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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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Activation of ERK5 potentiates while inhibition of ERK5 attenuates Neurog1-stimulated neurogenesis.For panels A–D, neurosphere assays. Freshly dissociated E13 cortical progenitors were co-infected with lentiviruses encoding Neurog1, constitutive active (ca) or dominant negative (dn) MEK5, or wild-type ERK5 as indicated. Cells infected with GFP-virus were used as a control. Neurospheres were allowed to form in culture for 5 d, and then transferred to PDL/laminin coated plates in bFGF-free medium to promote spontaneous differentiation for 3 d. Neurospheres infected with lentiviruses were identified by GFP expression. Neurons were identified by the pan-neuronal marker, β-III tubulin. A, Representative images of neurospheres infected with either GFP control virus (control) or wild-type Neurog1, and immunostained for β-III tubulin (red) and GFP (green). B, Effect of Neurog1 and ERK5 on the percentage of non-neuron spheres, defined as those neurospheres containing ≤10% neurons per sphere. C, Activation of ERK5 signaling potentiates the neurogenic effect of Neurog1. Data show distribution of the percentage of neurons per neurosphere. Data were collected from three independent experiments (n = 3). D, Inhibition of ERK5 signaling by dnMEK5 abolishes the neurogenic effect of Neurog1. E, Representative images of a progenitor cell clone in an adherent culture clonal assay, which allows us to specifically follow the cell fate of a single LeX+ cortical progenitor cell (Liu et al., 2006). Progenitor cells infected with lentiviruses were identified by GFP expression. Cells were immunostained for GFP (green) and β-III tubulin (red). F, Expression of dnMEK5 or dnERK5 suppresses the pro-neural effect of Neurog1 using the adherent culture clonal assay.
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pone-0005204-g001: Activation of ERK5 potentiates while inhibition of ERK5 attenuates Neurog1-stimulated neurogenesis.For panels A–D, neurosphere assays. Freshly dissociated E13 cortical progenitors were co-infected with lentiviruses encoding Neurog1, constitutive active (ca) or dominant negative (dn) MEK5, or wild-type ERK5 as indicated. Cells infected with GFP-virus were used as a control. Neurospheres were allowed to form in culture for 5 d, and then transferred to PDL/laminin coated plates in bFGF-free medium to promote spontaneous differentiation for 3 d. Neurospheres infected with lentiviruses were identified by GFP expression. Neurons were identified by the pan-neuronal marker, β-III tubulin. A, Representative images of neurospheres infected with either GFP control virus (control) or wild-type Neurog1, and immunostained for β-III tubulin (red) and GFP (green). B, Effect of Neurog1 and ERK5 on the percentage of non-neuron spheres, defined as those neurospheres containing ≤10% neurons per sphere. C, Activation of ERK5 signaling potentiates the neurogenic effect of Neurog1. Data show distribution of the percentage of neurons per neurosphere. Data were collected from three independent experiments (n = 3). D, Inhibition of ERK5 signaling by dnMEK5 abolishes the neurogenic effect of Neurog1. E, Representative images of a progenitor cell clone in an adherent culture clonal assay, which allows us to specifically follow the cell fate of a single LeX+ cortical progenitor cell (Liu et al., 2006). Progenitor cells infected with lentiviruses were identified by GFP expression. Cells were immunostained for GFP (green) and β-III tubulin (red). F, Expression of dnMEK5 or dnERK5 suppresses the pro-neural effect of Neurog1 using the adherent culture clonal assay.

Mentions: Our previous studies established that ERK5 is necessary and sufficient to promote neuron fate specification of cortical progenitors [18]. Because ERK5 is a MAP kinase that can phosphorylate and regulate the activity of several transcription factors, and Neurog1 can direct cortical progenitors to commit to a neuronal fate, we postulated that the pro-neural activity of ERK5 may be due to ERK5 regulation of Neurog1. To test this hypothesis, we performed a neurosphere assay to determine if ERK5 regulates Neurog1-stimulated neurogenesis in vitro (Fig. 1). Freshly dissociated embryonic day (E) 13 rat cortical progenitor cells were infected with lentiviral stocks encoding Neurog1, wild-type (wt) ERK5, constitutive active (ca) or dominant negative (dn) MEK5, an upstream kinase of ERK5. These genes were coupled to GFP through an internal ribosomal entry site (IRES) so that virus-infected cells can be easily identified by GFP expression [18]. Lentiviral infection was carried out 3 h after initial plating when the cells were still at the single-cell level in suspension. Neurons were identified by immunostaining of β-III tubulin, a marker expressed in immature neurons (Fig. 1 A). Those neurospheres with less than 10 β-III tubulin+ cells were defined as non-neuron spheres. Quantification of the data demonstrated that ectopic expression of Neurog1 significantly reduced the total number of non-neuron spheres compared to control GFP-infected spheres (Neurog1 15%; GFP 73%) (Fig. 1 B). This is consistent with other reports that ectopic expression of Neurog1 is sufficient to induce neurogenesis [6], [7]. Similar results were obtained with ectopic ERK5 activation (caMEK5+wtERK5), consistent with our previous report [18]. Co-expression of Neurog1 with caMEK5+wtERK5 generated no non-neuron spheres. Furthermore, the neurogenic effect of Neurog1 was reversed by co-expression of dnMEK5 which blocks ERK5 activation.


