Morphogenetic events are driven by cell-generated physical forces and complex cellular dynamics. To improve our capacity to predict developmental effects from cellular alterations, we built a multi-cellular agent-based model in CompuCell3D that recapitulates the cellular networks and collective cell behavior underlying growth and fusion of the mammalian secondary palate. The model incorporated multiple signaling pathways (TGF?, BMP, FGF, EGF, SHH) in a biological framework to recapitulate morphogenetic events from palatal outgrowth through midline fusion. It effectively simulated higher-level phenotypes (e.g., midline contact, medial edge seam (MES) breakdown, mesenchymal confluence, fusion defects) in response to genetic or environmental perturbations. Perturbation analysis of various control features revealed model functionality with respect to cell signaling systems and feedback loops for growth and fusion, diverse individual cell behaviors and collective cellular behavior leading to physical contact and midline fusion, and quantitative analysis of the TGF/EGF switch that controls MES breakdown – a key event in morphogenetic fusion. The virtual palate model was then executed with theoretical chemical perturbation scenarios to simulate switch behavior leading to a disruption of fusion following chronic (e.g., dioxin) and acute (e.g., retinoic acid, hydrocortisone) toxicant exposures. This computer model adds to similar systems models toward a ‘virtual embryo’ for simulation and quantitative prediction of adverse developmental outcomes following genetic perturbation and/or environmental. This dataset is associated with the following publication: Hutson, S., M. Leung, N. Baker, R. Spencer, and T. Knudsen. (CHEMICAL RESEARCH IN TOXICOLOGY) Computational Model of Secondary Palate Fusion and Disruption. CHEMICAL RESEARCH IN TOXICOLOGY. American Chemical Society, Washington, DC, USA, 30(4): 965-979, (2017).

A data set of 500 chemicals evaluated for their ability to induce cleft palate in animal prenatal developmental studies was compiled from Toxicity Reference Database and the biomedical literature, which included 63 cleft palate active and 437 inactive chemicals. To characterize the potential molecular targets for chemical‐induced cleft palate, we mined the ToxCast high‐throughput screening database for patterns and linkages in bioactivity profiles and chemical structural descriptors. The following datasets can be obtained via the links and files in the Data section: Phase II ToxCast assay data results (Judson et al., 2010); The Gene Score data set derived from ToxCast; ToxRefDB version 1 (Knudsen et al., 2009; Martin, Judson, et al., 2009); The ToxPrint chemotypes (Yang et al., 2015). This dataset is associated with the following publication: Baker, N., N. Sipes, J. Franzosa, D. Belair, B. Abbott, R. Judson, and T. Knudsen. Characterizing cleft palate toxicants using ToxCast data, chemical structure, and the biomedical literature. Birth Defects Research. John Wiley & Sons, Inc., Hoboken, NJ, USA, 1-21, (2019).

Organogenesis in the embryo involves cell differentiation and organization events that are unique to each tissue and organ and are susceptible to developmental toxicants. Animal models are the gold standard for identifying putative teratogens, but the limited throughput of developmental toxicological studies in animals coupled with the limited concordance between animal and human teratogenicity motivates a different approach. In vitro organoid models can mimic the cellular architecture and phenotype of many tissues and organs, and the three-dimensional (3D) architecture of organoids presents an opportunity to study developmental human toxicology. Common themes during development like the involvement of epithelial-mesenchymal transition and tissue fusion present an opportunity to develop in vitro models to study cell and tissue morphogenesis. We previously described organoids composed of human stem and progenitor cells that recapitulated the cellular features of palate fusion, and here we further characterized the model by examining pharmacological inhibitors targeting known palatogenesis and epithelial morphogenesis pathways as well as twelve cleft palate teratogens identified from rodent models. Organoid survival was dependent on signaling through EGF, IGF, HGF, and FGF pathways, and organoid fusion was disrupted by inhibition of BMP signaling. We observed concordance between the effects of EGF, FGF, and BMP inhibitors on organoid fusion and epithelial cell migration in vitro, suggesting that organoid fusion is dependent on epithelial morphogenesis. Three of the twelve putative cleft palate teratogens studied here significantly disrupted in vitro fusion, including theophylline, triamcinolone, and valproic acid. Tributyltin chloride and all-trans retinoic acid (ATRA) were cytotoxic to fusing organoids. The study herein demonstrates the utility of the in vitro fusion assay for identifying chemicals that disrupt human organoid survival and morphogenesis in a scalable format amenable to toxicology screening. This dataset is associated with the following publication: Belair, D., C. Wolf, S. Moorefield, C. Wood, C. Becker, and B. Abbott. A Three-Dimensional Organoid Culture Model to Assess the Influence of Chemicals on Morphogenetic Fusion.. TOXICOLOGICAL SCIENCES. Society of Toxicology, RESTON, VA, 394-408, (2018).

