{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,2,20]],"date-time":"2026-02-20T04:32:33Z","timestamp":1771561953760,"version":"3.50.1"},"reference-count":52,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2021,2,9]],"date-time":"2021-02-09T00:00:00Z","timestamp":1612828800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000066","name":"National Institute of Environmental Health Sciences","doi-asserted-by":"publisher","id":[{"id":"10.13039\/100000066","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000139","name":"U.S. Environmental Protection Agency","doi-asserted-by":"publisher","id":[{"id":"10.13039\/100000139","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100008637","name":"Center for Strategic Scientific Initiatives, National Cancer Institute","doi-asserted-by":"publisher","id":[{"id":"10.13039\/100008637","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Cell Dev. Biol."],"abstract":"<jats:p>Paracrine signaling in the tissue microenvironment is a central mediator of morphogenesis, and modeling this dynamic intercellular activity<jats:italic>in vitro<\/jats:italic>is critical to understanding normal and abnormal development. For example, Sonic Hedgehog (Shh) signaling is a conserved mechanism involved in multiple developmental processes and strongly linked to human birth defects including orofacial clefts of the lip and palate. SHH ligand produced, processed, and secreted from the epithelial ectoderm is shuttled through the extracellular matrix where it binds mesenchymal receptors, establishing a gradient of transcriptional response that drives orofacial morphogenesis. In humans, complex interactions of genetic predispositions and environmental insults acting on diverse molecular targets are thought to underlie orofacial cleft etiology. Consequently, there is a need for tractable<jats:italic>in vitro<\/jats:italic>approaches that model this complex cellular and environmental interplay and are sensitive to disruption across the multistep signaling cascade. We developed a microplate-based device that supports an epithelium directly overlaid onto an extracellular matrix-embedded mesenchyme, mimicking the basic tissue architecture of developing orofacial tissues. SHH ligand produced from the epithelium generated a gradient of SHH-driven transcription in the adjacent mesenchyme, recapitulating the gradient of pathway activity observed<jats:italic>in vivo<\/jats:italic>. Shh pathway activation was antagonized by small molecule inhibitors of epithelial secretory, extracellular matrix transport, and mesenchymal sensing targets, supporting the use of this approach in high-content chemical screening of the complete Shh pathway. Together, these findings demonstrate a novel and practical microphysiological model with broad utility for investigating epithelial-mesenchymal interactions and environmental signaling disruptions in development.<\/jats:p>","DOI":"10.3389\/fcell.2021.621442","type":"journal-article","created":{"date-parts":[[2021,2,9]],"date-time":"2021-02-09T08:18:09Z","timestamp":1612858689000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":8,"title":["A Microphysiological Approach to Evaluate Effectors of Intercellular Hedgehog Signaling in Development"],"prefix":"10.3389","volume":"9","author":[{"given":"Brian P.","family":"Johnson","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ross A.","family":"Vitek","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Molly M.","family":"Morgan","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Dustin M.","family":"Fink","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Tyler G.","family":"Beames","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Peter G.","family":"Geiger","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"David J.","family":"Beebe","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Robert