Abstract
We have previously established a protocol for the neural differentiation of mouse embryonic stem cells (mESCs) as an efficient tool to evaluate the neurodevelopmental toxicity of environmental chemicals. Here, we described a multivariate bioinformatic approach to identify the stage-specific gene sets associated with neural differentiation of mESCs. We exposed mESCs (B6G-2 cells) to 10−8 or 10−7 M of retinoic acid (RA) for 4 days during embryoid body formation and then performed morphological analysis on day of differentiation (DoD) 8 and 36, or genomic microarray analysis on DoD 0, 2, 8, and 36. Three gene sets, namely a literature-based gene set (set 1), an analysis-based gene set (set 2) using self-organizing map and principal component analysis, and an enrichment gene set (set 3), were selected by the combined use of knowledge from literatures and gene information selected from the microarray data. A gene network analysis for each gene set was then performed using Bayesian statistics to identify stage-specific gene expression signatures in response to RA during mESC neural differentiation. Our results showed that RA significantly increased the size of neurosphere, neuronal cells, and glial cells on DoD 36. In addition, the gene network analysis showed that glial fibrillary acidic protein, a neural marker, remarkably up-regulates the other genes in gene set 1 and 3, and Gbx2, a neural development marker, significantly up-regulates the other genes in gene set 2 on DoD 36 in the presence of RA. These findings suggest that our protocol for identification of developmental stage-specific gene expression and interaction is a useful method for the screening of environmental chemical toxicity during neurodevelopmental periods.
Highlights
Humans are exposed to environmental chemicals on a daily basis; many effects of these chemicals on human health are unclear
We have previously reported a mouse embryonic stem cell neural differentiation protocol and showed that it could be used as an efficient tool to evaluate the toxic effects of environmental chemicals on neurodevelopment (Nagano et al, 2012)
Nanog as a marker of undifferentiated embryonic stem cells (ESCs) and, Nestin, Map2, and Gfap as markers of neural cells were differentially expressed by retinoic acid (RA) treatments at differential doses during the neural differentiation of mouse embryonic stem cells (mESCs), suggesting that our protocol could detect the effects of RA on neuronal differentiation (Figure 2B)
Summary
Humans are exposed to environmental chemicals on a daily basis; many effects of these chemicals on human health are unclear. Brain development during embryonic stages is an important period when microstructures are formed and axon guidance and synapse formation are induced by neuronal signaling (Lamoury et al, 2006; Ligon et al, 2006). These processes are regulated by stage-specific gene expression during embryonic development. It is necessary to develop a more comprehensive and efficient system to identify the stage-specific gene expression signatures in embryonic development and to evaluate the toxicity of environmental chemicals on neural development
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