The authors have designated this AOP as all rights reserved. Re-use in any form requires advanced permission from the authors.
AOP: 460
Title
Antagonism of Smoothened receptor leading to orofacial clefting
Short name
Graphical Representation
Point of Contact
Contributors
- Jacob Reynolds
Coaches
- Judy Choi
Status
Handbook Version | OECD status | OECD project |
---|---|---|
v2.0 |
This AOP was last modified on May 25, 2023 08:32
Revision dates for related pages
Page | Revision Date/Time |
---|---|
Antagonism, Smoothened receptor | July 28, 2022 12:17 |
Decrease, GLI1/2 translocation to nucleus | October 28, 2022 15:22 |
Decrease, GLI1/2 target gene expression | March 22, 2023 10:38 |
Decrease, Cell proliferation | December 07, 2020 06:55 |
Decrease, palatal shelf outgrowth | May 16, 2023 14:37 |
Orofacial clefting | May 16, 2023 14:39 |
Decrease, Sonic Hedgehog second messenger production | March 22, 2023 12:09 |
Decrease, Smoothend relocation and activation | October 27, 2022 09:14 |
Apoptosis | December 20, 2022 08:33 |
Antagonism Smoothened leads to OFC | May 12, 2023 11:05 |
Antagonism Smoothened leads to Decrease, SMO relocation | May 17, 2023 09:12 |
Decrease, SMO relocation leads to Decrease, GLI1/2 translocation | May 22, 2023 09:22 |
Decrease, GLI1/2 translocation leads to Decrease, GLI1/2 target gene expression | May 22, 2023 09:46 |
Decrease, GLI1/2 target gene expression leads to Decrease, SHH second messenger production | May 22, 2023 09:56 |
Decrease, SHH second messenger production leads to Decrease, Cell proliferation | February 10, 2023 09:43 |
Decrease, Cell proliferation leads to Decrease, outgrowth | May 22, 2023 10:59 |
Decrease, outgrowth leads to OFC | May 22, 2023 12:36 |
Apoptosis leads to Decrease, outgrowth | April 11, 2023 10:25 |
Decrease, GLI1/2 target gene expression leads to Apoptosis | May 01, 2023 14:43 |
Vismodegib | July 14, 2022 13:04 |
Cyclopamine | August 03, 2022 09:21 |
SANT-1 | October 04, 2022 13:17 |
SANT-2 | October 04, 2022 13:17 |
SANT-3 | October 04, 2022 13:17 |
SANT-4 | October 04, 2022 13:18 |
Piperonyl butoxide | March 23, 2023 10:19 |
Abstract
This Adverse Outcome Pathway (AOP) describes the linkage between antagonism of the Smoothened (SMO) receptor and orofacial clefts (OFCs). The Sonic Hedgehog (SHH) is a major signaling pathway of intercellular signaling during embryonic development. Disruption of SHH during critical periods of development can lead to OFCs. In canonical SHH signaling, the SHH ligand binds to the Patched1 (PTCH1) receptor and relieves its’ suppression of the SMO receptor. Antagonism of SMO results in disruption the downstream SHH signaling cascade. Disruption to the signaling cascade causes a decrease in the translocation of the GLI1/2 transcription factors to the nucleus resulting in a decrease in expression of the GLI1/2 target genes. This decrease in gene expression which causes an increase in apoptosis and a decrease in production of SHH secondary messengers, namely Fgf10 and members of the BMP family. This reduction in secondary messengers leads to a decrease in cellular proliferation in the palatal shelves. This increase in apoptosis and reduction in cellular proliferation leads to a decrease in palatal shelf outgrowth which ultimately results in a cleft. This AOP is intended to serve as a tool for risk assessment for drug and chemical exposures during embryonic development when disruption to SHH through antagonism of SMO occurs.
