A Brn2–Zic1 axis specifies the neuronal fate of retinoic-acid-treated embryonic stem cells

Abstract

Mouse embryonic stem cells (ESCs) treated with all-trans retinoic acid differentiate into a homogenous population of glutamatergic neurons. Although differentiation is initiated through activation of target genes by the retinoic acid receptors, the downstream transcription factors specifying neuronal fate are less well characterised. Here, we show that the transcription factor Brn2 (also known as Pou3f2) is essential for the neuronal differentiation programme. By integrating results from RNA-seq following Brn2 silencing with results from Brn2 ChIP-seq, we identify a set of Brn2 target genes required for the neurogenic programme. Further integration of Brn2 ChIP-seq data from retinoicacid-treated ESCs and P19 cells with data from ESCs differentiated into neuronal precursors by Fgf2 treatment and that from fibroblasts trans-differentiated into neurons by ectopic Brn2 expression showed that Brn2 occupied a distinct but overlapping set of genomic loci in these differing conditions. However, a set of common binding sites and target genes defined the core of the Brn2-regulated neuronal programme, among which was that encoding the transcription factor Zic1. Small hairpin RNA (shRNA)-mediated silencing of Zic1 prevented ESCs from differentiating into neuronal precursors, thus defining a hierarchical Brn2-Zic1 axis that is essential to specify neural fate in retinoic-acid-treated ESCs.

Distinct retinoic acid receptor (RAR) isotypes control differentiation of embryonal carcinoma cells to dopaminergic or striatopallidal medium spiny neurons

“…Our data confirm original reports on the possibility of generating GABAergic neurons from EC cells 38 , 56 and extend such observations at several levels. First, by applying retinoid treatment during embryoid body formation 35 , we show that the rate of neurogenesis (88%) and homogeneity of obtained GABAergic neurons (90% of neurons) are much higher than previously reported (30–70% for neurogenesis and 60% for GABAergic neuron enrichment) 38 . Accordingly, we observed only rare astrocytes and no microglial cells, in contrast to previous reports, as confirmed by a comparative analysis of cells differentiated through the protocols reported by Staines et al .…”

“…To address the role of ATRA in generation of specific neuronal types and subtypes, we used a differentiation protocol applied recently to dissect genetic programs underlying ATRA-induced neurogenesis in EC cells 35 . Differentiation of EC aggregates was induced by ATRA, or selective agonists of RARα (BMS753), RARβ (BMS641), or RARγ (CD666).…”

NTF3 Is a Novel Target Gene of the Transcription Factor POU3F2 and Is Required for Neuronal Differentiation

“…3 ), sequence analysis of the NTF3 promoter region ranging from nt − 1390 to nt − 524 also revealed several putative binding sites for some important differentiation-related transcription factors such as CTCF, YY1, GATA-3, and Sp-1 [ 31 ]. In addition, the regulatory role of Zic 1 in neuronal differentiation has been addressed in embryonic mouse models [ 16 ]. We found that POU3F2 knockdown resulted in decreased Zic1 expression in NT2D1 cells with or without neuronal induction (Supplementary Fig.…”

“…Forced expression of POU3F2 and other neuronal transcription factors (ASCL1, MYT1L, and NEUROD1) has been shown to induce neuronal fate in pluripotent stem cells and to convert fetal and postnatal human fibroblasts or somatic cells into functional neurons [ 11 – 15 ]. More recently, Urban and colleagues demonstrated that POU3F2 was essential for retinoic acid-induced neuronal differentiation from mouse embryonic stem cells by regulating a set of target genes via binding to the genomic locus Zic 1 [ 16 ]. These results indicate that POU3F2 is a critical transcription factor in the conversion of embryonic stem cells into neurons and glial cells; however, little is known about the neurogenic genes it regulates.…”

Gene regulatory networks in neural cell fate acquisition from genome-wide chromatin association of Geminin and Zic1

“…Similar to Gmnn’s ability to promote neural fate acquisition of ES cells, Zic1 is required for specification of ES cells as neuronal precursor cells in response to retinoic acid37. In mouse models, Zic1 and Zic3 are required together to regulate forebrain development and cooperate in promoting neural progenitor expansion, while inhibiting neuronal differentiation38, while Zic1 also promotes neural precursor formation and maintenance throughout the neural tube39.…”