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Broad Impacts (3 pages)
Overview
Living cells can differentiate from one cellular state to another during growth, development, and regeneration. In higher organisms such as mammals, differentiation and proliferation create cells each with unique competence to either self-renew as stem/progenitor cells to maintain organismal/tissue homeostasis with desirable spatial patterns such as that in a stratified epidermis, or transiently proliferate and terminally differentiate to meet the needs of growth and regeneration of various cell types that carry out specialized biological functions at different spatial locations. How are proliferation and differentiation coordinately regulated? Are there regulatory themes that are common to all organisms in order to engineer desired cell differentiations and their spatial patterns?
This interdisciplinary proposal represents a joint effort between a computational mathematician with extensive collaborative experience in experimental biology, and an experimental biologist with prior collaborative experience in mathematical modeling. Specifically, we propose to use a systems biology approach integrating experiments with modeling/simulation to study the development of mammalian epidermal stem/progenitor cells during embryogenesis. The objective of the epidermal morphogenic process is to produce a stratified epidermis of a desired size composed of multiple cell types in proper proportion and spatial position from a single-layered primitive progenitor cells. A successful execution of the stratification process entails a precise and robust control of proliferation, differentiation, and migration, and must involve complex interactions between the extra-cellular environment and the intra-cellular regulatory networks. We will address how these interactions are integrated and disseminated at molecular and cellular levels to generate epidermis with a desirable size and spatial pattern.
One of our overall hypotheses is that a network of a small number of molecular and cellular components and regulations might be able to produce epidermis with correct size and patterns; however, the complexity of a network arises in order to achieve robustness (e.g. with respect to internal or environmental perturbations) of the development and growth process. Our work centers around Ovol1 and Ovol2, two homologous transcription factors with important roles in mammalian epidermal morphogenesis. We will develop new modeling and computational tools, delving into the known and proposed molecular networks encompassing Ovol1 and Ovol2, with the ultimate goal of rising above molecular details to unravel critical regulatory mechanisms that are likely to be generally applicable to multiple organisms and differentiation/developmental processes. We have two specific aims:
Aim 1: To investigate the role of Ovol1-Ovol2 mutual repression in controlling epidermal size and cell type proportion. Although expressed in different cellular compartments in developing epidermis, both Ovol1 and Ovol2 have a proliferation-suppressive role. They directly repress the expression of c-Myc, a proto-oncogene that promotes the proliferation of epidermal cells and has to be suppressed for terminal differentiation. Our preliminary modeling and experimental results suggest that a mutual repression between Ovol1 and Ovol2 produces bi-stability in c-Myc expression. We hypothesize that an Ovol1-Ovol2 mutual repression mechanism is required for robust epidermal morphogenesis by balancing proliferation with growth arrest, which is a prerequisite for terminal differentiation, and will perform modeling and experiments to test this hypothesis.
Aim 2: To investigate the complex molecular interactions that robustly control the spatial stratification of epidermis. Proliferation and differentiation are intimately linked to epidermal stratification. Although morphogen gradients may act globally through the proliferation module that contains the Ovol1-Ovol2 circuit to provide spatial information to cells for the stratification of epidermis, preliminary experimental data suggest that Notch signaling also plays an important role in epidermal stratification. We hypothesize that a cross-talk between the Ovol1-Ovol2-encompassing proliferation module network and a stratification module network is critical for the epidermal system to achieve several key performance objectives in spatial stratification, and will perform modeling and experiments to test this hypothesis.
Education, Outreach, and Diversity
For research at the interface between disciplines, education and outreach are particularly important and challenging. It is not just mathematics and biology that must be communicated to students, researchers, policy-makers and the public, it is also the range of possibilities that arise at the intersection of these fields that must be made known. Achieving these goals requires educational activities at all levels. Outreach must also be conducted to build the capacity of such training programs, and make effective use of the diversity in our nations population. Indeed, the investigators are active participants of many existing education and outreach activities, and some of them are exactly at the interface of mathematics and biology.
Nie is currently the acting director and has been the associate director of an interdisciplinary Ph.D. program on Mathematical and Computational Biology (MCB) at UCI (HYPERLINK "http://mcsb.bio.uci.edu"http://mcsb.bio.uci.edu). He is also a regular instructor for one of the two core sequences of the MCB program. Dai is an actively participant of the MCB program, and has mentored mathematically trained MCB rotation students in their wet laboratory experience, as well as a biologically trained graduate student in her mathematic modeling collaboration. Recently, Nie gave a public lecture for 300 7th8th graders and their parents in Math Count, an annual Orange County mathematics contest, with a title of his presentation Is Mathematics a New Microscope of Biology?. Dai presented scientific posters at Workshops during the Sally Ride Science Festival at UCI, designed to inspire and support 5th-8th grade girls interests in science and math, and served as a judge at the Irvine Unified School District Science Fair. Both Nie and Dai have provided research opportunity for high school seniors and mentored undergraduate student research in their labs. Nie recently supervised an 11th grader student, Brandon Sim, for a research project on modeling of cell lineages. The project won the Science Division of the 2010 Southern California Science and Humanities Symposium (HYPERLINK "http://www.cfep.uci.edu/jshs"http://www.cfep.uci.edu/jshs).
The unique combination of the researchers, students, and the research agenda in this proposal will present more and special opportunities for developing new activities in education and outreach at the interface of mathematics and biology.
Outreach to 4th-6th graders - Effective mathematics and science education has been recognized as a national need. A topic that combines mathematics, an abstract subject, and biology, a subject more easily accessible to students, may be particularly suitable and interesting for students. The PI and Co-PI have been frequent volunteers for classes of 4th-6th graders at a local public school, Turtle Rock Elementary School. We propose to give joint lectures to those 4th-6th graders, starting by helping to organize mathematics and science clubs at the school. We will also take the students to tour our wet and computing laboratories to further engage their interests in mathematics and science. We will transform these activities, if they show any initial success, into a more organized forum involving more researchers for a middle school and a high school in the same community.
