Retinoic-acid-concentration-dependent acquisition of neural cell identity during in vitro differentiation of mouse embryonic stem cells

Abstract

Retinoic acid (RA) is one of the most important morphogens, and its embryonic distribution correlates with neural differentiation and positional specification in the developing central nervous system. To investigate the concentration-dependent effects of RA on neural differentiation of mouse embryonic stem cells (ES cells), we investigated the precise expression profiles of neural and regional specific genes by ES cells aggregated into embryoid bodies (EBs) exposed to various concentrations of RA or the BMP antagonist Noggin. RA promoted both neural differentiation and caudalization in a concentration-dependent manner, and the concentration of RA was found to regulate dorso-ventral identity, i.e., higher concentrations of RA induced a dorsal phenotype, and lower concentrations of RA induced a more ventral phenotype. The induction of the more ventral phenotype was due to the higher expression level of the N-terminus of sonic hedgehog protein (Shh-N) when treated with low concentration RA, as it was abrogated by an inhibitor of Shh signaling, cyclopamine. These findings suggest that the concentration of RA strictly and simultaneously regulates the neuralization and positional specification during differentiation of mouse ES cells and that it may be possible to use it to establish a strategy for controlling the identity of ES-cell-derived neural cells.

Single Cell Analysis Reveals Concomitant Transcription of Pluripotent and Lineage Markers During the Early Steps of Differentiation of Embryonic Stem Cells

“…The process was accelerated through the addition of 10 nM all-trans retinoic acid to the differentiation medium, as shown on Supporting Information Figure S3A-S3C and as reported before [25][26][27][28]. As measured by the proportion of neural precursors at early time points (see below), the efficiency of differentiation was comparable to the ones reported previously [23,24].…”

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).…”

AP2γ regulates neural and epidermal development downstream of the BMP pathway at early stages of ectodermal patterning

“…Mouse monoclonal antibodies included: anti-GFP (1:400, MP Biomedicals), anti-Oct4 (1:200, Santa Cruz Biotechnology), anti-nestin (1:200, Upstate), anti-Tuj1 (1:500, Sigma), anti-MAP2 (1:500, Sigma), anti-AP2γ (1:500, Abcam), anti-cytokeratin18 (1:200, Abcam) and anti-cytokeratin14 (1:50, Abcam). Rabbit polyclonal antibodies were anti-GFP (1:400, Santa Cruz Biotechnology), an anti-Sox1/(2)/3 (1:100) that has a preference for Sox1 and Sox3 over Sox2 [78,79]. Fluorescein isothiocyanate-and Cy3-conjugated secondary antibodies were used (1:200 and 1:500, Jackson ImmunoResearch).…”