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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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Laminins rescue the phenotypic effects of Rest ablation on the neural differentiation of ES cells.Control (REST-100) and 4 Rest mutants ES cells were grown under neural-differentiating conditions on laminin coated glass coverslips. After 4 days of differentiation some cultures were fixed and labelled with the NSC markers Nestin (red) and Sox1 (green) (A–E). The results show that laminins restored the ability to generate Nestin+ cells in Rest mutants (C–E, compared with Fig. 3C–3E). Sister cultures were allowed to differentiate for 14 days to analyse neuronal differentiation using NeuN (red) and Map2 (green) (F–J and in higher power K–O). Laminins rescued the neuronal phenotypes derived from REST- ES cells, which included elaboration processes, neurite outgrowth and migration (see arrows in M and N) similar to that seen in neurons derived from control ES cells. Nuclei were stained with DAPI (blue) in all. Scale bars: 20 µm (A to E and K to O) and 100 µm (F to J).
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pone-0003656-g005: Laminins rescue the phenotypic effects of Rest ablation on the neural differentiation of ES cells.Control (REST-100) and 4 Rest mutants ES cells were grown under neural-differentiating conditions on laminin coated glass coverslips. After 4 days of differentiation some cultures were fixed and labelled with the NSC markers Nestin (red) and Sox1 (green) (A–E). The results show that laminins restored the ability to generate Nestin+ cells in Rest mutants (C–E, compared with Fig. 3C–3E). Sister cultures were allowed to differentiate for 14 days to analyse neuronal differentiation using NeuN (red) and Map2 (green) (F–J and in higher power K–O). Laminins rescued the neuronal phenotypes derived from REST- ES cells, which included elaboration processes, neurite outgrowth and migration (see arrows in M and N) similar to that seen in neurons derived from control ES cells. Nuclei were stained with DAPI (blue) in all. Scale bars: 20 µm (A to E and K to O) and 100 µm (F to J).

Mentions: REST- defects in cell adhesion may be the cause of its phenotypic effects on Nestin+ NSC production and neuronal differentiation, because cell adhesion defects caused aberrant NSC and neuron development [20]–[21]. Thus, we further examined the causes of defective adhesion seen in the REST- mutant. We and others have previously reported that several Rest target genes encode cell adhesion molecules or components of the extracellular matrix (ECM), particularly laminin subunits [7], [11]. Accordingly, we considered the notion that dysregulation of the ECM by Rest ablation might be responsible for this aspect of the phenotype. To test this hypothesis, we examined whether we could rescue the adhesion defect and any other phenotypic effects caused by Rest ablation by pre-treatment with ECM components. We plated ES cells from all groups on to either plastic or glass pre-treated with EHS laminins (which contain predominantly Laminin 1 (α1β1γ1)) [22] and subsequently subjected them to neural differentiation. On the glass surface pre-treated with laminins, but not those pre-treated with gelatin, the Rest mutant ES cells behaved like control ES cells, both of which adhered firmly and proliferated well. After 4-days of differentiation (NSC stage), cells from all groups plated onto glass surfaces pre-treated with laminins showed greater survival and significantly less apoptosis than those on untreated surfaces (Fig. 4K–4O). There was no significant difference in cell growth among the treated groups (the control and mutants). The surviving cells were highly proliferative as adjudged by BrdU incorporation (data not shown). Laminin pre-treatment had an even more profound effect after 14-days of differentiation (neuronal stage). No, or very few, apoptotic cells were detected in either the laminin-treated or control cells without laminin-treatment (Figs. 4F and 4P–4T), whereas significant cell death was found in the Rest mutants in the absence of laminin (Fig. 4H–4J). These data suggested that laminins prevented cell death in the REST- cells. Intriguingly, the laminin rescue also extended to the specification of NSCs. We reported above that the REST- ES cells were able to generate early Sox1+/Nestin− NSCs but were unable to produce normal numbers of late Sox1+/Nestin+ NSCs when they were differentiated on a gelatinised glass surface in the absence of laminins (Fig. 3C–3D). This deficiency was rescued by laminin-treatment. Under these conditions, REST- mutants, like control ES cells, generated equivalent numbers of both early NSCs (Sox+/Nestin−) and late NSCs (Sox+/Nestin+) (Fig. 5A–5E; Table 1). Furthermore, laminins rescued the production of NPCs and neurons from the REST- mutant (Table 1). Neurons derived from REST- ES cells showed widespread aggregation with extensive fasciculation (Fig. 5H–5I and 5M–5N) compared with those produced on glass in the absence of laminins (Fig. 3H–3I and 3M–3N).


