33718689 PMCID: PMC7945718 DOI: 10.1016/j.cobme.2020.100250] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
Understanding how cells self-organize into functional higher-order structures is of great interest, both towards deciphering animal development, as well as for our ability to predictably build custom tissues to meet research and therapeutic needs. The proper organization of cells across length-scales results from interconnected and dynamic networks of molecules and cells. Optogenetic probes provide dynamic and tunable control over molecular events within cells, and thus represent a powerful approach to both dissect and control collective cell behaviors. Here we emphasize the breadth of the optogenetic toolkit and discuss how these methods have already been used to reverse-engineer the design rules of developing organisms. We also offer our perspective on the rich potential for optogenetics to power forward-engineering of tissue assembly towards the generation of bespoke tissues with user-defined properties.
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Affiliation(s)
- Thomas R. Mumford
- University of Pennsylvania, Department of Bioengineering, 240 Skirkanich Hall, 210 South 33 Street, Philadelphia, Pennsylvania, 19104, United States
| | - Lee Roth
- University of Pennsylvania, Department of Bioengineering, 240 Skirkanich Hall, 210 South 33 Street, Philadelphia, Pennsylvania, 19104, United States
| | - Lukasz J. Bugaj
- University of Pennsylvania, Department of Bioengineering, 240 Skirkanich Hall, 210 South 33 Street, Philadelphia, Pennsylvania, 19104, United States
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Life, death, and self: Fundamental questions of primitive cognition viewed through the lens of body plasticity and synthetic organisms. Biochem Biophys Res Commun 2020; 564:114-133. [PMID: 33162026 DOI: 10.1016/j.bbrc.2020.10.077] [Citation(s) in RCA: 31] [Impact Index Per Article: 6.2] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/20/2020] [Revised: 10/25/2020] [Accepted: 10/28/2020] [Indexed: 12/16/2022]
Abstract
Central to the study of cognition is being able to specify the Subject that is making decisions and owning memories and preferences. However, all real cognitive agents are made of parts (such as brains made of cells). The integration of many active subunits into a coherent Self appearing at a larger scale of organization is one of the fundamental questions of evolutionary cognitive science. Typical biological model systems, whether basal or advanced, have a static anatomical structure which obscures important aspects of the mind-body relationship. Recent advances in bioengineering now make it possible to assemble, disassemble, and recombine biological structures at the cell, organ, and whole organism levels. Regenerative biology and controlled chimerism reveal that studies of cognition in intact, "standard", evolved animal bodies are just a narrow slice of a much bigger and as-yet largely unexplored reality: the incredible plasticity of dynamic morphogenesis of biological forms that house and support diverse types of cognition. The ability to produce living organisms in novel configurations makes clear that traditional concepts, such as body, organism, genetic lineage, death, and memory are not as well-defined as commonly thought, and need considerable revision to account for the possible spectrum of living entities. Here, I review fascinating examples of experimental biology illustrating that the boundaries demarcating somatic and cognitive Selves are fluid, providing an opportunity to sharpen inquiries about how evolution exploits physical forces for multi-scale cognition. Developmental (pre-neural) bioelectricity contributes a novel perspective on how the dynamic control of growth and form of the body evolved into sophisticated cognitive capabilities. Most importantly, the development of functional biobots - synthetic living machines with behavioral capacity - provides a roadmap for greatly expanding our understanding of the origin and capacities of cognition in all of its possible material implementations, especially those that emerge de novo, with no lengthy evolutionary history of matching behavioral programs to bodyplan. Viewing fundamental questions through the lens of new, constructed living forms will have diverse impacts, not only in basic evolutionary biology and cognitive science, but also in regenerative medicine of the brain and in artificial intelligence.
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18
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19
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Gheorghiu M, Stanica L, Polonschii C, David S, Ruckenstein A, Popescu O, Badea T, Gheorghiu E. Modulation of Cellular Reactivity for Enhanced Cell-Based Biosensing. Anal Chem 2020; 92:806-814. [PMID: 31751507 PMCID: PMC7641241 DOI: 10.1021/acs.analchem.9b03217] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
Cell-based sensing platforms provide functional information on cellular effects of bioactive or toxic compounds in a sample. Current challenges concern the rather extended length of the assays as well as their limited reproducibility and sensitivity. We present a biosensing method capable of appraising, on a short time scale and with exquisite sensitivity, the occurrence and the magnitude of cellular alterations induced by low levels of a bioactive/toxic compound. Our method is based on integrating optogenetic control of non-electrogenic human cells, modified to express light sensitive protein channels, into a non-invasive electro-optical analytical platform enabling quantitative assessment of the stimulus dependent, dynamical cellular response. Our system exploits the interplay between optogenetic stimulation and time lapse fast impedance assays in boosting the platform sensitivity when exposing cells to a model exogenous stimulus, under both static and flow conditions. The proposed optogenetically modulated cell-based sensing platform is suitable for in field applications and provides a new paradigm for impedance-based sensing.
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Affiliation(s)
- Mihaela Gheorghiu
- International Centre of Biodynamics, Intrarea Portocalelor 1 B, 060101 Bucharest, Romania
- Faculty of Biology, University of Bucharest, 91-95 Splaiul Independentei, Bucharest 050095, Romania
| | - Luciana Stanica
- International Centre of Biodynamics, Intrarea Portocalelor 1 B, 060101 Bucharest, Romania
- Faculty of Biology, University of Bucharest, 91-95 Splaiul Independentei, Bucharest 050095, Romania
| | - Cristina Polonschii
- International Centre of Biodynamics, Intrarea Portocalelor 1 B, 060101 Bucharest, Romania
| | - Sorin David
- International Centre of Biodynamics, Intrarea Portocalelor 1 B, 060101 Bucharest, Romania
| | - Andrei Ruckenstein
- Department of Physics, Boston University, 590 Commonwealth Avenue, Boston, Massachusetts 02215, United States
| | - Octavian Popescu
- Institute for Interdisciplinary Research in Bio-Nano-Sciences, Molecular Biology Center, Babes-Bolyai-University, 400084 Cluj-Napoca, Romania
- Institute of Biology Bucharest, Romanian Academy, 296 Splaiul Independentei, 060031 Bucharest, Romania
| | - Tudor Badea
- Retinal Circuit Development & Genetics Unit, Neurobiology Neurodegeneration & Repair Laboratory, National Eye Institute of the National Institutes of Health (N-NRL/NEI/NIH), 6 Center Drive, Bethesda 20892, Maryland, United States
| | - Eugen Gheorghiu
- International Centre of Biodynamics, Intrarea Portocalelor 1 B, 060101 Bucharest, Romania
- Faculty of Biology, University of Bucharest, 91-95 Splaiul Independentei, Bucharest 050095, Romania
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20
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Levin M, Selberg J, Rolandi M. Endogenous Bioelectrics in Development, Cancer, and Regeneration: Drugs and Bioelectronic Devices as Electroceuticals for Regenerative Medicine. iScience 2019; 22:519-533. [PMID: 31837520 PMCID: PMC6920204 DOI: 10.1016/j.isci.2019.11.023] [Citation(s) in RCA: 35] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/28/2019] [Revised: 10/15/2019] [Accepted: 11/12/2019] [Indexed: 12/21/2022] Open
Abstract
A major frontier in the post-genomic era is the investigation of the control of coordinated growth and three-dimensional form. Dynamic remodeling of complex organs in regulative embryogenesis, regeneration, and cancer reveals that cells and tissues make decisions that implement complex anatomical outcomes. It is now essential to understand not only the genetics that specifies cellular hardware but also the physiological software that implements tissue-level plasticity and robust morphogenesis. Here, we review recent discoveries about the endogenous mechanisms of bioelectrical communication among non-neural cells that enables them to cooperate in vivo. We discuss important advances in bioelectronics, as well as computational and pharmacological tools that are enabling the taming of biophysical controls toward applications in regenerative medicine and synthetic bioengineering.
