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Tcf7l2/Tcf4 Transcriptional Repressor Function Requires HDAC Activity in the Developing Vertebrate CNS

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ABSTRACT

The generation of functionally distinct neuronal subtypes within the vertebrate central nervous system (CNS) requires the precise regulation of progenitor gene expression in specific neuronal territories during early embryogenesis. Accumulating evidence has implicated histone deacetylase (HDAC) proteins in cell specification, proliferation, and differentiation in diverse embryonic and adult tissues. However, although HDAC proteins have shown to be expressed in the developing vertebrate neural tube, their specific role in CNS neural progenitor fate specification remains unclear. Prior work from our lab showed that the Tcf7l2/Tcf4 transcription factor plays a key role in ventral progenitor lineage segregation by differential repression of two key specification factors, Nkx2.2 and Olig2. In this study, we found that administration of HDAC inhibitors (Valproic Acid (VPA), Trichostatin-A (TSA), or sodium butyrate) in chick embryos in ovo disrupted normal progenitor gene segregation in the developing neural tube, indicating that HDAC activity is required for this process. Further, using functional and pharmacological approaches in vivo, we found that HDAC activity is required for the differential repression of Nkx2.2 and Olig2 by Tcf7l2/Tcf4. Finally, using dominant-negative functional assays, we provide evidence that Tcf7l2/Tcf4 repression also requires Gro/TLE/Grg co-repressor factors. Together, our data support a model where the transcriptional repressor activity of Tcf7l2/Tcf4 involves functional interactions with both HDAC and Gro/TLE/Grg co-factors at specific target gene regulatory elements in the developing neural tube, and that this activity is required for the proper segregation of the Nkx2.2 (p3) and Olig2 (pMN) expressing cells from a common progenitor pool.

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HDAC activity is required for Tcf repression of Gli2A-induced Nkx2.2 expression in vivo.(A-E) Sections through E3 chick embryos electroporated with Gli2A (at 2.0 μg/μl) and Gli2A+Tcf4R (at 2.0 μg/μl) in the presence of increasing concentrations of VPA. Right side was transfected in all cases. Note that the number of induced Nkx2.2+ cells increases with increasing VPA, indicating that Tcf4R antagonism becomes less effective at higher concentrations. (A’-E’) Olig2 expression in embryos transfected with Gli2A and Gli2A+TcfR in the presence of increasing concentrations of VPA. No effect is seen on Olig2 expression. (F) Quantification of data in A-E’. Inset at bottom right indicates corresponding bar chart shading. *p<0.001.
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pone.0163267.g002: HDAC activity is required for Tcf repression of Gli2A-induced Nkx2.2 expression in vivo.(A-E) Sections through E3 chick embryos electroporated with Gli2A (at 2.0 μg/μl) and Gli2A+Tcf4R (at 2.0 μg/μl) in the presence of increasing concentrations of VPA. Right side was transfected in all cases. Note that the number of induced Nkx2.2+ cells increases with increasing VPA, indicating that Tcf4R antagonism becomes less effective at higher concentrations. (A’-E’) Olig2 expression in embryos transfected with Gli2A and Gli2A+TcfR in the presence of increasing concentrations of VPA. No effect is seen on Olig2 expression. (F) Quantification of data in A-E’. Inset at bottom right indicates corresponding bar chart shading. *p<0.001.

Mentions: We next tested whether the ability of Tcf4 to block Gli2-mediated induction of ectopic Nkx2.2 (Lei et al., 2006) required HDAC activity. For this, we co-expressed an N-terminally truncated Gli2 cDNA (Gli2A) that encodes a constitutive transcriptional activator [10], and an N-terminally truncated Tcf4 cDNA (Tcf4R) encodes a constitutive transcriptional repressor, together in the presence of increasing concentrations of VPA, and assayed the number of induced ectopic Nkx2.2+ cells. As the concentration of VPA increased, so did the number of Nkx2.2+ cells (Fig 2A–2F). Similar results were obtained with Trichostatin-A and sodium butyrate (S1 Fig). Importantly, VPA had no effect on Gli2A induction of Nkx2.2 (S3 Fig), indicating that its affect on patterning cannot be explained by changes in Gli1 acetylation (which would be predicted to have a positive effect on its transcriptional activity based on the results from a prior study [18]). Finally, no effect on Olig2 expression was detected at any VPA concentration (Fig 2A’–2F), consistent with our prior data showing that Tcf4R does not effectively repress Olig2 induction in these assays (Lei et al., 2006), and work from other labs [19]. Together, these data support the idea that Tcf4 repression of ectopic Nkx2.2 involves HDAC in the spinal cord.


