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Relationship: 2691

Title

A descriptive phrase which clearly defines the two KEs being considered and the sequential relationship between them (i.e., which is upstream, and which is downstream). More help

Decrease, sox9 expression leads to Altered, Cardiovascular development/function

Upstream event

The causing Key Event (KE) in a Key Event Relationship (KER). More help

Decrease, sox9 expression

Downstream event

The responding Key Event (KE) in a Key Event Relationship (KER). More help

Altered, Cardiovascular development/function

Key Event Relationship Overview

The utility of AOPs for regulatory application is defined, to a large extent, by the confidence and precision with which they facilitate extrapolation of data measured at low levels of biological organisation to predicted outcomes at higher levels of organisation and the extent to which they can link biological effect measurements to their specific causes.Within the AOP framework, the predictive relationships that facilitate extrapolation are represented by the KERs. Consequently, the overall WoE for an AOP is a reflection in part, of the level of confidence in the underlying series of KERs it encompasses. Therefore, describing the KERs in an AOP involves assembling and organising the types of information and evidence that defines the scientific basis for inferring the probable change in, or state of, a downstream KE from the known or measured state of an upstream KE. More help

AOPs Referencing Relationship

AOP Name	Adjacency	Weight of Evidence	Quantitative Understanding	Point of Contact	Author Status	OECD Status
Aryl hydrocarbon receptor activation leading to early life stage mortality via sox9 repression induced cardiovascular toxicity	adjacent	Moderate	Moderate	Prarthana Shankar (send email)	Under development: Not open for comment. Do not cite	WPHA/WNT Endorsed

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) that help to define the biological applicability domain of the KER.In general, this will be dictated by the more restrictive of the two KEs being linked together by the KER. More help

Term	Scientific Term	Evidence	Link
zebrafish	Danio rerio	High	NCBI
mouse	Mus musculus	High	NCBI
human	Homo sapiens	High	NCBI
Salmo salar	Salmo salar	Moderate	NCBI
chicken	Gallus gallus	Moderate	NCBI

Sex Applicability

An indication of the the relevant sex for this KER. More help

Sex	Evidence
Unspecific	High

Life Stage Applicability

An indication of the the relevant life stage(s) for this KER. More help

Term	Evidence
Embryo	High
Development	High

Key Event Relationship Description

Provides a concise overview of the information given below as well as addressing details that aren’t inherent in the description of the KEs themselves. More help

Sox9 is an important transcriptional regulator that has been implicated in several functions including cardiovascular development (Akiyama et al., 2004).

Additionally, exposure of different animals to relevant environmental pollutants leads to a significant decrease of sox9 expression (Garcia et al., 2017; Shi et al., 2017; Tussellino et al., 2016).

This KER provides lines of evidence linking the sox9 repression to alterations in cardiovascular system development and function.

Evidence Collection Strategy

Include a description of the approach for identification and assembly of the evidence base for the KER. For evidence identification, include, for example, a description of the sources and dates of information consulted including expert knowledge, databases searched and associated search terms/strings. Include also a description of study screening criteria and methodology, study quality assessment considerations, the data extraction strategy and links to any repositories/databases of relevant references.Tabular summaries and links to relevant supporting documentation are encouraged, wherever possible. More help

Evidence Supporting this KER

Addresses the scientific evidence supporting KERs in an AOP setting the stage for overall assessment of the AOP. More help

KER 2691 concordance table: https://aopwiki.org/system/dragonfly/production/2022/10/20/39ueqelreb_Concordance_Table_sox9_to_cardiovascular_clean.pdf

Biological Plausibility

Addresses the biological rationale for a connection between KEupstream and KEdownstream. This field can also incorporate additional mechanistic details that help inform the relationship between KEs, this is useful when it is not practical/pragmatic to represent these details as separate KEs due to the difficulty or relative infrequency with which it is likely to be measured. More help

Several studies in different organisms such as rodents, chicken, frogs, and fish, have identified both sox9 mRNA and protein spatiotemporal expressions in the developing heart (Gallina and Lincoln 2019; Guo et al., 2010; Lee and Saint-Jeannet 2009; Liu et al., 2007; Montero et al., 2002; Ng et al., 1997; Plavicki et al., 2014; Rahkonen et al., 2003; Zhao et al., 2007).