Organogenesis in the embryo involves cell differentiation and organization events that are unique to each tissue and organ and are susceptible to developmental toxicants. Animal models are the gold standard for identifying putative teratogens, but the limited throughput of developmental toxicological studies in animals coupled with the limited concordance between animal and human teratogenicity motivates a different approach. In vitro organoid models can mimic the cellular architecture and phenotype of many tissues and organs, and the three-dimensional (3D) architecture of organoids presents an opportunity to study developmental human toxicology. Common themes during development like the involvement of epithelial-mesenchymal transition and tissue fusion present an opportunity to develop in vitro models to study cell and tissue morphogenesis. We previously described organoids composed of human stem and progenitor cells that recapitulated the cellular features of palate fusion, and here we further characterized the model by examining pharmacological inhibitors targeting known palatogenesis and epithelial morphogenesis pathways as well as twelve cleft palate teratogens identified from rodent models. Organoid survival was dependent on signaling through EGF, IGF, HGF, and FGF pathways, and organoid fusion was disrupted by inhibition of BMP signaling. We observed concordance between the effects of EGF, FGF, and BMP inhibitors on organoid fusion and epithelial cell migration in vitro, suggesting that organoid fusion is dependent on epithelial morphogenesis. Three of the twelve putative cleft palate teratogens studied here significantly disrupted in vitro fusion, including theophylline, triamcinolone, and valproic acid. Tributyltin chloride and all-trans retinoic acid (ATRA) were cytotoxic to fusing organoids. The study herein demonstrates the utility of the in vitro fusion assay for identifying chemicals that disrupt human organoid survival and morphogenesis in a scalable format amenable to toxicology screening. This dataset is associated with the following publication: Belair, D., C. Wolf, S. Moorefield, C. Wood, C. Becker, and B. Abbott. A Three-Dimensional Organoid Culture Model to Assess the Influence of Chemicals on Morphogenetic Fusion.. TOXICOLOGICAL SCIENCES. Society of Toxicology, RESTON, VA, 394-408, (2018).

The data presented here support the application of the Stemina devTOXqP platform for predictive toxicology and further demonstrate its value in ToxCast as a novel resource that can generate testable hypotheses aimed at characterizing potential pathways for teratogenicity and HTS prioritization of environmental chemicals for an exposure-based assessment of developmental hazard. The dataset from the Stemina (STM) assay is annotated in the ToxCast portfolio as STM. Major findings from the analysis of ToxCast_STM dataset include (1) 19% of 1065 chemicals yielded a prediction of developmental toxicity, (2) assay performance reached 79%-82% accuracy with high specificity (> 84%) but modest sensitivity (< 67%) when compared with in vivo animal models of human prenatal developmental toxicity, (3) sensitivity improved as more stringent weights of evidence requirements were applied to the animal studies, and (4) statistical analysis of the most potent chemical hits on specific biochemical targets in ToxCast revealed positive and negative associations with the STM response, providing insights into the mechanistic underpinnings of the targeted endpoint and its biological domain. The results of this study will be useful to improving our ability to predict in vivo developmental toxicants based on in vitro data and in silico models. This dataset is associated with the following publication: Zurlinden, T., K. Saili, N. Rush, P. Kothiya, R. Judson, K. Houck, E. Hunter, N. Baker, J. Palmer, R. Thomas, and T. Knudsen. Profiling the ToxCast Library With a Pluripotent Human (H9) Stem Cell Line-Based Biomarker Assay for Developmental Toxicity. TOXICOLOGICAL SCIENCES. Society of Toxicology, RESTON, VA, 174(2): 189-209, (2020).