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Affiliation(s)
- Michael Levin
- Allen Discovery Center at Tufts University, Medford, MA 02155, USA.
| | - John Selberg
- Electrical and Computer Engineering Department, University of California, Santa Cruz, CA 95064, USA
| | - Marco Rolandi
- Electrical and Computer Engineering Department, University of California, Santa Cruz, CA 95064, USA
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21
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Pai VP, Adams DS. Preventing Ethanol-Induced Brain and Eye Morphology Defects Using Optogenetics. Bioelectricity 2019; 1:260-272. [PMID: 32685918 DOI: 10.1089/bioe.2019.0008] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
Background: Embryonic exposure to the teratogen ethanol leads to dysmorphias, including eye and brain morphology defects associated with fetal alcohol spectrum disorder (FASD). Exposure of Xenopus laevis embryos to ethanol leads to similar developmental defects, including brain and eye dysmorphism, confirming our work and the work of others showing Xenopus as a useful system for studies of the brain and eye birth defects associated with FASD. Several targets of ethanol action have been hypothesized, one being regulation of Kir2.1 potassium channel. Endogenous ion fluxes and membrane voltage variation (bioelectric signals) have been shown to be powerful regulators of embryonic cell behaviors that are required for correct brain and eye morphology. Disruptions to these voltage patterns lead to spatially correlated disruptions in gene expression patterns and corresponding morphology. Materials and Methods: Here, we use controlled membrane voltage modulation to determine when and where voltage modulation is sufficient to rescue ethanol-induced brain and eye defects in Xenopus embryos. Results: We found (1) that modulating membrane voltage using light activation of the channelrhodopsin-2 variant D156A rescues ethanol exposed embryos, resulting in normal brain and eye morphologies; (2) hyperpolarization is required for the full duration of ethanol exposure; (3) hyperpolarization of only superficial ectoderm is sufficient for this effect; and(4) the rescue effect acts at a distance. Conclusions: These results, particularly the last, raise the exciting possibility of using bioelectric modulation to treat ethanol-induced brain and eye birth defects, possibly with extant ion channel drugs already prescribed to pregnant women. This may prove to be a simple and cost-effective strategy for reducing the impact of FASD.
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Affiliation(s)
- Vaibhav P Pai
- Department of Biology, Tufts Center for Regenerative and Developmental Biology, Tufts University, Medford, Massachusetts
| | - Dany Spencer Adams
- Department of Biology, Tufts University, Medford, Massachusetts.,Ion Diagnostics LLC, Watertown, Massachusetts
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22
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Mondal P, Krishnamurthy VV, Sharum SR, Haack N, Zhou H, Cheng J, Yang J, Zhang K. Repurposing Protein Degradation for Optogenetic Modulation of Protein Activities. ACS Synth Biol 2019; 8:2585-2592. [PMID: 31600062 DOI: 10.1021/acssynbio.9b00285] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
Non-neuronal optogenetic approaches empower precise regulation of protein dynamics in live cells but often require target-specific protein engineering. To address this challenge, we developed a generalizable light-modulated protein stabilization system (GLIMPSe) to control the intracellular protein level independent of its functionality. We applied GLIMPSe to control two distinct classes of proteins: mitogen-activated protein kinase phosphatase 3 (MKP3), a negative regulator of the extracellular signal-regulated kinase (ERK) pathway, and a constitutively active form of MEK (CA MEK), a positive regulator of the same pathway. Kinetics study showed that light-induced protein stabilization could be achieved within 30 min of blue light stimulation. GLIMPSe enables target-independent optogenetic control of protein activities and therefore minimizes the systematic variation embedded within different photoactivatable proteins. Overall, GLIMPSe promises to achieve light-mediated post-translational stabilization of a wide array of target proteins in live cells.
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Affiliation(s)
| | | | | | | | | | | | - Jing Yang
- Department of Comparative Biosciences, University of Illinois at Urbana−Champaign, 2001 S Lincoln Avenue, Urbana, Illinois 61802, United States
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23
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Toward Decoding Bioelectric Events in Xenopus Embryogenesis: New Methodology for Tracking Interplay Between Calcium and Resting Potentials In Vivo. J Mol Biol 2019; 432:605-620. [PMID: 31711960 DOI: 10.1016/j.jmb.2019.10.029] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/24/2019] [Revised: 10/07/2019] [Accepted: 10/15/2019] [Indexed: 12/16/2022]
Abstract
Although chemical signaling during embryogenesis is readily addressed by a plethora of available techniques, the developmental functions of ionic signaling are still poorly understood. It is increasingly realized that bioelectric events in nonneural cells are critical for pattern regulation, but their study has been hampered by difficulties in monitoring and manipulating them in vivo. Recent developments in visualizing electrical signaling dynamics in the field of neuroscience have facilitated functional experiments that reveal instructive developmental bioelectric signals. However, there is a pressing need for additional tools to explore time-dependent ionic signaling to understand complex endogenous dynamics. Here, we present methodological advances, including 4D imaging and data analysis, for improved tracking of calcium flux in the Xenopus laevis embryo, lowering the barrier for in vivo physiology work in this important model system. Using these techniques, we investigated the relationship between bioelectric ion channel activity and calcium, finding that cell hyperpolarization and depolarization both induce persistent static elevation of cytoplasmic calcium levels that fade over developmental time. These calcium changes correlate with increased cell mobility in early embryos and abnormal craniofacial morphology in later embryos. We thus highlight membrane potential modulation as a tractable tool for modulation of signaling cascades that rely on calcium as a transduction mechanism. The methods we describe facilitate the study of important novel aspects of developmental physiology, are extendable to numerous classes of existing and forthcoming fluorescent physiological reporters, and establish highly accessible, inexpensive protocols for their investigation.
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Tuszynski J, Tilli TM, Levin M. Ion Channel and Neurotransmitter Modulators as Electroceutical Approaches to the Control of Cancer. Curr Pharm Des 2019; 23:4827-4841. [PMID: 28554310 PMCID: PMC6340161 DOI: 10.2174/1381612823666170530105837] [Citation(s) in RCA: 29] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/13/2017] [Revised: 05/17/2017] [Accepted: 05/23/2017] [Indexed: 11/22/2022]
Abstract
The activities of individual cells must be tightly coordinated in order to build and maintain complex 3-dimensional body structures during embryogenesis and regeneration. Thus, one way to view cancer is within systems biology as a network disorder affecting the ability of cells to properly interact with a morphodynamic field of instructive signals that keeps proliferation and migration orchestrated toward the anatomical needs of the host or-ganism. One layer of this set of instructive microenvironmental cues is bioelectrical. Voltage gradients among all somatic cells (not just excitable nerve and muscle) control cell behavior, and the ionic coupling of cells into networks via electrochemical synapses allows them to implement tissue-level patterning decisions. These gradients have been increasingly impli-cated in the induction and suppression of tumorigenesis and metastasis, in the emerging links between developmental bioelectricity to the cancer problem. Consistent with the well-known role of neurotransmitter molecules in transducing electrical activity to downstream cascades in the brain, serotonergic signaling has likewise been implicated in cancer. Here, we review these recent data and propose new approaches for manipulating bioelectric and neurotransmitter pathways in cancer biology based on a bioelectric view of cancer. To sup-port this methodology, we present new data on the effects of the SSRI Prozac and its analog (ZINC ID = ZINC06811610) on survival of both cancer (MCF7) and normal (MCF10A) breast cells exposed to these compounds. We found an IC50 concentration (25 μM for Pro-zac and 100 μM for the Prozac analog) at which these compounds inhibited tumor cell sur-vival and proliferation. Additionally, at these concentrations, we did not observe alterations in a non-tumoral cell line. This constitutes a proof-of-concept demonstration for our hy-pothesis that the use of both existing and novel drugs as electroceuticals could serve as an alternative to highly toxic chemotherapy strategies replacing or augmenting them with less toxic alternatives. We believe this new approach forms an exciting roadmap for future bio-medical advances.