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)

Laminins rescue the phenotypic effects of Rest ablation on the neural differentiation of ES cells.Control (REST-100) and 4 Rest mutants ES cells were grown under neural-differentiating conditions on laminin coated glass coverslips. After 4 days of differentiation some cultures were fixed and labelled with the NSC markers Nestin (red) and Sox1 (green) (A–E). The results show that laminins restored the ability to generate Nestin+ cells in Rest mutants (C–E, compared with Fig. 3C–3E). Sister cultures were allowed to differentiate for 14 days to analyse neuronal differentiation using NeuN (red) and Map2 (green) (F–J and in higher power K–O). Laminins rescued the neuronal phenotypes derived from REST- ES cells, which included elaboration processes, neurite outgrowth and migration (see arrows in M and N) similar to that seen in neurons derived from control ES cells. Nuclei were stained with DAPI (blue) in all. Scale bars: 20 µm (A to E and K to O) and 100 µm (F to J).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2573962&req=5

pone-0003656-g005: Laminins rescue the phenotypic effects of Rest ablation on the neural differentiation of ES cells.Control (REST-100) and 4 Rest mutants ES cells were grown under neural-differentiating conditions on laminin coated glass coverslips. After 4 days of differentiation some cultures were fixed and labelled with the NSC markers Nestin (red) and Sox1 (green) (A–E). The results show that laminins restored the ability to generate Nestin+ cells in Rest mutants (C–E, compared with Fig. 3C–3E). Sister cultures were allowed to differentiate for 14 days to analyse neuronal differentiation using NeuN (red) and Map2 (green) (F–J and in higher power K–O). Laminins rescued the neuronal phenotypes derived from REST- ES cells, which included elaboration processes, neurite outgrowth and migration (see arrows in M and N) similar to that seen in neurons derived from control ES cells. Nuclei were stained with DAPI (blue) in all. Scale bars: 20 µm (A to E and K to O) and 100 µm (F to J).
Mentions: REST- defects in cell adhesion may be the cause of its phenotypic effects on Nestin+ NSC production and neuronal differentiation, because cell adhesion defects caused aberrant NSC and neuron development [20]–[21]. Thus, we further examined the causes of defective adhesion seen in the REST- mutant. We and others have previously reported that several Rest target genes encode cell adhesion molecules or components of the extracellular matrix (ECM), particularly laminin subunits [7], [11]. Accordingly, we considered the notion that dysregulation of the ECM by Rest ablation might be responsible for this aspect of the phenotype. To test this hypothesis, we examined whether we could rescue the adhesion defect and any other phenotypic effects caused by Rest ablation by pre-treatment with ECM components. We plated ES cells from all groups on to either plastic or glass pre-treated with EHS laminins (which contain predominantly Laminin 1 (α1β1γ1)) [22] and subsequently subjected them to neural differentiation. On the glass surface pre-treated with laminins, but not those pre-treated with gelatin, the Rest mutant ES cells behaved like control ES cells, both of which adhered firmly and proliferated well. After 4-days of differentiation (NSC stage), cells from all groups plated onto glass surfaces pre-treated with laminins showed greater survival and significantly less apoptosis than those on untreated surfaces (Fig. 4K–4O). There was no significant difference in cell growth among the treated groups (the control and mutants). The surviving cells were highly proliferative as adjudged by BrdU incorporation (data not shown). Laminin pre-treatment had an even more profound effect after 14-days of differentiation (neuronal stage). No, or very few, apoptotic cells were detected in either the laminin-treated or control cells without laminin-treatment (Figs. 4F and 4P–4T), whereas significant cell death was found in the Rest mutants in the absence of laminin (Fig. 4H–4J). These data suggested that laminins prevented cell death in the REST- cells. Intriguingly, the laminin rescue also extended to the specification of NSCs. We reported above that the REST- ES cells were able to generate early Sox1+/Nestin− NSCs but were unable to produce normal numbers of late Sox1+/Nestin+ NSCs when they were differentiated on a gelatinised glass surface in the absence of laminins (Fig. 3C–3D). This deficiency was rescued by laminin-treatment. Under these conditions, REST- mutants, like control ES cells, generated equivalent numbers of both early NSCs (Sox+/Nestin−) and late NSCs (Sox+/Nestin+) (Fig. 5A–5E; Table 1). Furthermore, laminins rescued the production of NPCs and neurons from the REST- mutant (Table 1). Neurons derived from REST- ES cells showed widespread aggregation with extensive fasciculation (Fig. 5H–5I and 5M–5N) compared with those produced on glass in the absence of laminins (Fig. 3H–3I and 3M–3N).

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