Genome-wide RNA tomography in the zebrafish embryo

Advancing our understanding of embryonic development is heavily dependent on identification of novel pathways or regulators. While genome-wide techniques such as RNA sequencing are ideally suited for discovering novel candidate genes, they are unable to yield spatially resolved information in embryos or tissues. Microscopy-based approaches, using for example in situ hybridization, can provide spatial information about gene expression, but are limited to analyzing one or a few genes at a time. Here, we present a method where we combine traditional histological techniques with low-input RNA sequencing and mathematical image reconstruction to generate a high-resolution genome-wide 3D atlas of gene expression in the zebrafish embryo at three developmental stages. We also demonstrate that our technique is suitable for spatially-resolved differential expression analysis in wildtype and Gli3 mutant mouse forelimbs. Importantly, our method enables searching for genes that are expressed in specific spatial patterns without manual image annotation. We envision broad applicability of RNA tomography as an accurate and sensitive approach for spatially resolved transcriptomics in whole embryos and dissected organs. To generate spatially-resolved RNA-seq data for zebrafish embryos (shield stage, 10 somites, 15 somites, 18 somites) and mouse forelimbs (E10.5), we cryosectioned samples, extracted RNA from the individual sections, and amplified and barcoded mRNA using the CEL-seq protocol (Hashimshony et al., Cell Reports, 2012) with a few modifications. Libraries were sequenced on Illumina HiSeq 2500 using 50bp paired end sequencing. Selected zebrafish libraries were sequenced on MiSeq 250bp paired-end to improve 3' annotations.深化对胚胎发育过程的认知，高度依赖于全新通路与调控因子的发掘。尽管全基因组技术（如RNA测序(RNA sequencing)）十分适用于新型候选基因的筛选，但无法获取胚胎或组织内的空间分辨率信息。基于显微镜的研究手段（例如原位杂交(in situ hybridization)）虽可提供基因表达的空间信息，但仅能单次分析单个或少数几个基因。本研究提出一种整合传统组织学技术、低起始量RNA测序与数学图像重构的方法，可构建三个发育阶段斑马鱼胚胎的全基因组高分辨率基因表达3D图谱。本研究同时证实，该技术可应用于野生型与Gli3突变小鼠前肢的空间分辨率差异表达分析。尤为关键的是，本方法无需人工图像注释，即可筛选出具有特定空间表达模式的基因。我们认为，RNA断层扫描(RNA tomography)作为一种精准灵敏的空间分辨率转录组学方法，可广泛应用于完整胚胎与解剖器官的相关研究。为获取斑马鱼胚胎（盾期、10体节、15体节、18体节）与小鼠前肢（E10.5）的空间分辨率RNA-seq数据，我们对样本实施冷冻切片，从单个切片中提取RNA，并通过稍加改良的CEL-seq实验方案（Hashimshony等, Cell Reports, 2012）对mRNA进行扩增与条形码标记。测序文库采用Illumina HiSeq 2500平台开展50bp双端测序。为优化3'端注释，部分斑马鱼测序文库采用MiSeq平台进行250bp双端测序。