Three distinct cell types are present within the 64-cell stage mouse blastocyst. We have investigated cellular development up to this stage using single-cell expression analysis of more than 500 cells. The 48 genes analyzed were selected in part based on a whole-embryo analysis of more than 800 transcription factors. We show that in the morula, blastomeres coexpress transcription factors specific to different lineages, but by the 64-cell stage three cell types can be clearly distinguished according to their quantitative expression profiles. We identify Id2 and Sox2 as the earliest markers of outer and inner cells, respectively. This is followed by an inverse correlation in expression for the receptor-ligand pair Fgfr2/Fgf4 in the early inner cell mass. Position and signaling events appear to precede the maturation of the transcriptional program. These results illustrate the power of single-cell expression analysis to provide insight into developmental mechanisms. The technique should be widely applicable to other biological systems.

Single-cell RNA sequencing (scRNA-seq) technologies are poised to reshape the current cell-type classification system. However, a transcriptome-based single-cell atlas has not been achieved for complex mammalian systems. Here, we developed Microwell-seq, a high-throughput and low-cost scRNA-seq platform using simple, inexpensive devices. Using Microwell-seq, we analyzed more than 400,000 single cells covering all of the major mouse organs and constructed a basic scheme for a mouse cell atlas (MCA). We reveal a single-cell hierarchy for many tissues that have not been well characterized previously. We built a web-based "single-cell MCA analysis" pipeline that accurately defines cell types based on single-cell digital expression. Our study demonstrates the wide applicability of the Microwell-seq technology and MCA resource.

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