Abstract
Studying chemical disturbances during neural differentiation of murine embryonic stem cells (mESCs) has been established as an alternative in vitro testing approach for the identification of developmental neurotoxicants. miRNAs represent a class of small non-coding RNA molecules involved in the regulation of neural development and ESC differentiation and specification. Thus, neural differentiation of mESCs in vitro allows investigating the role of miRNAs in chemical-mediated developmental toxicity. We analyzed changes in miRNome and transcriptome during neural differentiation of mESCs exposed to the developmental neurotoxicant sodium valproate (VPA). A total of 110 miRNAs and 377 mRNAs were identified differently expressed in neurally differentiating mESCs upon VPA treatment. Based on miRNA profiling we observed that VPA shifts the lineage specification from neural to myogenic differentiation (upregulation of muscle-abundant miRNAs, mir-206, mir-133a and mir-10a, and downregulation of neural-specific mir-124a, mir-128 and mir-137). These findings were confirmed on the mRNA level and via immunochemistry. Particularly, the expression of myogenic regulatory factors (MRFs) as well as muscle-specific genes (Actc1, calponin, myosin light chain, asporin, decorin) were found elevated, while genes involved in neurogenesis (e.g. Otx1, 2, and Zic3, 4, 5) were repressed. These results were specific for valproate treatment and―based on the following two observations―most likely due to the inhibition of histone deacetylase (HDAC) activity: (i) we did not observe any induction of muscle-specific miRNAs in neurally differentiating mESCs exposed to the unrelated developmental neurotoxicant sodium arsenite; and (ii) the expression of muscle-abundant mir-206 and mir-10a was similarly increased in cells exposed to the structurally different HDAC inhibitor trichostatin A (TSA). Based on our results we conclude that miRNA expression profiling is a suitable molecular endpoint for developmental neurotoxicity. The observed lineage shift into myogenesis, where miRNAs may play an important role, could be one of the developmental neurotoxic mechanisms of VPA.
Highlights
Exposures to xenobiotics during embryonic, fetal, and perinatal periods are of particular concern since scientific evidence suggest that the developing central nervous system (CNS) is much more vulnerable to chemicals than the adult CNS
The results suggest that the induction of myogenic miRNA expression by VPA might be at least partially mediated by histone deacetylase (HDAC) inhibition
VPA-specific miRNA expression patterns were further confirmed at the level of mRNA by demonstrating the induction of muscle-specific genes and the accompanied inhibition of genes involved in neurogenesis (Fig. 4, 6, 7), and phenotypically by immunofluorescent staining (Fig. 8)
Summary
Exposures to xenobiotics during embryonic, fetal, and perinatal periods are of particular concern since scientific evidence suggest that the developing central nervous system (CNS) is much more vulnerable to chemicals than the adult CNS. Exposure to drugs and environmental chemicals during critical developmental stages is likely to contribute to the increasing incidence of neurodevelopmental disorders in children [1,2,3,4,5]. There is a critical deficiency of knowledge when it comes to the developmental neurotoxicity of drugs and chemicals. Only very few compounds have been identified as developmental neurotoxicants [1], but this might not reflect the actual prevalence of neurotoxicants in the human environment, since only a minor portion of the more than 80,000 chemicals used worldwide have been tested to determine their potential to trigger developmental neurotoxicity (DNT) in vivo. There is a critical need for the development of alternative non-animal, highthroughput methods for DNT assessment to ensure the safety of chemicals and drugs
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