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Transcriptional regulation induced by cAMP elevation in mouse Schwann cells.

Schmid D, Zeis T, Schaeren-Wiemers N - ASN Neuro (2014)

Bottom Line: One of the strongest differentially regulated gene transcripts was the transcription factor Olig1 (oligodendrocyte transcription factor 1), whose mRNA expression levels were reduced in treated Schwann cells.Olig1 protein was localized in myelinating and nonmyelinating Schwann cells within the sciatic nerve as well as in primary Schwann cells, proposing it as a novel transcription factor of the Schwann cell lineage.Data analysis further revealed that a number of differentially expressed genes in forskolin-treated Schwann cells were associated with the ECM (extracellular matrix), underlining its importance during Schwann cell differentiation in vitro.

View Article: PubMed Central - PubMed

Affiliation: *Neurobiology, Department of Biomedicine, University Hospital Basel, University of Basel, Hebelstrasse 20, CH-4031 Basel, Switzerland.

ABSTRACT
In peripheral nerves, Schwann cell development is regulated by a variety of signals. Some of the aspects of Schwann cell differentiation can be reproduced in vitro in response to forskolin, an adenylyl cyclase activator elevating intracellular cAMP levels. Herein, the effect of forskolin treatment was investigated by a comprehensive genome-wide expression study on primary mouse Schwann cell cultures. Additional to myelin-related genes, many so far unconsidered genes were ascertained to be modulated by forskolin. One of the strongest differentially regulated gene transcripts was the transcription factor Olig1 (oligodendrocyte transcription factor 1), whose mRNA expression levels were reduced in treated Schwann cells. Olig1 protein was localized in myelinating and nonmyelinating Schwann cells within the sciatic nerve as well as in primary Schwann cells, proposing it as a novel transcription factor of the Schwann cell lineage. Data analysis further revealed that a number of differentially expressed genes in forskolin-treated Schwann cells were associated with the ECM (extracellular matrix), underlining its importance during Schwann cell differentiation in vitro. Comparison of samples derived from postnatal sciatic nerves and from both treated and untreated Schwann cell cultures showed considerable differences in gene expression between in vivo and in vitro, allowing us to separate Schwann cell autonomous from tissue-related changes. The whole data set of the cell culture microarray study is provided to offer an interactive search tool for genes of interest.

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Expression of Olig1 in sciatic nerves and cultured Schwann                            cells                        Immunofluorescent stainings of transversal (A) and longitudinal (C)                            tissue sections of sciatic nerves from P7 mice revealed Olig1                            immunofluorescence in Remak bundles (arrows), identified by a bundle of                            unmyelinated (MBP-negative) small diameter axons, and in a subset of                            myelinating Schwann cells (arrowheads). (B) Remak bundle localization                            was confirmed by the expression of p75NTR. (D) A progressive                            increase of Olig1 mRNA expression levels was detected during peripheral                            nerve development by qRT–PCR. Data were normalized to the                            expression of 60s, and values at P0 were set to 1. Each data point                            represent the mean value of at least eight experimental samples, and the                            error bars indicate the S.D. (E) In vitro,                            Olig1 expression was predominantly detected in the cytoplasm of cultured                            Schwann cells (E, arrows), whereas nuclear staining was significantly                            weaker (E, arrowheads). NF: neurofilament. Bar: A:                            100 μm; B, C, E: 20 μm.
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Figure 2: Expression of Olig1 in sciatic nerves and cultured Schwann cells Immunofluorescent stainings of transversal (A) and longitudinal (C) tissue sections of sciatic nerves from P7 mice revealed Olig1 immunofluorescence in Remak bundles (arrows), identified by a bundle of unmyelinated (MBP-negative) small diameter axons, and in a subset of myelinating Schwann cells (arrowheads). (B) Remak bundle localization was confirmed by the expression of p75NTR. (D) A progressive increase of Olig1 mRNA expression levels was detected during peripheral nerve development by qRT–PCR. Data were normalized to the expression of 60s, and values at P0 were set to 1. Each data point represent the mean value of at least eight experimental samples, and the error bars indicate the S.D. (E) In vitro, Olig1 expression was predominantly detected in the cytoplasm of cultured Schwann cells (E, arrows), whereas nuclear staining was significantly weaker (E, arrowheads). NF: neurofilament. Bar: A: 100 μm; B, C, E: 20 μm.