AOP Development Strategy
Context
Orofacial clefts (OFCs), encompassing cleft lip with or without palate (CL/P), and cleft palate only (CPO) represent the second most common birth defect in humans with a prevalence of 1-2/1,000 births (Lidral, Moreno et al. 2008). The etiology of OFCs is complex with approximately 50% of CPO and 70% of CL/P considered non-syndromic (2011). SHH signaling is required for normal facial development and plays a critical role in the growth of the facial processes that form the upper palate and lip (Bush and Jiang 2012, Kurosaka 2015). The epithelial derived SHH drives orofacial development through an induced gradient in the underlying mesenchyme (Lan and Jiang 2009, Kurosaka 2015). This gradient of SHH induces cellular proliferation and outgrowth of the mesenchyme (Lan and Jiang 2009). The SHH pathway is sensitive to chemical disruption and can be disrupted at multiple places along the signaling cascade during critical windows for exposure and has been shown to cause OFCs (Lipinski and Bushman 2010, Heyne, Melberg et al. 2015). The targets of this disruption include ligand modification, ligand secretion, downstream sensing, and signal transduction (Jeong and McMahon 2002, Lauth, Bergström et al. 2007, Petrova, Rios-Esteves et al. 2013). Chemical modulators of the SHH pathway have been identified including the natural alkaloid cyclopamine, both natural and synthetic pharmaceuticals, and a chemical commonly found in pesticides (Lipinski, Dengler et al. 2007, Lipinski, Song et al. 2010, Wang, Lu et al. 2012, Everson, Sun et al. 2019, Rivera-González, Beames et al. 2021).
Strategy
This AOP was developed as part of a larger network of AOPs linking disruption of SHH signaling with OFCs (EAGMST workplan project 1.101.). Orofacial clefts (OFCs) are one of the most common human birth defects and occur in approximately 1 in 700 live births (Mossey, Little et al. 2009, Dixon, Marazita et al. 2011). Early orofacial development involves epithelial ectoderm derived SHH ligand driving tissue outgrowth through an induced gradient of SHH dependent transcription in the underlying mesenchyme, which is thought to drive mesenchymal proliferation (Lan and Jiang 2009, Kurosaka 2015). The SHH pathway is sensitive to chemical disruption at multiple molecular targets along the signaling cascade, with exposure during critical windows in development leading to OFCs (Lipinski and Bushman 2010, Heyne, Melberg et al. 2015). The molecular targets of this disruption include SHH ligand modification with cholesterol and palmitoylate, ligand secretion, mesenchymal reception, and signal transduction (Jeong and McMahon 2002, Lauth, Bergström et al. 2007, Petrova, Rios-Esteves et al. 2013). This AOP focuses on the disruption to SHH signaling resulting in antagonism of the SMO receptor. To select the key events for the AOP, we used existing knowledge of the pathway along with reviews of the SHH pathway to assemble a path that was physiologically plausible. Care was taken to select events that would be of direct regulatory relevance (i.e. a method to quantify exists). To identify sources and data for each KER, Pubmed was used. Initially results were screened for relevance off title/abstract and any of suspected relevance were reviewed in full to determine their applicability for the KER. Each KER includes a table of relevant search information (date, search terms, citations, etc). The search terms and sources identifed appear below in Table 1. It is the hope of the authors that this AOP is used as a tool for risk assessment for drug and chemical exposures during embryonic development when disruption to SHH through antagonism of SMO occurs.