Tcf7l2/Tcf4 Transcriptional Repressor Function Requires HDAC Activity in the Developing Vertebrate CNS
HDAC activity is required for Tcf repression of Gli2A-induced Nkx2.2 expression in vivo.(A-E) Sections through E3 chick embryos electroporated with Gli2A (at 2.0 μg/μl) and Gli2A+Tcf4R (at 2.0 μg/μl) in the presence of increasing concentrations of VPA. Right side was transfected in all cases. Note that the number of induced Nkx2.2+ cells increases with increasing VPA, indicating that Tcf4R antagonism becomes less effective at higher concentrations. (A’-E’) Olig2 expression in embryos transfected with Gli2A and Gli2A+TcfR in the presence of increasing concentrations of VPA. No effect is seen on Olig2 expression. (F) Quantification of data in A-E’. Inset at bottom right indicates corresponding bar chart shading. *p<0.001.
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pone.0163267.g002: HDAC activity is required for Tcf repression of Gli2A-induced Nkx2.2 expression in vivo.(A-E) Sections through E3 chick embryos electroporated with Gli2A (at 2.0 μg/μl) and Gli2A+Tcf4R (at 2.0 μg/μl) in the presence of increasing concentrations of VPA. Right side was transfected in all cases. Note that the number of induced Nkx2.2+ cells increases with increasing VPA, indicating that Tcf4R antagonism becomes less effective at higher concentrations. (A’-E’) Olig2 expression in embryos transfected with Gli2A and Gli2A+TcfR in the presence of increasing concentrations of VPA. No effect is seen on Olig2 expression. (F) Quantification of data in A-E’. Inset at bottom right indicates corresponding bar chart shading. *p<0.001.
Mentions: We next tested whether the ability of Tcf4 to block Gli2-mediated induction of ectopic Nkx2.2 (Lei et al., 2006) required HDAC activity. For this, we co-expressed an N-terminally truncated Gli2 cDNA (Gli2A) that encodes a constitutive transcriptional activator [10], and an N-terminally truncated Tcf4 cDNA (Tcf4R) encodes a constitutive transcriptional repressor, together in the presence of increasing concentrations of VPA, and assayed the number of induced ectopic Nkx2.2+ cells. As the concentration of VPA increased, so did the number of Nkx2.2+ cells (Fig 2A–2F). Similar results were obtained with Trichostatin-A and sodium butyrate (S1 Fig). Importantly, VPA had no effect on Gli2A induction of Nkx2.2 (S3 Fig), indicating that its affect on patterning cannot be explained by changes in Gli1 acetylation (which would be predicted to have a positive effect on its transcriptional activity based on the results from a prior study [18]). Finally, no effect on Olig2 expression was detected at any VPA concentration (Fig 2A’–2F), consistent with our prior data showing that Tcf4R does not effectively repress Olig2 induction in these assays (Lei et al., 2006), and work from other labs [19]. Together, these data support the idea that Tcf4 repression of ectopic Nkx2.2 involves HDAC in the spinal cord.

View Article: PubMed Central - PubMed

ABSTRACT

The generation of functionally distinct neuronal subtypes within the vertebrate central nervous system (CNS) requires the precise regulation of progenitor gene expression in specific neuronal territories during early embryogenesis. Accumulating evidence has implicated histone deacetylase (HDAC) proteins in cell specification, proliferation, and differentiation in diverse embryonic and adult tissues. However, although HDAC proteins have shown to be expressed in the developing vertebrate neural tube, their specific role in CNS neural progenitor fate specification remains unclear. Prior work from our lab showed that the Tcf7l2/Tcf4 transcription factor plays a key role in ventral progenitor lineage segregation by differential repression of two key specification factors, Nkx2.2 and Olig2. In this study, we found that administration of HDAC inhibitors (Valproic Acid (VPA), Trichostatin-A (TSA), or sodium butyrate) in chick embryos in ovo disrupted normal progenitor gene segregation in the developing neural tube, indicating that HDAC activity is required for this process. Further, using functional and pharmacological approaches in vivo, we found that HDAC activity is required for the differential repression of Nkx2.2 and Olig2 by Tcf7l2/Tcf4. Finally, using dominant-negative functional assays, we provide evidence that Tcf7l2/Tcf4 repression also requires Gro/TLE/Grg co-repressor factors. Together, our data support a model where the transcriptional repressor activity of Tcf7l2/Tcf4 involves functional interactions with both HDAC and Gro/TLE/Grg co-factors at specific target gene regulatory elements in the developing neural tube, and that this activity is required for the proper segregation of the Nkx2.2 (p3) and Olig2 (pMN) expressing cells from a common progenitor pool.

No MeSH data available.


Related in: MedlinePlus