COSMOS - The California State Summer School for Mathematics and Science (http://HYPERLINK "http://www.cosmos.uci.edu"www.cosmos.uci.edu) is a month-long residential program for over 150 talented students in grades 8-12, taught by UCI faculty and scientists. The investigators will construct and implement a three-day Cluster on Mathematical Modeling and Computation for Epidermis for COSMOS. The graduate students and postdoctoral fellows participating in the proposed research projects will serve as tutors or instructors for this cluster.
The UCI Minority Science Programs and UCI Undergraduate Research Opportunities Program (UCOP) - The UCI Minority Science Programs, winner of the 2005 Presidents Award for Excellence in Science Math and Engineering Mentoring, provides an outstanding model for how to motivate and mentor undergraduate students from underrepresented groups. The UCOP is a longstanding program that provides opportunities for undergraduates to participate in research throughout the year, and it publishes The UCI Undergraduate Research Journal for undergraduate research results. The PI and co-PI are actively working with both programs to recruit and support minority students and undergraduate students in research. This grant will provide new research opportunities at the interface of mathematics and biology. The graduate students and postdoctoral fellows participating in the proposed research will assist the two investigators to mentor undergraduate students from both programs.
Graduate course - We propose to jointly teach one lecture on stem cells and epidermis for a core course of the MCB graduate program: Critical thinking in systems biology (Development Cell 203, HYPERLINK "http://lander-office.bio.uci.edu/CirticalThinking" http://lander-office.bio.uci.edu/CirticalThinking). This special 10-week discussion course is structured to cultivate the ability of independent and critical assessment of published research, and to expose students to cross-disciplinary perspectives that will facilitate interdisciplinary collaborations. Each week, the students are assigned a pair of papers carefully selected from the Systems Biology literature. The papers are chosen primarily for their didactic value, and may be new or old. Prior to the two-and-half hour class meeting each week, the students are instructed to work in teams to decipher and critique the weeks papers. The graduate students and postdoctoral fellows participating in the proposed research will also attend this lecture.
Journal club/seminar - We propose to develop a journal club on Modeling and Computation of Stem Cells and Epidermal Systems. The journal club will be organized by two graduate students and postdoctoral fellows: one from Mathematics and one from Biology. In addition to the bi-weekly journal club activity, each year the club will invite three leading experts in the area of stem cells and epidermal systems to lecture at UCI.
Diversity - Nie supervised one female Ph.D. student, and currently is supervising three female postdoctoral fellows and two female Ph.D. students. Dai supervised two female Ph.D. students, three female M.S. students, and is currently supervising two female Ph.D. students and one female postdoctoral fellow. Both Dai lab and Nie lab have had minority rotation students. Dai has served multiple times as a faculty panel member at the UCI Minority Graduate Research Conference. The investigators are committed to diversity in their research groups, and will continue to be proactive in recruiting and supporting female and underrepresented minority students. The University of California at Irvine is a Minority Institution according to U.S. Department of Education criteria.
Data sharing - We will establish a database for new models and computational tools on stem cells, cell lineages and epidermal systems developed for the proposed research. We will assemble them with existing models sharing similar features, providing open access to the broader research community. The database for models and computational tools will be classified in terms of functions, scales, and critical features such as deterministic/stochastic, discrete/continuous, and spatial/non-spatial. Several software developed by PI Nie for studying spatial dynamics of development and growth has already been uploaded in a portal website for systems biology (HYPERLINK "http://systems-biology.org/resources/"http://systems-biology.org/resources/) organized by a group of laboratories at the Centers of Excellence for Systems Biology that include UCI Center for Complex Biological Systems.
Results from Prior NSF Support
Qing Nie (PI) was supported by NSF-DMS051169 (PI, 7/1/05-6/30/09). The major goal of the grant was to develop computational methods for interface dynamics. This grant resulted in seven publications ADDIN EN.CITE ADDIN EN.CITE.DATA (1-7). In ADDIN EN.CITE ADDIN EN.CITE.DATA (1-3), we developed a new class of efficient temporal schemes for stiff reaction-diffusion systems arising from modeling biological systems. This new method can be directly applied to models for diffusing molecules in this proposal. In ADDIN EN.CITE ADDIN EN.CITE.DATA (4-6), we developed accurate and fast front tracking methods for solving nonlinear PDEs with moving boundaries. The continuum models for epidermal growth proposed in this application involve moving boundaries, and our past experiences on computational tools for PDEs with moving boundaries will greatly benefit the proposed study in this application.
Xing Dai (Co-PI) has been support by NIH and other non-NSF funding sources during the past five years.
Intellectual Merit (12 Pages)
SIGNIFICANCE
Epidermal systems The skin epidermis is an excellent system to dissect the complex yet dynamic regulatory processes of tissue morphogenesis because: 1) it is easily accessible to experimental interrogations; 2) it has a well-characterized spatial layout, with different cellular stages expressing different but well-characterized protein markers and occupying specific locations (e.g. K14 marks the stem/early progenitor cells present in the basal layer; K1 marks the late progenitor/early differentiating cells in the spinous layers of the suprabasal compartment; loricrin marks the late differentiating cells present in the suprabasal granular layers) (Fig. 1).