Summary of the AOP
Events:
Molecular Initiating Events (MIE)
Key Events (KE)
Adverse Outcomes (AO)
Type | Event ID | Title | Short name |
---|
MIE | 2027 | Antagonism, Smoothened receptor | Antagonism Smoothened |
KE | 2044 | Decrease, Smoothend relocation and activation | Decrease, SMO relocation |
KE | 2028 | Decrease, GLI1/2 translocation to nucleus | Decrease, GLI1/2 translocation |
KE | 2040 | Decrease, GLI1/2 target gene expression | Decrease, GLI1/2 target gene expression |
KE | 1262 | Apoptosis | Apoptosis |
KE | 2043 | Decrease, Sonic Hedgehog second messenger production | Decrease, SHH second messenger production |
KE | 1821 | Decrease, Cell proliferation | Decrease, Cell proliferation |
KE | 2041 | Decrease, palatal shelf outgrowth | Decrease, outgrowth |
AO | 2042 | Orofacial clefting | OFC |
Relationships Between Two Key Events (Including MIEs and AOs)
Title | Adjacency | Evidence | Quantitative Understanding |
---|
Antagonism Smoothened leads to Decrease, SMO relocation | adjacent | Moderate | Low |
Decrease, SMO relocation leads to Decrease, GLI1/2 translocation | adjacent | Moderate | Low |
Decrease, GLI1/2 translocation leads to Decrease, GLI1/2 target gene expression | adjacent | Low | Low |
Decrease, GLI1/2 target gene expression leads to Decrease, SHH second messenger production | adjacent | Low | Low |
Decrease, SHH second messenger production leads to Decrease, Cell proliferation | adjacent | Low | Low |
Decrease, Cell proliferation leads to Decrease, outgrowth | adjacent | Low | Low |
Decrease, outgrowth leads to OFC | adjacent | Moderate | Low |
Apoptosis leads to Decrease, outgrowth | adjacent | Low | Low |
Decrease, GLI1/2 target gene expression leads to Apoptosis | adjacent | Low | Low |
Antagonism Smoothened leads to OFC | non-adjacent | High | Moderate |
Network View
Prototypical Stressors
Life Stage Applicability
Life stage | Evidence |
---|---|
Embryo | High |
Taxonomic Applicability
Term | Scientific Term | Evidence | Link |
---|---|---|---|
mouse | Mus musculus | NCBI |
Sex Applicability
Sex | Evidence |
---|---|
Unspecific | High |
Overall Assessment of the AOP
Domain of Applicability
Domain(s) of Applicability
Chemical: This AOP applies to antagonists of the SMO receptor. Chemical modulators of the SHH pathway have been identified including the natural alkaloid cyclopamine, both natural and synthetic pharmaceuticals (e.g. Vismodegib) , and a widely used pesticide synergist (PBO) with established human exposures (Lipinski, Dengler et al. 2007, Lipinski, Song et al. 2010, Wang, Lu et al. 2012, Everson, Sun et al. 2019, Rivera-González, Beames et al. 2021).
Sex: This AOP is unspecific to sex.
Life Stages: The relevant life stage for this AOP is embryonic development. More specifically, the development of the craniofacial region which occurs between GD 10.0 and GD 14.0 in the mouse.
Taxonomic: At present, the assumed taxonomic applicability domain of this AOP is mouse (mus musculus). Most of the toxicological data that this AOP is based on has used mice as their model. Mice are a good analog of human craniofacial development and undergo similar signaling by SHH.
Essentiality of the Key Events
Essentiality of the Key Events To date, few studies have addressed the essentiality of the proposed sequence of key events. Evidence linking SHH disruption through a decrease in proliferation exists. The hypothesized sequence of events has a high temporal concordance for canonical SHH signaling pathway and orofacial development.
- Studies have shown that SHH signaling is required for normal facial development and plays a critical role in the growth of the facial processes that form the upper palate and lip (Bush and Jiang 2012, Kurosaka 2015).
- The epithelial derived SHH drives orofacial development through an induced gradient in the underlying mesenchyme (Lan and Jiang 2009, Kurosaka 2015). This gradient of SHH induces cellular proliferation and outgrowth of the mesenchyme (Lan and Jiang 2009).
- OFCs caused by disruption to SHH are believed to be due to a reduction in epithelial induced proliferation and the subsequent decrease in tissue outgrowth and the failure of the facial processes to meet and fuse (Lipinski, Song et al. 2010, Heyne, Melberg et al. 2015).
