Stanislav Y. Shvartsman - US grants

Muratov
0211864
Shvartsman
0211755
In this collaborative project the investigators combine
mechanistic modeling, computational analysis, and experimental
techniques of developmental genetics to analyze cell
communication networks in the development of the Drosophila egg
(oogenesis). They focus on the patterning events mediated by the
Epidermal Growth Factor Receptor (EGFR), during which a localized
source of the EGFR ligand is modulated in space and time by a
distributed network of autocrine loops to produce a biochemical
blueprint specifying the formation of a pair organ. The
investigators develop mechanistic models of EGFR signaling in
Drosophila oogenesis. These models are necessary to directly test
consistency of the proposed regulatory mechanisms, to make the
experimentally verifiable predictions, and to guide the design of
future experiments. The models should explicitly account for the
key components of the EGFR system: the receptor, four of its
ligands, ligand processing proteins, and intracellular signaling
cascades. The nonlinear reaction-transport models of spatially
distributed EGFR signaling networks are analyzed using a
combination of numerical simulations, asymptotic techniques, and
bifurcation analysis. The tests of model-based predictions rely
on experimental advantages of Drosophila genetics.
Signaling through the Epidermal Growth Factor Receptor EGFR
is essential in a number of developmental processes across
species, from fruitflies to humans, and is extensively studied at
the molecular level. The main goal of the project is to develop
modeling and computational tools necessary to describe
reaction-transport processes in developing epithelial layers. In
the context of Drosophila, the investigators aim to capture a
large number of phenotypic transitions in eggshell morphology
that have been observed following quantitative manipulations in
the doses of the regulatory genes. This leads to a class of
mathematical problems that are also relevant in other biological
and physico-chemical settings. Given the highly conserved nature
of EGFR systems, it is possible that the proposed analysis of
patterning events in Drosophila oogenesis may be used to
understand the role of EGFR in the formation of branched
epithelial structures in the development of higher organisms. The
project has a significant educational component: it brings
together and trains students and postdocs in biology, engineering
and mathematics.

0448919
Shvartsman

The proposed work aims to examine the fundamental mechanisms of cell communication and tissue development using the epidermal growth factor receptor (EGFR) as a model network and Drosophila as the model organism. Using mathematical modeling, genetic experiments, and genomic analyses, the Principal Investigator will quantify the spatial gradient of EGFR activation in vivo, characterize the genome-wide transcriptional response to this gradient, and analyze how it is modulated by feedback loops. Such work generally aims at elucidating how biological systems construct ordered structures using chemical and physical signals. The work is applicable to embryo development and tissue regeneration. An innovative undergraduate course entitled "Patterns of Biological Design" will be developed. This course will cover how key engineering concepts (e.g., feedback, redundancy, robustness) manifest in biological and more traditional chemical and electrical systems. An exchange program with the University of Puerto Rico at Mayaguez will also be established.

DESCRIPTION (provided by applicant): Embryonic development is an intrinsically multiscale phenomenon that requires a highly coordinated processes at all levels of biological organization, from genes, to proteins, to cells, to tissues and, eventually, to the whole organism. Thus, multiscale approaches are indispensable for understanding the mechanisms responsible for development. This project proposes to combine experiments and modeling to study the mechanisms by which developing epithelia are patterned by the Epidermal Growth Factor Receptor (EGFR), an evolutionary conserved regulator of tissues in animals from worms to humans. Using Drosophila melanogaster as the experimental system, the PIs will quantify the transcriptional response to EGFR signaling, develop models of EGFR-mediated cell communication in epithelial layers, and use these models to analyze the EGFR system in Drosophila oogenesis, spanning the scales from genes to organs. The experiments will combine developmental genetics, genomics, and transcriptional profiling experiments. The computational approaches will include asymptotic, homogenization, and model reduction techniques. This integrative research will lead to the first experimentally validated model of EGFR signaling in tissues. The success of this effort relies on the combined expertise of the PI at bench experiments, modeling, and analysis across developmental scales. EGFR is essential for normal tissue development, but deregulated EGFR signaling has been associated with numerous diseases, including many types of human cancers. Hence, an integrative understanding of EGFR action in tissues is of direct relevance to a wide range of medical problems. In addition to addressing the fundamental questions of cell fate diversification in development, this work will lead to computational and data integration tools for a wide range of epithelial patterning problems. First, the PIs will develop Virtual Epithelium (VE), a publicly available software for the computational analysis of epithelial patterning systems. Second, the PIs will develop and make publicly available the Database of Drosophila Oogenesis (DODO) that will combine the heterogeneous datasets generated by their experiments with bioinformatics and biostatistics tools. VE will enable systematic modeling and exploration of the spatiotemporal dynamics of cell communication by diffusible chemical signals. DODO will complement the existing database of gene expression patterns in the embryo and form a starting point for a multiscale analysis of epithelial patterning in a large number of developmental contexts. The project will bring together researchers in biology, engineering and mathematics in an interdisciplinary research program aimed at bringing about new understanding of EGFR system.

