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Spatiotemporal changes in Cx30 and Cx43 expression during neuronal differentiation of P19 EC and NT2/D1 cells.

Wan CK, O'Carroll SJ, Kim SL, Green CR, Nicholson LF - Cell Biol Int Rep (2010) (2013)

Bottom Line: Cx43 was observed intracellularly and on cell surfaces, while Cx30 was seen as puncta.In P19 cells Cx30 was localized on clusters of cells surrounded by MAP2- and doublecortin-positive processes.The expression pattern of Cx30 indicates a role in neuronal differentiation; the nature of that role warrants future investigation.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Medical and Health Sciences, Department of Anatomy with Radiology and Centre for Brain Research, The University of Auckland Auckland, 92019, New Zealand.

ABSTRACT

While connexins (Cxs) are thought to be involved in differentiation, their expression and role has yet to be fully elucidated. We investigated the temporal expression of Cx30, Cx36 and Cx43 in two in vitro models of neuronal differentiation: human NT2/D1 and murine P19 cells, and the spatial localisation of Cx30 and Cx43 in these models. A temporal Cx43 downregulation was confirmed in both cell lines during RA-induced neuronal differentiation using RT-PCR (P < 0.05) preceding an increase in neuronal doublecortin protein. RT-PCR showed Cx36 was upregulated twofold in NT2/D1 cells (P < 0.05) and sixfold in P19 cells (P < 0.001) during neuronal differentiation. Cx30 exhibited a transient peak in expression midway through the timecourse of differentiation increasing threefold in NT2/D1 cells (P < 0.001) and eightfold in P19 cells (P < 0.01). Qualitative immunocytochemistry was used to examine spatiotemporal patterns of Cx protein distribution alongside neuronal differentiation markers. The temporal immunolabelling pattern was similar to that seen using RT-PCR. Cx43 was observed intracellularly and on cell surfaces, while Cx30 was seen as puncta. Spatially Cx43 was seen on doublecortin-negative cells, which may indicate Cx43 downregulation is requisite for differentiation in these models. Conversely, Cx30 puncta were observed on doublecortin-positive and -negative cells in NT2/D1 cells and examination of the Cx30 peak showed puncta also localized to nestin-positive cells, with few puncta on MAP2-positive cells. In P19 cells Cx30 was localized on clusters of cells surrounded by MAP2- and doublecortin-positive processes. The expression pattern of Cx30 indicates a role in neuronal differentiation; the nature of that role warrants future investigation.

No MeSH data available.


Temporal expression of Cx30, Cx36 and Cx43 mRNA during differentiation of the NT2/D1 and P19 EC cell models, as assessed by RT-PCR. Experiments were performed in triplicate independent cultures. Statistically significant changes relative to undifferentiated cells are denoted by asterisks (*P < 0.05; **P < 0.01; ***P < 0.001). (A) Representative images of the expression pattern of Cx30, Cx36 and Cx43 subtypes, and beta-actin, during the timecourse of NT2/D1 (A1) and P19 EC (A2) neuronal differentiation. A similar pattern of expression for the three Cx subtypes was seen in both cell lines. (B) Changes in Cx43 mRNA expression. In the NT2/D1 cell line, Cx43 decreased between day 0 and day 14 (P < 0.05) and remained downregulated at days 21 (P < 0.01) and 28 of RA treatment (P < 0.05) (B1). Cx43 significantly decreased in the P19 EC cell line by day 4 post-RA treatment and remained downregulated throughout the remainder of the timecourse (P < 0.001) (B2). (C) Changes in Cx36 mRNA expression. A significant increase in Cx36 expression was observed at 14 and 28 days RA treatment of NT2/D1 cells (P < 0.05) (C1), while in the P19 EC model a significant increase in Cx36 expression was observed at 6 (P < 0.001) and 8 days post-RA (P < 0.05) (C2). (D) Changes in Cx30 mRNA expression. In the NT2/D1 model, a threefold increase in expression of Cx30 was observed at the 14 days RA (P < 0.001), and the subtype was also elevated at days 21 (P < 0.05) and 28 (P < 0.01) (D1). A greater than ninefold increase in expression of Cx30 was observed at 6 days post-RA (P < 0.001) relative to undifferentiated P19 EC cells (D2).
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fig01: Temporal expression of Cx30, Cx36 and Cx43 mRNA during differentiation of the NT2/D1 and P19 EC cell models, as assessed by RT-PCR. Experiments were performed in triplicate independent cultures. Statistically significant changes relative to undifferentiated cells are denoted by asterisks (*P < 0.05; **P < 0.01; ***P < 0.001). (A) Representative images of the expression pattern of Cx30, Cx36 and Cx43 subtypes, and beta-actin, during the timecourse of NT2/D1 (A1) and P19 EC (A2) neuronal differentiation. A similar pattern of expression for the three Cx subtypes was seen in both cell lines. (B) Changes in Cx43 mRNA expression. In the NT2/D1 cell line, Cx43 decreased between day 0 and day 14 (P < 0.05) and remained downregulated at days 21 (P < 0.01) and 28 of RA treatment (P < 0.05) (B1). Cx43 significantly decreased in the P19 EC cell line by day 4 post-RA treatment and remained downregulated throughout the remainder of the timecourse (P < 0.001) (B2). (C) Changes in Cx36 mRNA expression. A significant increase in Cx36 expression was observed at 14 and 28 days RA treatment of NT2/D1 cells (P < 0.05) (C1), while in the P19 EC model a significant increase in Cx36 expression was observed at 6 (P < 0.001) and 8 days post-RA (P < 0.05) (C2). (D) Changes in Cx30 mRNA expression. In the NT2/D1 model, a threefold increase in expression of Cx30 was observed at the 14 days RA (P < 0.001), and the subtype was also elevated at days 21 (P < 0.05) and 28 (P < 0.01) (D1). A greater than ninefold increase in expression of Cx30 was observed at 6 days post-RA (P < 0.001) relative to undifferentiated P19 EC cells (D2).