J.","family":"Lipinski","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1965","published-online":{"date-parts":[[2021,2,9]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"2912","DOI":"10.3791\/2912","article-title":"A high throughput in situ hybridization method to characterize mRNA expression patterns in the fetal mouse lower urogenital tract","volume":"19","author":"Abler","year":"2011","journal-title":"J. Vis. Exp"},{"key":"B2","doi-asserted-by":"publisher","first-page":"10476","DOI":"10.1073\/pnas.162356199","article-title":"Sonic hedgehog rescues cranial neural crest from cell death induced by ethanol exposure","volume":"99","author":"Ahlgren","year":"2002","journal-title":"Proc. Natl. Acad. Sci. U. S. A."},{"key":"B3","doi-asserted-by":"publisher","first-page":"dev191064","DOI":"10.1242\/dev.191064","article-title":"Gene-environment interactions: aligning birth defects research with complex etiology","volume":"147","author":"Beames","year":"2020","journal-title":"Development"},{"key":"B4","doi-asserted-by":"publisher","first-page":"394","DOI":"10.1093\/toxsci\/kfy207","article-title":"A Three-dimensional organoid culture model to assess the influence of chemicals on morphogenetic fusion","volume":"166","author":"Belair","year":"2018","journal-title":"Toxicol. Sci"},{"key":"B5","doi-asserted-by":"publisher","first-page":"e0184155","DOI":"10.1371\/journal.pone.0184155","article-title":"Engineering human cell spheroids to model embryonic tissue fusion in vitro","volume":"12","author":"Belair","year":"2017","journal-title":"PLoS ONE"},{"key":"B6","doi-asserted-by":"publisher","first-page":"499","DOI":"10.1002\/bdra.23021","article-title":"Environmental and genetic contributors to hypospadias: a review of the epidemiologic evidence","volume":"94","author":"Carmichael","year":"2012","journal-title":"Birth Defects Res. Part A Clin. Mol. Teratol"},{"key":"B7","doi-asserted-by":"publisher","first-page":"436","DOI":"10.1038\/s41587-019-0085-3","article-title":"Specification of positional identity in forebrain organoids","volume":"37","author":"Cederquist","year":"2019","journal-title":"Nat. Biotechnol"},{"key":"B8","doi-asserted-by":"publisher","first-page":"14071","DOI":"10.1073\/pnas.182542899","article-title":"Small molecule modulation of Smoothened activity","volume":"99","author":"Chen","year":"2002","journal-title":"Proc. Natl. Acad. Sci. U. S. A."},{"key":"B9","doi-asserted-by":"publisher","first-page":"1192","DOI":"10.1194\/jlr.M200082-JLR200","article-title":"Limb malformations of rat fetuses exposed to a distal inhibitor of cholesterol biosynthesis","volume":"43","author":"Chevy","year":"2002","journal-title":"J. Lipid Res"},{"key":"B10","doi-asserted-by":"publisher","first-page":"328","DOI":"10.1159\/000066748","article-title":"Hindbrain and cranial nerve dysmorphogenesis result from acute maternal ethanol administration","volume":"24","author":"Dunty","year":"2002","journal-title":"Dev. Neurosci"},{"key":"B11","doi-asserted-by":"publisher","first-page":"2082","DOI":"10.1242\/dev.149930","article-title":"Sonic hedgehog regulation of Foxf2 promotes cranial neural crest mesenchyme proliferation and is disrupted in cleft lip morphogenesis","volume":"144","author":"Everson","year":"2017","journal-title":"Development"},{"key":"B12","doi-asserted-by":"publisher","first-page":"107006","DOI":"10.1289\/EHP5260","article-title":"Developmental toxicity assessment of piperonyl butoxide exposure targeting sonic hedgehog signaling and forebrain and face morphogenesis in the mouse: an in vitro and in vivo study","volume":"127","author":"Everson","year":"2019","journal-title":"Environ. Health Perspect"},{"key":"B13","doi-asserted-by":"publisher","first-page":"3961","DOI":"10.1210\/en.2004-0079","article-title":"Hedgehog signaling promotes prostate xenograft tumor