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Affiliation(s)
- Jack Tuszynski
- Department of Oncology, Faculty of Medicine & Dentistry, University of Alberta, Edmonton, Alberta. Canada
| | - Tatiana M Tilli
- Laboratory of Biological System Modeling, National Institute for Science and Technology on Innovation in Neglected Diseases (INCT/IDN), Center for Technological Development in Health (CDTS), Oswaldo Cruz Foundation (Fiocruz), Rio de Janeiro. Brazil
| | - Michael Levin
- Biology Department, and Allen Discovery Center, Tufts University, Medford, MA, 02155. United States
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25
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Levin M, Pietak AM, Bischof J. Planarian regeneration as a model of anatomical homeostasis: Recent progress in biophysical and computational approaches. Semin Cell Dev Biol 2019; 87:125-144. [PMID: 29635019 PMCID: PMC6234102 DOI: 10.1016/j.semcdb.2018.04.003] [Citation(s) in RCA: 45] [Impact Index Per Article: 7.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/05/2018] [Revised: 04/03/2018] [Accepted: 04/06/2018] [Indexed: 12/22/2022]
Abstract
Planarian behavior, physiology, and pattern control offer profound lessons for regenerative medicine, evolutionary biology, morphogenetic engineering, robotics, and unconventional computation. Despite recent advances in the molecular genetics of stem cell differentiation, this model organism's remarkable anatomical homeostasis provokes us with truly fundamental puzzles about the origin of large-scale shape and its relationship to the genome. In this review article, we first highlight several deep mysteries about planarian regeneration in the context of the current paradigm in this field. We then review recent progress in understanding of the physiological control of an endogenous, bioelectric pattern memory that guides regeneration, and how modulating this memory can permanently alter the flatworm's target morphology. Finally, we focus on computational approaches that complement reductive pathway analysis with synthetic, systems-level understanding of morphological decision-making. We analyze existing models of planarian pattern control and highlight recent successes and remaining knowledge gaps in this interdisciplinary frontier field.
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Affiliation(s)
- Michael Levin
- Allen Discovery Center at Tufts University, Medford, MA 02155, United States; Biology Department, Tufts University, Medford, MA 02155, United States.
| | - Alexis M Pietak
- Allen Discovery Center at Tufts University, Medford, MA 02155, United States
| | - Johanna Bischof
- Allen Discovery Center at Tufts University, Medford, MA 02155, United States; Biology Department, Tufts University, Medford, MA 02155, United States
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Ferenc NN, Levin M. Effects of Ivermectin Exposure on Regeneration of D. dorotocephala Planaria: Exploiting Human-Approved Ion Channel Drugs as Morphoceuticals. Macromol Biosci 2018; 19:e1800237. [PMID: 30485697 DOI: 10.1002/mabi.201800237] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/22/2018] [Revised: 10/22/2018] [Indexed: 01/19/2023]
Abstract
Transformative applications in regenerative medicine await increased control of processes implementing repair and remodeling of complex living structures. Recent work reveals ion channel drugs as a powerful toolkit for modulating endogenous bioelectric circuits that control growth and form in vivo and in vitro. It is therefore especially important to develop assays in model systems that will enable the testing of these "morphoceuticals"-compounds with predictable effects on anatomical structure. The regenerative planaria are an ideal model system for this purpose. Several studies have shown a role for bioelectric signaling in planarian regeneration, but these have focused on Dugesia japonica and Schmidtea mediterranea. It is not known how the alterations of ion channel activity would affect regeneration in other species of planaria-an important aspect of building robust computational models of bioelectric circuits. Here, the effect of ivermectin (IVM), a chloride channel opener drug commonly used to combat heartworm is tested, on regeneration in a new species of planaria: Dugesia dorotocephala. Exposure to IVM during regeneration results in patterning abnormalities, such as bifurcated tails with partial heads, as well as delayed regeneration. These data extend our understanding of the effects of human-approved ion channel drugs on regenerative processes.
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Affiliation(s)
- Nina N Ferenc
- Chemistry Department, Westfield High School, Chantilly, VA, 20151, USA
| | - Michael Levin
- Allen Discovery Center at Tufts University, 200 Boston Ave., Medford, MA, 02155, USA
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27
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Bioelectrical coupling in multicellular domains regulated by gap junctions: A conceptual approach. Bioelectrochemistry 2018; 123:45-61. [DOI: 10.1016/j.bioelechem.2018.04.013] [Citation(s) in RCA: 43] [Impact Index Per Article: 6.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/06/2018] [Revised: 04/13/2018] [Accepted: 04/17/2018] [Indexed: 12/16/2022]
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28
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Pietak A, Levin M. Bioelectrical control of positional information in development and regeneration: A review of conceptual and computational advances. PROGRESS IN BIOPHYSICS AND MOLECULAR BIOLOGY 2018; 137:52-68. [PMID: 29626560 PMCID: PMC10464501 DOI: 10.1016/j.pbiomolbio.2018.03.008] [Citation(s) in RCA: 29] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/10/2018] [Revised: 03/23/2018] [Accepted: 03/26/2018] [Indexed: 12/16/2022]
Abstract
Positional information describes pre-patterns of morphogenetic substances that alter spatio-temporal gene expression to instruct development of growth and form. A wealth of recent data indicate bioelectrical properties, such as the transmembrane potential (Vmem), are involved as instructive signals in the spatiotemporal regulation of morphogenesis. However, the mechanistic relationships between Vmem and molecular positional information are only beginning to be understood. Recent advances in computational modeling are assisting in the development of comprehensive frameworks for mechanistically understanding how endogenous bioelectricity can guide anatomy in a broad range of systems. Vmem represents an extraordinarily strong electric field (∼1.0 × 106 V/m) active over the thin expanse of the plasma membrane, with the capacity to influence a variety of downstream molecular signaling cascades. Moreover, in multicellular networks, intercellular coupling facilitated by gap junction channels may induce directed, electrodiffusive transport of charged molecules between cells of the network to generate new positional information patterning possibilities and characteristics. Given the demonstrated role of Vmem in morphogenesis, here we review current understanding of how Vmem can integrate with molecular regulatory networks to control single cell state, and the unique properties bioelectricity adds to transport phenomena in gap junction-coupled cell networks to facilitate self-assembly of morphogen gradients and other patterns. Understanding how Vmem integrates with biochemical regulatory networks at the level of a single cell, and mechanisms through which Vmem shapes molecular positional information in multicellular networks, are essential for a deep understanding of body plan control in development, regeneration and disease.
