Aop: 436

Title

Each AOP should be given a descriptive title that takes the form “MIE leading to AO”. For example, “Aromatase inhibition [MIE] leading to reproductive dysfunction [AO]” or “Thyroperoxidase inhibition [MIE] leading to decreased cognitive function [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

Inhibition of RALDH2 causes reduced all-trans retinoic acid levels, leading to transposition of the great arteries

Short name
A short name should also be provided that succinctly summarises the information from the title. This name should not exceed 90 characters. More help
RALDH2 and cardiovascular developmental defects

Graphical Representation

A graphical summary of the AOP listing all the KEs in sequence, including the MIE (if known) and AO, and the pair-wise relationships (links or KERs) between those KEs should be provided. This is easily achieved using the standard box and arrow AOP diagram (see this page for example). The graphical summary is prepared and uploaded by the user (templates are available) and is often included as part of the proposal when AOP development projects are submitted to the OECD AOP Development Workplan. The graphical representation or AOP diagram provides a useful and concise overview of the KEs that are included in the AOP, and the sequence in which they are linked together. This can aid both the process of development, as well as review and use of the AOP (for more information please see page 19 of the Users' Handbook).If you already have a graphical representation of your AOP in electronic format, simple save it in a standard image format (e.g. jpeg, png) then click ‘Choose File’ under the “Graphical Representation” heading, which is part of the Summary of the AOP section, to select the file that you have just edited. Files must be in jpeg, jpg, gif, png, or bmp format. Click ‘Upload’ to upload the file. You should see the AOP page with the image displayed under the “Graphical Representation” heading. To remove a graphical representation file, click 'Remove' and then click 'OK.'  Your graphic should no longer be displayed on the AOP page. If you do not have a graphical representation of your AOP in electronic format, a template is available to assist you.  Under “Summary of the AOP”, under the “Graphical Representation” heading click on the link “Click to download template for graphical representation.” A Powerpoint template file should download via the default download mechanism for your browser. Click to open this file; it contains a Powerpoint template for an AOP diagram and instructions for editing and saving the diagram. Be sure to save the diagram as jpeg, jpg, gif, png, or bmp format. Once the diagram is edited to its final state, upload the image file as described above. More help

Authors

List the name and affiliation information of the individual(s)/organisation(s) that created/developed the AOP. In the context of the OECD AOP Development Workplan, this would typically be the individuals and organisation that submitted an AOP development proposal to the EAGMST. Significant contributors to the AOP should also be listed. A corresponding author with contact information may be provided here. This author does not need an account on the AOP-KB and can be distinct from the point of contact below. The list of authors will be included in any snapshot made from an AOP. More help

R.H. Mennen, S. Mitchell-Ryan

Health and Environmental Sciences Institute (HESI), Washington DC, USA

Point of Contact

Indicate the point of contact for the AOP-KB entry itself. This person is responsible for managing the AOP entry in the AOP-KB and controls write access to the page by defining the contributors as described below. Clicking on the name will allow any wiki user to correspond with the point of contact via the email address associated with their user profile in the AOP-KB. This person can be the same as the corresponding author listed in the authors section but isn’t required to be. In cases where the individuals are different, the corresponding author would be the appropriate person to contact for scientific issues whereas the point of contact would be the appropriate person to contact about technical issues with the AOP-KB entry itself. Corresponding authors and the point of contact are encouraged to monitor comments on their AOPs and develop or coordinate responses as appropriate.  More help
Arthur Author   (email point of contact)

Contributors

List user names of all  authors contributing to or revising pages in the AOP-KB that are linked to the AOP description. This information is mainly used to control write access to the AOP page and is controlled by the Point of Contact.  More help
  • Gina Mennen
  • Arthur Author

Status

The status section is used to provide AOP-KB 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. “Author Status” is an author defined field that is designated by selecting one of several options from a drop-down menu (Table 3). The “Author Status” field should be changed by the point of contact, as appropriate, as AOP development proceeds. See page 22 of the User Handbook for definitions of selection options. More help
Author status OECD status OECD project SAAOP status
Open for comment. Do not cite
This AOP was last modified on May 08, 2022 11:33
The date the AOP was last modified is automatically tracked by the AOP-KB. The date modified field can be used to evaluate how actively the page is under development and how recently the version within the AOP-Wiki has been updated compared to any snapshots that were generated. More help

Revision dates for related pages

Page Revision Date/Time
Inhibition of ALDH1A (RALDH) November 11, 2021 14:48
Decreased all-trans retinoic acid (atRA) concentration February 27, 2022 10:55
Disruption, Progenitor cells of second heart field February 15, 2022 10:39
Neural crest cell migration, reduced December 20, 2018 04:10
transposition of the great arteries February 15, 2022 10:47
ALDH1A (RALDH), inhibition leads to decreased, atRA concentration November 11, 2021 15:32
decreased, atRA concentration leads to Disruption, Progenitor cells of second heart field February 15, 2022 10:51
Disruption, Progenitor cells of second heart field leads to Reduced neural crest cell migration February 10, 2022 03:33
Reduced neural crest cell migration leads to Transposition of the great arteries February 15, 2022 10:53
4-diethylaminobenzaldehyde (DEAB) February 15, 2022 10:55
Disulfiram August 03, 2017 10:53
WIN18466 February 15, 2022 10:56
nitrofen May 22, 2019 05:18
Ethanol April 05, 2018 06:38
All-trans retionic acid February 15, 2022 11:14
Bisdiamine May 22, 2019 05:19
Vitamin A February 15, 2022 11:15
BMS-89453 February 15, 2022 10:46