The mature epidermis arises from a single-layer of basally attached (to basement membrane) primitive progenitor cells during mid-embryogenesis. During epidermal development, these progenitor cells choose between distinct cellular paths, namely proliferating symmetrically in a plane parallel to the basement membrane, dividing asymmetrically in a plane perpendicular to the basement membrane, cell cycle arrest, detachment from the basement membrane and migration upwards, and terminal differentiation ADDIN EN.CITE Fuchs20021561561564Fuchs, E.Raghavan, S.Howard Hughes Medical Institute, Department of Molecular Genetics and Cell Biology, The University of Chicago, Chicago, Illinois 60637, USA. lain@midway.uchicago.eduGetting under the skin of epidermal morphogenesisNat Rev Genet199-209332002Mar 111972157http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=11972157(8). The epidermis regenerates itself throughout postnatal life due to the presence of stem cells capable of recapitulating what happens during embryonic development. Despite the identification of important signaling and transcriptional activities that control epidermal stem/progenitor cell proliferation and differentiation, important questions remain. How does an epidermal stem cell integrate different molecular activities to ensure a state of long-term self-renewal and to prevent premature entry into terminal differentiation? How then are various regulatory modules integrated to allow the otherwise proliferating progenitor cells to exit cell cycle and terminally differentiate? How are these molecular and cellular events orchestrated at a systems level to produce a stratified epidermis? While experimental approaches are continuously needed to clarify known regulations and to uncover additional control modules, a systems approach is imperative and timely to integrate multiple regulatory modules, derive systems-level behavioral principles, and predict novel regulations that function to enhance systems performance.
Ovol genes and epidermal development The Dai laboratory has a long-standing history of studying two related transcription factors that regulate epidermal development: Ovol1 and Ovol2. Ovol1 and Ovol2 are homologs that share a highly conserved zinc finger DNA-binding domain, and bind to nearly identical cognate DNA sequences ADDIN EN.CITE ADDIN EN.CITE.DATA (9). Both encode transcriptional repressors that are able to recruit histone deacetylase complexes to target genes and hence suppress transcription ADDIN EN.CITE ADDIN EN.CITE.DATA (10). Despite similar biochemical activities, the two proteins are expressed at distinct cellular locations in the developing epidermis Ovol1 is predominantly expressed in the suprabasal spinous layers, and Ovol2 predominantly in the basal layer ADDIN EN.CITE ADDIN EN.CITE.DATA (11, 12). In vitro experiments using cultured keratinocytes (isolated epidermal cells) confirmed that Ovol1 is up-regulated as cells are induced to undergo cell cycle arrest and terminally differentiate, whereas Ovol2 expression is down-regulated upon differentiation ADDIN EN.CITE ADDIN EN.CITE.DATA (11, 13).
Studies using knockout mouse and cell culture models have elucidated a functional requirement for Ovol1 in the cell cycle exit of proliferating progenitor cells ADDIN EN.CITE ADDIN EN.CITE.DATA (10). Consequently, the developing e p i d e r m i s i n O v o l 1 - d e f i c i e n t m i c e i s t h i c k e r t h a n n o r m a l , a n d c o n t a i n s a n e x p a n d e d s p i n o u s c o m p a r t m e n t ( F i g . 2 ) . I n t e r e s t i n g l y , t h i s f u n c t i o n i s d o w n s t r e a m o f a v e r y i m p o r t a n t g r o w t h i n h i b i t o r y s i g n a l i n g p a t h w a y , n a m e l y T G F - b. O v o l 1 g e n e e x p r e s s i o n i s i n d u c e d u p o n T G F - b t r e a t m e n t , a n d O v o l 1 - d e f i c i e n t c e l l s f a i l t o u n d e r g o g r o w t h a r r e s t i n r e s p o n s e t o T G F - b A D D I N E N . C I T E A D D I N E N . C I T E . D A T A ( 1 0 , 1 4 ) . W h i l e i n v i t r o k e r a t i n o c y t e e x p e r i m e n t s h a v e u n c o v e r e d a c r i t i c a l f u n c t i o n f o r O v o l 2 i n m a i n t a i n i n g t h e long-term proliferative potential of epidermal progenitor cells (see below), germline knockout of Ovol2 in mice results in early lethality before epidermal development ADDIN EN.CITE Mackay200638038038017Mackay, D. R.Hu, M.Li, B.Rheaume, C.Dai, X.Department of Biological Chemistry, College of Medicine, D250 Med Sci I, University of California, Irvine, CA 92697-1700, USA.The mouse Ovol2 gene is required for cranial neural tube developmentDev BiolDev Biol38-5229112006Mar 116423343http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=16423343(15) whereas epidermis-specific knockout of Ovol2 yielded no remarkable epidermal defects (data not shown). In striking contrast, the overexpression of Ovol2 in its habitual basal compartment leads to reduced basal proliferation and a significant thinning of the epidermal spinous layers (Fig. 3). Mechanistically, both Ovol proteins repress the transcription of c-Myc ADDIN EN.CITE ADDIN EN.CITE.DATA (10, 16), the expression level of which is proposed to govern whether a stem/progenitor cell should exit into a transient amplification (TA) mode, how well a TA cell proliferates, or whether it should terminally differentiate ADDIN EN.CITE Watt200843343343317Watt, F. M.Frye, M.Benitah, S. A.Cancer Research UK Cambridge Research Institute, Li Ka Shing Centre, Robinson Way, Cambridge CB2 0RE, UK. fiona.watt@cancer.org.ukMYC in mammalian epidermis: how can an oncogene stimulate differentiation?Nat Rev Cancer234-42832008/02/23AnimalsCell AdhesionCell DifferentiationCell ProliferationChromatin/metabolismEpidermis/*metabolism*Gene Expression Regulation*Gene Expression Regulation, NeoplasticHumansKeratinocytes/cytology/metabolismMiceModels, Biological*OncogenesProto-Oncogene Proteins c-myc/*metabolismSkin Neoplasms/metabolism2008Mar1474-1768 (Electronic)
1474-175X (Linking)18292777http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=182927772494614nrc2328 [pii]
10.1038/nrc2328eng(17).
