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受精卵产生的所有分子 分化成胚胎的三个初级胚层。40年的创新胚胎学 操作，一次一个基因的基因功能测试，以及最近的下一代测序， 鉴定了多种转录本和丰富的蛋白质，这些蛋白质对脊椎动物的模式化至关重要， 胚胎然而，很少有人知道的总阵列的蛋白质及其翻译后修饰 这有助于形成的胚层，几乎没有什么是已知的贡献小 分子（称为代谢物）对这些过程的影响。到目前为止，系统生物学已经定义了空间和 mRNA的时间变化，丰富的蛋白质，和整个胚胎的代谢产物，但它一直是 技术上不可能利用高分辨率质谱（HRMS），黄金标准技术 对于小分子来说，研究单个胚胎细胞中成百上千的代谢物和蛋白质， 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技术来了解细胞分子过程， 有助于形成三个胚层，这三个胚层是脊椎动物青蛙成功形成图案所必需的。 非洲爪蟾（Xenopus laevis）胚胎，细胞/发育生物学中最喜欢的模型。最近，单细胞团 光谱分析发现了能够改变胚胎细胞正常细胞命运的代谢物， 完整的分子参与者尚未完全确定或理解胚层诱导。的 一项拟议的研究计划将确定在理解分子机制方面的这一缺失环节 控制着脊椎动物的发育这项工作将整合定量单细胞质谱，细胞命运 跟踪和基因敲除实验，以确定一组靶向小分子反应 影响背轴规格所需的信号中心的形成。这个结果 跨学科的方法将有助于阐明蛋白质组和代谢组的作用， 这些重要的前体。由于这些分子过程在脊椎动物中高度保守， 从非洲爪蟾收集的数据可能与人类结构性出生缺陷高度相关。新 生物化学信息，将在个体胚胎细胞及其后代（细胞谱系）中获得， 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 (e.g.,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.