growth","volume":"145","author":"Fan","year":"2004","journal-title":"Endocrinology"},{"key":"B14","doi-asserted-by":"crossref","first-page":"41","DOI":"10.1242\/dev.103.Supplement.41","article-title":"Palate development","volume":"103","author":"Ferguson","year":"1988","journal-title":"Development"},{"key":"B15","doi-asserted-by":"publisher","first-page":"340","DOI":"10.1067\/moe.2000.107360","article-title":"Establishment of a human polyclonal oral epithelial cell line","volume":"90","author":"Gilchrist","year":"2000","journal-title":"Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endod"},{"key":"B16","doi-asserted-by":"publisher","first-page":"865","DOI":"10.1002\/bdra.20193","article-title":"Gene-environment interactions in rare diseases that include common birth defects","volume":"73","author":"Graham","year":"2005","journal-title":"Birth Defects Res. Part A Clin. Mol. Teratol"},{"key":"B17","doi-asserted-by":"publisher","first-page":"2364","DOI":"10.1039\/C5LC00234F","article-title":"Micromilling: a method for ultra-rapid prototyping of plastic microfluidic devices","volume":"15","author":"Guckenberger","year":"2015","journal-title":"Lab Chip"},{"key":"B18","doi-asserted-by":"publisher","first-page":"1307","DOI":"10.1242\/dmm.026328","article-title":"Gli2 gene-environment interactions contribute to the etiological complexity of holoprosencephaly: evidence from a mouse model","volume":"9","author":"Heyne","year":"2016","journal-title":"Dis. Model. Mech"},{"key":"B19","doi-asserted-by":"publisher","first-page":"e0120517","DOI":"10.1371\/journal.pone.0120517","article-title":"Definition of critical periods for Hedgehog pathway antagonist-induced holoprosencephaly, cleft lip, and cleft palate","volume":"10","author":"Heyne","year":"","journal-title":"PLoS ONE"},{"key":"B20","first-page":"368","article-title":"A simple and reliable method for early pregnancy detection in inbred mice","volume":"54","author":"Heyne","year":"","journal-title":"J. Am. Assoc. Lab. Anim. Sci"},{"key":"B21","doi-asserted-by":"publisher","first-page":"e60351","DOI":"10.7554\/eLife.60351.sa2","article-title":"Mutation and fetal alcohol converge on Nodal signaling in a mouse model of holoprosencephaly","volume":"9","author":"Hong","year":"2020","journal-title":"Elife"},{"key":"B22","doi-asserted-by":"publisher","first-page":"567","DOI":"10.1242\/dev.114835","article-title":"A dynamic Shh expression pattern, regulated by SHH and BMP signaling, coordinates fusion of primordia in the amniote face","volume":"142","author":"Hu","year":"2015","journal-title":"Development"},{"key":"B23","doi-asserted-by":"publisher","first-page":"965","DOI":"10.1021\/acs.chemrestox.6b00350","article-title":"Computational model of secondary palate fusion and disruption","volume":"30","author":"Hutson","year":"2017","journal-title":"Chem. Res. Toxicol"},{"key":"B24","doi-asserted-by":"publisher","first-page":"3069","DOI":"10.1089\/scd.2012.0155","article-title":"A stable cranial neural crest cell line from mouse","volume":"21","author":"Ishii","year":"2012","journal-title":"Stem Cells Dev"},{"key":"B25","doi-asserted-by":"publisher","first-page":"591","DOI":"10.1172\/JCI0216506","article-title":"Cholesterol modification of Hedgehog family proteins","volume":"110","author":"Jeong","year":"2002","journal-title":"J. Clin. Invest"},{"key":"B26","doi-asserted-by":"publisher","first-page":"1152","DOI":"10.1002\/dvdy.20646","article-title":"Development of the upper lip: morphogenetic and molecular mechanisms","volume":"235","author":"Jiang","year":"2006","journal-title":"Dev. Dyn"},{"key":"B27","doi-asserted-by":"publisher","first-page":"63","DOI":"10.1002\/bdra.20430","article-title":"Mouse genetic models of cleft lip with or without cleft palate","volume":"82","author":"Juriloff","year":"2008","journal-title":"Birth Defects Res. Part A Clin. Mol. Teratol"},{"key":"B28","doi-asserted-by":"publisher","first-page":"e2000825","DOI":"10.1002\/adhm.202000825","article-title":"Engineered perineural vascular plexus for modeling developmental toxicity","volume":"9","author":"Kaushik","year":"2020","journal-title":"Adv. Healthc. Mater"},{"key":"B29","doi-asserted-by":"publisher","first-page":"1586","DOI":"10.1177\/1535370217717697","article-title":"Programming microphysiological systems for children's health protection","volume":"242","author":"Knudsen","year":"2017","journal-title":"Exp. Biol. Med."