An integrated experimental and computational approach While experimental studies portrait the details of isolated Ovol regulatory circuits, mathematical modeling and computation are needed to examine how Ovol proteins fit into the complex regulatory network involving multiple factors and how they serve to enhance the performance objectives of epidermal morphogenesis. The Ovol proteins are excellent candidates for a systems biology approach because they are nodal points that collect and interpret upstream signals (e.g., TGF- b s i g n a l i n g ) , a n d t r a n s l a t e t h e s e s i g n a l s i n t o d i f f e r e n t i a l l e v e l s o f c r i t i c a l d o w n s t r e a m e f f e c t o r s ( e . g . , c - M y c ) t h a t e x e c u t e t h e a c t u a l c h a n g e s i n g r o w t h a n d d i f f e r e n t i a t i o n . A m o n g m a n y i n t e r e s t i n g q u e s t i o n s c o n c e r n i n g O v o l 1 / O v o l 2 , o n e w o n d e r s w h y t w o d i stinct homologs are needed in different cellular compartments to perform seemingly similar cellular/molecular functions. What is the significance of this in epidermal morphogenesis?
The Nie lab has joined force with the Dai lab using a systems biology approach to study Ovol1/Ovol2 function in epidermis, resulting in three joint publications ADDIN EN.CITE ADDIN EN.CITE.DATA (16, 18, 19). First, we established a mathematical framework for epidermis system using multi-scale and continuum models, and found that homeostasis can be achieved if the differentiation is regulated ADDIN EN.CITE Cai200971717117Cai, A. Q.Peng, Y.Wells, J.Dai, X.Nie, Q.Multi-scale modelling for threshold dependent differentiationMath. Model of Nat. Phenom103-117442009(18). Next, through an integrated experimental and theoretical study ADDIN EN.CITE Wells200943443443417Wells, J.Lee, B.Cai, A. Q.Karapetyan, A.Lee, W. J.Rugg, E.Sinha, S.Nie, Q.Dai, X.Department of Biological Chemistry, School of Medicine, University of California, Irvine, California 92697, USA.Ovol2 suppresses cell cycling and terminal differentiation of keratinocytes by directly repressing c-Myc and Notch1J Biol Chem29125-35284422009/08/25AnimalsCell CycleCell DifferentiationCell Line, TumorCell Nucleus/metabolismCell Separation*Gene Expression RegulationHumansKeratinocytes/*cytologyMiceProto-Oncogene Proteins c-myc/*metabolismReceptor, Notch1/*metabolismStem Cells/cytologyTranscription Factors/*metabolism/physiology2009Oct 161083-351X (Electronic)
0021-9258 (Linking)19700410http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=197004102781457M109.008847 [pii]
10.1074/jbc.M109.008847eng(16), we found that Ovol2 participates in the regulation of proliferation potential and proliferation rate via c-Myc. This work allows us to add Ovo l 2 t o t h e l i s t o f k n o w n r e g u l a t o r s o f e p i d e r m a l p r o g e n i t o r c e l l p r o l i f e r a t i o n , i n c l u d i n g t h e c - M y c a n d T G F - a s i g n a l i n g p a t h w a y . I m p o r t a n t l y , o u r e x p e r i m e n t s a l s o d e m o n s t r a t e t h a t O v o l 2 s u p p r e s s e s t e r m i n a l d i f f e r e n t i a t i o n b y r e p r e s s i n g t h e e x p r e s s i o n o f N o tch1 ADDIN EN.CITE Wells200943443443417Wells, J.Lee, B.Cai, A. Q.Karapetyan, A.Lee, W. J.Rugg, E.Sinha, S.Nie, Q.Dai, X.Department of Biological Chemistry, School of Medicine, University of California, Irvine, California 92697, USA.Ovol2 suppresses cell cycling and terminal differentiation of keratinocytes by directly repressing c-Myc and Notch1J Biol Chem29125-35284422009/08/25AnimalsCell CycleCell DifferentiationCell Line, TumorCell Nucleus/metabolismCell Separation*Gene Expression RegulationHumansKeratinocytes/*cytologyMiceProto-Oncogene Proteins c-myc/*metabolismReceptor, Notch1/*metabolismStem Cells/cytologyTranscription Factors/*metabolism/physiology2009Oct 161083-351X (Electronic)
0021-9258 (Linking)19700410http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=197004102781457M109.008847 [pii]
10.1074/jbc.M109.008847eng(16), which is known to promote the stem/progenitor-TD switch, manifested as a switch from a basal fate to a suprabasal spinous fate ADDIN EN.CITE Dotto200830930930917Dotto, GPNotch tumor suppressor functionOncogeneOncogene5115-51232738200811954515625023603632related:sINji5_45qUJhttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=11954515625023603632related:sINji5_45qUJ(20). Motivated by these findings and the fact that Notch signaling occurs through adjacent cell-cell interactions, we recently developed a discrete cell model in 3D that allows the incorporation of Notch signaling for studying epidermal stratification ADDIN EN.CITE Christley201025925925917Scott ChristleyBriana LeeXing DaiQing NieIntegrative multicellular biological modeling: a case study of 3D epidermal development using GPU algorithmsBMC Systems Biology107412010(19).
In this application, we propose to extend beyond a purely molecular modeling of the system to a multi-scale approach, incorporating the interactions within intracellular molecular networks while following the spatial and temporal dynamics of cells, such that the modeling outputs can be better related to experimental data. In a typical experiment for epidermal stem cell dynamics, one probes the intra-cellular molecular components, measures growth of different types of cells with best available methods and markers, correlates the observations, and finally infers the role of the molecular components in the whole system. Analysis of models consisting of experimentally measurable components as well as components that cannot be directly measured and manipulated using current methodology will lead to a better understanding of the overall behavior of the system.
