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Michael Levin
Michael Levin, PhD
Senior Research Investigator
The Forsyth Center for Regenerative and Developmental Biology
Levin lab | Forsyth Center for Regenerative and Developmental Biology
email: mlevin@forsyth.org
Tufts University, B.S., 1992, Computer Science/Biology
Harvard Medical School, Ph.D., 1996, Developmental Biology
The capacity to generate a complex organism from the single cell of a fertilized egg is one of the most amazing qualities of multicellular animals. 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. Through experimental approaches and mathematical modeling, we examine the processes governing large-scale pattern formation and biological information storage during animal embryogenesis. 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 and regeneration, with a particular focus 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 lossof-function techniques to specifically modulate cells' ion flow we have the ability to regulate large-scale morphogenetic events relevant to limb formation, eye induction, etc. We believe this information will result in important clinical advances through harnessing the biophysical controls of cell behavior.
Understanding the biophysical factors controlling regeneration
Regeneration is a fascinating example of pattern formation, and has important biomedical implications. 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: planaria and Xenopus. Planarian flatworms have an impressive capacity for regeneration. They are able to regenerate large parts of the body, and are continuously maintained by 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? 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. 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 control of stem cell populations by ion flow. While vertebrate regeneration is usually much more limited, the Xenopus tadpole is able to regenerate its tail. The tail is a complex appendage containing spinal neurons, muscle, skin, etc. We identified three electrogenic proteins whose activity is required for the production of a depolarization zone that underlies regeneration in the blastema. We are currently working on inducing regeneration in normally non-regenerating species by providing the appropriate bioelectric signals to the cells at the wound site.
Left-Right Asymmetry
The vertebrate body plan is basically bilaterally-symmetrical; however, consistent and well-conserved asymmetries of the brain and visceral organs are superimposed upon the fundamental structure. Asymmetries in the left-right axis present a number of deep puzzles which link evolutionary biology, clinical medicine of birth defects, biochemistry, embryology, and perhaps even quantum parity violations. We are working to understand the mechanisms by which the embryo aligns the left-right axis with respect to the other two axes, and imposes this spatial information on macroscopic cell fields.
Gap Junctions in Pattern Formation
While asymmetrically expressed genes have been identified in several vertebrate systems, many critical questions remain. The upstream mechanisms directing consistent expression of the first asymmetric gene and the means for coordinating the left-right axis with the dorsoventral and antero-posterior axes are currently unknown. We have identified a dependence of asymmetric gene expression on early large-scale 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. We have shown that gap junctions are crucially involved in L-R patterning in early embryos of Xenopus and chicks. 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. We are also investigating other molecules besides the familiar connexins, such as ductin, which may underlie GJC in a number of patterning contexts.
Bioelectric Aspects of Very Early Left-Right Patterning
L-R asymmetry can only be derived from junctional movement of determinants that is directionally biased. In Xenopus, we have evidence for the existence of unidirectional junctions. One-way junctions are formed by two or more different connexin family members associating across a cell boundary. Through microinjection of wild-type and dominant negative constructs of connexin family members, we can create unidirectional junctions in any spatial pattern and are examining possible roles of specific connexins in early Xenopus embryos. However, the laws of thermodynamics require an energetic mechanism to drive this chiral flow.
We designed a methodology which uses pharmacological blockers and activators to rapidly screen for electrogenic targets involved in asymmetry. Our inverse drug screen rapidly implicated just four specific ion transporters in embryonic L-R asymmetry, and these targets were validated using specific molecular gain- and loss-of-function approaches. Examination of their localization patterns revealed numerous very early L-R asymmetries and suggested the presence of novel subcellular localization mechanisms. These electrogenic proteins are necessary for asymmetry in chick, frog, and zebrafish embryos upstream of asymmetric gene expression, and reveal some of the early physiological events which dictate asymmetry. We hypothesize that the net result of the coordinated ion transport at the zone of junctional isolation may be a source of voltage gradients that drive charged small molecule determinants in a preferred direction across gap junctions. Using misexpression of constructs and pH- and voltage-sensitive fluorescent dyes in vivo, we are dissecting the individual contribution of each electrogenic protein, testing several possible models of the relationships between ion channels, gap junctions, and L-R asymmetry, pursuing array approaches to locate early response genes linking cell membrane voltage changes to downstream gene cascades, and attempting to synthesize all of the data into a predictive, quantitative model of early embryonic physiology.
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 gradient and regulates the development of laterality.
The Properties of Memory Formation Flatworms can learn in a variety of behavioral paradigms
We have built a computer-controlled 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 and asking how and where information is encoded and how it can be imprinted upon the regenerating brain by other tissues. A scaled-up version of this technology will allow high-throughput screening of small molecule libraries for complex neuroactive effects (such as increasing learning rate, modulating addiction, etc.).
Mathematical Modeling and Physiomics 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. 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.
Selected Publications
Morokuma J, Blackiston D, Adams DS, Seebohm G, Trimmer B, Levin M. 2008. Modulation of potassium channel function confers a hyperproliferative invasive phenotype on embryonic stem cells. Proc Natl Acad Sci U S A. 28;105(43):16608-13
Oviedo NJ, Pearson BJ, Levin M, Sánchez Alvarado A. 2008. Planarian PTEN homologs regulate stem cells and regeneration through TOR signaling. Dis Model Mech. 1(2-3):131-43
Tseng AS, Levin M. 2008. Tail Regeneration in Xenopus laevis as a Model for Understanding Tissue Repair. J Dent Res. 87(9):806-16.
Morokuma J, Blackiston D, Levin M. 2008. KCNQ1 and KCNE1 K Channel Components are Involved in Early Left-Right Patterning in Xenopus laevis Embryos. Cell Physiol Biochem. 2008;21(5-6):357-372.
