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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 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 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,
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:
"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
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 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
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, 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 LR asymmetry, and these targets
were validated using specific molecular gain- and loss-of-function approaches.
Examination of their localization patterns revealed numerous very early LR 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:
"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. 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.
"If we knew what we were doing, it wouldn't be called research, would it?" -- Albert Einstein