INNOVATION
An important innovative aspect of the proposed research is a tight integration of mathematical modeling with experimentation. Also innovative, and we believe a strength of this proposal, is that the goal of modeling here is not just to test whether a particular mechanistic scheme is correct by fitting observations to models, but also to explore whether a given mechanistic scheme is capableover any plausible range of parametersof serving as a strategy for performing a certain type of job (e.g. achieving robust development or regeneration of the tissue in response to perturbations). In addition, the modeling and computational techniques proposed here combine complex intra-cellular interactions with extra-cellular environment and morphogens in both discrete and continuous set-up, and hence are challenging, innovative, and will likely provide an exemplary paradigm for studies of other biological systems. This proposal is also innovative in terms of the hypotheses that are put forth. For example, our hypothesis that a specific regulatory circuitry in epidermal development exists to control the correct size of the tissue and some other regulatory circuitries exist for the purpose of specifying stratification is novel. Likewise, our approach of dividing the complex network into one proliferation module and one stratification module, and our hypothesis that cross-talks between the two modules exist in order to achieve robustness are entirely novel. This hypothesis, if validated, has the potential to change the way biologists think about key signaling pathways and regulatory networks.
APPROACH
Aim 1: Investigate the role of Ovol1-Ovol2 mutual repression in controlling epidermal size and cell type proportion
Rational Intuitively, during epidermal morphogenesis, both positive cross-talks and negative feedback mechanisms must exist between proliferation and differentiation to achieve tissue growth and maturation while put a brake in the system to prevent excessive or over-accelerated growth. Only so the tissue can reach, but does not exceed its desired final size. Under pathological conditions such as embryonic wound healing, positive cross-talks might be further enhanced whereas negative feedback diminished. While Ovol1 and Ovol2 both negatively regulate proliferation (via repressing c-Myc), Ovol2 also directly impinges on differentiation via repressing Notch1 and its downstream signaling. Hence, these two factors influence both short and long-term growth dynamics of a cell population, and their interactions may be exploited by the systems to achieve differential regulations of proliferation and differentiation under normal as well as perturbed situations.
We consider specifically a model of mutual repression between Ovol1 and Ovol2. Such a strategy can be particularly useful when it comes to two homologous genes as it provides a means for one homolog to sense the state of the other (in biological systems the loss of one often causes an up-regulation of another as part of a compensatory mechanism). More importantly, mutual repression between different genes can drive cellular differentiation where the strength of each repression dictates the final outcome of genes being up-regulated or down-regulated ADDIN EN.CITE Cinquin200569696917Cinquin, O.Demongeot, J.CoMPLEX, University College London, Gower Street, London WC1E 6BT, UK. o.cinquin@ucl.ac.ukHigh-dimensional switches and the modelling of cellular differentiationJ Theor Biol391-41123332005/01/18AnimalsCell Differentiation/genetics*Cell Physiology*Genes, Switch*Models, Genetic2005Apr 70022-5193 (Print)15652148http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15652148S0022-5193(04)00499-0 [pii]
10.1016/j.jtbi.2004.10.027eng(21). We have previously shown that Ovol1 represses Ovol2 ADDIN EN.CITE ADDIN EN.CITE.DATA (12). We reason that Ovol2 might also repress Ovol1, and that Ovol1 represses c-Myc expression more efficiently than Ovol2. This will enable Ovol2 to counteract Ovol1 repression of c-Myc, allowing c-Myc to be expressed at a low level unless there is a high level of extra-cellular stimuli (e.g., TGF- b) . I n e s s e n c e , T G F - b l e v e l s g o v e r n t h e s w i t c h b e t w e e n O v o l 1 a n d O v o l 2 d o m i n a n t s t a t e s , a n d t h e i n t e r a c t i o n b e t w e e n t h e m i n t u r n g o v e r n s c - M y c l e v e l s a n d c o n s e q u e n t l y g r o w t h a r r e s t ( s e e F i g . 4 a - b ) .
O v e r a l l h y p o t h e s i s A n O v o l 1 - O v o l 2 m u t u a l r e p r e s s i on mechanism is required for cellular differentiation so that growth arrest occurs and the actual process of terminal differentiation begins, resulting in robust epidermal morphogenesis. The mutual repression mechanism allows the cell population to reach a desired, homeostatic final size relatively faster compared to a single regulatory element (e.g Ovol1 or Ovol2 only). While Ovol1 and Ovol2 have a similar function at molecular/cellular levels, their mutual repression together with different strengths in proliferation and differentiation regulation in cells allows the cell population to efficiently reach homeostasis during normal morphgenesis, or regeneration after perturbations.
Hypothesis 1 Ovol2 directly represses Ovol1 expression and Ovol1 represses the c-Myc promoter more efficiently than Ovol2.
Preliminary modeling and experimental data To explore the notion that Ovol2 represses Ovol1 expression, we experimentally depleted Ovol2 from cultured keratinocytes using the siRNA knockdown technology, and examined Ovol1 expression. A 1.6-fold increase (p value < 0.0001) in Ovol1 mRNA was observed in the knockdown cells. Moreover, we performed luciferase reporter assays, and found that exogenously added Ovol2 protein represses the activity of Ovol1 promoter (which drives luciferase activity) in a dosage-dependent manner (Fig. 4c). Together, our experimental data support an Ovol1-Ovol2 mutual repression.
