Molecular profiling of single cells has advanced our knowledge of the molecular basis of development. However, current approaches mostly rely on dissociating cells from tissues, thereby losing the crucial spatial context of regulatory processes. Here, we apply an image-based single-cell transcriptomics method, sequential fluorescence in situ hybridization (seqFISH), to detect mRNAs for 387 target genes in tissue sections of mouse embryos at the 8–12 somite stage. By integrating spatial context and multiplexed transcriptional measurements with two single-cell transcriptome atlases, we characterize cell types across the embryo and demonstrate that spatially resolved expression of genes not profiled by seqFISH can be imputed. We use this high-resolution spatial map to characterize fundamental steps in the patterning of the midbrain–hindbrain boundary (MHB) and the developing gut tube. We uncover axes of cell differentiation that are not apparent from single-cell RNA-sequencing (scRNA-seq) data, such as early dorsal–ventral separation of esophageal and tracheal progenitor populations in the gut tube. Our method provides an approach for studying cell fate decisions in complex tissues and development.

单细胞的分子谱分析提高了我们对发育分子基础的认识。然而，目前的方法主要依赖于从组织中分离细胞，从而失去了调节过程的关键空间背景。在这里，我们应用基于图像的单细胞转录组学方法，即顺序荧光原位杂交 (seqFISH)，在 8-12 体节阶段检测小鼠胚胎组织切片中 387 个靶基因的 mRNA。通过将空间背景和多重转录测量与两个单细胞转录组图谱相结合，我们表征了整个胚胎的细胞类型，并证明可以估算未通过 seqFISH 分析的基因的空间分辨表达。我们使用这张高分辨率空间图来描述中脑-后脑边界 (MHB) 和发育中的肠管形成模式的基本步骤。我们发现了从单细胞 RNA 测序 (scRNA-seq) 数据中不明显的细胞分化轴，例如肠管中食管和气管祖细胞群的早期背腹分离。我们的方法提供了一种研究复杂组织和发育中细胞命运决定的方法。