Abstract

In the abstract section, authors should provide a concise and informative summation of the AOP under development that can stand-alone from the AOP page. Abstracts should typically be 200-400 words in length (similar to an abstract for a journal article). Suggested content for the abstract includes the following: The background/purpose for initiation of the AOP’s development (if there was a specific intent) A brief description of the MIE, AO, and/or major KEs that define the pathway A short summation of the overall WoE supporting the AOP and identification of major knowledge gaps (if any) If a brief statement about how the AOP may be applied (optional). The aim is to capture the highlights of the AOP and its potential scientific and regulatory relevance More help

The aim was to describe a linear AOP of the relationship between inhibition of retinaldehyde dehydrogenase (RALDH), an all-trans retinoic acid (ATRA) synthesizing enzyme, and the subsequent disturbance of ATRA levels that can influence impaired positioning of the great arteries as the adverse outcome (AO) during cardiovascular development. The selected molecular initiating event (MIE) is inhibition of RALDH. This MIE was selected based on robust available literature. Intermediate key events (KE) are decreased ATRA concentration, disruption of progenitor cells of the second heart field (SHF), and reduced neural crest cell (NCC) migration. Evidence for this AOP is mainly generated in chick and mouse ablation or gene mutation studies. Human evidence is limited, but comparable mechanisms between vertebrates have been reported, thereby establishing relevance due to similarities in cardiovascular development. Impaired great artery positioning is related to reduced cardiovascular function that can result in fetal/embryonic preterm deaths or require surgical intervention to correct defects in newborns. Various factors can influence the formation of the cardiovascular system and the interplay between the pharyngeal endoderm, the splanchnic mesoderm (second heart field) and the neural crest is of importance for proper development. This AOP describes the influence of the secondary heart field (SHF) on cardiac neural crest cell migration and functioning, which in turn is essential for development of the great arteries.  

Background (optional)

This optional subsection should be 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. Examples of potential uses of the optional background section are listed on pages 24-25 of the User Handbook. More help

Vertebrate embryo-fetal cardiovascular development involves multiple steps. The importance of all-trans retinoic acid (ATRA) in this process is well documented and has been the subject of multiple review papers (Brade et al., 2018; Duong & Waxman, 2021; Nakajima, 2019; Perl & Waxman, 2019; Stefanovic & Zaffran, 2017; S. Wang & Moise, 2019).

Cardiac progenitors

During early gastrulation at E6.5 in mice, the first cardiac progenitors are formed around the primitive streak and migrate anterior laterally to form the cardiac crescent to become the first heart field (FHF) at E7-7.5 (Stefanovic & Zaffran, 2017). An evolutionary approach to cardiac development related to the vertebrate heart was reviewed by Pérez-Pomares et al. in 2009 (Pérez-Pomares et al., 2009).

To make the cardiac crescent and primary heart tube, weak or no ATRA signaling is sufficient (Nakajima, 2019). At this stage of cardiogenesis, ATRA controls the cardiac progenitor pool size (Keegan et al., 2005; S. Wang & Moise, 2019). This early requirement of ATRA is conserved across species and restricts ventricular and atrial specification within the cardiac progenitor pool (Duong & Waxman, 2021). Before the primitive heart tube is formed, ATRA signaling is symmetrical (Nakajima, 2019).

Tube formation and anterior/posterior heart segments before looping

The heart tube is derived from the FHF at E8 in mice or after three weeks in human development (Brade et al., 2018). As the cardiac crescent folds, it will form the primitive heart tube and consequently fusion and systemic circulation can emerge (S. Wang & Moise, 2019). The FHF mainly will give rise to the left ventricle. The linear heart tube expands by cell proliferation and recruitment of additional cells that contribute to the arterial and venous poles of the heart tube and are derived from the second heart field (SHF) (Brade et al., 2018). The SHF mainly contributes to the outflow tract (OFT), the right ventricle, and part of the atria (Brade et al., 2018).

The posterior mesoderm and cardiac precursors produce ATRA, which at this stage is patterned in a caudo-rostral gradient determining the inflow-outflow poles of the heart tube (S. Wang & Moise, 2019).

Specification Second Heart Field

The SHF is specified by ISL1 (Islet-1, transcription factor and SHF specifier) positive cells and contributes to the sub-pulmonary myocardium (S. Wang & Moise, 2019).

Higher levels of ATRA promote posterior precursors of the FHF and SHF committing to the inflow tract (Hochgreb et al., 2003; Niederreither et al., 2001; Ryckebusch et al., 2008a; Sirbu et al., 2008; S. Wang & Moise, 2019). Subsequently, ATRA restricts SHF boundaries and stimulates a SHF sublineage giving rise to the outflow tract (OFT) (P. Li et al., 2010; Ma et al., 2016; Nakajima, 2019; S. Wang & Moise, 2019).

Heart tube looping / outflow tract development

As the heart tube grows, looping will occur at E9 in mice (S. Wang & Moise, 2019). During this process the straight heart tube will be remodeled by forming a coiling loop to eventually form a multichambered heart.

ATRA receptors (RARs) have been shown to be essential for cardiac looping, left-right patterning, and inflow tract development (Perl & Waxman, 2019). For proper OFT development in mice, Hox genes are necessary which are responsive to ATRA (Perl & Waxman, 2019).

Chamber formation

Chamber septation emerges at E10 in mice (S. Wang & Moise, 2019). Interaction of cardiomyocytes with epicardial, endocardial and cardiac neural crest cells (Brade et al., 2018).

ATRA is needed for the posterior heart segment to become the primitive atrium and sinus venosus (Nakajima, 2019). At later stages, ATRA stimulates growth and maturation of the ventriculi (Nakajima, 2019).

Proepicardium / Coronary vessel development

The proepicardial cells are formed between E9.5 and E11.5 in mice, migrate from a location near the sinus venosus to cover the primitive heart tube, and will form the epicardium which is the outer layer of the heart (Brade et al., 2018; S. Wang & Moise, 2019).

