Identification of Stage-Specific Gene Expression Signatures in Response to Retinoic Acid during the Neural Differentiation of Mouse Embryonic Stem Cells

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.

Molecular Analysis of Stromal Cells-Induced Neural Differentiation of Mouse Embryonic Stem Cells

“…Our results are consistent with gene expression profiling of differentiating embryonic stem cells during neural development using bayesian networks in terms of identifying and differentiating temporal changes of markers of pluripotency and stage-specific neural cells. [57] A similar approach of studying temporal kinetics of cell surface markers and transcription factors expression during neutrophil differentiation of human iPSCs showed stage specific expression profiles that recapitulated embryonic hematopoiesis and revealed unexpected molecular events that could help enhance in vitro protocols for iPSC hematopoiesis. [58]…”

Retinoic acid signaling and neuronal differentiation

“…Multiple genomics-based studies have elucidated some genes that respond to RA under a variety of differentiation conditions [175,176,[190][191][192][193][194][195][196]. A subset of the genes identified in these analyses will be candidate neurogenic genes regulated by RAR.…”

Evidence‐Based Theory for Integrated Genome Regulation of Ontogeny—An Unprecedented Role of Nuclear FGFR1 Signaling

“…Within a few hours, nFGFR1 accumulates in the nuclei of ESC,s as the cells exit from the cell cycle, and neurogenic and neuronal genes are upregulated. By 24 h, the cells display a neuronal morphology (including long neurites and growth‐cone endings), and express neuron‐specific β‐III tubulin (B), MAP2, neurofilament L, TH (Lee et al, 2012), as well as glutamate and acetylcholine receptors (Guan et al, 2001; Okada et al, 2004; Akanuma et al, 2012; Lee et al, 2012). After 96 h of at‐RA treatment, only single cells displayed characteristic glial morphology and GFAP immunoreactivity (B), (Lee et al, 2012).…”