Limits...
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.

Show MeSH

Related in: MedlinePlus

Laminins attenuate cell apoptosis caused by Rest ablation during neural differentiation of ES cells.The control REST-100 and 4 Rest mutant ES cells were either plated on gelatinised glass coverslips (A–J) or on glass coverslips coated with laminins (K–T) and subjected to standard neural differentiation. Cultures were fixed after 4 days of differentiation (A–E and K–O), the peak of NSC generation or fixed after 14 days of differentiation (F–J and P–T), when neurons were prevalent, and cell death in the cultures was assessed using TUNEL staining (green). In all cases cell nuclei were identified by DAPI staining (blue). As expected, cell death was observed when NSCs were generated in all groups (A–E). However, a marked reduction in cell death was observed in all cases when ES cells were plated and differentiated on laminin substrates (compare A–E with K–O). After 14 days of differentiation on gelatinised glass coverslips, markedly more cell death was observed in REST- than in REST-100 and REST/KD-50 (compare H with F and G). A similar level of death was observed in REST-+MTV (I). REST-+REST exhibited less cell apoptosis (J). Laminin-treatment reduced apoptosis in all Rest mutants (compare H–J with R–T). Scale bar: 20 µm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2573962&req=5

pone-0003656-g004: Laminins attenuate cell apoptosis caused by Rest ablation during neural differentiation of ES cells.The control REST-100 and 4 Rest mutant ES cells were either plated on gelatinised glass coverslips (A–J) or on glass coverslips coated with laminins (K–T) and subjected to standard neural differentiation. Cultures were fixed after 4 days of differentiation (A–E and K–O), the peak of NSC generation or fixed after 14 days of differentiation (F–J and P–T), when neurons were prevalent, and cell death in the cultures was assessed using TUNEL staining (green). In all cases cell nuclei were identified by DAPI staining (blue). As expected, cell death was observed when NSCs were generated in all groups (A–E). However, a marked reduction in cell death was observed in all cases when ES cells were plated and differentiated on laminin substrates (compare A–E with K–O). After 14 days of differentiation on gelatinised glass coverslips, markedly more cell death was observed in REST- than in REST-100 and REST/KD-50 (compare H with F and G). A similar level of death was observed in REST-+MTV (I). REST-+REST exhibited less cell apoptosis (J). Laminin-treatment reduced apoptosis in all Rest mutants (compare H–J with R–T). Scale bar: 20 µm.

Mentions: Either on plastic or glass surfaces, we found that REST- ES cells generated fewer cells during neural differentiation than those from REST-100 and REST/KD-50 ES cells. Accordingly, we examined proliferation and cell death during the course of neurogenesis. Using BrdU incorporation, no differences were observed in the proliferation of either Sox1+/Nestin− early NSCs derived from control and all Rest mutants or in Sox1+/Nestin+ late NSCs derived from the control, REST/KD-50 and REST-+REST ES cells. However, it was difficult to evaluate the Sox1+/Nestin+ NSCs from REST- and REST-+MTV ES mutants, because of their low number and the ease with which they were lost from coverslips during the staining process. We then used TUNEL staining to assess the degree of apoptosis at 4 and 14-days of differentiation, i.e. at the peaks of NSC and neuron generation respectively. The ES monolayer culture system used in this study does not employ mitogens to induce neural differentiation [19], and accordingly, significant cell death occurs during NSC formation, especially during days 3 through 6 of differentiation, but thereafter this becomes much less marked as neuronal differentiation proceeds. At the NSC stage, cell death was equally prevalent in all samples derived from either control cells or Rest mutants (Fig. 4A–4E). However, at the neuronal stage, there was markedly more cell death in REST- cells than those in the control and REST/KD-50 cells (Fig. 4F–4H), which is correlated with the ailing look of neurons derived from REST- ES cells (Fig. 3H and 3I). Our results indicate that Rest ablation did not impair the proliferation of Sox1+/Nestin− NSCs; however it is difficult to assess the proliferation of Sox1+/Nestin+ NSCs, due to the paucity of Nestin+ NSCs derived from REST- ES cells. Moreover, Rest levels had to be reduced by more than 92% before an increase in cell death at the neuronal stage was observed.