Using chip-seq, the sox9 protein has been found to interact with the genomic regions of proliferation genes as well as important transcription factors involved in mouse heart development (Garside et al., 2015), making it conceivable that the loss of sox9 can have a significant impact on cardiovascular development.

Zebrafish exposed to 1ng/mL TCDD have reduced sox9b (one of two paralogs of sox9 in zebrafish) expression in the heart (Hofsteen et al., 2013).

In a mouse embryonic study, the loss of kruppel-like factor 2 (part of the zinc-finger family of transcription factors) led to obvious heart malformations, while also repressing expression of sox9 (Chiplunkar et al., 2013).

Empirical Evidence

Provides specific (citable) evidence that supports the idea of a change in the upstream KE (KEupstream) leading to, or being associated with, a subsequent change in the downstream KE (KEdownstream), assuming the perturbation of KEupstream is sufficient. More help

Empirical evidence and essentiality of KE_up for KE_downto occur

Mutations in the sox9 gene locus as well as in the gene regulatory region in humans have been associated with chronic and congenital heart diseases (Gong et al., 2022; Sanchez-Castro et al., 2013) suggesting the importance of normal sox9 expression levels for normal cardiovascular development. These results were also recapitulated in goats, where a 4bp deletion in the 3’UTR of the sox9 gene altered the girth of the developing hearts (He et al., 2020).

Cardiomyocyte-specific dominant negative sox9b led to several abnormalities in zebrafish cardiovascular structure and function, including reduced end diastolic volume and epicardium formation (Gawdzik et al., 2018). Inhibition of sox9b expression also led to downstream changes in several cardiac development-related genes such as nkx2.5, nkx2.7, myl7, and c-fos. Another study found that sox9b morpholino knockdown led to prevention of epicardium progenitors and formation of valve cushions and leaflets, as well as the zebrafish having apparent cardiovascular deformities such as pericardial edema (Hofsteen et al., 2013).

Multiple studies investigating the impact of the loss of sox9 in mice, including one conditional inactivation study in endothelial cells and another using cre-lox generated sox9 mutants, show the significant effects on cardiovascular development and functioning in the absence of sox9 (Akiyama et al., 2004; Lincoln et al., 2007).

Developing zebrafish exposed to a concentration range of TCDD show trends of repressing sox9b gene expression from 0.125 ng/mL, while zebrafish have apparent pericardial edema at 0.25 ng/mL TCDD exposure (Garcia et al., 2018).

Salmon larvae exposed to the dioxin-like PCB-77 repressed sox9 at 500 day degrees at both exposure concentrations (1 or 10 ng/L), while also inducing cardiac edema and an arrhythmic effect on the heart (Olufsen and Arukwe 2011).

Uncertainties and Inconsistencies

Addresses inconsistencies or uncertainties in the relationship including the identification of experimental details that may explain apparent deviations from the expected patterns of concordance. More help

Important to point out that not all chemicals that induce developmental cardiovascular toxicity induce sox9 expression. For example, developmental zebrafish exposed to the fungicide, procymidone, significantly increased sox9b expression despite the fish having significant pericardial edema (Wu et al., 2018).

One study investigated sox9b expression (on a microarray) in heart tissue from zebrafish exposed to 1ng/mL TCDD and did not detect sox9b repression, despite the same study identifying sox9b repression in the zebrafish jaws (Xiong et al. 2008). The resolution of the microarray experiment might not have been good enough to detect sox9b repression which has been identified in other studies (Hofsteen et al., 2013).