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Affiliation(s)
| | - Michael Levin
- Allen Discovery Center at Tufts, USA; Center for Regenerative and Developmental Biology, Tufts University, Medford, MA, USA
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29
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Sullivan KG, Levin M. Inverse Drug Screening of Bioelectric Signaling and Neurotransmitter Roles: Illustrated Using a Xenopus Tail Regeneration Assay. Cold Spring Harb Protoc 2018; 2018:pdb.prot099937. [PMID: 29437995 DOI: 10.1101/pdb.prot099937] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
Abstract
Xenopus embryos and larvae are an ideal model system in which to study the interplay between genetics, physiology, and anatomy in the control of structure and function. An important emerging field is the study of bioelectric signaling, the exchange of ion- and neurotransmitter-mediated messages among all types of cells (not just nerve and muscle cells), in the regulation of growth and form during embryogenesis, regeneration, and cancer. To facilitate the mechanistic investigation of bioelectric events in vivo, it is necessary to identify the endogenous signaling machinery involved in any patterning process of interest. This protocol uses the tail regeneration assay in Xenopus to perform an inverse drug screen; tiers of known compounds are used to probe the involvement of increasingly specific classes of bioelectric and neurotransmitter machinery. By using a hierarchical approach, large classes of targets are ruled out in early rounds, focusing attention on progressively narrower sets of proteins. Such a screen avoids many of the limitations of a molecular-genetic targeting approach and provides a rapid and efficient way to focus on specific targets. Usually, <10 experiments are needed to determine whether bioelectrics and/or neurotransmitter signaling are involved in the process of interest. This protocol describes the strategy in the context of a semiquantitative analysis of tail regeneration but can be applied to any assay in Xenopus or other small aquatic model system (e.g., zebrafish). Given the ever-increasing toolkit of chemical genetics, such screens represent a powerful and versatile methodology for probing the physiological circuits underlying pattern regulation.
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Affiliation(s)
- Kelly G Sullivan
- Biology Department, and Allen Discovery Center at Tufts University, Medford, Massachusetts 02155
| | - Michael Levin
- Biology Department, and Allen Discovery Center at Tufts University, Medford, Massachusetts 02155
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Levin M, Martyniuk CJ. The bioelectric code: An ancient computational medium for dynamic control of growth and form. Biosystems 2018; 164:76-93. [PMID: 28855098 PMCID: PMC10464596 DOI: 10.1016/j.biosystems.2017.08.009] [Citation(s) in RCA: 120] [Impact Index Per Article: 17.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/14/2017] [Revised: 08/20/2017] [Accepted: 08/22/2017] [Indexed: 12/19/2022]
Abstract
What determines large-scale anatomy? DNA does not directly specify geometrical arrangements of tissues and organs, and a process of encoding and decoding for morphogenesis is required. Moreover, many species can regenerate and remodel their structure despite drastic injury. The ability to obtain the correct target morphology from a diversity of initial conditions reveals that the morphogenetic code implements a rich system of pattern-homeostatic processes. Here, we describe an important mechanism by which cellular networks implement pattern regulation and plasticity: bioelectricity. All cells, not only nerves and muscles, produce and sense electrical signals; in vivo, these processes form bioelectric circuits that harness individual cell behaviors toward specific anatomical endpoints. We review emerging progress in reading and re-writing anatomical information encoded in bioelectrical states, and discuss the approaches to this problem from the perspectives of information theory, dynamical systems, and computational neuroscience. Cracking the bioelectric code will enable much-improved control over biological patterning, advancing basic evolutionary developmental biology as well as enabling numerous applications in regenerative medicine and synthetic bioengineering.
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Affiliation(s)
- Michael Levin
- Allen Discovery Center at Tufts University, Biology Department, Tufts University, 200 Boston Avenue, Suite 4600 Medford, MA 02155, USA.
| | - Christopher J Martyniuk
- Department of Physiological Sciences and Center for Environmental and Human Toxicology, University of Florida Genetics Institute, Interdisciplinary Program in Biomedical Sciences Neuroscience, College of Veterinary Medicine, University of Florida, Gainesville, FL, 32611, USA
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31
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Use of genetically encoded, light-gated ion translocators to control tumorigenesis. Oncotarget 2017; 7:19575-88. [PMID: 26988909 PMCID: PMC4991402 DOI: 10.18632/oncotarget.8036] [Citation(s) in RCA: 77] [Impact Index Per Article: 9.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/22/2015] [Accepted: 02/11/2016] [Indexed: 01/01/2023] Open
Abstract
It has long been known that the resting potential of tumor cells is depolarized relative to their normal counterparts. More recent work has provided evidence that resting potential is not just a readout of cell state: it regulates cell behavior as well. Thus, the ability to control resting potential in vivo would provide a powerful new tool for the study and treatment of tumors, a tool capable of revealing living-state physiological information impossible to obtain using molecular tools applied to isolated cell components. Here we describe the first use of optogenetics to manipulate ion-flux mediated regulation of membrane potential specifically to prevent and cause regression of oncogene-induced tumors. Injection of mutant-KRAS mRNA induces tumor-like structures with many documented similarities to tumors, in Xenopus tadpoles. We show that expression and activation of either ChR2D156A, a blue-light activated cation channel, or Arch, a green-light activated proton pump, both of which hyperpolarize cells, significantly lowers the incidence of KRAS tumor formation. Excitingly, we also demonstrate that activation of co-expressed light-activated ion translocators after tumor formation significantly increases the frequency with which the tumors regress in a process called normalization. These data demonstrate an optogenetic approach to dissect the biophysics of cancer. Moreover, they provide proof-of-principle for a novel class of interventions, directed at regulating cell state by targeting physiological regulators that can over-ride the presence of mutations.
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Fields C, Levin M. Multiscale memory and bioelectric error correction in the cytoplasm-cytoskeleton-membrane system. WILEY INTERDISCIPLINARY REVIEWS-SYSTEMS BIOLOGY AND MEDICINE 2017; 10. [DOI: 10.1002/wsbm.1410] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/21/2017] [Revised: 08/19/2017] [Accepted: 10/04/2017] [Indexed: 12/12/2022]
Affiliation(s)
- Chris Fields
- 21 Rue des Lavandiéres, 11160 Caunes Minervois; France
| | - Michael Levin
- Allen Discovery Center at Tufts University; Medford MA USA
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Zahn N, Levin M, Adams DS. The Zahn drawings: new illustrations of Xenopus embryo and tadpole stages for studies of craniofacial development. Development 2017; 144:2708-2713. [PMID: 28765211 PMCID: PMC5560046 DOI: 10.1242/dev.151308] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/23/2023]
Abstract
The embryos and tadpoles of the frog Xenopus are increasingly important subjects for studies of the development of the head and face - studies that are providing novel and crucial insight into the causes and prevention of a suite of devastating birth defects, as well as basic evolutionary and developmental biology. However, many studies are conducted on a range of embryonic stages that are not fully represented in the beloved Xenopus resource, Nieuwkoop and Faber's classic Normal Table of Xenopus laevis (Daudin) The lack of standardized images at these stages acts as a barrier to the efficient and accurate representation and communication of experimental methodology and expression data. To fill this gap, we have created 27 new high-quality illustrations. Like their oft-used predecessors from Nieuwkoop and Faber, these drawings can be freely downloaded and used, and will, we hope, serve as an essential resource for this important model system.
