Keywords: asymmetry, embryogenesis, evolution, biological information, gap junctions, ion channels, mathematical modeling, morphogenesis, pattern formation, vertebrate development, ion current, voltage gradients, ion channels and pumps, regeneration, memory, learning

“Do not follow where the path may lead. Go instead where there is no path, and leave a trail.” -- Ralph Waldo Emerson

Summary:
     The capacity to generate a complex organism from the single cell of a fertilized egg is one of the most amazing qualities of multicellular creatures. The processes involved in laying out a basic body plan and defining the structures that will ultimately be formed depend upon a constant flow of information between cells and tissues. The Levin laboratory studies the molecular mechanisms cells use to communicate with one another in the 4-dimensional dynamical system known as the developing embryo, and the flow of information necessary for an injured system to recognize what structures must be rebuilt. Through experimental approaches and mathematical modeling, we examine the processes governing large-scale pattern formation and biological information storage during animal embryogenesis and regeneration. Our investigations are directed toward understanding the mechanisms of signaling between cells and tissues that allows a biological system to reliably generate and maintain a complex morphology. We study these processes in the context of embryonic development, regeneration and cancer, with a particular emphasis on the biophysics of cell behavior. In contrast to other groups focusing on gene expression networks and biochemical signaling factors, we are pursuing, at a molecular level, the roles of endogenous voltages, pH gradients, and ion fluxes as epigenetic carriers of morphological information. Using gain- and loss-of-function techniques to specifically modulate cells' ion flow we have the ability to regulate large-scale morphogenetic events relevant to limb formation, eye induction, craniofacial and neural patterning, etc. While our focus is on the fundamental mechanisms of pattern regulation, this information will also result in important clinical advances through harnessing bioelectrical controls of cell behavior in regenerative medicine.

Bioelectrical controls of vertebrate appendage regeneration:

"If you want to build a ship, don't herd people together to collect wood, and don't assign them tasks and work, but teach them to long for the endless immensity of the sea."         -- Antoine de Saint-Exupery,            "Wisdom of the Sands"

      Regeneration is a fascinating example of pattern regulation, and has important biomedical implications. A regenerating system must not only recognize damage, but pursue a goal-directed process of restoring the missing structures (and crucially, know to stop when this process is complete, thus avoiding cancerous overgrowth). Interestingly, systems with high regenerative ability have low susceptibility to neoplasm, contrary to the simple view in which cellular plasticity and propensity for proliferation should go together in cancer and regeneration. Instead, data suggest that the morphogenetic controls imposed during regeneration can prevent cells from ignoring the patterning cues of the host (as occurs in many cancers). What is the mechanistic nature of these controls? Our lab studies the role of important but poorly-understood biophysical factors (such as pH and voltage gradients) in the induction of regeneration and the imposition of correct morphology on the restored tissue. We use two model systems to understand these processes: Xenopus laevis tadpoles and planarian flatworms. An important part of our mission is the development of molecular reagents, protocols, databases, transgenic animals, and conceptual (modeling) tools to facilitate others' study of bioelectrical signals in many aspects of morphogenesis in diverse model systems.

    While vertebrate regeneration is considered to be imited, the Xenopus tadpole is able to regenerate its tail - a complex appendage containing spinal neurons, muscle, skin, and vasculature. We identified three electrogenic proteins whose activity is required for the production of a depolarization zone that underlies regeneration in the blastema and demonstrated that a proton flux from the wound epithelium is necessary and sufficient to drive the downstream events of regeneration, including cell proliferation, innervation, and expression of regeneration-specific markers. We are currently working on inducing regeneration of limbs, eyes, tails, and craniofacial structures in normally non-regenerating species by providing the appropriate bioelectric signals to the cells at the wound site. Strikingly, while ion transporters control low-level cell behaviors such as migration, differentiation, and proliferation, bioelectric signals appear to function as master regulators in many contexts: a simple signal can induce complex, highly orchestrated, self-limiting downstream morphogenetic cascades. For example, an unmodulated flux of protons can induce the formation of a complete tail of the right rise and tissue composition. Inducing the host to form structures it already knows how to make is a very desirable property for regenerative medicine approaches since it is not feasible to micromanage (directly bioengineer) the creation of complex organs and appendages. We are also pursuing novel long-range sources of patterning information for regeneration.