DESCRIPTION (provided by applicant): Morphogenetic signaling constitutes one of the key cell fate diversification mechanisms whereby a gradient of a diffusible signal specifies multiple fates in a field of naive cells. While the extra cellular regulation of morphogen gradients and of the immediate receptor and signaling events are becoming progressively characterized, our understanding of the ways by which morphogens induce cell fates is still very incomplete. Analysis of the mechanisms of cell fate induction by morphogen gradients is the main goal of this proposal. We propose to combine experimental and modeling approaches to investigate how two signaling pathways interact in patterning of a developing tissue. We will study how the joint activities of the Epidermal Growth Factor (EGF) receptor and Bone Morphogenetic Protein (BMP) receptor systems pattern the follicular epithelium in the developing Drosophila egg, an established model of developmental pattern formation. First, we will carry out a multivariable analysis of transcriptional responses in this system, using three different transcriptional profiling assays (micro arrays, quantitative real-time PCR, and in situ hybridization). Second, we will use the results of these experiments to computationally explore the regulation of discovered targets of signaling crosstalk by extra cellular signals. Third, we will formulate mechanistic models for the initial patterning events in this system and use these models to explore the regulation of Pipe, the gene essential for the induction of the dorsoventral embryonic axis. We expect that, as a result of these experimental, modeling, and computational studies, the follicular epithelium will become one of the best-characterized pattern formation systems. Deregulated EGFR and BMP signaling are associated with severe developmental defects and a large number of human diseases. Given the highly conserved nature of these signaling pathways, our results will provide quantitative insights into the mechanisms of signaling crosstalk in developing and adult tissues.

Embryonic development is driven by highly regulated spatial and temporal patterns of gene expression. These patterns emerge in a sequential process, with simple patterns providing inputs to molecular networks, which, in turn, transform these inputs into more complex outputs. Due to the recent advances in the molecular studies of development, these networks can now be modeled at mechanistic level. Exploring complex mechanistic models is critically dependent on the use of computational techniques; at the same time, the behavior of large-scale models can often be understood through analysis of simplified models, providing a "bridge" between computational and real experiments. Rigorous mathematical techniques for analyzing the dynamics of signal processing in developmental patterning are yet to be developed. To this end, the PIs propose to pursue a number of analytical approaches for studying the spatiotemporal dynamics in patterning networks. These approaches, which rely heavily on the techniques from the calculus of variations, will be applied in the context of epithelial pattern formation in the Drosophila egg, an established experimental model for studying how simple inputs establish complex spatial patterns in development. The proposed analytical and computational work will build on the modeling and experimental results obtained by the PIs during the previous funding period. Within the framework of this application, the PIs will focus on signal processing by two-dimensional patterning networks with positive feedback loops.

The proposed research is closely linked to the experimental work on pattern formation mediated by the Epidermal Growth Factor Receptor (EGFR), a key regulator of epithelial tissues across species. Given the highly conserved nature of EGFR signaling and the generality of inductive patterning events, the work of the PIs will provide insights into a large class of development problems. In addition, the mathematical techniques developed in this proposal will be applicable to a wide range of problems modeled by nonlinear parabolic partial differential equations. This research is strongly aligned with the educational effort of the PIs that involves further development of undergraduate and graduate courses on mathematical biology of cell communication systems and interdisciplinary training of mathematics and engineering graduate and undergraduate students at NJIT and Princeton.

The project describes a series of theoretical and experimental studies of morphogen gradients, defined as the concentration fields of chemical substances that control spatial patterns of cell differentiation in developing tissues. Molecular studies of embryogenesis have identified morphogen gradients in systems as diverse as body axes specification in insects and patterning of mammalian neocortex. Current studies of morphogen gradients move in an increasingly quantitative direction and demand the development of a rigorous theoretical framework that can be used to interpret experimental results and guide systems-level analyses of pattern formation mechanisms. This project develops such a framework, emphasizing the dynamics of morphogen gradient formation. This research combines rigorous mathematical and computational analysis of a general class of reaction-diffusion models of morphogen gradient formation and application of these models to a specific pattern formation event in Drosophila embryo.

Broader impacts of proposed work include the development of a general mathematical framework for a highly conserved biological process. One of the main outcomes of proposed activity is a set of analytical results that can be used for the back-of-the-envelope analysis of pattern formation dynamics in a wide range of developmental systems. The proposed theoretical and experimental studies go hand-in-hand with the development of educational program that provides interdisciplinary training in the emerging field of developmental systems biology.

