Birth defects are the most common cause of death in children under one year of age in the United States. The best way to reduce the morbidity and mortality of specific birth defects is to develop strategies to prevent them from occurring, as exemplified by the decrease of neural tube defects due to folic acid supplementation. Hirschsprung Disease (HSCR) is a birth defect of the enteric nervous system that occurs when enteric neural crest-derived cells (ENCCs) do not migrate all the way to the end of the bowel. The resulting distal bowel aganglionosis leads to a functional obstruction, which causes vomiting, abdominal distension and predisposes to premature death. There are many proteins important for normal ENCC migration and mutations in these genes increase the risk of HSCR, but none of these mutations cause disease in all affected children. Therefore, children who have HSCR must have additional factors that further increase their HSCR risk. To develop strategies to decrease HSCR occurrence, it is important to identify alterable factors, such as maternal diet or medications that could in combination with genetic defects cause disease in a child who would otherwise have been born healthy. Ibuprofen, a non-steroidal anti-inflammatory drug (NSAID) commonly used in pregnancy, inhibited ENCC migration in a zebrafish drug screen, causing a HSCR-like distal bowel aganglionosis in affected fish. Experiments in mammalian primary culture confirmed that ibuprofen slows ENCC migration and appears to disrupt actin cytoskeletal and focal adhesion regulation needed for efficient migration. The goals of this proposal are to determine the biochemical mechanism of ibuprofen's inhibition of ENCC migration and to determine if ibuprofen increases the likelihood of HSCR in genetically predisposed individuals. The biochemical mechanisms that ibuprofen disrupts to inhibit ENCC migration will be investigated using cutting edge time-lapse live cell imaging of fluorescently-tagged proteins. These investigations will allow for in depth analysis of ENCC actin protrusions and the dynamics of focal adhesions. In addition, ENCCs from transgenic animals with null mutations in Cox1 and Cox2, enzymes inhibited by ibuprofen, will be analyzed by immunohistochemistry and time-lapse imaging to determine if ibuprofen inhibits ENCC migration via a Cox- dependent mechanism. I will also determine if mice with null mutations in Cox1 and Cox2 have HSCR-like distal bowel aganglionosis. Finally, mice genetically predisposed to HSCR due to mutations in genes important for normal ENCC migration will be exposed to ibuprofen in utero during the critical period of ENS development. These experiments will examine if ibuprofen could be a common and avoidable exposure during pregnancy that increases HSCR risk by reducing the efficiency of ENCC migration.

Public Health Relevance

Birth defects are the most common cause of death for infants under the age of one in the United States. If we can identify environmental factors that increase the risk of specific birth defects, especially in families already known to be at a higher genetic risk for these problems, we can reduce exposure to these factors during pregnancy and therefore decrease the occurrence of these birth defects. In this proposal, I will determine how the over-the-counter medicine ibuprofen slows enteric nervous system precursor migration, a change that should increase the risk of Hirschsprung disease, a potentially deadly birth defect of the nervous system in the gut.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Individual Predoctoral NRSA for M.D./Ph.D. Fellowships (ADAMHA) (F30)
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Special Emphasis Panel (ZDK1)
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Podskalny, Judith M,
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Washington University
Schools of Medicine
Saint Louis
United States
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Schill, Ellen Merrick; Lake, Jonathan I; Tusheva, Olga A et al. (2016) Ibuprofen slows migration and inhibits bowel colonization by enteric nervous system precursors in zebrafish, chick and mouse. Dev Biol 409:473-88
Avetisyan, Marina; Schill, Ellen Merrick; Heuckeroth, Robert O (2015) Building a second brain in the bowel. J Clin Invest 125:899-907