Mesenchymal subepicardial cells and epicardial derived cells will merge and differentiate to the coronary plexus around E11.5 in mice and subsequently coronary vessels form around the ventricle until E13.5 (Brade et al., 2018).

Several lines of evidence indicate that precision in ATRA levels and timing is important in coronary vasculature development and are reviewed by Wang and Moise (S. Wang & Moise, 2019). Coronary vasculature defects resulting from perturbed ATRA homeostasis usually concurs with myocardial expansion, vasculature development and fetal erythropoiesis (S. Wang & Moise, 2019).

myocardial expansion (ventricular wall expansion)

The FHF contributes primarily to the myocardium (S. Wang & Moise, 2019). ATRA signaling in epicardial(-derived) cells stimulates a signal promoting myocardium growth (Brade et al., 2011; S. Wang & Moise, 2019). The embryonic liver is thought to contribute to myocardial expansion through erythropoietin (EPO) secretion, which is a direct target of ATRA in the liver, and in turn induces insulin growth factor 2 (IGF2) release from the epicardium (Brade et al., 2011; S. Wang & Moise, 2019). Inactivation of EPO or its receptor resulted in ventricular hypoplasia (S. Wang & Moise, 2019; Wu et al., 1999).

Cardiac neural crest orientation and positioning

Cardiac neural crest cells (cNCCs) mainly provide patterning signals that contribute to the aortic arteries, OFT/aortopulmonary development and septation, and formation of a functional myocardium (Brade et al., 2018; Stefanovic & Zaffran, 2017; S. Wang & Moise, 2019). Additionally, cNCCs contribute to the heart valves and parasympathetic innervation (Brade et al., 2018; Stefanovic & Zaffran, 2017; S. Wang & Moise, 2019). Furthermore, preotic cNCC derived precursors contribute to vascular smooth muscle cell formation and eventually to proximal coronary arteries (Stefanovic & Zaffran, 2017). ATRA influences the cNCC migration and differentiation, which contribute to the aorticopulmonary septum / OFT (el Robrini et al., 2016; Ma et al., 2016; S. Wang & Moise, 2019).

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 stressor and the biological system) of an AOP. More help
Key Events (KE)
This table summarises all of the KEs of the AOP. This table is populated in the AOP-Wiki as KEs are added to the AOP. Each table entry acts as a link to the individual KE description page.  More help
Adverse Outcomes (AO)
An AO is a specialised KE that represents the end (an adverse outcome of regulatory significance) of an AOP.  More help
Sequence Type Event ID Title Short name
MIE 1880 Inhibition of ALDH1A (RALDH) ALDH1A (RALDH), inhibition
KE 1881 Decreased all-trans retinoic acid (atRA) concentration decreased, atRA concentration
KE 1682 Disruption, Progenitor cells of second heart field Disruption, Progenitor cells of second heart field
KE 1557 Neural crest cell migration, reduced Reduced neural crest cell migration
AO 1970 transposition of the great arteries Transposition of the great arteries

Relationships Between Two Key Events (Including MIEs and AOs)

This table summarises 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.To add a key event relationship click on either Add relationship: events adjacent in sequence or Add relationship: events non-adjacent in sequence.For example, if the intended sequence of KEs for the AOP is [KE1 > KE2 > KE3 > KE4]; relationships between KE1 and KE2; KE2 and KE3; and KE3 and KE4 would be defined using the add relationship: events adjacent in sequence button.  Relationships between KE1 and KE3; KE2 and KE4; or KE1 and KE4, for example, should be created using the add relationship: events non-adjacent button. This helps to both organize the table with regard to which KERs define the main sequence of KEs and those that provide additional supporting evidence and aids computational analysis of AOP networks, where non-adjacent KERs can result in artifacts (see Villeneuve et al. 2018; DOI: 10.1002/etc.4124).After clicking either option, the user will be brought to a new page entitled ‘Add Relationship to AOP.’ To create a new relationship, select an upstream event and a downstream event from the drop down menus. The KER will automatically be designated as either adjacent or non-adjacent depending on the button selected. The fields “Evidence” and “Quantitative understanding” can be selected from the drop-down options at the time of creation of the relationship, or can be added later. See the Users Handbook, page 52 (Assess Evidence Supporting All KERs for guiding questions, etc.).  Click ‘Create [adjacent/non-adjacent] relationship.’  The new relationship should be listed on the AOP page under the heading “Relationships Between Two Key Events (Including MIEs and AOs)”. To edit a key event relationship, click ‘Edit’ next to the name of the relationship you wish to edit. The user will be directed to an Editing Relationship page where they can edit the Evidence, and Quantitative Understanding fields using the drop down menus. Once finished editing, click ‘Update [adjacent/non-adjacent] relationship’ to update these fields and return to the AOP page.To remove a key event relationship to an AOP page, under Summary of the AOP, next to “Relationships Between Two Key Events (Including MIEs and AOs)” click ‘Remove’ The relationship should no longer be listed on the AOP page under the heading “Relationships Between Two Key Events (Including MIEs and AOs)”. More help

Network View

The AOP-Wiki automatically generates a network view of the AOP. This network graphic is based on the information provided in the MIE, KEs, AO, KERs and 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

Stressors

The stressor field is a structured data field that can be used to annotate an AOP with standardised terms identifying stressors known to trigger the MIE/AOP. Most often these are chemical names selected from established chemical ontologies. However, depending on the information available, this could also refer to chemical categories (i.e., groups of chemicals with defined structural features known to trigger the MIE). It can also include non-chemical stressors such as genetic or environmental factors. Although AOPs themselves are not chemical or stressor-specific, linking to stressor terms known to be relevant to different AOPs can aid users in searching for AOPs that may be relevant to a given stressor. More help