Evidence Assessment
Evidence Assessment
- KER ID-Title-[Adjacency], [Evidence], [Quantitative Understanding
- KER 2734-Antagonism Smoothened leads to Decrease, SMO relocation-[Adjacent], [Moderate], [Low]-There is a high biological plausibility of this relationship and SMO localization to the primary cilia is essential for proper SHH signaling in vertebrates (Corbit, Aanstad et al. 2005, Rohatgi, Milenkovic et al. 2007, Rohatgi, Milenkovic et al. 2009). There is good evidence that the SANT compounds block the localization of SMO to the tip of the primary cilia. Contradictory in vivo data was found regarding whether cyclopamine blocks SMO relocation to the primary cilia. Further work is required to determine if SMO antagonism via cyclopamine results in decrease in SMO relocation.
- KER 2735- Decrease, SMO relocation leads to Decrease, GLI1/2 translocation-[Adjacent], [Moderate], [Low]- Moderate evidence is presented to support that a loss of the primary cilia leads to a significant decrease in GLI1. GLI1 requires activation prior to nuclear translocation.
- KER 2721-Decrease,GLI1/2 translocation leads to Decrease, GLI1/2 target gene-[Adjacent], [Low], [Low]- There is high biological plausibility of this relationship but to date few studies were found to explore the relationship.
- KER 2731-Decrease, GLI1/2 target gene expression leads to Decrease, SHH second messenger production-[Adjacent], [Low], [Low]-Coordinated signaling is paramount for proper embryonic development and the GLI signaling cascade drives feedback/forward loops with FGF and BMP signaling pathways. Support was found for SHH having a feedforward loop with FGF10 and BMP4 however further investigation into the interaction of these pathways and their crosstalk is required.
- KER 2732-Decrease, SHH second messenger production leads to Decrease, Cell proliferation-[Adjacent], [Low], [Low]- SHH is a known mitogen and drives proliferation through its’ secondary messengers. SHH was found to induce proliferation and FGF10 in vivo. In FGF10 deficient models SHH was found to be reduced.
- KER 2724-Decrease, Cell proliferation leads to Decrease, outgrowth-[Adjacent], [Low], [Low]-SHH is a known mitogen that helps to drive the proper development of the face which includes the outgrowth of the facial prominences. To date, few studies have measured by outgrowth of the facial prominences and proliferation. Hypoplasia of pharyngeal arch 1 was found in SHH-/- embryos supporting that outgrowth is driven by proliferation and is reduced when proliferation is decreased.
- KER 2726-Decrease, outgrowth leads to OFC-[Adjacent], [Moderate], [Low]- OFCs caused by disruption to SHH are believed to be due to a reduction in epithelial induced proliferation and the subsequent decrease in tissue outgrowth and the failure of the facial processes to meet and fuse (Lipinski, Song et al. 2010, Heyne, Melberg et al. 2015). Mice with disrupted SHH signaling are found to have palatal shelves that are spaced apart supporting that the cleft results from an EMi dependent, but epithelial-mesenchyme transition (Emt) independent manner.
- KER 2792-Apoptosis leads to Decrease, Outgrowth-[Adjacent], [Low], [Low]- SHH signaling is known to be associated with cell survival and there is a high biological plausibility that increasing apoptosis would cause a decrease in outgrowth. Supporting evidence is offered with increases in apoptosis in the mandibular arch seen in SHH signaling disrupted mice that exhibit decreased outgrowth.
- KER 2882-Decrase, GLI1/2 target gene expression leads to Apoptosis-[Adjacent], [Low], [Low]- To date few studies have examined the relationship of GLI1/2 target gene expression. There is a high biological plausibility that SHH plays a role in cell survival and death through GLI1/2 target gene expression. Decreased GLI1/2 target gene expression is seen in RA exposed dams alongside increased apoptosis on the CNCC.
