Molecular mechanisms of cell fate specification

在以前的工作中，我们已经证明Six3基因是胚胎中所有神经细胞发育所必需的。因此，我们进行了morpholino oligonucleotide loss-of-function筛选，以确定在胚胎发生过程中依赖Six3表达的基因，其中一个是SoxC。Morpholino敲除SoxC功能同样抑制了所有神经元的分化，使其位于Six3的下游和神经前体的上游。事实上，SoxC是多种神经元类型发育所必需的，这表明它不是它们末梢分化的直接驱动因素。与这一观点一致的是，SoxC mRNA在表达末梢分化标记物synaptotagmin B （SynB）的成熟神经细胞中未被检测到。因此，我们进行了RNA-Seq筛选，以鉴定soxc依赖性基因。从受影响的基因集中，我们进一步检测了编码Z167和Brn1/2/4两个转录因子的基因。双标记原位杂交显示，相应的mrna在一些携带SoxC信息的细胞和一些表达SynB的细胞中共表达，表明这些因子可能将SoxC功能与终端分化联系起来。Z167的表达局限于胚胎的前极区，在功能缺失实验中，我们发现它是仅在该区域形成的血清素能神经元分化所必需的。Morpholino敲低Brn1/2/4是致命的，这表明该基因对胚胎具有单独的关键早期功能。与此一致的是，对Brn1/2/4 mRNA表达的分析显示，Brn1/2/4 mRNA在母体中均匀积累。合成Brn1/2/4 mRNA的错误表达产生了与胚胎中所有信号传导被阻断时所观察到的相似的显著表型：内胚层和中胚层无法分化，胚胎由一个上皮球组成，其中含有过量的表达synb的神经元。这表明Brn1/2/4的一个早期功能可能在调节外胚层、内胚层和中胚层命运沿动物-植物胚胎轴分配的途径中（例如，见Range等人http://www.ncbi.nlm.nih.gov/pubmed/23335859）。我们正在测试一个模型，其中Six3和SoxC参与神经前体的承诺，而Brn1/2/4和Z167在神经元对血清素能和其他神经元命运的承诺中发挥下游作用。

In previous work we had shown that the Six3 gene is required for development of all nerve cells in the embryo. Therefore, we carried out a morpholino oligonucleotide loss-of-function screen to identify genes dependent on Six3 for their expression during embryogenesis, one of which was SoxC. Morpholino knockdown of SoxC function similarly repressed differentiation of all neurons, placing it downstream from Six3 and upstream of commitment of neural precursors. The fact that SoxC was required for development of multiple neuronal types suggested that it is not the immediate driver of their terminal differentiation. Consistent with this idea, SoxC mRNA was not detectable in mature nerve cells expressing the terminal differentiation marker synaptotagmin B (SynB). Therefore, an RNA-Seq screen was carried out to identify SoxC-dependent genes. From the set of affected genes, we further tested those encoding two transcription factors, Z167 and Brn1/2/4. Double-labeled in situ hybridization revealed that the corresponding mRNAs were co-expressed in some cells with SoxC message and also in some cells expressing SynB, suggesting that these factors could link SoxC function to terminal differentiation. Z167 expression was confined to the anterior pole domain of the embryo and, in a loss-of-function experiment, we found that it is required for differentiation of serotonergic neurons that form only in this region. Morpholino knockdown of Brn1/2/4 was lethal, suggesting that this gene has a separate critical early function for the embryo. Consistent with this, analysis of Brn1/2/4 mRNA expression showed significant levels accumulate maternally and uniformly in the embryo. Misexpression of synthetic Brn1/2/4 mRNA produced a dramatic phenotype similar to that observed when all signaling is blocked in the embryo: endoderm and mesoderm fail to differentiate and the embryo consists of an epithelial ball with an excess of SynB-expressing neurons. This suggests that one early function of Brn1/2/4 could be in the pathways that regulate allocation of ectoderm, endoderm and mesoderm fates along the animal-vegetal embryonic axis (for example, see Range et al. http://www.ncbi.nlm.nih.gov/pubmed/23335859). We are testing a model in which Six3 and SoxC are involved in commitment of neural precursors, while Brn1/2/4 and Z167 function downstream in commitment of neurons to serotonergic and other neuronal fates.