Life Stage Applicability

Identify the life stage for which the KE is known to be applicable. More help
Life stage Evidence
Fetal 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 in relation to this KE. More help
Term Scientific Term Evidence Link
chicken Gallus gallus High NCBI
mouse Mus musculus High NCBI
Vertebrates Vertebrates Moderate NCBI

Sex Applicability

The authors must select from one of the following: Male, female, mixed, asexual, third gender, hermaphrodite, or unspecific. More help
Sex Evidence
Mixed High

Overall Assessment of the AOP

This section addresses the relevant biological domain of applicability (i.e., in terms of taxa, sex, life stage, etc.) and WoE for the overall AOP as a basis to consider appropriate regulatory application (e.g., priority setting, testing strategies or risk assessment). The goal of the overall assessment is to provide a high level synthesis and overview of the relative confidence in the AOP and where the significant gaps or weaknesses are (if they exist). Users or readers can drill down into the finer details captured in the KE and KER descriptions, and/or associated summary tables, as appropriate to their needs.Assessment of the AOP is organised into a number of steps. Guidance on pages 59-62 of the User Handbook is available to facilitate assignment of categories of high, moderate, or low confidence for each consideration. While it is not necessary to repeat lengthy text that appears elsewhere in the AOP description (or related KE and KER descriptions), a brief explanation or rationale for the selection of high, moderate, or low confidence should be made. More help

  

Domain of Applicability

The relevant biological domain(s) of applicability in terms of sex, life-stage, taxa, and other aspects of biological context are defined in this section. Biological domain of applicability is informed by the “Description” and “Biological Domain of Applicability” sections of each KE and KER description (see sections 2G and 3E for details). In essence the taxa/life-stage/sex applicability is defined based on the groups of organisms for which the measurements represented by the KEs can feasibly be measured and the functional and regulatory relationships represented by the KERs are operative.The relevant biological domain of applicability of the AOP as a whole will nearly always be defined based on the most narrowly restricted of its KEs and KERs. For example, if most of the KEs apply to either sex, but one is relevant to females only, the biological domain of applicability of the AOP as a whole would be limited to females. While much of the detail defining the domain of applicability may be found in the individual KE and KER descriptions, the rationale for defining the relevant biological domain of applicability of the overall AOP should be briefly summarised on the AOP page. More help

This AOP regarding ATRA homeostasis and cardiovascular development applies to both male and female sexes. The life stage in which the processes described as KEs and KERs are applicable is during fetal development. Strongest evidence on cardiovascular development regulation used to generate this AOP is from chicken and mouse studies with also some evidence from rat and zebrafish. Evidence exists that similar mechanisms are available in vertebrate species and therefore this AOP also applies to humans, although the evidence is not as strong.

Essentiality of the Key Events

An important aspect of assessing an AOP is evaluating the essentiality of its KEs. 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.When assembling the support for essentiality of the KEs, authors should organise relevant data in a tabular format. The objective is to summarise briefly the nature and numbers of investigations in which the essentiality of KEs has been experimentally explored either directly or indirectly. See pages 50-51 in the User Handbook for further definitions and clarifications.  More help

The essentiality of each KE was determined by the impact of the upstream modified MIE or KE on the downstream KEs or AO. The level of support for essentiality of MIE and KEs within the overall AOP is high.

MIE: RALDH inhibition

The essentiality for the MIE is high because knockout and/or inhibition studies show perturbed ATRA concentrations (KE1), patterning defects in the second heart field (KE2), restricted contribution of neural crest cells (KE3), and various cardiovascular defects including transposition great arteries (AO).

Indirect evidence for essentiality of the MIE was identified as a large impact on the MIE (i.e. knockout studies) associated with an increased frequency of the downstream AO of the transposition of the great arteries. Evidence from stressors for RALDH related to cardiovascular developmental defects is also available. For example, WIN18446 (or bis-(dichloroacetyl)diamine) is an irreversible RALDH inhibitor (RALDH1, -2, and -3), that also can cause abnormal development of the aortic arch and outflow tract, coronary arteries, and the myocardium and also it can cause syndromes as tetralogy of Fallot in rats, mice and chick (Fujino et al., 2005; Kise et al., 2005; Kuribayashi & Roberts, 1993; Nishijima et al., 2000; Okamoto et al., 2004; Okishima et al., 1992; Tasaka et al., 1991; S. Wang et al., 2018).

RALDH2 is important for almost all ATRA synthesis in the heart as was shown by knockdown studies (Stefanovic & Zaffran, 2017). Mutations of the Raldh2 gene result in phenotypes characterized by prominent myocardial defects also resulting in embryonic lethality (Brade et al., 2011; el Robrini et al., 2016; Merki et al., 2005; Niederreither et al., 1999, 2001; Sorrell & Waxman, 2011; Stefanovic & Zaffran, 2017; S. Wang & Moise, 2019). Raldh2 null mice mutants that were rescued from embryo lethality showed abnormal cNCC migration (Niederreither et al., 2003). Rescued Raldh2 mouse mutant embryos also showed a disruption of the posterior limit of the SHF starting at E7.5 (Duong & Waxman, 2021; Ryckebusch et al., 2008a; Sirbu et al., 2008).

Next to RALDH, other enzymes are also involved in the formation of ATRA but also in the breakdown of ATRA.

KE1: ATRA concentration

The essentiality for the KE of disturbed ATRA levels is high as it all affects KE2, KE3 and AO.

