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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 effect of Rest ablation on the expression of stage-specific neural markers.The ability of control (REST-100) and Rest mutant ES cells (REST/KD-50 and REST-) to generate the different neural cell populations was assessed by real time-PCR using the following markers: NSCs Pax6, Msi1 and Nestin (A–C); NPCs Mash1 and Ngn1 (D–E); early neurons Tubb3 (F) and mature neurons, Scn2a and L1cam (G–H). Data are represented as mean±SEM. *P<0.05 and **P<0.01, significantly different from REST-100 and REST/KD-50.
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pone-0003656-g002: The effect of Rest ablation on the expression of stage-specific neural markers.The ability of control (REST-100) and Rest mutant ES cells (REST/KD-50 and REST-) to generate the different neural cell populations was assessed by real time-PCR using the following markers: NSCs Pax6, Msi1 and Nestin (A–C); NPCs Mash1 and Ngn1 (D–E); early neurons Tubb3 (F) and mature neurons, Scn2a and L1cam (G–H). Data are represented as mean±SEM. *P<0.05 and **P<0.01, significantly different from REST-100 and REST/KD-50.

Mentions: Next we examined the effects of Rest ablation on the development of NSCs and NPCs. The control REST-100, REST/KD-50 and REST- ES cells, which express 100%, 50% and 0% wild-type Rest levels respectively (Fig. S1B and S1D), were differentiated into NSCs and then into NPCs identified using an array of NSC and NPC markers (Fig. 1A). We assessed the effects of Rest ablation on gene and protein expression of these markers using quantitative Real-time PCR, FACS and immunocytochemical analysis. In REST-100 ES cells, the expression profiles of Pax6, Msi1 and Nestin were similar to one another, with expression peaking around 4 days and thereafter gradually declining (Fig. 2A–2C). REST/KD-50 ES cells showed no significant difference in the expression level and pattern of these genes as compared to the control REST-100 cells (Fig. 2A–2C). Similarly, no change in the number of Sox1+/Nestin+ NSCs was seen (Table 1 and Fig. S3B–3C). However, in REST- ES cells, the peak expression of Pax6 and Msi1 was significantly (P<0.01) reduced to 40% of control levels whilst expression of Nestin was reduced to 60% of control levels (Fig. 2A–2C). This mutant generated significantly (P<0.01) fewer Sox1+/Nestin+ NSCs (52%) as compared to the REST-100 (77%) and REST/KD-50 ES cells (76%), but generated double the number of Sox1+/ Nestin− NSCs compared with the control cells (P<0.01) (Table 1 and Fig. S3D). Moreover, REST- ES cells produced lower levels of Mash1 and Ngn1 expression (P<0.01) and this was reflected in a parallel reduction in the number of Mash1+ NPCs (∼50%) as compared to the control (87%) (P<0.05) (Fig. 2D–2E; Table 1; Fig. S4B and 4D). Our results suggest that a 50% depletion of Rest shows no discernible effect on the production of NSCs and NPCs from ES cells. In fact, the production of early Sox1+/Nestin− NSCs from REST- ES cells remained unaffected but formation of late Sox1+/Nestin+ NSCs and subsequent production of NPCs was inhibited.


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 effect of Rest ablation on the expression of stage-specific neural markers.The ability of control (REST-100) and Rest mutant ES cells (REST/KD-50 and REST-) to generate the different neural cell populations was assessed by real time-PCR using the following markers: NSCs Pax6, Msi1 and Nestin (A–C); NPCs Mash1 and Ngn1 (D–E); early neurons Tubb3 (F) and mature neurons, Scn2a and L1cam (G–H). Data are represented as mean±SEM. *P<0.05 and **P<0.01, significantly different from REST-100 and REST/KD-50.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2573962&req=5

pone-0003656-g002: The effect of Rest ablation on the expression of stage-specific neural markers.The ability of control (REST-100) and Rest mutant ES cells (REST/KD-50 and REST-) to generate the different neural cell populations was assessed by real time-PCR using the following markers: NSCs Pax6, Msi1 and Nestin (A–C); NPCs Mash1 and Ngn1 (D–E); early neurons Tubb3 (F) and mature neurons, Scn2a and L1cam (G–H). Data are represented as mean±SEM. *P<0.05 and **P<0.01, significantly different from REST-100 and REST/KD-50.
Mentions: Next we examined the effects of Rest ablation on the development of NSCs and NPCs. The control REST-100, REST/KD-50 and REST- ES cells, which express 100%, 50% and 0% wild-type Rest levels respectively (Fig. S1B and S1D), were differentiated into NSCs and then into NPCs identified using an array of NSC and NPC markers (Fig. 1A). We assessed the effects of Rest ablation on gene and protein expression of these markers using quantitative Real-time PCR, FACS and immunocytochemical analysis. In REST-100 ES cells, the expression profiles of Pax6, Msi1 and Nestin were similar to one another, with expression peaking around 4 days and thereafter gradually declining (Fig. 2A–2C). REST/KD-50 ES cells showed no significant difference in the expression level and pattern of these genes as compared to the control REST-100 cells (Fig. 2A–2C). Similarly, no change in the number of Sox1+/Nestin+ NSCs was seen (Table 1 and Fig. S3B–3C). However, in REST- ES cells, the peak expression of Pax6 and Msi1 was significantly (P<0.01) reduced to 40% of control levels whilst expression of Nestin was reduced to 60% of control levels (Fig. 2A–2C). This mutant generated significantly (P<0.01) fewer Sox1+/Nestin+ NSCs (52%) as compared to the REST-100 (77%) and REST/KD-50 ES cells (76%), but generated double the number of Sox1+/ Nestin− NSCs compared with the control cells (P<0.01) (Table 1 and Fig. S3D). Moreover, REST- ES cells produced lower levels of Mash1 and Ngn1 expression (P<0.01) and this was reflected in a parallel reduction in the number of Mash1+ NPCs (∼50%) as compared to the control (87%) (P<0.05) (Fig. 2D–2E; Table 1; Fig. S4B and 4D). Our results suggest that a 50% depletion of Rest shows no discernible effect on the production of NSCs and NPCs from ES cells. In fact, the production of early Sox1+/Nestin− NSCs from REST- ES cells remained unaffected but formation of late Sox1+/Nestin+ NSCs and subsequent production of NPCs was inhibited.

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