The NSF award by the Office of Emerging Frontiers in Research and Innovation supports work designed to address a fundamental question of developmental biology: what controls the spatial and temporal patterns of cell differentiation? Rigorous studies of this problem are necessary to understand basic principles of embryogenesis, to elucidate origins of developmental disorders, and to provide rational guidelines for tissue engineering and regenerative medicine. An increasing number of engineering and synthetic biology approaches invoke the notion of morphogen gradients, defined as concentration profiles of molecules that provide dose-dependent control of gene expression. Experimentally, this paradigm was first established in the early Drosophila embryo, which we use as our model system. We propose to study how morphogens pattern the dorsoventral axis of the embryo, subdividing it into the territories that give rise to the muscle, nerve, and skin tissues. By understanding this system at a deeper, quantitative level, we will elucidate general principles underlying the operation of genetic and multicellular networks that drive development. The broader impacts of our work are in the areas of developing high-throughput and broadly available research technology, in the establishment of publicly available databases, and in the interdisciplinary training of students and postdoctoral fellows. Students and postdoctoral fellows funded by this award will receive interdisciplinary training that will prepare them for independent careers in the rapidly emerging field of Developmental Systems Biology. As part of training in this important field, we will develop graduate and undergraduate courses that use early embryonic development to introduce some the key ideas of genetic, computational, and engineering studies of tissue development and regulation. Rigorous training provided by our studies will contribute to the new generation of scientists and engineers working on both molecular and systems-level aspects of multicellular signaling.

DESCRIPTION (provided by applicant): The Mitogen Activated Protein Kinase (MAPK) signaling pathway is a critical regulator of cellular processes in adult and developing tissues. Deregulated MAPK signaling is associated with a number of diseases, which makes it a key drug target in multiple therapeutic areas. Given a large number of components and levels of regulation within this important pathway, understanding and controlling its function is essentially impossible without quantitative experiments, mathematical modeling, and computational analysis. The terminal patterning system in the early Drosophila embryo is ideally suited for this purpose because of its relative anatomical simplicity and the availability of a large number of genetic tools for the manipulation of MAPK regulators and substrates. We have developed quantitative assays for the in vivo analysis of MAPK phosphorylation and signaling in the terminal patterning system. Based on these assays, in our recently published work we formulated a model according to which the spatial pattern of MAPK signaling in the early embryo is controlled by an enzyme-substrate competition network. Specifically, we proposed that MAPK substrates compete among themselves and with the MAPK phosphatase for binding to the activated MAPK. In addition, we proposed that MAPK substrate competition influences not only the MAPK pathway, but also its interaction with other signaling systems. The work described in this application will provide molecular and functional characterization of the substrate competition mechanism. The main innovation of our proposal is in synthesizing modeling, genetic, and biochemical approaches to developmental signal transduction. By combining our strengths in modeling, genetics, and biochemistry, we are uniquely positioned to formulate and experimentally test systems-level descriptions of MAPK signaling. Going beyond the early Drosophila embryo and MAPK pathway, we propose that substrate competition provides a general signal integration strategy in biomolecular networks where enzymes, such as MAPK, interact with their multiple regulators and substrates.

DESCRIPTION: We propose to combine computational and experimental approaches to investigate the mechanisms of epithelial morphogenesis, defined as the set of processes that transform sheets of cells into three-dimensional (3d) structures of tissues and organs. Studies of epithelial morphogenesis are important for understanding of normal development and for elucidating the origins of developmental abnormalities, such as neural tube defects. We will focus on a type of epithelial morphogenesis that involves only cell shape changes and rearrangements and does not depend on cell divisions or death. Among the examples of this type of morphogenesis are the early stages of heart development in fish, the formation of optic cups in birds, and mesoderm invagination in insects. In our experimental system (Drosophila oogenesis), a sheet of nondividing cells gives rise to an elaborate 3d shape. This transformation is induced by well-understood chemical signals, which specify a fate map, a correspondence between positions of cells within the sheet and their ultimate positions within the 3d structure. Our goal is to understand how the two-dimensional (2d) fate map is transformed into a 3d structure. Answering this question is important in multiple developmental systems, from simple metazoans to humans. Our hypothesis is that 3d epithelial morphogenesis during Drosophila oogenesis is driven by the 2d distribution of mechanical tensions within the patterned cell sheet. We will test this hypothesis by computational modeling and live imaging of epithelial dynamics and by direct analysis of mechanical tensions in patterned epithelial sheets. Our work will lead to an experimentally validated computational framework for 3d epithelial morphogenesis. Given the highly conserved nature of processes involved in epithelial morphogenesis, the results of or studies may shed light on epithelial dynamics in wide range of developmental systems.

