Non-cell autonomous Otx2 homeoprotein regulates visual cortex plasticity through Gadd45b

A better understanding of how Otx2 regulates plasticity in the visual cortex requires that its non-cell autonomous transcription targets be identified. We dissected layer IV of the visual cortex and used RNA-sequencing to analyze gene expression at postnatal day 30 (P30) and P100 in wild-type (WT) and Otx2+/GFP heterozygotes mice. The rationale is that CP plasticity is opened at P30 in WT but not in Otx2+/GFP mice, given that genetic deletion delays CP opening (Sugiyama et al., 2008), and that the CP is closed at P100 in WT mice and not yet in Otx2+/GFP mice. Thus, genes with similar expression at P30 in Otx2+/GFP and at P100 in WT mice but with a different level of expression during the critical period (P30 in WT or P100 Otx2+/GFP mice) were considered as potential genes involved in plasticity. Examination of genes expression at P30 and P100 in wild-type and Otx2 (+/GFP) mice. 4 mice were pooled per condition and experiment was done in duplicate.要更深入地解析Otx2对视觉皮层可塑性的调控机制，需明确其非细胞自主性转录靶标。我们通过解剖视觉皮层第四层，利用RNA测序（RNA-sequencing）分析了野生型（wild-type，WT）与Otx2+/GFP杂合小鼠在出生后第30天（P30）及第100天（P100）的基因表达水平。本研究的设计逻辑如下：野生型小鼠的关键期（critical period, CP）可塑性在P30时被激活，而Otx2+/GFP杂合小鼠则无此现象——已有研究表明，基因敲除会延迟关键期的开启（Sugiyama等，2008）；此外，野生型小鼠在P100时关键期已闭合，而Otx2+/GFP杂合小鼠的关键期仍未关闭。据此，我们将在Otx2+/GFP小鼠的P30与野生型小鼠的P100时表达水平相近，但在关键期内（野生型小鼠的P30或Otx2+/GFP小鼠的P100）表达水平存在显著差异的基因，认定为参与视觉皮层可塑性调控的潜在靶基因。本研究针对野生型与Otx2+/GFP杂合小鼠在P30及P100时的基因表达情况开展了检测。每个实验条件下混合4只小鼠的样本，且实验设置两次生物学重复。