Innovative Research for a Sustainable Future www.epa.gov/research Tom Knudsen | knudsen.thomas@epa.gov l 919-541-9776 Computational Modeling of Limb-Bud Dysmorphogenesis: Predicting Cellular Dynamics and Key Events in Developmental Toxicity with a Multicellular Systems Model BK Ahir1, ES Hunter2, NC Baker3, RM Spencer3, RS Dewoskin4, TB Knudsen1 U.S. Environmental Protection Agency, Office of Research and Development 1National Center for Computational Toxicology, 2National Health and Environmental Effects Research Laboratory, 3Lockheed Martin, 4National Center for Environmental Assessment 4. TOXICODYNAMICS: predicting key events 5. PRACTICAL APPLICATIONS OF A MULTICELLULAR SIMULATION o Predictive Toxicology :- new way to integrate masses of HTS data into knowledge of a system o Parameter Sweeps :- high-throughput hypothesis testing to inform experimental design o Mechanistic Models :- track lesion propagation through higher levels of biological organization o Temporal Analysis :- pinpoint key events and mine for quantitative relationships o Dose Predictivity :- compare diverse exposure scenarios across dose, time, duration & mixtures DISCLAIMER: does not reflect EPA policy GENETIC PERTURBATION: hindlimb-bud shown at MCS intervals 10k, 20k, 30k & 40k with SHH field for normal and ‘virtual knockouts’ disabled for Shh or Gli3 genes (wild-type contour traced magenta). Apical Ectodermal Ridge (AER) and Zone of Polarizing Activity (ZPA) are major signaling centers; pivotal signaling pathways fibroblast growth factor (FGF), sonic hedgehog (SHH), bone morphogenetic protein (BMP) and retinoic acid (RA) were modeled in CellDesigner (www.celldesigner.org/). 1. COMPUTATIONAL EMBRYOLOGY & PREDICTIVE TOXICOLOGY HYPOTHESIS: a computer model that simulates cellular function in a growing embryo can be used to predict the potential impact of chemical exposure during early limb development. 2. SIGNALING NETWORK: spatial information processing Query of Mouse Genome Informatics database (www.informatics.jax.org/) by ‘abnormal limb bud morphology’ (MP:0005650) returned genes for 132 relevant genotypes. An e-Library developed from these genes (31,701 PubMed records annotated by MeSH terms for limb development) returned 81 genes regulating early limb morphogenesis. CONTROL NETWORK CELL AGENT-BASED MODEL (ABM): multicellular and signaling dynamics were modeled in CompuCell3D (www.compucell3d.org/); the small working prototype simulated mouse hindlimb-bud development between Theiler stages 16-19 (~42h) in ~42,000 Monte Carlo Steps (MCS). 3. CELLULAR DYNAMICS: translation of spatial information o Disabling Shh in the model simulates undergrowth of limb-bud o SHH: posterior regulation of digit number & identity o Disabling Gli3 in the model leads to precocious overgrowth o GLI3R: blocked by SHH but negatively regulates Shh-expression o Future Goal: Model the emergence of digital rays Wild-type Shh-null Gli3-null Rate of elongation (n=5) Shh cell lineage (n=5) Wild-type Shh-null Gli3-null Predicted outcomes digital patterns inferred from the literature; not yet implemented in the model exposure Adhesion Apoptosis Differentiation Migration Mitosis Shape Size AER ZPA Cellular behaviors Signals Simulated outgrowth Shh cell lineage (n=10) control excess apoptosis mitotic arrest mixed effect exposed at MCS 32-38k Excessive apoptosis (n=10) CONTROL APOPTOSIS MITOTIC MIXED scan QR codes to run simulation on mobile device CHEMICAL DISRUPTION: How might local effects predicted by in vitro high-throughput screening (HTS) data such as ToxCast™propagate through the pivotal SHH cell lineage in silico to predict, therefore, a key event in vivo? EXAMPLE: 5-Fluorouracil, a teratogen that disrupts digit formation, perturbed 13 of 650 ToxCast HTS assays at ≤15 µM: impaired differentiation and increased cell loss (excessive apoptosis); p53- induction, mitotic arrest and cell death. These effects can be fed into the model for translation into predicted outcomes. o Model reac