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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.


Cell type localisation of Cx30 at the peak timepoint of expression during differentiation of NT2/D1 and P19 EC cells. Scale bar = 20 µm. (A) Representative images of Cx30 (green), Hoescht (blue) and cell marker (red) double-labelling in the NT2/D1 cell line at day 14 of RA treatment. Nestin-positive cells formed a dense underlying layer. Interspersed amongst this layer of cells were DCX-positive differentiating neurons. Cx30 was predominantly seen as punctate labelling forming a cobblestone pattern around nestin- and DCX-positive cells in this underlying layer. A more superficial layer of MAP2-positive postmitotic neurons was also observed, with few sparse Cx30 puncta. (B) Representative images of Cx30 (green), Hoescht (blue) and cell-type marker (red) double-labelling in P19 EC cell line at 6 days post-RA treatment. Unlike the NT2/D1 cells, strong nestin immunolabelling was not observed, however, DCX-positive and MAP2-positive neurons were present. Cx30 puncta appeared to predominantly be expressed on clusters of underlying cells surrounded by DCX- and MAP2-positive cell bodies and neuritic extensions.
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fig03: Cell type localisation of Cx30 at the peak timepoint of expression during differentiation of NT2/D1 and P19 EC cells. Scale bar = 20 µm. (A) Representative images of Cx30 (green), Hoescht (blue) and cell marker (red) double-labelling in the NT2/D1 cell line at day 14 of RA treatment. Nestin-positive cells formed a dense underlying layer. Interspersed amongst this layer of cells were DCX-positive differentiating neurons. Cx30 was predominantly seen as punctate labelling forming a cobblestone pattern around nestin- and DCX-positive cells in this underlying layer. A more superficial layer of MAP2-positive postmitotic neurons was also observed, with few sparse Cx30 puncta. (B) Representative images of Cx30 (green), Hoescht (blue) and cell-type marker (red) double-labelling in P19 EC cell line at 6 days post-RA treatment. Unlike the NT2/D1 cells, strong nestin immunolabelling was not observed, however, DCX-positive and MAP2-positive neurons were present. Cx30 puncta appeared to predominantly be expressed on clusters of underlying cells surrounded by DCX- and MAP2-positive cell bodies and neuritic extensions.

Mentions: In the NT2/D1 model, nestin-positive cells were observed in abundance at day 14 forming a dense underlying layer of cells. Punctate Cx30 staining was localised in a cobblestone pattern at the borders of these nestin-positive cells (Figure 3A). Conversely, MAP2-positive cells were found in clusters of cells in a more superficial layer. DCX-positive cells were observed both superficially and interspersed with the underlying layer of cells. Cx30 puncta were observed predominantly on DCX-positive cells within the underlying layer. Sparse puncta were also observed in the superficial MAP2-positive cell layer.


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)

Cell type localisation of Cx30 at the peak timepoint of expression during differentiation of NT2/D1 and P19 EC cells. Scale bar = 20 µm. (A) Representative images of Cx30 (green), Hoescht (blue) and cell marker (red) double-labelling in the NT2/D1 cell line at day 14 of RA treatment. Nestin-positive cells formed a dense underlying layer. Interspersed amongst this layer of cells were DCX-positive differentiating neurons. Cx30 was predominantly seen as punctate labelling forming a cobblestone pattern around nestin- and DCX-positive cells in this underlying layer. A more superficial layer of MAP2-positive postmitotic neurons was also observed, with few sparse Cx30 puncta. (B) Representative images of Cx30 (green), Hoescht (blue) and cell-type marker (red) double-labelling in P19 EC cell line at 6 days post-RA treatment. Unlike the NT2/D1 cells, strong nestin immunolabelling was not observed, however, DCX-positive and MAP2-positive neurons were present. Cx30 puncta appeared to predominantly be expressed on clusters of underlying cells surrounded by DCX- and MAP2-positive cell bodies and neuritic extensions.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4255794&req=5

fig03: Cell type localisation of Cx30 at the peak timepoint of expression during differentiation of NT2/D1 and P19 EC cells. Scale bar = 20 µm. (A) Representative images of Cx30 (green), Hoescht (blue) and cell marker (red) double-labelling in the NT2/D1 cell line at day 14 of RA treatment. Nestin-positive cells formed a dense underlying layer. Interspersed amongst this layer of cells were DCX-positive differentiating neurons. Cx30 was predominantly seen as punctate labelling forming a cobblestone pattern around nestin- and DCX-positive cells in this underlying layer. A more superficial layer of MAP2-positive postmitotic neurons was also observed, with few sparse Cx30 puncta. (B) Representative images of Cx30 (green), Hoescht (blue) and cell-type marker (red) double-labelling in P19 EC cell line at 6 days post-RA treatment. Unlike the NT2/D1 cells, strong nestin immunolabelling was not observed, however, DCX-positive and MAP2-positive neurons were present. Cx30 puncta appeared to predominantly be expressed on clusters of underlying cells surrounded by DCX- and MAP2-positive cell bodies and neuritic extensions.
Mentions: In the NT2/D1 model, nestin-positive cells were observed in abundance at day 14 forming a dense underlying layer of cells. Punctate Cx30 staining was localised in a cobblestone pattern at the borders of these nestin-positive cells (Figure 3A). Conversely, MAP2-positive cells were found in clusters of cells in a more superficial layer. DCX-positive cells were observed both superficially and interspersed with the underlying layer of cells. Cx30 puncta were observed predominantly on DCX-positive cells within the underlying layer. Sparse puncta were also observed in the superficial MAP2-positive cell layer.

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.