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)

Activation of ERK5 potentiates while inhibition of ERK5 attenuates Neurog1-stimulated neurogenesis.For panels A–D, neurosphere assays. Freshly dissociated E13 cortical progenitors were co-infected with lentiviruses encoding Neurog1, constitutive active (ca) or dominant negative (dn) MEK5, or wild-type ERK5 as indicated. Cells infected with GFP-virus were used as a control. Neurospheres were allowed to form in culture for 5 d, and then transferred to PDL/laminin coated plates in bFGF-free medium to promote spontaneous differentiation for 3 d. Neurospheres infected with lentiviruses were identified by GFP expression. Neurons were identified by the pan-neuronal marker, β-III tubulin. A, Representative images of neurospheres infected with either GFP control virus (control) or wild-type Neurog1, and immunostained for β-III tubulin (red) and GFP (green). B, Effect of Neurog1 and ERK5 on the percentage of non-neuron spheres, defined as those neurospheres containing ≤10% neurons per sphere. C, Activation of ERK5 signaling potentiates the neurogenic effect of Neurog1. Data show distribution of the percentage of neurons per neurosphere. Data were collected from three independent experiments (n = 3). D, Inhibition of ERK5 signaling by dnMEK5 abolishes the neurogenic effect of Neurog1. E, Representative images of a progenitor cell clone in an adherent culture clonal assay, which allows us to specifically follow the cell fate of a single LeX+ cortical progenitor cell (Liu et al., 2006). Progenitor cells infected with lentiviruses were identified by GFP expression. Cells were immunostained for GFP (green) and β-III tubulin (red). F, Expression of dnMEK5 or dnERK5 suppresses the pro-neural effect of Neurog1 using the adherent culture clonal assay.
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Related In: Results  -  Collection

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pone-0005204-g001: Activation of ERK5 potentiates while inhibition of ERK5 attenuates Neurog1-stimulated neurogenesis.For panels A–D, neurosphere assays. Freshly dissociated E13 cortical progenitors were co-infected with lentiviruses encoding Neurog1, constitutive active (ca) or dominant negative (dn) MEK5, or wild-type ERK5 as indicated. Cells infected with GFP-virus were used as a control. Neurospheres were allowed to form in culture for 5 d, and then transferred to PDL/laminin coated plates in bFGF-free medium to promote spontaneous differentiation for 3 d. Neurospheres infected with lentiviruses were identified by GFP expression. Neurons were identified by the pan-neuronal marker, β-III tubulin. A, Representative images of neurospheres infected with either GFP control virus (control) or wild-type Neurog1, and immunostained for β-III tubulin (red) and GFP (green). B, Effect of Neurog1 and ERK5 on the percentage of non-neuron spheres, defined as those neurospheres containing ≤10% neurons per sphere. C, Activation of ERK5 signaling potentiates the neurogenic effect of Neurog1. Data show distribution of the percentage of neurons per neurosphere. Data were collected from three independent experiments (n = 3). D, Inhibition of ERK5 signaling by dnMEK5 abolishes the neurogenic effect of Neurog1. E, Representative images of a progenitor cell clone in an adherent culture clonal assay, which allows us to specifically follow the cell fate of a single LeX+ cortical progenitor cell (Liu et al., 2006). Progenitor cells infected with lentiviruses were identified by GFP expression. Cells were immunostained for GFP (green) and β-III tubulin (red). F, Expression of dnMEK5 or dnERK5 suppresses the pro-neural effect of Neurog1 using the adherent culture clonal assay.
Mentions: Our previous studies established that ERK5 is necessary and sufficient to promote neuron fate specification of cortical progenitors [18]. Because ERK5 is a MAP kinase that can phosphorylate and regulate the activity of several transcription factors, and Neurog1 can direct cortical progenitors to commit to a neuronal fate, we postulated that the pro-neural activity of ERK5 may be due to ERK5 regulation of Neurog1. To test this hypothesis, we performed a neurosphere assay to determine if ERK5 regulates Neurog1-stimulated neurogenesis in vitro (Fig. 1). Freshly dissociated embryonic day (E) 13 rat cortical progenitor cells were infected with lentiviral stocks encoding Neurog1, wild-type (wt) ERK5, constitutive active (ca) or dominant negative (dn) MEK5, an upstream kinase of ERK5. These genes were coupled to GFP through an internal ribosomal entry site (IRES) so that virus-infected cells can be easily identified by GFP expression [18]. Lentiviral infection was carried out 3 h after initial plating when the cells were still at the single-cell level in suspension. Neurons were identified by immunostaining of β-III tubulin, a marker expressed in immature neurons (Fig. 1 A). Those neurospheres with less than 10 β-III tubulin+ cells were defined as non-neuron spheres. Quantification of the data demonstrated that ectopic expression of Neurog1 significantly reduced the total number of non-neuron spheres compared to control GFP-infected spheres (Neurog1 15%; GFP 73%) (Fig. 1 B). This is consistent with other reports that ectopic expression of Neurog1 is sufficient to induce neurogenesis [6], [7]. Similar results were obtained with ectopic ERK5 activation (caMEK5+wtERK5), consistent with our previous report [18]. Co-expression of Neurog1 with caMEK5+wtERK5 generated no non-neuron spheres. Furthermore, the neurogenic effect of Neurog1 was reversed by co-expression of dnMEK5 which blocks ERK5 activation.

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