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Affiliation(s)
| | - Michael Levin
- Department of Biology and Tufts Center for Regenerative and Developmental Biology, Tufts University, Medford, MA 02155, USA.,Allen Discovery Center at Tufts University, Medford, MA 02155, USA
| | - Dany Spencer Adams
- Department of Biology and Tufts Center for Regenerative and Developmental Biology, Tufts University, Medford, MA 02155, USA
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Herrera-Rincon C, Pai VP, Moran KM, Lemire JM, Levin M. The brain is required for normal muscle and nerve patterning during early Xenopus development. Nat Commun 2017; 8:587. [PMID: 28943634 PMCID: PMC5610959 DOI: 10.1038/s41467-017-00597-2] [Citation(s) in RCA: 30] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/05/2017] [Accepted: 07/10/2017] [Indexed: 01/20/2023] Open
Abstract
Possible roles of brain-derived signals in the regulation of embryogenesis are unknown. Here we use an amputation assay in Xenopus laevis to show that absence of brain alters subsequent muscle and peripheral nerve patterning during early development. The muscle phenotype can be rescued by an antagonist of muscarinic acetylcholine receptors. The observed defects occur at considerable distances from the head, suggesting that the brain provides long-range cues for other tissue systems during development. The presence of brain also protects embryos from otherwise-teratogenic agents. Overexpression of a hyperpolarization-activated cyclic nucleotide-gated ion channel rescues the muscle phenotype and the neural mispatterning that occur in brainless embryos, even when expressed far from the muscle or neural cells that mispattern. We identify a previously undescribed developmental role for the brain and reveal a non-local input into the control of early morphogenesis that is mediated by neurotransmitters and ion channel activity.Functions of the embryonic brain prior to regulating behavior are unclear. Here, the authors use an amputation assay in Xenopus laevis to demonstrate that removal of the brain early in development alters muscle and peripheral nerve patterning, which can be rescued by modulating bioelectric signals.
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Affiliation(s)
- Celia Herrera-Rincon
- Biology Department and Allen Discovery Center, Tufts University, 200 Boston Avenue, suite 4600, Medford, MA, 02155-4243, USA
| | - Vaibhav P Pai
- Biology Department and Allen Discovery Center, Tufts University, 200 Boston Avenue, suite 4600, Medford, MA, 02155-4243, USA
| | - Kristine M Moran
- Biology Department and Allen Discovery Center, Tufts University, 200 Boston Avenue, suite 4600, Medford, MA, 02155-4243, USA
| | - Joan M Lemire
- Biology Department and Allen Discovery Center, Tufts University, 200 Boston Avenue, suite 4600, Medford, MA, 02155-4243, USA
| | - Michael Levin
- Biology Department and Allen Discovery Center, Tufts University, 200 Boston Avenue, suite 4600, Medford, MA, 02155-4243, USA.
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Pietak A, Levin M. Bioelectric gene and reaction networks: computational modelling of genetic, biochemical and bioelectrical dynamics in pattern regulation. J R Soc Interface 2017; 14:20170425. [PMID: 28954851 PMCID: PMC5636277 DOI: 10.1098/rsif.2017.0425] [Citation(s) in RCA: 56] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/08/2017] [Accepted: 08/31/2017] [Indexed: 12/17/2022] Open
Abstract
Gene regulatory networks (GRNs) describe interactions between gene products and transcription factors that control gene expression. In combination with reaction-diffusion models, GRNs have enhanced comprehension of biological pattern formation. However, although it is well known that biological systems exploit an interplay of genetic and physical mechanisms, instructive factors such as transmembrane potential (Vmem) have not been integrated into full GRN models. Here we extend regulatory networks to include bioelectric signalling, developing a novel synthesis: the bioelectricity-integrated gene and reaction (BIGR) network. Using in silico simulations, we highlight the capacity for Vmem to alter steady-state concentrations of key signalling molecules inside and out of cells. We characterize fundamental feedbacks where Vmem both controls, and is in turn regulated by, biochemical signals and thereby demonstrate Vmem homeostatic control, Vmem memory and Vmem controlled state switching. BIGR networks demonstrating hysteresis are identified as a mechanisms through which more complex patterns of stable Vmem spots and stripes, along with correlated concentration patterns, can spontaneously emerge. As further proof of principle, we present and analyse a BIGR network model that mechanistically explains key aspects of the remarkable regenerative powers of creatures such as planarian flatworms. The functional properties of BIGR networks generate the first testable, quantitative hypotheses for biophysical mechanisms underlying the stability and adaptive regulation of anatomical bioelectric pattern.
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Affiliation(s)
- Alexis Pietak
- Allen Discovery Center, Tufts University, Medford, MA, USA
| | - Michael Levin
- Allen Discovery Center, Tufts University, Medford, MA, USA
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Jayaram DT, Luo Q, Thourson SB, Finlay AH, Payne CK. Controlling the Resting Membrane Potential of Cells with Conducting Polymer Microwires. SMALL (WEINHEIM AN DER BERGSTRASSE, GERMANY) 2017; 13:10.1002/smll.201700789. [PMID: 28556571 PMCID: PMC5560653 DOI: 10.1002/smll.201700789] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/09/2017] [Revised: 04/11/2017] [Indexed: 05/11/2023]
Abstract
All cells have a resting membrane potential resulting from an ion gradient across the plasma membrane. The resting membrane potential of cells is tightly coupled to regeneration and differentiation. The ability to control this parameter provides the opportunity for both biomedical advances and the probing of fundamental bioelectric pathways. The use of poly(3,4-ethylenedioxythiophene): polystyrene sulfonate (PEDOT:PSS) conducting polymer microwires to depolarize cells is tested using E. coli cells loaded with a fluorescent dye that is pumped out of the cells in response to depolarization; a more positive membrane potential. Fluorescence imaging of the cells in response to a conducting-polymer-microwire applied voltage confirms depolarization and shows that the rate of depolarization is a function of the applied voltage and frequency. Microwire activity does not damage the cells, demonstrated with a propidium iodide assay of membrane integrity. The conducting polymer microwires do not penetrate the cell, or even come into contact with the cell; they only need to generate a minimum electric field, controlled by the placement of the wires. It is expected that these microwires will provide a new, noninvasive, cellular-scale tool for the control of resting membrane potential with high spatial precision.
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Affiliation(s)
- Dhanya T Jayaram
- School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA, 30332, USA
| | - Qingjie Luo
- School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA, 30332, USA
| | - Scott B Thourson
- Interdisciplinary Program in BioEngineering and George W. Woodruff School of Mechanical Engineering, Georgia Institute of Technology, Atlanta, GA, 30332, USA
| | - Adam H Finlay
- School of Chemical and Biomolecular Engineering, Georgia Institute of Technology, Atlanta, GA, 30332, USA
| | - Christine K Payne
- School of Chemistry and Biochemistry and Petit Institute for Bioengineering and Biosciences, Georgia Institute of Technology, Atlanta, GA, 30332, USA
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Levin M, Pezzulo G, Finkelstein JM. Endogenous Bioelectric Signaling Networks: Exploiting Voltage Gradients for Control of Growth and Form. Annu Rev Biomed Eng 2017; 19:353-387. [PMID: 28633567 PMCID: PMC10478168 DOI: 10.1146/annurev-bioeng-071114-040647] [Citation(s) in RCA: 161] [Impact Index Per Article: 20.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/16/2023]
Abstract
Living systems exhibit remarkable abilities to self-assemble, regenerate, and remodel complex shapes. How cellular networks construct and repair specific anatomical outcomes is an open question at the heart of the next-generation science of bioengineering. Developmental bioelectricity is an exciting emerging discipline that exploits endogenous bioelectric signaling among many cell types to regulate pattern formation. We provide a brief overview of this field, review recent data in which bioelectricity is used to control patterning in a range of model systems, and describe the molecular tools being used to probe the role of bioelectrics in the dynamic control of complex anatomy. We suggest that quantitative strategies recently developed to infer semantic content and information processing from ionic activity in the brain might provide important clues to cracking the bioelectric code. Gaining control of the mechanisms by which large-scale shape is regulated in vivo will drive transformative advances in bioengineering, regenerative medicine, and synthetic morphology, and could be used to therapeutically address birth defects, traumatic injury, and cancer.