The systems map described in this manuscript for neurulation will set the stage for constructing mathematical models and computer simulation of neural tube closure for human-relevant AOPs and predictive toxicology of neural tube defects such as spina bifida. This dataset is not publicly accessible because: The data for the systems map has been obtained from comprehensive literature review. It can be accessed through the following means: Contact the corresponding author Harm J. Heusinkveld, Centre for Health Protection, National Institute for Public Health and the Environment (RIVM), P.O.Box 1, 3720BA, Bilthoven, The Netherlands. Email: harm.heusinkveld@rivm.nl. Format: Not availabie. This dataset is associated with the following publication: Heusinkveld, H., Y. Staal, N. Baker, G. Daston, T. Knudsen, and A. Piersma. An ontology for developmental processes and toxicities of neural tube closure. REPRODUCTIVE TOXICOLOGY. Elsevier Science Ltd, New York, NY, USA, 99: 160-167, (2021).

The systems map described in this manuscript for neurulation will set the stage for constructing mathematical models and computer simulation of neural tube closure for human-relevant AOPs and predictive toxicology of neural tube defects such as spina bifida. This dataset is not publicly accessible because: The data for the systems map has been obtained from comprehensive literature review. It can be accessed through the following means: Contact the corresponding author Harm J. Heusinkveld, Centre for Health Protection, National Institute for Public Health and the Environment (RIVM), P.O.Box 1, 3720BA, Bilthoven, The Netherlands. Email: harm.heusinkveld@rivm.nl. Format: Not availabie. This dataset is associated with the following publication: Heusinkveld, H., Y. Staal, N. Baker, G. Daston, T. Knudsen, and A. Piersma. An ontology for developmental processes and toxicities of neural tube closure. REPRODUCTIVE TOXICOLOGY. Elsevier Science Ltd, New York, NY, USA, 99: 160-167, (2021).

Cleft palate (CP) is a common birth defect, occurring in an estimated 1 in 1000 births worldwide. The secondary palate is formed by paired palatal shelves that grow toward each other, appose, attach and fuse. CP can result from disruption of any of these processes. The palatal shelves basically consist of a mesenchymal tissue core covered with a layer of epithelial cells. One of the mechanisms that can cause CP is failure of fusion, i.e., failure to remove the epithelial seam between the palatal shelves to allow the mesenchyme to merge and form a continuous palate. This process requires complex interactions between mesenchymal and epithelial cells, and signaling components such as growth factors. Epidermal growth factor (EGF) plays an important role in palate growth and differentiation, while it may impede fusion. We developed a 3D organotypic model using human mesenchymal and epithelial stem cells to mimic human embryonic palatal shelves, and tested its functional relevance by monitoring the effects of human EGF (hEGF) on proliferation and fusion. Spheroids were generated from human umbilical-derived mesenchymal stem cells (hMSCs) directed down an osteogenic lineage by culture medium and evaluated for osteogenic differentiation. Heterotypic spheroids, or organoids, were constructed by coating hMSC spheroids with MaxGel™ extracellular matrix solution followed by a layer of human progenitor epithelial keratinocytes (hPEK). Organoids were incubated in co-culture medium with or without hEGF and assessed for cell proliferation and spheroid pairs were assessed for time to fusion. Osteogenic differentiation in hMSC spheroids was highest by day 13. hEGF delayed fusion of heterotypic organoids after 12 and 18 hours of contact. hEGF increased proliferation in organoids at 4 ng/ml, and proliferation was detected in hPEKs alone on microcarrier beads, suggesting a potential mechanism for delayed fusion by hEGF. Our results show that this model of human palatal fusion consisting of a core of differentiated hMSCs with a hPEK outer layer appropriately mimics the morphology of the developing human palate and responds to hEGF as expected. Future studies will focus on using the organoid model to evaluate the effects of teratogenic chemicals on palatal fusion, and validating the results. This dataset is associated with the following publication: Wolf, C., D. Belair, C. Becker, K. Das, J. Schmid, and B. Abbott. Development of an organotypic stem cell model for the study of human embryonic palatal fusion. BIRTH DEFECTS RESEARCH PART B: DEVELOPMENTAL AND REPRODUCTIVE TOXICOLOGY. John Wiley & Sons, Ltd., Indianapolis, IN, USA, 1322-1334, (2018).

