Sip1 regulates sequential fate decisions through feedback signalling from postmitotic neurons to progenitor cells.

In the developing cerebral cortex different types of neurons and glial cells are born through a precisely controlled sequence of events. The fate of cortical progenitors, in turn, is determined by an elusive conundrum of temporally and spatially regulated signalling mechanisms. We found the DNA-binding transcription factor Sip1 (also known as Zfhx1b) to be produced at high levels in postmitotic neurons of the cerebral cortex. Conditional deletion of Sip1 in young neocortical neurons was found to induce premature and increased production of upper layer neurons at the expense of deep layer neurons. Furthermore, it caused precocious and increased generation of glial precursors during late corticogenesis, leading subsequently to enhanced astrocytogenesis at early postnatal stages. Expression profiling analysis indicated that the temporal shift in upper layer production coincides with overexpression of the neurotrophin-3 (NT3) gene and altered growth factor signalling in progenitors, while the premature gliogenesis is preceded by upregulation of fibroblast growth factor-9 (Fgf9) gene expression. Chromatin immunoprecipitation and in situ hybridization validates NT3 as a direct target of Sip1 in the cortex and confines the transcriptional repression by Sip1 to postmitotic neurons. Moreover, we show that exogenous application of Fgf9 in solution or via coated beads to wild-type cortical slices induces premature and excessive generation of glial precursors in the germinal zone. In conclusion, our data suggest that throughout corticogenesis Sip1 acts to restrain the level of production of secreted signalling factors in postmitotic neurons. These factors feed back to progenitor cells in order to regulate the timing of cell fate switch and the numbers of neurons and glial cells produced in the developing cerebral cortex. In a first series of arrays, we examined differential gene expression in neocortex and hippocampal tissue from 2 control (WT|Nestin) and 2 mutant (Sip1|Nestin) E18.5 littermate mice, each taken from 2 different litters (x|Nestin1 and x|Nestin2). In a separate experiment, we tested differential gene expression in E14.5 neocortex and hippocampal tissue from 2 control (WT|NEX) and 2 mutant (Sip1|NEX) littermate mice. RNA from mouse neocortex and hippocampus tissues was isolated using RNeasy kit according to the manufacturer’s protocol (QIAGEN). Total RNA was controlled for integrity and purity using an Agilent Bioanalyzer and a NanoDrop spectrophotometer, respectively. All samples were of similar RNA quality. Starting with 1ug of total RNA, the RNA amplification was performed by in vitro transcription (IVT) with a biotin labeling reaction during the IVT, according to the recommendations of the manufacturer (Amersham Biosciences). The probes were purified and analyzed again for yield (> 20 ug) and purity (260:280 nm and 260:230 nm >1.8). Ten micrograms of the resulting antisense RNA was fragmented according to the recommendations of the manufacturer (Amersham Biosciences) and resuspended in 260 ml of hybridization buffer. Codelink Mouse Whole Genome array is a single array representing 35,000 transcripts. The gene array chips were hybridized in a shaker-incubator at 37°C at 300 rpm for 18 hours and washed and stained with Cy5-Streptavidin according to the recommendations of the manufacturer (Amersham Biosciences). The DNA Microarray scanner of Agilent was used for scanning and image analysis was performed with the Codelink Expression Analysis 4.1 software在发育中的大脑皮层（cerebral cortex）中，不同类型的神经元与神经胶质细胞（glial cells）通过一系列精密调控的事件诞生。而皮层祖细胞（cortical progenitors）的命运，则由时空动态调控的信号通路构成的复杂谜题所决定。我们发现DNA结合转录因子（transcription factor）Sip1（亦称为Zfhx1b）在大脑皮层的有丝分裂后神经元（postmitotic neurons）中高表达。对新生新皮层（neocortex）神经元进行Sip1条件性敲除（conditional deletion）后，会诱导上层神经元（upper layer neurons）提前且过量产生，同时以深层神经元（deep layer neurons）的生成为代价。此外，该操作会在皮层发生（corticogenesis）后期提前并增加胶质前体细胞的生成，进而在出生后早期促进星形胶质发生（astrocytogenesis）。表达谱分析显示，上层神经元生成的时序偏移与神经营养因子3（neurotrophin-3, NT3）基因的过表达以及祖细胞中生长因子信号通路的改变相吻合；而提前发生的胶质生成则伴随着成纤维细胞生长因子9（fibroblast growth factor-9, Fgf9）基因表达的上调。染色质免疫沉淀（chromatin immunoprecipitation）与原位杂交（in situ hybridization）实验证实，NT3是Sip1在皮层中的直接靶标，并将Sip1的转录抑制作用限定在有丝分裂后神经元中。此外，我们证实，向野生型皮层脑片施加溶液中或包被在磁珠上的外源性Fgf9，会诱导生发区（germinal zone）中的胶质前体细胞提前且过量生成。综上，我们的数据表明，在整个皮层发生过程中，Sip1可通过抑制有丝分裂后神经元中分泌型信号因子的表达水平，这些因子会反馈作用于祖细胞，以调控细胞命运转换的时序以及发育中大脑皮层内神经元与神经胶质细胞的生成数量。 第一组芯片实验中，我们检测了2只对照（野生型|Nestin）与2只突变（Sip1|Nestin）同窝E18.5小鼠的新皮层与海马组织的差异基因表达，每只小鼠均来自2个不同的同窝（x|Nestin1与x|Nestin2）。在另一项独立实验中，我们检测了2只对照（WT|NEX）与2只突变（Sip1|NEX）同窝E14.5小鼠的新皮层与海马组织的差异基因表达。 使用RNeasy试剂盒（RNeasy kit）按照制造商凯杰（QIAGEN）的操作规程，从小鼠新皮层与海马组织中分离总RNA。分别使用安捷伦生物分析仪（Agilent Bioanalyzer）与NanoDrop分光光度计（NanoDrop spectrophotometer）对总RNA的完整性与纯度进行质控，所有样本的RNA质量均一致。取1 μg总RNA作为起始材料，按照制造商安玛西亚生物科技（Amersham Biosciences）的建议，通过体外转录（in vitro transcription, IVT）进行RNA扩增，并在IVT过程中加入生物素标记反应。对扩增得到的探针进行纯化后，再次检测其产量（>20 μg）与纯度（260 nm/280 nm、260 nm/230 nm比值均>1.8）。取10 μg纯化后的反义RNA，按照制造商安玛西亚生物科技（Amersham Biosciences）的建议进行片段化，并重悬于260 mL杂交缓冲液中。Codelink小鼠全基因组芯片（Codelink Mouse Whole Genome array）可代表35000个转录本。将基因芯片置于摇床培养箱中，于37℃、300 rpm条件下杂交18小时，随后按照制造商安玛西亚生物科技（Amersham Biosciences）的建议进行洗涤与Cy5-链霉亲和素（Cy5-Streptavidin）染色。使用安捷伦基因芯片扫描仪（Agilent DNA Microarray scanner）进行扫描，并通过Codelink表达分析4.1软件（Codelink Expression Analysis 4.1 software）完成图像分析。