Mentions: The transcription factor Olig1 was identified to be strongly down-regulated in forskolin-treated mouse Schwann cells. Although Olig1 is known to play an important functional role in differentiation of oligodendrocyte precursor cells (Li et al., 2007), its expression in the Schwann cell lineage is not known. For this reason, the expression of Olig1 protein was investigated on sciatic nerves of P7 mice. Colocalization analysis revealed Olig1 immunofluorescent signal primarily in MBP-negative areas consisting of small diameter axons identified by neurofilament (Figure 2B, inset and Figure 2D, arrows). These areas are reminiscent of Remak bundles evident by the expression of p75NTR, a marker for nonmyelinating Schwann cells (Figure 2B and Supplementary Figure S2; available at http://www.asnneuro.org/an/006/an006e142add.htm). In addition, also a subset of myelinating Schwann cells were identified to be Olig1 positive (Figures 2A and 2C, arrowheads). To determine Olig1 transcription during peripheral nerve development, its mRNA expression levels were investigated by qRT-PCR in sciatic nerves at P0, P3, P9, P21 and in adult mice (Figure 2D). Expression analysis of Olig1 mRNA levels during peripheral nerve development revealed progressively increased transcription correlating with Schwann cell maturation. In contrast to Mpz, Olig1 mRNA expression levels remained high in adult nerves. Confocal immunofluorescence microscopy identified Olig1 localization in the cytoplasm as well as in the nucleus of Schwann cells (Figure 2E). Quantification of the average immunofluorescent signal revealed a significant higher intensity in the cytoplasm compared with the nucleus (results not shown). From our analysis, we conclude that Olig1 is expressed in Schwann cells although at lower levels than in the CNS (central nervous system) (results not shown).


Transcriptional regulation induced by cAMP elevation in mouse Schwann cells.

Schmid D, Zeis T, Schaeren-Wiemers N - ASN Neuro (2014)