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Affiliation(s)
- Michael Levin
- Biology Department, Tufts University, Medford, Massachusetts 02155-4243;
- Allen Discovery Center, Tufts University, Medford, Massachusetts 02155;
| | - Giovanni Pezzulo
- Institute of Cognitive Sciences and Technologies, National Research Council, Rome 00185, Italy;
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Paré JF, Martyniuk CJ, Levin M. Bioelectric regulation of innate immune system function in regenerating and intact Xenopus laevis. NPJ Regen Med 2017; 2:15. [PMID: 29302351 PMCID: PMC5677984 DOI: 10.1038/s41536-017-0019-y] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/19/2016] [Revised: 02/27/2017] [Accepted: 04/02/2017] [Indexed: 02/07/2023] Open
Abstract
Two key inputs that regulate regeneration are the function of the immune system, and spatial gradients of transmembrane potential (Vmem). Endogenous bioelectric signaling in somatic tissues during regenerative patterning is beginning to be understood, but its role in the context of immune response has never been investigated. Here, we show that Vmem levels modulate innate immunity activity in Xenopus laevis embryos. We developed an assay in which X. laevis embryos are infected with a uropathogenic microorganism, in the presence or absence of reagents that modify Vmem, prior to the ontogenesis of the adaptive immune system. General depolarization of the organism's Vmem by pharmacological or molecular genetic (ion channel misexpression) methods increased resistance to infection, while hyperpolarization made the embryos more susceptible to death by infection. Hyperpolarized specimens harbored a higher load of infectious microorganisms when compared to controls. We identified two mechanisms by which Vmem mediates immune function: serotonergic signaling involving melanocytes and an increase in the number of primitive myeloid cells. Bioinformatics analysis of genes whose transcription is altered by depolarization revealed a number of immune system targets consistent with mammalian data. Remarkably, amputation of the tail bud potentiates systemic resistance to infection by increasing the number of peripheral myeloid cells, revealing an interplay of regenerative response, innate immunity, and bioelectric regulation. Our study identifies bioelectricity as a new mechanism by which innate immune response can be regulated in the context of infection or regeneration. Vmem modulation using drugs already approved for human use could be exploited to improve resistance to infections in clinical settings.
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Affiliation(s)
- Jean-François Paré
- Biology Department, and Allen Discovery Center at Tufts, Tufts University, Medford, MA USA
| | - Christopher J. Martyniuk
- Center for Environmental and Human Toxicology and Department of Physiological Sciences, University of Florida Genetics Institute, College of Veterinary Medicine, University of Florida, Gainesville, FL USA
| | - Michael Levin
- Biology Department, and Allen Discovery Center at Tufts, Tufts University, Medford, MA USA
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Durant F, Morokuma J, Fields C, Williams K, Adams DS, Levin M. Long-Term, Stochastic Editing of Regenerative Anatomy via Targeting Endogenous Bioelectric Gradients. Biophys J 2017; 112:2231-2243. [PMID: 28538159 PMCID: PMC5443973 DOI: 10.1016/j.bpj.2017.04.011] [Citation(s) in RCA: 92] [Impact Index Per Article: 11.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/20/2017] [Revised: 03/30/2017] [Accepted: 04/14/2017] [Indexed: 12/22/2022] Open
Abstract
We show that regenerating planarians' normal anterior-posterior pattern can be permanently rewritten by a brief perturbation of endogenous bioelectrical networks. Temporary modulation of regenerative bioelectric dynamics in amputated trunk fragments of planaria stochastically results in a constant ratio of regenerates with two heads to regenerates with normal morphology. Remarkably, this is shown to be due not to partial penetrance of treatment, but a profound yet hidden alteration to the animals' patterning circuitry. Subsequent amputations of the morphologically normal regenerates in water result in the same ratio of double-headed to normal morphology, revealing a cryptic phenotype that is not apparent unless the animals are cut. These animals do not differ from wild-type worms in histology, expression of key polarity genes, or neoblast distribution. Instead, the altered regenerative bodyplan is stored in seemingly normal planaria via global patterns of cellular resting potential. This gradient is functionally instructive, and represents a multistable, epigenetic anatomical switch: experimental reversals of bioelectric state reset subsequent regenerative morphology back to wild-type. Hence, bioelectric properties can stably override genome-default target morphology, and provide a tractable control point for investigating cryptic phenotypes and the stochasticity of large-scale epigenetic controls.
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Affiliation(s)
- Fallon Durant
- Allen Discovery Center at Tufts University, and Department of Biology, Tufts University, Medford, Massachusetts
| | - Junji Morokuma
- Allen Discovery Center at Tufts University, and Department of Biology, Tufts University, Medford, Massachusetts
| | | | - Katherine Williams
- Allen Discovery Center at Tufts University, and Department of Biology, Tufts University, Medford, Massachusetts
| | - Dany Spencer Adams
- Allen Discovery Center at Tufts University, and Department of Biology, Tufts University, Medford, Massachusetts
| | - Michael Levin
- Allen Discovery Center at Tufts University, and Department of Biology, Tufts University, Medford, Massachusetts.
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Pitcairn E, Harris H, Epiney J, Pai VP, Lemire JM, Ye B, Shi NQ, Levin M, McLaughlin KA. Coordinating heart morphogenesis: A novel role for hyperpolarization-activated cyclic nucleotide-gated (HCN) channels during cardiogenesis in Xenopus laevis. Commun Integr Biol 2017; 10:e1309488. [PMID: 28702127 PMCID: PMC5501196 DOI: 10.1080/19420889.2017.1309488] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/16/2017] [Accepted: 03/16/2017] [Indexed: 12/18/2022] Open
Abstract
Hyperpolarization-activated cyclic-nucleotide gated channel (HCN) proteins are important regulators of both neuronal and cardiac excitability. Among the 4 HCN isoforms, HCN4 is known as a pacemaker channel, because it helps control the periodicity of contractions in vertebrate hearts. Although the physiological role of HCN4 channel has been studied in adult mammalian hearts, an earlier role during embryogenesis has not been clearly established. Here, we probe the embryonic roles of HCN4 channels, providing the first characterization of the expression profile of any of the HCN isoforms during Xenopus laevis development and investigate the consequences of altering HCN4 function on embryonic pattern formation. We demonstrate that both overexpression of HCN4 and injection of dominant-negative HCN4 mRNA during early embryogenesis results in improper expression of key patterning genes and severely malformed hearts. Our results suggest that HCN4 serves to coordinate morphogenetic control factors that provide positional information during heart morphogenesis in Xenopus.