Bioelectrical, non-local controls of regenerative polarity in planaria

"Wonderful as are the laws and phenomena of electricity when made evident to us in inorganic or dead matter, their interest can bear scarcely any comparison with that which attaches to the same force when connected with the nervous system and with life."       -- Michael Faraday, 1839

        Planarian flatworms have an impressive capacity for regeneration. They are able to regenerate large parts of the body, and are continuously maintained by a well-characterized resident population of adult stem cells. Upon cutting, these organisms are able to regenerate the head and tail at their appropriate locations. What mechanisms determine the polarity and allow tissue re-patterning to take place?  Consider: after bisection, cells on one side of the cut (in the head fragment's posterior end) will form a tail, while cells which were their immediate neighbors before the cut will make a head (the tail fragment's anterior half).  Our data suggest that the mechanism by which blastema cells polls the rest of the host (to determine where the wound is located and what other tissues already exist in the fragment and thus don't need to be recreated) is mediated by physiological signals passing through nerves and long-range gap junctional paths. We have identified endogenous ion fluxes and voltage gradients maintained by specific ion pumps which are crucial for the determination of anterior-posterior polarity during regeneration; manipulating these signals allows us to specify tissue identity and thus control the anatomical structure of regenerating worms. Through studying the roles of electrical polarity (maintained by ion channel and gap junction systems) in planarian regeneration we are gaining insight into the control of regeneration and morphogenesis by endogenous ion fluxes and into the mechanisms by which stem cell differentiation is integrated into functional organ/tissue systems within the organism. Most importantly, we've recently shown that a rapid, transient alteration of the physiological signals underlying morphostasis and regeneration is maintained in perpetuity! That is, worms forming 2 heads (one at each end) because of a 2-day disruption of gap junctional signals will continue to form 2 heads through subsequent months of amputation or fission in normal conditions. These data illustrate how information embedded in physiological networks can be solidified into permanent alteration of the large-scale structure bodyplan. More broadly, this work identifies a molecular glimpse of how the "target morphology" of an animal (the form towards which regeneration regulates) can be permanently reset, and reveals that a drastic change in body structure and behavior can be maintained across a complex metazoan's organism's normal mode of reproduction without any change in DNA sequence.

Control of Stem Cell Behavior within the Host

        Achieving the differentiation of stem cells into specific tissues is an important goal but is only the beginning for regenerative medicine, since restoration of damaged, aging, and malformed organs requires not only the ability to induce specific cell types, but also to integrate them into extremely complex 3-dimensional patterns (e.g., limbs, eyes, craniofacial structures, etc.) while avoiding the production of tumors or unpatterned overgrowth. Thus, we are interested in understanding how the host directs adult stem cell behavior and coordinates their migration, differentiation, and proliferation into morphogenetic programs. In the frog embryo, we have shown that depolarizing small cell groups has drastic consequences for the neural crest (an important embryonic stem cell population): melanocyte derivatives of the neural crest overproliferate, migrate to inappropriate locations (colonizing blood vessels and other tissues), and achieve an arborized cell shape (key features of melanoma and other cancers). This revealed the transmembrane potential of nearby cells as a novel aspect of the microenvironment that can trigger the stem cell -> neoplastic-like cell transition. Similarly, in collaboration with David Kaplan's group, we have shown the bioelectrical control of human mesenchymal stem cell differentiation.

Left-Right Asymmetry:

"The source of all understanding is from the left side."              -- The Talmud "I am shocked not so much by the fact that the Lord prefers the left hand as by the fact that he still appears to be left-right symmetric when he expresses himself strongly."              -- Wolfgang Pauli, 1957

Gap Junctions in Pattern Formation:
     While asymmetrically expressed genes have been identified in several vertebrate systems, many critical questions remain about how cellular polarity is synchronized and amplified across embryonic fields to allow cells to ascertain their position with respect to the midline. We identified a dependence of asymmetric gene expression on early communication between left and right sides in the chick and frog. For example, expression of left-sided markers depends on events occurring on the right side, during very early stages, suggesting that the two sides need to coordinate their decision with respect to the L-R identity of each. One mechanism for communicating between cells and tissues involves gap junctions: multimers of connexin proteins form channels between cells and pass small molecules, subject to complex regulation by various signals. Using misexpression of Connexin proteins and their mutants to disrupt and induce long-range gap junctonal paths in early chick and frog embryos, we showed that gap junctions are crucially involved in L-R patterning in early embryos of Xenopus and chick. The data suggest the presence of a unidirectional circumferential flow of small molecules through gap junctions across the whole embryonic field during blastula/early gastrula stages. Our research focuses on understanding the mechanisms upstream and downstream of specific gap junction communications (GJC) in embryos, as they relate to pattern formation and growth control, and on identifying the small molecule morphogens that traverse junctions. More broadly, we focus on gap junctions as a bioelectric patterning element that sets up domains of isopotential cell fields during morphogenesis.

