Molecular mechanisms of cell fate specification

在以前的工作中，我们已经证明Six 3基因是胚胎中所有神经细胞发育所必需的。 因此，我们进行了吗啉代寡核苷酸功能丧失筛选，以鉴定在胚胎发生过程中依赖Six 3表达的基因，其中之一是SoxC。 类似地，SoxC功能的吗啉代敲低抑制了所有神经元的分化，将其置于Six 3的下游和神经前体的承诺的上游。 SoxC是多种神经元类型发育所必需的，这一事实表明它不是它们终末分化的直接驱动因素。 与这一观点一致，在表达终末分化标记物突触结合蛋白B（SynB）的成熟神经细胞中检测不到SoxC mRNA。 因此，进行RNA-Seq筛选以鉴定SoxC依赖性基因。 从一组受影响的基因中，我们进一步测试了编码两个转录因子Z167和Brn 1/2/4的基因。 双标记原位杂交结果显示，在SoxC信号转导细胞和SynB信号转导细胞中，SoxC和SynB的mRNA同时表达，提示SoxC和SynB可能与细胞的终末分化有关。 Z167的表达仅限于胚胎的前极区域，在功能丧失实验中，我们发现它是仅在该区域形成的多巴胺能神经元分化所必需的。 Brn 1/2/4的Morpholino敲低是致命的，这表明该基因对胚胎具有单独的关键早期功能。 与此一致，Brn 1/2/4 mRNA表达的分析表明，显着水平的积累母亲和均匀的胚胎。合成Brn 1/2/4 mRNA的错误表达产生了一个戏剧性的表型，类似于在胚胎中所有信号传导被阻断时观察到的表型：内胚层和中胚层未能分化，胚胎由上皮球和过量的SynB表达神经元组成。 这表明Brn 1/2/4的一个早期功能可能是在调节外胚层、内胚层和中胚层命运沿着动物-植物胚胎轴的分配的途径中（例如，参见Range等人http：//www.ncbi.nlm.nih.gov/pubmed/23335859）。 我们正在测试一个模型，其中Six 3和SoxC参与神经前体的定型，而Brn 1/2/4和Z167在神经元向神经元能和其他神经元命运的定型中起下游作用。

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.