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Affiliation(s)
- Emily Pitcairn
- Department of Biology and Allen Discovery Center at Tufts University, Medford, MA, USA
| | - Hannah Harris
- Department of Biology and Allen Discovery Center at Tufts University, Medford, MA, USA
| | - Justine Epiney
- Department of Biology and Allen Discovery Center at Tufts University, Medford, MA, USA
| | - Vaibhav P Pai
- Department of Biology and Allen Discovery Center at Tufts University, Medford, MA, USA
| | - Joan M Lemire
- Department of Biology and Allen Discovery Center at Tufts University, Medford, MA, USA
| | - Bin Ye
- Department of Medicine, University of Wisconsin-Madison, Madison, WI, USA
| | - Nian-Qing Shi
- Department of Medicine, University of Wisconsin-Madison, Madison, WI, USA
| | - Michael Levin
- Department of Biology and Allen Discovery Center at Tufts University, Medford, MA, USA
| | - Kelly A McLaughlin
- Department of Biology and Allen Discovery Center at Tufts University, Medford, MA, USA
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Abstract
The central nervous system (CNS) underlies memory, perception, decision-making, and behavior in numerous organisms. However, neural networks have no monopoly on the signaling functions that implement these remarkable algorithms. It is often forgotten that neurons optimized cellular signaling modes that existed long before the CNS appeared during evolution, and were used by somatic cellular networks to orchestrate physiology, embryonic development, and behavior. Many of the key dynamics that enable information processing can, in fact, be implemented by different biological hardware. This is widely exploited by organisms throughout the tree of life. Here, we review data on memory, learning, and other aspects of cognition in a range of models, including single celled organisms, plants, and tissues in animal bodies. We discuss current knowledge of the molecular mechanisms at work in these systems, and suggest several hypotheses for future investigation. The study of cognitive processes implemented in aneural contexts is a fascinating, highly interdisciplinary topic that has many implications for evolution, cell biology, regenerative medicine, computer science, and synthetic bioengineering.
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Affiliation(s)
- František Baluška
- Department of Plant Cell Biology, IZMB, University of Bonn Bonn, Germany
| | - Michael Levin
- Biology Department, Tufts Center for Regenerative and Developmental Biology, Tufts University Medford, MA, USA
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Adams DS, Uzel SGM, Akagi J, Wlodkowic D, Andreeva V, Yelick PC, Devitt-Lee A, Pare JF, Levin M. Bioelectric signalling via potassium channels: a mechanism for craniofacial dysmorphogenesis in KCNJ2-associated Andersen-Tawil Syndrome. J Physiol 2016; 594:3245-70. [PMID: 26864374 PMCID: PMC4908029 DOI: 10.1113/jp271930] [Citation(s) in RCA: 106] [Impact Index Per Article: 11.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/19/2015] [Accepted: 02/01/2016] [Indexed: 12/21/2022] Open
Abstract
KEY POINTS Xenopus laevis craniofacial development is a good system for the study of Andersen-Tawil Syndrome (ATS)-associated craniofacial anomalies (CFAs) because (1) Kcnj2 is expressed in the nascent face; (2) molecular-genetic and biophysical techniques are available for the study of ion-dependent signalling during craniofacial morphogenesis; (3) as in humans, expression of variant Kcnj2 forms in embryos causes a muscle phenotype; and (4) variant forms of Kcnj2 found in human patients, when injected into frog embryos, cause CFAs in the same cell lineages. Forced expression of WT or variant Kcnj2 changes the normal pattern of Vmem (resting potential) regionalization found in the ectoderm of neurulating embryos, and changes the normal pattern of expression of ten different genetic regulators of craniofacial development, including markers of cranial neural crest and of placodes. Expression of other potassium channels and two different light-activated channels, all of which have an effect on Vmem , causes CFAs like those induced by injection of Kcnj2 variants. In contrast, expression of Slc9A (NHE3), an electroneutral ion channel, and of GlyR, an inactive Cl(-) channel, do not cause CFAs, demonstrating that correct craniofacial development depends on a pattern of bioelectric states, not on ion- or channel-specific signalling. Using optogenetics to control both the location and the timing of ion flux in developing embryos, we show that affecting Vmem of the ectoderm and no other cell layers is sufficient to cause CFAs, but only during early neurula stages. Changes in Vmem induced late in neurulation do not affect craniofacial development. We interpret these data as strong evidence, consistent with our hypothesis, that ATS-associated CFAs are caused by the effect of variant Kcnj2 on the Vmem of ectodermal cells of the developing face. We predict that the critical time is early during neurulation, and the critical cells are the ectodermal cranial neural crest and placode lineages. This points to the potential utility of extant, ion flux-modifying drugs as treatments to prevent CFAs associated with channelopathies such as ATS. ABSTRACT Variants in potassium channel KCNJ2 cause Andersen-Tawil Syndrome (ATS); the induced craniofacial anomalies (CFAs) are entirely unexplained. We show that KCNJ2 is expressed in Xenopus and mouse during the earliest stages of craniofacial development. Misexpression in Xenopus of KCNJ2 carrying ATS-associated mutations causes CFAs in the same structures affected in humans, changes the normal pattern of membrane voltage potential regionalization in the developing face and disrupts expression of important craniofacial patterning genes, revealing the endogenous control of craniofacial patterning by bioelectric cell states. By altering cells' resting potentials using other ion translocators, we show that a change in ectodermal voltage, not tied to a specific protein or ion, is sufficient to cause CFAs. By adapting optogenetics for use in non-neural cells in embryos, we show that developmentally patterned K(+) flux is required for correct regionalization of the resting potentials and for establishment of endogenous early gene expression domains in the anterior ectoderm, and that variants in KCNJ2 disrupt this regionalization, leading to the CFAs seen in ATS patients.
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Affiliation(s)
- Dany Spencer Adams
- Department of Biology and Tufts Centre for Regenerative and Developmental Biology, Tufts University, 200 Boston Avenue, Medford, MA, 02155, USA
| | - Sebastien G M Uzel
- Department of Mechanical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA, 02139, USA
| | - Jin Akagi
- School of Applied Sciences, RMIT University, Melbourne, Australia
| | - Donald Wlodkowic
- School of Applied Sciences, RMIT University, Melbourne, Australia
| | - Viktoria Andreeva
- Department of Orthodontics, Division of Craniofacial and Molecular Genetics, Tufts University School of Dental Medicine, Boston, MA 02111, USA
| | - Pamela Crotty Yelick
- Department of Orthodontics, Division of Craniofacial and Molecular Genetics, Tufts University School of Dental Medicine, Boston, MA 02111, USA
| | - Adrian Devitt-Lee
- Department of Biology and Tufts Centre for Regenerative and Developmental Biology, Tufts University, 200 Boston Avenue, Medford, MA, 02155, USA
| | - Jean-Francois Pare
- Department of Biology and Tufts Centre for Regenerative and Developmental Biology, Tufts University, 200 Boston Avenue, Medford, MA, 02155, USA
| | - Michael Levin
- Department of Biology and Tufts Centre for Regenerative and Developmental Biology, Tufts University, 200 Boston Avenue, Medford, MA, 02155, USA
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Durant F, Lobo D, Hammelman J, Levin M. Physiological controls of large-scale patterning in planarian regeneration: a molecular and computational perspective on growth and form. REGENERATION (OXFORD, ENGLAND) 2016; 3:78-102. [PMID: 27499881 PMCID: PMC4895326 DOI: 10.1002/reg2.54] [Citation(s) in RCA: 43] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 12/15/2015] [Revised: 02/18/2016] [Accepted: 02/22/2016] [Indexed: 12/12/2022]
Abstract
Planaria are complex metazoans that repair damage to their bodies and cease remodeling when a correct anatomy has been achieved. This model system offers a unique opportunity to understand how large-scale anatomical homeostasis emerges from the activities of individual cells. Much progress has been made on the molecular genetics of stem cell activity in planaria. However, recent data also indicate that the global pattern is regulated by physiological circuits composed of ionic and neurotransmitter signaling. Here, we overview the multi-scale problem of understanding pattern regulation in planaria, with specific focus on bioelectric signaling via ion channels and gap junctions (electrical synapses), and computational efforts to extract explanatory models from functional and molecular data on regeneration. We present a perspective that interprets results in this fascinating field using concepts from dynamical systems theory and computational neuroscience. Serving as a tractable nexus between genetic, physiological, and computational approaches to pattern regulation, planarian pattern homeostasis harbors many deep insights for regenerative medicine, evolutionary biology, and engineering.
