Molecular mechanisms of cell fate specification

在以前的工作中，我们已经证明了Six3基因是胚胎中所有神经细胞发育所必需的。因此，我们开展了吗啉寡核苷酸介导的功能丧失筛选，以确定依赖于Six3在胚胎发生过程中表达的基因，SoxC就是其中之一。SoxC功能的吗啡敲除类似地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。SoxC是多种神经元发育所必需的，这一事实表明，SoxC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素B(SynB)的成熟神经细胞中未检测到SoxC mRNA。因此，进行了RNA-Seq筛选以鉴定SoxC依赖的基因。从这组受影响的基因中，我们进一步检测了编码两个转录因子Z167和Brn1/2/4的基因。双标记原位杂交显示，相应的mRNAs在一些带有SoxC信息的细胞中共表达，在一些表达泛神经元标记SynB的细胞中也有表达，表明这些因子可能将SoxC的功能与终末分化联系起来。Z167的表达局限于胚胎的前极域，在功能丧失的实验中，我们发现它是仅在该区域形成的5-羟色胺能神经元分化所必需的。Brn1/2/4的吗啉基因敲除存在剂量依赖性表型。在较高浓度下，它是致命的，这表明该基因对胚胎具有关键的早期功能。与此相一致的是，Brn1/2/4mRNA的表达分析表明，在早期胚胎中，Brn1/2/4mRNA的表达水平显著地在母体中积累，并且是均匀的。在适当的低浓度下，胚胎存活，SynB信号和5-羟色胺信号均显著减弱。因此，Brn1/2/4在SoxC下游发挥作用，是所有神经元分化所必需的。人工合成的Brn1/2/4mRNA的错误表达产生了一个戏剧性的表型，类似于当所有信号在胚胎中被阻断时观察到的表型：内胚层和中胚层无法分化，胚胎由一个上皮球组成，上面有过量表达SynB的神经元。这表明Brn1/2/4的早期功能之一可能是调节外胚层、内胚层和中胚层在动植物胚轴上的命运分配。用抗组蛋白H3抗体标记表达SoxC的细胞，发现部分表达SoxC的细胞仍在分裂，提示SoxC阳性细胞处于神经元前体状态。在相邻的两个分裂细胞中的一个细胞中罕见地观察到SoxC信息进一步表明，两个子细胞可能采取交替的命运，其中一个细胞保持在前体状态，而姐妹细胞正在分化。我们提出了一个模型，其中Six3参与神经外胚层区域的指定，SoxC参与神经前体的承诺，而Brn1/2/4和Z167在神经元对5-羟色胺能和非5-羟色胺能神经元命运的承诺中起下游作用。 在与拉里·塔巴克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。采用吗啉基因敲除的方法，我们发现UDP-GalNAc：多肽N-乙酰半乳糖胺转移酶SpGalNAc-T13在原肠胚骨形成间充质细胞(SMCs)中通过整体原位杂交检测到表达。当SpGalNAc-T13被吗啉寡核苷酸注射击倒时，胚胎不能分泌正常由SMC产生的骨针，神经元也不能分化。进一步的研究表明，在注射SpGalNAc-T13的胚胎中，SoxC的表达降低，这表明神经元的基因调控途径上游受阻。外胚层神经元的发育受到影响，但没有检测到SpGalNac-T13的mRNA，这表明该mRNA在低水平广泛表达，或者它在母体中表达，SpGalNac-T13蛋白在母体中供应。

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.