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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.


Spatial expression pattern of Cx43 and Cx30 protein with DCX as a marker of differentiating neurons in the NT2/D1 and P19 EC cell models. Scale bar = 20 ¡m. (A) Representative images of ICC labelling of Cx43 (green) and DCX (red) in undifferentiated NT2/D1 cells, cells at day 14 of RA treatment and mature hNT neuronal cultures. Intracellular and punctate labelling of Cx43 was observed in undifferentiated cells, however, immunolabelling for Cx43 was much less prevalent at day 14 (arrows) and appeared to largely be on cells without strong DCX staining rather than DCX-positive cells (arrowheads). In mature hNT neurons, Cx43 was not observed, while strong DCX was present in both cell bodies and neurites. (B) Representative images of ICC labelling of Cx30 (green) and DCX (red) in undifferentiated NT2/D1 cells, cells at day 14 of RA treatment and mature hNT neuronal cultures. Cx30 was not observed in undifferentiated NT2/D1 cells, however puncta became widespread peaking at day 14 of RA treatment, before decreasing again. Cx30 was not observed in cultures of hNT neurons. Cx30 puncta (arrows) were visible on DCX-positive cells (arrowheads) and on cells absent of DCX staining at day 14, with no obvious preferential localisation. (C) Representative images of ICC labelling of Cx43 (green) and DCX (red) in undifferentiated P19 EC cells, cells at day 4 post-RA treatment and day 10 post-RA treatment. Similar to NT2/D1 cells, Cx43 was observed as both intracellular and punctate labelling. Cx43 was initially ubiquitously expressed, however, both punctate and intracellular labelling decreased after RA treatment by day 4 post-RA treatment and remained low at day 10 post-RA treatment. (D) Representative images of ICC labelling of Cx30 (green) and DCX (red) in undifferentiated P19 EC cells, cells at day 6 post-RA treatment and day 10 post-RA treatment. Cx30 was not observed in undifferentiated P19 cells. At the 6-day timepoint post-RA treatment, Cx30 puncta were observed (arrows) at the centre of large clusters of cells with DCX-positive neurites (arrowheads) extending from them at their periphery. This peak in expression was transient as Cx30 decreased between day 6 and day 10 post-RA treatment.
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fig02: Spatial expression pattern of Cx43 and Cx30 protein with DCX as a marker of differentiating neurons in the NT2/D1 and P19 EC cell models. Scale bar = 20 ¡m. (A) Representative images of ICC labelling of Cx43 (green) and DCX (red) in undifferentiated NT2/D1 cells, cells at day 14 of RA treatment and mature hNT neuronal cultures. Intracellular and punctate labelling of Cx43 was observed in undifferentiated cells, however, immunolabelling for Cx43 was much less prevalent at day 14 (arrows) and appeared to largely be on cells without strong DCX staining rather than DCX-positive cells (arrowheads). In mature hNT neurons, Cx43 was not observed, while strong DCX was present in both cell bodies and neurites. (B) Representative images of ICC labelling of Cx30 (green) and DCX (red) in undifferentiated NT2/D1 cells, cells at day 14 of RA treatment and mature hNT neuronal cultures. Cx30 was not observed in undifferentiated NT2/D1 cells, however puncta became widespread peaking at day 14 of RA treatment, before decreasing again. Cx30 was not observed in cultures of hNT neurons. Cx30 puncta (arrows) were visible on DCX-positive cells (arrowheads) and on cells absent of DCX staining at day 14, with no obvious preferential localisation. (C) Representative images of ICC labelling of Cx43 (green) and DCX (red) in undifferentiated P19 EC cells, cells at day 4 post-RA treatment and day 10 post-RA treatment. Similar to NT2/D1 cells, Cx43 was observed as both intracellular and punctate labelling. Cx43 was initially ubiquitously expressed, however, both punctate and intracellular labelling decreased after RA treatment by day 4 post-RA treatment and remained low at day 10 post-RA treatment. (D) Representative images of ICC labelling of Cx30 (green) and DCX (red) in undifferentiated P19 EC cells, cells at day 6 post-RA treatment and day 10 post-RA treatment. Cx30 was not observed in undifferentiated P19 cells. At the 6-day timepoint post-RA treatment, Cx30 puncta were observed (arrows) at the centre of large clusters of cells with DCX-positive neurites (arrowheads) extending from them at their periphery. This peak in expression was transient as Cx30 decreased between day 6 and day 10 post-RA treatment.

Mentions: ICC was used to examine the spatiotemporal pattern of Cx43 and Cx30 protein expression in NT2/D1 cells during the timecourse of neuronal differentiation (Figures 2A and 2B). Cx43 immunolabelling was observed as punctate staining between cells along apposed cellular membranes indicative of gap junction plaques, but globular labelling was also observed within the cytoplasm of cell bodies (Figure 2A). Intracellular labelling was observed predominantly in cultures of undifferentiated cells, prior to any treatment with RA, and at earlier timepoints during the timecourse, where localisation of labelling was largely perinuclear.


