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Rest-mediated regulation of extracellular matrix is crucial for neural development.

Sun YM, Cooper M, Finch S, Lin HH, Chen ZF, Williams BP, Buckley NJ - PLoS ONE (2008)

Bottom Line: Neural development from blastocysts is strictly controlled by intricate transcriptional programmes that initiate the down-regulation of pluripotent genes, Oct4, Nanog and Rex1 in blastocysts followed by up-regulation of lineage-specific genes as neural development proceeds.Here, we demonstrate that the expression pattern of the transcription factor Rest mirrors those of pluripotent genes during neural development from embryonic stem (ES) cells and an early abrogation of Rest in ES cells using a combination of gene targeting and RNAi approaches causes defects in this process.Specifically, Rest ablation does not alter ES cell pluripotency, but impedes the production of Nestin(+) neural stem cells, neural progenitor cells and neurons, and results in defective adhesion, decrease in cell proliferation, increase in cell death and neuronal phenotypic defects typified by a reduction in migration and neurite elaboration.

View Article: PubMed Central - PubMed

Affiliation: Centre for the Cellular Basis of Behaviour, The James Black Centre, Institute of Psychiatry, King's College London, London, UK. yuh-man.sun@iop.kcl.ac.uk

ABSTRACT
Neural development from blastocysts is strictly controlled by intricate transcriptional programmes that initiate the down-regulation of pluripotent genes, Oct4, Nanog and Rex1 in blastocysts followed by up-regulation of lineage-specific genes as neural development proceeds. Here, we demonstrate that the expression pattern of the transcription factor Rest mirrors those of pluripotent genes during neural development from embryonic stem (ES) cells and an early abrogation of Rest in ES cells using a combination of gene targeting and RNAi approaches causes defects in this process. Specifically, Rest ablation does not alter ES cell pluripotency, but impedes the production of Nestin(+) neural stem cells, neural progenitor cells and neurons, and results in defective adhesion, decrease in cell proliferation, increase in cell death and neuronal phenotypic defects typified by a reduction in migration and neurite elaboration. We also show that these Rest- phenotypes are due to the dysregulation of its direct or indirect target genes, Lama1, Lamb1, Lamc1 and Lama2 and that these aberrant phenotypes can be rescued by laminins.

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The effects of Rest ablation on the gene expression of laminin subunits during neural differentiation.The expression patterns of Lama1 (A), Lamb1 (B), Lamc1 (C) and Lama2 (D) were analysed throughout ES cell-derived neural differentiation by real time-PCR. Data are represented as mean±SEM. *P<0.05 and **P<0.01, significantly different from REST-100 and REST/KD-50. #P<0.05 and ¥P<0.01, significantly different from REST- and REST-+MTV.
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pone-0003656-g006: The effects of Rest ablation on the gene expression of laminin subunits during neural differentiation.The expression patterns of Lama1 (A), Lamb1 (B), Lamc1 (C) and Lama2 (D) were analysed throughout ES cell-derived neural differentiation by real time-PCR. Data are represented as mean±SEM. *P<0.05 and **P<0.01, significantly different from REST-100 and REST/KD-50. #P<0.05 and ¥P<0.01, significantly different from REST- and REST-+MTV.

Mentions: The laminin rescue of the REST- phenotype indicated that laminins are downstream effectors of Rest. To test this idea, we determined whether the expression of laminins during neurogenesis was impaired by Rest ablation. Laminins have over 15 isoforms, each consisting of a combination of α, β, and γ subunits. In this experiment, we only focused on the effect of Rest deficiency on the expression of the genes encoding α1 (Lama1), β1 (Lamb1), γ1 (Lamc1) and α2 (Lama2) subunits since the EHS laminins used in the rescue experiments are composed mainly of laminin 1 (α1β1γ1). Furthermore α2 is a known Rest target gene [11] and a major component of the basement membrane in the embryonic CNS that is known to be involved in NSC development [23]. Each of these 4 laminin subunits exhibited distinct expression patterns during neural differentiation of ES cells (Fig. 6). In REST-100 and REST/KD-50 cells, the expression pattern of Lama1 closely followed the time course of NSC and neuron development. Expression of Lamb1 and Lamc1 both showed a very similar pattern with high expression levels in ES cells followed by an initial decline during NSC differentiation and a subsequent gradual increase as neuronal differentiation proceeded. These expression patterns of Lama1, b1 and c1 are similar to those reported in an earlier in vitro study [24]. Intriguingly, the expression pattern of Lama2 correlated closely with the time course of NSC formation, peaking at 4-days of differentiation, at a similar developmental stage as expression of α2 in vivo [23]. In contrast, REST- cells exhibited significantly (P<0.01) decreased expression levels of Lama1 throughout neurogenesis (Fig. 6A). Remarkably, raising the Rest level to 8% (REST-+REST) restored Lama1 levels to 40–50% of those of the control, although the levels remained significantly (P<0.01) lower than those seen in control cells. A similar change was also seen in the expression patterns of Lamb1, c1 and a2. Rest ablation reduced the levels of Lamb1, c1 and a2 to below 40% of the level seen in control cells throughout neurogenesis, while raising the Rest level to 8% restored, at least partially, the expression levels of Lamb1 and a2 (Fig. 6B–6D). Taken together, our results suggest that Rest ablation impairs expression of laminins 1 and 2 (α2β1γ1) during neurogenesis, which leads to defects in cell adhesion, expansion and fasciculation.