Known modulating factors

This table captures specific information on the MF, its properties, how it affects the KER and respective references.1.) What is the modulating factor? Name the factor for which solid evidence exists that it influences this KER. Examples: age, sex, genotype, diet 2.) Details of this modulating factor. Specify which features of this MF are relevant for this KER. Examples: a specific age range or a specific biological age (defined by...); a specific gene mutation or variant, a specific nutrient (deficit or surplus); a sex-specific homone; a certain threshold value (e.g. serum levels of a chemical above...) 3.) Description of how this modulating factor affects this KER. Describe the provable modification of the KER (also quantitatively, if known). Examples: increase or decrease of the magnitude of effect (by a factor of...); change of the time-course of the effect (onset delay by...); alteration of the probability of the effect; increase or decrease of the sensitivity of the downstream effect (by a factor of...) 4.) Provision of supporting scientific evidence for an effect of this MF on this KER. Give a list of references. More help

Quantitative Understanding of the Linkage

Captures information that helps to define how much change in the upstream KE, and/or for how long, is needed to elicit a detectable and defined change in the downstream KE. More help

Response-response Relationship

Provides sources of data that define the response-response relationships between the KEs. More help

Time-scale

Information regarding the approximate time-scale of the changes in KEdownstream relative to changes in KEupstream (i.e., do effects on KEdownstream lag those on KEupstream by seconds, minutes, hours, or days?). More help

Known Feedforward/Feedback loops influencing this KER

Define whether there are known positive or negative feedback mechanisms involved and what is understood about their time-course and homeostatic limits. More help

Domain of Applicability

A free-text section of the KER description that the developers can use to explain their rationale for the taxonomic, life stage, or sex applicability structured terms. More help

The KER is likely relevant for all vertebrate species.

References

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

Akiyama H, Chaboissier MC, Behringer RR, Rowitch DH, Schedl A, Epstein JA, de Crombrugghe B. 2004. Essential role of sox9 in the pathway that controls formation of cardiac valves and septa. Proc Natl Acad Sci U S A. 101(17):6502-6507.

Chiplunkar AR, Lung TK, Alhashem Y, Koppenhaver BA, Salloum FN, Kukreja RC, Haar JL, Lloyd JA. 2013. Kruppel-like factor 2 is required for normal mouse cardiac development. Plos One. 8(2).

Gallina D, Lincoln J. 2019. Dynamic expression profiles of sox9 in embryonic, post natal, and adult heart valve cell populations. Anat Rec (Hoboken). 302(1):108-116.

Garcia GR, Goodale BC, Wiley MW, La Du JK, Hendrix DA, Tanguay RL. 2017. In vivo characterization of an ahr-dependent long noncoding rna required for proper sox9b expression. Mol Pharmacol. 91(6):609-619.

Garcia GR, Shankar P, Dunham CL, Garcia A, La Du JK, Truong L, Tilton SC, Tanguay RL. 2018. Signaling events downstream of ahr activation that contribute to toxic responses: The functional role of an ahr-dependent long noncoding rna (slincr) using the zebrafish model. Environ Health Perspect. 126(11):117002.

Garside VC, Cullum R, Alder O, Lu DY, Vander Werff R, Bilenky M, Zhao Y, Jones SJ, Marra MA, Underhill TM et al. 2015. Sox9 modulates the expression of key transcription factors required for heart valve development. Development. 142(24):4340-4350.

Gawdzik JC, Yue MS, Martin NR, Elemans LMH, Lanham KA, Heideman W, Rezendes R, Baker TR, Taylor MR, Plavicki JS. 2018. Sox9b is required in cardiomyocytes for cardiac morphogenesis and function. Sci Rep. 8(1):13906.

Gong L, Wang C, Xie H, Gao J, Li T, Qi S, Wang B, Wang J. 2022. Identification of a novel heterozygous sox9 variant in a chinese family with congenital heart disease. Mol Genet Genomic Med. 10(5):e1909.

Guo X, Yan J, Liu S, Xiang B, Liu Y. 2010. Isolation and expression analyses of the sox9a gene in triploid crucian carp. Fish Physiol Biochem. 36(2):125-133.