Bioelectric Aspects of Very Early Left-Right Patterning
     L-R asymmetry can only be derived from gap-junctional movement of determinants that is directionally biased. In frog and chick embryos, we have identified a set of four ion transporters which reliably establish a voltage gradient along the midline. This gradient is required for normal asymmetry, and our current quantitative models suggest that it is sufficient to redistribute small molecule morphogens from left to right through the gap junctional paths. These transporters establish a battery across the midline, and in Xenopus, this occurs by the second cell cleavage. Molecular localization of ion channel/pump proteins, and direct detection of asymmetric ion flows (H+ and K+ ions) reveal that these embryos know their left from their right within about 2 hours of fertilization. What establishes the right-sided ion flow? Our latest work has focused on the cytoskeleton, and we showed that the early protein localization machinery is consistently right-biased, allowing kinesin and dynein motors to deliver maternal ion transporter cargo to the right ventral blastomere (thus establishing the battery and electromotive force for the trans-junctional morphogen(s)). We are currently characterizing the intracellular microtubule organizing center whose chirality is likely to be the ultimate origin of asymmetry, as its orientation with the other two axes is established during fertilization. We have also pursued roles for planar cell polarity in spreading LR information across a blastoderm.

The Role of Serotonin in Embryogenesis:
     The importance of serotonin in neuronal function is well established. Interestingly, it also has roles in early embryogenesis, long before nerve systems appear. This is probably indicative of evolutionarily early systems of cell signaling which became co-opted by neurons when they arose. Taking advantage of the well-characterized pharmacology and genetics of many steps in the serotonin signaling pathway, we are studying how serotonin signaling is used in information exchange between cells in processes such as L-R patterning and control of timing and cell movement during gastrulation. We have shown that serotonin is utilized by both chick and frog embryos, at very early stages, as a small molecule signal which is transported in a left-right gradient and regulates the development of laterality. Indeed, we now know that the early frog embryo is literally an electrophoresis chamber, which uses voltage potentials to generate consistently biased left-right gradients in serotonin in an epigenetic process not dependent on zygotic gene expression. We have modeled this process quantitatively, and characterized novel intracellular serotonin-binding proteins which directly activate asymmetric gene expression after their rightward movement, linking an early biophysical process to transcriptional regulation via chromatin modification pathways.

The Properties of Memory Storage and Transmission in Tissue:

"The great field for new discoveries ... is always the unclassified residuum. Round about the accredited and orderly facts of every science there ever floats a sort of dust-cloud of exceptional observations ... Any one will renovate his science who will steadily look after the irregular phenomena."         -- William James, 1890

        Flatworms can learn in a variety of behavioral paradigms and are a unique model system in which regeneration and memory can be studied in the same animal. In partnership with Wireless Techniques, we have designed and built a computer-controlled automated learning and testing chamber that allows the training of a set of worms in a consistent environment (removing sources of error such as experimenter bias, and greatly increasing the efficiency of the learning process). By combining a robust learning/memory response with experiments feasible only in this highly-regenerative model system, we are investigating the molecular basis of memory. Using quantitative, automated behavior analysis techniques we are asking how and where information is encoded and how it can be imprinted upon the regenerating brain by other tissues. Our parallelized machine vision and environmental control platform is a unique next-generation system allowing not only quantitative characterization of animal behavior (worm, tadpole, and zebrafish) but real-time reward/punishment feedback to each individual subject independently, allowing powerful learning and instrumental/classical conditioning assays. A scaled-up version of this technology will allow high-throughput screening of small molecule libraries for complex neuroactive effects (such as nootropic compounds that increase learning rate, modulate addiction, etc.). In vertebrate systems such as tadpoles, we are investigating the cognitive consequences of laterality inversion and brain plasticity under sensory augmentation and pharmacological/genetic CNS perturbation.

Mathematical Modeling and Physiomics:

"With many calculations, one can win; with few, one cannot." --Sun Tzu, "The Art of War"   "Beyond all questions of quantity there lie questions of pattern, which are essential for the understanding of nature." --Alfred North Whitehead (1934)   "A model is a lie that helps you see the truth." -- Howard Skipper

      Molecular biology and genomics are revealing a constantly expanding amount of information about genes, their products, and the way they interact. It is notoriously difficult to control or make predictions about systems involving mutual interactions of even a few components because of feedback loops and the basic results of dynamical systems theory. Indeed, looking at a high-resolution mechanistic pathway, such as painstakingly elucidated in many recent studies, is insufficient for knowing what biological pattern this transcriptional network results in. It is essential to develop constructive, synthetic models of morphogenesis which integrate 3-dimensional shape from the function of molecular components and pathways. To fully understand the implications of information coming from genome projects and biochemical analyses of gene activities for morphogenesis, a synthesis is needed. We are attempting to use the mathematical and computer modeling tools of chaos, information, and complexity theories to understand large-scale patterning and control properties of bioelectrical mechanisms and small molecule transport among cell groups. Our main efforts along these lines are directed towards (1) development of a formalization for morphogenetic processes (bioinformatics of shape, beyond gene/protein sequence, and automated model discovery), (2) testing the hypothesis that cell behavior can be understood as the segregation and movement of cell states through a multi-dimensional state space with axes defined by bioelectrical parameters such as membrane voltage, K+ content, pH, nuclear membrane potential, etc., and (3) developing quantitative models integrating physiology and genetics of ion transporter function during early left-right asymmetry.

"If we knew what we were doing, it wouldn't be called research, would it?" -- Albert Einstein