Adams DS, Masi A, Levin M. 2007 H+ pump-dependent changes in membrane voltage are an early mechanism necessary and sufficient to induce Xenopus tail regeneration. Development. 134(7):1323-35.
Ovideo NJ, Levin M. 2007 Gap junctions provide new links in left-right patterning. Cell 129(4):645-7.
Levin M, Palmer AR. 2007. Left-right patterning from the inside out: widespread evidence for intracellular control. Bioessays. 29(3):271-87.
Adams DS, Levin M. 2006. Inverse drug screens: a rapid and inexpensive method for implicating molecular targets. Genesis. 44(11):530-40.
Levin M. 2006. Gap junctional signaling in morphogenesis. Prog. Biophys. Mol. Biol. In press.
Hibino T, Ishii Y, Levin M, Nishino A. 2006. Ion flow regulates left-right asymmetry in sea urchin development. Dev. Genes Evol. 21 6(3):265–276.
Shimeld SM, Levin M. 2006. Evidence for the regulation of left-right asymmetry in Ciona intestinals by ion flux. Dev. Dyn. 235(6):1543–1553.
Esser AT, Smith KC, Weaver JC, Levin M. 2006. A mathematical model of morphogen electrophoresis through gap unctions. Dev. Dyn. 235(8)2144–2159.
Hicks C, Sorocco D, Levin M. 2006. Automated analysis of behavior: A computer-controlled system for drug screening and the investigation of learning. J. Neurobiol. 66(9):977–990.
Adams DS, Robinson KR, Fukumoto S, Yuan S, Albertson RC, Yelick P, Kuo L, McSweeney M, Levin M. Early, 2006. H+-V-ATPase-dependent proton flux is necessary for consistent left-right patterning of non-mammalian vertebrates. Development 133(9):1657–1671.
Levin M, Lauder J, Buznikov G. 2006. Of minds and embryos: Serotonin signaling as a pre-nervous morphogenetic mechanism. Dev. Neurosci. 28(3):171– 185.
Levin M. 2005. Left-right asymmetry in embryonic development: A comprehensive review. Mech. Dev. 122(1):3–25.
Adams D, Levin M. 2005. Ion fluxes and gap junctional communication: Probingbiophysical epigenetic factors in Xenopus patterning. In: Whitman M, Sater.
AK. (eds). Analysis of Growth Factor Signaling in Embryos. Methods in Signal Transduction Series. CRC Press.
Nogi T, Yuan Y, Sorocco D, Perez-Tomas R, Levin M. 2005. Eye regeneration assay reveals an invariant functional left-right asymmetry in the early bilaterian, Dugesia japonica. Laterality 10(3):193–205.
Fukumoto T, Kema I, Levin M. 2005. Serotonin signaling is a very early step in patterning of the left-right axis in chick and frog embryos. Curr. Biol. 15(9):794–803.
Qiu D, Cheng SM, Wozniak L, McSweeney M, Perrone E, Levin M. 2005. Localization and loss-of-function implicates ciliary proteins in early, cytoplasmic roles in left-right asymmetry. Dev. Dyn. 234 (1):176–189.
Fukumoto T, Blakely R, Levin M. 2005. Serotonin transporters are a conserved, early mechanism in left-right patterning. Dev. Neurosci. 27(6):349–363.
Nogi T, Levin M. 2005. Characterization of innexin gene expression and functional roles of gap-junctional communication in planarian regeneration. Dev. Biol. 287(2):314–335.
Levin M. 2004. A novel immunohistochemical method for evaluation of antibody specificity and detection of labile targets in biological tissue. J. Biophys. Biochem. Methods 58(1):85–96.
Adams DS, Levin M. 2004. Early Patterning of the Left/Right Axis. In: Stern CD. (ed). Gastrulation From Cells to Embryo, pp. 403–417. Cold Spring Harbor, New York.
Bunney TD, De Boer AH, Levin M. 2003. Fusicoccin signaling reveals 14-3-3 protein function as a novel step in left-right patterning during amphibian embryogenesis. Development 130(20) :4847–4858.
Levin M. 2003. Electromagnetic fields in morphogenesis. Bioelectromagnetics 24(5):295–315.
Levin M. 2003. Motor protein control of ion flux is an early step in embryonic left-right asymmetry. BioEssay 25(10) :1002–1010.
Levin M, Thorlin T, Robinson K, Nogi T, Mercola M . 2002. H+/K+-ATPase activity comprises an early step of left-right asymmetry during development. Cell 111(1):77–89.
Levin M. 2002. Gap-junctional communication and embryonic development. J. Membr. Biol. 185 (3) :177–192.
Mercola M, Levin M. 2001. Left-right asymmetry determination in vertebrates. Annu. Rev. Cell Dev. Biol. 17(1):779–805.
Levin M, Mercola M. 1999. Gap junction-mediated left-right patterning signals in the early chick blastoderm is upstream of Shh asymmetry. Development 1 26(21):4703–4714.
Levin M, Roberts D, Holmes L, Tabin C. 1996. Laterality defects in conjoined twins. Nature 384(6607):321.
Levin M. 1995. A genetic algorithm model of the evolution of animal communication. BioSystems 36(3):167–178.
Levin M, Johnson RL, Stern CD, Kuehn M, Tabin C. 1995. A molecular pathway determining left-right asymmetry in chick embryogenesis. Cell 82(5) :803–814.
Levin M, Ernst SG. 1995. AC magnetic field effects on early sea urchin development. Bioelectromagnetics 1 6(4):231–240.
Levin M. 1994. A Julia set model of field-directed morphogenesis. 1994. Comput. Appl. Biosci. 10(2) :85–103.
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