He L, Bi Y, Wang R, Pan C, Chen H, Lan X, Qu L. 2020. Detection of a 4 bp mutation in the 3'utr region of goat sox9 gene and its effect on the growth traits. Animals (Basel). 10(4).

Hofsteen P, Plavicki J, Johnson SD, Peterson RE, Heideman W. 2013. Sox9b is required for epicardium formation and plays a role in tcdd-induced heart malformation in zebrafish. Mol Pharmacol. 84(3):353-360.

Lee YH, Saint-Jeannet JP. 2009. Characterization of molecular markers to assess cardiac cushions formation in xenopus. Dev Dynam. 238(12):3257-3265.

Lincoln J, Kist R, Scherer G, Yutzey KE. 2007. Sox9 is required for precursor cell expansion and extracellular matrix organization during mouse heart valve development. Developmental Biology. 305(1):120-132.

Liu J, Liu S, Tao M, Li W, Liu Y. 2007. Isolation and expression analysis of testicular type sox9b in allotetraploid fish. Mar Biotechnol (NY). 9(3):329-334.

Montero JA, Giron B, Arrechedera H, Cheng YC, Scotting P, Chimal-Monroy J, Garcia-Porrero JA, Hurle JM. 2002. Expression of sox8, sox9 and sox10 in the developing valves and autonomic nerves of the embryonic heart. Mech Dev. 118(1-2):199-202.

Ng LJ, Wheatley S, Muscat GE, Conway-Campbell J, Bowles J, Wright E, Bell DM, Tam PP, Cheah KS, Koopman P. 1997. Sox9 binds DNA, activates transcription, and coexpresses with type ii collagen during chondrogenesis in the mouse. Dev Biol. 183(1):108-121.

Olufsen M, Arukwe A. 2011. Developmental effects related to angiogenesis and osteogenic differentiation in salmon larvae continuously exposed to dioxin-like 3,3',4,4'-tetrachlorobiphenyl (congener 77). Aquat Toxicol. 105(3-4):669-680.

Plavicki JS, Baker TR, Burns FR, Xiong KM, Gooding AJ, Hofsteen P, Peterson RE, Heideman W. 2014. Construction and characterization of a sox9b transgenic reporter line. Int J Dev Biol. 58(9):693-699.

Rahkonen O, Savontaus M, Abdelwahid E, Vuorio E, Jokinen E. 2003. Expression patterns of cartilage collagens and sox9 during mouse heart development. Histochem Cell Biol. 120(2):103-110.

Sanchez-Castro M, Gordon CT, Petit F, Nord AS, Callier P, Andrieux J, Guerin P, Pichon O, David A, Abadie V et al. 2013. Congenital heart defects in patients with deletions upstream of sox9. Hum Mutat. 34(12):1628-1631.

Shi G, Cui Q, Pan Y, Sheng N, Sun S, Guo Y, Dai J. 2017. 6:2 chlorinated polyfluorinated ether sulfonate, a pfos alternative, induces embryotoxicity and disrupts cardiac development in zebrafish embryos. Aquat Toxicol. 185:67-75.

Tussellino M, Ronca R, Carotenuto R, Pallotta MM, Furia M, Capriglione T. 2016. Chlorpyrifos exposure affects fgf8, sox9, and bmp4 expression required for cranial neural crest morphogenesis and chondrogenesis in xenopus laevis embryos. Environ Mol Mutagen. 57(8):630-640.

Wu Y, Zuo Z, Chen M, Zhou Y, Yang Q, Zhuang S, Wang C. 2018. The developmental effects of low-level procymidone towards zebrafish embryos and involved mechanism. Chemosphere. 193:928-935.

Xiong KM, Peterson RE, Heideman W. 2008. Aryl hydrocarbon receptor-mediated down-regulation of sox9b causes jaw malformation in zebrafish embryos. Mol Pharmacol. 74(6):1544-1553.

Zhao B, Etter L, Hinton RB, Jr., Benson DW. 2007. Bmp and fgf regulatory pathways in semilunar valve precursor cells. Dev Dyn. 236(4):971-980.