To investigate the functional significance of this mutual repression, we used classical populatio n d y n a m i c s m o d e l s t o c o m p a r e t h e g r o w t h d y n a m i c s b e t w e e n 1 ) a c e l l p o p u l a t i o n c o n s i s t i n g o f t w o r e v e r s i b l e s u b p o p u l a t i o n s ( O v o l 1 a n d O v o l 2 d o m i n a n t c e l l s ) a n d 2 ) a h o m o g e n e o u s p o p u l a t i o n ( F i g . 5 ) . I n b o t h m o d e l s w e l e t t h e c e l l s e c r e t e d T G F - b t o f e e d b a c k to repress cell growth. We found that, in some parameter regimes, the population with two subpopulations can obtain a faster speed to reach homeostasis than the homogeneous population (Fig. 5). This simple, preliminary model suggests that populational growth arrest is possible through TGF- b i n d u c t i o n o f O v o l 1 , l e a d i n g t o a n O v o l 1 d o m i n a n t p o p u l a t i o n , a n d t h a t O v o l 1 i s l i k e l y t o b e a s t r o n g e r r e p r e s s o r o f c - M y c t h a n O v o l 2 .
I t h a s b e e n h y p o t h e s i z e d t h a t d i f f e r e n t i a l p r o l i f e r a t i o n p o t e n t i a l s e x i s t w i t h i n t h e e p i d e r m i s w i t h s l o w b u t l o n g - t e r m proliferating stem cells and fast but transiently proliferating TA cells. Recent challenges to this paradigm is the model that the epidermis consists of a single progenitor pool of TA-like cells ADDIN EN.CITE Clayton200712212212217Clayton, E.Doup, D.Klein, A.Winton, D.Simons, B.Jones, P.MRC Cancer Cell Unit, Hutchison-MRC Research Centre, Cambridge CB2 0XZ, UK.A single type of progenitor cell maintains normal epidermisNature185-944671322007Mar 817330052http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=17330052(22) in homeostatic conditions. Given our finding that regeneration time can be improved by differential proliferation between two interacting populations, we propose that the epidermis consists of cell populations that can make reversible transitions (unlike the irreversible stem-TA transition) and that homeostasis consists mostly of a single dominant population. Such reversible fate switches will help reconcile the seemingly contradictory experimental findings and might be an important novel aspect for regulating cell population growth. The effects of having two subpopulations are most striking in conditions away from homeostasis. In particular when cell density is much smaller than homeostasis, e.g. a wound, it gives rise to the transient growth that is dominated by the fast proliferating subpopulation ADDIN EN.CITE Jones200834634634617Jones, PhilipSimons, Benjamin DPhilip Jones is at the MRC Cancer Cell Unit, Hutchison/MRC Research Centre, Cambridge, CB2 2XZ, UK. phj20@hutchison-mrc.cam.ac.ukEpidermal homeostasis: do committed progenitors work while stem cells sleep?Nat Rev Mol Cell Biol82-8912008Jan 117987044http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=17987044(23).
Plan We will continue to study and expand the intra-cellular network model (Fig. 5) to uncover and analyze conditions for fast regeneration of a homeostatic state. We will perform similar analytical studies and computational explorations as previously used for the study of olfactory epithelial regeneration ADDIN EN.CITE Lo200975757517Lo, W.CChou, C. S.Gokoffski, K.Calof, A.LWan, F.Y.MLander, A. D.Nie, Q.Feedback regulation in multistage cell lineagesMathematical Biosciences and EngineeringMathematical Biosciences and Engineering59-82612009(24) ADDIN EN.CITE Lander200935935935917Lander, ADGokoffski, KKWan, FYMNie, QCalof, ALCell lineages and the logic of proliferative controlPLoS Biole100001571200910009334599233938262related:VqNdSclM6IoJhttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=10009334599233938262related:VqNdSclM6IoJ(25). In particular, we will compare the time scale of the reversible switching between Ovol1 and Ovol2 states, and the proliferation rates of Ovol1 or Ovol2 dominate cells and how they affect the total population size and the proportion between Ovol1 and Ovol2 subpopulations. Because of the existence of multiple steady-states of the models, we will explore parameter regimes to search for insight into how the relative proportions of Ovol1 and Ovol2 dominant populations are regulated to achieve a unique homeostatic state consistent with experimental observations.
Experimentally, 1) we will test whether the repression of Ovol1 promoter by Ovol2 is a direct effect. We will repeat the luciferase reporter assays, but this time using deletion fragments of, or point mutations in, the Ovol1 promoter to delineate the Ovol2-reponsive element. Moreover, we will perform chromatin immunoprecipitation (ChIP) experiments, a method to detect occupancy of a transcription factor at its target promoter, to test if Ovol2 physically binds to the Ovol1 promoter at the predicted site(s). ChIP experiments will be performed using primary keratinocytes isolated from wild-type as well as Ovol2-deficient newborn mice, as the latter serves as a useful specificity control. 2) We will compare the repression and binding strengths of Ovol1 and Ovol2 on c-Myc promoter using luciferase reporter and ChIP assays, respectively. Since Ovol1 and Ovol2 antibodies differ in their affinities for their cognate proteins, we will generate constructs expressing Ovol1 or Ovol2 but each fused to a common antigen tag (e.g., Flag). These constructs will be transiently transfected into cultured keratinocytes for reporter assays, and stably transfected into these cells for ChIP assays. Relative repression strength will be calculated as fold repression per microgram of protein expressed (detected using an anti-Flag antibody). Relative binding strength will be calculated as ChIP signal per microgram of protein expressed (both detected using the anti-Flag antibody). Using this method, we will be able to directly compare the repression or binding strength of Ovol1 or Ovol2 on c-Myc promoter.
