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ERK5 MAP kinase regulates neurogenin1 during cortical neurogenesis.

Cundiff P, Liu L, Wang Y, Zou J, Pan YW, Abel G, Duan X, Ming GL, Englund C, Hevner R, Xia Z - PLoS ONE (2009)

Bottom Line: We identified S179/S208 as putative ERK5 phosphorylation sites in Neurog1.Mutations of S179/S208 to alanines inhibited the transcriptional and pro-neural activities of Neurog1.Our data identify ERK5 phosphorylation of Neurog1 as a novel mechanism regulating neuronal fate commitment of cortical progenitors.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Washington, Seattle, Washington, United States of America.

ABSTRACT
The commitment of multi-potent cortical progenitors to a neuronal fate depends on the transient induction of the basic-helix-loop-helix (bHLH) family of transcription factors including Neurogenin 1 (Neurog1). Previous studies have focused on mechanisms that control the expression of these proteins while little is known about whether their pro-neural activities can be regulated by kinase signaling pathways. Using primary cultures and ex vivo slice cultures, here we report that both the transcriptional and pro-neural activities of Neurog1 are regulated by extracellular signal-regulated kinase (ERK) 5 signaling in cortical progenitors. Activation of ERK5 potentiated, while blocking ERK5 inhibited Neurog1-induced neurogenesis. Furthermore, endogenous ERK5 activity was required for Neurog1-initiated transcription. Interestingly, ERK5 activation was sufficient to induce Neurog1 phosphorylation and ERK5 directly phosphorylated Neurog1 in vitro. We identified S179/S208 as putative ERK5 phosphorylation sites in Neurog1. Mutations of S179/S208 to alanines inhibited the transcriptional and pro-neural activities of Neurog1. Our data identify ERK5 phosphorylation of Neurog1 as a novel mechanism regulating neuronal fate commitment of cortical progenitors.

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Neurog1-induced neurogenesis is suppressed by mutations at S179 and S208 and inhibition of ERK5 signaling in organotypic slice cultures.Cortical slices were transfected and cultured as in Figure 7. A, B, Representative deconvolution images of cortical slices immunostained for GFP (green) and Tbr1 or NeuN (red), respectively. Images were captured using a 20× objective lens. Scale bar: 50 µm. C,D, Representative high magnification (63×) images of GFP+ cells co-labeled with Tbr1 or NeuN, respectively. E–H Quantification of cells double-immunostained for GFP and Tbr1 (panels E and G) or NeuN (panels F and H) in total GFP+ cells. Vector: vector control. The data were obtained from at least three sections each from three independent experiments.
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pone-0005204-g008: Neurog1-induced neurogenesis is suppressed by mutations at S179 and S208 and inhibition of ERK5 signaling in organotypic slice cultures.Cortical slices were transfected and cultured as in Figure 7. A, B, Representative deconvolution images of cortical slices immunostained for GFP (green) and Tbr1 or NeuN (red), respectively. Images were captured using a 20× objective lens. Scale bar: 50 µm. C,D, Representative high magnification (63×) images of GFP+ cells co-labeled with Tbr1 or NeuN, respectively. E–H Quantification of cells double-immunostained for GFP and Tbr1 (panels E and G) or NeuN (panels F and H) in total GFP+ cells. Vector: vector control. The data were obtained from at least three sections each from three independent experiments.

Mentions: We utilized ex vivo electroporation coupled to organotypic slice culture to examine the effect of SA179/208 mutations on Neurog1's pro-neural activity. The organotypic slice cultures maintain some of the anatomy and cell-cell interactions of the intact cortex [21]. Plasmid DNA encoding vector control, wt Neurog1 and the Neurog1 SA179/208 mutant were injected into the lateral ventricles of dissected E15 rat brains. A GFP plasmid was co-injected as a marker to identify transfected cells. The electrodes were placed in a way to consistently favor plasmid DNA electroporation into the dorsolateral cortex. The cortices were sliced into 300 µm sections and cultured ex vivo. The cellular phenotype of the transfected cells (GFP+) was identified by immunostaining for PCNA (Fig. 7 A), a marker for cells in early G1/S phase, or the T-box brain (Tbr) 2 (Fig. 7 B), a transcription factor and marker for cells actively proliferating in the upper layer of the ventricular zone (VZ) and the sub-ventricular zone (SVZ) [22], [23]. The slices were also stained with Tbr1 (Fig. 8 A) or NeuN (Fig. 8 B), markers for post-mitotic neurons in the cortical plate during development [22], [24], [25] and mature neurons, respectively. Co-labeling of cells immunopositive for GFP and PCNA (Fig. 7 C), Tbr2 (Fig. 7 D), Tbr1 (Fig. 8 C), or NeuN (Fig. 8 D) was confirmed using de-convolution imaging under high magnification.


