Single-cell Metabolomics and Proteomics: The Missing Link to Understanding Vertebrate Embryonic Patterning

摘要 了解胚胎发育需要了解作为受精卵产生的所有分子。 分化为胚胎的三个初级胚层。创新胚胎学四十年 操作，一次测试一个基因的基因功能，最近，下一代测序 鉴定了脊椎动物模式形成所必需的多种转录本和丰富的蛋白质 胚胎。然而，人们对蛋白质的全部序列及其翻译后修饰知之甚少 对胚层的形成有贡献，而对小细菌的贡献几乎一无所知 分子(称为代谢物)对这些过程起作用。到目前为止，系统生物学定义了空间和 整个胚胎中mRNAs、丰富的蛋白质和代谢物的时间变化，但它已经 在技术上不可能利用高分辨率质谱仪(HRMS)，这是黄金标准技术 对于小分子，研究单个胚胎细胞中数百到数千种代谢物和蛋白质 脊椎动物的胚胎。拟议的研究计划填补了这一巨大的知识和技术空白。 通过利用新的单细胞质谱学技术来了解细胞分子过程 有助于形成脊椎动物青蛙成功构图所需的三个胚层 非洲爪哇(Xenopus Laevis)胚胎，细胞/发育生物学中最受欢迎的模式。最近，单细胞质量 光谱分析发现了能够改变胚胎细胞正常细胞命运的代谢产物，这表明 对于胚层的诱导，完整的分子参与者还没有完全被识别或理解。这个 拟议的研究计划将确定分子机制理解中的这一缺失环节 管理脊椎动物的发育。这项工作将把定量的单细胞质谱学、细胞命运 跟踪和基因敲除实验来确定一组有针对性的小分子反应 影响背轴规范所需信号中心的形成。这样做的结果是 跨学科的方法将有助于阐明蛋白质组和代谢组在建立 这些重要的前兆。因为这些分子过程在脊椎动物中高度保守，所以 从非洲爪哇收集的数据可能与人类结构性出生缺陷有很高的相关性。新的 将在单个胚胎细胞及其后代(细胞谱系)中获得的生化信息 几个关键的发育时间点也将推进涉及细胞分化的其他研究领域 (例如，干细胞)和成人疾病的发育起源。

Abstract Understanding embryonic development requires knowledge of all the molecules produced as the zygote differentiates into the three primary germ layers of the embryo. Four decades of innovative embryological manipulations, testing of gene functions one gene at a time, and recently, Next-Generation Sequencing have identified multiple transcripts and abundant proteins that are essential to the patterning of the vertebrate embryo. However, very little is known about the total array of proteins and their post-translational modifications that contribute to the formation of the germ layers, and next to nothing is known about the contribution of small molecules (called metabolites) to these processes. To date, systems biology has defined the spatial and temporal changes of mRNAs, abundant proteins, and metabolites in the whole embryo, but it has been technologically impossible to utilize high-resolution mass spectrometry (HRMS), the gold standard technology for small molecules, to study hundreds-to-thousands of metabolites and proteins in single embryonic cells in the vertebrate embryo. The proposed research program fills this enormous knowledge and technological gap by utilizing novel single-cell mass spectrometry technologies to understand cell molecular processes that contribute to the formation of the three germ layers required for the successful patterning of the vertebrate frog (Xenopus laevis) embryo, a favorite model in cell/developmental biology. Most recently, single-cell mass spectrometry discovered metabolites capable of altering the normal cell fates of embryonic cells, suggesting that the complete molecular players are not yet fully identified or understood for germ layer induction. The proposed research program will determine this missing link in the understanding of molecular mechanisms governing vertebrate development. This work will integrate quantitative single-cell mass spectrometry, cell fate tracking, and gene knock-down experiments to determine how a targeted set of small-molecular reactions impact the formation of signaling centers required for dorsal axis specification. The outcomes of this interdisciplinary approach will help illuminate the role of the proteome and metabolome for the establishment of these important precursors. Because these molecular processes are highly conserved across vertebrates, the data collected from Xenopus are likely to have high relevance to human structural birth defects. The new biochemical information that will be obtained in individual embryonic cells and their progeny (cell lineage) at several critical developmental time points will also advance other research fields that involve cell differentiation (e.g., of stem cells) and the developmental origins of adult disease.