},{"key":"B30","doi-asserted-by":"publisher","first-page":"569","DOI":"10.1016\/bs.ctdb.2015.12.010","article-title":"Gene\u2013environment interactions and the etiology of birth defects","volume":"116","author":"Krauss","year":"2016","journal-title":"Curr. Top. Dev. Biol"},{"key":"B31","doi-asserted-by":"publisher","first-page":"901041","DOI":"10.1155\/2015\/901041","article-title":"The Roles of Hedgehog signaling in upper lip formation","volume":"2015","author":"Kurosaka","year":"2015","journal-title":"Biomed Res. Int."},{"key":"B32","doi-asserted-by":"publisher","first-page":"1387","DOI":"10.1242\/dev.028167","article-title":"Sonic hedgehog signaling regulates reciprocal epithelial-mesenchymal interactions controlling palatal outgrowth","volume":"136","author":"Lan","year":"2009","journal-title":"Development"},{"key":"B33","doi-asserted-by":"publisher","first-page":"8455","DOI":"10.1073\/pnas.0609699104","article-title":"Inhibition of GLI-mediated transcription and tumor cell growth by small-molecule antagonists","volume":"104","author":"Lauth","year":"2007","journal-title":"Proc. Natl. Acad. Sci. U.S.A."},{"key":"B34","doi-asserted-by":"publisher","first-page":"246","DOI":"10.1002\/ajmg.c.31381","article-title":"Genetics of cleft lip and cleft palate","author":"Leslie","year":"2013","journal-title":"Am. J. Med. Genet. C Semin. Med. Genet."},{"key":"B35","doi-asserted-by":"publisher","first-page":"543","DOI":"10.1126\/science.aao0645","article-title":"Morphogen gradient reconstitution reveals Hedgehog pathway design principles","volume":"360","author":"Li","year":"2018","journal-title":"Science"},{"key":"B36","doi-asserted-by":"publisher","first-page":"6548","DOI":"10.1073\/pnas.0600124103","article-title":"Cholesterol modification restricts the spread of Shh gradient in the limb bud","volume":"103","author":"Li","year":"2006","journal-title":"Proc. Natl. Acad. Sci. U. S. A."},{"key":"B37","doi-asserted-by":"publisher","first-page":"103","DOI":"10.1053\/j.sodo.2008.02.002","article-title":"Genetic factors and orofacial clefting","volume":"14","author":"Lidral","year":"2008","journal-title":"Semin. Orthod"},{"key":"B38","doi-asserted-by":"publisher","first-page":"1404","DOI":"10.1016\/j.tiv.2010.04.011","article-title":"Identification of Hedgehog signaling inhibitors with relevant human exposure by small molecule screening","volume":"24","author":"Lipinski","year":"2010","journal-title":"Toxicol. In Vitro"},{"key":"B39","doi-asserted-by":"publisher","first-page":"456","DOI":"10.1093\/toxsci\/kfm222","article-title":"Identification and characterization of several dietary alkaloids as weak inhibitors of hedgehog signaling","volume":"100","author":"Lipinski","year":"2007","journal-title":"Toxicol. Sci"},{"key":"B40","doi-asserted-by":"publisher","first-page":"232","DOI":"10.1002\/bdra.20656","article-title":"Cleft lip and palate results from Hedgehog signaling antagonism in the mouse: phenotypic characterization and clinical implications","volume":"88","author":"Lipinski","year":"2010","journal-title":"Birth Defects Res. Part A Clin. Mol. Teratol"},{"key":"B41","doi-asserted-by":"publisher","first-page":"248","DOI":"10.1034\/j.1399-0004.2002.610402.x","article-title":"Gene\/environment causes of cleft lip and\/or