ERK5 MAP kinase regulates neurogenin1 during cortical neurogenesis.

Cundiff P, Liu L, Wang Y, Zou J, Pan YW, Abel G, Duan X, Ming GL, Englund C, Hevner R, Xia Z - PLoS ONE (2009)

Neurog1-induced neurogenesis is suppressed by mutations at S179 and S208 and inhibition of ERK5 signaling in organotypic slice cultures.Cortical slices were transfected and cultured as in Figure 7. A, B, Representative deconvolution images of cortical slices immunostained for GFP (green) and Tbr1 or NeuN (red), respectively. Images were captured using a 20× objective lens. Scale bar: 50 µm. C,D, Representative high magnification (63×) images of GFP+ cells co-labeled with Tbr1 or NeuN, respectively. E–H Quantification of cells double-immunostained for GFP and Tbr1 (panels E and G) or NeuN (panels F and H) in total GFP+ cells. Vector: vector control. The data were obtained from at least three sections each from three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2664926&req=5

pone-0005204-g008: Neurog1-induced neurogenesis is suppressed by mutations at S179 and S208 and inhibition of ERK5 signaling in organotypic slice cultures.Cortical slices were transfected and cultured as in Figure 7. A, B, Representative deconvolution images of cortical slices immunostained for GFP (green) and Tbr1 or NeuN (red), respectively. Images were captured using a 20× objective lens. Scale bar: 50 µm. C,D, Representative high magnification (63×) images of GFP+ cells co-labeled with Tbr1 or NeuN, respectively. E–H Quantification of cells double-immunostained for GFP and Tbr1 (panels E and G) or NeuN (panels F and H) in total GFP+ cells. Vector: vector control. The data were obtained from at least three sections each from three independent experiments.
Mentions: We utilized ex vivo electroporation coupled to organotypic slice culture to examine the effect of SA179/208 mutations on Neurog1's pro-neural activity. The organotypic slice cultures maintain some of the anatomy and cell-cell interactions of the intact cortex [21]. Plasmid DNA encoding vector control, wt Neurog1 and the Neurog1 SA179/208 mutant were injected into the lateral ventricles of dissected E15 rat brains. A GFP plasmid was co-injected as a marker to identify transfected cells. The electrodes were placed in a way to consistently favor plasmid DNA electroporation into the dorsolateral cortex. The cortices were sliced into 300 µm sections and cultured ex vivo. The cellular phenotype of the transfected cells (GFP+) was identified by immunostaining for PCNA (Fig. 7 A), a marker for cells in early G1/S phase, or the T-box brain (Tbr) 2 (Fig. 7 B), a transcription factor and marker for cells actively proliferating in the upper layer of the ventricular zone (VZ) and the sub-ventricular zone (SVZ) [22], [23]. The slices were also stained with Tbr1 (Fig. 8 A) or NeuN (Fig. 8 B), markers for post-mitotic neurons in the cortical plate during development [22], [24], [25] and mature neurons, respectively. Co-labeling of cells immunopositive for GFP and PCNA (Fig. 7 C), Tbr2 (Fig. 7 D), Tbr1 (Fig. 8 C), or NeuN (Fig. 8 D) was confirmed using de-convolution imaging under high magnification.

Bottom Line: We identified S179/S208 as putative ERK5 phosphorylation sites in Neurog1.Mutations of S179/S208 to alanines inhibited the transcriptional and pro-neural activities of Neurog1.Our data identify ERK5 phosphorylation of Neurog1 as a novel mechanism regulating neuronal fate commitment of cortical progenitors.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Washington, Seattle, Washington, United States of America.

ABSTRACT
The commitment of multi-potent cortical progenitors to a neuronal fate depends on the transient induction of the basic-helix-loop-helix (bHLH) family of transcription factors including Neurogenin 1 (Neurog1). Previous studies have focused on mechanisms that control the expression of these proteins while little is known about whether their pro-neural activities can be regulated by kinase signaling pathways. Using primary cultures and ex vivo slice cultures, here we report that both the transcriptional and pro-neural activities of Neurog1 are regulated by extracellular signal-regulated kinase (ERK) 5 signaling in cortical progenitors. Activation of ERK5 potentiated, while blocking ERK5 inhibited Neurog1-induced neurogenesis. Furthermore, endogenous ERK5 activity was required for Neurog1-initiated transcription. Interestingly, ERK5 activation was sufficient to induce Neurog1 phosphorylation and ERK5 directly phosphorylated Neurog1 in vitro. We identified S179/S208 as putative ERK5 phosphorylation sites in Neurog1. Mutations of S179/S208 to alanines inhibited the transcriptional and pro-neural activities of Neurog1. Our data identify ERK5 phosphorylation of Neurog1 as a novel mechanism regulating neuronal fate commitment of cortical progenitors.

Show MeSH
Related in: MedlinePlus