Supplementary MaterialsReporting summary. supporting the findings of this study are available from the corresponding author on affordable request. Abstract During gastrulation, cell types from all three germ layers are specified and the basic body plan is usually established1. However, molecular analysis of this key developmental stage has been hampered by limited cell numbers and a paucity of markers. Single cell RNA sequencing circumvents these problems, but has so far been limited to specific organ systems2. Here we report single-cell transcriptomic characterisation of over 20000 cells immediately following gastrulation at E8.25 of mouse development. We identify 20 major cell types, which frequently contain sub-structure, including three distinct signatures in early foregut cells. Pseudospace ordering of somitic progenitor cells identifies dynamic waves of transcription and candidate regulators, which are validated by molecular characterisation of spatially resolved regions of the embryo. Within the endothelial populace, cells that transition from haemogenic endothelial to erythro-myeloid progenitors specifically express and its co-factor and for endoderm3, and (and for ectoderm5. We also identified germ layer specific genes that have not been described in the context of embryo development including and which show specific expression in ectoderm (Supplementary Fig. 1F). Furthermore, many other genes showed restricted expression to one or a few of our defined cell types (Supplementary Fig. 1E), providing valuable candidate markers for defining and potentially programming Has2 populations of cells toward specific lineages (for visualisation see http://marionilab.cruk.cam.ac.uk/organogenesis/). Closer inspection of specific clusters revealed that most exhibited additional, subtle sub-structure. We hypothesised that such sub-structure could shed light on early regulatory processes that drive fine-grained specification of cell fate. For example, between E8.0 and E9.0 the endoderm undergoes a series of morphogenetic changes that turn it from a flat sheet into a tube where the domains of major organs like the liver and lung arise6. While ventral folding and formation of the foregut pouch is already induced at E8.256, the earliest stages of foregut endoderm diversification remain ill-defined at the molecular level. To explore this further, we considered cells in the foregut cluster (Fig. 1C) and used a diffusion map approach7 to visualise three sub-clusters (Fig. 2A and Supplementary Fig. 2A). We then identified differentially expressed genes (Fig. 2B; Supplementary Table 3) and contrasted these with in situ images Alvimopan monohydrate from the literature to assign cluster identities. The red cluster expressed markers of early endodermal cells including and and and is a homeodomain transcription factor involved in the development of the forebrain and the pituitary gland14; in our data, it is restricted to the early endoderm cluster suggesting a possible role in regulating foregut development. Overall, our analysis illustrates how domain name specific knowledge can be used to allocate biological identity in the context of sparse scRNA-seq data. The molecular processes driving differentiation Alvimopan monohydrate cannot be readily studied in human embryos. This poses troubles for the validation of protocols that aim to produce authentic cell types from human induced pluripotent stem cells. We compared the transcriptome of human foregut progenitor cells C induced from human pluripotent stem cells (Methods) C to our mouse data. We used the reference that can be used to Alvimopan monohydrate assess the identity of derived cell populations. As a snapshot measure, scRNA-seq data seems ill-suited to recover dynamic information on cell fate specification. However, when entry into a defined differentiation program is usually desynchronised across a cell sub-population, dynamic information can be recovered through the chromatographic segregation of the molecular profile. Motivated by this, we focused on the process of somitogenesis, which involves the segmentation of the developing embryonic body axis into somites and is guided by oscillating genes, which create waves of expression that travel across the presomitic mesoderm (PSM) from posterior to anterior16 (Fig. 3A). Upon arrival of a wave at the PSM’s anterior end, Alvimopan monohydrate a new somite is usually formed. The posterior end of the PSM is usually marked by high levels of Wnt and FGF signalling while somites show high levels of retinoic acid (RA)16 (Fig. 3A). Open in a separate windows Fig. 3 Oscillating patterns of gene expression during somitogenesis can be inferred from scRNA-seq data.A) Schematic of mouse somitogenesis,.