- KER 2894-Antagonism Smoothened leads to OFC-[Non-adjacent], [High], [Moderate]- multiple studies have demonstrated in vivo that administration of SMO antagonists during critical windows of exposure leads to birth defects including OFC in a dose-dependent fashion.
Biological Plausibility Biological plausibility refers to the structural and/or functional relationship that exists between the key events based on our understanding of normal biology. SHH signaling is largely conserved in mammals and is required for normal facial development and plays a critical role in the growth of the facial processes that form the upper palate and lip (Bush and Jiang 2012, Kurosaka 2015). Multiple antagonists of the SMO receptor have been identified through binding studies for including cyclopamine, vismodegib, PBO, and the SANT compounds. While the level of support for most of the KERs is low, there is high support for the non-adjacent relationship linking antagonism of SMO and OFC.
Concordance of dose-response relationships
There are a limited number of studies in which multiple key events were assessed in the same study following exposure to known SMO antagonists. These studies form the basis of the dose-response concordance of this AOP. A summary of the dose-concordance can be found in table 1. Many studies were found to use a single exposure.
The concentration-dependence of the key event responses regarding concentration of known in vitro and/or in vivo for some of the KEs in this AOP.
- Concentration dependent clefting with cyclopamine exposure (Omnell, Sim et al. 1990)
- Concentration dependent neural tube malformations with cyclopamine exposure (Incardona, Gaffield et al. 1998)
- Dose dependent binding to SMO (Chen, Taipale et al. 2002)
- Concentration dependent decrease in SMO-ciliary accumulation in vitro for vismodegib exposure (Wang, Arvanites et al. 2012)
Temporal concordance
Temporal concordance refers to the degree to which the data supports the hypothesized sequence of MIE leading to the AO through a series of KEs. The SHH pathway is a well-known developmental pathway that plays a role in embryogenesis including the development of the face. The SHH pathway is sensitive to chemical disruption at multiple molecular targets along the signaling cascade including antagonism of the SMO receptor, with exposure during critical windows in development leading to OFCs (Lipinski and Bushman 2010, Heyne, Melberg et al. 2015). Chemical modulators of the SHH pathway have been identified including the natural alkaloid cyclopamine, both natural and synthetic pharmaceuticals, and a widely used pesticide synergist (PBO) with established human exposures (Lipinski, Dengler et al. 2007, Lipinski, Song et al. 2010, Wang, Lu et al. 2012, Everson, Sun et al. 2019, Rivera-González, Beames et al. 2021). Canonical SHH signaling through PTCH-SMO-GLI is well understood and our AOP remains consistent with the pathway. SHH signaling is required for normal facial development and plays a critical role in the growth of the facial processes that form the upper palate and lip (Bush and Jiang 2012, Kurosaka 2015). The epithelial derived SHH drives orofacial development through an induced gradient in the underlying mesenchyme (Lan and Jiang 2009, Kurosaka 2015). This gradient of SHH induces cellular proliferation and outgrowth of the mesenchyme (Lan and Jiang 2009). The hypothesized sequence of events is supported by the existing data and follow the field’s current understanding of the canonical SHH signaling pathway.
Consistency
The AO is not specific to this AOP. Many of the events is this AOP will overlap with AOPs linking disruption of SHH to OFC and some are expected to overlap with AOPs linking other developmental signaling pathways to OFCs.
Uncertainties, inconsistencies, and data gaps
This AOP would be strengthened by studies examining the dose-response and time-course relationships for these KERs. The main data gaps for this AOP exist in the lack of studies that have examined the relationship in the context of dose response or time course.
Does cyclopamine block SMO relocation-Rohatgi et al used NIH 3T3s cell and found that cyclopamine did not inhibit the accumulation of SMO in the cilia even when dosed at 5-10um (>10 fold above kd). All three antagonists inhibited SHH pathway transduction and target gene expression (Rohatgi, Milenkovic et al. 2009). Corbit et al used a renal epithelial MDCK (Madin-Darby canine kidney) line was engineered to express Myc-tagged SMO. Following culture for 1hr in SHH conditioned media SMO presence in the primary cilium is upregulated while cells cultured in the presence of cyclopamine see a downregulation of SMO in the primary cilia (Corbit, Aanstad et al. 2005). Further work is required to determine if SMO antagonism via cyclopamine results in decrease in SMO relocation.
