Elsevier

Differentiation

Volume 77, Issue 1, January 2009, Pages 19-28
Differentiation

NRSF downregulation induces neuronal differentiation in mouse embryonic stem cells

https://doi.org/10.1016/j.diff.2008.09.001Get rights and content

Abstract

Differentiation of embryonic stem (ES) cells into neurons is accompanied by global changes in transcriptional programs. One transcription factor that has been shown to be involved in neuronal differentiation is neuron restrictive silencing factor/RE1-silencing transcription factor (NRSF/REST). NRSF is a transcriptional repressor that silences the transcription of a large number of neuronal genes by binding to a 21-bp consensus DNA sequence, the RE1 binding site/neuron-restrictive silencer elements (RE1/NRSE), present in the regulatory regions of neuronal genes. The goal of the current study was to examine the role of NRSF during differentiation of ES cells into neurons. To do this, ShRNA construct was used to downregulate NRSF in undifferentiated ES cells. Our results show that although control ES cells required induction by retinoic acid (RA) to differentiate efficiently into neurons, downregulation of NRSF was sufficient to drive the ES cells down the neuronal lineage even in the absence of RA. This downregulation also led to increased expression of mature neuronal markers, and concomitantly decreased glial fibrillary acidic protein (GFAP) expression. The results suggest that NRSF downregulation increases the population of mature neurons at the expense of GFAP-positive cells.

Introduction

The current model for neuronal differentiation of embryonic stem (ES) cells is that it proceeds via a series of commitment steps whereby pluripotent embryonic stem (ES) cells give rise to more restricted multipotent neural stem (NS) cells, which in turn generate differentiated neurons (Bibel et al., 2004; Cazillis et al., 2006). The differentiation steps are correlated with global changes in transcriptional programs and patterns of gene expression that involve both positive and negative regulatory loops (Falk et al., 2007; Mikkelsen et al., 2007; Ma, 2006).

One key factor that has been shown to negatively regulate neuronal differentiation program is the Krüppel-type zinc-finger transcription factor, RE1-silencing transcription factor (REST; also known as the neuron-restrictive silencing factor, NRSF) (Schoenherr and Anderson, 1995; Schoenherr et al., 1996; Chong et al., 1995). NRSF is a global transcriptional repressor that silences the transcription of a large number of neuronal differentiation genes by binding to a 21-bp consensus DNA sequence, the RE1 binding site/neuron-restrictive silencer elements (RE1/NRSE), present in the regulatory regions of a number of neuron-specific genes. These include genes that code for ion channels, neurotransmitter receptors and proteins essential for synaptic function (Schoenherr et al., 1996).

NRSF knockouts die early in development and loss of NRSF in vivo leads to de-repression of neuronal genes in non-neuronal tissue (Chen et al., 1998). Conversely, constitutive expression of NRSF in differentiating neurons disrupts neuronal gene expression and causes axon pathfinding errors in vivo (Paquette et al., 2000). In the developing nervous system, NRSF is expressed at higher levels in the ventricular zone where progenitors are actively dividing than in the marginal zone in which cells have exited the cell cycle and begun differentiating (Chen et al., 1998; Chong et al., 1995; Schoenherr and Anderson, 1995). NRSF is also expressed in neuroblastoma cells and levels of NRSF decrease during their differentiation (Nishimura et al., 1996). Together, these data suggest that NRSF might function to repress neuronal gene expression in both non-neuronal tissues and neural precursors. They also imply that downregulation of NRSF during neurogenesis may be important for proper neuronal differentiation since NRSF could also suppress transcriptional activators of neuronal genes (Bergsland et al., 2006). Although during development NRSF plays a primary and important role in the appropriate activation of neuron-specific genes, the fact that the RE1 sequence is found in non-neuronal genes and its expression is also detected in mature neurons in the adult tissue points to a more complex role for NRSF that depends upon its cellular and physiological environment (Brivanlou, 1998; Palm et al., 1998; Scholl et al., 1996).

Previous studies have shown that NRSF is expressed in MH-1 embryonic stem cell and in ES-derived neural stem cells and downregulated upon differentiation into neurons (Cheong et al., 2005; Sun et al., 2005). In addition, immunoprecipitation-based cloning strategy has shown that there are many NRSF target genes that are expressed in ES cells, suggesting that regulation of NRSF expression may be important during lineage determination during ES cell differentiation (Sun et al., 2005). When ES cells were exposed to RA to allow directed differentiation into neuronal progenitors, NRSF downregulation took place at the level of post-translational control rather than at the transcriptional level. This post-translational regulation of NRSF was similar to that seen in vivo in cortical progenitor cells (Ballas et al., 2005).

Given the importance of NRSF in the control of neuronal identity (Lorincz et al., 2004), in this study we directly investigate whether inhibition of NRSF in murine ES cells using RNA interference would lead to the differentiation of ES cells into neurons in the absence of a neural inducer. We found that downregulation of NRSF using RNAi gave rise to neurons in the absence of RA. In addition, we show a significant reduction in glial differentiation following NRSF inhibition. This suggests that while RA leads to production of bipotential nestin-positive precursors that can give rise to neurons as well as glia, NRSF downregulation leads to the production of uni-potential nestin cells that differentiate and produce neuronal cells at the expense of glial differentiation.

Section snippets

Design and cloning of ShRNA

Four 21 nucleotide stretches within the coding region of NRSF cDNA were identified, which were ∼50% GC rich and unique in the genome using siRNA converter software (Ambion) and siRNA target designer-1.5 (Promega). Four 55-nucleotide DNA template oligonucleotides were designed separately to produce 21-nucleotide siRNAs. Each complementary 55-nucleotide oligonucleotide was mixed in equimolar amounts (150–300 ng/μl), heated for 5 min at 95 °C, then gradually cooled to room temperature in annealing

NRSF expression during neuronal differentiation of mouse ES cells with RA

First, the regulation of NRSF during ES cell differentiation was studied by real-time PCR. Results showed that NRSF was expressed in ES cells and downregulated in +6 d EB and in terminally differentiated cells (EP) (Fig. 1A, ***P<0.001). The differentiation of ES cells was confirmed by the increase in expression of GAP-43, a neuronal marker that showed the reverse expression pattern by real-time PCR to that of NRSF (Fig. 1B, ***P<0.001). ICC for GAP-43 and NRSF confirmed the results obtained by

Discussion

In this study, we show that inhibition of NRSF expression in undifferentiated ES-D3 cells by RNA interference enhanced the differentiation of these cells into mature neurons in the absence of RA. Further we show that inhibition of NRSF led to an increase in the number of nestin-positive neuroepithelial (NEP) cells that were obtained after culturing under conditions that lead to an expansion of this precursor population. Finally, we show that while treatment with RA gave rise to both

Acknowledgements

This work was supported by an ICMR-INSERM grant award (SM and PG) and a CSIR fellowship award (SKG). We gratefully acknowledge the help of Manoj Kumar Mishra with the FACS analysis.

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