Vertebrate embryo-fetal cardiovascular development involves multiple steps and great knowledge is available including the importance of all-trans retinoic acid (ATRA) which has been reviewed in multiple papers (Brade et al., 2018; Duong & Waxman, 2021; Nakajima, 2019; Perl & Waxman, 2019; Stefanovic & Zaffran, 2017; S. Wang & Moise, 2019). As different processes in embryodevelopment benefit from varying levels of ATRA, an ATRA gradient exists which is generated by multiple enzymes that synthesize and degrade ATRA to maintain the preferred balance (Kedishvili, 2013; Menegola et al., 2021; Tonk & Pennings, 2015). This gradient is important in SHF patterning (Nakajima, 2019).

There is also some evidence that mutants of the retinoid receptor partially revealed the mechanism of cNCC involved in cardiovascular development (Kubalak et al., 2002).

Vitamin A deficiency in embryos resulted in heart developmental defects such as septal defects, abnormalities to the inflow and outflow tract, aortic arch abnormalities and coronary malformations in quail and rat (Dersch & Zile, 1993; Heine et al., 1985; Wilson & Warkany, 1949, 1950).

KE2: SHF patterning

The essentiality is high for correct SHF patterning since different parts within the SHF are responsible for correct formation of the different anatomical positions in the developed heart as was reviewed by Nakajima in 2019 (Nakajima, 2019). Impaired development of the SHF causes cardiovascular developmental defects (Nakajima, 2019).

KE3: cNCC contribution

The contribution of cardiac neural crest cells to the development of the heart is highly essential because neural crest cell ablation studies show multiple cardiovascular developmental defects including transposed great arteries such as of the aortic arch (Plein et al., 2015).

AO: transposition great arteries

Normal cardiovascular development including normal patterning of the great arteries is essential as disruptions can lead to fetal death or teratogenic defects upon birth that hampers functioning of the newborn.

Evidence Assessment

The biological plausibility, empirical support, and quantitative understanding from each KER in an AOP are assessed together.  Biological plausibility of each of the KERs in the AOP is the most influential consideration in assessing WoE or degree of confidence in an overall hypothesised AOP for potential regulatory application (Meek et al., 2014; 2014a). Empirical support entails consideration of experimental data in terms of the associations between KEs – namely dose-response concordance and temporal relationships between and across multiple KEs. It is examined most often in studies of dose-response/incidence and temporal relationships for stressors that impact the pathway. While less influential than biological plausibility of the KERs and essentiality of the KEs, empirical support can increase confidence in the relationships included in an AOP. For clarification on how to rate the given empirical support for a KER, as well as examples, see pages 53- 55 of the User Handbook.  More help

Biological plausibility

The overall biological plausibility of the AOP was assessed as high. The role for ALDH1A2 in the synthesis of ATRA is well established as an essential component of regulating regional ATRA expression during development. The role of ATRA within cardiovascular development is highly plausible including its involvement in SHF patterning and signaling, which is important for consequent predispositioning to specific anatomical parts/occurrences in cardiovascular development.

After the initial patterning stages, the size of the cardiac progenitor pool is controlled within the anterior lateral plate mesoderm, ATRA signaling then divides the anterior and posterior SHFs (Keegan et al., 2005; S. Wang & Moise, 2019). High ATRA signaling defines the posterior boundary of the murine second heart field (Ryckebusch et al., 2008a; Sirbu et al., 2008). This is exemplified by the ATRA producing enzyme RALDH, which is expressed in posterior SHF progenitors in mice (Stefanovic et al., 2020).

The biological plausibility of the relationship between SHF signaling and cNCC contribution is moderate because the relationship between the two KEs still contain unknowns in terms of mechanistic connections. Additionally, there is an interdependent relationship between the SHF and cNCC, but also the pharyngeal endoderm plays an indispensable role in this tripartite connection (Diman et al., 2011).

There is strong biological plausibility for a link between ATRA synthesis and normal positioning of the great arteries during early development, mainly determined by mouse RALDH knouckout studies. Because of evolutionary consistencies between vertebrates (Pérez-Pomares et al., 2009), the relationship is regarded biologically plausible also in humans. No direct human evidence is available and therefore the weight of evidence is not as strong. 

Empirical evidence

Dose-concordance

The quantitative understanding of dose-response relationships is limited in this AOP. 0.25-0.5 mg/ml ATRA administration specific to the anterior heart field using beads in chick embryo culture, resulted in disturbed SHF specific gene expression levels and transposition of the great arteries (Narematsu et al., 2015).

ATRA is a morphogen and the required ATRA levels are dependent on the location within the fetus for patterning and varies depending on the developmental stage (Piersma et al., 2017).

Temporal-concordance

The temporal sequence of events is strong as they are based on the biological process that are taking place. The critical period for chemical perturbations that apply to this AOP is during fetal life. The sequence in time starts with inhibition of RALDH leading to reduced ATRA synthesis. Consequently, the ATRA gradient in the SHF is disrupted resulting in an impaired pattering of the progenitors within the SHF. The progenitors within the SHF differ in signaling properties and an impaired progenitor patterning also impairs signaling to the cNCCs, which in turn fail to contribute to correct great artery development.

The relationship between the SHF and cNCC occurs at similar timepoints as they are interdependent and at the same time also communicate with the pharyngeal endoderm to be able to contribute to cardiovascular development.

Uncertainties

In mice, there is strong evidence to support the view that ATRA is important in cardiovascular development including the positioning of the great arteries. Evidence from RALDH knockout studies is also consistent. The migration and development of cNCCs not solely depends on SHF signaling. The pharyngeal endoderm also plays an important role in the maintenance and deployment of cNCCs through signaling of sonic hedgehog (Shh) (Goddeeris et al., 2007; Vincent & Buckingham, 2010). In the absence of Shh, the development of proper pharyngeal arches and OFT is affected (Vincent & Buckingham, 2010). Additionally, a possible feedback loop exists between SHF signaling to cNCCs, since ablation of cNCCs results in SHF over proliferation because of excessive Fgf8 signaling (Rochais et al., 2009). Furthermore, NCC deletion of Smad4 leads to abnormal SHF patterning and a shorter OFT. Lastly, Tbx3 loss in NCCs and pharyngeal endoderm also resulted in SHF over proliferation and a shorter OFT (Rochais et al., 2009).