Mentions: The temporal expression patterns of Cx30, Cx36 and Cx43 mRNA were examined using RT-PCR, and semi-quantitative analysis was performed relative to the expression of the constitutive housekeeping gene, beta-actin, to provide a mean integrated density in arbitrary units (AU). The changes in Cx subtype expression levels were assessed relative to undifferentiated cells and the changes in expression level denoted in fold-change relative to undifferentiated cells (Figure 1A1 for NT2/D1 cells; Figure 1A2 for P19 EC cells).


Spatiotemporal changes in Cx30 and Cx43 expression during neuronal differentiation of P19 EC and NT2/D1 cells.

Wan CK, O'Carroll SJ, Kim SL, Green CR, Nicholson LF - Cell Biol Int Rep (2010) (2013)

Temporal expression of Cx30, Cx36 and Cx43 mRNA during differentiation of the NT2/D1 and P19 EC cell models, as assessed by RT-PCR. Experiments were performed in triplicate independent cultures. Statistically significant changes relative to undifferentiated cells are denoted by asterisks (*P < 0.05; **P < 0.01; ***P < 0.001). (A) Representative images of the expression pattern of Cx30, Cx36 and Cx43 subtypes, and beta-actin, during the timecourse of NT2/D1 (A1) and P19 EC (A2) neuronal differentiation. A similar pattern of expression for the three Cx subtypes was seen in both cell lines. (B) Changes in Cx43 mRNA expression. In the NT2/D1 cell line, Cx43 decreased between day 0 and day 14 (P < 0.05) and remained downregulated at days 21 (P < 0.01) and 28 of RA treatment (P < 0.05) (B1). Cx43 significantly decreased in the P19 EC cell line by day 4 post-RA treatment and remained downregulated throughout the remainder of the timecourse (P < 0.001) (B2). (C) Changes in Cx36 mRNA expression. A significant increase in Cx36 expression was observed at 14 and 28 days RA treatment of NT2/D1 cells (P < 0.05) (C1), while in the P19 EC model a significant increase in Cx36 expression was observed at 6 (P < 0.001) and 8 days post-RA (P < 0.05) (C2). (D) Changes in Cx30 mRNA expression. In the NT2/D1 model, a threefold increase in expression of Cx30 was observed at the 14 days RA (P < 0.001), and the subtype was also elevated at days 21 (P < 0.05) and 28 (P < 0.01) (D1). A greater than ninefold increase in expression of Cx30 was observed at 6 days post-RA (P < 0.001) relative to undifferentiated P19 EC cells (D2).
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Related In: Results  -  Collection

