Sox9bIs Required for Epicardium Formation and Plays a Role in TCDD-Induced Heart Malformation in Zebrafish

Abstract

Activation of the transcription factor aryl hydrocarbon receptor by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) prevents the formation of the epicardium and leads to severe heart malformations in developing zebrafish (Danio rerio). The downstream genes that cause heart malformation are not known. Because TCDD causes craniofacial malformations in zebrafish by downregulating the sox9b gene, we hypothesized that cardiotoxicity might also result from sox9b downregulation. We found that sox9b is expressed in the developing zebrafish heart ventricle and that TCDD exposure markedly reduces this expression. Furthermore, we found that manipulation of sox9b expression could phenocopy many but not all of the effects of TCDD at the heart. Loss of sox9b prevented the formation of epicardium progenitors comprising the proepicardium on the pericardial wall, and prevented the formation and migration of the epicardial layer around the heart. Zebrafish lacking sox9b showed pericardial edema, an elongated heart, and reduced blood circulation. Fish lacking sox9b failed to form valve cushions and leaflets. Sox9b is one of two mammalian Sox9 homologs, sox9b and sox9a. Knock down of sox9a expression did not cause cardiac malformations, or defects in epicardium development. We conclude that the decrease in sox9b expression in the heart caused by TCDD plays a role in many of the observed signs of cardiotoxicity. We find that while sox9b is expressed in myocardial cells, it is not normally expressed in the affected epicardial cells or progenitors. We therefore speculate that sox9b is involved in signals between the cardiomyocytes and the nascent epicardial cells.

Histological and Transcriptomic Changes in Male Zebrafish Testes Due to Early Life Exposure to Low Level 2,3,7,8-Tetrachlorodibenzo-p-Dioxin

“…46,47 Although it remains unknown whether sox9b downregulation occurs within testicular tissue acutely after TCDD exposure, and then has implications for testes development, sox9b upregulation may support our hypothesis that some of the transcriptomic changes may be due to long-term feedback/compensatory mechanism(s) in response to disruptions in testes homeostasis.…”

Dioxin disrupts cranial cartilage and dermal bone development in zebrafish larvae

“…To test this we made use of a newly derived reporter line Tg(−2421/+29 sox9b:EGFP ) that expresses EGFP from sox9b promoter elements (Plavicki et al, 2014; Hofsteen et al, 2013), and co-stained for collagen 2a1 to mark the chondrocytes. Consistent with previous reports that sox9b is expressed in the pharyngeal endoderm (Dalcq et al, 2012; Yan et al, 2005), we found EGFP expressed in perichondrial cells surrounding the immunostained collagen 2a1-expressing craniofacial cartilages, (Fig.…”

“…We know that TCDD repression of sox9b is a key element in producing craniofacial malformations (Xiong et al, 2008). We have recently constructed a sox9b:EGFP reporter fish, Tg(−2421/+29 sox9b :EGFP) that aids the investigation of jaw development and understanding the effects of TCDD (Plavicki et al, 2014; Hofsteen et al, 2013)…”

Involvement of COX2–thromboxane pathway in TCDD-induced precardiac edema in developing zebrafish

“…Recent studies reported that TCDD blocked development of the epicardium, which is crucial to heart development, through inhibition of proepicardium formation in developing zebrafish (Plavicki et al, 2013). The same studies further clarified the role of Sox9b in epicardial formation and pericardial edema using Sox9b-morphant and -null mutant, suggesting partial involvement of Sox-9b in TCDD-induced edema (Hofsteen et al, 2013). Thus, mechanisms of the circulation failure, particularly pericardial edema, caused by TCDD are only beginning to be understood in developing zebrafish as well as other fish.…”