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，我们进行了吗啉代谷胱甘肽介导的功能丧失筛选，以确定在胚胎发生过程中依赖于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，SoxC信号在某些细胞中同时表达，而SynB信号在某些细胞中也同时表达，提示这些因子可能与SoxC功能和终末分化有关。 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的吗啉代敲低产生剂量依赖性表型。 在更高的浓度下，它是致命的，这表明该基因对胚胎具有关键的早期功能。与此相一致，Brn 1/2/4 mRNA表达的分析显示，在早期胚胎中，显着水平的积累是母系和均匀的。在经过良好调整的较低浓度下，胚胎存活，SynB信号和5-羟色胺信号都大大降低。 因此，Brn 1/2/4在SoxC下游起作用，并且是所有神经元分化所必需的。 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过程中。通过用抗磷酸化组蛋白H3抗体标记SoxC表达细胞，我们发现一些SoxC表达细胞仍在分裂，表明SoxC阳性细胞处于神经元前体状态。在两个相邻的分裂细胞中的一个中罕见地观察到SoxC信息进一步表明，两个子细胞可以采取交替的命运，其中一个细胞保持在前体状态，而姐妹细胞分化。 我们提出了一个模型，其中Six 3参与神经外胚层结构域的特化，SoxC参与神经前体的定型，而Brn 1/2/4和Z167在神经元向多巴胺能和非多巴胺能神经元命运的定型中起下游作用。 在与Larry Tabak的实验室合作研究海胆胚胎中蛋白质糖基化的发育功能时，我们确定了13个SpGalNAc-T基因。 采用morpholino knockdown的方法，我们发现UDP-GalNAc：多肽N-乙酰氨基半乳糖转移酶SpGalNAc-T13的表达是通过整体原位杂交在早期原肠胚的成骨间充质细胞（SMCs）中检测到的。当SpGalNAc-T13被吗啉寡核苷酸注射敲低时，胚胎不能分泌正常由SMC产生的骨骼骨针，神经元不能分化。 进一步的研究表明，在SpGalNAc-T13注射的胚胎中，SoxC表达减少，表明神经元的基因调控途径上游被阻断。外胚层中神经元的发育受到影响，但在那里没有检测到SpGalNac-T13 mRNA的事实表明，mRNA以低水平广泛表达，或者备选地，它是母体表达的，SpGalNac-T13蛋白质是母体提供的。

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-mediated 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 the pan-neuronal marker 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 produced dosage-dependent phenotypes. At a higher concentration it was lethal, suggesting that this gene has a 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 early embryo. At a well-tuned lower concentration, the embryos survived and both SynB signal and serotonin signal were greatly reduced. Therefore Brn1/2/4 functions downstream of SoxC and is required for differentiation of all neurons. 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 processes that regulate allocation of ectoderm, endoderm and mesoderm fates along the animal-vegetal embryonic axis. By labeling SoxC-expressing cells with anti-phospho-histone H3 antibody we found that some SoxC expressing cells were still dividing suggesting that SoxC-positive cells are in a neuronal precursor state. The rare observation of SoxC message in one of two adjacent dividing cells further suggested that the two daughter cells could take alternative fates with one cell remaining in the precursor state and the sister cell differentiating. We proposed a model in which Six3 is involved in neuro-ectoderm domain specification and SoxC is involved in commitment of neural precursors, while Brn1/2/4 and Z167 function downstream in commitment of neurons to serotonergic and non-serotonergic neuronal fates. In a collaboration with Larry Tabak's lab to study developmental functions of protein glycosylation in sea urchin embryos, we identified 13 SpGalNAc-T genes. Using morpholino knockdown methods we showed that UDP-GalNAc:Polypeptide N-Acetylgalactosaminyltransferase SpGalNAc-T13 expression was detected by whole mount in situ hybiridzation in skeletogenic mesenchyme cells (SMCs) of early gastrulae. When SpGalNAc-T13 was knocked down by morpholino oligo injection, embryos failed to secrete skeletal spicules normally produced by SMCs and neurons failed to differentiate. Further study revealed that SoxC expression was reduced in SpGalNAc-T13 injected embryos, suggesting a block upstream in the gene regulatory pathway for neurons. The fact that development of neurons in ectoderm is affected, but SpGalNac-T13 mRNA was not detected there suggests that the mRNA is expressed broadly at low levels, or alternatively that it is expressed maternally and SpGalNac-T13 protein is supplied maternally.