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fig01: Temporal expression of Cx30, Cx36 and Cx43 mRNA during differentiation of the NT2/D1 and P19 EC cell models, as assessed by RT-PCR. Experiments were performed in triplicate independent cultures. Statistically significant changes relative to undifferentiated cells are denoted by asterisks (*P < 0.05; **P < 0.01; ***P < 0.001). (A) Representative images of the expression pattern of Cx30, Cx36 and Cx43 subtypes, and beta-actin, during the timecourse of NT2/D1 (A1) and P19 EC (A2) neuronal differentiation. A similar pattern of expression for the three Cx subtypes was seen in both cell lines. (B) Changes in Cx43 mRNA expression. In the NT2/D1 cell line, Cx43 decreased between day 0 and day 14 (P < 0.05) and remained downregulated at days 21 (P < 0.01) and 28 of RA treatment (P < 0.05) (B1). Cx43 significantly decreased in the P19 EC cell line by day 4 post-RA treatment and remained downregulated throughout the remainder of the timecourse (P < 0.001) (B2). (C) Changes in Cx36 mRNA expression. A significant increase in Cx36 expression was observed at 14 and 28 days RA treatment of NT2/D1 cells (P < 0.05) (C1), while in the P19 EC model a significant increase in Cx36 expression was observed at 6 (P < 0.001) and 8 days post-RA (P < 0.05) (C2). (D) Changes in Cx30 mRNA expression. In the NT2/D1 model, a threefold increase in expression of Cx30 was observed at the 14 days RA (P < 0.001), and the subtype was also elevated at days 21 (P < 0.05) and 28 (P < 0.01) (D1). A greater than ninefold increase in expression of Cx30 was observed at 6 days post-RA (P < 0.001) relative to undifferentiated P19 EC cells (D2).
Mentions: The temporal expression patterns of Cx30, Cx36 and Cx43 mRNA were examined using RT-PCR, and semi-quantitative analysis was performed relative to the expression of the constitutive housekeeping gene, beta-actin, to provide a mean integrated density in arbitrary units (AU). The changes in Cx subtype expression levels were assessed relative to undifferentiated cells and the changes in expression level denoted in fold-change relative to undifferentiated cells (Figure 1A1 for NT2/D1 cells; Figure 1A2 for P19 EC cells).

Bottom Line: Cx43 was observed intracellularly and on cell surfaces, while Cx30 was seen as puncta.In P19 cells Cx30 was localized on clusters of cells surrounded by MAP2- and doublecortin-positive processes.The expression pattern of Cx30 indicates a role in neuronal differentiation; the nature of that role warrants future investigation.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Medical and Health Sciences, Department of Anatomy with Radiology and Centre for Brain Research, The University of Auckland Auckland, 92019, New Zealand.

ABSTRACT

While connexins (Cxs) are thought to be involved in differentiation, their expression and role has yet to be fully elucidated. We investigated the temporal expression of Cx30, Cx36 and Cx43 in two in vitro models of neuronal differentiation: human NT2/D1 and murine P19 cells, and the spatial localisation of Cx30 and Cx43 in these models. A temporal Cx43 downregulation was confirmed in both cell lines during RA-induced neuronal differentiation using RT-PCR (P < 0.05) preceding an increase in neuronal doublecortin protein. RT-PCR showed Cx36 was upregulated twofold in NT2/D1 cells (P < 0.05) and sixfold in P19 cells (P < 0.001) during neuronal differentiation. Cx30 exhibited a transient peak in expression midway through the timecourse of differentiation increasing threefold in NT2/D1 cells (P < 0.001) and eightfold in P19 cells (P < 0.01). Qualitative immunocytochemistry was used to examine spatiotemporal patterns of Cx protein distribution alongside neuronal differentiation markers. The temporal immunolabelling pattern was similar to that seen using RT-PCR. Cx43 was observed intracellularly and on cell surfaces, while Cx30 was seen as puncta. Spatially Cx43 was seen on doublecortin-negative cells, which may indicate Cx43 downregulation is requisite for differentiation in these models. Conversely, Cx30 puncta were observed on doublecortin-positive and -negative cells in NT2/D1 cells and examination of the Cx30 peak showed puncta also localized to nestin-positive cells, with few puncta on MAP2-positive cells. In P19 cells Cx30 was localized on clusters of cells surrounded by MAP2- and doublecortin-positive processes. The expression pattern of Cx30 indicates a role in neuronal differentiation; the nature of that role warrants future investigation.

No MeSH data available.