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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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Time course of stage-specific marker expression during neural differentiation of HM1 embryonic stem (ES) cells.(A) Summary of neural stage-specific markers used in this study. NSC: neural stem cells; NPC: neural progenitor cells. (B) Down-regulation of Oct4 and Rest was observed as neural differentiation proceeded. (C) From day 2–8, the expression of NSC markers, Nestin and Pax6 is observed. (D) NPC markers, Mash1 and Ngn1, appeared in an overlapping but slightly later wave than NSC markers. (E) After 10 days of differentiation, markers of early (Tubb3) and mature neurons (Syn1 and L1cam) appeared.
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pone-0003656-g001: Time course of stage-specific marker expression during neural differentiation of HM1 embryonic stem (ES) cells.(A) Summary of neural stage-specific markers used in this study. NSC: neural stem cells; NPC: neural progenitor cells. (B) Down-regulation of Oct4 and Rest was observed as neural differentiation proceeded. (C) From day 2–8, the expression of NSC markers, Nestin and Pax6 is observed. (D) NPC markers, Mash1 and Ngn1, appeared in an overlapping but slightly later wave than NSC markers. (E) After 10 days of differentiation, markers of early (Tubb3) and mature neurons (Syn1 and L1cam) appeared.

Mentions: We investigated the role of Rest in neural development using an in-vitro ES cell-derived neural differentiation model, which recapitulates events during neural development in vivo. ES cells firstly differentiate into neuroepithelial cells (early NSCs), which peak around 4–6 days of differentiation and express Sox1 and Nestin (about 80% of population), and then differentiate further into more restricted NSCs that peak around 10 days of differentiation and express either Ngn1 or Mash1 (about 80% of population) (Fig. 1C–D and Fig. S2B–C). In this paper, we refer to early NSCs as NSCs and late more restricted NSCs as NPCs. To establish the time course during which NSCs, NPCs and neurons are formed from HM1 and 46C ES cells, we examined gene expression patterns for NSC markers (Nestin and Pax6), NPC markers (Mash1 and Ngn1) and neuronal markers (Tubb3, Syn1 and L1cam) during ES cell-derived neural differentiation (Fig. 1A). We found that Rest expression mirrored that of the pluripotent ES cell marker Oct4, and was expressed at highest level in ES cells with its expression level declining as differentiation proceeded; reaching its lowest level 4 days after differentiation just before NSC production reached its peak (Fig. 1B and Fig. S2A). Rest levels were maintained at low levels throughout neuron formation. Conversely, the expression patterns of Nestin and Pax6 reciprocated those of Rest and Oct4 indicating that NSC production started 1- or 2-days after differentiation, reached a peak around 6 days, and thereafter declined (Fig. 1C and Fig. S2B). Mash1 and Ngn1 expression indicated that NPCs started to be produced after 6-days of differentiation and peaked between 6 and 10 days (Fig. 1D and Fig. S2C). The early neuronal marker Tubb3 was observed around the same time but did not peak until 12 days of differentiation whereas the peak of mature neuron marker expression (Syn1 and L1cam) occurred at 14–16 days of differentiation (Fig. 1E and Fig. S2D). This time course closely recapitulates the sequential generation of NSCs, NPCs and neurons observed in vivo (Fig. 1A).


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)

Time course of stage-specific marker expression during neural differentiation of HM1 embryonic stem (ES) cells.(A) Summary of neural stage-specific markers used in this study. NSC: neural stem cells; NPC: neural progenitor cells. (B) Down-regulation of Oct4 and Rest was observed as neural differentiation proceeded. (C) From day 2–8, the expression of NSC markers, Nestin and Pax6 is observed. (D) NPC markers, Mash1 and Ngn1, appeared in an overlapping but slightly later wave than NSC markers. (E) After 10 days of differentiation, markers of early (Tubb3) and mature neurons (Syn1 and L1cam) appeared.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2573962&req=5

pone-0003656-g001: Time course of stage-specific marker expression during neural differentiation of HM1 embryonic stem (ES) cells.(A) Summary of neural stage-specific markers used in this study. NSC: neural stem cells; NPC: neural progenitor cells. (B) Down-regulation of Oct4 and Rest was observed as neural differentiation proceeded. (C) From day 2–8, the expression of NSC markers, Nestin and Pax6 is observed. (D) NPC markers, Mash1 and Ngn1, appeared in an overlapping but slightly later wave than NSC markers. (E) After 10 days of differentiation, markers of early (Tubb3) and mature neurons (Syn1 and L1cam) appeared.
Mentions: We investigated the role of Rest in neural development using an in-vitro ES cell-derived neural differentiation model, which recapitulates events during neural development in vivo. ES cells firstly differentiate into neuroepithelial cells (early NSCs), which peak around 4–6 days of differentiation and express Sox1 and Nestin (about 80% of population), and then differentiate further into more restricted NSCs that peak around 10 days of differentiation and express either Ngn1 or Mash1 (about 80% of population) (Fig. 1C–D and Fig. S2B–C). In this paper, we refer to early NSCs as NSCs and late more restricted NSCs as NPCs. To establish the time course during which NSCs, NPCs and neurons are formed from HM1 and 46C ES cells, we examined gene expression patterns for NSC markers (Nestin and Pax6), NPC markers (Mash1 and Ngn1) and neuronal markers (Tubb3, Syn1 and L1cam) during ES cell-derived neural differentiation (Fig. 1A). We found that Rest expression mirrored that of the pluripotent ES cell marker Oct4, and was expressed at highest level in ES cells with its expression level declining as differentiation proceeded; reaching its lowest level 4 days after differentiation just before NSC production reached its peak (Fig. 1B and Fig. S2A). Rest levels were maintained at low levels throughout neuron formation. Conversely, the expression patterns of Nestin and Pax6 reciprocated those of Rest and Oct4 indicating that NSC production started 1- or 2-days after differentiation, reached a peak around 6 days, and thereafter declined (Fig. 1C and Fig. S2B). Mash1 and Ngn1 expression indicated that NPCs started to be produced after 6-days of differentiation and peaked between 6 and 10 days (Fig. 1D and Fig. S2C). The early neuronal marker Tubb3 was observed around the same time but did not peak until 12 days of differentiation whereas the peak of mature neuron marker expression (Syn1 and L1cam) occurred at 14–16 days of differentiation (Fig. 1E and Fig. S2D). This time course closely recapitulates the sequential generation of NSCs, NPCs and neurons observed in vivo (Fig. 1A).

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