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Affiliation(s)
- Fallon Durant
- Department of Biology, Allen Discovery Center at Tufts University, Tufts Center for Regenerative and Developmental BiologyTufts UniversityMA02155USA
| | - Daniel Lobo
- Department of Biological SciencesUniversity of MarylandBaltimore County, 1000 Hilltop CircleBaltimoreMD21250USA
| | - Jennifer Hammelman
- Department of Biology, Allen Discovery Center at Tufts University, Tufts Center for Regenerative and Developmental BiologyTufts UniversityMA02155USA
| | - Michael Levin
- Department of Biology, Allen Discovery Center at Tufts University, Tufts Center for Regenerative and Developmental BiologyTufts UniversityMA02155USA
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Lienkamp SS. Using Xenopus to study genetic kidney diseases. Semin Cell Dev Biol 2016; 51:117-24. [PMID: 26851624 DOI: 10.1016/j.semcdb.2016.02.002] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/30/2015] [Accepted: 02/01/2016] [Indexed: 10/22/2022]
Abstract
Modern sequencing technology is revolutionizing our knowledge of inherited kidney disease. However, the molecular role of genes affected by the rapidly rising number of identified mutations is lagging behind. Xenopus is a highly useful, but underutilized model organism with unique properties excellently suited to decipher the molecular mechanisms of kidney development and disease. The embryonic kidney (pronephros) can be manipulated on only one side of the animal and its formation observed directly through the translucent skin. The moderate evolutionary distance between Xenopus and humans is a huge advantage for studying basic principles of kidney development, but still allows us to analyze the function of disease related genes. Optogenetic manipulations and genome editing by CRISPR/Cas are exciting additions to the toolbox for disease modelling and will facilitate the use of Xenopus in translational research. Therefore, the future of Xenopus in kidney research is bright.
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Affiliation(s)
- Soeren S Lienkamp
- Renal Division, Department of Medicine, University of Freiburg Medical Center, Hugstetter Straße 55, 79106 Freiburg, Germany; Center for Biological Signaling Studies (BIOSS), Albertstraße 19, 79104 Freiburg, Germany.
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Pai VP, Martyniuk CJ, Echeverri K, Sundelacruz S, Kaplan DL, Levin M. Genome-wide analysis reveals conserved transcriptional responses downstream of resting potential change in Xenopus embryos, axolotl regeneration, and human mesenchymal cell differentiation. ACTA ACUST UNITED AC 2015; 3:3-25. [PMID: 27499876 PMCID: PMC4857752 DOI: 10.1002/reg2.48] [Citation(s) in RCA: 50] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/14/2015] [Revised: 08/20/2015] [Accepted: 08/25/2015] [Indexed: 12/14/2022]
Abstract
Endogenous bioelectric signaling via changes in cellular resting potential (Vmem) is a key regulator of patterning during regeneration and embryogenesis in numerous model systems. Depolarization of Vmem has been functionally implicated in dedifferentiation, tumorigenesis, anatomical re‐specification, and appendage regeneration. However, no unbiased analyses have been performed to understand genome‐wide transcriptional responses to Vmem change in vivo. Moreover, it is unknown which genes or gene networks represent conserved targets of bioelectrical signaling across different patterning contexts and species. Here, we use microarray analysis to comparatively analyze transcriptional responses to Vmem depolarization. We compare the response of the transcriptome during embryogenesis (Xenopus development), regeneration (axolotl regeneration), and stem cell differentiation (human mesenchymal stem cells in culture) to identify common networks across model species that are associated with depolarization. Both subnetwork enrichment and PANTHER analyses identified a number of key genetic modules as targets of Vmem change, and also revealed important (well‐conserved) commonalities in bioelectric signal transduction, despite highly diverse experimental contexts and species. Depolarization regulates specific transcriptional networks across all three germ layers (ectoderm, mesoderm, and endoderm) such as cell differentiation and apoptosis, and this information will be used for developing mechanistic models of bioelectric regulation of patterning. Moreover, our analysis reveals that Vmem change regulates transcripts related to important disease pathways such as cancer and neurodegeneration, which may represent novel targets for emerging electroceutical therapies.
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Affiliation(s)
- Vaibhav P Pai
- Biology Department and Center for Regenerative and Developmental Biology Tufts University Medford Massachusetts 02155 USA
| | - Christopher J Martyniuk
- Center for Environmental and Human Toxicology and Department of Physiological Sciences UF Genetics Institute, University of Florida Gainesville Florida 32611 USA
| | - Karen Echeverri
- Department of Genetics, Cell Biology and Development University of Minnesota Minneapolis Minnesota 55455 USA
| | - Sarah Sundelacruz
- Department of Biomedical Engineering Tufts University Medford Massachusetts 02155 USA
| | - David L Kaplan
- Department of Biomedical Engineering Tufts University Medford Massachusetts 02155 USA
| | - Michael Levin
- Biology Department and Center for Regenerative and Developmental Biology Tufts University Medford Massachusetts 02155 USA
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Pezzulo G, Levin M. Re-membering the body: applications of computational neuroscience to the top-down control of regeneration of limbs and other complex organs. Integr Biol (Camb) 2015; 7:1487-517. [PMID: 26571046 DOI: 10.1039/c5ib00221d] [Citation(s) in RCA: 105] [Impact Index Per Article: 10.5] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
A major goal of regenerative medicine and bioengineering is the regeneration of complex organs, such as limbs, and the capability to create artificial constructs (so-called biobots) with defined morphologies and robust self-repair capabilities. Developmental biology presents remarkable examples of systems that self-assemble and regenerate complex structures toward their correct shape despite significant perturbations. A fundamental challenge is to translate progress in molecular genetics into control of large-scale organismal anatomy, and the field is still searching for an appropriate theoretical paradigm for facilitating control of pattern homeostasis. However, computational neuroscience provides many examples in which cell networks - brains - store memories (e.g., of geometric configurations, rules, and patterns) and coordinate their activity towards proximal and distant goals. In this Perspective, we propose that programming large-scale morphogenesis requires exploiting the information processing by which cellular structures work toward specific shapes. In non-neural cells, as in the brain, bioelectric signaling implements information processing, decision-making, and memory in regulating pattern and its remodeling. Thus, approaches used in computational neuroscience to understand goal-seeking neural systems offer a toolbox of techniques to model and control regenerative pattern formation. Here, we review recent data on developmental bioelectricity as a regulator of patterning, and propose that target morphology could be encoded within tissues as a kind of memory, using the same molecular mechanisms and algorithms so successfully exploited by the brain. We highlight the next steps of an unconventional research program, which may allow top-down control of growth and form for numerous applications in regenerative medicine and synthetic bioengineering.
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Affiliation(s)
- G Pezzulo
- Institute of Cognitive Sciences and Technologies, National Research Council, Rome, Italy
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