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)

Spatial expression pattern of Cx43 and Cx30 protein with DCX as a marker of differentiating neurons in the NT2/D1 and P19 EC cell models. Scale bar = 20 ¡m. (A) Representative images of ICC labelling of Cx43 (green) and DCX (red) in undifferentiated NT2/D1 cells, cells at day 14 of RA treatment and mature hNT neuronal cultures. Intracellular and punctate labelling of Cx43 was observed in undifferentiated cells, however, immunolabelling for Cx43 was much less prevalent at day 14 (arrows) and appeared to largely be on cells without strong DCX staining rather than DCX-positive cells (arrowheads). In mature hNT neurons, Cx43 was not observed, while strong DCX was present in both cell bodies and neurites. (B) Representative images of ICC labelling of Cx30 (green) and DCX (red) in undifferentiated NT2/D1 cells, cells at day 14 of RA treatment and mature hNT neuronal cultures. Cx30 was not observed in undifferentiated NT2/D1 cells, however puncta became widespread peaking at day 14 of RA treatment, before decreasing again. Cx30 was not observed in cultures of hNT neurons. Cx30 puncta (arrows) were visible on DCX-positive cells (arrowheads) and on cells absent of DCX staining at day 14, with no obvious preferential localisation. (C) Representative images of ICC labelling of Cx43 (green) and DCX (red) in undifferentiated P19 EC cells, cells at day 4 post-RA treatment and day 10 post-RA treatment. Similar to NT2/D1 cells, Cx43 was observed as both intracellular and punctate labelling. Cx43 was initially ubiquitously expressed, however, both punctate and intracellular labelling decreased after RA treatment by day 4 post-RA treatment and remained low at day 10 post-RA treatment. (D) Representative images of ICC labelling of Cx30 (green) and DCX (red) in undifferentiated P19 EC cells, cells at day 6 post-RA treatment and day 10 post-RA treatment. Cx30 was not observed in undifferentiated P19 cells. At the 6-day timepoint post-RA treatment, Cx30 puncta were observed (arrows) at the centre of large clusters of cells with DCX-positive neurites (arrowheads) extending from them at their periphery. This peak in expression was transient as Cx30 decreased between day 6 and day 10 post-RA treatment.
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Related In: Results  -  Collection

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fig02: Spatial expression pattern of Cx43 and Cx30 protein with DCX as a marker of differentiating neurons in the NT2/D1 and P19 EC cell models. Scale bar = 20 ¡m. (A) Representative images of ICC labelling of Cx43 (green) and DCX (red) in undifferentiated NT2/D1 cells, cells at day 14 of RA treatment and mature hNT neuronal cultures. Intracellular and punctate labelling of Cx43 was observed in undifferentiated cells, however, immunolabelling for Cx43 was much less prevalent at day 14 (arrows) and appeared to largely be on cells without strong DCX staining rather than DCX-positive cells (arrowheads). In mature hNT neurons, Cx43 was not observed, while strong DCX was present in both cell bodies and neurites. (B) Representative images of ICC labelling of Cx30 (green) and DCX (red) in undifferentiated NT2/D1 cells, cells at day 14 of RA treatment and mature hNT neuronal cultures. Cx30 was not observed in undifferentiated NT2/D1 cells, however puncta became widespread peaking at day 14 of RA treatment, before decreasing again. Cx30 was not observed in cultures of hNT neurons. Cx30 puncta (arrows) were visible on DCX-positive cells (arrowheads) and on cells absent of DCX staining at day 14, with no obvious preferential localisation. (C) Representative images of ICC labelling of Cx43 (green) and DCX (red) in undifferentiated P19 EC cells, cells at day 4 post-RA treatment and day 10 post-RA treatment. Similar to NT2/D1 cells, Cx43 was observed as both intracellular and punctate labelling. Cx43 was initially ubiquitously expressed, however, both punctate and intracellular labelling decreased after RA treatment by day 4 post-RA treatment and remained low at day 10 post-RA treatment. (D) Representative images of ICC labelling of Cx30 (green) and DCX (red) in undifferentiated P19 EC cells, cells at day 6 post-RA treatment and day 10 post-RA treatment. Cx30 was not observed in undifferentiated P19 cells. At the 6-day timepoint post-RA treatment, Cx30 puncta were observed (arrows) at the centre of large clusters of cells with DCX-positive neurites (arrowheads) extending from them at their periphery. This peak in expression was transient as Cx30 decreased between day 6 and day 10 post-RA treatment.
Mentions: ICC was used to examine the spatiotemporal pattern of Cx43 and Cx30 protein expression in NT2/D1 cells during the timecourse of neuronal differentiation (Figures 2A and 2B). Cx43 immunolabelling was observed as punctate staining between cells along apposed cellular membranes indicative of gap junction plaques, but globular labelling was also observed within the cytoplasm of cell bodies (Figure 2A). Intracellular labelling was observed predominantly in cultures of undifferentiated cells, prior to any treatment with RA, and at earlier timepoints during the timecourse, where localisation of labelling was largely perinuclear.

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.