Known Modulating Factors
Modulating Factor (MF) | Influence or Outcome | KER(s) involved |
---|---|---|
Quantitative Understanding
Assessment of quantitative understanding of the AOP:
The quantitative understanding for this AOP except for the non-adjacent relationship between Antagonism Smoothened leads to OFC is low. The majority of the data found through the literature was obtained from doses at a single dose and was not conducted with dose-response or time-course in mind. For Antagonism Smoothend leads to OFC several studies with dose response data showing a dose-dependent incidence of clefting were found. This AOP would benefit from the generation of additional data that addresses these relationships in a dose response and time course methodology to allow for an increased quantitative understanding of the linkage.
Considerations for Potential Applications of the AOP (optional)
Considerations for potential applications of the AOP
The intended use of this AOP from a regulatory standpoint is to improve predictive potential of developmental hazards as they relate to the SHH pathway and OFCs. It is hoped that this AOP can be applied to data from in silico and in vitro high-throughput screening assays (HTS) to guide selection of agents for further investigation in more representative models of orofacial development. Disruption of the Sonic Hedgehog pathway has broader outcomes than just OFCs and SHH is known to play a role in many aspects of embryonic development including patterning of many systems and limb and digit development. This AOP can be used as part of an integrated assessment of toxicity and can help to guide risk assessment for potential exposures during development.
References
(2011). "Prevalence at birth of cleft lip with or without cleft palate: data from the International Perinatal Database of Typical Oral Clefts (IPDTOC)." Cleft Palate Craniofac J 48(1): 66-81.
Bush, J. O. and R. Jiang (2012). "Palatogenesis: morphogenetic and molecular mechanisms of secondary palate development." Development 139(2): 231-243.
Chen, J. K., J. Taipale, M. K. Cooper and P. A. Beachy (2002). "Inhibition of Hedgehog signaling by direct binding of cyclopamine to Smoothened." Genes Dev 16(21): 2743-2748.
Corbit, K. C., P. Aanstad, V. Singla, A. R. Norman, D. Y. R. Stainier and J. F. Reiter (2005). "Vertebrate Smoothened functions at the primary cilium." Nature 437(7061): 1018-1021.
Dixon, M. J., M. L. Marazita, T. H. Beaty and J. C. Murray (2011). "Cleft lip and palate: understanding genetic and environmental influences." Nature Reviews Genetics 12(3): 167-178.
Everson, J. L., M. R. Sun, D. M. Fink, G. W. Heyne, C. G. Melberg, K. F. Nelson, P. Doroodchi, L. J. Colopy, C. M. Ulschmid, A. A. Martin, M. T. McLaughlin and R. J. Lipinski (2019). "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." Environ Health Perspect 127(10): 107006.
Heyne, G. W., C. G. Melberg, P. Doroodchi, K. F. Parins, H. W. Kietzman, J. L. Everson, L. J. Ansen-Wilson and R. J. Lipinski (2015). "Definition of critical periods for Hedgehog pathway antagonist-induced holoprosencephaly, cleft lip, and cleft palate." PLoS One 10(3): e0120517.
Incardona, J. P., W. Gaffield, R. P. Kapur and H. Roelink (1998). "The teratogenic Veratrum alkaloid cyclopamine inhibits sonic hedgehog signal transduction." Development 125(18): 3553-3562.
Jeong, J. and A. P. McMahon (2002). "Cholesterol modification of Hedgehog family proteins." The Journal of Clinical Investigation 110(5): 591-596.