Hypothesis 2 The Ovol1-Ovol2 mutual repression is an important strategy for efficient reversible fate switches. Specifically, the Ovol1-Ovol2 mutual repression may form a bi-stable switch, and TGF-( can induce a unidirectional switch from Ovol2-dominant to Ovol1-dominant expression in cells. How cells might achieve reversible expression between Ovol1 and Ovol2 is unclear. Stochastic gene expression can induce reversible switching ADDIN EN.CITE Macarthur200946646646617Macarthur, Ben DMa'ayan, AviLemischka, Ihor RSystems biology of stem cell fate and cellular reprogrammingNat Rev Mol Cell Biol1-102009Sep 9http://dx.doi.org/10.1038/nrm2766(26). Our preliminary data suggest that the interaction between cell division and a mutual inhibition motif also allow for bi-directional switching ADDIN EN.CITE Cai46846846817Cai, A. Q.Wang, L.Nie, QMutual Inhibition Promotes Stable Bimodal Distributionpreprint(27). We hypothesize that a synergy among stochasticity in gene expression, mutual inhibition, and cell division is critical for achieving the correct portions of different cell types (e.g. a bimodal population distribution in Ovol1 and Ovol2) and for maintaining robust homeostasis.
Preliminary modeling data We have developed multi-scale models that couple cell population with gene expressions based on our previous work ADDIN EN.CITE Cai200971717117Cai, A. Q.Peng, Y.Wells, J.Dai, X.Nie, Q.Multi-scale modelling for threshold dependent differentiationMath. Model of Nat. Phenom103-117442009(18). Such type of model allows straightforward inclusion of stochastic effects (e.g. stochastic participation of intra-cellular components during cell division) and direct comparison with experimental data (e.g. FACS data which display cell number as a distribution of gene expression level). Our preliminary simulations suggest that the feedback through TGF-( repression of c-Myc and its induction of Ovol1 is critical for reaching homeostasis (Fig. 6). The bidirectional switching between Ovol1 and Ovol2 dominant states inside cells are due to stochastic partition of molecules to daughter cells after division.
Plan First, we will explore the conditions of the multi-scale model that allow for a robust bimodal cell distribution in Ovol1- and Ovol2-expressing states as observed in experiments. Next, we will study and uncover the conditions in which the system can reach homeostasis after wounding. Clearly, the capability of bidirectional switching in a bi-stable system is important in this regard. We will incorporate the stochastic dynamics of molecules ADDIN EN.CITE Friedman200631531531517Friedman, NirCai, LongXie, X SunneyDepartment of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, USA.Linking stochastic dynamics to population distribution: an analytical framework of gene expressionPhys. Rev. Lett.16830297162006Oct 2017155441http://prl.aps.org/abstract/PRL/v97/i16/e168302(28) ADDIN EN.CITE Cai200628228228217Cai, LongFriedman, NirXie, X SunneyDepartment of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, Massachusetts 02138, USA.Stochastic protein expression in individual cells at the single molecule levelNature358-6244070822006Mar 1616541077http://www.nature.com/nature/journal/v440/n7082/full/nature04599.html(29) into our current multi-scale model to study effect of noise in maintaining homeostasis. The analysis and simulation from the model will show whether the epidermal development system is constrained by the stochasticities, or instead exploits them to reach performance objectives such as fast regeneration to a unique homeostatic state.
Experimentally, we will examine whether Ovol1 and Ovol2 are required for a speedy recovery after wounding. Specifically, we will use dermal punches to create wounds in the backskin of newborn Ovol1-/- and Ovol2 skin-specific knockout mice. One wound will be generated per mouse, and three mice will be used per genotype. The wound front will be excised at different time points after wounding (up to 14 days, when wound is normally closed). Hematoxylin/Eosin staining will be performed on skin sections to compare the kinetics of wound closure in wild-type and Ovol1 or Ovol2 mutant skin. The final size and morphology of the regenerated skin will also be assessed. Moreover, the expression of proliferation and differentiation (different layers) markers will be examined to monitor the proliferative activity as well as distribution of different cell types in the regenerated skin. All reagents and methods have been established in the Dai laboratory. If time permits, we will perform similar experiments on skin-specific knockout mice of Smad4, a common effector of TGF- b s i g n a l i n g t h e l o s s o r r e d u c t i o n o f w h i c h w i l l r e s u l t i n c o m p r o m i s e d T G F - b s i g n a l i n g A D D I N E N . C I T E A D D I N E N . C I T E . D A T A ( 3 0 ) . A l t h o u g h p r e v i o u s s t u d i e s h a v e d e m o n s t r a t e d t h e i n v o l v e m e n t o f S m a d 4 i n w o u n d h e a l i n g A D D I N E N . C I T E A D D I N E N . C I T E . D A T A (31), our focus will be on analyzing the healing kinetics and the relative presence of Ovol1- and Ovol2-expressing epidermal cells in the healing wound.
