Hardwick Laura J A, Philpott Anna
Wellcome-MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge, CB2 1QR, UK.
Department of Oncology, University of Cambridge, Cambridge, CB2 0XZ, UK.
Wellcome Open Res. 2018 Oct 1;3:129. doi: 10.12688/wellcomeopenres.14848.1. eCollection 2018.
From the relatively simple nervous system of to the elaborate mammalian cortex, neurogenesis requires exceptional spatial and temporal precision to co-ordinate progenitor cell proliferation and subsequent differentiation to a diverse range of neurons and glia. A limited number of transiently expressed proneural basic-helix-loop-helix (bHLH) transcription factors, for example and in and the vertebrate homologues Ascl1 and Neurogenin2 (Ngn2), are able to orchestrate the onset of neuronal determination, context-dependent subtype selection and even influence later aspects of neuronal migration and maturation. Within the last decade, two models have emerged to explain how the temporal activity of proneural determination factors is regulated by phosphorylation at distinct sites. One model describes how cell-cycle associated phosphorylation on multiple sites in the N and C termini of vertebrate proneural proteins limits neuronal differentiation in cycling progenitor cells. A second model describes phosphorylation on a single site in the bHLH domain of that acts as a binary switch, where phosphorylation terminates proneural activity. Here we combine activating mutations of phosphorylation sites in the N- and C- termini with an inhibitory phospho-mimetic mutation in the bHLH domain of Ascl1 and Ngn2 proteins, and test their functions using embryos to determine which mode of phospho-regulation dominates. Enhancing activity by preventing N- and C terminal phosphorylation cannot overcome the inhibitory effect of mimicking phosphorylation of the bHLH domain. Thus we have established a hierarchy between these two modes of proneural protein control and suggest a model of temporal regulation for proneural protein activity.
从相对简单的神经系统到复杂的哺乳动物皮层，神经发生需要极高的空间和时间精度来协调祖细胞增殖以及随后分化为多种神经元和神经胶质细胞。有限数量的瞬时表达的神经源性碱性螺旋-环-螺旋（bHLH）转录因子，例如果蝇中的Achaete和Scute以及脊椎动物同源物Ascl1和神经生成素2（Ngn2），能够协调神经元决定的起始、依赖于背景的亚型选择，甚至影响神经元迁移和成熟的后期阶段。在过去十年中，出现了两种模型来解释神经源性决定因子的时间活性如何通过不同位点的磷酸化来调节。一种模型描述了脊椎动物神经源性蛋白N端和C端多个位点上与细胞周期相关的磷酸化如何限制循环祖细胞中的神经元分化。第二种模型描述了果蝇Achaete-Scute复合物bHLH结构域中单个位点的磷酸化，该磷酸化作为一个二元开关，磷酸化终止神经源性活性。在这里，我们将Ascl1和Ngn2蛋白N端和C端磷酸化位点的激活突变与bHLH结构域中的抑制性磷酸模拟突变相结合，并使用斑马鱼胚胎测试它们的功能，以确定哪种磷酸调节模式占主导。通过阻止N端和C端磷酸化来增强活性并不能克服模拟bHLH结构域磷酸化的抑制作用。因此，我们建立了这两种神经源性蛋白控制模式之间的层次关系，并提出了神经源性蛋白活性的时间调节模型。
