Hotchkiss Brain Institute and Alberta Children's Hospital Research Institute, Department of Biochemistry, University of Calgary, Calgary, Alberta T2N 4N1, Canada.
J Neurosci. 2012 Jun 6;32(23):7791-805. doi: 10.1523/JNEUROSCI.1309-12.2012.
The neocortex is comprised of six neuronal layers that are generated in a defined temporal sequence. While extrinsic and intrinsic cues are known to regulate the sequential production of neocortical neurons, how these factors interact and function in a coordinated manner is poorly understood. The proneural gene Neurog2 is expressed in progenitors throughout corticogenesis, but is only required to specify early-born, deep-layer neuronal identities. Here, we examined how neuronal differentiation in general and Neurog2 function in particular are temporally controlled during murine neocortical development. We found that Neurog2 proneural activity declines in late corticogenesis, correlating with its phosphorylation by GSK3 kinase. Accordingly, GSK3 activity, which is negatively regulated by canonical Wnt signaling, increases over developmental time, while Wnt signaling correspondingly decreases. When ectopically activated, GSK3 inhibits Neurog2-mediated transcription in cultured cells and Neurog2 proneural activities in vivo. Conversely, a reduction in GSK3 activity promotes the precocious differentiation of later stage cortical progenitors without influencing laminar fate specification. Mechanistically, we show that GSK3 suppresses Neurog2 activity by influencing its choice of dimerization partner, promoting heterodimeric interactions with E47 (Tcfe2a), as opposed to Neurog2-Neurog2 homodimer formation, which occurs when GSK3 activity levels are low. At the functional level, Neurog2-E47 heterodimers have a reduced ability to transactivate neuronal differentiation genes compared with Neurog2-Neurog2 homodimers, both in vitro and in vivo. We thus conclude that the temporal regulation of Neurog2-E47 heterodimerization by GSK3 is a central component of the neuronal differentiation "clock" that coordinates the timing and tempo of neocortical neurogenesis in mouse.
新皮层由六层神经元组成，这些神经元按特定的时间顺序产生。虽然已知外在和内在的线索可以调节新皮层神经元的顺序产生，但这些因素如何相互作用并协调发挥作用还知之甚少。神经调节基因 Neurog2 在皮质发生过程中在祖细胞中表达，但仅需要指定早期出生的深层神经元身份。在这里，我们研究了神经元分化以及 Neurog2 功能在小鼠新皮层发育过程中是如何受到时间控制的。我们发现，Neurog2 神经调节活性在皮质发生后期下降，与 GSK3 激酶的磷酸化有关。相应地，GSK3 活性（受经典 Wnt 信号的负调控）随着发育时间的增加而增加，而 Wnt 信号相应地减少。当异常激活时，GSK3 会抑制培养细胞中的 Neurog2 介导的转录和体内的 Neurog2 神经调节活性。相反，GSK3 活性的降低促进了后期皮质祖细胞的早熟分化，而不影响层状命运特化。从机制上讲，我们表明 GSK3 通过影响其二聚化伙伴的选择来抑制 Neurog2 活性，促进与 E47（Tcfe2a）的异二聚体相互作用，而不是发生在 GSK3 活性水平较低时的 Neurog2-Neurog2 同源二聚体形成。在功能水平上，与 Neurog2-Neurog2 同源二聚体相比，Neurog2-E47 异二聚体在体外和体内激活神经元分化基因的能力降低。因此，我们得出结论，GSK3 对 Neurog2-E47 异二聚体形成的时间调节是协调小鼠新皮层神经发生时间和节奏的神经元分化“时钟”的核心组成部分。