Rest-mediated regulation of extracellular matrix is crucial for neural development.

Sun YM, Cooper M, Finch S, Lin HH, Chen ZF, Williams BP, Buckley NJ - PLoS ONE (2008)

The effects of Rest ablation on the gene expression of laminin subunits during neural differentiation.The expression patterns of Lama1 (A), Lamb1 (B), Lamc1 (C) and Lama2 (D) were analysed throughout ES cell-derived neural differentiation by real time-PCR. Data are represented as mean±SEM. *P<0.05 and **P<0.01, significantly different from REST-100 and REST/KD-50. #P<0.05 and ¥P<0.01, significantly different from REST- and REST-+MTV.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2573962&req=5

pone-0003656-g006: The effects of Rest ablation on the gene expression of laminin subunits during neural differentiation.The expression patterns of Lama1 (A), Lamb1 (B), Lamc1 (C) and Lama2 (D) were analysed throughout ES cell-derived neural differentiation by real time-PCR. Data are represented as mean±SEM. *P<0.05 and **P<0.01, significantly different from REST-100 and REST/KD-50. #P<0.05 and ¥P<0.01, significantly different from REST- and REST-+MTV.
Mentions: The laminin rescue of the REST- phenotype indicated that laminins are downstream effectors of Rest. To test this idea, we determined whether the expression of laminins during neurogenesis was impaired by Rest ablation. Laminins have over 15 isoforms, each consisting of a combination of α, β, and γ subunits. In this experiment, we only focused on the effect of Rest deficiency on the expression of the genes encoding α1 (Lama1), β1 (Lamb1), γ1 (Lamc1) and α2 (Lama2) subunits since the EHS laminins used in the rescue experiments are composed mainly of laminin 1 (α1β1γ1). Furthermore α2 is a known Rest target gene [11] and a major component of the basement membrane in the embryonic CNS that is known to be involved in NSC development [23]. Each of these 4 laminin subunits exhibited distinct expression patterns during neural differentiation of ES cells (Fig. 6). In REST-100 and REST/KD-50 cells, the expression pattern of Lama1 closely followed the time course of NSC and neuron development. Expression of Lamb1 and Lamc1 both showed a very similar pattern with high expression levels in ES cells followed by an initial decline during NSC differentiation and a subsequent gradual increase as neuronal differentiation proceeded. These expression patterns of Lama1, b1 and c1 are similar to those reported in an earlier in vitro study [24]. Intriguingly, the expression pattern of Lama2 correlated closely with the time course of NSC formation, peaking at 4-days of differentiation, at a similar developmental stage as expression of α2 in vivo [23]. In contrast, REST- cells exhibited significantly (P<0.01) decreased expression levels of Lama1 throughout neurogenesis (Fig. 6A). Remarkably, raising the Rest level to 8% (REST-+REST) restored Lama1 levels to 40–50% of those of the control, although the levels remained significantly (P<0.01) lower than those seen in control cells. A similar change was also seen in the expression patterns of Lamb1, c1 and a2. Rest ablation reduced the levels of Lamb1, c1 and a2 to below 40% of the level seen in control cells throughout neurogenesis, while raising the Rest level to 8% restored, at least partially, the expression levels of Lamb1 and a2 (Fig. 6B–6D). Taken together, our results suggest that Rest ablation impairs expression of laminins 1 and 2 (α2β1γ1) during neurogenesis, which leads to defects in cell adhesion, expansion and fasciculation.

Bottom Line: Neural development from blastocysts is strictly controlled by intricate transcriptional programmes that initiate the down-regulation of pluripotent genes, Oct4, Nanog and Rex1 in blastocysts followed by up-regulation of lineage-specific genes as neural development proceeds.Here, we demonstrate that the expression pattern of the transcription factor Rest mirrors those of pluripotent genes during neural development from embryonic stem (ES) cells and an early abrogation of Rest in ES cells using a combination of gene targeting and RNAi approaches causes defects in this process.Specifically, Rest ablation does not alter ES cell pluripotency, but impedes the production of Nestin(+) neural stem cells, neural progenitor cells and neurons, and results in defective adhesion, decrease in cell proliferation, increase in cell death and neuronal phenotypic defects typified by a reduction in migration and neurite elaboration.

View Article: PubMed Central - PubMed

Affiliation: Centre for the Cellular Basis of Behaviour, The James Black Centre, Institute of Psychiatry, King's College London, London, UK. yuh-man.sun@iop.kcl.ac.uk

ABSTRACT
Neural development from blastocysts is strictly controlled by intricate transcriptional programmes that initiate the down-regulation of pluripotent genes, Oct4, Nanog and Rex1 in blastocysts followed by up-regulation of lineage-specific genes as neural development proceeds. Here, we demonstrate that the expression pattern of the transcription factor Rest mirrors those of pluripotent genes during neural development from embryonic stem (ES) cells and an early abrogation of Rest in ES cells using a combination of gene targeting and RNAi approaches causes defects in this process. Specifically, Rest ablation does not alter ES cell pluripotency, but impedes the production of Nestin(+) neural stem cells, neural progenitor cells and neurons, and results in defective adhesion, decrease in cell proliferation, increase in cell death and neuronal phenotypic defects typified by a reduction in migration and neurite elaboration. We also show that these Rest- phenotypes are due to the dysregulation of its direct or indirect target genes, Lama1, Lamb1, Lamc1 and Lama2 and that these aberrant phenotypes can be rescued by laminins.

Show MeSH
Related in: MedlinePlus