Quantitative Understanding

Some proof of concept examples to address the WoE considerations for AOPs quantitatively have recently been developed, based on the rank ordering of the relevant Bradford Hill considerations (i.e., biological plausibility, essentiality and empirical support) (Becker et al., 2017; Becker et al, 2015; Collier et al., 2016). Suggested quantitation of the various elements is expert derived, without collective consideration currently of appropriate reporting templates or formal expert engagement. Though not essential, developers may wish to assign comparative quantitative values to the extent of the supporting data based on the three critical Bradford Hill considerations for AOPs, as a basis to contribute to collective experience.Specific attention is also given to how precisely and accurately one can potentially predict an impact on KEdownstream based on some measurement of KEupstream. This is captured in the form of quantitative understanding calls for each KER. See pages 55-56 of the User Handbook for a review of quantitative understanding for KER's. More help

This AOP is of qualitative understanding. Quantitative data between chemical potency and perturbations are insufficient. This relates to the mainly strong evidence of the MIE coming from gene knockout studies. Furthermore, ATRA is a morphogen and the required ATRA levels are dependent on the location within the fetus for patterning and varies depending on the developmental stage (Piersma et al., 2017).

Considerations for Potential Applications of the AOP (optional)

At their discretion, the developer may include in this section discussion of the 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. While it is challenging to foresee all potential regulatory application of AOPs and any application will ultimately lie within the purview of regulatory agencies, potential applications may be apparent as the AOP is being developed, particularly if it was initiated with a particular application in mind. This optional section is intended to provide the developer with an opportunity to suggest potential regulatory applications and describe his or her rationale.To edit the “Considerations for Potential Applications of the AOP” section, on an AOP page, in the upper right hand menu, click ‘Edit.’ This brings you to a page entitled, “Editing AOP.” Scroll down to the “Considerations for Potential Applications of the AOP” section, where a text entry box allows you to submit text. In the upper right hand menu, click ‘Update AOP’ to save your changes and return to the AOP page or 'Update and continue' to continue editing AOP text sections.  The new text should appear under the “Considerations for Potential Applications of the AOP” section on the AOP page. More help

References

List the bibliographic references to original papers, books or other documents used to support the AOP. More help

Brade, T., Kumar, S., Cunningham, T. J., Chatzi, C., Zhao, X., Cavallero, S., Li, P., Sucov, H. M., Ruiz-Lozano, P., & Duester, G. (2011). Retinoic acid stimulates myocardial expansion by induction of hepatic erythropoietin which activates epicardial Igf2. Development, 138(1), 139–148. https://doi.org/10.1242/dev.054239

Brade, T., Pane, L. S., Moretti, A., Chien, K. R., & Laugwitz, K.-L. (2018). Embryonic Heart Progenitors and Cardiogenesis. 1–18. https://doi.org/10.1101/cshperspect.a013847

Dersch, H., & Zile, M. H. (1993). Induction of normal cardiovascular development in the vitamin A-deprived quail embryo by natural retinoids. Developmental Biology, 160(2), 424–433. https://doi.org/10.1006/dbio.1993.1318

Diman, N. Y. S. G., Remacle, S., Bertrand, N., Picard, J. J., Zaffran, S., & Rezsohazy, R. (2011). A retinoic acid responsive Hoxa3 transgene expressed in embryonic pharyngeal endoderm, cardiac neural crest and a subdomain of the second heart field. PloS One, 6(11). https://doi.org/10.1371/JOURNAL.PONE.0027624

Duong, T. B., & Waxman, J. S. (2021). Patterning of vertebrate cardiac progenitor fields by retinoic acid signaling. Genesis (New York, N.Y. : 2000), e23458. https://doi.org/10.1002/dvg.23458

el Robrini, N., Etchevers, H. C., Ryckebüsch, L., Faure, E., Eudes, N., Niederreither, K., Zaffran, S., & Bertrand, N. (2016a). Cardiac outflow morphogenesis depends on effects of retinoic acid signaling on multiple cell lineages. Developmental Dynamics, 245(3), 388–401. https://doi.org/10.1002/dvdy.24357

el Robrini, N., Etchevers, H. C., Ryckebüsch, L., Faure, E., Eudes, N., Niederreither, K., Zaffran, S., & Bertrand, N. (2016b). Cardiac outflow morphogenesis depends on effects of retinoic acid signaling on multiple cell lineages. Developmental Dynamics, 245(3), 388–401. https://doi.org/10.1002/dvdy.24357

Fujino, H., Nakagawa, M., Nishijima, S., Okamoto, N., Hanato, T., Watanabe, N., Shirai, T., Kamiya, H., & Takeuchi, Y. (2005). Morphological differences in cardiovascular anomalies induced by bis-diamine between Sprague-Dawley and Wistar rats. Congenital Anomalies, 45(2), 52–58. https://doi.org/10.1111/j.1741-4520.2005.00063.x

Goddeeris, M. M., Schwartz, R., Klingensmith, J., & Meyers, E. N. (2007). Independent requirements for Hedgehog signaling by both the anterior heart field and neural crest cells for outflow tract development. Development (Cambridge, England), 134(8), 1593–1604. https://doi.org/10.1242/DEV.02824

Heine, U. I., Roberts, A. B., Munoz, E. F., Roche, N. S., & Sporn, M. B. (1985). Effects of retinoid deficiency on the development of the heart and vascular system of the quail embryo. Virchows Archiv. B, Cell Pathology Including Molecular Pathology, 50(2), 135–152. https://doi.org/10.1007/BF02889897

