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Development
2012 May 01;13910:1718-23. doi: 10.1242/dev.077552.
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Post-translational modification of Ngn2 differentially affects transcription of distinct targets to regulate the balance between progenitor maintenance and differentiation.
???displayArticle.abstract??? Neurogenin 2 (Ngn2) controls neuronal differentiation cell-autonomously by transcriptional activation of targets such as NeuroD, while simultaneously controlling progenitor maintenance non-cell-autonomously by upregulating Delta expression and Notch signalling. Reduction in Cdk-dependent multisite phosphorylation of Ngn2 enhances its promoter binding affinity. This leads specifically to an increase in neuronal differentiation without an apparent increase in progenitor maintenance via Delta-Notch signalling, although the mechanism underlying this imbalance remains unclear. Here we show in Xenopus embryos and mouse P19 cells that the NeuroD promoter is substantially more sensitive to the phosphorylation status of Ngn2 than the Delta promoter, and that this can be attributed to differences in the ease of promoter activation. In addition, we also show that the phosphorylation status of Ngn2 regulates sensitivity to Notch signalling. These observations explain how Ngn2 post-translational modification in response to changes in the cell cycle kinase environment results in enhanced neuronal differentiation upon cell cycle lengthening.
Fig. 3. The NeuroD promoter requires greater Ngn2 availability for activation than the Delta promoter. (A) The half-life of Ngn2 proteins, calculated using first-order rate kinetics (errors calculated using s.e.m.). In vitro translated 35S-methionine-labelled xNgn2, UbxNgn2, 9S-A xNgn2 or Ub9S-AxNgn2 were incubated in Xenopus mitotic egg extracts with unlabelled in vitro translated GFP or E12, and the rate of degradation was determined. (B) Xenopus embryos were injected in one of two cells with 20 pg xNgn2, UbxNgn2, 9S-A xNgn2 or Ub9S-A xNgn2 mRNA and subject to in situ hybridisation at stage 15 for neural β-tubulin expression (n≥76) (injected side, left). (C) Semi-quantitative analysis of in situ hybridisation data (n≥76). (D) qPCR analysis of NeuroD and Delta in Xenopus embryos. Embryos were injected at the one-cell stage with 20 pg xNgn2, UbxNgn2, 9S-A xNgn2 or Ub9S-A xNgn2 mRNA and harvested at stage 15. Average fold increase in mRNA expression compared with the GFP control, normalised to house keeping gene expression (ODC), with mean normalised values calculated together with the s.e.m. *, P≤0.05; **, P≤0.01; ***, P≤0.005.
Fig. 4. 9S-A Ngn2 is less sensitive than Ngn2 to Notch signalling. (A) Xenopus embryos were injected in one of two cells with 20 pg mRNA for GFP, xNgn2 or 9S-A xNgn2 and with 1 ng mRNA for GFP or Notch intracellular domain (NICD) as indicated (injected side, left). Stage 15 embryos were subject to in situ hybridisation for neural β-tubulin expression. (B) Semi-quantitative analysis of in situ hybridisation data (see supplementary material Fig. S2 for scoring schematic); n=23-33. (C) Model to explain why phosphorylated Ngn2 favours progenitor maintenance, whereas un(der)phosphorylated Ngn2 favours neuronal differentiation. See text for description. cdkis, cyclin-dependent kinase inhibitors.
Fig. S2. Examples of the embryo scoring system used in Fig. 3. Semi-quantitative criteria were used to compare neural β-tubulin expression following in situ hybridization on the injected side and the uninjected side, and assigning a score of: 0, no difference; +1, slight increase in neurogenesis within the neural plate on the injected side; +2, significant increase in neurogenesis with ectopic expression of neural β-tubulin on the injected side; +3, very large increase in neurogenesis with ectopic expression of neural β-tubulin on the injected side; −1, slight decrease in endogenous neurogenesis on the injected side; −2, substantial decrease in endogenous neurogenesis on the injected side; or −3, no neurons on the injected side.
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