Gene expression profile at single cell level of whole Drosophila melanogaster embryos at three developmental stages. Gene expression profile at single cell level of whole Drosophila melanogaster embryos at three developmental stages

Embryonic development results in the production of distinct tissue types, and different cell types within each tissue. A major goal of developmental biology is to uncover the “parts list” of cell types that comprise each organ. Here we perform single cell RNA sequencing (scRNA-seq) of the Drosophila embryo to identify the genes that characterize different cell and tissue types during development. We assay three different timepoints, revealing a coordinated change in gene expression within each tissue. Interestingly, we find that the elav and mhc genes, whose protein products are widely used as markers for neurons and muscles, respectively, show broad pan-embryonic expression, indicating the importance of post-transcriptional regulation. We next focus on the central nervous system (CNS), where we identify genes characterizing each stage of neuronal differentiation: from neural progenitors, called neuroblasts, to their immediate progeny ganglion mother cells (GMCs), followed by new-born neurons, young neurons, and the most mature neurons. Finally, we ask whether the clonal progeny of a single neuroblast (NB7-1) share a similar transcriptional identity. Surprisingly, we find that clonal identity does not lead to transcriptional clustering, showing that neurons within a lineage are diverse, and that neurons with a similar transcriptional profile (e.g. motor neurons, glia) are distributed among multiple neuroblast lineages. Although each lineage consists of diverse progeny, we were able to identify a previously uncharacterized gene, Fer3, as an excellent marker for the NB7-1 lineage. Within the NB7-1 lineage, transcriptional clusters are identifiable in neuroblasts and neurons, and each cluster is composed of current temporal transcription factor (e.g. Hunchback, Kruppel, Pdm, and Castor), novel temporal factors, and/or targets of the temporal transcription factors. In conclusion, we have characterized the embryonic transcriptome for all major tissue types and for three stages of development, as well as the first transcriptomic analysis of a single, identified neuroblast lineage, finding a lineage-enriched transcription factor. Overall design: Whole embryos were staged according to developmental windows of interest, dissociated and run through the 10X 3' mRNA seq pipeline Please note that processed data was generated from both Lane 1&2 samples and is linked to the corresponding *Lane 1* sample records.

胚胎发育可产生多种不同的组织类型，且每种组织内部又包含各异的细胞类型。发育生物学的核心目标之一，便是揭示构成每一器官的细胞类型组成清单。本研究对果蝇（Drosophila）胚胎开展单细胞RNA测序（single-cell RNA sequencing, scRNA-seq），以鉴定发育过程中表征不同细胞与组织类型的特征基因。我们选取三个不同的发育时间点进行实验，揭示了每种组织内基因表达的协同变化规律。有趣的是，我们发现elav与mhc基因的蛋白产物分别被广泛用作神经元与肌肉的标记物，而这两个基因在全胚胎范围内均有广泛表达，这提示转录后调控的重要性。随后我们将研究重点聚焦于中枢神经系统（central nervous system, CNS），在此处鉴定出了表征神经元分化各阶段的特征基因：从被称为成神经细胞（neuroblasts）的神经祖细胞，到其直接子代神经节母细胞（ganglion mother cells, GMCs），再到新生神经元、年轻神经元以及成熟度最高的神经元。最后，我们探究单个成神经细胞（NB7-1）的克隆子代是否具有相似的转录同一性。令人意外的是，我们发现克隆身份并不会导致转录聚类，这表明同一谱系内的神经元具有多样性，且具有相似转录特征（例如运动神经元、胶质细胞）的神经元分布于多个成神经细胞谱系之中。尽管每个谱系均包含多样的子代细胞，但我们成功鉴定出一个此前未被表征的基因Fer3，可作为NB7-1谱系的优秀标记物。在NB7-1谱系中，我们可在成神经细胞与神经元中鉴定出转录聚类簇，每个聚类簇均由当前已知的时序转录因子（temporal transcription factor）（例如Hunchback、Kruppel、Pdm及Castor）、新型时序因子以及/或时序转录因子的靶基因构成。综上，本研究已对所有主要组织类型以及三个发育阶段的胚胎转录组完成表征，同时完成了首个针对单个已鉴定成神经细胞谱系的转录组分析，并发现了一个谱系富集的转录因子。
实验整体设计：根据目标发育窗对完整胚胎进行分期，将其解离后采用10X 3'端mRNA测序流程进行建库测序。请注意，处理后的数据由Lane 1与Lane 2的样本共同生成，并与对应的*Lane 1*样本记录相关联。