From the Cover: Embryonic Exposure to TCDD Impacts Osteogenesis of the Axial Skeleton in Japanese medaka,Oryzias latipes

Abstract

Recent studies from mammalian, fish, and in vitro models have identified bone and cartilage development as sensitive targets for dioxins and other aryl hydrocarbon receptor ligands. In this study, we assess how embryonic 2,3,7,8-tetrachlorochlorodibenzo-p-dioxin (TCDD) exposure impacts axial osteogenesis in Japanese medaka (Oryzias latipes), a vertebrate model of human bone development. Embryos from inbred wild-type Orange-red Hd-dR and 3 transgenic medaka lines (twist:EGFP, osx/sp7:mCherry, col10a1:nlGFP) were exposed to 0.15 nM and 0.3 nM TCDD and reared until 20 dpf. Individuals were stained for mineralized bone and imaged using confocal microscopy to assess skeletal alterations in medial vertebrae in combination with a qualitative spatial analysis of osteoblast and osteoblast progenitor cell populations. Exposure to TCDD resulted in an overall attenuation of vertebral ossification characterized by truncated centra, and reduced neural and hemal arch lengths. Effects on mineralization were consistent with modifications in cell number and cell localization of transgene-labeled osteoblast and osteoblast progenitor cells. Endogenous expression of osteogenic regulators runt-related transcription factor 2 (runx2) and osterix (osx/sp7), and extracellular matrix genes osteopontin (spp1), collagen type I alpha I (col1), collagen type X alpha I (col10a1), and osteocalcin (bglap/osc) was significantly diminished at 20 dpf following TCDD exposure as compared with controls. Through global transcriptomic analysis more than 590 differentially expressed genes were identified and mapped to select pathological states including inflammatory disease, connective tissue disorders, and skeletal and muscular disorders. Taken together, results from this study suggest that TCDD exposure inhibits axial bone formation through dysregulation of osteoblast differentiation. This approach highlights the advantages and sensitivity of using small fish models to investigate how xenobiotic exposure may impact skeletal development.

Vitamin D deficiency serves as a precursor to stunted growth and central adiposity in zebrafish

“…This cycle was followed by a dissociation stage which ensured primer specificity and confirmed the absence of primer dimerization: (4) 95 °C for 15 s, 60 °C for 1 min, 95 °C for 15 s, 60 °C for 1 min. Individual threshold cycle values (C t ) were determined for each reaction by the ABI 7300 System SDS Software and relative fold change differences for each gene across each sample was calculated according to the ΔΔC t method 56 , 57 . Gene expression was normalized to efla as the housekeeping gene.…”

Mechanisms of Developmental Toxicity of Dioxins and Related Compounds

“…In addition, endodermal perichondrial cells that cover chondrocytes are affected by TCDD due to which they develop a reduced proliferation ability [31]. Furthermore, TCDD reduces ossification of craniofacial osteoblasts [31], which is consistent with TCDD-induced ossification failure in another fish model, medaka ( Oryzias latipes ) [110]. These findings indicate that chondrocytes and the peripheral cells are sensitive to TCDD insult, leading to malformations.…”

“…A reporter gene for sox9b promoter activity shows expression in perichondrial cells surrounding craniofacial cartilages [31]. In addition, TCDD-induced reduction in both sox9b expression and abnormality in jaw structure are also observed in medaka ( Oryzias latipes ) [110], suggesting a common role of sox9b among fish species. The extent to which reduction in sox9b expression affects TCDD-induced craniofacial malformations remains to be uncovered; the craniofacial malformations are observed at a TCDD dose (0.25 ng/mL) at which a reduction in sox9b mRNA level in the whole embryo is not evident [114].…”

Early life co-exposures to a real-world PAH mixture and hypoxia result in later life and next generation consequences in medaka (Oryzias latipes)

“…Individual PAHs or 2,3,7,8-Tetrachlorodibenzo- p -dioxin (TCDD) caused skeletal deformities in adult fish (Baker et al, 2013; Dong et al, 2012; Incardona et al, 2004) as well as transgenerational bone impairments (Baker et al, 2014; Corrales et al, 2014; Seemann et al, 2015). These compounds are associated with the aryl hydrocarbon receptor (AhR) in perichordal ossification of vertebrae in medaka (Watson et al, 2016) and endochondrial bone formation via mediation of Sonic hedgehog (Shh) by the AhR signaling pathway in rockfish ( Sebastiscus marmoratus ) (He et al, 2011). ERSE and hypoxia resulted in different effects on the spinal column.…”