Brain Research Institute, ETH Zurich/University of Zurich, 8057 Zurich, Switzerland.
Neural Dev. 2014 Oct 29;9:23. doi: 10.1186/1749-8104-9-23.
Neural stem cell (NSC) differentiation is a complex multistep process that persists in specific regions of the postnatal forebrain and requires tight regulation throughout life. The transcriptional control of NSC proliferation and specification involves Class II (proneural) and Class V (Id1-4) basic helix-loop-helix (bHLH) proteins. In this study, we analyzed the pattern of expression of their dimerization partners, Class I bHLH proteins (E-proteins), and explored their putative role in orchestrating postnatal subventricular zone (SVZ) neurogenesis.
Overexpression of a dominant-negative form of the E-protein E47 (dnE47) confirmed a crucial role for bHLH transcriptional networks in postnatal neurogenesis by dramatically blocking SVZ NSC differentiation. In situ hybridization was used in combination with RT-qPCR to measure and compare the level of expression of E-protein transcripts (E2-2, E2A, and HEB) in the neonatal and adult SVZ as well as in magnetic affinity cell sorted progenitor cells and neuroblasts. Our results evidence that E-protein transcripts, in particular E2-2 and E2A, are enriched in the postnatal SVZ with expression levels increasing as cells engage towards neuronal differentiation. To investigate the role of E-proteins in orchestrating lineage progression, both in vitro and in vivo gain-of-function and loss-of-function experiments were performed for individual E-proteins. Overexpression of E2-2 and E2A promoted SVZ neurogenesis by enhancing not only radial glial cell differentiation but also cell cycle exit of their progeny. Conversely, knock-down by shRNA electroporation resulted in opposite effects. Manipulation of E-proteins and/or Ascl1 in SVZ NSC cultures indicated that those effects were Ascl1 dependent, although they could not solely be attributed to an Ascl1-induced switch from promoting cell proliferation to triggering cell cycle arrest and differentiation.
In contrast to former concepts, suggesting ubiquitous expression and subsidiary function for E-proteins to foster postnatal neurogenesis, this work unveils E-proteins as being active players in the orchestration of postnatal SVZ neurogenesis.
神经干细胞（NSC）分化是一个复杂的多步骤过程，存在于新生脑后特定区域，并需要终生进行严格调控。NSC 增殖和特化的转录控制涉及 II 类（神经前体细胞）和 V 类（Id1-4）碱性螺旋-环-螺旋（bHLH）蛋白。在这项研究中，我们分析了其二聚体伴侣 I 类 bHLH 蛋白（E 蛋白）的表达模式，并探讨了它们在协调产后侧脑室下区（SVZ）神经发生中的潜在作用。
过表达显性失活形式的 E 蛋白 E47（dnE47）通过显著阻止 SVZ NSC 分化，证实了 bHLH 转录网络在产后神经发生中的关键作用。原位杂交与 RT-qPCR 相结合，用于测量和比较新生和成年 SVZ 以及磁亲和细胞分选祖细胞和神经母细胞中 E 蛋白转录本（E2-2、E2A 和 HEB）的表达水平。我们的结果表明，E 蛋白转录本，特别是 E2-2 和 E2A，在产后 SVZ 中富集，随着细胞向神经元分化，表达水平增加。为了研究 E 蛋白在协调谱系进展中的作用，我们在体外和体内进行了单个 E 蛋白的功能获得和功能丧失实验。E2-2 和 E2A 的过表达不仅促进了放射状胶质细胞的分化，还促进了其后代的细胞周期退出，从而促进了 SVZ 神经发生。相反，通过 shRNA 电穿孔敲低导致了相反的效果。在 SVZ NSC 培养物中操纵 E 蛋白和/或 Ascl1 表明，这些效应依赖于 Ascl1，但不能仅仅归因于 Ascl1 诱导的从促进细胞增殖到触发细胞周期停滞和分化的转变。
与以前的概念相反，这些概念表明 E 蛋白普遍表达且具有辅助功能，以促进产后神经发生，本工作揭示了 E 蛋白是协调产后 SVZ 神经发生的活跃参与者。