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 attenuate cell apoptosis caused by Rest ablation during neural differentiation of ES cells.The control REST-100 and 4 Rest mutant ES cells were either plated on gelatinised glass coverslips (A–J) or on glass coverslips coated with laminins (K–T) and subjected to standard neural differentiation. Cultures were fixed after 4 days of differentiation (A–E and K–O), the peak of NSC generation or fixed after 14 days of differentiation (F–J and P–T), when neurons were prevalent, and cell death in the cultures was assessed using TUNEL staining (green). In all cases cell nuclei were identified by DAPI staining (blue). As expected, cell death was observed when NSCs were generated in all groups (A–E). However, a marked reduction in cell death was observed in all cases when ES cells were plated and differentiated on laminin substrates (compare A–E with K–O). After 14 days of differentiation on gelatinised glass coverslips, markedly more cell death was observed in REST- than in REST-100 and REST/KD-50 (compare H with F and G). A similar level of death was observed in REST-+MTV (I). REST-+REST exhibited less cell apoptosis (J). Laminin-treatment reduced apoptosis in all Rest mutants (compare H–J with R–T). Scale bar: 20 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0003656-g004: Laminins attenuate cell apoptosis caused by Rest ablation during neural differentiation of ES cells.The control REST-100 and 4 Rest mutant ES cells were either plated on gelatinised glass coverslips (A–J) or on glass coverslips coated with laminins (K–T) and subjected to standard neural differentiation. Cultures were fixed after 4 days of differentiation (A–E and K–O), the peak of NSC generation or fixed after 14 days of differentiation (F–J and P–T), when neurons were prevalent, and cell death in the cultures was assessed using TUNEL staining (green). In all cases cell nuclei were identified by DAPI staining (blue). As expected, cell death was observed when NSCs were generated in all groups (A–E). However, a marked reduction in cell death was observed in all cases when ES cells were plated and differentiated on laminin substrates (compare A–E with K–O). After 14 days of differentiation on gelatinised glass coverslips, markedly more cell death was observed in REST- than in REST-100 and REST/KD-50 (compare H with F and G). A similar level of death was observed in REST-+MTV (I). REST-+REST exhibited less cell apoptosis (J). Laminin-treatment reduced apoptosis in all Rest mutants (compare H–J with R–T). Scale bar: 20 µm.
Mentions: Either on plastic or glass surfaces, we found that REST- ES cells generated fewer cells during neural differentiation than those from REST-100 and REST/KD-50 ES cells. Accordingly, we examined proliferation and cell death during the course of neurogenesis. Using BrdU incorporation, no differences were observed in the proliferation of either Sox1+/Nestin− early NSCs derived from control and all Rest mutants or in Sox1+/Nestin+ late NSCs derived from the control, REST/KD-50 and REST-+REST ES cells. However, it was difficult to evaluate the Sox1+/Nestin+ NSCs from REST- and REST-+MTV ES mutants, because of their low number and the ease with which they were lost from coverslips during the staining process. We then used TUNEL staining to assess the degree of apoptosis at 4 and 14-days of differentiation, i.e. at the peaks of NSC and neuron generation respectively. The ES monolayer culture system used in this study does not employ mitogens to induce neural differentiation [19], and accordingly, significant cell death occurs during NSC formation, especially during days 3 through 6 of differentiation, but thereafter this becomes much less marked as neuronal differentiation proceeds. At the NSC stage, cell death was equally prevalent in all samples derived from either control cells or Rest mutants (Fig. 4A–4E). However, at the neuronal stage, there was markedly more cell death in REST- cells than those in the control and REST/KD-50 cells (Fig. 4F–4H), which is correlated with the ailing look of neurons derived from REST- ES cells (Fig. 3H and 3I). Our results indicate that Rest ablation did not impair the proliferation of Sox1+/Nestin− NSCs; however it is difficult to assess the proliferation of Sox1+/Nestin+ NSCs, due to the paucity of Nestin+ NSCs derived from REST- ES cells. Moreover, Rest levels had to be reduced by more than 92% before an increase in cell death at the neuronal stage was observed.

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