palate","volume":"61","author":"Murray","year":"2002","journal-title":"Clin. Genet"},{"key":"B42","doi-asserted-by":"publisher","first-page":"811","DOI":"10.1002\/bies.20804","article-title":"Microfluidics meet cell biology: bridging the gap by validation and application of microscale techniques for cell biological assays","volume":"30","author":"Paguirigan","year":"2008","journal-title":"Bioessays"},{"key":"B43","doi-asserted-by":"publisher","first-page":"247","DOI":"10.1038\/nchembio.1184","article-title":"Inhibitors of Hedgehog acyltransferase block Sonic Hedgehog signaling","volume":"9","author":"Petrova","year":"2013","journal-title":"Nat. Chem. Biol"},{"key":"B44","doi-asserted-by":"publisher","first-page":"2132","DOI":"10.1039\/b903043c","article-title":"Biological implications of polydimethylsiloxane-based microfluidic cell culture","volume":"9","author":"Regehr","year":"2009","journal-title":"Lab Chip"},{"key":"B45","doi-asserted-by":"publisher","first-page":"128414","DOI":"10.1016\/j.chemosphere.2020.128414","article-title":"Examining the developmental toxicity of piperonyl butoxide as a Sonic hedgehog pathway inhibitor","volume":"264","author":"Rivera-Gonz\u00e1lez","year":"2021","journal-title":"Chemosphere"},{"key":"B46","doi-asserted-by":"publisher","first-page":"13424","DOI":"10.1073\/pnas.2235734100","article-title":"Loss-of-function mutations in the human GLI2 gene are associated with pituitary anomalies and holoprosencephaly-like features","volume":"100","author":"Roessler","year":"2003","journal-title":"Proc. Natl. Acad. Sci. U. S. A."},{"key":"B47","doi-asserted-by":"publisher","first-page":"676","DOI":"10.1038\/nmeth.2019","article-title":"Fiji: an open-source platform for biological-image analysis","volume":"9","author":"Schindelin","year":"2012","journal-title":"Nat. Methods"},{"key":"B48","doi-asserted-by":"publisher","first-page":"1005","DOI":"10.1038\/35023008","article-title":"Effects of oncogenic mutations in Smoothened and Patched can be reversed by cyclopamine","volume":"406","author":"Taipale","year":"2000","journal-title":"Nature"},{"key":"B49","doi-asserted-by":"publisher","first-page":"119","DOI":"10.1177\/154405910808700202","article-title":"Unraveling human cleft lip and palate research","volume":"87","author":"Vieira","year":"2008","journal-title":"J. Dent. Res"},{"key":"B50","doi-asserted-by":"publisher","first-page":"517","DOI":"10.1093\/toxsci\/kfs165","article-title":"The insecticide synergist piperonyl butoxide inhibits Hedgehog signaling: assessing chemical risks","volume":"128","author":"Wang","year":"2012","journal-title":"Toxicol. Sci"},{"key":"B51","doi-asserted-by":"publisher","first-page":"450","DOI":"10.1016\/j.devcel.2020.09.017","article-title":"Hedgehog Pathway activation requires coreceptor-catalyzed, lipid-dependent relay of the Sonic Hedgehog ligand","volume":"55","author":"Wierbowski","year":"2020","journal-title":"Dev. Cell"},{"key":"B52","doi-asserted-by":"publisher","first-page":"1055","DOI":"10.1097\/01.scs.0000183470.31202.c9","article-title":"Gene expression changes of sonic hedgehog signaling cascade in a mouse embryonic model of fetal alcohol syndrome","volume":"16","author":"Yamada","year":"2005","journal-title":"J. Craniofacial Surg."}],"container-title":["Frontiers in Cell and Developmental Biology"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fcell.2021.621442\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2022,12,15]],"date-time":"2022-12-15T22:21:20Z","timestamp":1671142880000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fcell.2021.621442\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,2,9]]},"references-count":52,"alternative-id":["10.3389\/fcell.2021.621442"],"URL":"https:\/\/doi.org\/10.3389\/fcell.2021.621442","relation":{},"ISSN":["2296-634X"],"issn-type":[{"value":"2296-634X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,2,9]]},"article-number":"621442"}}