Summary Across the animal kingdom, gastrulation represents a key developmental event during which embryonic pluripotent cells diversify into lineage-specific precursors that will generate the adult organism. Here we report the transcriptional profiles of 116,312 single cells from mouse embryos collected at nine sequential time-points ranging from 6.5 to 8.5 days post-fertilisation. We reconstruct a molecular map of cellular differentiation from pluripotency towards all major embryonic lineages, and explore the complex events involved in the convergence of visceral and primitive streak-derived endoderm. Furthermore, we demonstrate how combining temporal and transcriptional information illuminates gene function by single-cell profiling of Tal1-/- chimeric embryos, with our analysis revealing defects in early mesoderm diversification. Taken together, this comprehensive delineation of mammalian cell differentiation trajectories in vivo represents a baseline for understanding the effects of gene mutations during development as well as a baseline for the optimisation of in vitro differentiation protocols for regenerative medicine.

SUMMARY Despite great advances in understanding the mechanisms underlying blood production, lineage specification at the level of multipotent progenitors (MPPs) remains poorly understood. Here, we show that MPP2 and MPP3 are distinct myeloid-biased MPP subsets that work together with lymphoid-primed MPP4 cells to control blood production. We find that all MPPs are produced in parallel by hematopoietic stem cells (HSCs), but with different kinetics and at variable levels depending on hematopoietic demands. We also show that the normally rare myeloid-biased MPPs are transiently overproduced by HSCs in regenerating conditions, hence supporting myeloid amplification to rebuild the hematopoietic system. This shift is accompanied by a reduction in self-renewal activity in regenerating HSCs and reprogramming of MPP4 fate towards the myeloid lineage. Our results support a dynamic model of blood development in which HSCs convey lineage specification through independent production of distinct lineage-biased MPP subsets that, in turn, support lineage expansion and differentiation.