Cleft palate (CP) is a common birth defect, occurring in an estimated 1 in 1000 births worldwide. The secondary palate is formed by paired palatal shelves that grow toward each other, appose, attach and fuse. CP can result from disruption of any of these processes. The palatal shelves basically consist of a mesenchymal tissue core covered with a layer of epithelial cells. One of the mechanisms that can cause CP is failure of fusion, i.e., failure to remove the epithelial seam between the palatal shelves to allow the mesenchyme to merge and form a continuous palate. This process requires complex interactions between mesenchymal and epithelial cells, and signaling components such as growth factors. Epidermal growth factor (EGF) plays an important role in palate growth and differentiation, while it may impede fusion. We developed a 3D organotypic model using human mesenchymal and epithelial stem cells to mimic human embryonic palatal shelves, and tested its functional relevance by monitoring the effects of human EGF (hEGF) on proliferation and fusion. Spheroids were generated from human umbilical-derived mesenchymal stem cells (hMSCs) directed down an osteogenic lineage by culture medium and evaluated for osteogenic differentiation. Heterotypic spheroids, or organoids, were constructed by coating hMSC spheroids with MaxGel™ extracellular matrix solution followed by a layer of human progenitor epithelial keratinocytes (hPEK). Organoids were incubated in co-culture medium with or without hEGF and assessed for cell proliferation and spheroid pairs were assessed for time to fusion. Osteogenic differentiation in hMSC spheroids was highest by day 13. hEGF delayed fusion of heterotypic organoids after 12 and 18 hours of contact. hEGF increased proliferation in organoids at 4 ng/ml, and proliferation was detected in hPEKs alone on microcarrier beads, suggesting a potential mechanism for delayed fusion by hEGF. Our results show that this model of human palatal fusion consisting of a core of differentiated hMSCs with a hPEK outer layer appropriately mimics the morphology of the developing human palate and responds to hEGF as expected. Future studies will focus on using the organoid model to evaluate the effects of teratogenic chemicals on palatal fusion, and validating the results. This dataset is associated with the following publication: Wolf, C., D. Belair, C. Becker, K. Das, J. Schmid, and B. Abbott. Development of an organotypic stem cell model for the study of human embryonic palatal fusion. BIRTH DEFECTS RESEARCH PART B: DEVELOPMENTAL AND REPRODUCTIVE TOXICOLOGY. John Wiley & Sons, Ltd., Indianapolis, IN, USA, 1322-1334, (2018).

Epithelial-mesenchymal interactions drive embryonic fusion events during development and upon perturbation can result in birth defects. Cleft palate and neural tube defects can result from genetic defects or environmental exposures during development, yet very little is known about the effect of chemical exposures on fusion defects in humans because of the lack of relevant and robust human in vitro assays of developmental fusion behavior. Given the etiology and prevalence of cleft palate and the relatively simple architecture and composition of the embryonic palate, we sought to develop a three-dimensional culture system that could be used to study fusion behavior in vitro using human cells. We engineered human Wharton’s Jelly stromal cell (HWJSC) spheroids of defined size and established that 7 days of culture in osteogenesis differentiation medium was sufficient to promote an osteogenic phenotype consistent with embryonic palatal mesenchyme. HWJSC spheroids supported the attachment of human epidermal keratinocyte progenitor cells on the outer spheroid surface likely through deposition of collagens I and IV, fibronectin, and laminin, and co-cultured spheroids exhibited fusion behavior that was dependent on epidermal growth factor signaling and fibroblast growth factor signaling in agreement with palate fusion literature. The method described here may broadly apply to the generation of three-dimensional epithelial-mesenchymal co-cultures to study developmental fusion events in a format that is amenable to predictive toxicology applications. This dataset is associated with the following publication: Belair, D., C. Wolf, C. Wood, H. Ren, R. Grindstaff, W. Padgett, A. Swank, D. MacMillan, A. Fisher, W. Winnik, and B. Abbott. Engineering human cell spheroids to model embryonic tissue fusion in vitro.. PLoS ONE. Public Library of Science, San Francisco, CA, USA, N/A, (2017).