Hochgreb, T., Linhares, V. L., Menezes, D. C., Sampaio, A. C., Yan, C. Y. I., Cardoso, W. v., Rosenthal, N., & Xavier-Neto, J. (2003). A caudorostral wave of RALDH2 conveys anteroposterior information to the cardiac field. Development, 130(22), 5363–5374. https://doi.org/10.1242/DEV.00750

Kedishvili, N. Y. (2013). Enzymology of retinoic acid biosynthesis and degradation. Journal of Lipid Research, 54(7), 1744–1760. https://doi.org/10.1194/JLR.R037028

Keegan, B. R., Feldman, J. L., Begemann, G., Ingham, P. W., & Yelon, D. (2005). Retinoic acid signaling restricts the cardiac progenitor pool. Science (New York, N.Y.), 307(5707), 247–249. https://doi.org/10.1126/SCIENCE.1101573

Kise, K., Nakagawa, M., Okamoto, N., Hanato, T., Watanabe, N., Nishijima, S., Fujino, H., Takeuchi, Y., & Shiraishi, I. (2005). Teratogenic effects of bis-diamine on the developing cardiac conduction system. Birth Defects Research Part A - Clinical and Molecular Teratology, 73(8), 547–554. https://doi.org/10.1002/bdra.20163

Kubalak, S. W., Hutson, D. R., Scott, K. K., & Shannon, R. A. (2002). Elevated transforming growth factor β2 enhances apoptosis and contributes to abnormal outflow tract and aortic sac development in retinoic X receptor α knockout embryos. Development, 129(3), 733–746. https://doi.org/10.1242/dev.129.3.733

Kuribayashi, T., & Roberts, W. C. (1993). Tetralogy of fallot, truncus arteriosus, abnormal myocardial architecture and anomalies of the aortic arch system induced by bis-diamine in rat fetuses. Journal of the American College of Cardiology, 21(3), 768–776. https://doi.org/10.1016/0735-1097(93)90111-D

Li, P., Pashmforoush, M., & Sucov, H. M. (2010). Retinoic Acid Regulates Differentiation of the Secondary Heart Field and TGFβ-Mediated Outflow Tract Septation. Developmental Cell, 18(3), 480–485. https://doi.org/10.1016/J.DEVCEL.2009.12.019/ATTACHMENT/B413E05D-04E2-4F76-9417-72BF1FD02515/MMC1.PDF

Ma, M., Li, P., Shen, H., Estrada, K. D., Xu, J., Kumar, S. R., & Sucov, H. M. (2016). Dysregulated endocardial TGFβ signaling and mesenchymal transformation result in heart outflow tract septation failure. Developmental Biology, 409(1), 272–276. https://doi.org/10.1016/J.YDBIO.2015.09.021

Menegola, E., Veltman, C. H. J., Battistoni, M., di Renzo, F., Moretto, A., Metruccio, F., Beronius, A., Zilliacus, J., Kyriakopoulou, K., Spyropoulou, A., Machera, K., van der Ven, L. T. M., & Luijten, M. (2021). An adverse outcome pathway on the disruption of retinoic acid metabolism leading to developmental craniofacial defects. Toxicology, 458. https://doi.org/10.1016/J.TOX.2021.152843

Merki, E., Zamora, M., Raya, A., Kawakami, Y., Wang, J., Zhang, X., Burch, J., Kubalak, S. W., Kaliman, P., Belmonte, J. C. I., Chien, K. R., & Ruiz-Lozano, P. (2005). Epicardial retinoid X receptor alpha is required for myocardial growth and coronary artery formation. Proceedings of the National Academy of Sciences of the United States of America, 102(51), 18455–18460. https://doi.org/10.1073/PNAS.0504343102

Nakajima, Y. (2019). Retinoic acid signaling in heart development. Genesis, 57(7). https://doi.org/10.1002/dvg.23300

Narematsu, M., Kamimura, T., Yamagishi, T., Fukui, M., & Nakajima, Y. (2015). Impaired development of left anterior heart field by ectopic retinoic acid causes transposition of the great arteries. Journal of the American Heart Association, 4(5). https://doi.org/10.1161/JAHA.115.001889

Niederreither, K., Subbarayan, V., Dolle, P., & Chambon, P. (1999). Embryonic retinoic acid synthesis is essential for early mouse post-implantation development. Nature Genetics, 21(4), 444–448. https://doi.org/10.1038/7788

Niederreither, K., Vermot, J., le Roux, I., Schuhbaur, B., Chambon, P., & Dollé, P. (2003). The regional pattern of retinoic acid synthesis by RALDH2 is essential for the development of posterior pharyngeal arches and the enteric nervous system. Development, 130(11), 2525–2534. https://doi.org/10.1242/dev.00463

Niederreither, K., Vermot, J., Messaddeq, N., Schuhbaur, B., Chambon, P., & Dollé, P. (2001). Embryonic retinoic acid synthesis is essential for heart morphogenesis in the mouse. Development (Cambridge, England), 128(7), 1019–1031. https://doi.org/10.1242/dev.128.7.1019

Nishijima, S., Nakagawa, M., Fujino, H., Hanato, T., Okamoto, N., & Shimada, M. (2000). Teratogenic effects of bis-diamine on early embryonic rat heart: An in vitro study. Teratology, 62(2), 115–122. https://doi.org/10.1002/1096-9926(200008)62:2<115::aid-tera8>3.0.co;2-%23

Okamoto, N., Nakagawa, M., Fujino, H., Nishijima, S., Hanato, T., Narita, T., Takeuchi, Y., & Imanaka-Yoshida, K. (2004). Teratogenic Effects of Bis-diamine on the Developing Myocardium. Birth Defects Research Part A - Clinical and Molecular Teratology, 70(3), 132–141. https://doi.org/10.1002/bdra.20001

