Journal: The Journal of Biological Chemistry

Article Title: The Carboxyl-terminal Domain of Connexin43 Is a Negative Modulator of Neuronal Differentiation *

doi: 10.1074/jbc.M109.058750

Figure Lengend Snippet: Altered sublayer thickness of embryonic forebrains from Cx43-null mice. A and B, epifluorescence images of embryonic (day 16) forebrain cryosections (14 μm) showing the distribution of nuclei of progenitors stained with DAPI in three cortical layers (CP, cortical plate; IZ, intermediate zone; VZ/SVZ, ventricular zone/subventricular zone) of wild-type (A) and Cx43-null (B) mice. Scale bar, 16 μm. C, quantitative analyses of layer thickness (fraction of total), based on DAPI staining, shown as bar histograms of mean ± S.E. (error bars) values that were obtained from 58–43 hemicoronal sections of 3 WT and 3 Cx43-null animals, respectively. The fractional thickness of VZ/SVZ of Cx43-null brains was larger whereas the CP was smaller than those of WT littermates. D, mean ± S.E. (error bars) fractional thickness values obtained for the three cortical layers measured from each of the 3 wild-type (white bars) and Cx43-null (black bars) animals individually. Images were acquired using a cooled-CCD HQ2 camera (Photometrics) attached to an epifluorescence inverted microscope (TE2000-E; Nikon) equipped with a 10× dry objective and UV filter set. Three different litters were used.

Article Snippet: Images were acquired using a cooled-CCD HQ2 camera (Photometrics) attached to an epifluorescence inverted microscope (TE2000-E; Nikon) equipped with a 10× dry objective and UV filter set.

Techniques: Staining, Inverted Microscopy

Journal: The Journal of Biological Chemistry

Article Title: The Carboxyl-terminal Domain of Connexin43 Is a Negative Modulator of Neuronal Differentiation *

doi: 10.1074/jbc.M109.058750

Figure Lengend Snippet: More substantial neuronal differentiation from Cx43-null-derived neurospheres than WT. A and B, examples of epifluorescence images of WT (A) and Cx43-null (B) neurospheres showing the expression of the neuronal marker β-III-tubulin in 3-day adherent progenitors. Note the extensive neurite projections from Cx43-null neurospheres and their complete absence in that of a WT littermate. Insets in B illustrate a β-III-tubulin-positive cell with three neurites (arrowhead) emanating from the cell body. Inset on the right was magnified twice that of the left. Scale bar, 50 μm. C and D, bar histograms of the mean ± S.E. (error bars) values of percent neurospheres with differentiated neurons (β-III-tubulin-positive cells with neurite projections) (C) and of percent neurons/ neurosphere (D) obtained from WT (white bars) and Cx43-null (black bars) mice. E–G, bar histograms of the mean ± S.E. (error bars) values of neurite length (E) and of the number of primary (F) and secondary (G) neurites measured from β-III-tubulin-positive progenitor cells derived from WT (white bars) and Cx43-null (black bars) neurospheres. Morphometric analyses of neurites from β-III-tubulin-positive cells were performed on reconstructed images of confocal z-sections (supplemental Fig. 2S) using NeuronJ software.

Article Snippet: Images were acquired using a cooled-CCD HQ2 camera (Photometrics) attached to an epifluorescence inverted microscope (TE2000-E; Nikon) equipped with a 10× dry objective and UV filter set.

Techniques: Derivative Assay, Expressing, Marker, Software

Journal: The Journal of Biological Chemistry

Article Title: The Carboxyl-terminal Domain of Connexin43 Is a Negative Modulator of Neuronal Differentiation *

doi: 10.1074/jbc.M109.058750

Figure Lengend Snippet: Neuronal differentiation is independent of gap junction-mediated coupling. A–C, epifluorescence images showing β-III-tubulin-positive cells in mock transfected (A), CBX-treated (B), and Cx43 siRNA-transfected WT neurospheres (C). Note the presence of extensive neuronal neurite projections after knockdown of Cx43 with siRNA from WT neurospheres compared with CBX- and mock-treated neurospheres. Scale bar, 50 μm. D, bar histograms showing the mean ± S.E. (error bars) values obtained for the percent WT neurospheres with differentiated neurons (β-III-tubulin-positive cells with neurites) after 48-h treatment with transfection reagents (Mock), gap junction channel blocker CBX (100 μm), and after transfection with Cx43 siRNA. Knockdown of Cx43 with siRNA significantly increased the number of neurospheres with differentiated neurons to levels similar to those found in Cx43-null neurospheres; neither mock transfection nor CBX treatment altered the number of WT neurospheres with differentiated neurons. For knockdown efficiency, see supplemental Figs. 4S and 5S.

Article Snippet: Images were acquired using a cooled-CCD HQ2 camera (Photometrics) attached to an epifluorescence inverted microscope (TE2000-E; Nikon) equipped with a 10× dry objective and UV filter set.

Techniques: Transfection, Knockdown

Journal: The Journal of Biological Chemistry

Article Title: The Carboxyl-terminal Domain of Connexin43 Is a Negative Modulator of Neuronal Differentiation *

doi: 10.1074/jbc.M109.058750

Figure Lengend Snippet: Cx43 carboxyl terminus modulates neuronal differentiation. A–D, examples of epifluorescence images showing β-III-tubulin-positive cells in Cx43-null neurospheres mock transfected (A), transfected with full-length Cx43 (B), transfected with Cx43 truncated at position 257 (Cx43-M257) (C), and transfected with the Cx43 carboxyl terminus itself (Cx43-CT) (D). Scale bar, 50 μm. E, bar histograms showing the mean ± S.E. (error bars) values obtained for the percent Cx43-null neurospheres with differentiated neurons (β-III-tubulin-positive cells with neurite projections) following 48 h treatment with transfection reagents alone (Mock) and after transfection with full-length Cx43, Cx43 lacking the carboxyl terminus (M257), and with Cx43 carboxyl terminus itself (CT). A significant decrease in the percent neurospheres with differentiated neurons was obtained after expression of full-length Cx43 and the CT itself, but not with Cx43 constructs lacking the CT. These two effective constructs significantly reduced the number of neurospheres displaying neuronal neurite projections to levels seen in WT neurospheres. Transfection efficiency and cellular distribution of Cx43 and its mutants are shown in supplemental Figs. 6S and 7S.

Article Snippet: Images were acquired using a cooled-CCD HQ2 camera (Photometrics) attached to an epifluorescence inverted microscope (TE2000-E; Nikon) equipped with a 10× dry objective and UV filter set.

Techniques: Transfection, Expressing, Construct