Multifactorial environmental inhibition of Sonic hedgehog signaling: Impact of chemical interactions on pathway activity and craniofacial development

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PROJECT SUMMARY Birth defects cause tremendous individual, familial, and societal burdens, and the development of targeted prevention strategies has been stymied by biological and etiological complexity. Exemplary of multifactorial birth defects thought to be substantially modulated by the environment are orofacial clefts (OFCs) and holoprosencephaly (HPE), common human malformations of the face and brain. The pathogenesis of both OFCs and HPE is directly linked to embryonic disruption of Sonic hedgehog (Shh) signaling in animal models, supporting a pathway-based investigation of environmental contributions to birth defect etiology. That Shh signaling comprises a multi-step process inherently sensitive to modulation across multiple steps of its signaling cascade makes the pathway especially germane for examining the impact of co-exposures on etiologically complex, multifactorial disease. The studies proposed in this application are designed to test the central hypothesis that structurally diverse Shh inhibitors synergistically interact to reduce pathway activity at the cellular level and exacerbate Shh-associated craniofacial malformations. To test this hypothesis, I have leveraged NIEHS- and EPA-supported high-throughput chemical screens to create a prioritized list of environmentally relevant, putative Shh pathway disruptors and developed a novel Shh pathway-complete cell culture system that is sensitive to inhibition throughout the Shh pathway. I will utilize this system and other mechanism-based in vitro assays to validate bona fide pathway antagonists, delineate molecular targets within the Shh pathway, and evaluate additive and synergistic interactions resulting from co-exposure to compounds with diverse pathway targets. The developmental toxicity of these compounds will then be examined, both individually and in combination, by targeting dose administration to critical periods of Shh pathway activity during craniofacial development. The known Shh antagonists cyclopamine, vismodegib, and piperonyl butoxide will be used throughout the proposed studies as positive controls for pathway-specific effects. These rigorously designed experiments are expected to reveal environmental factors that adversely impact development and elucidate mechanisms of Shh pathway disruption that promote synergistic interactions. In completing the proposed studies, the applicant’s professional development will be advanced through the establishment of proficiency in experimental design, evaluating the toxicity of mixtures, utilizing animal models of developmental toxicology, and effectively communicating scientific concepts and results. These works will also facilitate the applicant’s goal of becoming an independent investigator at a government agency within the United States.