Okishima, T., Takamura, K., Matsuoka, Y., Ohdo, S., & Hayakawa, K. (1992). Cardiovascular anomalies in chick embryos produced by bis‐diamine in dimethylsulfoxide. Teratology, 45(2), 155–162. https://doi.org/10.1002/tera.1420450209

Pérez-Pomares, J. M., González-Rosa, J. M., & Muñoz-Chápuli, R. (2009). Building the vertebrate heart - An evolutionary approach to cardiac development. International Journal of Developmental Biology, 53(8–10), 1427–1443. https://doi.org/10.1387/IJDB.072409JP

Perl, E., & Waxman, J. S. (2019). Reiterative Mechanisms of Retinoic Acid Signaling during Vertebrate Heart Development. Journal of Developmental Biology, 7(2). https://doi.org/10.3390/jdb7020011

Piersma, A. H., Hessel, E. v., & Staal, Y. C. (2017). Retinoic acid in developmental toxicology: Teratogen, morphogen and biomarker. Reproductive Toxicology, 72, 53–61. https://doi.org/10.1016/J.REPROTOX.2017.05.014

Plein, A., Fantin, A., & Ruhrberg, C. (2015). Neural crest cells in cardiovascular development. In Current Topics in Developmental Biology (1st ed., Vol. 111). Elsevier Inc. https://doi.org/10.1016/bs.ctdb.2014.11.006

Rochais, F., Mesbah, K., & Kelly, R. G. (2009). Signaling pathways controlling second heart field development. Circulation Research, 104(8), 933–942. https://doi.org/10.1161/CIRCRESAHA.109.194464

Ryckebusch, L., Wang, Z., Bertrand, N., Lin, S. C., Chi, X., Schwartz, R., Zaffran, S., & Niederreither, K. (2008). Retinoic acid deficiency alters second heart field formation. Proceedings of the National Academy of Sciences of the United States of America, 105(8), 2913–2918. https://doi.org/10.1073/PNAS.0712344105

Sirbu, I. O., Zhao, X., & Duester, G. (2008). Retinoic acid controls heart anteroposterior patterning by down-regulating Isl1 through the Fgf8 pathway. Developmental Dynamics : An Official Publication of the American Association of Anatomists, 237(6), 1627–1635. https://doi.org/10.1002/DVDY.21570

Sorrell, M. R. J., & Waxman, J. S. (2011). Restraint of Fgf8 signaling by retinoic acid signaling is required for proper heart and forelimb formation. Developmental Biology, 358(1), 44–55. https://doi.org/10.1016/J.YDBIO.2011.07.022

Stefanovic, S., Laforest, B., Desvignes, J. P., Lescroart, F., Argiro, L., Maurel-Zaffran, C., Salgado, D., Plaindoux, E., de Bono, C., Pazur, K., Théveniau-Ruissy, M., Béroud, C., Puceat, M., Gavalas, A., Kelly, R. G., & Zaffran, S. (2020). Hox-dependent coordination of mouse cardiac progenitor cell patterning and differentiation. ELife, 9, 1–32. https://doi.org/10.7554/ELIFE.55124

Stefanovic, S., & Zaffran, S. (2017a). Mechanisms of retinoic acid signaling during cardiogenesis. Mechanisms of Development, 143, 9–19. https://doi.org/10.1016/j.mod.2016.12.002

Stefanovic, S., & Zaffran, S. (2017b). Mechanisms of retinoic acid signaling during cardiogenesis. Mechanisms of Development, 143, 9–19. https://doi.org/10.1016/j.mod.2016.12.002

Tasaka, H., Takenaka, H., Okamoto, N., Onitsuka, T., Koga, Y., & Hamada, M. (1991). Abnormal development of cardiovascular systems in rat embryos treated with bisdiamine. Teratology, 43(3), 191–200. https://doi.org/10.1002/tera.1420430303

Tonk, E. C. M., & Pennings, J. L. A. (2015). An adverse outcome pathway framework for neural tube and axial defects mediated by modulation of retinoic acid homeostasis. Reproductive Toxicology, 55, 104–113. https://doi.org/10.1016/J.REPROTOX.2014.10.008

Vincent, S. D., & Buckingham, M. E. (2010). How to make a heart. The origin and regulation of cardiac progenitor cells. Current Topics in Developmental Biology, 90(C), 1–41. https://doi.org/10.1016/S0070-2153(10)90001-X

Wang, S., Huang, W., Castillo, H. A., Kane, M. A., Xavier-Neto, J., Trainor, P. A., & Moise, A. R. (2018). Alterations in retinoic acid signaling affect the development of the mouse coronary vasculature. Developmental Dynamics, 247(8), 976–991. https://doi.org/10.1002/dvdy.24639

Wang, S., & Moise, A. R. (2019). Recent insights on the role and regulation of retinoic acid signaling during epicardial development. Genesis, 57(7). https://doi.org/10.1002/dvg.23303

WILSON, J. G., & WARKANY, J. (1949). Aortic-arch and cardiac anomalies in the offspring of vitamin A deficient rats. The American Journal of Anatomy, 85(1), 113–155. https://doi.org/10.1002/AJA.1000850106

WILSON, J. G., & WARKANY, J. (1950). Cardiac and aortic arch anomalies in the offspring of vitamin A deficient rats correlated with similar human anomalies. Pediatrics, 5(4), 708–725. https://doi.org/10.1542/peds.5.4.708

Wu, H., Lee, S. H., Gao, J., Liu, X., & Iruela-Arispe, M. L. (1999). Inactivation of erythropoietin leads to defects in cardiac morphogenesis. Development, 126(16), 3597–3605. https://doi.org/10.1242/DEV.126.16.3597