Modeling and computational challenges Our proposed multi-scale model is based on population balance framework which uses conservation of mass to describe the cell distributions in terms of the intra-cellular components ADDIN EN.CITE Mantzaris200666666617Mantzaris, N. V.Department of Chemical and Biomolecular Engineering and Bioengineering Department, Rice University, Houston, TX 77005, USA. nman@rice.eduStochastic and deterministic simulations of heterogeneous cell population dynamicsJ Theor Biol690-70624132006/02/21AlgorithmsAnimalsCell Division/*physiologyFeedback/physiologyLac Operon/physiology*Models, BiologicalMonte Carlo Method*Population DynamicsStochastic Processes2006Aug 70022-5193 (Print)16487980http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=16487980S0022-5193(06)00007-5 [pii]
10.1016/j.jtbi.2006.01.005eng(32). In this framework, the model couples intra-cellular regulations described in a system of ODEs with cell population and cell division in terms of hyperbolic PDEs (Fig. 6a). Previously, we applied such framework to c-Myc-dependent population growth and differentiation ADDIN EN.CITE Cai71717117Cai, A. Q.Peng, Y.Wells, J.Dai, X.Nie, Q.Multi-scale modelling for threshold dependent differentiationMath. Model of Nat. Phenom103-117442009(18), and the modeling study provided many insights on population distributions in terms of c-Myc and regulations needed to maintain homeostasis. The output of such modeling approach is directly related to experimental observations, and it can be easily used for testing a specific hypothesis. This approach has been less explored for systems involving more than one intra-cellular component partly because it requires solving hyperbolic PDEs in high dimensions. We propose to use higher order finite difference Weighted Essentially Non-oscillatory (WENO) schemes ADDIN EN.CITE Jiang199684848417Jiang, G. S.Shu, C. W.Brown Univ,Div Appl Math,Providence,Ri 02912Efficient implementation of weighted ENO schemesJournal of Computational PhysicsJournal of Computational Physics202-2281261shock-capturing schemesflow1996Jun0021-9991ISI:A1996UQ16400017<Go to ISI>://A1996UQ16400017English(33) to solve these types of models. The WENO method, which evaluates the numerical flux dimension by dimension in rectangular domains, and has computational complexity proportional to the total number of grid points, is particularly suitable for the multi-scale models based on population balance.
Aim 2: Investigate the complex molecular interactions that robustly control the spatial stratification of epidermis
Rational Control of proliferation and differentiation is intimately linked to epidermal stratification. Intuitively, the ultimate spatial pattern should be jointly governed by cell division rates and differentiation probability, size and shape of the cell populations, and their interactions with the environment and other cells to provide the spatial information that controls cellular behavior. The objective of this Aim is to investigate and to experimentally validate additional molecular interactions and control strategies that are used in combination with the molecular circuitry described in Aim 1 to robustly form the stratified layers of different cell types in the mature epidermis.
Some of the key performance objectives that we know about epidermal stratification are: 1) there is a short patterning range as the mature epidermis is a very thin structure of only 6-8 live cell diameters (excluding cornified layers); 2) there are sharp boundaries of gene expression (K1, K14, etc.) between layers; 3) the basal layer is only a single cell thickness; 4) proliferation occurs almost exclusively in the basal layer; 5) patterning should be robust despite of the likely presence of gene expression noise, cell variability, extracellular noise, and variation in environment to a population of heterogeneous cells; and 6) the original tissue structure needs to be regenerated after severe perturbations such as one or more layers being removed. These objectives are quite strict and have to be satisfied by the molecular circuitry, which, as indicated above, also has to satisfy the proliferatio n a n d d i f f e r e n t i a t i o n p e r f o r m a n c e o b j e c t i v e s f r o m A i m 1 .
I n A i m 2 a , w e w i l l s t a r t w i t h t h e b a s i c c o r e O v o l 1 / O v o l 2 m o d u l e f r o m A i m 1 a n d i n t r o d u c e e x t r a c e l l u l a r m o r p h o g e n g r a d i e n t s o f T G F - a a n d T G F - b a s t h e m e c h a n i s m t o a c h i e v e s t r a t i f i c a t i o n ( F i g . 8 ; p r o l iferation module in Fig. 11). As one adds more performance objectives to a system, in this case requiring the Ovol1/Ovol2 proliferation module to also control spatial stratification, then new constraints are created which may require tradeoffs in the ability of the system to satisfy its other proliferation objectives. One of our goals is to uncover these new constraints and the corresponding tradeoffs, thus providing us with knowledge about the effectiveness and limitations of morphogen gradient systems in achieving stratification.
Our expectation is that the morphogen gradients will be sufficient to specify stratification of the epidermal layers, but they may not be robust enough to jointly satisfy all performance objectives for proliferation, differentiation and stratification. For example, the basal layer is just a single cell layer thick, so the gradients must have a very short length scale over a couple cell diameters thus suggesting either an extreme value for the decay rate of the morphogens, an upper limit on the diffusion rate of the morphogens, or both. Furthermore, our preliminary experimental findings (Figs. 2, 3) that loss of Ovol1 and overexpression of Ovol2 result in overall a thicker and thinner epidermis with more or fewer spinous layers, respectively, but do not destroy stratification per se, suggest that the Ovol1/Ovol2 module does have some input into the stratification process but additional control circuit(s) is used to initiate stratification.
In Aim 2b, we study the role of cell-cell contact-mediated communication via the Notch signaling pathway. We postulate that Notch signaling constitutes a separate molecular circuit, independent from the Ovol1/Ovol2 circuit for proliferation and differentiation, to achieve epidermal stratification (Fig. 9; stratification module in Fig. 11). Indeed, experimental evidence indicates that Notch signaling is a key component in epidermal stratification where it functions as a differentiation switch for basal cells as they move into the spinous layer ADDIN EN.CITE ADDIN EN.CITE.DATA (34, 35). Interestingly, Ovol2 has been experimentally shown to inhibit Notch1 gene expression, which can be taken as evidence to suggest that there are direct interactions between the proliferation and stratification modules. Is this stratification module more capable of achieving the performance objectives of stratification than morphogen gradients? Which objectives can be better achieved with mechanisms utilizing local (Notch signaling) versus global (TGF- a a n d T G F -b g r a d i e n t s ) s p a t i a l i n f o r m a t i o n ? W e w i l l s t u d y t h e s e q u e s t i o n s b o t h c o m p u t a t i o n a l l y a n d e x p e r i m e n t a l l y .
I n A i m 2 c , w e i n c l u d e p 6 3 - a k n o w n m a s t e r r e g u l a t o r c o n t r o l l i n g b o t h s t r a t i f i c a t i o n a n d p r o l i f e r a t i o n A D D I N E N . C I T E <