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AOP: 460

Title

A descriptive phrase which references both the Molecular Initiating Event and Adverse Outcome.It should take the form “MIE leading to AO”. For example, “Aromatase inhibition leading to reproductive dysfunction” where Aromatase inhibition is the MIE and reproductive dysfunction the AO. In cases where the MIE is unknown or undefined, the earliest known KE in the chain (i.e., furthest upstream) should be used in lieu of the MIE and it should be made clear that the stated event is a KE and not the MIE.  More help

Antagonism of Smoothened receptor leading to orofacial clefting

Short name
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Anatagonsim SMO leads to OFC

Graphical Representation

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Click to download graphical representation template Explore AOP in a Third Party Tool

Authors

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Jacob I. Reynolds1 , Brian P. Johnson1,2 

1Department of Biomedical Engineering, Institute for Quantitative Health Science and Engineering, Michigan State University, East Lansing, MI

2Department of Pharmacology and Toxicology, Michigan State University, East Lansing, MI

Point of Contact

The user responsible for managing the AOP entry in the AOP-KB and controlling write access to the page by defining the contributors as described in the next section.   More help
Jacob Reynolds   (email point of contact)

Contributors

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  • Jacob Reynolds

Coaches

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  • Judy Choi

Status

Provides users with information concerning how actively the AOP page is being developed, what type of use or input the authors feel comfortable with given the current level of development, and whether it is part of the OECD AOP Development Workplan and has been reviewed and/or endorsed. OECD Status - Tracks the level of review/endorsement the AOP has been subjected to. OECD Project Number - Project number is designated and updated by the OECD. SAAOP Status - Status managed and updated by SAAOP curators. More help
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

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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

Used to provide background information for AOP reviewers and users that is considered helpful in understanding the biology underlying the AOP and the motivation for its development.The background should NOT provide an overview of the AOP, its KEs or KERs, which are captured in more detail below. More help

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

Provides a description of the approaches to the identification, screening and quality assessment of the data relevant to identification of the key events and key event relationships included in the AOP or AOP network.This information is important as a basis to support the objective/envisaged application of the AOP by the regulatory community and to facilitate the reuse of its components.  Suggested content includes a rationale for and description of the scope and focus of the data search and identification strategy/ies including the nature of preliminary scoping and/or expert input, the overall literature screening strategy and more focused literature surveys to identify additional information (including e.g., key search terms, databases and time period searched, any tools used). More help

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.

Table 1: Literature search AOP 460

Summary of the AOP

This section is for information that describes the overall AOP.The information described in section 1 is entered on the upper portion of an AOP page within the AOP-Wiki. This is where some background information may be provided, the structure of the AOP is described, and the KEs and KERs are listed. More help

Events:

Molecular Initiating Events (MIE)
An MIE is a specialised KE that represents the beginning (point of interaction between a prototypical stressor and the biological system) of an AOP. More help
Key Events (KE)
A measurable event within a specific biological level of organisation. More help
Adverse Outcomes (AO)
An AO is a specialized KE that represents the end (an adverse outcome of regulatory significance) of an AOP. More help
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)

This table summarizes all of the KERs of the AOP and is populated in the AOP-Wiki as KERs are added to the AOP.Each table entry acts as a link to the individual KER description page. More help

Network View

This network graphic is automatically generated based on the information provided in the MIE(s), KEs, AO(s), KERs and Weight of Evidence (WoE) summary tables. The width of the edges representing the KERs is determined by its WoE confidence level, with thicker lines representing higher degrees of confidence. This network view also shows which KEs are shared with other AOPs. More help

Prototypical Stressors

A structured data field that can be used to identify one or more “prototypical” stressors that act through this AOP. Prototypical stressors are stressors for which responses at multiple key events have been well documented. More help

Life Stage Applicability

The life stage for which the AOP is known to be applicable. More help
Life stage Evidence
Embryo High

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) can be selected.In many cases, individual species identified in these structured fields will be those for which the strongest evidence used in constructing the AOP was available. More help
Term Scientific Term Evidence Link
mouse Mus musculus NCBI

Sex Applicability

The sex for which the AOP is known to be applicable. More help
Sex Evidence
Unspecific High

Overall Assessment of the AOP

Addressess the relevant biological domain of applicability (i.e., in terms of taxa, sex, life stage, etc.) and Weight of Evidence (WoE) for the overall AOP as a basis to consider appropriate regulatory application (e.g., priority setting, testing strategies or risk assessment). More help

Domain of Applicability

Addressess the relevant biological domain(s) of applicability in terms of sex, life-stage, taxa, and other aspects of biological context. More help

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

The essentiality of KEs can only be assessed relative to the impact of manipulation of a given KE (e.g., experimentally blocking or exacerbating the event) on the downstream sequence of KEs defined for the AOP. Consequently, evidence supporting essentiality is assembled on the AOP page, rather than on the independent KE pages that are meant to stand-alone as modular units without reference to other KEs in the sequence. The nature of experimental evidence that is relevant to assessing essentiality relates to the impact on downstream KEs and the AO if upstream KEs are prevented or modified. This includes: Direct evidence: directly measured experimental support that blocking or preventing a KE prevents or impacts downstream KEs in the pathway in the expected fashion. Indirect evidence: evidence that modulation or attenuation in the magnitude of impact on a specific KE (increased effect or decreased effect) is associated with corresponding changes (increases or decreases) in the magnitude or frequency of one or more downstream KEs. More help

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

Addressess the biological plausibility, empirical support, and quantitative understanding from each KER in an AOP. More help

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.

Table 2: Concordance table

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.

  1. Concentration dependent clefting with cyclopamine exposure (Omnell, Sim et al. 1990)
  2. Concentration dependent neural tube malformations with cyclopamine exposure (Incardona, Gaffield et al. 1998)
  3. Dose dependent binding to SMO (Chen, Taipale et al. 2002)
  4. 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 factors (MFs) may alter the shape of the response-response function that describes the quantitative relationship between two KES, thus having an impact on the progression of the pathway or the severity of the AO.The evidence supporting the influence of various modulating factors is assembled within the individual KERs. More help
Modulating Factor (MF) Influence or Outcome KER(s) involved
     

Quantitative Understanding

Optional field to provide quantitative weight of evidence descriptors.  More help

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)

Addressess potential applications of an AOP to support regulatory decision-making.This may include, for example, possible utility for test guideline development or refinement, development of integrated testing and assessment approaches, development of (Q)SARs / or chemical profilers to facilitate the grouping of chemicals for subsequent read-across, screening level hazard assessments or even risk assessment. More help

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

List of the literature that was cited for this AOP. More help

(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.

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