Expression of Olig1 in sciatic nerves and cultured Schwann                            cells                        Immunofluorescent stainings of transversal (A) and longitudinal (C)                            tissue sections of sciatic nerves from P7 mice revealed Olig1                            immunofluorescence in Remak bundles (arrows), identified by a bundle of                            unmyelinated (MBP-negative) small diameter axons, and in a subset of                            myelinating Schwann cells (arrowheads). (B) Remak bundle localization                            was confirmed by the expression of p75NTR. (D) A progressive                            increase of Olig1 mRNA expression levels was detected during peripheral                            nerve development by qRT–PCR. Data were normalized to the                            expression of 60s, and values at P0 were set to 1. Each data point                            represent the mean value of at least eight experimental samples, and the                            error bars indicate the S.D. (E) In vitro,                            Olig1 expression was predominantly detected in the cytoplasm of cultured                            Schwann cells (E, arrows), whereas nuclear staining was significantly                            weaker (E, arrowheads). NF: neurofilament. Bar: A:                            100 μm; B, C, E: 20 μm.
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Figure 2: Expression of Olig1 in sciatic nerves and cultured Schwann cells Immunofluorescent stainings of transversal (A) and longitudinal (C) tissue sections of sciatic nerves from P7 mice revealed Olig1 immunofluorescence in Remak bundles (arrows), identified by a bundle of unmyelinated (MBP-negative) small diameter axons, and in a subset of myelinating Schwann cells (arrowheads). (B) Remak bundle localization was confirmed by the expression of p75NTR. (D) A progressive increase of Olig1 mRNA expression levels was detected during peripheral nerve development by qRT–PCR. Data were normalized to the expression of 60s, and values at P0 were set to 1. Each data point represent the mean value of at least eight experimental samples, and the error bars indicate the S.D. (E) In vitro, Olig1 expression was predominantly detected in the cytoplasm of cultured Schwann cells (E, arrows), whereas nuclear staining was significantly weaker (E, arrowheads). NF: neurofilament. Bar: A: 100 μm; B, C, E: 20 μm.
Mentions: The transcription factor Olig1 was identified to be strongly down-regulated in forskolin-treated mouse Schwann cells. Although Olig1 is known to play an important functional role in differentiation of oligodendrocyte precursor cells (Li et al., 2007), its expression in the Schwann cell lineage is not known. For this reason, the expression of Olig1 protein was investigated on sciatic nerves of P7 mice. Colocalization analysis revealed Olig1 immunofluorescent signal primarily in MBP-negative areas consisting of small diameter axons identified by neurofilament (Figure 2B, inset and Figure 2D, arrows). These areas are reminiscent of Remak bundles evident by the expression of p75NTR, a marker for nonmyelinating Schwann cells (Figure 2B and Supplementary Figure S2; available at http://www.asnneuro.org/an/006/an006e142add.htm). In addition, also a subset of myelinating Schwann cells were identified to be Olig1 positive (Figures 2A and 2C, arrowheads). To determine Olig1 transcription during peripheral nerve development, its mRNA expression levels were investigated by qRT-PCR in sciatic nerves at P0, P3, P9, P21 and in adult mice (Figure 2D). Expression analysis of Olig1 mRNA levels during peripheral nerve development revealed progressively increased transcription correlating with Schwann cell maturation. In contrast to Mpz, Olig1 mRNA expression levels remained high in adult nerves. Confocal immunofluorescence microscopy identified Olig1 localization in the cytoplasm as well as in the nucleus of Schwann cells (Figure 2E). Quantification of the average immunofluorescent signal revealed a significant higher intensity in the cytoplasm compared with the nucleus (results not shown). From our analysis, we conclude that Olig1 is expressed in Schwann cells although at lower levels than in the CNS (central nervous system) (results not shown).

Bottom Line: One of the strongest differentially regulated gene transcripts was the transcription factor Olig1 (oligodendrocyte transcription factor 1), whose mRNA expression levels were reduced in treated Schwann cells.Olig1 protein was localized in myelinating and nonmyelinating Schwann cells within the sciatic nerve as well as in primary Schwann cells, proposing it as a novel transcription factor of the Schwann cell lineage.Data analysis further revealed that a number of differentially expressed genes in forskolin-treated Schwann cells were associated with the ECM (extracellular matrix), underlining its importance during Schwann cell differentiation in vitro.

View Article: PubMed Central - PubMed

Affiliation: *Neurobiology, Department of Biomedicine, University Hospital Basel, University of Basel, Hebelstrasse 20, CH-4031 Basel, Switzerland.

ABSTRACT
In peripheral nerves, Schwann cell development is regulated by a variety of signals. Some of the aspects of Schwann cell differentiation can be reproduced in vitro in response to forskolin, an adenylyl cyclase activator elevating intracellular cAMP levels. Herein, the effect of forskolin treatment was investigated by a comprehensive genome-wide expression study on primary mouse Schwann cell cultures. Additional to myelin-related genes, many so far unconsidered genes were ascertained to be modulated by forskolin. One of the strongest differentially regulated gene transcripts was the transcription factor Olig1 (oligodendrocyte transcription factor 1), whose mRNA expression levels were reduced in treated Schwann cells. Olig1 protein was localized in myelinating and nonmyelinating Schwann cells within the sciatic nerve as well as in primary Schwann cells, proposing it as a novel transcription factor of the Schwann cell lineage. Data analysis further revealed that a number of differentially expressed genes in forskolin-treated Schwann cells were associated with the ECM (extracellular matrix), underlining its importance during Schwann cell differentiation in vitro. Comparison of samples derived from postnatal sciatic nerves and from both treated and untreated Schwann cell cultures showed considerable differences in gene expression between in vivo and in vitro, allowing us to separate Schwann cell autonomous from tissue-related changes. The whole data set of the cell culture microarray study is provided to offer an interactive search tool for genes of interest.

Show MeSH