Kurosaka, H. (2015). "The Roles of Hedgehog Signaling in Upper Lip Formation." Biomed Res Int 2015: 901041.
Lan, Y. and R. Jiang (2009). "Sonic hedgehog signaling regulates reciprocal epithelial-mesenchymal interactions controlling palatal outgrowth." Development 136(8): 1387-1396.
Lauth, M., A. Bergström, T. Shimokawa and R. Toftgård (2007). "Inhibition of GLI-mediated transcription and tumor cell growth by small-molecule antagonists." Proc Natl Acad Sci U S A 104(20): 8455-8460.
Lidral, A. C., L. M. Moreno and S. A. Bullard (2008). "Genetic Factors and Orofacial Clefting." Semin Orthod 14(2): 103-114.
Lipinski, R. J. and W. Bushman (2010). "Identification of Hedgehog signaling inhibitors with relevant human exposure by small molecule screening." Toxicol In Vitro 24(5): 1404-1409.
Lipinski, R. J., E. Dengler, M. Kiehn, R. E. Peterson and W. Bushman (2007). "Identification and characterization of several dietary alkaloids as weak inhibitors of hedgehog signaling." Toxicol Sci 100(2): 456-463.
Lipinski, R. J., C. Song, K. K. Sulik, J. L. Everson, J. J. Gipp, D. Yan, W. Bushman and I. J. Rowland (2010). "Cleft lip and palate results from Hedgehog signaling antagonism in the mouse: Phenotypic characterization and clinical implications." Birth Defects Res A Clin Mol Teratol 88(4): 232-240.
Lipinski, R. J., C. Song, K. K. Sulik, J. L. Everson, J. J. Gipp, D. Yan, W. Bushman and I. J. Rowland (2010). "Cleft lip and palate results from Hedgehog signaling antagonism in the mouse: Phenotypic characterization and clinical implications." Birth defects research. Part A, Clinical and molecular teratology 88(4): 232-240.
Mossey, P. A., J. Little, R. G. Munger, M. J. Dixon and W. C. Shaw (2009). "Cleft lip and palate." The Lancet 374(9703): 1773-1785.
Omnell, M. L., F. R. Sim, R. F. Keeler, L. C. Harne and K. S. Brown (1990). "Expression of Veratrum alkaloid teratogenicity in the mouse." Teratology 42(2): 105-119.
Petrova, E., J. Rios-Esteves, O. Ouerfelli, J. F. Glickman and M. D. Resh (2013). "Inhibitors of Hedgehog acyltransferase block Sonic Hedgehog signaling." Nat Chem Biol 9(4): 247-249.
Rivera-González, K. S., T. G. Beames and R. J. Lipinski (2021). "Examining the developmental toxicity of piperonyl butoxide as a Sonic hedgehog pathway inhibitor." Chemosphere 264: N.PAG-N.PAG.
Rohatgi, R., L. Milenkovic, R. B. Corcoran and M. P. Scott (2009). "Hedgehog signal transduction by Smoothened: pharmacologic evidence for a 2-step activation process." Proc Natl Acad Sci U S A 106(9): 3196-3201.
Rohatgi, R., L. Milenkovic and M. P. Scott (2007). "Patched1 regulates hedgehog signaling at the primary cilium." Science 317(5836): 372-376.
Wang, J., J. Lu, R. A. Mook, Jr., M. Zhang, S. Zhao, L. S. Barak, J. H. Freedman, H. K. Lyerly and W. Chen (2012). "The insecticide synergist piperonyl butoxide inhibits hedgehog signaling: assessing chemical risks." Toxicol Sci 128(2): 517-523.
Wang, Y., A. C. Arvanites, L. Davidow, J. Blanchard, K. Lam, J. W. Yoo, S. Coy, L. L. Rubin and A. P. McMahon (2012). "Selective identification of hedgehog pathway antagonists by direct analysis of smoothened ciliary translocation." ACS Chem Biol 7(6): 1040-1048.