Expression data from chick embryos overexpressing NEUROG2 or NEUROG2AQ in the neural tube. Gallus gallus

The proneural NEUROG2 is essential for neuronal commitment, cell cycle exit and neuronal differentiation. Characterizing genes networks regulated downstream of NEUROG2 is therefore of prime importance. To identify NEUROG2 early response genes, we combined gain of function in the neural tube with a global detection of modified transcripts using microarrays. We included in our study a mutant form of NEUROG2 (NEUROG2AQ) that cannot bind DNA and cannot trigger neurogenesis. Using this approach, we identified 942 genes modified at the onset of NEUROG2 activation. The global analysis of functions regulated by NEUROG2 allowed unmasking its rapid impact on cell cycle control. We found that NEUROG2 specifically represses a subset of cyclins acting at the G1 and S phases of the cell cycle, thereby impeding S phase re-entry. This repression occurs before modification of p27kip1, indicating that the decision to leave the cell cycle precedes the activation of this Cyclin-dependant Kinase Inhibitor. Moreover, NEUROG2 down-regulates only one of the D-type cyclins, cyclinD1, and maintaining cyclinD1 blocks the ability of the proneural to trigger cell cycle exit, without altering its capacity to trigger neuronal differentiation. The fact that NEUROG2 represses a subset but not all cell cycle regulators indicates that cell cycle exit is not an indirect consequence of neuronal differentiation but is precisely controlled by NEUROG2. Altogether our findings indicate that NEUROG2, by specifically repressing G1 and S cyclins, allows committed neuronal precursors to perform their last mitosis but blocks their re-entry in the cell cycle, thus favouring cell cycle exit. Overall design: Stage HH10-11 embryos (11 to 15 somites) were electroporated with a control vector (pGIG-GFP), a NEUROG2-expressing vector (pCIGNEUROG2-GFP), or a NEUROG2AQ-expressing vector (pCIGNEUROG2AQ-GFP). For each biological replicate, neural tubes from 20 embryos were pooled for GFP+ cells collection. GFP+ cells were collected 6h later using FACS sorting (Epics Altra HSS cell sorter, Toulouse Rio platform) and processed for RNA probe preparation and hybridization on Affymetrix microarrays. For each experimental condition, four biological replicates were processed.

成神经因子NEUROG2对于神经元定向、细胞周期退出以及神经元分化至关重要。因此，解析NEUROG2下游调控的基因网络具有核心意义。
为鉴定NEUROG2的早期应答基因，本研究将神经管中的功能过表达实验与基于微阵列（microarrays）的全局转录本变化检测相结合。本研究纳入了一种无法结合DNA、亦无法触发神经发生的NEUROG2突变体（NEUROG2AQ）。
通过该实验策略，我们共鉴定出942个在NEUROG2激活初期发生表达变化的基因。对NEUROG2调控的功能进行全局分析后，我们揭示了其对细胞周期调控的快速影响。研究发现，NEUROG2可特异性抑制细胞周期G1期与S期发挥功能的细胞周期蛋白（cyclins），从而阻碍细胞重新进入S期。
该抑制作用发生于p27kip1表达调控之前，这表明细胞周期退出的决定早于该细胞周期蛋白依赖性激酶抑制剂（Cyclin-dependent Kinase Inhibitor）的激活。此外，NEUROG2仅下调一类D型细胞周期蛋白——cyclinD1；若维持cyclinD1的表达，则会阻断成神经因子触发细胞周期退出的能力，但不会影响其诱导神经元分化的功能。
NEUROG2仅抑制部分而非全部细胞周期调控因子，这一现象表明细胞周期退出并非神经元分化的间接结果，而是由NEUROG2精准调控的。
综合以上研究结果，我们认为NEUROG2通过特异性抑制G1期与S期细胞周期蛋白，使已定向的神经元前体能够完成其最后一次有丝分裂，同时阻断其再次进入细胞周期，最终促进细胞周期退出。
实验设计：将处于HH10-11期（11~15体节）的鸡胚分别电转染对照载体（pGIG-GFP）、表达NEUROG2的载体（pCIGNEUROG2-GFP）以及表达NEUROG2AQ的载体（pCIGNEUROG2AQ-GFP）。
每个生物学重复均收集20枚胚胎的神经管以富集GFP阳性细胞；6小时后，利用荧光激活细胞分选术（FACS，Epics Altra HSS细胞分选仪，图卢兹Rio平台）收集GFP阳性细胞，随后进行RNA探针制备，并在Affymetrix微阵列上进行杂交。
每个实验条件均设置4个生物学重复。