? DESCRIPTION (provided by applicant): Our work is designed to provide fundamental understanding of RASopathies, a large class of developmental abnormalities caused by the germline mutations in components of the RAS/MAPK pathway. Patients with these conditions display a broad spectrum of phenotypes, including heart defects, short stature, facial dysmorphisms, and neurocognitive delays. Whole genome sequencing of RASopathies provides new sequence variants in the well characterized components of the RAS pathway, but the functional consequences of these sequence variations is poorly understood. We will quantitatively characterize the functional and phenotypic consequences of the activating mutations in MEK, a core component of the RAS pathway. Focusing on the same group of mutations, we will first determine their effects on the regulation and enzymatic activity of MEK (Aim 1). This will be done using mass spectrometry-based kinetic assays with purified proteins. Next, we will quantify the consequences of activating mutations for the kinetics of RAS signaling in vivo (Aim 2). This will be done using a quantitative imaging approach, with which we will monitor RAS signaling in Drosophila embryos. Finally, we will determine how the same mutations influence RAS-dependent tissue morphogenesis (Aim 3). This will be done using live imaging experiments in zebrafish, focusing on heart development as a model of a morphogenetic event that is commonly affected in RASopathies. Our studies should provide mechanistic insights into the biochemical and morphogenetic effects of mutations identified in a large group of human developmental abnormalities and highlight the importance of using multiple models and quantitative approaches for answering a specific biological question.

Understanding the complex set of signals that control communication between cells in a multicellular organism is a challenging problem that requires a diverse set of tools to solve. This project will use methods from developmental biology, applied mathematics and computer science to uncover the complex signaling patterns that regulate tissue formation in the developing fruit fly (Drosophila) embryo. The quantitative, computational and visualization tools to be developed in this study will be applicable to a broad range of signaling mechanisms in complex three-dimensional tissues or organisms, thereby providing methods of general applicability in biology. In addition, this project will provide interdisciplinary training for students from chemical and biological engineering, molecular biology, and computer science departments, as well as for postdoctoral fellows with applied mathematics and life sciences backgrounds.

Alterations in the activation of receptor tyrosine kinases (RTKs) have been implicated in multiple developmental abnormalities, motivating quantitative studies of developmental RTK signaling. Signaling systems involved in embryogenesis have been highly conserved in evolution, which implies that studies in model organisms, such as Drosophila, yield broadly applicable insights. The early Drosophila embryo provides unique opportunities for high-throughput quantitative experiments, and this project will focus on signaling by the Epidermal Growth Factor Receptor (EGFR), a key regulator of developing tissues in many species. EGFR signaling in the early embryo is accurately described as a temporal pulse that leads to a stable pattern of gene expression, and this project will examine the molecular mechanisms controlling the quantitative parameters of this pulse and its function, as well as establishing experimentally testable models of EGFR signaling in vivo. This project will also develop methods to combine information from different experimental assays that address different aspects of developmental dynamics in different embryos to generate a stereotypical developmental trajectory.

This award is funded jointly by the Systems and Synthetic Biology Program in the Division of Molecular and Cellular Biosciences and the Biomedical Engineering Program in the Division of Chemical, Bioengineering, Environmental and Transport Systems.

PROJECT SUMMARY The proposed research will advance the mechanistic understanding of dynamics in cell clusters with stable intercellular cytoplasmic bridges. This class of multicellular systems is thought to have played a role during the emergence of multicellularity and serves critical functions in present day organisms. Intercellular transport through cytoplasmic bridges gives rise to a wealth of functionally significant collective effects. One key obstacle in understanding the origins of these effects is the current lack of tractable experimental systems that can be studied quantitatively. Our research will use Drosophila oogenesis, an experimental system that provides unmatched opportunities for quantitative studies. This system relies on two types of cell clusters with cytoplasmic bridges: a germline-derived cluster containing the future oocyte and 15 nurse cells, and somatic cell clusters in the epithelium that envelops the germline cluster. Our research identified collective dynamics in both tissue types. First, we discovered that cells in the germline cluster grow in groups defined by the cluster's connectivity. Aim 1 tests the hypothesis that this growth pattern depends on intercellular transport within the cluster and reflects synchronized dynamics of endoreplication cell cycles. Second, we showed that somatic cell clusters display strong clonal dominance, a commonly observed, yet poorly understood effect during developmental growth. Aim 2 tests the hypothesis that clonal dominance emerges as a consequence of spatiotemporal coordination of mitotic cell cycles. Our work uses live imaging and computational modeling to investigate emergent dynamics in an important class of multicellular systems. Given the ubiquitous nature of cell clusters with stable cytoplasmic bridges, results of our studies will have broad impact.